Taxonomic assessment and distribution of common toads (Bufo bufo and B. verrucosissimus) in Turkey based on morphological and molecular data

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  • 1 Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdoğan University, Rize, Turkey
  • 2 Department of Biology, Faculty of Arts and Sciences, Aydın Adnan Menderes University, Aydın, Turkey
  • 3 Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, Trabzon, Turkey

Abstract

The Bufo bufo species group includes four species distributed in the western Palearctic: B. bufo, B. eichwaldii, B. spinosus and B. verrucosissimus. Both B. bufo and B. verrucosissimus are known to occur in Turkey, but their range boundaries and the taxonomic status of B. verrucosissimus are still uncertain. In this study, we analyzed the variation in a set of morphological characters and in two mitochondrial and two nuclear DNA markers to address these questions. Phylogenetic analyses of sequence data support two main clades of common toads in Turkey, corresponding to B. bufo and B. verrucosissimus. The latter is subdivided into two allopatric subclades including populations along the Mediterranean and Black Sea coast, respectively. Discriminant analysis of morphological data showed separation among groups as defined by molecular analyses. We discuss these results and their implications for the evolutionary history of common toads in Turkey.

Abstract

The Bufo bufo species group includes four species distributed in the western Palearctic: B. bufo, B. eichwaldii, B. spinosus and B. verrucosissimus. Both B. bufo and B. verrucosissimus are known to occur in Turkey, but their range boundaries and the taxonomic status of B. verrucosissimus are still uncertain. In this study, we analyzed the variation in a set of morphological characters and in two mitochondrial and two nuclear DNA markers to address these questions. Phylogenetic analyses of sequence data support two main clades of common toads in Turkey, corresponding to B. bufo and B. verrucosissimus. The latter is subdivided into two allopatric subclades including populations along the Mediterranean and Black Sea coast, respectively. Discriminant analysis of morphological data showed separation among groups as defined by molecular analyses. We discuss these results and their implications for the evolutionary history of common toads in Turkey.

Introduction

The Bufo bufo species group consists of four species distributed in the western Palearctic: B. bufo (Linnaeus, 1758) from the larger part of Europe, B. eichwaldii Litvinchuk, Borkin, Skorinov and Rosanov, 2008 from the Talysh mountains of Azerbaijan and Iran, B. spinosus (Daudin, 1803) from North Africa, Iberia and much of France, and B. verrucosissimus (Pallas, 1814) from the Caucasus and Turkey (fig. 1). B. spinosus has traditionally been seen as a circum-Mediterranean subspecies (Mertens and Wermuth, 1960; Litvinchuk et al., 2008; Sinsch et al., 2009) but has been more recently shown to be restricted to the western Mediterranean (Recuero et al., 2012). For a summary of the taxonomic history of the B. bufo species group see Frost (2019).

Figure 1.
Figure 1.

A distribution of common toad species in the western Palearctic, with Bufo spinosus in red (Arntzen et al., 2018), B. bufo in blue (Agasyan et al., 2009), B. verrucosissimus (this study) in orange, and B. eichwaldi (AmphibiaWeb., 2016) in green. Insets – haplotype networks of each nDNA gene (B: RPL3 gene; C: RAG1 gene) and the concatenated mtDNA sequences (D).

Citation: Amphibia-Reptilia 41, 3 (2020) ; 10.1163/15685381-bja10009

Previous studies have documented the presence of both B. bufo and B. verrucosissimus in Turkey, but their range boundaries are not well delimited. Recuero et al. (2012) studied toad samples from four localities in Turkey (Samsun, Tokat, Karabük, and Artvin), and based on phylogenetic analyses of mitochondrial and nuclear genes, they assigned the population from Artvin to B. verrucosissimus and the others to B. bufo. In a study published in the same year, Garcia-Porta et al. (2012) also analyzed samples from Turkey. They assigned individuals from Alanya to their Caucasus clade (corresponding to B. verrucosissimus), and those from other localities in Turkey (Anayurt, Kayabaşı, Bursa, and Bafra) to their European clade (corresponding to B. bufo) based on mitochondrial DNA. In addition, they analyzed variation in allozymes in samples from Abant Lake that were correspondingly assigned to B. verrucosissimus.

In a subsequent study, Arntzen et al. (2013a) reconciled the results of the Garcia-Porta et al. (2012) and Recuero et al. (2012) papers, which arrived at contrasting taxonomic arrangements regarding the status of B. spinosus and B. verrucosissimus, considered at the subspecies level by Garcia-Porta et al. (2012) and as full species by Recuero et al. (2012). Arntzen et al. (2013a) reanalyzed the allozyme dataset of Garcia-Porta et al. (2012) and found no conclusive evidence for hybridization between B. bufo and B. verrucosissimus, including at the locality of Lake Abant, where individual-based genetic clustering analysis showed the simultaneous presence of the two species with no admixture. Accordingly, they called for maintaining species status for B. verrucosissimus

In this study, we present new morphological and molecular data pertaining to the identification and spatial delineation of the Turkish Bufo species. Specifically, we aimed to provide and evaluate criteria for species identification and to further delineate species distributions of the two common toad species in Turkey.

Materials and methods

Sampling and morphometric analyses

A total of 193 adult Bufo toads (96 females, 97 males) were sampled during the spring breeding periods of 2015, 2016 and 2017 (supplementary table S1) in 25 Turkish localities (fig. 2). Prior to measuring, the toads were anesthetized with 250 mg/l MS222 and after processing they were released at the place of capture. The following 27 external morphological characters were measured following Castellano and Giacoma (1998), Orlova and Tuniyev (1989) and Arntzen et al. (2013b): snouth vent length (SVL), length of the head (LHEAD), width of the head (WHEAD), minimum distance between the nostrils (INTNOS), distance between the nostril and the tip of snout (NOSTIP), minimum distance from the nostril to the anterior corner of the eye (NOSEYE), eye-tympanum distance (EYETYM), horizontal diameter of the eye (DEYE), diameter of the tympanum (DTYM), length of the parotoid (LPAR), distance between the elbows with humerus kept perpendicular to the body axis (WGRASP), radioulna length (RADUL), length of the hand (LHAND), length of the first finger (L1FING), length of the femur (LFEM), length of the tibia (LTIB), length of the tarsus (LTARS), length of the foot (LFOOT), minimum distance from the distal extremity of the inner metatarsal tubercle and the web between the third and fourth digit (WEB), length of the metatarsal tubercle (LMET), interorbital distance (IOD), anterior parotoid distance (PDA), posterior parotoid distance (PDP), left parotoid width (LPW), right parotoid width (RPW), length of the inner metatarsal tubercle (LIMT) and width of the inner metatarsal tubercle (WIMT). Measurements were taken with digital calipers to the nearest 0.01 mm. To avoid inter-observer variation all measurements were done by the same researcher. We also studied the character parotoid angle (PA) and three derived characters, namely parotoid divergence (PD = PDA/PDP), MTSIZE (LIMT/SVL) and MTSHAPE (WIMT/LIMT) as referenced by Arntzen et al. (2013b). Finally, tissue samples (toe clips) were collected for molecular analyses. Samples were preserved in Eppendorf tubes containing 96% ethanol and kept in a freezer at −20 °C until DNA extraction.

Figure 2.
Figure 2.

Detailed information on sampling locations in Turkey. Blue triangles represent Bufo bufo and orange triangles represent Bufo verrucosissimus. Locality numbers as in supplementary table S1.

Citation: Amphibia-Reptilia 41, 3 (2020) ; 10.1163/15685381-bja10009

Molecular data and phylogenetic analyses

Genomic DNA was extracted from 74 ethanol-preserved tissue samples using the NucleoSpin Tissue Kit (Macherey-Nagel) following the manufacturer’s instructions. Two mitochondrial (16S and cytochrome b) and two nuclear (RAG1 and RPL3) gene fragments were amplified by polymerase chain reaction (PCR) with the primer pairs 16Sar-16Sbr (16S, Palumbi et al., 1991), Amp-RAG1 F-Amp-RAG1 R1 (RAG1, San Mauro et al., 2004), and Cyt Bufo F-Cyt Bufo R (Cytb) and RPL3buF1-RPL3buR1 (RPL3, Recuero et al., 2012). PCRs were run in a T100 thermal cycler (BioRad) with 50 μl final volume, following protocols by Goebel et al. (1999) for 16S, Recuero et al. (2012) for Cytb and RPL3, and San Mauro et al. (2004) for RAG1. Amplified products were sequenced by Macrogen Europe Inc. (Amsterdam, The Netherlands).

We reconstructed haplotype networks using the median-joining algoritm for nuclear and mitochondrial gene fragments using PopArt 1.7 (Leigh and Bryant, 2015). Phylogenetic analyses were carried out on all DNA sequences listed in supplementary table S1, including outgroup sequences from the related toad species B. gargarizans Cantor, 1842, B. bankorensis Barbour, 1908 and B. japonicus Temminck and Schlegel, 1838. Sequences were aligned with ClustalW in BioEdit v7.2.5 (Hall, 1999). Inferred insertion/deletion polymorphisms (indels) were coded as informative characters with Fastgap v1.2 (Borchsenius, 2009). The best fitting nucleotide substitution model was selected with MrModelTest v2.3 for each gene separately, under the Akaike Information Criterion (Nylander, 2004). The SYM + G was the best-fit nucleotide evolution model for all genes and applied where appropriate. Phylogenetic trees were reconstructed with Bayesian (BI) and maximum likelihood methods (ML). We used MrBayes 3.2.6 (Ronquist and Huelsenbeck, 2003) for BI analyses. Four independent analyses were run for 106 generations (1.5 × 106 generations for concatenated data sets), after which the standard deviation of split frequencies was below 0.01 and the analysis was terminated. Each run included four chains (one cold and three heated), sampling one tree every 1000 generations. The first quarter of trees from each run was considered as burn-in and discarded. The remaining results were used to generate a 50% majority-rule consensus tree. The results were inspected with TRACER 1.7.1 (Rambaut et al., 2018) to check for convergence and for effective sample size values > 200 for each parameter in the model. ML analyses were performed with RaxML v8.0 (Stamatakis, 2014) with one thousand bootstrap replicates, under the GTRCAT model. Phylogenetic trees were visualized and edited with FigTree v 1.4.0 (Rambaut, 2012).

Lastly, we applied the species tree reconstruction method implemented in *BEAST under BEAST2 (Bouckaert et al., 2019) to assess phylogenetic relationships in the Bufo bufo species group. Samples of Bufo species from Turkey were assigned to species based on their mtDNA. We also included published sequences of B. spinosus, B. eichwaldi and Bufo gargarizans as sequential outgroups. The concatenated dataset (mtDNA + nDNA) was considered as four independent partitions and the nuclear dataset as two independent partitions. Independent models of each partition were automatically selected using bModelTest (Bouckaert et al., 2017) integrated in BEAST2. Since all the mitochondrial genes are effectively linked due to a lack of recombination, 16S and Cytb genes were set up to use the same linked tree. The topologies and support values were built under the birth-death tree prior and relaxed lognormal clock. One independent analysis was run for 12 × 107 MCMC generations, sampling every 5000 steps. Convergence and ESS were checked using Tracer 1.7.1. (Rambaut et al., 2018) to confirm parameters were >200. Afterwards, a Maximum Clade Credibility (MCC) tree was generated in TreeAnnotator with %20-burn-in and visualized using FigTree v.1.4.0 (Rambaut et al., 2012).

To strengthen the *BEAST results, Bayesian Factor Delimitation (BFD) analysis was further conducted with different species arrangements. BFD approach is allowing to compare models that contain different numbers of species especially useful to test predefined species delimitation models or competing taxonomies, ranking models by marginal likelihood estimations (Baele et al., 2012), and using Bayes factors (Kass and Raftery, 1995) to assess support for model rankings. In order to run BDF analysis, a total of 28 samples were selected representing B. bufo species group and two different species arrangement models were constituted: 1) Current taxonomy was taken as reference model considering B. bufo and B. verrucosissimus as seperate species (Recuero et al., 2012); 2) B. verrucosissimus was supposed to a subspecies of B. bufo (Garcia-Porta et al., 2012). The models were tested by calculating the marginal likelihood estimates (MLE) using stepping-stone sampling (Xie et al., 2011). Chain lengths were selected 106, number of steps 100 and alpha value 0.3 and burn-in %10. After obtaining MLE values, the models were compared to calculate Bayes factor (BF) with the formula: 2 × (model1 − model2) and crosschecked using the framework of BF support values (see Leache and Bouckaert, 2018).

Statistical analyses of morphological data

Prior to multivariate analyses, linear measurements were ln-transformed to reduce deviations from normality, the effect of variation in individual size, and to increase the fit to the requirements for such analyses (Sokal and Rohlf, 1981). As male and female toads differed markedly in body size, the two sexes were analyzed separately. We used discriminant analysis to quantify the morphometric differences among the genetically defined groups (see Results), herein referred to as B. bufo, ‘verrucosissimus_North’ and ‘verrucosissimus_South’. We also applied cross-validation to estimate the predictive accuracy of morphological classification. To allow the application of diagnostic character states in the field, measurements were analyzed with Student’s t-test on selected untransformed data. Three derived characters of Arntzen et al. (2013b) were only used in univariate analyses. All statistical analyses were carried out with IBM SPSS v.21 (IBM Corp., 2012). Results were visualized with PAST 3.25 (Hammer et al., 2001).

Results

Molecular phylogenetics

For all 74 individuals studied we obtained 547 homologous base pairs for 16S, 786 bp for Cytb, 937 bp for RAG1 and 522 bp for RPL3. All newly generated DNA sequences have been deposited in GenBank (supplementary table S1). Aligning the sequences was straightforward but included a 25 bp indel for RPL3 that separated all individuals from localities 64-71 (Hatay, Mersin and Osmaniye) from the remaining ones, thus representing a putative molecular synapomorphy of the B. verrucosissimus South clade. For 16S, we found eight different haplotypes distributed in two groups separated by five substitutions; these groups represent B. bufo (n=6, B1-B6) and B. verrucosissimus (n=2, V1 and V2). For Cytb, we found 23 different haplotypes. These also classified in two groups that were separated by nine substitutions and represent B. bufo (n=18, B7-B23 and B56), and B. verrucosissimus (n=5, V3-V7). The haplotype network based on concatenated mitochondrial genes (fig. 1D) showed a clear separation between the two groups.

We found four different haplotypes for RPL3 and RAG1 nuclear genes that did not show full separation between species. The haplotype networks for RPL3 and RAG1 genes are shown in fig. 1B, C. The specimens of B. verrucosissimus from Antalya shared haplotype B54 with B. bufo, Artvin specimens shared haplotype V16 and the rest of B. verrucosissimus specimes from Mediterranean Region shared V17 haplotype for RPL3 gene. But Isparta specimens of B. bufo shared haplotype V15 with Mediterranean B. verrucosissimus specimens for RAG1 gene. Finally, Artvin specimens of B. verrucosissimus shared haplotype B51 with B. bufo.

We recovered consistent topologies in BI and ML analyses of mtDNA sequences (fig. 3), with two major clades corresponding to B. bufo and B. verrucosissimus. According to these results, B. verrucosissimus is found in two disjunct regions: in the northeast of Turkey (Artvin) and along the Mediterranean coast in the south (Osmaniye, Hatay, Mersin and Antalya), while B. bufo is present at the remaining localities sampled. Within the B. verrucosissimus clade, a separate group including samples from Russia and the north of Georgia has strong support. Stronger support was found for two further subclades, referred to as verrucosissimus_North (including the V08 haplotype from Artvin and samples from the southwest of Georgia in localities Adjara and Borjomi) and verrucosissimus_South (including haplotypes V09-V12, found in the Turkish Mediterranean localities Osmaniye, Hatay, Mersin and Antalya).

Figure 3.
Figure 3.

Phylogenetic tree based on Bayesian analysis of concatenated mtDNA sequences of the Bufo bufo species group. Colour codes as in fig. 1. Support values for internal nodes are on the zero to one scale for BI-analyses and from zero to 100 for ML-analyses. Hyphen (-) denotes <95% BPP and <70% ML bootstrap support values. Haplotype V08 represents B. verrucosissimus North while haplotypes V09-V012 represent B. verrucosissimus South.

Citation: Amphibia-Reptilia 41, 3 (2020) ; 10.1163/15685381-bja10009

The species trees obtained from the *BEAST analyses revealed generally fully resolved topologies and represented topological congruencies compared with the mitochondrial gene tree. The inferred species trees support the monophyly of B. bufo and B. verrucosissimus with high supported, with branch values having BPP ⩾ 95 (fig. 4). Furthermore, the concatenated species tree constructed from all loci produced identical multilocus species tree topology with approximate branch support values as in Recuero et al. (2012). The nuclear species tree also revealed similar results, except for a weakly supported B. eichwaldi branch, probably because of a lack of RAG1 data available for this taxon.

Figure 4.
Figure 4.

The species tree constructed using *BEAST. A: Concatenated dataset (all loci); B: Nuclear loci. Posterior probabilities are shown with support values.

Citation: Amphibia-Reptilia 41, 3 (2020) ; 10.1163/15685381-bja10009

In the stepping-stone analysis, the MLE was found to be −5848.289 for model 1 (current taxonomy, i.e. considering B. bufo and B. verrucosissimus as seperate species) and −5867.362 for model 2 (considering B. verrucosissimus as a subspecies of B. bufo). Subsequently, the BF calculation was made against the current taxonomy model (model 1) and the value was calculated as +39 (BF > 10, decisive). The positive BF value indicated strong support for model 1, distinguishing B. bufo and B. verrucosissimus as distinct species.The result also matched with species trees those obtained in the *BEAST analysis.

Multivariate morphological differentiation

Discriminant functions separate the three genetically identified groups to various degrees. For females, the largest contributions to the first discriminant axis are by characters SVL, LPW, RADUL, and LHEAD, PDP and IOD, with negative and positive factor loads, respectively. At the second axis, the characters with a higher contribution are WHEAD, WGRASP and PDP (negative), and LTIB and EYETYM (positive). Scores of correct group classification were 97.9% for the original grouping and 86.5% for cross validation (supplementary table S2). For males, the largest contributions to the first axis are by characters RPW and SVL (negative) and LTARS (positive). At the second axis the characters contributing most are RPW, EYETYM, WHEAD (positive), and LPW and IOD (negative). Scores of correct group classification were 97.9% for the original grouping and 81.4% for cross validation (supplementary table S2). The results are summarized in fig. 5.

Figure 5.
Figure 5.

Bivariate plots of factor loadings on the first and second axes of the discriminant analysis based on morphological data for the three groups as identified by molecular analyses (fig. 2). A: Females; B: Males.

Citation: Amphibia-Reptilia 41, 3 (2020) ; 10.1163/15685381-bja10009

Univariate morphological differentiation of northern and southern B. verrucosissimus

The investigated character states followed a normal distribution (Kolmogorov-Smirnov test, P>0.05). Student’s t-tests identified some morphological differences between the two different groups of B. verrucosissimus. Descriptive statistics are presented in table 1: verrucosissimus_South toads have wider and more divergent parotoids than verrucosissimus_North toads, and the PA of verrucosissimus_South toads is smaller, congruently with PD for both sexes. Moreover, in males (but not in females), in parallel with PD, PDA and especially PDP of verrucosissimus_South members are longer.

Discussion

Previous studies have shown B. verrucusissimus to occur in at least three different regions in Turkey (García-Porta et al., 2012; Recuero et al., 2012; Arntzen et al., 2013a). First, García-Porta et al. (2012) reported the occurrence of B. verrucosissimus at Lake Abant, in the northwestern of Turkey, based on allozyme data (no mtDNA sequences were available). The reanalysis of this allozyme dataset by Arntzen et al. (2013a) suggested that the sample of Lake Abant analyzed by Garcia-Porta et al. (2012) was composed of one B. verrucosissimus and three B. bufo, with no evidence for admixture. Our study, based on both mtDNA and nDNA sequences, only found evidence for the presence of B. bufo in this area (figs 2 and 3). Considering the closest B. verrucosissimus populations are very distant (750 km, see fig. 3), we suggest that the reported presence of B. verrucosissimus in Lake Abant in previous studies may result from an error in sample labelling or processing.

Excluding the isolated Lake Abant record, B. verrucosissimus is present in two disjunct regions in Turkey. A first group is present in the South, along the Mediterranean coast, as first reported by Kutrup et al. (2006) based on the analysis of samples from Mersin. Later, Garcia-Porta et al. (2012) reported the presence of B. verrucosissimus in Alanya (their locality 167) based on both mitochondrial DNA sequences and allozymes (García-Porta et al., 2012). Our study further extends the range of B. verrucosissimus in southern Turkey with new locality records (supplementary table S1). This group of populations seems to be isolated by the Taurus Mountains to the north (fig. 1). These mountains have provided a complex topographic and microclimatic setting that has functioned as a historical refugium for the persistence of many amphibians (Gvozdik et al., 2010; Gül, 2013; Plötner et al., 2015; van Riemsdijk et al., 2017) and reptiles (Kapli et al., 2013; Tamar et al., 2015; Candan et al., 2016; Skourtanioti et al., 2016; Stümpel et al., 2016; Kotsakiozi et al., 2018). These populations of B. verrucosissimus seem to be isolated from those in Syria and Lebanon (see Jablonski and Sadek, 2019).

A second group of populations of B. verrucosissimus occupies northeastern Turkey and the Caucasus. Kutrup et al. (2006) did not find significant differences in mtDNA 16S sequences in samples from the Black Sea Region, Italy and Greece, including those from Artvin in Turkey, which were later assigned to B. verrucosissimus by Recuero et al. (2012), in agreement with our study. García-Porta et al. (2012) analyzed a sample from a single locality from northeastern Turkey (Anayurt near Trabzon), which was assigned to B. bufo (García-Porta et al., 2012). This raises the possibility of the existence of a hybrid zone between B. bufo and B. verrucosissimus in this region, since according to Tuniyev et al. (2014), the range of B. verrucosissimus in Turkey extends from the west of Kaçkar Mountains to the boundary of Şavşat district, Artvin. Future studies should focus on this area and quantify patterns of reproductive isolation under a cline analysis framework as implemented in van Riemsdijk et al. (2019).

Table 1.
Table 1.

Descriptive statistics of morphological variables (see text for abbreviations) and results of Student’s t-test for differences between B. verrucosissimus North and B. verrucosissimus South.

Citation: Amphibia-Reptilia 41, 3 (2020) ; 10.1163/15685381-bja10009

The disjunct distribution of B. verrucosissimus in Turkey, with populations in the Mediterranean and in the Caucasus parallels observations in other taxa, notably reptiles (Fritz et al., 2009; Jandzik et al., 2013; Milucek et al., 2013; Tamar et al., 2016), but also amphibians (van Riemsdijk et al., 2017; Dufresnes et al., 2019). A major biogeographic event potentially related with this peculiar pattern is the emergence of the Anatolian Diagonal, an important geographic barrier associated with changes in species diversity and composition (Davis, 1971; Nilson et al., 1990; Yiğit et al., 2012; Gül, 2013; Mohammadin et al., 2017; Şeker et al., 2018; Kočiš et al., 2018). The combination of mountain orogenesis and climatic changes during the Pleistocene may have driven the disjunct evolution of the two lineages of B. verrucosissimus, a divergence which seems recent, in view of the incomplete sorting of some nuclear alleles that are shared between the two populations as well as with some specimen of its sister taxon B. bufo.

Other amphibian taxa show diverged lineages along the Black Sea shore, as is observed in Bufo. For instance, Triturus karelinii is represented with three distinct gene pool groups along the Black Sea region (Wielstra and Arntzen, 2011; Wielstra et al., 2013a). Further studies revealed that these groups represent different species: T. karelinii sensu stricto, T. anatolicus and T. ivanbureschi (Wielstra et al., 2013b, 2014; Wielstra and Arntzen, 2016). In the north-east of Turkey, T. karelinii and T. anatolicus show a distribution gap. The genus Lissotriton shows a (wider) distribution gap in Anatolia as well, with L. kosswigi occurring in the west and L. lantzi protruding in the east (Wielstra et al., 2015, 2018). The occurrence of B. bufo and verrucosissimus_North along the Black Sea coast suggest a potential parallel biogeograhphic history. While there are no localities known where the two Bufo taxa meet, the distance between known B. bufo and verrucosissimus_North is smaller than observed in the two newt genera and it is possible there is a secondary contact zone.

Discriminant analysis of morphological data showed separation among groups as defined by molecular analyses. The characters involved are related to variation in the parotoids, inner metatarsal tubercles and snouth-venth length. Differences in parotoids and the inner metatarsal tubercle were also used as diagnostic characters to separate B. bufo and B. spinosus in previous studies (Arntzen et al., 2013b, 2014, 2016, 2017, 2018; Trujillo et al., 2017). We also found some differences between the two major B. verrucosissimus groups, with verrucosissimus_South individuals having more divergent parotoids than verrucosissimus_North. Observed differences, taking into account sexual dimorphism, may facilitate species assignment of adult toads in the field.

Through the combination of our comprehensive sampling, using different loci and Genbank data, the species status of B. bufo and B. verrucosissimus was supported. B. verrucosissimus was previously considered as a subspecies of B. bufo (Garcia-Porta et al., 2012), whereas following studies presented it as a separate species (Recuero et al., 2012; Arntzen et al., 2013b). A recent paper (van Riemsdijk et al, 2019; preprint on BioRxiv) shows that restriction-site associated DNA (RAD) sequencing data works well for the Bufo complex. So future genome-scale analysis (such as RADseq) would be a valuable next step for working on contact zones. In addition, more detailed analyses focusing on reproductive isolation in contact zones (both in north and south of Turkey) using other markers (like microsatellites or SNPs) and under a cline analysis framework, need to be carried out to settle the issue.

*

Corresponding author; e-mail: nurhayat.ozdemir@erdogan.edu.tr

Acknowledgements

This work was supported by the TUBİTAK with grant 114Z823. Sampling was carried out under permission of the local ethics committee for animal experiments (Republic of Turkey Recep Tayyip Erdoğan University Local Ethics Committee for Animal Experiments, approval reference number: 2014/4). We thank Ö. Özkan and E. Kızılhan for help in the laboratory and Dr. M. Z. Yıldız for providing specimens from Hatay. We lastly thank J. W. Arntzen for help the while writing manuscript.

Supplementary material

Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.12129777

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Footnotes

Associate Editor: Matthias Stöck.

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    A distribution of common toad species in the western Palearctic, with Bufo spinosus in red (Arntzen et al., 2018), B. bufo in blue (Agasyan et al., 2009), B. verrucosissimus (this study) in orange, and B. eichwaldi (AmphibiaWeb., 2016) in green. Insets – haplotype networks of each nDNA gene (B: RPL3 gene; C: RAG1 gene) and the concatenated mtDNA sequences (D).

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    Detailed information on sampling locations in Turkey. Blue triangles represent Bufo bufo and orange triangles represent Bufo verrucosissimus. Locality numbers as in supplementary table S1.

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    Phylogenetic tree based on Bayesian analysis of concatenated mtDNA sequences of the Bufo bufo species group. Colour codes as in fig. 1. Support values for internal nodes are on the zero to one scale for BI-analyses and from zero to 100 for ML-analyses. Hyphen (-) denotes <95% BPP and <70% ML bootstrap support values. Haplotype V08 represents B. verrucosissimus North while haplotypes V09-V012 represent B. verrucosissimus South.

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    The species tree constructed using *BEAST. A: Concatenated dataset (all loci); B: Nuclear loci. Posterior probabilities are shown with support values.

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    Bivariate plots of factor loadings on the first and second axes of the discriminant analysis based on morphological data for the three groups as identified by molecular analyses (fig. 2). A: Females; B: Males.

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    Descriptive statistics of morphological variables (see text for abbreviations) and results of Student’s t-test for differences between B. verrucosissimus North and B. verrucosissimus South.

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