Borkin et al. (2005) Mass polyploidy in the hybridogenic complex Rana esculenta

Borkin L. Ya., Zinenko A. I., Korshunov A. V., Lada G. A., Lytvynchuk S. N., Rozanov Yu. M., Shabanov D. A. Mass polyploidy in the hybridogenetic complex Rana esculenta (Ranidae, Anura, Amphibia) in eastern Ukraine // Materials of the First Conference of the Ukrainian Herpetological Society – K.: Zoological Museum of the NNP of the NAS of Ukraine, 2005. – pp. 23–26.  Mass polyploidy in the hybridogenetic complex Rana esculenta (Ranidae, Anura, Amphibia) in eastern Ukraine L. Ya. Borkin1, A. I. Zinenko2, A. V. Korshunov3, G. A. Lada1,4, S. N. Lytvynchuk5, Yu. M. Rozanov5, D. A. Shabanov3 1 Zoological Institute of the Russian Academy of Sciences, Universitetskaya nab., 1, St. Petersburg, 199034. E‑mail: lacerta@zin.ru 2 Nature Museum of Kharkiv National University named after V. N. Karazin, Trinklera St., 8, Kharkiv, 61022. E‑mail: zinenkoa@yahoo.com 3 V. N. Karazin Kharkiv National University, Svobody Pl., 4, Kharkiv, 61077. E‑mail: d_sh@list.ru 4 Tambov State University, Internatsionalnaya St., 33, Tambov, 392622. E‑mail: esculenta@mail.ru 5 Institute of Cytology of the Russian Academy of Sciences, Tikhoretsky pr., 4, St. Petersburg, 194064. E‑mail: slitvinchuk@yahoo.com Mass triploidy in hybrid Rana esculenta was revealed by means of DNA flow cytometry in 19 localities of eastern Ukraine (Kharkiv, Donetsk, and Luhansk provinces). Triploids consist of two groups in terms of parental species genomes: LLR and LRR. Both groups were recorded in population systems of the E and R‑E types. Two tetraploids were found as well. All polyploids were distributed along the middle part of the Seversky Donets River basin (above 450 km). The nearest localities with mass occurrence of triploidy were known at a distance of 1000 km (Poland) or 1500 km (Hungary). In eastern Europe, green frogs (Rana esculenta complex) are represented by three taxa: the lake form (Rana ridibunda Pallas, 1771), the pond form (Rana lessonae Camerano, 1882) and the edible form (Rana esculenta Linnaeus, 1758). The latter arises from hybridisation of the two former species. This complex is characterised by unusual speciation, whose mechanisms include hybridisation, semi‑clonal (or meroclonal) inheritance, polyploidy and diversity of population‑system composition, where R. esculenta may be represented by one or two sexes (see Günther, 1990, 1991; Vinogradov et al., 1990). Thus, the R. esculenta complex can be regarded as one of the models of reticulate (hybridogenetic) speciation in animals (Borkin, Darevsky, 1980). Despite the wide distribution of R. esculenta (from France to the Volga), triploidy in hybrids has been found only in western and central Europe. It is most frequently encountered from north‑west France to Sweden and Poland, and also in Slovakia and Hungary (Borkin et al., 2004). Long‑term studies carried out by us and other authors since the late 1970s in the former USSR using various methods (protein electrophoresis, karyotyping, DNA cytometry) did not reveal polyploids, except for a 2n individual from Kaliningrad region and a 4n individual from Latvia (Borkin, 2001). In this context, the discovery of mass polyploidy in a whole series of R. esculenta populations in eastern Ukraine (Borkin et al., 2004) was an unexpected result. Our investigations of green frogs in this region by flow DNA cytometry began already in 1989. The first triploid was detected in 1996 (Ishkov pond, village Haidary, Zmiiv district, Kharkiv region). However, the most intensive collections and material analysis were performed in 2002–2005. In total we examined 813 specimens from 48 sites. According to our data, all three species of green frogs inhabit the territories of Kharkiv, Donetsk and Luhansk oblasts. R. lessonae is the rarest (about 2 %) and was recorded only in six sites of Kharkiv oblast: in the north‑west (settlements Kozievka, Horodne, Mykhaylivske, Haidary, Velyka Homolshcha; the first two are represented by museum collections) and in the south‑west (the “Russian Orchik” nature reserve). In Haidary and Velyka Homolshcha only single year‑old individuals of R. lessonae were found, which may result from their segregation when males and females of R. esculenta interbreed. The majority of examined individuals belonged to R. esculenta (63 %), the remainder to R. ridibunda (35 %). Among R. esculenta, diploids comprised 76 % (354 of 467 individuals), and polyploids 24 % (113). The latter were found in the vicinity of 16 settlements: Izbytske, Zhadankivka, Haidary, Velyka Homolshcha, Sukha Homolshcha, Kreydyanka, Chervona Husarivka, Olkhovatka, Chervonyi Shakhtar, Zadonetske, Lyman, Balakliia and Studenok in Kharkiv oblast; Sviatohirsk in Donetsk oblast; as well as Novokondrashivka and Stanychno‑Luhanske in Luhansk oblast. Among the polyploids, 111 individuals were 3 n and 2 specimens were 4 n. The proportion of triploids among hybrids (in samples containing at least 10 hybrids) averaged 41 % of hybrids in a local sample, ranging from 9 to 76 %. According to genome size, triploids split into two clearly defined classes. Individuals with a relatively smaller genome were assigned by us to the LLR type (two haploid genomes from R. lessonae and one from R. ridibunda; 54.8 %), and those with a relatively larger genome to the RRL type (41.5 %). 3.7 % of triploids had intermediate genome sizes and were not assigned to either of the above types. Both triploid types are similarly widespread and represented by both males and females. The occurrence of triploids in samples and the share of LLR‑type hybrids increase downstream along the Seversky Donets River. The proportion of triploid individuals sharply declines with distance from the river. Triploids were found both in population systems consisting of R. esculenta and R. ridibunda (the so‑called R‑E type) and in systems consisting only of R. esculenta (pure E type). Comparative analysis of DNA content in somatic (blood) and germ cells by flow DNA cytometry showed that in the sample from the vicinity of Haidary (R‑E type, n = 47) triploid RRL‑type males transmit to offspring, most likely, only haploid gametes containing a single ridibunda genome. The situation with diploid R. esculenta males proved more complex. It turned out that 35 % of males produced haploid gametes containing only the ridibunda genome, and 17 % only the lessonae genome. A third type of males was also discovered, carrying both genome lines, i.e., in the same male some gametes bore the ridibunda genome and others the lessonae genome, with the ratio of such haploid gametes varying markedly among individuals (from approximate equality to a clear predominance of one genome). The appearance of triploids appears to be linked to the formation of non‑reduced diploid eggs in hybrid females. Tetraploids (vicinity of Velyka Homolshcha, Kharkiv oblast) were found in a mixed R‑E system together with LLR and LRR triploids, as well as diploid R. esculenta. This finding, together with previously identified tetraploids from Latvia and Sweden (Borkin, 2001), confirms the reality of spontaneous tetraploidy among hybrids, which may serve as another confirmation of the reticulate evolution concept based on the interplay of hybridisation, clonality and polyploidy (Borkin, Darevsky, 1980). Diploid R. esculenta were detected by us in many regions of the European part of the former USSR, from Kaliningrad region and Transcarpathia to Udmurtia and Samara oblast, including northern forest and forest‑steppe zones of Ukraine (Borkin et al., 2004: Fig. 4). In contrast, the mass distribution of triploids, based on our data, is limited to a narrow strip along the middle course of the Seversky Donets River. Nevertheless, it covers a fairly large area of more than 450 km—from the settlement Izbytske in the north of Kharkiv oblast to the settlement Stanychno‑Luhanske in the east of Luhansk oblast. The latter finding suggests the possible existence of triploids in the adjacent south‑west Russian territory in the lower part of the Seversky Donets. It should be noted, however, that we have not found them in the Russian part of the Seversky Donets basin (Lada et al., 1995). Mass polyploidy in R. esculenta in eastern Ukraine is also interesting from a geographical point of view. The nearest areas where a similar phenomenon is known are quite distant, roughly 1000–1500 km away. These are the western part of Hungary (Tunner, Tunner‑Heppich, 1992) and Poland (Rybacki, Berger, 2001), respectively. According to the latter authors, there is a clear trend of decreasing triploid frequency from eastern Germany southward and eastward. In this respect, the high occurrence of 3 n hybrids found by us in geographic isolation in eastern Ukraine represents an intriguing puzzle. The work was carried out with partial support from the Russian Foundation for Basic Research (grants RFBR 05‑04‑48403 and 05‑04‑48815). Borkin L. Ya. Speciation, hybridisation and polyploidy in Palaearctic amphibians // Herpetology Issues. — Pushchino; Moscow, 2001. — pp. 46‑48. Borkin L. Ya., Darevsky I. S. Reticulate (hybridogenetic) speciation in vertebrates // J. Gen. Biol. — 1980. — 41, No. 4. — pp. 485‑506. Borkin L. J., Korshunov A. V., Lada G. A. et al. Mass occurrence of polyploid green frogs (Rana esculenta complex) in eastern Ukraine // Russ. J. Herpetol. — 2004. — 11, No. 3. — pp. 203‑222. Gunther R. Die Wasserfrösche Europas (Anura — Froschlurche). — Wittenberg, Lutherstadt: A. Ziemsen, 1990, — 288 p. — (Die Neue‑Brehm‑Biicherei, 600). Gunther R. Europäische Wasserfrösche (Anura, Ranidae) und biologisches Artkonzept // Mitt. Zool. Mus. Berlin, — 1991. — 67, H. 1. — pp. 39‑53. Lada G. A., Borkin L. J., Vinogradov A. E. Distribution, population systems and reproductive behaviour of green frogs (hybridogenetic Rana esculenta complex) in the Central Chernozem Territory of Russia // Russ. J. Herpetol. — 1995. — 2, No. 1. — pp. 46‑57. Rybacki M., Berger L. Types of water frog populations (Rana esculenta complex) in Poland // Mitt. Mus. Naturkunde Berlin, Zool. Reihe, — 2001. — 77, H. 1. — pp. 51‑57. Tunner H. G., Heppich‑Tunner S. A new population system of water frogs discovered in Hungary // Korsys Z., Kiss I. (eds.), Proceedings of the 6th Ordinary General Meeting of the Societas Europaea Herpetologica. 19‑23 August 1991. Budapest, Hungary. — Budapest, 1992. — pp. 453‑460. Vinogradov A. E., Borkin L. J., Gunther R., Rosanov J. M. Genome elimination in diploid and triploid Rana esculenta males: cytological evidence from DNA flow cytometry // Genome. — 1990. — 33, No. 5. — pp. 619‑627.