DNA Barcodes Reveal 68 Amphibian and Reptile Species in Georgia and Expose Taxonomic Inflation Across the Caucasus

A landmark taxonomic audit has delivered the most transparent picture yet of Georgia’s herpetofauna, cataloguing 12 amphibian and 56 reptile species while simultaneously challenging decades of species descriptions across the Caucasus Biodiversity Hotspot. The new annotated checklist, published in the journal Caucasiana, pairs traditional faunistic synthesis with DNA barcoding data generated through the Caucasus Barcoding of Life (CaBOL) project, and it arrives at a moment when the very definition of what constitutes a species in the region has become a matter of heated scientific debate.

The checklist is far more than a simple species tally. For each taxon, the authors provide scientific and vernacular names in English and Georgian, historical synonymy, nomenclatural sources, infraspecific variation, and critically, a biological validation status that distinguishes between biologically confirmed species, candidate species whose status requires further validation, and rejected taxa. This three-tier framework represents a deliberate departure from recent regional treatments that have elevated geographically distinct populations to species rank based primarily on mitochondrial divergence, a practice the authors argue has contributed to taxonomic inflation across the Caucasus and beyond.

At the heart of the paper lies a methodological argument grounded in the Integrative Species Concept introduced by De Queiroz in 2007, which considers species as taxonomic units with separate evolutionary pathways validated through differences at multiple independent loci. The authors, drawing on Wüster’s 2025 review, distinguish between a critical approach that disqualifies taxonomic status when firm evidence shows no species-level differences, and a confirmatory approach that merely notes insufficient support without rejecting a hypothesis outright. They argue that most recent species descriptions have used molecular and morphological data to confirm rather than to critically test, and that the decisive test—whether phenotypic or genomic markers show a stepped cline rather than gradual change across contact zones—has rarely been performed for Caucasian taxa.

The DNA barcoding component of the study is technically rigorous. Tissue samples were processed using Quick-DNA miniprep Plus Kits, and partial sequences of cytochrome oxidase subunit I (COI) were amplified by polymerase chain reaction using the primer pair LCOI490-JJ and HCO2198-JJ. Thermal cycling involved a two-stage touchdown protocol: an initial 15-cycle set with annealing temperatures decreasing from 55°C by 1°C per cycle, followed by a 25-cycle set at a constant 45°C annealing temperature. Sequencing was conducted at the Beijing Genomics Institute in Hong Kong, with extracted DNA deposited in the scientific collections of Ilia State University in Tbilisi and aliquots destined for the LIB Biobank at Museum Koenig in Bonn, Germany. The resulting sequences, ranging from 500 to 658 base pairs, were submitted to the Barcode of Life Data System (BOLD) and compared against both BOLD and NCBI databases to identify Barcode Index Numbers (BINs) and nearest neighbours.

The results reveal striking inconsistencies in how genetic distance maps onto taxonomic status. Among nominally conspecific individuals from different geographic areas, COI differences reach 8 to 12 percent in several cases. Sequences of the Caucasian Salamander (Mertensiella caucasica) from central Georgia and northeastern Türkiye differ by 11.9 percent, aligning with earlier findings that identified an undescribed but genetically isolated evolutionary lineage in the Borjomi Gorge. The steppe-runner lizard (Eremias velox) from Georgia and western China differs by 8.4 percent, while the snake-eyed lizard (Ophisops elegans) from Georgia and Türkiye differs by 11.9 percent. These profound infraspecific divergences suggest possible genetic isolation between formally conspecific populations and may warrant future taxonomic revision.

Conversely, some nominally different species show remarkably little genetic separation. The Caucasian Toad (Bufo verrucosissimus) from Georgia differs from the nominal Common Toad (Bufo bufo) from Central Europe by less than 3 percent in COI, and mitochondrial Cytochrome b differences are below 2 percent. The European Marsh Turtle (Emys orbicularis) from Georgia and E. trinacris from Sicily differ by only 1.6 percent. The Eastern Treefrog (Hyla orientalis) from Georgia and H. molleri from the Iberian Peninsula, despite a broad geographic gap, reach only 4.3 percent divergence. The steppe viper (Vipera renardi eriwanensis) from Georgia and V. renardi from Central Asia differ by just 1.8 percent, leading the authors to treat the Georgian form as a subspecies rather than a separate species.

The checklist documents several biologically confirmed species with deep evolutionary histories. The Caucasian Salamander is separated from its closest relative, Chioglossa lusitanica of the Iberian Peninsula, by an estimated 40 to 70 million years of divergence, making it an Eocene relict endemic to the western Lesser Caucasus. The Caucasian Parsley Frog (Pelodytes caucasicus) diverged from its relatives since the Eocene and is endemic to the western Caucasus ecoregion. The Northern Banded Newt (Ommatotriton ophryticus) shows no introgression with its closest relative based on genomic data analysis, and the Eastern Crested Newt (Triturus karelinii) builds a monophyletic group with two allopatric congeners separated by a narrow hybrid zone.

The paper also addresses hybrid parthenogenesis among Caucasian rock lizards of the genus Darevskia, a phenomenon first described by Ilya Darevsky in the 1960s. The parthenogenetic forms D. armeniaca and D. dahli are hybrid origin species, with D. armeniaca supposedly arising from males of D. valentini and females of D. mixta, and D. dahli from D. mixta and D. portschinskii. The mitochondrial genomes of these parthenogens are shared with their matrilineal ancestors, explaining the very high sequence similarity among these species in the barcode data. The authors note that D. alpina may itself be a hybrid species between the caucasica and saxicola complexes.

Among the candidate species flagged for further validation are the Caucasian Smooth Newt (Lissotriton lantzi), which represents the oldest isolated lineage within the L. vulgaris complex but whose isolation time of approximately 6.4 million years may not suffice for postzygotic isolation; the rock lizard Darevskia obscura, elevated from within the D. rudis complex based primarily on mitochondrial phylogeny without evidence for or against gene flow across its contact zone with D. rudis; and several vipers within the Vipera kaznakovi group, including V. dinniki and V. darevskii, whose genetic differences from V. kaznakovi are modest and whose contact zones remain insufficiently studied with nuclear genomic data.

The authors conclude that the presented species list is not final and that nomenclature will likely change as genomic and population genetic studies either confirm or reject the true species status of formally described taxa. They emphasize that both under-describing and over-describing species diversity can be counterproductive from a conservation standpoint. While bureaucratic bodies prefer to include species rather than infraspecific units in conservation lists, ill-founded taxonomic inflation compromises species integrity and diverts attention from widespread taxa that remain unprotected across the most important parts of their range. The authors highlight a paradox: subspecies of several widespread amphibians and reptiles included in Annex II of the Bern Convention were re-described as separate species in recent decades, and as a result, none of those Caucasian forms are now prioritized in conservation programs supported by the European Commission because they no longer appear in the relevant annex.

Subject of Research: An annotated checklist of Georgian amphibians and reptiles with DNA barcoding data and critical taxonomic evaluation

Article Title: An Annotated Checklist of the Amphibians and Reptiles of Georgia with DNA Barcoding Data and Taxonomic Remarks

Article References: Tarkhnishvili, D., Iankoshvili, G., Seropian, A., Kachlishvili, N., & Todua, M. (2026). An Annotated Checklist of the Amphibians and Reptiles of Georgia with DNA Barcoding Data and Taxonomic Remarks. Caucasiana, 5, 59-138. https://doi.org/10.3897/caucasiana.5.e189214

Image Credits: AI Generated

DOI: 10.3897/caucasiana.5.e189214

Keywords: Georgia, Caucasus, herpetology, DNA barcoding, taxonomy, biodiversity, Annotated, Checklist, Amphibians, Reptiles, Barcoding, Data