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Speciation history and widespread introgression in the Europeanshort-call tree frogs (Hyla arborea sensu lato, H. intermedia and  H. sarda)

Václav Gvož dík a,b,⇑, Daniele Canestrelli c, Mario García-París d, Jiř í Moravec b, Giuseppe Nascetti c,Ernesto Recuero e, José Teixeira f ,g, Petr Kotlík a

a Laboratory of Molecular Ecology, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburská 89, 277 21 Libě  chov, Czech Republic b Department of Zoology, National Museum, Cirkusová 1740, 193 00 Prague, Czech Republic c Department of Ecology and Biology, Tuscia University, Largo dell’Università s.n.c., I-01100 Viterbo, Italyd Museo Nacional de Ciencias Naturales, C.S.I.C., c/Jose Gutierrez Abascal, 2, 28006 Madrid, Spaine Departamento de Ecología de la Biodiversidad, Instituto de Ecología, Universidad Nacional Autónoma de México, Ap. Postal 70-275, Ciudad Universitaria, México DF 04510, Mexico

f CIBIO/InBio – Research Centre in Biodiversity and Genetic Resources, Campus Agrario de Vairao, 4485-661 Vairao, Portugalg CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, n.289, 4050-123 Porto, Portugal

a r t i c l e i n f o

 Article history:

Received 23 July 2014Revised 10 November 2014Accepted 22 November 2014Available online 4 December 2014

Keywords:

Cryptic species complexBayesian species delimitationGene flow

Species treePhylogeographySystematics

a b s t r a c t

European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form sixgenetically differentiated but morphologically cryptic taxa,   H. arborea sensu stricto,  H. orientalis  and H.molleri from across Europe to western Asia (together referred to as  H. arborea sensu lato), two putativetaxa within H. intermedia (Northern and Southern) fromthe Italian Peninsula and Sicily, and H. sarda fromSardinia and Corsica. Here, we assess species limits and phylogenetic relationships within these ‘short-call tree frogs’ based on mitochondrial DNA and nuclear protein-coding markers. The mitochondrialand nuclear genes show partly incongruent phylogeographic patterns, which point to a complex historyof gene flow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogsand to infer the species tree, we used coalescent-based approaches. The monophyly of  H. arborea  sensu

lato is supported by the mtDNA as well as by the all-gene species tree. The Northern and Southern lin-eages of  H. intermedia have been connected by nuclear gene flow (despite their deep mtDNA divergence)and should be treated as conspecific. On the contrary, the parapatric taxa within  H. arborea  sensu latoshould be considered distinct species (H. arborea, H. orientalis, H. molleri) based on thecoalescent analysis,although signs of hybridization were detected between them (H. arborea H. orientalis; H. arborea H.molleri). A mitochondrial capture upon secondary contact appears to explain the close mtDNA relation-ship between the geographically remote Iberian  H. molleri  and H. orientalis  from around the Black Sea.Introgressive hybridization occurred also between the Balkan H. arborea and northern Italian H. interme-dia, and between the Minor Asiatic H. orientalis and Arabian H. felixarabica (the latter belonging to a dif-ferent acoustic group/clade). Our results shed light on the species limits in the European short-call treefrogs and show that introgression played an important role in the evolutionary history of the short-calltree frogs and occurred even between taxa supported as distinct species.

 2014 Elsevier Inc. All rights reserved.

1. Introduction

Western Palearctic tree frogs were considered to represent asingle species  Hyla arborea  until the end of the 1960s. Later on,two taxa, H. meridionalis and H. savignyi from the western and east-

ern Mediterranean, respectively, were elevated from subspecieslevel to full species based on differences in advertisement calls(Paillette, 1967; Schneider, 1968, 1974; Schneider and Nevo,1972). Advertisement calls are an important behavioral feature inanurans with a critical role in maintaining reproductive barriersbetween species (Schneider, 1977; Schneider and Sinsch, 2007).In the Western Palearctic tree frogs, the advertisement calls havebeen thoroughly studied (reviews in Schneider, 1974, 2004a). Thecall segments are formed by pulse-groups (notes) and interveningquiet intervals. Advertisement calls of the Western Palearctic treefrogs might be divided into three categories according to the

http://dx.doi.org/10.1016/j.ympev.2014.11.012

1055-7903/ 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Present address: Institute of Vertebrate Biology, Acad-emy of Sciences of the Czech Republic, Kvě tná 8, 603 65 Brno, Czech Republic.

E-mail addresses:   vaclav.gvozdik@ivb.cz (V. Gvož dík), canestrelli@unitus.it (D.Canestrelli),  mparis@mncn.csic.es   (M. García-París),   jiri_moravec@nm.cz  (J. Mor-avec),  nascetti@unitus.it (G. Nascetti),   ernestorecuerogil@gmail.com (E. Recuero), jteixeira@cibio.up.pt (J. Teixeira), kotlik@iapg.cas.cz (P. Kotlík).

Molecular Phylogenetics and Evolution 83 (2015) 143–155

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Molecular Phylogenetics and Evolution

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http://dx.doi.org/10.1016/j.ympev.2014.11.012mailto:vaclav.gvozdik@ivb.czmailto:canestrelli@unitus.itmailto:mparis@mncn.csic.esmailto:jiri_moravec@nm.czmailto:nascetti@unitus.itmailto:ernestorecuerogil@gmail.commailto:jteixeira@cibio.up.ptmailto:kotlik@iapg.cas.czhttp://dx.doi.org/10.1016/j.ympev.2014.11.012http://www.sciencedirect.com/science/journal/10557903http://www.elsevier.com/locate/ympevhttp://www.elsevier.com/locate/ympevhttp://www.sciencedirect.com/science/journal/10557903http://dx.doi.org/10.1016/j.ympev.2014.11.012mailto:kotlik@iapg.cas.czmailto:jteixeira@cibio.up.ptmailto:ernestorecuerogil@gmail.commailto:nascetti@unitus.itmailto:jiri_moravec@nm.czmailto:mparis@mncn.csic.esmailto:canestrelli@unitus.itmailto:vaclav.gvozdik@ivb.czhttp://dx.doi.org/10.1016/j.ympev.2014.11.012http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.ympev.2014.11.012&domain=pdfhttp://-/?-

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pulse-group properties (Fig. 1; grouping made according to theresults of   Grach et al., 2007; Gvož dík et al., 2010; Schneider,1974, 2004a). The ‘long-call tree frogs’ are characterized by longpulse-groups composed of a high number of pulses (34–56 pulses;210–610 ms), ‘short-call tree frogs’ by short pulse-groups with alow number of pulses (6–12 pulses; 50–110 ms), and ‘medium-calltree frogs’ by intermediate values (13–25 pulses; 85–190 ms).

The Tyrrhenian tree frog, H. sarda

 from the islands of Sardinia,Corsica, Elba and surrounding islets, was distinguished from   H.arborea on the basis of the differentiation at allozyme loci (Lanza,1983; Nascetti et al., 1985), but the advertisement call of this spe-cies was not found to be substantially differentiated from  H. arbo-rea  (Schneider, 1974, 2004a). This is also true for the Italian treefrog,  H. intermedia (Schneider, 2004a,2004b), which also was dis-tinguished from   H. arborea  on the basis of allozyme differences(Nascetti et al., 1995). With the expansion of DNA sequencing,new hypotheses on the taxonomy of the Western Palearctic treefrogs emerged (Gvož dík et al., 2010; Stöck et al., 2008, 2012).Besides the description of the new species   H. felixarabica  fromthe Arabian Peninsula (Gvož dík et al., 2010), deeply divergent lin-eages were uncovered within H. meridionalis (south-eastern part of the range) and in   H. intermedia   (northern part of the range)(Canestrelli et al., 2007a,b; Gvož dík, 2009; Gvož dík et al., 2007;Recuero et al., 2007; Stöck et al., 2008, 2012), which may each rep-resent a separate (as yet unnamed) taxon (Stöck et al., 2008). Fur-thermore,   H. arborea  was suggested to represent three speciesaccording to the mitochondrial and single nuclear-gene phyloge-nies:  H. orientalis in the east,  H. molleri in the west, and  H. arboreasensu stricto (s.s.) in the central part of the former H. arborea sensulato (s.l.) range (Stöck et al., 2008).

For the Iberian  H.  (arborea)  molleri, this new taxonomy corre-sponds to the former morphology-based intraspecific taxonomyof  H. arborea. In contrast, the genetic break between the central(H. arborea  s.s.) and eastern (H. orientalis) populations does notmatch the limits and distributions of the morphology-based sub-species,  H. arborea kretensis (Crete, southern Balkans, Aegean, wes-

tern Asia Minor; e.g.   Stumpel, 1997),   H. a. schelkownikowi(Caucasus; e.g.  Kuzmin, 1999),   H. a. gumilevskii   (Talysh Mts. inTranscaucasia; Litvinchuk et al., 2006) and H. a. arborea in the restof the range (except the Iberian Peninsula; e.g. Stumpel, 1997). [Forhistorical overview of the taxonomy of the Western Palearctic treefrogs see Fig. 1.] This incongruence between molecular phylogeniesand morphology-based taxonomy, which discouraged wideracceptance of the revised taxonomy (e.g. Grosse, 2009; Özdemiret al., 2012; Sillero et al., 2014; Speybroeck et al., 2010), is further

fostered by the unexpected and surprisingly close sister-clade rela-tionship between the eastern (near the Black Sea; H. orientalis) andwestern (on Iberian Peninsula;  H. molleri) populations, currentlygeographically separated by the range of  H. arborea s.s. (Gvož dík,2009; Gvož dík et al., 2007; Stöck et al., 2008, 2012). As yet, nostudy has provided explanation for this biogeographic pattern.

The clade containing H. arborea s.l.,  H. intermedia and  H. sarda

(all considered H. arborea

  till the 1990s; e.g.  Stumpel, 1997) hasconsistently received high support from molecular phylogeneticanalyses (Gvož dík, 2009; Stöck et al., 2008, 2012), while relation-ships within the clade have remained ambiguous (Stöck et al.,2008, 2012). The exceptions are the sister-clade relationships of H. orientalis and  H. molleri  and of the Northern (N) and Southern(S) lineages of   H. intermedia   (Gvož dík, 2009; Stöck et al., 2008,2012). However, using different mitochondrial markers (12S and16S rRNA) than in Stöck et al. (2008, 2012), our preliminary datashowed high support also to H. arborea s.l., i.e. H. orientalis, H. mol-leri and H. arborea s.s. forming a clade (Gvož dík, 2009). In additionto the unresolved internal topology of the clade, species limits inthe short-call tree frogs are an intriguing issue, especially giventhe following evidence: (i) similar advertisement calls of  H. arbo-rea,  H. orientalis,  H. molleri,   H. intermedia and H. sarda  (Fig. 1; e.g.Castellano et al., 2002; Schneider, 1974, 2000, 2004a, 2004b); (ii)morphological similarity of   H. arborea,   H. orientalis   (Gvož díket al., 2008; bothas H. arborea), H. molleri (Terentjev, 1960) and lar-gely also  H. intermedia   (Nascetti et al., 1995; Rosso et al., 2004;Terentjev, 1960); (iii) incongruence in phylogeographic patternsbetween different nuclear genes across the Balkans and northernItaly (H. orientalis/H. arborea/H. intermedia   N), especially alongthe western Balkan coast (Gvož dík et al., 2007; distinct Balkan/Adriatic lineage was detected also by   Stöck et al., 2008   andDufresnes et al., 2013). The Western Palearctic short-call tree frogsthus seem to represent an assemblage of morphologically andacoustically cryptic taxa, and the extent of gene flow betweenthem needs to be properly evaluated.

Current advances in molecular phylogenetics have introduced

new methods, including multilocus coalescent-based species treeinference (Knowles and Kubatko, 2010) and coalescent-based spe-cies delimitation, which permit probabilistic tests with an assump-tion of no recent admixture between species and of bipartitions of individuals in gene trees that are shared across loci (Yang andRannala, 2010). In this study, we focus on the short-call tree frogs(H. arborea, H. orientalis, H. molleri, two lineages of  H. intermedia, H.sarda) using new molecular markers and a multilocus coalescent-based species-tree approach and a Bayesian species delimitation

Fig. 1.  Historical overview of the taxonomy of the Western Palearctic tree frogs and assignment of the taxa into groups according to the advertisement-call properties. Redbox highlights ingroup species considered in this study. Example oscillograms of a 600 ms long portion of the advertisement call on right. Numbers in circles stand for the

number of species recognized in the corresponding time period. Color dotscorrespond to the colors in Figs. 2and 3. For the status of  H. heinzsteinitzi see discussion in Werner(2010) and Stöck et al. (2010). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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method to test (1) the monophyly of  H. arborea s.l. (H. arborea,  H.orientalis, H. molleri); (2) sister-clade relationship of the geograph-ically disjunctive  H. orientalis and H. molleri; and (3) species-leveldistinction of the taxa, including of the northern (N) and southern(S) lineages of H. intermedia, and of the western (W) Balkan H. arbo-rea/H. orientalis populations.

2. Materials and methods

 2.1. Taxon sampling and laboratory procedures

We obtained sequence data for altogether 169 specimens from95 localities distributed throughout the geographic range of   H.arborea   s.l., including one specimen from GenBank (DQ055835,loc. 36; Smith et al., 2005), 19 specimens from 8 localities of   H.intermedia  and 10 specimens from 6 localities of  H. sarda, whichtotaled 198 ingroup samples from 109 localities (Fig. 2, Table S1).Due to the lack of reliable diagnostic morphological characters,the material of  H. arborea  s.l. could not be  a priori  sorted into theputative species, i.e.   H. arborea   s.s.,   H. orientalis   and   H. molleri(sensu Stöcket al., 2008). The H. orientalis material from Asia Minor

and Caucasus-Caspian region used in a previous study (Gvož díket al., 2010) was reanalyzed in the context of the new data fromthe European part of the range. The outgroup species H. meridional-is data were taken from the previous study (Gvož dík et al., 2010)and those for a second outgroup,  H. japonica, were retrieved fromGenBank (AY843633, AY844615, AY844078;   Faivovich et al.,2005). The outgroup status of these two species relative to theshort-call tree frogs was demonstrated by, e.g. Hua et al. (2009).

Genomic DNA was extracted from tissue samples using com-mercial kits following the manufacturers’ protocols. Two frag-ments of mitochondrial DNA (mtDNA), 12S rRNA (12S , 355aligned bp) and 16S rRNA (16S , 546 aligned bp), and two nucleargenes (nDNA), tyrosinase precursor exon 1 (Tyr , 496 bp), and rho-dopsin exon 1 (Rhod, 276 bp) were targeted. The nDNA markers

were sequenced for a subset of samples (ca. 43%) representing allmain mitochondrial groups and covering the entire geographicrange (Table S1). Primers and PCR protocols followed the previousstudy (Gvož dík et al., 2010). PCR amplicons were purified anddirectly sequenced with the PCR primers using a commercialsequencing service (Macrogen Inc.).

 2.2. DNA alignment and phasing of autosomal genotypes

Alignments of nucleotide sequences were prepared with Clu-stalW (Thompson et al., 1994) as implemented in BioEdit 7.2(Hall, 1999). Mitochondrial  12S  and  16S  fragments were concate-nated. Heterozygous sites in nDNA were identified according toelectropherograms when two different nucleotides were present

at the same position with the lower peak reaching at least a half of the higher peak. Gametic haplotypes were inferred by the coa-lescent-based Bayesian algorithm of Phase 2.1 (Stephens et al.,2001; Stephens and Scheet, 2005) as implemented in DnaSP 5.10(Librado and Rozas, 2009). The analyses were run without out-group species and were repeated five times for each nuclear-genedataset with different seeds for the random number generator tocheck if the phase estimates are consistent across the runs accord-ing to goodness-of-fit values. Each run was conducted under theparent-independent mutation model with a burn-in-period of 100 iterations, which was followed by 1000 iterations. Results witha probability > 0.70 were accepted. Two samples (H. intermediaHD03A, Tyr ;  H. arborea HJ55,  Rhod) contained heterozygous posi-tions whose phases were not resolved (mean probability 0.50),

and one sample (H. intermedia   HD04B,   Tyr ) had one positionweakly resolved (mean probability 0.69). In these cases, both

possible gametic phases and their phylogenetic positions wereconsidered in haplotype networks, which were constructed usingthe parsimony approach under the 95% parsimony limit as imple-mented in TCS 1.21 (Clement et al., 2000). The alternative gameticphases did not change the networks substantially (see haplotypesR arb3, Tint 7, Tint 8, Tint 9, Tint 10 in Fig. 2). No stop codons weredetected in the haplotypes as checked by translation with the stan-

dard genetic code using BioEdit 7.2 (Hall, 1999). All new uniquemitochondrial haplotypes and new nuclear alleles or nucleotidesequences with the standard IUPAC ambiguity codes, if heterozy-gous positions remained unresolved, have been deposited in Gen-Bank (KP109551–KP109674;   Table S2). Genetic distances werecalculated from   16S   haplotypes (a marker commonly used inamphibian DNA barcoding; Vences et al., 2012) and estimated as p-distances averaged between and within taxa in MEGA 6.0(Tamura et al., 2013). Incomplete mtDNA sequences covering onlythe 12S  fragment (from localities 36 and 50) were used in the mito-chondrial gene tree only.

 2.3. Gene trees

For gene-tree estimation, mtDNA and phased   Tyr   and   Rhodsequences were sorted into distinct haplotypes using Collapse 1.2(Posada, 2006). Gene trees were reconstructed by Bayesian infer-ence (BI) and maximum likelihood criterion (ML). For BI analysesperformed by MrBayes v3.2.2 (Ronquist et al., 2012), best-fit parti-tioning schemes and nucleotide substitution models according tothe Bayesian information criterion (BIC) were selected using Parti-tionFinder v1.1.0 (Lanfear et al., 2012): mtDNA, 12S and 16S treatedas onepartition (SYM+G); Tyr , 1st + 2nd/3rd codon position (K80+I/HKY+G); Rhod, 1st + 2nd/3rd codon position (JC/K80). Each datasetwas analyzed with two runs of four Markov chains, which were runfor 6 106 generations and samples saved every 100th generation.That the analyses reached stationarity was verified by examiningthe plots of log-likelihood scores using Tracer v1.5 [Rambaut andDrummond, 2009; all parameters had effective sample size

(ESS) 200], and the chain convergence was confirmed by theconvergence diagnostics (average standard deviation of split fre-quencies, potential scale reduction factor) comparing the twosimultaneous runs against each other. From the sampled trees,the first third was discarded as a burn-in and a 50% majority-ruleconsensus tree was produced from the remaining trees that weretaken as representing the posterior distribution. For ML analysesperformed by PhyML v3.0 (Guindon et al., 2010), best-fit modelsof sequence evolution were selected using jModelTest v2.1.4(Darriba et al., 2012) according to the BIC: mtDNA, TIM2ef+I;  Tyr ,TrNef+I;  Rhod, JC. The best option of a combination of the nearestneighbour interchange and the subtree pruning and regraftingalgorithm of tree improvement and optimization of the topologyand branch lengths were applied. The nodal support was assessed

by 100 bootstrap pseudoreplicates. Clades supported with BI pos-terior probability (pp) valuesP 0.95 and ML bootstrap val-uesP 70 were considered highly supported (Huelsenbeck andRannala, 2004).

 2.4. Data preparation for coalescent-based applications

Since the coalescent-based species-tree and species-delimita-tion analyses rely on, and are sensitive to, the assignment of eachindividual/allele to a taxonomic unit (Leaché et al., 2014; Olaveet al., 2014), we carefully inspected distribution of haplotypesacross different genes to identify specimens with inconsistent phy-logenetic signal and thus identify potential hybrids (TableS1). Suchpotential hybrids or possible cases of allelic introgression (H. arbo-

rea–orientalis; 10 individuals from the Balkans and Poland) wereomitted from the species-tree analyses. Similarly, contrasting  Tyr 

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alleles, such as in a sample from Brittany (loc. 4) or  H. felixarabica-like alleles of six individuals of  H. orientalis   from south-westernTurkey (haplotypes T fel3, Tori5, Tori6, Tori7, Tori9; see Fig. 2 andGvož dík et al., 2010), were also omitted from the analyses (see Sec-tion 3 for details). Remaining samples were allocated to the follow-ing taxonomic units according to their provenance and geneticidentity:   H. arborea   s.s. (n = 64),   H. orientalis   (n = 74),   H. molleri(n = 14), H. intermedia  South (n = 16), H. intermedia  North (n = 3),and  H. sarda   (n = 10). Beside these ‘standard’ units (Stöck et al.,

2012) we also tested a position of the western Balkan tree frogs(W Balkan H. arborea, loc. 33–35, 37, 38 in Slovenia, Croatia, Mon-tenegro, Greece; n  = 5), which carry inconsistent phylogenetic sig-nal across the studied genes (Fig. 2), as noted earlier by Stöck et al.(2008). Coalescent-based model assumptions include an absence of recombination (Castillo-Ramírez et al., 2010; Lavretsky et al., 2014;but see Lanier and Knowles, 2012). Thus, the minimum numbers of recombination events (Hudson and Kaplan, 1985) in nuclear locifor different subsets of tree-frog species were estimated in DnaSP

Fig. 2.  Geographic pattern of variation across three different molecular markers (mtDNA:  12S /16S ; and two nuclear genes:  Tyr  and Rhod) for the short-call tree frogs. Color

shading corresponds to the approximate ranges of  H. arborea s.l. (yellow),  H. intermedia  (blue) and H. sarda  (purple) following IUCN (2013). Phylogenetic relationships arerepresented by maximum-parsimony haplotype networks for nuclear genes. For mitochondrial phylogenetic relationships see Figs. 3 and4. In haplotype networks the size of the circles corresponds to the haplotype frequency, and the smallest circles represent hypothetic, unobserved haplotypes. Dashed linesreflect two possibilities of a haplotypeconnection in cases when haplotypes were phased with low support. Samples classified as W Balkan   H. arborea   in the coalescent analyses are marked with ‘‘B’’, whilepotential hybrids (or introgressed alleles) omitted from the coalescent analyses are marked with ‘‘’’ on the maps. Light green in  Tyr  stands for  H. felixarabica-like allelesintrogressed to  H. orientalis (in south-western Asia Minor). For more details see Table S1. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

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5.10 (Librado and Rozas, 2009), with no recombination detected inRhod, and a minimum of one (H. intermedia,   H. sarda), seven (H.arborea   s.l.) and ten (all ingroup taxa) recombination eventsdetected in Tyr . Traces of recombination were removed from theTyr  dataset (not containing   H. felixarabica-like alleles) using IMgc(Woerner et al., 2007), and a recombination-filtered dataset wasproduced by the approach of favoring the retention of individuals

(a

 = 1.0; 423 bp and 86% samples kept; hereafter as Tyr 

IMgc1 data-set; see also Table S1 and Fig. S1).

 2.5. Species trees and divergence dating 

A species tree was inferred using   ⁄BEAST (Heled andDrummond, 2010) based on the dataset including all genes, mito-chondrial 12S  and 16S , the nuclear Rhod and the recombination-fil-tered   Tyr   (Tyr IMgc1). Alignments of all individuals (withoutpotential ‘hybrids’; see above) including one outgroup (H. meridio-nalis) for each gene were uploaded into BEAUti v1.8.0, a part of theBEAST package (Drummond et al., 2012), where they wereassigned separate and unlinked substitution, clock and tree mod-els. Hyla meridionalis was set as outgroup by enforcing monophylyof the remaining taxa. Best-fit partitioning schemes and sequence-evolution models were set according to the results from Partition-Finder v1.1.0 (Lanfear et al., 2012) following the BIC [mtDNA,  12S and 16S  treated as one partition (HKY+G); Tyr IMgc1 without parti-tions (K80);   Rhod, 1st + 2nd/3rd codon position (K80/TrNef)].Ploidy of mtDNA was set to mitochondrial (haploid), and the othertwo loci to autosomal nuclear (diploid). The Yule species-tree priorwas set and UPGMA starting tree used for each gene and a strictmolecular clock assumed. The clock rate for mtDNA was inferredfrom a calibration based on an assumption of   H. sarda  being adescendant of a radiation that had occurred during the Late Mio-cene (Stöck et al., 2012), particularly when the land bridgebetween the Sardo–Corsican block and continent was submergedat the end of the Messinian salinity crisis when the MediterraneanBasin was re-flooded at ca. 5.33 mya (Bisconti et al., 2011a; Hsü

et al., 1973; Krijgsman et al., 1999; Rouchy and Caruso, 2006). Nor-mal distribution prior was assigned with a mean of 5.33 (mya) andstandard deviation of 0.16 in order to accommodate uncertainty inthis estimate. Clock rates for  Tyr  and  Rhod  were estimated frommtDNA using 1/x prior distribution. Five independent   ⁄BEAST runswere performed, each for 200 million generations, sampling every20,000th generation to obtain a posterior sample of 10,000 trees.The likelihoods were inspected using Tracer v1.5 (Rambaut andDrummond, 2009) and convergence checked using ESS values(200) after discarding the 10% burn-in. The post burn-in samplesof the five runs were combined in the BEAST module LogCombinerv1.8.0. The output of 45,000 sampled trees was uploaded toanother BEAST module, TreeAnnotator v1.8.0, to infer the final spe-cies tree as a maximum clade credibility tree (MCCT) with node

ages represented by median heights and confidence intervals with95% highest posterior densities (HPD). The set of all 45 thousandtrees was superimposed and visualized as a ‘cloudogram’ withDensiTree v2.01 (Bouckaert, 2010) to show the probability distri-bution of topologies, and the three most frequent ‘consensus trees’(sensu Bouckaert, 2010) were inspected. To assess the influence of the mtDNA dataset on the species-tree topology, the nuclear data-set was also employed in a separate species-tree analysis with vir-tually the same settings but without molecular-clock calibrationand run for 500 million generations.

 2.6. Species delimitation

Coalescent-based Bayesian species delimitation (BSD) analyses

were conducted in the program Bayesian Phylogeny and Phyloge-ography (BP&P v2.2; Rannala and Yang, 2003; Yang and Rannala,

2010) to test whether the operational taxonomic units fit theassumption of no post-divergence gene flow, which is inherent tothe biological species concept (Yang and Rannala, 2010). Thismethod accommodates the species phylogeny and accounts forlineage sorting due to the ancestral polymorphism using thereversible-jump Markov chain Monte Carlo (rjMCMC) algorithmthat successively splits or joins nodes on the guide species tree,

and employs population-size parameters h

 and species divergencetimes s in the coalescent model (Rannalaand Yang, 2013; Yang andRannala, 2010). Numerous possible species-tree topologies (guidetrees) were tested using different datasets with individual allelescarefully assigned to putative species (see Olave et al., 2014): allmarkers (mtDNA and phased nDNA), nDNA only,  Rhod  only, andTyr  only;   Tyr  was used in both ways – including recombinationand recombination-filtered (Tyr IMgc1); all individuals (withoutH. felixarabica-like Tyr  alleles), with or without ‘hybrids’, and con-sidering W Balkan populations as a discrete unit or part of  H. arbo-rea   (Table S3). A gamma prior   G(2,1000) was applied on thepopulation-size parameters (hs). Also the age of the rootin the spe-cies tree (s0) was assigned the gamma prior  G(2,1000), while theother divergence-time parameters were assigned the Dirichletprior (Yang and Rannala, 2010). When both mtDNA and autosomalnuclear data were involved in an analysis, the parameters ‘hered-ity’ (allows   h  to vary among loci) and ‘locusrate’ (allows variablemutation rates among loci) were estimated with a gamma  G(4,4)and Dirichlet prior, respectively. The algorithm 0 was used withseveral values for the fine-tuning parameter  e  to ensure that therjMCMC mixed properly. BP&P analyses were run for 105 genera-tions with first 50% of samples treated as a burn-in and discarded.If necessary, fine-tuning variables were adjusted to display propor-tion values close to 0.3–0.4 (within the interval 0.15–0.7) as rec-ommended in the manual (Yang, 2013). Each analysis was runrepeatedly to check consistency between runs. To control for a pos-sible influence of the settings, the all-gene and nDNA MCCT spe-cies-tree topologies were used as guide trees also for runs withalternative prior settings of    hs and   s0   [G(1,10)/G(1,10);

G(2,2000)/G(2,2000);   G(1,10)/G(2,2000)], as recommended byLeaché and Fujita (2010), and runs employing the rjMCMC algo-rithm 1, with fine-tuning parameters  a = 2 and m = 1 (Yang, 2013).

3. Results

 3.1. DNA polymorphism

The mitochondrial DNA dataset contained 198 ingroupsequences, which collapsed into 77 haplotypes (891 bp excludinginsertion/deletion polymorphisms), contained the number of poly-morphic sites S  = 126 (89 parsimony informative), and showed thehaplotype diversity h = 0.922 ± 0.014 (SD) and nucleotide diversityp = 1.91 ± 0.09%. The two nuclear genes were considerablyless var-iable. Phased ingroup   Tyr  dataset (166 sequences, 496 bp) con-tained 47 haplotypes, with   S  = 38(34),   h = 0.900 ± 0.016, andp = 0.90 ± 0.04%, while the   H. felixarabica-like- and recombina-tion-filtered  Tyr IMgc1 (133 sequences, 423 bp) contained 22 hap-lotypes, with   S  = 26(23),   h = 0.820 ± 0.025, and   p = 0.88 ± 0.05%.Gametic-phased ingroup   Rhod   dataset (174 sequences, 276 bp)contained 15 haplotypes, with   S  = 14(7),   h = 0.779 ± 0.022, andp = 0.60 ± 0.03%. The mitochondrial marker has a roughly twotimes higher polymorphism than Tyr  and three times higher thanRhod.

 3.2. Gene trees

The mitochondrial DNA phylogeny had an almost identicaltopology based on the BI and ML approaches (Fig. 3). Most of the

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main clades are highly supported with   H. sarda   in a sister-clade

position to all other taxa (Bayesian pp 1.00/ML bootstrap 63).These are grouped into two clades corresponding to  H. intermedia

and H. arborea  s.l. The latter is highly supported (1.00/75), while

the support for a clade containing two highly divergent lineagesof H. intermedia (Northern/Southern) is weaker (0.94/56), reflecting

Fig. 3.  Haplotype gene trees of the mitochondrial DNA fragment (12S /16S ) and nuclear Tyr  and Rhod as represented by maximum-likelihood trees. Numbers along branchescorrespond to ML bootstrap support values and Bayesian posterior probabilities if higher than 50/0.50. Colors are in accordance to Fig. 2. Boxed haplotypes in H. arborea s.s.are present in individuals constituting the W Balkan operational unit as defined based on the incongruence in phylogenetic signal between the two studied nuclear markers(see also Fig. 2 and Table S1). Taxa of special interest due to their dual placements in nuclear phylogenies are highlighted. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

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the deep mitochondrial differentiation of the two geographicallyclose lineages. Despite the geographically remote distributions,the Iberian H. molleri  and Eastern European/Western Asian H. ori-entalis  together form a highly supported clade (1.00/88), whilethe support for each of the two taxa is rather low (H. molleri0.85/60;  H. orientalis  0.94/68).  Hyla arborea  s.s. is well differenti-ated (0.97/78) and is supported as the sister clade to H. molleri + H.orientalis

  (1.00/75). Mitochondrial DNA (16S 

) genetic distancesamong taxa are given in Table 1.The two nuclear-gene phylogenies (Fig. 3) have generally low

nodal support likely caused by the relatively low variation. How-ever, both nuclear markers,  Tyr  and Rhod, possess variation suffi-cient to show clear structure (Figs. 2 and 3). The   Tyr  phylogenydiffers from mtDNA in terms of the detection of major lineages/species in three main aspects: (i)  H. arborea  s.s. forms two mainclusters. One contains predominantly samples from the westernBalkans, while the second the remaining samples. (ii)  H. orientalisalso forms two main groups, which do not form a clade. Oneaccommodates most samples, while the other is restricted onlyto south-western Asia Minor. In the latter group the most commonhaplotype of  H. felixarabica  (T fel3) was recorded (plus four haplo-types closely related to it), as documented earlier (Gvož dík et al.,2010). (iii) H. intermedia, Northern lineage is not clearly detectableand most alleles are in the Southern lineage. In the Rhod haplotypephylogenetic relationships, the most striking difference from themtDNA phylogeny is the position of W Balkan H. arborea samples,which do not carry ‘typical’  H. arborea Rhod  haplotypes, but havethe most-common  H. orientalis  haplotype (R ori1) or a haplotypederived from it (R ori6) (Fig. 3). Similarly, the majority of samples(5/6) of the Northern lineage  H. intermedia carries the most com-mon H. orientalis haplotype (Fig. 3).

 3.3. Geographic distribution of haplotypes and hybrid location

Intraspecific phylogeographic relationships based on mitochon-

drial DNA are presented in Fig. 4. The south-western endemics,  H.sarda and H. molleri do not demonstrate clear phylogeographic pat-tern, with an exception of the presence of one relatively distanthaplotype of  H. molleri in Galicia. The Southern lineage of the Ital-ian endemic H. intermedia  forms two subgroups, southernmost andcentral. The most common H. arborea s.s. haplotype and its single-mutational (or indel) derivatives, arranged in a star-like pattern,have been recorded from over much of Europe, from Greece inthe south to Denmark in the north (Fig. 4). On the contrary, inthe southern Balkans a more diversified haplotype group is pres-ent, predominantly recorded from Greece but found also in north-ern Croatia and the Czech Republic. One relatively distanthaplotype was detected on Pag, a Croatian island in the northernAdriatic Sea. The highest diversity was found in the eastern taxon,H. orientalis. Two main groups (weakly supported clades) are rep-resented by the Caucasus-Caspian populations and by all the otherpopulations, respectively. Besides the Caucasus-Caspian group,

further structuring in the second group is also detectable. Mostdiversified is the Asia Minor population with haplotypes presentin several different subgroups, but with less clear geographic struc-turing, which is true also for the remaining Eastern European pop-ulations, which have clear affinities to the north-western AsiaMinor population.

For the nuclear Tyr  and Rhod markers, the distribution of haplo-

types generally agrees with the pattern in mtDNA, with the excep-tion of the cases described above (Section  3.2.). However, therehave been also several striking findings at particular locations.Based on the combination of alleles across different loci, potentialhybrids were detected in Brittany (H. arborea H. molleri), Poland,and across the Balkans in Greece, Bulgaria and Romania (H. arbo-rea H. orientalis) (Fig. 2 and Table S1).

 3.4. Species trees and divergence time estimate

The maximum clade credibility species tree based on the mito-chondrial and nuclear loci inferred the same topology as themtDNA gene tree:   H. sarda  being the sister lineage to all othershort-call tree frogs, and  H. intermedia  being the sister lineage to

H. arborea s.l., and with H. orientalis forming a clade with H. molleriand H. arborea s.s. closely related to the W Balkan H. arborea group(Fig. 5). However, high support is present only for the crown sister-clade relationships (H. intermedia N – H. intermedia S; H. orientalis –H. molleri; H. arborea – W Balkan). Three most frequent topologiesoccurred with the posterior frequency of 38.7%, 33.2%, 11.6% – firstwith the same topology as the MCCT, second differing by the pres-ence of a clade  H. intermedia   (N+S) + H. sarda   in the sister-cladeposition to  H. arborea s.l., and third interchanging positions of  H.intermedia (N+S) and  H. sarda in comparison to the MCCT. All thethree topologies, accounting for almost 84% posterior trees, hadthe H. arborea  s.l. taxa as a clade. On the contrary, if we consideronly the two nuclear loci the MCCT (Fig. S2) as well as the threemost frequent topologies (22.6%, 15.1%, 13.8%) form two mainclades: (i) H. arborea s.s. (+ W Balkan) + H. sarda; (ii) all other taxa,i.e. H. intermedia (N+S), H. molleri, H. orientalis. The molecular clockbased on all loci estimated that the main radiation within theshort-call tree frogs occurred in the Pliocene (considering 95%highest posterior densities, HPD, 5.4–1.9 mya), with only the mostrecent splits occurring during the Pleistocene. The split between H.orientalis and H. molleri is dated close to that between the Southernand Northern  H. intermedia, ca. 1.4 mya (HPD 2.8–0.4), while thestructuring within H. arborea with respect to the W Balkan popula-tion dates to the Middle to Early Pleistocene ca. 200 kya (HPD 370–70 kya).

 3.5. Species delimitation

Bayesian species delimitation supported all the taxa as specieswithout a history of gene flow (speciation probability 1.00;Fig. 5), assuming various guide tree topologies, datasets, and con-

 Table 1

Genetic distances (uncorrected  p; expressed as percentages) averaged among and within taxa based on the fragment of mitochondrial 16S rRNA gene.

H. arborea H. orientalis H. mol leri H. intermedia S   H. intermedia N   H . sard a H . meridiona lis H. jap on ica

H. arborea   0.4H. orientalis   3.3 0.5H. molleri   3.2 0.9 0.4H. intermedia S 3.8 3.8 3.4 0.3H. intermedia N 3.9 4.5 4.0 2.9 0.2H. sarda   4.9 4.5 4.1 4.0 4.0 0.3

OutgroupH. meridionalis   6.1 5.7 6.0 6.8 6.3 6.6 –H. japonica   7.3 7.8 8.0 6.8 6.1 6.2 5.8 –

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ditions with respect to inclusion of potential ‘hybrids’, recombina-

tion in Tyr , or considering W Balkan populations as a discrete unitor as a part of  H. arborea (see overview of results in Table S3). The

alternative prior settings and type of rjMCMC algorithm also did

not affect the results of high speciation probability for any node.Only when the  Tyr  dataset was evaluated independently was the

Fig. 4.  Phylogeographic pattern of the short-call tree frogs based on mtDNA (12S /16S ) variation. Phylogenetic relationships are shown using sections of the ML tree (Fig. 3)and maximum-parsimony haplotype networks where ellipsoid size mirrors the haplotype frequency and the small dots are theoretical, undetected haplotypes. Numbers inellipsoids correspond to the locality numbers as in the maps and Table S1. Different colors indicate different haplogroups with clear geographic affinities within individualspecies and are not fully concordant to the colors in Figs. 1–3 (only the most widespread haplogroups are encoded by the same colors like the species in  Figs. 1–3). (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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speciation probability always low (0.08–0.60) for the Northern andSouthern lineage of   H. intermedia, both for the dataset containingrecombination and the recombination-filtered dataset. There was

also low speciation probability for the node H. arborea – W Balkanin   Tyr   (0.32), but the probability increased substantially whenrecombination was filtered out (0.97). Interestingly, when the nodeW Balkan –  H. orientalis  was tested in  Rhod where there is a highlevel of haplotype sharing (R ori1; frequency 4/10 in W Balkan),the speciation probability was also high (0.99). This was likely aconsequence of the R ori6 haplotype (frequency 5/10 in W Balkan)being common in the northern Adriatic.

4. Discussion

4.1. Species tree

The short-call tree frogs are phenotypically very uniform(Grosse, 2009; Gvož dík, 2009; Gvož dík et al., 2008; Kaya, 2001;

Pisanets and Matvyeyev, 2012; Rosso et al., 2004).   Hyla sarda   isthe only species that is substantially differentiated in morphology,and it was the first taxon to be distinguished from H. arborea at the

species level (Lanza, 1983; Nascetti et al., 1985), followed by   H.intermedia, which was elevated to the species rank some ten yearslater (Nascetti et al., 1995). All the remaining taxa have beenincluded under   H. arborea   until recently (e.g.   Grosse, 2009;Schneider and Grosse, 2009; Sillero et al., 2014; Speybroecket al., 2010). Stöck et al. (2008, 2012) found H. arborea s.l. paraphy-letic with respect to H. intermedia (Stöck et al., 2012) or to both H.intermedia and H. sarda (Stöck et al., 2008). However, the previousstudies did not obtain high support for the inferred topologiesexcept for the sister-clade relationship of the two H. intermedia lin-eages (Northern and Southern), and of   H. orientalis and H. molleri.By contrast, in our analyses,  H. arborea  s.l. was highly supportedin the mtDNA phylogeny (Fig. 3) and its monophyly was confirmedby the species tree analysis (Fig. 5) by the presence of the H. arbo-

rea s.l. clade in the three most common all-gene species-tree topol-ogies and the overall frequency of 83.6% among the posterior trees.

Fig. 5.  Species tree of the short-call tree frogs (H. meridionalis outgroup) as inferred in ⁄

BEAST based on two mitochondrial and two nuclear loci (tree a and trees b on left) orsolely on nuclear loci (trees b on right). The upper tree (a) is the maximum clade credibility tree with node ages (in mya; numbers below branches) represented by theirmedian heights and confidence intervals with 95% highest posterior densities (horizontal bars). Black dots on branches represent highly supported clades (pp = 1.00);otherwise pp values are above branches, while numbers in labels above branchesstand for speciation probabilities (=1.00 for all nodes) as inferred fromthe BP&P analysis (formore results see Table S3). The bottom trees/‘cloudograms’ (b) are outputs from DensiTree showing all posterior trees. Incongruence in topology and/or branch length formslight webs, while highly congruent trees constitute darker clusters (well-supported clades). Three most frequent ‘consensus trees’ (sensu Bouckaert, 2010) are shown as boldlines in different colors, with branch lengths as averages over all posterior trees with identical topology. On the bottom (c) is a graphic representation of temperaturefluctuations during the Plio-Pleistocene as inferred from the Mediterranean planktonic oxygen isotope ratio (redrawn after  Colleoni et al., 2012). The arrow marks thesubstantial thermal decrease in the Calabrian (Early Pleistocene) at around 1.4 mya when the split between the Northern and Southern  H. intermedia   is dated. The splitbetween H. orientalis and H. molleri is similarly dated in this period; however, in this case the divergence estimate is likely biased by the capture of  H. orientalis mtDNA in H.molleri (see also Fig. S2). Note also the external morphological similarity of the short-call tree-frog species. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)

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This discrepancy in mtDNA phylogenies between the studies mightbe a result of different markers utilized by Stöck et al. (2008, 2012).On the other hand, our nuclear-only species tree does not corre-spond to the all-gene topology in supporting a sister-clade rela-tionship between  H. arborea  and H. sarda  (64.5% of the posteriortrees), which has not been documented in any of the previouslypublished phylogenies (Hua et al., 2009; Pyron and Wiens, 2011;

Stöck et al., 2008, 2012). The H. arborea

 s.l. clade was present inthe nDNA species tree with very low frequency of only 1.1%.

4.2. Mitochondrial DNA capture and autosomal gene introgression

We further assessed the striking sister-clade relationship inmtDNA of the geographically remote, eastern  H. orientalis and thewestern/Iberian  H. molleri, which are currently separated by morethan 1500 km, while their genetic distance (16S ) is only 0.9%. Thisclade was strongly supported in the all-gene species tree (Fig. 5a),including its presence in the three most common topologies fromthe posterior trees (Fig. 5b, left). The clade was present in 99.9%of the posterior trees. On the contrary, in the nuclear-only speciestree (Fig. 5b, right), the sister-clade relationship of  H. orientalis + H.molleri was present only in one of the three most common topolo-gies, with the overall frequency of only 23.2% in the posterior sam-ple. In the nuclear-only species tree there was a clear tendency fora clade to show up that included the two taxa together with   H.intermedia  (59.3%), but besides the highly supported subclade  H.intermedia   N + H. intermedia   S (99.8%), the other relationshipswithin the clade were distributed among the posterior trees withrelatively equal and lower frequencies. Thus, it seems that thereare two plausible explanations for the observed pattern: (1) H. ori-entalis  –  H. molleri  split is relatively young (1.4 mya dated in ourstudy), of the age when a substantial thermal drop is evidencedin the Mediterranean (Colleoni et al., 2012; Fig. 5), probably caus-ing range restrictions and isolation of the two lineages. Or alterna-tively, and perhaps more likely, assuming the nDNA species tree iscorrect, then (2) H. orientalis –  H. molleri  split occurred much ear-

lier, and a mitochondrial introgression and capture (probably froman ancestral population of  H. orientalis   to  H. molleri) could havehappened during a secondary contact in a climatically suitable/warm period of the Early Pleistocene, which allowed range exten-sions. This or similar event(s) could also be responsible for  Rhodintrogression from H. orientalis  to W Balkan H. arborea and to theNorthern lineage of  H. intermedia  (see also Verardi et al., 2009),for Tyr  introgression from  H. felixarabica   to southern Asia Minorpopulation of  H. orientalis, and probably also for the genetic consti-tution of the Breton tree frogs (loc. 4), where   H. molleri  allelesappear to have introgressed into H. arborea.

4.3. Species delimitation

Coalescent-based BSD supported species-level distinction of alltaxa, including the W Balkan operational unit (Fig. 5), for a varietyof guide trees and datasets (Table S3). Only  Tyr  does not presentsignatures of completed speciation within  H. intermedia, implyingthe presence of some gene flow (detected previously byCanestrelli et al., 2007b). These results suggest that the two taxo-nomic units within H. intermedia should be treated as conspecific,despite the relatively high mtDNA distance (2.9% in  16S ). The  H.intermedia   N + H. intermedia   S clade was present in 99.9% and99.8% posterior species trees (all-gene and nDNA only, respec-tively). Similarly, signatures of speciation were not found in  Tyr for the split between H. arborea and W Balkan (the clade was pres-ent in the posterior species-tree sample of the all-gene and nDNAapproach with frequencies of 100% and 99.8%, respectively). None-

theless, when recombination is removed from the Tyr  dataset, thespeciation probability becomes significant in the latter case, sug-

gesting that the recombined alleles carry the signal of geneexchange among H. arborea populations. In addition, when recom-bination is filtered out the cluster of alleles originated from thewestern Balkans (not directly corresponding to the W Balkangroup; Fig. 2) is no more obvious (Tyr IMgc1 dataset; Fig. S1). Geneflow followed by recombination between  H. arborea  and H. orien-talis  alleles thus probably account for the presence of the W Bal-

kans allele cluster in the non-filtered Tyr 

 data (Fig. 2), despite thefact that BSD analysis of the filtered data suggests species-level dis-tinction of the W Balkan tree frogs. The lack of monophyly and of private alleles, together with the cryptic phenotype (Gvož díket al., 2008), support retaining the W Balkans populations in   H.arborea.

4.4. New insights into the phylogeographic history

The intraspecific genetic structuring was largely formed duringthe climatic oscillations in the Middle and Late Pleistocene as evi-denced in the case of  H. arborea–W Balkan split (Fig. 5), which cor-responds well to the conclusion of   Dufresnes et al. (2013). Thephylogeographic patterns of mtDNA (which shows higher diversityand thus geographic resolution than nDNA; Fig. 4) recovered in ourstudy extend the previous findings in several ways, but we provideseveral important novel insights.

4.4.1. Hyla arborea sensu stricto

We found three main haplogroups, similar to the study of Dufresnes et al. (2013). One of them is found in the northern Adri-atic (W Balkans) and we detected it on Pag Island, located betweenIstria and the Croatian coast where it was found previously(Dufresnes et al., 2013). This highlights the Adriatic region as apotential glacial refugium for   H. arborea   s.s. Another refugiumwas probably located in the southern Balkans, which is predomi-nantly occupied by the second haplogroup harboring a relativelyhigh genetic variation. One haplotype of this south-Balkan haplo-group was found at some northerly-located places like the north-

ern inland Croatia (see also   Dufresnes et al., 2013) andsurprisingly also as far north as the Czech Republic. This distribu-tion suggests multiple routes of northward expansion from theBalkan Peninsula. The range of the third, most widespread haplo-group is centered in central and north-western Europe, but itpartly overlaps with the other two haplogroups. Its star-like pat-tern suggests a recent expansion (see also Dufresnes et al., 2013).

4.4.2. Hyla orientalis

For this species we uncovered a somewhat different patterncompared to Stöck et al. (2012). First of all, the Caucasus-Caspianpopulation forms only a single haplogroup, distributed from thewestern forelands of the Caucasus in the Krasnodar region to thesouth-eastern Caspian coast in Iran. The most common haplotype

is widespread throughout the area, including regions of the typelocality of  H. arborea schelkownikowi  and H. a. gumilevskii, whichsupports their status as synonyms, currently under the name   H.orientalis (Gvož dík et al., 2010; Stöck et al., 2008). On the contrary,Stöck et al. (2012) found a population from the Talysh region insouth-eastern Azerbaijan as a distinct clade in a sister-clade posi-tion to the Caucasian population. This discordance might beexplained by differences in the level of variation in the mtDNAmarkers used in our study and that of  Stöck et al. (2012). It mightalso be due to the fact that the Caspian population of  Stöck et al.(2012) was represented solely by samples originating from a rela-tively small area of the Talysh region, while Iranian samples weremissing from their study. Interestingly, despite the distribution gapin the steppes of northern Crimea, the southern Crimean popula-

tion of H. orientalis is a part of the predominantly Eastern-Europeanhaplogroup, not of the geographically close Caucasus group. This

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Eastern-European haplogroup is distributed to the north and eastof the Carpathians from Poland, Ukraine, Romania and southwardsto Bulgaria and Turkey, and it is characterized by a complexgeneticvariation. This includes further sub-structuring, which is well evi-dent in Asia Minor.

4.4.3. Hyla molleri, H. intermedia and H. sarda

Our sampling of the Iberian, Italian and Tyrrhenian tree frogswas not designed as a detailed phylogeographic study, whichwas performed in those regions earlier (Barth et al., 2011;Bisconti et al., 2011a,b; Canestrelli et al., 2007a,b). However, wefound a distinct haplotype of  H. molleri in Galicia, suggesting thatthis region could play a role as a glacial refugium, while the restof the range of this species is inhabited by a widespread haplo-group (Barth et al., 2011). The Southern H. intermedia was split intotwo haplogroups, corresponding to the southernmost (Calabria,Sicily) and central part of the Italian Peninsula, highlighting theseregions as presumed glacial refugia (Canestrelli et al., 2007a). TheNorthern H. intermedia is highly diverged from the Southern line-age in mtDNA (2.9% in 16S ), but its nuclear genes bear signaturesof gene flow to/from the Southern   H. intermedia   (Tyr ; see also

Canestrelli et al., 2007b) and H. arborea/H. orientalis (Rhod; see alsoVerardi et al., 2009). Our limited sampling of   H. sarda   did notrecover a clear phylogeographic pattern, which was addressed indetail elsewhere (Bisconti et al., 2011a,b).

4.5. Hybridization

A case of hybridization between  H. orientalis  and H. savignyi insouthern Asia Minor was mentioned by Stöck et al. (2012). How-ever, that might have been a misinterpretation of the signaturesof introgression, not from   H. savignyi   but from the allopatric   H. felixarabica.  Such introgression characterizes the population of  H.orientalis   in south-western Asia Minor, as was discussed aboveand briefly mentioned by Gvož dík et al. (2010). The nearest local-

ities of the Arabian tree frog, H. felixarabica, are known from south-ern Syria, about 800 kmaway (Gvož dík et al., 2010), but the specieslikely had a secondary contact with H. orientalis during Pleistoceneperiods favorable to range extension (Gvož dík et al., 2010). To date,there is no unequivocal evidence for ongoing hybridizationbetween the parapatric short-call H. orientalis and the medium-callH. savignyi (cf. Gül et al., 2012; Gvož dík et al., 2010). However, thepresent study found hybrids in two different pairsof short-call spe-cies, i.e.  H. orientalis H. arborea s.s. in Greece, Bulgaria, Romaniaand Poland, and   H. arborea   s.s. H. molleri   in Brittany, France(see Table S1). It is not trivial to distinguish introgression fromretention of ancestral polymorphism due to incomplete lineagesorting. However, clear hybrids, including putative F1 hybrid, werefound in southern Poland near Krakow (loc. 48;   Fig. 2   andTable S1). This is in line with Stöck et al. (2012) who found hybridsin northern Poland and postulated a hypothesis that the geo-graphic border between  H. arborea  s.s. and  H. orientalis  approxi-mately follows the course of the Vistula River.

5. Conclusion

We conclude that the phylogenetic signal of mtDNA had stronginfluence on the reconstructed species tree of the short-call treefrogs, to some extent overwhelming the signal of the nucleargenes. However, the results strongly suggest that the western Bal-kan H. arborea does not represent a distinct species, as do neitherNorthern nor Southern lineage of  H. intermedia, which should betreated as conspecific despite their mtDNA divergence. However,

all other taxa should be considered species as no gene flow wasdetected between them by coalescent, regardless of some phyloge-

netic incongruence among the markers. A mitochondrial captureupon secondary contact is a plausible explanation for the closemtDNA relationship between the geographically remote  H. orien-talis   and   H. molleri. Our study thus provided several importantnovel insights into the speciation history and systematics of theshort-call tree frogs, despite the fact that some findings await con-firmation using novel markers.

 Acknowledgments

We are grateful to many friends and colleagues who providedsamples or helped in the field, namely to E. Albert, P. Benda, J. Č er-venka, L. Choleva, D. Cogălniceanu, S. Dubey, G. Evanno, W.-R.Grosse, D. Jablonski, J. Jaquiery, U. Joger, C. Klütsch, H. Kulíková,Z. Lajbner, S. N. Litvinchuk, R. Luč an, I. Martínez-Solano, M. Šander-a, C. Settanni, P. Široký, J. Šmíd, B. Švecová, J. M. Szymura, D. Tarkh-nishvili, M. Velenský, P. Velenský, G. Velo-Antón. We also thank A.Larson (Washington University in St. Louis) and two anonymousreviewers for their comments, which improved the previous ver-sion of the manuscript. The study was supported by the Ministryof Culture of the Czech Republic (DKRVO 2014/14, NationalMuseum, 00023272). ER is supported by a DGAPA-UNAM postdoc-toral fellowship.

 Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at   http://dx.doi.org/10.1016/j.ympev.2014.11.012.

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