ISSN 0095 4527, Cytology and Genetics, 2010, Vol. 44, No. 4, pp. 212–216. © Allerton Press, Inc., 2010. Original Russian Text © S.V. Mezhzherin, S.Yu. Morozov Leonov, O.V. Rostovskaya, D.A. Shabanov, L.Yu. Sobolenko, 2010, published in Tsitologiya i Genetika, 2010, Vol. 44, No. 4, pp. 23–28. The Ploidy and Genetic Structure of Hybrid Populations of Water Frogs Pelophylax esculentus Complex (Amphibia, Ranidae) of Ukraine Fauna S. V. Mezhzherina, S. Yu. Morozov Leonova, O. V. Rostovskayaa, D. A. Shabanovb, and L. Yu. Sobolenkoc a Shmal’gauzen Institute of Zoology, Ukrainian National Academy of Sciences, Kiev, Ukraine e mail: mezh@izan.kiev.ua b Karazin Kharkov National University, Ukraine c Pavel Tichina Umansk State Teachers’ University, Ukraine Received March 24, 2009 Abstract—The complex study, including allozyme variability and cytometry of hybrid populations of green frogs Pelophylax esculentus (L., 1758) complex has confirmed that the only region of Ukraine where allodip loid are encountered frequently is the Severski Donets basin (9% of all hybrids). In other areas, only two poly ploidy hybrids (0.9%) and one probably autopolyploid individual of each parental species have been regis tered. According to allozyme specters, all three polyploidy hybrids from the Severski Donets basin were males and belonged to biotype P. esculentus (=lessonae) – 2 ridibundus, and their population in this region has halved during the past decade. DOI: 10.3103/S0095452710040043 INTRODUCTION The complex of western palaearctic water frogs Pelophylax esculentus (L., 1758) s.lato where no less than eight new species have been described [1] is inter esting not only because of its hidden genetic variety but also due to peculiarities of interspecies relations, including the exchange of genetic information [2–5]. The hybridization of the two most dominant and widespread species of lake P. ridibundus (Pallas, 1811) and pond P. esculentus (L., 1758) (=lessonae (Camer ano, 1882) frogs is at the base of these processes that result in the creation of allodiploid hybrids P. ridibun dus esculentus, which are usually considered as the specific taxon, clade P. kl. esculentus [6, 7]. These hybrids are fertile but reproduce only by crossbreeding because F2 hybrids are not vital [8–10]. Their gameto genesis occurs in two ways: with the creation of hap loid or sometimes diploid gametes. The first type of gematogenesis in green frogs occurring in most cases is usually called hybridogene sis when the elimination of one of the parental genomes in a hybrid occurs during gametogenesis [11–13]. As a result, the diploid generation appears at backcrossing: either this is characteristic of one of the parental species in whose genome there is an intro gression of the genetic material of the second parental species [2–5], or hybrids allodiploids with the genetic structure of a F1 hybrid or hybrids with a recombina tion of genetic material. Since descendants obtain one nonrecombinant genome from one of the parental species, the method of sexual reproduction was defined as polyclonal reproduction. The ameiosis reproduction of hybrids P. ridibun dus esculentus is unique and can be found in single geographical populations whose hybrids produce hap loid gametes. Parthenogenesis is absent but allotrip loid hybrids appear as the result of the fusion of diploid hybrid gamete and haploid parental species [14–16]. It should be mentioned that the character of gameto genesis can be indeterminate and therefore hybrids from the same reservoir can produce only haploid gametes and others only diploid gametes [17] or both diploid and triploid gametes. There are cases described when the females produce only haploid gametes and males divide into two groups, producers of haploid and diploid gametes, respectively [18]. The geographical distribution of triploid hybrids has a mosaic character. Mostly they can be found in the reservoirs of Western, Northern, and Central Europe: in Germany [12–14], Poland [15, 16, 18], Denmark [17], France [19], Sweden [20], Hungary [21], Slovakia [22] and the Netherlands [23]. In these areas, the frequency of polyploids vary within a wide range (from 4 to 100%) and in case the polyploids are dominant, it is thought that hybrids [17] are able to carry out autonomous reproduction without the involvement of parental species. There are few exam ples of all hybrid populations and their stability over time has not been studied. In the greater part of East ern Europe (Belarus, Russia, Ukraine, Latvia, and 212 THE PLOIDY AND GENETIC STRUCTURE OF HYBRID POPULATIONS OF WATER FROGS 213 Estonia), polyploids are not found [11–14]. The single exception is Severski Donets [24, 25], which is the southeastern border of the combined habitation of parental species. The polyploids within the Ukrainian part of the basin of this river were on average 20% of the studied individuals [24] that allowed the conclu sion on the occurrence of mass polyploidy. Because of the disjunctive location of polyploid populations of green frogs, the question on the pres ence of triploid hybrids within Ukraine, whose popu lations are the link connecting polyploid settlements of northwestern Europe and Eastern Ukraine, is still not clear. At the same time, one cannot say that studies of the genetic structure of hybrids on the territory of Ukraine were not undertaken. However, most of these were carried out without specific analysis of the ploidy and the conclusions on diploid state of the hybrids in these works were made based on the character of elec trophoresis specters, which, as is well known [26], has a clear effect on the dose gene in triploid frogs. This method of the identification of polyploids is reliable in case of a series of polyploids, while single cases can be skipped. Other studies [27] aimed to determine ploidy but were undertaken without gene marking and with out consideration of the genetic structure of hybrids. Moreover, conclusions based only on cytometry stud ies are not reliable because their resolution for the sep aration of triploid biotypes is not high [28]. Thus, there is a demand for an integrated study of the genetic structure of the green frog of Ukrainian fauna, includ ing both gene marking as well as the analysis of the ploidy of the genome. MATERIAL AND METHODS The series of frogs collected during 2007–2008 in reservoirs of Ukraine and covering almost the whole territory where there is hybridization of pond and lake frogs served as the subject of the study. In total, they include 34 samples (table) including 1070 individuals. Analysis of the allozyme variability of some enzymes encoded by proper loci was carried out in all species and included: aspartate aminotransferase (Aat 1, Aat 2), analyzed in muscles; lactate dehydro genase (Ldh B) in muscles or buds; and nonspecific esterases (Es 1) and albumin (Alb), identified in tis sues of buds and liver. It is certain that the loci Ldh B, Alb, and Alb 2 were species specific. The latter locus was rarely used compared to the former two because of often its specters combined with specters of polymor phic locus Aat 1. Loci Aat 1 and Es 1 were polymor phous and diagnostically valuable only for populations from western regions. Electrophoresis was carried out in 7.5% polyacrylamide gel in a tris EDTA borate sys tem of buffers [29]. Ploidy was determined by measurement of the erythrocyte size according to the method tested by Schemeller et al. [19] and used for the mass analysis of CYTOLOGY AND GENETICS Vol. 44 No. 4 2010 40 30 20 10 0 289 317 344 371 399 426 454 481 508 536 563 590 Distribution of green frog species (hybrids and parental species) according to the average area of erythrocytes: the horizontal axis is the average square of the erythrocytes, µm2, and the vertical axis is the number of species. materials in population studies [15]. The size of eryth rocytes in single individuals was determined by the calculation of the average obtained after the measure ment of 20 randomly selected morphologically normal cells. RESULTS The distribution of frogs according to average indi vidual sizes of erythrocytes is shown in the figure. Obviously, in this case, there are two different distribu tions according to volume. The first one includes over 98% of the studied samples and includes frogs with the erythrocytes covering an area from 264 to 458 µm, with an average area of 359 ± 1.3 µm. This group includes, as individuals, two parental types as hybrids. The second distribution includes only 17 individuals with the erythrocytes’ size in a diapason ranging from 500 to 609 µm, with an average value of 553 ± 9.4 µm and includes mostly hybrids and single individuals of the parental species. The average values of these two distributions is expressed as a ratio 1 : 1.5, which com plies with an increase of the area of the erythrocytes in triploid individuals in comparison with diploid indi viduals, which is usually not less than 33% [19]. The single species with an average value of 472 µm accord ing to its specter and genotype Ldh B64/77 100 is the hybrid P. esculentus–2 ridibundus. It was probably aneuploid. As a result of the biochemical gene marking, 16 biotypes of green frogs were identified, including parental species, and various hybrid and different ploid forms (table). The lake frog P. ridibundus, a spe cies that has recently been actively widening its area had the largest mass. This area includes about 56% of all species. The share of the second parental species P. esculentus was lower and amounted to about 10%. Hybrids P. esculentus × P. ridibundus made up 34% of frogs. This value is greater for the P. esculentus species and shows primarily the possibility of autonomous The composition of studied selections of green frogs in Ukraine P. ridibundus Coordinates Lat/Long 2n R ᭺ 81 1 1 12 1 ᭺ 9 51 12 31 40 46 54 1 1 4 1 2 1 4 1 1 1 1 1 3 2 1 3 1 10 2 1 10 8 16 1 7 6 2 11 12 12 7 8 1 1 Ri 3n ᭺ + h ? ? ᭺ + ᭺ + ᭺ ᭺ + ? 2n F1 B Rec P. esculentus–ridibundus P. esculentus– 2 ridibundus 3n P. esculentus 214 Selection 19 26 10 16 17 17 27 12 1 29 MEZHZHERIN et al. 4 1 5 CYTOLOGY AND GENETICS 18 40 1 1 3 1 10 1 5 1 22 11 8 20 8 18 56 26 26 4 19 12 541 1 3 13 20 1 230 32 5 9 4 1 Vol. 44 Merla river Gaidari village Luganka river Sukhoi Torets river Stanichno Luganskoe Gomol’sha Krasnokutsk town Vorskla river Niznii Dnepr Erchiki village Feofaniya Teterev river Samara river Novobelichi Romen river Sivorotka river Bogdanovka village Irsha river Pushcha Voditsa Sluch river Seim river Shevchenko village (canal) Oster river Berezani town Zhukov village Podol’e Kamenka river Nizhnii Dunai Manevichi village Shatskie lakes Gorin’ river Total 1 1 38 36 1 8 1 13 1 1 1 50.0/35.0 49.6/36.3 48.0/37.7 48.8/37.6 48.6/39.4 49.6/36.3 50.0/35.1 49.6/34.5 49.6/34.5 46.6/32.7 50.0/29.6 50.6/31.0 50.2/28.6 48.77/35.31 50.4/30.3 50.7/33.4 50.8/34.9 48.5/36.1 50.5/28.4 50.6/30.4 51.3/32.9 51.1/33.2 51.0/31.9 49.4/24.9 49.5/24.9 48.9/25.5 48.7/30.2 45.4/29.6 51.3/25.5 51.4/23.9 50.6/26.7 No. 4 104 1 2010 Note: R – Pelophylax esculentus; Ri – Pelophylax esculentus with introgressions; F1 – hybrids of first generation; B – backcrosses; Rec – hybrids recombinants; h – hermaphrodite; ? – sex is not determined due to young age. THE PLOIDY AND GENETIC STRUCTURE OF HYBRID POPULATIONS OF WATER FROGS 215 reproduction for hybrids and, second, that hybridiza tion with the lake frog can be a reason of its gradual disappearance within the south area. Hybrid species appeared as the result of reverse breeding with species of parental species (lake frogs with introgressions of genetic material from pond frog and allodiploids with recombinations of genetic material), and in total made up 5% of the number of studied species (table). Fifteen of seventeen triploid frogs were hybrids and two were parental species. The species identified as the P. ridibundus was characterized with an average size of erythrocytes 502 µm and did not have any introgres sions of P. ridibundus genes. Another triploid species with average sizes of erythrocytes of 501 µm in alloz ymes was determined as P. esculentus. Thus, the share of polyploids among the studied frogs was 1.6%, and their share among hybrids was 4% and among parental species their share was 0.3%. Most of the polyploid hybrids (13 species) were identified in Severskii Donets, one in Nizhnii Dnepr, and one in the lakes of Volin. The peculiarities of specters in a particular locus, Ldh B, which provides us with reliable and reproducible results, and also Aat 1 confirm that all frogs belong to biotype P. esculentus–2 ridibundus, or, as they are usually interpreted by researchers of green frogs [16], are hybrids of the RRL type. The hybrid species are characterized either by triheterozygous specters of Ldh B64/77 100, where the products of two alleles inherent to P. ridibundus were represented, or by genotypes with double products of one of the genes often belonging to this species, the Ldh B64/77 100, and less often, the Ldh B64/77 77. The hybrid triploid from Volin whose genotype carries alleles Ldh B88 and Ldh B100 and the dose gene in its specter cannot be deter mined due to the close mobility of alleles’ products when its biotypical diagnostic was carried out in locus Aat 1, whose specter had a clear prevalence of dose genes specific for P. ridibundus. All triploid hybrids were found in populations inhabited together with hybrids, which were mostly males, only by the lake frog (table). It indirectly shows that triploid hybrids are created by the backcrossing of allodiploids, producing diploid sperm, with females P. ridibundus. Triploid species of parental species, which occur infrequently, are probably of autoplyploid origin. DISCUSSION The study of the genetic structure of hybrid popu lations of frogs, including the analysis of allozymes and cytometry, confirms the results obtained previ ously. First, the rarity and scarcity of the hybrid poly ploid in populations of green frogs of Eastern Europe and, second, the relatively high number of hybrid trip loids of frogs in the Severski Donets basin. The popu lations of the Dnieper, Dniester, Zapadnii Bug, and Danube included 0.9% polyploids from the total amount of analyzed hybrids, while in the Severski CYTOLOGY AND GENETICS Vol. 44 No. 4 2010 Donets basin their share was about 9%, although this value is half the value that was noted previously for this region [24]. This should draw attention to the absence in our material of individuals of biotypes P. 2 esculen tus–idibundus whose frequency, as has been previously established by the same researchers [24] was not less than in biotypes P. esculentus–2 ridibundus. It should be mentioned that the equal representation of two alternative biotypes in this region is doubtful because the second parental species P. esculentus in the flood plain of Severski Donets was always small and there fore the backcrossing of hybrid males with females of this species was a rare event and there should only be a few of these hybrids. The reverse breeding of hybrid males with females of P. ridibundus are normal and the only way of reproduction for hybrids in populations of Severki Donets, therefore in case of the production of a small number of diploid sperm by the hybrids, the triploid P. esculentus–2 ridibundus can be produced. The estimations obtained in this experiment on the distribution of polyploidy and the correlation of trip loid biotypes in Servski Donets can differ from the results of previous studies [24] due to two reasons. The first reason is the insufficient sensitivity of the previ ously used method of flow cytometry that does not allow us to divide two alternative triploid biotypes according to genome size, which results in an artifi cially increased number of hybrids of P. 2 esculentus ridibundus in this river system. This aspect has already been discussed by other researchers [24]. The second reason, explaining the decrease in the number of poly ploid species is related to the drastic alteration of floodplain stations, drying wetlands, and salinity of lakes that has occurred in Severskii Donetsk over the past decade. As a result, the pond frog, which is rare in the floodplain of this river and vulnerable at the south ern border area, has disappeared and thus hybridiza tion has terminated. Besides, hybrids, which are more suited to life on land than lake frogs, having found themselves in drought conditions, have also disap peared. 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