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


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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.
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Taxonomic revisions in Pelobates 131
Call a spade a spade: taxonomy and distribution of
Pelobates, with description of a new Balkan endemic
Christophe Dufresnes1,2,3, Ilias Strachinis4, Elias Tzoras5,
Spartak N. Litvinchuk6,7, Mathieu Denoël8
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 Sheeld, Alfred Denny Building, Western Bank, S10 2TN Sheeld, United Kingdom 4 School of
Biology, Aristotle University of essaloniki 54124 essaloniki, Greece 5 26442 Patra, Achaia, Greece 6 Insti-
tute 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 (
Academic editor: Angelica Crottini|Received 4 February 2019|Accepted 10 June 2019|Published 2 July 2019
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.
e genomic era contributes to update the taxonomy of many debated terrestrial vertebrates. In an ac-
companying 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 conrmed
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 specic level. ird, we evidenced cryptic speciation and diversication 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.
ZooKeys 859: 131–158 (2019)
doi: 10.3897/zookeys.859.33634
Copyright Christophe Dufresnes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
Amphibian, Palearctic, Pelobates balcanicus, Pelobates balcanicus chloeae, Pelobates vespertinus, Pelobatidae,
phylogenomics, phylogeography, spadefoot toad
e revolution initiated by high-throughput sequencing techniques has reached the
eld 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). ese 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 ossied skull (to dig themselves out) (Székely
et al. 2017; Dufresnes 2019). ey 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 con-
tractions 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). ird, 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 conrmed the old
split between P. cultripes and P. varaldii, previously identied 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 conclu-
sions of Litvinchuk et al. (2013) that P. fuscus and P. vespertinus deserve a specic status.
ird, 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 spe-
cies feature deep intraspecic divergence, worthy of subspecic status. is is the case
between Levantine and Anatolian/Caucasian populations in P. syriacus, and between
the northern Balkans and Peloponnese in P. balcanicus.
Taxonomic revisions in Pelobates 133
In this paper, we integrate these recent ndings 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 subspe-
cies, we examined nomina available in the literature. To this end, we rst referred to
the Amphibian Species of the World online database (Frost 2019) and subsequently
reviewed all the original references available.
We reviewed phenotypic (coloration, morphology) and genetic (genome size, karyo-
type, and sequence divergence) variation of the considered taxa. Coloration is illustrat-
ed 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
Figure 1. Phylogeny and distribution of Pelobates taxa. e 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).
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
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 dierences among taxa and sex by a two
way analysis of variance (ANOVA) in R. We then performed comparisons between spe-
cies using a Tukey test. Finally, we tested sex-specic dierences within taxa for which
measures of both sexes were available in at least ve populations, by paired t-tests.
We briey described the karyotype of each taxon based on the literature and fur-
ther report nuclear DNA content as a proxy to genome size, obtained from ow cy-
tometry. In addition, sequence divergence, available from our phylogeographic study
(Dufresnes et al. 2019b), are provided between each pair of taxa, based on mitochon-
drial (cyt-b + 16S, 1.2 kb) and nuclear DNA (63.5 kb of RAD tags).
We detailed the distribution of each Pelobates taxon, based on available literature, i.e.
national and regional atlas, as well as scientic 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. Dis-
tribution layers were originally obtained from the IUCN Red List of reatened Spe-
cies (, 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 con-
ducted a short eldwork expedition to the only recently conrmed locality of this tax-
on, Strofylia meadows in Peloponnese (38.1239°N, 21.3858°E) on December 2018.
Collection of live animals was authorized by permit ΑΔΑ: ΩΣΜ34653Π8-9ΣΟ 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. e 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.
Taxonomic revisions in Pelobates 135
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 conrmed 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 conrmed 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). e
remaining intraspecic 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 signicant global eect of species (P < 0.001)
but not of sex (P = 0.08), neither of their interaction (P = 0.42) (two way ANOVA).
e species eect was mainly due to dierences 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 signicant (P < 0.001), but none within
groups (P > 0.05) (Tukey test). Females were signicantly 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.
e following present accounts for each taxon. We could successfully access the
original literature for all but one description, and herein report the primary informa-
tion as it was published. e 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. e largest Pelobates species, P. cultripes diers from the other Eurasian spa-
defoots by metatarsal spades being entirely black and a at skull. Sizes largely overlap
between sexes although males are generally smaller than females (Fig. 2). e back-
ground 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). e karyotype consists of six large and seven small (i.e. <
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
6% of total length) pairs of two-armed chromosomes (Morescalchi 1967, 1971; Mores-
calchi 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). e 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 lo-
cality: “prope Malagam” (near Malagam), probably Malaga, Spain; type(s): not men-
tioned. 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. e 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 Med-
iterranean 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 (irion
and Cheylan 2012). IUCN status: Near reatened (Beja et al. 2009).
Diversity. Combining mtDNA and microsatellite data, Gutiérrez-Rodriguez et
al. (2017) identied 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 ge-
netic 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) diering by a few phenotypic fea-
tures. Unlike P. cultripes, the black coloration of the spades is often concentrated on
the edges (Pasteur and Bons 1959; Busack et al. 1985). e cranial braincase is high
and narrow in P. varaldii, while it is low and wide in P. cultripes (Pasteur and Bons
1959; Roček 1981). e 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). Aver-
age SVL = 53 mm (range: 36–66 mm) for females (n = 4 populations) and 51 mm
Taxonomic revisions in Pelobates 137
(33–65mm) for males (n = 4 populations) (Suppl. material 1, Table S1; Fig. 2). e
karyotype includes six large and seven small pairs of two-armed chromosomes. Large
centromeric C-bands appears 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). e nuclear DNA content averages 7.3 pg (Litvinchuk et al. 2013). As shown
Figure 2. Between-population variation of average size (snout–vent length – SVL) for each Pelobates
species, measured separately for females (pink) and males (blue). is 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.
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
in Table 1, P. varaldii diers from P. cultripes by 6.0% at mtDNA and 0.40% at nuclear
DNA (Dufresnes et al. 2019b).
Taxonomy. e 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. e ancient split of P. varaldii,
dating back to the Messinian Salinity Crisis (5.3 My), supports its specic 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 phylogeo-
graphic or population genetic work.
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).
Taxonomic revisions in Pelobates 139
Pelobates fuscus (Laurenti, 1768)
Common spadefoot
Diagnosis. Small spadefoot characterized by pale grayish metatarsal spades and a
domed skull. e webbing of the hindfeet is well developed. Males are smaller than
females (Fig. 2). e species can be found in a spectrum of gray, brown, or yellow-
ish 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 diers 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). e 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). e nuclear DNA content (calculated from ow cytometry)
averages 8.7–9.0pg (Litvinchuk et al. 2013).
Taxonomy. Originally described as Bufo fuscus Laurenti, 1768; type locality: not
specically 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 specically 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 amplexed 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).
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
e 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 dierentiation
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 Eu-
rope (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-Paz 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 Swe-
den, as well as the coastline of the Baltic Sea from Germany to Estonia, and east-
ward until it reaches P. vespertinus in Russia (Kuzmin 1999; Nyström et al. 2007;
Litvinchuk et al. 2013; Sillero et al. 2014). e 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). e 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 east-
ern Austria around the Alps (Cabela et al. 2001), and southern Germany (Nöllert
and Gunther 1996). e 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 west-
ern Bulgaria (around Soa: 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; Egg-
ert et al. 2006).
Diversity. e phylogeographic work by Crottini et al. (2007) and Litvinchuk
et al. (2013) characterized two refugial groups for this species (as the “western line-
age of P. fuscus”), based on shallow mtDNA divergence and allozyme dierentiation:
in the Balkans/northern Italy and on the western shores of the Black Sea coast. is
seems supported by weak genomic dierentiation among Central-European samples
(Dufresnes et al. 2019b). e 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).
Taxonomic revisions in Pelobates 141
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. e coloration also spans the gray-yellowish-
brownish spectrum, including reddish individuals (Fig. 3); orange dots can be heavily
marked or absent (Fig. 3). It diers 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). e 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). e nuclear DNA content averages 9.2–9.4 pg (Lit-
vinchuk et al. 2013). As shown in Table 1, P. vespertinus diers from P. fuscus by 2.5%
at mtDNA and 0.13% at nuclear DNA (Dufresnes et al. 2019b). e 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 specically designated, but the author mentioned this taxon in Zarbay Creek
(“Bach Sarbei”, Samara oblast), Russia, which can be considered as the type local-
ity; type(s): not mentioned. ree junior synonyms. Pelobates fuscus var. orien-
talis Severtsov, 1855; type locality: “Voronezhskaya Gubernia” (Voronezh gover-
norate), 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 speci-
men nor a type locality were designated (Zagorodniuk 2003). Pelobates vesperti-
nus was previously considered a subspecies of the common spadefoot, as Pelobates
fuscus vespertinus (Crochet and Dubois 2004). e signicant 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 dierences
(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). e southernmost
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
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).
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 Martinez (Israël) e IS (Limnos, Greece) f SNL (Eu-
ropean Turkey) g IS (Limnos, Greece) h A Nöllert (Dagestan, Russia) i MD (Danube Delta, Romania)
jIS (race, Greece) k IS (Macedonia, Greece) l IS (Evia, Greece) m–o IS (Peloponnese, Greece) p CD
(Peloponnese, Greece).
Taxonomic revisions in Pelobates 143
Pelobates syriacus Boettger, 1889
Eastern spadefoot
Diagnosis. Large spadefoot with whitish metatarsal spades and a at 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). e
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). e 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: “Haia in Syrien” (Haifa), Israel; type: SMF 1437.1a (Boettger
1892), subsequently designated as lectotype SMF 1722 (Mertens 1967). Other nomi-
na 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;
Soanidou 2012) (Fig. 1). e 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; Soanidou 2012). It may be extinct from western Jordan (Agasy-
an et al. 2009b; Disi and Amr 2010). e 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. Alter-
natively, the latter populations could belong to P. balcanicus, notably along the Maritsa
River, and identication is pending molecular analyses. e presence of P. syriacus is
also documented on the Greek islands of Limnos, Lesbos, and Kos (Soanidou 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. e
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).
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
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 dierentiated despite their
present-day fragmentation (see also Munwes et al. 2010 for P. s. syriacus). Populations
from the Caucasus (P. s. boettgeri) diers from Anatolian ones at nuclear, but not
mitochondrial markers. In the Lesser Caucasus and southern Turkey, P. s. boettgeri
features traces of past gene ow 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
diers from P. s. syriacus by 1.7% at mtDNA and 0.01% at nuclear DNA (Dufresnes
et al. 2019b).
Taxonomy. e 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: “Tiis” (Tbilisi),
Georgia; types: ten syntypes, nine at ZISP, and one at ZIK (Amph A5/A (2164)). Sub-
species 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 sub-
species in Armenia, Turkey (Antalya region), and Israel argue against a specic 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 at 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 dierentiated
from Asia Minor (P. syriacus); yet P. syriacus populations from European Turkey
(Edirne, genetically conrmed by Dufresnes et al. 2019b) and southeastern Bulgaria
(Primorsko) grouped with P. balcanicus (Uğurtas et al. 2002). Roček (1981) only found
one cranial dierence: the processus posterior parasphenoidei is present in P.syriacus but
Taxonomic revisions in Pelobates 145
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). e 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). e
nuclear DNA content (calculated from ow cytometry) averages 7.9 pg (Litvinchuk et
al. 2013; data from P. b. balcanicus). As shown in Table 1, P. balcanicus diers 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). is 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). erefore, 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). ere 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 popula-
tions (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, Soanidou (2012) reported the spe-
cies 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
Table 1. Pairwise % of genetic dierences between Pelobates taxa (from the data of Dufresnes et al.
2019b). e estimates below diagonal correspond to mitochondrial DNA (cyt-b + 16S, 1.2 kb); the esti-
mates above diagonal correspond to nuclear DNA (63.5 kb of RAD tags).
P. cultripes P. varaldii P. fuscus P. vespertinus P. s. syriacus P. s. boettgeri P. b. balcanicus P. b. chloeae
P. cultripes 0.40 0.66 0.75 0.72 0.70 0.74 0.73
P. varaldii 6.0 – 0.83 0.92 0.89 0.88 0.92 0.90
P. fuscus 10.1 10.0 0.13 0.63 0.62 0.65 0.64
P. vespertinus 9.7 9.6 2.5 0.71 0.70 0.74 0.73
P. s. syriacus 9.1 8.6 9.1 8.9 0.01 0.32 0.30
P. s. boettgeri 9.2 8.9 9.2 9.0 1.7 0.31 0.29
P. b. balcanicus 9.2 8.6 8.5 8.5 7.2 7.0 0.02
P. b. chloeae 9.2 8.2 8.5 8.6 7.7 7.7 2.8
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
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 race (Džukić et al. 2008; Soanidou,
2012). e 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
identied: a rst one in the eastern ranges, from the Carpathians to the Black Sea
and as south as Greek race; 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). e
eastern and western lineages widely admix. Populations from central Greece are yet
to be examined.
Pelobates balcanicus chloeae ssp. nov.
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, 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); mitochon-
drial 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 at, as viewed from the side, with large interorbital; tym-
panum 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 ngers; palm tubercles visible, oval. Ventral and dor-
sal 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.
Taxonomic revisions in Pelobates 147
Paratype. NHMC, 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); mito-
chondrial 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 at 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. e
dorsum coloration is generally light gray, sometimes yellow, covered with dark green-
brown reticulate patches and variable orange dots (Fig. 4). From our own observa-
tions, the color patterns seem to slightly dier 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).
e ventrum and limbs are glossy and slightly bluish (rather pale whitish in the nomi-
nal subspecies). Moreover the snout of P. b. chloeae appears shorter and blunter than
the nominal subspecies. ese 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). e mating call and the tadpole are yet to be
described and diagnosed. e karyotype has not been documented. As shown in Table
1, P. b. chloeae diers from the nominal subspecies by 2.8% at mtDNA and 0.02% at
nuclear DNA (Dufresnes et al. 2019b).
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.
Strofylia population Holotype NMHC Paratype NMHC
SVL 71.5 ± 3.4 78.7 74.1
HW 23.7 ± 1.1 26.6 25.5
HL 21.8 ± 0.9 23.4 23.1
ED 7.4 ± 0.24 7.2 7.1
EE 15.9 ± 0.7 16.7 17.3
NN 4.4 ± 0.2 4.6 4.2
EN 6.0 ± 0.3 6.7 6.0
ML 6.1 ± 0.4 7.1 6.5
MH 2.6 ± 0.1 2.6 2.8
HLL 83.7 ± 3.6 92 90
TTL 64.2 ± 3.1 72 69
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
Figure 5. Description of Pelobates balcanicus chloeae. To p live photograph of the holotype, NHMC (CD, taken on December 10th 2018); middle dorsal and lateral views of the type specimens
(left NHMC; right NHMC post-mortem (IS); bottom Strofylia meadows, the
type locality in Peloponnese, Greece (ET).
Taxonomic revisions in Pelobates 149
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 mi-
tochondrial phylogenetic data, but refrain a specic 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 refer-
ence 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.
e 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 Pelo-
ponnese in southern Greece (Dufresnes et al. 2019b) (Fig. 1); it was so far genetically
conrmed 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; Soanidou 2012). Nowadays, the two known Pelobates localities
are restricted to the central (Tripoli) and north-western (Strofylia) areas. Conse-
quently, 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 (essaly, Greece)
bore trace of introgression by P. b. chloeae, suggesting past or present contact (Du-
fresnes 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, at, 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 diver-
sity recorded across the entire ranges of P. balcanicus and P. syriacus (Dufresnes et al.
2019b). is 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 evalu-
ated 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).
Christophe Dufresnes et al. / ZooKeys 859: 131–158 (2019)
Identication 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 dierences, 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–Mos-
cow imaginary line .................................................................................P. fuscus
ree 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 chloeae
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 intraspecic diversity into subspecic divisions, P. s. syriacus, P. s. boettgeri,
P.b. balcanicus, and P. b. chloeae, the latter as a newly described taxon. eir 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.
We thank M. Pajković for translating publications from Serbo-Croatian, P. Lymberakis
(NHMC) for processing the type series of P. b. chloeae, A. Nöllert, G. Haimovitch, G.
Taxonomic revisions in Pelobates 151
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 sci-
entique (FNRS). is study was founded by a grant from the Swiss National Science
Foundation (SNSF) to Nicolas Perrin (no. 31003A_166323), and a SNSF fellowship
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Supplementary material 1
Average snout-vent length in Pelobates populations
Authors: Christophe Dufresnes, Ilias Strachinis, Elias Tzoras, Spartak N. Litvinchuk,
Mathieu Denoël
Data type: measurement
Copyright notice: is dataset is made available under the Open Database License
( e 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.
... Podvrstu je opisao Karaman na osnovu primeraka koje je našao u oblasti Starog Dojrana u Makedoniji (Karaman, 1928), što je predstavljalo i prvi nalaz sirijske češnjarke (sensu lato) u Evropi. Na osnovu molekularnih podataka, ovaj takson podignut je na nivo vrste i predstavlja balkanski endemit (Dufresnes et al., 2019a). Severnu granicu njenog rasprostranjenja predstavlja tok Dunava, od južnog Banata do Crnog mora, a zapadnu granicu predstavlja tok Velike Morave (Džukić et al., 2008). ...
... Severnu granicu njenog rasprostranjenja predstavlja tok Dunava, od južnog Banata do Crnog mora, a zapadnu granicu predstavlja tok Velike Morave (Džukić et al., 2008). Severni deo areala odvojen je od južnog, koji čine populacije u Severnoj Makedoniji, istočnoj Albaniji, jugozapadnoj Bugarskoj i Grčkoj, gde naseljava oblast oko Korintskog zaliva, Peloponez, severne i severoistočne delove kopnene Grčke i obalu Egejskog mora do Trakije (Džukić et al., 2008;Dufresnes et al., 2019a). U Srbiji prati tok Dunava istočno od Beograda, ima je u južnom Banatu, u oblasti ušća Velike Morave, čiji tok prati sve do Južne Morave, a izolovane populacije nalaze se u oblasti Ključa, sa velikim jazom u zoni Đerdapa i Karpatskih planina (Džukić et al., 2008;Crnobrnja-Isailović i sar., 2012;Vukov i sar., 2013). ...
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Vodozemci smederevskog kraja-pregled dosadašnjih istraživanja, distribucija i biogeografska pripadnost Apstrakt: Rad se bavi faunom vodozemaca opštine Smede-revo. Iznosi prikaz publikovanih nalaza vodozemaca na teri-toriji Smedereva, kao i nove, do sada neobjavljene nalaze sa terena. Smederevo predstavlja tranzicionu zonu između pa-nonskih i balkanskih ekoregiona, što se ogleda i u relativnom bogatstvu faune vodozemaca. Prisustvo različitih horotipa svedoči o mešanju fauna vodozemaca severa, istoka i juga. Smederevo je tokom proteklih decenija pretrpelo značajne antropogene izmene usled urbanizacije, industrijalizaci-je i klimatskih promena, a naročito su se na udaru izmena našle površinske vode. Zbog toga je izmenjen ili izgubljen veliki broj prirodnih staništa vodozemaca. Takođe, nalazi pojedinih vrsta-podunavskog velikog mrmoljka, šarenog daždevnjaka i balkanske češnjarke, postoje samo u literaturi odnosno nisu potvrđeni na terenu. Izostanak potvrde nala-za u prethodne dve ili više decenija može biti posledica izo-stanka sistematskih istraživanja ovih vrsta koje se odlikuju skrovitim načinom života. Drugi razlog mogu biti izmene i gubici adekvatnih staništa usled gorepomenutih antropoge-nih faktora. Buduća faunistička istraživanja trebalo bi kon-centrisati upravo na ponovnu potvrdu prisustva tih vrsta. Preostala netaknuta staništa vodozemaca na području opšti-ne Smederevo predstavljaju konzervacioni prioritet.
... Table 3. Checklist of amphibians and reptiles recorded at Lake Karla's Plain during field surveys, along with literature mentions (Chondropoulos, 1989;Gordinho, 2009;Sofianidou, 2012 Pelobates balcanicus (Karaman, 1928) -Balkan spadefoot.-The Balkan spadefoot has a single record without details in the literature, from the former Lake Karla area, mentioned as Pelobates syriacus (Sofianidou, 2012) -but see Dufresnes et al. (2019) for the current name of the taxon (P. balcanicus) who's distribution area includes the area of Lake Karla. ...
... Further specialised searching in the area is considered necessary. In Greece the species is distributed in the eastern parts of the mainland, along the Aegean Sea shores, western Macedonia, Peloponnese and Euboea Island (Dufresnes et al., 2019). ...
The distribution of herpetofauna in large parts of mainland Greece has not been studied extensively. This study covers a poorly-known herpetological area in Thessaly Region - Lake Karla’s Plain. We conducted 59 field surveys during 12 consecutive years (2008–2019) and recorded a total of 26 herpetofauna species: 4 anuran amphibians and 22 reptiles (5 tortoises and terrapins, 7 lizards and 10 snakes). Our study brings updates regarding the presence, distribution and the main threats for the herpetofauna of Lake Karla’s Plain, with 17 species being recorded here for the first time, including an alien freshwater turtle, Trachemys scripta
... 19 Bufo spinosus -The Western Ligurian populations of the genus Bufo, from the French border up to Calice Ligure, have been recently assigned to this species based on genetic evidence (Recuero et al., 2012;Arntzen et al., 2020). 20 Bufotes boulengeri siculus -Subspecific status according to Speybroeck et al. (2020) and Dufresnes et al. (2019). 21 Bufotes viridis balearicus -We follow Speybroeck et al. (2020) to consider B. balearicus as a subspecies of B. viridis, due to the wide hybrid zone in north-east Italy (Dufresnes et al., 2014). ...
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This paper represents an update of the list of Italian amphibians and reptiles published 15 years ago by Razzetti et al. (2006) and of the checklist published in 1993 by the late Benedetto Lanza. At present, the Italian herpetofauna includes 100 species (41 amphibians and 60 reptiles) and an amphibian taxon of hybrid origin. Seven species and one subspecies are allochthonous and became naturalized within the last century. Since the last published list, a new species has been described (Vipera walser), five taxa have been raised to species rank (Salamandrina perspicillata, Speleomantes sarrabusensis, Zootoca carniolica, Malpolon insignitus and Natrix helvetica) while three taxa have been downgraded to subspecies. All the relevant taxonomic changes based upon new research have been discussed, including tentative revisions and controversial taxa. Nine species reported or listed dubitatively in Lanza’s 1993 list are excluded here.
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The 2020 spring processes in the European Russia were developing according to the type of false spring. The absence or weak development of snow cover, as well as its early descent in the last decade of February, determined the rapid warming of the soil profile. A certain complex of meteorological factors led to an abnormally early start of spawning migrations of the common spadefoot toad (Pelobates fuscus (Laurenti, 1768)) and Pallas's spadefoot toad (Pelobates vespertinus (Pallas, 1771)) over a vast territory which included the northwestern, western, central and southeastern parts of the species habitat in the region. Analysis of the snow cover dynamics and the temperature course according to the data of the network of meteorological stations made it possible to assess the phenology of these species of anuran amphibians within this region using the method of reconstructing the reproductive period events of the spadefoot toads. The duration of the period between the start dates of false and true spawning migrations was more than 40 days in the west, in the center and in the south-east of the European Russia. The abnormally early formation of the 2020 false spring phenomenon in the spawning migrations of spadefoot toads is currently registered as a precedent which may have a significant impact on the reproduction success of the species of this genus.
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The development of genomics in recent years has led to updates in the taxonomy of many terrestrial vertebrates. Following the most recent taxonomic revisions (Dufresnes et al., 2019a, b) of the anuran family Pelobatidae Bonaparte, 1850 (genus Pelobates Wagler, 1830), three species of spadefoot toad are now recognised on the territory of Bulgaria: Pelobates fuscus (Laurenti, 1768), P. syriacus Boettger, 1889, and P. balcanicus Karaman, 1928. According to Dufresnes et al. (2019a), the Balkan Spadefoot Toad has two subspecies: P. b. balcanicus and Pelobates b. chloeae Dufresnes et al., 2019, of which only the former is found in Bulgaria.
Genomic resources for amphibians are still hugely underrepresented in vertebrate genomic research, despite being a group of major interest for ecology, evolution and conservation. Amphibians constitute a highly threatened group of vertebrates, present a vast diversity in reproductive modes, are extremely diverse in morphology, occupy most ecoregions of the world, and present the widest range in genome sizes of any major group of vertebrates. We combined Illumina, Nanopore and Hi-C sequencing technologies to assemble a chromosome-level genome sequence for an anuran with a moderate genome size (assembly span 2.8 Gb); Pelobates cultripes, the Western Spadefoot toad. The genome has an N50 length of 330 Mb with 98.6% of the total sequence length assembled into 14 super scaffolds, and 87.7% complete BUSCO genes. We use published transcriptomic data to provide annotations, identifying 32,684 protein-coding genes. We also reconstruct the P. cultripes phylome and identify 2527 gene expansions. We contribute the first draft of the genome of the Western Spadefoot toad, Pelobates cultripes. This species represents a relatively basal lineage in the anuran tree with an interesting ecology and a high degree of developmental plasticity, and thus is an important resource for amphibian genomic research.
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The frogs inhabiting the rivers and marshes of Mesopotamia were a common part of the fauna between the Euphrates and Tigris rivers. Figurative amulets and parts of the animal were used to heal, and frogs served as a substitute animal. In a variety of early societies frogs were regarded as symbols of fertility, rebirth, healing, and transformation due to their metamorphosis and their ability to live in water and on land. Additionally, frogs' link to water and rain often lead to associations with cleansing. This paper cross-references the textual evidence, collected, and commented on by A. Bácskay, with herpetological insights into the medical use of frogs and toads. Using the descriptions given in Bácskay 2018, four anuran species were identified to have been present in the Mesopotamian marshlands that could have been used as medicinal treatment and as substitute animals. We provide a short summary on their habitat and give insights into their ecology and possible medical value. Together with the presentation of figurative frog amulets from Mesopotamia, dating from the 3rd to 1st millennium BCE, including their materials and archaeological contexts, the authors give a comprehensive overview of the magico-religious perception of frogs in Mesopotamia.
Anurans are a highly diverse amphibian order with a biphasic lifestyle. The larva, better known as tadpole, and the adult frog of one species typically occupy different habitats and exploit different resources. Therefore, both are subject of largely independent selection and are able to evolve stage-specific adaptations. Among others, the musculoskeletal features of the larval and adult head of anurans and the description of their specific traits are important to gain a deeper understanding of the evolution and the phylogeny of this highly diverse group. Here we present an overview of the larval and adult cranial morphology of the common spadefoot toad Pelobates fuscus. We provide a detailed description of the cranial musculoskeletal structures based on micro-computed tomography and following 3D reconstruction of two long-term stored specimens of P. fuscus and refer to striking adaptations of this species. P. fuscus inhabits a wide spectrum of habitats including mixed forests, steppes, fields and sandy areas with soft soils in close proximity to permanent water bodies. Adults have a semi-fossorial lifestyle and they are able to dig themselves in using their hindlimbs whereas the tadpoles are the largest of all European anurans. Derived larval features are the presence of an adrostral cartilage within the lower jaw, the presence of three portions of the M. subarcualis rectus I, the presence of the M. diaphragmatopraecordialis and a bipartite suprarostral cartilage. The presence of an enlarged prehallux and the co-ossification of the frontoparietal, as well as a pterygoid that borders the orbit ventrally and rostrally are striking adult features and adaptation to the semi-fossorial lifestyle. With the described morphological features, we want to underline their importance for several adaptations which may have facilitated and stimulated the evolution of anurans and provide baseline data for further investigations.
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First evidence on the occurence of Greek Newt Lissotriton graecus and the Aesculapian snake Zamenis longissimus on Evia Island, Greece - Parnassiana Archives 9: 115-117
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Aphanius fasciatus is a small fish occurring in Mediterranean brackish environments. In Cyprus it is known from three localities separated by long stretches of coast. The genetic diversity of these populations was evaluated using fragments of two mitochondrial genes. A comparison with the other available data showed that Cyprus populations represent a distinct lineage. The other lineages are concentrated in a relatively small area between the Strait of Sicily and the Western Ionian Sea, while all other areas include a subset of these lineages, suggesting that the aforementioned area might have acted as a glacial refugium. Landlocked North-African populations diverge from all other populations, suggesting that they might have originated in the Late Pleistocene, during transgression events of the Mediterranean Sea in North-African inland water bodies. The genetic diversity of A. fasciatus varied across different Cyprus populations, with a pattern mirroring the degree of environmental degradation, which likely affected population genetic variability through demographic reductions. The three Cyprus populations showed genetic uniqueness, suggesting the need of population-based management practices; the low genetic diversity of two populations, and the number of threats affecting them, suggest that the species should be considered endangered at national level and deserves protection measures.
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Cryptic phylogeographic diversifications are unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecology. Yet, to date very few comparative studies were attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (RAD-seq and mtDNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species-level cryptic divergences (>5My) between populations distributed in the Near-East (hereafter P. syriacus sensu stricto) and south-eastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10My), parapatry with highly-restricted hybridization (P. balcanicus and P. syriacus s. s., >5My), narrow hybrid zones (~15km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3My), to extensive admixture between Pleistocene and refugial lineages (≤2My). This full spectrum empirically supports a gradual build-up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.
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Current approaches to biodiversity conservation are largely based on geographic areas, ecosystems, ecological communities, and species, with less attention on genetic diversity and the evolutionary continuum from populations to species. Conservation management generally rests on discrete categories, such as identified species, and, for threated taxa, intraspecific units. Species, in particular, provide a common measure of biodiversity yet in both theory and nature, speciation is typically a protracted process progressing from connected populations to unambiguous species with variable rates of phenotypic, ecological and genetic divergence. Thus, most recognized species are not genetically uniform and are sometimes highly structured into historically isolated populations worthy of consideration as intraspecific units that represent unique genetic diversity for conservation. Genome screens offer unprecedented resolution of structure across taxonomic boundaries in species complexes, and have the potential to oversplit species if not interpreted conservatively. This highlights the blurred line between populations and species, and can confound simple dichotomies of “species” vs. “not species.” At the same time, like plants, there is increasing evidence that even distantly related animal species can hybridize and exchange genes. A review of conservation legislation reveals that legal definitions of “species” are quite flexible and can accommodate a range of infra-specifictaxa and divergent populations, as well as taxonomically recognized species. For example, the legislative definition of a species around the world can include: species, subspecies, varieties, and geographically and/or genetically distinct populations. In principle, this flexibility allows for protection of genetic diversity and maintenance of evolutionary processes at a broad range of infra-specific levels. However, evolutionary biologists often fail to adequately justify and then translate their evidence for genetically defined units into categories suited to assessment under local legislation. We recommend that (i) genomic data should be interpreted conservatively when formally naming species, (ii) concomitantly, there should be stronger impetus and a more uniform approach to identifying clearly justified intraspecific units, (iii) guidelines be developed for recognizing and labeling intraspecific data that align with best scientific practice, and (iv) that the more nuanced view of species and speciation emerging from genomic analyses is communicated more effectively by scientists to decision makers.
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Despite increasing appreciation of the “speciation continuum”, delimiting and describing new species is a major yet necessary challenge of modern phylogeography to help optimize conservation efforts. In amphibians, the lack of phenotypic differences between closely-related taxa, their complex, sometimes unresolved phylogenetic relationships, and their potential to hybridize all act to blur taxonomic boundaries. Here we implement a multi-disciplinary approach to evaluate the nature of two deeply-diverged mitochondrial lineages previously documented in Italian tree frogs (Hyla intermedia s. l.), distributed north and south of the Northern Apennine Mountains. Based on evidence from mitochondrial phylogenetics, nuclear phylogenomics, hybrid zone population genomics, niche modelling analyses, and biometric assessments, we propose that these lineages be considered distinct, cryptic species. Both mitochondrial and nuclear data affirm that they belong to two monophyletic clades of Pliocene divergence (~3.5 My), only admixing over a relatively narrow contact zone restricted to the southeast of the Po Plain (50-100km). These characteristics are comparable to similarly-studied parapatric amphibians bearing a specific status. Inferred from their current geographic distribution, the two Italian tree frogs feature distinct ecological niches (<15% of niche overlap), raising questions regarding potential adaptive components contributing to their incipient speciation. However, we found no diagnostic morphological and bioacoustic differences between them. This system illustrates the speciation continuum of Western-Palearctic tree frogs and identifies additional cryptic lineages of similar divergence to be treated as separate species (H. cf. meridionalis). We recommend combined approaches using genomic data as applied here for the future taxonomic assessment of cryptic diversity in alloparapatric radiations of terrestrial vertebrates, especially in controversial taxa. Finally, we formally described the northern Italian tree frogs as a new species, Hyla perrini, sp. nov.
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New data about distribution of Pelobates fuscus and P. vespertinus in Ukraine are presented. They are based on genome size data. The location of a contact zone of these species were studied. Both species were found on the territory of Kherson and Zaporozhye region. The nearest localities belonging to the different species were Mayachka village (P. vespertinus) and Energodar Town (P. fuscus) in Zaporozhye region. The distance between these localities was about 50 km. In the south of Ukraine, P. fuscus inhabit flood plain of Dnieper River and west of it, and P. vespertinus lives east of the river. The sintopic populations of the species was not revealed. A presumed hybrid of these species was found in Podgornoe village of Zaporozhye region. Both species occupy a wide range of biotopes, but prefer sandy soils. The body length and mass of P. vespertinus were larger than P. fuscus. The color of dorsal surface of body of P. vespertinus, as a rule, was striped, but P. fuscus was spotted.
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Based on known literature data and data collected during field research in 2014 and 2015, we present updated distribution map of the common spadefoot toad (Pelobates fuscus) in Western Balkans (Bosnia and Herzegovina and Croatia). Pelobates fuscus is listed as least concern on the global IUCN Red List, data deficient in Croatian Red Book and the populations are in constant decline. Until year 2014 this species was only suspected to inhabit Bosnia and Herzegovina. Today we have confirmed localities in Posavina region (western, central and eastern part) presented with precise coordinates and elevation. In this paper we also present first findings of tadpoles, more precisely, the reproductive sites of the common spadefoot toad in the Bosnia and Herzegovina. In Croatia, this species is found along the rivers Mura, Drava and Sava, including most lowland areas up to 300 m above sea level. Historical records of Pelobates fuscus in Adriatic region are discussed and compared with distribution area of Italian isolated population.
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The rise of high-throughput sequencing techniques provides the unprecedented opportunity to analyse controversial phylogenetic relationships in great depth, but also introduce the risk of being misinterpreted by high node support values influenced by unevenly distributed missing data or unrealistic model assumptions. Here, we use three largely independent phylogenomic data sets to reconstruct the controversial phylogeny of true salamanders of the genus Salamandra. a group of amphibians providing an intriguing model to study the evolution of aposematism and viviparity. We used RNA sequencing (RNAseq) and restriction-associated sequencing (RADseq) to obtain for all six Salamandra species, and for two species in its sister genus Lyciasalamandra as outgroup, data sets of (1) 3070 nuclear protein-coding genes from RNAseq; (2) 7440 loci obtained by RADseq; and (3) full mitochondrial genomes. The RNAseq and RADseq data sets retrieved fully congruent topologies when each of them was analysed in a concatenation approach, with high support from bootstrap respectively Bayesian posterior probabilities for: (1) S. infraimmaculata being sister group to all other Salamandra species; (2) S. algira being sister to S. salamandra; (3) these two species being the sister group of a clade containing S. atra, S. corsica and S. lanzai; and (4) the alpine species S. atra and S. lanzai being sister groups. The phylogeny inferred from the mitochondrial genome sequences differed from these results, most notably by strongly supporting a clade containing S. atra and S. corsica as sister taxa. A different placement of S. corsica was also retrieved when analysing the RNAseq and RADseq data under species tree approaches. Closer examination of gene trees derived from RNAseq revealed that only a low number of them supported each of the alternative placements of S. atra. Furthermore, gene jackknife support for the S. atra - S. lanzai node in the concatenated analysis stabilized only with very large concatenated data sets. The phylogeny of true salamanders thus provides a compelling example of how classical node support metrics such as bootstrap and Bayesian posterior probability can provide high confidence values in a phylogenomic topology even if the phylogenetic signal for some nodes is spurious, highlighting the importance of complementary approaches such as gene jacknifing. Yet, the general congruence among the topologies recovered from the RNAseq and RADseq data sets increases our confidence in the results, and validates the use of phylotranscriptomic approaches for reconstructing shallow relationships among closely related taxa. We hypothesize that the evolution of Salamandra has been characterized by episodes of introgressive hybridization, which would explain the difficulties of fully reconstructing their evolutionary relationships.
Reconstructing reliable timescales for species evolution is an important and indispensable goal of modern biogeography. However, many factors influence the estimation of divergence times, and uncertainty in the inferred time trees remains a major issue that is often insufficiently acknowledged. We here focus on a fundamental problem of time tree analysis: the combination of slow-evolving (nuclear DNA) and fast-evolving (mitochondrial DNA) markers in a single time tree. Both markers differ in their suitability to infer divergences at different time scales (the 'genome-timescale-dilemma'). However, strategies to infer shallow and deep divergences in a single time tree have rarely been compared empirically. Using Mediterranean amphibians as model system that is exceptional in its geographic and taxonomic completeness of available genetic information, we analyze 202 lineages of western Palearctic amphibians across the entire Mediterranean region. We compiled data of four nuclear and five mitochondrial genes and used twelve fossil calibration points widely acknowledged for amphibian evolution. We reconstruct time trees for an extensive lineage-level data set and compare the performances of the different trees: the first tree is based on primary fossil calibration and mitochondrial DNA, while the second tree is based on a combination of primary fossil and on secondary calibrations taken from a nuclear tree using mitochondrial DNA (two-step protocol). Focusing on a set of nodes that are most likely explained by vicariance, we statistically compare the reconstructed alternative time trees by applying a biogeographical plausibility test. Our two-step protocol outperformed the alternative approach in terms of spatial and temporal plausibility. It allows us to infer scenarios for Mediterranean amphibian evolution in eight geographic provinces. We identified several tectonic and climatic events explaining the majority of Mediterranean amphibian divergences, with Plio-Pleistocene climatic fluctuations being the dominant driver for intrageneric evolution. However, often more than one event could be invoked for a specific split. We give recommendations for the use of secondary calibrations in future molecular clock analyses at the community level.
Comparative molecular studies emphasized a new biogeographic paradigm for the terrestrial fauna of North Africa, one of the last uncharted ecoregions of the Western Palearctic: two independent east-west divisions across the Maghreb. Through a comprehensive phylogeography, we assessed how this model suits the genetic diversification documented for the tree frog Hyla meridionalis sensu lato. Analyses of mtDNA variation and thousands of nuclear loci confirmed the old split (low-Pliocene) between Tunisian and Algerian populations. These lineages meet but barely admix in the eastern Maghreb (Algerian-Tunisian border), a sign of putatively advanced reproductive isolation. In the western Maghreb, we report a Pleistocene divergence between Moroccan and Algerian populations. Tree frogs thus follow both predictions: a double east-west break that gave rise to two suture zones characteristic of North-African phylogeography. Moreover, some intraspecific mtDNA variation is not mirrored by the nuclear data, emphasizing that evolutionary units should always be designated by multilocus approaches. Last but not least, we describe the Tunisian lineage as a new species endemic to Africa.
The Balkan Peninsula constitutes a biodiversity hotspot with high levels of species richness and endemism. The complex geological history of the Balkans in conjunction with the climate evolution are hypothesized as the main drivers generating this biodiversity. We investigated the phylogeography, historical demography, and population structure of closely related wall-lizard species from the Balkan Peninsula and southeastern Europe to better understand diversification processes of species with limited dispersal ability, from Late Miocene to the Holocene. We used several analytical methods integrating genome-wide SNPs (ddRADseq), microsatellites, mitochondrial and nuclear DNA data, as well as species distribution modelling. Phylogenomic analysis resulted in a completely resolved species level phylogeny, population level analyses confirmed the existence of at least two cryptic evolutionary lineages and extensive within species genetic structuring. Divergence time estimations indicated that the Messinian Salinity Crisis played a key role in shaping patterns of species divergence, whereas intraspecific genetic structuring was mainly driven by Pliocene tectonic events and Quaternary climatic oscillations. The present work highlights the effectiveness of utilizing multiple methods and data types coupled with extensive geographic sampling to uncover the evolutionary processes that shaped the species over space and time.