Speciation History and Widespread Introgression

Molecular Phylogenetics and Evolution 83 (2015) 143155
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev
Speciation history and widespread introgression in the European

short-call tree frogs (Hyla arborea sensu lato, H. intermedia and H. sarda)
Vclav Gvozdk a,b,, Daniele Canestrelli c, Mario Garca-Pars d, Jir Moravec b, Giuseppe Nascetti c,
Ernesto Recuero e, Jos Teixeira f,g, Petr Kotlk a
a
Laboratory of Molecular Ecology, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumbursk 89, 277 21 Libechov, Czech Republic
Department of Zoology, National Museum, Cirkusov 1740, 193 00 Prague, Czech Republic
Department of Ecology and Biology, Tuscia University, Largo dellUniversit 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 Ecologa de la Biodiversidad, Instituto de Ecologa, Universidad Nacional Autnoma de Mxico, Ap. Postal 70-275, Ciudad Universitaria, Mxico 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
b
c
a r t i c l e
i n f o
Article history:
Received 23 July 2014
Revised 10 November 2014
Accepted 22 November 2014
Available online 4 December 2014
Keywords:
Cryptic species complex
Bayesian species delimitation
Gene ow
Species tree
Phylogeography
Systematics
a b s t r a c t
European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form six
genetically 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 putative
taxa within H. intermedia (Northern and Southern) from the Italian Peninsula and Sicily, and H. sarda from
Sardinia and Corsica. Here, we assess species limits and phylogenetic relationships within these shortcall tree frogs based on mitochondrial DNA and nuclear protein-coding markers. The mitochondrial
and nuclear genes show partly incongruent phylogeographic patterns, which point to a complex history
of gene ow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogs
and 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 lineages of H. intermedia have been connected by nuclear gene ow (despite their deep mtDNA divergence)
and should be treated as conspecic. 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 H. orientalis; H. arborea 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.
2014 Elsevier Inc. All rights reserved.
1. Introduction
Western Palearctic tree frogs were considered to represent a
single species Hyla arborea until the end of the 1960s. Later on,
two taxa, H. meridionalis and H. savignyi from the western and east Corresponding author. Present address: Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Kvetn 8, 603 65 Brno, Czech Republic.
E-mail addresses: vaclav.gvozdik@ivb.cz (V. Gvozdk), canestrelli@unitus.it (D.
Canestrelli), mparis@mncn.csic.es (M. Garca-Pars), jiri_moravec@nm.cz (J. Moravec), nascetti@unitus.it (G. Nascetti), ernestorecuerogil@gmail.com (E. Recuero),
jteixeira@cibio.up.pt (J. Teixeira), kotlik@iapg.cas.cz (P. Kotlk).
http://dx.doi.org/10.1016/j.ympev.2014.11.012
1055-7903/ 2014 Elsevier Inc. All rights reserved.
ern Mediterranean, respectively, were elevated from subspecies

level 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 in
anurans with a critical role in maintaining reproductive barriers
between species (Schneider, 1977; Schneider and Sinsch, 2007).
In the Western Palearctic tree frogs, the advertisement calls have
been thoroughly studied (reviews in Schneider, 1974, 2004a). The
call segments are formed by pulse-groups (notes) and intervening
quiet intervals. Advertisement calls of the Western Palearctic tree
frogs might be divided into three categories according to the
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V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155
pulse-group properties (Fig. 1; grouping made according to the

results of Grach et al., 2007; Gvozdk et al., 2010; Schneider,
1974, 2004a). The long-call tree frogs are characterized by long
pulse-groups composed of a high number of pulses (3456 pulses;
210610 ms), short-call tree frogs by short pulse-groups with a
low number of pulses (612 pulses; 50110 ms), and medium-call
tree frogs by intermediate values (1325 pulses; 85190 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 species was not found to be substantially differentiated from H. arborea (Schneider, 1974, 2004a). This is also true for the Italian tree
frog, H. intermedia (Schneider, 2004a,2004b), which also was distinguished 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 tree
frogs emerged (Gvozdk et al., 2010; Stck et al., 2008, 2012).
Besides the description of the new species H. felixarabica from
the Arabian Peninsula (Gvozdk et al., 2010), deeply divergent lineages 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; Gvozdk, 2009; Gvozdk et al., 2007;
Recuero et al., 2007; Stck et al., 2008, 2012), which may each represent a separate (as yet unnamed) taxon (Stck et al., 2008). Furthermore, H. arborea was suggested to represent three species
according to the mitochondrial and single nuclear-gene phylogenies: H. orientalis in the east, H. molleri in the west, and H. arborea
sensu stricto (s.s.) in the central part of the former H. arborea sensu
lato (s.l.) range (Stck et al., 2008).
For the Iberian H. (arborea) molleri, this new taxonomy corresponds to the former morphology-based intraspecic taxonomy
of H. arborea. In contrast, the genetic break between the central
(H. arborea s.s.) and eastern (H. orientalis) populations does not
match the limits and distributions of the morphology-based subspecies, H. arborea kretensis (Crete, southern Balkans, Aegean, western Asia Minor; e.g. Stumpel, 1997), H. a. schelkownikowi
(Caucasus; e.g. Kuzmin, 1999), H. a. gumilevskii (Talysh Mts. in
Transcaucasia; Litvinchuk et al., 2006) and H. a. arborea in the rest
of the range (except the Iberian Peninsula; e.g. Stumpel, 1997). [For
historical overview of the taxonomy of the Western Palearctic tree
frogs see Fig. 1.] This incongruence between molecular phylogenies
and morphology-based taxonomy, which discouraged wider
acceptance of the revised taxonomy (e.g. Grosse, 2009; zdemir
et al., 2012; Sillero et al., 2014; Speybroeck et al., 2010), is further
fostered by the unexpected and surprisingly close sister-clade relationship between the eastern (near the Black Sea; H. orientalis) and
western (on Iberian Peninsula; H. molleri) populations, currently
geographically separated by the range of H. arborea s.s. (Gvozdk,
2009; Gvozdk et al., 2007; Stck et al., 2008, 2012). As yet, no
study 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) has
consistently received high support from molecular phylogenetic
analyses (Gvozdk, 2009; Stck et al., 2008, 2012), while relationships within the clade have remained ambiguous (Stck 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 (Gvozdk, 2009; Stck et al., 2008,
2012). However, using different mitochondrial markers (12S and
16S rRNA) than in Stck et al. (2008, 2012), our preliminary data
showed high support also to H. arborea s.l., i.e. H. orientalis, H. molleri and H. arborea s.s. forming a clade (Gvozdk, 2009). In addition
to the unresolved internal topology of the clade, species limits in
the short-call tree frogs are an intriguing issue, especially given
the following evidence: (i) similar advertisement calls of H. arborea, 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 (Gvozdk
et al., 2008; both as H. arborea), H. molleri (Terentjev, 1960) and largely also H. intermedia (Nascetti et al., 1995; Rosso et al., 2004;
Terentjev, 1960); (iii) incongruence in phylogeographic patterns
between different nuclear genes across the Balkans and northern
Italy (H. orientalis/H. arborea/H. intermedia N), especially along
the western Balkan coast (Gvozdk et al., 2007; distinct Balkan/
Adriatic lineage was detected also by Stck et al., 2008 and
Dufresnes et al., 2013). The Western Palearctic short-call tree frogs
thus seem to represent an assemblage of morphologically and
acoustically cryptic taxa, and the extent of gene ow between
them needs to be properly evaluated.
Current advances in molecular phylogenetics have introduced
new methods, including multilocus coalescent-based species tree
inference (Knowles and Kubatko, 2010) and coalescent-based species delimitation, which permit probabilistic tests with an assumption of no recent admixture between species and of bipartitions of
individuals in gene trees that are shared across loci (Yang and
Rannala, 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 coalescentbased 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 Stck et al. (2010). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
method to test (1) the monophyly of H. arborea s.l. (H. arborea, H.

orientalis, H. molleri); (2) sister-clade relationship of the geographically disjunctive H. orientalis and H. molleri; and (3) species-level
distinction of the taxa, including of the northern (N) and southern
(S) lineages of H. intermedia, and of the western (W) Balkan H. arborea/H. orientalis populations.
2. Materials and methods
2.1. Taxon sampling and laboratory procedures
We obtained sequence data for altogether 169 specimens from
95 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, which
totaled 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 the
putative species, i.e. H. arborea s.s., H. orientalis and H. molleri
(sensu Stck et al., 2008). The H. orientalis material from Asia Minor
and Caucasus-Caspian region used in a previous study (Gvozdk
et al., 2010) was reanalyzed in the context of the new data from
the European part of the range. The outgroup species H. meridionalis data were taken from the previous study (Gvozdk et al., 2010)
and those for a second outgroup, H. japonica, were retrieved from
GenBank (AY843633, AY844615, AY844078; Faivovich et al.,
2005). The outgroup status of these two species relative to the
short-call tree frogs was demonstrated by, e.g. Hua et al. (2009).
Genomic DNA was extracted from tissue samples using commercial kits following the manufacturers protocols. Two fragments of mitochondrial DNA (mtDNA), 12S rRNA (12S, 355
aligned bp) and 16S rRNA (16S, 546 aligned bp), and two nuclear
genes (nDNA), tyrosinase precursor exon 1 (Tyr, 496 bp), and rhodopsin exon 1 (Rhod, 276 bp) were targeted. The nDNA markers
were sequenced for a subset of samples (ca. 43%) representing all
main mitochondrial groups and covering the entire geographic
range (Table S1). Primers and PCR protocols followed the previous
study (Gvozdk et al., 2010). PCR amplicons were puried and
directly sequenced with the PCR primers using a commercial
sequencing service (Macrogen Inc.).
2.2. DNA alignment and phasing of autosomal genotypes
Alignments of nucleotide sequences were prepared with ClustalW (Thompson et al., 1994) as implemented in BioEdit 7.2
(Hall, 1999). Mitochondrial 12S and 16S fragments were concatenated. Heterozygous sites in nDNA were identied according to
electropherograms 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 coalescent-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 outgroup species and were repeated ve times for each nuclear-gene
dataset with different seeds for the random number generator to
check if the phase estimates are consistent across the runs according to goodness-of-t values. Each run was conducted under the
parent-independent mutation model with a burn-in-period of
100 iterations, which was followed by 1000 iterations. Results with
a probability > 0.70 were accepted. Two samples (H. intermedia
HD03A, Tyr; H. arborea HJ55, Rhod) contained heterozygous positions whose phases were not resolved (mean probability 0.50),
and one sample (H. intermedia HD04B, Tyr) had one position
weakly resolved (mean probability 0.69). In these cases, both
145
possible gametic phases and their phylogenetic positions were

considered in haplotype networks, which were constructed using
the parsimony approach under the 95% parsimony limit as implemented in TCS 1.21 (Clement et al., 2000). The alternative gametic
phases did not change the networks substantially (see haplotypes
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
mitochondrial haplotypes and new nuclear alleles or nucleotide
sequences with the standard IUPAC ambiguity codes, if heterozygous positions remained unresolved, have been deposited in GenBank (KP109551KP109674; Table S2). Genetic distances were
calculated from 16S haplotypes (a marker commonly used in
amphibian 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 only
the 12S fragment (from localities 36 and 50) were used in the mitochondrial gene tree only.
2.3. Gene trees
For gene-tree estimation, mtDNA and phased Tyr and Rhod
sequences were sorted into distinct haplotypes using Collapse 1.2
(Posada, 2006). Gene trees were reconstructed by Bayesian inference (BI) and maximum likelihood criterion (ML). For BI analyses
performed by MrBayes v3.2.2 (Ronquist et al., 2012), best-t partitioning schemes and nucleotide substitution models according to
the Bayesian information criterion (BIC) were selected using PartitionFinder v1.1.0 (Lanfear et al., 2012): mtDNA, 12S and 16S treated
as one partition (SYM+G); Tyr, 1st + 2nd/3rd codon position (K80+I/
HKY+G); Rhod, 1st + 2nd/3rd codon position (JC/K80). Each dataset
was analyzed with two runs of four Markov chains, which were run
for 6 106 generations and samples saved every 100th generation.
That the analyses reached stationarity was veried by examining
the plots of log-likelihood scores using Tracer v1.5 [Rambaut and
Drummond, 2009; all parameters had effective sample size
(ESS) 200], and the chain convergence was conrmed by the
convergence diagnostics (average standard deviation of split frequencies, potential scale reduction factor) comparing the two
simultaneous runs against each other. From the sampled trees,
the rst third was discarded as a burn-in and a 50% majority-rule
consensus tree was produced from the remaining trees that were
taken as representing the posterior distribution. For ML analyses
performed by PhyML v3.0 (Guindon et al., 2010), best-t models
of 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 nearest
neighbour interchange and the subtree pruning and regrafting
algorithm of tree improvement and optimization of the topology
and branch lengths were applied. The nodal support was assessed
by 100 bootstrap pseudoreplicates. Clades supported with BI posterior probability (pp) values P 0.95 and ML bootstrap values P 70 were considered highly supported (Huelsenbeck and
Rannala, 2004).
2.4. Data preparation for coalescent-based applications
Since the coalescent-based species-tree and species-delimitation analyses rely on, and are sensitive to, the assignment of each
individual/allele to a taxonomic unit (Leach et al., 2014; Olave
et al., 2014), we carefully inspected distribution of haplotypes
across different genes to identify specimens with inconsistent phylogenetic signal and thus identify potential hybrids (Table S1). Such
potential hybrids or possible cases of allelic introgression (H. arboreaorientalis; 10 individuals from the Balkans and Poland) were
omitted from the species-tree analyses. Similarly, contrasting Tyr
146
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 reect two possibilities of a haplotype
connection in cases when haplotypes were phased with low support. Samples classied 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 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 gure legend, the reader is
referred to the web version of this article.)
alleles, such as in a sample from Brittany (loc. 4) or H. felixarabicalike alleles of six individuals of H. orientalis from south-western
Turkey (haplotypes Tfel3, Tori5, Tori6, Tori7, Tori9; see Fig. 2 and
Gvozdk et al., 2010), were also omitted from the analyses (see Section 3 for details). Remaining samples were allocated to the following taxonomic units according to their provenance and genetic
identity: 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 (Stck et al.,
2012) we also tested a position of the western Balkan tree frogs

(W Balkan H. arborea, loc. 3335, 37, 38 in Slovenia, Croatia, Montenegro, Greece; n = 5), which carry inconsistent phylogenetic signal across the studied genes (Fig. 2), as noted earlier by Stck et al.
(2008). Coalescent-based model assumptions include an absence of
recombination (Castillo-Ramrez 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 loci
for different subsets of tree-frog species were estimated in DnaSP
5.10 (Librado and Rozas, 2009), with no recombination detected in

Rhod, and a minimum of one (H. intermedia, H. sarda), seven (H.
arborea s.l.) and ten (all ingroup taxa) recombination events
detected in Tyr. Traces of recombination were removed from the
Tyr dataset (not containing H. felixarabica-like alleles) using IMgc
(Woerner et al., 2007), and a recombination-ltered dataset was
produced by the approach of favoring the retention of individuals
(a = 1.0; 423 bp and 86% samples kept; hereafter as TyrIMgc1 dataset; see also Table S1 and Fig. S1).
2.5. Species trees and divergence dating
A species tree was inferred using BEAST (Heled and
Drummond, 2010) based on the dataset including all genes, mitochondrial 12S and 16S, the nuclear Rhod and the recombination-ltered Tyr (TyrIMgc1). Alignments of all individuals (without
potential hybrids; see above) including one outgroup (H. meridionalis) for each gene were uploaded into BEAUti v1.8.0, a part of the
BEAST package (Drummond et al., 2012), where they were
assigned separate and unlinked substitution, clock and tree models. Hyla meridionalis was set as outgroup by enforcing monophyly
of the remaining taxa. Best-t partitioning schemes and sequenceevolution models were set according to the results from PartitionFinder v1.1.0 (Lanfear et al., 2012) following the BIC [mtDNA, 12S
and 16S treated as one partition (HKY+G); TyrIMgc1 without partitions (K80); Rhod, 1st + 2nd/3rd codon position (K80/TrNef)].
Ploidy of mtDNA was set to mitochondrial (haploid), and the other
two loci to autosomal nuclear (diploid). The Yule species-tree prior
was set and UPGMA starting tree used for each gene and a strict
molecular clock assumed. The clock rate for mtDNA was inferred
from a calibration based on an assumption of H. sarda being a
descendant of a radiation that had occurred during the Late Miocene (Stck et al., 2012), particularly when the land bridge
between the SardoCorsican block and continent was submerged
at the end of the Messinian salinity crisis when the Mediterranean
Basin was re-ooded at ca. 5.33 mya (Bisconti et al., 2011a; Hs
et al., 1973; Krijgsman et al., 1999; Rouchy and Caruso, 2006). Normal distribution prior was assigned with a mean of 5.33 (mya) and
standard deviation of 0.16 in order to accommodate uncertainty in
this estimate. Clock rates for Tyr and Rhod were estimated from
mtDNA using 1/x prior distribution. Five independent BEAST runs
were performed, each for 200 million generations, sampling every
20,000th generation to obtain a posterior sample of 10,000 trees.
The likelihoods were inspected using Tracer v1.5 (Rambaut and
Drummond, 2009) and convergence checked using ESS values
(200) after discarding the 10% burn-in. The post burn-in samples
of the ve runs were combined in the BEAST module LogCombiner
v1.8.0. The output of 45,000 sampled trees was uploaded to
another BEAST module, TreeAnnotator v1.8.0, to infer the nal species tree as a maximum clade credibility tree (MCCT) with node
ages represented by median heights and condence intervals with
95% highest posterior densities (HPD). The set of all 45 thousand
trees was superimposed and visualized as a cloudogram with
DensiTree v2.01 (Bouckaert, 2010) to show the probability distribution of topologies, and the three most frequent consensus trees
(sensu Bouckaert, 2010) were inspected. To assess the inuence of
the mtDNA dataset on the species-tree topology, the nuclear dataset was also employed in a separate species-tree analysis with virtually the same settings but without molecular-clock calibration
and run for 500 million generations.
147
2010) to test whether the operational taxonomic units t the

assumption of no post-divergence gene ow, which is inherent to
the biological species concept (Yang and Rannala, 2010). This
method accommodates the species phylogeny and accounts for
lineage sorting due to the ancestral polymorphism using the
reversible-jump Markov chain Monte Carlo (rjMCMC) algorithm
that successively splits or joins nodes on the guide species tree,
and employs population-size parameters h and species divergence
times s in the coalescent model (Rannala and Yang, 2013; Yang and
Rannala, 2010). Numerous possible species-tree topologies (guide
trees) were tested using different datasets with individual alleles
carefully assigned to putative species (see Olave et al., 2014): all
markers (mtDNA and phased nDNA), nDNA only, Rhod only, and
Tyr only; Tyr was used in both ways including recombination
and recombination-ltered (TyrIMgc1); all individuals (without
H. felixarabica-like Tyr alleles), with or without hybrids, and considering W Balkan populations as a discrete unit or part of H. arborea (Table S3). A gamma prior G(2, 1000) was applied on the
population-size parameters (hs). Also the age of the root in the species tree (s0) was assigned the gamma prior G(2, 1000), while the
other divergence-time parameters were assigned the Dirichlet
prior (Yang and Rannala, 2010). When both mtDNA and autosomal
nuclear data were involved in an analysis, the parameters heredity (allows h to vary among loci) and locusrate (allows variable
mutation rates among loci) were estimated with a gamma G(4, 4)
and Dirichlet prior, respectively. The algorithm 0 was used with
several values for the ne-tuning parameter e to ensure that the
rjMCMC mixed properly. BP&P analyses were run for 105 generations with rst 50% of samples treated as a burn-in and discarded.
If necessary, ne-tuning variables were adjusted to display proportion values close to 0.30.4 (within the interval 0.150.7) as recommended in the manual (Yang, 2013). Each analysis was run
repeatedly to check consistency between runs. To control for a possible inuence of the settings, the all-gene and nDNA MCCT species-tree topologies were used as guide trees also for runs with
alternative 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 by
Leach and Fujita (2010), and runs employing the rjMCMC algorithm 1, with ne-tuning parameters a = 2 and m = 1 (Yang, 2013).
3. Results
3.1. DNA polymorphism
The mitochondrial DNA dataset contained 198 ingroup
sequences, which collapsed into 77 haplotypes (891 bp excluding
insertion/deletion polymorphisms), contained the number of polymorphic sites S = 126 (89 parsimony informative), and showed the
haplotype diversity h = 0.922 0.014 (SD) and nucleotide diversity
p = 1.91 0.09%. The two nuclear genes were considerably less variable. Phased ingroup Tyr dataset (166 sequences, 496 bp) contained 47 haplotypes, with S = 38 (34), h = 0.900 0.016, and
p = 0.90 0.04%, while the H. felixarabica-like- and recombination-ltered TyrIMgc1 (133 sequences, 423 bp) contained 22 haplotypes, 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, and
p = 0.60 0.03%. The mitochondrial marker has a roughly two
times higher polymorphism than Tyr and three times higher than
Rhod.
2.6. Species delimitation

3.2. Gene trees
Coalescent-based Bayesian species delimitation (BSD) analyses
were conducted in the program Bayesian Phylogeny and Phylogeography (BP&P v2.2; Rannala and Yang, 2003; Yang and Rannala,
The mitochondrial DNA phylogeny had an almost identical

topology based on the BI and ML approaches (Fig. 3). Most of the
148
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 dened 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
gure legend, the reader is referred to the web version of this article.)
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 lineages
of H. intermedia (Northern/Southern) is weaker (0.94/56), reecting
the deep mitochondrial differentiation of the two geographically

close lineages. Despite the geographically remote distributions,
the Iberian H. molleri and Eastern European/Western Asian H. orientalis together form a highly supported clade (1.00/88), while
the support for each of the two taxa is rather low (H. molleri
0.85/60; H. orientalis 0.94/68). Hyla arborea s.s. is well differentiated (0.97/78) and is supported as the sister clade to H. molleri + H.
orientalis (1.00/75). Mitochondrial DNA (16S) genetic distances
among 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. However, both nuclear markers, Tyr and Rhod, possess variation sufcient to show clear structure (Figs. 2 and 3). The Tyr phylogeny
differs from mtDNA in terms of the detection of major lineages/
species in three main aspects: (i) H. arborea s.s. forms two main
clusters. One contains predominantly samples from the western
Balkans, while the second the remaining samples. (ii) H. orientalis
also forms two main groups, which do not form a clade. One
accommodates most samples, while the other is restricted only
to south-western Asia Minor. In the latter group the most common
haplotype of H. felixarabica (Tfel3) was recorded (plus four haplotypes closely related to it), as documented earlier (Gvozdk et al.,
2010). (iii) H. intermedia, Northern lineage is not clearly detectable
and most alleles are in the Southern lineage. In the Rhod haplotype
phylogenetic relationships, the most striking difference from the
mtDNA phylogeny is the position of W Balkan H. arborea samples,
which do not carry typical H. arborea Rhod haplotypes, but have
the most-common H. orientalis haplotype (Rori1) or a haplotype
derived from it (Rori6) (Fig. 3). Similarly, the majority of samples
(5/6) of the Northern lineage H. intermedia carries the most common H. orientalis haplotype (Fig. 3).
3.3. Geographic distribution of haplotypes and hybrid location
Intraspecic phylogeographic relationships based on mitochondrial DNA are presented in Fig. 4. The south-western endemics, H.
sarda and H. molleri do not demonstrate clear phylogeographic pattern, with an exception of the presence of one relatively distant
haplotype of H. molleri in Galicia. The Southern lineage of the Italian endemic H. intermedia forms two subgroups, southernmost and
central. The most common H. arborea s.s. haplotype and its singlemutational (or indel) derivatives, arranged in a star-like pattern,
have been recorded from over much of Europe, from Greece in
the south to Denmark in the north (Fig. 4). On the contrary, in
the southern Balkans a more diversied haplotype group is present, predominantly recorded from Greece but found also in northern Croatia and the Czech Republic. One relatively distant
haplotype was detected on Pag, a Croatian island in the northern
Adriatic Sea. The highest diversity was found in the eastern taxon,
H. orientalis. Two main groups (weakly supported clades) are represented by the Caucasus-Caspian populations and by all the other
populations, respectively. Besides the Caucasus-Caspian group,
149
further structuring in the second group is also detectable. Most

diversied is the Asia Minor population with haplotypes present
in several different subgroups, but with less clear geographic structuring, which is true also for the remaining Eastern European populations, which have clear afnities to the north-western Asia
Minor population.
For the nuclear Tyr and Rhod markers, the distribution of haplotypes generally agrees with the pattern in mtDNA, with the exception of the cases described above (Section 3.2.). However, there
have been also several striking ndings at particular locations.
Based on the combination of alleles across different loci, potential
hybrids were detected in Brittany (H. arborea H. molleri), Poland,
and across the Balkans in Greece, Bulgaria and Romania (H. arborea H. orientalis) (Fig. 2 and Table S1).
3.4. Species trees and divergence time estimate
The maximum clade credibility species tree based on the mitochondrial and nuclear loci inferred the same topology as the
mtDNA gene tree: H. sarda being the sister lineage to all other
short-call tree frogs, and H. intermedia being the sister lineage to
H. arborea s.l., and with H. orientalis forming a clade with H. molleri
and H. arborea s.s. closely related to the W Balkan H. arborea group
(Fig. 5). However, high support is present only for the crown sisterclade relationships (H. intermedia N H. intermedia S; H. orientalis
H. molleri; H. arborea W Balkan). Three most frequent topologies
occurred with the posterior frequency of 38.7%, 33.2%, 11.6% rst
with the same topology as the MCCT, second differing by the presence of a clade H. intermedia (N+S) + H. sarda in the sister-clade
position to H. arborea s.l., and third interchanging positions of H.
intermedia (N+S) and H. sarda in comparison to the MCCT. All the
three topologies, accounting for almost 84% posterior trees, had
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
most frequent topologies (22.6%, 15.1%, 13.8%) form two main
clades: (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 clock
based on all loci estimated that the main radiation within the
short-call tree frogs occurred in the Pliocene (considering 95%
highest posterior densities, HPD, 5.41.9 mya), with only the most
recent splits occurring during the Pleistocene. The split between H.
orientalis and H. molleri is dated close to that between the Southern
and Northern H. intermedia, ca. 1.4 mya (HPD 2.80.4), while the
structuring within H. arborea with respect to the W Balkan population dates to the Middle to Early Pleistocene ca. 200 kya (HPD 370
70 kya).
Bayesian species delimitation supported all the taxa as species
without a history of gene ow (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.
H.
H.
H.
H.
H.
arborea
orientalis
molleri
intermedia S
intermedia N
sarda
Outgroup
H. meridionalis
H. japonica
H. arborea
H. orientalis
H. molleri
H. intermedia S
H. intermedia N
H. sarda
0.4
3.3
3.2
3.8
3.9
4.9
0.5
0.9
3.8
4.5
4.5
0.4
3.4
4.0
4.1
0.3
2.9
4.0
0.2
4.0
0.3
6.1
7.3
5.7
7.8
6.0
8.0
6.8
6.8
6.3
6.1
6.6
6.2
H. meridionalis
H. japonica
5.8
150
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 afnities within individual
species and are not fully concordant to the colors in Figs. 13 (only the most widespread haplogroups are encoded by the same colors like the species in Figs. 13). (For
interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
ditions with respect to inclusion of potential hybrids, recombination in Tyr, or considering W Balkan populations as a discrete unit
or 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
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 condence 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
uctuations 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 gure legend,
the reader is referred to the web version of this article.)
speciation probability always low (0.080.60) for the Northern and

Southern lineage of H. intermedia, both for the dataset containing
recombination and the recombination-ltered dataset. There was
also low speciation probability for the node H. arborea W Balkan
in Tyr (0.32), but the probability increased substantially when
recombination was ltered out (0.97). Interestingly, when the node
W Balkan H. orientalis was tested in Rhod where there is a high
level of haplotype sharing (Rori1; frequency 4/10 in W Balkan),
the speciation probability was also high (0.99). This was likely a
consequence of the Rori6 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; Gvozdk, 2009; Gvozdk et al., 2008; Kaya, 2001;
Pisanets and Matvyeyev, 2012; Rosso et al., 2004). Hyla sarda is

the only species that is substantially differentiated in morphology,
and it was the rst 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 years
later (Nascetti et al., 1995). All the remaining taxa have been
included under H. arborea until recently (e.g. Grosse, 2009;
Schneider and Grosse, 2009; Sillero et al., 2014; Speybroeck
et al., 2010). Stck et al. (2008, 2012) found H. arborea s.l. paraphyletic with respect to H. intermedia (Stck et al., 2012) or to both H.
intermedia and H. sarda (Stck 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 lineages (Northern and Southern), and of H. orientalis and H. molleri.
By contrast, in our analyses, H. arborea s.l. was highly supported
in the mtDNA phylogeny (Fig. 3) and its monophyly was conrmed
by the species tree analysis (Fig. 5) by the presence of the H. arborea s.l. clade in the three most common all-gene species-tree topologies and the overall frequency of 83.6% among the posterior trees.
152
This discrepancy in mtDNA phylogenies between the studies might

be a result of different markers utilized by Stck et al. (2008, 2012).
On the other hand, our nuclear-only species tree does not correspond to the all-gene topology in supporting a sister-clade relationship between H. arborea and H. sarda (64.5% of the posterior
trees), which has not been documented in any of the previously
published phylogenies (Hua et al., 2009; Pyron and Wiens, 2011;
Stck et al., 2008, 2012). The H. arborea s.l. clade was present in
the 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 in
mtDNA of the geographically remote, eastern H. orientalis and the
western/Iberian H. molleri, which are currently separated by more
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),
including its presence in the three most common topologies from
the posterior trees (Fig. 5b, left). The clade was present in 99.9%
of the posterior trees. On the contrary, in the nuclear-only species
tree (Fig. 5b, right), the sister-clade relationship of H. orientalis + H.
molleri was present only in one of the three most common topologies, with the overall frequency of only 23.2% in the posterior sample. In the nuclear-only species tree there was a clear tendency for
a 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 relationships
within the clade were distributed among the posterior trees with
relatively equal and lower frequencies. Thus, it seems that there
are two plausible explanations for the observed pattern: (1) H. orientalis H. molleri split is relatively young (1.4 mya dated in our
study), of the age when a substantial thermal drop is evidenced
in the Mediterranean (Colleoni et al., 2012; Fig. 5), probably causing range restrictions and isolation of the two lineages. Or alternatively, and perhaps more likely, assuming the nDNA species tree is
correct, then (2) H. orientalis H. molleri split occurred much earlier, and a mitochondrial introgression and capture (probably from
an ancestral population of H. orientalis to H. molleri) could have
happened during a secondary contact in a climatically suitable/
warm period of the Early Pleistocene, which allowed range extensions. This or similar event(s) could also be responsible for Rhod
introgression from H. orientalis to W Balkan H. arborea and to the
Northern lineage of H. intermedia (see also Verardi et al., 2009),
for Tyr introgression from H. felixarabica to southern Asia Minor
population of H. orientalis, and probably also for the genetic constitution of the Breton tree frogs (loc. 4), where H. molleri alleles
appear to have introgressed into H. arborea.
Coalescent-based BSD supported species-level distinction of all
taxa, including the W Balkan operational unit (Fig. 5), for a variety
of guide trees and datasets (Table S3). Only Tyr does not present
signatures of completed speciation within H. intermedia, implying
the presence of some gene ow (detected previously by
Canestrelli et al., 2007b). These results suggest that the two taxonomic units within H. intermedia should be treated as conspecic,
despite the relatively high mtDNA distance (2.9% in 16S). The H.
intermedia N + H. intermedia S clade was present in 99.9% and
99.8% posterior species trees (all-gene and nDNA only, respectively). Similarly, signatures of speciation were not found in Tyr
for the split between H. arborea and W Balkan (the clade was present in the posterior species-tree sample of the all-gene and nDNA
approach with frequencies of 100% and 99.8%, respectively). Nonetheless, when recombination is removed from the Tyr dataset, the
speciation probability becomes signicant in the latter case, sug-
gesting that the recombined alleles carry the signal of gene

exchange among H. arborea populations. In addition, when recombination is ltered out the cluster of alleles originated from the
western Balkans (not directly corresponding to the W Balkan
group; Fig. 2) is no more obvious (TyrIMgc1 dataset; Fig. S1). Gene
ow followed by recombination between H. arborea and H. orientalis alleles thus probably account for the presence of the W Balkans allele cluster in the non-ltered Tyr data (Fig. 2), despite the
fact that BSD analysis of the ltered data suggests species-level distinction of the W Balkan tree frogs. The lack of monophyly and of
private alleles, together with the cryptic phenotype (Gvozdk
et al., 2008), support retaining the W Balkans populations in H.
arborea.
4.4. New insights into the phylogeographic history
The intraspecic genetic structuring was largely formed during
the climatic oscillations in the Middle and Late Pleistocene as evidenced in the case of H. arboreaW Balkan split (Fig. 5), which corresponds well to the conclusion of Dufresnes et al. (2013). The
phylogeographic patterns of mtDNA (which shows higher diversity
and thus geographic resolution than nDNA; Fig. 4) recovered in our
study extend the previous ndings in several ways, but we provide
several 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 Adriatic (W Balkans) and we detected it on Pag Island, located between
Istria and the Croatian coast where it was found previously
(Dufresnes et al., 2013). This highlights the Adriatic region as a
potential glacial refugium for H. arborea s.s. Another refugium
was probably located in the southern Balkans, which is predominantly occupied by the second haplogroup harboring a relatively
high genetic variation. One haplotype of this south-Balkan haplogroup was found at some northerly-located places like the northern inland Croatia (see also Dufresnes et al., 2013) and
surprisingly also as far north as the Czech Republic. This distribution suggests multiple routes of northward expansion from the
Balkan Peninsula. The range of the third, most widespread haplogroup is centered in central and north-western Europe, but it
partly overlaps with the other two haplogroups. Its star-like pattern suggests a recent expansion (see also Dufresnes et al., 2013).
4.4.2. Hyla orientalis
For this species we uncovered a somewhat different pattern
compared to Stck et al. (2012). First of all, the Caucasus-Caspian
population forms only a single haplogroup, distributed from the
western forelands of the Caucasus in the Krasnodar region to the
south-eastern Caspian coast in Iran. The most common haplotype
is widespread throughout the area, including regions of the type
locality of H. arborea schelkownikowi and H. a. gumilevskii, which
supports their status as synonyms, currently under the name H.
orientalis (Gvozdk et al., 2010; Stck et al., 2008). On the contrary,
Stck et al. (2012) found a population from the Talysh region in
south-eastern Azerbaijan as a distinct clade in a sister-clade position to the Caucasian population. This discordance might be
explained by differences in the level of variation in the mtDNA
markers used in our study and that of Stck et al. (2012). It might
also be due to the fact that the Caspian population of Stck et al.
(2012) was represented solely by samples originating from a relatively small area of the Talysh region, while Iranian samples were
missing from their study. Interestingly, despite the distribution gap
in the steppes of northern Crimea, the southern Crimean population of H. orientalis is a part of the predominantly Eastern-European
haplogroup, not of the geographically close Caucasus group. This
Eastern-European haplogroup is distributed to the north and east

of the Carpathians from Poland, Ukraine, Romania and southwards
to Bulgaria and Turkey, and it is characterized by a complex genetic
variation. This includes further sub-structuring, which is well evident in Asia Minor.
4.4.3. Hyla molleri, H. intermedia and H. sarda
Our sampling of the Iberian, Italian and Tyrrhenian tree frogs
was not designed as a detailed phylogeographic study, which
was performed in those regions earlier (Barth et al., 2011;
Bisconti et al., 2011a,b; Canestrelli et al., 2007a,b). However, we
found a distinct haplotype of H. molleri in Galicia, suggesting that
this region could play a role as a glacial refugium, while the rest
of the range of this species is inhabited by a widespread haplogroup (Barth et al., 2011). The Southern H. intermedia was split into
two haplogroups, corresponding to the southernmost (Calabria,
Sicily) and central part of the Italian Peninsula, highlighting these
regions as presumed glacial refugia (Canestrelli et al., 2007a). The
Northern H. intermedia is highly diverged from the Southern lineage in mtDNA (2.9% in 16S), but its nuclear genes bear signatures
of gene ow to/from the Southern H. intermedia (Tyr; see also
Canestrelli et al., 2007b) and H. arborea/H. orientalis (Rhod; see also
Verardi et al., 2009). Our limited sampling of H. sarda did not
recover a clear phylogeographic pattern, which was addressed in
detail elsewhere (Bisconti et al., 2011a,b).
4.5. Hybridization
A case of hybridization between H. orientalis and H. savignyi in
southern Asia Minor was mentioned by Stck et al. (2012). However, that might have been a misinterpretation of the signatures
of 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 above
and briey mentioned by Gvozdk et al. (2010). The nearest localities of the Arabian tree frog, H. felixarabica, are known from southern Syria, about 800 km away (Gvozdk et al., 2010), but the species
likely had a secondary contact with H. orientalis during Pleistocene
periods favorable to range extension (Gvozdk et al., 2010). To date,
there is no unequivocal evidence for ongoing hybridization
between the parapatric short-call H. orientalis and the medium-call
H. savignyi (cf. Gl et al., 2012; Gvozdk et al., 2010). However, the
present study found hybrids in two different pairs of short-call species, i.e. H. orientalis H. arborea s.s. in Greece, Bulgaria, Romania
and Poland, and H. arborea s.s. H. molleri in Brittany, France
(see Table S1). It is not trivial to distinguish introgression from
retention of ancestral polymorphism due to incomplete lineage
sorting. However, clear hybrids, including putative F1 hybrid, were
found in southern Poland near Krakow (loc. 48; Fig. 2 and
Table S1). This is in line with Stck et al. (2012) who found hybrids
in northern Poland and postulated a hypothesis that the geographic border between H. arborea s.s. and H. orientalis approximately follows the course of the Vistula River.
5. Conclusion
We conclude that the phylogenetic signal of mtDNA had strong
inuence on the reconstructed species tree of the short-call tree
frogs, to some extent overwhelming the signal of the nuclear
genes. However, the results strongly suggest that the western Balkan H. arborea does not represent a distinct species, as do neither
Northern nor Southern lineage of H. intermedia, which should be
treated as conspecic despite their mtDNA divergence. However,
all other taxa should be considered species as no gene ow was
detected between them by coalescent, regardless of some phyloge-
153
netic incongruence among the markers. A mitochondrial capture

upon secondary contact is a plausible explanation for the close
mtDNA relationship between the geographically remote H. orientalis and H. molleri. Our study thus provided several important
novel insights into the speciation history and systematics of the
short-call tree frogs, despite the fact that some ndings await conrmation using novel markers.
Acknowledgments
We are grateful to many friends and colleagues who provided
samples or helped in the eld, namely to E. Albert, P. Benda, J. Cervenka, L. Choleva, D. Cogalniceanu, S. Dubey, G. Evanno, W.-R.
Grosse, D. Jablonski, J. Jaquiery, U. Joger, C. Kltsch, H. Kulkov,
Z. Lajbner, S. N. Litvinchuk, R. Lucan, I. Martnez-Solano, M. andera, C. Settanni, P. iroky, J. md, B. vecov, J. M. Szymura, D. Tarkhnishvili, M. Velensky, P. Velensky, G. Velo-Antn. We also thank A.
Larson (Washington University in St. Louis) and two anonymous
reviewers for their comments, which improved the previous version of the manuscript. The study was supported by the Ministry
of Culture of the Czech Republic (DKRVO 2014/14, National
Museum, 00023272). ER is supported by a DGAPA-UNAM postdoctoral fellowship.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.20
14.11.012.
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Supplementary data
Speciation history and widespread introgression in the European short-call tree frogs
(Hyla arborea sensu lato, H. intermedia and H. sarda)
Vclav Gvodk, Daniele Canestrelli, Mario Garca-Pars, Ji Moravec, Giuseppe Nascetti,

Ernesto Recuero, Jos Teixeira, Petr Kotlk
Molecular Phylogenetics and Evolution
Table S1. Specimens examined.

Haplotype
Map Taxon: Hyla
1
2a
2b
2b
3
3
3
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea (x molleri )
5
6
7
8
8
8
8
9
10
11
12
13
14
15
15
16
16
16
17
17
18
19
20
21
21
21
22
22
22
22
22
23
23
24
24
25
26
27
28
28
29
29
30
31
32
33
34
35
35
36
37
38
39
40
40
41
41
42
42
42
43
44
44
44
45
45
45
46
47
48
48
49
49
50
51
51
52
52
52
53
54
55
55
55
56
56
56
57
57
57
58
59
60
60
61
62
63
64
64
64
64
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea x orientalis
b
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
65
orientalis
65
orientalis
65
66
orientalis
orientalis
67
orientalis
67
67
68
orientalis
orientalis
orientalis
69
orientalis
69
69
orientalis
orientalis
Species tree Species delimitation

Sample ID Tyr IMgc1a Country
ID
ID (/alternative)
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
(Tyr omitted)
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
- ('hybrid')
arborea
arborea
W Balkan
W Balkan
W Balkan
arborea
W Balkan
W Balkan
arborea
- ('hybrid')
arborea
- ('hybrid')
- ('hybrid')
- ('hybrid')
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
- ('hybrid')
- (hybrid)
- (hybrid)
orientalis
- (hybrid)
orientalis
- (hybrid)
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
(Tyr omitted)
orientalis
(Tyr omitted)
orientalis
orientalis
orientalis
(Tyr omitted)
orientalis
orientalis
orientalis
orientalis
(Tyr omitted)
orientalis
orientalis
arborea
arborea
arborea
arborea
arborea
arborea
arborea
H488
HJ66
HCE1
HCE2
H485
H486
H487
arborea
HM1
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea /- ('hybrid')
arborea
arborea
W Balkan/arborea
W Balkan/arborea
W Balkan/arborea
arborea
W Balkan/arborea
W Balkan/arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea
arborea /- (hybrid)
arborea /- (hybrid)
orientalis
orientalis /- (hybrid)
orientalis
orientalis /- (hybrid)
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
(Tyr omitted)
orientalis
(Tyr omitted)
orientalis
orientalis
orientalis
(Tyr omitted)
orientalis
orientalis
orientalis
orientalis
(Tyr omitted)
orientalis
orientalis
HJ47
HJ46
HJ45
H278
H279
H280
H281
HJ55
HJ42
HJ44
HJ43
HJ57
HJ56
H546
H545
H269
H270
H271
H383
H384
H551
H146
H550
H272
H273
H274
H221
H222
H223
H224
H226
H211
H212
H500
H501
HJ39
H463
H541
H290
H291
H538
H539
H389
HJ41
HJ40
HJ64
H537
H459
H460
DQ055835
HJ54
H142
H489
H289
H288
HD13A
HD13B
H524
H523
H525
HJ49
HD20A
HD20B
HD20C
HD11A
HD11B
HD11C
H549
HJ48
H548
H547
H543
H544
H542
H536
H535
H208
H209
H210
H462
H267
H470
H471
H472
H363
H364
H365
H213
H214
H215
H268
H207
HLi48
HLi49
H492
HJ50
HJ51
H156
H157a
H158a
H159
Tyr B
Tyr B
Tyr B
Tyr B
Tyr B
Tyr B
Tyr AB
Tyr B
Tyr B
Tyr AB
Tyr AB
Tyr B
Tyr B
Tyr A
Tyr B
Locality
Voucher/Reference
Tyr B
Rhod A
Rhod B
Switzerland
Switzerland
Switzerland
Switzerland
France
France
France
Aargau region
Lake Geneva, Aubonne
Lake Geneva, near Lausanne
Lake Geneva, near Lausanne
Villars-les-Dombes
Villars-les-Dombes
Villars-les-Dombes
47.49 08.22 46.48 06.40 46.01 04.94 -
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb FR1
mtarb CH1
mtarb CH1
Tarb 1
Tarb 1
-
Tarb 1
Tarb 1
-
Rarb 1
Rarb 1
Rarb 1
-
Rarb 1
Rarb 1
Rarb 1
-
France
Monterfil, Bretagne
48.05 -01.97 -
mtarb FR2
Tmol 1
Tmol 6
Rarb 1
Rarb 1
Germany
Germany
Germany
Germany
Germany
Germany
Germany
Netherlands
Denmark
Denmark
Denmark
Poland
Poland
Poland
Poland
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Czech Rep.
Austria
Hungary
Slovakia
Slovakia
Slovakia
Romania
Romania
Romania
Croatia
Croatia
Slovenia
Croatia
Croatia
Croatia
Croatia
Montenegro
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Greece
Poland
Poland
Poland
Poland
Poland
Bulgaria
Bulgaria
Bulgaria
Bulgaria
Bulgaria
Bulgaria
Bulgaria
Romania
Romania
Romania
Romania
Romania
Romania
Romania
Romania
Romania
Ukraine
Ukraine
Ukraine
Ukraine
Ukraine
Greece
Greece
Turkey
Turkey
Turkey
Turkey
Munich
Fabrikschleichach, Bavaria
Schmittenhhe, Koblenz
Papitz, Leipzig
Papitz, Leipzig
Papitz, Leipzig
Papitz, Leipzig
Lichtenvoorde
Vejle, Jutland
Lolland Island
Bornholm Island
Cybinka
Pruszowice, Wroclaw
Zebrzydowice
Zebrzydowice
Slavkov, Opava
Slavkov, Opava
Slavkov, Opava
Borovec, Nov Jin
Borovec, Nov Jin
lutava, Otrokovice
Oen, Brno
Nov Hubenov, Jihlava
Vesel nad Lunic
Vesel nad Lunic
Vesel nad Lunic
Nalovice
Nalovice
Nalovice
Nalovice
Nalovice
Brnk
Brnk
Luh nad Svatavou
Luh nad Svatavou
Hrndlwald, Wien
Kiskunflegyhza
Murnska Lehota, Revca
Streda nad Bodrogom
Streda nad Bodrogom
Finatale Clujuluj
Finatale Clujuluj
Armenis
Zdenci, Orahovica
Crna Mlaka, Jastrebarsko
Povir
Krk Island
Pag Island
Pag Island
Donja Korita, Dalmatia
Tivat
Korfu Island, Vatos
Korfu Island, Korision
Zakynthos Island
Zakynthos Island
Ilis, Peloponnese
Ilis, Peloponnese
Gialova, Peloponnese
Crete, Agia Lake, Kydonias
Crete, Agios Georgios
Axios
Axios
Axios
Kalamitsi, Sithonia, Chalkidiki
Imeros, Komotini
Spytkowice
Spytkowice
Wodzisaw Maopolski
Wodzisaw Maopolski
Borwna
Vidin
Vidin
Montana
Montana
Montana
Sadovo
elezino, Ivajlovgrad
Insula Mic a Brilei
Insula Mic a Brilei
Insula Mic a Brilei
Cetatea Histria, Istria
Nufaru
Nufaru
Nufaru
Gerojske
Rybal'e
Balakleya (Iskov prud), Kharkov
Balakleya (Iskov prud), Kharkov
Crimean Mts.
Lesbos Island, Vatoussa
Rhodos Island, Maritsa
Havsa
Havsa
Havsa
Havsa
49.15
49.92
50.28
51.37
11.58
10.55
07.45
12.23
52.00
55.65
54.77
55.07
52.20
51.18
49.88
06.47
09.50
11.52
14.93
14.78
17.13
18.65
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb PL1
mtarb PL2
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb GR7
mtarb CH1
mtarb CZ2
mtarb CH1
mtarb CH1
mtarb CZ1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CZ3
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb HU1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb CH1
mtarb HU1
mtarb GR7
mtarb CH1
mtarb HR2
mtarb HR1
mtarb HR1
DQ055835
mtarb CH1
mtarb GR10
mtarb GR9
mtarb GR2
mtarb GR4
mtarb GR12
mtarb GR11
mtarb GR8
mtarb GR2
mtarb GR2
mtarb GR5
mtarb GR5
mtarb GR5
mtarb GR6
mtarb CH1
mtarb GR1
mtarb CH1
mtarb GR3
mtarb GR7
mtarb CH1
mtori UA2
mtori UA2
mtori UA2
12Sori PL1
mtori BG4
mtori BG3
mtori BG1
mtori TR14
mtori BG2
mtori TR14
mtori UA2
mtori TR14
mtori TR14
mtori TR14
mtori TR14
mtori RO3
mtori TR14
mtori TR14
mtori RO1
mtori RO2
mtori UA2
mtori UA2
mtori UA1
mtori UA1
mtori UA2
mtori GR1
mtori GR2
mtori TR18
mtori TR18
mtori TR19
mtori TR20
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 2
Tarb 2
Tarb 12
Tarb 1
Tarb 6
Tarb 5
Tarb 1
Tarb 1
Tarb 1
Tarb 8
Tarb 13
Tarb 3
Tarb 1
Tarb 4
Tarb 7
Tarb 7
Tori 1
Tarb 1
Tarb 1
Tori 1
Tori 10
Tori 1
Tori 1
Tori 1
Tori 10
Tori 1
-
Tarb 1
Tarb 1
Tarb 1
Tarb 15
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 1
Tarb 2
Tarb 1
Tarb 1
Tarb 1
Tarb 2
Tarb 14
Tarb 14
Tarb 2
Tarb 14
Tarb 14
Tarb 6
Tarb 9
Tarb 11
Tarb 1
Tarb 10
Tarb 8
Tarb 11
Tarb 10
Tarb 16
Tarb 3
Tarb 7
Tori 1
Tarb 1
Tarb 1
Tori 11
Tori 1
Tori 2
Tori 1
Tori 1
Tori 10
Tori 10
Tori 1
-
Rarb 2
Rarb 1
(Rarb 3) c
Rarb 1
Rarb 1
Rarb 1
Rarb 1
Rarb 1
Rarb 2
Rarb 2
Rarb 1
Rarb 1
Rarb 2
Rarb 2
Rarb 2
Rarb 1
Rarb 1
Rarb 2
Rarb 2
Rori 6
Rori 6
Rori 1
Rori 1
Rarb 2
Rori 1
Rori 1
Rori 1
Rarb 2
Rarb 1
Rarb 2
Rarb 2
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 7
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
-
Rarb 1
Rarb 1
c
(Rarb 1?2)
Rarb 1
Rarb 1
Rarb 1
Rarb 1
Rarb 1
Rarb 2
Rarb 1
Rarb 1
Rarb 1
Rarb 2
Rarb 1
Rarb 1
Rori 1
Rarb 1
Rarb 1
Rori 6
Rori 6
Rori 6
Rori 1
Rori 1
Rori 1
Rori 1
Rarb 2
Rori 1
Rarb 2
Rarb 1
Rarb 2
Rarb 2
Rori 1
Rori 1
Rarb 1
Rori 1
Rori 1
Rori 6
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
49.92 17.83
49.63 18.10
49.20
49.28
49.40
49.18
17.48
16.60
15.47
14.70
49.70 14.37
49.98 14.90
50.21 12.61
48.17
46.71
48.73
48.37
16.25
19.85
20.03
21.77
46.83 23.62
45.20
45.59
45.61
45.70
45.03
44.45
22.32
17.95
15.73
13.95
14.55
15.06
43.70
42.43
39.61
39.44
37.78
16.81
18.70
19.79
19.94
20.88
37.87 21.37
36.95 21.70
35.47 23.93
35.13 24.70
40.62 22.71
39.98 23.98
40.93 25.28
49.98 19.50
50.52 20.18
49.90 20.53
43.98 22.87
43.38 23.22
42.13 24.93
41.48 26.01
44.90 27.83
44.57 28.72
45.15 28.92
46.52 31.90
46.48 32.19
49.65 36.27
44.44
39.23
36.38
41.55
34.05
26.05
28.10
26.82
NMP6V 71523
NMP6V 72920
NMP6V 72696
Smith et al. (2005)
NMP6V 71879
NMP6V 73809
NMP6V 74296
NMP6V 72533/1
NMP6V 72533/2
NMP6V 72533/3
-
mtDNA
Tyr A
H160a
Tyr AB
Turkey
Seluk - Efes
37.95 27.37 NMP6V 72534/1
mtori TR1
Tfel 3
Tori 5
Rori 1
H204
Tyr AB
Turkey
Seluk - Efes
NMP6V 72534/2
mtori TR1
Tori 5
Tori 7
Rori 1
Rori 1
Turkey
Turkey
Seluk - Efes
Bafa Gl
NMP6V 72534/3
37.47 27.50 -
mtori TR1
mtori TR14
Turkey
Marmaris
36.87 28.27 -
mtori TR16
Tori 8
Tori 9
Rori 1
Rori 5
Turkey
Turkey
Turkey
Marmaris
Marmaris
Dalaman
36.77 28.80 NMP6V 70833
mtori TR14
mtori TR17
mtori TR9
Turkey
Syedra, 15 km E Alanya
36.48 32.12 NMP6V 72535/1
mtori TR2
Tori 6
Tfel 3
Rori 1
Rori 1
Turkey
Turkey
mtori TR3
mtori TR2
H205
H461
HD14A
Tyr AB
HD14B
HD14C
H388
H161a
H162
H163
Tyr AB
70
orientalis
70
orientalis
71
orientalis
orientalis
(Tyr omitted)
orientalis
orientalis
(Tyr omitted)
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia N
intermedia N
intermedia N
sarda
sarda
sarda
sarda
sarda
sarda
sarda
sarda
sarda
sarda
orientalis
(Tyr omitted)
orientalis
orientalis
(Tyr omitted)
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
orientalis
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
molleri
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia S
intermedia N
intermedia N
intermedia N
sarda
sarda
sarda
sarda
sarda
sarda
sarda
sarda
sarda
sarda
H164a
Tyr AB
H165
H166a
Tyr AB
Turkey
Gazipaa
36.27 32.32 NMP6V 72536/1
mtori TR4
Tfel 3
Tfel 3
Rori 1
Rori 1
Turkey
Gazipaa
NMP6V 72536/2
mtori TR5
Turkey
72 orientalis
H446
Turkey
72 orientalis
H447
Turkey
72 orientalis
H448
Turkey
73 orientalis
H429
Turkey
73 orientalis
H430
Turkey
73 orientalis
H431
Turkey
74 orientalis
H428
Turkey
75 orientalis
H216
Turkey
75 orientalis
H217
Turkey
75 orientalis
H218
Turkey
75 orientalis
H219
Turkey
75 orientalis
H220
Turkey
76 orientalis
H420
Georgia
77 orientalis
H421
Georgia
77 orientalis
H422
Georgia
77 orientalis
H423
Georgia
78 orientalis
H407
Georgia
78 orientalis
H408
Georgia
78 orientalis
H409
Georgia
79 orientalis
H392
Azerbaijan
79 orientalis
H393
Azerbaijan
79 orientalis
H394
Azerbaijan
80 orientalis
HJ62
Russia
81 orientalis
HJ63
Russia
82 orientalis
H257
Iran
82 orientalis
H258
Iran
82 orientalis
H259
Iran
83 orientalis
H390
Iran
84 orientalis
H251
Iran
84 orientalis
H252
Iran
84 orientalis
H253
Iran
85 orientalis
H362a
Iran
86 molleri
HJ60
Tyr B
Portugal
87 molleri
HJ58
Portugal
88 molleri
HE098A
Spain
88 molleri
HE098B
Spain
- molleri
HC097
Spain
89 molleri
HE092A
Spain
89 molleri
HE092B
Spain
90 molleri
HD18A
Spain
90 molleri
HD18B
Spain
91 molleri
HE095A
Spain
91 molleri
HE095B
Spain
92 molleri
HE096
Spain
93 molleri
HE093A
Spain
93 molleri
HE093B
Spain
94 intermedia S
HD01A
Italy
94 intermedia S
HD01B
Italy
94 intermedia S
HD01C
Italy
95 intermedia S
H563
Italy
96 intermedia S
H562
Italy
97 intermedia S
H560
Italy
97 intermedia S
H561
Italy
98 intermedia S
HD02A
Italy
98 intermedia S
HD02B
Italy
98 intermedia S
HD02C
Italy
99 intermedia S
HD03A
Tyr B
Italy
99 intermedia S
HD03B
Italy
99 intermedia S
HD03C
Italy
100 intermedia S
HD07A
Italy
100 intermedia S
HD07B
Italy
100 intermedia S
HD07C
Italy
101 intermedia N
HD04A
Italy
101 intermedia N
HD04B
Tyr B
Italy
101 intermedia N
HD04C
Italy
102 sarda
HE097
France
103 sarda
H458
Italy
104 sarda
HD06A
Italy
104 sarda
HD06B
Italy
104 sarda
HD06C
Italy
105 sarda
H497a
Italy
106 sarda
HD05A
Italy
106 sarda
HD05B
Italy
106 sarda
HD05C
Italy
- sarda
HC112
Italy
- meridionalis
HE101A
Spain
- japonica
Japan
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
Melle
36.07 32.70 -
mtori TR6
Tfel 3
Tfel 3
Rori 1
Rori 1
nebolu
nebolu
nebolu
Sinop
Sinop
Sinop
Kumru, 5 km NE
Hopa
Hopa
Hopa
Hopa
Hopa
Kutaisi
Tsodoreti Lake, 10 km W Tbilisi
Telavi
Telavi
Telavi
Ki, ki
Ki, ki
Ki, ki
Malyj Utrish, Anapa
Guzeripl', Adygea Rep.
Motalla Sara-ye Lemir
Chayjan, Gilan
Tonekabon
Tonekabon
Tonekabon
Babol Sar
Carvalhal, Odeceixe
Mindelo
Pontevedra
Pontevedra
NW Spain
Chilln, Ciudad Real
Chilln, Ciudad Real
Toledo
Toledo
Villaverde, Albacete
Villaverde, Albacete
Buenache, Cuenca
Lares, Huesca
Lares, Huesca
Sicily, Gibilmanna
Sicily, Gibilmanna
Sicily, Gibilmanna
Sicily, Nebrodi Mts., pool 1
Sicily, Nebrodi Mts., pool 2
Sicily, Lentini
Sicily, Lentini
Pizzo, Calabria
Pizzo, Calabria
Pizzo, Calabria
San Giovanni Rotondo
Firenze
Firenze
Firenze
Novara
Novara
Novara
Corsica, Bocca de l'Oru
Sardinia, Badesi at Valledoria
Sardinia, Lago di Posada
Sardinia, Marina di Torre Grande
San Pietro Island
San Pietro Island
San Pietro Island
Sardinia
Tenerife Island, Puerto de la Cruz
Yasufutuichi-cho, Aito, Hiroshima
41.97 33.76 NMP6V 73700/2

NMP6V 73700/1
42.03 35.16 NMP6V 73698/2
NMP6V 73698/1
NMP6V 73698/3
40.87 37.26 NMP6V 73697
41.41 41.44 NMP6V 72688/1
NMP6V 72688/2
NMP6V 72688/3
42.26 42.72 41.76 44.65 41.92 45.49 NMP6V 73713/2
NMP6V 73713/1
NMP6V 73713/3
41.26 47.19 NMP6V 73709/1
NMP6V 73709/2
NMP6V 73709/3
44.72 37.47 43.83 40.20 38.20 48.87 NMP6V 72677/2
NMP6V 72677/3
NMP6V 72677/1
37.01 50.50 NMP6V 72842
36.81 50.88 NMP6V 72676/4
36.68 52.65 37.48 -08.75 41.32 -08.73 42.43 -08.63 38.80 -04.87 39.87 -04.03 38.80 -02.37 39.65 -02.17 42.52 -00.85 37.92 14.02 37.94 14.67 37.95 14.72 NMP6V 74149
37.30 14.98 NMP6V 74148/1
NMP6V 74148/2
38.73 16.17 41.72 15.72 43.82 11.47 45.48 08.65 41.55 09.28 40.97 08.88 NMP6V 70598
40.63 09.75 39.90 08.53 39.15 08.28 28.38 -16.55 Faivovich et al. (2005)
mtori TR14
mtori TR15
mtori TR15
mtori TR11
mtori TR12
mtori TR13
mtori TR10
mtori TR7
mtori TR8
mtori TR8
mtori TR8
mtori TR8
mtori TR8
mtori TR8
mtori TR8
mtori GE1
mtori TR8
mtori TR8
mtori TR8
mtori AZ1
mtori AZ1
mtori TR8
mtori RU1
mtori TR8
mtori TR8
mtori TR8
mtori TR8
mtori TR8
mtori IR1
mtori IR2
mtori IR3
mtori IR2
mtmol 1
mtmol 6
mtmol 7
mtmol 7
mtmol 4
mtmol 2
mtmol 2
mtmol 5
mtmol 1
mtmol 1
mtmol 1
mtmol 2
mtmol 2
mtmol 3
mtint S2
mtint S1
mtint S1
mtint S1
mtint S1
mtint S1
mtint S4
mtint S3
mtint S5
mtint S3
mtint S7
mtint S8
mtint S6
mtint S8
mtint S10
mtint S9
mtint N1
mtint N2
mtint N1
mtsar 1
mtsar 1
mtsar 4
mtsar 5
mtsar 4
mtsar 6
mtsar 2
mtsar 2
mtsar 3
mtsar 7
mtmer ES1
AY843633
Tori 1
Tori 4
Tori 1
Tori 1
Tori 3
Tori 1
Tori 1
Tori 1
Tori 1
Tori 1
Tori 1
Tori 1
Tmol 1
Tmol 1
Tmol 3
Tmol 2
Tmol 2
Tmol 1
Tint 1
Tint 1
Tint 1
Tint 1
Tint 3
Tint 2
c
(Tint 7)
Tint 2
Tint 1
d
(Tint 1?10)
Tint 5
Tsar 3
Tsar 2
Tsar 1
Tmer 1
AY844078
Tori 1
Tori 4
Tori 1
Tori 4
Tori 3
Tori 3
Tori 1
Tori 1
Tori 1
Tori 1
Tori 1
Tori 2
Tmol 5
Tmol 1
Tmol 3
Tmol 4
Tmol 2
Tmol 1
Tint 1
Tint 1
Tint 2
Tint 1
Tint 4
Tint 2
(Tint 8) c
Tint 2
Tint 1
(Tint 9) d
Tint 6
Tsar 4
Tsar 3
Tsar 1
Tmer 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rori 1
Rori 1
Rori 1
Rsar 1
Rsar 1
Rsar 1
Rmer 1
AY844615
Rori 1
Rori 1
Rori 4
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 1
Rori 3
Rori 2
Rori 2
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rmol 1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rint S1
Rori 1
Rori 1
Rint N1
Rsar 1
Rsar 2
Rsar 1
Rmer 1
Table S2. GenBank accession numbers. Haplotype codes in parentheses mark nucleotide sequences
with the standard IUPAC ambiguity codes when heterozygous positions remained unresolved.
mtDNA
haplotype
GenBank
(composite 12S /16S )
Rhod
haplotype
GenBank
mtarb CH1
mtarb CZ1
mtarb CZ2
mtarb CZ3
mtarb FR1
mtarb FR2
mtarb GR1
mtarb GR2
mtarb GR3
mtarb GR4
mtarb GR5
mtarb GR6
mtarb GR7
mtarb GR8
mtarb GR9
mtarb GR10
mtarb GR11
mtarb GR12
mtarb HR1
mtarb HR2
mtarb HU1
mtarb PL1
mtarb PL2
mtori AZ1
mtori BG1
mtori BG2
mtori BG3
mtori BG4
mtori GE1
mtori GR1
mtori GR2
mtori IR1
mtori IR2
mtori IR3
mtori RO1
mtori RO2
mtori RO3
mtori RU1
mtori TR1
mtori TR2
mtori TR3
mtori TR4
mtori TR5
mtori TR6
mtori TR7
mtori TR8
mtori TR9
mtori TR10
mtori TR11
mtori TR12
mtori TR13
mtori TR14
mtori TR15
mtori TR16
mtori TR17
mtori TR18
mtori TR19
mtori TR20
mtori UA1
mtori UA2
12Sori PL1
mtmol 1
mtmol 2
mtmol 3
mtmol 4
mtmol 5
mtmol 6
mtmol 7
mtint S1
mtint S2
mtint S3
mtint S4
mtint S5
mtint S6
mtint S7
mtint S8
mtint S9
mtint S10
mtint N1
mtint N2
mtsar 1
mtsar 2
mtsar 3
mtsar 4
mtsar 5
mtsar 6
mtsar 7
mtmer ES1
KP109551 / KP109580
KP109551 / KP109581
KP109551 / KP109582
KP109552 / KP109580
KP109551 / KP109583
KP109551 / KP109584
KP109551 / KP109585
KP109553 / KP109580
KP109553 / KP109586
KP109554 / KP109580
KP109553 / KP109587
KP109553 / KP109588
KP109553 / KP109589
KP109555 / KP109590
KP109556 / KP109591
KP109553 / KP109592
KP109553 / KP109593
KP109553 / KP109594
KP109557 / KP109595
KP109551 / KP109596
KP109558 / KP109580
KP109559 / KP109580
KP109551 / KP109597
GQ916749 / GQ916803
GQ916746 / KP109598
GQ916746 / KP109599
KP109560 / GQ916792
GQ916746 / KP109600
GQ916751 / GQ916798
KP109561 / KP109601
GQ916746 / GQ916802
GQ916749 / GQ916799
GQ916749 / GQ916800
GQ916749 / GQ916801
GQ916746 / KP109602
GQ916746 / KP109603
KP109562 / GQ916792
GQ916749 / KP109604
GQ916745 / GQ916792
GQ916746 / GQ916793
GQ916746 / GQ916794
GQ916746 / GQ916795
GQ916747 / GQ916793
GQ916748 / GQ916796
GQ916746 / GQ916797
GQ916749 / GQ916798
GQ916750 / GQ916802
GQ916746 / GQ916804
GQ916746 / GQ916805
GQ916746 / GQ916806
GQ916746 / GQ916807
GQ916746 / GQ916792
GQ916746 / GQ916808
GQ916752 / GQ916809
GQ916750 / GQ916809
KP109563 / KP109605
GQ916746 / KP109606
GQ916746 / KP109607
GQ916746 / KP109608
GQ916746 / KP109609
KP109564 / -------------KP109565 / KP109610
KP109565 / KP109611
KP109565 / KP109612
KP109565 / KP109613
KP109566 / KP109610
KP109567 / KP109614
KP109568 / KP109615
KP109569 / KP109616
KP109569 / KP109617
KP109570 / KP109616
KP109571 / KP109616
KP109572 / KP109616
KP109573 / KP109618
KP109574 / KP109616
KP109575 / KP109619
KP109574 / KP109619
KP109575 / KP109620
KP109576 / KP109621
KP109576 / KP109622
KP109577 / KP109623
KP109577 / KP109624
KP109577 / KP109625
KP109578 / KP109623
KP109579 / KP109626
KP109577 / KP109627
KP109577 / KP109628
GQ916753 / GQ916810
Rarb 1
Rarb 2
(Rarb 3)
Rori 1
Rori 2
Rori 3
Rori 4
Rori 5
Rori 6
Rori 7
Rmol 1
Rint S1
Rint N1
Rsar 1
Rsar 2
Rmer 1
KP109629
KP109630
KP109631
GQ916815
GQ916816
GQ916817
GQ916818
GQ916819
KP109632
KP109633
KP109634
KP109635
KP109636
KP109637
KP109638
GQ916820
Tyr
haplotype
GenBank
Tarb 1
Tarb2
Tarb 3
Tarb 4
Tarb 5
Tarb 6
Tarb 7
Tarb 8
Tarb 9
Tarb 10
Tarb 11
Tarb 12
Tarb 13
Tarb 14
Tarb 15
Tarb 16
Tori 1
Tori 2
Tori 3
Tori 4
Tori 5
Tori 6
Tori 7
Tori 8
Tori 9
Tori 10
Tori 11
Tfel 3
Tmol 1
Tmol 2
Tmol 3
Tmol 4
Tmol 5
Tmol 6
Tint 1
Tint 2
Tint 3
Tint 4
Tint 5
Tint 6
(Tint 7/8)
(Tint 9/10)
Tsar 1
Tsar 2
Tsar 3
Tsar 4
Tmer 1
KP109639
KP109640
KP109641
KP109642
KP109643
KP109644
KP109645
KP109646
KP109647
KP109648
KP109649
KP109650
KP109651
KP109652
KP109653
KP109654
GQ916713
GQ916714
GQ916715
GQ916716
GQ916717
GQ916718
GQ916719
GQ916720
GQ916721
KP109655
KP109656
GQ916708
KP109657
KP109658
KP109659
KP109660
KP109661
KP109662
KP109663
KP109664
KP109665
KP109666
KP109667
KP109668
KP109669
KP109670
KP109671
KP109672
KP109673
KP109674
GQ916722
Table S3. Results of the Bayesian species delimitation (BSD) analyses conducted in BP&P.
Dataset
Tree topology and BSD result (#speciation probability)
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 (Tyr IMgc1) ((((ori, mol)'#1.00', arb)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
mtDNA + nDNA (Tyr IMgc1) ((((ori, arb)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
mtDNA + nDNA (Tyr IMgc1) ((((ori, arb)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
nDNA
(((arb, Bal)'#1.00', sar)'#1.00', (((intN, intS)'#1.00', mol)'#1.00', ori)'#1.00')'#1.00';
nDNA
((((ori, mol)'#1.00', (arb, Bal)'#1.00')'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
nDNA
((((ori, Bal)'#1.00', (arb, mol)'#1.00')'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
nDNA
((((ori, mol)'#1.00', arb)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
nDNA
((((ori, arb)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
nDNA
((((arb, mol)'#1.00', ori)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
nDNA
(((((ori, mol)'#1.00', arb)'#1.00', intN)'#1.00', intS)'#1.00', sar)'#1.00';
nDNA
(((((ori, arb)'#1.00', mol)'#1.00', intN)'#1.00', intS)'#1.00', sar)'#1.00';
nDNA
(((ori, mol)'#1.00', arb)'#1.00', ((intN, intS)'#1.00', sar)'#1.00')'#1.00';
nDNA
(((((ori, intN)'#1.00', mol)'#1.00', arb)'#1.00', intS)'#1.00', sar)'#1.00';
nDNA
((((arb, ori)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)'#1.00';
nDNA
((((arb, mol)'#1.00', ori)'#1.00', (intS, intN)'#1.00')'#1.00', sar)#1.00';
nDNA (Tyr IMgc1)
((((arb, ori)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)#1.00';
nDNA (Tyr IMgc1)
((((arb, ori)'#1.00', mol)'#1.00', (intS, intN)'#1.00')'#1.00', sar)#1.00';
Rhod
Rhod
Tyr
Tyr
Tyr IMgc1
Tyr IMgc1
Tyr IMgc1
Tyr IMgc1
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
~ Species tree (data)
Conditions a
mtDNA + nDNA
+ 'hybrids'; - recombination
+ 'hybrids'; - recombination; without W Balkan
- 'hybrids'; + recombination
+ 'hybrids'; + recombination
+ 'hybrids'; + recombination
- 'hybrids'
- 'hybrids'
- 'hybrids'; - recombination
- 'hybrids'; - recombination
nDNA
mtDNA + nDNA
mtDNA + nDNA
mtDNA + nDNA
mtDNA + nDNA
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
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 ( = 1.0; ~1 in TyrIMgc1). 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
Fig. S2. Species tree (maximum clade credibility tree) based on (a) mtDNA (12S, 16S) and (b) nDNA
(Rhod, TyrIMgc1) as inferred in *BEAST. Numbers along branches are posterior probabilities. Note
the relationship between H. orientalis and H. molleri, which is in the mtDNA phylogeny likely affected
by a mitochondrial capture of H. orientalis mtDNA in H. molleri. The trees are scaled according to
the split of H. sarda.
1.00
mtDNA
0.96
0.46
0.88
sensu stricto
H. arborea W Balkans
0.91
species tree
H. arborea
H. orientalis
mtDNA
H. molleri
H. intermedia North
0.84
H. intermedia South
H. sarda
H. meridionalis
0.01 substitution/site
1.00
sensu stricto
H. arborea W Balkans
0.64
nDNA
H. arborea
species tree
H. sarda
1.0
1.00
0.40
0.59
H. intermedia North
H. intermedia South
H. molleri
H. orientalis
H. meridionalis
0.001 substitution/site

Speciation History and Widespread Introgression

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Speciation History and Widespread Introgression

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Molecular Phylogenetics and Evolution 83 (2015) 143155

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

Speciation history and widespread introgression in the European

ern Mediterranean, respectively, were elevated from subspecies

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

pulse-group properties (Fig. 1; grouping made according to the

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

method to test (1) the monophyly of H. arborea s.l. (H. arborea, H.

possible gametic phases and their phylogenetic positions were

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

2012) we also tested a position of the western Balkan tree frogs

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

5.10 (Librado and Rozas, 2009), with no recombination detected in

2010) to test whether the operational taxonomic units t the

2.6. Species delimitation

The mitochondrial DNA phylogeny had an almost identical

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

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

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

the deep mitochondrial differentiation of the two geographically

further structuring in the second group is also detectable. Most

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

alternative prior settings and type of rjMCMC algorithm also did

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

speciation probability always low (0.080.60) for the Northern and

Pisanets and Matvyeyev, 2012; Rosso et al., 2004). Hyla sarda is

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

This discrepancy in mtDNA phylogenies between the studies might

gesting that the recombined alleles carry the signal of gene

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

Eastern-European haplogroup is distributed to the north and east

netic incongruence among the markers. A mitochondrial capture

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

Grosse, W.-R., 2009. Laubfrsche. Europa. Mittelmeerregion. Kleinasien. Frankfurt

Posada, D., 2006. Collapse: Describing Haplotypes from Sequence Alignments.

V. Gvozdk et al. / Molecular Phylogenetics and Evolution 83 (2015) 143155

Vclav Gvodk, Daniele Canestrelli, Mario Garca-Pars, Ji Moravec, Giuseppe Nascetti,

Molecular Phylogenetics and Evolution

Table S1. Specimens examined.

Species tree Species delimitation

47.49 08.22 46.48 06.40 46.01 04.94 -

37.95 27.37 NMP6V 72534/1

36.77 28.80 NMP6V 70833

36.48 32.12 NMP6V 72535/1

36.27 32.32 NMP6V 72536/1

41.97 33.76 NMP6V 73700/2

Tree topology and BSD result (#speciation probability)

~ Species tree (data)

arb = Hyla arborea

Fig. S1. Maximum-parsimony haplotype network of the recombination-filtered TyrIMgc1 dataset

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