DNA barcoding is a molecular identification technique that uses a short, standardized fragment of DNA as a species label, much like a universal library card for life. For animals, the standard barcode is a 648-base-pair fragment of the mitochondrial gene Cytochrome C Oxidase Subunit I, known simply as COI. Because COI evolves quickly enough to differ between closely related species while remaining recognizable within species, comparing a specimen’s sequence against a curated reference database allows researchers to identify known species and flag candidates for new ones. The approach has proven highly efficient and reliable across many animal groups and is regularly used for biodiversity assessments, conservation planning, and species discovery.

The choice of study area matters. The Caucasus region is recognized as one of the 36 global biodiversity hotspots, and Rize sits within a spectacularly rich corner of it. Cool summers, mild winters, and rain throughout the year sustain lush forests and endemic fauna and flora, and some habitats—particularly in the Kaçkar Mountains—are included among protected ecoregions declared by the IUCN. Remarkably, Rize province hosts approximately 25 percent of the amphibian species distributed throughout Türkiye, yet barcoding studies for vertebrate taxa in the Caucasus remain scarce, and much of the region’s biodiversity data is outdated and incomplete. The new study set out to fill part of that gap by sampling 16 individuals from different localities across Rize.

The technical workflow followed the standard barcoding playbook. During fieldwork, the team collected small muscle tissue samples by toe-clipping and preserved them in 95 percent alcohol. Total DNA was extracted using a commercial isolation kit, and a universal amphibian primer mix designed by Che and colleagues in 2012 was used to amplify the COI fragment by polymerase chain reaction. The reaction protocol involved an initial denaturation at 95 degrees Celsius, 36 amplification cycles, and a final extension, with amplicons confirmed by agarose gel electrophoresis. Sequences were checked and edited in the software BioEdit and then submitted to BOLD, the open-access repository that supports the recording, storage, and publication of barcoded DNA from around the world.

To infer species boundaries from the sequences, the researchers deployed several complementary computational methods. The BOLD platform’s Refined Single Linkage (RESL) analysis, a stepwise clustering process that assigns records to operational taxonomic units, produced four distinct barcode index numbers belonging to members of three different families. Two additional delimitation tests—Assemble Species by Automatic Partitioning (ASAP), which uses hierarchical clustering of pairwise genetic distances, and the Automatic Barcode Gap Discovery (ABGD) method, which searches for a threshold distance separating intra- from interspecific divergence—both yielded a more conservative partition of three OTUs. The barcode gap distance in the ABGD analysis was 0.12, and the sequence partition with the lowest ASAP score distributed the sequences into three OTUs with a threshold distance value of approximately 0.116. Pairwise p-distances among species and genera were calculated in MEGA X, and haplotype networks were reconstructed to visualize the genetic relationships among localities.

The toad results proved the most genetically intriguing. Specimens of Bufo from the Çat plateau in the Kaçkar Mountains of Çamlıhemşin and from the İyidere region were assigned different BIN numbers in BOLD, indicating the existence of two distinct mitochondrial lineages. The Çat specimens clustered with material from Georgia and were identified in the BOLD identification system as B. verrucosissimus, a close relative of the common toad affiliated with South Caucasian populations. For this lineage, genetic distances between specimens from Türkiye, Georgia, and Russia were low—0.017 between Rize and Russia and 0.003 between Türkiye and Georgia. The İyidere sequences, by contrast, were assigned to B. bufo, and within this lineage the mean genetic distance was 0.038, ranging from 0.03 to 0.05, with a distance of 0.03 in all cases between İyidere specimens and BOLD records from Ukraine, Russia, Austria, Germany, and Norway.

Haplotype network analysis added nuance to the toad picture. Although the İyidere and Çamlıhemşin specimens showed moderate genetic differentiation, they appeared in the same position on the network, and haplotypes of specimens from Georgia and Türkiye sit close together. The researchers found 17 haplotypes across the two mitochondrial lineages of B. bufo when combining their data with BOLD records. This discordance—moderate sequence divergence but shared haplotype placement—echoes earlier findings that mitochondrial DNA tends to oversplit species in the Bufo species complex, particularly in hybrid zones, a phenomenon attributed to asymmetric mitochondrial introgression. Previous work estimated genetic differences between the Çamlıhemşin and İyidere toads at 0.023 to 0.026 using the Cytochrome b gene; the COI marker used here revealed an even higher distance of 0.038, consistent with the observation that mitochondrial markers yield more variable results than nuclear DNA in this group.

The treefrogs and wood frogs told cleaner stories. Within Rana macrocnemis, genetic distances ranged from 0.018 to 0.020: 0.018 between the Çamlıhemşin population and records from Kars and Hakkari in Türkiye, and 0.020 between Çamlıhemşin and Armenia. Notably, the Çamlıhemşin population shared no haplotypes with any other locality. Genetic divergence in this species is attributed to the last glaciation period, with gene flow among populations restricted by physical barriers and climate, and the new distances are higher than previously estimated values of 0.002 to 0.016 within Türkiye. In Hyla orientalis, distances ranged from 0.01 to 0.02—0.01 between Rize and Azerbaijan, 0.02 between Rize and Kastamonu, and 0.01 between Rize and Ukraine—showing that Rize treefrogs are genetically more similar to specimens from Ukraine and Azerbaijan than to those from Kastamonu, despite the latter being within the same country. No shared haplotypes were found among the localities, and the low mitochondrial divergence contrasts with the high genetic differentiation this species shows using nuclear markers in the Caucasian region.

The study’s broader significance lies in what it contributes to the global reference infrastructure. All generated sequences were submitted to BOLD, with the majority representing the first barcoding data from Rize in the database, and the work presents the initial barcoding data for two mitochondrial lineages of Bufo bufo from Türkiye. The authors emphasize that BOLD’s coverage remains incomplete—limited coverage of barcode variation or entirely missing species can hinder accurate identification—and that interpreting results can be challenging when reference data are sparse. Researchers have repeatedly recommended more local efforts to enrich the global list of barcoded species, and this study answers that call for a province whose amphibian fauna had never been barcoded before.

DNA barcoding is a vital tool for biodiversity research and conservation planning, offering promise for rapid species identification and biodiversity assessments in hotspot areas like the Caucasus. By revealing four distinct barcode index numbers among three familiar frog species and documenting genetic distances that hint at hidden lineages, the Rize study demonstrates that even well-known European amphibians can harbor unexpected genetic diversity in mountain refugia. The findings indicate that COI sequencing is a useful tool for identifying the anuran species present in Rize, and the authors encourage extending DNA barcoding to monitor genetic diversity and resources across a wider geographic range and in specimens throughout Türkiye—turning a short stretch of mitochondrial DNA into a map of the hidden connections linking Black Sea frogs to their cousins across the Caucasus and beyond.

Subject of Research: DNA barcoding of anuran amphibians from Rize province, Türkiye, using the COI gene marker

Article Title: DNA barcoding of some Anura species from Rize

Article References: Gür, B., & Kalaycı, T. E. (2025). DNA barcoding of some Anura species from Rize. Caucasiana, 4, 15-24. https://doi.org/10.3897/caucasiana.4.e144670

Image Credits: AI Generated

DOI: 10.3897/caucasiana.4.e144670

Keywords: DNA barcoding, COI gene, Anura, Bufo bufo, Hyla orientalis, Rana macrocnemis, Rize, Caucasus biodiversity hotspot, BOLD database, amphibians, Türkiye, species delimitation