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Cryptic species in Iphisa elegans Gray, 1851 (Squamata:Gymnophthalmidae) revealed by hemipenialmorphology and molecular data

PEDRO M. SALES NUNES1*, ANTOINE FOUQUET1, FELIPE F. CURCIO1,PHILIPPE J. R. KOK2,3 and MIGUEL TREFAUT RODRIGUES1

1Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Caixa Postal11.461, CEP 05422-970, São Paulo, SP, Brazil2Biology Department, Unit of Ecology and Systematics, Vrije Universiteit Brussel, 2 Pleinlaan,B-1050 Brussels, Belgium3Department of Vertebrates, Royal Belgian Institute of Natural Sciences, 29 rue Vautier, B-1000Brussels, Belgium

Received 7 November 2011; revised 17 May 2012; accepted for publication 17 May 2012

Iphisa elegans Gray, 1851 is a ground-dwelling lizard widespread over Amazonia that displays a broadly conservedexternal morphology over its range. This wide geographical distribution and conservation of body form contrastswith the expected poor dispersal ability of the species, the tumultuous past of Amazonia, and the previouslydocumented prevalence of cryptic species in widespread terrestrial organisms in this region. Here we investigatethis homogeneity by examining hemipenial morphology and conducting phylogenetic analyses of mitochondrial(CYTB) and nuclear (C-MOS) DNA sequence data from 49 individuals sampled across Amazonia. We detectedremarkable variation in hemipenial morphology within this species, with multiple cases of sympatric occurrenceof distinct hemipenial morphotypes. Phylogenetic analyses revealed highly divergent lineages corroborating thepatterns suggested by the hemipenial morphotypes, including co-occurrence of different lineages. The degrees ofgenetic and morphological distinctness, as well as instances of sympatry among mtDNA lineages/morphotypeswithout nuDNA allele sharing, suggest that I. elegans is a complex of cryptic species. An extensive and integrativetaxonomic revision of the I. elegans complex throughout its wide geographical range is needed.

© 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 361–376.doi: 10.1111/j.1096-3642.2012.00846.x

ADDITIONAL KEYWORDS: C-MOS – cryptic diversity – CYTB – hemipenis – prezygotic isolation.

INTRODUCTION

Biodiversity is unevenly distributed on Earth withtropical forests sheltering more than 50% of the livingspecies known to science (Wilson, 1992; Gaston &Williams, 1996; Myers et al., 2000). Despite its recog-nition as a megadiverse biome, the species richness ofAmazonia remains superficially known and patternsof diversification throughout its area are poorlyunderstood (Bush, 1994; Noonan & Wray, 2006).

Many small terrestrial vertebrates apparently similarin body form and external characters have surprisingwidespread distributions if we consider the extent oftheir range, their putative low vagility, and the highlycomplex climatic and geological history of the region(Antonelli et al., 2010; Hoorn et al., 2010). Studiesaddressing patterns of genetic diversity within suchspecies are scarce but most have revealed ancient andwell-geographically structured lineages, suggestingcomplexes of cryptic species (Chek et al., 2001;Noonan & Wray, 2006; Fouquet et al., 2007;Amézquita et al., 2009; Mott & Vieites, 2009; Geurgas*Corresponding author. E-mail: [email protected]

Zoological Journal of the Linnean Society, 2012, 166, 361–376. With 3 figures

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© 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 361–376 361

& Rodrigues, 2010; Funk, Caminer & Ron, 2012).DNA sequences have indeed been decisive in therecognition of hidden diversity in morphologicallysimilar forms, from the African elephants (Vogel,2001) to the tiny Amazonian sphaerodactylid geckosof the genus Chatogekko (Geurgas & Rodrigues, 2010;Gamble et al., 2011). Moreover, the adoption of a moreintegrative taxonomy as a framework to integratediverse lines of evidence such as molecular and mor-phological data has greatly improved the reliabilityand efficiency of species delineation (Padial et al.,2010; Miralles et al., 2010).

The monotypic genus Iphisa Gray, 1851 (Gymnoph-thalmidae) is one such widespread species that ispresent throughout Amazonia (Dixon, 1974; Peters &Donoso-Barros, 1986). It is a small (maximum snout–vent length around 62 mm) leaf litter-dwelling speciesshowing little morphological variation along this area.It was erected to allocate I. elegans, after a specimencollected by Wallace and Bates with an imprecise typelocality comprising a vast area around the region ofBelém, Pará State, northern Brazil (Gray, 1851; Dixon,1974). The only revision of the genus supported Iphisaas comprising a single species and recognized thepopulations from Peru and Bolivia as a distinct sub-species (I. e. soinii) diagnosed by lack of prefrontalsand presence of a higher average number of femoral-preanal pores (25.0 vs 19.3 in the typical form) (Dixon,1974). However, as Dixon’s (1974) scheme was basedon a restricted sample (50 specimens, mainly fromnorthern and western Amazonia) it was never followed(Hoogmoed, 1973; Ávila-Pires, 1995). Moreover, thestatus of I. e. soinii with respect to the nominal formhas never been properly examined on the basis of amore representative sampling.

In the course of a broad study of gymnophthalmidhemipenial morphology (Nunes, 2011) we detected asurprising hemipenial variation in the organs ofI. elegans. The hemipenis of squamates is directlyinvolved in copulation and differences in hemipenialmorphology can be considered a physical mechanismof reproductive isolation (Pope, 1941; Arnold,1986a). Therefore, characteristics of this organ areexpected to be highly informative for phylogeneticstudies and species characterization. In fact, formore than a century hemipenial morphology hasbeen efficiently used to understand the relationshipsamong squamates.

The relevance of hemipenial data for phylogeneticstudies has been widely debated in the literature.According to some authors, the evolution of this organwould be less subjected to ecological and environmen-tal constraints than features of external morphology,and thus would be phylogenetically more informativethan other structures (Dowling, 1967; Arnold, 1986a;Keogh, 1999). However, evidence of intraspecific

hemipenial polymorphism (McDowell, 1979; Cole &Hardy, 1981; Zaher & Prudente, 1999; Inger & Marx,1962) apparently contradicts such a hypothesis, sug-gesting that hemipenial characters are not distinc-tively more conservative, accurate, or informativethan any other morphological features (Myers, 1974;Zaher & Prudente, 1999). In fact, the importance ofvariation in traits that mediate pre-zygotic isolation,such as call advertisement in frogs, is clear and oftenrepresents a crucially relevant source of charactersfor taxonomic approaches (Padial et al., 2010).

The rare reports of discrete populational variationsin hemipenial morphology refer to shape, ornamenta-tion, and/or size (Myers, 1974; Cole & Hardy, 1981).Inger & Marx (1962) detected four remarkablydistinct hemipenial morphotypes in the snakeCalamaria lumbricoidea; nonetheless, despite thegeographical proximity among the populations inves-tigated, none of the patterns was found in sympatry.Zaher & Prudente (1999) also reported remarkablydifferent hemipenial morphotypes, including sympat-ric occurrence, in the widespread Neotropical forestsnake Siphlophis compressus (Pseudoboini). Althoughthe studies above have interpreted cases of hemipe-nial polymorphism as intraspecific variation, hemipe-nial differences between populations that areotherwise largely homogeneous may also indicate theexistence of cryptic species, as demonstrated by Pru-dente & Passos (2010). Moreover, there have as yetbeen no attempts to explore genetic information insuch cases of morphological variation. Interpretingthe evolutionary significance of these particularexamples of morphological plasticity is far from easy,and molecular data may greatly contribute to theclarification of such complex patterns.

Oriented by the astonishing hemipenial variationobserved in I. elegans, a lizard that is otherwise mor-phologically extremely conservative, we (1) investi-gate its morphological and genetic homogeneityexamining hemipenis (49 specimens from 32 differentlocalities) and DNA sequences (22 samples from 16localities; nuclear and mitochondrial genes) acrossAmazonia in order to (2) explore hemipenial variabil-ity among populations and (3) discuss the morpho-logical diversity in the light of DNA sequences ofnuclear and mitochondrial genes, suggesting hypoth-eses for the origin of the hemipenial variation foundin I. elegans throughout Amazonia.

MATERIAL AND METHODSTAXON SAMPLING

We examined the hemipenis of 49 specimens of I. el-egans from 32 different localities in Brazil, Ecuador,French Guiana, Guyana, and Peru (Appendix 1).

362 P. M. S. NUNES ET AL.

© 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 361–376

Vouchers are deposited in the following institutions(acronyms in parentheses): American Museum ofNatural History, New York, USA (AMNH); InstitutoNacional de Pesquisas da Amazônia, Manaus, Brazil(INPA); Museu de Zoologia, Universidade de SãoPaulo, São Paulo, Brazil (MZUSP); National Museumof Natural History, Smithsonian Institution, Wash-ington, DC, USA (USNM); Natural History Museum,University of Kansas, Lawrence, USA (KU); InstitutRoyal des Sciences Naturelles de Belgique, Brussels,Belgium (IRSNB); Museo de Historia Natural, Uni-versidad Nacional Mayor San Marcos, Lima, Peru(MHNSM); and Centro de Ornitología y Biodiver-sidad, Lima, Peru (CORBIDI).

For the DNA-based phylogenetic analyses, weselected tissue samples (liver) of 22 individuals ofI. elegans from 16 localities; 12 of these samples cor-responded to males for which we had hemipenialpreparations. To use the closest taxa as outgroups, wealso analysed DNA sequences of one individual ofeach genus within Heterodactylini and Iphisiiniaccording to Rodrigues et al. (2009).

HEMIPENIAL PREPARATIONS

Hemipenis preparation followed the proceduresdescribed by Manzani & Abe (1988), as modified byPesantes (1994) and Zaher (1999) for snake organs. Inaddition, we used an alcohol solution of Alizarin Redto stain ornamenting calcareous structures in anadaptation of the procedures used by Uzzell (1973)and Harvey & Embert (2008). Terminology for hemi-penial characters follows Dowling & Savage (1960),Uzzell (1973), Zaher (1999), and Myers, Rivas Fuen-mayor & Jadin (2009).

MOLECULAR METHODS

Genomic DNA was extracted using the Promega DNAextraction kit. One fragment of the mitochondrialgene (mtDNA) Cytochrome b (CYTB) and one frag-ment of the nuclear gene (nuDNA) oocyte maturationfactor Mos (C-MOS) were amplified by standard PCRtechniques. Primers and PCR conditions used foramplification were as described by Bickham, Wood &Patton (1995) and Kocher et al. (1989) for CYTB andby Godinho et al. (2006) and Saint et al. (1998) forC-MOS (Appendix 2). PCR products were purifiedusing EXOI (Exonuclease I) and SAP (Shrimp Alka-line Phosphatase) techniques. Sequencing was per-formed using ABI Big Dye v3.1 (ABI, Foster City, CA,USA) and resolved on an automated sequencer atInstituto de Química da Universidade de São Paulo(IQUSP – São Paulo, Brazil) and Genomic Engen-haria (São Paulo). Sequences were edited and alignedwith Codon Code Aligner v.3.5.2. New sequences weredeposited in GenBank (Appendix 1). Some CYTB

sequences of gymnophtalmid species used as outgroupused remained slightly shorter (~150 bp).

ALIGNMENT AND SEQUENCE DATA ANALYSIS

Alignments were verified by eye and trimmed toremove the most incomplete data, leading to 752aligned base pairs (bp) of the CYTB gene and 532 bpof the C-MOS gene.

We used the software MrModeltest version 2.3(Nylander, 2004) to select the substitution models thatbest fit each codon position of CYTB and the fragmentas a whole according to the Akaike Information Cri-terion (Akaike, 1974). The resulting three models foreach codon position were used in a partitioned Baye-sian analysis (Appendix 3) performed with MrBayes3.1 (Huelsenbeck & Ronquist, 2001). Bayesian analy-sis consisted of two independent runs of 1.0 ¥ 107

generations with random starting trees and tenMarkov chains (one cold) sampled every 1000 genera-tions. Adequate burn-in was determined by examininga plot of the likelihood scores of the heated chains forconvergence on stationarity as well as the effectivesample size of values in Tracer 1.5 (Rambaut & Drum-mond, 2003). We also performed maximum-likelihood(ML) and maximum-parsimony (MP) analyses withPAUP 4.0b10 (Swofford, 1993). The ML analysis wasconducted using the best fitting model estimated forthe entire CYTB fragment. We computed 100 non-parametric bootstrap pseudoreplicates (Efron, 1979;Felsenstein, 1985) with the heuristic search option,tree bisection-reconnection (TBR) branch swappingand ten random taxon addition replicates per pseu-doreplicate. Support for proposed clades using MP wasassessed via 10 000 non-parametric bootstrap pseu-doreplicates (Efron, 1979; Felsenstein, 1985) with theheuristic search option, TBR branch swapping and tenrandom taxon addition replicates per pseudoreplicate.In total, 275 characters were parsimony informative,and 55 variable characters were not parsimony infor-mative. We considered relationships with posteriorprobabilities �0.95 and/or bootstrap percentages�70% (Hillis & Bull, 1993) to be strongly supported.Trees were rooted on Heterodactylini (Caparaonia +Heterodactylus + Colobodactylus) according to Rod-rigues et al. (2009).

To determine the most probable alleles for individu-als recovered as heterozygous on the C-MOS frag-ment we used PHASE (Stephens, Smith & Donnelly,2001; Stephens & Donnelly, 2003) implemented inDnaSP 5 (Librado & Rozas, 2009). We used defaultconditions, including 500 iterations (which were suf-ficient to reach stationarity), a burn-in of 100, and athinning interval of 1. To improve reliability, we ranthe algorithm multiple times with a different randomnumber of seeds. We chose the run with the highest

CRYPTIC SPECIES IN IPHISA ELEGANS GRAY, 1851 363


average value for the goodness of fit. One individualremained with ambiguous phasing (MHNC 10082).Statistical parsimony network was calculated for thephased C-MOS alignment using TCS 1.21 (Clement,Posada & Crandall, 2000), with a 95% connectionlimit.

RESULTSHEMIPENIAL MORPHOLOGY

Analysis of hemipenial morphology revealed aremarkable variation among I. elegans populations; inone case, variations were also detected among speci-mens of the same population (i.e. specimens belongingto the same locality). We recognized five distincthemipenial morphotypes (Fig. 1) based on hemipenialbody shape, position and number of calcareous spi-cules, and size and form of lobes. We describe the fivehemipenial morphotypes, explicitly associating eachone with their respective localities of occurrence.

Morphotype 1 (Figs 1A, 2A: Candidate species 1)Localities of occurrence: BRAZIL: Mato Grosso:Apiacás; Aripuanã; PERU: Loreto: Rio Ampiyacu.

General description: Hemipenes slender and cylindri-cal; hemipenial body covered by numerous calcifiedspicules; lobes short, apexes ornamented by smallpapillate folds.

Variation: Populations from Aripuanã and Rio Ampiy-acu (the latter not sampled in the molecular analyses)have a narrow bare area on the central region of theasulcate face, whereas the hemipenial bodies of theremaining specimens are entirely covered by spicules.

Morphotype 2 (Figs 1B, 2A: Candidate species 2)Localities of occurrence: BRAZIL: Amazonas: AltoRio Aripuanã; Interfluve of Rivers Madeira–Purus;Campo Catuquira; Campo Tupana; São Sebastião (RioAbacaxis); Igarapé-Açu (Rio Abacaxis); Mato Grosso:Juruena; Rondônia: Rio Machado.

General description: Base of hemipenial body dis-tinctly wider than apex (‘pear-shaped’ organ); largecalcified spines present on lateral surface of hemi-

penial body and on the asulcate face; spines of asul-cate face organized in two rows, converging towardsapex into a single row attaining level of lobularcrotch; flounces lacking calcified spicules; long and

Figure 1. Hemipenial morphotypes. A, morphotype 1(MZUSP 82662: Aripuanã, Mato Grosso, Brazil); B, mor-photype 2 (MZUSP 82428: Juruena, Mato Grosso, Brazil);C, morphotype 3 (MZUSP 100257: Igarapé-Açu, Amazo-nas, Brazil); D, morphotype 4 (IRSNB 17069: KaieteurNational Park, Guyana); E, morphotype 5 (MHNSM16718: Distrito Genaro Herrera, Provincia Requena,Peru). Scale bars = 3 mm.

�



Figure 2. A, phylogram hypothesized from Bayesian analysis using 752 bp of mtDNA (CYTB). For Bayesian analysis weused a partitioned model of evolution combining one model, estimated using MrModeltest 2, for each codon position. Nodesupports are indicated with 1, posterior probability*100 (10 000 000 generations sampled every 1000; 10 chains); 2,maximum likelihood bootstrap support (n = 100); maximum parsimony bootstrap support (n = 10 000). Posterior prob-abilities equal to 1 and 0.99 and bootstrap values equal to 100 and 99 are indicated with ‘*’ while ‘–’ indicates bootstrapsupport <50%. Relationships that remained poorly supported are indicated in red. Hemipenial morphotypes are illustratedbeside each of their corresponding clades. Localities for which molecular and hemipenial data are available are indicatedby ‘°’. B, statistical parsimony network based on phased C-MOS alleles (n = 44). Colours correspond to the major cladesillustrated in the mtDNA-based phylogenic tree reconstruction with the size of the circles being proportional to thefrequency of the allele as also indicated in the circles. Each C-MOS allele has been coded with a letter (a–l).



slender finger-shaped appendices present on apexesof lobes; sulcus spermaticus ending on apex oflobular appendix.

Variation: Specimens from Campo Catuquira andCampo Tupana (both not sampled in the molecularanalyses) lack spines on lateral surface of hemipenialbody, and the spines of asulcate face are not arrangedin convergent rows, but scattered throughout itscentral region.

Morphotype 3 (Figs 1C, 2A: Candidate species 3)Localities of occurrence: BRAZIL: Amazonas:Manaus; São Sebastião (Rio Abacaxis); Igarapé-Açu(Rio Abacaxis).

General description: Hemipenes cylindrical; lobessmall, apices ornamented by papillate flounces;lobular appendices absent; lateral surfaces covered byflounces with calcified spicules; central area of asul-cate face ornamented by two simple rows of spinesoriginating at base, converging until midpoint of bodyand then assuming centrifugal orientation towardslobe base.

Variation: In contrast to the pattern described above,the spine rows present in the central area of theasulcate face are not simple in one specimen fromManaus (MZUSP 8354), but composed of irregularlyaligned sets of spines.

Morphotype 4 (Figs 1D, 2A: Candidate species 4)Localities of occurrence: BRAZIL: Amapá: IgarapéCamaipi, Rio Maracá; Rondônia: UHE Jirau;FRENCH GUIANA: Paracou, Sinnamary; GUYANA:Kaieteur National Park.

General description: Hemipenial body cylindrical;lobes small, lobular appendices lacking; lateral sur-faces covered by flounces with calcified spicules;central area of asulcate face mostly bare, ornamentedby two parallel spine rows running in roughly sagitalposition.

Variation: One specimen from Igarapé Camaipi has astripe of scattered spines along the midline of asul-cate face reaching proximal two-thirds of hemipenialbody; distally from this point, only two parallel rowsremain towards apex with a bare area between them.

Morphotype 5 (Figs 1E, 2A: Candidate species 5)Localities of occurrence: ECUADOR: Morona-Santiago: Rampon; Napo: Puerto Libre, Rio Aguarico;PERU: Amazonas: Rio Cenepa, Rio Marañon valley;Cuzco: Pagoreni; San Martin; Distrito GenaroHerrera, Provincia Requena; Loreto: Rio Corrientes.

General description: Hemipenial body cylindrical;lateral surfaces of body and lateral margins of asul-cate face covered by rows of flounces adorned bycalcified spines; central area of asulcate face orna-mented by a wide stripe of scattered spines thatbecome increasingly numerous towards apex; lobessmall, lobular appendices absent.

Variation: The number and size of spines on thecentral area of the asulcate face vary among thelocalities sampled.

MOLECULAR PHYLOGENETIC ANALYSES

The topology recovered from mtDNA is relatively wellresolved, with the ingroup (genus Iphisa) having 12 of21 nodes strongly supported by posterior probability(PP � 0.95) and eight of 21 nodes strongly supportedby maximum parsimony bootstrap (bootstrap support� 95%) (Fig. 2A). Although levels of support are het-erogeneous among the methods used, there are noconflicts among topologies. Iphisa elegans was unam-biguously recovered as monophyletic and, althoughpoorly resolved, the relationships among gymnoph-talmids match the topology of Rodrigues et al. (2009).

The genetic diversity recovered within Iphisa isstriking and is broadly congruent with the hemipenialpatterns (Fig. 2A). Each population harbours a highlydivergent lineage, the only exception being the threesouthern populations of clade 1A from the localitiesof UHE Guaporé, Apiacás, and Montenegro-Cacaulândia. As a matter of comparison, the degree ofdivergence (maximum p distance = 0.152) withinIphisa is comparable with the divergence amongrelated Iphisiini genera, which have remarkably dis-tinct general morphology (Appendix 4).

The analyses recovered two major clades withinIphisa: Clade 1 occurs in southern Amazonia (fromRio Abacaxis to Guaporé), and Clade 2 occurs over theremaining territory of Amazonia (from the GuianaShield to Peru and Rondônia). Each of these cladesincludes highly divergent and strongly supported sub-clades. Clade 1 displays a clear structure, although itstwo subclades show wide geographical overlap overthe Abacaxis and the Aripuanã basins. Clade 2 and itssubdivisions are not well supported. Nevertheless,given the geographical distribution of the subclades(Peru vs. Guiana Shield and Rondônia) we consideredthese two groups in latter analyses. The genetic sub-divisions detected are so pronounced that each ofthese subclades (1A, 1B, 2A, and 2B) is itself dividedinto multiple highly divergent lineages. Such geneticstructure reveals that our sampling remains insuffi-cient to estimate the actual distribution of these lin-eages so that the degree of geographical overlapamong them may in fact be far more important.



Nevertheless, it is noteworthy that, in one case, twolineages (1Ba and 1Bb) within subclade 1B are syn-topic and that at least subclades 1A and 1B appearlargely sympatric and do not share any C-MOS allele(Fig. 2B). Clade 2A appears to cover a wide area fromthe Guiana Shield to Rondônia, but again the differ-ent lineages involved show high degrees of geneticdivergence. Even the two populations sampled at veryclose localities in Peru (EEBB Pithecia and Ham-burgo, in Loreto) are highly divergent (CYTB p dis-tance = 0.11; Appendix 4).

Interestingly, the nuDNA data provide a structurethat is concordant with the most basal mtDNA splitswithin Iphisa. Subclade 1A displays a fixed substitutionand within subclade 1B the two groups do not share anyallele. The only instances of allele sharing are amonggeographically distant populations and correspond tothe central haplotype, which probably represents anancestral state. Therefore, our data provide no evidenceof gene flow among genetically differentiated popula-tions occurring in geographical proximity.

DISCUSSIONCRYPTIC SPECIES

Inaccuracy in the evaluation of diversity has impor-tant ramifications, and thus a precise delimitation ofspecies is essential to many disciplines such as bio-geography, ecology, macroevolution, biodiversityassessment, and conservation given that species arebasic units of analysis. However, delineating speciesremains a challenge. Cryptic species are detectedwhen one recognizes two or more distinct speciespreviously classified as a single species due to overallmorphological similarity that prevents immediateobvious distinction (Bickford et al., 2007; Pfenninger& Schwenk, 2007; Trontelj & Fišer, 2009). RecentDNA-based studies (e.g. Fouquet et al., 2007; Kochet al., 2009; Mott & Vieites, 2009; Geurgas & Rod-rigues, 2010; Oliver, Adams & Doughty, 2010;Hekkala et al., 2011; Morin et al., 2011; Wu et al.,2011) have demonstrated the existence of consider-ably divergent lineages ignored by taxonomic systemssolely based on morphological grounds, revealingdegrees of genetic divergence that reflect millions ofyears of evolutionary history. Although morphologymay be of little use in revealing important historicaldivergences among cryptic species (Elmer, Dávila &Lougheed, 2007; Koch et al., 2009; Geurgas & Rod-rigues, 2010), morphological evidence remains crucialin their description and precise diagnoses (Hillis &Wiens, 2000). Therefore, species limits are undoubt-edly better understood through the combination ofdifferent kinds of information (Padial et al., 2010)such as DNA and morphology. Another crucial point

in delimiting cryptic species lies in distinguishingbetween broad-admixture, narrow contact zones withrestricted hybridization and complete isolation (Wake& Jockusch, 2000), allowing assessment of the inde-pendence of evolutionary trajectories of the entities.Thus, delineation of species not only requires the useof a combination of multiple lines of evidence, but alsoa thorough sampling to provide an accurate descrip-tion of the biodiversity.

The striking congruence between different types ofcharacters (hemipenial, mitochondrial, and nucleardata) in I. elegans provides strong evidence support-ing the existence of distinct species (Dayrat, 2005;DeSalle, Egan & Siddal, 2005; Padial et al., 2010)that remained masked by overall homogeneous exter-nal morphology. In addition, some of these candidatespecies distributions overlap geographically, withsyntopy observed in at least one locality (SãoSebastião, Rio Abacaxis). Broader sampling through-out the range of the genus will probably reveal moreinstances of co-occurrence of distinct entities. Thegeographically overlapping mtDNA-based groups donot share any nuclear alleles, suggesting that theselineages are reproductively isolated. On the otherhand, the candidate species that are undistinguish-able on the basis of nuDNA and have more similarhemipenial morphology were found in distant loca-tions and are probably geographically isolated. There-fore, concordance between the criteria of coalescenceand isolation, as coined by de Queiroz (1998), as wellas two independent lines of evidence (DNA and mor-phology) suggest that several cryptic species existunder the name I. elegans and indicate the need ofreformulations towards a more informative taxonomicsystem for the genus Iphisa.

Our results indicate the existence of five majorgroups supported by molecular and hemipenial data,contrasting with Dixon’s (1974) conclusion that thegenus is monotypic with no more than two distin-guishable varieties (i.e. subspecies). However, Dixon’s(1974) conclusions were based on a rather limitedsampling and strictly on morphology.

Within the clades recovered here, the Peruvianlineage designated as clade 2B corresponds to speci-mens that morphologically fit Dixon’s (1974) descrip-tion of I. e. soinii. Unfortunately, specimens from thetype locality of I. e. elegans [300-mile radius of Pará,Brazil, sensu Gray (1851); by Pará, Dixon (1974)probably refers to the municipality of Belém, ParáState] were not available for our analyses. However,in our sample, the specimens from the closest locali-ties to the Belém region (Pará state) are from theleft bank of the Amazon River and appear nested inclade 2A. The imprecise locality of the holotype andthe limited number of samples available for thisstudy prevent any conclusion regarding the actual



correspondence between the nominal form of I. e.elegans and any of the lineages detected herein.Therefore, we believe that any nomenclatural actionsregarding the I. elegans complex depend on a com-prehensive taxonomic revision with more extensivesampling, as well as more detailed morphologicaland molecular analyses.

PREMATING REPRODUCTIVE ISOLATION

The hemipenial variation within I. elegans is unusualfor squamate species showing otherwise homogeneousoverall morphology (e.g. Uzzell, 1966, 1973; Arnold,1986a, b; Zaher, 1999). Regarding gymnophthalmids,such a level of variation could be compared with thevariation observed between different related genera(Presch, 1978; Nunes, 2011). Thus, it is striking thatthese hemipenial morphotypes are sympatric, with atleast four lineages occurring in very close geographi-cal proximity, or even in syntopy [specimens belong-ing to clades 1Ba and 1Bb are syntopic on the samebank of Rio Abacaxis, but are remarkably distinctwith respect to hemipenial structure (see Figs 2A, 3)].In contrast, the allopatric lineages constituting clade2 display more similar hemipenes (Fig. 2).

Such results suggest that hemipenial morphologymay be directly linked to the origin of this pattern. Areasonable preliminary hypothesis for the origin ofsuch pattern could rely in a speciation process occur-ring through reinforcement of premating isolation as aconsequence of secondary contact between lineages(Dobzhansky, 1940, 1951; Butlin, 1987; Liou & Price,1994; Hoskin et al., 2005). Although the basis of thisprocess has been seriously questioned in recentdecades (Butlin, 1987, 1995, 2004), there remainsstrong support (Hoskin et al., 2005; Lemmon, 2009).Speciation by reinforcement is based on prezygoticisolation between two or more populations previouslyhybridizing, enhancing characters that decrease geneflow between them. According to Servedio & Noor(2003), if two populations have diverged to such anextent that they produce unfit hybrids, one mustexpect that more successful offspring will result fromindividuals belonging to the same population; there-fore, those characters increasing assortative matingwill be favoured until full speciation eventually takesplace (Butlin, 1987). Nevertheless, in the case of Iphisathe ecological differences or characteristics favouringassortative mating remain uncertain, as habitat isapparently similar among groups. Therefore, thishypothesis implies that the differences in hemipenialgeneral conformation may prevent hybridizationamong cryptic species exposed to secondary contactand ultimately favoured speciation and range overlap.

Similar patterns of hemipenial variation concor-dant with genetic variability may be expected for

other squamate groups. Molecular investigations onpreviously documented cases of intraspecific hemipe-nial morphological variation [e.g. Calamaria lumbri-coidea (Inger & Marx, 1962)] and Siphlophiscompressus (Zaher & Prudente, 1999)] may alsoreveal cryptic species and thus provide additionalsupport for our interpretation regarding the presenceof a complex of species under the name I. elegans.

ACKNOWLEDGMENTS

We are grateful to D. Frost and D. Kizirian (AMNH),K. de Queiroz, R. McDiarmid, R. Heyer and G. Zug(USNM), W. Duellman and L. Trueb (KU), J. C. Chap-arro (MHNC), P. Venegas (CORBIDI), J. H. C. Santa-Gádea and J. Suárez (MHNSM), S. M. Souza and R.Vogt (INPA), and H. Zaher and C. Castro-Mello(MZUSP) for providing access to specimens and tissuesamples under their care. We are also grateful to P.Hayward and two anonymous reviewers for the sug-gestions and criticism of the manuscript. P.J.R.K.’sfieldwork in Guyana was made possible with thefinancial support of the Belgian Directorate-Generalof Development Cooperation and the help and supportof the Prime Minister of Guyana, the HonorableSamuel Hinds. P.J.R.K. thanks G. Seegobin, P. Ben-jamin, H. Sambhu, F. Marco, R. Williams, and I.Roopsind for field assistance and M. Kalamandeen, K.Holder, and C. Bernard (University of Guyana) forhelp in obtaining export permits. P.J.R.K.’s researchpermits in Guyana (180604BR011, 030605BR006)and export permits (031204SP017, 191205SP011 and040406SP014) were issued by the Guyana Environ-mental Protection Agency. We are grateful to M.Antunes, M. Concistré, M. Sena, and S. Baroni forsupport during laboratory procedures and D. Pavan,G. Skuk, J. Cassimiro, J. M. Ghellere, M. A. Sena, M.Teixeira Jr, R. Recoder, S. M. Souza, V. Verdade, V.Xavier, and E. M. Freire for help in collecting speci-mens. J. Cassimiro kindly revised an early version ofthe manuscript. A.F., F.F.C., M.T.R. and P.M.S.N.were supported by Fundação de Amparo à Pesquisado Estado de São Paulo (FAPESP) and ConselhoNacional de Desenvolvimento Científico e Tecnológico(CNPq) (FAPESP: A.F. – grant 2009/51931–9, M.T.R.– grant 2003/10335–8, P.M.S.N. – grant 2007/00811–8; CNPq: F.F.C. – grant 2009.1.812.41.2).

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APPENDIX 1

Locality information and specimens of Iphisa elegans and outgroups sampled in this study. Locality numberscorrespond to Fig. 3 and clade numbers correspond to Figs 2 and 3B. Type of data is represented by: G (genetic)and M# (morphological and the respective hemipenial morphotype). Voucher numbers are represented by fieldand/or institutional identification numbers. Accession numbers of the previously published sequences refer tospecific individuals and are denoted by an asterisk, and the accession numbers of specimens analysed in thisstudy refer to the alleles. Brazilian state abbreviations are as follows: AM, Amazonas; AP, Amapá; BA, Bahia;CE, Ceará; GO, Goiás; MT, Mato Grosso; PA. Pará; RO, Rondônia; SP, São Paulo.

Species Locality/CoordinatesTypeof data

Voucher

Clade Cytb CmosField no. Institutional no.

Iphisa e. elegans (1) Apiacás, MT, Brazil(09°34′10″S, 57°21′04″W)

G/M1 968293 MZUSP 81634 1A JX079892 a/a JX079870

Iphisa e. elegans (2) Aripuanã, MT, Brazil(10°19′00″S, 59°27′34″W)

M1 – MZUSP 82662 –M1 – MZUSP 82666 –G/M1 977413 MZUSP 82655 1A JX079894 a/a JX079872G 977426 MZUSP 82656 1A JX079895 a/a JX079873G/M1 977669 MZUSP 82669 1A JX079896 a/a JX079874

(3) Cachoeira das Pombas,AM, Brazil (06°24′00″S,60°21′00″W)

G MTR 10210 1Ba JX079902 JX079880

(4) Cachoeirinha, RioMadeira, AM, Brazil(05°29′16″S, 68°48′23″W)

G RCV 2225 – 1A JX079907 a/a JX079885

(5) Campo Catuquira, AM,Brazil (04°54′16″S,61°06′46″W)

M2 SMS 197 INPA 20313 –M2 SMS 208 INPA 20326 –M2 SMS 209 INPA 20298 –

Iphisa e. elegans (6) Campo Tupana, AM,Brazil (04°09′37″S,60°07′51″W)

M2 SMS 092 INPA 20302 –

Iphisa eleganssoinii

(7) Centro deInvestigaciones del IIAP,Distrito Genaro Herrera,Provincia Requena, Peru(04°54′17″S, 73°39′11″W)

M5 – MHNSM 16718 –

Iphisa e. elegans (8) Comunidade Projó, AltoRio Aripuanã, AM, Brazil(07°37′01″S, 60°40′54″W)

M2 SMS 028 INPA 18445 –

Iphisa e. elegans M2 SMS 022 INPA 18432 –



APPENDIX 1 Continued


Voucher


Iphisa e. elegans (9)Montenegro-Cacaulândiaborder, RO, Brazil(10°19′53″S, 63°05′24″W)

G Uniban1704

– 2B JX079910 a/a JX079888


(10) EEBB Pithecia, RioSamiria, Dist. Parinari,Loreto, Peru (05°13′51″S,74°37′40″W)

G MHNC10115

– 2B JX079901 b/dJX079879

Iphisa e. elegans (11) Essequibo Co.,Kartabo, KuyuwiniLanding, Guyana(06°21′00″ N 58°41′00″W)

M4 – AMNH 21294 –


(12) Hamburgo, RioSamiria, Dist. Parinari,Loreto, Peru (05°14′26″S,75°07′09″W)

G MHNC10082

– 2B JX079900 e/f JX079878

Iphisa e. elegans (13) Igarapé Camaipi, AP,Brazil (00°01′00″S,51°42′00″W)

G/M4 LG 1786 MZUSP 88464 2A JX079898 b/b JX079876

Iphisa e. elegans (14) Igarapé-Açu, RioAbacaxis, AM, Brazil(04°20′39″S, 58°38′06″W)

M2 MTR 12758 MZUSP 100251 –G/M3 MTR 12772 MZUSP 100256 1Bb JX079911 j/k JX079889M3 MTR 12867 MZUSP 100257 –G/M3 MTR 12892 MZUSP 100258 1Bb JX079913 i/j JX079891M3 MTR 12896 MZUSP 100259 –M2 MTR 12913 MZUSP 100260 –

Iphisa e. elegans (15) Interfluve of RiosMadeira and Purus, AM,Brazil (04°59′30″S,61°07′09″W)

M2 SMS 201 INPA 20331 –

Iphisa e. elegans (16) Itapinima, AM, Brazil(05°24′37″S, 60°43′16″W)

G RCV 2247 – 1A JX079908 a/a JX079886

Iphisa e. elegans (17) Juruena, MT, Brazil(10°19′05″S, 58°21′32″W)

G/M2 976915 MZUSP 82428 1Ba JX079893 JX079871

Iphisa e. elegans (18) Kaieteur NationalPark, Guyana(05°11′00″S, 59°28′00″W)

M4 – IRSNB 17069 –Iphisa e. elegans G/M4 PK 1412 – 2A JX079905 b/b JX079883Iphisa e. elegans G/M4 PK 1413 – 2A JX079906 b/b JX079884Iphisa e. elegans M4 PK 1564 – –Iphisa e. elegans (19) Left margin Rio

Machado, RO, Brazil(08°10′37″S, 62°48′56″W)

M2 SMS 008 INPA 18458 –

Iphisa e. elegans (20) Manaus, AM, Brazil(03°06′24″S, 60°01′32″W)

M3 – MZUSP 8354 –

Iphisa e. elegans (21) mouth Rio Cenepa,Peru (04°35′00″S,72°12′00″W)

M5 – AMNH 56223 –


(22) Pagoreni, DistritoEcharate, Provincia LaConvencion, Cuzco, Peru(11°47′09″S, 72°42′05″W)

M5 – MHNSM 29541 –

Iphisa e. elegans (23) Paracou, FrenchGuiana (05°16′31″N,52°55′25″W)

M4 – AMNH 139958 –

Iphisa e. elegans (24) Puerto Libre, RioAguarico, Ecuador(00°04′50″N, 76°47′30″W)

M5 – KU 122173 –



APPENDIX 1 Continued


Voucher


Iphisa e. elegans (25) Rampon, nearChiguaza, Ecuador(02°01′00″S, 77°58′00″W)

M5 – USNM 196107 –

Iphisa e. elegans (26) Rio Ampiyacu,Estirón, Peru(03°19′03″S, 71°51′00″W)

M1 – MZUSP 13964 –

Iphisa e. elegans (27) Rio Corrientes ,Loreto, Peru (03°03′54″S,75°49′35″W)

M5 – CORBIDI 2684 –

Iphisa e. elegans (28) San Martin, Cuzco,Peru (11°47′08″S,72°41′57″W)

M5 – USNM 538401 –

Iphisa e. elegans (29) São Sebastião, AM,Brazil (04°18′32″S,58°38′11″W)

G/M2 MTR 12750 MZUSP 100249 1Ba JX079903 g/g JX079881G MTR 12785 MZUSP 100252 1Bb JX079912 i/l JX079890G/M2 MTR 12821 MZUSP 100253 1Ba JX079904 g/h

JX079882Iphisa e. elegans (30) UHE Guaporé, MT,

Brazil (15°07′00″S,58°58′00″W)

G RGL 1781 – 1A JX079909 a/a JX079887

Iphisa e. elegans (31) UHE Jirau, RO,Brazil (09°37′38″S, 65°26′46″W)

G H503 MZUSP 100247 2A JX079897 b/b JX079875

Iphisa e. elegans (32) Vai-Quem-Quer, PA,Brazil (01°30′00″S,55°50′00″W)

G LG 744 – 2A JX079899 b/c JX079877

Acratosauramentalis

Morro do Chapéu, BA,Brazil (11°33′00″S,41°09′22″W)

G MTR906448

– OG JX079915

Alexandresauruscamacan

Ilhéus, BA, Brazil(14°47′20″S, 39°02′58″W)

G MD304 – OG JX079917

Caparaoniaitaiquara

Parque Nacional Caparaó,MG, Brazil (20°28′00″S,41°49′00″W)

G MTR 10852 – OG JX079916

Colobodactylusdalcyanus

Campos do Jordão, SP,Brazil (22°44′22″S,45°35′29″W)

G LG761 – OG JX079918

Colobodactylustaunayi

PE Ilha do Cardoso, SP,Brazil (25°07′50″S,48°58′12″W)

G 3444 – OG JX079919

Colobosauramodesta

Serra da Mesa, GO, Brazil(13°50′02″S, 48°18′10″W)

G LG1145 – OG JX079920

Heterodactylusimbricatus

Serra da Cantareira, SP,Brazil (23°26′00″S,46°38′47″W)

G 1504 – OG JX079921

Stenolepis ridleyi Ibiapaba, CE, Brazil(05°02′58″S, 40°55′20″W)

G LG2123 – OG JX079914



APPENDIX 2

Details of the primers used with names, sequence and original reference.

Sequence Reference

CytbH15149 TGCAGCCCCTCAGAATGATATTTGTCCTCA Kocher et al. (1989)CYB-02H AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA Kocher et al. (1989)CYB-05L GCCAACGGCGCATCCTTCTTCTT Meyer (1993)LGL765 GAAAAACCAYCGTTGTWATTCAACT Bickham et al., 1995

CmosLSCH1 CTCTGGKGGCTTTGGKKCTGTSTACAAGG Godinho et al. (2006)LSCH2 GGTGATGGCAAARGAGTAGATGTCTGC Godinho et al. (2006)G73 GCGGTAAAGCAGGTGAAGAAA Saint et al. (1998)G74 TGAGCATCCAAAGTCTCCAATC Saint et al. (1998)

APPENDIX 3

Models used for each partition in the Bayesian analysis.

Partition Model Base frequencies Nst Rmat Rates Shape Pinvar

1 Cytb GTR + I + G 0.2912 0.2506 0.2084 6 3.5044 33.3230 3.57752.2249 74.1059

Gamma 0.4434 0.4986

2 Cytb HKY + I 0.1970 0.2623 0.1474 2 TRatio = 2.9587 Equal NA 0.81133 Cytb GTR + I + G 0.4140 0.3034 0.0539 6 0.2352 9.3226 0.1672

0.3785 4.7455Gamma 2.1566 0.0071

Total HKY + I + G 0.3408 0.2957 0.0937 2 TRatio = 6.7942 Gamma 1.3596 0.5238



AP

PE

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10.

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227

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206

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198

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192

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208

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20.

192

0.20

50.

203

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20.

174

0.18

40.

142

0.17

80.

189

0.18

1



cryptic species in iphisa elegans gray, 1851 (squamata ... · cryptic species in iphisa elegans...

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