Speciation History and Widespread Introgression in the European Short-Call Tree Frogs (Hyla Arborea Sensu Lato, H

Molecular Phylogenetics and Evolution 83 (2015) 143–155

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Molecular Phylogenetics and Evolution

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Speciation history and widespread introgression in the European short-call tree frogs (Hyla arborea sensu lato, H. intermedia and H. sarda) ⇑ Václav Gvozˇdík a,b, , Daniele Canestrelli c, Mario García-París d, Jirˇí 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 Libeˇchov, 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, Italy d Museo Nacional de Ciencias Naturales, C.S.I.C., c/Jose Gutierrez Abascal, 2, 28006 Madrid, Spain e 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, Portugal g CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, n.289, 4050-123 Porto, Portugal article info abstract

Article history: European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form six Received 23 July 2014 genetically differentiated but morphologically cryptic taxa, H. arborea sensu stricto, H. orientalis and H. Revised 10 November 2014 molleri from across Europe to western Asia (together referred to as H. arborea sensu lato), two putative Accepted 22 November 2014 taxa within H. intermedia (Northern and Southern) from the Italian Peninsula and Sicily, and H. sarda from Available online 4 December 2014 Sardinia 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 mitochondrial Keywords: and nuclear genes show partly incongruent phylogeographic patterns, which point to a complex history Cryptic species complex of gene flow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogs Bayesian species delimitation Gene flow and to infer the species tree, we used coalescent-based approaches. The monophyly of H. arborea sensu Species tree lato is supported by the mtDNA as well as by the all-gene species tree. The Northern and Southern lin- Phylogeography eages of H. intermedia have been connected by nuclear gene flow (despite their deep mtDNA divergence) Systematics and should be treated as conspecific. On the contrary, the parapatric taxa within H. arborea sensu lato should be considered distinct species (H. arborea, H. orientalis, H. molleri) based on the coalescent analysis, although signs of hybridization were detected between them (H. arborea Â H. orientalis; H. arborea Â H. molleri). A mitochondrial capture upon secondary contact appears to explain the close mtDNA relationship 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. intermedia, and between the Minor Asiatic H. orientalis and Arabian H. felixarabica (the latter belonging to a different acoustic group/clade). Our results shed light on the species limits in the European short-call tree frogs and show that introgression played an important role in the evolutionary history of the short-call tree frogs and occurred even between taxa supported as distinct species. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction ern Mediterranean, respectively, were elevated from subspecies level to full species based on differences in advertisement calls Western Palearctic tree frogs were considered to represent a (Paillette, 1967; Schneider, 1968, 1974; Schneider and Nevo, single species Hyla arborea until the end of the 1960s. Later on, 1972). Advertisement calls are an important behavioral feature in two taxa, H. meridionalis and H. savignyi from the western and east- anurans with a critical role in maintaining reproductive barriers between species (Schneider, 1977; Schneider and Sinsch, 2007).

⇑ Corresponding author. Present address: Institute of Vertebrate Biology, Acad- In the Western Palearctic tree frogs, the advertisement calls have emy of Sciences of the Czech Republic, Kveˇtná 8, 603 65 Brno, Czech Republic. been thoroughly studied (reviews in Schneider, 1974, 2004a). The E-mail addresses: [email protected] (V. Gvozˇdík), [email protected] (D. call segments are formed by pulse-groups (notes) and intervening Canestrelli), [email protected] (M. García-París), [email protected] (J. Mor- quiet intervals. Advertisement calls of the Western Palearctic tree avec), [email protected] (G. Nascetti), [email protected] (E. Recuero), frogs might be divided into three categories according to the [email protected] (J. Teixeira), [email protected] (P. Kotlík). http://dx.doi.org/10.1016/j.ympev.2014.11.012 1055-7903/Ó 2014 Elsevier Inc. All rights reserved. 144 V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155 pulse-group properties (Fig. 1; grouping made according to the fostered by the unexpected and surprisingly close sister-clade rela- results of Grach et al., 2007; Gvozˇdík et al., 2010; Schneider, tionship between the eastern (near the Black Sea; H. orientalis) and 1974, 2004a). The ‘long-call tree frogs’ are characterized by long western (on Iberian Peninsula; H. molleri) populations, currently pulse-groups composed of a high number of pulses (34–56 pulses; geographically separated by the range of H. arborea s.s. (Gvozˇdík, 210–610 ms), ‘short-call tree frogs’ by short pulse-groups with a 2009; Gvozˇdík et al., 2007; Stöck et al., 2008, 2012). As yet, no low number of pulses (6–12 pulses; 50–110 ms), and ‘medium-call study has provided explanation for this biogeographic pattern. tree frogs’ by intermediate values (13–25 pulses; 85–190 ms). The clade containing H. arborea s.l., H. intermedia and H. sarda The Tyrrhenian tree frog, H. sarda from the islands of Sardinia, (all considered H. arborea till the 1990s; e.g. Stumpel, 1997) has Corsica, Elba and surrounding islets, was distinguished from H. consistently received high support from molecular phylogenetic arborea on the basis of the differentiation at allozyme loci (Lanza, analyses (Gvozˇdík, 2009; Stöck et al., 2008, 2012), while relation- 1983; Nascetti et al., 1985), but the advertisement call of this spe- ships within the clade have remained ambiguous (Stöck et al., cies was not found to be substantially differentiated from H. arbo- 2008, 2012). The exceptions are the sister-clade relationships of rea (Schneider, 1974, 2004a). This is also true for the Italian tree H. orientalis and H. molleri and of the Northern (N) and Southern frog, H. intermedia (Schneider, 2004a,2004b), which also was dis- (S) lineages of H. intermedia (Gvozˇdík, 2009; Stöck et al., 2008, tinguished from H. arborea on the basis of allozyme differences 2012). However, using different mitochondrial markers (12S and (Nascetti et al., 1995). With the expansion of DNA sequencing, 16S rRNA) than in Stöck et al. (2008, 2012), our preliminary data new hypotheses on the taxonomy of the Western Palearctic tree showed high support also to H. arborea s.l., i.e. H. orientalis, H. mol- frogs emerged (Gvozˇdík et al., 2010; Stöck et al., 2008, 2012). leri and H. arborea s.s. forming a clade (Gvozˇdík, 2009). In addition Besides the description of the new species H. felixarabica from to the unresolved internal topology of the clade, species limits in the Arabian Peninsula (Gvozˇdík et al., 2010), deeply divergent lin- the short-call tree frogs are an intriguing issue, especially given eages were uncovered within H. meridionalis (south-eastern part of the following evidence: (i) similar advertisement calls of H. arbo- the range) and in H. intermedia (northern part of the range) rea, H. orientalis, H. molleri, H. intermedia and H. sarda (Fig. 1; e.g. (Canestrelli et al., 2007a,b; Gvozˇdík, 2009; Gvozˇdík et al., 2007; Castellano et al., 2002; Schneider, 1974, 2000, 2004a, 2004b); (ii) Recuero et al., 2007; Stöck et al., 2008, 2012), which may each rep- morphological similarity of H. arborea, H. orientalis (Gvozˇdík resent a separate (as yet unnamed) taxon (Stöck et al., 2008). Fur- et al., 2008; both as H. arborea), H. molleri (Terentjev, 1960) and lar- thermore, H. arborea was suggested to represent three species gely also H. intermedia (Nascetti et al., 1995; Rosso et al., 2004; according to the mitochondrial and single nuclear-gene phyloge- Terentjev, 1960); (iii) incongruence in phylogeographic patterns nies: H. orientalis in the east, H. molleri in the west, and H. arborea between different nuclear genes across the Balkans and northern sensu stricto (s.s.) in the central part of the former H. arborea sensu Italy (H. orientalis/H. arborea/H. intermedia N), especially along lato (s.l.) range (Stöck et al., 2008). the western Balkan coast (Gvozˇdík et al., 2007; distinct Balkan/ For the Iberian H. (arborea) molleri, this new taxonomy corre- Adriatic lineage was detected also by Stöck et al., 2008 and sponds to the former morphology-based intraspeciﬁc taxonomy Dufresnes et al., 2013). The Western Palearctic short-call tree frogs of H. arborea. In contrast, the genetic break between the central thus seem to represent an assemblage of morphologically and (H. arborea s.s.) and eastern (H. orientalis) populations does not acoustically cryptic taxa, and the extent of gene ﬂow between match the limits and distributions of the morphology-based sub- them needs to be properly evaluated. species, H. arborea kretensis (Crete, southern Balkans, Aegean, wes- Current advances in molecular phylogenetics have introduced tern Asia Minor; e.g. Stumpel, 1997), H. a. schelkownikowi new methods, including multilocus coalescent-based species tree (Caucasus; e.g. Kuzmin, 1999), H. a. gumilevskii (Talysh Mts. in inference (Knowles and Kubatko, 2010) and coalescent-based spe- Transcaucasia; Litvinchuk et al., 2006) and H. a. arborea in the rest cies delimitation, which permit probabilistic tests with an assump- of the range (except the Iberian Peninsula; e.g. Stumpel, 1997). [For tion of no recent admixture between species and of bipartitions of historical overview of the taxonomy of the Western Palearctic tree individuals in gene trees that are shared across loci (Yang and frogs see Fig. 1.] This incongruence between molecular phylogenies Rannala, 2010). In this study, we focus on the short-call tree frogs and morphology-based taxonomy, which discouraged wider (H. arborea, H. orientalis, H. molleri, two lineages of H. intermedia, H. acceptance of the revised taxonomy (e.g. Grosse, 2009; Özdemir sarda) using new molecular markers and a multilocus coalescent- et al., 2012; Sillero et al., 2014; Speybroeck et al., 2010), is further 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. Red box 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 dots correspond to the colors in Figs. 2 and 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.) V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155 145 method to test (1) the monophyly of H. arborea s.l. (H. arborea, H. possible gametic phases and their phylogenetic positions were orientalis, H. molleri); (2) sister-clade relationship of the geograph- considered in haplotype networks, which were constructed using ically disjunctive H. orientalis and H. molleri; and (3) species-level the parsimony approach under the 95% parsimony limit as imple- distinction of the taxa, including of the northern (N) and southern mented in TCS 1.21 (Clement et al., 2000). The alternative gametic (S) lineages of H. intermedia, and of the western (W) Balkan H. arbo- phases did not change the networks substantially (see haplotypes rea/H. orientalis populations. Rarb3, Tint7, Tint8, Tint9, Tint10 in Fig. 2). No stop codons were detected in the haplotypes as checked by translation with the standard genetic code using BioEdit 7.2 (Hall, 1999). All new unique 2. Materials and methods mitochondrial haplotypes and new nuclear alleles or nucleotide sequences with the standard IUPAC ambiguity codes, if heterozy- 2.1. Taxon sampling and laboratory procedures gous positions remained unresolved, have been deposited in Gen- Bank (KP109551–KP109674; Table S2). Genetic distances were We obtained sequence data for altogether 169 specimens from calculated from 16S haplotypes (a marker commonly used in 95 localities distributed throughout the geographic range of H. amphibian DNA barcoding; Vences et al., 2012) and estimated as arborea s.l., including one specimen from GenBank (DQ055835, p-distances averaged between and within taxa in MEGA 6.0 loc. 36; Smith et al., 2005), 19 specimens from 8 localities of H. (Tamura et al., 2013). Incomplete mtDNA sequences covering only intermedia and 10 specimens from 6 localities of H. sarda, which the 12S fragment (from localities 36 and 50) were used in the mito- totaled 198 ingroup samples from 109 localities (Fig. 2, Table S1). chondrial gene tree only. Due to the lack of reliable diagnostic morphological characters, the material of H. arborea s.l. could not be a priori sorted into the 2.3. Gene trees putative species, i.e. H. arborea s.s., H. orientalis and H. molleri (sensu Stöck et al., 2008). The H. orientalis material from Asia Minor For gene-tree estimation, mtDNA and phased Tyr and Rhod and Caucasus-Caspian region used in a previous study (Gvozˇdík sequences were sorted into distinct haplotypes using Collapse 1.2 et al., 2010) was reanalyzed in the context of the new data from (Posada, 2006). Gene trees were reconstructed by Bayesian infer- the European part of the range. The outgroup species H. meridional- ence (BI) and maximum likelihood criterion (ML). For BI analyses is data were taken from the previous study (Gvozˇdík et al., 2010) performed by MrBayes v3.2.2 (Ronquist et al., 2012), best-fit parti- and those for a second outgroup, H. japonica, were retrieved from tioning schemes and nucleotide substitution models according to GenBank (AY843633, AY844615, AY844078; Faivovich et al., the Bayesian information criterion (BIC) were selected using Parti- 2005). The outgroup status of these two species relative to the tionFinder v1.1.0 (Lanfear et al., 2012): mtDNA, 12S and 16S treated short-call tree frogs was demonstrated by, e.g. Hua et al. (2009). as one partition (SYM+G); Tyr, 1st + 2nd/3rd codon position (K80+I/ Genomic DNA was extracted from tissue samples using com- HKY+G); Rhod, 1st + 2nd/3rd codon position (JC/K80). Each dataset mercial kits following the manufacturers’ protocols. Two frag- was analyzed with two runs of four Markov chains, which were run ments of mitochondrial DNA (mtDNA), 12S rRNA (12S, 355 for 6 Â 106 generations and samples saved every 100th generation. aligned bp) and 16S rRNA (16S, 546 aligned bp), and two nuclear That the analyses reached stationarity was verified by examining genes (nDNA), tyrosinase precursor exon 1 (Tyr, 496 bp), and rho- the plots of log-likelihood scores using Tracer v1.5 [Rambaut and dopsin exon 1 (Rhod, 276 bp) were targeted. The nDNA markers Drummond, 2009; all parameters had effective sample size were sequenced for a subset of samples (ca. 43%) representing all (ESS) 200], and the chain convergence was confirmed by the main mitochondrial groups and covering the entire geographic convergence diagnostics (average standard deviation of split fre- range (Table S1). Primers and PCR protocols followed the previous quencies, potential scale reduction factor) comparing the two study (Gvozˇdík et al., 2010). PCR amplicons were purified and simultaneous runs against each other. From the sampled trees, directly sequenced with the PCR primers using a commercial the first third was discarded as a burn-in and a 50% majority-rule sequencing service (Macrogen Inc.). consensus tree was produced from the remaining trees that were taken as representing the posterior distribution. For ML analyses 2.2. DNA alignment and phasing of autosomal genotypes performed by PhyML v3.0 (Guindon et al., 2010), best-fit models of sequence evolution were selected using jModelTest v2.1.4 Alignments of nucleotide sequences were prepared with Clu- (Darriba et al., 2012) according to the BIC: mtDNA, TIM2ef+I; Tyr, stalW (Thompson et al., 1994) as implemented in BioEdit 7.2 TrNef+I; Rhod, JC. The best option of a combination of the nearest (Hall, 1999). Mitochondrial 12S and 16S fragments were concate- neighbour interchange and the subtree pruning and regrafting nated. Heterozygous sites in nDNA were identified according to algorithm of tree improvement and optimization of the topology electropherograms when two different nucleotides were present and branch lengths were applied. The nodal support was assessed at the same position with the lower peak reaching at least a half by 100 bootstrap pseudoreplicates. Clades supported with BI pos- of the higher peak. Gametic haplotypes were inferred by the coa- terior probability (pp) values P 0.95 and ML bootstrap val- lescent-based Bayesian algorithm of Phase 2.1 (Stephens et al., ues P 70 were considered highly supported (Huelsenbeck and 2001; Stephens and Scheet, 2005) as implemented in DnaSP 5.10 Rannala, 2004). (Librado and Rozas, 2009). The analyses were run without outgroup species and were repeated five times for each nuclear-gene 2.4. Data preparation for coalescent-based applications dataset with different seeds for the random number generator to check if the phase estimates are consistent across the runs accord- Since the coalescent-based species-tree and species-delimita- ing to goodness-of-fit values. Each run was conducted under the tion analyses rely on, and are sensitive to, the assignment of each parent-independent mutation model with a burn-in-period of individual/allele to a taxonomic unit (Leaché et al., 2014; Olave 100 iterations, which was followed by 1000 iterations. Results with et al., 2014), we carefully inspected distribution of haplotypes a probability > 0.70 were accepted. Two samples (H. intermedia across different genes to identify specimens with inconsistent phy- HD03A, Tyr; H. arborea HJ55, Rhod) contained heterozygous posi- logenetic signal and thus identify potential hybrids (Table S1). Such tions whose phases were not resolved (mean probability 0.50), potential hybrids or possible cases of allelic introgression (H. arbo- and one sample (H. intermedia HD04B, Tyr) had one position rea–orientalis; 10 individuals from the Balkans and Poland) were weakly resolved (mean probability 0.69). In these cases, both omitted from the species-tree analyses. Similarly, contrasting Tyr 146 V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155

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 are represented by maximum-parsimony haplotype networks for nuclear genes. For mitochondrial phylogenetic relationships see Figs. 3 and 4. In haplotype networks the size of the circles corresponds to the haplotype frequency, and the smallest circles represent hypothetic, unobserved haplotypes. Dashed lines reflect two possibilities of a haplotype connection in cases when haplotypes were phased with low support. Samples classified as W Balkan H. arborea in the coalescent analyses are marked with ‘‘B’’, while potential hybrids (or introgressed alleles) omitted from the coalescent analyses are marked with ‘‘Â’’ on the maps. Light green in Tyr stands for H. felixarabica-like alleles introgressed 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 is referred to the web version of this article.) alleles, such as in a sample from Brittany (loc. 4) or H. felixarabica- 2012) we also tested a position of the western Balkan tree frogs like alleles of six individuals of H. orientalis from south-western (W Balkan H. arborea, loc. 33–35, 37, 38 in Slovenia, Croatia, Mon- Turkey (haplotypes Tfel3, Tori5, Tori6, Tori7, Tori9; see Fig. 2 and tenegro, Greece; n = 5), which carry inconsistent phylogenetic sig- Gvozˇdík et al., 2010), were also omitted from the analyses (see Sec- nal across the studied genes (Fig. 2), as noted earlier by Stöck et al. tion 3 for details). Remaining samples were allocated to the follow- (2008). Coalescent-based model assumptions include an absence of ing taxonomic units according to their provenance and genetic recombination (Castillo-Ramírez et al., 2010; Lavretsky et al., 2014; identity: H. arborea s.s. (n = 64), H. orientalis (n = 74), H. molleri but see Lanier and Knowles, 2012). Thus, the minimum numbers of (n = 14), H. intermedia South (n = 16), H. intermedia North (n = 3), recombination events (Hudson and Kaplan, 1985) in nuclear loci and H. sarda (n = 10). Beside these ‘standard’ units (Stöck et al., for different subsets of tree-frog species were estimated in DnaSP V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155 147

5.10 (Librado and Rozas, 2009), with no recombination detected in 2010) to test whether the operational taxonomic units fit the Rhod, and a minimum of one (H. intermedia, H. sarda), seven (H. assumption of no post-divergence gene flow, which is inherent to arborea s.l.) and ten (all ingroup taxa) recombination events the biological species concept (Yang and Rannala, 2010). This detected in Tyr. Traces of recombination were removed from the method accommodates the species phylogeny and accounts for Tyr dataset (not containing H. felixarabica-like alleles) using IMgc lineage sorting due to the ancestral polymorphism using the (Woerner et al., 2007), and a recombination-filtered dataset was reversible-jump Markov chain Monte Carlo (rjMCMC) algorithm produced by the approach of favoring the retention of individuals that successively splits or joins nodes on the guide species tree, (a = 1.0; 423 bp and 86% samples kept; hereafter as TyrIMgc1 data- and employs population-size parameters h and species divergence set; see also Table S1 and Fig. S1). times s in the coalescent model (Rannala and Yang, 2013; Yang and Rannala, 2010). Numerous possible species-tree topologies (guide 2.5. Species trees and divergence dating trees) were tested using different datasets with individual alleles carefully assigned to putative species (see Olave et al., 2014): all A species tree was inferred using ⁄BEAST (Heled and markers (mtDNA and phased nDNA), nDNA only, Rhod only, and Drummond, 2010) based on the dataset including all genes, mito- Tyr only; Tyr was used in both ways – including recombination chondrial 12S and 16S, the nuclear Rhod and the recombination-fil- and recombination-filtered (TyrIMgc1); all individuals (without tered Tyr (TyrIMgc1). Alignments of all individuals (without H. felixarabica-like Tyr alleles), with or without ‘hybrids’, and con- potential ‘hybrids’; see above) including one outgroup (H. meridio- sidering W Balkan populations as a discrete unit or part of H. arbo- nalis) for each gene were uploaded into BEAUti v1.8.0, a part of the rea (Table S3). A gamma prior G(2,1000) was applied on the BEAST package (Drummond et al., 2012), where they were population-size parameters (hs). Also the age of the root in the spe- assigned separate and unlinked substitution, clock and tree mod- cies tree (s0) was assigned the gamma prior G(2,1000), while the els. Hyla meridionalis was set as outgroup by enforcing monophyly other divergence-time parameters were assigned the Dirichlet of the remaining taxa. Best-fit partitioning schemes and sequence- prior (Yang and Rannala, 2010). When both mtDNA and autosomal evolution models were set according to the results from Partition- nuclear data were involved in an analysis, the parameters ‘hered- Finder v1.1.0 (Lanfear et al., 2012) following the BIC [mtDNA, 12S ity’ (allows h to vary among loci) and ‘locusrate’ (allows variable and 16S treated as one partition (HKY+G); TyrIMgc1 without parti- mutation rates among loci) were estimated with a gamma G(4,4) tions (K80); Rhod, 1st + 2nd/3rd codon position (K80/TrNef)]. and Dirichlet prior, respectively. The algorithm 0 was used with Ploidy of mtDNA was set to mitochondrial (haploid), and the other several values for the fine-tuning parameter e to ensure that the two loci to autosomal nuclear (diploid). The Yule species-tree prior rjMCMC mixed properly. BP&P analyses were run for 105 genera- was set and UPGMA starting tree used for each gene and a strict tions with first 50% of samples treated as a burn-in and discarded. molecular clock assumed. The clock rate for mtDNA was inferred If necessary, fine-tuning variables were adjusted to display propor- from a calibration based on an assumption of H. sarda being a tion values close to 0.3–0.4 (within the interval 0.15–0.7) as rec- descendant of a radiation that had occurred during the Late Mio- ommended in the manual (Yang, 2013). Each analysis was run cene (Stöck et al., 2012), particularly when the land bridge repeatedly to check consistency between runs. To control for a pos- between the Sardo–Corsican block and continent was submerged sible influence of the settings, the all-gene and nDNA MCCT spe- at the end of the Messinian salinity crisis when the Mediterranean cies-tree topologies were used as guide trees also for runs with

Basin was re-flooded at ca. 5.33 mya (Bisconti et al., 2011a; Hsü alternative prior settings of hs and s0 [G(1,10)/G(1,10); et al., 1973; Krijgsman et al., 1999; Rouchy and Caruso, 2006). Nor- G(2,2000)/G(2,2000); G(1,10)/G(2,2000)], as recommended by mal distribution prior was assigned with a mean of 5.33 (mya) and Leaché and Fujita (2010), and runs employing the rjMCMC algo- standard deviation of 0.16 in order to accommodate uncertainty in rithm 1, with fine-tuning parameters a = 2 and m =1(Yang, 2013). this estimate. Clock rates for Tyr and Rhod were estimated from mtDNA using 1/x prior distribution. Five independent ⁄BEAST runs 3. Results were performed, each for 200 million generations, sampling every 20,000th generation to obtain a posterior sample of 10,000 trees. 3.1. DNA polymorphism The likelihoods were inspected using Tracer v1.5 (Rambaut and Drummond, 2009) and convergence checked using ESS values The mitochondrial DNA dataset contained 198 ingroup (200) after discarding the 10% burn-in. The post burn-in samples sequences, which collapsed into 77 haplotypes (891 bp excluding of the five runs were combined in the BEAST module LogCombiner insertion/deletion polymorphisms), contained the number of poly- v1.8.0. The output of 45,000 sampled trees was uploaded to morphic sites S = 126 (89 parsimony informative), and showed the another BEAST module, TreeAnnotator v1.8.0, to infer the final spe- haplotype diversity h = 0.922 ± 0.014 (SD) and nucleotide diversity cies tree as a maximum clade credibility tree (MCCT) with node p = 1.91 ± 0.09%. The two nuclear genes were considerably less var- ages represented by median heights and confidence intervals with iable. Phased ingroup Tyr dataset (166 sequences, 496 bp) con- 95% highest posterior densities (HPD). The set of all 45 thousand tained 47 haplotypes, with S = 38(34), h = 0.900 ± 0.016, and trees was superimposed and visualized as a ‘cloudogram’ with p = 0.90 ± 0.04%, while the H. felixarabica-like- and recombina- DensiTree v2.01 (Bouckaert, 2010) to show the probability distri- tion-filtered TyrIMgc1 (133 sequences, 423 bp) contained 22 hap- bution of topologies, and the three most frequent ‘consensus trees’ lotypes, with S = 26(23), h = 0.820 ± 0.025, and p = 0.88 ± 0.05%. (sensu Bouckaert, 2010) were inspected. To assess the influence of Gametic-phased ingroup Rhod dataset (174 sequences, 276 bp) the mtDNA dataset on the species-tree topology, the nuclear data- contained 15 haplotypes, with S = 14(7), h = 0.779 ± 0.022, and set was also employed in a separate species-tree analysis with vir- p = 0.60 ± 0.03%. The mitochondrial marker has a roughly two tually the same settings but without molecular-clock calibration times higher polymorphism than Tyr and three times higher than and run for 500 million generations. Rhod. 2.6. Species delimitation 3.2. Gene trees Coalescent-based Bayesian species delimitation (BSD) analyses were conducted in the program Bayesian Phylogeny and Phyloge- The mitochondrial DNA phylogeny had an almost identical ography (BP&P v2.2; Rannala and Yang, 2003; Yang and Rannala, topology based on the BI and ML approaches (Fig. 3). Most of the 148 V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155

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 branches correspond 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 this figure legend, the reader is referred to the web version of this article.) main clades are highly supported with H. sarda in a sister-clade and H. arborea s.l. The latter is highly supported (1.00/75), while position to all other taxa (Bayesian pp 1.00/ML bootstrap 63). the support for a clade containing two highly divergent lineages These are grouped into two clades corresponding to H. intermedia of H. intermedia (Northern/Southern) is weaker (0.94/56), reflecting V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155 149 the deep mitochondrial differentiation of the two geographically further structuring in the second group is also detectable. Most close lineages. Despite the geographically remote distributions, diversified is the Asia Minor population with haplotypes present the Iberian H. molleri and Eastern European/Western Asian H. ori- in several different subgroups, but with less clear geographic struc- entalis together form a highly supported clade (1.00/88), while turing, which is true also for the remaining Eastern European pop- the support for each of the two taxa is rather low (H. molleri ulations, which have clear affinities to the north-western Asia 0.85/60; H. orientalis 0.94/68). Hyla arborea s.s. is well differenti- Minor population. ated (0.97/78) and is supported as the sister clade to H. molleri + H. For the nuclear Tyr and Rhod markers, the distribution of haplo- orientalis (1.00/75). Mitochondrial DNA (16S) genetic distances types generally agrees with the pattern in mtDNA, with the excep- among taxa are given in Table 1. tion of the cases described above (Section 3.2.). However, there The two nuclear-gene phylogenies (Fig. 3) have generally low have been also several striking findings at particular locations. nodal support likely caused by the relatively low variation. How- Based on the combination of alleles across different loci, potential ever, both nuclear markers, Tyr and Rhod, possess variation suffi- hybrids were detected in Brittany (H. arborea Â H. molleri), Poland, cient to show clear structure (Figs. 2 and 3). The Tyr phylogeny and across the Balkans in Greece, Bulgaria and Romania (H. arbo- differs from mtDNA in terms of the detection of major lineages/ rea Â H. orientalis)(Fig. 2 and Table S1). species in three main aspects: (i) H. arborea s.s. forms two main clusters. One contains predominantly samples from the western 3.4. Species trees and divergence time estimate Balkans, while the second the remaining samples. (ii) H. orientalis also forms two main groups, which do not form a clade. One The maximum clade credibility species tree based on the mito- accommodates most samples, while the other is restricted only chondrial and nuclear loci inferred the same topology as the to south-western Asia Minor. In the latter group the most common mtDNA gene tree: H. sarda being the sister lineage to all other haplotype of H. felixarabica (Tfel3) was recorded (plus four haplo- short-call tree frogs, and H. intermedia being the sister lineage to types closely related to it), as documented earlier (Gvozˇdík et al., H. arborea s.l., and with H. orientalis forming a clade with H. molleri 2010). (iii) H. intermedia, Northern lineage is not clearly detectable and H. arborea s.s. closely related to the W Balkan H. arborea group and most alleles are in the Southern lineage. In the Rhod haplotype (Fig. 5). However, high support is present only for the crown sister- phylogenetic relationships, the most striking difference from the clade relationships (H. intermedia N–H. intermedia S; H. orientalis – mtDNA phylogeny is the position of W Balkan H. arborea samples, H. molleri; H. arborea – W Balkan). Three most frequent topologies which do not carry ‘typical’ H. arborea Rhod haplotypes, but have occurred with the posterior frequency of 38.7%, 33.2%, 11.6% – first the most-common H. orientalis haplotype (Rori1) or a haplotype with the same topology as the MCCT, second differing by the pres- derived from it (Rori6) (Fig. 3). Similarly, the majority of samples ence of a clade H. intermedia (N+S) + H. sarda in the sister-clade (5/6) of the Northern lineage H. intermedia carries the most com- position to H. arborea s.l., and third interchanging positions of H. mon H. orientalis haplotype (Fig. 3). intermedia (N+S) and H. sarda in comparison to the MCCT. All the three topologies, accounting for almost 84% posterior trees, had 3.3. Geographic distribution of haplotypes and hybrid location the H. arborea s.l. taxa as a clade. On the contrary, if we consider only the two nuclear loci the MCCT (Fig. S2) as well as the three Intraspecific phylogeographic relationships based on mitochon- most frequent topologies (22.6%, 15.1%, 13.8%) form two main drial DNA are presented in Fig. 4. The south-western endemics, H. clades: (i) H. arborea s.s. (+ W Balkan) + H. sarda; (ii) all other taxa, sarda and H. molleri do not demonstrate clear phylogeographic pat- i.e. H. intermedia (N+S), H. molleri, H. orientalis. The molecular clock tern, with an exception of the presence of one relatively distant based on all loci estimated that the main radiation within the haplotype of H. molleri in Galicia. The Southern lineage of the Ital- short-call tree frogs occurred in the Pliocene (considering 95% ian endemic H. intermedia forms two subgroups, southernmost and highest posterior densities, HPD, 5.4–1.9 mya), with only the most central. The most common H. arborea s.s. haplotype and its single- recent splits occurring during the Pleistocene. The split between H. mutational (or indel) derivatives, arranged in a star-like pattern, orientalis and H. molleri is dated close to that between the Southern have been recorded from over much of Europe, from Greece in and Northern H. intermedia, ca. 1.4 mya (HPD 2.8–0.4), while the the south to Denmark in the north (Fig. 4). On the contrary, in structuring within H. arborea with respect to the W Balkan popula- the southern Balkans a more diversified haplotype group is pres- tion dates to the Middle to Early Pleistocene ca. 200 kya (HPD 370– ent, predominantly recorded from Greece but found also in north- 70 kya). ern Croatia and the Czech Republic. One relatively distant haplotype was detected on Pag, a Croatian island in the northern 3.5. Species delimitation Adriatic Sea. The highest diversity was found in the eastern taxon, H. orientalis. Two main groups (weakly supported clades) are rep- Bayesian species delimitation supported all the taxa as species resented by the Caucasus-Caspian populations and by all the other without a history of gene flow (speciation probability 1.00; populations, respectively. Besides the Caucasus-Caspian group, 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. molleri H. intermedia S H. intermedia N H. sarda H. meridionalis H. japonica H. arborea 0.4 H. orientalis 3.3 0.5 H. molleri 3.2 0.9 0.4 H. intermedia S 3.8 3.8 3.4 0.3 H. intermedia N 3.9 4.5 4.0 2.9 0.2 H. sarda 4.9 4.5 4.1 4.0 4.0 0.3 Outgroup H. 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 – 150 V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155

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 in ellipsoids correspond to the locality numbers as in the maps and Table S1. Different colors indicate different haplogroups with clear geographic afﬁnities within individual species 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). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) ditions with respect to inclusion of potential ‘hybrids’, recombina- alternative prior settings and type of rjMCMC algorithm also did tion in Tyr, or considering W Balkan populations as a discrete unit not affect the results of high speciation probability for any node. or as a part of H. arborea (see overview of results in Table S3). The Only when the Tyr dataset was evaluated independently was the V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155 151

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) or solely 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 their median 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 branches stand for speciation probabilities (=1.00 for all nodes) as inferred from the BP&P analysis (for more results see Table S3). The bottom trees/‘cloudograms’ (b) are outputs from DensiTree showing all posterior trees. Incongruence in topology and/or branch length forms light webs, while highly congruent trees constitute darker clusters (well-supported clades). Three most frequent ‘consensus trees’ (sensu Bouckaert, 2010) are shown as bold lines in different colors, with branch lengths as averages over all posterior trees with identical topology. On the bottom (c) is a graphic representation of temperature fluctuations during the Plio-Pleistocene as inferred from the Mediterranean planktonic oxygen isotope ratio (redrawn after Colleoni et al., 2012). The arrow marks the substantial 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 split between 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.) speciation probability always low (0.08–0.60) for the Northern and Pisanets and Matvyeyev, 2012; Rosso et al., 2004). Hyla sarda is Southern lineage of H. intermedia, both for the dataset containing the only species that is substantially differentiated in morphology, recombination and the recombination-filtered dataset. There was and it was the first taxon to be distinguished from H. arborea at the also low speciation probability for the node H. arborea – W Balkan species level (Lanza, 1983; Nascetti et al., 1985), followed by H. in Tyr (0.32), but the probability increased substantially when intermedia, which was elevated to the species rank some ten years recombination was filtered out (0.97). Interestingly, when the node later (Nascetti et al., 1995). All the remaining taxa have been W Balkan – H. orientalis was tested in Rhod where there is a high included under H. arborea until recently (e.g. Grosse, 2009; level of haplotype sharing (Rori1; frequency 4/10 in W Balkan), Schneider and Grosse, 2009; Sillero et al., 2014; Speybroeck the speciation probability was also high (0.99). This was likely a et al., 2010). Stöck et al. (2008, 2012) found H. arborea s.l. paraphy- consequence of the Rori6 haplotype (frequency 5/10 in W Balkan) letic with respect to H. intermedia (Stöck et al., 2012) or to both H. being common in the northern Adriatic. intermedia and H. sarda (Stöck et al., 2008). However, the previous studies did not obtain high support for the inferred topologies except for the sister-clade relationship of the two H. intermedia lin- 4. Discussion eages (Northern and Southern), and of H. orientalis and H. molleri. By contrast, in our analyses, H. arborea s.l. was highly supported 4.1. Species tree in the mtDNA phylogeny (Fig. 3) and its monophyly was confirmed by the species tree analysis (Fig. 5) by the presence of the H. arbo- The short-call tree frogs are phenotypically very uniform rea s.l. clade in the three most common all-gene species-tree topol- (Grosse, 2009; Gvozˇdík, 2009; Gvozˇdík et al., 2008; Kaya, 2001; ogies and the overall frequency of 83.6% among the posterior trees. 152 V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155

This discrepancy in mtDNA phylogenies between the studies might gesting that the recombined alleles carry the signal of gene be a result of different markers utilized by Stöck et al. (2008, 2012). exchange among H. arborea populations. In addition, when recom- On the other hand, our nuclear-only species tree does not corre- bination is filtered out the cluster of alleles originated from the spond to the all-gene topology in supporting a sister-clade rela- western Balkans (not directly corresponding to the W Balkan tionship between H. arborea and H. sarda (64.5% of the posterior group; Fig. 2) is no more obvious (TyrIMgc1 dataset; Fig. S1). Gene trees), which has not been documented in any of the previously flow followed by recombination between H. arborea and H. orien- published phylogenies (Hua et al., 2009; Pyron and Wiens, 2011; talis alleles thus probably account for the presence of the W Bal- Stöck et al., 2008, 2012). The H. arborea s.l. clade was present in kans allele cluster in the non-filtered Tyr data (Fig. 2), despite the the nDNA species tree with very low frequency of only 1.1%. fact that BSD analysis of the filtered data suggests species-level distinction of the W Balkan tree frogs. The lack of monophyly and of 4.2. Mitochondrial DNA capture and autosomal gene introgression private alleles, together with the cryptic phenotype (Gvozˇdík et al., 2008), support retaining the W Balkans populations in H. We further assessed the striking sister-clade relationship in arborea. mtDNA of the geographically remote, eastern H. orientalis and the western/Iberian H. molleri, which are currently separated by more 4.4. New insights into the phylogeographic history than 1500 km, while their genetic distance (16S) is only 0.9%. This clade was strongly supported in the all-gene species tree (Fig. 5a), The intraspecific genetic structuring was largely formed during including its presence in the three most common topologies from the climatic oscillations in the Middle and Late Pleistocene as evi- the posterior trees (Fig. 5b, left). The clade was present in 99.9% denced in the case of H. arborea–W Balkan split (Fig. 5), which cor- of the posterior trees. On the contrary, in the nuclear-only species responds well to the conclusion of Dufresnes et al. (2013). The tree (Fig. 5b, right), the sister-clade relationship of H. orientalis + H. phylogeographic patterns of mtDNA (which shows higher diversity molleri was present only in one of the three most common topolo- and thus geographic resolution than nDNA; Fig. 4) recovered in our gies, with the overall frequency of only 23.2% in the posterior sam- study extend the previous findings in several ways, but we provide ple. In the nuclear-only species tree there was a clear tendency for several important novel insights. a clade to show up that included the two taxa together with H. intermedia (59.3%), but besides the highly supported subclade H. 4.4.1. Hyla arborea sensu stricto intermedia N+H. intermedia S (99.8%), the other relationships We found three main haplogroups, similar to the study of within the clade were distributed among the posterior trees with Dufresnes et al. (2013). One of them is found in the northern Adri- relatively equal and lower frequencies. Thus, it seems that there atic (W Balkans) and we detected it on Pag Island, located between are two plausible explanations for the observed pattern: (1) H. ori- Istria and the Croatian coast where it was found previously entalis – H. molleri split is relatively young (1.4 mya dated in our (Dufresnes et al., 2013). This highlights the Adriatic region as a study), of the age when a substantial thermal drop is evidenced potential glacial refugium for H. arborea s.s. Another refugium in the Mediterranean (Colleoni et al., 2012; Fig. 5), probably caus- was probably located in the southern Balkans, which is predomi- ing range restrictions and isolation of the two lineages. Or alterna- nantly occupied by the second haplogroup harboring a relatively tively, and perhaps more likely, assuming the nDNA species tree is high genetic variation. One haplotype of this south-Balkan haplo- correct, then (2) H. orientalis – H. molleri split occurred much ear- group was found at some northerly-located places like the north- lier, and a mitochondrial introgression and capture (probably from ern inland Croatia (see also Dufresnes et al., 2013) and an ancestral population of H. orientalis to H. molleri) could have surprisingly also as far north as the Czech Republic. This distribu- happened during a secondary contact in a climatically suitable/ tion suggests multiple routes of northward expansion from the warm period of the Early Pleistocene, which allowed range exten- Balkan Peninsula. The range of the third, most widespread haplo- sions. This or similar event(s) could also be responsible for Rhod group is centered in central and north-western Europe, but it introgression from H. orientalis to W Balkan H. arborea and to the partly overlaps with the other two haplogroups. Its star-like pat- Northern lineage of H. intermedia (see also Verardi et al., 2009), tern suggests a recent expansion (see also Dufresnes et al., 2013). for Tyr introgression from H. felixarabica to southern Asia Minor population of H. orientalis, and probably also for the genetic consti- 4.4.2. Hyla orientalis tution of the Breton tree frogs (loc. 4), where H. molleri alleles For this species we uncovered a somewhat different pattern appear to have introgressed into H. arborea. compared to Stöck et al. (2012). First of all, the Caucasus-Caspian population forms only a single haplogroup, distributed from the 4.3. Species delimitation western forelands of the Caucasus in the Krasnodar region to the south-eastern Caspian coast in Iran. The most common haplotype Coalescent-based BSD supported species-level distinction of all is widespread throughout the area, including regions of the type taxa, including the W Balkan operational unit (Fig. 5), for a variety locality of H. arborea schelkownikowi and H. a. gumilevskii, which of guide trees and datasets (Table S3). Only Tyr does not present supports their status as synonyms, currently under the name H. signatures of completed speciation within H. intermedia, implying orientalis (Gvozˇdík et al., 2010; Stöck et al., 2008). On the contrary, the presence of some gene flow (detected previously by Stöck et al. (2012) found a population from the Talysh region in Canestrelli et al., 2007b). These results suggest that the two taxo- south-eastern Azerbaijan as a distinct clade in a sister-clade posi- nomic units within H. intermedia should be treated as conspecific, tion to the Caucasian population. This discordance might be despite the relatively high mtDNA distance (2.9% in 16S). The H. explained by differences in the level of variation in the mtDNA intermedia N+H. intermedia S clade was present in 99.9% and markers used in our study and that of Stöck et al. (2012). It might 99.8% posterior species trees (all-gene and nDNA only, respec- also be due to the fact that the Caspian population of Stöck et al. tively). Similarly, signatures of speciation were not found in Tyr (2012) was represented solely by samples originating from a rela- for the split between H. arborea and W Balkan (the clade was pres- tively small area of the Talysh region, while Iranian samples were ent in the posterior species-tree sample of the all-gene and nDNA missing from their study. Interestingly, despite the distribution gap approach with frequencies of 100% and 99.8%, respectively). None- in the steppes of northern Crimea, the southern Crimean popula- theless, when recombination is removed from the Tyr dataset, the tion of H. orientalis is a part of the predominantly Eastern-European speciation probability becomes significant in the latter case, sug- haplogroup, not of the geographically close Caucasus group. This V. Gvozˇdík et al. / Molecular Phylogenetics and Evolution 83 (2015) 143–155 153

Eastern-European haplogroup is distributed to the north and east netic incongruence among the markers. A mitochondrial capture of the Carpathians from Poland, Ukraine, Romania and southwards upon secondary contact is a plausible explanation for the close to Bulgaria and Turkey, and it is characterized by a complex genetic mtDNA relationship between the geographically remote H. orien- variation. This includes further sub-structuring, which is well evi- talis and H. molleri. Our study thus provided several important dent in Asia Minor. novel insights into the speciation history and systematics of the short-call tree frogs, despite the fact that some findings await con- 4.4.3. Hyla molleri, H. intermedia and H. sarda firmation using novel markers. Our sampling of the Iberian, Italian and Tyrrhenian tree frogs was not designed as a detailed phylogeographic study, which Acknowledgments was performed in those regions earlier (Barth et al., 2011; Bisconti et al., 2011a,b; Canestrelli et al., 2007a,b). However, we We are grateful to many friends and colleagues who provided found a distinct haplotype of H. molleri in Galicia, suggesting that samples or helped in the field, namely to E. Albert, P. Benda, J. Cˇer- this region could play a role as a glacial refugium, while the rest venka, L. Choleva, D. Coga˘lniceanu, S. Dubey, G. Evanno, W.-R. of the range of this species is inhabited by a widespread haplo- Grosse, D. Jablonski, J. Jaquiery, U. Joger, C. Klütsch, H. Kulíková, group (Barth et al., 2011). The Southern H. intermedia was split into Z. Lajbner, S. N. Litvinchuk, R. Lucˇan, I. Martínez-Solano, M. Šander- two haplogroups, corresponding to the southernmost (Calabria, a, C. Settanni, P. Široky´ , J. Šmíd, B. Švecová, J. M. Szymura, D. Tarkh- Sicily) and central part of the Italian Peninsula, highlighting these nishvili, M. Velensky´ , P. Velensky´ , G. Velo-Antón. We also thank A. regions as presumed glacial refugia (Canestrelli et al., 2007a). The Larson (Washington University in St. Louis) and two anonymous Northern H. intermedia is highly diverged from the Southern line- reviewers for their comments, which improved the previous ver- age in mtDNA (2.9% in 16S), but its nuclear genes bear signatures sion of the manuscript. The study was supported by the Ministry of gene flow to/from the Southern H. intermedia (Tyr; see also of Culture of the Czech Republic (DKRVO 2014/14, National Canestrelli et al., 2007b) and H. arborea/H. orientalis (Rhod; see also Museum, 00023272). ER is supported by a DGAPA-UNAM postdoc- Verardi et al., 2009). Our limited sampling of H. sarda did not toral fellowship. recover a clear phylogeographic pattern, which was addressed in detail elsewhere (Bisconti et al., 2011a,b). Appendix A. Supplementary material

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

Václav Gvoždík, Daniele Canestrelli, Mario García-París, Jiří Moravec, Giuseppe Nascetti, Ernesto Recuero, José Teixeira, Petr Kotlík

Molecular Phylogenetics and Evolution Table S1. Specimens examined.

Haplotype Species tree Species delimitation Map Taxon: Hyla Sample ID Tyr IMgc1a Country Locality N E Voucher/Reference ID ID (/alternative) mtDNA Tyr A Tyr B Rhod A Rhod B

1 arborea arborea arborea H488 Switzerland Aargau region 47.49 08.22 - mtarb CH1 Tarb 1 Tarb 1 Rarb 1 Rarb 1 2a arborea arborea arborea HJ66 Switzerland Lake Geneva, Aubonne 46.48 06.40 - mtarb CH1 - - - - 2b arborea arborea arborea HCE1 Switzerland Lake Geneva, near Lausanne - - - mtarb CH1 - - Rarb 1 Rarb 1 2b arborea arborea arborea HCE2 Switzerland Lake Geneva, near Lausanne - mtarb CH1 - - - - 3 arborea arborea arborea H485 France Villars-les-Dombes 46.01 04.94 - mtarb FR1 Tarb 1 Tarb 1 Rarb 1 Rarb 1 3 arborea arborea arborea H486 France Villars-les-Dombes - mtarb CH1 - - - - 3 arborea arborea arborea H487 France Villars-les-Dombes - mtarb CH1 - - - - arborea 4 arborea (x molleri ) arborea HM1 Tyr B France Monterfil, Bretagne 48.05 -01.97 - mtarb FR2 Tmol 1 Tmol 6 Rarb 1 Rarb 1 (Tyr omitted) 5 arborea arborea arborea HJ47 Germany Munich 49.15 11.58 - mtarb CH1 - - - - 6 arborea arborea arborea HJ46 Germany Fabrikschleichach, Bavaria 49.92 10.55 - mtarb CH1 - - - - 7 arborea arborea arborea HJ45 Germany Schmittenhöhe, Koblenz 50.28 07.45 - mtarb CH1 Tarb 1 Tarb 1 Rarb 2 Rarb 1 8 arborea arborea arborea H278 Germany Papitz, Leipzig 51.37 12.23 - mtarb CH1 Tarb 1 Tarb 1 Rarb 1 Rarb 1 8 arborea arborea arborea H279 Germany Papitz, Leipzig - mtarb CH1 - - - - 8 arborea arborea arborea H280 Germany Papitz, Leipzig - mtarb CH1 - - - - 8 arborea arborea arborea H281 Germany Papitz, Leipzig - mtarb CH1 - - - - 9 arborea arborea arborea HJ55 Netherlands Lichtenvoorde 52.00 06.47 - mtarb CH1 Tarb 1 Tarb 1 (Rarb 3) c (Rarb 1?2) c 10 arborea arborea arborea HJ42 Denmark Vejle, Jutland 55.65 09.50 - mtarb CH1 - - - - 11 arborea arborea arborea HJ44 Denmark Lolland Island 54.77 11.52 - mtarb CH1 - - - - 12 arborea arborea arborea HJ43 Denmark Bornholm Island 55.07 14.93 - mtarb CH1 - - Rarb 1 Rarb 1 13 arborea arborea arborea HJ57 Tyr B Poland Cybinka 52.20 14.78 - mtarb PL1 Tarb 1 Tarb 15 Rarb 1 Rarb 1 14 arborea arborea arborea HJ56 Poland Pruszowice, Wroclaw 51.18 17.13 - mtarb PL2 - - - - 15 arborea arborea arborea H546 Poland Zebrzydowice 49.88 18.65 - mtarb CH1 Tarb 1 Tarb 1 Rarb 1 Rarb 1 15 arborea arborea arborea H545 Poland Zebrzydowice - mtarb CH1 - - - - 16 arborea arborea arborea H269 Czech Rep. Slavkov, Opava 49.92 17.83 - mtarb CH1 Tarb 1 Tarb 1 Rarb 1 Rarb 1 16 arborea arborea arborea H270 Czech Rep. Slavkov, Opava - mtarb CH1 - - - - 16 arborea arborea arborea H271 Czech Rep. Slavkov, Opava - mtarb CH1 - - - - 17 arborea arborea arborea H383 Czech Rep. Borovec, Nový Jičín 49.63 18.10 - mtarb CH1 - - - - 17 arborea arborea arborea H384 Czech Rep. Borovec, Nový Jičín - mtarb GR7 - - - - 18 arborea arborea arborea H551 Czech Rep. Žlutava, Otrokovice 49.20 17.48 - mtarb CH1 Tarb 1 Tarb 1 Rarb 1 Rarb 1 19 arborea arborea arborea H146 Czech Rep. Ořešín, Brno 49.28 16.60 NMP6V 71523 mtarb CZ2 - - - - 20 arborea arborea arborea H550 Czech Rep. Nový Hubenov, Jihlava 49.40 15.47 - mtarb CH1 - - - - 21 arborea arborea arborea H272 Czech Rep. Veselí nad Lužnicí 49.18 14.70 - mtarb CH1 - - - - 21 arborea arborea arborea H273 Czech Rep. Veselí nad Lužnicí - mtarb CZ1 - - - - 21 arborea arborea arborea H274 Czech Rep. Veselí nad Lužnicí - mtarb CH1 - - - - 22 arborea arborea arborea H221 Czech Rep. Nalžovice 49.70 14.37 - mtarb CH1 Tarb 1 Tarb 1 Rarb 2 Rarb 2 22 arborea arborea arborea H222 Czech Rep. Nalžovice - mtarb CH1 - - - - 22 arborea arborea arborea H223 Czech Rep. Nalžovice - mtarb CH1 - - - - 22 arborea arborea arborea H224 Czech Rep. Nalžovice - mtarb CH1 - - - - 22 arborea arborea arborea H226 Czech Rep. Nalžovice - mtarb CH1 - - - - 23 arborea arborea arborea H211 Czech Rep. Brník 49.98 14.90 - mtarb CZ3 Tarb 1 Tarb 1 Rarb 2 Rarb 1 23 arborea arborea arborea H212 Czech Rep. Brník - mtarb CH1 - - - - 24 arborea arborea arborea H500 Czech Rep. Luh nad Svatavou 50.21 12.61 - mtarb CH1 Tarb 1 Tarb 1 Rarb 1 Rarb 1 24 arborea arborea arborea H501 Czech Rep. Luh nad Svatavou - mtarb CH1 - - - - 25 arborea arborea arborea HJ39 Austria Hörndlwald, Wien 48.17 16.25 - mtarb CH1 Tarb 1 Tarb 2 Rarb 1 Rarb 1 26 arborea arborea arborea H463 Hungary Kiskunfélegyháza 46.71 19.85 NMP6V 72920 mtarb HU1 Tarb 1 Tarb 1 Rarb 2 Rarb 2 27 arborea arborea arborea H541 Slovakia Muránska Lehota, Revúca 48.73 20.03 - mtarb CH1 - - - - 28 arborea arborea arborea H290 Slovakia Streda nad Bodrogom 48.37 21.77 - mtarb CH1 - - - - 28 arborea arborea arborea H291 Slovakia Streda nad Bodrogom - mtarb CH1 - - - - 29 arborea arborea arborea H538 Romania Finatale Clujuluj 46.83 23.62 - mtarb CH1 Tarb 1 Tarb 1 Rarb 2 Rarb 1 29 arborea arborea arborea H539 Romania Finatale Clujuluj - mtarb CH1 Tarb 1 Tarb 1 Rarb 2 Rarb 1 30 arborea - ('hybrid') arborea /- ('hybrid') H389 Romania Armenis 45.20 22.32 NMP6V 72696 mtarb CH1 Tarb 1 Tarb 2 Rarb 1 Rori 1 31 arborea arborea arborea HJ41 Tyr B Croatia Zdenci, Orahovica 45.59 17.95 - mtarb HU1 Tarb 1 Tarb 14 Rarb 1 Rarb 1 32 arborea arborea arborea HJ40 Tyr B Croatia Crna Mlaka, Jastrebarsko 45.61 15.73 - mtarb GR7 Tarb 2 Tarb 14 Rarb 2 Rarb 1 33 arborea W Balkan W Balkan/arborea HJ64 Slovenia Povir 45.70 13.95 - mtarb CH1 Tarb 2 Tarb 2 Rarb 2 Rori 6 34 arborea W Balkan W Balkan/arborea H537 Tyr B Croatia Krk Island 45.03 14.55 - mtarb HR2 Tarb 12 Tarb 14 Rori 6 Rori 6 35 arborea W Balkan W Balkan/arborea H459 Tyr B Croatia Pag Island 44.45 15.06 - mtarb HR1 Tarb 1 Tarb 14 Rori 6 Rori 6 35 arborea arborea arborea H460 Croatia Pag Island - mtarb HR1 - - - - 36 arborea - - DQ055835 Croatia Donja Korita, Dalmatia 43.70 16.81 Smith et al. (2005) DQ055835 - - - - 37 arborea W Balkan W Balkan/arborea HJ54 Montenegro Tivat 42.43 18.70 - mtarb CH1 Tarb 6 Tarb 6 Rori 1 Rori 1 38 arborea W Balkan W Balkan/arborea H142 Tyr AB Greece Korfu Island, Vatos 39.61 19.79 NMP6V 71879 mtarb GR10 Tarb 5 Tarb 9 Rori 1 Rori 1 39 arborea arborea arborea H489 Greece Korfu Island, Korision 39.44 19.94 - mtarb GR9 - - - - 40 arborea - ('hybrid') arborea /- ('hybrid') H289 Tyr B Greece Zakynthos Island 37.78 20.88 - mtarb GR2 Tarb 1 Tarb 11 Rarb 2 Rori 1 40 arborea arborea arborea H288 Greece Zakynthos Island - mtarb GR4 - - - - 41 arborea - ('hybrid') arborea /- ('hybrid') HD13A Greece Ilis, Peloponnese 37.87 21.37 - mtarb GR12 Tarb 1 Tarb 1 Rori 1 Rori 1 41 arborea - ('hybrid') arborea /- ('hybrid') HD13B Tyr B Greece Ilis, Peloponnese - mtarb GR11 Tarb 1 Tarb 10 Rori 1 Rarb 2 42 arborea - ('hybrid') arborea /- ('hybrid') H524 Tyr AB Greece Gialova, Peloponnese 36.95 21.70 - mtarb GR8 Tarb 8 Tarb 8 Rori 1 Rori 1 42 arborea arborea arborea H523 Tyr AB Greece Gialova, Peloponnese - mtarb GR2 Tarb 13 Tarb 11 Rarb 2 Rarb 2 42 arborea arborea arborea H525 Greece Gialova, Peloponnese - mtarb GR2 - - - - 43 arborea arborea arborea HJ49 Tyr B Greece Crete, Agia Lake, Kydonias 35.47 23.93 - mtarb GR5 Tarb 3 Tarb 10 Rarb 1 Rarb 1 44 arborea arborea arborea HD20A Tyr B Greece Crete, Agios Georgios 35.13 24.70 - mtarb GR5 Tarb 1 Tarb 16 Rarb 2 Rarb 2 44 arborea arborea arborea HD20B Greece Crete, Agios Georgios - mtarb GR5 - - - - 44 arborea arborea arborea HD20C Greece Crete, Agios Georgios - mtarb GR6 - - - - 45 arborea arborea arborea HD11A Tyr A Greece Axios 40.62 22.71 - mtarb CH1 Tarb 4 Tarb 3 Rarb 2 Rarb 2 45 arborea arborea arborea HD11B Greece Axios - mtarb GR1 - - - - 45 arborea arborea arborea HD11C Greece Axios - mtarb CH1 - - - - 46 arborea - ('hybrid') arborea /- ('hybrid') H549 Greece Kalamitsi, Sithonia, Chalkidiki 39.98 23.98 - mtarb GR3 Tarb 7 Tarb 7 Rori 1 Rori 1 47 arborea x orientalis - (hybrid) arborea /- (hybrid) HJ48 Greece Imeros, Komotini 40.93 25.28 - mtarb GR7 Tarb 7 Tori 1 Rori 1 Rori 1 48 arborea x orientalis b - (hybrid) arborea /- (hybrid) H548 Poland Spytkowice 49.98 19.50 - mtarb CH1 Tori 1 Tarb 1 Rori 1 Rarb 1 48 orientalis orientalis orientalis H547 Poland Spytkowice - mtori UA2 - - - - 49 arborea x orientalis - (hybrid) orientalis /- (hybrid) H543 Poland Wodzisław Małopolski 50.52 20.18 - mtori UA2 Tarb 1 Tarb 1 Rori 1 Rori 1 49 orientalis orientalis orientalis H544 Poland Wodzisław Małopolski - mtori UA2 - - - - 50 orientalis - - H542 Poland Borówna 49.90 20.53 - 12Sori PL1 - - - - 51 arborea x orientalis - (hybrid) orientalis /- (hybrid) H536 Tyr B Bulgaria Vidin 43.98 22.87 - mtori BG4 Tarb 1 Tori 11 Rori 1 Rori 1 51 orientalis orientalis orientalis H535 Bulgaria Vidin - mtori BG3 Tori 1 Tori 1 Rori 1 Rori 6 52 orientalis orientalis orientalis H208 Bulgaria Montana 43.38 23.22 - mtori BG1 Tori 10 Tori 2 Rori 7 Rori 1 52 orientalis orientalis orientalis H209 Bulgaria Montana - mtori TR14 - - - - 52 orientalis orientalis orientalis H210 Bulgaria Montana - mtori BG2 - - - - 53 orientalis orientalis orientalis H462 Bulgaria Sadovo 42.13 24.93 - mtori TR14 - - - - 54 orientalis orientalis orientalis H267 Bulgaria Železino, Ivajlovgrad 41.48 26.01 NMP6V 73809 mtori UA2 - - - - 55 orientalis orientalis orientalis H470 Romania Insula Micā a Brāilei 44.90 27.83 - mtori TR14 - - - - 55 orientalis orientalis orientalis H471 Romania Insula Micā a Brāilei - mtori TR14 - - - - 55 orientalis orientalis orientalis H472 Romania Insula Micā a Brāilei - mtori TR14 - - - - 56 orientalis orientalis orientalis H363 Romania Cetatea Histria, Istria 44.57 28.72 - mtori TR14 - - - - 56 orientalis orientalis orientalis H364 Romania Cetatea Histria, Istria - mtori RO3 - - - - 56 orientalis orientalis orientalis H365 Romania Cetatea Histria, Istria - mtori TR14 - - - - 57 orientalis orientalis orientalis H213 Romania Nufaru 45.15 28.92 - mtori TR14 Tori 1 Tori 1 Rori 1 Rori 1 57 orientalis orientalis orientalis H214 Romania Nufaru - mtori RO1 - - - - 57 orientalis orientalis orientalis H215 Romania Nufaru - mtori RO2 - - - - 58 orientalis orientalis orientalis H268 Ukraine Gerojske 46.52 31.90 - mtori UA2 - - - - 59 orientalis orientalis orientalis H207 Ukraine Rybal'če 46.48 32.19 - mtori UA2 Tori 1 Tori 1 Rori 1 Rori 1 60 orientalis orientalis orientalis HLi48 Ukraine Balakleya (Iskov prud), Kharkov 49.65 36.27 - mtori UA1 - - - - 60 orientalis orientalis orientalis HLi49 Ukraine Balakleya (Iskov prud), Kharkov - mtori UA1 Tori 1 Tori 10 Rori 1 Rori 1 61 orientalis orientalis orientalis H492 Ukraine Crimean Mts. 44.44 34.05 NMP6V 74296 mtori UA2 Tori 10 Tori 10 Rori 1 Rori 1 62 orientalis orientalis orientalis HJ50 Greece Lesbos Island, Vatoussa 39.23 26.05 - mtori GR1 - - - - 63 orientalis orientalis orientalis HJ51 Greece Rhodos Island, Maritsa 36.38 28.10 - mtori GR2 - - - - 64 orientalis orientalis orientalis H156 Turkey Havsa 41.55 26.82 NMP6V 72533/1 mtori TR18 - - - - 64 orientalis orientalis orientalis H157a Turkey Havsa NMP6V 72533/2 mtori TR18 - - - - 64 orientalis orientalis orientalis H158a Turkey Havsa NMP6V 72533/3 mtori TR19 Tori 1 Tori 1 Rori 1 Rori 1 64 orientalis orientalis orientalis H159 Turkey Havsa - mtori TR20 - - - - orientalis orientalis 65 orientalis H160a Tyr AB Turkey Selçuk - Efes 37.95 27.37 NMP6V 72534/1 mtori TR1 Tfel 3 Tori 5 Rori 1 Rori 1 (Tyr omitted) (Tyr omitted) orientalis orientalis 65 orientalis H204 Tyr AB Turkey Selçuk - Efes NMP6V 72534/2 mtori TR1 Tori 5 Tori 7 Rori 1 Rori 1 (Tyr omitted) (Tyr omitted) 65 orientalis orientalis orientalis H205 Turkey Selçuk - Efes NMP6V 72534/3 mtori TR1 - - - - 66 orientalis orientalis orientalis H461 Turkey Bafa Gölü 37.47 27.50 - mtori TR14 - - - - orientalis orientalis 67 orientalis HD14A Tyr AB Turkey Marmaris 36.87 28.27 - mtori TR16 Tori 8 Tori 9 Rori 1 Rori 5 (Tyr omitted) (Tyr omitted) 67 orientalis orientalis orientalis HD14B Turkey Marmaris - mtori TR14 - - - - 67 orientalis orientalis orientalis HD14C Turkey Marmaris - mtori TR17 - - - - 68 orientalis orientalis orientalis H388 Turkey Dalaman 36.77 28.80 NMP6V 70833 mtori TR9 - - - - orientalis orientalis 69 orientalis H161a Tyr AB Turkey Syedra, 15 km E Alanya 36.48 32.12 NMP6V 72535/1 mtori TR2 Tori 6 Tfel 3 Rori 1 Rori 1 (Tyr omitted) (Tyr omitted) 69 orientalis orientalis orientalis H162 Turkey Syedra, 15 km E Alanya - mtori TR3 - - - - 69 orientalis orientalis orientalis H163 Turkey Syedra, 15 km E Alanya - mtori TR2 - - - -

2 orientalis orientalis 70 orientalis H164a Tyr AB Turkey Gazipaşa 36.27 32.32 NMP6V 72536/1 mtori TR4 Tfel 3 Tfel 3 Rori 1 Rori 1 (Tyr omitted) (Tyr omitted) 70 orientalis orientalis orientalis H165 Turkey Gazipaşa NMP6V 72536/2 mtori TR5 - - - - orientalis orientalis 71 orientalis H166a Tyr AB Turkey Melleç 36.07 32.70 - mtori TR6 Tfel 3 Tfel 3 Rori 1 Rori 1 (Tyr omitted) (Tyr omitted) 72 orientalis orientalis orientalis H446 Turkey İnebolu 41.97 33.76 NMP6V 73700/2 mtori TR14 Tori 1 Tori 1 Rori 1 Rori 1 72 orientalis orientalis orientalis H447 Turkey İnebolu NMP6V 73700/1 mtori TR15 - - - - 72 orientalis orientalis orientalis H448 Turkey İnebolu - mtori TR15 - - - - 73 orientalis orientalis orientalis H429 Turkey Sinop 42.03 35.16 NMP6V 73698/2 mtori TR11 - - - - 73 orientalis orientalis orientalis H430 Turkey Sinop NMP6V 73698/1 mtori TR12 - - - - 73 orientalis orientalis orientalis H431 Turkey Sinop NMP6V 73698/3 mtori TR13 - - - - 74 orientalis orientalis orientalis H428 Turkey Kumru, 5 km NE 40.87 37.26 NMP6V 73697 mtori TR10 Tori 4 Tori 4 Rori 1 Rori 1 75 orientalis orientalis orientalis H216 Turkey Hopa 41.41 41.44 - mtori TR7 Tori 1 Tori 1 Rori 1 Rori 4 75 orientalis orientalis orientalis H217 Turkey Hopa NMP6V 72688/1 mtori TR8 - - - - 75 orientalis orientalis orientalis H218 Turkey Hopa NMP6V 72688/2 mtori TR8 - - - - 75 orientalis orientalis orientalis H219 Turkey Hopa NMP6V 72688/3 mtori TR8 - - - - 75 orientalis orientalis orientalis H220 Turkey Hopa - mtori TR8 - - - - 76 orientalis orientalis orientalis H420 Georgia Kutaisi 42.26 42.72 - mtori TR8 Tori 1 Tori 4 Rori 1 Rori 1 77 orientalis orientalis orientalis H421 Georgia Tsodoreti Lake, 10 km W Tbilisi 41.76 44.65 - mtori TR8 - - - - 77 orientalis orientalis orientalis H422 Georgia Tsodoreti Lake, 10 km W Tbilisi - mtori TR8 - - - - 77 orientalis orientalis orientalis H423 Georgia Tsodoreti Lake, 10 km W Tbilisi - mtori GE1 - - - - 78 orientalis orientalis orientalis H407 Georgia Telavi 41.92 45.49 NMP6V 73713/2 mtori TR8 Tori 3 Tori 3 Rori 1 Rori 1 78 orientalis orientalis orientalis H408 Georgia Telavi NMP6V 73713/1 mtori TR8 - - - - 78 orientalis orientalis orientalis H409 Georgia Telavi NMP6V 73713/3 mtori TR8 - - - - 79 orientalis orientalis orientalis H392 Azerbaijan Kiş, Şəki 41.26 47.19 NMP6V 73709/1 mtori AZ1 Tori 1 Tori 3 Rori 1 Rori 1 79 orientalis orientalis orientalis H393 Azerbaijan Kiş, Şəki NMP6V 73709/2 mtori AZ1 - - - - 79 orientalis orientalis orientalis H394 Azerbaijan Kiş, Şəki NMP6V 73709/3 mtori TR8 - - - - 80 orientalis orientalis orientalis HJ62 Russia Malyj Utrish, Anapa 44.72 37.47 - mtori RU1 Tori 1 Tori 1 Rori 1 Rori 1 81 orientalis orientalis orientalis HJ63 Russia Guzeripl', Adygea Rep. 43.83 40.20 - mtori TR8 Tori 1 Tori 1 Rori 1 Rori 1 82 orientalis orientalis orientalis H257 Iran Motalla Sara-ye Lemir 38.20 48.87 NMP6V 72677/2 mtori TR8 Tori 1 Tori 1 Rori 1 Rori 1 82 orientalis orientalis orientalis H258 Iran Motalla Sara-ye Lemir NMP6V 72677/3 mtori TR8 - - - - 82 orientalis orientalis orientalis H259 Iran Motalla Sara-ye Lemir NMP6V 72677/1 mtori TR8 - - - - 83 orientalis orientalis orientalis H390 Iran Chayjan, Gilan 37.01 50.50 NMP6V 72842 mtori TR8 Tori 1 Tori 1 Rori 1 Rori 3 84 orientalis orientalis orientalis H251 Iran Tonekabon 36.81 50.88 NMP6V 72676/4 mtori IR1 Tori 1 Tori 1 Rori 1 Rori 2 84 orientalis orientalis orientalis H252 Iran Tonekabon - mtori IR2 - - - - 84 orientalis orientalis orientalis H253 Iran Tonekabon - mtori IR3 - - - - 85 orientalis orientalis orientalis H362a Iran Babol Sar 36.68 52.65 - mtori IR2 Tori 1 Tori 2 Rori 1 Rori 2 86 molleri molleri molleri HJ60 Tyr B Portugal Carvalhal, Odeceixe 37.48 -08.75 - mtmol 1 Tmol 1 Tmol 5 Rmol 1 Rmol 1 87 molleri molleri molleri HJ58 Portugal Mindelo 41.32 -08.73 - mtmol 6 Tmol 1 Tmol 1 Rmol 1 Rmol 1 88 molleri molleri molleri HE098A Spain Pontevedra 42.43 -08.63 - mtmol 7 - - - - 88 molleri molleri molleri HE098B Spain Pontevedra - mtmol 7 Tmol 3 Tmol 3 Rmol 1 Rmol 1 - molleri molleri molleri HC097 Spain NW Spain --- mtmol 4 - - - - 89 molleri molleri molleri HE092A Spain Chillón, Ciudad Real 38.80 -04.87 - mtmol 2 - - Rmol 1 Rmol 1 89 molleri molleri molleri HE092B Spain Chillón, Ciudad Real - mtmol 2 - - - - 90 molleri molleri molleri HD18A Spain Toledo 39.87 -04.03 - mtmol 5 Tmol 2 Tmol 4 Rmol 1 Rmol 1 90 molleri molleri molleri HD18B Spain Toledo - mtmol 1 - - - - 91 molleri molleri molleri HE095A Spain Villaverde, Albacete 38.80 -02.37 - mtmol 1 - - - - 91 molleri molleri molleri HE095B Spain Villaverde, Albacete - mtmol 1 - - Rmol 1 Rmol 1 92 molleri molleri molleri HE096 Spain Buenache, Cuenca 39.65 -02.17 - mtmol 2 Tmol 2 Tmol 2 Rmol 1 Rmol 1 93 molleri molleri molleri HE093A Spain Larúes, Huesca 42.52 -00.85 - mtmol 2 Tmol 1 Tmol 1 Rmol 1 Rmol 1 93 molleri molleri molleri HE093B Spain Larúes, Huesca - mtmol 3 - - - - 94 intermedia S intermedia S intermedia S HD01A Italy Sicily, Gibilmanna 37.92 14.02 - mtint S2 Tint 1 Tint 1 Rint S1 Rint S1 94 intermedia S intermedia S intermedia S HD01B Italy Sicily, Gibilmanna - mtint S1 - - - - 94 intermedia S intermedia S intermedia S HD01C Italy Sicily, Gibilmanna - mtint S1 - - - - 95 intermedia S intermedia S intermedia S H563 Italy Sicily, Nebrodi Mts., pool 1 37.94 14.67 - mtint S1 Tint 1 Tint 1 Rint S1 Rint S1 96 intermedia S intermedia S intermedia S H562 Italy Sicily, Nebrodi Mts., pool 2 37.95 14.72 NMP6V 74149 mtint S1 Tint 1 Tint 2 Rint S1 Rint S1 97 intermedia S intermedia S intermedia S H560 Italy Sicily, Lentini 37.30 14.98 NMP6V 74148/1 mtint S1 Tint 1 Tint 1 Rint S1 Rint S1 97 intermedia S intermedia S intermedia S H561 Italy Sicily, Lentini NMP6V 74148/2 mtint S4 Tint 3 Tint 4 Rint S1 Rint S1 98 intermedia S intermedia S intermedia S HD02A Italy Pizzo, Calabria 38.73 16.17 - mtint S3 Tint 2 Tint 2 Rint S1 Rint S1 98 intermedia S intermedia S intermedia S HD02B Italy Pizzo, Calabria - mtint S5 - - - - 98 intermedia S intermedia S intermedia S HD02C Italy Pizzo, Calabria - mtint S3 - - - - 99 intermedia S intermedia S intermedia S HD03A Tyr B Italy San Giovanni Rotondo 41.72 15.72 - mtint S7 (Tint 7) c (Tint 8) c Rint S1 Rint S1 99 intermedia S intermedia S intermedia S HD03B Italy San Giovanni Rotondo - mtint S8 - - - - 99 intermedia S intermedia S intermedia S HD03C Italy San Giovanni Rotondo - mtint S6 - - - - 100 intermedia S intermedia S intermedia S HD07A Italy Firenze 43.82 11.47 - mtint S8 Tint 2 Tint 2 Rint S1 Rint S1 100 intermedia S intermedia S intermedia S HD07B Italy Firenze - mtint S10 - - - - 100 intermedia S intermedia S intermedia S HD07C Italy Firenze - mtint S9 - - - - 101 intermedia N intermedia N intermedia N HD04A Italy Novara 45.48 08.65 - mtint N1 Tint 1 Tint 1 Rori 1 Rori 1 101 intermedia N intermedia N intermedia N HD04B Tyr B Italy Novara - mtint N2 (Tint 1?10) d (Tint 9) d Rori 1 Rori 1 101 intermedia N intermedia N intermedia N HD04C Italy Novara - mtint N1 Tint 5 Tint 6 Rori 1 Rint N1 102 sarda sarda sarda HE097 France Corsica, Bocca de l'Oru 41.55 09.28 - mtsar 1 Tsar 3 Tsar 4 Rsar 1 Rsar 1 103 sarda sarda sarda H458 Italy Sardinia, Badesi at Valledoria 40.97 08.88 NMP6V 70598 mtsar 1 - - - - 104 sarda sarda sarda HD06A Italy Sardinia, Lago di Posada 40.63 09.75 - mtsar 4 Tsar 2 Tsar 3 Rsar 1 Rsar 2 104 sarda sarda sarda HD06B Italy Sardinia, Lago di Posada - mtsar 5 - - - - 104 sarda sarda sarda HD06C Italy Sardinia, Lago di Posada - mtsar 4 - - - - 105 sarda sarda sarda H497a Italy Sardinia, Marina di Torre Grande 39.90 08.53 - mtsar 6 - - - - 106 sarda sarda sarda HD05A Italy San Pietro Island 39.15 08.28 - mtsar 2 Tsar 1 Tsar 1 Rsar 1 Rsar 1 106 sarda sarda sarda HD05B Italy San Pietro Island - mtsar 2 - - - - 106 sarda sarda sarda HD05C Italy San Pietro Island - mtsar 3 - - - - - sarda sarda sarda HC112 Italy Sardinia --- mtsar 7 - - - - - meridionalis HE101A Spain Tenerife Island, Puerto de la Cruz 28.38 -16.55 - mtmer ES1 Tmer 1 Tmer 1 Rmer 1 Rmer 1 - japonica Japan Yasufutuichi-cho, Aito, Hiroshima Faivovich et al. (2005) AY843633 AY844078 AY844615 a omitted haplotype is listed b possibly F1 hybrid c gametic haplotypes inferred with mean probability 0.50 in one from two heterozygous sites d gametic haplotypes inferred with mean probability 0.69 in one from three heterozygous sites NMP6V, National Museum, Department of Zoology, Prague

3 Table S2. GenBank accession numbers. Haplotype codes in parentheses mark nucleotide sequences with the standard IUPAC ambiguity codes when heterozygous positions remained unresolved.

mtDNA GenBank Rhod Tyr GenBank GenBank haplotype (composite 12S /16S ) haplotype haplotype mtarb CH1 KP109551 / KP109580 Rarb 1 KP109629 Tarb 1 KP109639 mtarb CZ1 KP109551 / KP109581 Rarb 2 KP109630 Tarb2 KP109640 mtarb CZ2 KP109551 / KP109582 (Rarb 3) KP109631 Tarb 3 KP109641 mtarb CZ3 KP109552 / KP109580 Rori 1 GQ916815 Tarb 4 KP109642 mtarb FR1 KP109551 / KP109583 Rori 2 GQ916816 Tarb 5 KP109643 mtarb FR2 KP109551 / KP109584 Rori 3 GQ916817 Tarb 6 KP109644 mtarb GR1 KP109551 / KP109585 Rori 4 GQ916818 Tarb 7 KP109645 mtarb GR2 KP109553 / KP109580 Rori 5 GQ916819 Tarb 8 KP109646 mtarb GR3 KP109553 / KP109586 Rori 6 KP109632 Tarb 9 KP109647 mtarb GR4 KP109554 / KP109580 Rori 7 KP109633 Tarb 10 KP109648 mtarb GR5 KP109553 / KP109587 Rmol 1 KP109634 Tarb 11 KP109649 mtarb GR6 KP109553 / KP109588 Rint S1 KP109635 Tarb 12 KP109650 mtarb GR7 KP109553 / KP109589 Rint N1 KP109636 Tarb 13 KP109651 mtarb GR8 KP109555 / KP109590 Rsar 1 KP109637 Tarb 14 KP109652 mtarb GR9 KP109556 / KP109591 Rsar 2 KP109638 Tarb 15 KP109653 mtarb GR10 KP109553 / KP109592 Rmer 1 GQ916820 Tarb 16 KP109654 mtarb GR11 KP109553 / KP109593 Tori 1 GQ916713 mtarb GR12 KP109553 / KP109594 Tori 2 GQ916714 mtarb HR1 KP109557 / KP109595 Tori 3 GQ916715 mtarb HR2 KP109551 / KP109596 Tori 4 GQ916716 mtarb HU1 KP109558 / KP109580 Tori 5 GQ916717 mtarb PL1 KP109559 / KP109580 Tori 6 GQ916718 mtarb PL2 KP109551 / KP109597 Tori 7 GQ916719 mtori AZ1 GQ916749 / GQ916803 Tori 8 GQ916720 mtori BG1 GQ916746 / KP109598 Tori 9 GQ916721 mtori BG2 GQ916746 / KP109599 Tori 10 KP109655 mtori BG3 KP109560 / GQ916792 Tori 11 KP109656 mtori BG4 GQ916746 / KP109600 Tfel 3 GQ916708 mtori GE1 GQ916751 / GQ916798 Tmol 1 KP109657 mtori GR1 KP109561 / KP109601 Tmol 2 KP109658 mtori GR2 GQ916746 / GQ916802 Tmol 3 KP109659 mtori IR1 GQ916749 / GQ916799 Tmol 4 KP109660 mtori IR2 GQ916749 / GQ916800 Tmol 5 KP109661 mtori IR3 GQ916749 / GQ916801 Tmol 6 KP109662 mtori RO1 GQ916746 / KP109602 Tint 1 KP109663 mtori RO2 GQ916746 / KP109603 Tint 2 KP109664 mtori RO3 KP109562 / GQ916792 Tint 3 KP109665 mtori RU1 GQ916749 / KP109604 Tint 4 KP109666 mtori TR1 GQ916745 / GQ916792 Tint 5 KP109667 mtori TR2 GQ916746 / GQ916793 Tint 6 KP109668 mtori TR3 GQ916746 / GQ916794 (Tint 7/8) KP109669 mtori TR4 GQ916746 / GQ916795 (Tint 9/10) KP109670 mtori TR5 GQ916747 / GQ916793 Tsar 1 KP109671 mtori TR6 GQ916748 / GQ916796 Tsar 2 KP109672 mtori TR7 GQ916746 / GQ916797 Tsar 3 KP109673 mtori TR8 GQ916749 / GQ916798 Tsar 4 KP109674 mtori TR9 GQ916750 / GQ916802 Tmer 1 GQ916722 mtori TR10 GQ916746 / GQ916804 mtori TR11 GQ916746 / GQ916805 mtori TR12 GQ916746 / GQ916806 mtori TR13 GQ916746 / GQ916807 mtori TR14 GQ916746 / GQ916792 mtori TR15 GQ916746 / GQ916808 mtori TR16 GQ916752 / GQ916809 mtori TR17 GQ916750 / GQ916809 mtori TR18 KP109563 / KP109605 mtori TR19 GQ916746 / KP109606 mtori TR20 GQ916746 / KP109607 mtori UA1 GQ916746 / KP109608 mtori UA2 GQ916746 / KP109609 12Sori PL1 KP109564 / ------mtmol 1 KP109565 / KP109610 mtmol 2 KP109565 / KP109611 mtmol 3 KP109565 / KP109612 mtmol 4 KP109565 / KP109613 mtmol 5 KP109566 / KP109610 mtmol 6 KP109567 / KP109614 mtmol 7 KP109568 / KP109615 mtint S1 KP109569 / KP109616 mtint S2 KP109569 / KP109617 mtint S3 KP109570 / KP109616 mtint S4 KP109571 / KP109616 mtint S5 KP109572 / KP109616 mtint S6 KP109573 / KP109618 mtint S7 KP109574 / KP109616 mtint S8 KP109575 / KP109619 mtint S9 KP109574 / KP109619 mtint S10 KP109575 / KP109620 mtint N1 KP109576 / KP109621 mtint N2 KP109576 / KP109622 mtsar 1 KP109577 / KP109623 mtsar 2 KP109577 / KP109624 mtsar 3 KP109577 / KP109625 mtsar 4 KP109578 / KP109623 mtsar 5 KP109579 / KP109626 mtsar 6 KP109577 / KP109627 mtsar 7 KP109577 / KP109628 mtmer ES1 GQ916753 / GQ916810

4 Table S3. Results of the Bayesian species delimitation (BSD) analyses conducted in BP&P.

Dataset Tree topology and BSD result (#speciation probability) ~ Species tree (data) Conditions a mtDNA + nDNA (Tyr IMgc1) ((((ori, mol)'#1.00', (arb, Bal)'#1.00')'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; mtDNA + nDNA + 'hybrids'; - recombination mtDNA + nDNA (Tyr IMgc1) ((((ori, mol)'#1.00', arb)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; + 'hybrids'; - recombination mtDNA + nDNA (Tyr IMgc1) ((((ori, arb)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; + 'hybrids'; - recombination mtDNA + nDNA (Tyr IMgc1) ((((ori, arb)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; + 'hybrids'; - recombination; without W Balkan nDNA (((arb, Bal)'#1.00', sar)'#1.00', (((intN, intS)'#1.00', mol)'#1.00', ori)'#1.00')'#1.00'; nDNA - 'hybrids'; + recombination nDNA ((((ori, mol)'#1.00', (arb, Bal)'#1.00')'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA ((((ori, Bal)'#1.00', (arb, mol)'#1.00')'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA ((((ori, mol)'#1.00', arb)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA ((((ori, arb)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA ((((arb, mol)'#1.00', ori)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA (((((ori, mol)'#1.00', arb)'#1.00', intN)'#1.00', intS)'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA (((((ori, arb)'#1.00', mol)'#1.00', intN)'#1.00', intS)'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA (((ori, mol)'#1.00', arb)'#1.00', ((intN, intS)'#1.00', sar)'#1.00')'#1.00'; - 'hybrids'; + recombination nDNA (((((ori, intN)'#1.00', mol)'#1.00', arb)'#1.00', intS)'#1.00', sar)'#1.00'; - 'hybrids'; + recombination nDNA ((((arb, ori)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; + 'hybrids'; + recombination nDNA ((((arb, mol)'#1.00', ori)'#1.00', (intS, intN)'#1.00')'#1.00', sar)#1.00'; + 'hybrids'; + recombination nDNA (Tyr IMgc1) ((((arb, ori)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)#1.00'; + 'hybrids'; - recombination nDNA (Tyr IMgc1) ((((arb, ori)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)#1.00'; + 'hybrids'; - recombination Rhod ((((ori, mol)'#1.00', (arb, Bal)'#1.00')'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; mtDNA + nDNA - 'hybrids' Rhod ((((ori, Bal)'#0.99', (arb, mol)'#1.00')'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00'; - 'hybrids' Tyr ((((ori, mol)'#1.00', (arb, Bal)'#0.32')'#1.00', (intS, intN)'#0.49')'#1.00', sar)'#1.00'; mtDNA + nDNA - 'hybrids'; + recombination Tyr ((((ori, Bal)'#1.00', (arb, mol)'#1.00')'#1.00', (intS, intN)'#0.60')'#1.00', sar)'#1.00'; - 'hybrids'; + recombination Tyr IMgc1 ((((ori, mol)'#1.00', (arb, Bal)'#0.97')'#1.00', (intS, intN)'#0.08')'#1.00', sar)'#1.00'; mtDNA + nDNA - 'hybrids'; - recombination Tyr IMgc1 ((((ori, Bal)'#1.00', (arb, mol)'#1.00')'#1.00', (intS, intN)'#0.08')'#1.00', sar)'#1.00'; - 'hybrids'; - recombination Tyr IMgc1 ((((ori, mol)'#1.00', (arb, Bal)'#1.00')'#1.00', (intS, intN)'#0.15')'#1.00', sar)'#1.00'; mtDNA + nDNA + 'hybrids'; - recombination Tyr IMgc1 ((((ori, Bal)'#1.00', (arb, mol)'#1.00')'#1.00', (intS, intN)'#0.15')'#1.00', sar)'#1.00'; + 'hybrids'; - recombination a without H. felixarabica -like Tyr alleles of H. orientalis in all cases W Balkan as discrete unit (otherwise W Balkan within H. arborea ) speciation probability < 0.95 arb = Hyla arborea Bal = Western Balkan Hyla arborea ori = Hyla orientalis mol = Hyla molleri intS = Hyla intermedia Southern lineage intN = Hyla intermedia Northern lineage sar = Hyla sarda

5 Fig. S1. Maximum-parsimony haplotype network of the recombination-filtered TyrIMgc1 dataset (without H. felixarabica-like alleles) as prepared with the IMgc software with the approach of favoring retention of individuals (α = 1.0; ~‘1’ inTyr IMgc1). See Fig. 2 for comparison to which extent the data variation was simplified by filtering the recombination out.

H. arborea

W Balkan group H. sarda H. orientalis

H. intermedia S+N

H. molleri

6 Fig. S2. Species tree (maximum clade credibility tree) based on (a) mtDNA (12S , 16S ) and (b) nDNA (Rhod , Tyr IMgc1) as inferred in *BEAST. Numbers along branches are posterior probabilities. Note the relationship between H. orientalisand H. molleri , which is in the mtDNA phylogeny likely affected by a mitochondrial capture of H. orientalis mtDNA inH. molleri . The trees are scaled according to the split ofH. sarda .

a H. arborea sensu stricto 1.00 mtDNA H. arborea ‘W Balkans’ species tree 0.91 H. orientalis 0.96 mtDNA 0.46 H. molleri

H. intermedia North 0.88 0.84 H. intermedia South

H. sarda

H. meridionalis

0.01 substitution/site

H. arborea sensu stricto b 1.00 nDNA 0.64 H. arborea ‘W Balkans’ species tree H. sarda

1.0 1.00 H. intermedia North

0.40 H. intermedia South

0.59 H. molleri

H. orientalis

H. meridionalis

0.001 substitution/site