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December

2016

Vol. 11 – N. 2

Acta Herpetologica ISSN 1827-9635

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Acta Herpetologica Acta Herpetologica è la rivista ufficiale della Societas Herpetologica Italica (S.H.I.), un’associazione scientifica che promuove la ricerca erpetologica di base e applicata, la divulgazione delle conoscenze e la protezione degli Anfibi e Rettili e dei loro habitat. Acta Herpetologica is the official journal of the Societas Herpetologica Italica (S.H.I.), a scientific association that promotes basic, applied, and conservation researches on Amphibians and Reptiles. Direttore responsabile (Editor): Sebastiano Salvidio, DISTAV – Università di Genova, C.so Europa 26, I-16132 Genova, Italia Redattori (Associate Editors): Aaron Bauer, Department of Biology, Villanova University, 800 Lancaster Avenue, Villanova, PA 19085, USA Adriana Bellati, Dipartimento di Scienze della Terra e dell’Ambiente Università degli Studi di Pavia, Italy Paolo Casale, Dept. of Biology and Biotechnologies “Charles, Darwin”, University of Rome “La Sapienza”, Viale dell’Università 32, I-00185 Roma, Italy Francesco Ficetola, Laboratoire d’Ecologie Alpine LECA, Université Grenoble-Alpes. F-38000 Grenoble, France Ernesto Filippi, via Aurelia 18, I-00040 Ariccia, Roma, Italy Uwe Fritz, Museum of Zoology, Senckenberg Dresden, A.B. Meyer Building, 01109 Dresden, Germany Fabio Maria Guarino, Dipartimento di Biologia, Università degli Studi di Napoli Federico II, Via Cinthia, Napoli, Italy Sandra Hochscheid, Stazione Zoologica Anton Dohrn, Villa Comunale 1, I-80121 Napoli, Italy Daniele Pellitteri-Rosa, Dipartimento di Scienze della Terra e dell’Ambiente, università degli Studi di Pavia, Italy Marco Sannolo, CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, da Universidade do Porto, e reside no Campus Agrário de Vairão, Vairão, Portugal Giovanni Scillitani, Dipartimento di Biologia, sezione di Biologia animale ed ambientale, Università degli studi Aldo Moro, Bari, Italy Rocco Tiberti, Parco Nazionale Gran Paradiso, Degioz 11, 1101 Valsavarenche, Aosta Consiglio direttivo S.H.I. (S.H.I. Council): Presidente onorario (Honorary President): Benedetto Lanza (Firenze) Presidente (President): Massimo Delfino (Torino) Vice Presidente (Vice-President): Roberto Sindaco (Torino) Segretario (Secretary): Dalila Giacobbe (Messina) Tesoriere (Treasurer): Stefano Vanni (Firenze) Consiglieri (Council members): Annarita Di Cerbo (Milano), Mario Lo Valvo (Palermo), Antonio Romano (Roma) Sito ufficiale S.H.I. (Official S.H.I. website): http://www.unipv.it/webshi Modalità di associazione Le nuove domande di associazione sono esaminate periodicamente dal Consiglio Direttivo; solo successivamente i nuovi soci riceveranno la comunicazione di accettazione con le modalità per regolarizzare l’iscrizione (ulteriori informazioni sul sito: http://www.unipv.it/webshi). La quota annuale di iscrizione alla S.H.I. è di € 35,00. I soci sono invitati a versare la quota di iscrizione sul conto corrente postale n. 62198205 intestato a: SHI Societas Herpetologica Italica. In alternativa è possibile effettuare un bonifico bancario sul Conto Corrente Postale: n. conto 62198205 intestatario: SHI Societas Herpetologica Italica IBAN: IT-54-K-07601-03200-000062198205. Membership The S.H.I. Council will examine periodically new applications to S.H.I.: if accepted, new Members will receive confirmation and payment information (for more information contact the official website: http:// www.unipv.it/webshi). Annual membership fee is € 35.00 (Euro). Payments are made on the postal account of SHI Societas Herpetologica Italica no. 62198205, or by bank transfer on postal account no. 62198205 IBAN: IT-54-K-07601-03200-000062198205 to SHI Societas Herpetologica Italica. Versione on-line: http://www.fupress.com/ah

Acta Herpetologica Vol. 11, n. 2 - December 2016

Firenze University Press

Referee list. In alphabetical order the scientists that have accepted to act as editorial board members of Acta Herpetologica vol. 11 (2016). Elenco dei revisori. In ordine alfabetico gli studiosi che hanno fatto parte del comitato editoriale di Acta Herpetologica vol. 11 (2016). Abdala Virginia, Altig Ronald, Andreone Franco, Andrews Robin, Avery Roger, Basile Marco, Bauer Aaron M., Bellati Adriana, Biancardi Carlo, Brusquetti Francisco, Carella Francesca, Carrettero Miguel A., Casale Paolo, Comas Mar, Cordero Gerardo, Costa Andrea, Correa Déciu Tadeu, Covaciu-Marcov Severus-Daniel, de Carvalho Thiago R., Delaugerre Michel-Jean, Ernst Raffael, Escoriza Daniel, Ficetola Francesco G., Filippi Ernesto, Fritz Uwe, Garey Michel, Gazzola Andrea, Gherghel Iulian, Grismer L. Lee, Guarino Fabio Maria, Hoschscheid Sandra, Krause Tobias, Lingnau Rodrigo, Lin Zhi-Hua, Manenti Raoul, Mangiacotti Marco, Marrone Federico, Mebs Dietrich, Lanfen Tom., Liwanag Heather, Macip-Ríos Rodrigo, Mebert Konrad, Meegaskumbura Madhava, Mihalca Andrei, Miranda Jivanildo P., Mollov Ivelin A., Moravec Jiří, Murphy Robert W., Ortega Zaida, Pasmans Frank, Pauwels Olivier S.G., Pearson Steven, Pellitteri-Rosa Daniele, Pérez-Cembranos Ana, Pokrant Felix, Razzetti Edoardo, Ribas Alexis, Rodriguez Ariel, Romano Antonio, Rovatsos Michail, Salvidio Sebastiano, Sannolo Marco, Santos Viviane G.T., Scrocchi Gustavo J., Stewart James, Russel Anthony P., Santoro Guillermo, Saviola Anthony J., Smith Geoffrey R., Scali Stefano, Schneeweiss Norbert, Schulte Ulrich, Scillitani Giovanni, Schmidt Benedikt, Sinsch Ulricht, Stuckas Heiko, Tiberti Rocco, Torres-Carvajal Omar, Tsuboi Masahito, Valenzuela Nicole, Vági Balázs, Vitt Laurie, Vogt Richard C., Walguarnery Justin, Warren Dan L., Zagar Anamarija, Zaher Hussam.

Acta Herpetologica 11(2): 101-109, 2016 DOI: 10.13128/Acta_Herpetol-18261

Predator-prey interactions between a recent invader, the Chinese sleeper (Perccottus glenii) and the European pond turtle (Emys orbicularis): a case study from Lithuania Vytautas Rakauskas1,*, Rūta Masiulytė1, Alma Pikūnienė2 1 Life Sciences Centre, Vilnius University, Saulėtekio Ave. 7, LT-10221 Vilnius, Lithuania. *Corresponding author. E-mail: vytucio@ gmail.com 2 Lithuanian Zoo, Radvilėnų str. 21, LT-50299 Kaunas, Lithuania

Submitted on May, 5th 2016; revised on June, 11th 2016; accepted on June, 12th 2016 Editor: Uwe Fritz

Abstract. The European pond turtle, Emys orbicularis, is a critically endangered species in most European countries. Habitat degradation and fragmentation are considered the main reasons for the decline of E. orbicularis. However, the spread of invasive species may also contribute to the disappearance of E.  orbicularis populations. We examined the range overlap and predator-prey interactions between the invasive Chinese sleeper, Perccottus glenii, and E. orbicularis through controlled experiments and in field studies. Field surveys showed that both species occupied similar habitats. Predator-prey experiments suggested that newly hatched turtles are resistant to P. glenii predation. Conversely, adults of E. orbicularis consumed juvenile P. glenii even when other food sources were available. Overall, these findings suggested that E. orbicularis is not among the potential prey organisms in the diet of the invasive P. glenii, and that this fish does not directly contribute to the decline of E. orbicularis in Europe. Keywords. Turtle, aquatic invasion, endangered species, inland waters, Lithuania.

INTRODUCTION

At present, there is no doubt that one of the main causes of the loss of biodiversity is the spread of introduced, invasive species. These species reduce local biodiversity through both indirect competitive and direct predatory impacts on resident native populations. The spread of invasive species is now considered to be an international problem, and local governments are actively working to reduce their spread and impacts (EU Regulation No 1143/2014). However, aquatic invasive species spread is on-going, and its impacts on local communities cannot be predicted (Sundseth, 2014). Understanding the pattern of invasive species interactions with native species can help us to predict those effects and build better strategies for protection of endangered species. From a ISSN 1827-9635 (print) ISSN 1827-9643 (online)

biodiversity management point of view, the impact of the invasive versatile predator Chinese sleeper, Perccottus glenii (Dybowski, 1877) on populations of the endangered native European pond turtle, Emys orbicularis (Linnaeus, 1758) is one such case in point. Perccottus glenii, a fish native to the Amur River basin, eastern Asia (Mori, 1936; Berg, 1949). This is one of the most widespread alien invasive freshwater fish in Eurasia (Reshetnikov, 2010; Reshetnikov and Ficetola, 2011). It is also a recent invader in the Baltic Sea region (Aleksejevs and Birzaks, 2011; Witkowski and Grabowska, 2012; Reshetnikov and Karyagina, 2015; Pupina et al., 2015; Rakauskas et al., 2016). In Europe P.  glenii occurs mostly in water bodies that either have weak current or are stagnant, with well developed aquatic vegetation. Such habitats include river flood plains, littoral zones © Firenze University Press www.fupress.com/ah

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of lakes, and swampy water bodies (Reshetnikov, 2010; Reshetnikov and Ficetola, 2011; Pupina et al., 2015). Shallow, well-vegetated and isolated lakes, ponds, drainage ditches and oxbows are important reserves of biological diversity for many groups of aquatic and semi-aquatic animals, including pond turtles. Colonisation of such water bodies by P. glenii leads to a dramatic simplification of ecosystem taxonomic structure. P.  glenii is a versatile predatory fish and represents a specific threat for macroinvertebrates and small fish and amphibians (Shlyapkin and Tikhonov, 2001; Reshetnikov, 2003; Reshetnikov, 2004; Koščo et al., 2008; Grabowska et al., 2009; Reshetnikov, 2013). Due to its generalist predatory habit, it has been assumed that P.  glenii preys upon hatchling E.  orbicularis turtles (Pupina et al., 2015). Adult turtles can deter predation by their size and bony shell, but hatchlings lack the adult’s size and shell strength. Populations of E. orbicularis hatchlings were assumed to be under P. glenii predation pressure until their first or second years. This paper addresses the predator-prey interactions between P.  glenii and locally endangered E.  orbicularis. Concerns about P.  glenii are based on observations that this fish (1) inhabits similar habitats as E.  orbicularis (Mitrus, 2004; Najbar, 2007; Briggs and Reshetylo, 2009; Reshetnikov, 2010; Reshetnikov and Ficetola, 2011); (2) is an opportunistic, competitive predator that rises to the top of the food chain in invaded ecosystems (Reshetnikov 2003; Reshetnikov, 2004; Reshetnikov, 2013); and (3) grows large enough to prey on newly hatched E.  orbicularis. This scenario, however, has not been well substantiated for interactions in European waters. To fill this knowledge gap, we performed predatorprey experiments between P. glenii and two aquatic turtle species. In laboratory experiments we tested the capacity of P. glenii to consume newly hatched Chinese pond turtles, Mauremys reevesii (Gray, 1831), when it is the only available prey. It was assumed that there would be no differences in P.  glenii feeding preferences for same-sized E.  orbicularis and M.  reevesii juveniles. Additionally, we tested the capacity of the adult E. orbicularis to consume 0+ age P.  glenii when several prey sources were available. As a complement to the laboratory studies, we analyzed the overlap of recent distributions of P.  glenii and E. orbicularis species in natural Lithuanian freshwaters. MATERIAL AND METHODS Study area Natural habitat overlap between P. glenii and E. orbicularis species was assessed for the inland waters of Lithuania. Lithua-

nia stands within the Baltic Sea drainage basin, along the southeastern shore of the Baltic Sea. The country has a territory of 64,800 km2, which is divided by seven main river basins (Kažys, 2013) (Fig. 1A). There are 2,850 lakes that have surface areas larger than 0.005 km2, and 3,150 smaller lakes, with a combined area of 913.6 km2. In addition, there are 1,132 reservoirs and more than 3,000 ponds in Lithuania (Kažys, 2013). Distribution of P. glenii and E. orbicularis The recent distribution of P.  glenii and E.  orbicularis in Lithuanian inland waters was calculated from widely published (Rakauskas et al., 2016) and specialty (Bastytė, 2015) publications. Additional data on the presence of P.  glenii within the local range of E.  orbicularis was provided by our own (to be published) surveys. The results of our surveys include data from 32 lentic water bodies, each with a surface area smaller than 0.3 km2 and each known to have been occupied by E. orbicularis (Fig. 1B). P. glenii populations were investigated by dip net (25 × 25 cm opening, 1.5 mm mesh size) sampling in water depths of 0.3-1.3 m in areas of the vegetated littoral zone (Pupina et al., 2015). From five to ten study sites were examined on each water body waving with a dip net for ten minutes at each study site. In general, if P. glenii was to be found in a body of water, it was caught within the first 5 minutes (Rakauskas pers. obs.). Experiment 1 (P. glenii vs. M. reevesii) Predator-prey experiments (P.  glenii vs. M.  reevesii) were conducted to determine if the invasive predator P. glenii recognize newly hatched aquatic turtles as potential prey, and if they consume them under experimental conditions. M.  reevesii was chosen as a model for E.  orbicularis as it is easy available, not protected, and is of similar size and coloration as E. orbicularis (Mitrus and Zemanek, 2000; Stephens and Wiens, 2003; Lovich et al., 2011; Lin et al., 2015). Previous studies have shown that E. orbicularis juveniles are similar length (approximate 26 mm) (Drobenkov, 2000; Mitrus and Zemanek, 2000; Zinenko, 2004; Najbar and Mitrus, 2013) when they first reach the water, as were the M.  reevesii juveniles used in these experiments (3.0 ± 0.1 mm). E. orbicularis is less available and is protected by European and local laws (Council Directive 92/43/EEC; Rašamavičius, 2007). Newly hatched (two week old) M.  reevesii were transported to the laboratory one week before the experiments from the local zoo-shop. The experiment turtles were maintained in twelve 10.6-L aquaria (22.5 cm deep and long × 21 cm wide) filled with tap water forming a closed circulation system with an ammonia filter (38.5 cm deep and wide × 49.0 cm long aquarium filled with expanded clay and JBL filter start (GmbH & Co, Germany)). During this period, daily rations of frozen midge larvae were provided. Adults of P.  glenii were collected from small lakes using electric fishing gear (Samus-725mp) in September 2015. For one week before conducting the experiments, all fish were accli-

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Predator-prey interactions of Perccottus glenii and Emys orbicularis mated separately in twelve 92.4-L rectangular tanks (49.0 cm deep and long × 38.5 cm wide) filled with tap water forming a closed circulation system with an ammonia filter. During this period, daily rations of live Rutilus rutilus (Linnaeus, 1758) fry were provided at ~4% of the fish’s body weight. The fish were starved for 48 h before the predation experiment. In total, 12 P.  glenii and M.  reevesii individuals were used in these experiments (Tables 1 and 2). Predator-prey experiments were performed in the same experimental aquaria where fish were acclimatised. Each experimental aquarium was filled with tap water to 44 cm depth resulting in an actual water volume of 83 L per aquarium, with water flow through each experimental aquarium of 3.0 L min-1. Animals were kept under a 15 hours per day photoperiod and at a temperature of 19 ºC throughout these studies. No substrate was added into the experimental aquaria. After acclimation period, a single individual of M.  reevesii was added to each aquarium. During the experiments, turtles were fed frozen midge larvae every second day. Overall, the fish were allowed to forage for one week, after which turtles were removed back to their acclimation aquaria and the conditions of all turtles in each experimental aquarium were recorded. Assessment of turtle condition was based on appearance and movement. Surviving turtles were crawling and no injuries were seen on their skin or carapace. These turtles were kept for three months after the experiments to confirm that they retained their normal viability and activity levels.

Experiment 2 (E. orbicularis vs. P. glenii) Predation experiments (E.  orbicularis vs. P.  glenii) were conducted to determine if E. orbicularis recognize P. glenii juveniles as prey and consumed them. Approximately 150 P.  glenii juveniles were collected from a small lake using a standard dip net (25 × 25 cm) in August 2015. All fish were small enough to be preyed upon by turtles and were selected for size similarity (body length of 3.3 ± 0.2 cm) to avoid cannibalism. For one week before conducting the experiments, all specimens were acclimated in six 92.4-L rectangular tanks (49.0 cm deep and long × 38.5 cm wide) filled with tap water forming a closed circulation system with an ammonia filter. Daily rations of live midge larvae were provided. Predator-prey experiments (E. orbicularis vs. P. glenii) were performed in August 2015. Three concrete water reservoirs were used for the experiments. Reservoirs were in cages under the natural outdoor conditions. Cages were made from stainless steel to protect the experiment from wild birds and animals. The first cage contained two identical reservoirs of approximate 5 m2 area (2.8 m long × 1.8 m wide, with a range of 5−45º slope and 0.1−0.6 m of water depth). The second cage contained one large rectangular reservoir of approximate 10 m2 area (4.5 m long × 2.3 m wide, with a range of 5-45º slope and 0.1-0.7 m of water depth). All reservoirs were filled with tap water. Woody shelters, stones and muddy gravel substrate were present in all experimental reservoirs. An average noonday temperature of

Table 1. Percottus glenii specimens used in predator-prey experiments. Values are means ± standard deviation.

Number of used specimens Range of total body length, cm Average of total body length, cm Range of total weight, g Average of total weight, g Range of P. glenii mouth diameter, cm Average of P. glenii mouth diameter, cm

Experiment I

Experiment II first cage

Experiment II second cage

12 17.0-24.7 20.3 ± 3.0 90.3-280.0 150.8 ± 62.1 3.0-4.8 3.7 ± 0.6

27 2.9-3.2 3.0 ± 0.2 0.9-1.5 1.2 ± 0.2 – –

33 3.3-3.7 3.5 ± 0.1 1.1-1.8 1.4 ± 0.2 – –

Table 2. Mauremys reevesii and Emys orbicularis specimens used in predator-prey experiments. Values are means ± standard deviation.

Number of specimens Range of carapace length, cm Average of carapace length, cm Range of carapace width, cm Average of carapace width, cm Range of plastron length, cm Average of plastron length, cm Range of total weight, g Average of total weight, g

Experiment I M. reevesii

Experiment II (1) E. orbicularis

Experiment II (2) E. orbicularis

12 2.9-3.1 3.0 ± 0.1 2.3-2.0 2.2 ± 0.1 2.7-2.9 2.8 ± 0.1 6.8-7.2 7.0 ± 0.1

10 9.5-12 10.8 ± 0.8 7.6-10.1 8.7 ± 0.7 7.8-11.5 9.8 ± 1.6 10.6-37.1 22.2 ± 9.8

8 15.2-18.1 16.9 ± 1.1 13.6-16.0 14.8 ± 0.9 12.0-18.5 15.5 ± 2.5 39.0-167.0 89.5 ± 42.6

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Table 3. Daily turtle diet rations of various food types per specimens during the experiments. Food type

quantity

Flesh fish Flesh meat Dried crustacean Beetle larvae (live) insects (cricket, cockroach) (live) Earthworm (live) Snail (live) Midge larvae (live) Cabbage

10 (g) 10 (g) 5 (ind.) 5 (ind.) 3 – 4 (ind.) 5 – 10 (ind.) 3 – 4 (ind.) 10 (ind.) 1 (g)

20.6 ± 3.2 ºC was observed. 18 E. orbicularis adults big enough to prey on these juveniles were divided, with ten smaller individuals closed in the first experimental cage and eight larger ones in the second (Table 2). After an acclimation period, 27 and 33 fish were transferred into the experimental reservoirs of the first and second cages respectively. In the first cage released individuals were equally divided (14 + 13) per each reservoir. Fish in the second cage were slightly bigger compare to the fish in the first one (Table 1). 60 fish were left as a control group in six acclimation tanks (ten individuals per tank) for the whole experiment period. Overall, E.  orbicularis were kept with P.  glenii juveniles for two weeks. The turtles and the fish were fed once a day with dried or live insects, earth worms, snails, and small pieces of meat during the experiments. Daily turtle food ratios are presented in Table 3. Moreover, there was lots of naturally breeding Culex pipiens (Linnaeus, 1758) larvae inside the reservoirs which were an alternative food source for the fish. The reservoirs were surveyed for dead fish every day during the experiments. After the experiment termination, reservoirs were pumped out, all remained fish were removed back to their acclimation aquaria and the condition of all fish in each reservoir was recorded. Assessment of fish condition was based on appearance and movement. Surviving fish were swimming and no external injuries were seen.

RESULTS

overlap of these two species. Both were found inhabiting one shallow water body (Fig. 1B). Four P. glenii individuals of body lengths ranging from 43 to 61 mm were captured in a water body inhabited by E.  orbicularis. Seven water bodies with well-known E.  orbicularis populations were occupied by Carassius carassius (Linnaeus, 1758). Experiment 1 (P. glenii vs. M. reevesii) Experiments showed that none of the tested P.  glenii adults were capable of ingesting or even injuring newly hatched M.  reevesii turtles, though tested fish appeared big enough to do so. The mouth diameter of tested fish was significantly larger than the carapace lengths of the turtles (Mann-Whitney U test: Z = 3.2, P < 0.002; Tables 1 and 2). However, only a few predation signs were observed during the first ten minutes after newly hatched turtles were offered for P. glenii predation. The largest tested fish specimens (TL > 22.0 cm and mouth diameter > 4.0 cm) repeatedly attacked turtles for the first few minutes. In these cases the prey was completely sucked in the fish mouth cavity and jaws were fully closed. However, after a few seconds the prey was expelled undamaged. Overall, none of the tested turtles were injured; all were alive and healthy three months after the experiments. Experiment 2 (E. orbicularis vs. P. glenii) Adult E.  orbicularis were found to consume P.  glenii juveniles in caged experiments. 15 individual P. glenii (55.6% of the cohort) were missing after 14 days in the first cage where smaller turtles were foraging. Similarly, 13 P.  glenii (39.4%) were missing in a cage with larger turtles. No dead or injured fishes were found during or after the experiment. Predatory behaviour was frequently observed. Turtles stalked and struck at P. glenii juveniles. Cannibalism was not observed among the fish, including in control tanks, where all 60 specimens left in tanks survived the experimental period.

Distribution of P. glenii and E. orbicularis In Lithuania less than 400 E.  orbicularis survive, mostly in waters of southern part of the country (Bastytė, 2015). Meanwhile P. glenii has been recorded in all of the country’s river basins (Rakauskas et al., 2016). Analysis of the recent distribution range of P. glenii and E. orbicularis in the inland waters showed these species overlapping on a regional scale. Both species were recently reported from the Nemunas River basin in southern Lithuania (Fig. 1A). Additionally, our surveys of P. glenii presence in well known E.  orbicularis habitats revealed the small-scale

DISCUSSION

The European pond turtle, E.  orbicularis is the most widely distributed freshwater turtle species in Europe (Fritz, 2001, 2003). The geographic range of E. orbicularis extends from North Africa over most of Europe to Latvia and to Lake Aral in the Middle East (Fritz, 1998; Fritz, 2001, 2003). However, the turtle is critically endangered in Lithuanian, and all other European waters (Council Directive 92/43/EEC; Fritz, 2001, 2003; Rašamavičius,

Predator-prey interactions of Perccottus glenii and Emys orbicularis

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Fig. 1. (A) Distribution of the European pond turtle, Emys orbicularis (squares) and the Chinese sleeper, Perccottus glenii (triangles) in the inland waters of Lithuania (followed by Bastytė, 2015; Rakauskas et al., 2016). Different river basins are marked by different shading. (B) P. glenii presence in water bodies inhabited by E. orbicularis: presence (opened circles), absence (closed circles).

2007). In Lithuania reproductive E.  orbicularis populations number less than 400 turtles and are found mostly in waters of the southern part of a country (Fritz and Günther, 1996; Meeske, 2008; Bastytė, 2015). Habitat degradation and fragmentation have been identified as the main reasons for the decline of E. orbicularis populations (Kovacs et al., 2004; Fritz and Chiari, 2013; Bastytė, 2015). However, the recent spread of invasive aquatic species may also negatively affect E.  orbicularis populations. The introduction of exotic Trachemys scripta (Schoepff, 1792) has been reported to have indirect effects on E.  orbicularis populations. Introduced T.  scripta competes against E.  orbicularis for habitat resources, and reduces the survival of the turtle (Cadi and Joly, 2004; Lacomba and Sancho, 2004). The invasive predatory fish Micropterus salmoides (Lacépède, 1802), is assumed to prey on E. orbicularis hatchlings and juveniles (Lacomba and Sancho, 2004; Ayres and Cordero, 2007). Correspondingly, direct and/ or indirect interactions between invasive predatory fish P.  glenii and E.  orbicularis could be expected. Both species prefer similar habitats and diets. However, the impact of P. glenii on the natural populations of E.  orbicularis in inland European waters remains largely undocumented. Habitat overlap Our review of the literature on P. glenii and E. orbicularis habitat preferences indicated that both species are commonly found in shallow waters with weak or with

no current, and with well-developed aquatic vegetation (Mitrus, 2004; Meeske et al. 2006; Najbar, 2007; Briggs and Reshetylo, 2009; Reshetnikov, 2010; Reshetnikov and Ficetola, 2011; Pupina et al., 2015). Both species usually forage in littoral zones of small shallow lakes or swampy water bodies. Both species are particularly abundant in small water bodies unsuitable for most ichthyophagous fishes. In these situations, they are the top predators (Meeske et al., 2006; Briggs and Reshetylo, 2009; Bastytė, 2015; Reshetnikov, 2003; Grabowska et al., 2009). Therefore these two species could be expected to compete against, and even attack each other in these environments. Our field surveys confirmed that P.  glenii and E.  orbicularis may settle in the same water bodies, although both species are relatively rare in the inland waters of Lithuania. The presence of both species in the same water body was also reported from Latvian inland waters (Pupina et al., 2015). However, we found P.  glenii only in one (3.1%) from all investigated water bodies settled by E. orbicularis. An increase in conflicts between these two species is expected in a future where both species expand their ranges within the inland waters of Lithuania. The rapid natural spread of P.  glenii is ongoing not only in Lithuanian (Rakauskas et al. 2016) but also in other European waters (Alexandrov et al., 2007; Grabowska et al., 2010; Mastitsky et al., 2010; Wolter and Röhr, 2010; Reshetnikov and Ficetola, 2011; Semenchenko et al., 2011; Movchan 2015). The fish pursues a profligate reproductive strategy, and exhibits resistance to adverse conditions

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(Reshetnikov, 2004). On the other hand, an intensive reestablishment program of E.  orbicularis populations is ongoing in Lithuanian waters. Up to 100 individuals are released annually to areas they formerly occupied (Pikūnienė unpub. data). Therefore, there is little doubt that direct and indirect interactions between these two species will increase. Predator-prey interactions In small lakes unsuitable for most ichthyophagous fish, P.  glenii can grow up to 25 cm in body length (Reshetnikov, 2003; Pupina et al., 2015) and become a top predator (Reshetnikov, 2003; Koščo et al., 2008; Grabowska et al., 2009). In such waters almost all links of the trophic network converge to P.  glenii. Although adult P.  glenii feed primarily on fish and large insects, they occasionally prey on newts, frogs or other larger animals (Shlyapkin and Tikhonov, 2001; Reshetnikov, 2003; Reshetnikov, 2004; Koščo et al., 2008; Grabowska et al., 2009; Reshetnikov, 2013). Due to this dietary breadth, it has been assumed that large P. glenii individuals are capable of ingesting and eating E. orbicularis hatchlings (Pupina et al., 2015). Newly hatched pond turtles are less than 3 cm in length (Najbar and Mitrus, 2013) and could be attacked by P. glenii, which has a wide gape (up to 5 cm) and forages in shallow waters. However, our experiments did not support the hypothesis that P.  glenii may directly, through the predator-prey interaction, threaten E. orbicularis populations. None of the tested specimens of P.  glenii consumed or injured newly hatched turtles, even though they were big enough to prey on them and even though no other food was available to them. Thus it was concluded that the presence of P. glenii would not directly threaten wild E. orbicularis populations. We used a different pond turtle species M.  reevesii instead of E.  orbicularis during the experiments. The applicability of our conclusions to E.  orbicularis is supported by the similarity of the behaviour of the two turtle species in the water and the similarities in their sizes and their camouflage (Mitrus and Zemanek, 2000; Stephens and Wiens, 2003; Lovich et al., 2011; Lin et al., 2015). Our results showed that only the largest P.  glenii specimens showed interest in preying on turtle juveniles. P.  glenii specimens shorter than 22 cm length did not even approach newly hatched turtles. We used P.  glenii specimens in a range of sizes, up to the largest found in European waters, 24.7 cm of a total body length (the biggest recorded specimens have been 25 cm in total body length; Reshetnikov, 2003; Pupina et al., 2015). Specimens over 20 cm were found only in 11 (14.6%) of a total 75

Vytautas Rakauskas et alii

lentic waters bodies inhabited by P.  glenii in Lithuania (Rakauskas unpub. data). Similar results were obtained from other countries where P. glenii was usually reported to live for about 5-7 years and reach up to 15 cm length (Grabowska et al., 2011; Nowak et al., 2008; Grabowska et al., 2009; Terlecki and Palka, 2012; Kutsokon et al., 2014). Our findings suggest that E.  orbicularis consumption by predatory P. glenii in natural environments where many of other food sources are available is not likely. Similar results were obtained with other pond turtle and predatory fish species. Britson and Gutzke (1993) revealed that although turtle hatchlings of T.  scripta and Chrysemys picta (Schneider, 1783) were attacked by predatory fish M.  salmoides they were subsequently rejected unharmed. It was concluded that hatchling behaviour such as clawing or biting may be harmful to the gill apparatus or digestive tract of fish and thus provides defence against predation (Britson and Gutzke, 1993). Our experiments revealed that adults of E. orbicularis preyed on P. glenii juveniles even when other food sources were available. Fish fry consumption by E.  orbicularis has been shown in other studies (Kotenko, 2000; Briggs and Reshetylo, 2009). No cannibalism cases were observed within a control fish group during our experiments, suggesting that the fish were consumed by the turtles. Our findings suggest that not only will P.  glenii not directly endanger European pond turtle populations, but that E.  orbicularis may even control P.  glenii populations where their ranges overlap. Concluding remarks The decline in European pond turtle populations likely results from a complex set of factors, linked to the modern decline in biodiversity worldwide. However, this study demonstrates that one factor is probably not the threat posed to these turtles by the invasive predatory fish P.  glenii. Conversely, we found that mature adults of E.  orbicularis can prey on P.  glenii juveniles. The turtle could possible come to control the invasive fish with the increase of the habitat overlap between the two species. P.  glenii may impact local E.  orbicularis populations indirectly, through depletion of available macroinvertebrate food sources in invaded water bodies (Reshetnikov, 2001; Reshetnikov, 2003). Benthic invertebrates usually dominate in both species diet (Lebboroni and Chelazzi, 1991; Kotenko, 2000; Ottonello et al., 2005; Ficetola and De Bernardi, 2006; Koščo et al., 2008; Grabowska et al., 2009). P.  glenii may also serve as a vector for E.  orbicularis parasite transfer (Pupina et al., 2015). The fish is a host for the E.  orbicularis parasitic nematode Spiroxys

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contortus (Rudolphi), whose paratenic hosts also include small fish, insect larvae, tadpoles and adult Anura (Hedrick, 1935, Moravec, 1994). Further investigation of the growth and physiology conditions of E.  orbicularis during the pre- and post-invasion periods of P.  glenii in various water bodies will be needed to sort out these multiple effects. ACKNOWLEDGEMENTS

We thank the Environmental Protection Agency of Lithuania, under the Ministry of Environment of the Republic of Lithuania, for the permits used to collect, breed and grow E.  orbicularis hatchlings and adults at the Lithuanian Zoo: permits No. (6)-A4-2084; LGF-1440; 032/2014. We also thank the Lithuanian Zoo for the permit to conduct predator-prey experiments within the Zoo: permit No. LZS-96/2015. The zoo shop “Zuvytes. com” is greatly appreciated for providing us with newly hatched M.  reevesii turtles. For the permission to catch P.  glenii we are also grateful to the Environmental Protection Agency under the Ministry of Environment of the Republic of Lithuania, permit No. 056/2015. Comments and suggestions of anonymous reviewers and Steve Daubert (University of California, Davis) greatly improved the manuscript. REFERENCES

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Acta Herpetologica 11(2): 111-118, 2016 DOI: 10.13128/Acta_Herpetol-18075

Effective thermoregulation in a newly established population of Podarcis siculus in Greece: a possible advantage for a successful invader Grigoris Kapsalas1, Ioanna Gavriilidi1, Chloe Adamopoulou2, Johannes Foufopoulos3, Panayiotis Pafilis1,* 1 Section of Zoology and Marine Biology, Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, GR-15784, Greece. *Corresponding author. E-mail: [email protected] 2 Zoological Museum, Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, GR-15784, Greece 3 School of Natural Resources and Environment, University of Michigan, Ann Arbor, MI 48109, USA

Submitted on 2016, 3rd March; revised on 2016, 15th June; accepted on 2016, 21th June Editor: Daniele Pellitteri-Rosa

Abstract. Temperature affects all aspects of reptilian biology. In order to colonize new habitats and support viable populations lizards have to successfully deal with their thermal environment. Podarcis siculus is a notorious example of a successful colonizer that has invaded numerous habitats outside its natural distribution range. Though certain features of its thermal biology have been assessed so far, the thermoregulatory abilities of the species remain poorly described. Here we investigated a recently discovered population in Greece and evaluated the effectiveness of thermoregulation measuring three main thermal parameters: set-point range, operative and field body temperatures. The Greek P. siculus appear to be accurate, precise and effective thermoregulators achieving E = 0.96. This effective thermoregulation may be used to explain, among other special characteristics, its spreading success. Keywords. Temperature, thermoregulation, invasive species, Italian wall lizard, Greece.

INTRODUCTION

Thermoregulation is crucial in ectotherms, shaping all features of their overall biology (Bartholomew, 1982). In small reptiles like lizards, thermoregulation is most times achieved behaviorally through appropriate movements between warmer and cooler microhabitat sites, shade and sun (Avery, 1982; Stevenson, 1985). Systematic research of reptilian thermal biology dates back to the mid-1940s (Cowles and Bogert, 1944). In 1976, Huey and Slatkin introduced a concise and detailed model to evaluate thermoregulation in ectotherms. This paradigm remained in use for the next two decades until Hertz and his partners (1993) fundamentally changed the way thermoregulation was perceived and proposed a thorough research protocol to answer a question of paramount importance: how effectively do lizards thermoregulate? ISSN 1827-9635 (print) ISSN 1827-9643 (online)

In order to answer this question, Hertz et al. (1993) took into account three main parameters: body temperatures (Tb, the temperature that animals achieve in the field), operative temperatures (Te, the temperature that a non-regulating animal would achieve in the field, measured with the use of models), and the species’ setpoint range (Tset, the temperature that animals select in a laboratory setting in the absence of any ecological constraints). Therefore Tb can be viewed as the result of the species’ mean Tset, which is considered a thermal utopia under ideal conditions (Sagonas et al., 2013a) and its interactions with a biotope’s Te. By considering these variables together, we are able to assess the effectiveness, accuracy and precision of thermoregulation of any given species (Hertz et al., 1993). The Italian Wall Lizard Podarcis siculus (RafinesqueSchmaltz, 1810) (Sauria, Lacertidae) is definitely not any © Firenze University Press www.fupress.com/ah

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given species. It is a small-bodied (snout-vent length, SVL, up to 90 mm; Corti and Lo Cascio, 2002), diurnal, heliothermic lacertid that feeds mainly on terrestrial invertebrates (Corti, 2006), although its diet may include unusual food resources such as rodents, geckos and even conspecifics (Capula and Aloise, 2011; Grano et al., 2011), revealing a flexible and opportunistic feeder (Zuffi and Giannelli, 2013), as most lacertids are (Scali et al., 2015). A native lizard of the Italian peninsula and north Adriatic coasts, P. siculus has recently expanded its distribution in many other Mediterranean countries by establishing numerous new populations (Crnobrnja Isailovic et al., 2009). Its excellent dispersal abilities (Vignoli et al., 2012) are underscored by the fact that it is the only Podarcis lizard that can be found in four continents: Europe, Asia, North America and Africa (Arnold and Ovenden, 2002; Kolbe et al., 2013; Tok et al., 2015). As such, P. siculus has been widely used as a model organism in numerous ecological, physiological, behavioral and phylogenetic studies (Fulgione et al., 2004; Podnar et al., 2005; Bonacci et al., 2008; Biaggini et al., 2009; Vervust et al., 2010; Raia et al., 2010). Several aspects of its thermal biology have been also studied (Avery, 1978; Ouboter, 1981; Van Damme et al., 1990; Tosini et al., 1992, 1996). However, its thermoregulation effectiveness (sensu Hertz et al., 1993) remains undetermined. In this study, we worked with a new, recently established population in Greece (Adamopoulou, 2015). We focused on the thermoregulatory capacity of P. siculus by measuring the three main thermal features, that is Tb, Te, and mean Tset. Furthermore, we compared the published data on set-point range temperatures from different sites within the species range. We hypothesized that since P. siculus is a highly successful colonizer capable of adapting easily to new habitats, it should achieve effective thermoregulation. MATERIALS AND METHODS The study site (Palaio Faliro) is located along the back side of a sandy beach in the Athens, Greece (37o55’9.38”N, 23o42’0.50”E). It is a heavily modified habitat planted with oleanders, tamarisks and yuccas delimited by a crowded beach on the front and a highway avenue on the back. The only other reptile recorded on site was the Ocellated skink (Chalcides ocellatus). The site represents the only known location of P. siculus in Greece (Adamopoulou, 2015), which probably originates from the Adriatic region (Silva-Rocha et al., 2014). In May 2015 we measured the operative temperatures of the site using 31 hollow, electroformed copper models that mimic the size, shape and reflectance of the species and were validated against live animals in the field (Bakken, 1992; Dzialowski, 2005). To simulate the heat capacity of the lizards, we

added 2.5-3 ml of water into each model, both ends of which were sealed with plasticine, except for a narrow opening where the probe of a data-logger (HOBO U12 4-Channel External Data Logger - U12-008) was inserted (Diaz, 1997; Grbac and Bauwens, 2001). The models were placed on various spots on site so as to cover all types of microhabitats available to lizards (Huey, 1991). We recorded Te every 15 minutes for two consecutive days (09:00-19:00). To ensure that the temperature responses between the copper models and the study animals were similar, we tested their cooling and heating rate (Lutterschmidt and Reinert, 2012). An adult male lizard and a copper model were placed side by side near a heating source (two 140 W lamps) for 45 minutes. Subsequently, the heating source was turned off and the subjects were left to cool down for 45 minutes, resulting in a total period of 90 minutes. During this period we recorded the temperature of the model and the lizard every five minutes with a Weber quick reading cloacal thermometer. A linear regression of Tb on Te suggested that there was a good fit between the model and the animal responses (regression statistics ± SE; slope = 1.305 ± 0.118, intercept = -7.290 ± 3.557, r2 = 0.928, P < 0.001). The body temperatures (Tb) of 30 individuals were measured on site during the same dates that Tes were sampled. Lizards were caught by hand or noose from all occupied microhabitats and their temperature was measured within 20 seconds using a quick-reading cloacal thermometer (T-4000, Miller & Weber, Inc., Queens, NY) to the nearest 0.1 °C. For every lizard caught we also measured SVL with digital calipers (Silverline 380244, accurate to 0.01 mm) while sex was determined by inspection of the femoral pores. Finally, 11 adult males (since sampling took place within the reproductive period we tried to avoid the impact of sex on set-point range temperatures; Carneiro et al., 2015) were transferred to the laboratory facilities of the Department of Biology at the University of Athens to measure Tset. The lizards were placed in a specially designed terrarium (100 x 25 x 25 cm) where a thermal gradient (10-60 °C) was achieved with the use of two incandescent heating lamps (100 W and 60 W) at one end and two ice bags on the opposite (Van Damme et al., 1991). Subsequently their body temperature was measured every 30 min for four consecutive hours (Castilla and Bauwens, 1991) using a quick-reading cloacal thermometer (T-4000, Miller & Weber, Inc., Queens, NY). We used the inter-quartile range of the body temperatures selected by lizards in the thermal gradient (Hertz et al., 1993). To evaluate the effectiveness of thermoregulation we used − − − the formula: Ε = 1 - (db / de ), where db represents the mean − deviation of field Tbs from mean Tset and de provides a measure of thermoregulation accuracy and the mean deviation of Tes from mean Tset, (Hertz et al., 1993). Mean db provides an index − of the accuracy of thermoregulation whereas de sketches out the thermal quality of the habitat. E may range from zero (perfect thermoconformers) to one (perfect thermoregulators). Complementary to the classical evaluation of the thermoregulatory effectiveness (Hertz et al., 1993), we also employed an alternative approach that quantifies the extent of departure from perfect thermoconformity (Blouin-Demers and Weatherhead, 2001). In the latter, positive values of the differ-

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Thermoregulatory effectiveness of Podarcis siculus − − ence between de and db describe thermoregulation, zero represents animals demonstrating perfect thermoconformity, and negative values describe animals avoiding habitats of high ther− − mal quality. The magnitude of the difference (de - db) provides an index of the thermoregulatory effectiveness (Blouin-Demers and Weatherhead, 2001).

RESULTS

Our results (male SVL = 74.3 ± 3.5 mm, n = 9; female SVL = 65.3 ± 7.0 mm, n = 20; t-test, t = -3.650, df = 27, P = 0.001) corroborated the typical pattern of sexual body size differences according to which male P. siculus are larger than females (Henle and Klaver, 1986). Males did not differ from females in their Tb (t-test, t = 0.758, df = 28, P = 0.455) and achieved similar body temperatures in the field (mean Tb for males = 33.2, mean Tb for females = 32.6). The mean value for the pooled Tb data was 32.8°C (Table 1). The majority of Tb fell within the spectrum of mean Tset while the diel variation of body temperatures was limited (Fig. 1). Operative

Table 1. The thermal variables used in this study: operative temperatures (Te), body temperatures in the field (Tb), set-point range (Tset), deviation of Te from Tset (de) and deviation of Tb from Tset (db). Variable

n

Te de Tb db Tset

31 31 30 30 11

Mean (°C) Range (°C) 31.7 3.2 32.8 0.1 33.8

19.5 – 53.4 0 – 16.4 26.8 – 35.8 0 – 3.2 30.0 – 37.0

SD

SE

7.10 3.30 1.94 0.59 2.25

0.22 0.10 0.35 0.11 0.30

temperatures ranged from 19.5 °C at 09:00 h (minimum) to 53.4 °C at 14:15 (maximum) and mean Te was 31.7 °C (Table 1, Fig. 1). Tset values ranged from 30.0°C to 37.0 °C (Table 1). Lizards achieved a mean set-point temperature of 33.8 °C (Table 1). The mean deviation of Tb from mean Tset was 0.1 °C while the mean deviation of Te from mean Tset was 3.2 °C (Table 1). These values were used to estimate the effectiveness of thermoregulation (sensu Hertz et al., 1993), that was E = 0.96. This value indicates that P. siculus is an active thermoregulator, as expected from the closeness of body temperatures to mean Tset. The complementary approach we used (Blouin-Demers and Weatherhead, 2001), also revealed high thermoregulatory effectiveness (d−e - d−b = 3.1) DISCUSSION

Fig. 1. Distribution of the mean body temperature in the field (Tb) and the mean operative temperature (Te). The shaded area indicates the set-point range (Tset) measured in the laboratory.

This study provides a comprehensive analysis of the thermoregulation effectiveness of the Italian wall lizard. In line with our first hypothesis, P. siculus proved to be an effective thermoregulator and able to achieve body temperatures within mean Tset. Body temperatures that lizards achieve in the field vary considerably among different biotopes (Avery, 1978; Van Damme et al., 1990; Tosini et al., 1992), in accordance with our second prediction. Operative temperatures at the study site revealed – at least during the study period – a benign habitat lacking extreme temperatures (Table 1). Nonetheless, the site offers the lizards the required thermal heterogeneity for behavioral thermoregulation. Mean de received a low value (3.2), indicating the high thermal quality of the habitat. In other words, lizards can easily achieve body temperatures within mean Tset. Set-point range temperatures are critically important in reptiles as they determine the optimal overall performance of the animal (Clusella-Trullas et al., 2007). Many factors, such as season, sex, age, reproductive status, body size and habitat may affect Tset (Andrews et al., 1999; Car-

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Table 2. Studies on mean set-point range (Tset) and effectiveness of thermoregulation (E) in Podarcis lizards. Species P. siculus P. liolepis P. milensis P. lilfordi P. gaigeae P. levendis P. muralis P. peloponnesiacus P. erhardii P. melisellensis P. tiliguerta P. bocagei P. tauricus P. vaucheri P. hispanicus P. hispanicus

Location

E

Tset

Reference

Athens, Greece Columbretes Island, Spain Milos Island, Greece Menorca Island, Spain Skyros Island, Greece Pori Island, Greece Cres, Croatia Peloponnese, Greece Andros Island, Greece Cres, Croatia Corsica, France Spain Peloponnese, Greece Ketama, Morocco Bellaterra, Spain Galera, Spain

0.96 0.95 0.95 0.88 0.87 0.84 0.81 0.76 0.66 0.63

33.8 34.2 33.4 35 33.7 33.9 31.9 34 35.1 33.5 35.47 35.15 33.8 33.43 33.07 31.65

This study Bauwens et al., 1996 Adamopoulou and Valakos, 2005 Ortega et al., 2014 Sagonas et al., 2013a Lymberakis et al., 2015 Grbac and Bauwens, 2001 Pafilis, 2003 Pafilis, 2003 Grbac and Bauwens, 2001 Van Damme et al., 1990 Bauwens et al., 1995 Pafilis, 2003 Verissimo and Carretero, 2009 Carretero et al., 2006 Carretero, 2012

retero et al., 2005; Carretero, 2008; Veríssimo and Carretero, 2009; Sagonas et al., 2013 a, b). The mean Tset for P. siculus was 1°C lower than the only other value reported in literature (Avery 1978) and falls within the upper percentile of the thermal range of other Podarcis species (Table 2). To the best of our knowledge there are three published studies that present data on the set-point range and body temperatures of P. siculus (Avery, 1978; Van Damme et al., 1990; Tosini et al., 1992). The Greek population achieved higher Tbs in the field than lizards in Pisa (32.04 °C, Tosini et al., 1992) but lower compared to the populations from Tuscany (35.16 °C, Avery, 1978) and Corsica (33.89 °C, Van Damme et al., 1990). In regard to set-point range temperatures the mean Tset in the Italian population is approximately 1 °C higher (34.79 °C, Avery, 1978) than the one estimated in the present study. An interesting finding was that Tbs did not vary considerably during the day, contrary to other Podarcis lizards (e.g., Adamopoulou and Valakos, 2005; Sagonas et al., 2013a), a feature that was also reported by previous studies (Van Damme et al., 1990; Tosini et al., 1992). The limited range of Tbs is an indication of high precision in thermoregulation (Hertz et al., 1993). As predicted by the low d−e that was mentioned above, an impressive 93% of all Tbs fell within the measured range of mean Tset. This corresponded to a very low mean db (0.1), which suggests high thermoregulation accuracy (Hertz et al., 1993). Thus, P. siculus appears to be not only a precise thermoregulator (Van Damme et al., 1990; Tosini et al., 1992) but also an accurate one.

The index of thermoregulation effectiveness took a high value (E = 0.96), which is among the highest that have been reported so far, not only for Podarcis lizards (Table 2) but among all lacertids. When animals are unable to thermoregulate, E will approach zero, whereas when they can successfully regulate their body temperature, E will be close to one (Herzt et al., 1993). According to our results, P. siculus, at least in the study ecosystem, is actively thermoregulating with great success. This is directly related to the small deviation of Tbs from mean Tset. At this point we have to stress out that the evaluation of E was based on mean Tset that was calculated from exclusively male individuals and also that a limited number of Tbs were obtained in a short period of time. In order to invade new ecosystems and establish viable populations, animals have to fullfill certain requirements (Kolar and Lodge, 2001). Ectotherms need to furthermore meet additional thermal demands: they have to adapt swiftly to the environmental temperatures of the new biotope while, at the same time, perform close to optimal levels (Kraus, 2009). Thermal specialists, which achieve optimal performance in a narrow range of temperatures, are less apt to invade new habitats than thermal generalists (Angilletta et al., 2003; Angilletta, 2009). The Italian wall lizard is a successful colonizer thanks to the quick acclimatization and adaptability to the environmental conditions of the new “home” and to the numerous ways of dispersal (Deichsel et al., 2010; Valdeón et al., 2010; Silva-Rocha et al., 2012, 2014). Our findings suggest that P. siculus is able to effectively, accurately and precisely regulate its body temperature. Thanks to this

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Thermoregulatory effectiveness of Podarcis siculus

effective thermoregulation, it may overcome the thermal challenges of new environments. As P. siculus settles new populations, there is strong evidence that it competes with native lacertids. Downes and Bauwens (2002) found that P. siculus is more aggressive and dominant than Podarcis melisellensis and occupies better thermal microhabitats. In laboratory experiments, P. siculus was also more aggressive and eventually suppressed activity levels in Podarcis tiliguerta (Vanhooydonck et al., 2000). Furthermore, P. siculus hybridizes with other endemic Podarcis lizards (Capula, 1993; Capula, 2002; Capula et al., 2002). The above considerations highlight the risk that P. siculus posses for autochthonous species, above all for Greece, which is home to seven endemic lacertids (Valakos et al., 2008). Since P. siculus has become established in Athens, it is only matter of time before it invades places that host endemic lizards (e.g., Peloponnese, Milos, Skyros, Crete). To eliminate the danger of further dispersal and the concomitant negative effects, the investigated Athens population of P. siculus needs to be exterminated. The Hellenic Herpetological Society inaugurated an eradication project in spring 2015. More than 150 individuals have been captured so far indicating that the initially estimated 50-60 animals (Adamopoulou, 2015) have multiplied to a much larger actual population in only a couple of years. This study, in spite of sampling flaws in field and lab temperature measurements, paves the way for delineating the particular features of ectotherms, which, like P. siculus, rapidly expand their distribution. Further research that will assess the thermoregulation pattern of P. siculus in diverse habitats throughout its range is badly required. Effectiveness of thermoregulation and mean Tset should be the focal points. Thereby it will be clarified whether the successful dispersal of the species is due to a standardized, conservative thermal pattern (the “static” view, Bogert, 1949) or just a response to environmental factors (the “labile” view, Huey, 1982). If E will receive equally high values and mean Tset will be similar to the studied population, then P. siculus will be indeed an effective thermoregulator with a conservative thermal physiology. Our results are the first step to this end. ACKNOWLEDGEMENTS

We are grateful to Natalia Gourgouliani, Ana Pereira, Kostantina Mitsi and Stratos Kafentzis for their valuable help with fieldwork. All work was conducted with permission from the Hellenic Ministry for Environment and Energy (permit code 61ΣΜ465ΦΘΗ-ΕΣ6). 

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Acta Herpetologica 11(2): 119-125, 2016 DOI: 10.13128/Acta_Herpetol-18009

The unexpectedly dull tadpole of Madagascar’s largest frog, Mantidactylus guttulatus Arne Schulze1,*, Roger-Daniel Randrianiaina2,3, Bina Perl3, Frank Glaw4, Miguel Vences3 1 Hessisches Landesmuseum Darmstadt (HLMD), Friedensplatz 1, 64283 Darmstadt, Germany. *Corresponding author. E-mail: arne. [email protected] 2 Département de Biologie Animale, Université d’Antananarivo, BP 906, Antananarivo 101, Madagascar 3 Division of Evolutionary Biology, Zoological Institute, Technical University of Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany 4 Zoologische Staatssammlung München (ZSM-SNSB), Münchhausenstr. 21, 81247 München, Germany

Submitted on 2016, 11th February; revised on 2016, 13th April; accepted on 2016, 9th July Editor: Marco Sannolo

Abstract. The Madagascar-endemic mantellid genus Mantidactylus contains one subclade with two described frog species characterized by very large body sizes. This subclade is classified as the subgenus Mantidactylus and is widespread in eastern and northern Madagascar, but their reproductive biology and larval stages are still unknown. We here provide a detailed description of the larvae of one species in this subgenus, M. guttulatus, on the basis of genetic assignment (16S DNA barcoding). The tadpoles were collected in the dry season from shallow waters near a stream in the Mahajanga Province in northwestern Madagascar. Their body and tail shape is remarkably generalized as typical for stream-adapted tadpoles, and the oral disc and labial keratodont row formula (4(2-4)/3(1)) are similar to those of other lotic mantellid frog larvae with generalized mouthparts like those in the subgenus Brygoomantis. The well-separated positions of these subgenera in the mantellid phylogeny suggest extensive homoplasy in the evolution of larval mouthpart morphology within Mantidactylus. Keywords. Amphibia, Madagascar, Mantellidae, Mantidactylus, generalized oral disc, tadpole morphology.

INTRODUCTION

Among Madagascar’s native frogs, the family Mantellidae is the most diverse clade with 212 named species (Amphibiaweb, 2016) and numerous undescribed species (Vieites et al., 2009; Perl et al., 2014). Mantellids are endemic to Madagascar and the Comoros and include a fascinating diversity in ecomorphology and reproductive modes. The largest mantellids are classified in a wellsupported subclade of the genus Mantidactylus (i.e., in the nominal subgenus Mantidactylus): Mantidactylus guttulatus, M. grandideri, and the candidate species M. sp. aff. grandideri “North”, although their alpha-taxonomy is in need of revision (see comments under Materials and ISSN 1827-9635 (print) ISSN 1827-9643 (online)

Methods). With up to 120 mm snout-vent length M. guttulatus is the largest frog in Madagascar and is common in rainforest streams of the northern and eastern part of the island (Glaw and Vences, 2007). Despite their size and local abundance, information on the reproduction of this frog species is scarce and basically limited to one report of a calling specimen (Vences et al., 2004). Because for decades no tadpoles could be assigned to Mantidactylus guttulatus or its close relatives, it was assumed that these species lack a larval phase or that the pre-metamorphic tadpoles develop in a nesting burrow (Glaw and Vences, 1994, 2007). Altig and McDiarmid (2006) described a tadpole with reduced oral structures from the Ranomafana region and tentatively © Firenze University Press www.fupress.com/ah

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assigned it as belonging to M. guttulatus. Randrianiaina et al. (2011) provided molecular evidence for an assignment of these tadpoles to M. majori whose juveniles are morphologically similar to those of M. guttulatus. During a survey in northwestern Madagascar, we obtained a small series of three Mantidactylus tadpoles that we initially identified by morphology as belonging into the subgenus Brygoomantis, despite being more elongated than other, syntopic Brygoomantis larvae. Molecular evidence demonstrated that these tadpoles instead belonged to Mantidactylus guttulatus, and we provide a detailed description of their morphology. MATERIALS AND METHODS Three tadpoles (field numbers ZCMV 13332, 13333 and 13334) were collected by R.D. Randrianiaina, F.M. Ratsoavina, A.S. Rasamison, A. Rakotoarison, D.R. Vieites, and M. Vences in the dry season on 29 June 2010. They were found in an opportunistic encounter survey near a large stream close to the Analamisondrotra mobile phone pylon, between 56-57 km along the national road N°31 from Bealanana to Antsohihy (14.72602°S, 048.55497°E; 1175 m a.s.l.) in the Mahajanga Province. Tadpoles were euthanized in a chlorobutanol solution shortly after collection. A tissue sample from the first third of the tail musculature of each tadpole was preserved in 99% ethanol. After tissue sampling, all specimens were preserved in 5% formalin and two of them were deposited in the Zoologische Staatssammlung München, Germany (ZSM; collection numbers ZSM 704/2010, ZSM 705/2010). Tadpoles were identified by DNA barcoding based on a fragment of the mitochondrial 16S rRNA gene (Thomas et al., 2005). The fragment of about 550 bp was amplified with primers 16Sar-L and 16Sbr-H from Palumbi et al. (1991) and standard protocols resolved on automated sequencers were compared to a nearly complete database of sequences of adult Malagasy frog species. DNA sequences were deposited in GenBank (accession numbers KX023902, KX023903, KX023904). For species names in the subgenus Mantidactylus, we here follow the taxonomy suggested by Glaw and Vences (2007) who defined M. guttulatus as the species with a rather tubercular dorsum occurring mostly in northern Madagascar, and M. grandidieri as the species with smooth skin widespread in the southern and central east of the island. This differs from the definition of Altig and McDiarmid (2006) who applied the name M. guttulatus to populations from the southern central east. However, it is obvious that this taxonomy is in need of revision and it is likely that the available names (Rana guttulata Boulenger, 1881; Mantidactylus grandidieri Mocquard, 1895; and Rana pigra Mocquard, 1900, currently a synonym of M. guttulatus) will have to be applied in a different way to the biological entities in the subgenus than in current practice. In fact, the tadpoles described herein might turn out to belong to a yet undescribed species. Independent from this taxonomic conundrum, however, the subgenus Mantidactylus is well defined and

the molecular data leave no doubt that the tadpoles described herein belong into this clade. A Canon DSLR with 100 mm 2.8L and MP-E 65 mm lenses mounted on an electronic-driven macro rail was used to obtain the digital images of the preserved specimens. A stack of 10-15 images was taken and merged with Helicon Pro software to achieve images with a wide depth of focus. Morphological descriptions and measurements were done on the basis of digital and scaled images of preserved tadpoles. Terminology of morphological characters follows Altig and McDiarmid (1999). Gosner’s (1960) classification was used to identify developmental stages. Structures of the oral apparatus were described according to Altig (1970), except for the term “keratodont,” which is used for the keratinized structures on the labia of the oral disc and presented as the labial keratodont row formula (LKRF). Marginal papillae are considered separately for the region of the upper labium, the lateral region, and the region of the lower labium and the “marginal papillae row formula” (MPRF) is provided according to Schulze et al. (2015). All morphological landmarks and distances considered for the description are described and specified in Table 1. Comparing measurements, we consider them as “almost equal” if ratios of the measured values are 95-96% or 104-105%, “equal” if they are in the range 97-103%, as almost “in the middle” if they are in the range 45-46% or 54-55% and “in the middle” if they are in the range 47-53% (Randrianiaina et al., 2011).

RESULTS

Three tadpoles identified by 16S DNA barcoding as Mantidactylus guttulatus were collected within rainforest and close (ca. 20 m) to a large stream of 25 m width. The tadpoles were in a seepage area of a small, very shallow puddle (1-2 cm deep) with a slow steady flow of water. The 16S rDNA sequences of these tadpoles were 99% identical to a reference sequence of an adult M. guttulatus from the Tsaratanana Massif (GenBank accession no. FJ559237). The following description refers to one of these tadpoles in Gosner stage 26 (field number ZCMV 13332 / ZSM 704/2010, body length 9.5 mm, tail length 30.7 mm; Figs. 1, 2; Tab. 1). In dorsal view body elliptical, maximal body width attained almost at mid-body length, snout narrowly rounded. In lateral view, body depressed, maximal body height attained between the 3/5 and 4/5 of the body length, snout rounded. Eyes moderately large, not visible from ventral view, positioned high laterally and directed anterolaterally, situated between the 2/10 and 3/10 of the body length. Distance between eyes wide. Nares rounded and small, marked with a marginal rim, positioned moderately high dorsolaterally and directed anterolaterally, situated closer to snout than to eye and lower than eye. Distance between nares wide. Spiracle sinistrally positioned and short, directed posteriorly, visible from ventral view, invisible from dorsal

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Table 1. Measurements of landmarks (in mm) and their ratios (in %) of the preserved tadpole specimen of Mantidactylus guttulatus (ZCMV 13332 / ZSM 704/2010) at Gosner stage 26: A1 = first upper keratodont row; BH = maximal body height; BL = body length; BW = maximal body width; DF = dorsal fin height at region of mid-tail; DG = size of the gap of marginal papillae in the region of the upper labium; DMTH = distance of maximal tail height from the tail-body junction; ED = eye diameter; HAB = height of the point where the axis of the tail myotomes contacts the body, measured from the lower curve of the belly; IND = inter-narial distance, measured from the centre of the eyes; IOD = inter-orbital distance; JW = maximal width of keratinized upper jaw sheath; MTH = maximal tail height; NH = naris height, measured from the lower curve of the belly to the centre of the naris; NP = naris-pupil distance; ODW = maximum width of opened oral disc; RN = rostro-narial distance, measured from the centre of the nares; SBH = distance between snout and the point of maximal body height; SBW = distance between snout and the point of maximal body width; SE = snout-eye distance, measured to the centre of the pupil; SH = spiracle height; SL = spiracle length, measured from its visible edges; SS = snout-spiracle distance, measured from the centre of the spiracle opening; TAL = tail length, measured from medium point of body-tail junction; TH = tail height at the body-tail junction; THM = tail height at mid-tail; TL = total length; TMH = tail muscle height at the body-tail junction; TMHM = tail muscle height at mid-tail; TMW = tail muscle width at the body-tail junction; VF = ventral fin height at mid-tail. Landmarks

mm

Ratio

%

BH BL BW DF DG DMTH ED EH HAB IND IOD JW MTH NH NP ODW RN SBH SBW SE SH SL SS TAL TH THM TL TMH TMHM TMW VF

4.1 9.5 4.8 0.9 1.4 7.5 1.0 2.1 2.8 2.3 3.4 1.2 4.8 1.9 1.7 2.5 0.8 6.5 5.4 2.4 1.4 0.8 5.0 20.3 3.6 4.8 30.7 2.6 2.9 2.6 1.1

SBW - BL BW - BH SBW - BL ED - BL SE - BL IOD - BW ND - BL NH - BH RN - NP IND - IOD SL - BL SS - BL SH - BH SH - HAB TAL - BL MTH - BH THM - BH THM - MTH TH - BH TMW - BW TMH - BH - MTH TMHM - THM and MTH DF - TMHM VF - TMHM DF - VF DMTH - TAL HAB - BH ODW - BW DG - ODW A1 - ODW JW - ODW

57 117 57 11 25 71 3 46 47 67 8 53 34 50 213 117 117 100 88 54 63 54 60 31 38 82 37 68 52 56 83 48

view and perceptible from lateral view; posterior third of inner wall free from body and formed that aperture is lateroposteriorly directed, its opening rounded, narrower than tube, situated between the 2/5 and 3/5 of the body length, located low on the body at the height of the hind

limb insertion. Long medial vent tube with dextral wall shorter than sinistral, causing a dextral directed opening, fully attached to ventral fin. Glands absent. Tail long, maximal tail height higher than body height, tail height at mid-tail higher than body height and as high as maxi-

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Fig. 1. Images of the living tadpole specimen of Mantidactylus guttulatus (ZCMV 13332 / ZSM 704/2010) at Gosner stage 26; a-c), in dorsal, lateral and ventral view (scale bar = 10 mm).

mal tail height, tail height at body-tail junction lower than body height. Caudal musculature well developed. Tail muscle reaches tail tip. Tail fins very low, dorsal fin slightly lower than ventral fin at mid-tail, but slightly higher in posterior third. Dorsal fin originates slightly behind dorsal body-tail junction, with shallow, gradually rising until the anterior 1/3 of the tail where it increases brusquely to attain its maximal height behind mid-tail and then continues gradually until the posterior 3/4 of the tail where it descends abruptly towards the tail tip. Ventral fin originates at the ventral terminus of the body, rises meticulously until the anterior 1/4 of the tail, and then remains almost parallel to the ventral border of the tail muscle until close to the tail tip. Maximal tail height located behind mid-tail, lateral line vein and myosepta imperceptible, point where the axis of the tail myotomes contacts the body located in the upper half of the body height, axis of the tail myotomes parallel with the axis of body length. Tail tip narrowly rounded. Moderately wide generalized oral disc, positioned almost ventrally and directed anteroventrally, clearly laterally emarginated. Oral disc not visible from dorsal view, upper labium as a continuation of snout. Marginal papillae uniseriate and interrupted by a wide gap on the upper labium, gap on the lower labium absent, total number of mar-

ginal papillae 48 (MPRF: (1)/1/1). Sixteen submarginal papillae present (8 on each side of the jaw sheaths folds). LKRF 4(2-4)/3(1), A1 keratodont row very long. Density of keratodonts varies from 20/mm to 71/mm, A1 59/mm (total 118). Gap in the A2 row narrow (>1% of A2 row) and distinctly wider in A3 and A4. Gap in the P1 row less than the width of three keratodonts. Alignment of anterior and posterior rows regular and nearly of same length. Distal keratodonts of same length as those in the centre; prominent space between marginal papillae and keratodont rows. Jaw sheaths partially keratinized, only the half section close to the edge coloured black; with finely pointed serrations. Upper jaw sheath moderately wide and slightly arched, with a very shallow medial concavity. Lower jaw sheath V-shaped, partially keratinized and partially hidden by the upper jaw sheath when closed. Colouration in life uniformly dark brownish. Dorsally, body covered by homogeneous dark brown melanophoric pigments. Laterally, area below eyes, flank, and abdominal region densely reticulated. Ventrally, oral disc and gular region reticulated, branchial regions reddish and spotted, beating heart visible; venter transparent, regularly spiralled intestinal coils visible. Tail musculature yellowish coloured, and coarsely reticulated. Fins patched with dark small spots with fringy edges.

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13333) was destroyed. The external morphology of the third voucher specimen (ZCMV 13334 / ZSM 705/2010; GS 25) from the same locality shows the same characters and an identical oral disc configuration as the described above. DISCUSSION

Fig. 2. Images of the preserved tadpole specimen of Mantidactylus guttulatus (ZCMV 13332 / ZSM 704/2010) at Gosner stage 26; a-c) in dorsal, lateral and ventral view (scale bar = 10 mm); d) wide open oral disc with anterior (A1-A4) and posterior (P1-P3) keratodont rows (white outline for better visibility, median gap in A1 row caused by preparation, scale bar = 1 mm); e) spiracle and f) vent tube in closer view (white outline for better visibility, scale bar = 1 mm).

Colouration in preservative uniformly brownish coloured. Brown melanic pigment in layers deeper than the skin covered the dorsum and flank, leaving laterally a slightly transparent area. Some dark brown blotches scattered on the dorsum skin, condensed to form dark patches above the brain and the vertebral region. Laterally, area below eyes and flank covered by dark brown reticulations, leaving out a perceptible transparent spiracle on the body wall. Lower part of the flank spotted. Tail musculature overlaid by dark brown spots which condensed in some area to form reticulations. Fins covered by brown spots. Ventrally, oral disc, gular and branchial regions reticulated; venter pale and spotted, intestinal coils visible with regular spiral shaped. In total, three tadpoles were captured, but due to a transportation problem, the second specimen (ZCMV

For decades, searches for the tadpole of Mantidactylus guttulatus and its relatives in the subgenus Mantidactylus have been unsuccessful, and scientists eventually hypothesized a nidicolous developmental mode for this species with a nest hidden very deep in the soil or even direct development (e.g., Glaw and Vences, 2007). During our tadpole surveys in many streams in Ranomafana National Park mainly during the rainy seasons between 2006 and 2009, no tadpole assignable to this subgenus was encountered (Strauß et al., 2013), even at sites where many adults were present. One possible explanation for the absence might be a shifted onset of their reproductive season. Contrary to many other species that start their reproductive efforts at the beginning of the warm-rainy season, the reproduction period of these frogs might peak at the end of each rainy season towards the beginning of the cool-dry season. The avoidance of reproductive competition with co-occurring species would be one benefit of this shift. An indication for this hypothesis is the early developmental stage of these tadpoles which suggests that they hatched in May. On the other hand, the single report of a calling individual from February (Vences et al., 2004) indicates that some reproductive activity occurs during the peak of the warm-rainy season. The noticeable fact that the tadpoles were found in very shallow water and, moreover, in the seepage area of a small water body could be seen as an indication of fossorial habits. However, morphological adaptions for fossoriality like a prominent tubular spiracle or particularly small eyes present in other fossorial tadpoles e.g. Otophryne robusta (Wassersug and Pyburn, 1987), Leptobrachella mjobergi (Haas et al., 2006) or Micrixalus herrei (Senevirathne et al., 2016) are absent in Mantidactylus guttulatus. Due to small sample size and the close vicinity of a large stream from which the tadpoles could have been washed away during a heavy rainfall this enigma requires further studies. Also, because we did not hypothesize these tadpoles would belong to M. guttulatus when encountering them in the wild we undertook no special efforts to further investigate the seepage area in which they occurred. For instance, we cannot exclude that upstream the seepage would originate from some kind of cavity, more suitable for such a large frog to deposit its eggs.

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Mantidactylus guttulatus tadpoles show the typical morphology of stream-adapted, Orton (1953) Type IV tadpoles with a large and muscular tail and low fins. According to Altig and Johnston (1989) they can be classified as lotic-benthic and thus assigned to the ecomorphological guild Section I, Guild 7. The tadpoles of M. guttulatus are similar to those of the subgenus Brygoomantis (Schmidt et al., 2009) which are considered as rather generalized lotic tadpoles. They share an oral disc with a large dorsal gap of marginal papillae, and a LKRF of 3-5 keratodont rows on the anterior labium with only the first being continuous and the others are interrupted by medial gaps, and three keratodont rows on the posterior labium of which the first usually has a very small medial gap. Instead, the larvae of several other subgenera have highly specialized mouthparts, such as funnelshaped structures (Chonomantis), poorly developed and reduced keratinized parts (Ochthomantis, Hylobatrachus) or a reduced number of keratodont rows in combination with unpigmented jaw sheaths (Maitsomantis) (Glaw and Vences, 1994; Vejarano et al., 2006; Grosjean et al., 2011; Randrianiaina et al., 2011). The well resolved phylogeny of Wollenberg et al. (2011) suggests that the subgenera with generalized mouthparts (Mantidactylus and Brygoomantis) are not sister clades. While the subgenus Mantidacylus branches off from the basal node of the Mantidactylus clade, Brygoomants is a sister clade to Chonomantis (Wollenberg et al., 2011). If these relationships are confirmed, it suggests extensive homoplasy in the evolution of tadpole mouthparts — either multiple independent evolution of specialized mouthparts, or reversal towards generalized mouthparts in the Brygoomantis clade. It is surprising that a frog like Mantidactylus guttulatus, whose reproductive mode has intrigued researchers for decades, has such a dull tadpole as described herein. The reproductive behaviour and the unusual microhabitat of the species still remains a mystery. Where the species deposits its eggs and whether it displays any kind of pre-hatching parental care requires being elucidated by future studies. ACKNOWLEDGEMENTS

We are grateful to F. M. Ratsoavina, A. S. Rasamison, A. Rakotoarison, and D. R. Vieites for assisting during fieldwork for this study. This study was carried out in the framework of a cooperation accord between the Département de Biologie Animale of the University of Antananarivo, Madagascar and the Technical University of Braunschweig. Permission for collection was granted by the Ministère des Eaux et Forêts, Service de la Gestion

Faune et Flore; Autorisation de Recherche 064/070/10/ MEF/SG/DGF/DCB.SAP/SLRSE, delivered 25 March 2010;  including the export permit 135N-EAO07/MG10, delivered 8 July 2010. Financial support was granted by the Volkswagen Foundation to MV, RDR, and AS, and by the Deutscher Akademischer Austauschdienst to RDR. REFERENCES

Altig, R. (1970): A key to the tadpoles of the continental United States and Canada. Herpetologica 26: 180-207. Altig, R., Johnston, G.F. (1989): Guilds of anuran larvae: relationships among developmental modes, morphologies, and habitats. Herp. Monogr. 3: 81-109. Altig, R., McDiarmid, R.W. (1999): Body plan. Development and morphology. In: Tadpoles: the Biology of Anuran Larvae, pp. 24-51. McDiarmid, R.W., Altig, R. Eds, Chicago University Press, Chicago. Altig, R., McDiarmid, R.W. (2006): Descriptions and biological notes on three unusual mantellid tadpoles (Amphibia: Anura: Mantellidae) from south-eastern Madagascar. Proc. Biol. Soc. Wash. 119: 418-425. AmphibiaWeb: Information on amphibian biology and conservation. [web application] (2016). Berkeley, California: AmphibiaWeb. Available: http://amphibiaweb. org/. (Accessed: 05 February 2016). Glaw, F., Vences, M. (1994): A Fieldguide to the Amphibians and Reptiles of Madagascar. Second Edition. Vences & Glaw Verlag, Köln. Glaw, F., Vences, M. (2007): A Field Guide to the Amphibians and Reptiles of Madagascar. Third Edition, Vences & Glaw Verlag, Köln. Gosner, K.L. (1960): A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: 183-190. Grosjean, S., Strauß, A., Glos, J., Randrianiaina, R.D., Ohler, A., Vences, M. (2011): Morphological and ecological uniformity in the funnel-mouthed tadpoles of Malagasy litter frogs, subgenus Chonomantis. Zool. J. Linn. Soc-Lond. 162: 149-183. Haas, A., Hertwig, S., Das, I. (2006): Extreme tadpoles: the morphology of the fossorial megophryid larva, Leptobrachella mjobergi. Zoology 109: 26-42. Orton, G.L. (1953): The systematics of vertebrate larvae. Syst. Biol. 2: 63-75. Palumbi, S.R., Martin, A., Romano, S., McMillian, W.O., Stine, L., Grabowski, G. (1991): The simple fool’s guide to PCR, v.2.0. Honolulu: Department Zoology, Kewalo Marine Laboratory, University of Hawaii. Perl, R.G.B., Nagy, Z.T., Sonet, G., Glaw, F., Wollenberg, K.C., Vences, M. (2014): DNA barcoding Madagas-

Tadpole of Mantidactylus guttulatus

car’s amphibian fauna. Amphibia-Reptilia 35: 197-206. Randrianiaina, R.D., Strauß, A., Glos, J., Glaw, F., Vences, M. (2011): Diversity, external morphology and “reverse taxonomy” in the specialized tadpoles of Malagasy river bank frogs of the subgenus Ochthomantis (genus Mantidactylus). Contrib. Zool. 80: 17-65. Schmidt, H., Strauß, A., Glaw, F., Teschke, M., Vences, M. (2009): Description of tadpoles of five frog species in the subgenus Brygoomantis from Madagascar (Mantellidae: Mantidactylus). Zootaxa 1988: 48-60. Schulze, A., Jansen, M., Köhler, G. (2015): Tadpole diversity of Bolivia’s lowland anuran communities: molecular identification, morphological characterisation, and ecological assignment. Zootaxa 4016: 1-111. Senevirathne, G., Garg, S., Kerney, R., Meegaskumbura, M., Biju, S.D. (2016): Unearthing the fossorial tadpoles of the Indian dancing frog family Micrixalidae. PLoS ONE 11: e0151781. Strauß, A., Randrianiaina, R.D., Vences, M., Glos, J. (2013): Species distribution and assembly patterns of frog larvae in rainforest streams of Madagascar. Hydrobiologia 702: 27-43. Thomas, M., Raharivololoniaina, L., Glaw, F., Vences, M., Vieites, D.R. (2005): Montane tadpoles in Madagas-

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car: molecular identification and description of the larval stages of Mantidactylus elegans, Mantidactylus madecassus, and Boophis laurenti from the Andringitra Massif. Copeia 2005: 174-183. Vejarano, S., Thomas, M., Glaw, F., Vences, M. (2006): Advertisement call and tadpole morphology of the clutch-guarding frog Mantidactylus argenteus from eastern Madagascar. African Zoology 41: 164-169. Vences, M., Andreone, F., Glaw, F. (2004): Voice of a giant: bioacoustic data for Mantidactylus guttulatus (Amphibia: Mantellidae). Amphibia-Reptilia 25: 112115. Vieites, D.R., Wollenberg, K.C., Andreone, F., Köhler, J., Glaw, F., Vences, M. (2009): Vast underestimation of Madagascar’s biodiversity evidenced by an integrative amphibian inventory. PNAS 106: 8267-8272. Wassersug, R., Pyburn, W. (1987): The biology of the Peret’ toad, Otophryne robusta (Microhylidae), with special consideration of its fossorial larva and systematic relationships. Zool. J. Linn. Soc-Lond. 91: 137-169. Wollenberg, K.C., Vieites, D.R., Glaw, F., Vences, M. (2011): Speciation in little: the role of range and body size in the diversification of Malagasy mantellid frogs. BMC Evol. Biol. 11: 217.

Acta Herpetologica 11(2): 127-133, 2016 DOI: 10.13128/Acta_Herpetol-18117

Thermal ecology of Podarcis siculus (Rafinesque-Schmalz, 1810) in Menorca (Balearic Islands, Spain) Zaida Ortega*, Abraham Mencía, Valentín Pérez-Mellado Department of Animal Biology, University of Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain .*Corresponding author. E-mail: [email protected] Submitted on 2016, 15th March; revised on 2016, 20th July; accepted on 2016, 25th July Editor: Sebastiano Salvidio

Abstract. We studied the thermal ecology of an introduced population of the Italian wall lizard, Podarcis siculus, in Menorca (Balearic Islands, Spain). We measured field body temperatures of adult lizards, as well as air and substrate temperatures at their capture places, during spring and summer. We assessed the relations between body and air temperatures, and between body and substrate temperatures, for both seasons. We studied the preferred temperature range of P. siculus in a laboratory thermal gradient. In addition, we recorded the operative temperatures of the habitat of the Italian wall lizard during summer. Then, we calculated the three indexes of behavioural thermoregulation for summer: thermal quality of the habitat, accuracy of thermoregulation, and effectiveness of thermoregulation. As expected, our results show that Italian wall lizards achieved significantly higher body temperatures during summer than during spring. Body temperatures were not significantly related to air temperatures in spring, but the correlation was significant in summer. In addition, body temperatures were not significantly related to substrate temperatures for any season. The preferred temperature range of the species was similar for males and females: 28.40-31.57 °C. Introduced Italian wall lizards of Menorca are effective thermoregulators, with an effectiveness of 0.82 during summer. Keywords. Thermal biology, behavioural thermoregulation, temperature, heliothermy, Lacertidae, Italian wall lizard, Podarcis siculus.

INTRODUCTION

Thermal ecology is a central point in the biology of squamate vertebrates. Their ability to exploit any resource is closely related to an effective control of their body temperature (Cowles and Bogert, 1974; Huey, 1974; Adolph and Porter, 1993). Themal ecology would cover two important traits: thermal sensitivity and thermoregulation (Angilletta, 2009). Thermal sensitivity is the dependence of physiological performance on temperature, which ranges from thermal specialists to generalists (Angilletta et al., 2002; Angilletta, 2009). Thermoregulation is the capacity to regulate body temperatures, which ranges from thermoconformers, whose body temperatures ISSN 1827-9635 (print) ISSN 1827-9643 (online)

would totally depend on ambient temperatures, to perfect thermoregulators, whose body temperatures would be constant, regardless of ambient temperatures (Huey, 1974; Hertz et al., 1993; Sears and Angilletta, 2015). Lizards mainly use three mechanisms to regulate their body temperature: adjusting activity periods (Hertz, 1992; Adolph and Porter, 1993), shuttling between different microhabitats (Heath, 1970; Bauwens et al., 1996), and adjusting their body posture (Bauwens et al., 1996). The combination of these strategies depends on the balance between costs and benefits, which in turn depends on different biotic and abiotic factors (Huey and Slatkin, 1976; Sears and Angilletta, 2015). Within lizards, Lacertids generally are effective thermoregulators and mostly © Firenze University Press www.fupress.com/ah

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heliothermic, which use to move between sunny and shade microhabitats for thermoregulation (Avery, 1976; Van Damme et al., 1990; Castilla et al., 1999; Ortega et al., 2016a). Our aim is to study the thermal ecology of an introduced population of the Italian wall lizard, Podarcis siculus, in Menorca (Balearic Islands, Spain). We measured body temperatures of active lizards, as well as air and substrate temperatures of the microhabitats occupied by lizards. In order to search for seasonal effects in the thermoregulation, we compared these measures for spring and summer. We hypothesized that lizards would achieve higher body temperatures in summer than in spring, as it is usual in lacertid lizards (e.g. Díaz and Cabezas-Díaz, 2004; Ortega et al., 2014). We also measured the thermal preferences of lizards in a thermal gradient. In addition, we recorded the operative temperatures of the habitat during summer. Finally, we studied the thermal quality of the habitat, the accuracy of thermoregulation, and the effectiveness of thermoregulation (Hertz et al., 1993) of the Italian wall lizard during summer. MATERIAL AND METHODS Study species and area The Italian wall lizard Podarcis siculus (RafinesqueSchmalz, 1810) is a robust ground-dwelling lacertid lizard. The original distribution covers Italy (continental Italy, Sardinia, Sicily and several coastal islets), Corsica (France) and the east coast of the Adriatic Sea, from Slovenia to Montenegro (Henle and Klaver, 1986). However, P. siculus has been introduced in many Mediterranean countries and in the United States (Corti et al., 2004). Here we studied the population of Menorca (Balearic Islands, Spain), which inhabits all kinds of habitats, from coastal dunes to forests and anthropogenic walls (PérezMellado, 1998; Pérez-Mellado, 2002), and would be introduced from Sicily and/or Sardinia (Silva-Rocha et al., 2012). The Italian wall lizard is a heliothermic lizard, which previously reported mean temperatures range between 29 °C in spring and approximately 33 °C in summer (Avery, 1978; Van Damme et al., 1990; Foà et al., 1992; Tosini et al., 1992). We studied the population of Es Canutells, in Southern Menorca (Spain), an almost undisturbed Mediterranean habitat of mixed woodland and scrubland (patches of pines and holm oaks, and patches of large shrubs, mainly Pistacia lentiscus), spotted with large rocks. The studied population exhibited a clear sexual size dimorphism, with larger (mean SVL males: 73.35 ± 1.22 mm, n = 20; mean SVL females: 64.97 ± 1.00 mm, n = 9; one-way ANOVA, F1, 27 = 18.417, P < 0.0001) and heavier (mean weight males: 10.28 ± 0.41 g, n = 20; mean weight females: 7.09 ± 0.41 g, n = 9; one-way ANOVA, F1, 27 = 22.061, P < 0.0001) males.

Field sampling We recorded field temperatures of Podarcis siculus between 27 May and 30 July 2013, in 12 sunny days of fieldwork (7 in spring and 5 in summer). We considered the natural seasons: the data obtained before the 21st of June have been considered as spring data, and those obtained after that date as summer data. We captured active adult lizards by noosing, during their daily activity period, from 07:00 to 17:00 h (GMT), 16 in spring (11 males and 5 females) and 15 in summer (11 males and 4 females). Immediately after capture (within 30 s), we measured cloacal body temperature (Tb) with a Testo® 925 digital thermometer, shadowing the probe, as well as air temperature (Ta) 1 cm above the capture point, and substrate temperature (Ts) of the capture point. We also recorded the type of substrate, the height of the perch (in cm), and the sunlight situation (full sun, filtered sun, or full shade). Finally, we measured wind speed with a Kestrel® 3000 anemometer, but during field work, its variation was almost insignificant (a mean of 0.15 ms-1). So, for this study, we discarded the wind as a possible variable affecting thermal behaviour of lizards. As a null hypothesis for thermoregulation, we recorded operative temperatures (Te). We recorded Te during the same days of the field sampling of summer (between 16 July 2013 and 30 July 2013) in the same area of study (Es Canutells), in order to control for potential variations in weather conditions. We used copper models as null Te models (Bakken and Angilletta, 2014). These models achieve similar temperatures to those of non-thermoregulating lizards. We placed one thermocouple probe into each hollow model and connected it to a data logger HOBO® H8 (Onset Computer Corporation), programmed to take a temperature record every five minutes. We randomly placed the copper models in different microhabitats and used the Te hourly mean of each microhabitat for analysis, since raw Te data could be autocorrelated. Based in observations of the behaviour of lizards, we selected four types of microhabitats: rock, soil, grass, and logs of Pistacia lentiscus; each of them was considered in the three sunlight situations (see above). Preferred temperature range (PTR) We measured selected body temperatures (Tsel) of P. siculus between 12 June 2013 and 14 June 2013 in a laboratory thermal gradient. We captured lizards from the same location of field sampling and immediately transported them to the laboratory in Es Castell (Menorca, Spain). There, we housed lizards on individual terraria and fed them with mealworms and crickets. Water was provided ad libitum during the length of the experiment. We built the thermal gradient in a glass terrarium (100 x 60 x 60 cm) with a 150 W infrared lamp over one of the sides, obtaining a gradient between 20 to 60 ºC. Then, we measured the selected temperature of a lizard each hour from 08:00 to 17:00 h (GMT) with a digital thermometer. We used 24 P. siculus adult lizards, 14 males and 10 females. We considered the 50% of the central values of selected body temperatures as the preferred temperatures range (PTR) in all analyses, as it is the more common procedure, although we also report the 80%

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Thermoregulation of Podarcis siculus in Menorca PTR, since some authors employ this range (Hertz et al., 1993; Blouin-Demers and Nadeau, 2005). We released lizards at their capture places immediately after the experiment. Data analysis To test the null hypothesis of thermoregulation, that is, if lizards use microhabitats randomly regarding temperature, we followed the protocol developed by Hertz et al. (1993), and calculated their three indexes of thermoregulation. The first is the − index of accuracy of thermoregulation (db ), that is the mean of absolute values of the deviations between each Tb from the preferred temperature range. Thus, the values of the index of accuracy of thermoregulation are counterintuitive: higher values of − db indicate lower accuracy of thermoregulation, and vice-versa. − The second is the index of thermal quality of habitat (de), calculated as the mean of absolute values of the deviations of each Te from the preferred temperature range. Accordingly, the values of the index of thermal quality of the habitat are also counter− intuitive: higher values of de indicate a lower thermal quality of the habitat, and vice-versa. The third is the index of effective− ness of thermoregulation (E), that is calculated as Ε = 1 - (db / − de ). Hence, values of E range from 0 to 1, meaning the higher effectiveness of thermoregulation the higher the value of E (see Hertz et al., 1993). Effectiveness of thermoregulation was calculated with THERMO, a Minitab module written by Richard Brown. THERMO uses three kinds of input data: Tb, Te and Tsel of the preferred temperature range, and was programed to perform bootstraps of 100 iterations, building pseudo-distributions of three kinds of output values: the arithmetic mean of − the index of accuracy of thermoregulation (db ), the arithmetic − mean of the index of thermal quality of the habitat (de), and the arithmetic mean of the index of effectiveness of thermoregulation (E). As we measured Te in summer, we only computed this protocol of study for the body temperatures of summer. We performed parametric statistics when data followed the assumptions of normality and variance homogeneity. When data did not fulfill these assumptions, even after log-transformations, we carried out non-parametric equivalent tests (Sokal and Rohlf, 1995; Crawley, 2012). We conducted all analyses on R, version 3.1.3 (R Core Team, 2015), and we computed posthoc comparisons of Kruskal-Wallis tests with Nemenyi test with the package PMCMR (Pohlert, 2014). We reported mean values of variables accompanied by standard errors. Significance level was α = 0.05.

RESULTS

Selected body temperatures (Tsel) were similar regarding sex (mean Tsel of males: 29.84 ± 0.41 °C, n = 14; mean Tsel of females: 30.15 ± 0.31 °C, n = 10; one-way ANOVA, F1, 22 = 0.301, P = 0.589). Thus, we pooled them in subsequent analyses, and considered a preferred temperature range (PTR) for this population. The 50% PTR is 28.40 31.57 °C, and the 80% PTR is 26.85-32.54 °C.

Body temperatures (Tb) were also similar regarding sex (mean Tb of males = 30.99 ± 0.53 °C, n = 22; mean Tb of females = 30.23 ± 1.09 °C, n = 9; one-way ANOVA, F1, 29 = 0.494, P = 0.488). Thus, also in this case, we pooled data from males and females for subsequent analyses. Body temperatures (Tb) of lizards (one-way ANOVA, F1, 29 = 7.996, P = 0.008), as well as air temperatures (oneway ANOVA, F1, 29 = 18,704, P < 0.0001) and substrate temperatures (Ts; one-way ANOVA, F1, 29 = 8.244, P = 0.008) were significantly higher in summer than in spring (Table 1). Although sample size for subsets of each sex within each season is small, we checked for potential differences in Tb between sexes, in order to confirm if males and females should be pooled together within each season. Results show similar Tb of males and females both in spring (one-way ANOVA, F1, 15 = 0.136, P = 0.718) and in summer (one-way ANOVA, F1, 14 = 0.267, P = 0.614). An ANCOVA test reveals that the linear relation between Tb and Ta significantly changed between spring and summer (Ta as a covariate; interaction season*Ta: F1, 27 = 5.590, P = 0.026). Thus, linear regressions must be studied separately regarding season. Correlation between Tb and Ta was not significant in spring (r = 0.209, P = 0.438, n = 16), but was significant in summer (r = 0.756, P = 0.001, n = 15). The linear regression slope of Ta on Tb was also not significant (β = 0.21, P = 0.438, n = 16; R2 = 0.044; Fig. 1) in spring, and was statistically significant in summer (β = -0.61, P = 0.001, n = 15; R2 = 0.571; Fig. 1). However, the slope of the linear regression of Ts on Tb was similar for both seasons (ANCOVA, Ts as covariate; interaction season*Ts: F1, 27 = 0.042, P = 0.839). The correlation coefficient was significant (r = 0.481, P = 0.003), as well as the regression coefficient (β = 0.38, P = 0.006, n = 31; R2 = 0.231; Fig. 1). The available microhabitats at the study site provided different operative temperatures (Kruskal-Wallis test, H = 222.525, P < 0.0001, n = 528, df = 12; see Table 2 and Fig. 2). Only grass and rock in full shade provided optimal temperatures for the thermoregulation of P. siculus (i.e., within the PTR) during all hourly periods of the day (Fig. 2). The index of thermal quality of the habitat (de) showed a mean of 8.07 ± 0.05, the index of thermal accu-

Table 1. Mean ± SE (sample size) body temperatures (Tb), air temperatures (Ta) and substrate temperatures (Ts) of Podarcis siculus at Menorca (Balearic Islands, Spain). Temperatures are in °C.

Tb Ta Ts

Spring

Summer

29.57 ± 0.56 (16) 25.77 ± 0.56 (16) 27.11 ± 0.76 (16)

32.05 ± 0.68 (15) 28.92 ± 0.45 (15) 30.27 ± 0.80 (15)

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Fig. 1. Slopes of the simple linear regressions models of body temperatures of Podarcis siculus lizards (Tb) against air temperatures (Ta; left plot) and of the simple linear regressions of Tb against substrate temperatures (Ts; right plot) in spring and summer. The regression Tb-Ta was not significant in spring, but was is significant in summer, and the regression Tb-Ts was significant and had a similar slope for both seasons (see results in the text).

Table 2. Mean values of the operative temperatures (Te) of the different microhabitats studied for Podarcis siculus at Menorca (Balearic Islands, Spain). Temperatures are in °C. The letters between brackets match the non-significant pairs of the Nemenyi post-hoc comparisons of the Kruskal-Wallis test (P > 0.05 in the paired comparisons). To avoid pseudoreplication, calculations are based in the hourly means of Te so sample size coincides with the hours of monitoring of each microhabitat at the study site.

Under rock (b, d) Rock Full sun (a) Rock Filtered sun (h, i) Rock Full shade Soil Full sun (a) Soil Filtered sun (f, g, h) Soil Full shade Grass Full sun (a) Grass Filtered sun (e, f, i) Grass Full shade Pistacia Full sun (b, c) Pistacia Filtered sun (c, d, e, g) Pistacia Full shade (b)

n

Te

SE

44 33 44 44 11 44 44 44 44 44 44 44 44

37.55 45.40 40.64 30.78 47.57 39.98 29.30 51.56 40.59 30.37 36.86 38.48 36.43

0.88 1.08 1.12 0.28 3.84 1.19 0.33 1.75 1.38 0.29 0.94 1.20 0.92

racy (db) was 1.41 ± 0.04, and the index of effectiveness of thermoregulation (E) of P. siculus in summer was 0.820 ± 0.005. DISCUSSION

The preferred temperature range of P. siculus, obtained in the late spring, ranges from 28.40 to 31.70 °C. This is lower than the preferred temperature range of the endemic lacertid lizard from Menorca, P. lilfordi, which showed a range between 31.78 and 35.68 °C during spring (unpublished data), and 32-36 °C during summer (Pérez-Mellado et al., 2013; Ortega et al., 2014). This is also lower than the preferred temperature range of the third lacertid lizard present in Menorca, Scelarcis perspicillata, which showed a range from 33.90 to 36.10 °C during summer (Ortega et al., 2016b). Thus, the precision of thermoregulation obtained for the Italian wall lizard was 3.3 °C, while the Balearic lizard exhibited 3.9 °C, and the Moroccan rock lizard 2.2 °C. The thermal preferences in a laboratory thermal gradient represent the optimal temperatures that lizards would intend to achieve in their habitats if there were no other ecological constraints than temperature (e.g., Dawson, 1975; Huey and Bennett,

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Fig. 2. Operative temperatures (Te) provided by the different microhabitats studied in Es Canutells (Menorca, Spain) for the Italian lizard, Podarcis siculus. The dotted lines comprise the preferred temperature range (PTR) of the species.

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1987). The thermal preferences are closely related with thermal sensitivity of performance (Angilletta et al., 2002; Martin and Huey, 2008). Our results suggest that P. siculus would perform better at lower temperatures than the other two diurnal lacertid lizards inhabiting Menorca. Our results were coherent with previous studies about body temperatures of P. siculus. Italian wall lizards showed mean body temperatures approximately 2 °C lower in June and July at Menorca than those recorded near Florence, in Italy (Avery, 1978). However, mean body temperatures found in summer in Menorca are similar to those found in summer near Pisa, (Tosini et al., 1992). Regarding spring thermoregulation, our data were similar to those found in Corsica (France) in May: mean Tb are ≈ 2 °C lower, mean Ta are ≈ 2 °C higher, and mean Ts are similar (Van Damme et al., 1990). In addition, the regression slope between body and air temperatures was very similar to the slope reported by Van Damme et al. (1990) for P. siculus of Corsica during spring, and was also not significantly different from zero. Our results also confirmed the conclusion of Van Damme et al. (1990) and Tosini et al. (1992) about the lack of a sexual effect on body temperatures of P. siculus. Mean body temperatures of the Italian wall lizard were significantly higher in summer than in spring, but approximately 3 °C lower, for each season, than the body temperatures of the Balearic lizard in the close islets of Aire and Colom (Ortega et al., 2014). Mean body temperatures were also approximately 2 °C lower than those of the Moroccan rock lizard in Menorca (Ortega et al., 2016b). During summer, the Italian wall lizard achieved a lower accuracy and effectiveness of thermoregulation (d−b ≈ 1.41 °C; E ≈ 0.82) than the Balearic lizard (d−b ≈ 0.50 °C; E ≈ 0.91; Ortega et al., 2014) and the Moroccan rock lizard (d−b ≈ 0.62 °C; E ≈ 0.88; Ortega et al., 2016b). However, our data shows that the Italian wall lizard is an effective thermoregulator lacertid, which seems well adapted to inhabit a wide range of microhabitats. A comparative study on the flexibility of thermal physiology and behavioural thermoregulation of P. siculus lizards and the species with which they coexist worldwide would help explain the possible causes of the remarkable ability of this species to adapt to different environments. ACKNOWLEDGEMENTS

We thank Mario Garrido and Ana Pérez-Cembranos for their company during fieldwork, and Mary Trini Mencía and Joe McIntyre for linguistic revision. We captured lizards under the licenses of the Government of the Balearic Islands. Zaida Ortega and Abraham Mencía had

financial support from predoctoral grants of the University of Salamanca. During the preparation of the manuscript, this work was supported by the research project CGL2015-68139-C2-2-P from the Spanish Ministry of Economy and Competitivity and FEDER European funds. All research was conducted in compliance with ethical standards and procedures of the University of Salamanca. REFERENCES

Adolph, S.C., Porter, W.P. (1993): Temperature, activity, and lizard life histories. Am. Nat. 142: 273-295. Angilletta, M.J. (2009): Thermal adaptation: A theoretical and empirical synthesis. Oxford University Press, Oxford. Angilletta, M.J., Niewairowski, P.H., Navas, C.A. (2002): The evolution of thermal physiology in ectotherms. J. Therm. Biol. 27: 249-268. Avery, R.A. (1976): Thermoregulation, metabolism and social behaviour in Lacertidae. In: Morphology and Biology of Reptiles, pp. 245-259. Bellairs, A. d’A., Cox, C.B., Eds, Academic Press, London. Avery, R.A. (1978): Activity patterns, thermoregulation and food consumption in two sympatric lizard species (Podarcis muralis and P. sicula) from central Italy. J. Anim. Ecol. 47: 143-158. Bakken, G.S., Angilletta, M.J. (2014): How to avoid errors when quantifying thermal environments. Funct. Ecol. 28: 96-107. Bauwens, D., Hertz, P.E., Castilla, A.M. (1996): Thermoregulation in a lacertid lizard: the relative contributions of distinct behavioral mechanisms. Ecology 77: 1818-1830. Blouin-Demers, G., Nadeau, P. (2005): The cost-benefit model of thermoregulation does not predict lizard thermoregulatory behaviour. Ecology 86: 560-566. Castilla, A.M., Van Damme, R., Bauwens, D. (1999): Field body temperatures, mechanisms of thermoregulation and evolution of thermal characteristics in lacertid lizards. Natura Croatica 8: 253-274. Corti, C., Nistri, A., Lanza, B., Vanni, S. (2004): Podarcis sicula (Rafinesque-Schmalz, 1810). In: Atlas of Amphibians and Reptiles in Europe. Reedition, pp. 294-295. Gasc, J.P. et al., Eds, Museum national d’Histoire Naturelle, Paris. Cowles, R.A., Bogert, C.M. (1974): A preliminary study of the thermal requeriments of desert reptiles. B. Am. Mus. Nat. His. 83: 261-296. Crawley, M.J. (2012): The R book. Wiley, Chichester, UK. Dawson, W.R. (1975): On the physiological significance of the preferred body temperatures of reptiles. In: Per-

Thermoregulation of Podarcis siculus in Menorca

spectives of biophysical ecology, pp. 443-473. Gates, D.M., Schmerl, R.B., Eds., Springer Berlin, Heidelberg. Díaz, J.A., Cabezas-Díaz, S. (2004): Seasonal variation in the contribution of different behavioural mechanisms to lizard thermoregulation. Funct. Ecol. 18: 867-875. Foà, A., Tosini, G., Avery, R. (1992): Seasonal and diel cycles of activity in the ruin lizard, Podarcis sicula. Herpetol. J. 2: 86-89. Heath, J.E. (1970): Behavioral regulation of body temperature in poikilotherms. Physiologist 13: 399-410. Henle, K., Klaver, C.J.J. (1986): Podarcis sicula (Rafinesque-Schmalz, 1810) - Ruineneidechse. In: Handbuch der Reptilien und Amphibien Europas. Band 2/II. Echsen (Sauria) III (Lacertidae III: Podarcis), pp. 254342. Böhme, W., Ed., Aula Verlag, Wiesbaden. Hertz, P.E. (1992): Temperature regulation in Puerto Rican Anolis lizards: a field test using null hypotheses. Ecology 73: 1405-1417. Hertz, P.E., Huey, R.B., Stevenson, R.D. (1993): Evaluating temperature regulation by field-active ectotherms: the fallacy of the inappropiate question. Am. Nat. 142: 796-818. Huey, R.B. (1974): Behavioral Thermoregulation in lizards: importance of associated costs. Science 184: 1001-1003. Huey, R.B., Bennett, A.F. (1987): Phylogenetic studies of coadaptation: preferred temperatures versus optimal performance temperatures of lizards. Evolution 41: 1098-1115. Huey, R.B., Slatkin, M. (1976): Costs and benefits of lizard thermoregulation. The Q. Rev. Biol. 51: 363-384. Martin, T.L., Huey, R.B. (2008): Why “suboptimal” is optimal: Jensen’s inequality and ectotherm thermal preferences. Am. Nat. 171: E102-E118. Ortega, Z., Pérez-Mellado, V., Garrido, M., Guerra, C., Villa-García, A., Alonso-Fernández, T. (2014): Seasonal changes in thermal biology of Podarcis lilfordi (Squamata, Lacertidae) consistently depend on habitat traits. J. Therm. Biol. 39: 32-39. Ortega, Z., Mencía, A., Pérez-Mellado, V. (2016a): The peak of thermoregulation effectiveness: thermal biology of the Pyrenean rock lizard, Iberolacerta bonnali (Squamata, Lacertidae). J. Therm. Biol. 56: 77-83. Ortega, Z., Mencía, A., Pérez-Mellado, V. (2016b): Sexual

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differences in behavioral thermoregulation of the lizard Scelarcis perspicillata. J. Therm. Biol. 61: 44-49. Pérez-Mellado, V. (1998): Podarcis sicula (Rafinesque, 1810). In: Fauna Ibérica. Reptiles, pp. 302-307. Salvador, A., Coord, Ramos, M.A. et al., Eds, Museo Nacional de Ciencias Naturales, Madrid. Pérez-Mellado, V. (2002): Podarcis sicula (Rafinesque, 1810). Lagartija italiana. In: Atlas y Libro Rojo de los Anfibios y Reptiles de España, pp.257-259. Pleguezuelos, J.M., Márquez, R., Lizana, M., Eds, Dirección General de Conservación de la Naturaleza-Asociación Herpetológica Española, Madrid. Pérez-Mellado, V., Alonso-Fernández, T., Garrido M., Guerra C., Ortega, Z., Villa-García, A. (2013): Biología térmica de la lagartija balear, Podarcis lilfordi (Günther, 1874) en dos poblaciones de Menorca. Revista de Menorca 92: 219-244. Pohlert. T. (2014): The Pairwise Multiple Comparison of Mean Ranks Package (PMCMR). R package. Accesible at https://cran.r-project.org/web/packages/PMCMR/ vignettes/PMCMR.pdf (Accessed: 20 July 2016). R Core Team (2015): R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Accesible at http:// www.R-project.org/ (Accessed: 20 July 2016). Sears, M.W., Angilletta, M.J. (2015): Costs and benefits of thermoregulation revisited: both the heterogeneity and spatial structure of temperature drive energetic costs. Am. Nat. 185: E94-E102. Silva-Rocha, I., Salvi, D., Carretero, M.A. (2012): Genetic data reveal a multiple origin for the populations of the Italian wall lizard Podarcis sicula (Squamata: Lacertidae) introduced in the Iberian Peninsula and Balearic islands. Ital. J. Zool. 79: 502-510. Sokal, R.R., Rohlf, F.J. (1995): Biometry: the principles and practice of statistics in biological research. State University of New York at Stony Brook, New York. Tosini, G., Foà, A., Avery, R. (1992): Body temperatures and exposure to sunshine of ruin lizards Podarcis sicula in central Italy. Amphibia-Reptilia 13: 169-175. Van Damme, R., Bauwens, D., Castilla, A.M., Verheyen, R.F. (1990): Comparative thermal ecology of the sympatric lizards Podarcis tiliguerta and Podarcis sicula. Acta Oecol. 11: 503-512.

Acta Herpetologica 11(2): 135-149, 2016 DOI: 10.13128/Acta_Herpetol-18695

Growth, longevity and age at maturity in the European whip snakes, Hierophis viridiflavus and H. carbonarius Sara Fornasiero1, Xavier Bonnet2, Federica Dendi1, Marco A.L. Zuffi1,* Museo di Storia Naturale, Università di Pisa, via Roma 79, I-56011 Calci (Pisa) Italy. *Corresponding authors: E-mail: marco.zuffi@ unipi.it 2 CEBC, CNRS University of La Rochelle (UMR 7372), France 1

Submitted on 2016, 27th July; revised on 2015, 25th August; accepted on 2016, 30th August Editor: Uwe Fritz

Abstract. Age and size at maturity are major life history traits, because they influence lifetime fecundity. They represent the outcome from complex interactions among environmental pressures (abiotic and biotic) and individual characteristics. They are also difficult to measure in natural populations and thus they are rarely appraised, especially in reptiles due to the elusive nature of juveniles. Using skeletochronology to circumvent these difficulties, this study aims to compare age structures, longevity, age-size relationships, growth curves, age and size at maturation and potential reproductive lifespan in three populations of the European whip snake (two Hierophis viridiflavus, one H. carbonarius). We measured the body size and counted the skeletal growth marks on 132 specimens, accidentally killed or from museum collections (72 from NW France [Chizé]; 28 from Tuscan Archipelago [Montecristo], Italy; 32 from S Italy [Calimera]). General patterns of age at maturity and longevity were consistent with previous studies based on recapture investigations. Strong differences among populations suggest local adaptation to contrasted environmental conditions. These results suggest that skeletochronology is a useful technique that can be applied opportunistically in snakes (e.g., using road-kills) in order to collect otherwise unavailable data that are essential to address fundamental questions regarding longevity, life-history traits and to perform population viability analyses. Keywords. Insularity, snakes, Hierophis, sexual maturity, skeletochronology.

INTRODUCTION

Iteroparous species with indeterminate growth (e.g., many fish, squamate reptiles) exhibit strong variations in most life history traits in response to environmental factors; body size is highly variable among and within populations for example (Wimberger, 1992; Madsen and Shine, 1993; Ryser, 1996; Rohr, 1997; Zuffi et al., 2007; Warner, 2014). Considering versatile life history traits, age at maturity is pivotal because fitness is particularly sensitive to changes in this trait (Stearns, 1992). Similarly, body size exerts strong effects on fecundity, survival, and thus on fitness (Shine, 1988, 1990). Age and body size at maturity are inextricably linked because ISSN 1827-9635 (print) ISSN 1827-9643 (online)

they both depend on juvenile growth rate which in turns depends on the trophic and climatic conditions experienced by individuals (Sinervo and Adolph, 1994; Webb et al., 2003). Fast juvenile growth promotes early maturation, large body size, and thus likely increases individual potential reproductive lifespan (Ryser, 1996; Day and Taylor, 1997; Bronikowski and Arnold, 1999). However, many examples suggest that there may be costs associated with fast growth; strong metabolic increase or reduced longevity in early-breeders for instance (Beaupre and Zaidan III, 2001; Blouin-Demers et al., 2002). Sexual maturity entails a shift in the allocation of resources used to sustain growth during juvenile phase to reproduction (and secondarily to growth in many © Firenze University Press www.fupress.com/ah

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reptiles) during adulthood (Shine and Charnov, 1992; Day and Taylor, 1997; Rohr, 1997; Wapstra et al., 2001; Stanford and King, 2004). There is a marked decrease of growth at maturity, although reduced growth may persist through life in many species with marked differences among individuals (Congdon et al., 2003). As a result, marked intra-population differences of body size set at maturity are usually maintained in individual later ageclasses (Madsen and Shine, 1993; Shine, 1994; Zuffi et al., 2011). Individuals that mature at a small size remain small for the rest of their life; thereby counter balancing the fecundity advantages of early maturation (Halliday and Verrell, 1988). Overall, it is expected that the physiological processes that determine maturity, and thus the average age and body size of adults, should maximize lifetime reproductive success through differential adjustments in response to environmental conditions (Stearns and Crandall, 1981; Stearns and Koella, 1986; Bernardo, 1993; Ford and Seigel, 1994; Wapstra et al., 2001). Studies that compare populations of the same species throughout different parts of its distribution range are valuable to understand how environmental factors shape age and size at maturity along with related lifehistory attributes (e.g., age-size relationship, longevity, population age structure; Mateo and Castanet, 1994; Nobili and Accordi, 1997; Lima et al., 2000; Miaud and Guillaume, 2005). Organisms that display important intra- and inter-population variations in body size are of particular interest because these variations may correlate with growth rate and age at maturity (Alcobendas and Castanet, 2000; Miaud et al., 2001; Kutrup et al., 2005). Unfortunately the relationships between growth rate, body size and age are not easily assessed in the field. Indeed, long-term mark-recapture surveys must be set up to collect the raw data necessary to calculate growth rate and to examine key life history traits. But in most reptile species juveniles escape observations (Pike et al., 2008; Bjorndal et al., 2013). Growth rate has been rarely measured accurately before sexual maturity (Bonnet et al., 2011) and the exact age of monitored individuals is seldom known in the field (Lagarde et al., 2001). This lack of information poses major difficulties to perform analyses. For example, for a given species a single body size for maturity is usually extracted from the literature, used to assign individuals into age categories, and then used to perform various analyses (e.g., regarding population viability, demography). Possible variations caused by inter-individual, geographic, and time heterogeneity are systematically neglected. These limitations apply with force in snakes due both to the extremely elusive nature of immature individuals (that precludes estimating accurately the age of individuals) and to the marked pheno-

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typic plasticity of these organisms (Madsen and Shine, 1993; Bronikowski, 2000). Skeletochronology offers a reliable alternative (Halliday and Verrell, 1988). This approach is based on counting the skeletal growth marks (SGM) that are successively deposited on the growing bone (Castanet et al., 1992). Although this method does not require long term population monitoring, major prerequisites are nonetheless important. Continuous growth and a lack of bone remodelling are essential characteristics. The relationships between age and SGM count is accurate in species where an active season precisely alternates with a period of inactivity (e.g., hibernation); it has been validated through mark-recapture in several reptiles (Lagarde et al., 2001). Many snakes from temperate climates fulfil the above conditions, are spread across a wide range of habitats, and thus they represent suitable candidates to examine the influence of environmental factors on the relationships between age and body size. The aim of this study was to compare the mean age, mean longevity, the age-size relationship, growth curves, age and size at maturation of individuals sampled in three populations of two closely related species of whip snakes (two populations Hierophis viridiflavus and in one population H. carbonarius; Mezzasalma et al., 2015). The three populations are situated in distant parts of the distribution range of the species characterised by contrasted environmental conditions (one forested site in temperate climate zone and two sites in the Mediterranean climate zone; one continental and one insular). We thus expected marked differences among populations in the traits observed. MATERIALS AND METHODS Study species and study sites The taxonomy of the European whip snake (Hierophis [Coluber] viridiflavus) has been recently revised (Rato et al. 2009; Mezzasalma et al., 2015). Depending upon the study and criteria, it has been suggested to differentiate several subspecies or species. The debate is not closed because the taxonomic boundaries between species and subspecies are often tenuous. We considered that we sampled three populations and two species, H. viridiflavus and H. carbonarius, of the European whip snake; but we emphasize that we could not rule out the possibility that one species and two subspecies were sampled. Thus, for conciseness (and cautiousness) we refer to three populations of the ‘European whip snake’ hereafter. The three populations studied are widely spread across the distribution range of the species: (1) Forest of Chizé, France (46°07’N, 00°25’W). The site is close to the northern limit of the distribution range of H. viridiflavus; (2) Montecristo Island, Tuscan Archipelago, Central Italy (42°19’54”N, 10°18’38”E) is

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Growth, longevity and age at maturity in the European whip snakes situated more than 900 km south-easterly. An isolated population of H. viridiflavus inhabits this rocky island; and (3) surroundings of Calimera, Lecce (Apulia, Southern Italy; 40°15’N, 18°16’E). This third site located more than 700km further south-easterly is in the southern distribution range of H. carbonarius. More than 1600 km separate the first and third populations whereas climatic and general environmental conditions strongly contrast among all populations. Climatological data were obtained from the three meteorological stations closer to the respective study sites: Niort (30 Km from the Forest of Chizé), Lecce (15 km from Calimera) and Gorgona island (65 km from Montecristo island; Elba island is closer to Montecristo, but Gorgona resembles much more Montecristo island in overall surface, vegetation and climatic conditions). While it was possible to obtain data for Lecce only for the period between 1961 and 1990, data for France and Montecristo are referred to the same time span, between 1998 and 2006. Average monthly temperatures (°C) and average monthly rainfalls (mm) for the over cited time intervals are reported in Fig. 1. Sampling snakes Our samples were based on individuals accidentally killed (e.g., road-kills) and museum specimens and no snake was intentionally killed for this study. Used animals derived from a quite short time range (less than 10 years), in order to avoid any bias and/or constraint due to a too large time gap that could prevent a proper analysis of age estimation. A total of 132 specimens were analysed during four years: 72 whip snakes from the Forest of Chizé (mainly road kills; Bonnet et al. 1999a), 28 from Montecristo Island (museum specimens) and 32 from Calimera (museum specimens). The specimens were all alcohol preserved and came from the herpetological collections of Florence (Museo Zoologico “La Specola”, Università di Firenze, Italy), Pisa (Museo di Storia Naturale, Università di Pisa, Italy), Frankfurt (Senkemberg Museum Frankfurt a.M., Germany), and Chizé lab. Preliminary analyses showed no effect of sampling date or duration of stay in alcohol before examination (i.e. alcohol did not destroy SGM, at least over several years). Each snake was sexed (except newborns and juveniles), and measured (SVL, Snout-to-Vent Length) to the nearest mm by stretching it along a measuring tape. Snakes were considered adult on the basis of external body coloration and using minimal SVL for maturity in each population (Fornasiero et al., 2007). Very small snakes with typical neonate colouration were considered as newborns; larger immature snakes were considered as juveniles. Skeletochronological analysis SGM (skeletal growth marks) reveal temporary variations in osteogenesis rate (Castanet et al., 1992). Three categories of SGM can be recognised: opaque layers (zones), translucent layers (annuli) and lines of arrested growth (LAG) (Castanet et al., 1993). Zones correspond to fast osteogenesis phases made of badly spatially structured bone matrix, rich in randomly

Fig. 1. Climatic features of the study sites. a) Chizé b) Montecristo c) Calimera. ( Average min. Temperature); ( Average Max. Temperature); ( Average monthly Temperature); (▶ Average monthly Rainfall).

•

distributed osteocytic lacunae. Due to their 3D structure, zones are more opaque than other marks and appear therefore dark when observed under transmitted light (Castanet et al., 1993). Annuli alternate with zones and correspond to periods of slow osteogenesis. Bone matrix is well structured (often being made of lamellar bone) and usually poor in osteocytes. When observed under transmitted light, annuli appear narrower and more translucent than adjacent zones (Castanet

138 et al., 1993). LAGs correspond to a temporary arrest of local osteogenesis. They are narrow, 1 not always visible, typically bordering annuli or, sometimes, appearing inside them (Castanet et al., 1993). Skeletochronology is based on the assumption that growth marks are the 2 histological expression of temporary and periodical variation in bone growth rate. SGMs may originate from endogenous rhythms, but they are influenced and synchronised by external seasonality, like the alternation of hibernation and active season in snakes from temperate climates (Castanet and Naulleau, 1974; Castanet et al., 1993). Until sexual maturity, when growth rate is high, annuli or LAGs are well separated by wide zones of fast-growing tissues, while the subsequent SGM appear narrower and more irregular. This pattern has been named “rapprochement” by Francillon-Vieillot et al. (1990). Although caudal vertebrae can be used for age estimation of living specimens (Waye and Gregory, 1998), two flat skull bones, the supra-angular and the ectopterygoid especially, are preferred in dead individuals (Hailey and Davies, 1987). In road kills, pairs of ectopterygoids and supra-angulars (four bones) were removed. In museum specimens, the bones were removed from one side of the head only in order to preserve the external morphology and scalation of the head. The bones were stored in fresh water until organic tissues (e.g., ligaments) became soft and carefully cleaned. Counting SGM SGM counting followed the procedure reported by Castanet et al. (1993): bones were observed in toto with a binocular microscope, under transmitted light. During the reading of SGM, the bone was kept under water, in order to enhance the contrast between different growth marks. Counting was performed by two different people (or by the same observer on separate occasions), always blind to sample identity. Parallel readings on the same sample were compared, SGM were counted again in case of discrepancy. If the problem persisted, the sample was discarded. In case of divergence between ectopterygoid and supra-angular counting, ectopterygoid counting was retained because it provides more reliable results (Hailey and Davies, 1987). Sample where strong bone remodelling occurred, obviating age estimation, were discarded. Several specious countings were not retained in the analyses.

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Gn + 1 =

Rn + 1 - Rn R1

where Gn+1 represents the increment of bone per year in relation to the increment of the first year, expressed in arbitrary units, and R (rings). The analysis was restricted to pictures where the basis of subsequent zones was clearly visible. Successive G were examined for the first 12 years in order to detect the expected drop associated with maturity (Francillon-Vieillot et al., 1990; Castanet et al., 1993). The last year before a sharp decrease in mean bone growth was considered to be the onset of sexual maturity. Analyses For most comparisons we used analysis of variance (ANOVA) when the distribution of the data did not deviate from normality (Kolmogorov-Smirnov tests) and U-Mann-Whitney tests otherwise. Mean age and longevity (maximum age recorded) were calculated for each population and each sex. The relationship between age and body size for each sex and each population was examined using Pearson’s correlations, possible sex effect was assessed using analysis of co-variance (ANCOVA). Growth patterns were estimated using Von Bertalanffy’s equation, SVLt = SVLasymp (1 - e-k(t-t0)) where t represents number of growing seasons (i.e., years), SVLt stands for body length at age t, SVLasymp represents the estimated asymptotic body size that can theoretically be reached, k is the growth coefficient, and t0 represents the intercept at the tempo-

Measure of “rapprochement” Ectopterygoids of adults presenting a clear sequence of SGM were photographed using a LEICA DC300F camera assembled with a WILD HEERBRUGG MAKROSKOP M 420 1.25×. Using an image-editing computer software, the distance from the basis of the first visible zone (corresponding to the first active season bone growth) and the basis of next zone was measured along a straight line broadly perpendicular to SGM and transecting LAGs (Fig. 2). The thickness of each zone + annulus complex represents one year of bone growth (G) expressed as follow:

Fig. 2. Parameters for the measure of “rapprochement” (see text for definition).

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Growth, longevity and age at maturity in the European whip snakes ral axis, thus hypothetical age at size 0. The parameters SVLasymp, k and t0 and their asymptotic confidence intervals were estimated using nonlinear least-squares regressions. The Von Bertalanffy equation was fitted to age and size data for sexes within a population and for each population. Data of newborns of undetermined gender were used to build both male and female curves in all the populations. Two estimated SVLasymp, k and t0 values were considered to be significantly different (at the 0.95 level) when their confidence intervals did not overlap. The potential reproductive lifespan was calculated for each sex and each population by subtracting the estimated age at maturity from the respective age of the oldest individual found (longevity). The estimated ages at maturity for each sex, within each population, were then substituted in the respective derived Bertalanffy’s equation to obtain size at maturity. Small sample sizes precluded performing several analyses (e.g. analyses of bone growth patterns were reliable results for Chizé and Montecristo populations only).

RESULTS

Body size, age, longevity and population age structures Chizé. The whole sample (n = 72) included 46 males, 24 females and 2 newborn specimens of undetermined sex. Sixty-five specimens out of 72 were adults, and, among these, 43 were males and 22 females. Adult body size was normally distributed (Kolmogorov-Smirnov test, Z = 0.632, P = 0.820) and adult males were significantly larger than females (t-test, t = 1.171, df = 58, P = 0.001). It was possible to reliably estimate the age of 90.3% (65 of 72; Fig. 3) of the whole sample. Mean estimated population age was 12.1, ranging from 0 to 24 years; additional details are provided in Table 1A. Montecristo. The overall Montecristo sample (n = 28) included 14 males, 13 females and 1 specimen of undetermined sex. All determined males and females in the population were adults, the only juvenile being of undetermined gender. Adult body size was normally distributed (Kolmogorov-Smirnov test, Z = 0.392, P = 0.998) and there was no significant difference in mean adult body length between the sexes (t-test, t = 0.995, df = 23, P = 0.330). It was possible to reliably estimate the age of 92.9% (26 of 28) of the whole sample. Estimated mean population age was 19.4, ranging from 0 to 29 years (details in Table 1B). Calimera. The Apulian sample (n = 32) included 21 males, 6 females and 5 newborns or juveniles of undetermined gender. All males were adults, while two out of the six females were subadults; on the whole the sample included therefore 25 adult specimens and seven subadults. Adult body size was normally distributed (Kolmogorov-Smirnov test, Z = 0.817, P = 0.517), however,

Fig. 3. Examples of SGM reading on ectopterygoids, Chizé sample. a) 0 yr; b) 12 yr; c) 20 yr; d) 13 yr; the arrow indicates a double zone, and white dots indicate the age in years. Table 1. Sample sizes, Mean, minimum and maximum age values estimated for Hierophis sample. Values are reported in years. A) Chizé

n

Mean age (± 1SD)

Min.

Max. (Longevity)

Whole population Adult males Adult females Subadults

65 39 19 7

12.06 ± 5.06 14.31 ± 3.11 11.42 ± 3.58 1.29 ± 1.98

0 10 6 0

24 24 20 5

26 13 12 1

19.35 ± 5.87 22 ± 4.58 18.08 ± 3.37 /

0 14 11 /

29 29 24 /

32 21 4 7

15.69 ± 9.31 19.9 ± 6.33 18.5 ± 3.70 1.43 ± 1.62

0 7 14 0

33 33 22 4

B) Montecristo Whole population Adult males Adult females Subadults C) Calimera Whole population Adult males Adult females Subadults

considering the small number of adult females in the sample (n = 4), mean SVL was compared between sexes using a Mann-Whitney U test. The analysis revealed that there was no significant difference in mean adult body

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Table 2. Results of statistical comparisons of age distributions between the different populations. Sex

Populations compared

n1

n2

Z

Asymp. Sig.

Exact Sig.

Males

Chizé - Montecristo Chizé - Calimera Montecristo - Calimera

39 39 13

13 21 21

2.242 2.260 0.540

< 0.001** < 0.001** 0.933

< 0.001** < 0.001** 0.788

Females

Chizé - Montecristo Chizé - Calimera Montecristo - Calimera

19 19 12

12 4 4

2.117 1.435 0.577

< 0.001** 0.033* 0.893

< 0.001** 0.016* 0.807

length between the sexes (U = 23.500, P = 0.201). This result could however be the consequence of the relatively small data-set of females. Although SGMs reading was difficult in many cases, as a consequence of bone remodelling and of “rapprochement” of outer SGMs, it was possible to estimate the age in 100% of the whole sample. Mean population age was 15.7, ranging from 0 to 33 years (details in Table 1C). In all the three populations, age values were normally distributed (Kolmogorov-Smirnov test. Chizé: Z = 0.969, P = 0.305; Montecristo: Z = 0.554, P = 0.919; Calimera: Z = 0.607, P = 0.855) and adult males were on average significantly older than females in Chizé and Montecristo (t-test, t. Chizé: 3.160, df = 56, P = 0.003; Montecristo: 2.417, df = 23, P = 0.024), not in Calimera (Mann-Whitney U test = 34.5, P = 0.577). In the three populations age distributions did not differ significantly between the sexes, when considering only adult specimens (Kolmogorov-Smirnov Z test. Chizé: 1.230, P = 0.097; Montecristo: 1.121, P = 0.162; Calimera: 0.611, P = 0.849). In Chizé, Exact test showed a significant tendency towards a leftshifted age distribution in adult females with respect to adult males (P = 0.033). However, in Montecristo and Calimera this pattern was not evident (Exact test. Montecristo: P = 0.093; Calimera: P = 0.653).

assumption was not met, but the ANOVA is quite robust to violations of this assumption, Zar, 1984). The Bonferroni Post-Hoc test revealed that Chizé females were larger than Montecristo females (P = 0.003). Mean estimated age was significantly different between adult males from the three populations (F2,70 = 19.195, P < 0.001). Bonferroni Post-Hoc multiple comparison test revealed that males from Chizé were significantly younger than males from Montecristo and from Apulia (both P < 0.001), while no differences were found between adult males from the two Italian populations (P = 0.575). Similarly, adult females from the three sites showed significant differences in mean estimated age (ANOVA, same design, F2,32 = 16.066, P < 0.001), females from Chizé being significantly younger than females from Montecristo and Apulia (Bonferroni Post-Hoc test, P < 0.001 and P = 0.003 respectively). The mean age between the two Italian female samples was not significantly different (P = 1). In Table 2 we reported results of Kolmogorov-Smirnov tests on differences in age distributions among sexes and sites: both age distributions of males and females from France are significantly shifted to the left (thus to younger ages) with respect to age distributions of Italian whip snakes. Moreover, no significant differences in age distributions were found between the two Italian populations in both sexes.

Inter-population comparisons

Relationship between age and body size

Mean adult male SVL differed significantly among the three populations (ANOVA, with SVL as the dependent variable and population as the factor, F2, 71 = 36.106, P < 0.001). A Post-Hoc test revealed that males from Chizé were larger compared to the males from the two other populations (Bonferroni Post-Hoc test, both multiple comparison P < 0.001), while males from Montecristo and from Apulia did not differ significantly in mean body size (Bonferroni Post-Hoc test, P = 0.086). The same analysis was then performed on adult females (ANOVA, with SVL as the dependent variable and population as the factor, F2,32 = 6.794, P = 0.003; homogeneity of variances

Body size was highly correlated with age both in the whole population (Pearson correlation. Chizé: r = 0.904, P < 0.001; Montecristo: r = 0.876, P < 0.001; Calimera: r = 0.936, P < 0.001) and considering each sex separately (Pearson correlation. Chizé: rmales = 0.882, Pmales < 0.001, rfemales = 0.838, Pfemales < 0.001; Montecristo rmales = 0.792, Pmales = 0.001, rfemales = 0.818, Pfemales = 0.001; Calimera: rmales = 0.820, Pmales < 0.001; rfemales = 0.921, Pfemales = 0.009). Differences in mean age between the sexes were then re-analyzed taking into account this relationship. An ANCOVA performed with age as the dependent variable, SVL as the covariate and sex as the factor revealed that

Growth, longevity and age at maturity in the European whip snakes

there was no difference between the sexes in age in Chizé and Calimera snakes, when body size was taken into account (Chizé: F1,55 = 0.216, P = 0.644; Calimera: F1,23 = 0.007, P = 0.935). ANCOVA on Montecristo snakes, on the contrary, revealed a significant effect of gender on age (F1,22 = 6.603, P = 0.017). It has to be noted, however, that in all the three populations, the assumption of homogeneity of slopes was not met (P < 0.001). A t-test was then performed comparing the unstandardized residuals obtained from the linear regression of the ln-transformed estimated age on the ln-transformed SVL. The result again highlighted the absence of any significant sexual difference in size-corrected age in Chizé and Calimera snakes (t-test. Chizé: t = 0.177, df = 22.486, P = 0.861; Calimera: t = −0.331, df = 24, P = 0.744). Males from Montecristo were on average older than females when estimated age was corrected for body size (t = 2.473, df = 23, P = 0.021). Inter-population comparisons Taking into account the positive correlation between age and body size, males from the three populations exhibited significant differences in estimated age (ANCOVA, with age as the dependent variable, SVL as the covariate and population as the factor, F2,69 = 89.752, P < 0.001). Bonferroni Post-Hoc multiple comparisons were all highly significant (all P ≤ 0.001), showing that, for similar SVL values, Montecristo males were the oldest, while males from Chizé were the youngest. However, the assumption of homogeneity of slopes was not met (P < 0.001). Yet the differences among populations were marked (Fig. 4a),

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suggesting that possible effect of violation of homogeneity assumption on our main conclusions was limited. The analysis was then performed by considering the unstandardized residuals obtained from the linear regression of the ln-transformed estimated age on the ln-transformed SVL. Results confirmed that population differences in mean age, among males, were not dependent on differences in mean body sizes (ANOVA, with residuals as the dependent variable and population as the factor, F2,68 = 191.326, P < 0.001; all Post-Hoc multiple comparisons P < 0.001; Fig. 5). Within females, age was significantly different among populations, when age-size correlation was considered (ANCOVA, with age as the dependent variable, SVL as the covariate and population as the factor, F2,32 = 41.042, P < 0.001). For similar SVL values, females from Chizé were significantly younger than females from the other two populations (Bonferroni Post-Hoc test, both P < 0.001), while no differences were found between Italian females from Montecristo and from Apulia, even if there was a trend towards older island females and the small Apulian sample size may have influenced the result (P = 0.074; Fig. 4b). Unstandardized residuals of the linear regression of the ln-transformed estimated age on the lntransformed SVL were furthermore analysed, and the over mentioned result was confirmed (ANOVA, with residuals as the dependent variable and population as the factor, F2, 33 = 34.870, P < 0.001). Females from Chizé were significantly younger than females from both Montecristo (PostHoc Dunnett test, P < 0.001) and Calimera (Post-Hoc Dunnett test, P = 0.05), while the difference was not significant between females from Italian populations (PostHoc Dunnett test, P = 0.149; Fig. 5).

Fig. 4. Relationship between age and body size in males (a) and females (b) of the three populations. (• Chizé); (▶ Montecristo); (o Calimera).

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Fig. 5. Intra population and intraspecific differences in size-corrected age. Grey=males; white=females.

Population growth curves In the three studied populations, derived values for SVL asymp, k and t0, their asymptotic confidence intervals and the coefficients of correlation of the Von Bertalanffy equation fitted separately on male and female data (Table 3). In the three populations, modelled curves fit-

ted well the relation between age and body size in both sexes (correlation coefficients in Table 4). Growth curves had similar shapes for males and females (Chize: Fig. 6a; Montecristo: Fig. 6b; Calimera: Fig. 6c): even if SVLasymp was higher in males and k was higher in females, these differences were not significant (confidence intervals widely overlap in all considered populations). In the snakes from Chizé, the estimated male SVLasymp was very close to actual maximum SVL observed in the field (1200 mm, n > 1,600 records; X. Bonnet, personal unpubl. data), while the Von Bertalanffy model slightly underestimated the asymptotic body length for females (maximum SVL observed in the field 1080 mm, n > 1,100 records; X. Bonnet, personal unpubl. data). In the snakes from Montecristo the model provided a satisfactory male asymptotic size (maximum SVL observed in the field 873 mm, n = 53) but slightly overestimated it in females (maximum SVL observed in the field 790 mm, n = 30). In the snakes from Calimera the model slightly overestimated asymptotic sizes, both in males (maximum SVL observed in the field 920 mm, n = 16) and in females (maximum SVL observed in the field 825 mm, n = 4). Inter-population comparisons of growth Comparisons of growth parameters are reported in Table 3. While the estimated k and t0 for males were

Table 3. Coefficients of correlation and parameters of the Von Bertalanffy’s estimated model for male and female H. viridiflavus from the three populations considered. 95% confidence intervals are in parentheses. Sex

SVLasymp

k

t0

R2

1216.97 (1032.86-1401.10) 1012.38 (811.47-1213.28)

0.086 (0.052-0.119) 0.123 (0.049-0.197)

−2.69 (−3.85-−1.52) −2.19 (−3.83-−0.55)

0.925 0.907

Montecristo Males Females

843.05 (686.61-999.50) 854.20 (637.19-1071.22)

0.068 (0.025-0.110) 0.071 (0.018-0.124)

−5.01 (−8.29-−1.75) −4.72 (−7.84-−1.60)

0.943 0.953

Calimera Males Females

965.60 (843.36-1087.82) 861.67 (690.74-1032.59)

0.073 (0.043-0.103) 0.095 (0.028-0.162)

−4.10 (−5.85-−2.34) −3.55 (−5.84-−1.27)

0.958 0.977

Chizé Males Females

Table 4. Coefficients of correlation and parameters of the Von Bertalanffy’s estimated models for the three populations considered. 95% confidence intervals are in parentheses. Population

SVLasymp

k

t0

R2

Chizé Montecristo Calimera

1178.22 (1028.90-1327.54) 820.81 (731.52-910.11) 948.26 (842.03-1054.50)

0.091 (0.060-0.122) 0.077 (0.047-0.107) 0.076 (0.048-0.105)

−2.55 (−3.66-−1.44) −4.55 (−6.92-−2.17) −3.94 (−5.56-−2.31)

0.901 0.907 0.954

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Fig. 7. Estimated Von Bertalanffy’s growth curves for Chizé (—), Montecristo (....) and Calimera (- - -) populations.

Fig. 6. Population growth curves for H. viridiflavus at a) Chizé; b) Montecristo; H. carbonarius at c) Calimera. (• males); (o females); (▶ juveniles).

comparable across populations with a strong overlap of the 95% confidence intervals, there was a limited overlap between confidence intervals for asymptotic SVL between Chizé and Calimera (5.05%) and no overlap with Monte-

cristo (see Table 3). This suggests that males from different populations attained different asymptotic body sizes following comparable growth rates. A different pattern was observed in females: k and t0 were relatively similar across populations with strongly overlapping of 95% confidence intervals, and SVLasymp confidence intervals were overlapping (even if estimated values were dissimilar between populations). We note however, that the small Calimera sample limited the power of this analysis. The absence of significant male-female differences for growth parameters within each sample allowed to estimate cumulative population growth curves by fitting the Von Bertalanffy equation to each population entire data set. Derived values for SVLasymp, k and t0, their asymptotic confidence intervals and the coefficients of correlation of the Von Bertalanffy equation fitted for each population are reported in Table 4. Growth curves for the three sites are plotted in Fig. 6. While the growth coefficient k and the estimated t0 had similar values throughout populations, and their 95% confidence intervals strongly overlapped, there was only a negligible overlap (e.g., 2.43%) between confidence intervals of asymptotic SVL of Chizé population and of Calimera population. There was no overlap between the French versus insular populations. Moreover, the asymptotic SVL estimated for the two Italian populations overlapped only marginally, suggesting that, even if less pronounced, differences in growth pattern also exist between these two populations (Fig. 7). These results suggested that whip snakes from the Forest of Chizé (H. viridiflavus) follow different growth trajectories compared to the two Italian populations (H. viridiflavus, H. carbonarius) and attain larger maximal size. Derived values of the two main growth parameters, SVLasymp and k, for males, females and the whole sample

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Fig. 8. Annual bone growth (Mean G) in males (a) and females (b) Hierophis viridiflavus from Chizé. Lines connect mean values, bars represent ±1SD. Age in years in the abscissa.

were inversely correlated within each population; this relationship was significant for the populations of Chizé and Calimera, while it was not for the population of Montecristo island (Pearson correlation, r = -0.999, P = 0.035, r = -1, P = 0.019 and r = -0.785, P = 0.425 respectively). “Rapprochement”: age and size at maturity and potential reproductive lifespan It was possible to clearly measure successive annual bone growth marks for 4 males and 6 females in Chizé, for 9 males and 7 females in Montecristo and for for 10 males and only 2 females in Calimera. In Chizé, the annual bone growth showed a sudden decrease between the seventh and the eighth year of age for males (see also Fig. 8a, an example for all the populations) and between the sixth and the seventh for females (Fig. 8b). It is therefore likely that males Hierophis viridiflavus of this population attain sexual maturity at 7 years of age, while females become reproductive when they are one year younger, at 6 years of age. In both sexes bone growth was particularly rapid during the second active season, and then it showed a decrease, followed by a subsequent increase and a slowing down until the sharp decrease in connection with sexual maturation. From these estimated values it emerged a potential reproductive lifespan of 17 years for males and 14 for females. Using the derived Bertalanffy’s equation for each sex, it emerged that male size at maturity is 687.9 mm, while females reach sexual maturity at a mean size of 642.6 mm. In Montecristo, annual bone growth in males showed the highest mean value during the second

year of life, then it decreased but remained at more or less constant values until the eighth year, when it showed a strong decrease. Therefore males Hierophis viridiflavus of this population likely reach sexual maturity when they are 8 years old. Females showed a bone growth pattern characterised by a decrease of growth after the second active season, followed by a subsequent increase and a slowing down until sharp decrease between the sixth and the seventh years. However, mean annual bone growth slowed down under initial values only after the eighth year. It is therefore likely that island females, as island males, attain sexual maturity at 8 years of age. Substituting these values in the derived Bertalanffy’s equation for each sex it emerged that male size at maturity is 495.1 mm, while females reach sexual maturity at a mean size of 507.9 mm. Calculated potential reproductive lifespan for males and females were, respectively, 21 and 16 years. In Calimera, unfortunately, as a consequence of bone remodelling, LAGs on ectopterygoids of this population were not so sharply differentiated on the bone surface and measurements of annual radius (e.g., Rn,) were sometimes performed quite subjectively. The following results are therefore only indicative. Male annual bone growth showed the highest mean value during the third year of life, and then it suddenly decreased. The last drop off was in correspondence of the sixth active season (when mean annual bone growth slowed down under initial values). Male Hierophis viridiflavus of Calimera may therefore attain sexual maturity during their sixth year of life, with an estimated potential reproductive lifespan of 27 years. Bone growth pattern was obtained only for two females; hence it was not possible to estimate age at maturity for females of this population. Sub-

Growth, longevity and age at maturity in the European whip snakes

stituting male estimated age at maturity the respective derived Bertalanffy’s equation it emerged that male size at maturity is 503.7 mm. Inter-population comparisons of age at maturity. Only Chizé and Montecristo populations with reliable estimated age at maturity were considered. Plotting estimated values for each sex we observed a positive trend (not significant, Pearson correlation, r = 0.827, P = 0.173), between the estimated age at maturity and longevity. Moreover, there was a clear positive relationship between the estimated SVL at maturity and asymptotic body length (SVLasymp). This correlation was statistically significant (Pearson correlation, r = 0.954, P = 0.046). DISCUSSION

Skeletochronology enabled us to estimate the age of most of the sampled individuals, providing novel information (otherwise unavailable) in both sexes and for wide spectrum of body sizes. Below we first examine broad patterns and then population divergences. Broad patterns In the European whip snake, estimated size at maturity ranged from 57% to 64% of the maximal body size (in Chizé males and Montecristo females respectively), in accordance with previous studies in snakes (Shine and Charnov, 1992). The positive correlations between estimated age at maturity and longevity, or between size at maturity and Bertalanffy’s asymptotic length, also conform to the general pattern described in ectotherms in general (Shine and Charnov, 1992) and in snakes more specifically (Parker and Plummer, 1987). These relationships represent trade-offs between maturation programmes, growth patterns, and costs versus benefits of large body size (Shine and Charnov, 1992). The congruence of our results with previous studies suggests that skeletochronology provided useful information. In the studied populations, we found a strong relationship between estimated age and body size (all R2 > 0.91). Following a fast growing juvenile phase, growth rate decreased with body size. Similar results have been documented in other reptile species monitored through capture-mark-recapture surveys (CMR); growth following asymptotic patterns derived from the von Bertalanffy curve (Dunham, 1978; James, 1991; El Mouden et al., 1999; Bonnet et al., 2011). However, despite a strong rela-

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tionship between age and size, there was a considerable variance in age-related body size, especially among older individuals. Our study confirms that SVL is not a reliable estimator of age in snakes, especially after maturity and in large specimens. Many idiosyncratic and environmental factors influence growth in snakes, notably after maturity, and can explain the strong inter-individual divergences of trajectories (Bronikowski, 2000; Madsen and Shine, 2000; Bonnet et al., 2002; Lelièvre et al., 2013). Sex is one of those important factors (Koos Slob and van der Werff Ten Bosch, 1975; Kuwamura et al., 1994). On average males were older and attained greater maximal longevity compared to females. In the three populations studied, male and female growth curves were not significantly different. Male Hierophis showed the highest values of SVLasymp and the smallest values of k within each population: this could means that i) the tendency for males to reach a larger maximum size approaching this asymptote at slower rate than females is overall present in the populations considered or that ii) males grow more rapidly than females after maturity. Yet, because they continue to grow they need longer time to reach their maximal size. In other words, the trend for a cessation of growth in females means that they reach earlier maximal SVL, but not at a faster rate. Consequently, the observed male-biased sexual size dimorphism (SSD; Fornasiero et al., 2007; Zuffi, 2007) was essentially attributable to a longer period of growth after maturity in males (King, 1989). Sexual bimaturation (sexes maturing at different ages) can also influence SSD (Shine, 1990, 1994): because growth decreases after maturity, earlier maturing sex tend to exhibit smaller mean adult sizes (e.g., Lagarde et al., 2001). Our results provide a partial support to this scenario. In the northern population (Chizé), males reached maturity one year after females, no difference was observed in the Montecristo population, and a small sample size precluded performing robust analyses in the Apulian population. Overall, a longer growth period after maturity in male European whip snakes may explain the larger mean body size in this sex. However, the respective contribution of possible underlying mechanisms remains unknown. Many males are killed during mate searching and suffer from a high-risk mortality compared to females (Bonnet et al., 1999a; Meek, 2009). This sex difference in mortality should induce a female biased SSD and thus does not fit well with the above results. However, survival in emaciated females after reproduction can be very low in snakes (Bonnet et al., 1999b). Examining the effect of sex and age on survival is necessary to clarify these issues (Bonnet et al., 2011). Beside fundamental morphological differences (Bonnet et al., 1998) various ecological and physiological factors may generate sexual divergences in growth rate.

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During the mating season male European whip snakes engage into vigorous ritual combats and display very high testosterone levels (Bonnet and Naulleau, 1996). In reptiles, this androgenic steroid stimulates sexual behaviours and growth, resulting into male biased SSD (Cox et al., 2009). Moreover, high reproductive investment controlled by high estradiol levels during vitellogenesis exhausts body reserves and can markedly hampers growth in females (Bonnet et al., 1994, 2011; Van Dyke and Beaupre, 2011). In species exhibiting male-to-male combats, selection for large body size of males tends to overrule selection for large body size to accommodate larger clutches in females (Shine, 1993, 1994). European whip snakes display all these traits; lower growth rate in females than in males is thus expected in this species but our results show no sex effect. Yet, other key factors may balance growth rates between sexes. For example, males tend to be anorexic during the mating season while females forage during the whole active period (Bonnet and Naulleau, 1996), females possess more developed attributes to process food (Bonnet et al., 1998), and thus females may assimilate greater amounts of food available for growth compared to males. Sex difference in prey selection can also influence body size (Zuffi et al., 2010). Overall, our results regarding age and size pose more questions than offering responses; CMR studies combined with eco-physiological investigations are necessary to obtain a general understanding of the determinants of SSD in free ranging snakes. Inter-population variations We found strong differences in mean adult body size, mean adult age, age distribution, longevity, growth rate, age and size at maturity among the three populations studied. These results are important to better interpret already documented inter population variations in body size and reproductive traits (Fornasiero et al., 2007; Zuffi et al., 2007). The most salient differences were observed between genetically close populations (H. viridiflavus [sub]species) respectively sampled in the northern and southern parts of the distribution range (Chizé versus Montecristo). The genetically distinct third population (H. carbonarius from Apulia) was relatively more similar to the island population (Montecristo). Thus genetic proximity did not translate into similarities in age/size life history traits. On average, the snakes from Chizé were markedly larger and younger compared to the two other populations. Less pronounced differences in body size were found between specimens from Montecristo and Apulia. Snakes from Chizé reached larger asymptotic size without

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difference in the Bertalanffy’s growth coefficients (k), and thus exhibited higher absolute growth rate than snakes from Montecristo and Apulia (Dunham, 1978; Wapstra et al., 2001; Stanford and King, 2004). Additionally, French specimens exhibited lower longevity compared to Italian snakes. Growth rate and mean body size should be higher at lower latitudes in ectotherms due to more favourable temperatures and longer activity period. On the contrary, we found a reverse trend, likely because Mediterranean, dry and arid habitats of the Italian populations offer less favourable trophic (Zuffi, 2007) and hydric conditions compared to more productive areas typical of mild oceanic climate. In fact, during drought periods snakes remain sheltered to maintain their hydro-mineral balance (Bonnet and Brischoux, 2008), even entering into a prolonged estivation period (Vipera aspis, M.A.L. Zuffi, unpublished data). Hierophis carbonarius from Calimera follows an intermediate growth trajectory between the opposite extremes represented by the insular and the northern population, being however closer in this pattern to the former one. Limits of the study Estimating age with skeletal marks does not pose major problems to assess broad patterns for comparisons among sexes and populations because possible methodological biases will apply equally in the different groups examined. However, absolute values should be considered with caution. Inferring the exact age at maturity and exact growth rates rely on a set of assumptions. Our results suggest that whip snakes from Montecristo Island mature at an estimated age of 8 yr, and at a minimum derived body size (SVL) of 495 and 507 mm in males and females respectively. But the smallest female with developed follicles from Montecristo measured 598 mm in SVL, leading to an estimated age of 12 yr according to our growth curves. This discrepancy might be due to insufficient sampling of reproductive females in the field (n = 18), to imprecision in the estimates (e.g., small sample size in juveniles hampered comparing linear versus nonlinear functions to select the best fitting equations between age and body size) or due to other factors (e.g., a lack of perfect correspondence between reduced bone growth and maturity). Indeed, 8yr is already an elevated age for maturity for snakes, 12yr would be a remarkable value. Our results in the Chizé population suggest an estimated age at maturity of 7 yr for males and 6 yr for females, with a derived minimum size at maturity of 687 and 642 mm in SVL respectively. The smallest reproductive female found in the Forest of Chizé measured 680 mm in SVL (X. Bonnet, unpubl. data), a value

Growth, longevity and age at maturity in the European whip snakes

in accord with skeletochronology. However, using our results, it also suggests an estimated age of 7 yr for maturity, a value that does not fit well with CMR data. Lelièvre et al. (2013) showed that juvenile growth rate averages 0.04 cm/day in Chizé, leading to an age for maturity of 3-4 years. As above, this discrepancy might be caused by inappropriate sampling (e.g., few road-kill juveniles were found intact) or to insufficient fitting of the growth models used. Further analyses based on larger number of juveniles are required to better calibrate skeletochronology to CMR data and to take into account marked interindividual differences in growth trajectories. Whatever the case, the strong differences of age at maturity observed between populations likely reflect adaptation to local conditions. The markedly slow growth, delayed maturity, smaller body size and higher longevity of Montecristo snakes are expected in a dry environment where the acquisition of trophic resources (see also Zuffi, 2001) is more challenging than under mild oceanic climate. Relative clutch size, an index of energetic investment per reproductive bout is lower in Montecristo snakes, in accordance with the notion that resources availability is limited in this island (Zuffi et al. 2007). Populations respond by shifting a set of traits along a slow-fast gradient (Stearns and Koella, 1986; Wapstra et al., 2001). Using dead snakes, our results suggest a strong plasticity of major traits driven by trophic and climatic conditions. Collecting opportunistically and examining dead snakes might be useful to address key questions. Major parameters, especially before maturity, can be inferred and implemented to calibrate models that aim to examine the impact of climatic changes. Indeed currently implemented mean values do not permit to encompass the wide range of variations observed among individuals and across populations. ACKNOWLEDGMENTS

We thank Jacques Castanet, Museum National d’Histoire Naturelle in Paris for support during SF stay in Paris. Gunther Köhler at Senkenberger Museum provided several Montecristo samples. Two anonymous referees and Hervé Lelièvre provided helpful comments on previous drafts and revision. REFERENCES

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Acta Herpetologica 11(2): 151-160, 2016 DOI: 10.13128/Acta_Herpetol-17874

Redescription of Cyrtodactylus fumosus (Müller, 1895) (Reptilia: Squamata: Gekkonidae), with a revised identification key to the benttoed geckos of Sulawesi Sven Mecke1,*,§, Lukas Hartmann1,2,§, Felix Mader3, Max Kieckbusch1, Hinrich Kaiser4 1  Department

of Animal Evolution and Systematics and Zoological Collection Marburg, Faculty of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Straße 8, 35032 Marburg, Germany. *Corresponding author. E-mail: [email protected] 2 Current address: Department of Ecology and Evolution, Johann Wolfgang Goethe-Universität – Biologicum, Max-von-Laue-Straße 13, 60438 Frankfurt am Main, Germany 3 Janusstraße 5, 93051 Regensburg, Germany 4 Department of Biology, Victor Valley College, 18422 Bear Valley Road, Victorville, California 92395, USA; and Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013, USA § Co-first authors Submitted on 2016, 27th January; revised on 2016, 16th August; accepted on 2016, 30th August Editor: Aaron M. Bauer

Abstract. The binominal Cyrtodactylus fumosus has frequently been used for populations of bent-toed geckos occurring on some Indonesian islands, including Java, Bali, Sulawesi, and Halmahera. Unfortunately, incorrect usage of this name for different geographic lineages has resulted in confusion about the true identity of C. fumosus. Examination of the type specimen and additional specimens from Rurukan and Mount Masarang, North Sulawesi Province, Indonesia, revealed that this population is distinct from other forms heretofore called ‘fumosus’ by a combination of unique morphological characters. In order to stabilize the taxonomy of C.  fumosus sensu stricto, and to prevent further confusion, we provide a comprehensive redescription of this species, whose distribution we herein restrict to North Sulawesi. Cyrtodactylus fumosus is one of the most distinctive species among the six bent-toed geckos recorded from Sulawesi, and it differs from Sulawesi congeners by the presence of (1) precloacofemoral scales, including three porebearing scales on each thigh, separated from 10 or 11 pore-bearing scales in the precloacal region by 9-11 interscales in males, (2) a precloacal groove in adult males, (3) flat dorsal tubercles in 4-7 irregularly arranged longitudinal rows at midbody, and (4) a distinct lateral fold lacking tubercles. We also provide a revised identification key to the benttoed gecko species of Sulawesi. Keywords. Cyrtodactylus fumosus, C. marmoratus, Lacertilia, bent-toed geckos, reptiles, North Sulawesi, Indonesia, morphology.

INTRODUCTION

The bent-toed gecko fauna of Sulawesi consists of six species, including Cyrtodactylus batik Iskandar et al., 2011; C.  fumosus (Müller, 1895); C.  hitchi Riyanto et al., 2016; C. jellesmae (Boulenger, 1897); C. spinosus Linkem et al., 2008; and C.  wallacei Hayden et al., 2008. Two of ISSN 1827-9635 (print) ISSN 1827-9643 (online)

these, C.  fumosus and C.  jellesmae have been reported from North Sulawesi Province, Indonesia (e.g., Boulenger, 1897; Koch et al., 2009; Iskandar et al., 2011; Koch, 2012). Cyrtodactylus fumosus was described by Müller (1895a) based on a single specimen (NMB-REPT 2662; adult female), collected by Paul Benedict Sarasin (1856-1929) and Karl Friedrich (“Fritz”) Sarasin (1859-1942) in the © Firenze University Press www.fupress.com/ah

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“Boelawa Mountains” (= Huidu Matabulawa) of northern Sulawesi. Following its original description, C. fumosus was also reported from localities outside of Sulawesi (e.g., De Rooij, 1915; Mertens, 1929, 1934; Manthey and Grossmann, 1997; McKay, 2006; Oliver et al., 2009; Das, 2010; Koch, 2012; De Lisle et al., 2013; Riyanto et al., 2013, 2015), leading to the perception of a wide distribution and a rather inconsistent or even erroneous definition of the taxon, since the name became applied to bent-toed gecko species not representing C. fumosus sensu stricto (see Hartmann et al., 2016). Boulenger (1897) was the only author who provided a detailed, though not entirely correct (see Hartmann et al., 2016: footnote 1), species account for C.  fumosus sensu stricto, based on specimens from North Sulawesi. The recent identification of new species from the Sunda Islands masquerading under the name C.  fumosus (Riyanto et al., 2015; Hartmann et al., 2016) and re-examination of C.  fumosus specimens from North Sulawesi, however, show that the taxonomy of C. fumosus is in disarray, and this makes it necessary to properly redescribe this conspicuous taxon based on a multitude of eidonomic characters, some of which have never been provided in the literature. Whereas Hartmann et al. (2016) already published remarks on the taxonomy of C.  fumosus and provided selected comparative morphological data for this species, a comprehensive redescription of C.  fumosus is necessary to stabilize the taxonomy of a species that has experienced prominent use in the literature, but whose identity has regularly been misconstrued. This redescription, featured below, can serve as solid basis for the delineation and description of additional new species of bent-toed geckos currently recognized as C. fumosus, and allows the correction of comparative literature data. MATERIALS AND METHODS Our redescription of Cyrtodactylus fumosus is based on the examination of four specimens of that taxon, including the holotype (NMB-REPT 2662) and three additional specimens (NMB-REPT 2663; BMNH 1895.2.27.7, 1896.12.9.3). For each specimen used in the redescription, we recorded data for 31 eidonomic characters (see Table 1 for definitions and abbreviations). Of these, 14 were metric and 16 meristic. We also calculated the following ratios: AxialL/SVL, ArmL/SVL, LegL/SVL, HeadL/SVL, HeadW/HeadL, SnoutL/HeadL, SnoutL/OrbD, and MentalH/MentalW. All measurements were taken to the nearest 0.1 mm using digital calipers. Scale counts and observations of external morphology were made using a dissection microscope. Characters occurring bilaterally were measured or counted on the right side of specimens, unless stated otherwise; for femoral pores, interscales, and labial scales, we provide counts for both sides of the body (the prefixes “R” and “L” are

used to distinguish characters counted on the right or left side, respectively). In our diagnosis, ranges are followed by means ± standard deviations. For descriptions of pattern and coloration we apply the terminology of Köhler (2012). Numbers in parentheses behind the respective capitalized color name refer to the coding therein. The terminology used to distinguish between different depressed precloacal areas follows Mecke et al. (2016). Drawings are based on photographs of ethanol-preserved specimens and were prepared using the program CorelDraw Graphics Suite X3. Museum abbreviations follow Sabaj Pérez (2014).

RESULTS

Cyrtodactylus fumosus (Müller, 1895) (Figs 1; 2) Gymnodactylus fumosus Müller, 1895a: 833 (holotype NMB-REPT 2662; type locality: “Boelawa Gebirge,” Sulawesi, elevation 1200 m) Gymnodactylus fumosus—Müller, 1895b: 865 Gymnodactylus fumosus—Boulenger, 1897: 204 Gymnodactylus fumosus (part.)—De Rooij, 1915: 16 Gymnodactylus fumosus—Brongersma, 1934: 168 Gymnodactylus fumosus—Brongersma, 1953: 172 Gymnodactylus fumosus—Kramer, 1979: 160 Cyrtodactylus fumosus (part.)—Manthey and Grossmann, 1997: 222 Cyrtodactylus fumosus (part.)—Grismer, 2005: 429 Cyrtodactylus fumosus (part.)—Grismer and Leong, 2005: 588 Cyrtodactylus fumosus (part.)—Biswas, 2007: 19 Cyrtodactylus fumosus (part.)—Hayden et al., 2008: 109 Cyrtodactylus fumosus (part.)—Rösler and Glaw, 2008: 14 Cyrtodactylus fumosus (part.)—Chan and Norhayati, 2010: 50 Cyrtodactylus fumosus (part.)—Das, 2010: 209 Cyrtodactylus fumosus (part.)—Iskandar et al., 2011: 65 Cyrtodactylus fumosus (part.)—Koch, 2012: 161 Cyrtodactylus fumosus—Hartmann et al., 2016: 556 Cyrtodactylus fumosus (part)—Riyanto et al., 2016: 69 Cyrtodactylus fumosus—Mecke et al., 2016: 356 Holotype: NMB-REPT 2662 (Fig. 1A and Table 2; Hartmann et al. 2016: Fig.  5): adult female (SVL = 77.8 mm) collected by Paul and Fritz Sarasin in 1894; terra typica: “Boelawa Gebirge” (= Huidu Matabulawa), corrected to “Bone Mountains” (= Pegunungan Bone, North Sulawesi Province, Indonesia) by Boulenger (1897). Referred specimens: NMB 2663 (Fig. 1B): Mount Masarang; BMNH 1895.2.27.7 (Fig. 1C; same specimen figured in Boulenger, 1897: Plate VII, Fig. 2), 1896.12.9.3 (Fig. 1D): Rurukan. Definition: Cyrtodactylus fumosus is a moderatelysized bent-toed gecko species (maximum SVL = 78 mm)

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Redescription of Cyrtodactylus fumosus Table 1. Metric and meristic characters with abbreviations used in this study. Character Snout-vent length Axial length Tail length Arm length Leg length Head length Head width Head height Snout length Orbit-Ear distance Orbital diameter Ear length Mental length Mental width

Abbreviation Definition SVL AxialL TailL ArmL LegL HeadL HeadW HeadH SnoutL OrbEarD OrbD EarL MentalL MentalW

Dorsal tubercle rows

DTR

Paravertebral tubercles

PVT

Ventral scales Precloacofemoral scales Femoral pores

VS PFS FP

Interscalesa

InterS

Precloacal pores Postcloacal tubercles

PP PCT

Subdigital lamellae under 4th toe

LT4

Supralabial scales 1

SupraLab1

Supralabial scales 2

SupraLab2

Infralabial scales

InfraLab

Internasal scales Supraciliar scales Interorbital scales Gular scales

InterNas SC IOS GulS

a

From tip of snout to cloaca From axilla to groin From cloaca to tip of tail From insertion of brachium into body wall to claw of longest finger From insertion of thigh into body wall to claw of longest toe From tip of snout to articulation of quadrate bone with lower jaw Measured at level of ear openings Measured at level of ear openings From tip of snout to anterior margin of orbit From posterior margin of orbit to anterior margin of ear opening From anterior to posterior margin of orbit From anterior to posterior margin of ear opening Maximum length of mental shield Maximum width of mental shield Number of longitudinal tubercle rows on dorsum at midbody, counted in one row between lateral folds Number of tubercles counted in a longitudinal row between posterior insertion of forelimb and anterior insertion of hindlimb Number of ventral scales at midbody, counted in one row between lateral folds Number of enlarged precloacofemoral scales, counted along lowest, pore-bearing series Number of femoral pores on enlarged scales on thigh Number of enlarged poreless scales between series of pore-bearing precloacal scales and series of pore-bearing femoral scales on thigh Number of precloacal pores situated in precloacal groove Number of postcloacal tubercles Number of subdigital scales under 4th toe, counted from first enlarged scale (lamellae) on lower side of toe to scale proximal to apical scale Number of labial scales of upper jaw, beginning with first enlarged scale bordering rostral shield, ending with last enlarged scale bordering labial angle Number of labial scales of upper jaw, beginning with first enlarged scale bordering rostral shield, ending with enlarged scale below anterior margin of eye Number of labial scales of lower jaw, beginning with first scale bordering mental shield, ending with last enlarged scale bordering labial angle Number of scales between rostronasals, bordering rostral Number of enlarged scales extending from anterior-ventral to posterior-dorsal edge of orbit Number of scales counted in a row between the medial edges of orbits across head Number of gular scales bordering pair of first postmentals

Rösler et al. (2007); Hartmann et al. (2016); and Mecke et al. (2016) referred to interscales as “infrascales.”

that can be readily distinguished from all other congeners occurring in the Greater and Lesser Sunda Islands, Sulawesi, and the Maluku Islands by the following combination of characters: (1) a single series of precloacofemoral scales, including three pore-bearing scales on each thigh, separated from 10 or 11 pore-bearing scales in the precloacal region by 9-11 interscales in males (Fig. 2A), (2) a precloacal groove in adult males (Fig. 2A), (3) posterior precloacal scales (Fig. 2A), (4) flat and smooth (unkeeled) dorsal tubercles in 4-7 irregularly arranged

longitudinal rows at midbody (Fig. 2B), (5) a distinct lateral fold lacking tubercles, (6) 37-50 longitudinal rows of ventral scales at midbody, (7) 17-23 scales under 4th toe, and (8) a horizontal slit-like ear opening. Comparisons: Characters distinguishing Cyrtodactylus fumosus from all other species of Cyrtodactylus occurring on the Sunda Islands and Sulawesi were provided by Mecke et al. (2016: Table 2). Here, our comparisons are limited to Sulawesi taxa, with characters of C.  fumosus provided in parentheses. Cyrtodactylus batik can be

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Fig. 2. Diagnostic characters of Cyrtodactylus fumosus. (A) Precloacofemoral region (with pore-bearing precloacal scales and groove shaded in grey) of a male specimen (BMNH 1896.12.9.3), showing precloacal and femoral pores. Scale bar equals 2 mm (B) Dorsum, showing tubercles (holotype NMB-REPT 2662). Scale bar equals 2 mm. (C) Ventral side of anterior part of head (holotype NMB-REPT 2662). Scale bar equals 1 mm. Drawings prepared by Felix Mader based on photographs by Sven Mecke. Fig. 1. Dorsal views of the known specimens of Cyrtodactylus fumosus: (A) NMB-REPT 2662 (holotype, adult female); (B) NMBREPT 2663 (subadult male); (C) BMNH 1895.2.27.7 (adult female); (D) BMNH 1896.12.9.3 (adult male). Photographs by Sven Mecke. BMNH 1895.2.27.7 is also figured (in dorsal view) in Boulenger (1897: Plate VII, Fig. 2).

distinguished from C.  fumosus by a larger size of adults with a maximum SVL of 115 mm (78 mm); the absence of PFS (PFS present); the absence of PP and FP in both sexes (PP and FP present in males); the absence of a precloacal depression in both sexes (precloacal groove present in males); 23-26 slightly keeled DTR (4-7 unkeeled DTR); the presence of tubercles on the lateral skin fold (tubercles on lateral skin fold absent); 24-27 LT4 (17-23 LT4); and the presence of transversely enlarged subcaudal scales in a single row (enlarged, paired median subcaudals) (Iskandar et al., 2011; Riyanto et al., 2016). Cyrtodactylus hitchi can be distinguished from C.  fumosus by the absence of PFS (PFS present); the absence of PP and FP in both sexes (PP and FP present in males); the absence of a precloacal depression in both sexes (precloacal groove present in males); the presence of 18-20 keeled DTR (4-7 unkeeled DTR); the presence of tubercles on the lateral skin fold (tubercles on lateral skin fold

absent); and the presence of transversely enlarged subcaudal scales in a single row (enlarged paired median subcaudals) (Riyanto et al., 2016). Cyrtodactylus jellesmae can be distinguished from C.  fumosus by the absence of PFS (PFS present); the absence of PP and FP in both sexes (PP and FP present in males); the absence of a precloacal depression in both sexes (precloacal groove present in males); the presence of 13-22 raised DTR (4-7 flat DTR); the presence of tubercles on the lateral skin fold (tubercles on lateral skin fold absent); and the absence of enlarged subcaudal scales (enlarged paired median subcaudals present) (Boulenger, 1897; Mecke et al., 2016, pers. obs.). Cyrtodactylus spinosus can be distinguished from C.  fumosus by the absence of a continuous series of enlarged precloacal and femoral scales (PFS present); by widely spaced femoral scales (femoral scales juxtaposed); the presence of a shallow precloacal pit in males (deep precloacal groove in males); the presence of lateral and caudal spines (spines absent); and the presence of a prehensile tail (tail not prehensile) (Linkem et al., 2008; Harvey et al., 2016). Cyrtodactylus wallacei can be distinguished from C. fumosus by a larger size of adults, reaching a maximum SVL of 114 mm (78 mm); the absence of PFS (PFS present); the absence of PP and FP in both sexes (PP and FP present in males); the absence of a pre-

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Redescription of Cyrtodactylus fumosus Table 2. Metric (in mm) and meristic data for the known specimens of Cyrtodactylus fumosus. Abbreviations are defined in Table 1. Characters occurring bilaterally were measured or counted on the right side of specimens, unless stated otherwise; for femoral pores, interscales, and labial scales we provide counts for both sides of the body (the prefixes “R” and “L” are used to distinguish characters counted on the right and left side, respectively). n/a = not applicable. Our metric data of BMNH 1895.2.27.7, the only known specimen with an original tail (TailL = 67.1), well agree with the measurements listed by Boulenger (1897), who also provided a drawing of a specimen (Plate VII, Fig. 2) identifiable as BMNH 1895.2.27.7. NMB-REPT NMB-REPT BMNH 2662 2663 1895.2.27.7 (holotype) Sex SVL AxialL ArmL LegL HeadL HeadW HeadH SnoutL OrbEarD OrbD EarL DTR PVT VS PFS FP InterS PP LT4 (proximal) LT4 (distal) LT4 SupraLab1 SupraLab2 InfraLab GulS

Female 77.8 35.2 35.7 43.9 21.3 14.2 9.2 8.8 6.6 5.2 1.2 5 13 38 46 0 n/a 0 7 10 17 R12 L12 R6 L5 R9 L11 9

Male 56.6 22.2 22.1 29.6 15.7 10.6 7.0 6.9 4.1 3.6 1.2 7 16 37 45 R3 L3 R10 L9 11 8 11 19 R13 L13 R6 L6 R10 L10 8

Female 60.7 28.3 24.9 32.9 16.8 11.9 6.7 7.7 4.3 4.0 2.0 4 14 47 46 0 n/a 0 10 13 23 R11 L12 R6 L6 R11 L10 7

BMNH 1896.12.9.3 Male 77.5 31.4 32.9 42.0 20.4 14.5 9.5 9.4 6.3 4.1 2.3 6 18 50 39 R3 L3 R10 L11 10 9 (L) 12 (L) 21 (L) R11 L12 R6 L6 R8 L8 8

cloacal depression in both sexes (precloacal groove present in males); and the presence of 23-25 slightly keeled, trihedral DTR (4-7 unkeeled and flat DTR) (Hayden et al., 2008). Description of the holotype. General habitus, metrics, and ratios: Adult female; SVL = 77.8 mm; AxialL = 35.2 mm; TailL (broken, only tail stump present) = 8.7 mm; ArmL = 35.7 mm; LegL = 43.9 mm; HeadL = 21.3 mm; HeadW = 14.2 mm; HeadH = 9.2 mm; SnoutL = 8.8 mm; OrbEarD = 6.6 mm; OrbD = 5.2 mm; EarL = 1.2

mm; head rather short (HeadL/SVL = 0.27) and wide (HeadW/HeadL = 0.67), clearly depressed between eyes, distinct from neck; snout rather elongate (SnoutL/HeadL = 0.41), longer than OrbD (SnoutL/OrbD = 1.69), canthus rostralis distinct; fore- and hindlimbs of moderate size (ArmL/SVL = 0.46; LegL/SVL = 0.56), without webbing between digits; relative length of fingers = IV > III > V > II > I; relative length of toes = IV > III > V > II > I; lateral skin fold distinct, lacking tubercles. Scalation: Dorsal scales granulate, interspersed with slightly enlarged, flat, roundish and irregularly arranged dorsal tubercles (Fig. 2B), 5 DTR; 13 PVT; tubercles on occiput, neck, and hindlimbs similar in shape to those on dorsum (no tubercles present on the forelimbs). Thirty-eight VS, distinctly larger than those on dorsum, juxtaposed; a single series of 46 poreless PFS; enlarged posterior precloacal scales present, arranged in a chevron-like shape consisting of five series of scales (from anterior to posterior: 10/ 8/ 7/ 6/ 2 scales); 2 flat PCT; number of lamellae under fingers: I 12, II 16, III 16, IV 18, V 16; number of lamellae under toes: I 13, II 15, III 17, IV 17, V 16. Rostral shield rectangular, 2.2 times as wide as high, partly divided by a median, vertical furrow, in contact with 1st SupraLab, 2 rostro-nasals and a single InterNas; naris surrounded by rostral, 1st SupraLab, a single rostronasal, and three post-nasals; R12 L12 SupraLab1, R6 L5 SupraLab2, separated from orbit by three rows of small granular scales; R9 L11 InfraLab; cephalic scales small, rounded, granulate and juxtaposed; tubercles on occiput and neck flat and unkeeled; 40 SC; 46 IOS; mental triangular, wider than long (MentalW/MentalL = 1.7); one pair of enlarged 1st postmentals, enlarged 2nd postmentals absent (Fig. 2C); pair of 1st postmentals bordered by mental, 1st InfraLab, and 9 GulS (Fig. 2C); scales on throat minute and rounded. Coloration: Natural color and pattern altered due to preservation. Ground color of dorsum CinnamonDrab (50); head darker than dorsum, Burnt Umber (48) in color, with indistinct Warm Sepia (40) stripe running from posterior border of orbits along neck, forming a collar at level of posterior margin of forelimbs; labial scales Buff (5), stippled with darker color, with stipples most concentrated at edges of some scales; dorsum with irregular, faint Dark Drab (45) blotches, not arranged in distinct pairs, most visible on vertebral region between forelimbs and on mid-dorsum; ground color of dorsal surface of limbs similar to ground color of dorsum; limbs with diffuse Dark Drab (45) markings; venter, throat and lower surface of limbs uniformly Smoke Grey (266), heavily dotted; color of dorsal and ventral surfaces of tail stump similar to dorsal and ventral ground color, respectively.

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Intraspecific variation: Our assessment of the variation is based on the holotype and three additional specimens from North Sulawesi (one adult and one subadult male, one adult female) unless stated otherwise. Measurements (in mm) are listed as range followed by mean ± standard deviation provided in parentheses: SVL = 56.677.8 (68.2 ± 11.1); AxialL = 22.2-35.2 (29.3 ± 5.5); TailL (original tail) = 67.1 (n = 1); ArmL = 22.1-35.7 (28.9 ± 6.4); LegL = 29.6-43.9 (37.1 ± 6.9); HeadL = 15.7-21.3 (18.6 ± 2.7); HeadW = 10.6-14.5 (12.8 ± 1.9); HeadH = 6.7-9.5 (8.1 ± 1.5); SnoutL = 6.9-9.4 (8.2 ± 1.1); OrbEarD = 4.1-6.6 (5.3 ± 1.3); OrbD = 3.6-5.2 (4.2 ± 0.7); EarL = 1.2-2.3 (1.7 ± 0.6). Ratios: AxialL/SVL = 0.39-0.47 (0.43 ± 0.03); ArmL/SVL = 0.39-0.46 (0.42 ± 0.03); LegL/SVL = 0.52-0.56 (0.54 ± 0.02); HeadL/SVL = 0.27-0.28 (0.27 ± 0.01); HeadW/HeadL = 0.67-0.71 (0.69 ± 0.02); SnoutL/ HeadL = 0.41-0.46 (0.44 ± 0.02); SnoutL/OrbD = 1.692.29 (1.96 ± 0.25); RostralW/RostralH = 1.53-2.18 (1.91 ± 0.28); MentalW/MentalL = 1.29-1.83 (1.64 ± 0.24). Scale counts are listed as range followed by mean ± standard deviation provided in parentheses: DTR = 4-7 (5.75 ± 1.3); PVT = 13-18 (15.25 ± 2.2); VS = 37-50 (43 ± 6.5); PFS = 39-46 (44 ± 3.4), only a single series present; enlarged posterior precloacal scales consisting of 5 or 6 series; PCT = 2-3, flat in shape; LT4 = 17-23 (19 ± 2.8); SupraLab1 = 11-13 on right side of head and 12-13 on left side of head; InfraLab = 8-11 on right side of head and 8-11 on left side of head; SC = 32-40 (33.5 ± 4.4); IOS = 45-49 (47.3 ± 2.1); GulS = 7-9. Furthermore, all specimens possess a distinct lateral skin fold lacking tubercles and a horizontal, slit-like ear opening. A distinctive row of 5 or 6 tubercles on the dorsal surface of the upper leg is present in three specimens (absent in the holotype). Specimens with unregenerated tails possess two strongly enlarged median subcaudal rows. Unlike female specimens, male specimens of Cyrtodactylus fumosus (n = 2) possess three pore-bearing scales on each thigh, separated from 10 or 11 pore-bearing precloacal scales by 9-11 InterS. A distinct precloacal groove is fully developed in adult males (n = 1) only. Data of measurements and scale counts for the main characters of the holotype and additional specimens used for the diagnosis are provided in Table 2. Ground color of dorsal surface of body, head, and tail varies considerably between the specimens available to us and appears to depend on the respective preservation method. Hence, ground color of dorsal surface varies from Cinnamon (255) over Cinnamon-Drab (50) to Drab (19), with the specimens housed in NMB being darker than the ones housed in BMNH; dorsum with 4-7, sometimes indistinct, Warm Sepia (40) blotches; original tail (n = 1) with six Warm Sepia (40) blotches; regener-

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ated tail of one specimen (BMNH 1896.12.9.3) possesses three indistinct, partially interrupted, Warm Sepia (40) lines, running from base to tip of tail; dorsal surface of limbs and head with diffuse Warm Sepia (40) or Dark Drab (45) markings; venter, lower surface of limbs, and throat uniformly Pale Buff (1) or Smoke Grey (266 and 267). See Fig. 1 for coloration and pattern of preserved specimens. Distribution and natural history: Although the name Cyrtodactylus fumosus has frequently been applied to bent-toed gecko populations from Java, Bali, Halmahera, and the entire island of Sulawesi (e.g., De Rooij, 1915; Grismer, 2005; Das, 2010; De Lisle et al., 2013; Riyanto and Mumpini, 2013; Riyanto et al., 2015), C.  fumosus sensu stricto is only known from the four specimens featured herein, all of which were collected in North Sulawesi (Müller, 1895a, b; Boulenger, 1897; see Fig. 3). The occurrence of C.  fumosus on Lembeh Island, off the coast of northern Sulawesi (Grismer, 2005: Appendix 1, Grimser and Leong, 2005: Appendix  1), appears to be based on misidentified specimens, since the data (including key characters for diagnosis) provided by Grismser (2005: Table 2) and Grismer and Leong (2005: Table 2) do not match those of C.  fumosus sensu stricto as reported herein. Moreover, the data provided by Grismer (2005) and Grismer and Leong (2005: Table 2) seem to be partly based on the erroneous description of C.  fumosus provided by De Rooij (1915) (see Hartmann et al., 2016). According to the data provided by Müller (1895a, b), specimens of Cyrtodactylus fumosus sensu stricto were collected at elevations 1200-1260 m, in a terrain that is, based on satellite images (Google Earth, viewed on 24 January 2016), covered with montane rainforest. Although there are only limited data available on the natural history of C.  fumosus, we believe the species to be restricted to montane rainforest habitats in North Sulawesi. The distribution of C.  fumosus, as currently known, overlaps with the range of C. jellesmae, the only other species of Cyrtodactylus known from North Sulawesi. Figure 3 shows the distribution of the six benttoed geckos currently known from Sulawesi. Remarks on the identity of Cyrtodactylus fumosus from Java: Hartmann et al. (2016) discussed the status of Cyrtodactylus fumosus populations outside of Sulawesi and came to the conclusion that these records were based on erroneous data provided in the literature (e.g., De Rooij, 1915) and/or misidentified specimens. Recently, Riyanto et al. (2015) applied the name C.  fumosus to populations of bent-toed geckos from Java, which are unequivocally identifiable as belonging to the C. marmoratus (Gray, 1831) complex. These authors largely based their

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Redescription of Cyrtodactylus fumosus

Fig. 3. Map of Sulawesi showing the distribution of the six species of Cyrtodactylus currently recognized from this island: Cyrtodactylus batik (inverted black triangle), C. fumosus (black star), C. hitchi (black circle), C. jellesmae (white circle), C. spinosus (black triangle), and C.  wallacei (black diamond). Records are based on specimens listed in the appendices and data provided in Hayden et al. (2008), Linkem et al. (2008), Iskandar et al. (2011), Wanger et al., (2011), Koch (2012), Riyanto et al., (2016). A white circle with a black dot represents a photo-voucher for C. jellesmae available to us. Base map modified from Wikipedia © Sadalmelik / Wikimedia Commons / CC-BY-SA-3.0 by Max Kieckbusch.

assumption on De Rooij (1915), who mainly distinguished between C.  fumosus and C.  marmoratus by a continuous or discontinuous pore series, respectively. However, De Rooij (1915) largely based her definition of C. fumosus on Boulenger (1897), who erroneously reported this species to have a continuous pore series, and her personal examination of specimens housed in the collections of BMNH and SMF, which are conspecific with C.  halmahericus (Mertens, 1929) (see Hartmann et al., 2016: Footnote 1). Cyrtodactylus halmahericus, unlike C. fumosus, possesses a continuous pore series in males (a redescription of C. halmahericus is currently underway). Whereas the lectotype of C.  marmoratus (RMNH. RENA 2710a.1; adult male), all other adult male paralectotypes housed in RMNH (RMNH.RENA 2710a.2-a.5, 2710.1-2), and several other adult male specimens we have examined personally, possess a continuous series of pores (precloacofemoral pores), this character may vary ontogenetically (Brongersma, 1953, pers. obs.), between sexes (Rösler et al.; 2007, Mecke et al., 2016), and between C.  marmoratus sensu stricto and morphologically similar species masquerading under this name.

Cyrtodactylus fumosus can be easily distinguished from C.  marmoratus as currently defined by the following characters: (1) a discontinuous series of precloacal (10 or 11) and femoral pores (three on each thigh) in males, (2) the absence of pores in females, (3) the presence of posterior precloacal scales, (4) the presence of widely scattered, roundish, flat, and smooth dorsal tubercles in 4-7 rows at midbody (11-19 in the type series of C.  marmoratus at RMNH), (5) 14-18 paravertebral tubercles (22-29 in in the type series of C.  marmoratus at RMNH), and enlarged paired median subcaudals (enlarged subcaudals absent in C. marmoratus). It is obvious that the male specimen (MZB.Lace 12903) identified as Cyrtodactylus fumosus by Riyanto et al. (2015) and depicted in their Fig. 4B is not conspecific with C.  fumosus, because it possesses a continuous pore series and lacks posterior precloacal scales. The precloacofemoral region of that specimen rather matches that of C. marmoratus sensu stricto (see Hartmann et al., 2016: Fig. 3H, Mecke et al., 2016: Fig. 1A). Since Riyanto et al. (2015) failed to properly identify C.  fumosus and C.  marmoratus, their comparative Table 3 should not be used for the identification of these taxa. The example well demonstrates the importance of examining relevant type specimens before taxonomic decisions are made. DISCUSSION

The phylogenetic affinities of Cyrtodactylus fumosus remain unclear. The presence of pores, a precloacal depression in males, and posterior precloacal scales are shared with other species of Cyrtodactylus from the region, e.g., C.  halmahericus (Halmahera) and C.  klakahensis Hartmann et al., 2016 (eastern Java), with which it may be closely allied1. By contrast, C. fumosus might represent an offshoot of an exclusive clade containing Sulawesi bent-toed geckos only. Results of studies on Sulawesi amphibians and reptiles suggest that this island is herpetogeographically complex, supporting taxa of both Sundaic and Australopapuan affinities (Koch, 2011, 2012), including endemics (e.g., How and Kitchener, 1997; Whitten et al., 2001; Koch, 2011, 2012). The restriction of Cyrtodactylus fumosus to Sulawesi underscores that this island holds a significant amount 1 Cyrtodactylus petani Riyanto et al., 2015 also shares with C.  fumosus the presence of pores and posterior precloacal scales. Riyanto et al. (2015) provided inconsistent data on whether a precloacal groove is present in male specimens of C.  petani. However, male C.  petani lack a precloacal groove or pit (Awal Riyanto, in litt.; Mecke et al., 2016).

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of endemism. The species is apparently only found in the mountains of North Sulawesi Province, and such a limited range exemplifies that isolated geographic features in this region (e.g., mountain ranges) may be the key locales for such endemism. According to Koch (2012: Table 11) more than 20 amphibians and reptiles (including candidate species) are endemic to northern Sulawesi. Most of these appear to be endemic to offshore islands, but we hypothesize that the North Sulawesi mountain ranges may harbor a higher number of endemic herpetofaunal taxa than generally assumed as well. We disagree with Iskandar et al. (2011), who considered that the Sulawesi herpetofauna is impoverished compared to other regions in Southeast Asia, largely due to natural factors alone. The high rate at which new amphibian and reptile species are being discovered on Sulawesi contradicts this hypothesis, and the relatively low diversity may simply reflect the limited amount of fieldwork conducted there to date. Since 2000, 16 reptile species have been described from Sulawesi (e.g., Tropidophorus baconi Hikida et al., 2003; Calamaria butonensis Howard and Gillespie, 2007; Rabdion grovesi Amarasinghe et al., 2015), a number that equals ~15% of the reptiles known from this island. The number of described species of Cyrtodactylus in Sulawesi alone increased by 200% during the last decade. Preliminary examination of preserved bent-toed geckos from Sulawesi in museum collections suggests that at least one undescribed species of bent-toed gecko is present on the island. Photographic images of specimens in life available to us indicate that a further three species of Cyrtodactylus from Sulawesi are yet to be described. Therefore we agree with e.g., Linkem et al. (2008), and Koch (2011, 2012), who considered the herpetological diversity of Sulawesi to be underestimated. KEY TO THE SPECIES OF THE GENUS CYRTODACTYLUS OF SULAWESI

This key is applicable to identify adult bent-toed geckos based on non-sexually dimorphic characteristics, although characters present in males only may accompany a choice. 1a Long spines on lateral fold and lateral portion of tail present; tail prehensile C. spinosus 1b Long spines on lateral fold and lateral portion of tail absent; tail not prehensile 2 2a Enlarged precloacofemoral scales present in both sexes, bearing a total number of 16 or 17 pores in males, 10 or 11 of which are precloacal pores and 3 of which are femoral pores; pore-bearing scales separated by

2b 3a 3b 4a 4b 5a 5b

9-11 enlarged interscales; precloacal groove present in males; no tubercles on lateral fold C. fumosus Enlarged precloacofemoral scales; pores; precloacal groove; and tubercles on lateral fold absent 3 Enlarged median subcaudals absent C. jellesmae Enlarged median subcaudals present 4 Enlarged subcaudals in multiple rows C. wallacei Enlarged subcaudals in a single row for most of the tail’s length 5 24-27 lamellae under 4th toe; SVL in adults 103-113 mm C. batik 18-21 lamellae under 4th toe; SVL in adults 62-79 mm C. hitchi ACKNOWLEDGEMENTS

The authors thank Denis Vallan and Urs Wüest (NMB), Patrick Campbell (BMNH), Esther Dondorp (RMNH), Raffael Ernst and Markus Auer (MTKD), Christopher J. Raxworthy, David A. Kizirian, David A. Dickey, and Lauren Vonnahme (AMNH), Joseph Martinez and José Rosado (MCZ), and Gunther Köhler and Linda Acker (SMF), for allowing examination of material in their care. We also thank Ka Schuster (PhilippsUniversität Marburg, Germany) for reading and commenting on a draft of this publication, and Olivier S.G. Pauwels (RBINS) and Lee L. Grismer (LSUHC) for their helpful reviews, which greatly improved this publication. This study was supported by an AMNH collection study grant to SM. REFERENCES

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Whitten, T., Henderson, G.S., Mustafa, M. (2001): Ecology of Sulawesi. Periplus Editions, Singapore. APPENDIX Specimens examined for diagnosis and comparison Cyrtodactylus fumosus.—Indonesia: North Sulawesi Province: Bone Mountains (Pegunungan Bone, 1200 m a.s.l.): NMB 2662 (holotype); Mount Masarang: NMB 2663; Rurukan: BMNH 1895.2.27.7, 1896.12.9.3. Cyrtodactylus halmahericus.—Indonesia: North Maluku Province: North Halmahera: MCZ Herp R-19279, SMF 8230 (paratype); Central Halmahera: Oba (Payahe): SMF 8232 (paratype); Soah Konorah (Soakonora): SMF 8233 (holotype). Cyrtodactylus jellesmae.—Indonesia: Central Sulawesi Province: Malakosa, Kuala Navusu: AMNH R142969-73; Tolai, Sungai River: AMNH R142974; North Sulawesi Province: Kema: NMB-REPT 2659 (paralectotype); Buol: NMB-REPT 2660 (lectotype); Mount Masarang: NMB-REPT 2661 (paralectotype); Pulau Biaro: MCZ 171466; South Sulawesi Province: Lowah (Muara Loa): MCZ 25337. Cyrtodactylus klakahensis.—Indonesia: Jawa Timur Province: Lumajang, Klakah: SMF 22476 (holotype); SMF 22477-79 (paratypes). Cyrtodactylus marmoratus.—Indonesia: Java: RMNH.RENA 2710.1-8 (paralectotypes), RMNH.RENA 2710a.1 (lectotype), RMNH.RENA 2710a.2-6 (paralectotypes), MTKD 8903-05, SMF 8218; West Java: RMNH.RENA 9847, ZMA.RENA 15387 (three specimens); Jawa Barat Province: Garoet (Garut Regency): RMNH.RENA 9846 (three specimens), RMNH.RENA 10114 (two specimens), Kamodjang (Kawah Kamojang): RMNH.RENA 9849; Jawa Tengah Province: “Goewa Djatidjadjar Jdjoe Bagelen” (= Gua Jatijajar, Kebumen); Karangpucung: SMF 92361; Jawa Timur Province: Malang: RMNH.RENA 9848 (two specimens). Cyrtodactylus petani.—Indonesia: Jawa Timur Province: Toelong Agoeng (Tulungagung Regency): ZMA.RENA 11353.

Acta Herpetologica 11(2): 161-169, 2016 DOI: 10.13128/Acta_Herpetol-18201

The castaway: characteristic islet features affect the ecology of the most isolated European lizard Petros Lymberakis1, Efstratios D. Valakos2, Kostas Sagonas2, Panayiotis Pafilis3,* 1 Natural

History Museum of Crete, University of Crete. Knossos Av., P.B. 2208, 71409, Irakleio, Crete, Greece Section of Animal and Human Physiology, Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, GR-15784, Greece 3 Section of Zoology and Marine Biology, Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, GR-15784, Greece. * Corresponding author. E-mail: [email protected] 2

Submitted on 2016, 27th April; revised on 2016, 3rd June; accepted on 2016, 20th July Editor: Adriana Bellati

Abstract. The ecological importance of islet endemics are in the front line of conservation efforts and thus the good knowledge of their biology is required. Podarcis levendis is a lacertid lizard, endemic to two rocky islets in the Cretan Sea, Greece, that was raised to specific level in 2008 and since then no data on its biology are available. Here we present the first ecological information on the species, focusing on population density, tail autotomy and feeding preferences. We recorded regenerated and damaged tails in the field and estimated population density with the transect method. We also dissected museum specimens and analyzed their stomach content. Regenerated tails were common and reached a considerable 71%. The latter finding could be attributed to the intense intraspecific competition due to high population density but also to the seasonal predation pressure by migratory birds. The diet of P. levendis coincides with that of other insular congenerics, including high percentages of plant material. Keywords. Islands, population density, intraspecific competition, feeding ecology, Lacertidae, Podarcis levendis.

INTRODUCTION

Deviations of island life from mainland norms have been repeatedly underlined (Van Valen, 1973; Adler and Levins, 1994). Studying the constellation of these departures, island biology attracted scientific interest and became a hot spot in ecological and evolutionary studies during the last 30 years (Carlquist, 1974; Losos and Ricklefs, 2009). Herpetological research is in the frontline of this general trend. Insular giants (Harlow et al., 2010) and dwarfs (Hedges and Thomas, 2001) and the impressive adaptations of island herpetofaunas (Herrel et al., 2008; Stuart et al., 2014) stimulate a growing body of literature. In this framework, Mediterranean islands lend themselves to understanding the particularities of insularity. Among the numerous endemic reptiles ISSN 1827-9635 (print) ISSN 1827-9643 (online)

and amphibians that live on the Mediterranean islands, Podarcis wall lizards (family Lacertidae) stand out. Podarcis is the largest genus of European lizards comprising 23 species that occur in Europe and North Africa (Uetz and Hošek, 2016). Though most Podarcis species have both mainland an insular populations, 10 of them are strictly endemic to Mediterranean islands. The overall biology of these species depart in many aspects from their mainland peers, including thermal biology (Grbac and Bauwens, 2001; Adamopoulou and Valakos, 2005), feeding ecology (Salvador, 1986; Capula and Luiselli, 1994), life history (Adamopoulou and Valakos, 2000; Castilla and Bauwens, 2000), digestive performance (Pafilis et al., 2007, in press), defensive tactics (Pafilis et al., 2009a; Li et al., 2014), and behavior (Pérez-Mellado et al., 2000; Traveset and Riera, 2005; Cooper et al., 2009; © Firenze University Press www.fupress.com/ah

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Brock et al., 2014a). Podarcis islanders differ in the limits of their range. Three of them live on very large islands (P. tiliguerta in Corsica and Sardinia, P. waglerianus in Sicily and P. cretensis in Crete), six on medium-sized islands (e.g., P. filfolensis in Malta, P. gaigeae in Skyros), while three are exclusively restricted on small islets: P. lilfordi lives on rocky islets off Menorca and Mallorca and the Cabrera Archipelago in Spain, P. raffonei inhabits three rocky islets and few isolated places on Vulcano Island at the Aeolian Islands in Italy, and P. levendis occurs only on two islets in the west Cretan Sea in Greece. Podarcis levendis was recognized as distinct species and separated from P. erhardii, which dominates the south and central Aegean islands, only recently (Lymberakis et al., 2008). The species is found on the islets Pori and Lagouvardos, north of Antikythira Island, between Crete and the Peloponnese (Fig. 1). This very restricted and remote range had two main consequences: the complete lack of knowledge on the biology of the species since its first description, and its categorization by IUCN as ‘vulnerable’. To the best of our knowledge, the only other paper referring to the Pori population is the first record of lizards (under the former name, P. erhardii) on the islet (Valakos et al., 1999).

Petros Lymberakis et alii

In order to effectively protect species that may face the threat of extinction we need to know their biology as best we can. In this study we provide the first ecological data for P. levendis. We assessed population density, body size (estimating also body condition, a proxy for fitness), frequencies of autotomized tails and feeding preferences of the Pori population. We made three hypotheses. First, we predicted that the population should be dense. Due to their small area, islets usually host very few (or even no) predators and, as a result, lizard densities are usually high (Pérez-Mellado et al., 2008). Alternatively high densities could be attributed to high food abundance through ‘marine subsidies’ (Polis and Hurd, 1996). Second, we expected high frequencies of broken tails. Intraspecific competition on islets could be intense due to high population density and these antagonistic interactions may induce caudal autotomy (Pafilis et al., 2008; Cooper et al., 2015). Furthermore Pori serves as a refueling station of migratory birds that increase predation pressure during their stay on the islet. Third, we hypothesized that plant material would constitute a significant part of stomach content as many microinsular lacertids demonstrate a clear shift towards herbivory (Van Damme, 1999).

Fig. 1. Map of the two islets hosting P. levendis in northwest Cretan Sea (Greece, NE Mediterranean Basin).

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Ecology of an islet lizard

MATERIALS AND METHODS Study system Levendis wall lizard (Podarcis levendis) is a well-built, medium sized lacertid lizard (snout vent length, SVL, 72.25 ± 3.44 mm). The vegetation on the islets Pori (0.317 km2, max altitude: 129 m) and Lagouvardos (0.0127 km2, max altitude: 19.3 m) consists of sparse phrygana shrubs among which Sarcopoterium spinosum and Euphorbia dendroides are the most common while several Olea europaea and Pistacea lentiscus are also present. The only other terrestrial reptile living on the two islets is the Kotschy’s gecko (Mediodactylus kotschyi). Fieldwork was conducted on Pori islet in May 2010. Lizards were captured by noose and then transferred to the laboratory facilities of the Department of Biology at the University of Athens. Animals were housed individually in vitreous terraria (80×30×40 cm) with sand and artificial shelters and were held at 30oC under a controlled photoperiod with fluorescent tube lighting (12 h light: 12 h dark). Additional incandescent lamps (60 W) allowed animals to thermoregulate for 8 h/day. Lizards had access to water ad libitum and were fed every other day with mealworms (Tenebrio molitor), coated with a powder containing vitamins and minerals supplements (TerraVit Powder, JBL GmbH & Co. KG). Morphological measurements For each lizard we recorded SVL and body mass using a digital caliper (Silverline 380244, accurate to 0.01 mm) and a digital scale (Ohaus, Scout-TM, accurate to 0.01g), respectively. To define body condition (BC) we included only lizards with intact or fully regenerated tail and non-gravid females. We estimated BC using the classical body mass index (mass divided by SVL), a standard measure for reptiles (Goodman, 2008; Battles et al., 2013; Damas-Moreira et al., 2014), and tested for differences between sexes using a Mann-Whitney test. Additionally, we examined the tail condition (intact or broken/regenerated) in the 22 adult lizards (16 males and 6 females) that were captured in the field and also in 44 museum specimens (18 males and 26 females) that are deposited to the Herpetological Collection of the Natural History Museum of Crete (collected in January 1992). Chi-square test was used to examine for differences between sexes.

Diet composition To assess the feeding preferences of the P. levendis we dissected 36 preserved specimens (15 males and 21 females) that were deposited to the Herpetological Collection of the Natural History Museum of Crete and removed the digestive tract to examine the prey remnants. Prey items were analyzed under a binocular dissecting microscope and identified to order level. We recorded the percentage of the total number of prey items found in the stomachs (%n) as well as the percentage of lizards that ate a given prey taxon (F). Spearman correlation was performed to test whether F is related to %n. We also recorded the consumption of plant material (estimated as frequency of presence). We used the Shannon-Wiener diversity index (Krebs, 1998): H’ = ∑pilnpi, to calculate the niche breadth (H’), where pi is the percentage of each prey item found in the stomachs. We conducted a t-test to search for differences between sexes regarding the diversity index. As a complementary approach to test for sexual differences in food composition and to control for the effects of the most abundant prey items, we used the Jaccard similarity coefficient (Jaccard, 1908) as implemented in PAST (Hammer et al., 2001):

J(A,B) =

X∩Y , X∪Y

where X and Y correspond to the sets of entities that occur at A and B groups, respectively. Finally, using the program EcoSim 7.0 (Gotelli and Entsminger, 2001), we employed the Pianka’s overlap index (Qjk) and estimated the food niche similarity between sexes (Pianka, 1975):

Q jk =

∑p p ∑p p

ij ik 2 ij

2 ik

,

where j and k refer to the two groups under comparison and pij and pik to the proportion of the food component i in each group.

RESULTS Population density We estimated population density using the line transect method (Lovich et al., 2012). Eight random line transects of 100 m were walked by the same observer (LP) and all lizards seen within 4-m wide belt (2 m on either side of the survey line; total area covered per trail 400 m2) were recorded. Transect surveys were made during morning hours when Podarcis lizards are active and line transects were chosen to cover as many different microhabitats as possible.

Morphological measurements and population density Males had significant longer SVL than females (Mann-Whitney test; Z = 2.469, P = 0.013) (Table 1). In addition, males were heavier compared to females, but these differences were not statistically significant (MannWhitney test; Z = 1.769, P = 0.077). Finally, we found no significant differences in body condition between sexes (Mann-Whitney test; Z = 0.589, P = 0.55) (Table 1).

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Table 1. Snout vent length (SVL; mm), body mass (BM; g) and body condition (BC) of males (n = 16) and females (n = 16) of P. levendis population at Pori islet. Table reports means ± SD values; ranges are also given (within brackets). Sexes males females

SVL

BM

BC

75.25 ± 2.11 (71.0-79.0) 66.00 ± 9.48 (54.0-76.0)

8.21 ± 0.78 (6.7-9.5) 7.07 ± 1.46 (5.0-8.8)

0.11 ± 0.01 (0.09-0.13) 0.11 ± 0.01 (0.09-0.12)

Table 2. Diet composition of P. levendis for males and females during summer. %n refers to the percentage of prey items in the stomachs, F refers to the proportion of lizards having eaten a specific prey category while H’ to the Shannon-Wiener diversity index. Sexes

Males

Females

F

%n

F

%n

Araneae Opilionidia Orthoptera Gastropoda Homoptera Diptera Isopoda Lepidoptera Coleoptera Hymenoptera Formicidae Insect larvae Plant material

0.40 0.00 0.07 0.20 0.00 0.07 0.13 0.13 0.67 0.07 0.20 0.33 0.13

23.08 0.00 1.92 7.69 0.00 1.92 3.85 3.85 28.85 3.85 7.69 17.31

0.29 0.14 0.05 0.24 0.14 0.00 0.14 0.10 0.48 0.00 0.43 0.19 0.14

18.18 4.55 1.52 9.09 4.55 0.00 10.61 3.03 24.24 0.00 16.67 7.58

Total Preys Specimens Prey/stomach H’

52 15 3.47 1.923

66 21 3.14 2.054

We examined tail condition in 66 adult lizards overall. 47 of them (71%) had regenerated or broken tails. We found no differences in the percentage of autotomized tails between males and females, either for lizards that we captured in the field (males: 75% and females: 67%; χ2 = 0.008, P = 0.275), or for museum specimens (males: 66% and females: 73%; χ2 = 0.006, P = 0.357). Pori islet hosts a rather dense population of approximately 262 lizards per hectare. Taking into account the area of the islet (31.74 ha) our finding regarding population density provides the first (though rough) estimation of the total population of the species on the islet, which should be less than 7,000 individuals (given that

the peripheral parts of the islands close to the sea are not available to lizards). Diet composition We found a significant correlation between the proportion of the number of prey taxa in the stomachs (%n) and the proportion of lizards that ate that given taxa (F) (Spearman test, r = 0.99, P < 0.05) (Table 2). The predominant prey groups in the diet of P. levendis were Coleoptera, Araneae and Formicidae (Table 2). We also found a considerable consumption of plant material, mostly leaves (five out of 36 lizards; 14%). Food niche breath was high for both males (H’ = 1.923) and females (H’ = 2.054) and the comparison between them revealed no differences (t-test; t = 1.042, P = 0.30). We found no statistically significant differences between males and females regarding the total number of prey items found in the stomachs (Mann-Whitney test; Z = 0.513, P = 0.608). It is worth noting that almost 20% of the specimens examined had empty stomachs. Finally, our results revealed a high food niche overlap between males and females (Qjk = 0.91), despite the relative low Jaccard similarity index (0.67). DISCUSSION

Our results suggest that the particular conditions on Pori islet shaped the ecological profile of P. levendis. Our initial hypotheses were verified: Levendis wall lizard enhances its diet with plant material and autotomizes its tail frequently. Population density was high, though not as high as in other islet Podarcis. Body condition of lizards was remarkably high, suggesting a sufficient energy flow. Population density was estimated at 262 individuals per hectare. This is a rather high value for eastern Mediterranean standards where insular lizards do not form very dense populations. Valakos (1990) reported 76 lizards/ha (P. erhardii, Naxos Island), Pafilis et al. (2009b) 95-185 lizards/ha (P. gaigeae, Skyros Island), Adamopoulou (1999) 395 lizards/ha (P. milensis, Milos Island) and Chondropoulos and Lykakis (1983) found densities that varied across islands from 118 to 247 lizards/ha (P. tauricus, numerous Ionian islands). A striking deviation comes from Diavates islet (off Skyros) that harbors 875 lizards/ha, the denser population on east Mediterranean islands (Pafilis et al., 2013). Lizard densities from western Mediterranean islands are much higher (Delaugerre and Cheylan, 1992; Scalerà et al., 2004), reaching even 8,000 lizards/ha (Pérez-Mellado et al., 2008).

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Ecology of an islet lizard

Insular populations are much denser than the mainland ones (Rodda et al., 2001; Buckley and Roughgarden, 2006) as a result of the relaxed predation and interspecific competition on the islands (Buckley and Jetz, 2007; Novosolov et al., 2016). This is the case for Pori islet where there is not a single terrestrial predator while the only other reptile is Kotshyi’s gecko (Mediodactylus kotschyi), which does not compete with Podarcis lizards for food or space (Valakos and Vlachopanos, 1989). Certain birds nest on the islet, such as falcons (Falco eleonorae), European shags (Phalacrocorax aristotelis) and yellow-legged gulls (Larus michahellis). These birds do not prey on Podarcis lizards (Walter, 1967; Pérez-Mellado et al., 2014). To the contrary, lizards seem to have developed a particular mutualism, at least with falcons, and benefit from their presence (Walter, 1967; Delaugerre et al., 2012). In addition, sea birds enhance islet ecosystems through ‘marine subsidies’ (Polis and Hurd, 1996) that fuel dense lizard populations (Barrett et al., 2005). Some Mediterranean islets also receive this sea-derived energy and thus may support high lizard abundances (Vidal et al., 2001; Pafilis et al., 2011). The frequencies of autotomized tails are typically considered to reflect predation pressure (Arnold, 1988). However, intraspecific competition through aggressive encounters may also end up to caudal autotomy (Pafilis et al., 2008; Pafilis et al., 2009b). The high lizard densities on Mediterranean islets have been proven to be the major factor inducing tail shedding (Cooper et al., 2015; Donihue et al., 2015). Predation pressure is minimal at Pori, so the high population density should account for the high percentages of regenerated and broken tails in the field. The observed 71% lies within the top percentile for other islet Podarcis (Pérez-Mellado et al., 1997; Pafilis et al., 2008; Pafilis et al., 2009a; Brock et al., 2014a). Nevertheless, we may not exclude the possibility of predation pressure resulting from bird predators during migration as the islets are on an important migratory route (Hellenic Ornithological Society, 2016). Food abundance on Mediterranean islands is quite restricted (Brown and Pérez-Mellado, 1994; Blondel et al., 2010). As a result insular lacertids widen their feeding preferences and thus adopt a wider food niche breadth (Pérez-Mellado and Corti, 1993; Sagonas et al., 2015a). Food scarcity is even more exacerbated on small islets (Pérez-Mellado and Corti, 1993; Castilla et al., 2008) where lizards adopt extreme feeding behaviors in order to survive (Castilla and Herrel, 2009; Brock et al., 2014b). Though our sample size was rather small, our data implied that P. levendis followed the general trophic profile of the genus feeding mainly on terrestrial invertebrates with a clear preference on insects (Arnold, 1987). The

predominant food groups were Coleoptera, Araneae and Formicidae (Table 2). Araneae and, much more, Coleoptera are the commonest prey groups in Podarcis lizards (Maragou et al., 1997; Carretero, 2004; Zuffi and Giannelli, 2013). Myrmecophagy, on the other hand, is a typical feeding strategy of island Podarcis, especially during late spring and summer when other food resources come in short supplies (Valakos, 1986; Pérez-Mellado and Corti, 1993; Valakos et al., 1997; Adamopoulou et al., 1999; Lo Cascio and Capula, 2011). Ants may be less rich in terms of nutrients and energy compared to beetles, but they compensate this disadvantage with their high numbers. Previous studies reported an important shift towards herbivory in most island lacertid populations (PérezMellado and Corti, 1993; Van Damme, 1999; Sagonas et al., 2015b). The percentage of plant material in the stomachs of P. levendis was not very high, though it seems to conform to this trend (14%). Islet populations follow the aforementioned pattern, demonstrating a clear preference for plant consumption (Lo Cascio and Pasta, 2006; Lo Cascio et al., 2006; Carretero et al., 2010; Pérez-Cembranos et al., 2016) that in some cases may be extreme and even induce morphological changes (Herrel et al., 2008; Vervust et al., 2010). Mediterranean islets are demanding habitats with limited food resources (Ouboter, 1981; Brown et al., 1992). Podarcis lizards living on islets share many common characteristics imposed by the particular conditions of these habitats: low predation pressure (Tsasi et al., 2009; Durand et al., 2012), high population densities (Pérez-Mellado et al., 2008) and strong intraspecific competition (Cooper et al., 2015), while they usually have high body condition (Van den Berg et al., 2015). Podarcis levendis conforms to this general pattern. The importance of such isolated populations, some of which have developped unique adaptations (Pérez-Mellado et al., 2000; Herrel et al., 2008; Raia et al., 2010), is of top priority in conservation biology (Capula et al., 2002). ACKNOWLEDGEMENTS

We are grateful to Grigoris Kapsalas for drawing the map. Fieldwork and lab measurements were conducted in accordance with the Greek National Legislation (Presidential Decree 67/81). REFERENCES

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Acta Herpetologica 11(2): 171-178, 2016 DOI: 10.13128/Acta_Herpetol-15109

Sources of calcium for the agamid lizard Psammophilus blanfordanus during embryonic development Jyoti Jee1, Birendra Kumar Mohapatra2, Sushil Kumar Dutta1, Gunanidhi Sahoo1,3,* Department of Zoology, North Orissa University, Baripada, Odisha 757 003, India. *Corresponding author. E-mail: gunanidhi.nou@ gmail.com 2 Institute of Mineral & Materials Technology, Bhubaneswar, Odisha 751 013, India 3 Current address: Post Graduate Department of Zoology, Utkal University, Bhubaneswar 751 004, India 1

Submitted on 2014, 26th October; revised on 2015, 14th May; accepted on 2016, 30th August Editor: Aaron M. Bauer

Abstract. We determined the sources of calcium for the developing embryo and the parallel changes in eggshell structure in the Indian agamid lizard Psammophilus blanfordanus. The developing eggs were opened at 0 (freshly laid), 10, 20, 30, 35, 38, and 40 days of incubation and at hatching (day 41) and subjected to chemical and structural analyses. The oval and flexible-shelled eggs had undergone significant changes in size (40% increase in length, 68% increase in breadth and 315% increase in weight) from laying to hatching. The fresh eggshell contained 2.76 mg (12.51%) calcium whereas the hatched eggshell had only 1.02 mg (7.20%), or a 63% reduction from its original content. The yolk + fluids fraction provides only 0.47 mg to the 1.76 mg of calcium in the hatchling, the rest being resorbed from the eggshell during development. The fresh eggshell (62 µm thick) had a rough granular structure in its calcareous layer with near uniform rectangular/polygonal fields made up of globules of varying sizes. The membrane layer had a multilayered mat of interwoven, irregularly oriented and bifurcated, fibres of uneven thickness. The spherical globules were absent at several places in the hatched eggshell as a result of eggshell calcium utilisation by the developing embryo. Hence, like that of most reptiles, the eggshell of Psammophilus blanfordanus also acts as a secondary source of calcium for the developing embryo. The embryo utilizes the eggshell calcium towards the end of development. Keywords. Psammophilus blanfordanus, embryonic development, eggshell, calcium, ultrastructure.

INTRODUCTION

Eggshell structure of reptiles is diverse, ranging from the small, flexible, parchment-shelled eggs of most squamates to the large, rigid-shelled eggs of crocodilians. It consists of an outer, inorganic layer underlain by an organic (shell) membrane comprised of multiple layers of fibres. The structural units in the calcareous layer are also diverse (Schleich and Kästle, 1988). The eggshell of reptiles plays a complex, but only partially understood, role in development, particularly as a source of inorganic ions (Packard et al., 1992). Some major functions of the eggshell are to accommodate permeability to gas and water, ISSN 1827-9635 (print) ISSN 1827-9643 (online)

provide mechanical stability, and to serve as a potential calcium reserve (Schleich and Kästle, 1988). The eggshells of most oviparous lepidosourians are fibrous and poorly calcified (Packard et al., 1982a, b; Packard and Demarco, 1991; Thompson et al., 2001).The inorganic (mineral) content of the shell is usually restricted to the outermost portion and is comprised mainly of calcium carbonate in the form of calcite (Packard et al., 1982a, b, Deeming, 1988). Yolk provides a considerable amount of calcium during embryonic growth for most oviparous reptiles whereas the eggshell calcium supplements that of yolk late in the incubation period (Packard et al., 1984, 1985; Shad© Firenze University Press www.fupress.com/ah

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rix et al., 1994; Packard and Clark, 1996; Stewart et al., 2004; Sahoo et al., 2009). As embryos of oviparous squamates depend heavily on the yolk as a source of calcium, their calcium mobilisation pattern has been proposed as the most appropriate model for functional characteristics of the common ancestor of oviparous amniotes (Packard and Seymour, 1997; Stewart et al., 2004). The morphological changes in eggshell structure are indeed caused by the mobilisation of eggshell calcium by the developing embryo. The present work describes the eggshell calcium utilization pattern and the parallel structural changes in the eggshell of an agamid lizard, Psammophilus blanfordanus, during various stages of embryonic development.

To 10 ml of the digestate, 20 ml distilled water, 2-3 ml NaOH solution and 3-4 drops of calcon were added to make the solution alkaline. This solution was titrated against 0.02 M EDTA. Samples with very low levels of calcium (e.g., egg components) were analyzed using an atomic absorption spectrophotometer (Perkinelmer, Analyst 200) with standard solutions of 1 ppm, 2 ppm, 4 ppm and 10 ppm of calcium (r = 0.995074). Magnesium in the digestate was analyzed by an atomic absorption spectrophotometer and potassium by a flame photometer (Systronics, Model K-III). Phosphorus was determined with a spectrophotometer (Varian, Model – Carry 100). The characteristic blue colour was developed with a phosphomolybdenum complex and ascorbic acid and the intensity of the colour was measured at 882 nm. Structural analysis

MATERIAL AND METHODS Five clutches of Psammophilus blanfordanus eggs (n= 12-14) were collected on campus of North Orissa University from nests just after oviposition in July, 2013 and brought to the laboratory. The eggs were measured, weighed and numbered. Two eggs from each clutch were reserved for chemical analyses. The remaining eggs of each clutch were incubated in separate plastic boxes at ambient temperature (33° C average, range = 28–38° C) with sandy soil as the medium of incubation. The soil medium was rehydrated at regular intervals. The development of two more clutches (n = 13 and 12) were also observed in their original nests from the time of oviposition. Laboratory eggs were maintained at similar hydric condition as those in the field. One egg from each clutch from both field and laboratory studies was removed on days 10, 20, 30, 35, 38, and 40 of incubation. Eggs (total of eight sample days – freshly laid, 10, 20, 30, 35, 38, 40 day and hatched) were washed in tap water; egg dimensions and weight were measured and eggs were separated into shell and egg components which included yolk, albumen and embryo including the extraembryonic membranes) and subjected to chemical and structural analyses. Chemical analysis Determination of inorganic composition requires conversion of the solid samples to liquid form through acid digestion. The samples (eggshell and egg components: yolk, albumen and embryo) were dried to constant weight at 80 ºC in an oven and digested following the method of Geisey and Weiner (1978) with a slight modification. Pre-weighed samples (about 1 g) were heated to 100˚C until dry with 20 ml of concentrated nitric acid, cooled and again heated until dry with 5 ml of the acid. After cooling, 10 ml of 30% hydrogen peroxide was added to the digestate and heated for 30 minutes until a clear solution was obtained. Distilled water was added to reconstitute the samples that were then filtered and diluted to 100 ml. Calcium estimation was done gravimetrically using calcon indicator (0.4 g solid calcon dissolved in 100 ml methanol).

The structure of eggshells (fresh, 10 day, 20 day, 30 day, 35 day, 38 day, 40 day) was determined with a scanning electron microscope (JEOL, JSM 35 CF) with working voltage of 5-15 kv at magnifications ranging from 100 to 1500x. The eggshell samples were air dried and broken into pieces suitable for microscopic observations. For SEM study, outer surface, radial section and sub-surface samples were mounted in stubs with doublesided adhesive carbon tape and coated with gold for 2 minutes by an Ion sputterer (JFC-1100). The thickness of the fiber and mineral layers was measured using the scale bar on the photomicrographs. Tests for homogeneity of variance, one way ANOVA and Tamhane’s post-hoc tests were conducted for comparing the differences in eggshell calcium concentration at all eight developmental stages using SPSS 15.0 (SPSS Inc., Chicago)

RESULTS

Physical characteristics The physical and chemical composition of Psammophilus blanfordanus eggs from both laboratory and in-situ field samples were similar and were therefore pooled for analysis (Table 1). The incubation period varied from 41-42 days (laboratory eggs) to 44 days (field eggs). At oviposition, eggs were 1.21 ± 0.46 cm long, 0.75 ± 0.27 cm in diameter, and weighted 0.393 ± 0.021 g, n =65); variation in linear dimensions and weight among clutches was not significant (one-way ANOVA and Tamhane’s post-hoc test, P < 0.05). The wet weight of eggshells ranged from 0.037 to 0.048 g (0.042 ± 0.004, n =10). The average wet weight of the egg contents was 0.351 g ± 0.019 (88.4%) whereas the eggshell formed 11.6% of the total egg weight. The fresh egg contained about 62.8% water. The total dry weight varied from 0.132 to 0.166 g (0.146 ± 0.007 g). The shell weight varied from 0.020 to 0.027 g and the egg content

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Sources of calcium for Psammophilus

Table 1. Changes in physical and chemical composition of Psammophilus blanfordanus eggs during embryonic development (n = 7 per sample date, field and laboratory eggs combined). Calcium content (dry weight) Wet weight (g)

Day of incubation

Freshly laid 10 day 20 day 30 day 35day 38 day 40 day Hatched

Dry weight (g)

Egg contents including embryo

Eggshell

Total wt.

Shell

Egg component

Total wt.

Shell

Egg component

mg

%

mg

%

0.393 0.526 0.603 1.120 1.454 1.586 1.623 1.631

0.042 0.044 0.051 0.269 0.211 0.127 0.176 0.087

0.351 0.482 0.602 0.851 1.243 1.459 1.447 1.544

0.146 0.145 0.144 0.122 0.139 0.159 0.167 0.102

0.022 0.022 0.021 0.020 0.017 0.015 0.014 0.014

0.124 0.123 0.123 0.102 0.122 0.143 0.153 0.088

2.76 2.19 2.04 1.73 1.61 1.05 1.03 1.02

12.51 9.95 9.71 9.63 9.42 7.48 7.30 7.20

0.47 0.47 0.55 0.59 0.74 1.38 1.54 1.76

0.31 0.31 0.41 0.43 0.62 1.69 1.80 1.91

Fig.1. Changes in the size of Psammophilus blanfordanus eggs as a function of the day of incubation.

Fig.1

from 0.113 to 0.147 g. On dry weight basis, the eggshell composed 15% and the egg components 85% of the total egg weight. The eggs of P. blanfordanus increased in size and weight from laying to hatching (Fig. 1). The eggs increased 40% in length (from 1.21 cm at laying to 1.69 cm at hatching); 68% in width (0.75 cm at laying to 1.26 cm at hatching); and 315% in weight (from 0.393 g at laying to 1.631 g at hatching) (Table 1). Calcium content of the eggshell and the egg components at different days of incubation are presented in Table 1 and Fig. 2. The amount of other minerals like

Fig. 2. Percentage calcium in eggshell and egg contents + embryo of Psammophilus blanfordanus eggs through incubation.

Fig. 2

magnesium (0.79 mg in fresh egg contents and 0.75 mg in hatched embryo) and potassium (2.68 mg in fresh egg contents and 2.23 mg in embryo) did not change during development. The fresh eggshell contained 2.76 mg of calcium that formed 12.51% of the total eggshell. The fresh egg components contained only 0.47 mg of calcium (0.31% of the total egg). Thus, 3.23 mg calcium was present in the freshly laid egg. The calcium content of 35 day incubated eggshell (1.61 mg, 9.42%) was comparable to that at 30 days. The calcium contents of 38, 40 day developed and that of the hatched eggshell was almost comparable (one-way ANOVA and Tamhane’s post-hoc test, P < 0.05 for pair wise comparisons between all stages, Table 1).

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The yolk fraction of the fresh egg contained 0.47 mg of calcium (0.3% of the egg content). The calcium content of the hatchling increased over the embryonic period. The freshly emerged hatchling contained 1.76 mg of calcium (1.91%). Calcium utilisation pattern The total calcium content of the freshly laid egg was 3.23 mg of which the eggshell contained 2.76 mg and the egg component 0.47 mg. The calcium in the egg component increased with the growth of the embryo. Significant alternations were observed in calcium content of the eggshell from laying to hatching (Fig. 2). The eggshell calcium content decreased slowly from day 10 to day 38 (Table 1). The mobilization of calcium from the eggshell was slow up to 35 day of incubation. However, a quantum jump was observed thereafter up to 40 days of incubation. A parallel gradual increase in calcium content of the egg component was also observed during development. The hatched eggshell (1.02 mg) contained about 63% less calcium than that of the fresh eggshell (2.76 mg). Structural characteristics The fresh eggshell Surface morphology: The outer surface of the eggshell exhibited a rough granular structure (Fig. 3A). The surface was organized into regular rectangular and/or polygonal fields of almost uniform size. The fields were arranged in a parallel fashion. The boundaries of such fields appeared to be elevated. The outer surface at higher magnification (Fig. 3B) also did not reveal any definite crystal structure. Rounded/spherical globules of varying sizes made up the entire thickness of the calcareous layer. Peripheral margins of the polygonal fields were formed by larger globules whereas the central area was formed by smaller ones. Cross Section: The thickness of the shell was about 62 µm (membrane layer 50 µm, calcareous layer 12 µm) (Fig. 3C). The calcareous layer did not show any discernible structure in this view. Figure 3D reveals the demarcations of the polygonal calcareous fields on the surface of the egg. The thick membrane layer appeared to have layers of fibers. Inner surface: The inner surface of the egg shell was bounded by a very thin smooth boundary layer (Fig. 3E and F). It revealed irregularly placed globules/spheres of varying sizes. The underlying fibres of the membrane layer were not visible because of a smooth covering layer.

Jyoti Jee et alii

The hatched eggshell Upper Surface: The smooth outer surface at lower magnification (Fig. 4A) revealed clear grooves arranged in a regular parallel pattern. The grooves were formed due to absence of larger globules from the boundaries of the polygonal areas that were present on the fresh eggshell. Fig. 4B depicts irregular absence of smaller globules from inside the fields. The absence of globules from the outer surface exposed the underlying membrane fibers. Inner surface: The inner surface with a thin shell membrane (Fig. 4C) revealed the underlying fibers to be distinctly visible showing their arrangement. The membrane layer contained a mat of interwoven, irregularly oriented uneven fibrils that were bifurcated at intervals (Fig. 4D-F). The fibrils were arranged into several layers. DISCUSSION

The eggshells of P. blanfordanus are typical of the flexible shelled eggs of squamates (Packard et al., 1982c). The calcareous layer is thin and the shell membrane contributes the majority of shell thickness. Calcium carbonate crystals are present as calcite, the form of calcium carbonate found in vast majority of lacertilian eggs (Packard et al., 1982a, 1982b; Packard and Hirsch, 1986). Also, the eggs increased in size throughout incubation with uptake of water from the substrate, which is typical of flexible shelled squamate eggs in favorable hydric environments (Packard et al., 1980, 1982a; Andrews and Sexton, 1981). Some of the eggs tripled their mass during incubation. Water uptake is essential for normal embryonic development and survival for many squamate embryos with flexible-shelled eggs (Muth, 1980; Tracy, 1980). The most important function of the egg is related to embryonic nutrition (Bellairs, 1964; Cook, 1968; Ackerman, 1991; Linville, 2010) for which it contains all the necessary materials. Any loss of egg components may seriously affect embryonic development within the egg and hatchling maintenance and growth. Embryos of oviparous lizards have two potential sources of calcium for developmental requirements; calcium sequestered in yolk during vitellogenesis and calcium deposited on the eggshell by oviducal secretions. Packard (1994) reviewed the calcium contribution of the eggshell to the embryos in various groups of oviparous, amniotic vertebrates (snakes, lizards, etc.). According to her, the degree of calcification of the eggshell is very important as it acts as a source of embryonic calcium in these groups of animals. Psammophilus blanfordanus eggshell is poor in calcium in comparison to other species (Stewart and Ecay, 2010) and has a thin calcareous

Sources of calcium for Psammophilus

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Fig. 3. Scanning electron micrographs of freshly laid eggshells (A & B upper surface, C & D cross section, E & F inner surface) of Psammophilus blanfordanus: (A) Upper surface at low magnification showing regular nearly polygonal fields on a smooth surface. (B) Part of calcareous layer removed to show the inner surface. (C) Much of the thickness of the shell is made up of membrane fibres with a thin calcareous layer. (D) The cross section slightly tilted showing the irregular field-like arrangement on the surface of the eggshell. (E & F) The inner surface is covered by a smooth inner membrane (boundary layer). Globules of varying sizes are embedded on this surface that faces the egg contents.

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Fig. 4. Scanning electron micrograph of hatched eggshell of P. blanfordanus. (A) Upper surface: Larger globules on the boundary of the irregular fields (see Fig.3A) are absent. This has made the formation of clear grooves on the surface. (B) Enlarged view of (A) smaller globules are also absent at places. (C) Inner surface: The thin boundary membrane present on the inner surface exposes the inner membrane fibres. (D–F) Inner surface, enlarged view showing the arrangement of ramfied and somewhat curved fibres of uneven thickness on the membrane layer.

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Sources of calcium for Psammophilus

layer, yet it provides a considerable proportion of calcium to the developing embryo. The freshly laid egg contains 3.23 mg of calcium (2.76 mg in shell and 0.47 mg in egg component), whereas the embryo needs 1.7 mg of calcium for its complete development. The embryo initially utilized the calcium of the yolk fraction for its development. This means the embryo required 1.29 mg more calcium from sources other than yolk. At the same time the eggshell had undergone 1.74 mg reduction in its calcium content over the entire embryonic period. This suggests that the embryo has utilized this much amount of calcium from the eggshell. Thus, the embryo used calcium from two sources: initially from the yolk followed by the eggshell. The calcium requirement of the embryo and loss of that of the eggshell was comparable. According to Abdel-Salam et al. (2006), concentration of calcium in the eggshell of reptiles is higher than that of sodium/magnesium and calcium distribution in the eggshell differs before and after hatching. In the present study, concentrations of minerals like potassium and magnesium in the egg contents of a freshly laid egg did not alter through incubation. The eggshell provides about 63% of its calcium to the developing embryo, the rest being supported by the yolk. The eggshell had undergone considerable structural changes from laying to hatching. The calcareous layer formed distinct grooves due to the absence of larger globules. Besides, smaller globules were also absent exposing the underlying membrane fibres. Absence of globules was caused due to the resorption of calcium by the developing embryo from the calcareous layer. However, no change was observed in the thickness of the shell membrane and in the arrangement of the membrane fibres. Embryos of most oviparous reptiles obtain calcium from both yolk and eggshell, but patterns of calcium mobilization vary (Packard and Packard, 1984; Packard, 1994). Oviparous squamate reptiles generally have lightly calcified eggshells and embryos mobilize most calcium from yolk, whereas embryonic turtles, crocodilians and birds are highly dependent on calcium from the eggshell. However, in P. blanfordanus, 63% of embryonic requirement is supplied by the eggshell. Thus, the eggshell composition and structure of Psammophilus blanfordanus fall within the range reported for other species in this group. The role of eggshell as a calcium source in lizards is little studied. Future research into this field is important to understand the complex role the eggshell played in development of the embryo. The broad similarities in chemical composition and structure of eggshells of agamid lizards (Packard and Demacro, 1991; Osborne and Thompson, 2005; Stewart and Ecay, 2010) suggest a conserved evolutionary strat-

egy in eggs of lizards, but more species must be studied to confirm such a conclusion. ACKNOWLEDGEMENTS

GS is thankful to his students for information regarding the laying of eggs by Psammophilus blanfordanus at various locations. Formal permission was not required for research on the species, as the species is listed as Least Concern as it is widely distributed and very common throughout its range. REFERENCES

Abdel-Salam,  Z.A., Abdou, A.M., Harith, M.A.  (2006): Elemental and ultrastructural analysis of the eggshell: Ca, Mg, and Na distribution during embryonic development via LIBS and SEM techniques.  Int. J. Poultry Sci. 5: 35-42. Ackerman, R.A. (1991): Physical factors affecting the water exchange of buried reptile eggs. In: Egg Incubation: Its Effect on Embryonic Development in Birds and Reptiles, pp. 193-211. Deemings, D.C., Ferguson, M.W.J., Eds, Cambridge University Press, Cambridge. Andrews, R.M., Sexton, O.J. (1981): Water relations of the eggs of Anolis auratus and Anolis limifrons. Ecology 62: 556-562. Bellairs, R. (1964): Biological aspects of the yolk of the hen’s egg. In: Advances in Morphogenesis, PP. 217272. Abercrombie, M., Brachet, J., Ed, Academic Press, New York, USA. Cook, W.H. (1968): Macromolecular components of egg yolk. In: Egg Quality: A Study of the Hen’s Egg, pp. 109-132. Carter, T.C., Ed, Oliver and Boyd, Edinburgh, United Kingdom. Deeming, D.C. (1988): Eggshell structure of lizards of two sub-families of the Gekkonidae. Herpetol. J. 1: 230-234. Giesey, J.P., Weiner, J.G. (1978): Frequency distribution of trace metal concentrations in five fresh water fishes. Am. Fishery Soc. 106: 393-397. Linville, B.J., Stewart, J.R., Tom, J.R., Ecay, T.W., Herbert, J.F., Parker, S.L., Thompson, M.B. (2010). Placental calcium provision in a lizard with prolonged oviductal egg retention. J. Comp. Physiol. B 180: 221-227. Muth, A. (1980): Physiological ecology of desert iguana (Dipsosaurus dorsalis) eggs: temperature and water relations. Ecology 61: 1335-1343.  Packard, M.J. (1994): Patterns of mobilization and deposition of calcium in embryos of oviparous, amniotic vertebrates. Israel J. Zool.40: 481-492.

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Packard, M.J., Burns, I.K., Packard, G.C. (1982a): Structure of shells from eggs of zebra-tailed lizards (Iguanidae: Callisaurus draconoides). Zool. J. Linn. Soc. 75: 297-311. Packard, M.J., Clark, N.B. (1996): Aspects of calcium regulation in embryonic lepidosaurians and chelonians and a review of calcium regulation in embryonic archosaurs. Physiol. Zool. 69: 435–466. Packard, M.J., Demarco, V.G. (1991): Eggshell structure and formation in eggs of oviparous reptiles. In: Egg Incubation: Its Effect on Embryonic Development in Birds and Reptiles, pp. 53-69. Deemings, D.C., Ferguson, M.W.J., Eds, Cambridge University Press, Cambridge, United Kingdom. Packard, M.J., Hirsch, K.F. (1986): Scanning electron microscopy of eggshells of contemporary reptiles. Scan. Electron Micro. 4: 1581-1590. Packard, M.J., Hirsch, K.F., Meyer-Rochow, V.B. (1982b): Structure of the shell from the eggs of the tuatara, Sphenodon punctatus. J. Morphol. 174: 197-205. Packard, M.J., Packard, G.C. (1984): Comparative aspects of calcium metabolism in embryonic reptiles and birds. In: Respiration and Metabolism of Embryonic Vertebrates, pp. 155-179. Seymour, R.S., Ed, Dr W Junk Publishers, Dordrecht, The Netherlands. Packard, M.J., Packard, G.C., Boardman, T.J. (1980): Water balance of the eggs of a desert lizard (Calusaurus draconoides). Canadian J. Zool. 58: 2051-2058. Packard, M.J., Packard, G.C., Boardman, T.J. (1982c): Structure of eggshells and water relations of reptilian eggs. Herpetologica 38: 136-155. Packard, M.J., Packard, G.C., Gutzke, W.H.N. (1984): Calcium metabolism in embryos of oviparous snake Coluber constrictor. J. Exp. Biol. 110: 99-112. Packard, M.J., Packard, G.C., Miller, J.D., Jones, M.E., Gutzke, W.H.N. (1985): Calcium mobilization, water

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balance and growth in embryos of the agamid lizard Amphibolurus barbatus. J. Exp. Zool. 235: 349-357. Packard, M.J., Phillips, J.A., Packard, G.C. (1992): Sources of mineral for green iguanas (Iguana iguana) developing in eggs exposed to different hydric environments. Copeia 1992: 851-858. Packard, M.J., Seymour, R.S. (1997): Evolution of the amniote egg. In: Amniote Orgins. pp. 265-290. Sumida, S.S., Martin, K.L.M., Eds, Academic Press, San Diego, USA. Sahoo, G. Mohapatra, B.K., Dutta, S.K. (2009): Structural changes in olive ridley turtle eggshells during embryonic development. Herpetol. J. 19: 143-149. Schleich, H.H., Kästle, W. (1988): Reptile Eggshells SEM Atlas. Gustav Fischer, Stuttgart, Germany. Shadrix, C.A., Grotzer, D.R., Mc Kinney, S.L., Stewart, J.R. (1994): Embryonic growth and calcium mobilization in oviposited eggs of the scincid lizard, Eumeces fasciatus. Copeia 1994: 493-498. Stewart, J.R., Ecay, T.W. (2010): Pattern of maternal provision and embryonic mobilization of calcium in oviparous and viviparous squamate reptiles. Herpetol. Conser. Biol. 5: 341-359. Stewart, J.R., Ecay, T.W., Blackburn D.G. (2004): Sources and timing of calcium mobilization during embryonic development of the corn snake, Pantherophis guttatus. Comp. Biochem. Physiol. A 139: 335-341. Thompson, M.B., Speake, B.K., Russell, K.J., McCartney, R.J. (2001): Utilization of lipids, protein, ions and energy during embryonic development of Australian oviparous skinks in the genus Lampropholis. Comp. Biochem. Physiol. A 129: 313-326. Tracy, C. R. (1980): On the water relations of parchmentshelled lizard (Sceloporus undulatus) eggs. Copeia 1980: 478-82.

Acta Herpetologica 11(2): 179-187, 2016 DOI: 10.13128/Acta_Herpetol-18176

Mediodactylus kotschyi in the Peloponnese peninsula, Greece: distribution and habitat Rachel Schwarz1,*, Ioanna-Aikaterini Gavriilidi2, Yuval Itescu1, Simon Jamison1, Kostas Sagonas3, Shai Meiri1, Panayiotis Pafilis2 Department of Zoology, Tel Aviv University, Tel Aviv 6997801, Israel. * Corresponding author. E-mail: [email protected] of Zoology and Marine Biology, School of Biology, University of Athens, Panepistimioupolis, Ilissia, Greece 3 Department of Human and Animal Physiology, School of Biology, University of Athens, Panepistimioupolis, Ilissia, Greece 1

2 Department

Submitted on 2016, 14th April; revised on 2016, 03th July; accepted on 2016, 20th August Editor: Marco Sannolo

Abstract. The gecko Mediodactylus kotschyi is considered rare in mainland Greece, yet it is very abundant on the Aegean islands. It has been thought to be saxicolous throughout much of its range. In a recent survey on the Peloponnese peninsula, however, we encountered it mainly on trees, and with higher frequency than previously reported. We combined our observations of localities in which we detected this gecko, and places where we failed to detect it, with data about its occurrence from the literature and museum collections. We posited two hypotheses as possible causes for the apparent relative scarcity of M. kotschyi in the Peloponnese: that it is associated with low precipitation and that it has an aversion to limestone rock. We predicted that M. kotschyi would be more likely to be found in arid places and where limestone is not the dominant type of rock, since it has been reported that this substrate is less suitable for this species. Moreover, we predicted that geckos occurring in limestone regions would be found on trees rather than under rocks. Geckos were indeed found mainly in the more arid parts of the Peloponnese, but not exclusively so. We found no evidence of limestone avoidance. We suggest that, because M. kotschyi is better known as being mostly saxicolous over most of its range, and exclusively so on the Greek islands, in the Peloponnese the search for this species has been restricted to a single habitat type, i.e., under rocks and not on trees. It may thus inhabit more localities in the Peloponnese and be more abundant there than has previously been thought. Keywords. Arboreality, habitat preferences, Mediodactylus kotschyi, Peloponnese, rock type.

INTRODUCTION

The genus Mediodactylus is predominantly Asian, with only one of 13 species being found in Europe. The Mediterranean thin-toed gecko, Mediodactylus kotschyi (Steindachner, 1870) (Reptilia: Gekkonidae), has a discontinuous distribution incorporating southern Italy, through to the Balkans and the Crimean peninsula, to Israel and Iran (Arnold and Ovenden, 2002; Sindaco and Jeremcenko, 2008). In Greece, M. kotschy is ubiquitous and highly abundant on the Sporades, Cyclades, and south Aegean islands as well as around Crete, where its distribution is ISSN 1827-9635 (print) ISSN 1827-9643 (online)

well documented (e.g., Wettstein, 1937; Beutler and Gruber, 1977; Beutler, 1981; Chondropoulos, 1986), and its diversity is high: 13 subspecies are currently recognized from the Greek islands (Karandinos and Paraschi, 1992; Kasapidis et al., 2005; Uetz and Hošek, 2016). However, it is considered to be rare on the Greek mainland and throughout the Balkans (Stojanov et al., 2011; Tomovic et al., 2014), and is often absent from Peloponnese species checklists (e.g., Werner, 1929; Cyrén, 1935; Bischoff and Bischoff, 1980; Henle, 1989; Pèrez-Mellado et al., 1999). The Peloponnese peninsula nonetheless forms a major part of M. kotschyi’s distribution on mainland Greece © Firenze University Press www.fupress.com/ah

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(Valakos et al., 2008). It has been recorded from several locations (e.g., Monemvasia, the terra typica of M. k. bibroni, Beutler and Gruber, 1977; Sparta and Kalamata, Stepánek, 1937), and is thought to be widespread in the western Peloponnese (Valakos et al. 2008). It is nonetheless considered rare, exhibiting a low population density almost everywhere on the Greek mainland (Ajtić, 2014). M. kotschyi is described as being mainly saxicolous across much of its range, being found under rocks and stone piles, on dry stone walls and even on the external walls of houses and other buildings (Beutler, 1981 and citations therein; Musters and In den Bosch, 1982; Arnold and Ovenden, 2002; Ajtic 2014). In Greece it is described as preferring dry areas with phrygana (=dwarf shrub steppe) vegetation, although it also inhabits cultivated areas (Beutler, 1981). Phrygana is common on the Aegean islands, but is rare on mainland Greece, and this has been claimed to be the main reason for its rarity on the mainland (Beutler, 1981). In Israel, in contrast, M. kotschyi is almost obligatorily arboreal, and its Hebrew name (“‫עצים‬ ‫“ = ”שממית‬tree-gecko”) reflects this (Werner, 1993; Bar and Haimovitch, 2012; and our pers. obs.). In Israel, southern Turkey and Iraq it can be found on some common Mediterranean trees such as oak, olive, fig, almond and carob, as well as on introduced species such as Eucalyptus (Weber, 1960; Beutler, 1981; and pers. obs.). Another major factor thought to influence the distribution of M. kotschyi is that of substrate. Like all members of its genus, this species lacks adhesive toe pads (Gamble et al., 2012). Many pad-less saxicolous species are associated with rough rock surfaces (Higham, 2015), such as sandstone (Russell et al., 2007), perhaps because they are able to attain a secure grip on these types of rocks compared to smoother ones. Beutler (1981) suggested that in the Cyclades, where M. kotschyi is very abundant, the ground is comprised mainly of slate, granite, mica, marble and volcanic rocks. However, the main rock type on the Greek mainland is limestone, which according to Beutler is less suitable for M. kotschyi. Consequently, he has claimed that the only mainland region where M. kotschyi is abundant is in the Taygetos mountain range south of Sparta, where the dominant rock types are shale and sandstone (Beutler, 1981). The range of precipitation for Mediterranean vegetation is ~250-800 mm, the lower limit of which corresponds to phrygana vegetation (Aschmann, 1973), which covers the Peloponnese peninsula’s coastline from south to east (Mavromatis, 1978). We hypothesized that, because M. kotschyi is thought to be strongly associated with sparse phrygana, it would be more common in the eastern Peloponnese, where the climate is drier due to the rain shadow cast by the central mountains (Kotini-

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Zabaka, 1983; Bringsoe, 1985). We further hypothesized that M. kotschyi would be rare in areas where the main type of rock is limestone (Beutler, 1981), and that in wet regions, and where limestone predominates, it would be more likely to occur on trees than among rocks. MATERIAL AND METHODS To test our predictions we constructed a presence and absence distribution map of M. kotschyi, incorporating 68 locations from across the entire Peloponnese. Sixteen of these observations (eight presences and eight absences, Table 1a, b) are derived from fieldwork we conducted in June and October 2015, including two locations from which the gecko had been previously reported (Maragou et al., 2015) as well as six new localities in both phrygana habitats and tree groves. The remaining 52 locations were sourced from publications, museum records and localities surveyed independently by KS (Table 2). We searched for M. kotschyi by turning over rocks and visually scanning tree trunks during daylight hours (M. kotschyi is mostly diurnal, active during all but the hottest hours of the day, Beutler, 1981; Valakos et al., 2008; Baier et al., 2009; and pers. obs.). In each locality two to four people searched for geckos for at least 30 minutes. Weather conditions were favourable for M. kotschyi activity throughout. If no individuals were observed in a locality we considered it to be absent, although we acknowledge that false absences are a possibility. We recorded the habitat and type of substrate on which the individuals were found. We recorded GPS coordinates of all locations surveyed, for both presence and absence of M. kotschyi, and assembled them on a map using ArcGIS (ESRI, 2011). Most literature-based locations (Table 2) are provided only as verbal descriptions (usually the name of a town). We digitized the coordinates of these using Google Maps (2015). To determine whether a connection exists between the distribution of M. kotschyi and aridity, we assigned mean annual precipitation data (from Worldclim, Hijmans et al., 2005) to sampled localities (presence and absence, Tables 1, 2). We also recorded rock type for all such locations using geological maps (Higgins and Higgins, 1996) in order to test for substrate preferences. We performed statistical tests of rock type associations only for the presence locations for which substrate data were specified. We used χ2 tests for goodness of fit to compare observations from wet and arid regions, Student’s t tests were applied to test for a connection between substrate type and precipitation, and to compare climatic conditions at sites with and without geckos. We used Fisher’s exact test to search for a connection between rock type and preferred substrate. All analyses were carried out using R version 3.0.1 (R Development Core Team, 2013).

RESULTS

During our 2015 survey we encountered geckos in eight locations but failed to encounter them in the remaining eight (Table 1). Most records of M. kotschyi

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Mediodactylus kotschyi in the Peloponnese peninsula

Table 1. Presence (a) and absence (b) of Mediodactylus kotschyi at sites surveyed during our field work in June and October 2015 in the Peloponnese. Data on substrate and rock type, annual precipitation (mm) and GPS coordinates were combined with the data presented in Table 2 for map construction. a. presence Location North west of Neapolis, Malea Peninsula, Laconia Neapolis, Malea Peninsula, Laconia

Habitat searched

Substrate

Rock type

Annual precipitation (mm)

No. individuals caught (rocks/ trees)

Latitude

Longitude

564

(2/7)

36.5631N

23.0040E

548

(2/4)

36.5339N

23.0421E

585

(1/0)

36.5690N

22.9890E

543

(3/2)

36.6867N

23.0368E

572

(0/17)

36.7291N

22.9802E

688

(0/5)

36.999N

22.722E

600

(0/5)

37.4396N

22.7370E

766

(4/2)

38.13N

21.37E

Sparse phrygana On carob and olive with carob trees and Limestone trees, and eucalypt logs eucalypt logs On olive and carob Olive grove trees and on a Alluvium building’s wall

North west of Neapoli, Malea Peninsula, Dense phrygana Under a rock Limestone Laconia Gefira, near Dense phrygana with Under rocks Monemvasia, Malea Limestone carob trees and on carob trees Peninsula, Laconia North west of Eucalypt, almond and On eucalypt, almond Monemvasia, Malea Phyillites carob grove and carob trees Peninsula, Laconia Phrygana with carob North east of Geraki, On carob and olive trees bordering an Alluvium Laconia trees olive grove Kato Vervena, Arcadia Olive grove On olive trees Alluvium Kalogria, south west of Eucalypt and pine Under rock piles and Alluvium Patras, Achaea forest on eucalypt trees b. absence Location Lagokili, Mani Peninsula South of Platsa, Mani Peninsula, Messenia Kardamyli, Mani Peninsula West of Prosilio, Mani Peninsula South east of Agii Anargiri, Laconia South west of Kosmas, Laconia East of Kalogria, Achaea Trochalia, Malea Peninsula, Laconia

Habitat searched

Rock type

Annual precipitation (mm)

Latitude

Longitude

Olive grove (both on trees and under rocks)

Limestone- marble

707

36.6502N

22.4017E

Stone piles

Limestone- marble

752

36.800N

22.318E

Eucalypt grove (trees only) Phrygana (rocks only) Phrygana (rocks only) Stream bed (rocks only) Phrygana (rocks only)

Neogene sediments

719

36.891N

22.233E

Limestone

770

36.9134N

22.2240E

Neogene sediments

676

37.0160N

22.6360E

Limestone- marble Limestone

849 793

37.0800N 38.1605N

22.7200E 21.3847E

Eucalypt grove (trees only)

Alluvium

546

36.6535N

23.0241E

are from the central and eastern parts of the peninsula (Fig. 1). The 800 mm isohyet divides the Peloponnese into roughly equal areas (above 800 mm: 10,134.45 km2; below 800 mm: 11,113.96 km2), and thus the null expectation would be for 10:11 number of observations from

arid and wet regions. Nevertheless, most records of presence (77%, 46 out of 60, Table 1 a) are from where annual mean precipitation is lower than 800 mm (χ2 goodness of fit test, χ2 = 14.21, n = 60, P = 0.0002). Fourteen locations where presence has been recorded are from regions

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Table 2. Locations, substrate and rock type, annual precipitation (mm), sources of data, and estimated GPS coordinates (longitude and latitude in decimal degrees) used for map construction. Location name

Substrate

Lachos, Mani Peninsula Unspecified Kokkala, Mani Peninsula Unspecified Kato Kastania, Malea Peninsula, Phrygana with Laconia many rocks Ano Kastania, Malea Peninsula, Unspecified Laconia Lira, Malea Peninsula, Laconia Unspecified Loutsa, Mani peninsula Unspecified Monemvasia, Malea Peninsula, Unspecified Laconia South of Agios Ioannis, Malea Unspecified Peninsula, Laconia 5 km north of Monemvasia, Building Malea Peninsula, Laconia Methoni, Messenia Unspecified Kastania, Kariofouni and Driopi Area with stone area, Laconia walls Between Saidona and Kastane, Unspecified Mani Peninsula On trees in Lakkos, Mani Peninsula forest Exochori (Taygetos), Mani Unspecified Peninsula Mandina near Kampos, Mani Unspecified Peninsula Kalamata, Messenia 2 km south of Gargalianoi, Messenia Mystras, Laconia 10 km west of Sparta, Laconia Pyrgos, Elis Sparta, Laconia 5 km north east of Kosmas, Arcadia Rouzaki, Messenia Agii Asomatoi, Arkadia Tegea, Arcadia Didima, Corinthia Tripoli, Arcadia 1 km south west of Mainalo, Arkadia Methanon, Malea Peninsula, Laconia

Rock type

Annual prec. (mm)

Latitude

Longitude

Source

Limestone-marble Limestone-marble

688 651

36.48N 36.52N

22.37E 22.47E

Valakos et al. 2008 Valakos et al. 2008

Limestone

596

36.52N

23.11E

Bringsoe 1985

Phyllites

606

36.537N

23.102E

Valakos et al. 2008

Phyllites Limestone-marble

637 711

36.640N 36.643N

22.964E 22.474E

Limestone

546

36.69N

23.05E

Valakos et al. 2008 Valakos et al. 2008 Beutler and Gruber 1977

Limestone

557

36.726N

23.007E

Valakos et al. 2008

Limestone

549

36.73N

23.02E

Bringsoe 1985

Alluvium

742

36.82N

21.704E

Limestone-marble

762

36.84N

Limestone-marble

797

36.87N

Neogene sediments

768

36.893N

Neogene sediments

768

36.90N

Flysch

781

36.93N

Alluvium

762

37.04N

813

37.049N

Unspecified

Limestone- marble

776

37.06N

Unspecified Near stone terraces Unspecified Eggs under a flat rock On trees in forest Under stones in maquis

Limestone-marble

913

37.064N

Valakos et al. 2008 Bauer 2004; Valakos et 22.35E al. 2008 Bringsoe 1985; Valakos 22.29E et al. 2008 Pers. obs. Kostas 22.259E Sagonas 2014 Werner 1937; Valakos et 22.26E al. 2008 Naturhistorisches 22.20E Museum Wien Stepánek 1937; Valakos 22.11E et al. 2008 Pers. obs. Kostas 21.634E Sagonas 2014 Beutler and Gruber 22.37E 1977; Stepánek 1937; Valakos et al. 2008 22.305E Valakos et al. 2008

Neogene sediments

839

37.07N

21.69E

Bringsoe 1985

Alluvium

712

37.071N

22.430E

Valakos et al. 2008

Limestone

715

37.12N

22.78E

Bringsoe 1985

Neogene sediments

790

37.236N

21.662E

Flysch

671

37.332N

22.699E

Unspecified

Alluvium

798

37.45N

22.41E

Unspecified Unspecified Under stones in maquis

Limestone Limestone

527 807

37.461N 37.507N

23.171E 22.371E

Limestone

836

37.529N

22.299E

Unspecified

Volcanic rocks

452

37.58N

23.39E

Unspecified

On the ground Peridotite and serpentinite in open field

Pers. obs. Kostas Sagonas 2014 Pers. obs. Kostas Sagonas 2014 Naturhistorisches Museum Wien Valakos et al. 2008 Valakos et al. 2008 Pers. obs. Kostas Sagonas 2014 Naturhistorisches Museum Wien

183

Mediodactylus kotschyi in the Peloponnese peninsula

Substrate

Rock type

Annual prec. (mm)

Latitude

Tiryntha, Argolis Peninsula

Unspecified

Alluvium

583

37.59N

Argos, Argolis Peninsula Palea Epidavros, Argolis Peninsula

Unspecified

Alluvium

603

37.632N

Beutler and Gruber 1977; Valakos et al. 2008 22.732E Valakos et al. 2008

Unspecified

Alluvium

502

37.635N

23.153E

Epidavros, Argolis Peninsula

Unspecified

Limestone

560

37.65N

23.14E

781

37.651N

21.618E

603 555

37.659N 37.675N

22.750E 23.126E

538

37.675N

23.134E

Location name

Longitude

Source

22.80E

Valakos et al. 2008

Levidi, Arcadia

Unspecified

Limestone

873

37.68N

22.29E

Kamenitsa, Laconia

Unspecified

Limestone

833

37.72N

22.19E

Tropaia, Arcadia 3 km north east of Sofiko, Corinthia 9 km east of Lampeia, Archaia Olympia Ano Tripotama, Achaea

Unspecified

Limestone

864

37.730N

21.954E

Beutler and Gruber 1977 Pers. obs. Kostas Sagonas 2014 Valakos et al. 2008 Valakos et al. 2008 Pers. obs. Kostas Sagonas 2014 Beutler and Gruber 1977; Valakos et al. 2008 Bringsoe 1985; Valakos et al. 2008 Valakos et al. 2008

Stone terrace

Limestone

639

37.81N

23.08E

Bringsoe 1985

Limestone

927

37.85N

21.91E

Bringsoe 1985

Limestone Neogene and Pleistocene sediments Neogene and Pleistocene sediments Alluvium Alluvium

887

37.857N

21.912E

Valakos et al. 2008

587

37.903N

22.882E

Valakos et al. 2008

566

37.936N

22.927E

Valakos et al. 2008

835 600

37.950N 37.966N

22.325E 22.779E

Alluvium

607

38.009N

22.442E

Koppitz 2013 Valakos et al. 2008 Pers. obs. Kostas Sagonas 2014

Unspecified

Limestone

883

38.010N

22.079E

Valakos et al. 2008

Unspecified Unspecified

Neogene sediments Neogene sediments

865 848

38.018N 38.08N

22.102E 22.17E

On a wall

Neogene sediments

728

38.172N

22.229E

Valakos et al. 2008 Stepánek 1937 Pers. obs. Kostas Sagonas 2014

Under stones in Alluvium phrygana Inachos, Corinthia Unspecified Alluvium Nea Epidavros, Argolis Peninsula Unspecified Peridotite and serpentinite Under stones in Nea Epidavros, Corinthia Peridotite and serpentinite phrygana Archea Olympia, Elis

4 eggs under a flat rock Unspecified

Archea Korinthos, Achaea

Unspecified

Korinthos, Achaea

Unspecified

Feneos, Corinthia Kokkoni, Achaea

On walls Unspecified On a wall in phrygana

2.5 km east of Karia, Corinthia 4 km south west of Kalavrita, Achaea 2 km south of Kalavrita, Achaea Mega Spilaio, Achaea Trapeza, Achaea

with > 800 mm precipitation annually (Fig. 1), although only seven of these are from areas with > 850 mm precipitation annually. In seven of the eight locations (Table 1a) in which we encountered the species in our 2015 fieldwork we found geckos on trees (in three of them exclusively on trees), and in five locations we encountered them under rocks (in one exclusively under rocks). Absence from both microhabitats was also recorded (Table 1b). Thirty-two percent of gecko localities in the Peloponnese are located in regions where limestone is the dominant rock type (combined data from Tables 1a and 2). We

found no connection between rock type and arboreality (Fisher’s exact test, two on trees and nine under rocks in limestone habitats, seven on trees and 11 under rocks in other rock types, P = 0.41), or between habitat type (rocks or trees) and precipitation (trees: 649 ± 35 mm, rocks: 692.1 ± 28 mm; t-test assuming unequal variances, t = 2.1, n = 29, P = 0.34). Twenty five percent of our absence findings were in localities in which limestone is the dominant rock type (although we did not try to identify the type of the rocks under which we searched for geckos), and all of them were for locations in which we searched for geckos under

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Rachel Schwarz et alii

Annual precipitation (mm)

1 2

Fig. 1. Presence and absence localities derived from the literature and our field observations plotted on an annual mean temperature map of the Peloponnese (adopted from Worldclim, Hijmans et al. 2005). Squares (☐) designate specimens found among rocks; triangles (∆) desig nate specimens found on trees; circles (o) designate specimens for which substrate was not specified; dots inside the symbols (•) represent published, museum and observational data (otherwise: our data); Blue: presence; Red: absence.

rocks (Table 1b). Precipitation was not significantly greater in localities where geckos were not present (Table 1b) than where they were encountered (Table 1a; presence: 690 ± 23 mm, absence: 727 ± 32 mm; t-test assuming unequal variances, t = 2.13, n = 33, P = 0.38). DISCUSSION

Our hypotheses were only partially supported. Geckos were indeed more common in the more arid areas of the Peloponnese. However, we did not find evidence of limestone avoidance. The high frequency of occurrence of M. kotschyi on trees in the Peloponnese contrasts with that on the Greek islands. During fieldwork in the Cyclades (once or twice from 2013 to 2015; e.g., Slavenko et al., 2015) we observed M. kotschyi on a tree trunk only

twice (on Ano Koufonisi, in May 2013, and on Kimolos Island, in May 2015, Fig. 2). All our other observations (~ 800, from 40 islands) of this species were on and under rocks, in stone piles, on dry stone walls and on low building walls, under various objects of refuse and in abandoned stone shelters (see also Arnold and Ovenden, 2002; Beutler, 1981; Musters and In den Bosch, 1982 and citations therein). During our 2015 survey in the Peloponnese, most specimens (81%) were found on trees, especially on almond, olive and eucalypts (Table 1a). In only three locations were specimens found under rocks (Table 1a), despite searching localities with apparently suitable phrygana habitats. Because our sole criterion for establishing absence was that we did not find the species following a search under what we considered to be suitable conditions for M. kotschyi, we are well aware that some absences may

185

Mediodactylus kotschyi in the Peloponnese peninsula

Fig. 2. Mediodactylus kotschyi on the bark of a tree on Kimolos Island, 34.79 N, 24.58 E, 7 May 2015. Photographed by SM.

very well be false-absences. This can only be supported (or refuted), however, by future surveys. That said, we have no reason to believe that reported absences are more likely for either the tree or for the rock microhabitat, or for different geographic locations, and thus false absences are unlikely to alter our conclusions. Werner (1993) described M. kotschyi as being a “paradoxical” species. He contended that, in Israel and Iraq, it lives mainly on large tree trunks with exfoliating bark, such as carob, eucalypts and oak (Werner, 1993), even though it lacks the characteristic adhesive toe pads of other arboreal geckos (Gamble et al., 2012). M. kotschyi is nonetheless superbly camouflaged against the background pattern of tree trunks (Werner, 1993; Baier et al., 2009; Bar and Haimovitch, 2012; see also Fig. 2), making it hard to dismiss the idea that it is well adapted to living on trees as well as on rocks. According to the most comprehensive phylogenies available (Pyron and Burbrink, 2014), the closest relatives of M. kotschyi are the arboreal Mediodactylus sagittifer and the saxicolous Mediodactylus heteropholis and Mediodactylus heterocercus (M. kotschyi is sister to a clade containing all three). More distantly-related allies (Pyron and Burbrink, 2014) include members of the mostly saxicolous and terrestrial genera Tenuidactylus and Cyrtopodion (note that Tenuidactylus caspius is described as arboreal, saxicolous and terrestrial, Rogner 1997), and the mostly terrestrial Bunopus, Agamura and Crossobamon. The ancestral state of M. kotschyi is thus most likely terrestrial or saxicolous although an arboreal ancestor cannot be ruled out. The fact that M. kotschyi is saxicolous over most of its distribution may imply that this species was originally saxicolous and later adapted to inhabit trees too.

Current data on the occurrence of this species in the Peloponnese (Tables 1 and 2) do not suggest a strong preference of M. kotschyi for a specific type of substrate, and we did not detect an aversion to limestone. The thintoed gecko does occur in places where the mean annual precipitation is greater than 850 mm, although it is probably scarce in such regions. It is certainly not obligatorily associated with phrygana, in contrast to what was previously suggested (Beutler, 1981). Our findings, along with our observations of this species on trees, lead us to suggest that M. kotschyi is highly flexible and adaptable in its habitat preference, which may have contributed to its successful establishment and broad range. Our observations indicate that M. kotschyi is relatively abundant on trees in the Peloponnese, whereas it is extremely abundant and conspicuous on and under rocks and on stone walls in the Cyclades. This might have led to the general misconception that it is purely saxicolous. Although it is certainly much more abundant on islands (as many lizards are, Novosolov et al., 2013), we suggest that M. kotschyi is more common in the Peloponnese than has previously been considered, because it was formerly sought mostly on and under rocks. ACKNOWLEDGEMENTS

This research was done under permit number 20305/824 from the Ministry of the Environment. We wish to thank Oliver Tallowin, Anat Feldman and Maria Novosolov for help with GIS construction. We wish to thank three anonymous referees for comments on a previous version of this manuscript. This study is funded by an ISF grant #1005/12 to SM. REFERENCES

Ajtić, R. (2014): Morphological, biogeographical and ecological characteristics of Kotschy’s gecko (Cyrtodactylus kotschyi Steindachner, 1870 Gekkonidae) from the mainland portion of its distribution range. Fauna Balkana 3: 1-70. Arnold, E.N., Ovenden, D. (2002): Amphibians and Reptiles of Britain and Europe. R. Collins Field Guide. Harper Collins Publishers, London. Aschmann, H. (1973): Distribution and peculiarity of Mediterranean ecosystems. In: Mediterranean type Ecosystems, Di Castro, F., Mooney, H.A. Eds. Springer-Verlag, New York, USA. Bar, A., Haimovitch, G. (2012): A Field Guide to Reptiles and Amphibians of Israel. Privately published, Herzlyia.

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Baier, F., Sparrow, D.J., Wiedl, H.J. (2009): The Amphibians and Reptiles of Cyprus. Edition Chimaira, Frankfurt am Main. Bauer, L. (2004): Een week op de Peloponnesus. Lacerta 63: 94-107. Beutler, A. (1981): Cyrtodactylus kotschyi (Steindachner, 1870)-Ägäischer Bogenfingergecko. Handbuch der Reptilien und Amphibien Europas. Ed W. Bohme Band 1. Akad. Verl. Wiesbaden: 53-74. Beutler, A., Gruber, U. (1977): Intraspezifische Untersuchungen an Cyrtodactylus kotschyi (Steindachner, 1870); Reptilia: Gekkonidae. Beitrag zu einer Mathematischen Definition des Begriffs Unterart. Spixiana 1: 165-202. Bischoff, W., Bischoff, U. (1980): Einige Bemerkungen zur Herpetofauna des Peloponnes. Herpetofauna 3: 17-22. Bringsoe, H. (1985): A check-list of Peloponnesian amphibians and reptiles, including new records from Greece. Ann. Mus. Goulandris 7: 271-318. Chondropoulos, B.P. (1986): A checklist of the Greek reptiles. I. The lizards. Amphibia-Reptilia 7: 217-235. Cyrén, O. (1935): Herpetologisches vom Balkan. Blät. Aqua. Terrar. -Kun., Stuttgart 46: 129-135. ESRI 2011. ArcGIS Desktop: Release 10. Redlands, CA: Environmental Systems Research Institute. Gamble, T., Greenbaum, E., Jackman, T.R., Russell, A.P., Bauer, A.M. (2012): Repeated origin and loss of adhesive toepads in geckos. PLoS One 7:   e39429. doi: 10.1371/ journal.pone.0039429. Google Maps (2015): Peloponnese, Greece. Retrieved from: https://www.google.co.il/maps/place/Peloponnes e,+Greece/@37.263957,21.9363472,9z/data=!3m1!4b1! 4m2!3m1!1s0x13602f6bdafa0f77:0x100bd2ce2b980c0? hl=en (Accessed: 8 November 2015). Henle, K. (1989): Herpetologische Beobachtungen in Griechenland. Herpetofauna 11: 6-10. Higgins, M.D., Higgins, R. (1996): A Geological Companion to Greece and the Aegean. Cornell University Press, Ithaca, NY. Higham, T.E. (2015): Bolting, bouldering, and burrowing: functional morphology and biomechanics of pedal specialisations in desert-dwelling lizards. In: All Animals are Interesting: A Festschrift in Honour of Anthony P. Russell, pp. 279-301. Bininda-Emonds, O.R.P., Powell, G.L., Jamniczky, H.A., Bauer, A.M., Theodor, J. Eds, BIS Verlag, Oldenburg, Germany. Hijmans, R.J., Cameron, S.E., Parra, J.L, Jones, P.G, Jarvis, A. (2005): Very high resolution interpolated climate surfaces for global land areas. Int. J. Climatol. 25: 1965-1978. Karandinos, M., Paraschi, L. (1992): The Red Data Book of Threatened Vertebrates of Greece. Hellenic Zoolog-

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and Central Asia. Edizioni Belvedere di Luigi Corsetti, Rome, Italy. Slavenko, A., Itescu, Y., Foufopoulos, J., Pafilis, P., Meiri, S. (2015): Clutch size variability in an ostensibly fixclutched lizard: effects of insularity on a Mediterranean gecko. Evol. Biol. 42: 129-136. Stepánek, O. (1937): Gymnodactylus kotschyi Steindachner und sein Rassenkreis. Arch. Naturgesch. 6: 258280. Stojanov, A.J., Tzankov, N., Naumov, B. (2011): Die Amphibien und Reptilien Bulgariens. Edition Chimaira, Frankfurt am Main. Tomovic, L., Ajtiz, R., Ljubisavljevic, K., Urosevic, A., Jovic, D., Krizmanic, I., Labus, N., Dordevic, S., Kalezic, M., Vukov, T., Dzukic, G. (2014): Reptiles in Serbia - distribution and diversity patterns. Bull. Nat. Hist. Mus. Belgrade. 7: 129-158. Uetz, P., Hošek, J. (2016): The Reptile Database, http:// www.reptile-database.org (Accessed: 17 April 2016). Valakos, E.D., Pafilis, P., Sotiropoulos, K., Lymberais, P.,

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Acta Herpetologica 11(2): 189-195, 2016 DOI: 10.13128/Acta_Herpetol-18616

Swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures Hong-Liang Lu, Qiong Wu, Jun Geng, Wei Dang* Hangzhou Key Laboratory for Animal Adaptation and Evolution, School of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, Zhejiang, China. *Corresponding author. E-mail: [email protected] Submitted on 2016, 11th July; revised on 2016, 20th October; accepted on 2016, 22nd October Editor: Rocco Tiberti

Abstract. Thermal acclimatory adjustments of locomotor performance and thermal tolerance occur commonly in ectothermic animals. However, few studies have investigated ontogenetic differences in these acclimatory responses, and thus, their causes remain unclear. In this study, juvenile and adult Chinese fire-bellied newts (Cynops orientalis) were acclimated to one of two temperatures (16 or 24 °C) for 4 weeks to examine ontogenetic differences in acclimation effect on burst swimming speed, and critical thermal minimum (CTMin) and maximum (CTMax). Swimming performance was thermally acclimated in both juvenile and adult C. orientalis. Adult newts had greater absolute swimming speeds than juveniles, which may simply result from their larger sizes. Cold acclimation enhanced low-temperature resistance, and warm acclimation enhanced high-temperature resistance in both juveniles and adults. Despite no ontogenetic difference in CTMin, adult newts had greater CTMax and acclimation response ratio than juveniles, indicating their greater abilities to withstand extreme high temperatures and manage rapid temperature shifts. Ontogenetic change in the thermal acclimatory responses of newts may be related to changes in the thermal environment they experience. Keywords. Salamandridae, ontogeny, thermal acclimatory response, swimming performance, thermal tolerance.

INTRODUCTION

Acclimation is the process that modulates the physiological and behavioural performance of organisms, allowing them to adjust to fluctuating environmental factors such as temperature, humidity, and salinity (Lagerspetz, 2006). Thermal acclimation of physiological and behavioural traits has been widely investigated in various organisms and has been shown to vary considerably among different species (Angilletta et al., 2002; Lagerspetz and Vainio, 2006). Due to the potential impact on determining resilience to future climate change, the thermal acclimatory ability of ectothermic species has attracted increasing attention in recent years (Gvoždík, 2012; Sandblom et al., 2014; Seebacher et al., 2015). Locomotor performance and thermal tolerance are fitness-related traits, and may determine the survival ISSN 1827-9635 (print) ISSN 1827-9643 (online)

of animals that are exposed to high predation pressures or extreme environmental temperatures (Arnold, 1983; Leroi et al., 1994; Willett, 2010). Consequently, the locomotor performance and thermal tolerance of animals acclimated to various environmental conditions have frequently been assessed (Wilson et al., 2000; Gvoždík et al., 2007; Měráková and Gvoždík, 2009; Grigaltchik et al., 2012; Xu et al., 2015). Such acclimation effects also vary at different ontogenetic stages (Brooks and Sassaman, 1965; Menke and Claussen, 1982; Wilson and Franklin, 2000; Wilson et al., 2000). Previous studies on anuran species have showed that thermal acclimatory ability on locomotor performance could easily be observed before metamorphosis, but was lost after metamorphosis, which was explained by an ontogenetic shift in the living environment (Wilson and Franklin, 2000; Wilson et al., 2000). Acclimatory ability on locomotor performance © Firenze University Press www.fupress.com/ah

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should be reduced when metamorphosed frogs migrate from thermally stable aquatic habitats to terrestrial habitats with large daily temperature fluctuations (Wilson and Franklin, 2000; Wilson et al., 2000). In most amphibian species, the critical thermal minimum (CTMin) and maximum (CTMax) generally increase with increasing acclimation temperatures (Floyd, 1983; Gvoždík et al., 2007; Shi et al., 2012), but sometimes warm-acclimated metamorphosing tadpoles do not necessarily have higher CTMax than those that are cold-acclimated (Cupp, 1980; Menke and Claussen, 1982). This might also be partly due to a shift in the thermal regime of a species (Cupp, 1980; Sherman, 1980; Menke and Claussen, 1982). However, studies on ontogenetic differences in the thermal acclimation of locomotor performance and thermal tolerance are still limited in amphibian species. Since the mechanisms underlying the ontogenetic change in thermal acclimatory response are still not completely understood, it is necessary to collect more extensive data. The Chinese fire-bellied newt, Cynops orientalis, is a small-sized (up to 80 mm snout-vent length, SVL) primarily aquatic newt that is widely distributed in central and eastern China, and can be commonly found in permanent ponds, rice terraces, and ditches (Fei et al., 2006). C. orientalis individuals are predominantly aquatic, but occasionally migrate short distances across land to other water bodies. Mating and oviposition occur between March and July when water temperature is between 15 and 23 °C (Yang and Shen, 1993). Although the histology, sexual behaviour, and breeding ecology of this species have been studied during the past decades (Yang and Shen, 1993; Sparreboom and Mouta Faria, 1997; Xie et al., 2012; Jin et al., 2016), none of these studies has focused on thermal physiological performance. Here, we acclimated juvenile and adult C. orientalis to two temperatures for 4 weeks to examine ontogenetic differences in thermal acclimatory performances. On the basis of results from previous studies on the thermal acclimatory responses of amphibian species, we predict the following: (1) the ability to thermally acclimate locomotor performance should not disappear, (2) and ability to withstand extreme temperatures should be enhanced from juvenile to adult stages in predominantly aquatic newts. MATERIALS AND METHODS All newts (16 metamorphosed juveniles and 24 adults) used in the present study were collected from Fuyang mountainous area (Hangzhou, Zhejiang, eastern China) in July 2015 and transferred to our laboratory at Hangzhou Normal University. Prior to thermal acclimation, animals were maintained in six 60 (L) × 50 (W) × 40 (H) cm3 aquaria (6−7 individuals per aquar-

ium) with a water depth of 15 cm at 20 °C and on an L:D 12:12 photoperiod for 2 weeks. Each aquarium was provided with pieces of tiles and some aquatic plants that served as refuges. The newts were then randomly divided into two groups (8 juveniles and 12 adults in each group), each of which was assigned to one temperature treatment: 16 or 24 °C. These temperatures were chosen because they may approximate the range of optimal temperatures for newt activity in the field (Yang and Shen, 1993). Each group of animals was housed in five identical aquaria (4 juveniles or 4 adults per aquarium) in one of two temperature-controlled rooms held at the experimental temperatures. Aquaria (photoperiod L:D 12:12) were placed on the same shelf to minimize water temperature difference among aquaria. Water temperature of each aquarium was confirmed multiple times using a UT-325 electronic thermometer (Uni-trend Group, Shanghai, China), and it varied less than 1 °C. Newts were maintained at the designated temperatures for 4 weeks. Throughout the experiment, newts were fed with Tubifex worms or fish meat. All newts were measured for burst swimming performances at test temperatures of 16 and 24 °C, and allowed to rest for 48 h between trials. During the resting period, newts were maintained in their aquaria at corresponding acclimation temperature. To avoid possible test sequence effects, newts were randomly assigned to different test orders (different acclimation and test temperatures). The test temperatures of newts were achieved by placing them into an incubator at the corresponding temperatures for approximately 1 h prior to each trial. Newts were placed into a racetrack (120 × 10 × 20 cm3) filled with water to a depth of 10 cm at the test temperature and then encouraged to swim by tapping the tails with a paintbrush. A Panasonic HDC-HS900 digital video camera (Panasonic Co., Japan) was positioned laterally to record the swimming performance of each newt. Each newt was tested twice with a minimum of 30 min rest between the trials. To minimise the possible diel and photophasic effects, measurements on any given day started at 13:00 and ended within 3 h. All video-clips were examined using MGI VideoWave III software (MGI Software Co., Canada) for maximal speed over 25 cm. In the following text, speed was expressed as two metrics: absolute speed (cm/s) and relative speed (the ratio of absolute speed to SVL for each individual, SVL/s). We used the dynamic method for determining the CTMin and CTMax of the newts (Kour and Hutchison, 1970; Lutterschmidt and Hutchison, 1997). Trials were conducted in water baths between 10:00 and 15:00. The newts were cooled or heated from their acclimation temperatures at a rate of 0.3 °C min-1 until individuals lost righting response, and their body temperatures were measured by inserting the probe of an electronic thermometer into the cloaca (Lutterschmidt and Hutchison, 1997; Xu et al., 2015). We ran tests at 1-week intervals to minimise possible interactions between CTMin and CTMax. The newts were maintained in their aquaria during the intervals between trials. The thermal resistance range (TRR) was calculated as the difference between CTMax and CTMin (van Berkum, 1988), and the acclimation response ratio (ARR) was calculated by dividing the tolerance change by the change in acclimation temperature (Claussen, 1977).

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RESULTS

There were no differences between groups in the body sizes of juveniles (SVL: 39.9 ± 1.1 mm vs 41.5 ± 0.8 mm, t = 1.21, df = 14, P = 0.246; mass: 1.89 ± 0.09 g vs 1.84 ± 0.11 g, t = 0.35, df = 14, P = 0.731) or adults1 (SVL: 64.2 ± 1.4 mm vs 65.4 ± 0.7 mm, t = 0.73, df = 22, P = 0.470; mass: 6.48 ± 0.49 g vs 7.00 ± 0.35 g, t = 0.86,2 df = 22, P = 0.400) prior to the beginning of the experiment. The absolute swimming speed of C. orientalis was significantly affected by acclimation, test temperature, and ontogeny (Table 1, Fig. 1A, B). Overall, newts that acclimated and tested at high temperature swam faster than those acclimated and tested at low temperature. Moreover, adults swam faster than juveniles (Fig. 1A, B). The absolute speeds of newts were positively related to their SVLs (linear regression analysis, all P < 0.05). With regard to relative speed, the differences between acclimation temperatures and between test temperatures were still evident, but not between adult and juvenile individuals (Table 1, Fig. 1C, D). The interaction between test temperature and acclimation temperature, and between ontogeny and acclimation temperature had no significant effects on relative speed of newts (Table 1). Both mean CTMin and CTMax of juvenile and adult newts significantly increased as acclimation tempera-

Juvenile

40

Adult

o

acclimated to 16 C o

Absolute speed (cm/s)

acclimated to 24 C

32

24

16

8

0

A

B

C

D

6

Relative speed (SVL/s)

We used Statistica 6.0 (StatSoft, Tulsa, USA) to analyse the data. Data were tested for normality using KolmogorovSmirnov tests, and for homogeneity of variances using Bartlett’s test. The primary analyses indicated that aquarium had no visible effects on swimming performance (mixed model ANOVAs with aquarium as the random factor, all P > 0.532), so repeatedmeasure ANOVAs were used to determine whether ontogeny, acclimation temperature and test temperature affected swimming performance. Two-way ANOVAs were used to determine whether ontogeny and acclimation temperature affected CTMin and CTMax.

5 4 3 2 1 0 16

24

16

24

Test temperature (oC)

Fig. 1. Mean values (+SE) for swimming performance (absolute and relative swimming speed) of juvenile and adult Cynops orientalis acclimated to different temperatures.

ture increased (Table 2, Fig. 2A, B). Overall, the mean CTMax of adults was significantly higher than that of juveniles (Fig. 2B), but there was no significant difference in CTMin between adults and juveniles (Table 2, Fig. 2A). The effect of thermal acclimation on CTMax differed significantly between adults and juveniles, but this effect on CTMin did not (Table 2). There was a significant increase in the CTMax of adults as acclimation temperature increased (t = 7.78, df = 22, P < 0.0001), but not in that of juveniles (t = 1.56, df = 14, P = 0.141) (Fig. 2B). Similarly, acclimation temperature significantly affected the TRR of newts (Table 2, Fig. 2C). The acclimation temperature effect differed between adult and juvenile individuals. The TRR of adults increased as acclimation

Table 1. Results of repeated-measures ANOVAs on swimming performance variables (absolute and relative speed) measured for juvenile and adult Cynops orientalis acclimated to two different temperatures. Swimming performance

Acclimation temperature Test temperature Ontogeny Acclimation × test temperature interaction Acclimation temperature × ontogeny interaction Test temperature × ontogeny interaction Acclimation × test temperature × ontogeny interaction

Absolute speed

Relative speed

F1, 36 = 4.73, P = 0.036 F1, 36 = 7.96, P = 0.008 F1, 36 = 13.43, P < 0.001 F1, 36 = 0.12, P = 0.730 F1, 36 = 0.22, P = 0.641 F1, 36 = 0.61, P = 0.439 F1, 36 = 0.01, P = 0.905

F1, 36 = 6.27, P = 0.017 F1, 36 = 10.31, P = 0.003 F1, 36 = 0.12, P = 0.726 F1, 36 = 0.13, P = 0.716 F1, 36 = 0.13, P = 0.720 F1, 36 = 0.03, P = 0.853 F1, 36 = 0.02, P = 0.900

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Table 2. Results of two-way ANOVAs on critical thermal minimum, critical thermal maximum, and thermal resistance range of juvenile and adult Cynops orientalis acclimated to two different temperatures.

Acclimation temperature Ontogeny Acclimation temperature × ontogeny interaction

Critical thermal minimum (oC)

2.0

Critical thermal minimum

Critical thermal maximum

Thermal resistance range

F1, 36 = 88.77, P < 0.0001 F1, 36 = 0.27, P = 0.606 F1, 36 = 3.07, P = 0.088

F1, 36 = 36.60, P < 0.0001 F1, 36 = 9.76, P = 0.004 F1, 36 = 12.51, P = 0.002

F1, 36 = 5.15, P = 0.029 F1, 36 = 11.64, P = 0.001 F1, 36 = 8.35, P = 0.006

o

acclimated to 16 C

temperature increased, but slightly decreased in juveniles (Table 2, Fig. 2C). The ARR values of CTMin and CTMax at acclimation temperatures between 16 and 24 °C were 0.08 and 0.07 for juveniles, and 0.12 and 0.26 for adults, respectively.

o

acclimated to 24 C

1.5

1.0

DISCUSSION 0.5

A

0.0

Critical thermal maximum (oC)

39

38

37

36

B

35

Thermal resistance range (oC)

38

37

36

35

C

34

1 2

Juvenile

Adult

Fig. 2. Mean values (+SE) for critical thermal minimum, critical thermal maximum, and thermal resistance range of juvenile and adult Cynops orientalis acclimated to different temperatures.

Our results showed that thermal acclimation significantly affected the locomotor performance of C. orientalis. Warm-acclimated newts appeared to have better locomotor performance than those that were cold-acclimated, which is not consistent with the beneficial acclimation hypothesis that predicts acclimation to a particular temperature should enhance an animal’s performance or fitness at that temperature (Leroi et al., 1994). The effect of thermal acclimation on locomotor performance has been shown to vary among different amphibian species. For example, constant temperature acclimation failed to affect aquatic and terrestrial locomotor performance in adult Ambystoma tigrinum nebulosum and Ichthyosaura alpestris (Else and Bennett, 1987; Šamajová and Gvoždík, 2010), or only had acclimatory capacity in terrestrial locomotion to warm temperatures in Triturus dobrogicus (Gvoždík et al., 2007), or in aquatic locomotion to cold temperatures in Eurycea guttolineata and Pseudotriton ruber (Marvin, 2003a, b). The fire-bellied newts living in permanent aquatic habitats in mountainous areas may experience limited temperature fluctuations at both juvenile and adult stages (Fei et al., 2006). Therefore, unlike those newts and salamanders that spend more than one-half of the year on land, such as A. tigrinum nebulosum and I. alpestris (Else and Bennett, 1987; Šamajová and Gvoždík, 2010), C. orientalis individuals do not lose the ability to acclimate their aquatic locomotor performance when they reach sexual maturity. This is consistent with our aforementioned prediction. In fact, the explanation proposed by Wilson and Franklin (2000) for the reduced acclimatory ability was based on the absence of thermal acclimatory responses of terrestrial locomotor performance rather than aquatic locomotor performance. Aquatic locomotor performance can still be thermally acclimated in adults of fully aquat-

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ic or semi-aquatic species (Wilson et al., 2000; Marvin, 2003a, b; Gvoždík et al., 2007; Wu et al., 2013; Mineo and Schaeffer, 2014; Xu et al., 2015). Inconsistent with the results of previous studies on one species of Triturus newt and two species of Ambystoma salamander (Shaffer et al., 1991; Wilson, 2005; Landberg and Azizi, 2010), adults swam faster than juveniles in C. orientalis. This might simply result from larger body size at adulthood because there was no significant ontogenetic difference in relative speed. The reduced swimming performance in adult urodeles amphibians is interpreted as a consequence of changes in tail shape, rather than a negative size effect on performance (Landberg and Azizi, 2010). The effect of ontogenetic change in tail shape on the swimming performance of C. orientalis should be investigated in future studies. The ability to withstand extreme temperatures may determine the survival of animals. The CTMin value for C. orientalis (0.5–1.5 °C) falls within the values reported for other fully aquatic or semi-aquatic urodeles (-1.9–3.9 °C for four Desmognathus, one Plethodon, and one Eurycea salamanders, Layne and Claussen, 1982a, b, 1987), whereas the CTMax for C. orientalis (36.2–38.3 °C) is similar to the values reported for most other urodeles, and is higher than those for some high-latitude or high-altitude species (Hutchison, 1961; Brooks and Sassaman, 1965; Sealander and West, 1969; Berkhouse and Fries, 1995; Gvoždík et al., 2007). Compared with anuran species, for C. orientalis, the CTMin (tadpoles: 7.4−8.9 °C for Fejervarya limnocharis, 8.7−11.7 °C for Microhyla ornata, Shi et al., 2012; but 0–1.6 °C for Rana catesbeiana, Menke and Claussen, 1982; adults: 2.1−5.1 °C for three Hyla treefrogs, Layne and Romano, 1985; 4.1−4.9 °C for Rhinella arenarum and Odontophrynus occidentalis, Sanabria et al., 2012, 2013) and CTMax (tadpoles: 37–43 °C, Cupp, 1980; Sherman, 1980; Navas et al., 2010; Shi et al., 2012; Simon et al. 2015; adults: 41.5–43.7 °C for two Hyla treefrogs, Blem et al., 1986; but 35.0–37.8 °C and 34.1–36.1 °C for R. arenarum and O. occidentalis, Sanabria et al., 2012, 2013) were lower than those for most frog and toad species. Therefore, thermal tolerance varies among amphibian species, and is believed to be correlated with habitat and geographic distribution (Hutchison, 1961). Moreover, adult C. orientalis had a greater CTMax than did juveniles, which was also found in other urodeles and anurans, such as E. nana, Notophthalmus viridescens, Bufo woodhousii fowleri, and Hoplobatrachus chinensis (Hutchison, 1961; Sherman, 1980; Berkhouse and Fries, 1995; Fan et al., 2012). As reported for other amphibian species (e.g., Brooks and Sassaman, 1965; Sealander and West, 1969; Menke and Claussen, 1982; Gvoždík et al., 2007; Shi et al., 2012), low-temperature resistance can be enhanced by cold

acclimation, whereas high-temperature resistance can be enhanced by warm acclimation in C. orientalis. Warmacclimated adult newts had a relatively wider TRR than those that were cold-acclimated, but this pattern was not observed in juveniles. Contrarily, the TRR of tadpoles decreased with increasing acclimation temperature (20, 25 and 30 °C) in two other anuran species, F. limnocharis and M. ornata (Shi et al., 2012). Although partially reflecting a difference in temperature treatment, the differential results from these studies may also reflect different optimal temperatures that enable animals to exhibit a high thermal resistance. Those thermal conditions resembling environmental temperatures in animals’ natural habitats may be propitious for enhancing their thermal resistance (Xu et al., 2015). The magnitude of the resistance response to thermal acclimation may reflect the ability to manage temperature shifts. It has been assumed that the species living in environments with large daily temperature variations should have a greater ability to withstand rapid temperature shifts than those living in thermally stable environments (Sandblom et al., 2014). Surprisingly, the ARR of CTMax for adult C. orientalis is greater than that of other semi-aquatic urodeles (0.02–0.17, Hutchison, 1961; Sealander and West, 1969; Gvoždík et al., 2007). Despite no significant ontogenetic difference in acclimation effect on CTMin, the ARRs of critical thermal limits in adult C. orientalis appeared to be greater than those of juveniles. Combined with the greater CTMax and TRR, our results indicate that adult C. orientalis have greater abilities to withstand extreme high temperatures and manage rapid temperature shifts than juveniles do. This is consistent with our second prediction. Such ontogenetic shifts in thermal resistance may be related to  changes  in the  thermal environments experienced by active newts. Animals living in warmer and more thermally variable environments are believed to have greater resistance abilities than those living in cooler, less variable environments (Brooks and Sassaman, 1965; Berkhouse and Fries, 1995). Adult C. orientalis can be active over a wider area, and occasionally migrate from aquatic environments to humid-land environments. Consequently, adult individuals are likely to be exposed to higher and more variable temperatures than are juveniles. ACKNOWLEDGMENTS

Our experimental procedures complied with the current laws on animal welfare and research in China, and were specifically approved by the animal welfare and ethics committee of Hangzhou Normal University (HZNU-

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201506-005). This work was supported by grants from the Natural Science Foundation of China (31670399) and Zhejiang Province (LQ12C03003, LY15C030006), and the China Scholarship Council. REFERENCES

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Acta Herpetologica 11(2): 197-212, 2016 DOI: 10.13128/Acta_Herpetol-18061

Olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the Circeo National Park (Central Italy) Antonio Romano1,*, Riccardo Novaga2, Andrea Costa1 1 Consiglio Nazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale, Via Salaria Km 29,300 I-00015 Monterotondo Scalo, Rome, Italy. * Corresponding author. E-mail: [email protected] 2 Viale dello Statuto 37, Latina, Italy.

Submitted on 2016, 27th February; revised on 2016, 27th June; accepted on 2016, 7th July Editor: Gentile Francesco Ficetola

Abstract. The Circeo National Park lies in a territory that was deeply shaped by human activity, and represents one of the few remaining patches of plain wetland habitat in Central Italy. In this study distribution and few demographic information of the amphibians in the Park were provided. Seven species and 25 bibliographic and 84 original breeding sites were recorded, and population size estimations were carried out for a population of these three species: Pelophylax sinkl esculentus, Bufo balearicus and Rana dalmatina. For the studied populations of pool frog and green toad the operational sex ratio and the demographic effective population size was also estimated. For Rana dalmatina, which is strictly associated to forest environment, a positive and significant correlation between the number of egg clutches and maximum depth of the swamps was found. The State plain forest is the most important habitat for amphibians’ conservation in the park. The occurrence of dangerous alien species was investigated and they are evaluated as the major threat for amphibians in the park, especially the crayfish Procambarus clarkii in the State plain forest. Index of Calling Survey were performed for anurans and the medians did not differ among species. The potential distribution of amphibians in the Park was evaluated by building a species distribution model. Finally, the absence of three species reported in literature in the 60’s of the last century (Bombina pachypus, Salamandrina perspicillata, Rana italica) is also discussed. Keywords. Alien species, Capture-Marking-Recapture, effective population size, Index of Calling Survey, land reclamation, Species Distribution Models, swamps.

INTRODUCTION

Until the early twenties of the past century, marshlands dominated the landscapes along the coast about 45 km southeast of Rome, from Anzio to Terracina towns between the  Tyrrhenian sea and the Volsci Chain, at inland distances from  15 to 25  km. The territory, which now belongs to the province of Latina (Latium region), was an extensive marsh at about sea level originated in an alluvial plain (e.g., Linoli, 2005). Forested swamps dominated above sea level while areas below the sea level were ISSN 1827-9635 (print) ISSN 1827-9643 (online)

covered by mud flats and pools. These so called “Paludi Pontine” (Pontine Marshes) were the subject of land reclamation works, performed periodically since the preRoman period, initially by the Italic tribe  of Latins, but with scarce success. Land reclamation was conducted extensively with considerable success by Fascist regime, starting in the 1920s and radically changing the landscape of the area, which was converted into an extensive agricultural plain and new towns were founded and built (Littoria, renamed Latina, Pontinia, Sabaudia, Aprila and Pomezia are the most important ones). Out of the origi© Firenze University Press www.fupress.com/ah

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nal 20,700 hectares of forest and swampland, about 3,200 were put under protection with the creation in 1934 of the Circeo National Park (CNP), which includes also other not forested areas. Among terrestrial vertebrates, amphibians are the class most strictly associated to wetlands. They are an important component of biodiversity and suffer a recent worldwide decline (Stuart et al., 2004; Wake and Vredenburg, 2008). In Italy protected areas play a key role in conservation of amphibians, and act as stepping stones in the face of climate change (D’Amen et al., 2011). The area where CNP falls is classified in the highest irreplaceability naturalistic values (Maiorano et al., 2006). However data on the fauna before the land reclamation was available only for mammals and birds (Lepri, 1935). Consequently, information on which herpetological species occurred in this area before land reclamation could be only deducted by current species distribution on wider areas (Bruno, 1973; Bruno 1981; Bologna et al., 2000), by specific works on the fauna of the CNP (Carpaneto, 1986; Ravenna, 2013; Cinquegranelli et al., 2015), and by herpetological census of surrounding areas which were also subjected to land reclamation in the 30’s (Novaga et al., 2013). In particular, the recent paper of Cinquegranelli et al. (2015) provided few updated data on distribution of amphibians in the Park. However Cinquegranelli an co-authors surveyed only 15 sites, and information on habitat use, occupancy level and species detection probability were the main goals of their work. The aim of our study was fourfold. First, by performing an extensive survey on aquatic habitats, we provide an updated species distribution of amphibians. Second, we estimated some demographic and abundance parameters. Third, we evaluated habitat preferences of amphibians and we provided potential distribution information. Finally, we detected relevant threats for species, population or habitats and we provided information on conservation measures. MATERIALS AND METHODS Study area The Circeo National Park covers 8,484 hectares of a coastal area of the Central Italy, and it consists of five main environments: the plain forest, four coastal lakes, the coastal dune area, the limestone massif of Mount Circeo (541 m asl, a promontory that marks the southwestern limit of the former  Pontine Marshes) and the island of Zannone. The plain State Forest covers about 3,190 hectares and mainly consists of deciduous woods. With many areas few meters below the sea level, most of the Park does not exceed the 30 meters a.s.l. The Park ranges from latitude 41°13’N to 41°24’N, and from longitude 12°50’E to 13°07’E. CNP is covered by wooded areas and semi-natural

habitats for the 56%, agricultural fields cover the 18%, water bodies the 13%, artificial territories the 11% and, finally, a small portion is covered by wetlands (2%; Giagnacovo et al., 2003). The climate falls in the Lower Mesomediterranean Thermotype, Upper Subhumid Ombrotype (Blasi, 1994). Mean temperature of this area is from 9.5 °C to 17.1 °C, and temperatures below zero are uncommon. Precipitation is mainly concentrated in autumn and early winter  (October-December); relative humidity is high all over the year, wind is frequent with a South-Western dominance (Padula , 1985). Distribution and species occurrence The data reported in literature, when they were provided with enough accuracy, were georeferenced. Field surveys (March-September 2015) were preceded by a careful analysis of the maps produced by the Istituto Geografico Militare (I.G.M, 1:25000) and by a analysis of satellite images to detect water bodies not reported in the maps (see Romano et al., 2012). Information on methods used to detect the occurrence of amphibians are reported in detail by Romano et al. (2010; 2012). To describe species rarity and their diffusion we used a method that weights both diffusion (W: wide; M: medium; L: limited) and density (C: common; F: frequent; R: rare) and it consists of a graph of the relationship between the coverage (%) of the UTM grid (we used a mesh of 2x2 km) and the mean number of observations for a square occupied by each species, according to the method proposed by Doria and Salvidio (1994) and used in other herpetological studies (Turrisi and Vaccaro, 2004; Romano et al., 2012). In the computation of the score to build the graph of rarity and diffusion we decided to use only a subset of meshes. We included only the meshes where at least one species record occurred (i.e., 35 meshes), excluding, for example, meshes in highly urbanised areas with no data. Ecology Breeding aquatic sites were assigned to the following typologies: (i) ponds and marshes; (ii) slow running waters: ditches, streams and artificial channels (iii) rheocrenic springs (which were checked s up to 50 meters from they source); (iv) forest swamps (including those whose filling is partially due to limnocrenic springs); (v) artificial tanks; (vi) brackish coastal lakes. Correspondence Analysis (CoA) was used to identify associations among amphibian species and aquatic habitats. Considering that the variance of the data was homogenous (Levene’s test for homogeneity, based on means, P = 0.758), the hypothesis that habitat categories may host different syntopic number of species was tested using one-way ANOVA. Correlation between the habitat availability and the number of species for each habitat type was tested using non-parametric Spearman’s rank correlation. Analyses concerning habitat typologies were performed both on original and bibliographic data, when the latter could be certainly assigned to a given habitat typology. Ecological analyses were performed in the statistical package PAST (Hammer et al., 2001).

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Amphibians of the Circeo National Park Population abundance estimation Populations estimates were performed using four different methods: Index of calling survey (ICS), capture-mark-recapture  (CMR), removal sampling (RES), and egg mass counts (EMC). For the ICS, which is the relative measure of calling density (Mossman et al., 1998; Weir and Mossman, 2005), surveys were performed as reported in Dorcas et al. (2009) and Romano et al. (2012). ICS may vary among 0 and 3. Considering that ICS provides scores as ordinal measures and with many ties, the scores were analysed using the median test as performed in Statistica® ver. 5.0 (Statistica package, Statsoft Inc., USA). Differences in calling survey scores among anuran species were compared using the non parametric Kruskall-Wallis test and relative post hoc comparisons. Using photo-identification, CMR analysis was applied on a population of Bufo balearicus breeding in an artificial tank in the inner of Sabaudia town; we used the software CAPTURE (Otis et al. 1978) to estimate adult population size (N) analysing data from the four sampling sessions performed in a short time range (23 September – 2 October; light rain, between 9 and 11 p.m.). RES was applied on a population of Pelophylax sinkl. esculentus breeding in the artificial pond of the headquarter of the Park (Sabaudia). We used the jackknife estimator of Pollock and Otto (1983) as performed in the program CAPTURE to estimate N on the basis of three removal sessions performed in about three hours. Both for Bufo balearicus and Pelophylax sinkl. esculentus populations, the operational sex ratio (calculated just on the number of males and females captured and not on population estimates), in accordance with Wilson and Hardy (2002), was expressed as the proportion of mature males, i.e. males/(males + females). The demographic effective population size (Ne) was estimated as Ne = (4*Nm*Nf )/( Nm+Nf ), where N is the number of mature males (m) or females (f) individuals (Wright, 1938), which is a widely used equation to obtain demographic estimates of Ne (e.g. Jehle et al., 2001; Schmeller and Merila, 2007). EMC was used in March to estimate breeding populations of Rana dalmatina in seven natural ponds in forest environment; egg counts were performed in a unique sampling session per pond; as R. dalmatina is an explosive breeder (Sofianidou and Kyriakopoulou-Sklavounou, 1983; Guarino and Bellini, 1993) and each female lays a single egg mass per season (Nollert and Nollert, 1992), consequently we considered the counts of egg masses as a good proxy of the minimum female population size (Griffiths and Raper, 1994; Grossenbacher et al., 2002). Surface areas of breeding ponds and swamps were estimated by walking the  perimeter  of each site with a GPS, automatically calculating the area inside the resulting shape. Where egg masses of R. dalmatina were counted, Spearman  rank  correlation was used to test the association between density of egg masses and size or maximum depth of the swamps. Correla-

tion between water surface and their maximum depth was also tested for all 15 swamps where R. dalmatina breed (correlations were performed in PAST; Hammer et al., 2001). Potential distribution The potential distribution of amphibians in the CNP was evaluated by building a species distribution model (SDM), using the algorithm of maximum entropy (Maxent, Phillips et al., 2006). Selection of environmental data layers, to be employed for these analysis, was based on availability and a priori expectation of influences on amphibian population. Considering that the Park area runs along the coast and it is a small area, precipitations and other climatic variables were considered homogenous in that area and were not included among variables. We used the Digital Elevation Model (DEM) with a spatial resolution of 90m to obtain other topographic variables. All variables were resampled in order to match the 90m resolution of the DEM. The environmental variables used were: Corine land cover data 2006; Landsat tree cover, representing the percentage of canopy cover of trees higher than 5 m; distance from forest swamps; distance from running waters; distance from lakes; topographic variables calculated from DEM were elevation, aspect (Northness or Eastness), valley depth, Topographic Wetness Index (TWI; Sörensen et al., 2006), Topographic Ruggedness Index (TRI; Riley et al., 1999), wind exposure, direct insolation (kw/h per square meter). To build the SDM, both original and bibliographic data were pooled together. The whole data set was split in two subgroups by random selection: 70% of points were used for building the model (training), while the remaining 30% of point-data were employed to evaluate its predictive power. This procedure was repeated 10 times for each species, generating an averaged prediction of amphibians’ distribution. Finally, the predictive power of the model was evaluated by calculating the area under the receiver operating characteristic curve (AUC). Analysis were conducted in software MaxEnt 3.3.3k and default software settings were used, with the exception of the employment of bootstrapping procedure and number of iterations (1000). All GIS processing to obtain the above mentioned layers was performed using software SAGA Gis. Threats Considering that in the Park there is a high-density road network and that road mortality may be considered as an additional factor contributing to the amphibian decline (Puky, 2006; Glista et al., 2008), roads were checked systematically. In particular we controlled the two parallel coastal road and waterfront roads (both about 25 km) after rains in spring and summer. The causes for amphibian declines are many (e.g., Collins, 2010) but habitat loss and alteration, predator alien species and emerging diseases are among the leading. Habitat loss and habitat alterations in progress was recorded and we searched for exotic animal species for which is well established that they are a threats for amphibians (i.e., predator fishes, crayfishes). Alien species were searched in every site where amphibians surveys

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were performed, both using visual surveys (using a binocular too) and blind dip netting (20-30 dip netting per site). Information on fish was also obtained by fishermen.

RESULTS

Distribution and species occurrence Distribution was the result of both bibliographic (when they could be georeferenced with a good approximation, i.e. 500 m; n = 25) and original data (n =84). All available literature (Bruno, 1973; Bruno 1981; Bologna et al., 2000; Carpaneto, 1986; Ravenna, 2013; Cinquegranelli et al., 2015) is consistent in reporting the following species in the park: Triturus carnifex (Laurenti, 1768), Lissotriton vulgaris (Linnaeus, 1758), Bufo balearicus Boettger, 1880, Bufo bufo (Linnaeus, 1758), Hyla intermedia Boulenger, 1882, Pelophylax sinkl. esculentus (Linnaeus, 1758) and Rana dalmatina Fitzinger in Bonaparte, 1838. The pool frog synklepton is formed by two entities: the parental species, P. lessonae (Camerano, 1882) and its hemiclonal hybrid, the klepton P. kl. easculentus (Linnaeus, 1758). Our research confirmed the occurrence of all these species. Conversely, the occurrence of Salamandrina perspicillata (Savi, 1821), Bombina pachypus (Bonaparte, 1838) and Rana italica Dubois, 1987 reported by Bruno (1981) in many localities of the park was not confirmed by our samplings, consistently with other researches (Carpaneto, 1986; Bologna et al., 2000). Species distributions are reported in Figs. 1 and 2 The graph related to diffusion and density (Fig. 3) showed that Triturus carnifex is the species having the most critical situation in the Park, with very limited diffusion and occurrence (i.e., low number of sites). No species are positioned in the upper right corner of the graph, however Rana dalmatina and Pelophylax sinkl. esculentus are of little concern. All the other species, considering their distribution and rarity, do not appear to be particularly threatened. For the particular situation and position in the Fig. 3 of Bufo balearicus, at the boundary between 4 quadrants, see Discussion. Ecology Fig. 4 shows aquatic habitat preferences of each species. Both newts occurred in a limited number of habitat typologies while the more ecologically plastic species was the tree frog that breeds in all the habitat typologies. Variation in amphibian species composition among habitats is highly explained (more than 80%) by the first two axes of the CoA scatter plot (Fig. 5). Associations were found

between the amphibian species and the various aquatic habitat typologies. The results show that all the species are quite different from each other for habitat preference. Few species are strictly associated to only one aquatic habitat typology: Rana dalamatina, Hyla intermedia and Pelophylax sinkl. esculentus are closely linked to forest swamps, ponds and slow running waters respectively. Bufo balearicus is associated with ponds and lakes while B. bufo seems to be associated with springs and artificial tanks. The newts display a moderate and comparable association with forest swamps, ponds and slow running waters. The number of syntopic species did not differ significantly among the six habitat categories (one-way ANOVA, F1,5 = 0.32, P = 0.90). Number of species in the different aquatic habitat typologies ranges between 3 (lakes) and 7 (ponds and slow running waters). Spearman’s  rank  correlation did not detect a significant association between the habitat availability and the number of species for each habitat type (r = 0.806; P = 0.083). Population abundance estimation Index of calling survey (ICS) was performed to several populations of B. balearicus (16 populations), H. intermedia (16), P. sinkl. esculentus (52) and B. bufo (16). For the three first species all the four ICS scores were recorded (0-3), but for the latter the highest score lacked (Fig. 6). The score 1 for both toads clearly exceeded other scores proportionally. Medians of ICS did not differ among species (χ2 = 5.678, df = 3, P = 0.128). Capture-Marking-Recapture (CMR) was applied on a population of Bufo balearicus living in a urban meadow (5800 m2) surrounding the breeding site (a concrete tank). During the four sampling sessions, we performed a total of 56 captures in which 26 different individuals were marked. Closure test confirmed that the population was closed (z = 1.89; p = 0.97). The recapture rate was high and the estimated population was 27 ± 1.32 adult toads (estimate ± SE; CI 95% = 27-33). Considering that the operational sex ratio was extremely balanced (0.46), the estimate of effective population size (Ne=25.8) was similar to that of the adult population census size (N). The N of P. sinkl. esculentus breeding in an artificial ponds (88 m2), was 60 ± 6 individuals (estimate ± SE; CI 95% = 53-78). The operational sex ratio was strongly male biased (0.94) and, as a result, effective population size was much lower than N (Ne=11.25). The seven swamps where egg mass counting (EMC) of R. dalmatina was performed had greatly variable water surface area (mean ± SD = 4637.43 ± 5691.36 m2; range = 124-15860 m2) and maximum depth (mean

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m Salamandrina Salamandrina perspicillata perspicillata

o

Bombina pachypus

pachypus o Bombina Rana italica Rana italica

Triturus carnifex

Lissotriton vulgaris

aa

cc

Bufo Bufobalearicus balearicus

bb

dd

Fig. 1. Distribution of amphibians in the Circeo National Park (Central Italy). a = distribution of three species reported just once in the literature but not recorded during further researches. For figs. b, c and d, circles = original data; triangles = bibliographic data. Grid reports 10x10 km UTM squares. Dashed line: Park boundary. Dotted lines: surface hydrography. Dotted areas: lakes. The urban area of Sabaudia is showed in grey.

Figure 1

± SD =64.28 ± 26.37 cm; range = 40-110 cm). Surface area and maximum depth were not significantly correlated (r=0.654; p=0.128). Correlation remained above the significance threshold even considering all the swamps (N = 15) where reproductive activity was recorded (i.e., pooling sites where egg counts was performed and these

where it was not performed) (r = 0.506; P = 0.053). Number and density of egg masses greatly varied among sites (Fig. S7). A total of 1419 egg masses were recorded (mean ± SD = 202.71 ± 199.09 SD; range 13-604) and their density ranged from 0.01 to 0.47 egg clutches per square meter (mean ± SD = 0.14/m2 ± 0.18). A signifi-

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Bufo bufo

yla intermedia

a

elophylax i

b

esculentus

Rana dalmatina

c

d

Fig. 2. Distribution of amphibians in the Circeo National Park (Central Italy). Circles = original data; triangles = bibliographic data. Grid reports 10x10 km UTM squares. Dashed line: Park boundary. Dotted lines: surface hydrography. Dotted areas: lakes. The urban area of Sabaudia is showed in grey.

Figure

cant correlation among eggs parameters and swamps features was detected only between the number of egg masses and maximum depth of the swamps (r = -0.509, P = 0.249; r = -0.055, P = 0.919, for egg density vs. swamp size or depth respectively; r = 0.643, P = 0.109; r = 0.836, P = 0.025 for egg number vs. swamp size or depth respectively. See Fig. 7).

Potential distribution Species distribution models (SDM) were built for all seven amphibian species occurring in the Park. The number of available data to be employed (both as training and test data) for model building was dependent on the species (see Tab. 1 for details) and ranged between

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Fig. 3. Relationship between percentage of amphibian species occurrence in UTM square grids (2x2 km) and mean number of observations per UTM square. Tricar = Triturus carnifex; Lisvul = Lissotriton vulgaris; Bufbuf = Bufo bufo; Bufbal = Bufo balearicus; Hylint = Hyla intermedia; Randal = Rana dalmatina; Pelesc = Pelophylax synkl. esculentus.

Fig. 5. Correspondence Analysis (CoA) scatter plot illustrating variations of amphibians species (black dots) distribution with aquatic habitat typologies (white squares). The percentages of variation explained by each axis are given in round brackets (codes of species are as reported in Fig.3).

an AUC value > 0.970 (ranging from 0.974 for Bufo balearicus to 0.997 for Rana dalmatina) with the exception of the models regarding Triturus carnifex, which shown a slightly lower predictive power (AUC = 0.947). As a rule, among all variables included in the analyses, the most important ones for the major part of the species are the distance from water bodies (both lakes and swamps), tree cover and insolation, altogether with many Corine categories. The detailed results, regarding the list of variable effect, together with variable percentage contribution, for each species, is reported in Tab. 1. Potential distribution maps are presented as supplementary materials (Figs. S1-S4) Threats Fig. 4. Habitat partitioning (number of sites on the left vertical axe) of amphibians in the Circeo National Park (Central Italy). Codes of species are as reported in Fig. 3. Figure 11 (Triturus carnifex) and 57 (Pelophylax sinkl. esculentus). All models shown a high predictive power, as revealed by AUC: indeed all averaged models received

In the Circeo National Park we found three ponds and a swamps which are breeding sites of Lissotriton vulgaris, Pelophylax sinkl. esculentus and Rana dalmatina that suffer progressive filling with soil, reduction of the depth and earlier dry up. In April 2015, remains of several R. dalmatina eggs desiccated before hatching were recorded in these ponds. The water body with highest danger of disappearing was the one in locality “Cerreto

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sites UTM (2x2 km) % UTM with amphibians (n=35)

20

15

10

Fig. 6. Percentage of Index of Calling Survey (ICS) scores for four anuran species in the Circeo National Park (code of species as in Fig. 3). N = number of sites for each species.

5

0 Trachemys scripta

Carassius auratus

Lepomis gibbosus

Gambusia affinis

Poracambarus clarkii

Alien species

Fig. 8. Number of sites and UTM mashes (2x2 km) where alien species were recorded in the Circeo National Park.

individuals of B. balearicus were found crushed in summer, after a rainy day, in a small stretch of the seafront road (loc. Bufalara, Sabaudia). Reproductive populations of five alien species, identified by the literature as threat to amphibians (see Discussion), were detected. In ascending order of threat to amphibians they were one reptile, three fishes and a crustacean: Trachmeys scripta; Carassius auratus, Lepomis gibbosus, Gambusia sp., Procambarus clarkii. On the whole they occurred at least in 14 UTM mashes (2x2 km), that is 32% of the total meshes occupied by the Park for at least 10% of their surface (n= 44; Fig. 8). Amphibian occurred in all 2x2 km UTM meshes where alien species were recorded. Original data concerning the distributions of these alien species are shown in supplementary materials (Figs S5-S6). Fig. 7. Trend of the relationship between the number of Rana dalmatina egg masses and maximum depth of seven swamps. Alphanumerical codes refer to swamps: VER (Piscina della Verdesca), BAG (Piscina delle Bagnature), CAR (Piscina del Carpino). Correlation was statistically significant (Spearman correlation, r = 0.836, P = 0.025).

Fontana”, a pond with surface area of 240 m2 and maximum depth of about 20 cm. No relevant evidence of road killing on amphibians were recorded and, during about 20 surveys, just few

DISCUSSION

Species occurrence and their actual and potential distribution In the 84 sites with amphibians, we found seven species in the Circeo National Park as reported in literature (Carpaneto, 1986; Cinquegranelli et al., 2015). However the occurrence ratios among species we found (L. vulgaris: 17%; T. carnifex 4%; B. balearicus: 18%; B. bufo: 25%, H. intermedia: 19%; P. synkl. esculentus: 62%;

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Table 1. Contribution to species distribution models of amphibians in of the Circeo National Park (central Italy). AUC, that may range from 0 to 1 (null-maximum predictive power of the model) is also shown. Lissotriton vulgaris Variable Distance from swamps Tree Cover Corine Triturus carnifex Variable Corine Distance from Swamps Distance from Lakes Bufo balearicus Variable Corine Distance from Lakes Insolation Bufo bufo Variable Corine Distance from Lakes TRI Hyla intermedia Variable Tree Cover Distance from Lakes Corine Distance from Swamps Pelophylax sinkl. esculentus Variable Distance from Swamps Tree Cover Distance from Lakes Rana dalmatina Variable Distance from Swamps Tree Cover

AUC = 0.995

Occurrence locations = 24

% Contribution Effect / Corine categories 54.1 Negative 13.1 Positive for values > 50% Agricultural areas with significant portions of natural vegetation, Forested areas – Scrubs 12.1 and herbaceous – Sclerophyllous vegetation, Inland water-bodies – Water courses AUC = 0.947

Occurrence locations = 11

% Contribution Effect / Corine categories Agricultural areas with significant portions of natural vegetation, Agro-Forestry areas, 50.3 Forested areas – Scrub and herbaceous – Sclerophyllous vegetation 24.3 Negative 14.2 Negative AUC = 0.974

Occurrence locations = 20

% Contribution Effect / Corine categories Agricultural areas with significant portions of natural vegetation, Forested areas, 42.6 Open spaces with little vegetation 36.1 Negative 11.2 Negative AUC = 0.993 % Contribution 25.7 21.9 20.3 AUC = 0.989 % Contribution 19.2 18.7 18 17.5 AUC = 0.981 % Contribution 44.4 15.6 14.4 AUC = 0.997

Occurrence locations = 24 Effect / Corine categories Forested areas Negative Positive Occurrence locations = 23 Effect / Corine categories Positive Negative Inland water-bodies - Wetlands, Forested areas Negative Occurrence locations = 57 Effect / Corine categories Negative Positive Negative Occurrence locations = 29

% Contribution Effect / Corine categories 75.1 Negative 10.7 Positive for intermediate values

R. dalmatina: 30%) differed, in some cases, from those reported by Cinquegranelli et al., 2015 (20%, 13%, 20%, 20%, 53%, 67%, 20%, respectively). These authors, performing 14 visit in each site, provide an interesting contribution testing the species detection probabilities (p),

misdetection rates (Mr) and minimum number of visits (Nm maximum=10.3) necessary to be 95 % certain that an unrecorded species is in fact absent from a given site. We performed 1-5 samplings in each site and the species presence we recorded is, obviously, affected by species

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detection probabilities. However in the 40% of the sites sampled by Cinquegranelli et al. (2015) we found at least one additional species (1-4). We think that this discrepancy among the species’ ratios could be probably due to the high difference in the number of sampling sites, to different sampling protocols, and to different years (difference in annual precipitation may affect, intuitively, amphibian species detectability) Our original data showed that urodelans have a more limited diffusion and occurrence than anurans. In particular, T. carnifex has been detected only in three sites during our survey, resulting as the rarest species in the Park (Fig. 3). Its distribution appeared associated with the deep swamps and slow running waters of the plain forest (Fig. 4; Tab. 1), and this datum agrees with the habitat preferences known for this species (Andreone and Marconi, 2006). However, data for the surrounding areas (Novaga et al., 2013) indicate that vernal ponds and marshes are often colonized by both T. carnifex and L. vulgaris, suggesting that the limited distribution of newts outside the State forest might also be affected by the occurrence of alien species (Fig. S5), especially P. clarkii (Fig. S6). Bufo balearicus was recorded in open habitat with sandy and clay soils, as typically showed by this species in other Italian areas (Balletto et al., 2007), around the coastal lakes (Fig. 1) where retrodunal ponds and marshes in grazing lands are the most preferred breeding sites (Fig 4; Tab. 1). The concentration of its elective habitats along the coastal areas explains the overall limited diffusion of this toad in the Park area. On the contrary, B. bufo appears widespread in different habitat typologies (Fig. 2), and uses a greater variety of breeding sites (Fig. 4). Hyla intermedia has been detected both in open and forest habitats but the ICS scores showed higher concentrations in the wooded habitats, as could be predicted for a semi-arboreal species. Rana dalmatina can be considered to be the most representative species of the hygrophilous plain forest and no breeding sites were found outside the forest boundary. Finally, P. sinkl. esculentus is the commonest species in the park (Fig. 2 and 3), as expected for a species which is ecologically plastic and tolerant to anthropic disturbance in a highly urbanised context. During our surveys we did not find S. perspicillata, R. italica and B.pachypus. The question is whether these species actually occurred in the 60s’ as reported by Bruno (1981) and became extinct in the last decades, or their records reported by this author have to be considered as erroneous. These three species are characterised by different ecological requirements, with Salamandrina and R. italica strictly associated to clean running waters, and mainly shady, cool and damp areas (Utzeri et al., 2004, Angelini et al., 2007). Conversly, Bombina pachypus has a

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realtively wide ecological niche, but it is an heliophilous and thermophilus species tipically linked to open, lentic and shallow waters (Guarino et al., 2007). While for the latter species suitbale habitat were actually identified in the park and its occurrence in past decades cannot be excluded, for the first two amphibians we did not find terrestrial and aquatic habitats matching their ecological requirements. Suitable breeding sites could presumably be available before land reclamation (20s’ of the XX century) in a restricted piedmont area on the northern slope of the Circeo massif (loc. “sorgente Mezzomonte” and “Rio Torto”). Ecology The full range of aquatic habitats available in the Park is largely exploited by toads, tree frog and pool frog, while the two newts and Rana dalmatina exhibited a narrow habitat niche (Fig. 4). Larger pools are generally deeper in environments similar to that of the CNP (Brooks, 2005) but in the forest we studied this correlation was not significant. Wet phase duration is generally correlated with both pond surface area and maximum depth (Schneider and Frost, 1996; Brooks and Hayashi, 2002) but, if these two features are considered independently, maximum water depth is generally the best predictor of hydroperiod (Calhoun et al., 2003; Skidds and Golet, 2005). The Habitat Suitability Index for R. dalmatina elaborated by Radiguet (2012) showed that the date of drying of the pond is one of the key components to make habitats highly suitable for this frog. The positive and significant correlation we found between the number of egg clutches of R. dalmatina and maximum depth of the swamps (Fig. 7) is probably related to wet phase duration. Furthermore, as in other Italian areas (e.g., Bernini et al., 2004), also in the CNP Rana dalmatina was strictly associated with swamps and eggs were preferentially spawned in water bodies with intermediate values of tree cover (Tab. 1; Fig. 4 and 5). Distance from swamps also negatively affected the occurrence of both newt species (Tab. 1; Fig. 5). Toads are associated with the environment surrounding the lakes (Tab. 1), but the two species differed in their canopy requirements, as the green toad was associated with open spaces with little vegetation, while the common toad was associated with forested areas. Population abundance estimation Abundance of B. bufo, B. balearicus, H. intermedia and P. sinkl. esculentus greatly varied among sites, as emerged from the ICS (Fig. 6); the maximum score (3) was recorded for almost all species (2-19% of the surveyed

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sites, see Fig. 6), except the common toad. The question of whether Index of Calling Survey (ICS) can be considered a good proxy of actual population size is controversial (see for instance Jansen, 2009 and Corn et al., 2011 for articulated discussions on this topic). The reliability of ICS depends on species and is higher for species with loud calls (i.e., higher detectability), as that we studied, and for tree frogs and pool frogs in particular (Pellet and Schmidt, 2005; Tanadini and Schimdt, 2011). For B. bufo, B. balearicus, H. intermedia and P. sinkl. esculentus in the Circeo National Park, ICS could be easily used in extensive monitoring programs with relatively low effort. Furthermore, monitoring data collected for ICS may be elaborated using a recent class of statistic models that provide abundance estimations and that consider detectability (N-mixture models; Royle, 2004; Royle and Link, 2005). Fort three species we elaborated demographic estimates. Capture-Marking-Recapture (CMR) method estimated about 27 green toads in a population, displaying  an even sex ratio; a population of pool frogs, using removal method, was estimated to consist of about 60 adults, with strongly male biased sex ratio. In several Italian populations of B. balearicus sex ratio is typically male biased and their Ne are highly variable (see table 1 in Giacoma, 1999). In 26 Italian populations demographic parameters are (range, mean ± s.d., 25th75th  percentile): N= 3-292; 89.46 ± 79.66, 28-144.5; sex ratio = 0.53-1, 0.82 ± 0.13, 0.73-0.94, Ne = 0-254.94, 50.27 ± 61.85, 12.84-64.05; (data elaboration from synoptic table 1 in Giacoma, 1999). These two anurans populations showed a contrasting situation if N and Ne are considered independently. One season of data collection provides the size of effectively breeding individuals (Nb) which is directly connected and derived by Ne because Nb times the generation time approximates Ne (Waples, 1990). Thus both Nb and Ne are connected to population’s persistence probability, and may be used as indicators of a population’s viability (e.g., Frankham et al., 2002). Considering both the high accessibility of the two sites and the ease of the sampling, these two populations of B. balearicus and P. sinkl. esculentus could monitored in be long term to corroborate our data and to assess populations trends. Rana dalmatina may be considered the most representative species of the Park, because this frog is strictly associated with the swamps in the State Forest which represents the residual environment of the pre-reclamation (Tab. 1). Although this frog has a limited distribution it is common (Fig. 3) and abundant (Figs 2d and 7) in its elective environment. The sex ratio in Rana dalmatina is male biased in 90% of the breeding ponds (Lodé et al., 2004; Lodé et al., 2005; Lodé 2009) ranging from about 0.8 to 3. Considering this range of sex ratio, in the seven

swamps we studied in the state forest, the whole population size might approximately range between 2550 and 5700 adults. Threats and conservation strategies Habitat loss and alteration. The CNP is among the most urbanized protected areas at national level. As a consequence, aquatic habitat loss and alteration (which are the main causes of amphibian decline at global level; see Collins, 2010) associated with land development, present the greatest challenge to the persistence of these habitats and their animals. However we did not find significant evidence of aquatic habitat loss due to current anthropic pressure. The aquatic habitat characterizing the PNC are swamps and ponds with a typical semiperennial or seasonal hydroperiod. They are lentic shallow water bodies that are deep, on average, about 60 cm (see results). The main conservation problem of swamps and ponds was an habitat evolution toward dryer situations in shorter time, which is largely due to the dramatic change in the hydrological regime resulting from the past land reclamation. An excavation to increase water depth and the hydroperiod duration was planned for the pond in the locality “Cerreto Fontana” where three amphibian species spawn and that is exposed to a fast drying (see results). Road mortality. Road mortality in the CNP does not seem to be a problem for amphibians and, probably, only one situation (loc. Bufalara) deserves further researches to estimate the actual impact on the green toad population. Alien species. Invasive alien species are among the key factors threatening biodiversity (EEA, 2012). We found five alien species that, considering the available literature, may be considered as threats to amphibians (Fig. 8, Fig. S5-S6). The red-eared  terrapin Trachemys scripta may have a large negative impact on amphibian populations (tadpoles; Polo-Cavia et al., 2010) in water bodies with high numbers of alien turtles. Trachemys scripta seems to be more diffused in the northern part of the Park (Fig. S5a). Its occurrence in the swamps of the State Forest was never recorded, even in the past (A. Romano pers. obs.). We did not find massive aggregations of Trachemys, although some sites (e.g., the surroundings of the Fogliano lake) support higher population density than southernmost areas. As a consequence of the apparently low density of Trachemys, we think that its management, for the conservation of amphibians, is less urgent than that of other aliens species. Three alien fishes were detected in small water basin too. The gold fish, Carassius auratus can strongly affect

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amphibian populations either by predation at different life stages (e.g., Monello and Wright, 2001) and influencing reproductive behavior (Winandy and Denoël, 2013). However when gold fish does not reach high demographic density, it seems that its presence is compatible with persistence of native amphibians (Hartel, 2004). Therefore to well understand the threat level to amphibians, the occurrence of gold fish in the Park should be evaluated both in the distribution and in population size. The presence data here reported (Fig. S5b), which shows a scattered and limited distribution, are the only one recorded during this study, but it should be considered that this fish is more widespread in the park (Zerunian, 1984; Zerunian and Leone, 1996). Gold fish did not occur in the water bodies of the State Forest, probably because these aquatic habitats have a seasonal hydroperiod. The pumpkinseed sunfish, Lepomis gibbosus, was introduced for the first time in Italy in the Varano Lake in 1900 (Central Italy) and experienced an impressive increase of distribution in these last years (Zerunian, 2002). Due to its relatively small size L. gibbosus mainly preys upon amphibian larvae and eggs, but can severely damage also the adults (Hartel et al., 2007). Information about its negative impact on amphibian populations are corroborated from experiments with  controlled conditions (Adams, 2000). L. gibbosus, that we recorded in several ditches, is probably widespread in this park characterised by a high connectivity among linear water bodies. It is worth to mention that L. gibbous was recorded at high density in a large concrete artificial tank (30x5 m, about 2 m depth) on the southern slope of the Circeo massif, (Fig. S5c), which is the area with lowest water habitat availability in the Park. We found a female of Bufo bufo in that tank but no breeding activity was recorded. The artificial tank is a potential habitat for at least three amphibians species, because B bufo, P. sinkl. esculentus, and H. intermedia breed in a similar tank 1.6 km away on the same slope (Fig. 2). The eradication of Lepomis gibbosus for these sites is planned and will be carried out in 2016 by the Park. Vredenburg (2004) demonstrated that removing introduced fish can enable amphibian populations to recover to pre-decline levels. The mosquitofishes of the genus Gambusia were introduced into natural or artificial water environments in many parts of the world as a  biocontrol  to mosquito populations, in particular where there are (or there were) malaria infections. The effectiveness of this fish in combating malaria is still debated and this discussion is outside the scope of this paper. By the way, it was imported for the first time in Italy, in Pontine marshes on 1922 by G.B. Grassi (Sella, 1926; Ronchetti, 1968) where, as the other Italian areas, is widespread. The introduction of

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Gambusia sp. strongly depresses all amphibian populations (Adams, 2000; Katz and Ferrer, 2003; Wells, 2007; Segev et al., 2009). The observations in the Park indicate widespread presence (Fig. S5d), as the mosquitofish occurs in a least in a quarter of aquatic sites that host amphibians (Fig. 8). The mosquitofish seems to be absent from the water bodies in the State Forest, probably because they have of a seasonal wet regime. Eradication of mosquitofish was planned and carried out in 2015, in an artificial ponds where only pool frogs breed. Around this aquatic site, located in a meadow garden, six rock piles were also placed to offer additional refugia to small vertebrates. After fish removal and the placing of the artificial shelters (November 2015), on February 2016, the pond was colonised by B. bufo, but it is a potential breeding site also for tree frogs that are in the surroundings. The pond is in the area devoted to tourist and visitor reception; information panels about the performed conservation action were also placed. The last alien species we recorded, and the most dangerous one, is the red swamp crayfish, Procambarus clarkii, which is an efficient predator of amphibian larvae of several European species (Gherardi et al, 2001; Cruz and Rebelo, 2005; Cruz et al., 2006a; Ficetola et al., 2011). The presence of this crayfish is a deterrent for the colonization of potentially suitable aquatic habitats by amphibians (Cruz et al., 2006a, b; Ficetola et al., 2011). Furthermore, P. clarkii is the cause of massive local extinction of amphibians, as happened for instance in a Portugal natural reserve where crayfish caused the disappearance of more than 50% of amphibian species (Cruz et al., 2006b), or, in Italy, the extinction of Rana latastei from one part of its already small distribution range (Mazzotti et al., 2007). It is also a vector of the pathogen Batrachochytrium dendrobatidis which is capable to depressing or to extinct of amphibian populations. In the CNP it is the alien species with more records and widest distribution (Figs 8 and S6) and its occurrence is likely underestimated. Procambarus clarkii is the only alien species reported within the State Forest (Fig. S6), so all amphibians occurring in the forest are severely threatened. Eradication of P. clarkii from the site in the State Forest, is an aim of the Park. To achieve this goal, an integrate strategy is under consideration, using both intensive removal (Hein et al., 2007) and biological control (Aquiloni et al., 2010). An annual monitoring to detect further invasions into other areas of the forest was also planned. A pilot study to test the effectiveness of active removal (i.e., the less expensive method) on the State Forest populations is currently in progress. An updated, large, and geo-referenced database of species distribution is the essential prerequisite for any protected area to effectively manage its resources and

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to plan appropriate conservation strategies. In the CNP seven species were recorded, few of them strictly associated with particular aquatic habitats and thus with limited distribution, as Rana dalamatina is. For this species the maintenance of swamps is an essential prerequisite for conservation. The debated issue about the presumed presence of Salamandrina perspicillata, Bombina pachypus and Rana italica can reasonably be regarded as solved: these species are absent in the Park and their closest sites are at least 10 km away, on the Volsci chain. No reliable information are available to assess if they occurred before land reclamation or not. Despite the highly urbanised territory, habitat loss and alteration seem to be limited, and few practical and rather simple actions can be made to improve the current situation. The main threats to amphibians in the park, in our opinion, are the spread of alien species. Particular concern deserves the invasion of the red swamp crayfish in the State plain forest which is the area with highest level of species richness. ACKNOWLEDGEMENT

This research was carried out under the project “Progetto di Sistema dei Parchi Nazionali Italiani; Action 6: Monitoraggio delle specie  di  ambiente umido  /  acquatico”, funded by the Italian Ministry of Environment (Direttiva MATTM ex cap. 1551). Capture permit and manipulation of individuals were approved by the Italian Ministry of Environment with the authorisation number PNM-2015-0016824/PNM. Ester Del Bove (PNC) and Alessandra Noel (Corpo Forestale dello Stato) greatly support this research; Luigi Loffredi contributed to field researches. Thanks to Marta Biaggini for her help in the field study on green toad. The budding naturalist Francesco Maria Romano has contributed with great enthusiasm to sampling activities. We are indebt with two anonymous reviewers who greatly improved the ms. SUPPLEMENTARY MATERIAL

Supplementary material associated with this article can be found at < http://www.unipv.it/webshi/appendix >. REFERENCES

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Acta Herpetologica 11(2): 213-219, 2016 DOI: 10.13128/Acta_Herpetol-17821

On the feeding ecology of Pelophylax saharicus (Boulenger 1913) from Morocco Zaida Ortega1,*, Valentín Pérez-Mellado1, Pilar Navarro2, Javier Lluch2 Department of Animal Biology, University of Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain. *Corresponding author. Email: [email protected] 2 Department of Zoology, University of Valencia, C/ Doctor Moliner, 50, 46100, Burjassot, Valencia, Spain 1

Submitted on 2016, 14th January; reviewed on 2016, 11th April; accepted on 2016, 7th May Editor: Sebastiano Salvidio

Abstract. The Sahara frog is the most common amphibian found in North Africa. However, the knowledge of its natural history is rather fragmentary. In the present work we studied the trophic ecology of Pelophylax saharicus at some areas of Morocco through the analysis of 130 gastric contents. We did not find any significant sexual dimorphism in body size of adult individuals. Consumed prey show similar sizes in both sexes, while bigger frogs normally eat larger prey. As in other Palearctic frogs, the diet is basically insectivorous, including terrestrial and aquatic prey. We found some differences in the diet of juveniles, with a higher proportion of flying prey, probably indicating a foraging strategy closer to ambush hunting. In the Atlas region, the high consumption of slow-moving terrestrial prey, as Gastropoda, stands out. Only in the Atlas region, the diet was similar to that described from other areas of North Africa, as Tunisia. Keywords. Trophic ecology, Ranidae, Green frogs, Morocco, Pelophylax saharicus.

The Sahara frog, Pelophylax saharicus, inhabits a large portion of North Africa, from South Sahara to the Mediterranean coast, through the Atlas Mountains (Pasteur and Bons, 1959; Amor et al., 2010), living at altitudes of more than 2600 m.a.s.l. Its distribution ranges from Morocco to Egypt, being the most common green frog of North Africa (Salvador, 1996; Amor et al., 2010). The species is strictly aquatic and is found both in natural and artificial permanent ponds, even when these are slightly eutrophized (Salvador, 1996). Pelophylax saharicus is currently considered a full species, following Bons and Géniez (1996) that summarized the discussion about the controversial status of Moroccan green frogs. Molecular studies support the specific status of P. saharicus (Plötner, 1998; Frost et al., 2006; Lymberakis et al., 2007; Lansari et al., 2015; Nicolas et al., 2015), ranging it as the sister group of Pelophylax perezi, apart from the other species of the genus. Both ISSN 1827-9635 (print) ISSN 1827-9643 (online)

males and females reach the sexual maturity in the second year of life, and are able to live as much as six years (e.g. Oromi et al., 2011). Previous studies concluded that P. saharicus does not show sexual dimorphism in the size of adult animals (Esteban et al., 1999). In a preliminary study of feeding ecology of Palearctic frogs, Smith (1951) described the diet of Rana ridibunda ridibunda. Then, Lizana et al. (1989) compared the feeding ecology of P. perezi with other Iberian amphibians and with trophic availability at an area of the central Iberian Peninsula. Subsequent studies have addressed the feeding ecology of other Pelophylax species (Çiçek et al., 2006; Sas et al., 2009; Mollov et al., 2010; Paunović et al., 2010; Bogdan et al., 2012, 2013; Plitsi et al., in press). A recent study assesses the effect of temperature, density and food in the growth and metamorphosis of P. saharicus tadpoles (Bellakhal et al., 2014). Regarding its trophic ecology, some data have been published about © Firenze University Press www.fupress.com/ah

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P. saharicus in the oases of Kettana, in Tunisia (Hassine and Nouira, 2009). Here we present the first data about trophic ecology of the Sahara frog in Morocco. All samples used in this study came from Moroccan areas within the semi-arid Mediterranean zone of North Africa (Le Houéron, 1989). Frogs were captured during 1996 in three different areas of Morocco: (1) The Western Plateau, an area of subhumid to semiarid climate and two localities were sampled: El Borj and Zwiat Cheikh, (2) Rif Mountains and adjacent areas (this is the most humid area of Morocco with more than 600 mm of annual rainfall), and (3) The Middle Atlas, with a climate of strong continental characteristics. Sample sizes were 9 males, 4 females and 5 juveniles for the Western Plateau, 26 males, 52 females and 6 juveniles for the Rif Mountains, and 11 males, 16 females and 1 juvenile for the Middle Atlas. Frogs were euthanized during the field work because they were captured to study helminthic parasites in the framework of a parasitological research (see Navarro and Lluch, 2006). Maturity and sex of the individuals were determined by direct examination of the gonads after dissection. The analysis included 130 gastric contents. Prey items were identified to Family or Order level. Prey size was measured from intact items with a micrometric ocular. Afterwards, absolute frequencies of each prey type and its percentage in the diet were calculated for each region, as well as the number of gastric contents in which such prey was present. We used Spearmann rank correlation and ANCOVA on prey size, with SVL (snout-vent length) as a covariate, to study the relation between body length of frogs and the size of consumed prey for each category (adult males, adult females, and juveniles). Then, we estimated and compared diet diversities using the approach proposed by Pallmann et al. (2012). Instead describing diet diversity through a given index as, for example, Simpson or Shannon indices, we converted these “raw” indices into “true” diversities. That is, regarding different measures as special cases of Hill’s general definition of diversity measures (Hill, 1973). To study differences in diversity between males, females and juveniles, we performed two-tailed tests for integral Hill numbers of orders -1 ≤ q ≤ 3. This selection includes the transformed versions of the three following indices: the species richness index, Hsr (q = 0), the Shannon entropy index, Hsh (q → 1) and the Simpson concentration index, Hsi (q = 2). All comparisons among diversities of groups were made with Tukey-like contrasts employing a resampling procedure. We did 5000 bootstrap replications so as to obtain reliable p-values (Westfall and Young, 1993). Methods described here are implemented in R package “simboot” (Scherer and Pallmann, 2014) and are fully described in Pallmann et al. (2012).

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All calculations were done in R version 3.0.3 (R Core Team, 2014). Finally, in order to visualize differences in the composition of the diet of adults of both sexes and juveniles, we conducted a discriminant function analysis. Box’s M test of equality of covariance matrix was not significant, so data were suitable for discriminant analysis. Only two variables (Dictyoptera larvae and Dermaptera larvae) failed the tolerance test, so were excluded from the analysis, the rest of the variables (Table 3) were suitable for analysis (tolerance test with P > 0.05). The diet of P. saharicus was mainly insectivorous and more varied in females than in males or juveniles. But, we did not find significant differences in the diversity values of males, females and juveniles (P > 0.05 in all pairwise comparisons, Table 2). Diptera were the most important prey item. The diet of juvenile individuals, principally dominated by Formicidae and other small Hymenoptera, was less diverse than that of adult males and females. We observed a high proportion of Hymenoptera in the diet of Western Plateau frogs, much higher than for Tunisian populations (Hassine and Nouira, 2009). This is principally due to the massive presence of this prey in five juvenile individuals, in which we found 95.58% (65 of 68 prey items) of all sampled Hymenoptera. In addition, all adult individuals of P. saharicus from the Plateau ate proportionally more Hymenoptera than those from the Atlas or Rif regions, suggesting a greater availability of such prey at the Plateau. Alternatively, these differences can be due to a different foraging behaviour in different areas. According to our results, there is no sexual dimorphism in adult individuals of P. saharicus (see also Esteban et al., 1996; Meddeb et al., 2007). SVL of juveniles was 51.00 ± 2.00 mm (mean ± SE, n = 8). We did not find significant differences in body size of adult males and females of P. saharicus (one-way ANOVA of log-transformed data, F = 0.304, P = 0.583, homogeneous variances, Levene test, P = 0.90; SVL of adult males, mean = 88.43 ± 4.96 mm, range = 48-97 mm, n = 44; adult females, mean = 92.51 ± 4.40 mm, range = 48-223, n = 74), even if females were slightly larger than males. We measured 803 prey items (mean = 5.26 ± 0.18 mm, range = 0.5-70 mm). We found a significant correlation between frog body size (SVL) and prey size (Spearmann Rank correlation, Rs = 0.510, P < 0.001, n = 803). This correlation was also maintained within adult individuals (Rs = 0.399, P < 0.001, n = 695; mean = 5.68 ± 0.20 mm, range = 0.5-70 mm). According to this result, we analysed the prey size in both genders employing the SVL as the covariate. Adult females ate prey of significantly larger size than adult males (ANCOVA analysis, F = 4.816, P = 0.029, with no significant differences in regression slopes, F = 1.055, P = 0.305; mean = 5.04

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Feeding ecology of Pelophylax saharicus

Table 1. Data from the analysis of 130 stomach contents of Pelophylax saharicus. Fi is the absolute frequency of each type of prey item group in the sample, % Fi the relative frequency of the group in the sample, P is the presence of each group (i.e. the number of stomach contents in which the group appears), and % P the percentage of the presence of the item in the sample. Group

Total

Juveniles

Adult males

Adult females

Fi

% Fi

P

%P

Fi

% Fi

P

%P

Fi

% Fi

P

%P

Fi

% Fi

P

%P

Gastropoda Araneae Acarina Ostracoda Isopoda Crustacea Diplopoda Chilopoda Diplura larvae Thysanura Odonata Ephemeroptera Plecoptera Plecoptera larvae Orthoptera Orthoptera larvae Dictyoptera Dictyoptera larvae Dermaptera Dermaptera larvae Phasmida Embioptera Thysanoptera Homoptera Homoptera larvae Heteroptera Heteroptera larvae Diptera Diptera larvae Trichoptera larvae Lepidoptera Lepidoptera larvae Coleoptera Coleoptera larvae Hymenoptera Formicidae Undet. Arthropoda Undet. Larvae Birds

48 21 2 5 7 1 1 6 1 2 1 9 4 6 28 1 16 1 4 1 7 1 4 28 3 45 1 245 19 1 9 1 149 29 155 74 5 18 1

5 2.18 0.21 0.52 0.73 0.1 0.1 0.62 0.1 0.21 0.1 0.94 0.41 0.62 2.92 0.1 1.66 0.1 0.41 0.1 0.73 0.1 0.41 2.92 0.31 4.69 0.1 25.52 1.98 0.1 0.94 0.1 15.52 3.02 16.14 7.71 0.52 1.87 0.1

21 17 1 2 6 1 1 3 1 2 1 1 4 4 21 1 5 1 4 1 1 1 4 25 3 29 1 87 14 1 7 1 65 11 55 42 5 14 1

16.2 13.1 0.8 1.5 4.6 0.8 0.8 2.3 0.8 1.5 0.8 0.8 3.1 2.3 16.2 0.8 3.8 0.8 3.1 0.8 0.8 0.8 3.1 19.2 2.3 22.3 0.8 66.9 10.8 0.8 5.4 0.8 50 8.5 42.3 32.3 3.8 10.8 0.8

2 0 2 0 0 0 0 0 1 0 0 0 0 0 0 0 2 0 0 0 0 0 2 1 0 2 0 18 1 0 0 0 6 6 68 2 0 2 0

1.74 0 1.74 0 0 0 0 0 0.87 0 0 0 0 0 0 0 1.74 0 0 0 0 0 1.74 0.87 0 1.74 0 15.65 0.87 0 0 0 5.22 5.22 59.13 1.74 0 1.74 0

2 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 2 1 0 2 0 7 1 0 0 0 3 3 8 2 0 2 0

18.18 0 9.09 0 0 0 0 0 9.09 0 0 0 0 0 0 0 9.09 0 0 0 0 0 18.18 9.09 0 18.18 0 63.64 9.09 0 0 0 27.27 27.27 72.73 18.18 0 18.18 0

9 13 0 0 1 1 1 0 1 1 1 9 1 2 5 1 2 1 1 1 0 0 0 12 2 20 1 97 5 0 5 0 50 2 27 30 1 7 0

2.90 4.19 0 0 0.32 0.32 0.32 0 0.32 0.32 0.32 2.90 0.32 0.64 1.61 0.32 0.64 0.32 0.32 0.32 0 0 0 3.87 0.64 6.45 0.32 31.29 1.61 0 1.61 0 16.13 0.64 8.71 9.68 0.32 2.26 0

6 11 0 0 1 1 1 1 0 1 1 1 1 2 5 1 2 1 1 1 0 0 0 12 2 12 1 33 4 0 4 0 23 2 17 15 1 6 0

13.64 25 0 0 2.27 2.27 2.27 2.27 0 2.27 2.27 2.27 2.27 4.54 11.36 2.27 5.54 2.27 2.27 2.27 0 0 0 27.27 4.54 27.27 2.27 75 9.09 0 9.09 0 52.27 4.54 38.64 34.09 2.27 13.64 0

37 8 0 5 6 0 0 5 0 1 0 0 3 4 23 0 12 0 3 0 7 1 2 15 1 23 0 129 13 1 4 1 93 21 60 42 4 9 1

6.93 1.50 0 0.94 1.12 0 0 0.94 0 0.19 0 0 0.56 0.75 4.31 0 2.25 0 0.56 0 1.31 0.19 0.37 2.81 0.19 4.31 0 24.16 2.43 0.19 0.75 0.19 17.42 3.93 11.24 7.86 0.75 1.68 0.19

13 6 0 2 5 0 0 2 0 1 0 0 3 2 16 0 2 0 3 0 1 1 2 12 1 15 0 47 9 1 3 1 39 6 30 25 4 6 1

17.57 8.11 0 2.70 6.76 0 0 2.70 0 1.35 0 0 4.05 2.70 21.62 0 2.70 0 4.05 0 1.35 1.35 2.70 16.22 1.35 20.27 0 63.51 12.16 1.35 4.05 1.35 52.70 8.11 40.54 33.78 5.40 8.11 1.35

Total

960

130

115

11

310

44

534

74

± 0.24, n = 268 for adult males, and mean = 6.08 ± 0.29 mm, n = 427 for adult females). For the discriminant analysis, the correlations between the variables and the two discriminant axes are

provided in Table 3. The discriminant function is able to correctly classify the 64.5% of individuals as adult males, adult females or juveniles according to their diet, so the goodness of fit is acceptable. Differences in the diet of

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Table 2. Simpson’s diversity values of the diet of males, females and juveniles of P. saharicus and p-values from pairwise comparisons of Hill’s numbers (see more details in the text)

diversity values Hill’s numbers q=0 q=1 q=2

Adult males

Adult females

Juveniles

0.8521 ± 1.80 x 10-4 males-females 0.8398 0.7928 0.8300

0.8818 ± 5.51 x 10-5 females-juveniles 0.9134 0.7014 0.7280

0.6273 ± 2.19 x 10-3 juveniles-males 0.6960 0.4258 0.4742

Fig. 1. Values of each dimension selected in the discriminant function analysis of the diet of Pelophylax saharicus are plotted for each studied frog. Individuals are marked regarding age and sex in order to visualize the age and sex differences in the trophic ecology of the Sahara frog.

males, females and juveniles are plotted in Figure 1. On one hand, the discriminant axes 1 somewhat divides diet of males (negative values) from diet of females (positive values), and it is mainly positively correlated with the presence of Formicidae and Coleoptera, and negatively correlated with the presence of Hymenoptera, larvae of Diplura, Acarina and Tysanoptera (Fig. 1, Table 3). On the other hand, the discriminant axes 2 divides the diet of juveniles (negative values) from the diet of adults (positive values), and it is mainly positively correlated with the presence of Orthoptera, Gastropoda, larvae of Isopoda, Ostracoda and larvae of Coleoptera, among others, and mainly negatively correlated with the pres-

Fig. 2. Values of each dimension selected in the discriminant function analysis of the diet of Pelophylax saharicus are plotted for each studied frog. Individuals are marked regarding the area of study: the Western Plateau, the Rif Mountains, and the Middle Atlas.

ence of Araneae, Ephemenoptera, larvae of Orthoptera larvae, Diplopoda, larvae of Dermaptera, or larvae of Dictyoptera, among others (Fig. 1, Table 3). Regarding the area of study, we did not detect with the discriminant analysis any clear pattern in the composition of the diet (Fig. 2). The diet of juvenile individuals is clearly different, being less diverse than the diet of adults (Table 2). Young frogs use to hunt smaller prey than adults, mainly small Hymenoptera. Hirai and Matsui (1999) found a significant correlation between SVL and prey size of Pelophylax nigromaculatus, as we observed in P. saharicus, suggesting that individuals of green frogs tend to eat larger prey as they grow.

217

Feeding ecology of Pelophylax saharicus Table 3. Pooled values of within-groups correlations between the discriminating variables (the prey items) and the standardized canonical discriminant functions (the two discriminant axes). Discriminating variables are ordered by absolute size of correlation within the discriminant axes 1. Group Hymenoptera Diplura larvae Acarina Thysanoptera Formicidae Coleoptera Plecoptera larvae Araneae Orthoptera Gastropoda Ephemenoptera Orthoptera larvae Diplopoda Dermaptera larvaea Dictyoptera larvaea Crustacea Odonata Heteroptera larvae Isopoda larvae Ostracoda Homoptera larvae Lepidoptera Coleoptera larvae Diptera Undet. Arthropoda Lepidoptera larvae Embioptera Trichoptera larvae Phasmida Bird Diptera larvae Diplura larvae Chilopoda Homoptera Dictyoptera Plecoptera Dermaptera Undet. Larvae Thysanura

Discriminant axes 1

Discriminant axes 2

-0.481* -0.302* -0.302* -0.281* 0.119* 0.088* 0.050* 0.144 0.109 0.049 0.041 0.041 0.041 0.041 0.041 0.041 0.041 0.041 0.061 0.032 0.061 0.084 -0.084 0.049 0.060 0.023 0.023 0.023 0.023 0.023 0.076 0.040 0.037 0.093 -0.020 0.055 0.055 -0.018 0.045

-0.019 -0.043 -0.043 0.087 -0.059 0.052 0.030 -0.319* 0.280* 0.211* -0.200* -0.200* -0.200* -0.200* -0.200* -0.200* -0.200* -0.200* 0.188* 0.174* -0.159* -0.140* 0.140* -0.139* 0.137* 0.125* 0.125* 0.125* 0.125* 0.125* -0.119* 0.110* 0.101* -0.095* 0.090* 0.089* 0.089* -0.069* -0.052*

* Largest absolute correlation between each variable and any discriminant function a This variable not used in the analysis.

Pelophylax saharicus has a similar feeding ecology composition that its sister taxon, P. perezi, from the Iberian Peninsula (Lizana et al., 1989), and other species of

the genus, as P. ridibundus (Çiçek et al., 2006; Mollov et al., 2010). Diptera predominates as the main prey item of adult individuals of both species, followed in abundance by Coleoptera prey (aquatic species mostly) and Hymenoptera, often Formicidae. The diet of P. saharicus in Morocco has some differences with the diet of other species of Pelophylax, as P. kurtmuelleri in Greece, which actively selects arachnids over other types of prey (Plitsi et al., in press). Nonetheless, we lack data about availability of prey in the habitat of Sahara frogs, which limits our results. Thus, our results about the differences in the diet of sexes and ages should be taken with caution, since it is possible that the electability of each type of prey would be similar to their availability in the environment. Therefore, future research in the diet of P. saharicus frogs, including the availability of prey in their habitats and seasonal comparisons would be useful to get deeper knowledge about the ecology of the species. Furthermore, the diet of P. kurtmuelleri frogs is highly influenced by their habitat (Plitsi et al., in press), and the diets of P. ridibundus and P. esculenta are also influenced by seasonality (Sas et al., 2009; Mollov et al., 2010) and weather conditions (Bogdan et al., 2012). Thus, we cannot exclude that P. saharicus could also employ a variable foraging strategy. Lizana et al. (1989) observed that the females of the Iberian green frog ate significantly larger prey than adult males, as we observed in P. saharicus. The ingestion of larger prey by females and their more diverse diet could be the reason of the slightly bigger parasite load of this sex. In this sense, and working with the same frogs, Navarro and Lluch (2006) found that females showed more diverse helminth infracommunities, even if differences with males were not statistically significant. The inclusion of a large amount of flying prey in the diet reinforces the hypothesis that P. saharicus is a sit-and-wait forager. Gastropoda were only important in the Rif sample, with a similar proportion as the reported for the Tunisian studied population (Hassine and Nouira, 2009). In P. ridibunda of Turkey no differences of diet regarding sex were found (Çiçek et al., 2006). The consumption of Formicidae is not higher in P. saharicus than in P. perezi of the Iberian Peninsula, and it is consistent with the diet of the Tunisian population (Hassine and Nouira, 2009). The consumption of ants and other prey groups could be due to a foraging behaviour near the water or at more terrestrial habitats. In fact, many rivers and natural ponds of Morocco scarcely have riverside edges, forcing the individuals to stand close to water shore. Sas et al. (2009) found that P. esculenta of Romania changes the proportion of aquatic and terrestrial preys along the year activity period, which, although unknown yet, would be also possible for P. saharicus of Morocco.

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ACKNOWLEDGEMENTS

Mohamed El Ayadi helped during field work in Morocco. Angélica Hernández-González and María Marquínez helped during laboratory work. Field work was possible thanks to collecting permits (“Ordres de Mission”) issued by Morrocan Government to Faculté des Sciences de l’Université Abdelmalek Essaadi. Laboratory work and data analysis were supported by the grants CGL2006-10893-CO2-02 and CGL2012-39850 from the Spanish Ministry of Science and Technology and Spanish Ministry of Economy and Competitiveness. REFERENCES

Amor, N., Velo-Antón, G., Farjallah, S., Said, K. (2010): Genetic variation across Tunisian populations of the anuran species Discoglossus pictus and Pelophylax saharicus. African Zool. 45: 121-128. Bellakhal, M., Neveu, A., Fartouna-Bellakhal M., Missaoui H., Aleya L. (2014): Effects of temperature, density and food quality on larval growth and metamorphosis in the north African green frog Pelophylax saharicus. J. Therm. Biol. 45: 81-86. Bogdan, H.V., Covaciu-Marcov, S.D., Gaceu, O., CicortLucaciu, A.S., Ferenţi, S., Sas-Kovács, I. (2013): How do we share food? Feeding of four amphibian species from an aquatic habitat in south-western Romania. Anim. Biodivers. Conserv. 36: 89-99. Bogdan, H.V., Covaciu-Marcov, S.D., Cupsa, D., CicortLucaciu, A.S., Sas, I. (2012): Food Composition of a Pelophylax ridibundus (Amphibia) population from a thermal habitat in Banat region (Southwestern Romania). Acta Zool. Bulgar. 64: 253-262. Bons, J., Géniez, P. (1996): Amphibiens et Reptiles du Maroc (Sahara Occidental compris), Atlas Biogéographique. Asociación Herpetológica Española, Barcelona. Çiçek, K., Mermer, A. (2006): Feeding biology of the marsh frog, Rana ridibunda Pallas 1771, (Anura, Ranidae) inTurkey’s Lake District. North-West. J. Zool. 2: 57-72. Esteban, M., García-París, M., Buckley, D., Castanet, J. (1999): Bone growth and age in Rana saharica, a water frog living in a desert environment. Ann. Zool. Fenn. 36: 53-62. Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Haddad, C.F.B., De Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C. (2006):

The Amphibian Tree of Life. Bull. Am. Mus. Nat. Hist. 297: 1-369. Hassine, B., Nouira, S. (2009): Diet of Discoglossus pictus Otth 1837 (Anura, Alytidae) and Pelophylax saharicus (Boulenger in Hartert, 1913) in the oases of Kettana (Gabes, Tunisia). B. Soc. Zool. Fr. 134: 321-332. Hill, M.O. (1973): Diversity and evenness: a unifying notation and its consequences. Ecology. 54:427-432. Hirai, T., Matsui, M. (1999): Feeding habits if the Pond Frog, Rana nigromaculata, inhabiting rice fields in Kyoto, Japan. Copeia 1999: 940-947. Hódar, J.A., Ruiz, I., Camacho, I. (1990): La alimentación de la Rana Común (Rana perezi Seoane, 1885) en el sureste de la Península Ibérica. Misc. Zool. 14: 145153. Lansari, A., Vences, M., Hauswaldt, S., Hendrix, R., Donaire, D., Bouazza, A., Joger, U., El Mouden, H., Slimani, T. (2015): The Atlas Massif separates a northern and a southern mitochondrial haplotype group of North African water frogs Pelophylax saharicus (Anura: Ranidae) in Morocco. Amphibia-Reptilia 36: 437-443. Le Houéron, H.N. (1989): Classification écologique des zones arides (s.l.) De l’Afrique du Nord. Ecol. Medit. 15: 95-144. Levins, R. (1968): Evolution in Changing Environments: Some Theoretical Explorations. Princeton University Press, New Jersey. Lizana, M., Ciudad, M.J., Pérez-Mellado, V. (1989): Uso de los recursos tróficos en una comunidad ibérica de anfibios. Rev. Esp. Herpetol. 1: 209-271. Lymberakis, P., Poulakakis, N., Manthalou, G., Tsigenopoulos C.S., Magoulas A., Mylonas M. (2007): Mitochondrial phylogeography of Rana (Pelophylax) populations in the Eastern Mediterranean region. Mol. Phylogenet. Evol. 44: 115-125. Meddeb, C., Nouira, S., Cheniti, T.L., Walsh, P.T., Downie J.R. (2007): Age structure and growth in two Tunisian populations of green water frogs Rana saharica: a skeletochronological approach. Herpetol. J. 17: 54-57. Mollov, I.A., Boyadzhiev, P., Donev, A. (2010): Trophic role of the marsh frog Pelophylax ridibundus (Pallas, 1771) (Amphibia: Anura) in the aquatic ecosystems. Bulg. J. Agric. Sci. 16: 298-306. Navarro, P., Lluch, J. (2006): Helminth communities of two green frogs (Rana perezi and Rana saharica) from both shores of the Alboran Sea. Parasite 13: 291-297. Nicolas, V., Mataame, A., Crochet, P.A., Geniez, P., Ohler, A. (2015): Phylogeographic patterns in North African water frog Pelophylax saharicus (Anura: Ranidae). J. Zool. Syst. Evol. Res. 53: 239-248. Oromi, N., Brunet, P., Taibi, K., Aït Hammou, M., Sanuy, D. (2011): Life-history traits in Pelophylax saharicus

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from Tiaret semiarid lands (northwestern Algeria). Herp. J. 21: 267-269. Pallmann, P., Schaarschmidt, F., Hothorn, L.A., Fischer, C., Nacke, H., Priesnitz, K.U. (2012): Assessing group differences in biodiversity by simultaneously testing a user-defined selection of diversity indices. Mol. Ecol Resour. 12:1068-1078. Pasteur, G., Bons, J. (1959): Les Batraciens du Maroc. Trav. Inst. Sci. Chérif., sér. Zool. 17: 1-241. Paunović, A., Bjeliċ-Čabrilo, O., Smiljka, Š. (2010): The diet of water frogs (Pelophylax esculentus ”complex”) from the Petrovaradiski Rit Marsh (Serbia). Arch. Biol. Sci. 62: 799-806. Plitsi, P., Koumaki, M., Bei, V., Pafilis, P., Polymeni, R.M. (2016): Feeding ecology of the Balkan Water frog (Pelophylax kurtmuelleri) in Greece with emphasis on habitat effect. North-West. J. Zool. (online first): art. e161502. Plötner, J. (1998): Genetic diversity in mitochondrial 12S rDNA of Western Palearctic water frogs (Anura, Ranidae) and implications for their systematics. J. Zoolog. Syst. Evol. Res. 36: 191-201.

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R Core Team. (2014): R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available from: http:// www.R-project.org/. Salvador, A. (1996): Amphibians of northwest Africa. Smithsonian Herp. Inf. Serv. 109: 1-43. Sas, I., Covaciu-Marcov, S.D., Strugariu, A., David, A., Ilea, C. (2009): Food Habit of Rana (Pelophylax) kl. esculenta Females in a New recorded E-System Population from a Forested Habitat in North-Western Romania. Turk. J. Zool. 33: 1-5. Scherer, R., Pallmann, P. (2014): simboot: Simultaneous inference for diversity indices. R package version 0.25. 2014. Available from: http://CRAN.R project.org/ package=simboot Smith, M.A. (1951): The feeding habits of the Marsh frog (Rana ridibunda ridibunda). Br. J. Herpetol. 1: 170172. Westfall, PH, Young, SS. (1993): Resampling-based multiple testing: Examples and methods for p-value adjustment. New York: John Wiley & Sons, Inc.

Acta Herpetologica 11(2): 221-225, 2016 DOI: 10.13128/Acta_Herpetol-17842

Notes on the reproductive ecology of the rough-footed mud turtle (Kinosternon hirtipes) in Texas, USA Steven G. Platt1, Dennis J. Miller2, Thomas R. Rainwater3,*, Jennifer L. Smith4 Department of Biology, Box C-64, Sul Ross State University, Alpine, TX 79832, USA Box 792, Alpine, Texas 79831, USA. Present address: Wildlife Conservation Society, Myanmar Program, Office Block C-1, Aye Yeik Mon 1st Street, Hlaing Township, Yangon, Myanmar 3 Tom Yawkey Wildlife Center & Belle W. Baruch Institute of Coastal Ecology and Forest Science, Clemson University, Georgetown, South Carolina 29440, USA. * Corresponding author. E-mail: [email protected] 4 Department of Biology, New Mexico State University-Alamogordo, Alamogordo, New Mexico 88310, USA 1 2

Submitted on 2016, 20th January; revised on 2016, 25th June; accepted on 2016, 27th June Editor: Paolo Casale

Abstract. Kinosternon hirtipes is among the least-studied North American turtles and little is known concerning reproduction. In the United States, K. hirtipes occurs at < 10 sites within a single creek drainage in Presidio County, Texas where it is considered a threatened species. We investigated the reproductive ecology of one of these populations at Plata Wetland Complex in 2007. We captured nine female K. hirtipes in wire mesh traps and hoop nets baited with fish. Eggs were obtained by injecting females with oxytocin. We recovered 19 eggs from six females captured in May and June. The smallest female that produced eggs was about 7.1 years old. Mean (± 1SD) clutch size, egg length, egg width, egg mass, and clutch mass were 3.1 ± 1.4 eggs, 28.7 ± 1.4 mm, 17.5 ± 0.9, 5.3 ± 0.6 g, and 17.5 ± 0.8 g, respectively. Relative egg mass and relative clutch mass were 0.035 and 0.011, respectively. There was a significant, positive linear relationship between female carapace length (CL) and egg width. The relationship between CL and relative egg mass was negative, and approached statistical significance. Relationships between CL and clutch size, egg length, and egg mass were not significant. Reproductive attributes of K. hirtipes in Texas are similar to those reported from a population in Mexico. Keywords. Clutch size, egg size, Kinosternon hirtipes, reproduction, threatened species, Texas.

The rough-footed mud turtle (Kinosternon hirtipes) is a highly variable species comprising six recognized subspecies distributed from west Texas, USA, southwards into Chihuahua, Mexico, and south and east on the Mexican Plateau to the Chapala, Zapotlán, San Juanico, Pátzcuaro, and Valle de México basins (Iverson, 1981; Legler and Vogt, 2013). Kinosternon hirtipes murrayi is the largest subspecies (straight-line carapace length [CL] to 196 mm; Smith et al., 2015) and has the most extensive distribution, occurring from west Texas and Chihuahua, south into northern Jalisco, northern Michoacan, and eastern Estado de Mexico (Iverson, 1981; Legler and Vogt, 2013). ISSN 1827-9635 (print) ISSN 1827-9643 (online)

The northernmost populations of K. hirtipes (sensu lato) occur in Presidio County, Texas where < 10 small, isolates are known from the Alamito Creek drainage (Ernst and Lovich, 2009; Platt and Medlock, 2015). Kinosternon hirtipes is considered a threatened species by Texas Parks and Wildlife Department (2013) owing to a limited geographic distribution within the state and on-going habitat degradation. Kinosternon hirtipes ranks among the least-studied North American turtles and many aspects of its natural history remain poorly known (Lovich and Ennen, 2013). In particular, there is a notable paucity of information on © Firenze University Press www.fupress.com/ah

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reproductive ecology. Iverson et al. (1991) investigated reproduction among a population in Chihuahua, Mexico, and laboratory studies indicate that hatchling sex is determined by incubation temperature (Ewert et al., 2004); otherwise the reproductive ecology of K. hirtipes has gone unreported. Importantly, published reports of reproduction among the remnant K. hirtipes populations in Texas are lacking, although such natural history data are critical for designing effective conservation measures (Dayton, 2003). We here report on the reproductive ecology of one of the few known K. hirtipes populations in Texas. Our study was conducted at Plata Wetland Complex (PWC; elevation = 1125 m), located on a private ranch in the Alamito Creek drainage approximately 56 km SE of Marfa in Presidio County, Texas. PWC consists of four livestock tanks (ponds): Railroad (612 m2), Crotalus (900 m2), Turner One (2520 m2), and Turner Two (3780 m2). Railroad Tank is fed by an artesian spring and linked to Crotalus Tank by a shallow drainage ditch about 244 m long. Turner One is located approximately 190 m from Railroad Tank and water is supplied by rainfall and a wind-driven pump. Turner Two is approximately 245 m from Turner Tank One and reliant on rainfall for water. Because Railroad and Crotalus Tanks are spring fed, water levels remain relatively stable throughout the year with a maximum depth of about 1.5 m. Water depth in Turner Tanks One and Two varies depending on seasonal rainfall (less so at Turner One); maximum depth in each is about 1.2 m. The environmental characteristics of the study site are described in greater detail elsewhere (Wilde and Platt, 2011; Platt et al., 2016). Regional climate is characterized by mild winters (rarely < 0 °C) and hot summers (> 40 °C) with highly variable annual rainfall (mean ca. 370 mm) (Powell, 1998). We trapped turtles at PWC from April through midJuly, and September 2007 as part of a larger population study of K. hirtipes conducted in the Alamito Creek drainage (2007-2010). We captured turtles using a combination of wire mesh funnel traps (1.0 m long × 50 cm diameter; mesh = 12.5 mm) and hoop nets (2.5 m long × 1.0 m diameter; mesh = 25 mm). Traps and hoop nets were baited with sardines (packed in oil or water) or fresh carcasses of locally captured sunfish (Lepomis). Wire mesh traps were set from mid-morning to early evening (ca. 1000-2030 hr) and checked at intervals of 1-2 hours. Hoop nets were deployed in mid-morning (ca. 1030) on one day and checked the following morning. Each captured turtle was permanently marked by shell notching (Cagle, 1939) and then measured (CL and plastron length [PL]) with dial (± 0.1 mm) or tree calipers (± 1.0 mm) depending on body size. Body mass (BM) was determined with spring scales (± 1.0 g). Turtles with a CL

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≥ 100 mm were sexed using external secondary sexual characteristics (Iverson 1985b). We were unable to reliably determine the sex of turtles below this size threshold. Male and juvenile turtles were processed in the field and released, while females were returned to the lab and held in plastic wading pools for 21-24 days to insure that oviducal eggs were fully shelled. We then induced oviposition by injecting oxytocin into the pectoral muscles at a dosage of 2.0 units/100 g of body mass (Ewert and Legler, 1978). Following the injection, each female was placed in a 38 liter tub half-filled with water and fitted with a wire-mesh grate positioned 5 cm above the bottom; this allowed eggs to fall through and prevented accidental trampling by the female (Platt et al., 2008; Legler and Vogt, 2013). A second injection was administered within 24 hours to insure deposition of the complete clutch. Most eggs were removed from the tub 10-20 minutes after deposition, although a few remained underwater for somewhat longer; all eggs were recovered < 1 hour after being deposited. Eggs were then weighed on an Ohaus triple beam balance (± 0.1 g), and measured (length and width) with dial calipers (± 0.1 mm). Following Iverson et al. (1991) we calculated relative clutch mass (RCM) [clutch mass / (gravid female body mass — clutch mass) × 100] and relative egg mass (REM) [RCM/clutch size]. Female turtles were released at their respective capture sites 24-48 hours after oviposition. We used linear regression to explore relationships between female body size and clutch and egg attributes. Statistical analyses were performed by program JMP (version 3.2, SAS Institute, Cary, North Carolina, USA). General statistical references are from Zar (1996). Mean values are presented as ± 1SD, and results were considered significant at P ≤ 0.05. We captured 87 turtles (25 males, nine females, and 53 juveniles) at PWC. We treated the females (mean CL = 142 ± 15 mm; range = 121-163 mm) captured in May-June with oxytocin, and recovered 19 eggs from six females (CL = 132-159 mm) in late June (Table 1). Based on von Bertalanffy growth models (Fabens, 1965) developed for this population (Smith, 2016), the smallest female in our sample that produced eggs was estimated to be 7.1 years old. There was a significant positive linear relationship between egg width (EW) and CL (R = 0.91; F1, 5 = 20.72; P = 0.0104; Fig. 1). The relationship between CL and relative egg mass (REM) was negative (REM = -0.0002CL + 0.04) and approached statistical significance (R = 0.76; F1, 5 = 5.52; P = 0.0785). The relationships between CL and clutch size (R = 0.03; P = 0.94), egg length (R = 0.02; P = 0.96), and egg mass (R = 0.68; P = 0.13) were not significant. Our data provide the only information on the reproductive ecology of K. hirtipes outside of Mexico (Iverson

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Kinosternon hirtipes reproduction Table 1. Attributes of clutches obtained from six female Kinosternon hirtipes collected in Presidio County, Texas, USA. *See text for discussion of potential methodological biases. Attribute

n

Mean ± 1 SD

Range

Clutch size* 6 Egg length (mm) 19 Egg width (mm) 19 Egg mass (g)* 19 Mean clutch mass (g)* 6 Relative clutch mass* 6 Relative egg mass* 6

3.1 ± 1.4 28.7 ± 1.4 17.5 ± 0.9 5.3 ± 0.6 17.5 ± 8.8 0.035 ± 0.019 0.011 ± 0.002

1–5 24.7–30.8 15.0–18.7 3.6–6.4 5.3–29.1 0.009–0.061 0.008–0.015

18.5

r2 = 0.83 p = 0.0104

Egg width (mm)

18.0

17.5

17.0

16.5

16.0 130

140

150

160

Carapace length (mm)

Fig. 1. Relationship between carapace length and egg width in a sample (n = 6) of female Kinosternon hirtipes murrayi from Presidio County, Texas, USA.

et al., 1991). The clutch size (mean and range) we report for K. hirtipes in Texas is almost identical to the clutch size estimated from corpora lutea counts in Chihuahua, Mexico (3.0 ± 0.8 eggs; range = 1-5 eggs; n = 34; Iverson et al. 1991). However, it should be noted that egg retention can occur after oxytocin injections (Congdon and Gibbons, 1985) and without taking radiographs of females (Gibbons and Greene, 1979), we cannot be certain that females deposited a complete clutch. Therefore, the clutch size we report is best considered a conservative estimate. That said, Tucker (2007) found that clutch size in a group of female red-eared sliders (Trachemys scripta elegans) treated with oxytocin was statistically equivalent to clutch size in natural nests. Although Iverson et al. (1991) found a positive correlation between clutch size and CL in K. hirtipes, no such relationship was evident in our study, possibly owing to the small number of females we sampled. Because K. hirtipes deposits multiple clutches during a single reproductive season (Iverson et

al., 1991), CL may be a more suitable predictor of total annual fecundity rather than individual clutch size. The values we report for egg mass (EM), egg length (EL), and egg width (EW) are similar to those from a much larger sample of K. hirtipes in Mexico (EM = 4.8 ± 1.0 g; range = 3.7-6.5 g; n = 11; EL = 28.8 ± 1.8 mm; range = 24.1-33.2 mm; n = 74; EW = 16.3 ± 0.8 mm; range = 14.6-18.5; n = 74; Iverson et al., 1991). Because females in our study deposited eggs directly into the water and these remained submerged for varying (but usually brief) periods, it is possible some eggs absorbed water and increased in mass (e.g., Wilgenbusch and Gantz, 2000). However, any increase in mass was probably minimal as most eggs were retrieved < 20 minutes after deposition. Clutch mass (CM) and relative clutch mass (RCM) were also similar to values reported by Iverson et al. (1991) among K. hirtipes in Mexico (CM = 14.4 ± 4.3 g; range = 6.7-29.4 g; n = 28; RCM = 0.071 ± 0.014; range = 0.048-0.105; n = 28). The negative relationship between REM and body size reported by Iverson et al. (1991) was likewise evident in our data, although of marginal statistical significance. This relationship suggests a decreased investment in each egg with increasing maternal body size (Iverson et al., 1991). Similar to our results, Iverson et al. (1991) found a significant positive relationship between female CL and mean egg width. This relationship is not unexpected if egg width is determined by the pelvic aperture diameter which in turn scales to body size (Congdon and Gibbons, 1987). However, evidence for pelvic aperture constraints on egg size in kinosternids is conflicting (Macip-Rios et al., 2013). Some studies suggest egg width is determined by pelvic morphology (Wilkinson and Gibbons, 2005; Macip-Rios et al., 2012, 2013), while others found no evidence for such constraints (Iverson, 1991; MacipRíos et al., 2009; Lovich et al., 2012; Macip-Rios et al., 2013). Larger female K. hirtipes are producing larger eggs and according to Iverson et al. (1991), this increase is achieved by increases in egg width rather than length, which is probably constrained by oviduct length. The two smallest mature females with corpora lutea found by Iverson et al. (1991) in Mexico had CLs of 99.4 and 97.4 mm, a body size very similar to the smallest females exhibiting secondary sexual characteristics in our study population. However, the smallest female from which we recovered eggs was considerably larger (CL = 132 mm). Despite this difference between the two populations, growth models suggest sexual maturity is attained at approximately the same age in Texas (7-8 years) and Mexico (6-8 years; Iverson et al., 1991). Obviously given our small sample size of reproductive females, further study is necessary to fully address this question.

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ACKNOWLEDGEMENTS

This research was conducted under a scientific research permit (SPR-0307-844) issued to SGP by the Texas Department of Parks and Wildlife, with the approval of the Sul Ross State University (SRSU) Institutional Animal Care and Use Committee. Support for this project was provided by Research Enhancement Grants from SRSU to SGP. We thank Lewis Medlock for field assistance and Hardrock Mining Company for allowing us to conduct research on their property. REFERENCES

Cagle, F.R. (1939): A system for marking turtles for future identification. Copeia 1939: 170-173. Congdon, J.D., Gibbons, J.W. (1985): Egg components and reproductive characteristics of turtles: relationships to body size. Herpetologica 41: 194-205. Congdon, J.D., Gibbons, J.W. (1987): Morphological constraints on egg size: a challenge to optimal egg size theory? Proc. Nat. Acad. Sci. 84: 41-45. Dayton, P.K. (2003): The importance of the natural sciences to conservation. Amer. Natur. 162: 1-13. Ernst, C.H., Lovich, J.E. (2009): Turtles of the United States and Canada. 2nd Edition. Johns Hopkins University Press, Baltimore. Ewert, M.A., Etchberger, C.R., Nelson, C.E. (2004): Turtle sex-determining modes and TSD patterns, and some TSD pattern correlates. In: Temperature-dependent Sex Determination in Vertebrates, pp 21-32. Valenzuela, N., Lance, V.A., Eds. Smithsonian Institution Press, Washington, D.C. Ewert, M.A., Legler, J.M. (1978): Hormonal induction of oviposition in turtles. Herpetologica 34: 314-318. Fabens, A.J. (1965): Properties and fitting of the von Bertalanffy growth curve. Growth 29: 265-289. Gibbons, J.W., Greene, J.L. (1979): X-ray photography: a technique to determine reproductive patterns of freshwater turtles. Herpetologica 31: 86-89. Iverson, J.B. (1981): Biosystematics of the Kinosternon hirtipes group (Testudines, Kinosternidae). Tulane Stud. Zool. Bot. 23: 1-74. Iverson, J.B. (1985): Geographic variation in sexual dimorphism in the Mud Turtle Kinosternon hirtipes. Copeia 1985: 388-395. Iverson, J.B. (1991): Life history and demography of the yellow mud turtle, Kinosternon flavescens. Herpetologica 47: 373-395. Iverson, J.B., Barthelmess, E.L., Smith, G.R., DeRivera, C. E. (1991): Growth and reproduction in the Mud Tur-

tle Kinosternon hirtipes in Chihuahua, México. J. Herpetol. 25: 64-72. Legler, J.M., Vogt, R.C. (2013): The Turtles of Mexico. University of California Press, Berkeley. Lovich, J.E., Ennen, J.R. (2013): A quantitative analysis of the state of the knowledge of turtles of the United States and Canada. Amphibia-Reptila 34: 11-23. Lovich, J.E., Madrak, S.V., Drost, C.A., Monatesti, A.J., Casper, D., Znari, M. (2012): Optimal egg size in a suboptimal environment: reproductive ecology of female Sonora mud turtles (Kinosternon sonoriense) in central Arizona, USA. Amphibia-Reptilia 33: 161-170. Macip-Ríos, R., Arias-Cisneros, M.L., Aguilar-Miguel, X.S., Casas-Andreu, G. (2009): Population ecology and reproduction of the Méxican mud turtle (Kinosternon integrum) in Tonatico, Estado de México. West. North Amer. Natur. 69: 501-510. Macip-Ríos, R., Brauer-Robleda, P., Casas-Andreu, G., Arias-Cisneros, M.L., Sustaita-Rodriguez, V.H. (2012): Evidence for the morphological constraint hypothesis and optimal offspring size theory in the Mexican mud turtle (Kinosternon integrum). Zool. Sci. 29: 60-65. Macip-Ríos, R., Sustaita-Rodriguez, V.H., Casas-Andreu, G. (2013): Evidence of pelvic and non-pelvic constraint on egg size in two species of Kinosternon from Mexico. Chelon. Conserv. Biol. 12: 218-226. Platt, S.G., Berezin, A.R., Miller, D.J., Rainwater, T.R. (2016): A dietary study of the rough-footed mud turtle (Kinosternon hirtipes) in Texas, USA. Herpetol. Conserv. Biol. 11: 142-149. Platt, S.G., Medlock, L. (2015): Kinosternon hirtipes (rough-footed mud turtle). Aerial basking, winter activity, habitat, and new locality. Herpetol. Rev. 46: 424-425. Platt, S.G., Sovannara, H., Kheng, L., Holloway, R., Stuart, B.L., Rainwater, T.R. (2008): Biodiversity, exploitation, and conservation of turtles in the Tonle Sap Biosphere Reserve, Cambodia, with notes on reproductive ecology of Malayemys subtrijuga. Chelon. Conserv. Biol. 7: 195-204. Powell, M.A. (1998): Trees and shrubs of the Trans-Pecos and Adjacent Areas. University of Texas Press, Austin. Smith, J. (2016): Habits and habitats of the rough-footed mud turtle, Kinosternon hirtipes, and outlook for its survival. Unpublished doctoral dissertation. New Mexico State University, Las Cruces. Smith, J.L., Platt, S.G., Boeing, W.J. (2015): Kinosternon hirtipes (rough-footed mud turtle). Maximum size and habitat. Herpetol. Rev. 46: 82-83. Texas Parks and Wildlife Department (2013): Species of Conservation Concern. Available: www.tpwd.state. tx.us (Accessed: 15 January 2016).

Kinosternon hirtipes reproduction

Tucker, J.K. (2007): Comparison of clutch size from natural nests and oxytocin induced clutches in the Redeared Slider, Trachemys scripta elegans. Herpetol. Rev. 38: 40. Wilde, M., Platt, S. (2011): A life-giving trail: documenting the environmental history of Alamito Creek. J. Big Bend Stud. 23: 85-106. Wilgenbusch, J.C., Gantz, D.T. (2000): The effects of hor-

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monally induced oviposition on egg viability in the Common Snapping Turtle Chelydra serpentina. Herpetologica 56: 1-7. Wilkinson, L.R., Gibbons, J.W. (2005): Patterns of reproductive allocation: clutch and egg size variation in three freshwater turtles. Copeia 2005: 868-879. Zar, J.H. (1996): Biostatistical Analysis. Prentice Hall, Saddle River.

Acta Herpetologica 11(2): 227-231, 2016 DOI: 10.13128/Acta_Herpetol-17922

Heavy traffic, low mortality - tram tracks as terrestrial habitat of newts Mikołaj Kaczmarski*, Jan M. Kaczmarek University of Life Sciences, Institute of Zoology, Wojska Polskiego 71c, 60-625 Poznań, Poland. * Corresponding author. E-mail: traszka. [email protected] Submitted on 2016, 3nd February; revised on 2016, 29th April; accepted on 2016, 10th June Editor: Adriana Bellati

Abstract. Amphibian mortality caused by rail traffic has not attracted much attention in comparison to road mortality. Density of railways in landscape, as well as traffic intensity, is usually much lower than in case of roads. As a consequence, their overall effect on amphibian populations is tacitly assumed to be less negative. To test whether very intensive rail traffic can cause substantial mortality in population of a small amphibian, we investigated a Smooth newt Lissotriton vulgaris population located in the city of Poznań, W Poland, where tram tracks border isolated breeding ponds. We performed controls during the peak of autumn migratory activity along the tracks. Less than 1% of all individuals found during the survey were killed by rail traffic. Observed mortality was very low despite large number of individuals present on the track and intensive tram traffic. As negative effects of traffic are low, rail or tram embankments can provide an important terrestrial habitat for small European newts. Keywords. Amphibian, Lissotriton vulgaris, mortality, railway, Smooth newt, traffic.

Amphibian mortality caused by rail transport has not attracted much attention in comparison to road mortality, which is investigated by a growing number of papers (Coffin, 2007; Elzanowski et al., 2009; Glista et al., 2009; Cayuela et al., 2015; Franch et al., 2015). Density of railways in landscape, as well as traffic intensity, is usually much lower than in case of roads. As a consequence, their overall effect on amphibian populations is tacitly assumed to be less negative. Despite that, amphibians crossing the railway line suffer from mortality by train collisions (Budzik and Budzik, 2013) or turbulence effect (Barandun, 1991). Railway infrastructure can block or hinder migration, especially in case of large newts or toads (Etienne et al., 2003). However, the structure of a typical railway track could enable small, crawling amphibians, like European Lissotriton newts, to move under the rails, therefore minimizing the risk of collision. On the other hand, in case of small species, dead individuals are more likely to disappear quickly after death, because of scavenging (Beckmann and Shine, 2014), dryISSN 1827-9635 (print) ISSN 1827-9643 (online)

ing (Budzik and Budzik, 2013) or physical destruction by traffic (Hels and Buchwald, 2001). As a consequence, rail traffic mortality rates for species like Lissotriton newts are likely to be underestimated, just as they are for road traffic (Elzanowski et al., 2009; Cayuela et al., 2015). To verify whether very intensive rail traffic can cause substantial mortality in populations of a small amphibian, we investigated a Smooth newt Lissotriton vulgaris Linnaeus, 1758 population located in the Poznań city, Poland (52°23’31.1”N, 16°58’33.5”E), where the breeding site is located near double tram tracks with very heavy traffic load. Smooth newt is considered the most widespread newt of the Old World (Arntzen et al., 2009; Sparreboom, 2014). Although L. vulgaris is a common species, detailed analyses show that it undergoes a broad decline in Western Europe, analogous to that of other newt species (Denoël, 2012; Denoël et al., 2013). It breeds in a vast diversity of water bodies and is generally associated with different kinds of wetlands (Bell, 1977). The breeding sites are two small ponds (about 700 m2 and 900 m2, © Firenze University Press www.fupress.com/ah

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Fig. 1. Situational map showing the area of the research.

respectively) completely overgrown by Phragmites australis (Cav.) Trin. ex Steud, 1841 within an intensively managed urban park (Fig. 1). Until 1960s, the studied area was the part of suburban farmland with a small river valley; urban development in 1970-1980s isolated L. vulgaris from surrounding populations. The park borders with housing estates and a forested buffer of an industrial area, containing a fish-inhabited pond that is the breeding site for common toads Bufo bufo Linnaeus, 1758. The double tram track is located along the southern border of the park. Daytime traffic load is ca. 20 trams/hour, peaking at late evening and early morning hours (over 40 trams/hour). The structure of the tram track is analogous to a typical railway track: wooden sleepers (Fig. 2C) on crushed stone aggregate (Fig. 2A, B). We performed 11 controls between 6th and 19th October 2014 (peak of autumn migratory activity). We searched for newts between 20:00 and 24:00 along the 300-m transect at the edge of the tram tracks in their section closest to the breeding ponds. We also searched for newts crossing pedestrian trails within the park and roads along park boundaries. In total, 303 individuals were noted along tram tracks in the whole survey period. We found individuals of both

sexes, as well as juveniles (Table 1). Captured individuals were measured (SVL, body mass) using digital caliper and electronic scale. However, we did not mark individuals, so numbers are given separately for each day, with the highest number of animals observed on October 15th (68 individuals; Fig. 3). Only 3 individuals killed by tram were found during the survey period. Dead newts were found only at pedestrian crossings. Newts were observed at the edge of the track as well as between the rails (Fig. 3). No individuals were found elsewhere (forested area, park trails or roads along the park boundaries). It has already been suggested that railway tracks do not necessarily block the migration efforts of newts. No significant genetic effects were found in populations of the Alpine newt Ichtyosaura alpestris Laurenti, 1768 at both sides of the 30-year old railway, which could have been a consequence of newt migration across the track (Prunier et al., 2014). In contrast, in case of North American salamander Ambystoma opacum Gravenhorst, 1807 a 100-years old railway led to total genetic isolation of two subpopulations divided by tracks (Bartoszek and Greenwald, 2009). Such difference could be ascribed to the time of isolation or the lesser mobility of Ambystoma opacum compared to European newts. Also, the popula-

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Tram tracks as terrestrial habitat of newts

Fig. 3. Number of individuals during each day of our survey with mortality (15th October, one female; 18th October, one male and one juvenile).

Fig. 2. (A) Male Smooth newt L. vulgaris during the searching for prey among crushed stone aggregate; (B) crushed stone aggregate as newts habitat with the rail in the background; (C) female Smooth newt found feeding on earthworm on the surface of a wooden sleeper (October, 2014). Table 1. Morphometric values of L. vulgaris from 14th October. x

min

max

SD

n

SVL [mm]

MM FF JUV

38.78 38.82 22.92

31.03 29.67 19.86

45.07 46.11 27.45

3.17 4.36 2.93

25 28 7

Body mass [g]

MM FF JUV

1.79 1.91 0.32

1.18 0.72 0.12

2.93 3.12 0.59

0.40 0.58 0.16

25 28 7

tion researched by Bartoszek and Greenwald (2009) was already isolated and had relatively low number of individuals, while Prunier et al. (2014) performed analysis on landscape scale with large number of interconnected populations. Another evidence for low level of isolation provided by railway lines is the work of Budzik and Budzik (2013), who found no dead newts along the 45-km transect along the railway line in SE Poland. Budzik and Budzik (2013) suggested that either newts disappear shortly after death due to their small body size, or they are not subject to mortality caused by rail traffic. Our results provide evidence for the latter hypothesis, as observed

mortality was low compared to high number of live specimens and great intensity of tram traffic, much exceeding the train traffic intensity in Budzik and Budzik (2013). Dead newts were found only at pedestrian crossings, where newt movement under the tracks is impossible, and animals are forced to move over instead of under the rails. We confirmed that, in late autumn, L. vulgaris from the urban population use the tram track as their terrestrial habitat, and are not subject to extensive mortality. The timing of the controls enabled us to search for dead individuals just after the evening peak of tram traffic (and, presumably, the peak of newt activity and mortality). Temperature and humidity during all controls were likely to prevent the dead individuals from quick drying. As dead individuals were collected during the night, the risk of scavenging by birds was minimal. We could not exclude the possibility of scavenging by mammals (e.g., hedgehogs, feral cats, foxes, rats), but we treat it unlikely as the controls were performed early in the night when tram and human traffic was still relatively intensive, restricting the mammalian activity. Thus, we feel that the detection probability of dead newts was higher than usually assumed for estimating road mortality (Hels and Buchwald, 2001; Elzanowski et al., 2009). Although the length of the transect was short compared to some other studies (e.g., Budzik and Budzik, 2013), we stress that it was enough to cover most of the terrestrial habitat of the investigated population, as the surrounding areas are either build-up or do not provide enough shelter for the newts (Kaczmarek et al., 2014.). Therefore, we suggest that the rail aggregate consisting of sleepers and stones is a crucial winter habitat for the investigated population (Fig. 2). A large number of shelters within rail aggregate, providing humid and prey-rich terrestrial habitat, enable the persistence of a large newt population, despite isolation and deteriorating quality of the breeding pond. Additionally, driving trams directly scare away large

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predators, thus reducing predation risk. Easily warming up stone aggregate could also affect newt activity pattern, as active individuals were observed until the end of December (pers. comm.). Presence of juvenile individuals (up to 38% in daysamples over 20 individuals) is interesting (Fig. 3). Malmgren (2002) showed that out of the pond movement of juvenile newts was more random than of the adult individuals, which is in accordance with theoretical framework presented by Pittman et al. (2014). However, the majority of juveniles in Malmgren (2002) chose similar direction as adults, i.e., towards the forest. Abundance of juvenile individuals on the track suggests that their movements are nonrandom. If juvenile newts used ‘normal’ cues, signaling the proximity of optimal terrestrial habitat (e.g., humidity gradient, shadow, slope axis), they should move along the old riverbed and remain within the park. Therefore, we suppose that at least some juvenile individuals follow the adults using olfactory cues (Hayward et al., 2000). In contrast to low rail traffic mortality, European newts are vulnerable to road traffic mortality (Denoël, 2012). For slow-moving newts, the probability of being killed is very high regardless of traffic intensity (Hels and Buchwald, 2001). In a setup similar to this study (500m transect along a local road, breeding newt population migrating from pond located 300 m from the transect), the road kills-to-spawners ratio for L. vulgaris was higher than for any other amphibian, with more than half of the population killed each year (Elzanowski et al., 2009). In such a context, rail traffic seems much less detrimental for newt populations, despite potential mortality caused by herbicide spraying (Brühl et al., 2013). It seems that in the studied population, where the managed urban greenery does not provide a sufficient number of high-quality hibernation sites, cavities within the rail aggregate act as the key wintering habitat. It requires further investigation whether the rail/tram aggregate and sleepers are a suitable all-year habitat for juvenile newts. Whereas it is indisputable that roads and railways negatively affect animals, some positive effects on birds and reptiles were also reported (Morelli et al., 2014). We argue that for small, crawling amphibians like L. vulgaris the benefits from new habitats created by railway infrastructure can outweigh the potential costs of traffic-induced mortality. ACKNOWLEDGEMENTS

Animal collection was performed according to RDOS permit no. WPN-II.6401.245.2014.AG. We are very thankful to Piotr Tryjanowski, Anna M. Kubicka,

and Katarzyna Pędziwiatr for encouragement and discussion during preparation of the manuscript. The authors thank M. Piasecka, M. Machura and other students for field assistance. We are also grateful to three anonymous reviewers whose comments greatly improved this paper. REFERENCES

Arntzen, J.W., Kuzmin, S., Beebee, T., Papenfuss, T., Sparreboom, M., Ugurtas, I.H., Anderson, S., Anthony, B., Andreone, F., Tarkhnishvili, D., Ishchenko, V., Ananjeva, N., Orlov, N., Tuniyev, B. (2009): Lissotriton vulgaris. The IUCN Red List of Threatened Species. Version 2014.3. (Accessed: 6 January 2015). Barandun, J. (1991): Amphibienschutz an Bahnlinien. Natur und Landschaft 66: 305. Bartoszek, J., Greenwald, K.R. (2009): A population divided: railroad tracks as barriers to gene flow in an isolated population of marbled salamanders (Ambystoma opacum). Herpetol. Conserv. Biol. 4: 191-197. Bell, G. (1977): The life of the smooth newt (Triturus vulgaris) after metamorphosis. Ecol. Monograph. 47: 279-299. Beckmann C., Shine, R. (2014): Do the numbers and locations of road-killed anuran carcasses accurately reflect impacts of vehicular traffic? J. Wildlife Manage. 79: 92-101. Brühl, C.A., Schmidt, T., Pieper, S., Alscher, A. (2013): Terrestrial pesticide exposure of amphibians: an underestimated cause of global decline? Sci. Rep. 3: 1135. Budzik, K.A., Budzik, K.M. (2013): A preliminary report of amphibian mortality patterns on railways. Acta Herpetol. 9: 103-107. Cayuela, H., Quay, L., Dumet, A., Léna, J.P., Miaud, C., Rivière, V. (2015): Intensive vehicle traffic impacts morphology and endocrine stress response in a threatened amphibian. Oryx 2015: 1-7. Coffin, A.W. (2007): From roadkill to road ecology: a review of the ecological effects of roads. J. Transp. Geogr. 15: 396-406. Denoël, M. (2012): Newt decline in Western Europe: highlights from relative distribution changes within guilds. Biodivers. Conserv. 21: 2887-2898. Denoël, M., Perez, A., Cornet, Y., Ficetola, G.F. (2013): Similar local and landscape processes affect both a common and a rare newt species. PLoS ONE 8: e62727. Elzanowski, A., Ciesiołkiewicz, J., Kaczor, M., Radwańska, J., Urban, R. (2009): Amphibian road mortality in

Tram tracks as terrestrial habitat of newts

Europe: a meta-analysis with new data from Poland. Eur. J. Wildlife Res. 55: 33-43. Etienne, R.S., Vos, C.C., Jansen, M.J. (2003): Ecological impact assessment in data-poor systems: a case study on metapopulation persistence. Environ. Manage. 32: 760-777. Franch, M., Silva, C., Lopes, G., Ribeiro, F., Trigueiros, P., Seco, L., Sillero, N. (2015): Where to look when identifying roadkilled amphibians? Acta Herpetol. 10: 103-110. Glista, D.J., DeVault, T.L., DeWoody, J.A. (2009): A review of mitigation measures for reducing wildlife mortality on roadways. Landsc. Urban Plan. 91: 1-7. Hayward, R., Oldham, R.S., Watt, P.J., Head, S.M. (2000): Dispersion patterns of young great crested newts (Triturus cristatus). Herpetol. J. 10: 129-136. Hels, T., Buchwald, E. (2001): The effect of road kills on amphibian populations. Biol. Conserv. 99: 331-340. Kaczmarek, J., Kaczmarski, M., Pędziwiatr, K. (2014): Changes in the batrachofauna in the city of Poznań over 20 years. In: Urban fauna. Animal, Man, and the City – Interactions and Relationships, pp. 169-178.

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Indykiewicz, P., Böhner, J., Eds, ARTStudio, Bydgoszcz. Malmgren, J.C. (2002): How does a newt find its way from a pond? Migration patterns after breeding and metamorphosis in great crested newts (Triturus cristatus) and smooth newts (T. vulgaris). Herpetol. J. 12: 29-35. Morelli, F., Beim, M., Jerzak, L., Jones, D., Tryjanowski, P. (2014): Can roads, railways and related structures have positive effects on birds? – A review. Transport. Res. D-Tr. E. 30: 21-31. Pittman, S.E., Osbourn, M.S., Semlitsch, R.D. (2014): Movement ecology of amphibians: A missing component for understanding population declines. Biol. Conserv. 169: 44-53. Prunier, J.G., Kaufmann, B., Léna, J.P., Fenet, S., Pompanon, F., Joly, P. (2014): A 40-year-old divided highway does not prevent gene flow in the alpine newt Ichthyosaura alpestris. Conserv. Genet. 15: 453-468. Sparreboom, M. (2014): Salamanders of the Old World. 1st edition. KNNV Publishing, Zeist.

Acta Herpetologica 11(2): 233-234, 2016 DOI: 10.13128/Acta_Herpetol-18614

Book Review: Sutherland, W.J., Dicks, L.V., Ockendon, N., Smith, R.K. (Eds). What works in conservation. Open Book Publishers, Cambridge, UK. http://dx.doi.org/10.11647/OBP.0060 Sebastiano Salvidio Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università di Genova, I 16132 Genova Italy. E-mail: [email protected]

The book “What works in conservation” is the product of the “Conservation Evidence” project (www.conservationevidence.com) which consists of four parts: i) a searchable database, ii) synopses of the evidence reported in the database for different species, habitats and conservation interventions, iii) the book itself and iv) the journal “Conservation Evidence”. In this open access journal, evidences of management actions and their post hoc monitoring are always reported on, usually by the comparison with a control or a previous situation. By the way, it is worth noticing that a recent special issue of the journal dedicated to amphibians has been recently published (Meredith et al., 2016). The volume “What works in conservation” consists of an short “Introduction” (pages 1-7) and seven chapters dedicated to different animal taxa, habitats or conservation interventions: 1) Amphibian conservation (pages 9-65); 2) Bat conservation (pages 67-93); 3) Bird conservation (pages 95-244); 4) Farmland conservation (pages 245-284); 5) Some aspects of control of freshwater invasive species (pages 285-292); 6) Some aspects of enhancing natural pest control (pages293-315) and finally 7) Enhancing soil fertility (pages 317-338). The interventions are listed according to IUCN categories, while worldwide conservation evidences were obtained by reviewing the available scientific literature in English and, when needed, also in other languages. Two criteria are requested to be included in the assessment: first the intervention was fully implemented in the field and second the effects of intervention were monitored sciISSN 1827-9635 (print) ISSN 1827-9643 (online)

entifically, to allow some kind of statistical inference about the results. Therefore, this approach excludes predictive species modelling and also correlative studies that are sometimes used to plan or realize conservation projects. The book is not descriptive or based on illustrated case studies, as is the case of conventional books on conservation (e.g., Sutherland, 2000), but is a synthetic guide intended to provide a rapid overview of the scientific evidence as obtained from specialized literature. Effectiveness and harmful effects of conservation actions or management interventions are assessed by a panel of experts cited in the first page of each chapter. Experts were asked to classify interventions in six categories from “Beneficial” to “Likely to be ineffective or harmful” (Table 1). The experts were asked to judge anonymously the evidence and the certainty for each intervention and to review their own judgment after seeing the overall scores and comments from the entire panel. Revision rounds were stopped after a large consensus among experts was achieved. This method, based on published data judged by experts, is a modified Delphi technique, which is now becoming a relevant decision tool in ecology and biodiversity conservation (Mukherjee et al., 2015). References to the reviewed literature are not reported within the book, but the link to the online literature database is always given and, therefore, the reader is bound to a web connection to retrieve citations and deepen each conservation outcome. In this review I will comment only on the first chapter regarding “Amphibian conservation”. The expert panel for amphibians was composed by 28 scientists and man© Firenze University Press www.fupress.com/ah

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Sebastiano Salvidio

Table 1. Synthesis of the categories for judging conservation interventions used in “What works in conservation”. Intervention result

Short definition

Beneficial Likely to be beneficial Trade-off between benefit and harms Unknown effectiveness Unlikely to be beneficial Likely to be ineffective or harmful

Evidence for high effectiveness and no harm Evidence for medium effectiveness and low harm Both effectiveness and harm present; to be evaluated according to local circumstances Insufficient or inadequate quality of data Lack of effectiveness not clearly demonstrated by data; no agreement among experts Ineffectiveness or harm clearly demonstrated by data

agers and at a first glance it is clearly European Union biased (14/28 = 50% of experts) with a large prevalence of UK experts (10 out of 14). Experts from USA constituted the 19% (5/28), Africans the 15% (4/28) and Asians only the 7% (2/28). In this panel, the scarcity of South American amphibian conservationists, represented by only one member, is also noticeable. Many different threats were assessed in the chapter “Amphibian conservation”: agriculture, urban development, transportation, collecting, logging and habitat modification. For each threat, a table with the final judgment of the experts on the conservation action is given, following the classification given in the “Introduction” (see also Table 1). Then, a short text explaining the scientific bases on how the consensus was reached and in particular the number of studies, countries in which the actions were implemented and their main results is shortly given. In addition, the experts scored “effectiveness”, “certainty” and “harms” related to the intervention, expressed as percentages. Going through the many different conservations actions assessed to reduce amphibian threats, some well-known interventions are confirmed to have large beneficial effects, with little or no harm at all, such as “Remove or control fish by drying out ponds”, “Deepen ponds to prevent desiccation” or “Create ponds”. On the other hand, there are some interesting responses to some long-debated conservation actions, such as “Commercially breed amphibians for the pet trade” or “Use amphibians sustainably”, for which no scientific evidence was found. Another example is the response about interventions to reduce population declines of amphibians crossing paved roads. Thus, the common practice to “Use humans to assist migrating amphibians across roads” (i.e., the use of volunteers to rescue toads on roads), was evaluated by the panel of experts as “Unlikely to be beneficial”. In this specific case, the best alternative conservation action should be

“Close roads during seasonal amphibian migration” or “Modify gully pots or kerns”. In short, the volume “What works in conservation” is an original, useful and practical tool for conservationists, managers, activists of non-governmental organizations and also for amphibian ecologists, All of them will obtain relevant information about conservation actions to be realized or eventually to be avoided, this latter information almost never discussed in classic conservation textbooks. The book should always be consulted before (and I stress the word “before”) planning any kind of conservation intervention to correctly evaluate, not only possible positive outcomes but, also non-desired and collateral harmful effects. It should also be used by local and national authorities that are charged to judge and fund biodiversity conservation actions. These actions are sometimes based not on scientific evidence but only on some self-assessed evaluation. The fact that “What works in conservation” is online and downloadable free of charges should facilitate its wide consultation by private and public entities working on the long-term conservation of amphibian populations. REFERENCES

Meredith, H.M.H., Van Buren, C., Antwis, R.E. (2016): Making amphibian conservation more effective. Conservation Evidence 13: 1-6. Mukherjee, N., Hugé, J., Sutherland, W.J., McNeill, J., Van Opstal, M., Dahdouh-Guebas, F., Koedam, N. (2015). The Delphi technique in ecology and biological conservation: applications and guidelines. Methods Ecol. Evol. 6: 1097-1109 Sutherland, W.J. (2000):The conservation handbook. Research, management and policy. Blackwell Science, Oxford, UK.

Finito di stampare da Logo s.r.l. – Borgoricco (PD) – Italia

Notes on the reproductive ecology of the rough-footed mud turtle (Kinosternon hirtipes) in Texas, USA STEVEN G. PLATT, DENNIS J. MILLER, THOMAS R. R AINWATER, JENNIFER L. SMITH Heavy traffic, low mortality - tram tracks as terrestrial habitat of newts MIKOŁAJ K ACZMARSKI, JAN M. K ACZMAREK

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Book Review Sutherland, W.J., Dicks, L.V., Ockendon, N., Smith, R.K. (Eds). What works in conservation. Open Book Publishers, Cambridge, UK SEBASTIANO SALVIDIO

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Cover: Rana dalmatina. Photo by A. Romano

© 2016 Firenze University Press Università degli Studi di Firenze Firenze University Press via Cittadella 7, 50144 Firenze, Italy http://www.fupress.com/ E-mail: [email protected]

Periodicità: semestrale ISSN 1827-9643 (online) ISSN 1827-9635 (print) Registrata al n. 5450 del 3.11.2005 del Tribunale di Firenze

acta herpetologica CONTENTS

December 2016 Vol. 11 – N. 2

Predator-prey interactions between a recent invader, the Chinese sleeper (Perccottus glenii) and the European pond turtle (Emys orbicularis): a case study from Lithuania VYTAUTAS R AKAUSKAS1,*, RūTA MASIULYTė, ALMA PIKūNIENė Effective thermoregulation in a newly established population of Podarcis siculus in Greece: a possible advantage for a successful invader G RIGORIS K APSALAS , I OANNA GAVRIILIDI , C HLOE A DAMOPOULOU, J OHANNES F OUFOPOULOS , PANAYIOTIS PAFILIS The unexpectedly dull tadpole of Madagascar’s largest frog, Mantidactylus guttulatus ARNE SCHULZE, ROGER-DANIEL R ANDRIANIAINA, B INA PERL, FRANK GLAW, MIGUEL VENCES

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Thermal ecology of Podarcis siculus (Rafinesque-Schmalz, 1810) in Menorca (Balearic Islands, Spain) Z AIDA ORTEGA, ABRAHAM MENCÍA , VALENTÍN PÉREZ-MELLADO

127

Growth, longevity and age at maturity in the European whip snakes, Hierophis viridiflavus and H. carbonarius SARA FORNASIERO, X AVIER BONNET, FEDERICA DENDI, MARCO A.L. ZUFFI

135

Redescription of Cyrtodactylus fumosus (Müller, 1895) (Reptilia: Squamata: Gekkonidae), with a revised identification key to the bent-toed geckos of Sulawesi SVEN MECKE, LUKAS HARTMANN, FELIX MADER, MAX KIECKBUSCH, HINRICH K AISER

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The castaway: characteristic islet features affect the ecology of the most isolated European lizard PETROS LYMBERAKIS, EFSTRATIOS D. VALAKOS, KOSTAS SAGONAS, PANAYIOTIS PAFILIS

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Sources of calcium for the agamid lizard Psammophilus blanfordanus during embryonic development JYOTI JEE, B IRENDRA KUMAR MOHAPATRA , SUSHIL KUMAR DUTTA, GUNANIDHI SAHOO

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Mediodactylus kotschyi in the Peloponnese peninsula, Greece: distribution and habitat R ACHEL SCHWARZ, IOANNA-AIKATERINI GAVRIILIDI, YUVAL ITESCU, SIMON JAMISON, KOSTAS SAGONAS, SHAI MEIRI, PANAYIOTIS PAFILIS

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Swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures HONG-LIANG LU, QIONG WU, JUN GENG, WEI DANG

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Olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the Circeo National Park (Central Italy) ANTONIO ROMANO, RICCARDO NOVAGA, ANDREA COSTA

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Short Note On the feeding ecology of Pelophylax saharicus (Boulenger 1913) from Morocco Z AIDA ORTEGA, VALENTÍN PÉREZ-MELLADO, PILAR NAVARRO, JAVIER LLUCH

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