Two new species of Euscorpius (Scorpiones: Euscorpiidae) from [PDF]

Zootaxa 3894 (1): 083–105 www.mapress.com /zootaxa / Copyright © 2014 Magnolia Press

Article

ISSN 1175-5326 (print edition)

ZOOTAXA

ISSN 1175-5334 (online edition)

http://dx.doi.org/10.11646/zootaxa.3894.1.7 http://zoobank.org/urn:lsid:zoobank.org:pub:DE6A56E8-DA80-4C1D-A5BA-634D0F53D92E

Two new species of Euscorpius (Scorpiones: Euscorpiidae) from Bulgaria, Serbia, and Greece VICTOR FET1, MATTHEW R. GRAHAM2, MICHAEL M. WEBBER3 & GERGIN BLAGOEV4 1

Department of Biological Sciences, Marshall University, Huntington, West Virginia, USA Department of Biology, Eastern Connecticut State University, Connecticut, USA 3 School of Life Sciences, University of Nevada, Las Vegas, Nevada, USA 4 Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada Corresponding author: Victor Fet ([email protected]) 2

Abstract Two new species of Euscorpius Thorell, 1876 (subgenus Euscorpius s.str.) (Scorpiones: Euscorpiidae) are described based on morphology and the COI DNA barcoding marker: E. deltshevi sp. n. from northern Bulgaria and neighbouring Serbia (formerly reported as E. carpathicus) and E. solegladi sp. n. from southwestern Bulgaria and neighbouring Greece (formerly reported as E. hadzii). Key words: Scorpions, Balkans, DNA barcoding

Introduction A recent review of Bulgarian scorpions of the genus Euscorpius Thorell, 1876 (Fet & Soleglad 2007) outlined issues and problems involved with delineation and understanding of species in these enigmatic arachnids. In the first thorough molecular study of Greek Euscorpius taxa, Parmakelis et al. (2013a) demonstrated that cryptic speciation among these scorpions appears to be dramatically underestimated. Here, we apply DNA barcoding, or sequencing of the mitochondrial gene coding for cytochrome c oxidase subunit I (COI), to clarify the phylogenetic position and taxonomic identity of two new, but common, species of Euscorpius inhabiting Bulgaria, and provide descriptions of each.

Material and Methods Material Studied. Thanks to the courtesy of Dr. Petar Beron, who loaned the entire Euscorpius collection of the National Museum of Natural History, Sofia (NMNHS) to V.F., we studied as many as 190 specimens of E. deltshevi sp. n. and 62 specimens of E. solegladi sp. n., which allowed us to gain a better understanding of morphological variation as well as broaden our geographic perspective. A detailed list of material with label data is provided below. Specimens used for DNA barcoding. Of 25 sequences of Euscorpius used for our phylogeny, 18 are reported here for the first time (listed below with GenBank accession numbers). Euscorpius (E.) tergestinus (C.L. Koch, 1837), SLOVENIA: under Nanos Mt., Vipava, 45°50'48"N, 13°57'47"E, 15.06.2001 (Univ. of Ljubljana), VF-0777 (AMSCO047-10, KM111249); CROATIA: Majorija, Senj, 44°59'25"N, 14°54'05"E, 550 m, 7.08.2000 (B. Sket), VF-0823 (AMSCO081-10, HM418305). Euscorpius (E.) hadzii Di Caporiacco, 1950, BOSNIA & HERZEGOVINA: Herzegovina, 15 km S Trebinje, 42°42'N, 18°20'E, 3.08.2000 (P. Trontelj), VF-0795 (AMSCO056-10, HM418290); Herzegovina, Čemerno, 43°14'14"N, 18°36'4"E, 8.09.2006 (D. Pavicevic), VF0796 (AMSCO057-10, HM418291); Republika Srpska, Lukavac, 44°46'13"N, 18°53'31"E, 8.09.2006 (D.

Accepted by Y. Marusik: 3 Oct. 2014; published: 11 Dec. 2014

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Pavicevic), VF-0797 (AMSCO058-10, HM418292). Euscorpius (E.) deltshevi sp. n., BULGARIA: Lovech Province, Teteven, 42°55′N, 24°16′E, 05.2007, VF-0811 (AMSCO070-10, HM418298); Montana Province, Chiprovtsi District, Gorna Laka, 43º27'N, 22º54'E, 406 m, 16.05.2005 (V. Fet & S. Fet), VF-0812 (AMSCO07110, HM418299); Sliven Province, Sliven, 42°41'N, 26°20'E, 2.05.2003, ca. 800 m (J.O. Rein), VF-0814 (AMSCO073-10, HM418300). Sofia Province, Beledie Han, 11 km N of Kostinbrod, 42º53'N, 23º09'E, 17.05.2005 (V. Fet & S. Fet), VF-0818 (AMSCO076-10, HM418302); Lakatnik Railway Station, 43°05'10"N, 23°23'01"E, 4.05.2005 (V. Fet & A. Popov, VF-0815 (AMSCO074-10, HM418301); Tserovo, 43.00ºN, 23.35ºE, 739 m, 4.05.2005 (V. Fet & A. Popov, VF-0817 (AMSCO075-10, KM111245); Vidin Province, Belogradchik, 43º37'38"N, 22º41'E, 538 m, 16.05.2005 (V. Fet & S. Fet), VF-0821 (AMSCO079-10, KM111243); Oreshets Railway Station, near Suhi Pech Cave, 43º29'26"N, 22º44'20"E, 16.05.2005 (V. Fet & S. Fet), VF-0822 (AMSCO080-10, KM111242); SERBIA: Nišava Province: Niš, Niška Banja, 15.04.2006 (I. Karaman), VF-0746 (AMSCO026-10, KM111244). Euscorpius solegladi sp. n., BULGARIA: Blagoevgrad Province, Rila Monastery, 42°08′N, 23°20'25"E, 1147 m, 5.06.1999 (V. Fet & V. Sakalian), VF-0799 (AMSCO060-10, HM418293); Blagoevgrad Province, Sandanska Bistritsa River, 41°34'N, 23°17'E, 26.05.2005 (V. Fet & E. Fet), VF-0801 (AMSCO062-10, KM111247); Pernik Province, Radomir District, Baikalsko, 844 m, 42°25.378'N, 22°48.862'E, 28.05.2005 (V. Fet & E. Fet), VF-0802 (AMSCO063-10, KM111246); Pernik Province, Tran District, Erma Gorge, 703 m, 42°51.679'N, 22°38.952'E, 28.05.2005 (V. Fet & E. Fet), VF-0813 (AMSCO072-10, KM111248). The remaining 7 barcodes were published recently (Graham et al. 2012a, b; Parmakelis et al. 2013a): Euscorpius (Alpiscorpius) gamma Di Caporiacco, 1950. AUSTRIA, Carinthia, Trögerner-Klamm, 46°27'28"N, 14°30'02"E, 13.06.1999 (V. Fet & B. Scherabon), VF-0732 (AMSCO014-10, HM418273). Euscorpius (E.) carpathicus (Linnaeus, 1767): ROMANIA, Caraş-Severin County, Băile Herculane, 44°52'43"N, 22°24'51"E, 4.06.2008 (F. Šťáhlavský), VF-0768 (AMSCO044-10, HM418284). Euscorpius (E.) sicanus (C.L. Koch, 1837): MALTA, Buskett Gardens, 35°51'41"N, 14°23'56"E, 17.09.2001 (P. Schembri), VF-0792 (AMSCO053-10, HM418288). ITALY, Sardinia, S. Niccolo Gerrei, near Grotta Saturru, 39.49816°N, 09.31503°E, 395 m, 04.2006 (A. v.d. Mejden), VF-0789 (AMSCO052-10, JX133089). TUNISIA, Zaghouan Governorate, Jebel Zaghouan Mts., along the trek, 36°22.423'N, 10°06'E to 36°22.924'N, 10°06.789'E, 650-780 m, mixed forest, 03.2008 (P. Stoev & N. Akkari), VF-0793 (AMSCO054-10, HM418289). Euscorpius (E.) hadzii, ALBANIA, Bjeshket e Nemuna (Prokletije) Mts., Maya e Shtegut (Theth), 21.07.2000 (T. Wraber), 189F (KC215665). Euscorpius (E.) solegladi sp. n. (as E. hadzii): BULGARIA, Blagoevgrad Province, Gorna Breznitsa, 41°45'N, 23°07'E, 27.05.2005 (V. Fet & D. Dobrev), VF-0798 (AMSCO059-10, HM880289). Depositories: FKCP, private collection of František Kovařík, Prague, Czech Republic; MNHN, Muséum national d’Histoire Naturelle, Paris, France; NHMW, Naturhistorisches Museum Wien, Vienna, Austria; NMNHS, National Museum of Natural History, Sofia, Bulgaria; NMPC, National Museum of Natural History, Prague, Czech Republic; SMF, Senckenberg-Museum, Frankfurt, Germany; VFPC, private collection of Victor Fet, Huntington, West Virginia, USA; ZMB, Zoologisches Museum Berlin, Humboldt-Universität, Berlin, Germany; ZMMSU, Zoological Museum, Moscow State University, Moscow, Russia. Morphology: Terminology and measurements (in mm) follow Stahnke (1970) and Sissom (1990); trichobothrial designations follow Fet & Soleglad (2002).

Molecular Analyses Phylogenetic Analyses. We assessed the phylogenetic position of the two new species with DNA barcoding, an approach that has recently been applied to other Euscorpius taxa (Graham et al. 2012a, b; Parmakelis et al. 2013a). All DNA work was performed in the University of Guelph and new barcodes were submitted to GenBank (Accession Nos. as listed above). Barcodes are also accessible through BOLD (http://www.boldsystems.org) (Ratnasingham & Hebert 2007) under project title “Scorpions of the Ancient Mediterranean 2c (AMSCU)”. Voucher specimens are in a private collection of V. Fet (VFPC). We imported the barcodes into Geneious v. 7.0.2 (available from http://www.geneious.com/) and aligned them with the software using MUSCLE (Edgar 2004). The best-fit model of nucleotide substitution was determined for each codon position with MEGA v. 5.2.2 (Tamura et al. 2011) based on the Akaike Information Criterion (Posada 2008). Using the appropriate models (HKY for codons 1 and 2, HKY+G for codon 3), we constructed a COI phylogeny using Bayesian inference (BI) implemented in MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003) ran

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through the Cyberinfrastructure for Phylogenetic Research cluster (CIPRES Gateway v 3.1) at the San Diego Supercomputer Center. We based our interpretations on runs of 20 million generations with trees sampled every 2000 generations. We set the temperature to 0.075 and discarded the first 20% of sampled trees as burn-in. Divergence Dating. We placed the COI phylogeny in a temporal context using a relaxed molecular clock implemented in BEAST v. 1.7.2. (Drummond & Rambaut 2007). We unlinked substitution models across each codon partition, but linked the tree and clock models, and ran the analysis with the same best-fit substitution models used in the MrBayes analysis. We excluded the outgroup used in the MrBayes analysis and instead rooted the chronogram by constraining a monophyletic taxon set that included all samples except the two E. tergestinus. This approach produced a midpoint root between the E. tergestinus and the clade containing all remaining samples, which was identical to the strongly supported branching pattern (1.0 pp) in the MrBayes analysis. We ran the program for 40 million generations, with trees sampled every 4000 generations. We used the uncorrelated lognormal-relaxed clock model and applied the Yule tree prior with a scorpion specific mutation rate of 0.007 substitutions/site/million years and a mean standard deviation of 0.003 (Gantenbein & Keightley 2004). Convergence was assessed using Tracer v. 1.6 (Rambaut et al. 2009). We used TreeAnnotator to construct a maximum clade credibility tree with the first 20% of trees discarded as burn-in.

FIGURE 1. Geographic distribution of two new species: Euscorpius deltshevi, sp. n., squares; E. solegladi, sp. n., circles; black squares and circles represent populations sampled for DNA; stars represent type localities (both sampled for DNA).

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FIGURE 2. (A) Majority rule (50%) consensus tree depicting results of Bayesian phylogenetic analysis of COI indicating the position of Euscorpius deltshevi, sp. n. and E. solegladi, sp. n., within subgenus Euscorpius s.str. Posterior probabilities greater than 0.5 are indicated above each node.

Systematics Family Euscorpiidae Laurie, 1893 Genus Euscorpius Thorell, 1876 Subgenus Euscorpius Thorell, 1876, s.str. NOTE. The traditional subgenus Euscorpius (sensu lato) is paraphyletic, as was recently confirmed by DNA phylogenies (Tropea 2013; Parmakelis et al. 2013a). A well-supported clade containing the type species, E. carpathicus, is addressed here as subgenus Euscorpius s.str. At the same time, a number of distant basal taxa, morphologically similar to those of Euscorpius s.str., group distantly in DNA phylogenies, and have to be placed as incertae sedis, such as E. aquilejensis (C.L. Koch, 1837), E. avcii Tropea et al. 2012, E. balearicus Di Caporiacco, 1950, E. tauricus (C.L. Koch, 1837), and a number of undescribed species (Graham et al. 2012b; Tropea 2013; Parmakelis et al. 2013a, b; Tropea et al., in prep.).

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FIGURE 2. (B) A rate-calibrated chronogram depicting the timing of divergence among focal taxa. Bars represent highest posterior densities (95%) around mean estimates.

Euscorpius deltshevi sp. n. (Figs 3–22, 43; Table I) Euscorpius carpathicus: Jurinich1904: 140 (in part; Bulgaria, in part); Valle 1975: 233 (in part, Bulgaria: Belogradchik). Euscorpius carpathicus “Subgroup A1” (in part; Bulgaria: Montana & Pleven Provinces): Fet 2000: 52. Euscorpius carpathicus “Subgroup A2” (in part; Bulgaria: Sofia & Veliko Tarnovo Provinces): Fet 2000: 52–53. Euscorpius sp. (“carpathicus” complex) (in part; “northern” group): Fet & Soleglad 2007: 413.

Type material: Holotype♂ (NMNHS), BULGARIA, Sofia Province: Tserovo, 43.00ºN, 23.35ºE, 739 m, 4.05.2005 (V. Fet & A. Popov). Paratypes: same label as holotype, 3♂, 8♀ (NMNHS), 1♂, 1♀ (NHMW 21.952–21.953); Tserovo (P. Beron), 1♂, 2♀ (NMNHS 36); between Tserovo and Iskrets, 1000 m, 25.09.1960 (V. Beshkov), 1♀ (NMNHS 235); Lakatnik Railway Station, 23.03.1930 (P. Drenski), 1♀ (NMNHS 313); Lakatnik Railway Station, 6.05.1934 (J. Zonkov), 2♂ sbad. (NMNHS 305); Lakatnik Railway Station, 20.08.1934 (G. Stoyanov), 1♀ (NMNHS 300); Lakatnik Railway Station, 10.07.1948 (I.A. Ivanov & P. Tranteev), 1♂ sbad., 2♀ sbad., 1♀ juv. (NMNHS 274); Lakatnik Railway Station, near Razhishkata Cave, 7.08.1948 (G. Rupev), 2♀, 1♀ juv. (NMNHS 281); Lakatnik Railway Station, 15.05.1997 (B. Petrov), 2♀ (NMNHS 220); Lakatnik Railway Station, near Temnata Dupka Cave, 24.07.1988 (P. Mitov), 1♂, 4♀ (VFPC); Lakatnik Railway Station, 43°05'10"N, 23°23'01"E, 4.05.2005 (V. Fet & A. Popov), 6♀ (VFPC), 1♀ (NHMW 21.954); Lakatnik Railway Station, 1970, 1♂, 2♀ (NMPC), 1♂ (FKPC); Opletnya near Lakatnik, 43°06'N, 23°25'60"E, 21.05.1994 (P. Stoev), 1♂, 2♀ (NMNHS 214); Svoge, 8.07.1934 (N. Miladinov), 1♀ (NMNHS 303); Svoge, 42º58'N, 23º21'E, 679 m, 4.05.2005 (V. Fet & A. Popov), 2♀ (VFPC); Tompsan Station, Iskar Valley, 29.06.1952 (A. Popov), 1♂ (NMNHS 532); Zanoge, 1100–1300 m, 2.05.1985 (P. Beron), 4♀ (NMNHS 114).

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TABLE I. Measurements (in mm) of male holotypes and female paratypes of Euscorpius deltshevi sp. n. and E. solegladi sp. n. L, length; W, width; D, depth; Dp, number of pectinal teeth; number of patellar trichobothria: Pv, ventral; et, external terminal; em, external median; eba, external basal-a; eb, external basal. Euscorpius deltshevi sp. n. Male Holotype

Female Paratype

Tserovo, Sofia Province, Bulgaria

Euscorpius solegladi sp. n. Male Holotype

Female Paratype

Sandanska Bistritsa, Blagoevgrad Province, Bulgaria

Total L

33.00

35.40

34.50

34.80

Carapace L

5.45

5.52

5.85

5.58

Carapace Posterior W

5.45

5.70

5.60

5.50

Metasoma segments: Met I L

2.41

1.97

2.13

1.90

Met I W

1.96

1.89

1.92

1.91

Met II L

2.58

2.33

2.43

2.11

Met II W

1.71

1.65

1.85

1.56

Met III L

2.43

2.37

2.88

2.39

Met III W

1.66

1.54

1.72

1.48

Met IV L

2.60

2.66

3.23

2.48

Met IV W

1.60

1.47

1.51

1.46

Met V L

5.24

4.50

5.64

4.76

Met V W

1.58

1.53

7.53

1.42

Vesicle L

4.16

3.31

4.48

3.02

Vesicle W

2.25

1.42

4.89

1.42

Vesicle D

2.38

1.59

2.58

1.44

Femur L

4.19

4.47

4.73

4.55

Femur W

1.67

1.85

1.84

1.93

Patella L

4.79

4.51

4.52

4.37

Patella W

1.87

1.88

1.84

1.97

Palm L

5.07

4.64

5.46

5.23

Palm W

4.04

3.72

4.28

3.72

Palm D

2.80

2.59

2.81

2.25

Movable finger L

5.50

5.07

5.71

5.42

Fixed finger L

4.10

3.94

4.16

4.18

Dp (Left/Right)

8/8

7/7

10/10

8/8

Middle lamellae

4/4

4/4

6/6

5/5

Pv

9/9

9/9

11/10

9/10

et

7/7

8/7

7/8

7/7

em

4/4

4/4

4/4

4/4

eba

4/4

4/4

5/5

5/5

eb

4/4

4/4

5/5

5/5

Trichobothria (Left/Right):

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FIGURES 3–4. Euscorpius deltshevi sp. n., adult male, general habitus: 3 dorsal view, 4 ventral view.

Other material studied. BULGARIA. Lovech Province: Thracian sanctuary (Asklepion) near Zlatna Panega Village, 4.08.1948 (Caving Brigade "T. Pavlov"), 4.08.1948, 2♂, 3♀ (NMNHS 340), 3♂, 1♀ (NMNHS 341); Karlukovo, 9.09.1923 (I. Buresch), 1♂ (NMNHS 297); Karlukovo, near Temnata Dupka Cave, 24.11.1935 (K. Popov), 1♂ (NMNHS 316); Lukovit District, Bezhanovo, Georgikovata Cave, 11.10.1973 (A. Petkova), 1♂ (NMNHS 157); Lukovit District, Bezhanovo, near entrance of Parnitsite Cave, 15.03.1998 (B. Petrov), 1♂ (NMNHS 203); Teteven, 42°55'N, 24°16'E, 05.2007 (VFPC); Troyan, 06.1919 (P. Drenski), 1♀, 1♀ sbad. (NMNHS 307). Montana Province: Chiprovtsi District, Beli Mel, 13.06.1973 (P. Beron), 1♂, 2♀ (NMNHS 49); Chiprovtsi District, Debelidelska Murtvina area, 1100–1300 m, 28.06.1998 (B. Petrov), 1♂, 1♀ juv. (NMNHS 204); Chiprovtsi District, Martinovo, 4.09.1998 (B. Petrov), 1♂, 2♀ (NMNHS 207); Chiprovtsi District, Gorna Laka, 43º27'N, 22º54'E, 406 m, 16.05.2005 (V. Fet & S. Fet), 2♂, 1♀ (VFPC); Diva Slatina, 21.06.1998 (B. Petrov), 2♀ juv. (NMNHS 205); Chiprovtsi District, Ravna, 1.06.1972 (P. Beron), 1♀ (NMNHS 348); Gorna Bela Rechka, Vartop Cave, 7.05.1909, 1♀ (NMNHS 503). Pernik Province: Radomir District, Golo Bardo Mts., 1000 m, 25.03.1937 (P. Drenski), 1♂, 2♀ (NMNHS 510). Pleven Province: Cherven Bryag District, Reselets, 20–27.07.1989 (P. Mitov), 1♂, 2♀ (VFPC); Cherven Bryag District, Deventsi, Haydushkata Cave, 24.08.1927 (H. Matrov), 1♂ (NMNHS 283); Dolny Dabnik District, Sadovets near Pleven, 29.05.1926 (H. Matrov), 1♀ (NMNHS 524); Pleven, 06.1912 (Klein), 1♀ (NMNHS 312). Ruse Province: Ruse (“Ruschuk”), 1♀ (ZMB 15263); Svalenik, Byalata Stena, "Rusenski Lom" Nature Park, 3.10.1999 (B. Petrov), 1♂, 1♀ (NMNHS 226); Pepelina, 4.10.1999

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(B. Petrov), 1♂ (NMNHS 225). Shumen Province: Preslav (now Veliki Preslav), Patleyna, 13.10.1933 (I. Grozev), 1♀ with juvs. (NMNHS 293), 3♂, 3♀ (NMNHS 523). Sliven Province: Sliven, 42°41'N, 26°20'E, 2.05.2003, ca. 800 m (J. O. Rein), 1♀ (VFPC); environs of Sliven, ca. 500 m, 06.1985 (G. Ribarov), 1♀, 1♀ juv. (NMNHS 343); Byala, 13.06.1927 (I. Julius & P. Drenski), 1♂ sbad. (NMNHS 311); Davidov Dol, 10.07.1934 (G. Kozarov), 2♀ (NMNHS 285); Katunishte, 10.1993 (T. Lerov), 1♂ (NMNHS 239); Kotel, 600 m (G. Ribarov), 2♀ juv. (NMNHS 240); "Ponor" near Kotel, 30.04.1924 (N. Radev), 1♀ juv. (NMNHS 509); Sinite Kamani National Park, 29.03.1983 (T. Petrov), 1♀ (NMNHS 241); Sinite Kamani National Park, between Karandila and Buchvata, 800–900 m, 25.05.2001 (B. Petrov & St. Beshkov), 1♂ (NMNHS 259). Sofia Province: Beledie Han, 5.10.1958 (A. Popov), 1♀ (NMNHS 549); Beledie Han, 11 km N of Kostinbrod, 42º53'N, 23º09'N, 17.05.2005 (V. Fet & S. Fet), 2♂, 4♀ (VFPC); Pravets District, Praveshka Lakavitsa, Aliova Dupka Cave, 5.02.1994 (B. Petrov), 1♂ (NMNHS 206); Botevgrad District, Lipnitsa Village, 575 m, 8.09.1997 (P. Mitov), 1♂ (VFPC); Bov Village, Mt. Izdremets, 1400 m, 14.09.1999 (B. Petrov, V. Beshkov & Yu. Gorelov), 1♂ (NMNHS 224); Chepan Hill, above Dragoman, 28.05.1932 (J. Zonkov), 1♀ sbad. (NMNHS 284); Goten Mt. above Buhovo Village (G. Stoyanov), 1♂ juv., 2♀ (NMNHS 513); Kremikovski Monastery, 23.04.1900 (I. Buresch), 2♀ (NMNHS 273); Novi Iskar, Kurilo, 6.05.1952 (A. Popov & I. Buresch), 1♂ juv. (NMNHS 517); Kurilo, 30.05.1952 (G. Stoyanov), 1♂, 1♀ (NMNHS 540); between Milanovo and Gorna Bela Rechka, 05.1911 (P. Drenski), 4♂, 2♀, 1♂ juv. (NMNHS 270); Ogoya, 12.06.1938 (G. Stoyanov), 5♀ (NMNHS 327); Rebrovo Railway Station, 10.06.1949 (G. Zagorov), 1♂ (NMNHS 233); Iskar Valley, Batuliya, Rebrovo Station, 23.05.1954 (I. Urumov & I. Buresch), 1♂, 2♀ (NMNHS 542); Rebrovo, 9.10.1980 (P. Beron), 1♀ (NMNHS 109); Rebrovo, 10.10.1985 (M. Kvartirnikov), 15♂, 39♀ (ZMMSU); Sedemte Prestola Monastery, 10.04.1988 (P. Beron), 1♂ juv. (NMNHS 232); Sofia (with firewood?), 1.06.1957 (A. Popov), 1♂ (NMNHS 516). Targovishte Province: valley near Targovishte, 43.26ºN, 26.59ºE, 207 m, 1.05.1962 (I. Buresch), 1♀ (NMNHS 539). Veliko Tarnovo Province: Arbanasi, Lyaskovskata Cave, 6.08.1968 (P. Beron), 1♀ (NMNHS 39); Preobrazhenski Monastery near Tarnovo, 25.07.1928 (Kr. Tuleschkov), 1♀ (NMNHS 538). Vidin Province: Belogradchik, Neprivetlivata Pothole, 6.06.1973 (P. Beron), 1♂ (NMNHS 333); Belogradchik, 43º37'38"N, 22º41'E, 538 m, 16.05.2005 (V. Fet & S. Fet), 5♀ (VFPC); Oreshets Railway Station, 17.10.1971 (P. Beron), 1♀ (NMNHS 339); Oreshets Railway Station, near Suhi Pech Cave, 43º29'26"N, 22º44'20"E, 16.05.2005 (V. Fet & S. Fet), 4♀ (VFPC). Vratsa Province: Vratsa town, 1.07.1924 (I. Buresch), 1♀ sbad. (NMNHS 291); environs of Vratsa, 2.06.1926 (H. Matrov), 1♂ sbad, 1♀ (NMNHS 278); Vratsa, 290 m, 8.05.1999 (S. Boev), 4♀ (VFPC); southern Vrachanska Mts., 880 m, 1.01.2005 (T. Ljubomirov), 1♀ (NMNHS); Chelopek (near Vratsa), Malata Yama pothole, 30 m deep, 1.07.1929 (N. Radev), 1♂ (NMNHS 304); Cherepishki Monastery, 1.05.1959 (A. Popov), 1♂ (NMNHS 528); Chiren Village, small cave near entrance of Ponora Cave, 22.04.1995 (T. Ivanova) (NMNHS 234); Roman District, Kunino, 1.05.1993 (T. Ivanova), 2♀ (NMNHS 186); near Dyavolska Vodenitsa Cave, near Kunino, Iskar Valley, 5.04.1924 (I. Buresch), 1♂ (NMNHS 529); near Ledenika Cave, 5.06.1933 (D. Papazov & N. Atanassov), 1♂ (NMNHS 289), 1♂ (NMNHS 294); Vrachanska Mts., Lyutadjik, 450 m, 1.04.2000 (B. Petrov & V. Beshkov), 1♀ (NMNHS 237); Vrachanska Mts., Matnishki Monastery, Cherniya Izvoz Cave, 3.04.1999 (B. Petrov), 1♀ (NMNHS 209); Vrachanska Mts., Parshevitsa Hut, 1000 m, 2.05.1994 (P. Stoev), 1♀ (NMNHS 216). Yambol Province: Yambol, 10.08.1934 (I. Tarpanov), 1♂ (NMNHS 302). SERBIA. Nišava Province: E of Niš, 1♀ (NHMW 11748); Niš, no date (I. Karaman), 1♀ (VFPC); Niš, Niška Banja, 15.04.2006 (I. Karaman), 1♀ (VFPC); Niš, Gornja Studena (“Grustudena”), 43º16'N, 22º05'E, 31.05–2.06.1954 (excursion Zool. Mus. Amsterdam), 2♀ (MNHN 6316–6317). Etymology. Named after our colleague and friend Christo Deltshev (Sofia, Bulgaria), a famous Bulgarian arachnologist. Geographic range. Known from Stara Planina Mts., Bulgaria, and adjacent areas of Bulgaria and Serbia (Fig. 1). Diagnosis. Medium sized species (33-35 mm), dark brown in overall coloration, mottling on carapace. Metasomal segments and chelae are somewhat robust in appearance. Dorsal patellar spur (DPS) is enlarged and spinoid. Pedipalp patellar external trichobothria numbers: eb = 4, eba = 4, esb = 2, em = 4, est = 4, and et = usually 6 to 7 (mean 6.46); ventral aspect of patella 8 to 9 (mean 8.82). Pectinal tooth counts: male usually 8 to 9 (mean 8.57), female usually 7. Male (Figs 3–4). Description based on holotype male. Measurements of male holotype and female paratype are provided in Table 1. All illustrations (except female chela in Fig. 9 and telson in Fig. 20) are based on a mature male specimen originating from Tserovo, Sofia Province, Bulgaria.

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FIGURES 5–6. Euscorpius deltshevi sp. n., adult female, general habitus: 5 dorsal view, 6 ventral view.

Coloration. Overall colour dark brown; legs orange, chelicerae light brown to orange with dark brown mottling, telson orange; pectines light orange to yellow. Pedipalp carinae dark brown to black. Carapace (Fig. 7). Slight median furrow extends entire length of carapace. Almost smooth with extremely fine granulation, more expressed granulation laterally behind lateral eyes; anterior edge straight with few fine setae. Two pairs of lateral eyes, anterior eye slightly larger; median eyes and tubercle situated slightly anterior of middle with following length and width ratios: 0.431 (anterior edge of the carapace to median tubercle centre/carapace length) and 0.161 (width of median tubercle/width of carapace at that point). Mesosoma. Dark mottling pattern is present. Tergites with extremely weak to absent carination on segments I–VII; sternites smooth and shiny, carinae absent on segment V with fine granulation; stigmata small, sub-oval. Metasoma. Generally stocky and slightly robust in proportions. Dorsal groove is moderately emarginate. Carination - Segments I–IV: Median carina small and granular; dorsolateral weak and composed of a few small rounded granules; intermediary obsolete; ventrolateral weak smooth and shiny on segments III and IV; ventral obsolete. Segment V (Figs 12–13): Median carina weak and granular; dorsolateral weak, round and granular; intermediary absent; ventrolateral rough and weakly serrate; ventral slightly rounded and serrate. Telson (Fig. 19). Vesicle with fine granulation, and swollen both laterally and ventrally. Aculeus forming a short conspicuous curve; 4 pairs of setae at vesicle/aculeus juncture. Pectines (Fig. 21). Pectinal tooth counts 8/8 and middle lamellae counts 4/4; fulcra well developed; numerous fine setae situated on anterior lamellae. Sensorial areas of teeth developed along approximately 2/3 their length. Basal piece anterior edge slightly concave. TWO NEW SPECIES OF EUSCORPIUS

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FIGURES 7–20. Euscorpius deltshevi sp. n. 7 carapace, 8 external view of the chela of adult male, 9 external view of the chela of adult female, 10 ventral view of the chela, 11 dorsal view of the chela, 12 lateral view of the metasomal segment V, 13 ventral view of the metasomal segment V, 14 external view of pedipalp patella, 15 dorsal view of pedipalp patella, 16 ventral view of pedipalp patella, 17 dorsal view of pedipalp femur (line indicates trichobothrium i), 18 ventral view of pedipalp femur, 19 telson of adult male, 20 telson of adult female.

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FIGURES 21–22. Euscorpius deltshevi sp. n., sternopectinal area: 21 adult male, 22 adult female.

Genital operculum. Separated most of length, genital papillae extend proximally. Sternum. Pentagonal, slightly wider than long, length/posterior width ratio 0.910. Chelicerae. Typical for the genus Euscorpius. Movable finger: dorsal distal denticles considerably shorter than ventral distal denticle; dorsal edge has five denticles, two of which are subdistal denticles; ventral edge smooth, lacking serrulae, and covered with heavy brush-like setae for most of its length. Fixed finger: four denticles, two of which are basal denticles conjoined on a common trunk. Pedipalps (Figs 8, 10–11, 14–18). Pedipalps with somewhat stocky and robust chelae exhibiting prominent scalloping at finger bases. Femur: dorsal and ventral internal and external carinae crenate to dentate; dorsal and ventral surfaces granulose, internal surface with numerous enlarged rounded to spinoid granules, and external surface contains numerous small rounded granules. Patella: dorsal internal and ventral internal carinae crenate, dorsal external smooth and weakly crenate, and exteromedian rounded and irregularly granulose. Dorsal surface finely granulose, ventral surface contains few weak rounded granules, prominent dorsal patellar spur (DPS) of medium to strong development, ventral patellar spur (VPS) very weak, represented as small granule. Chela carinae: digital strong and smooth; subdigital in relief, represented by weak granulation; dorsal secondary nearly obsolete with few weak granules; dorsal marginal weak and rounded, dorsal internal smooth and rounded; ventroexternal strong, smooth and extending to external condyle of finger, external to trichobothrium Et1. Ventromedian essentially obsolete with sparse minute granules; ventrointernal small, rounded and smooth; and external secondary strong and irregularly granulose. Chelal finger dentition: Median denticle row straight; 7/6 inner denticles, 5/6 outer denticles, and 5/5 inner accessory denticles for fixed and movable fingers respectively. Trichobothria patterns. Type C, neobothriotaxic (major additive) on patella. Femur (Fig. 17): trichobothrium d positioned slightly proximal in relation to i, and e is situated on dorsoexternal carina, distal in relation to both d and i. Patella (Figs 13–14): ventral series number 9/9 and external series number eb = 4/4, eba = 4/4, esb = 2/2, em = 4/4, est = 4/4, and et = 7/8. Chela (Figs 8–10): trichobothria on ventral surface is 4/4 (V1–3 + Et1), V4 on external surface set in small dimple but removed from ventroexternal carina. Legs. One pair of pedal spurs present, ungues medium length with average curve. Tarsus ventral median spinule row is formed by variable number of elongated spinules usually terminating distally in a single pair; one or two basal spinules sometimes offset; spinule counts are: tarsus I, 1+10+2/1+9+2; tarsus II, 1+8+2/1+8+2; tarsus III, 1+10+2/2+10+2; tarsus IV, 1+10+2/2+10+2 (Fig. 43). Female (Figs 5–6). Granulation of carapace, metasoma and pedipalps same as in male. Chela scalloping/notch combination less expressed in female (Fig. 9) than in male (Fig. 8). The considerably inflated telson vesicle of sexually mature males (Fig. 19) is quite conspicuous when compared to the thinner shaped telson of the female (Fig. 20). The pectines are more prominent on the male, whose teeth are longer and greater in number (Figs 21–22

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FIGURES 23–24. Euscorpius solegladi sp. n., adult male, general habitus: 23 dorsal view, 24 ventral view.

Variation. We scored 190 specimens of E. deltshevi sp. n. from Bulgaria for trichobothrial variation, of which 175 specimens (63♂, 112♀) were scored for pectinal teeth variation. Variation was as follows: Pectinаl teeth number in males (Dp): 7/7 (2), 8/? (1), 8/8 (21), 8/9 (5), 9/8 (5), 9/9 (26), 9/10 (1), 10/9 (2), 10/10 (1); in total: 7 (4), 8 (53), 9 (69), and 10 (5); mean 8.57, SD 0.63 [n=125]. Bimodal, with Dp=8 (42.4%) or 9 (55.2%). Pectinаl teeth number in females (Dp): 6/6 (9), 6/7 (3), ?/7 (1), 7/6 (7), 7/7 (74), 7/8 (4), 8/7 (4), 8/6 (1), 8/ 8 (5), 8/9 (1), 9/8 (2), 9/9 (1), in total: 6 (29), 7 (167), 8 (22) and 9 (5); mean 7.01, SD 0.57 [n=223]. Unimodal, with Dp=7 in 74.9%. Number of ventral patellar trichobothria (Pv): 7/? (1), 7/7 (3), 7/8 (4), 8/8 (32), 8/9 (12), 9/8 (8), 8/10 (1), 9/? (1), 9/7 (1), 9/9 (91), 9/10 (13), 10/9 (6), 10/? (1), 10/10 (12); in total: 7 (16), 8 (85), 9 (227), and 10 (45); mean 8.75; SD 0.72 [n=369]. Bimodal, with Pv=8 (23.0%) or 9 (61.5%). Number of external terminal patellar trichobothria (et): 5/5 (2), 5/6 (4), 6/5 (2), 6/? (2), 6/6 (89), 6/7 (12), 7/6 (15), 7/7 (69), 7/8 (2), 8/7 (2), 8/8 (1); in total: 5 (5), 6 (200), 7 (167), and 8 (6); mean 6.43, SD 0.57 [n=378]. Bimodal, with et =6 (52.9%) or 7 (44.2%). Number of external subterminal (est=4), median (em=4), suprabasal (esb=2), basal-a (eba=4) and basal (eb=4) patellar trichobothria was constant in all specimens, except one aberrant specimen from Ogoya (NMNHS 327) that had em=3/4.

Notes

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1. Bayesian phylogenetic analyses of the COI data (Fig. 2) place this species as a sister group to E. carpathicus, s.str. from Romania; see Fet & Soleglad (2002) for a redescription of this classical species (including a study of the Linnaean holotype). So far E. deltshevi sp. n. is the most closely related species to E. carpathicus that has been discovered during recent revisions. Morphologically, the most obvious difference is a rare, clearly derived trichobothrial reduction in the external median patellar series, em=3 in E. carpathicus as opposed to em=4 in all other related species such as E. deltshevi sp. n., E. tergestinus, E. concinnus (C.L. Koch, 1837). Two other variable trichobothrial series numbers also differed, being considerably higher in the Bulgarian species: Pv=8 (25.4%) or 9 (57.8%) in E. deltshevi sp. n. compared to Pv=8 (76.7%) in E. carpathicus; and et =6 (53.5%) or 7 (42.5%) in E. deltshevi sp. n. compared to et=6 (73.3%) in E. carpathicus. Pectinal teeth counts, on the other hand, were close in two species, with mean male Dp=8.56 and female 7.01 in E. deltshevi sp. n., and Dp=8.57 and 7.08, respectively, in E. carpathicus. 2. The Bulgarian populations of this species have been first documented by Valle (1975) (as E. carpathicus from Belogradchik, NW Bulgaria), and then outlined by Fet (2000). E. deltshevi sp. n. comprises the "northern" group of populations reported from Bulgaria by Fet & Soleglad (2007) as Euscorpius sp. (“carpathicus” complex). The status of other populations reported from Bulgaria by Fet & Soleglad (2007) is under current investigation. We suspect that several more undescribed species inhabit southern Bulgaria, most of them unrelated to E. deltshevi sp. n. Some of these (two populations from the Rhodopes; Tropea et al. (in prep.)) were included in the molecular phylogeny of Parmakelis et al. (2013a). 3. Narrowly localised records from eastern Serbia (Nišava Province) fall within western Stara Planina Mts. that extend to the country. The scorpion fauna of Serbia has not yet been assessed with modern taxonomic criteria, but Nišava populations were listed already by Hadži (1931) as his “E. carpathicus oligotrichus” and “E. c. mesotrichus” (both invalid names).

Euscorpius solegladi sp. n. (Figs 23–42, 44; Table I) Euscorpius carpathicus: Valle, 1975: 232 (in part, Bulgaria: Sanda[n]ski, Blago[j]evgrad). Euscorpius carpathicus “Subgroup B1”: Fet 2000: 54; Beron 2001 : 63. Euscorpius hadzii (in part: Bulgaria): Fet & Soleglad 2002: 24, 30; Fet & Soleglad 2007: 406; Kaltsas et al. 2008 : 223; Vignoli & Salomone 2008: 201; Graham et al. 2012a: 19.

Type Material: Holotype♂, BULGARIA, Blagoevgrad Province: Sandanska Bistritsa River, 41°34'N, 23°17'E, 26.05.2005 (V. Fet & E. Fet) (NMNHS). Paratypes: same label as holotype, 2♀ (NMNHS), 1♂, 1♀ (NHMW 21.955–21.956); Sandanska Bistritsa River, 26.05.1997 (D. Hoferek), 1♀ (FKCP); Kresna District, Gorna Breznitsa, 41°45'N, 23°07'E, 27.05.2005 (V. Fet & D. Dobrev), 2♂, 4♀ (NMNHS); Kresna, 30.04.1983 (P. Beron & K. Marincheva), 1♂, 2♀ (NMNHS 87); waterfall near Kresna station, 14.05.1981 (P. Beron & S. Andreev), 2♂, 1♀ (NMNHS 3); Stara Kresna, 6–7.04.1988 (M. Langourov, 1♀ (VFPC); Blagoevgrad, 14.04.1955 (G. Radev), 1♂ (NMNHS 534); Belasitsa Mts. near Petrich, 14.05.1995 (P. Beron), 1♀ (NMNHS 268); Belasitsa Mts., Klyuch Village, 300–400 m, 8.07.1989 (P. Mitov), 2♀ sbad. (VFPC); Gotse Delchev District, Breznitsa, 25.06.1937 (J. Zonkov), 1♂, 2♀ (NMNHS 543); Kresna Gorge, 9.04.1922 (I. Buresch, 1♀ (NMNHS 526); Kresna Gorge, 8.06.1966 (A. Popov), 2♀ (NMNHS 547); Kresna Gorge, Krupnik Village, Stara Kresna, 300 m, 25.04.1999 (P. Mitov), 1♀ (VFPC); Kresna Gorge, Krupnik, Stara Kresna Railway Station, 300 m, 27.04.1996 (P. Mitov), 2♀ (NMNHS); Kresna Gorge, 21.03.1994 (B. Petrov), 1♂ juv (NMNHS 194); Kresna Gorge, near Sheitandere, 2–3.07.1997 (B. Petrov), 1♂, 1♀ (NMNHS 198); Maleshevska Mts., Ilyina Cheshma, 14.06.1992 (T. Ljubomirov), 1♀ (NMNHS 342); Maleshevska Mts., W from Gorna Breznitsa, soil traps, dense forest of Platanus orientalis, 41°45'N, 23°07'E, 14.08–2.10.2003 (B. Guéorguiev), 1♂, 1♀ (NMNHS); Melnik, 30.06.1935 (Kr. Tuleschkov), 1♂, 1♀ (NMNHS 317); Melnik, 1.08.1983 (K. Marincheva), 1♀ (NMNHS 112); Melnik, 7.06.1989 (S. Becvar), 2♀ (VFPC); Melnik, 41º31'N, 23º24'E, 380 m, 3.06.1999 (V. Fet & V. Sakalian), 4♀ (VFPC); Ograzhden Mts., Markovi Kladentsi Peak, 1500 m, 26.05.1996 (B. Petrov), 1♂ juv. (NMNHS 197); Parangalitsa, 1400 m, 12.07.1931 (N. Fenenko), 1♂, 1♀ (NMNHS 271); Petrich, 29.07.1983 (K. Marincheva), 2♀ (NMNHS 111); Strumyani District, Pirin Mts. above Ilindentsi, near entrance of Sharaliiskata Cave, 1600 m, 3.05.1999 (B. Petrov), 1♂ (NMNHS 208); “Sandanski Pirin”, 15.05.1970 (J. Horák), 1♂, 1♀ (NMPC); Sandanski District, Ploski, Zandana Cave, under stones in guano, 31.05.2000 (B. TWO NEW SPECIES OF EUSCORPIUS

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Petrov & L. Nissen), 1♀ (NMNHS 244); Ribnitsa, 31.07.1983 (K. Marincheva), 1♂, 2♀ (NMNHS 110); Samuilovo (P. Beron), 2♂, 3♀ (NMNHS 6); Slavyanka (Alibotush) Mts., Summer Post 10, 28.06.1937 (J. Zonkov), 1♀ (NMNHS 501), 1♀ (NMNHS 506).

FIGURES 25–26. Euscorpius solegladi sp. n., adult female, general habitus: 25 dorsal view, 26 ventral view.

Other Material Studied: BULGARIA. Kyustendil Province: near Kyustendil, 27.05.1937 (Capt. Bandarski), 1♂, 1♂ juv., 2♀ (NMNHS 514); Osogovska Mts., Stradalovo, 10.08.1994 (P. Stoev), 1♀ (NMNHS 215); Rila town, 862 m, 42°07.459’N, 23°14.776'E, 6.08.2005, sieving, beech-conifer forest (P. Jäger & D. Kunz), 2♂, 3♀ (SMF); Rila Mts., below Rilski Monastery, Pastra, 500 m, 14–15.04.1928 (P. Drenski), 1♂, 7♀, 1♀ juv. (NMNHS 518); Rila Mts., above Rilski Monastery, 1450 m, 5.07.1939 (N. Atanassov), 1♀ (NMNHS 318); Rila Mts., road to Kalin Peak, above Pastra, 1100 m, 24.08.1997 (B. Petrov & P. Stoev), 2♂, 1♀ (NMNHS 201); Rilski Monastery, 42°08'N, 23° 20'25''E, 1147 m, 5.06.1999 (V. Fet & V. Sakalian), 2♂, 4♀ (VFPC); Rila, 1970, 1♀ (NMPC); Tsurvaritsa, Gabra Reserve, 800-1000 m, 6.06.2001 (B. Petrov & G. Stoyanov), 1♀, 1♂ juv., 1♀ juv. (NMNHS 258). Pazardzhik Province: Belovo, 9.04.1909 (P. Drenski), 1♂ (NMNHS 525); Gabrovitsa, left bank of Maritsa, stream Dalbochitsa, 6.04.1986, 450 m (P. Beron), 1♂, 2♀ (NMNHS 117). Pernik Province: Radomir District, Baikalsko, 844 m, 42°25.378'N, 22°48.862'E, 28.05.2005 (V. Fet & E. Fet), 5♀ (VFPC); Tran District, Erma Gorge, 703 m, 42°51.679 N, 22°38.952 E, 28.05.2005 (V. Fet & E. Fet), 4♀ (VFPC); Zemen Gorge, pitfall traps,

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15.08–15.09.1997, 3♂ (VFPC). Smolyan Province: Devin, 22.06.1924 (P. Drenski), 1♀, 2♀ juv. (NMNHS 288). Sofia Province: Kostenets District, Trajan’s Gate Pass (“Trajans Pforte”), 10.05.1961 (Schweiger), 1♀ (NHMW); Ihtiman District, Ihtimanska Sredna Gora Mts., Muhovo, 16.08.1997 (D. Milcheva), 2♂, 1♀ (NMNHS); Sofia (brought with firewood), 1.10.1931 (I. Buresch) (NMNHS 314). GREECE. Central Macedonia: Thessaloniki, Hortiatis Mt., 2.07.1939 (D. Papazov), 1♂, 1♀ (NMNHS); Thessaloniki, Rentina, 100 m, under Platanus bark, 19.09.2000 (B. Petrov, P. Stoev & St. Beschkov), 1♂, 1♀ (NMNHS 250). Etymology. Named after our colleague and friend Michael E. Soleglad (California, USA), one of the most preeminent scorpion scholars or our time. Geographic range. Known from southwestern Bulgaria and northeastern Greece (Fig. 1). Diagnosis. Medium sized species (34-35 mm), dark brown in overall coloration, no distinct patterns present. Metasomal segments and chelae somewhat robust in appearance. Dorsal patellar spur (DPS) is enlarged and spinoid. Pedipalp patellar external trichobothria numbers: eb = 5, eba = 5–6 (mean 5.41), esb = 2, em = 4, est = 4, and et = 6 to 7 (mean 6.91); ventral aspect of patella 9–10 (mean 9.59). Pectinal tooth counts: male 9 to 10 (mean 8.94), female 7 to 8 (mean 7.66). Male (Figs 23–24). Description based on holotype male. Measurements of male holotype and female paratype is provided in Table 1. All illustrations (except female chela in Fig. 28 and telson in Fig. 40) are based on a male specimen originating from Sandanska Bistritsa River, Blagoevgrad Province, Bulgaria. Coloration. Overall colour dark brown; legs light brown, chelicerae light brown with dark brown mottling, telson orange; pectines light brown to yellow. Pedipalp carinae black. Carapace (Fig. 27). Almost smooth with extremely fine granulation and shiny at 10x; anterior edge straight with extremely fine setae. Two pairs of lateral eyes, anterior eye slightly larger; median eyes and tubercle situated slightly anterior of middle with following length and width ratios: 0.429 (anterior edge of the carapace to median tubercle centre/carapace length) and 0.158 (width of median tubercle/width of carapace at that point). Mesosoma. Tergites with extremely weak carination on segments I–VII; sternites smooth and shiny, carinae absent on segment V with fine granulation; stigmata small, sub-oval. Metasoma. Generally stocky and somewhat robust in proportions. Dorsal groove is deeply emarginated. Carination: Segments I–IV: median carina granular, smooth and crenulate; dorsolateral weak to absent; intermediary nearly obsolete; ventrolateral absent; ventral obsolete. Segment V (Figs 32–33): Median carina small, round and granular; dorsolateral weak granular and crenulate; Intermediary smooth; ventrolateral rough and serrate; ventral serrate to dentate. Telson (Fig. 35). Vesicle with fine granulation, swollen both laterally and ventrally. Aculeus forming a short conspicuous curve; 4–5 pairs of setae at vesicle/aculeus juncture. Pectines (Fig. 41). Pectinal tooth counts 10/10 and middle lamellae counts 6/6; fulcra well developed; numerous fine setae situated on anterior lamellae. Sensorial areas of teeth developed along approximately one-half to two-thirds of their length. Basal piece anterior edge slightly concave. Genital operculum (Fig. 41). Separated most of length, genital papillae extend proximally. Sternum. Pentagonal, slightly wider than long, length/posterior width ratio 0.918. Chelicerae. Typical for the genus Euscorpius. Movable finger: dorsal distal denticles considerably shorter than ventral distal denticle; dorsal edge has five denticles, two of which are subdistal denticles; ventral edge smooth, lacking serrulae, and covered with heavy brush-like setae for most of its length. Fixed finger: four denticles, two of which are basal denticles conjoined on a common trunk. Pedipalps (Figs 29–31, 34–38). Pedipalps with somewhat stocky and robust chelae exhibiting prominent scalloping at finger bases. Femur: dorsal and ventral internal and external carinae crenate to serrulate; dorsal and ventral surfaces granulose, internal surface with numerous enlarged rounded granules, and external surface contains small rounded granules. Patella: dorsal internal crenate and ventral internal carinae slightly dentate, dorsal external smooth and weakly crenate, and exteromedian rounded and irregularly granulate. Dorsal and ventral surface finely granulose, prominent dorsal patellar spur (DPS) of medium to strong development, ventral patellar spur (VPS) very weak, represented as small granule. Chela carinae: digital very strong and smooth; subdigital in relief, represented by 2 granules; dorsal secondary obsolete; dorsal marginal small and rounded, dorsal internal smooth, minute and rounded; ventroexternal strong, smooth and extending to external condyle of finger, external to trichobothrium Et1. Ventromedian carina essentially obsolete with sparse minute granules; ventrointernal rounded and smooth; and external secondary strong and irregularly granular. Chelal finger dentition: Median denticle row straight; 6/5 inner denticles, 5/6 outer denticles, and 4/4 inner accessory denticles for fixed and movable fingers respectively.

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FIGURES 27–40. Euscorpius solegladi sp. n. 27 carapace, 28 external view of the chela of adult female, 29 external view of the chela of adult male, 30 ventral view of the chela, 31 dorsal view of the chela, 32 lateral view of the metasomal segment V, 33 ventral view of the metasomal segment V, 34 external view of pedipalp patella, 35 dorsal view of pedipalp patella, 36 ventral view of pedipalp patella, 37 dorsal view of pedipalp femur (line indicates trichobothrium i), 38 ventral view of pedipalp femur, 39 telson of adult male, 40 telson of adult female.

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FIGURES 41–42. Euscorpius solegladi sp. n., sternopectinal area: 41 adult male, 42 adult female.

Trichobothria patterns. Type C, neobothriotaxic (major additive) on patella. Femur (Fig. 38): trichobothrium d positioned slightly proximal in relation to i; trichobothrium e is situated on dorsoexternal carina, distal in relation to both d and i. Patella (Figs 35–36): ventral series number 11/10, and external series number eb = 5/5, eba = 5/5, esb = 2/2, em = 4/4, est = 4/4, and et = 7/7. Chela (Figs 28–30): number of trichobothria on ventral surface is 4/4 (V1–3 + Et1), V4 on external surface set in small dimple but removed from ventroexternal carina. Legs. One pair of pedal spurs present, ungues medium length with average curve. Tarsus ventral median spinule row is formed by variable number of elongated spinules usually terminating distally in a single pair; the basal spinule is sometimes offset; spinule counts are: tarsus I, 7+2/7+2; tarsus II, 8+2/9+1; tarsus III, 1+7+2/ 1+8+2; tarsus IV, 1+9+2/1+9+2 (Fig. 44). Female (Figs 25–26). Granulation of carapace, metasoma and pedipalps same as in male. Chela scalloping/ notch combination less expressed in female (Fig. 28) than in male (Fig. 29). The considerably inflated telson vesicle of sexually mature males is quite conspicuous (Fig. 39) when compared to the thinner “teardrop” shaped telson of the female (Fig. 40). Genital operculum/genital papillae: On the female, the genital operculum is connected for its entire length by a membrane, whereas on males, it is separated for most of its length, exposing protruding genital papillae. The pectines are more prominent on the male, whose teeth are longer and greater in number (Figs 41–42). Middle lamellae counts in female 5/5. Variation. We scored 62 specimens of E. solegladi sp. n. from Bulgaria for trichobothrial variation, of which 59 specimens (18♂, 41♀) were scored for pectinal teeth variation. Variation was as follows: Pectinаl teeth number in males (Dp): 7/8 (2), 8/8 (1), 8/9 (1), 9/8 (1), 9/? (1), 9/9 (8), 10/10 (4); in total: 7 (2), 8 (6), 9 (19), 10 (8); mean 8.94, SD 0.80 [n=33]. Bimodal, with Dp=9 (57.6%) or 10 (24.2%). Pectinаl teeth number in females (Dp): 7/7 (9), 7/8 (8), 8/7 (3), 8/8 (20), 8/9 (1); in total: 7 (29), 8 (52) and 9 (1); mean 7.66, SD 0.50 [n=82]. Bimodal, with Dp=7 (35.4%) or 8 (63.4%). Number of ventral patellar trichobothria (Pv): 8/9 (2), 9/8 (3), 9/? (1), 9/9 (12), 9/10 (8), 10/ 9 (5), 9/11 (1), 10/10 (28), 10/11 (1), 11/10 (1); in total: 8 (5), 9 (44), 10 (71), 11 (3); mean 9.59, SD 0.61 [n=123]. Bimodal, with Pv=9 (35.8%) or 10 (57.7%). Number of external terminal patellar trichobothria (et): 4/7 (1), 6/6 (5), 6/7 (6), 7/6 (1), 7/? (1), 7/7 (41), 8/6 (1), 7/8 (2), 8/7 (1), 8/8 (3); in total: 4 (1), 6 (18), 7 (94), 8 (10); mean 6.91, SD 0.54 [n=123]. Unimodal, with et=7 (76.4%). Number of external basal-a patellar trichobothria (eba): 4/4 (2), 4/5 (1), 5/5 (27), 5/6 (5), 6/5 (4), 6/6 (19), 7/5

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(1), 6/7 (1), 7/6 (1); in total: 4 (5), 5 (66), 6 (49) and 7 (3); mean 5.41; SD 0.62 [n=123]. Bimodal, with eba=5 (53.7%) or 6 (39.8%). Number of external basal patellar trichobothria (eb) was always 5, except in one aberrant specimen from Kresna (NMHNS 3) with eb=4/5. Number of external median patellar trichobothria (em) was always 4, except in two aberrant specimens from Kresna (NMNHS 198) with em=3/4, and Rila (SMF) with em=?/5. (Note that this number is em=5 in some West Balkan E. hadzii). Number of external subterminal (est=4) and suprabasal (esb=2) patellar trichobothria was constant in all specimens.

FIGURES 43–44. Tarsal spination, legs I–IV (top to bottom): 43 Euscorpius deltshevi sp. n.

Notes In the COI phylogenies (Figs 2A–B), all Bulgarian populations form a monophyletic clade, which is placed as a sister group to E. hadzii from Prokletije Mts. of Albania. The latter area has been designated as a terra typica for E.

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hadzii by Fet & Soleglad (2002) who designated a neotype of this species. Three additional sampled populations of E. hadzii (all from Bosnia & Herzegovina) group considerably outside of the clade from Albania and Bulgaria. It appears that E. hadzii is not monophyletic, but rather a complex of species, whose remaining populations from exYugoslavia (for which at this moment little material is available) have to be further addressed. The relationship between E. hadzii and the large “E. sicanus” complex also needs to be reassessed (see also Parmakelis et al. 2013a).

FIGURES 43–44. Tarsal spination, legs I–IV (top to bottom): 44 E. solegladi sp. n.

The Bulgarian populations of this species have been first documented by Valle (1975) (as E. carpathicus from Sandanski, with trichobothrial index B3=10 to 12, i.e. eba=5 to 6), and then outlined by Fet (2000) as “Group B1”. TWO NEW SPECIES OF EUSCORPIUS

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Fet & Soleglad (2007) studied morphological trends within E. hadzii, comparing Bulgarian populations to all material available from the Western Balkans (i.e. Albania and ex-Yugoslavia). E. solegladi sp. n. has the lowest (presumably plesiomorphic) number of external basal (eb=5), basal-a (eba=5–6), and median (em=4) patellar trichobothria compared to western populations; see Fet & Soleglad (2007, Figs 3–12, table 1) for detailed analysis. In our opinion, the species status for the Bulgarian population is supported by both the COI data and morphology. Two new records from Greece (both near Thessaloniki) expand the range of E. solegladi sp. n. southward; this species probably will be also found in neighboring Macedonia (FYROM) and Serbia. Four available specimens from Greece have eba=6 (n=6) or 5 (n=2). Note that E. hadzii s.str. (or an unnamed “western” species of this complex) is also found in Greece but in its western portion (Epirus as well as Zakynthos Island) (Fet & Soleglad 2002). Trichobothrial pattern eba=6, eb=5 found in many E. solegladi sp. n. is also present in some populations of E. hadzii from Albania and Herzegovina (our data, unpublished). Trichobothrial pattern eba=eb=5 (but not eba=6), independently of E. solegladi sp. n., is also present in many Greek (Thessaly, Peloponnese) and some Italian (Gargano) populations of “E. sicanus complex” (Fet et al. 2003; Fet et al., in progress). A multilocus DNA phylogeny of several Greek populations of “E. sicanus complex” has been recently published by Parmakelis et al. (2013a).

Results of Molecular Analyses Phylogenetic Analysis COI alignments consisted of 649 bp for 25 ingroup taxa and one outgroup. The BI analysis (Fig. 2A) strongly supported the monophyly of the ingroup, with E. tergestinus forming a basal lineage. Remaining species formed a monophyletic group with strong support containing two strongly supported clades; one clade comprising E. carpathicus and E. deltshevi sp. n., and one consisting of E. hadzii, E. sicanus, and E. solegladi sp. n. Romanian E. carpathicus formed a basal lineage sister to a clade containing all E. deltshevi sp. n. samples with low support (0.62 pp). Four E. hadzii samples were polyphyletic, with two specimens from Bosnia & Herzegovina forming a monophyletic group sister to a clade containing the E. hadzii sample from Albania and E. solegladi sp. n. The remaining E. hadzii sample from Bosnia & Herzegovina and a strongly supported clade containing E. sicanus occurred as basal lineages that formed a polytomy (Fig. 2A).

Divergence Dating The topology produced by the BEAST analysis (Fig. 2B) was identical to the MrBayes phylogeny except that E. hadzii were paraphyletic with respect to E. solegladi sp. n. rather than polyphyletic with E. solegladi sp. n. and E. sicanus. Mean estimates for the common ancestor of all samples and for the clade containing all samples except E. tergestinus were in the late Miocene. Subsequent diversification resulted in four nodes with mean estimates in the Pliocene and 17 nodes with estimates in the Pleistocene.

Discussion From a biogeographic perspective, the two new species represent two different ancient elements of the northern and southwestern Balkans. A western Balkan, or Dinaric (Guéorguiev 2007) element, E. solegladi sp. n. is the easternmost member of its clade (“E. hadzii” complex), widespread in the Balkans from Albania in the west across ex-Yugoslavia. Fet & Soleglad (2007) reviewed the distribution of this species (as E. hadzii) in Bulgaria. E. solegladi sp. n. is found only in the southwestern corner of the country (Fig. 1) where it is common along the Struma River Valley, extending to the Rila Mts. and Mesta Valley. It is found along the Macedonian border from Belasitsa in the southwest to Ograzhden Mts. and Maleshevska Mts. and as far toward north as Kraishte (Erma Gorge), Konyavska Mts. (Baikalsko) and Osogovo Mts. The E. solegladi sp. n. record from Kyustendil represents

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the westernmost for scorpions in Bulgaria. In the southeastern direction E. solegladi sp. n. reaches the Slavyanka (Alibotush) Mts. but there is only a single record from the Western Rhodopes (Devin). Three other disjunct records show that E. solegladi sp. n. is also found in the Maritsa Valley. The sole record from Pirin is undoubtedly due to poor representation of this area in collections. The highest altitudes at which E. solegladi sp. n. has been found in Bulgaria are 1600 m in Western Pirin, 1500 m in Ograzhden, and 1450 m, in Rila (Fet & Soleglad 2007). Our COI phylogeny did not reveal any internal structure among five Bulgarian localities sampled (from south to north: Sandanska Bistritsa, Gorna Breznitsa, Rila, Baikalsko, Erma), which formed a strongly supported clade with origins in the late Pleistocene (Fig. 2B). In contrast, E. deltshevi sp. n. appears to be a northeastern Balkan species, widespread across the Stara Planina (=Balkan) mountain system and some adjacent areas in the northwestern corner of the country in the mountains bordering Serbia (the northernmost record in Bulgaria is Belogradchik), and in numerous localities of Western Stara Planina (especially Iskar Gorge) (Fig. 1). Data from Central North Bulgaria (Pleven, Veliko Tarnovo) and Central Stara Planina (Sliven) are scarce (Fet & Soleglad 2007). Scorpions have not been collected on the lowlands of the Danubian Plain, so there is no evidence that the Bulgarian range of Euscorpius comes close to that of Romanian E. carpathicus, its sister species. Few scorpions were recorded from the east and northeast of Bulgaria: populations near Sliven, Yambol, and Shumen are known. At this moment, we choose to treat E. deltshevi sp. n. as a single species. Our COI phylogeny, however, shows considerable genetic and geographic structure among nine localities sampled, with divergence time estimates well before the Pleistocene (3 Mya). We propose that E. deltshevi sp. n. could be a northern (Carpathian) faunal element and potentially represents additional cryptic Bulgarian species (Guéorguiev 2007). In the COI phylogeny, eastern Bulgarian populations (Teteven and the easternmost outlying locality of Sliven) are separated from well-sampled populations of Iskar Valley (represented by Lakatnik and Tserovo, the type locality). Remaining four Bulgarian populations from Western Stara Planina (Beledie Han, Gorna Laka, Oreshets, and Belogradchik) expectedly cluster with the westernmost sample from eastern Serbia. Interestingly, the two new species are allopatric as their ranges come close in the Pernik Province in western Bulgaria but do not appear to overlap. At the same time, other Euscorpius species in the Balkans are often sympatric. For example, E. solegladi sp. n. is sympatric in southern Bulgaria (Melnik, Pirin, Slavyanka) with an undescribed Euscorpius sp. belonging to a distantly related clade (Parmakelis et al. 2013a). With two newly described species, the Bulgarian scorpion fauna comprises no less than six species: Mesobuthus gibbosus (Brullé, 1832) (Buthidae), Euscorpius (Euscorpius) solegladi sp. n., E. (E.) deltshevi sp. n., E. (Alpiscorpius) sp. (Pirin Mts.), and at least two undescribed Euscorpius sp. from Pirin, Slavyanka, and the Rhodopes (Fet & Soleglad 2007; Parmakelis et al. 2013a; Tropea et al. in prep.). Further studies will be directed at clarifying the Euscorpius fauna in the mountains of southern Bulgaria.

Acknowledgements We are grateful to all colleagues who kindly loaned and shared types and comparative material with us, and helped in field collection and laboratory procedures, including (but not limited to) Michael Adams, Julia Altmann, Petar Beron, Michael Brewer, Christo Deltshev, Dobrin Dobrev, Elizabeth Fet, Galina Fet, Simon Fet, Jürgen Gruber, Christoph Hörweg, Natalia Ivanova, Peter Jäger, Dimitris Kaltsas, Ivo Karaman, František Kovařík, Mihail Kvartirnikov, Wilson Lourenço, W. Brett Mattingly, Kirill Mikhailov, Plamen Mitov, Moysis Mylonas, Ivan Pandourski, Aristeidis Parmakelis, Boyan Petrov, Alexi Popov, Jan Ove Rein, Vladimir Sakalian, Michael Salcido, Boris Sket, Michael Soleglad, Verena Stagl, Iasmi Stathi, František Šťáhlavský, Pavel Stoev, and Milen Vassilev. Special thanks are to Michael Soleglad who kindly provided a map (Fig. 1), and to Alexi Popov who meticulously checked and corrected Bulgarian toponyms. The DNA barcoding conducted for this project was performed at the Canadian Centre of DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, with administrative support from P.D.N. Hebert, and funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (2008-OGIICI-03). V.F.'s initial travel to Bulgaria in 1999 was supported by a COBASE (Cooperation in Basic Science and Engineering) grant from the National Research Council, Washington, DC, USA. More extended V.F.'s travel to Bulgaria in 2005 was supported by the Fulbright Scholar Award 04-11-08 from CIES (Council of International

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Exchange of Scholars), Washington, DC, USA, which allowed Victor and Galina Fet to travel and live in Bulgaria in January–May 2005. Help, hospitality, and friendship of many Bulgarian colleagues, especially Petar Beron, Gergin Blagoev, Christo Deltshev, Dobrin Dobrev, Ivan Pandourski, Lyubomir Penev, Alexi Popov, Vladimir Sakalian, and Milen Vassilev, made the 1999 and 2005 visits productive and enjoyable. We thank Yuri Marusik and three anonymous reviewers for their kind and detailed comments on this manuscript.

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