New Multiplex PCR Method for Identification of East European Green Frog Species and Their Hybrids

O. Ermakov, A. Ivanov, S. Titov, A. Svinin, S. N. Litvinchuk

Abstract


A molecular multiplex PCR method for identification of East European green frog species (Pelophylax ridibundus, P. cf. bedriagae and P. lessonae) and their hybrids was developed. This simple and rapid method can be used for identification of species-specific mitochondrial and nuclear DNA. The method is based on species-specific differences in primary structure of the subunit 1 of the mitochondrial cytochrome C oxidase gene (COI) and the intron-1 of the nuclear serum albumin gene (SAI-1). Based on the method, we analyzed numerous individuals of these species and their hybrids from East European Plain, the Crimea, the Caucasus, the Ural, as well as introduced populations from Western Siberia and the Kamchatka. In all cases, identification of species performed by use of the multiplex PCR method coincided with results of study of primary nucleotide sequences.

Keywords


Pelophylax ridibundus; Pelophylax cf. bedriagae; Pelophylax lessonae; Pelophylax esculentus; rapid species identification; water frogs; cytochrome C oxidase; serum albumin

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References


Aljanabi S. M. and Martinez I. (1997), «Universal and rapid salt-extraction of high genomic DNA for PCR-based techniques», Nucleic Acids Res., 25, 4692 – 4693.

Arioli M., Jakob C., and Reyer H.-U. (2010), «Genetic diversity in water frog hybrids (Pelophylax esculentus) varies with population structure and geographic location», Mol. Ecol., 19(9), 1814 – 1828.

Dufresnes C., Di Santo L., Leuenberger J., Schuerch J., Mazepa G., Grandjean N., Canestrelli D., Perrin N., and Dubey S. (2016) «Cryptic invasion of Italian pool frogs (Pelophylax bergeri) across Western Europe unraveled by multilocus phylogeography», Biol. Invasions, 19(5), 1407 – 1420.

Dufresnes C., Denoel M., Di Santo L., and Dubey S. (2017), «Multiple uprising invasions of Pelophylax water frogs, potentially inducing a new hybridogenetic complex», Sci. Rep., 7(1), 6506.

Dubey S. and Dufresnes C. (2017), «An extinct vertebrate preserved by its living hybridogenetic descendant», Sci. Rep., 7(1), 12768.

Dubey S., Leuenberger J., and Perrin N. (2014) «Multiple origins of invasive and ‘native’ water frogs (Pelophylax spp.) in Switzerland», Biol. J. Linn. Soc., 112(3), 442 – 449.

Ermakov O. A., Fayzulin A. I., Zaks M. M., Kaybeleva E. I., and Zaripova F. F. (2014), «Distribution of «western» and «eastern» forms of the marsh frog Pelophylax ridibundus s. l. in the Samara and Saratov regions (on data of analysis of mtDNA and nDNA)», Izv. Samar NTs RAN, 16[5(1)], 409 – 412 [in Russian].

Hauswaldt J. S., Hoeer M., Ogielska M., Christiansen D. G., Dziewulska-Szwajkowska D., Czernicka E., and Vences M. (2012), «A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax)», Mol. Ecol. Res., 12(5), 797 – 805.

Herczeg D., Vörös J., Christiansen D. G., Benovics M., and Mikulíček P. (2017), «Taxonomic composition and ploidy level among European water frogs (Anura: Ranidae: Pelophylax) in eastern Hungary», J. Zool. Syst. Evol. Res., 55(2), 129 – 137.

Hofman S., Pabijan M., Dziewulska-Szwajkowska D., and Szymura J. M. (2012), «Mitochondrial genome organization and divergence in hybridizing central European water frogs of the Pelophylax esculentus complex (Anura, Ranidae)», Gene, 491(1), 71 – 80.

Hofman S., Pabijan M., Osikowski A., Litvinchuk S. N., and Szymura J. M. (2016), «Phylogenetic relationships among four new complete mitogenome sequences of Pelophylax (Amphibia: Anura) from the Balkans and Cyprus», Mitochondrial DNA Pt A, 27(5), 3434 – 3437.

Holsbeek G. and Jooris R. (2010), «Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (Pelophylax esculentus complex)», Biol. Invasions, 12(1), 1 – 13.

Holsbeek G., Mergeay J., Hotz H., Plötner J., Volckaert F., and De Meester L. (2008), «A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation», Mol. Ecol., 17(23), 5023 – 5035.

Holsbeek G., Maes G. E., De Meester L., and Volckaert A. M. (2009), «Conservation of the introgressed European water frog complex using molecular tools», Mol. Ecol., 18, 1071 – 1087.

Holsbeek G., Mergeay J., Volckaert F., and De Meester L. (2010), «Genetic detection of multiple exotic water frog species in Belgium illustrates the need for monitoring and immediate action», Biol. Invasions, 12(6), 1459 – 1463.

Leuenberger J., Gander A., Schmidt B. R., and Perrin N. (2014), «Are invasive marsh frogs (Pelophylax ridibundus) replacing the native P. lessonae/P. esculentus hybridogenetic complex in Western Europe? Genetic evidence from a field study», Conserv. Genet., 15(4), 869 – 878.

Lyapkov S. M., Ermakov O. A., and Titov S. V. (2018), «Distribution and origin of two forms of the marsh frog Pelophylax ridibundus complex (Anura, Ranidae) from the Kamchatka based on mitochondrial and nuclear DNA data», Biol. Byull., 45(7), 83 – 89.

Patrelle C., Ohst T., Picard D., Pagano A., Sourice S., Dallay M.-G., and Plötner J. (2011), «A new PCR-RFLP-based method for an easier systematic affiliation of European water frogs», Mol. Ecol. Res., 11, 200 – 205.

Plötner J., Köhler F., Uzzell T., Beerli P., Schreiber R., Guex G-D., and Hotz H. (2009), «Evolution of serum albumin intron-1 is shaped by a 5’ truncated non-long terminal repeat retrotransposon in western Palearctic water frogs (Neobatrachia)», Mol. Phylogen. Evol., 53, 784 – 791.

Plötner J., Baier F., Akın C., Mazepa G., Schreiber R., Beerli P., Litvinchuk S. N., Bilgin C., Borkin L., and Uzzell T. (2012), «Genetic data reveal that water frogs of Cyprus (genus Pelophylax) are an endemic species of Messinian origin», Zoosyst. Evol., 88(2), 261 – 283.

Stakh V. O., Strus Iu. M., and Khamar I. S. (2018), «Genetic diversity in population systems of green frogs (Pelophylax esculentus complex) in waterbodies of Western Ukraine», Studia Biol., 12(3 – 4), 17 – 26.

Svinin A. O., Ivanov A. Yu., Zaks M. M., Litvinchuk S. N., Borkin L. J., Rosanov J. M., and Ermakov O. A. (2015), «Distribution of the «eastern» and «western» forms of the marsh frog, Pelophylax ridibundus, and their participation in the origin of hemiclonal hybrids, P. esculentus in Mari El Republic», Curr. Stud. Herpetol., 15(3 – 4), 120 – 129 [in Russian].

Zamaletdinov R. I., Pavlov A. V., Zaks M. M., Ivanov A. Y., and Ermakov O. A. (2015), «Molecular-genetic characteristic of Pelophylax esculentus complex from the eastern range of distribution (Volga region, Tatarstan Republic)», Tomsk. Gos. Univ. Zh. Biol., 3(31), 54 – 66 [in Russian].




DOI: https://doi.org/10.30906/1026-2296-2019-26-6-367-370

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