# Call a spade a spade: taxonomy and distribution of Pelobates, with description of a new Balkan endemic

Christophe Dufresnes, Ilias Strachinis, Elias Tzoras, Spartak N Litvinchuk, Mathieu Denoël

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Christophe Dufresnes, Ilias Strachinis, Elias Tzoras, Spartak N Litvinchuk, Mathieu Denoël. Call a spade a spade: taxonomy and distribution of Pelobates, with description of a new Balkan endemic. Zookeys, 2019, 859, pp.131-158. 10.3897/zookeys.859.33634. hal-04887136

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# Call a spade a spade: taxonomy and distribution of *Pelobates*, with description of a new Balkan endemic

Christophe Dufresnes<sup>1,2,3</sup>, Ilias Strachinis<sup>4</sup>, Elias Tzoras<sup>5</sup>,
Spartak N. Litvinchuk<sup>6,7</sup>, Mathieu Denoël<sup>8</sup>

1 Laboratory for Conservation Biology, University of Lausanne, 1015 Lausanne, Switzerland 2 Hintermann & Weber SA, Avenue des Alpes 25, 1820 Montreux, Switzerland 3 Department of Animal and Plant Sciences, University of Sheffield, Alfred Denny Building, Western Bank, S10 2TN Sheffield, United Kingdom 4 School of Biology, Aristotle University of Thessaloniki 5 26442 Patra, Achaia, Greece 6 Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. 194064 Petersburg, Russia 7 Department of Zoology and Physiology, Dagestan State University, Gadzhiyev str. 43-a, 336700 Makhachkala, Dagestan, Russia 8 Laboratory of Fish and Amphibian Ethology, Behavioural Biology Group, Freshwater and OCEanic science Unit of reSearch (FOCUS), University of Liège, Liège, Belgium

Corresponding author: Christophe Dufresnes (christophe.dufresnes@hotmail.fr)

Academic editor: Angelica Crottini | Received 4 February 2019 | Accepted 10 June 2019 | Published 2 July 2019

http://zoobank.org/5E2B8623-A309-4EF6-9123-B95F04C5A88E

**Citation:** Dufresnes C, Strachinis I, Tzoras E, Litvinchuk SN, Denoël M (2019) Call a spade a spade: taxonomy and distribution of *Pelobates*, with description of a new Balkan endemic. ZooKeys 859: 131–158. https://doi.org/10.3897/zookeys.859.33634

## Abstract

The genomic era contributes to update the taxonomy of many debated terrestrial vertebrates. In an accompanying work, we provided a comprehensive molecular assessment of spadefoot toads (*Pelobates*) using genomic data. Our results call for taxonomic updates in this group. First, nuclear phylogenomics confirmed the species-level divergence between the Iberian *P. cultripes* and its Moroccan relative *P. varaldii*. Second, we inferred that *P. fuscus* and *P. vespertinus*, considered subspecies until recently, feature partial reproductive isolation and thus deserve a specific level. Third, we evidenced cryptic speciation and diversification among deeply diverged lineages collectively known as *Pelobates syriacus*. Populations from the Near East correspond to the Eastern spadefoot toad *P. syriacus* sensu stricto, which is represented by two subspecies, one in the Levant (*P. s. syriacus*) and the other in the rest of the range (*P. s. boettgeri*). Populations from southeastern Europe correspond to the Balkan spadefoot toad, *P. balcanicus*. Based on genetic evidence, this species is also polytypic: the nominal *P. b. balcanicus* inhabits the Balkan Peninsula; a new subspecies *P. b. chloeae* ssp. nov. appears endemic to the Peloponnese. In this paper, we provide an updated overview of the taxonomy and distribution of all extant *Pelobates* taxa and describe *P. b. chloeae* ssp. nov.
**Keywords**

Amphibian, Palearctic, *Pelobates balcanicus*, *Pelobates balcanicus chloeae*, *Pelobates vespertinus*, Pelobatidae, phylogenomics, phylogeography, spadefoot toad

**Introduction**

The revolution initiated by high-throughput sequencing techniques has reached the field of phylogeography (Coates et al. 2018), where it lifts the veil on cryptic species and solves long-term taxonomic issues (e.g. Rodriguez et al. 2017; Psonis et al. 2018; Dufresnes et al. 2018, 2019a). We conducted such study in spadefoot toads from the monotypic family Pelobatidae Bonaparte, 1850 (genus *Pelobates* Wagler, 1830) endemic to the Western Palearctic (Dufresnes et al. 2019b). These grassland species typically inhabit soft (e.g. sandy) soils with freshwater ponds for breeding and have a semi-fossorial lifestyle, thanks to well-known adaptations such as metatarsal spades (to dig themselves in) and a strongly ossified skull (to dig themselves out) (Székely et al. 2017; Dufresnes 2019). They are threatened in many parts of their fragmented ranges due to land-use changes, wetland destruction, pollution, species introduction, and ongoing changes in climate, which already led to population extinctions and contractions of geographic ranges (Nyström et al. 2002, 2007; Džukić et al. 2005; Eggert et al. 2006). Mediterranean regions, where most of the diversity is located (Litvinchuk et al. 2013; Dufresnes et al. 2019b), could be particularly threatened (Iosif et al. 2014).

Until recently, *Pelobates* included four recognized extant species. First, the sister taxa *P. cultripes* (Cuvier, 1829) and *P. varaldii* Pasteur & Bons, 1959 are found north and south of the Strait of Gibraltar, respectively (Busack et al. 1985). Second, the western and eastern sister taxa *P. fuscus* (Laurenti, 1768) and *P. vespertinus* (Pallas, 1771) were long considered subspecies (e.g. Crottini et al. 2007), but their narrow transition is rather consistent with a species level (Litvinchuk et al. 2013). Third, Mediterranean populations from the Near East and the Balkans are commonly referred to as *P. syriacus* Boettger, 1889 and split as two subspecies: *P. syriacus syriacus* in Asia Minor and *P. syriacus balcanicus* Karaman, 1928 in the Balkans, based on morphological (Uğurtas et al. 2002) and scattered phylogenetic data (Veith et al. 2006; Litvinchuk et al. 2013; Ehl et al. 2019).

Our accompanying paper (Dufresnes et al. 2019b) revisits the evolution of this group, with several taxonomic implications. First, phylogenomics confirmed the old split between *P. cultripes* and *P. varaldii*, previously identified with mtDNA (Garcia-Paris et al. 2003; Veith et al. 2006; Crottini et al. 2007) and allozyme markers (Busack et al. 1985; Litvinchuk et al. 2013). Second, hybrid zone analyses support the conclusions of Litvinchuk et al. (2013) that *P. fuscus* and *P. vespertinus* deserve a specific status. Third, *P. syriacus* represents two cryptic species respectively distributed in the Near East and the Balkans, then corresponding to *P. syriacus* and *P. balcanicus*. Fourth, these species feature deep intraspecific divergence, worthy of subspecific status. This is the case between Levantine and Anatolian/Caucasian populations in *P. syriacus*, and between the northern Balkans and Peloponnese in *P. balcanicus*.
<figure><img src="image_2.png" /></figure>

**Figure 1.** Phylogeny and distribution of *Pelobates* taxa. The tree is adapted from the phylogenomic analysis of Dufresnes et al. (2019b), and the map was built from known records updated with genetic data (see accounts). Note that the distribution of *P. vespertinus* extends further east to Kazakhstan and Siberia. Photo credits: CD (*P. cultripes*, *P. b. chloeae*), SNL (*P. s. boettgeri*), IS (*P. b. balcanicus*), A Sanchez Vialas (*P. varaldii*), A Nöllert (*P. fuscus*), N Suriadna (*P. vespertinus*).

In this paper, we integrate these recent findings into an updated overview of the *Pelobates* radiation, including comparative diagnosis, current taxonomy, distribution, and diversity of the resulting eight extant taxa (Fig. 1). Last but not least, we describe the newly discovered clade from Peloponnese as a subspecies of *P. balcanicus*.

# Material and methods

## Nomenclatural search

In order to attribute names to the newly documented *Pelobates* species and subspecies, we examined nomina available in the literature. To this end, we first referred to the Amphibian Species of the World online database (Frost 2019) and subsequently reviewed all the original references available.

## Diagnosis

We reviewed phenotypic (coloration, morphology) and genetic (genome size, karyotype, and sequence divergence) variation of the considered taxa. Coloration is illustrated by high-quality photographs of known geographic origins, taken by us and credited photographers. Besides detailing general characteristics, we compiled a dataset of snout-vent length (SVL) from published studies (Suppl. material 1, Table S1), consisting of average SVL (computed separately for males and females) from 82 populations, totaling
6,004 individuals at least, and representing all taxa expect the narrowly distributed *P. s. syriacus* and *P. b. chloeae* (Suppl. material 1, Table S1). We report the ranges (minimum-maximum) and average values for each sex separately, and illustrate interpopulation variation by boxplots. We statistically tested differences among taxa and sex by a two way analysis of variance (ANOVA) in *R*. We then performed comparisons between species using a Tukey test. Finally, we tested sex-specific differences within taxa for which measures of both sexes were available in at least five populations, by paired *t*-tests.

We briefly described the karyotype of each taxon based on the literature and further report nuclear DNA content as a proxy to genome size, obtained from flow cytometry. In addition, sequence divergence, available from our phylogeographic study (Dufresnes et al. 2019b), are provided between each pair of taxa, based on mitochondrial (*cyt-b* + 16S, 1.2 kb) and nuclear DNA (63.5 kb of RAD tags).

## Distribution

We detailed the distribution of each *Pelobates* taxon, based on available literature, i.e. national and regional atlas, as well as scientific articles informative of distribution. Boundaries between cryptic taxa were inferred from genetic studies, and thus remain unclear for parapatric ranges for which no molecular survey has been conducted. Distribution layers were originally obtained from the IUCN Red List of Threatened Species (https://www.iucnredlist.org/), and meticulously reshaped with the drawing tools of ArcMap 10.3.

## Description of *Pelobates balcanicus chloeae* sp. nov.

In order to describe the new *P. balcanicus* subspecies from southern Greece, we conducted a short fieldwork expedition to the only recently confirmed locality of this taxon, Strofylia meadows in Peloponnese (38.1239°N, 21.3858°E) on December 2018. Collection of live animals was authorized by permit ΑΔΑ: ΩΣΜ34653[8-9ΣΟ issued by the Greek Ministry of Environment, Energy and Climate Change. *Pelobates* usually breed during spring (March–April) in this area but are active all-year round providing proper weather conditions. A total of 18 individuals could be captured in the evening of December 10th, under heavy rains. The largest 12 individuals (putatively adults) were measured for 11 standard morphometric variables, i.e. SVL: snout-vent length; HW: head width; HL: head length; ED: eye diameter; EE: inter-eye distance; NN: inter-nostril distance; EN: eye-nostril distance; ML: metatarsal tubercle length; MH: metatarsal tubercle height; HLL: hind leg length; TTL: tibia + tarsus length. HLL and TTL were measured with a ruler (1 mm precision); all other variables were measured with a digital caliper (0.1 mm precision). For the sake of comparison, only one of us (IS) measured all individuals. Note that we did not discriminate the sex of individuals as it was unclear whether all specimens were adults.
Toads were subsequently released at their place of capture, except for two females that were chosen as holotype and paratype, sent to the Natural History Museum of Crete (NHMC). Our choice for a small type series was bounded by the rarity of this taxon, so far confirmed from a single locality, with unknown population trends.

**Results and discussion**

We updated the distributions and taxonomy of Eurasian spadefoot toads (genus *Pelobates*). Following recent molecular results (Dufresnes et al. 2019b), a total of eight extant clades are distinguished. Six of them correspond to species level divergences, given their confirmed or putative reproductive isolation, as inferred from hybrid zone analyses, which make ad hoc tests to evaluate where two lineages stand along the speciation continuum (Singhal and Moritz 2013; Dufresnes et al. 2019b). The remaining intraspecific lineages are accordingly treated as subspecies, because they featured extensive admixture and thus seem to lack reproductive barriers.

From our SVL dataset, there was a significant global effect of species (*P* < 0.001) but not of sex (*P* = 0.08), neither of their interaction (*P* = 0.42) (two way ANOVA). The species effect was mainly due to differences between the large *P. cultripes*, *P. syriacus*, and *P. balcanicus* versus the smaller *P. varaldii*, *P. fuscus*, and *P. vespertinus*: all pairwise comparisons between these two groups were significant (*P* < 0.001), but none within groups (*P* > 0.05) (Tukey test). Females were significantly larger than males in *P. cultripes* (*P* = 0.002, *n* = 16 populations with both sexes), *P. fuscus* (*P* < 0.001, *n* = 21), but not in *P. balcanicus* (*P* = 0.58, *n* = 15) (paired *t*-test). Sample size precluded similar analyses in the remaining taxa.

The following present accounts for each taxon. We could successfully access the original literature for all but one description, and herein report the primary information as it was published. The only exception is *Pelobates praefuscus* Khosatzky, 1985, and we rely on Frost (2019) for its information. Phylogeny and distributions of extant *Pelobates* are shown in Figure 1, sizes and color variation are displayed in Figure 2, and Figures 3 and Figures 4, respectively.

*Pelobates cultripes* (Cuvier, 1829)

Western spadefoot

**Diagnosis.** The largest *Pelobates* species, *P. cultripes* differs from the other Eurasian spadefoots by metatarsal spades being entirely black and a flat skull. Sizes largely overlap between sexes although males are generally smaller than females (Fig. 2). The background coloration can be yellow, gray, or brown, reticulated by dark patches; it typically lacks orange spots (Fig. 3). Average SVL = 74 mm (range: 32–105 mm) for females (*n* = 16 populations) and 71 mm (34–93 mm) for males (*n* = 17 populations) (Suppl. material 1, Table S1; Fig. 2). The karyotype consists of six large and seven small (i.e. <
6% of total length) pairs of two-armed chromosomes (Morescalchi 1967, 1971; Morescalchi et al. 1977; Schmid et al. 1987; Herrero and Talavera 1988). Large centromeric C-bands appears in pairs 1, 2, 4, 9, and 12; pericentric bands in the short arm of pair 1 and the long arm of pair 8; telomeric bands in the long arms of pairs 1, 2, and 11; the short arm of pair 7 is almost heterochromatic (Herrero and Talavera 1988). Nucleolus organizers (NORs) are in the short arm of pair 7 (Schmid et al. 1987). The nuclear DNA content averages 7.4 pg (Litvinchuk et al. 2013).

**Taxonomy.** First named *Rana cultripes* Cuvier, 1829; holotype: MNHNP 0.4554; type locality: “notre midi”, corresponds to southern France, as noted by Mertens and Müller (1928). Two junior synonyms. *Rana calcarata* Michahelles, 1830; type locality: “prope Malagam” (near Malagam), probably Malaga, Spain; type(s): not mentioned. *Cultripes provincialis* Müller, 1832; type locality: “Provence” (meridional France), France; type(s): not designated, but the author refers to *Rana cultripes* from Paris (MNHN). First mentioned as *Pelobates cultripes* by Tschudi (1838).

**Distribution.** The species inhabits south-western Europe (0–1770 m elevation a.s.l.) (Sillero et al. 2014; Beja et al. 2009) (Fig. 1). Its main distribution spans across the Iberian Peninsula, where it occurs roughly everywhere in suitable habitats south of the Cantabrian Mountains and Pyrenees (Lizana 1997; Malkmus 2004). It is yet absent from the south-eastern tip of Spain (Lizana 1997). In France, it is present only along the Atlantic coast, from the Landes region to the Loire River, and along the Mediterranean Sea, from the Spanish border to the Var Department, reaching the area of Valence in the Rhone Valley. Some isolates exist also in south-western France (Thirion and Cheylan 2012). IUCN status: Near Threatened (Beja et al. 2009).

**Diversity.** Combining mtDNA and microsatellite data, Gutiérrez-Rodriguez et al. (2017) identified three closely-related mtDNA haplogroups (see also Crottini et al. 2010) in the southern, western / northwestern, and northeastern parts of the range, which are mirrored by equivalent nuclear clusters that widely admix. Most of the genetic diversity of this species is found in southern ranges, where climate conditions remained stable through the last ice ages (Gutiérrez-Rodriguez et al. 2017).

*Pelobates varaldii Pasteur & Bons, 1959*

Moroccan spadefoot

**Diagnosis.** A smaller version of *P. cultripes* (Fig. 2) differing by a few phenotypic features. Unlike *P. cultripes*, the black coloration of the spades is often concentrated on the edges (Pasteur and Bons 1959; Busack et al. 1985). The cranial braincase is high and narrow in *P. varaldii*, while it is low and wide in *P. cultripes* (Pasteur and Bons 1959; Roček 1981). The background coloration can be yellow, gray, and brown, with dark reticulate patches, and the dorsal surface is abundantly covered by orange dots, most pronounced on the eyelids (usually absent in *P. cultripes*; Pasteur and Bons 1959; Beukema et al. 2013; Fig. 3). Males are usually smaller than females (Fig. 2). Average SVL = 53 mm (range: 36–66 mm) for females (n = 4 populations) and 51 mm
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Between-population variation of average size (snout–vent length – SVL) for each *Pelobates* species, measured separately for females (pink) and males (blue). This compiles average size-data from 82 populations, representing at least 6,004 individuals (Suppl. material 1, Table S1). For *P. balcanicus*, it only includes populations from the nominal *P. b. balcanicus*. For *P. syriacus*, it only includes populations from *P. s. boettgeri*.</figcaption></figure>

(33–65 mm) for males (n = 4 populations) (Suppl. material 1, Table S1; Fig. 2). The karyotype includes six large and seven small pairs of two-armed chromosomes. Large centromeric C-bands appear in the pairs 1, 2, 4, 9, and 12; pericentric bands in the short arms of pair 1 and long arm of pair 8; telomeric bands in the long arms of pairs 1, 2, and 11; the short arm of pair 7 is almost heterochromatic (Herrero and Talavera 1988). The nuclear DNA content averages 7.3 pg (Litvinchuk et al. 2013). As shown
P. cultripes

<figure><img src="image_4.png" /></figure>

a

<figure><img src="image_5.png" /></figure>

b

<figure><img src="image_6.png" /></figure>

c

<figure><img src="image_7.png" /></figure>

d

P. varaldii

<figure><img src="image_8.png" /></figure>

e

<figure><img src="image_9.png" /></figure>

f

<figure><img src="image_10.png" /></figure>

g

<figure><img src="image_11.png" /></figure>

h

P. fuscus

<figure><img src="image_12.png" /></figure>

i

<figure><img src="image_13.png" /></figure>

j

<figure><img src="image_14.png" /></figure>

k

<figure><img src="image_15.png" /></figure>

l

P. vespertinus

<figure><img src="image_16.png" /></figure>

m

<figure><img src="image_17.png" /></figure>

n

<figure><img src="image_18.png" /></figure>

o

<figure><img src="image_19.png" /></figure>

p

**Figure 3.** Color variation in *Pelobates cultripes*, *P. varaldii*, *P. fuscus* and *P. vespertinus*. Photo credits and origins as follows **a** CD (Hérault, France) **b, c** CD (Algarve, Portugal) **d** A Sanchez Vialas (Spain) **e** G Martinez (Kenitra, Morocco) **f–h** A Sanchez Vialas (Tanger, Morocco) **i, j** N Suriadna (Ukraine) **k** CD (Wojewodztwo podkarpackie, Poland) **l** A Nöllert (Burgenland, Austria) **m–p** N Suriadna (Ukraine).

in Table 1, *P. varaldii* differs from *P. cultripes* by 6.0% at mtDNA and 0.40% at nuclear DNA (Dufresnes et al. 2019b).

**Taxonomy.** The nomen *Pelobates varaldii* Pasteur & Bons, 1959 is the only one ever proposed for the Moroccan populations of spadefoot toads; holotype: MNHN-RA-1959.1; type locality: Merja Samora, Morocco. The ancient split of *P. varaldii*, dating back to the Messinian Salinity Crisis (5.3 My), supports its specific distinction from *P. cultripes* (Busack et al. 1985; Crottini et al. 2007).

**Distribution.** It is endemic to north-western Morocco (0–350 m elevation a.s.l.), found along the Atlantic coast, from the south of Tanger to Oualidia, where it is rare (de Pous et al. 2012; Beukema et al. 2013; Frost 2019). IUCN status: Endangered (Salvador et al. 2009).

**Diversity.** To our knowledge, *P. varaldii* has not been the focus of any phylogeographic or population genetic work.
*Pelobates fuscus* (Laurenti, 1768)

Common spadefoot

**Diagnosis.** Small spadefoot characterized by pale grayish metatarsal spades and a domed skull. The webbing of the hindfeet is well developed. Males are smaller than females (Fig. 2). The species can be found in a spectrum of gray, brown, or yellowish colors, but rarely greenish (P. Székely pers. comm.), and features patterns such as stripes or blotches of varying sizes; variable presence of orange dots, from almost absent to very abundant (Fig. 3). In Eastern Europe, it differs from its sister species *P. vespertinus* by most individuals having numerous dark rounded spots on a light dorsum (Suriadna et al. 2016) and lacking a dark stripe between the eyes (Lada et al. 2005). Average SVL = 54 mm (range: 37–78 mm) for females (n = 21 populations) and 47 mm (36–65 mm) for males (n = 21 populations) (Suppl. material 1, Table S1; Fig. 2). The karyotype consists of seven large and six small pairs of two-armed chromosomes (Mészáros 1973; Schmid et al. 1987; Manilo and Radchenko 2004; Manilo and Manuilova 2013; Suriadna 2014). Centromeric C-bands are obvious in pairs 2, 6, and 7–13 (Schmid et al. 1987). NORs are in the short arm of pair 7 (Schmid 1980, 1982). The nuclear DNA content (calculated from flow cytometry) averages 8.7–9.0 pg (Litvinchuk et al. 2013).

**Taxonomy.** Originally described as *Bufo fuscus* Laurenti, 1768; type locality: not specifically designated (“in paludibus, rarissime hospitantur in continenti”, in swamps, rarely on the land); type(s): the specimens depicted by Rösel von Rosenhof (1758: pls XVII, XVIII), expressively cited by Laurenti (1768); although controversial (see Nöllert et al. 2012; Frost 2019), the additional mention of pl. XV (p. 122), a drawing of a dissected *Pelophylax*, could simply be an error. Rösel depicted the amphibians of Germany, and Shaw (1802) accordingly mentioned that Rösel found his specimens in the neighborhood of “Nurenberg” (Nürnberg), Germany, which could then apply as the type locality. Seven junior synonyms. *Rana alliacea* Shaw, 1802; type locality: not specifically designated, but Shaw (1802) refers to Rösel’s toads from Nürnberg, Germany; type(s): the toad illustrated by the author (pl. 41), which may very well corresponds to the amplexd female on the top right of pl. XVII in Rösel von Rosenhof (1758), of identical posture and color patterns. *Bombinator marmorata* Sturm, 1828; type locality: near Penig, Germany; holotype: the frog illustrated by the author. *Cultripes minor* Müller, 1832; type locality: “unbekannt” (unknown); type(s): not mentioned. *Pelobates fuscus* var. *lividis* Koch, 1872: type locality: “von den Wiesen in der Nähe des Röder-Wäldchens bei Frankfurt” (the meadows in the Röder groove near Frankfurt), Germany; type(s): not mentioned; *Pelobates insubricus* Cornalia, 1873; type locality: nearby Milano, Italy; type(s): not mentioned, most likely deposited at MSNM, but presumably lost since (Blackburn and Scali 2014). *Pelobates latifrons* Herón-Royer, 1888; type locality: “environ de Turin” (nearby Torino), Italy; type(s): not mentioned. *Pelobates praefuscus* Khosatzky, 1985; type locality: Etuliya, Moldova; holotype: ZISP 21N RNA M-1, a Pliocene fossil (according to Frost 2019).
The Italian populations, for long considered as a subspecies *P. f. insubricus*, have been a matter of debate until recently because they bear private mtDNA haplotypes (Crottini et al. 2007). Litvinchuk et al. (2013) synonymized this taxon with *P. fuscus*, given the weak divergence of these haplotypes, together with the lack of differentiation of allozyme and genome content. As it stands, *P. fuscus* should thus be considered a monotypic taxon.

**Distribution.** Widespread distribution in western, central and eastern Europe (0–810 m elevation a.s.l.), but absent from the northern European countries and most of southern Europe (Sillero et al. 2014; Nöllert at al. 2012) (Fig. 1). In the west, it reaches the eastern edge of the Netherlands (Creemers and Van Delft 2009), the eastern part of Flanders in Belgium (Bauwens and Claus 1996), the western parts of Nordrhein-Westfalens and the south-east of Rheinland-Pflaz in Germany (Bitz et al. 1996; Chmela and Kronshage 2011), the north-eastern side of France (particularly along the Rhine River, Eggert and Vacher 2012). In the north, it extends to northern Netherlands (Creemers and Van Delft 2009), the North Sea coastline of Germany (Nöllert and Günther 1996) and Denmark, the south of Sweden, as well as the coastline of the Baltic Sea from Germany to Estonia, and eastward until it reaches *P. vespertinus* in Russia (Kuzmin 1999; Nyström et al. 2007; Litvinchuk et al. 2013; Sillero et al. 2014). The contact zone with the latter is well delineated from the Kursk region in Russia to the Black Sea coast (Dufresnes et al. 2019b). From there, it is present westward along the Black Sea coast of Ukraine to north-eastern Bulgaria (Kuzmin 1999; Stojankov et al. 2011). The southern edges extend along the Danube at the borders of Romania and Bulgaria (Stojankov et al. 2011) and across Serbia (Vukov et al. 2013), eastern Croatia, northern Bosnia and Herzegovina, Slovenia (Džukić et al. 2008, Curić et al. 2018), northern and eastern Austria around the Alps (Cabela et al. 2001), and southern Germany (Nöllert and Günther 1996). The species is also present in a large area of northern Italy, especially in the Po Valley (Andreone 2006). Last, isolated populations persist in central France (Indre, Loiret, Indre-et-Loire: Eggert and Vacher 2012) and western Bulgaria (around Sofia: Stojankov et al. 2011). IUCN Status: Not Evaluated, considered Least Concern when grouped with *P. vespertinus* (Agasyan et al. 2009a). Declines have been reported for more than a century in various parts of Europe, which have caused a regression of the distribution limits (Džukić et al. 2005; Eggert et al. 2006).

**Diversity.** The phylogeographic work by Crottini et al. (2007) and Litvinchuk et al. (2013) characterized two refugial groups for this species (as the “western lineage of *P. fuscus*”), based on shallow mtDNA divergence and allozyme differentiation: in the Balkans/northern Italy and on the western shores of the Black Sea coast. This seems supported by weak genomic differentiation among Central-European samples (Dufresnes et al. 2019b). The refugial areas bear nearly all the genetic diversity of the species, which was lost in the derived northern populations, following post-glacial colonizations (Eggert et al. 2006).
*Pelobates vespertinus* (Pallas, 1771)

Pallas' spadefoot

**Diagnosis.** Morphologically close to its sister species *P. fuscus*, it similarly features pale metatarsal spades and a domed skull. The coloration also spans the gray-yellowish-brownish spectrum, including reddish individuals (Fig. 3); orange dots can be heavily marked or absent (Fig. 3). It differs from *P. fuscus* by most individuals having three light longitudinal stripes on the dorsum (Suriadna et al. 2016), as well as a dark stripe between the eyes (Lada et al. 2005). Sexes of similar size (Fig. 2). Average SVL = 47 mm (range: 29–59 mm) for females (*n* = 3 populations) and 48 mm (29–61 mm) for males (*n* = 12 populations) (Suppl. material 1; Table S1, Fig. 2). The karyotype consists of seven large and six small pairs of two-armed chromosomes (Manilo and Manuilova 2013; Suriadna 2014). NORs (secondary constrictions) are in the short arm of pair 7 (Manilo and Radchenko 2008). The nuclear DNA content averages 9.2–9.4 pg (Litvinchuk et al. 2013). As shown in Table 1, *P. vespertinus* differs from *P. fuscus* by 2.5% at mtDNA and 0.13% at nuclear DNA (Dufresnes et al. 2019b). The genome of *P. vespertinus* is about 5% larger than *P. fuscus* (Borkin et al. 2001; Litvinchuk et al. 2013; Suriadna 2014).

**Taxonomy.** Originally named *Rana vespertina* Pallas, 1771; type locality: not specifically designated, but the author mentioned this taxon in Zarbay Creek (“Bach Sarbei”, Samara oblast), Russia, which can be considered as the type locality; type(s): not mentioned. Three junior synonyms. *Pelobates fuscus* var. orientalis Severtsov, 1855; type locality: “Voronezhskaya Gubernia” (Voronezh governorate), Russia; type(s): not mentioned. *Pelobates campestris* Severtsov, 1855; type locality: between Bityug, Don and Ikorets rivers in today’s Voronezh province, Russia; type(s): not mentioned. *Pelobates borkini* Zagorodniuk, 2003; proposed for the eastern form of *P. fuscus* but nomen nudum because neither a type specimen nor a type locality were designated (Zagorodniuk 2003). *Pelobates vespertinus* was previously considered a subspecies of the common spadefoot, as *Pelobates fuscus vespertinus* (Crochet and Dubois 2004). The significant divergence (~2–3 My) and restricted admixture with *P. fuscus*, consistent with reproductive isolation, both support the distinction of *P. vespertinus* as a separate species (Litvinchuk et al. 2013; Dufresnes et al. 2019b), as also proposed from genome size differences (Suriadna 2014).

**Distribution.** A lowland species (0–830 m elevation a.s.l.) widespread from the contact zone with *P. fuscus*, to western Siberia and Kazakhstan, and along the Ural River (Kuzmin 1999) (Fig. 1). However, the exact limits with *P. fuscus* are not known in the northern 700 km of the distribution range. Detailed genetic data showed that the transition extends between the Kursk region to southern Ukraine (Litvinchuk et al. 2013; Dufresnes et al. 2019b). In the south, it is present along the Sea of Azov coast to the northern Caucasus (Kuzmin 1999; Suriadna et al. 2016). Spadefoot populations in the Crimea are attributed to *P. vespertinus* (Litvinchuk et al. 2013). The southernmost
P. s. syriacus

<figure><img src="image_20.png" /></figure>

P. b. balcanicus

<figure><img src="image_21.png" /></figure>

P. s. boettgeri

<figure><img src="image_22.png" /></figure>

P. b. chloeae

<figure><img src="image_23.png" /></figure>

**Figure 4.** Color variation in *Pelobates syriacus* and *P. balcanicus*. Photo credits and origins as follows: **a, b** G Hamoivitch (Israël) **c** R Winkler (Israël) **d** G Martínez (Israël) **e** IS (Limnos, Greece) **f** SNL (European Turkey) **g** IS (Limnos, Greece) **h** A Nöllert (Dagestan, Russia) **i** MD (Danube Delta, Romania) **j** IS (Thrace, Greece) **k** IS (Macedonia, Greece) **l** IS (Evia, Greece) **m–o** IS (Peloponnese, Greece) **p** CD (Peloponnese, Greece).

populations are in Dagestan, where it is sympatric with *P. syriacus* (Mazanaeva and Askenderov 2007). IUCN Status: Not Evaluated, as *P. vespertinus* was previously included in the *P. fuscus* assessment.

**Diversity.** Crottini et al. (2007) and Litvinchuck et al. (2013) provided detailed phylogeographic accounts for this species (as the “eastern lineage of *P. fuscus*”), which consists of a homogenous clade that expanded from a single glacial refugia located in the eastern shores of the Sea of Azov. *Pelobates vespertinus* forms a narrow hybrid zone (< 20 km) with *P. fuscus* in eastern Ukraine/western Russia (Litvinchuk et al. 2013; Dufresnes et al. 2019b).
*Pelobates syriacus* Boettger, 1889

Eastern spadefoot

**Diagnosis.** Large spadefoot with whitish metatarsal spades and a flat skull. Webbing of the hind feet less developed than in *P. fuscus* and *P. vespertinus*. Sexes of similar size (Fig. 2). Coloration can be gray, yellow, greenish but rarely brown; orange dots often present, but not as abundant and marked as in some individuals of *P. fuscus*, *P. vespertinus*, or *P. balcanicus* (Fig. 4). Based on populations of *P. syriacus boettgeri*, average SVL = 68 mm (range: 40–92) for females (n = 5 populations) and 69 mm (57–83 mm) for males (n = 4 populations) (Suppl. material 1, Table S1; Fig. 2). The karyotype consists of seven large and six small pairs of two-armed chromosomes (Uğurtaş et al. 2001, from *P. s. boettgeri*). Centromeric C-bands are obvious in pairs 8 and 10 and telomeric Q-bands in the long arms of pairs 9 and 10 (Schmid 1979; Schmid et al. 1987). NORs are in the short arm of pair 7 (Schmid 1982; Schmid et al. 1987). The nuclear DNA content averages 8.2 pg (Litvinchuk et al. 2013; data from *P. s. boettgeri*).

**Taxonomy.** Described from the Levant region as *Pelobates syriacus Boettger, 1889*; type locality: “Haiffa in Syrien” (Haifa), Israel; type: SMF 1437.1a (Boettger 1892), subsequently designated as lectotype SMF 1722 (Mertens 1967). Other nomina proposed apply to *P. s. boettgeri* and *P. balcanicus* (see below).

**Distribution.** Scattered distribution; mainly present in the Middle East with 0–2000 m elevation a.s.l. (Agasyan et al. 2009b; Uğurtas 2001; Džukić et al. 2008; Sofianidou 2012) (Fig. 1). The nominate subspecies *P. syriacus syriacus* inhabits the southern part of the distribution in the Levant, from the Syrian coast at the border of Lebanon to the southern Israeli coast, as well as in south-western Syria (Boettger 1889; Munwes et al. 2010; Sofianidou 2012). It may be extinct from western Jordan (Agasyan et al. 2009b; Disi and Amr 2010). The subspecies *P. syriacus boettgeri* occupies the remaining ranges. In the west, it is present in western Turkey and along the Aegean coastline. It also occurs in European Turkey and probably southeastern Bulgaria. Alternatively, the latter populations could belong to *P. balcanicus*, notably along the Maritsa River, and identification is pending molecular analyses. The presence of *P. syriacus* is also documented on the Greek islands of Limnos, Lesbos, and Kos (Sofianidou 2012; Strachinis and Roussos 2016). Its central distribution is poorly known and therefore not well delineated, with several isolates described in Turkey, both along the Black and Mediterranean sea coasts, as well as the central parts of Anatolia. In the northeast, *P. syriacus* reaches the southern slopes of the Caucasus, from Georgia to Azerbaijan. The northernmost records are in Dagestan, on the west coast of the Caspian Sea, where it meets *P. vespertinus* (Mazanaeva and Askenderov 2007). Further east, it is present along the southern shores of the Caspian Sea in Iran (eastern limit in Golestan; Kamali and Malekzadeh 2013). IUCN status: Not Evaluated; considered Least Concern when grouped with *P. balcanicus* (Agasyan et al. 2009b).
**Diversity.** Using mtDNA and genomic data, Dufresnes et al. (2019b) evidenced a Pleistocene split between the Levant (*P. s. syriacus*) and the rest of the range (*P. s. boettgeri*; see below). Within both subspecies, populations are weakly differentiated despite their present-day fragmentation (see also Munwes et al. 2010 for *P. s. syriacus*). Populations from the Caucasus (*P. s. boettgeri*) differs from Anatolian ones at nuclear, but not mitochondrial markers. In the Lesser Caucasus and southern Turkey, *P. s. boettgeri* features traces of past gene flow with *P. s. syriacus*. Iranian populations have not been examined with genetic tools and could bear cryptic diversity.

**Pelobates syriacus boettgeri Mertens, 1923**

Anatolian spadefoot

**Diagnosis.** Similar to the nominal subspecies, notably in terms of cranial characters (Roček 1981) and coloration patterns (Fig. 4). Most biometric data on *P. syriacus* come from populations of *P. s. boettgeri* (Fig. 2, see above). As shown in Table 1, *P. s. boettgeri* differs from *P. s. syriacus* by 1.7% at mtDNA and 0.01% at nuclear DNA (Dufresnes et al. 2019b).

**Taxonomy.** The oldest nomen available for Anatolian/Caucasian spadefoots is *Pelobates syriacus boettgeri Mertens, 1923*; type locality: Belesuwar, southeastern Azerbaijan; holotype: SMF 1725 (originally 1437.2a, Mertens 1923). A single junior synonym. *Pelobates transcaucasicus Delwig, 1928*; type locality: “Tiflis” (Tbilisi), Georgia; types: ten syntypes, nine at ZISP, and one at ZIK (Amph A5/A (2164)). Subspecies level of *P. s. boettgeri* is granted by its phylogenetic divergence from *P. s. syriacus*, but the recent split (~1 My) and the widespread traces of admixture between both subspecies in Armenia, Turkey (Antalya region), and Israel argue against a specific status.

**Distribution and diversity.** See the accounts for *P. syriacus*.

**Pelobates balcanicus Karaman, 1928**

Balkan spadefoot

**Diagnosis.** Resembling *P. syriacus* with which it was previously considered a synonym (Frost 2019). Large toad with whitish metatarsal spades and a flat skull. Sexes of similar size (Fig. 2). Various motifs with gray, yellow or greenish colors, but rarely brown (unlike the sympatric *P. fuscus*, P. Székely pers. comm.); frequently specked with orange dots, sometimes heavily (perhaps more than in *P. syriacus*) (Fig. 4). Based on 25 biometric characters, Uğurtas et al. (2002) showed that the *P. balcanicus* populations from the Balkans are morphologically very variable and differentiated from Asia Minor (*P. syriacus*); yet *P. syriacus* populations from European Turkey (Edirne, genetically confirmed by Dufresnes et al. 2019b) and southeastern Bulgaria (Primorsko) grouped with *P. balcanicus* (Uğurtas et al. 2002). Roček (1981) only found one cranial difference: the *processus posterior parasphenoidei* is present in *P. syriacus* but
<table><caption>**Table 1.** Pairwise % of genetic differences between *Pelobates* taxa (from the data of Dufresnes et al. 2019b). The estimates below diagonal correspond to mitochondrial DNA (*cyt-b* + 16S, 1.2 kb); the estimates above diagonal correspond to nuclear DNA (63.5 kb of RAD tags).</caption><thead><tr><th></th><th><i>P. cultripes</i></th><th><i>P. varaldii</i></th><th><i>P. fuscus</i></th><th><i>P. vespertinus</i></th><th><i>P. s. syriacus</i></th><th><i>P. s. boettgeri</i></th><th><i>P. b. balcanicus</i></th><th><i>P. b. chloeae</i></th></tr></thead><tbody><tr><th><i>P. cultripes</i></th><td>—</td><td>0.40</td><td>0.66</td><td>0.75</td><td>0.72</td><td>0.70</td><td>0.74</td><td>0.73</td></tr><tr><th><i>P. varaldii</i></th><td>6.0</td><td>—</td><td>0.83</td><td>0.92</td><td>0.89</td><td>0.88</td><td>0.92</td><td>0.90</td></tr><tr><th><i>P. fuscus</i></th><td>10.1</td><td>10.0</td><td>—</td><td>0.13</td><td>0.63</td><td>0.62</td><td>0.65</td><td>0.64</td></tr><tr><th><i>P. vespertinus</i></th><td>9.7</td><td>9.6</td><td>2.5</td><td>—</td><td>0.71</td><td>0.70</td><td>0.74</td><td>0.73</td></tr><tr><th><i>P. s. syriacus</i></th><td>9.1</td><td>8.6</td><td>9.1</td><td>8.9</td><td>—</td><td>0.01</td><td>0.32</td><td>0.30</td></tr><tr><th><i>P. s. boettgeri</i></th><td>9.2</td><td>8.9</td><td>9.2</td><td>9.0</td><td>1.7</td><td>—</td><td>0.31</td><td>0.29</td></tr><tr><th><i>P. b. balcanicus</i></th><td>9.2</td><td>8.6</td><td>8.5</td><td>8.5</td><td>7.2</td><td>7.0</td><td>—</td><td>0.02</td></tr><tr><th><i>P. b. chloeae</i></th><td>9.2</td><td>8.2</td><td>8.5</td><td>8.6</td><td>7.7</td><td>7.7</td><td>2.8</td><td>—</td></tr></tbody></table>

not developed in *P. balcanicus*. Morphometric assessments associated to genetic data are needed. Based on populations of *P. balcanicus balcanicus*, average SVL = 67 mm (48–100 mm) for females (n = 16 populations) and 68 mm (46–94 mm) for males (n = 15 populations) (Suppl. material 1, Table S1; Fig. 2). The karyotype (*P. b. balcanicus*) consists of six large and seven small pairs of two-armed chromosomes. NORs (secondary constrictions) are in the short arm of pair 7 (Belcheva et al. 1977). The nuclear DNA content (calculated from flow cytometry) averages 7.9 pg (Litvinchuk et al. 2013; data from *P. b. balcanicus*). As shown in Table 1, *P. balcanicus* differs from *P. syriacus* by ~7.4% at mtDNA and ~0.31% at nuclear DNA (Dufresnes et al. 2019b).

**Taxonomy.** Originally described as a subspecies of the Eastern spadefoot, *Pelobates syriacus balcanicus Karaman, 1928*; type locality: Dojran Lake, North Macedonia; type(s): most likely include the skeleton described by Karaman (1928), deposited at MMNH (Skopje, North Macedonia), but destroyed in an earthquake in 1963 (V. Sidorovska pers. comm.); the MMNH currently hosts one specimen from the type locality, MMNH-A-699 (collected in 2001). This taxon represents a distinct species from *P. syriacus*, due its old divergence (>6 My) and the absence of contemporary introgression at their area of contact in European Turkey, consistent with advanced reproductive isolation (Dufresnes et al. 2019b). Therefore, we herein remove *P. balcanicus* from its previous synonymy with *P. syriacus*.

**Distribution.** *Pelobates balcanicus* is restricted to the Balkan Peninsula, 0–920 m a.s.l. (Džukić et al. 2008) (Fig. 1). In the north, it is present in northern Serbia and northwestern Romania. It follows the Danube River from Serbia to the Black Sea in Romania (Székely et al. 2013; Ţeran et al. 2017). There are yet some possible gaps along the Danube (e.g. around the Iron Gate: Vukov et al. 2013; Ţeran et al. 2017). In the north-west, the Great Morova River in Serbia marks its western margin (Džukić et al. 2008). Northern ranges are currently disconnected from the southern populations (Vukov et al. 2013) of North Macedonia, eastern Albania (a single location), south-west Bulgaria (Strimon River), and Greece (Džukić et al. 2008; Mollov et al. 2006; Szabolcs and Mizsei 2017). In the 1980s, Sofianidou (2012) reported the species along the western coastline of the Adriatic Sea and the northern coastline of the Gulf of Corinth (Greece), but there is no recent observation in this region. Elsewhere
in Greece, it is present in Peloponnese (*P. balcanicus chloeae* ssp. nov., see below), in the eastern parts of the mainland, and along the Aegean Sea shores, from Sterea Ellas to the Evros River, until it reaches *P. syriacus* in Thrace (Džukić et al. 2008; Sofianidou, 2012). The spadefoots known from the Maritsa (Evros) River in southern Bulgaria, and along the western coasts of the Black Sea, may correspond to *P. syriacus* (Stojanov et al. 2011; Dufresnes et al. 2019b). IUCN status: Not Evaluated; previously included in *P. syriacus* assessment.

**Diversity.** Using mtDNA and genomic data, Dufresnes et al. (2019b) evidenced a Pleistocene split (~2 My) for spadefoots from the Peloponnese (*P. balcanicus chloeae* ssp. nov.). In the rest of the range, at least three glacial lineages (<1 My) were identified: a first one in the eastern ranges, from the Carpathians to the Black Sea and as south as Greek Thrace; a second one in western ranges from Serbia to northern Greece; and a third one on the coastal island of Evia (north-east of Peloponnese). The eastern and western lineages widely admix. Populations from central Greece are yet to be examined.

**Pelobates balcanicus chloeae ssp. nov.**

http://zoobank.org/A1C08645-8307-49EF-A2EB-7F09D7BCC89D
Chloe's spadefoot

**Type locality.** Strofylia meadows, near the village of Metochi, Peloponnese, Greece (38.1239°N, 21.3858°E, 1 m a.s.l.). Coastal sandy meadows with shallow ponds (Fig. 5).

**Holotype.** NHMC 80.2.15.10, adult female captured on December 10th 2018 by CD, IS and ET at Strofylia meadows, Greece (38.1239°N, 21.3858°E, 1 m a.s.l.); subsequently deposited at the Natural History Museum of Crete (NHMC); mitochondrial *cyt-b* haplotype BAL19 (Dufresnes et al. 2019b). Full measurements are available in Table 2 and photographs in Figure 5. Large specimen (SVL = 78.7 mm) with the head narrower than the body, ending by a rounded snout; nostrils closer to each other's than from the eyes; forehead flat, as viewed from the side, with large interorbital; tympanum invisible; vomerine teeth present. Large, bulging eyes (7.2 mm of diameter) with vertical pupil and a dark-golden iris. Legs relatively short (92 mm), 1.2 times the size of the body. Five partially webbed toes; webbing formula: I 1-1+ II 1-2 III 1-2+ IV 3-1+ V; relative lengths from inner to outer toes: 4>3>5>2>1; large and long rounded (blade-shaped) metatarsal tubercle (“spade”), whitish; subarticular tubercles indistinct. Strong arms with four unwebbed fingers; palm tubercles visible, oval. Ventral and dorsal skins smooth, although the latter bears scattered warts. Coloration in life: ventrum glossy white, bluish near the limbs; dorsum light gray with prominent green-brown reticulated patches featuring orange dots, notably at the armpits; head darker, with a horizontal brown line running between the eyes. Changes of coloration in ethanol: dorsum less contrasted; fainted orange dots.
<table><caption>**Table 2.** Morphometric measurements (mm) of *Pelobates balcanicus chloeae* at the type locality (Strofylia meadows), based on 12 adults (both sexes combined), and detailed for the type specimens. SVL: snout-vent length; HW: head width; HL: head length; ED: eye diameter; EE: inter-eye distance; NN: inter-nostril distance; EN: eye-nostril distance; ML: metatarsal tubercle length; MH: metatarsal tubercle height; HLL: hind leg length; TTL: tibia + tarsus length.</caption><thead><tr><th></th><th>Strofylia population</th><th>Holotype NMHC 80.2.15.10</th><th>Paratype NMHC 80.2.15.11</th></tr></thead><tbody><tr><td>SVL</td><td>71.5 ± 3.4</td><td>78.7</td><td>74.1</td></tr><tr><td>HW</td><td>23.7 ± 1.1</td><td>26.6</td><td>25.5</td></tr><tr><td>HL</td><td>21.8 ± 0.9</td><td>23.4</td><td>23.1</td></tr><tr><td>ED</td><td>7.4 ± 0.24</td><td>7.2</td><td>7.1</td></tr><tr><td>EE</td><td>15.9 ± 0.7</td><td>16.7</td><td>17.3</td></tr><tr><td>NN</td><td>4.4 ± 0.2</td><td>4.6</td><td>4.2</td></tr><tr><td>EN</td><td>6.0 ± 0.3</td><td>6.7</td><td>6.0</td></tr><tr><td>ML</td><td>6.1 ± 0.4</td><td>7.1</td><td>6.5</td></tr><tr><td>MH</td><td>2.6 ± 0.1</td><td>2.6</td><td>2.8</td></tr><tr><td>HLL</td><td>83.7 ± 3.6</td><td>92</td><td>90</td></tr><tr><td>TTL</td><td>64.2 ± 3.1</td><td>72</td><td>69</td></tr></tbody></table>

**Paratype.** NHMC 80.2.15.11, adult female captured on December 10th 2018 by CD, IS and ET at Strofylia meadows, Greece (38.1239°N, 21.3858°E, 1 m a.s.l.); subsequently deposited at the Natural History Museum of Crete (NHMC); mitochondrial *cyt-b* haplotype BAL20 (Dufresnes et al. 2019b). Full measurement and post-mortem pictures are provided in Table 2 and Figure 5, respectively.

**Diagnosis.** Supposedly similar morphologically to the nominal subspecies and reliably diagnosed only by molecular data. So far studied from the type locality only (Strofylia). Like the nominal subspecies, *Pelobates balcanicus chloeae* is a large spadefoot with whitish metatarsal spades, a flat skull and incomplete webbing on the hind feet (Fig. 4). It also shares general characteristics of the genus, i.e. stocky built, smooth skin and vertical pupil; males bear oval protuberances on the arms, absent in females. The dorsum coloration is generally light gray, sometimes yellow, covered with dark green-brown reticulate patches and variable orange dots (Fig. 4). From our own observations, the color patterns seem to slightly differ from the nominal subspecies (Fig. 4). In *P. b. chloeae*, the green patches are small and numerous (fewer but larger patches in the nominal subspecies); dots are usually orange (more reddish in the nominal subspecies) and located inside the green patches (randomly distributed in the nominal subspecies). The ventrum and limbs are glossy and slightly bluish (rather pale whitish in the nominal subspecies). Moreover the snout of *P. b. chloeae* appears shorter and blunter than the nominal subspecies. These suspicions will need to be assessed by formal phenotypic analyses. At the type locality, the SVL of adults averaged 71.5 mm (range: 62–84; *n* = 12 individuals, both sexes combined). The mating call and the tadpole are yet to be described and diagnosed. The karyotype has not been documented. As shown in Table 1, *P. b. chloeae* differs from the nominal subspecies by 2.8% at mtDNA and 0.02% at nuclear DNA (Dufresnes et al. 2019b).
<figure><img src="image_24.png" /><figcaption>**Figure 5.** Description of *Pelobates balcanicus chloeae*. **Top** live photograph of the holotype, NHMC 80.2.15.10 (CD, taken on December 10th 2018); **middle** dorsal and lateral views of the type specimens (**left** NHMC 80.2.15.10; **right** NHMC 80.2.15.11) post-mortem (IS); **bottom** Strofylia meadows, the type locality in Peloponnese, Greece (ET).</figcaption></figure>
**Taxonomic status.** Following Dufresnes et al. (2019b), we raise the population(s) from the Peloponnese as a distinct *P. balcanicus* subspecies based on nuclear and mitochondrial phylogenetic data, but refrain a specific status from current data, due to the relatively young evolutionary divergence (~2 My) and potential introgression with the nominal subspecies.

**Etymology.** No name is available for spadefoots from the Peloponnese or Greece in general. We hence attribute it a new nomen, *Pelobates balcanicus chloeae*, as a reference to the young daughter of CD, Chloé, who played a decisive role in guiding his research towards European biogeography and herpetology. Moreover, “Chloé” is an ancient Greek name (“Χλόη”) designating the young green grass spurring from the ground in spring, reminiscent of spadefoots unearthing themselves to breed in mass. The name is also associated with Dimitra (Δήμητρα), the Ancient Greek goddess of agriculture who protected traditional farmlands in which so many amphibians used to thrive.

**Distribution.** From current knowledge, this subspecies is endemic to the Peloponnese in southern Greece (Dufresnes et al. 2019b) (Fig. 1); it was so far genetically confirmed from its type locality only. Historically (1980s), there were records of spadefoots all over the Peloponnese, except in the three southern peninsulas (Böhme 1975; Eiselt 1988; Sofianidou 2012). Nowadays, the two known *Pelobates* localities are restricted to the central (Tripoli) and north-western (Strofylia) areas. Consequently, it is likely that there are only few populations left for this subspecies. It is not excluded that its range extends to Central Greece, where potential populations have not been examined; one sample from Kallithea Elassonos (Thessaly, Greece) bore trace of introgression by *P. b. chloeae*, suggesting past or present contact (Dufresnes et al. 2019b).

**Ecology.** Never studied as such, but this subspecies most likely shares a similar ecology as the nominal subspecies (*P. b. balcanicus*). Inhabits open, flat, lowland areas with soft sandy soil near shallow ponds or ditches with aquatic vegetation for breeding, as described for *P. balcanicus* (Dufresnes 2019). Mostly nocturnal and semi-fossorial: comes out of the ground for foraging and breeding during / right after heavy rains. Hence it can be observed in high numbers during winter-spring showers; ET counted >70 individuals (mostly juveniles) in 15 min of search in late-October 2018 at the type locality; usually active around 13-20 °C, but also as low as 7 °C (ET pers. obs.).

**Diversity.** Our *P. b. chloeae* samples featured the lowest nuclear genetic diversity recorded across the entire ranges of *P. balcanicus* and *P. syriacus* (Dufresnes et al. 2019b). This implies that the Strofylia population and perhaps the subspecies as a whole have been heavily bottlenecked. Two mtDNA haplotypes co-occur (Dufresnes et al. 2019b). Genetic studies are urgently needed to assess the range and diversity of this regional endemic.

**Conservation Status** – Ioannidis and Mebert (2011) mentioned road casualties at the type locality of this taxon, one of few extant populations. Although not evaluated yet, this taxon is clearly threatened according to IUCN criteria; given the narrow extent of occurrence (EOO), it should be listed as Critically Endangered (CR).
## Identification key

Based on our updated overview of the taxonomy and distribution of *Pelobates*, we hereby provide a key to summarize the main discriminating features within this group. Because several taxa are cryptic and lack diagnostic phenotypic differences, geographic origin remains an essential information.

1 Black spades on the hind legs..................................................................................2

– Spades of light coloration..................................................................................3

2 Large body (6–9 cm) without orange dots, spades entirely black; Spain, Portugal and southern France.................................................................................. *P. cultripes*

– Small body (<6 cm) with orange dots, spades bordered with black; Morocco .................................................................................................................. *P. varaldii*

3 Domed skull, developed webbing, and small body (<6 cm)........................................4

– Flat skull, partial webbing, and large body (6–8 cm)........................................5

4 Dorsum stripes rare; Central and northwestern Europe, west of a Crimea–Moscow imaginary line.................................................................................. *P. fuscus*

– Three dorsum stripes often present; Eastern Europe and Central Asia, east of a Crimea–Moscow imaginary line.................................................................. *P. vespertinus*

5 Levantine region (Israel, Lebanon, and Syria).................................. *P. syriacus syriacus*

– Caucasus and Caspian Sea shores, Anatolia, and European Turkey.................................................................................................................. *P. syriacus boettgeri*

– Balkan Peninsula, except Peloponnese.................................. *P. balcanicus balcanicus*

– Peloponnese.................................................................................. *P. balcanicus chloae*

## Conclusions

Our phylogeographic analyses of *Pelobates* (Dufresnes et al. 2019b) called for a taxonomic reassessment of this threatened amphibian group. We reviewed the evidence for distinct Moroccan (*P. varaldii*), Iberian (*P. cultripes*), Central (*P. fuscus*), and Eastern European (*P. vespertinus*) species. Furthermore, we revised the taxonomy of *P. syriacus* by distinguishing two cryptic species, *P. syriacus* and *P. balcanicus*, and by considering their strong intraspecific diversity into subspecific divisions, *P. s. syriacus*, *P. s. boettgeri*, *P. b. balcanicus*, and *P. b. chloae*, the latter as a newly described taxon. Their variation in size and coloration are detailed and illustrated, based on a literature review and high-quality photographs, respectively. Finally, our paper provides up-to-date whole-range distribution maps for all extant *Pelobates* taxa.

## Acknowledgements

We thank M. Pajković for translating publications from Serbo-Croatian, P. Lymberakis (NHMC) for processing the type series of *P. b. chloae*, A. Nöllert, G. Haimovitch, G.
Martinez, and A. Sanchez Vialas for sharing their pictures, P.-A. Crochet for taxonomic advices and Nicolas Perrin for support. We are also grateful to T. Vukov, O. Zinenko, A. Ohler, L. Ceriaco, and A. Crottini, as well as an anonymous reviewer, for their expertise and useful feedback. M.D. is Research Director at Fonds de la Recherche scientifique (FNRS). This study was founded by a grant from the Swiss National Science Foundation (SNSF) to Nicolas Perrin (no. 31003A_166323), and a SNSF fellowship to CD (no. P2LAP3_171818).

## References

Agasyan A, Avci A, Tuniyev B, Isailovic JC, Lymberakis P, Andrén C, Cogălniceanu D, Wilkinson J, Ananjeva N, Uzüm N (2009a) *Pelobates fuscus*. The IUCN Red List of Threatened Species: eT16498A5951455. https://doi.org/10.2305/IUCN.UK.2009.RLTS.T16498A5951455.en

Agasyan A, Tuniyev B, Crnobrnja-Isailovic J, Lymberakis P, Andrén C, Cogălniceanu D, Wilkinson J, Ananjeva N, Üzüm N, Orlov N, Podloucky R, Tuniyev S, Kaya U (2009b) *Pelobates syriacus*. The IUCN Red List of Threatened Species 2009: eT58053A11723660. https://doi.org/10.2305/IUCN.UK.2009.RLTS.T58053A11723660.en

Andreone F (2006) *Pelobates fuscus* (Laurenti, 1768). In: Sindaco R, Doria G, Razzetti E, Bernini F (Eds) Atlanti degli anfibi e dei rettili d’Italia Atlas of Italian amphibians and reptiles. Edizioni Polistampa, Firenze, 292–297.

Bauwens D, Claus K (1996) Verspreiding van Amfibieën en Reptielen in Vlaanderen. De Wielewaal Natuurvereniging, Turnhout, 192 pp.

Belcheva R, Ilieva H, Beshkov V (1977) On the karyotype of *Pelobates syriacus balcanicus* Karman (Amphibia, Pelobatidae, Anura). Annuaire de l'Université de Sofia, Faculté de Biologie, Livre 1 – Zoologie 68: 15–18.

Beja P, Bosch J, Tejedo M, Lizana M, Martinez Solano I, Salvador A, García París M, Recuero Gil E, Pérez Mellado V, Díaz-Paniagua C (2009) *Pelobates cultripes*. The IUCN Red List of Threatened Species 2009: eT58052A86242868. https://doi.org/10.2305/IUCN.UK.2009.RLTS.T58052A11722636.en

Beukema W, De Pous P, Donaire-Barroso D, Bogaerts S, Garci-Port J, Escoriza D, Arribas OJ, El Mouden EH, Carranza S (2013) Review of the systematics, distribution, biogeography and natural history of Moroccan amphibians. Zootaxa 3661: 1–60. https://doi.org/10.11646/zootaxa.3661.1.1

Bitz A, König H, Simon L (1996) Knaublauchkröte – *Pelobates fuscus* (Laurenti, 1768). In: Bitz A, Fischer K, Simon L, Thiele R, Veith M (Eds) Die Amphibien und Reptilien in Rheinland-Pfalz. GNOR, Landau, 165–182.

Blackburn DC, Scali S (2014) An annotated catalog of the type specimens of Amphibia in the collection of the Museo Civico di Storia Naturale, Milan, Italy. Herpetological Monographs 28: 24–45. https://doi.org/10.1655/HERPETOLOGICA-D-13-00008

Boettger O (1889) Ein neuer *Pelobates* aus Syrien. Zoologischer Anzeiger 12: 144–147.

Boettger O (1892) Katalog der Batrachier-Sammlung im Museum der Senckenbergischen Naturforschenden Gesellschaft in Frankfurt am Main. Gebrüder Knauer, Frankfurt am Main, 48.
Böhme W (1975) Zur Vorkommen von *Pelobates syriacus* in Griechenland. Senckenbergiana Biologica 56: 199–202.

Borkin LJ, Litvinchuk SN, Rosanov JM, Milto KD (2001) Cryptic speciation in *Pelobates fuscus* (Anura, Pelobatidae): evidence from DNA flow cytometry. Amphibia-Reptilia 22: 387–396. https://doi.org/10.1023/A:1018806900399

Busack SD, Maxson LR, Wilson MA (1985) *Pelobates varaldii* (Anura: Pelobatidae): A morphologically conservative species. Copeia 1985: 107–112. https://doi.org/10.2307/1444797

Cabela A, Grillitsch H, Tiedemann F (2001) Atlas zur Verbreitung und Ökologie der Amphibien und Reptilien in Österreich. Umweltbundesamt, Wien, 880 pp.

Chmela C, Kronshage A (2011) Knoblauchkröte – *Pelobates fuscus*. In: Hachtel M, Schlüpmann M, Weddeling K, Thiesmeier B, Geiger A, Willigalla C (Eds) Handbuch der Amphibien und Reptilien Nordrhein-Westfalens Band 1. Laurenti Verlag, Bielefeld, 543–582.

Coates DJ, Byrne M, Moritz C (2018) Genetic diversity and conservation units: dealing with the species-population continuum in the age of genomics. Frontiers in Ecology & Evolution 6: 165. https://doi.org/10.3389/fevo.2018.00165

Cornalia EBM (1873) Observazioni sul *Pelobates fuscus* e sulla *Rana agilis* trovate in Lombardia. Atti della Societa Italiana di Scienze Naturali e del Museo Civico di Storia Naturale di Milano 16: 97–107.

Creemers RCM, van Delft JJCW (2009) De amfibieën en reptielen van Nederland. Nationaal Natuurhistorisch Museum Naturalis, Leiden, 476 pp.

Crochet P-A, Dubois A (2004) Recent changes in the taxonomy of European amphibians and reptiles. In: Gasc J-P, Cabela A, Crnobrnja-Isailovic J, Dolmen D, Grossenbacher K, Haffner P, Lescure J, Martens H, Martínez Rica JP, Maurin H, Oliveira ME, Sofianidou TS, Veith M, Zuiderwijk A (Eds) Atlas of Amphibians and Reptiles in Europe (2nd edn). Societas Herpetologica Europeae and Museum National Histoire Naturelle, Paris, 495–516.

Crottini A, Andreone F, Kosuch J, Borkin L, Litvinchuk SN, Eggert C, Veith M (2007) Fossorial but widespread: the phylogeography of the common spadefoot toad (*Pelobates fuscus*), and the role of the Po Valley as a major source of genetic variability. Molecular Ecology 16: 2734–2754. https://doi.org/10.1111/j.1365-294X.2007.03274.x

Crottini A, Galan P, Vences M (2010) Mitochondrial diversity of western spadefoot toads, *Pelobates cultripes*, in northwestern Spain. Amphibia-Reptilia 31: 443–448. https://doi.org/10.1163/156853810791769527

Curić A, Zimić A, Bogdanović T, Jelić D (2018) New data and distribution of common spadefoot toad *Pelobates fuscus* (Laurenti, 1768) (Anura: Pelobatidae) in Western Balkans. North-Western Journal of Zoology 14: 50–59.

Cuvier G (1829) Le règne animal distribué d'après son organisation, pour servir de base à l'histoire naturelle des animaux et d'introduction à l'anatomie comparée. Vol. 2. Déterville, Paris, 406 pp.

de Pous P, Beukema W, Dingemans D, Donaire D, Geniez P, El Mouden EH (2012) Distribution review, habitat suitability and conservation of the endangered and endemic Moroccan spadefoot toad (*Pelobates varaldii*). Basic and Applied Herpetology 26: 57–71. https://doi.org/10.11160/bah.11015
Delwig, W (1928) Eine neue Art der Gattung *Pelobates* Wagl. aus dem zentralen Transkaukasus. Zoologischer Anzeiger 75: 24–31.

Disi AM, Amr ZS (2010) Morphometrics, distribution and ecology of the amphibians in Jordan. Vertebrate Zoology 60: 147–162.

Dufresnes C (2019) Amphibians of Europe, North Africa and the Middle East. Bloomsbury, London, 224 pp.

Dufresnes C, Mazepa G, Rodrigues N, Brelsford A, Litvinchuk SN, Sermier R, Lavanchy G, Betto-Colliard C, Blaser O, Borzée A, Cavoto E, Fabre G, Ghali K, Grossen C, Horn A, Leuenberger J, Philips BC, Saunders PA, Savary R, Maddalena T, Stöck M, Dubey S, Canestrelli D, Jeffries DL (2018) Genomic evidence for cryptic speciation in tree frogs from the Apennine Peninsula, with description of *Hyla perrini* sp. nov. Frontiers in Ecology and Evolution 6: 144. https://doi.org/10.3389/fevo.2018.00144

Dufresnes C, Beddek M, Skorinov DV, Fumagalli L, Perrin N, Crochet P-A, Litvinchuk SN (2019a) Diversification and speciation in tree frogs from the Maghreb (*Hyla meridionalis sensu lato*), with description of a new African endemic. Molecular Phylogenetics and Evolution 134: 291–299. https://doi.org/10.1016/j.ympev.2019.02.009

Dufresnes C, Strachinis I, Suriadna N, Mykytynets G, Cogălniceanu D, Székely P, Vukov T, Arntzen JW, Wielstra B, Lymberakis P, Geffen E, Gafny S, Kumlutas Y, Ilgaz C, Candan K, Mizsei E, Szabolcs M, Kolenda K, Smirnov N, Geniez P, Lukanov S, Crochet P-A, Dubey S, Perrin N, Litvinchuk S, Denoël M (2019b) Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (*Pelobates*). Molecular Ecology. https://doi.org/10.1111/mec.15133

Džukić G, Beškov V, Sidorovska V, Cogălniceanu D, Kalezić M (2005) Historical and contemporary ranges of the spadefoot toads *Pelobates* spp. (Amphibia: Anura) in the Balkan Peninsula. Acta Zoologica Cracoviensia 48A: 1–9. https://doi.org/10.3409/173491505783995699

Džukić G, Beškov V, Sidrovska V, Cogălniceanu D, Kalezić ML (2008) Contemporary chorology of the spadefoot toads (*Pelobates* spp.) in the Balkan Peninsula. Zeitschrift für Feldherpetologie 15: 61–78.

Eggert C, Cogălniceanu D, Veith M, Džukić G, Taberlet P (2006) The declining spadefoot toad, *Pelobates fuscus* (Pelobatidae): paleo and recent environmental changes as a major influence on current population structure and status. Conservation Genetics 7: 185–195. https://doi.org/10.1007/s10592-006-9124-y

Eggert C, Vacher J-P (2012) *Pelobates fuscus* (Laurenti, 1768). Pélobate brun. In: Lescure J, de Massary JC (Eds) Atlas des amphibiens et reptiles de France. Biotope, Mèze and Muséum national d'Histoire naturelle, Paris, 102–103.

Ehl S, Vences M, Veith M (2019) Reconstructing evolution at the community level: a case study on Mediterranean amphibians. Molecular Phylogenetics & Evolution 134: 211–225. https://doi.org/10.1016/j.ympev.2019.02.013

Eiselt J (1988) Krötenfrösche (*Pelobates* gen., Amphibia Salienta) in Türkisch-Thrakien und Griechenland. Annalen des Naturhistorischen Museums in Wien 90: 51–59.

Frost D (2019) Amphibian Species of the World: an Online Reference. Version 6.0 (30 January 2019). American Museum of Natural History, New York. http://research.amnh.org/herpetology/amphibia/index.html [Accessed on: 2019-3-1]
Garcia-Paris M, Buchholz DR, Parra-Olea G (2003) Phylogenetic relationships of Pelobatidae re-examined using mtDNA. Molecular Phylogenetics and Evolution 28: 12–23. https://doi.org/10.1016/S1055-7903(03)00036-8

Gutiérrez-Rodriguez J, Barbosa AM, Martinez-Solano I (2017) Present and past climatic effects on the current distribution and genetic diversity of the Iberian spadefoot toad (*Pelobates cultripes*): an integrative approach. Journal of Biogeography 44: 245–258. https://doi.org/10.1111/jbi.12791

Herón-Royer L-F (1888) Description du *Pelobates latifrons* des environs de Turin, et d'une conformation particulière de l'ethmoide chez le batraciens. Bulletin de la Société Zoologique de France 13: 85-91. https://doi.org/10.5962/bhl.part.8060

Herrero P, Talavera RR (1988) Cytotaxonomic studies on Iberian and Moroccan *Pelobates* (Anura: Pelobatidae). Acta Zoologica Cracoviensia 31: 505–508.

Ioannidis Y, Mebert KJM (2011) Habitat preferences of *Natrix tessellata* at Strofylia, northwestern Peloponnese, and comparison to syntopic *N. natrix*. Mertensiella 18: 302–310.

Iosif R, Papes M, Samoila C, Cogălniceanu D (2014) Climate-induced shifts in the niche similarity of two related spadefoot toads (genus *Pelobates*). Organisms Diversity and Evolution 14: 397–408. https://doi.org/10.1007/s13127-014-0181-7

Kamali K, Malekzadeh S (2013) A record of *Pelobates syriacus* Boettger, 1889 (Anura: Pelobatidae) in northern Iran. Russian Journal of Herpetology 20: 238–240.

Karaman S (1928) III Prilog herpetologii Jugoslavije – Contribution a l’Herpètologie de la Jugoslavija. Bulletin de la Société Scientifique de Skoplje, Section des Sciences Naturelles 4: 129–143.

Khosatzky LI (1985) Novy' vyd chesnochnyls iz Pliocena Modavii. In: Negadaev-Niknov KN (Ed.) [Fauna and Flora of Late Caenozoic Moldavia]. Akademia Nauk Moldavkoi SSR Shitiintsa, 59-72.

Koch C (1872) Formen und Wandlungen der ecaudaten Batrachier des Unter-Main und Lahn-Gebietes. Bericht der Senckenbergischen Naturforschenden Gesellschaft in Frankfurt am Main 1871-72: 122-183.

Kuzmin SL (1999) The amphibians of the former Soviet Union. Pensoft Publisher, Sofia, 538 pp.

Lada GA, Borkin LJ, Litvinchuk SN (2005) Morphological variation in two cryptic forms of the common spadefoot toad (*Pelobates fuscus*) from eastern Europe. In: Ananjeva N, Tsinenko O (Eds), Herpetologia Petropolitana, St. Petersburg, 53-56.

Laurenti JN (1768) Specimen medicum exhibens synopsin reptilium emendatam cum experimentis circa venena et antidota reptilium austriacorum. Johann Thomas von Trattner, Vienne, 214 pp. https://doi.org/10.5962/bhl.title.5108

Litvinchuk SN, Crottini A, Federici S, De Pous P, Donaire D, Andreone F, Kalezić ML, Džukić G, Lada GA, Borkin LJ, Rosanov JM (2013) Phylogeographic patterns of genetic diversity in the common spadefoot toad, *Pelobates fuscus* (Anura: Pelobatidae), reveals evolutionary history, postglacial range expansion and secondary contact. Organisms Diversity and Evolution, 13: 433–451. https://doi.org/10.1007/s13127-013-0127-5

Lizana M (1997) *Pelobates cultripes* (Cuvier, 1829). In: Pleguezelos JM (Ed.) Distribucion y biogeografia de los anfibios y reptiles en España y Portugal. Editorial Universidad de Granada, Granada, 140-142.
Malkmus R (2004) Amphibians and reptiles of Portugal, Madeira and the Azores-Archipelago. Ganter Verlag K.G., Ruggell, 448 pp.

Manilo VV, Radchenko VI (2004) Comparative karyological research of the «western» form of the common spadefoot, *Pelobates fuscus* (Amphibia, Pelobatidae), from Kyivska, Chernigivska and Zakarpatska Region of Ukraine. Vestnik Zoologii 38: 91–94.

Manilo VV, Radchenko VI (2008) Karyological research of ecaudatas amphibians faunae of Ukraine. Proceeding of the Ukrainian Herpetological Society 1: 29–38.

Manilo VV, Manuilova ON (2013) Comparative karyological research of two species of spadefoot toads *Pelobates fuscus* and *P. vespertinus* (Pelobatidae, Anura, Amphibia) from territory of Ukraine. Zbirnyk Prac’ Zoologichnogo Muzeyu 44: 109–115.

Mazanaeva LF, Askenderov AD (2007) New data on the distribution of eastern spadefoot, *Pelobates syriacus* Boettger, 1889 and common spadefoot, *Pelobates fuscus*, Laurenti, 1768 in Dagestan (the North Caucasus). Russian Journal of Herpetology 14: 161–166.

Mertens R (1923) Beiträge zur Kenntnis der Gattung *Pelobates* Wagler. Senckenbergiana, 118–128.

Mertens R, Müller L (1928) Liste der Amphibien und Reptilien Europas. Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft 41: 1–62.

Mertens, R (1967) Die herpetologische Sektion des Natur-Museums und Forschungs-Institutes Senckenberg in Frankfurt a. M. nebst einem Verseichnis ihrer Typen. Senckenbergiana Biologica 48: 1–106.

Mészáros B (1973) Critical studies on karyotypes of eight anuran species from Hungary and some problems concerning the evolution of the Order. Acta Biologica Debrecina 10–11: 151–161.

Michahelles, C (1830) Neue südeuropäische Amphibien. Isis von Oken 23: 806–809.

Milto KD, Barabanov AV (2011) An annotated catalogue of the amphibian types in the collection of the Zoological Institute, Russian Academy of Sciences, St. Petersburg. Russian Journal of Herpetology 18: 137–153.

Mollov I, Popgeorgiev G, Naumov B, Georgiev D (2006) New data on the distribution of the Syrian Spadefoot (*Pelobates syriacus* balcanicus Karaman, 1928) in Bulgaria. Scientific Researches of the Union of Scientists in Bulgaria-Plovdiv, Series B Natural Sciences and the Humanities 8: 132–136. [in Bulgarian]

Morescalchi A (1967) The close karyological affinities between a *Ceratophrys* and *Pelobates* (Amphibia, Salientia). Experientia 23: 1071–1072. https://doi.org/10.1007/BF02136458

Morescalchi A (1971) Comparative karyology of the Amphibia. Bolletino di Zoologia 38: 317–320. https://doi.org/10.1080/11250007109429162

Morescalchi A, Olmo E, Stingo V (1977) Trends of karyological evolution in pelobatoid frogs. Experientia 33: 1577–1578. https://doi.org/10.1007/BF01934007

Müller J (1832) Ueber drey verschiedene Familien der froschartigen Thiere nach dem Bau der Gehörwerkzeuge. Isis von Oken 25: 536–539.

Munwes I, Geffen E, Roll U, Friedmann A, Daya A, Tikochinski Y, Gafny S (2010) The change in genetic diversity down the core-edge gradient in the eastern spadefoot toad (*Pelobates syriacus*). Molecular Ecology 19: 2675–2689. https://doi.org/10.1111/j.1365-294X.2010.04712.x

Nöllert A, Günther R (1996) Knoblauchkröte – *Pelobates fuscus* (Laurenti, 1768). In: Günther R (Ed.) Die Amphibien und Reptilien Deutschlands. Gustav Fisher, Jena, 252–274.
Nöllert A, Grossenbacher K, Laufer H (2012) *Pelobates fuscus* (Laurenti, 1768) – Syrische
Schaufelkröte. In: Grossenbacher K (Ed.) Handbuch der Reptilien und Amphibien Eu-
ropas Bd 51 Froschlurche I. Aula, Wiebelsheim, 465–562.

Nyström P, Birkedal L, Dahlberg C, Brönmark C (2002) The declining spadefoot toad *Pelobates fuscus*: calling site choice and conservation. Ecography 25: 488–498. https://doi.org/10.1034/j.1600-0587.2002.250411.x

Nyström P, Hansson J, Månsson J, Sundstedt M, Reslow C, Broström A (2007) A documented amphibian decline over 40 years: possible causes and implications for species recovery. Biological Conservation 138: 399–411. https://doi.org/10.1016/j.biocon.2007.05.007

Pallas PS (1771) Reise durch verschiedene Provinzen des russischen Reichs. Gedruckt bey der Kayserlichen Academie der Wissenschaften, St. Petersburg, 504 pp.

Pasteur G, Bons J (1959) Les batraciens du Maroc. Travaux de l'Institut Scientifique Chérifien. Série Zoologique 17, Rabat, 241 pp.

Psonis N, Antoniou A, Karameta E, Leaché AD, Kotsakiozi P, Darriba D, Kozlov A, Stamatakis A, Poursanidis D, Kukushkin O, Jablonski D, Crnobrnja-Isailovic J, Gherghel I, Lymberakis P, Poulakakis, N (2018) Resolving complex phylogeography patterns in the Balkan Peninsula using closely related wall-lizard species as a model system. Molecular Phylogenetics and Evolution 125: 100–115. https://doi.org/10.1016/j.ympev.2018.03.021

Roček Z (1981) Cranial anatomy of the frogs of the family Pelobatidae Stannius, 1856, with outlines of their phylogeny and systematics. Acta Univesitatis Carolinae – Biologica 1980: 164 pp.

Rodriguez A, Burgon JD, Lyra M, Irisarri I, Baurain D, Blaustein L, Göcmen B, Künzel S, Mable BK, Nolte AW, Veith M, Steinfartz S, Elmer KR, Philippe H, Vences M (2017) Inferring the shallow phylogeny of true salamanders (*Salamandra*) by multiple phylogenomic approaches. Molecular Phylogenetics and Evolution 115: 16–26. https://doi.org/10.1016/j.ympev.2017.07.009

Rösel von Rosenhof AJ (1758) Historia Naturalis Ranarum Nostratium in Qua Omnes earum Proprietates Præsertim quæ ad Generationem Ipsarum Pertinent, Fusius Enarrantur / Die natürliche Historie der Frösche hiesigen Landes, Albrecht von Haller, Nürnberg, 115 pp. https://doi.org/10.5962/bhl.title.149946

Salvador A, Donaire-Barroso D, Slimani T, El Mouden EH, Geniez P (2009) *Pelobates varaldii*. The IUCN Red List of Threatened Species 2009: eT58054A11723994. https://doi.org/10.2305/IUCN.UK.2004.RLTS.T58054A11723994.en

Schmid M (1979) On the arrangement of chromosomes in the elongated sperm nuclei of Anura (Amphibia). Chromosoma 75: 215–234. https://doi.org/10.1007/BF00292209

Schmid M (1980) Chromosome banding in Amphibia. IV. Differentiation of GC- and AT-Rich chromosome regions in Anura. Chromosoma 77: 83–103. https://doi.org/10.1007/BF00292043

Schmid M (1982) Chromosome banding in Amphibia. VII. Analysis of the structure and variability of NORs in Anura. Chromosoma 87: 327–344. https://doi.org/10.1007/BF00327634

Schmid M, Vitelli I, Batistoni R (1987) Chromosome banding in Amphibia. XI. Constitutive heterochromatin, nucleolus organizers, 18S + 28S and 5S ribosomal RNA genes in
Ascaphidae, Pipidae, Discoglossidae and Pelobatidae. Chromosoma 95: 271–284. https://doi.org/10.1007/BF00294784

Severtsov N (1855) Periodic Phenomena in the Life of Animals of Voronezh Governorate. Moscow, 385-386. [in Russian]

Shaw G (1802) General Zoology or Systematic Natural History. Volume III, Part 1. Amphibia. London: Thomas Davison.

Sillero N, Campos J, Bonardi A, Corti C, Creemers R, Crochet P-A, Crnobrnja Isailović J, Denoël M, Ficetola GF, Gonçalves J, Kuzmin S, Lymberakis P, de Pous P, Rodríguez A, Sindaco R, Speybroeck J, Toxopeus B, Vieites DR, Vences M (2014) Updated distribution and biogeography of amphibians and reptiles of Europe. Amphibia-Reptilia 35: 1–31. https://doi.org/10.1163/15685381-00002935

Singhal S, Moritz C (2013) Reproductive isolation between phylogeographic lineages scales with divergence. Proceedings of the Royal Society B: Biological Sciences 280: 20132246. https://doi.org/10.1098/rspb.2013.2246

Sofianidou T (2012) *Pelobates syriacus* (Boetger, 1899)—Syrische Schaufelkröte. In: Grossenbacher K (Ed.) Handbuch der Reptilien und Amphibien Europas Bd 51 Froschlurche I. Aula, Wiebelsheim, 563–620.

Stojankov A, Tzankov N, Naumov B (2011) Die Amphibien und Reptilien Bulgariens. Chimalaira, Frankfurth am Main, 588 pp.

Strachinis I, Roussos SA (2016) Terrestrial herpetofauna of Limnos and Agios Efstratios (North-ern Aegean, Greece), including new species records for *Malpolon insignitus* (Geoffroy Saint-Hilaire, 1827) and *Pelobates syriacus* Boettger, 1889. Herpetology Notes 9: 237–248.

Sturm J (1828) Deutschlands Fauna. III. Abtheilung. Die Amphibien. Nürnberg, 36

Suriadna NN (2014) Comparative analysis of karyotypes of two cryptic species of pelobatid frogs (Amphibia, Anura) of Ukraine. Vestnik Zoologii 48: 511–520. https://doi.org/10.2478/vzoo-2014-0061

Suriadna NN, Mikitinets GI, Rozanov YuM, Litvinchuk SN (2016) Distribution, morphological variability and peculiarities of biology of spadefood toads (Amphibia, Anura, Pelobatidae) in the south of Ukraine. Zbirnyk Prac’ Zoologichnogo Muzeyu 47: 67–87.

Szabolcs IM, Mizsei E (2017) First record of the eastern spadefoot toad (*Pelobates syriacus*) Boettger, 1889) in Albania. NorthWestern Journal of Zoology 13: 175–176.

Székely P, Iosif R, Székely D, Stănescu F, Cogălniceanu D (2013) Range extension for the Eastern spadefoot toad *Pelobates syriacus* (Boettger, 1889) (Anura: Pelobatidae). Herpetology Notes 6: 481–484.

Székely D, Cogălniceanu D, Székely P, Denoël M (2017) Out of the ground: two coexisting fossorial toad species differ in their emergence and movement patterns. Zoology 121: 49–55. https://doi.org/10.1016/j.zool.2016.12.003

Țeran L, Sos T, Hegyeli Z, Gábos E, Fülöp T, Marton A (2017) New data on the distribution of Eastern spadefoot toad *Pelobates syriacus* Boettger, 1889 (Anura: Pelobatidae) in the Pannonian Plain. North-Western Journal of Zoology 13: 179–182.

Thirion J-M, Cheylan M (2012) *Pelobates cultripes* (Cuvier, 1829). Pélobate cultripède. In: Lescure J, de Massary JC (Eds) Atlas des amphibiens et reptiles de France. Biotope – Muséum national d’Histoire naturelle, Mèze & Paris, France, 100–101.
Tschudi JJ (1838) Classification der Batrachier mit Berücksichtigung der fossilen Thiere dieser Abtheilung der Reptilien. Neuchâtel: Petitpierre, 99 pp. https://doi.org/10.5962/bhl.tiele.4883

Uğurtas IH (2001) Variation in *Pelobates syriacus* of Turkey. Asiatic Herpetological Research 9: 139–141.

Uğurtaş IH, Tunca B, Aydemir N, Bilaloğlu R (2001) A cytogenetic study on the *Pelobates syriacus* (Amphibia, Anura) in Bursa-Turkey. Turkish Journal of Zoology 25: 159–161. https://doi.org/10.1560/UMHA-6J3N-FUG9-D7EV

Uğurtas IH, Ljubusavljevic K, Sidorovska V, Kalezic ML, Džukić G (2002) Morphological differentiation of eastern spadefoot toad (*Pelobates syriacus*) populations. Israel Journal of Zoology 48: 13–32.

Veith M, Fromhage L, Kosuch J, Vences M (2006) Historical biogeography of Western Palearctic pelobatid and pelodytid frogs: a molecular phylogenetic perspective. Contribution to Zoology 75: 109–120. https://doi.org/10.1163/18759866-0750304001

Vukov T, Kalezić ML, Tomović L, Krizmanić I, Jović D, Labus N, Džukić G (2013) Amphibians in Serbia: distribution and diversity patterns. Bulletin of the Natural History Museum 6: 90–112. https://doi.org/10.5937/bnhmb1306090V

Zagorodniuk I (2003) Species of the lower tetrapodes from Ukraine: in the nature and on the paper. Visnyk of Lviv University, Biology Series, Lviv, 33: 80–90. [in Ukrainian]

**Supplementary material I**

**Average snout-vent length in *Pelobates* populations**

Authors: Christophe Dufresnes, Ilias Strachinis, Elias Tzoras, Spartak N. Litvinchuk, Mathieu Denoël

Data type: measurement

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: https://doi.org/10.3897/zookeys.859.33634.suppl1