Biological Journal of the Linnean Society, 2008, 95, 583–597. With 7 figures
Species limits and hybridization zones in Icterus cayanensis–chrysocephalus group (Aves: Icteridae)
1
Programa de Pós-Graduação em Zoologia, Museu Paraense Emílio Goeldi, C. P. 3999, 66017-970, Belém, Pará, Brazil 2 Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, 05508-090, São Paulo, São Paulo, Brazil 3 Conservation International, Avenida Governador José Malcher 652, 2o. Andar, 66035-100, Belém, Pará, Brazil Received 1 November 2007; accepted for publication 18 January 2008
One of the best examples of differentiation and hybridization among South American passerine birds is exhibited by Icterus cayanensis (Epaulet Oriole) and Icterus chrysocephalus (Moriche Oriole). Icterus chrysocephalus is a monotypic species restricted to northern South America. Icterus cayanensis is a polytypc species that ranges from Suriname and French Guyana to northern Argentina. Five subspecies are recognized to I. cayanensis. Hybrid zones are known between I. cayanensis and I. chrysocephalus as well as between subspecies of I. cayanenis, even though character variation has never been adequately assessed and mapped. Although molecular data support the hypothesis that I. cayanensis and I. chrysocephalus form a monophyletic group, they do not support the species limits currently recognized within this group. We analysed the geographic variation of plumage characters along the range of this group to map the geographic variation of individual plumage characters and identify the populations that have uniform phenotypic character expression and therefore represent genuine phylogenetic species. We also used molecular data to investigate the phylogenetic relationships among these species. Geographic variation of plumage characters, habitat preferences and molecular data identified four species within I. cayanensis–chrysocephalus clade: an Amazonian species group, formed by I. cayanensis and I. chrysocephalus and a Southern species group composed of I. pyrrhopterus and I. tibialis. The Amazonian species are separated by a relatively narrow hybrid zone along the Amazon valley, whereas the Southern species are separated by a hybrid zone that is larger than the ranges of the two species individually. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 583–597.
ADDITIONAL KEYWORDS: diversification – epaulet oriole – geographic variation – moriche oriole – neotropics – oriole – speciation – systematics.
INTRODUCTION Species are the basic units of systematic, ecological, biogeographical, and macroevolutionary studies and the documentation and demarcation of their boundaries is a critical step towards a broad understanding of the biodiversity patterns and processes at all spatial scales (Cracraft, 1983; Brooks & McLennan,
*Corresponding author. E-mail:
[email protected]
1991; Lomolino, Brown & Riddle, 2005). In addition, species are the currency for global biodiversity assessments, definition of conservation priorities, and establishment of innovative legislation or conventions, making them especially important from a conservation viewpoint (Agapow et al., 2004; Brooks et al., 2006). The delimitation of species boundaries is not always a clear-cut process (Sites & Marshall, 2004). There are at least two major reasons for that. The first is that the criteria applied to delimitate a species
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FERNANDO MENDONÇA D’HORTA1,2*, JOSÉ MARIA CARDOSO DA SILVA1,3 and CAMILA CHEREM RIBAS2
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F. MENDONÇA D’HORTA ET AL. cayanensis (Linnaeus, 1766), from Suriname, French Guyana, Brazilian Amazonia south of the Amazon, eastern Peru, northern Bolivia; Icterus cayanensis tibialis Swainson, 1837, from northeastern Brazil; Icterus cayanensis valenciobuenoi Ihering, 1902, from central and southeastern Brazil; Icterus cayanensis periporphyrus (Bonaparte, 1850), from northeastern Bolivia and western Brazil; and Icterus cayanensis pyrrhopterus (Vieillot, 1819), from southeastern Bolivia, Paraguay, southwestern Brazil, Uruguay and northern Argentina. Hybrids have been described between I. cayanensis and I. chrysocephalus (Blake, 1968; Haverschmidt, 1968) as well as between subspecies of I. cayanensis (Hellmayr, 1937; Pinto, 1944; Sick, 1984, 1997). The current taxonomy was criticized by Sick (1984, 1997), although he did not present any data for supporting his argument. Species limits currently recognized within this group are not supported by molecular data because some populations of I. cayanensis are more closely related to I. chrysocephalus than to other populations of I. cayanensis (Omland et al., 1999). Because species and subspecies’ limits within this monophyletic group were defined without any detailed analyses of the character variation across the entire distribution of the group, it is not clear whether all units that were taxonomically recognized represent distinctive phylogenetic species (Cracraft, 1989). In the present study, we present the first analysis of the geographic variation of plumage characters in I. cayanenss and I. chrysocephalus, and test the hypothesis that current taxonomy does not accurately describe the diversification patterns within it (Sick, 1984, 1997). We document the character variation and define the ranges of the phylogenetic species, the evolutionary relationships among them, and the location and size of the hybrid zones within this group. By mapping the ranges of the phylogenetic species as well as the hybrid zones among them, we provide for the first time a geographical framework upon which the nature and dynamics of these hybrid zones can be assessed in more detailed studies.
MATERIAL AND METHODS MORPHOLOGICAL ANALYSIS Museum specimens We examined and measured 350 male adult specimens and 246 female adult specimens in five museum collections: Museu Paraense Emílio Goeldi (MPEG), Belém, Brazil; Museu de Zoologia da Universidade de São Paulo (MZUSP), São Paulo, Brazil; Museu Nacional do Rio de Janeiro (MNRJ), Rio de Janeiro, Brazil; Field Museum of Natural History (FMNH),
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depend upon the species concept that is used and there are currently at least 20 species concepts available in the literature (Mayden, 1997). The second is that the speciation process creates fuzzy boundaries under which all methods proposed to demarcate species boundaries will occasionally fail or be discordant with each other. This is an unavoidable consequence of the many combinations of deterministic and stochastic processes associated with any speciation event (Sites & Marshall, 2004). Despite those problems, an understanding of evolutionary processes requires that we attempt to define objectively the arena within and across which these processes can act, with rigorous empirical methods (Cracraft, 2000; Wiens & Servedio, 2000; Sites & Marshall, 2004). Geographically widespread monophyletic clades composed of two or more diagnosable sets of populations separated by hybrid zones are the most challenging ones to have their species clearly delimited because of the dynamic nature of hybrid zones (Barton & Hewitt, 1985; Sites & Marshall, 2004). This general pattern of differentiation appears to be common among terrestrial vertebrates (Mayr, 1969; Haffer, 1986). However, it is especially well documented among continental birds (Haffer, 1974, 1992; Newton, 2003), possibly because natural hybrids are common among birds, and hybrids are more easily detected in birds than in other groups of vertebrates (Grant & Grant, 1992). The characteristics of the hybrid zones differ according to latitudinal gradient: broad and dynamic hybrid zones are more typical of higher latitudes whereas the tropics are generally characterized by sharp replacement and limited introgression zones (Price, 2008). Although hybrid zones are a challenge for taxonomists (Newton, 2003), they are genuine natural laboratories for studies of selection, dispersal, competition, mate choice, and the nature of genetic control of phenotypic characters (Endler, 1977; Barton & Hewitt, 1985; Rohwer & Wood, 1998). The clade composed by Icterus cayanensis and Icterus chrysocephalus is one of the most remarkable anecdotic examples of a complex pattern of differentiation and hybridization among Neotropical birds (Blake, 1968; Sick, 1984, 1997), even though no formal analysis of character variation within this group has been carried out. Molecular phylogenetic analyses indicate that this clade is monophyletic (Omland, Lanyon & Sabine, 1999). It is currently composed of two biological species: I. chrysocephalus (Linnaeus, 1766), a monotypic species restricted to northern South America, and I. cayanensis (Linnaeus, 1766), a polytypic species that ranges from Suriname and French Guyana to northern Argentina. Five subspecies are currently recognized as I. cayanensis (Blake, 1968; Sick, 1984, 1997): Icterus cayanensis
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Figure 1. Number of specimens studied per locality.
Chicago, USA; and American Museum of Natural History (AMNH), New York, USA. These specimens, the vast majority of known skins of these taxa in New World museums, represented 276 localities that encompass a large portion of the range of the study group (Fig. 1). The locality for each specimen was taken from the specimen label and its longitude and latitude were obtained (when available) from ornithological gazetteers for each country (Paynter, 1982, 1989, 1992, 1993, 1995, 1997; Paynter & Traylor, 1991; Stephens & Traylor, 1983, 1985; Vanzolini, 1992) or directly from the specimen label. Each locality was entered into a geographic information system and mapped. The list of specimens examined is available from the authors. Plumage variation and body measurements We scored all feather patches that varied markedly in coloration across the epaulet-moriche clade. We defined a feather patch as a continuous region of feathers with similar coloration and structure (Omland & Lanyon, 2000). For most of the feather patches (crown, under-wing coverts, carpal-base region, rump, vent, and thighs), we scored whether they were: (0) black, (1) intermediate (i.e. present a mixture of yellow and black feathers) or (2) yellow. The only exception was the epaulets that we scored as: (0) dark, ranging from reddish black or dark reddish brown; (1) intermediate, ranging from dark brown to yellowish brown; and (2) yellow (colours described according to Munsell Soil Colour Charts (2000).
Identification of phylogenetic species and hybrid zones We adopted the phylogenetic species concept proposed by Nixon & Wheeler (1990) to guide all analyses. According to this concept, a ‘species is the smallest aggregation diagnosable cluster of populations (sexual) or lineages (asexual) diagnosable by a unique combination of character states in comparable individuals (semaphoronts)’. We used this concept because it is fully operational and has been proposed specifically as a framework for the discovery of basal terminals for phylogenetic and evolutionary studies (Davis & Nixon, 1992). We used the method of population aggregation analysis (PAA) proposed by Davis & Nixon (1992) to delimitate species boundaries and identify the location and extension of the hybridization zones. The PAA is based on two principles: (1) all individuals sampled from a local population are assumed to be conspecific and (2) identical character attributes shared among individuals drawn from two or more populations provide evidence for conspecificity (Davis & Nixon, 1992). The PAA requires a summary of character states for all individuals in a sample (= locality) to estimate a population profile for those states, and then it combines all samples with identical profiles for all character states. This process is continued iteratively until the only remaining sample aggregates are those separated from each other by fixed character state differences. During the application of PAA, we gave special attention to the identification of the localities that present one or more specimens with intermediate plumage characters because they provide clues about the location and extension of hybrid zones. Populations presenting any evidence of intermediate character states are classified as representing hybrid zones, except when they are represented by a few specimens dispersed within a large region dominated by individuals with pure character states. We used this criterion to avoid underestimating hybridization between phylogenetic
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We took the following measurements (to the nearest 0.1 mm) by using digital callipers according to the methods described in Baldwin, Oberholser & Worley (1931): length of total culmen from base to tip of maxilla (CL); length of bill from nostril to tip of maxilla (BL); height of bill at nostril (HB); width of bill at gape (WB); length of tail (LT); and tarsus length, from junction of tibiotarsus and tarsometatarsus to distal junction of hind toe and tarsometatarsus (TL). Wing length (WL), from bend of folded wing to tip of longest primary feather, was measured (nearest millimetre) with a ruler to minimize damage to the specimens. All measurements were taken by only one of the authors (F.H.).
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Table 1. Individuals studied, collection localities, voucher numbers, and GenBank accession numbers Phenotype/identification
Locality
Voucher
GenBank cyt b/ND2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Intermediate Intermediate Intermediate Intermediate Icterus cayanensis Icterus cayanensis Icterus cayanensis Icterus cayanensis Icterus tibialis Icterus tibialis Icterus tibialis Icterus tibialis Icterus pyrrhopterus Icterus pyrrhopterus Icterus chrysocephalus Icterus cayanensis Icterus bonana Icterus laudabilis
Brazil, Tocantins, Peixe Brazil, Tocantins, Peixe Brazil, Tocantins, Peixe Brazil, Tocantins, Peixe Brazil, Pará, Caxiuanã Brazil, Pará, Caxiuanã Brazil, Pará, Caxiuanã Brazil, Pará, Caxiuanã Brazil, Sergipe, Serra de Itabaiana Brazil, Sergipe, Serra de Itabaiana Brazil, Piauí, Serra das Confusões Brazil, Piauí, Serra das Confusões Bolivia, Sta Cruz, Chiquitos Bolivia, Sta Cruz, Chiquitos Venezuela, Sucre, Guanoco Brazil, Rondonia, Rio JiParana Martinique, Fond Baron St Lucia, Anse la Sorciere
23 55 149 151 229 231 233 237 20 717 20 718 co160 co206 FMNH 334608 FMNH 334609 FMNH 339734 MPEG 40357 STRI MA-IBO2 STRI SL-ILA4
AF099280/AF099319 AF089028/AF099315 AF099279/ AF089027/AF099316 AF099278/AF099313 AF099298/AF099338
Numbers in the first column correspond to numbers in all figures. cyt b, cytochrome b.
species because individuals from backcrosses can resemble individuals of pure states. We combined males and females in this analysis because there is no difference between them in plumage. We used ARCVIEW, version 3, to map the ranges of species and their hybrid zones as well as to estimate their sizes. We used body measurements to analyse if there are sexual differences within each phylogenetic species as well as to evaluate if these measurements can be also used to distinguish between pairs of the units diagnosed by plumage characters. To evaluate sexual differences, we used univariate tests (t-test). We considered that body measurements are useful to diagnose pairs of taxonomic units if and only if the ranges of measurements of the two samples do not overlap. Sexes were analyzed separetaly. We used BIOESTAT (Ayres et al., 2003) to compute all statistical tests.
MOLECULAR
ANALYSIS
Taxon sampling and genes chosen We obtained sequences from 12 specimens collected throughout the range of Icterus cayanensis– chrysocephalus group, and from four specimens available in GenBank (Table 1), representing all the evolutionary units identified in the morphological analysis. We selected as outgroups Icterus laudabilis and Icterus bonana, which were shown to be relatively closely related to I. cayanensis and I.
chrysocephalus clade by Omland et al. (1999). Outgroup sequences were also obtained from GenBank (Table 1). We selected the mitochondrial genes cytochrome b (cyt b) and NADH dehydrogenase subunit 2 (ND2) wich have been shown to provide good resolution in studies involving closely related taxa (Hackett, 1996; Omland et al., 1999; Ribas et al., 2005). DNA extraction, amplification, and sequencing DNA was extracted from tissue samples according to Bruford et al. (1992). The primers used for amplificatios were: N5L14750 (Ribas et al., 2005), CBH 15422 (Ribas et al., 2005), CBL15298 (Cheng, Higuchi & Stoneking, 1994), CBH15764 (Miyaki et al., 1998), CBL15507 (J. Feinstein, pers. comm.), HB20 (J. Feinstein, pers. comm.), for cyt b; and LMet (J. Groth, pers. comm.), H5776 (Omland et al., 1999), modified from primer designed by K. Johnson), L5758 (Johnson & Sorenson, 1998), H6313 (Sorenson et al., 1999), for ND2. Amplifications were performed in 10-ml reactions containing 0.8 mM dNTP, 1 mM of each primer, 0.5 U Taq polymerase (Pharmacia), buffer 1¥ (Pharmacia), and 1 ml of template DNA. Thermal cycling conditions were: denaturation step of 95 °C for 5 min followed by 40 cycles of 95 °C for 40 s, 52–54 °C for 40 s, 72 °C for 1 min, and a final incubation of 72 °C. Purified polymerase chain reaction products were used as templates for Big Dye (Perkin Elmer) terminator cycle sequencing reactions follow-
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#
SPECIES LIMITS AND HYBRID ZONES IN ORIOLES ing the manufacturer’s recommendations. Sequences were read with an ABI 377 automated sequencer.
RESULTS PLUMAGE
VARIATION
Four patterns of geographic variation were found for the seven plumage characters examined. The first one includes crown, vent, and rump. These characters distinguish two major clusters of populations: one in northern of South America and another encompassing all remaining localities (Fig. 2). No specimen presented a rump classified as intermediate. Thus, the change between the two population clusters is sharp and well defined (Fig. 2C). By contrast, specimens with intermediate crown and vent were found (Fig. 2A, B). Two specimens from Manaus (AMNH 238755 and 248840), near the Amazon River, have both crown and vent intermediate, composed of yellow and black feathers (Fig. 2B, C). Specimens from Oiapoque (MPEG 15265) and Vila Velha do Caciporé (MPEG 23263), Amapá, northern Brazil show an intermediate crown. The second pattern is exhibited by the carpal-base region. It distinguishes two major population clusters: one in northeastern and eastern Brazil and another including most of the remaining localities (Fig. 3). There is a broad zone with intermediate specimens separating these two pure population clusters right across central Brazil. Some specimens (Venezuela, Guanoco, AMNH 521583; Peru, Cuzco, Consuelo, FMNH 311740; Peru, Sarayacu, AMNH 238701; Brasil, Santarém, AMNH 50211; Suriname, Paramaribo, AMNH 521577) presented an intermediate carpal-base region within the region dominated by pure black individuals (Fig. 3). Because these specimens are few and far apart, they possibly cannot be used as evidence of hybridization between the two major population clusters in these specific sites. Similarly, some individuals with pure yellow carpal-base regions are also found isolated among pure black and intermediate individuals close to the boundary between Brazil and Paraguay (Fig. 3). The third pattern is exhibited by two characters: thigh and under-wing coverts (Fig. 4A, B). They distinguish three major pure population clusters: (1) one dominated by yellow feathers in northern South America; (2) one dominated by yellow feathers in northeastern Brazil; and (3) one dominated by black feathers in the remaining regions (Fig. 4A, B). No specimen with intermediate thigh was found between the black population and the yellow population from northern South America, but several intermediate specimens were found along a very narrow belt surrounding north-eastern Brazil (Fig. 4A, B). By contrast, many specimens with intermediate under-wing coverts were found along the boundary (the Amazon valley) between the black population and the yellow one in northern South America as well as between the
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Phylogenetic analyses Heavy and light chains were compared and corrected using Sequencher, version 4.1.2 (GeneCodes Corp.). Alignments were performed manually using MacClade, version 4.0 (Maddison & Maddison, 1999). Uncorrected pairwise divergence values were computed using Mega (Kumar, Tamura & Nei, 2001). Saturation was evaluated for each region by plotting the absolute numbers of transitions against p-distances for all pairwise comparisons. The partition homogeneity test (Farris et al., 1995) was implemented in PAUP*. Phylogenetic analyses were implemented in PAUP*4.0 b10 (Swofford, 1998) using the maximum parsimony (MP) and maximum likelihood (ML) methods. Bayesian Inference analyses were implemented in MrBayes, version 3.0 b4 (Huelsenbeck & Ronquist, 2001). MP analyses were performed using unordered and equally weighted characters, with heuristic tree searches, tree bissection–reconnection (TBR) branch swapping and 100 random-addition sequence replications. Bootstrap (1000 replications) and decay indices (Bremer, 1994) were used to determine the relative support for inferred monophyletic groupings. The likelihood-ratio test as implemented in MODELTEST, version 3.6 (Posada & Crandall, 1998) was used to select the simplest model of molecular evolution yielding a significantly higher likelihood than others. The model selected was used for the ML analyses, which were performed using heuristic tree search, TBR branch swapping and ten random addition replicates. The robustness of the trees found was determined by 100 bootstrap replications. Another likelihood ratio test (Huelsenbeck & Rannala, 1997) was applied to the cyt b, ND2, and combined datasets to compare ML trees constructed with or without a molecular clock constraint. Bayesian inference of phylogeny was implemented following the ML models that were selected by MODELTEST for each dataset (cyt b and ND2). The analysis was run using partitioned likelihood, so that the parameters could vary independently for the two genes in the combined analysis. Four simultaneous chains were run for 10 000 000 generations with trees being sampled every 1000 generations for a total of 10 000 trees. The ML scores became stable (stopped improving) around the 1000th tree, so that burnin was completed by the 1000th tree, and 9000 trees were kept in each analysis. Three independent analyses were performed for the combined dataset and the 27 000 sampled trees obtained in each analysis were used to compute the posterior probabilities of each node.
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A
Figure 2. Geographic variation of crown (A), vent (B), and rump (C). Solid circles denote median values = 0; half-solid circles = 1; and open circles = 2.
black population and the yellow one in north-eastern Brazil. In both transitions, the change is sharp and the zone is narrow. The fourth pattern is exhibited only by epaulets. This character groups the localities into two major population clusters: (1) one with yellow feathers that encompasses most of the localities in the
northern part of the range and (2) one dominated by individuals with dark shoulders that are centred in Paraguay, Bolivia and Argentina (Fig. 5). Between these two clusters, there is a broad zone composed of localities with specimens exhibiting intermediate shoulders. This zone is located in central and eastern Brazil.
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C
SPECIES LIMITS AND HYBRID ZONES IN ORIOLES
IDENTIFICATION
OF PHYLOGENETIC SPECIES
By combining population profiles of all localities and identifying those regions whose specimens can be diagnosed by a unique combination of character
A
states, four phylogenetic species were identified (Fig. 6). The first phylogenetic species (A) is characterized by having all six plumage characters analysed yellow, except the carpal-base region. It is restricted to northern South America and its range includes Colombia, Venezuela, Guyana, northwestern Brazil to the Amazon valley, Ecuador and northern Peru. The second species (B) is diagnosable because it has all plumage characters black, except for the yellow epaulets. It includes populations from eastern Peru and northern Brazil, south of the Amazon, to the Araguaia River. The third species (C) is diagnosable because it has three plumage characters black (crown, vent and rump) and four (all other characters) yellow. It is restricted to northeastern Brazil. Finally, the fourth species (D) can be put apart because it has all plumage characters black, except the epaulets that vary from darkbrown to yellowish brown. Phylogenetic species D is restricted to the central and southern Bolivia, Paraguay, southwestern Brazil, Uruguay and northern Argentina. These four phylogenetic species are diagnosed only by plumage characters (Table 2), because body size measurements of these populations present considerable overlap for all measurements in both sexes (see Appendix, Table A1). Sexual dimorphism in body measurements differs among these four phylogenetic species, although, on average, males are larger than females in all measurements (see Appendix, Table A1). Males and
B
Figure 4. Geographic variation of infra-wings coverts (A) and thighs (B). Solid circles denote median values = 0; half-solid circles = 1; and open circles = 2. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 583–597
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Figure 3. Geographic variation of carpal-base region. Solid circles denote median values = 0; half-solid circles = 1; and open circles = 2.
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females of population A differ significantly in both WL and TL; in population B in WL and TC; and in population C in WL and TL (Table 3). Population D is by far the population in which sexual differences in size are strongly marked, as males and females differ significantly in WL, TL, TC and LB (Table 3).
RANGES
AND HABITAT PREFERENCES
EVOLUTIONARY Figure 5. Geographic variation of epaulets. Solid circles denote median values = 0; half-solid circles = 1; and open circles = 2.
RELATIONSHIPS AMONG
PHYLOGENETIC SPECIES
We obtained sequences of the whole cyt b (1143 bp) and ND2 (1041 bp) genes for 12 individuals.
Table 2. Plumage characteristics of taxonomic units Characters Taxonomic units
Crown
Vent
Rump
Thighs
Infra-wing coverts
Carpal-base region
Epaulets
A B C D
Yellow Black Black Black
Yellow Black Black Black
Yellow Black Black Black
Yellow Black Black Black
Yellow Black Yellow Black
Black Black Yellow Black
Yellow Yellow Yellow Brown
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The four phylogenetic species differ in the size of their ranges and habitat preferences. The range of the phylogenetic species A is estimated in 2 913 000 km2. It encompasses riverine forests along the rivers Branco and Negro, the savannas of RoraimaRupununi, the Llanos and forests along the rivers Solimões and Marañon (Fig. 6). Along its range, this species is somewhat local and seldom numerous. It inhabits humid forest borders, gallery forest, savanna
with scattered trees and moriche palm (Mauritia sp.) groves (Ridgely & Greenfield, 2001; Hilty, 2003). It shows dependence on the presence of moriche palms but is frequently registered well away from them (Jaramillo & Burke, 1999; Hilty, 2003). The range of the phylogenetic species B is estimated in 3 303 000 km2. This species is restricted to Amazonian forests south of the Amazon, ranging from the Ucayali River eastwards to the Tocantins-Araguaia Rivers (Fig. 6). It is not a common species across its entire range and appears to be quite local (Ridgely & Greenfield, 2001). This uncommon to locally common species inhabits the canopy and the edge of terra firme forest, even in disturbed environments (Jaramillo & Burke, 1999). This species is frequently registered in pairs or solitary and in mixed-species assemblage at fruiting or flowering trees. The range of the phylogenetic species C is estimated in 847 000 km2 and is the smallest one. It is mostly coincident with Caatinga, a dry vegetation that covers the shallow soils in northeastern Brazil, but it was recorded along the adjacent Atlantic Forest as well. It is not possible to determine if records along the Atlantic Forest are a consequence of recent expansion of the species toward this region caused by the replacement of the forests by agriculture fields. Within Caatinga, it is an uncommon species found in tall dry forests as well as along riverine forests and forest edges. The range of the phylogenetic species D is estimated in 2 259 000 km2 and encompasses four very distinctive ecological regions, including Pantanal, Chaco, Bolivian dry forests, and western Atlantic Forest. This common bird inhabits chaco woodlands, forest borders, gallery forest, and open vegetation with scattered trees (Jaramillo & Burke, 1999). Like species C, it is common even in urban gardens (Sick, 1997).
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Table 3. Differences in body size measurements between sexes of the taxonomic units Differences between sexes Characters Populations
Wing length
Tail length
Total Culmen
Length of bill from nostril
Width of bill at gape
Height of bill at nostrils
Tarsus
A B C D
P < 0.01 P < 0.05 P < 0.01 P < 0.01
P < 0.01 NS NS P < 0.01
NS P < 0.05 NS P < 0.05
NS NS NS P < 0.01
NS NS NS NS
NS NS NS NS
NS NS P < 0.05 NS
NS, not significant.
Sequences from additional individuals were obtained from GenBank. Among these, four lacked the first 98 bp of cyt b and two lacked the first 141 bp; these six sequences also lacked around 120 bp in the end of cyt b. ND2 sequences were complete in both ends. Genetic distances are low among ingroup taxa: the highest pairwise distance for cyt b was 1.74% and for
ND2 1.35%. Distances between the ingroup and the outgroups ranged from 4.46% to 5.33% for cyt b and from 3.49% to 5.00% for ND2. No saturation was detected in the dataset. Because the partition homogeneity test did not indicate conflict of phylogenetic signal between the two genes. we combined the datasets and obtained a
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Figure 6. Taxonomic units (dark gray) and hybrid zones (gray). Northern South America unit (A); Southern Amazon unit (B); Northeastern-Brazil unit (C); and Southwestern South America unit (D).
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matrix with 2184 bp and 18 individuals. MP analysis of this matrix resulted in one most parsimonious tree with 183 steps (Fig. 7). The topology recovers two well-supported clades, the first one uniting the phylogenetic species A and B and the second uniting the phylogenetic species C, D and individuals with intermediate phenotype collected in central Brazil. ML analyses were implemented using Tamura–Nei model of evolution with proportion of invariable sites and equal rates for all sites as selected by Modeltest. The topology obtained has -lnL = 3956.97 and is equal to the MP topology. Bootstrap support for uniting species A and B was also high (93%), but the support for the node uniting C and D was lower (66%). Overall bootstrap support was lower in the ML analysis than in MP analysis. Bayesian analyses also resulted in the same topology obtained in MP and ML analyses, but posterior probabilities were generally low. The likelihood ratio test showed that sequences are evolving in a clock-like manner (P > 0.05).
IDENTIFICATION
OF HYBRID ZONES
Two major hybrid zones were identified (Fig. 6). The first hybrid zone lies between populations A and B and its size is estimated in 873 600 km2. It is narrow along the Amazon valley until near Manaus (around 50–100 km wide), but becomes broader after this point and incorporates a large portion of Suriname, French Guyana and the Amapá State, northern Brazil. This hybrid zone is characterized by populations which are composed by individuals that exhibit intermediate phenotype as well as parental ones. The second hybrid zone is located between populations C and D. It is very wide (approximately 2 915 400 km2) and comprises a large region that includes distinctive ecological regions such as Cerrado, part of the Atlantic Forest, and Pantanal. The populations that form this hybrid zone, in turn, are composed, exclusively, by individuals that exhibit intermediate phenotype.
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Figure 7. Maximum-likelihood (ML) tree constructed using sequences of cytochrome b and ND2 genes (2184 bp). A maximum parsimony tree had identical topology for supported nodes. Numbers as terminal taxa represent the samples showed in map. Numbers on nodes are MP bootstrap proportions for 1000 replicates per decay index (up) and ML bootstrap proportions for 100 replicates per posterior probability (down).
SPECIES LIMITS AND HYBRID ZONES IN ORIOLES
DISCUSSION THE
PHYLOGENETIC SPECIES AND THEIR NAMES
I. cayanensis–chrysocephalus group leads to an increase of 100% in the number of species formally recognized. Instead of two paraphyletic biological species (Omland et al., 1999), we are proposing four phylogenetic species that can be used as terminals in phylogenetic studies, providing a more accurate assessment of intra- and intercladal diversity patterns.
RANGES
AND HABITAT PREFERENCES
It is possible to distinguish two species groups in relation to habitat preferences. The first group is composed of cayanensis and chrysocephalus. Their ranges are centered in Amazonia and both species live in the canopy and along the edges of forest environments. Although the overall habitat of these species looks similar, chrysocephalus presented a higher dependence of moriche palms than cayanensis. The second group of species is composed of tibialis and pyrrhopterus. Their ranges encompass large ecological regions in which forests are not the dominant vegetation type. Although these species have been recorded in the region previously occupied by Atlantic Forest, it is not clear if these species have always lived there or if they are recent colonists that followed the widespread replacement of forests by humanmade open landscapes during the last five centuries (Silva & Casteleti, 2003). No difference in habitat preferences of tibialis and pyrrhopterus was found. Both phylogenetic species as well as the populations from the intergradation zone between them have been recorded in a variety of open habitats such as forest edges, woodlands, open areas with scattered trees, and even heavily disturbed environments like urban gardens.
PHYLOGENETIC
RELATIONSHIPS AND HYBRID ZONES
The molecular data support the hypothesis (Omland et al., 1999) that all four phylogenetic species of I. cayanensis–chrysocephalus are included in a wellsupported monophyletic group. This clade can be further divided into two species groups: an Amazonian group composed of cayanensis and chrysocephalus, and a southern group, formed by tibialis and pyrrhopterus. This phylogenetic division matches well the division proposed independently based only on habitat preferences, indicating that divergence between these two species groups was associated with a marked habitat change (Brooks & McLennan, 1991). The mean genetic distance between Amazonian and Southern species groups ranges from 1.0% (ND2) to 1.3% (cyt b), indicating that this group has diversified quite recently (i.e. in the last 800 000 years) if
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The hypothesis that the current taxonomy does not correspond to the pattern of plumage variation within the I. cayanensis–chrysocephalus clade is fully supported. The absence of a formal study of morphological variation in this group has produced an equivocated taxonomic interpretation, generating a inflation of subspecific taxa. Even under the biological species concept the taxonomy needs to be changed. As noted by Price (2008), the subespecies category has been abused, with different intergrading population sometimes given subspecies rank. From the six taxa currently recognized for this group (Blake, 1968; Jaramillo & Burke, 1999), only four can be regarded as distinctive phylogenetic species (see Supporting Information, Doc. 1). The phylogenetic species ‘A’ matches the plumage characters described for I. chrysocephalus (Linnaeus, 1766), a species based on ‘Xanthornis icterocephalus americanus’ from Brisson. Berlepsch & Hartert (1902) suggested ‘Cayenne’ as type locality of I. chrysocephalus. The plumage characters of the phylogenetic species ‘B’ corresponds to the description of I. cayanensis (Linnaeus, 1766), whose description was based on ‘Pica alis flavis’ from Edwards and ‘Xanthornus cayanensis’ from Brisson. I. cayanensis has also ‘Cayenne’ as type locality. Because the hybrid zone between I. chrysocephalus and I. cayanensis includes French Guyana (Fig. 6), specimens with both phenotypes can be collected there. The characteristics of the phylogenetic species ‘C’ fits well with I. tibialis Swainson, 1837, a taxon whose description was based on specimens labelled only ‘Brazil’ and that possibly were from north-eastern Brazil (possibly the state of Pernambuco). Finally, the combination of the characters that defines the phylogenetic species ‘D’ corresponds well to that described for I. pyrrhopterus (Vieillot, 1819) that was originally described based on specimens collected in Paraguay. Two taxa currently recognized within the I. cayanensis–chrysocephalus clade are not distinctive phylogenetic species and, therefore, do not deserve formal taxonomic recognition. I. c. valenciobuenoi Ihering, 1902, that was described based on specimens collected in Piracicaba and Jaboticabal, São Paulo, Brazil, corresponds to an intermediate population between I. tibialis and I. pyrrhopterus, a hypothesis that had already been advanced by Sick (1984, 1997). Icterus c. periporphyrus (Bonaparte, 1850), a subspecies of I. cayanensis described with basis on specimens from Chiquitos, Bolívia, represents an individual variation of I. pyrrhopterus and, therefore, should be synonymized with this taxon. The application of the phylogenetic species concept to describe the pattern of differentiation within the
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ACKNOWLEDGEMENTS We thank John Allen, Kevin Omland, and the anonymous referees who reviewed and improved the manuscript, and Jose Tello and Alexandre Aleixo for their helpful comments about the first manuscript. We are indebted to the following curators, staff and their institutions, for permission to examine specimens under their care: Alexandre Aleixo and Fátima Lima (MPEG); Mario de Vivo (MZUSP), Marcos Raposo and Jorge Nacinovic (MNRJ) John Bates and David Willard (FMNH), Joel Cracraft and Paul Sweet (AMNH). FMH research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Frank Chapmann Fund (American Museum of Natural History) and Visiting Scholar Fund (Field Museum of Natural History). JMCS research is supported by Conservation International, Gordon and
Betty Moore Foundation and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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we assume a cyt b rate of molecular evolution of 1.6% divergrnce per million year (Fleischer, Mcintosh & Tarr, 1998). Hybrid zones within the I. cayanensis– chrysocephalus clade occur between sister species rather than between nonsister species. No evidence of gene flow between Amazonian and Southern species groups was found. Intermediate individuals recorded along the boundary between tibialis and cayanensis are better interpreted as representing a hybrid zone between tibialis and pyrrhopterus than between tibialis and cayanensis or between pyrrhopterus and cayanensis. This hypothesis is supported by the fact that the region in which putative hybrids have been recorded is covered by cerrado vegetation (IBGE, 1988), a savana-like vegetation much more similar to habitats in which tibialis and pyrrhopterus were recorded than to habitats in which cayanensis were observed. Amazonian and Southern species groups differ significantly in terms of their range sizes and extension of their hybrid zones. Amazonian species have ranges quite similar in size (the ratio between the large/ small range is only 1.3) and the size of the hybrid zones between them is only 14% of the size of the combined range of the two species. By contrast, Southern species have ranges that differ largely in size (the ratio large/small range is 3.7) and the size of the hybrid zone between them is 93.8% of the size of the combined range of the two species. The hybrid zone between tibialis and pyrrhopterus is larger than the individual ranges of each of these two species and, even though there are still very few quantitative studies estimating the size of hybrid zones, it is one of the largest hybrid zones ever recorded for birds (Barton & Hewitt, 1985; Ford, 1987; Haffer, 1992; Newton, 2003).
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Additional Supporting Information may be found in the online version of this article: Doc. 1. Diagnose and distribution of the phylogenetic species. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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SUPPORTING INFORMATION
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 583–597
Male Female Total Male Female Total Male Female Total Male Female Total
Icterus cayanensis
Icterus pyrrhopterus
Icterus tibialis
Icterus chrysocephalus
Sex
Taxon
11 08 19 37 25 62 25 18 43 120 91 211
1.33 1.28 1.58 1.39 1.22 1.58 1.25 1.30 1.30 1.83 1.80 1.86
8.25 7.89 8.10 8.05 8.15 8.09 7.39 7.30 7.35 7.09 7.06 7.08
0.43 0.41 0.45 0.38 0.35 0.37 0.31 0.38 0.34 0.37 0.39 0.38
Standard deviation
22.27 11.96 22.27 13.12 15.27 17.80 21.45 12.32 21.45 21.68 18.87 23.89
100.20 97.61 99.28 97.90 95.14 96.84 95.28 93.42 94.64 94.03 90.84 92.74
6.70 4.54 5.97 3.27 3.36 3.54 5.02 3.29 4.54 3.74 4.05 4.16
10 08 18 38 28 66 26 19 45 119 90 209
11 08 19 29 22 51 24 14 38 88 70 158
0.77 0.94 0.94 1.32 1.56 1.56 1.36 1.40 1.46 1.55 1.15 1.55
Amplitude
Number of specimens
Amplitude
Mean
09 05 14 35 22 57 23 12 35 111 76 187
Number of specimens
5.40 4.34 5.76 3.18 2.39 3.60 4.24 3.09 4.21 3.42 2.75 3.86
Length of bill from nostril (cm)
5.91 5.83 5.88 5.93 5.87 5.90 5.39 5.38 5.39 5.29 5.21 5.25
Mean
2.15 2.96 3.10 3.69 4.75 4.75 4.43 4.04 4.43 4.79 5.34 5.51
0.23 0.35 0.28 0.30 0.36 0.33 0.36 0.35 0.35 0.37 0.30 0.34
Standard deviation
23.89 22.94 23.47 24.48 24.13 24.33 20.75 20.53 20.66 20.25 19.93 20.11
10 08 18 38 27 65 25 19 44 120 92 212
08 07 15 35 24 59 17 08 25 114 87 201
Number of specimens
Tarsus (cm)
0.78 0.99 0.98 0.82 1.17 0.99 1.00 0.99 0.99 0.94 0.99 0.97
Mean 22.16 22.14 22.15 22.34 22.06 22.23 22.52 21.94 22.33 22.14 21.92 22.04
2.20 2.01 2.38 2.93 3.10 3.16 2.04 1.41 2.24 3.67 4.19 4.38
14.88 14.31 14.63 15.40 15.25 15.34 12.75 12.67 12.71 12.54 12.24 12.41
Amplitude
2.01 2.92 3.42 2.76 4.22 4.23 2.02 2.42 2.65 5.18 4.33 5.50
0.75 0.87 0.77 0.69 0.69 0.70 0.64 0.51 0.66 0.78 0.80 0.79
Standard deviation
0.67 0.97 0.84 0.60 0.90 0.74 0.57 0.73 0.63 0.83 0.77 0.82
Standard Number of Standard Number of Standard deviation specimens Amplitude Mean deviation specimens Amplitude Mean deviation
Height of bill at nostrils (cm)
102.85 97.69 101.11 103.20 98.76 101.27 92.50 88.75 90.97 90.74 86.20 88.74
Standard Number of deviation specimens Amplitude Mean
Total culmen (cm)
Width of bill at gape (cm)
18.50 12.00 18.50 17.00 11.50 20.50 18.00 11.50 18.00 15.50 16.00 19.50
Number of specimens Amplitude Mean
Male 10 Female 08 Total 18 Icterus Male 38 chrysocephalus Female 29 Total 67 Icterus tibialis Male 26 Female 18 Total 44 Icterus Male 120 pyrrhopterus Female 95 Total 215
Icterus cayanensis
Sex
Tail length (cm)
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Taxon
Wing length (cm)
Table A1. Body measurements
APPENDIX
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