Genetic Diversity of Pelophylax cerigensis-like Populations on the Anatolian Mainland in Türkiye (Amphibia: Anura: Ranidae)

Abstract

Genetic diversity is increasingly used as a vital component in planning appropriate conservation strategies. Water frogs in the eastern Mediterranean consist of several endemic species. The Critically Endangered Karpathos water frog (Pelophylax cerigensis) is one such species, restricted to Karpathos Island, but recently P. cerigensis specific haplotypes were also found in Rhodes and southwestern Türkiye. Since geologically Karpathos and Rhodes have been separated from the Anatolian mainland millions years ago, the genetic diversity of P. cerigensis-like populations on the Anatolian mainland are not known. Here, we aim to evaluate the genetic diversity of this mainland populations (N=52) in southwestern Anatolia by using 5 polymorphic microsatellite loci. According to results, a total of 38 alleles which five loci exhibited a moderate level of genetic diversity (observed heterozygosity, HO=0.423). The population has not gone through a bottleneck anytime soon; however, signs of inbreeding were determined (Fıs=0.401). Due to restricted occurrence from Antalya to Aydın provinces in southwestern Türkiye and a moderate level of genetic diversity, they should be considered a third Management Unit (MU) of P. cerigensis populations in addition to previous Karpathos and Rhodes MUs. This approach is very crucial to formulate suitable management strategies for conservation of these threatened populations.

Keywords

• genetic diversity
• water frog
• P. cerigensis
• Anatolia

References

1. Lacy RC. Loss of genetic diversity from managed populations: interacting effects of drift, mutation, immigration, selection, and population subdivision. Conservation Biology. 1987;1:143-158.
2. Amos W, Balmford A. When does conservation genetics matter? Heredity. 2001;8:7:257.
3. Hoban S, Bruford MW, Chris Funk W, Galbusera P, Patrick Griffith M, Grueber CE, et al. Global commitments to conserving and monitoring genetic diversity are now necessary and feasible. BioScience. 2021;71(9):964–976.
4. Conservation International. Biodiversity Hotspots. 2021. Biodiversity Hotspots. viewed 31 August 2021, fromhttps://www.conservation.org/priorities/biodiversity-hotspots.
5. Gür H. The future impact of climate and land-use changes on Anatolian ground squirrels under different scenarios. Ecological Informatic. 2022;70:1-12.
6. Plötner J, Uzzell T, Beerli P, Akın Ç, Bilgin C, Haefeli C, et al. Genetic divergence and evolution of reproductive isolation in eastern Mediterranean water frogs. In: Glaubrecht M (ed.). Evolution in Action. Berlin, Heidelberg: Springer. 2010; p. 373–403.
7. Akın Ç, Can Bilgin C, Beerli P, Westaway R, Ohst T, Litvinchuk SN, et al. Phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs were determined by geological processes and climate change in the late Cenozoic. J. Biogeogr. 2010;37:2111-2124.
8. Beerli P, Hotz H, Tunner HG, Heppich S, Uzzell T. Two new water frog species from the Aegean islands Crete and Karpathos (Amphibia, Salientia, Ranidae). Not. Nat. 1994;470:1-9.

9. Plötner J, Baier F, Akın C, Mazepa G, Schreiber R, Beerli P, et al. Genetic data reveal that water frogs of Cyprus (genus Pelophylax) are an endemic species of Messinian origin. Zoosyst. Evol. 2012;88:261-283.
10. Beerli P, Uzzell T, Lymberakis P. Pelophylax cerigensis. The IUCN Red List of Threatened Species, 2009: e. T58567A11787309. https://amphibiaweb.org/species/5000.
11. IUCN, SSC Amphibian Specialist Group. Pelophylax cerigensis (errata version published in 2022). The IUCN Red List of Threatened Species 2022: e.T58567A89696593. [cited 2023 Decem 1] Available from: https://dx.doi.org/10.2305/IUCN. UK.2020-3.RLTS.T58567A89696593.en.
12. Pafilis P, Deimezis-Tsikoutas A, Kapsalas G, Maragou P. Action Plan for Pelophylax cerigensis. LIFEIP 4 NATURA project: integrated actions for the conservation and management of Natura 2000 sites, species, habitats and ecosystems in Greece (LIFE16 IPE/GR/000002). Deliverable Action A.1. Ministry of Environment and Energy, Athens. 2020;43 pages- Annexes, 40 pp.
13. Meulenkamp JE. Aspects of the late Cenozoic evolution of the Aegean region. In: Stanley, D. J. and F. C. Wezel (Eds.): Geological evolution of the Mediterranean basin. Springer, New York. 1985;307-321.
14. Lymberakis P, Poulakakis N, Manthalou G, Tsigenopoulos C, Magoulas A, Mylonas M. Mitochondrial phylogeography of Rana (Pelophylax) populations in the eastern Mediterranean region, Mol. Phylogen. Evol. 2007;44:115-125.
15. Toli E, Siarabi S, Bounas A, Pafilis P, Lymperakis P, Sotiropoulos K. New insights on the phylogenetic position and population genetic structure of the critically endangered Karpathos marsh frog Pelophylax cerigensis (Amphibia: Anura: Ranidae). Acta Herpetol. 2018;13:117-123.
16. Toli EA, Bounas A, Christopoulos A, Pafilis P, Lymperakis P, Sotiropoulos K. Phylogenetic analysis of the critically endangered Karpathos water frog (Anura, Amphibia): conservation insights from complete mitochondrial genome sequencing. Amphibia-Reptilia. 2023;44(3):277-287.
17. Akın Pekşen Ç. Molecular evolution and phylogeography of the eastern Mediterranean water frog (Pelophylax) complex. Ph.D dissertation. Middle East Technical University. 2015;342 pp.
18. Martínez-Cruz B, Godoy JA. Genetic evidence for a recent divergence and subsequent gene flow between Spanish and Eastern imperial eagles. BMC Evolutionary Biology. 2007;7:1-8.
19. Lopes F, Hoffman JI, Valiati VH, Bonatto SL, Wolf JBW, Trillmich F. et al. Fine-scale matrilineal population structure in the Galapagos fur seal and its implications for conservation management. Conserv. Genet. 2015;16:1099–1113.
20. Doyle JM, Katzner TE, Bloom PH, Roemer GW, Cain JW, Millsap B, et al. Genetic structure and viability selection in the Golden Eagle (Aquila chrysaetos), a vagile raptor with a Holarctic distribution. Conserv. Genet. 2016;17:1–16.
21. Zeisset I, Rowe G, Beebee TJC. Polymerase chain reaction primers for microsatellite loci in the north European water frogs Rana ridibunda and R. lessonae. Molecular Ecology. 2000;9:1173–1174.
22. Garner TWJ, Gautschi B, Rothlisberger S, Reyer HU. A set of CA repeat microsatellite markers derived from the pool frog, Rana lessonae. Molecular Ecology, 2000;9,2173–2175.
23. Chapuis MP, Estoup A. Microsatellite null alleles and estimation of population differentiation. Mol. Biol. Evol. 2006;24:621–631.
24. Van Oosterhout C, Hutchinson WF, Wills DP, Shipley P. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Resour, 2004; 4:535–538.
25. Goudet J. FSTAT; a program to estimate and test gene diversities and fixation indices version. 2001;2.9. 3. Available at: https://www2.unil.ch/popgen/softwares/fstat.htm (accessed 23 Sep 2023).
26. Marshall T, Slate J, Kruuk L, Pemberton J. Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology. 1998;7:639–655.
27. Kalinowski ST, Taper ML, Marshall TC. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology. 2007;16:1099–1106.
28. Peakall R, Smouse P. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research – an update. Bioinformatics. 2012;28:2537–2539.
29. Raymond M, Rousset F. GENEPOP (Version 1.2): population genetics software for exact tests and ecumenicalism. Journal of Heredity. 1995;86: 248–249.
30. Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics. 1980;32:314.
31. Cornuet JM, Luikart G. Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics. 1996;144:2001–2014.
32. Piry S, Luikar G, Cornuet JM. Computer note. BOTTLENECK: a computer program for detecting recent reductions in the effective size using allele frequency data. Journal of Heredity. 1999;90:502–503.
33. Luikart G, Allendorf F, Cornuet, J, Sherwin W. Distortion of allele frequency distributions provides a test for recent population bottlenecks. Journal of Heredity. 1998;89:238–247.
34. Ganapathi P, Rajendran R, Kathiravan P. Detection of occurrence of a recent genetic bottleneck event in Indian hill cattle breed Bargur using microsatellite markers. Trop. Anim. Health Prod. 2012;44:2007–2013.
35. Holsbeek G, Maes GE, De Meester L, Volckaert FAM. Conservation of the introgressed European water frog complex using molecular tools. Molecular Ecology. 2009;18:1071–1087.
36. Leuenberger J, Gander A, Schmidt BR, Perrin N. Are invasive marsh frogs (Pelophylax ridibundus) replacing the native P. lessonae/P. esculentus hybridogenetic complex in Western Europe? Genetic evidence from a field study. Conservation Genetics. 2014;15:869–878.
37. De Meeûs T, Chan CT, Ludwig JM, Tsao JI, Patel J, Bhagatwala J, et al. Deceptive combined effects of short allele dominance and stuttering: An example with Ixodes scapularis, the main vector of Lyme disease in the USA. Peer Community Journal. 2021;1.
38. Coltman DW, Slate J. Microsatellite measures of inbreeding: A Meta‐Analysis. Evolution. 2003;57(5):971–983.
39. Hale ML, Burg TM, Steeves TE. Sampling for microsatellite based population genetic studies: 25 to 30 individuals per population is enough to accurately estimate allele frequencies. PLoS ONE. 2012;7: e45170.
40. Pemberton J. Measuring inbreeding depression in the wild: the old ways are the best. Trends Ecol Evol. 2004;19(12)613-5.
41. Brzeski KE, Rabon DRJr, Chamberlain MJ, Waits LP, Taylor SS. Inbreeding and inbreeding depression in endangered red wolves (Canis rufus). Molecular Ecology. 2014;23(17):4241-55.
42. Hewitt GM. Speciation, hybrid zones and phylogeography – or seeing genes in space and time. Mol.Ecol. 2001;10:537-549.
43. Mutz T, Steinfartz S. Mertensiella luschani flavimembris ssp. n., Eine Neue Unterart des Lykischen Salamanders aus der Turkei (Caudata: Salamandridae). Salamandra. 1995;31:137-148.
44. Başoğlu M, Atatür MK. The subspecific division of the Lycian salamander, Mertensiella luschani (Steindachner) in Southwestern Anatolia. İstanbul Üniversitesi Fen Fakültesi Mecmuası Seri B. 1974;39(3-4):147-155.
45. Kornilios P, Kumlutaş Y, Lymberakis P, Ilgaz Ç. Cryptic diversity and molecular systematics of the Aegean Ophiomorus skinks (Reptilia: Squamata), with the description of a new species. Journal of Zoological Systematics and Evolutionary Research. 2018;56(3):364-381.
46. Baran İ, Avcı A, Kumlutaş Y, Olgun K, Ilgaz Ç. Türkiye Amfibi ve Sürüngenleri. Palme Press, Ankara. 2021;236 pp.
47. Boger H, Dermitzakis MD. Neogene paleogeography in the Central Aegean region. In: VIIIth International Neogene Congress, Budapest. 1985; vol.70.
48. Lymberakis P, Poulakakis N. Three continents claiming an archipelago: the evolution of Aegean’s herpetofaunal diversity. Diversity. 2010;2:233-255.
49. Plötner, J, Akın Pekşen Ç, Baier F, Uzzell T, Bilgin CC. Genetic evidence for human-mediated introduction of Anatolian water frogs (Pelophylax cf. bedriagae) to Cyprus (Amphibia: Ranidae). Zoology in the Middle East,.2015;61:125–132.