Bombina variegata rhodopensis Lukanov, Denoël, Jablonski & Dufresnes, 2025

Dufresnes, Christophe, Lukanov, Simeon, Gippner, Sven, Ambu, Johanna, Strachinis, Ilias, Arsovski, Dragan, Monod-Broca, Benjamin, Cayuela, Hugo, Lymberakis, Petros, Canestrelli, Daniele, Cogălniceanu, Dan, Poyarkov, Nikolay A., Litvinchuk, Spartak N., Suchan, Tomasz, Denoël, Mathieu & Jablonski, Daniel, 2025, Historical biogeography and systematics of yellow-bellied toads (Bombina variegata), with the description of a new subspecies from the Balkans, Vertebrate Zoology 75, pp. 1-30 : 1-30

Bombina variegata rhodopensis Lukanov, Denoël, Jablonski & Dufresnes subsp. nov.

Identity.

Previously identified as a divergent mitochondrial (cyt b) lineage, attributed to the subspecies Bombina variegata scabra by Hofman et al. (2007) and Fijarczyk et al. (2011) and labeled as the “ Rhodopean ”, “ R ” or “ RD ”, in reference to its distribution in the Rhodope Mountains of southeastern Bulgaria and northeastern Greece. This new taxon corresponds to the Rhodope lineage (mtDNA R) that is purple-coded in our figures. Given its level of divergence (see Discussion), we describe this lineage as a new subspecies, Bombina variegata rhodopensis subsp. nov.

Holotype.

BG-IBER-VER-000010561 , adult male collected on 23 April 2024 by SL, MD, DJ and CD in a water fountain at the northeastern exit of Kostino , Kardzhali Municipality, Kardzhali Province, Bulgaria (41.7039 ° N, 25.3028 ° E; elevation: 563 m a. s. l.), and curated at the Institute of Biodiversity and Ecosystem Research of the Bulgarian Academy of Sciences ( IBER-BAS), Sofia, Bulgaria . Measurements in mm: L. 42.7, F. 16.3, T. 17.3, Lt. c. 12.8, Sp. p. 4.8, L. o. 4.4, Sp. n. 2.4. The holotype and the type locality are depicted on Fig. 10 .

Paratypes.

BG-NMNHS-HER-000000000552 (L. 41.6 mm), adult male collected on 23 April 2024 by SL, MD, DJ and CD at the type locality and curated at the National Museum of Natural History of the Bulgarian Academy of Sciences ( NMNHS-BAS), Sofia, Bulgaria ; MNHN -RA-2024.0001 (L. 46.0 mm), adult female collected on 24 April 2024 by SL, MD, DJ and CD in Lisitsite , Kardzhali Municipality, Kardzhali Province, Bulgaria (41.6108 ° N, 25.4543 ° E; elevation: 233 m a. s. l.), and curated at MNHN . NHMW 41962 (L. 39.7 mm), adult male collected on 24 April 2024 by SL, MD, DJ and CD in Panichkovo , Chernoochene Municipality, Kardzhali Province, Bulgaria (41.8565 ° N, 25.1517 ° E; elevation: 753 m a. s. l.) and curated at NHMW . These three specimens are depicted in Fig. 11 .

Diagnosis.

General characteristics similar to those of the yellow-bellied toad B. variegata . It is the sister taxon of the Balkan subspecies B. v. scabra , from which it is distinguished by substantial mitochondrial, nuclear (especially phylogenomic) and morphological divergence. Specifically, B. v. rhodopensis subsp. nov. differs from B. v. scabra by 1.3 % of sequence divergence at 16 S, 1.6 % of sequence divergence at cox 1, and 2.4 % of sequence divergence at cyt b (Table 1 ). Based on the mitochondrial time tree, the two taxa initiated their divergence during the Early Pleistocene, either around 2.1 or 1.4 Mya, depending on the calibration (Fig. 2 ). On the cyt b gene, the following nucleotides distinguish B. v. rhodopensis subsp. nov. from any other B. variegata subspecies: a “ G ” in the site 469, a “ G ” in the site 726 and an “ A ” in the site 801 (positions relative to the full gene sequence). The new subspecies also features a unique combination of alleles at the nuclear genes ncx 1, rag- 1, rag- 2 and rho (Figs 3 , S 3), as well as at 4759 RAD loci (Fig. 4 ). Externally, B. v. rhodopensis subsp. nov. differs from B. v. scabra by having on average a shorter femur, tibia, and first toe, as well as bigger eyes, noting that these rely on a small sample of B. v. rhodopensis subsp. nov. specimens (Table 2 ). Body sizes are on average similar (43.6 mm in B. v. rhodopensis subsp. nov. vs. 43.3 mm in B. v. scabra ), with higher variation between the sexes in B. v. rhodopensis subsp. nov. (Fig. 5 ). From the examined specimens, the sexes appear morphologically dimorphic (Table 2 , Fig. 5 ), as also seen in B. v. scabra ( Radojičić et al. 2002; Fig. 5 ). Compared to B. v. scabra , B. v. rhodopensis subsp. nov. features a higher average proportion of yellow vs. dark coloration on the ventral side, although with wide overlap (Fig. 7 ).

Etymology.

The name rhodopensis is a Latin toponymic adjective given in reference to the Rhodope Mountains in the southeastern part of the Balkan Peninsula ( Bulgaria and Greece) where the new taxon is mostly distributed. It spotlights a rare case of Rhodope endemism in vertebrates – Rhodope endemics are so far known only from plants and invertebrates.

Vernacular names.

Rhodope yellow-bellied toad (English), Родопска жълтокоремна бумка (Bulgarian), Κιτρινομπομπίνα της Ροδόπης (Greek), Rodop Sarılı Kurbağa (Turkish), Sonneur à ventre jaune des Rhodopes (French), Kunka žltobruchá rodopská (Slovak).

Distribution.

Bombina v. rhodopensis subsp. nov. is essentially restricted to the Rhodope Mountains and their foothills (from sea level up to 1600 m a. s. l.) in southeastern Bulgaria, northeastern Greece, and the adjacent part of Turkish Thrace ( Global Biodiversity Information Facility 2024). In Greece, it was documented eastward up to the National Forest Park of Dadia – Lefkimi – Soufli close to Evros (Maritsa) River which makes the border with Turkey ( Petrov 2004; Valakos et al. 2008; Kret and Poirazidis 2015; Pafilis and Maragkou 2020; Strachinis 2024). In Bulgaria, population isolates exist east of the Rhodopes, namely in Sakar Mountain and perhaps Strandzha Mountain near the Black Sea ( Boev et al. 2008; Stojanov et al. 2011). In Turkey, it is very rare and restricted to Karacahasan Mountain (Enez District), close to the Evros River ( Kariş et al. 2017). The eastern and northern margins of B. v. rhodopensis subsp. nov. correspond to the shifts from forest hills towards open lowland habitats colonized by the fire-bellied toad ( B. bombina ), where they probably form hybrid zones. The southern boundary of the range follows the coastal foothills of the Rhodopes (Valakos et al. 2008). According to the mtDNA barcoding, the transition with B. v. scabra in the west might follow the Nestos (Mesta) River valley, which separates the Rhodopes from the Rila massif in the north, and from the Pirin massif in the west; the mtDNA of both subspecies were reported in the middle part of the Nestos River in Greece (Platanias-Pteleas). Nevertheless, many sampling gaps remain in the Pirin, western Rhodopes and southern Rila, so the exact subspecies boundaries shall be fine-tuned by multilocus genotyping.

Natural history.

The new subspecies inhabits similar habitats as B. v. scabra , being found in various aquatic sites such as mountain brooks, rivers, ponds, natural and artificial lakes or water-filled ruts and puddles ( Petrov 2004). The type series was found in drinking throughs, a valuable habitat for this subspecies as it is for B. v. scabra in northwestern Greece ( Denoël 2004). The breeding season starts in March and lasts until late July. Reports of several thousand eggs laid at the bottom of a slow-flowing stream in Eastern Rhodopes suggested possibly up to 200 per female ( Stojanov et al. 2011). Toads are most active during the day and at dusk, and hibernation occurs on land. Diet studies indicate that Bulgarian populations are mostly insectivorous, preferring water beetles and winged insects, with arachnids and snails having a minor share ( Donev 1984). Bombina v. rhodopensis subsp. nov. displays the anti-predator defense posture (“ Unkenreflex ”), well-known in Bombina toads (Baijger 1980), and which we could observe in Bulgaria (see also Kariş et al. 2017 for Turkey).

Conservation.

Bombina v. rhodopensis subsp. nov. was reported widespread over the eastern Rhodopes of Greece and Bulgaria ( Petrov 2004), but due to its much smaller distribution than that of B. v. scabra , it may be more vulnerable. In Greece, it is abundant above 200 m ( Strachinis 2024). In Turkey, it subsists only in a few isolated localities with potentially small population sizes (e. g., Kariş et al. 2017), and any potential pressure (habitat change, collection) might threaten its persistence in the country. Informed conservation management would benefit from assessing the continuity of the distribution of B. v. rhodopensis subsp. nov. across its range, in respect to known occurrence data (e. g., Valakos et al. 2008; Global Biodiversity Information Facility 2024) and from quantifying habitat loss and threats. As B. variegata is listed in the European Union Habitats Directive Annex 2 (= “ Natura 2000 ”), dedicated protected areas could be declared for the conservation of B. v. rhodopensis subsp. nov.

Figures

Figure 2. Bayesian phylogeny obtained with BEAST based on full mitogenomes (17,240 kb). Nodes are annotated with dating estimates and their 95 % HPD (node bars) obtained from two calibrations (dark grey: calibration I; light grey: calibration II). Sequence labels indicate accession numbers, geographic origins, taxa and mitochondrial lineages. Asterisks denote full branch support (posterior probabilities of 1.0).

Figure 3. Cloudogram of the nuclear species trees obtained with * BEAST based on four gene fragments (3715 bp) and distinguishing samples representative of the different B. variegata mitochondrial lineages. Node labels indicate Bayesian posterior probabilities; sequence labels indicate taxa and mitochondrial lineages; the thick and thin lines show the average tree root and all the sampled trees, respectively.

Figure 4. Maximum-likelihood phylogeny obtained with IQ-TREE of 4759 concatenated RAD loci (553,608 bp). Colors distinguish the different B. variegata mitochondrial lineages. Sequence labels indicate sample names, geographic origins, taxa and mitochondrial lineages. Asterisks denote robust branch support (bootstrap > 95). The tree is rooted by B. bombina.

Figure 5. Morphological comparison of specimens attributed to the two lineages of B. v. scabra. A PCA on 11 characters corrected by body length distinguishing the males (triangles) and females (circles) of each lineage; B boxplots showing variation in body length (L.) among sexes and lineages. For the list of characters, see Methods (section Morphometric analyses in Bombina variegata scabra).

Figure 7. Proportion of color / dark coloration on the ventral side of specimens previously attributed to B. v. scabra. Bracket indicates significant difference (Kruskal-Wallis test) at P < 0.05. The body area analyzed is emphasized in the two colored (top) and dark (bottom) examples.

Figure 10. The holotype and type locality of Bombina variegata rhodopensis subsp. nov. Credit: CD (top and middle) and DJ (bottom).

Figure 11. The paratypes of Bombina variegata rhodopensis subsp. nov. Credit: CD.