and the adjacent regions =, CAUCASIANA Journal on the biodiversity of the Caucasus Caucasiana 3: 31-39 (2024) DOI: 10.3897/caucasiana.3.e117215 Research Article Which species of water frogs inhabit ponds of the Caucasus? Taxonomic mess with Pelophylax ridibundus species complex David Tarkhnishvili'®, Mariami Todua™, Giorgi lankoshvili'® 1 Institute of Ecology, Ilia State University, Tbilisi, Georgia Corresponding author: David Tarkhnishvili (david_tarkhnishvili@iliauni.edu.ge) OPEN Qrceess Academic editor: Levan Mumladze Received: 11 December 2023 Accepted: 20 February 2024 Published: 21 March 2024 ZooBank: https://zoobank. org/20B68FF3-5CD7-492A-A488- 8EB6CC881651 Citation: Tarkhnishvili D, Todua M, lankoshvili G (2024) Which species of water frogs inhabit ponds of the Caucasus? Taxonomic mess with Pelophylax ridibundus species complex. Caucasiana 3: 31-39. https://doi.org/10.3897/ caucasiana.3.e11/215 Copyright: © Tarkhnishvili et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Two nominal species of water frogs of the genus Pelophylax, P. ridibundus and P. bedria- gae, are found in Central and Eastern Europe, the Middle East, Western Asia, Western Ka- zakhstan, and Siberia. So far, the taxonomic status of Pelophylax in most of the Caucasus has remained unknown. Sequencing of the Cytochrome Oxidase 1 mitochondrial gene attributed the frogs throughout Georgia to the P. ‘bedriagae’ lineage, bringing them very close to the specimens from Kazakhstan and Greece. Simultaneously, the current nomen- clature of water frogs appears to be formally incorrect since western Kazakhstan, the type locality for P. ridibundus, has frogs genetically closer to nominal P. bedriagae, than to nom- inal P. ridibundus from Europe. Because there is no evidence that the frogs from Central Europe, Kazakhstan, and the Caucasus, as well as from Anatolia and Iran, are biological species with individual evolutionary pathways, we suggest a conservative approach and synonymize nominal P. bedriagae from most of West Asia with P. ridibundus. Key words: mitochondrial DNA, Pelophylax ridibundus, Pelophylax bedriagae, water frog taxonomy Introduction During most of the XX century, the Western Palaearctic water frogs were at- tributed to two species: small-bodied Rana lessonae from Western Europe and large-bodied Rana ridibunda from Central and Eastern Europe, Central and Western Asia, and Northern Africa; the hybrid form between R. ridibunda and R. lessonae, R. esculenta, was originally described as a separate species (Hell- mich 1962; Bannikov et al. 1977; Kuzmin 1999). Based on the analysis of en- zyme profiles, Berger (1983) suggested the presence of three other species of the group, R. perezi from the Iberian Peninsula and two species with a limit- ed distribution — from Italy and Greece. The taxonomic revolution exploded in the early 1990s. First, the polyphyletic origin of the nominal genus Rana was shown (Wiens et al. 2009), and Palaearctic water frogs were attributed to the genus name Pelophylax; hence, R. ridibunda was renamed Pelophylax ridibun- dus. Then, the nominal P. ridibundus has been split into several geographically distinct taxa. Schneider et al. (1992) suggested a species status for frogs from West Asia and named it Pelophylax bedriagae (see also Sinsch and Schneider 1999). Plotner et al. (2001) suggested the presence of two additional cryptic 31 Tarkhnishvili et al.: Water frogs from Georgia species in Anatolia and the Middle East, P caralitanus and P. cypriensis, al- though the former one was recently synonymized with P. bedriagae (Sinsch et al. 2023). Later, a few other species were described, including R. saharica, synonymized with R. perezi (Steinwarz and Schneider 1991; see the recent dis- cussion in Dufresnes et al. (2024)). Materials and methods Samples (toe phalanges) of frogs were collected from five Georgian locations (Fig. 1), representing different parts of the country, from both the Black and Caspian Sea basins and the Greater and Lesser Caucasus Mountain systems. Tissue samples were stored in 95% alcohol for further DNA extraction. DNA analyzes DNA purification was processed using the Quick-DNA Miniprep Plus Kit (Zymo Research). Partial sequences of cytochrome oxidase subunit | (COI) were am- plified by polymerase chain reaction (PCR) using the primer pairs LCOI490-JJ and HCO2198-JJ (Astrin and Stiben 2008). Thermal conditions included dena- turation at 95 °C for 1 min, followed by the first cycle set (15 cycles): 94 °C for 30 s, annealing at 55 °C for 1 min (—1 °C per cycle), and extension at 72 °C for 1:30 min. Second cycle set (25 cycles): 94 °C for 35 s, 45 °C for 1 min, 72 °C for 1:30 min, followed by 1 cycle at 72 °C for 3 min, and the final extension step at 72 °C for 5 min. PCR amplicons were visualized on 1% agarose gels using 1.7 ul of PCR product. The unpurified PCR products were sequenced in both directions at the Beijing Genomics Institute (Hong Kong, CN) using the amplification prim- ers. Sequence analysis was performed using Geneious Prime 2022.1.1 (http:// www.geneious.com). Extracted DNA was deposited in the scientific collections of Ilia State University, Tbilisi, Georgia, and aliquots will be deposited at the LIB - . i : sg E ORG Churia G Figures 1. Sampling locations of Pelophylax sp. in Georgia. Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 32 Tarkhnishvili et al.: Water frogs from Georgia Biobank at Museum Koenig, Bonn, Germany. At the same time, the sequences have been submitted to Barcode of Life Data System (BOLD) databases. The newly obtained DNA barcodes of COI sequences were checked out against the BOLD systems and BLAST database (http://www.boldsystems.org/, https:// blast.ncbi.nlm.nih.gov/Blast.cgi). In the analysis, we used six obtained CO1 mitochondrial gene sequences of Pelophylax from throughout Georgia and 89 sequences of water frogs down- loaded from NCBI GenBank (Benson et al. 2012) from Turkey, Kazakhstan, the Balkan Peninsula, Central, and Western Europe that belong to the nominal spe- cies Pelophylax ridibundus, P. bedriagae, P. kurtmuelleri, P. caralitanus, P. pe- rezi, P. lessonae, P. mongolius, and a few non-identified Pelophylax. The access numbers, origin of the samples, and nominal species names are shown in the Suppl. material 1. Results Pairwise genetic distances between Georgian samples and green frogs from throughout West Eurasia, based on the analysis of Cytochrome Oxidase 1 sequences (COl), are shown in Suppl. material 2. The distances between the 545 bp long fragment of COI of samples from Georgia and Kazakhstan vary between 0-4% (3.4%), between samples from Georgia and Greece — between 1-5.5% (2.0%), between Georgia and Central Europe (excluding P. lessonae, but including nominal P. kurtmuelleri) — 5-6% (5.7%) and between Georgian sam- ples and P. caralitanus — 1-1.5%. The differences between the nominal P. ridi- bundus and P. bedriagae on one side and P. lessonae — 14-15%, between the nominal R. ridibundus and R. perezi — 15 %. In general, sequence differences between geographic populations of the nominal P. ridibundus + P. bedriagae groups vary between 0-6% and are proportional to the geographic distance be- tween the populations. In contrast, sequence divergence between this group and P. lessonae, as well as P. perezi and P. mongolius, is qualitatively higher and usually exceeds 14%. The Maximum Likelihood tree of the obtained haplotypes is shown in Fig. 2. The Georgian mitochondrial sequences of the nominal P. ridibundus belong to a clade shared with the nominal P. ridibundus from Kazakhstan and the nominal P. bedriagae from Greece. The differences between the haplotypes of frogs from Georgia are minor but still exceed those between some individuals from Georgia and Kazakhstan and Georgia and Greece. In general, based on the sim- ilarity of mitochondrial haplotypes, there is no evidence of the non-conspecific status of frogs from the entire Caucasus, west and north of the Caspian Sea Basin, Anatolia, and the Balkan Peninsula. Discussion Several publications tried to infer the phylogeny of Pelophylax, based on the analysis of mitochondrial genes (Plétner et al. 2001; Lymberakis et al. 2007; Akin et al. 2010; Hofman et al. 2016; Dufresnes et al. 2017, 2024; Ualiyeva et al. 2022; Papezik et al. 2023). There is a consensus suggesting monophyly of a widespread subclade including nominal P. ridibundus, P. bedriagae, P. kurt- muelleri, P. caralitanus, P. cerigensis, and P. cypriensis (although the separate Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 33 Tarkhnishvili et al.: Water frogs from Georgia Kazakhstan Kazakhstan 1 Balkans 88 Central Europe 85 804 Kazakhstan2 - P. caralitanus (Anatolia) Anatolia 99,-Kazakhstan ON796912 | 96/9,Greece MT881774 Greece Western Georgia ‘||! Skuri CaBOL1027452 Tabatskuri CaBOL1007162 ca| 91 Kazakhstan ON7eones Eastern Georgia ON796820 ON /96894 Anaklia CaBOL1027451 7g Kazakhstan ON796845 Tetrobi CaBOL1027454 Kazakhstan ON79697_ 90 Tabatskuri CaBOL100716: Kazakhstan ON796829 66)Chili Lake CaBOL1027453 Kazakhstan ON 72683) Kazakhstan ON796894 Greece MT881771 72 Central Europe 79 87 B lessonae MN993180 P. bergeri MK014013 98 P. lessonae P. perezi MK14019 994 P. mongolius : Figures 2. Maximum Likelihood tree of water frogs from Europe and West Asia (see also Suppl. material 1), including six samples from Georgia. Bootstrap values are shown at the nodes. species status of P caralitanus and P. cerigensis is not supported by genet- ic data - see Limberakis et al. (2007), Akin et al. (2010), Sinsch et al. (2023) and Dufresnes et al. (2024)). The phylogenetic pattern within this subclade de- pends on the sampling areas. Dufresnes et al. (2017) suggested that P. bedria- gae and P. ridibundus/P. kurtmuelleri are sister, reciprocally monophyletic taxa. Their results are in line with studies by Lymberakis et al. (2007) and Papezik et al. (2023), although the latter authors suggested the ingroup status for P Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 34 Tarkhnishvili et al.: Water frogs from Georgia cypriensis within this subclade. However, these findings contradict the results of Plotner et al. (2001) and Ualiyeva et al. (2022), who rejected the reciprocal monophyly of P. ridibundus and P. bedriagae. The situation with these two species remained unclear. There are no fixed morphological characters that could distinguish between P. ridibundus and P. bedriagae (Sinsch and Schneider 1999), but there are obvious differences be- tween mating calls of water frogs from Europe and the Middle East, including southern Anatolia (Schneider et al. 1992). Mating call analysis of frogs from Armenia and Kazakhstan placed them closer to European P. ridibundus than to Middle Eastern P. bedriagae (Schneider and Sinsch 1999). The mitochondrial haplogroup of the frogs from the Levant does not cluster with those from most of Anatolia, and it has a sister status to the clade found throughout Europe, most of Anatolia, and the Caspian area (Plotner et al. 2001; Ualiyeva et al. 2022). Simultaneously, the latter clade contains two reciprocally monophyletic hap- logroups, one from the Balkans, Anatolia, the Caucasus, and Western Kazakh- stan, and another one from Central Europe. As Schneider and Sinsch (1999) fairly stated, frogs from Western Kazakhstan are undoubtedly P. ridibundus, considering that this is Terra Typica for this species and mating calls of the frogs from this area do not differ from those of P. ridibundus from Central Eu- rope, and the same applies to the frogs from Armenia. However, later research- ers routinely attributed frogs from the Balkans and Western Asia to P. bedriag- ae. Plotner et al. (2001) used the name P. bedriagae for all frogs from Western Asia. Lymberakis et al. (2007) classified all frogs from the southern Balkans, Anatolia, and the Middle East as P. bedriagae. Ualiyeva et al. (2022) attribut- ed the mitochondrial haplotypes of water frogs from western Kazakhstan to P. bedriagae because they were clustered with the sequences from Anatolia (but not with P. bedriagae from the Levant) and not with the haplogroup of P. ridibundus from Central Europe. Dufresnes et al. (2024) also used the name P bedriagae for frogs from Central Asia. Our results support the reciprocal monophyly of mitochondrial haplogroups of marsh frogs from (1) the Balkans, the Caucasus, Western and Central Asia, and (2) Central Europe. However, we suggest that the name Pelophylax bed- riagae can only be applied to the frogs from the Levant, which Schneider et al. described as a separate species back in 1992. Applying this name to the frogs from most of Anatolia, the Caucasus, and Central Asia, including Kazakhstan, is incorrect because (a) western Kazakhstan, and specifically the Atyrau district, is Terra Typica for P. ridibundus (Pallas 1771). By definition, they should be at- tributed to P. ridibundus, assuming that all marsh frogs from this area are con- specific; (b) the vocalization of frogs from western Kazakhstan, Central Europe, and the Caucasus (Armenia) is similar as opposed to the frogs from the Middle East (Schneiderand Sinsch 1999). The frogs from the northern (Ermakov et al. 2016) and the southern (this paper) Caucasus, the region that is on the way between Anatolia and the Caspian Area/Kazakhstan, also have the haplogroup widespread in the Balkans and Anatolia. Ualiyeva et al. (2022) showed that the nuclear gene SAI of frogs from the P ridibundus group is separated into three haplogroups. They did not show recip- rocal monophyly between nominal P ridibundus and P. bedriagae. In the Terra typica of P ridibundus, two nuclear haplogroups are present, one closer to the Anatolian and the other to the Central European frogs. Finally, a genetic study Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 30 Tarkhnishvili et al.: Water frogs from Georgia of invasive marsh frogs in Belgium showed introgressive hybridization between nominal P. ridibundus and P. ‘bedriagae’, suggesting the absence of reproduc- tive barriers between these two (Holsbeek et al. 2009). In conclusion, the presence of distinct mitochondrial haplogroups of marsh frogs from the north-west and south-east of their western Eurasian range re- flects a long period of isolation between the European and West Asian or Bal- kan populations. However, it is not sufficient to attribute them to different spe- cies. There are no vocalization differences between the frogs from Kazakhstan, the Caucasus, and Central Europe, and no data is available for those Anatolian populations, which belong to the mitochondrial lineage widespread in Western Asia and the Caucasus. Nuclear haplotypes more common in Central Europe and Anatolia are admixed in the Terra Typica of P. ridibundus and are occasion- ally found in the same population of Western Kazakhstan; finally, there is no ev- idence of reproductive isolation between these populations. The proportion of the sequence differences cannot be taken as evidence of the effective isolation of "different evolutionary pathways" (in the sense of DeQueiroz 2007). There is no evidence of a limited gene flow or the presence of tension zones between the eastern and western geographic populations or marsh frogs. While reserv- ing the name R. bedriagae for frogs from some populations from the Levant and southern Anatolia, marsh frogs from most of Anatolia, the Balkans, the Cauca- sus, and the Caspian area should all be attributed to the species P ridibundus. Acknowledgements We appreciate Armen Seropian and Lasha-Giorgi Japaridze for collecting the samples, and four anonymous reviewers, which made valuable comments on the first version of the Manuscript. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding The study was funded within the framework of the project by the Federal Ministry of Ed- ucation and Research of Germany (BMBF) under grant number 01DK20014A (CaBOL). Author contributions GI planned field work and collected samples, MT performed genetic analysis and tree building, DT wrote the text with assistance of GI and MT, all authors participated in final data analysis. Author ORCIDs David Tarkhnishvili © https://orcid.org/0000-0003-1479-9880 Mariami Todua © https://orcid.org/0000-0003-1870-3030 Giorgi lankoshvili © https://orcid.org/0000-0002-0429-1488 Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 36 Tarkhnishvili et al.: Water frogs from Georgia Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Astrin JJ, Stiben PE (2008) Phylogeny in cryptic weevils: molecules, morphology and new genera of western Palaearctic Cryptorhynchinae (Coleoptera: Curculionidae). In- vertebrate Systematics 22(5): 503-522. https://doi.org/10.1071/IS07057 Bannikov AG, Darevsky IS, Ishchenko VG, Rustamov AK, Szczerbak NN (1977) Guide to Amphibians and Reptiles of the USSR Fauna. Prosveshchenie, Moscow, 414 pp. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi |, Lipman DJ, Ostell J, Sayers EW (2012) GenBank. Nucleic Acids Research 41(D1): D36—D42. https://doi.org/10.1093/ nar/gks1195 Berger L (1983) Western Palearctic water frogs (Amphibia, Ranidae): systematics, genetics and population compositions. Experientia 39(2): 127-130. https://doi. org/10.1007/BF01958859 De Queiroz K (2007) Species concepts and species delimitation. Systematic Biology 56(6): 879-886. https://doi.org/10.1080/10635150701 701083 Dufresnes C, Denoél M, Di Santo L, Dubey S (2017) Multiple uprising invasions of Pelo- phylax water frogs, potentially inducing a new hybridogenetic complex. Scientific Re- ports 7(1): 6506. https://doi.org/10.1038/s41598-017-06655-5 Dufresnes C, Monod-Broca B, Bellati A, Canestrelli D, Ambu J, Wielstra B, Dubey S, Cro- chet PA, Denoél M, Jablonski D (2024) Piecing the barcoding puzzle of Palearctic wa- ter frogs (Pelophylax) sheds light on amphibian biogeography and global invasions. Global Change Biology 30(3): e17180. https://doi.org/10.1111/gcb.17180 Ermakov OA, Fayzulin Al, Askenderov AD, Ivanov AJu (2016) Molecular-genetic charac- teristics of marsh frog from the republic of Dagestan (based on mitochondrial and nuclear DNA data). Proceedings of Samara Research Center of the Russian Academy of Sciences 18: 94-97. Frost DR (2023) Amphibian Species of the World: an Online Reference. Version 6.2. American Museum of Natural History, New York. https://doi.org/10.5531/db.vz.0001 Hofman S, Pabijan M, Osikowski A, Litvinchuk SN, Szymura JM (2016) Phylogenetic rela- tionships among four new complete mitogenome sequences of Pelophylax (Amphib- ia: Anura) from the Balkans and Cyprus. Mitochondrial DNA Part A 27(5): 3434-3437. https://doi.org/10.3109/19401736.2015.1025266 Holsbeek G, Maes GE, De Meester L, Volckaert FAM (2009) Conservation of the intro- gressed European water frog complex using molecular tools. Molecular Ecology 18: 1071-1087. https://doi.org/10.1111/j.1365-294X.2009.04087.x Kuzmin SL (1999) The Amphibians of the Former Soviet Union. Pensoft Publishers, Bul- garia, 544 pp. Hellmich W (1962) Reptiles and Amphibians of Europe. Blanford Press, London, 160 pp. IUCN SSC Amphibian Specialist Group (2022) Pelophylax bedriagae. The IUCN Red List of Threatened Species 2022: e.T58893474A173829632. https://doi.org/10.2305/ IUCN.UK.2022-1.RLTS.T58893474A173829632.en [Accessed on 03 December 2023] Linnaeus C (1758) Systema naturae (Vol. 1, p. 824). Laurentii Salvii, Stockholm. Lymberakis P, Poulakakis N, Manthalou G, Tsigenopoulos CS, Magoulas A, Mylonas M (2007) Mitochondrial Phylogeography of Rana (Pelophylax) Populations in the East- ern Mediterranean Region. Molecular Phylogenetics and Evolution 44(1): 115-125. Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 37 Tarkhnishvili et al.: Water frogs from Georgia Pallas PS (1771) Reise durch verschiedene Provinzen des russischen Reichs (Vol. 1). Kayserliche Akademie der Wissenschaften. Papezik P Mikulic¢ek P Benovics M, Balogova M, Choleva L, Dolezalkova-Kastankova M, Lymberakis P Mizsei E, Papezikova S, Poulakakis N, Sag¢danaku E (2023) Compara- tive mitochondrial phylogeography of water frogs (Ranidae: Pelophylax spp.) from the southwestern Balkans. Vertebrate Zoology 73: 525-544. https://doi.org/10.3897/ vz.73.e€95220 Plotner J, Ohst T, Bohme W, Schreiber R (2001) Divergence in Mitochondrial DNA of Near Eastern Water Frogs with Special Reference to the Systematic Status of Cypriote and Anatolian populations (Anura, Ranidae). Amphibia-Reptilia 22(4): 397-412. https:// doi.org/10.1163/15685380152770363 Schneider H, Sinsch U, Nevo E (1992) The lake frogs in Israel represent a new species. Zoologischer Anzeiger 228(1-2): 97-106. Schneider H, Sinsch U (1999) Taxonomic reassessment of Middle Eastern water frogs: Bioacoustic variation among populations considered as Rana ridibunda, R. bedriag- ae or R. levantina. Journal of Zoological Systematics and Evolutionary Research 37: 57-66. https://doi.org/10.1046/j.1439-0469.1999.372098.x Sinsch U, Schneider H (1999) Taxonomic reassessment of Middle Eastern water frogs: Morphological variation among populations considered as Rana ridibunda, R. bed- riagae or R. levantina. Journal of Zoological Systematics and Evolutionary Research 37(2): 67-74. https://doi.org/10.1046/j.1439-0469.1999.372099.x Steinwarz D, Schneider H (1991) Distribution and bioacoustic of "Rana perezi" seoane, 1885 (Amphibia, Anura, Ranidae) in Tunisia. Zoologisches Forschungsinstitut und Museum Alexander Koenig 42(3-4): 283-297. Ualiyeva D, Ermakov OA, Litvinchuk SN, Guo X, Ivanov AY, Xu R, Li J, Xu F, Arifulova II, Kaptyonkina AG, Khromov VA (2022) Diversity, phylogenetic relationships and dis- tribution of marsh frogs (the Pelophylax ridibundus complex) from Kazakhstan and Northwest China. Diversity 14(10): 869. https://doi.org/10.3390/d14100869 Wiens JJ, Sukumaran J, Pyron RA, Brown RM (2009) Evolutionary and biogeographic origins of high tropical diversity in Old World frogs (Ranidae). Evolution 63(5): 1217- 1231. https://doi.org/10.1111/j.1558-5646.2009.00610.x Supplementary material 1 Samples and downloaded sequences used in the study Authors: Tarkhnishvili D, Todua M, lankoshvili G Data type: GenBank identification numbers Explanation note: Samples and downloaded sequences (NCBI identification numbers) used in the study. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/caucasiana.3.e117215.suppl1 Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 38 Tarkhnishvili et al.: Water frogs from Georgia Supplementary material 2 Genetic distances (proportion of differences) between the studied sequences Authors: Tarkhnishvili D, Todua M, lankoshvili G Data type: Genetic distance matrix Explanation note: Genetic distances (proportion of differences) between the studied se- quences of CO1 mitochondrial gene of Pelophylax. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/caucasiana.3.e117215.suppl2 Caucasiana 3: 31-39 (2024), DOI: 10.3897/caucasiana.3.e117215 39