Shabanov et al. (2006) Study of population systems of green frogs in Kharkiv region
Although the article presented here retains some interest, at least for its authors, the terminology requires correction. Ideas about the possible pathways of change in population systems have been refined, and certain concepts and even species names have changed. In particular, the term "...
Although the article presented here retains some interest, at least for its authors, the terminology requires correction. Ideas about the possible pathways of change in population systems have been refined, and certain concepts and even species names have changed. In particular, the term "meroclonal inheritance" had to be abandoned in favour of "hemiclonal inheritance" (more details here). The PDF of the article in its original form is available here. In this online publication, "meroclonality" has been corrected to "hemiclonality," with the edits highlighted in colour. Shabanov D.A., Zinenko A.I., Korshunov A.V., Kravchenko M.A., Mazepa G.A. Study of population systems of green frogs (Rana esculenta complex) in Kharkiv region: history, current state, and prospects // Visnyk of V.N. Karazin Kharkiv National University. Series: Biology. – 2006. – Issue 3 (No. 729). UDC 597.851(477.54) Study of population systems of green frogs (Rana esculenta complex) in Kharkiv region: history, current state, and prospects D.A. Shabanov, A.I. Zinenko, A.V. Korshunov, M.A. Kravchenko, G.A. Mazepa V.N. Karazin Kharkiv National University (Kharkiv, Ukraine) The article presents a review of Central European green frogs (Rana esculenta complex). The merohemiclonal inheritance (transmission of one of the genomes without recombination) characteristic of their interspecific hybrids is described. The main methods for studying this phenomenon are described, as well as the history of the study of green frogs in the Kharkiv region. Different types of population systems of green frogs have been registered in the Kharkiv region, including those involving di- and polyploid hybrids. It is suggested that the composition of population systems of green frogs reflects the result of their local evolution and frequency-dependent selection of hybrid lines producing different types of gametes. A scheme describing possible transformations of population systems during their evolution is proposed. Promising directions for further study of the diversity of green frogs in the Kharkiv region are formulated. Keywords: Rana esculenta complex, triploids, tetraploids, hybridogenesis, merohemiclonal inheritance, population systems, Kharkiv region. D.A. Shabanov, O.I. Zinenko, O.V. Korshunov, M.O. Kravchenco, G.A. Mazepa. The study of population systems of green frogs (Rana ecsulenta complex) in Kharkiv region: history, modern condition and prospects. The article presents a review of Central European green frogs, which includes: a specific way of interspecies hybrids reproduction, during which one genome inherited without recombination – merohemiclonal inheritance; description of the main research methods of this phenomenon and the history of studying of green frogs in Kharkiv region. Different types of green frogs' population systems have been registered in Kharkiv region, which also include di- and polyploid hybridogenetic frogs. There is an assumption that the combination of frogs' population systems is the result of their local evolution and frequency-dependent selection of hybridogenetic lines, producing different types of gametes. A scheme of possible ways of population systems transformation is suggested. The prospective ways of further studying of green frogs' diversity are also represented. Keywords: Rana esculenta complex, triploids, tetraploids, hybridogenesis, merohemiclonal inheritance, populations, Kharkiv region. D.A. Shabanov, O.I. Zinenko, O.V. Korshunov, M.O. Kravchenko, G.A. Mazepa. Study of population systems of green frogs (Rana esculenta complex) in Kharkiv region: history, current state and prospects. The article presents a review of Central European green frogs (Rana esculenta complex). Merohemiclonal inheritance (transmission of one of the genomes without recombination), which is characteristic of their interspecific hybrids, is described. The main methods for studying this phenomenon are described, as well as the history of studying green frogs in the territory of Kharkiv region. Different types of population systems of green frogs have been registered in Kharkiv region, including those that involve di- and polyploid hybrids. It is suggested that the composition of population systems of green frogs reflects the result of their local evolution and frequency-dependent selection of hybrid lines producing different types of gametes. A scheme describing possible transformations of population systems during their evolution is proposed. Promising directions for further study of the diversity of green frogs in Kharkiv region are formulated. Keywords: Rana esculenta complex, triploids, tetraploids, hybridogenesis, merohemiclonal inheritance, population systems, Kharkiv region. Merohemiclonal inheritance in Rana esculenta The complex of Central European green frogs (Rana esculenta complex) comprises three principal forms: the pool frog (R. lessonae Camerano, 1882), the marsh frog (R. ridibunda Pallas, 1771), and the edible frog (R. esculenta Linnaeus, 1758). All of them occur in the Kharkiv region. Green frogs belong to the life-form of shore-leaping water frogs, to which the emergence of the principal characteristics of the order Anura is linked. Owing to their wide distribution, high abundance, and relative accessibility, green frogs are among the best studied and most familiar animals, as well as widely used experimental subjects. Nevertheless, prior to the second half of the twentieth century the taxonomic status of the forms included in this group was repeatedly revised. This is connected both with the considerable morphological variability of these animals and with the characteristic phenomenon of interspecific hybridisation, which was discovered only in the course of experiments by the Polish hydrobiologist L. Berger (Berger, 1964 and subsequent works). Thus, Berger found that when R. lessonae and R. ridibunda are crossed, the offspring consist of individuals that should be referred to as R. esculenta. When such hybrids were crossed with R. lessonae, the offspring also consisted exclusively of R. esculenta. The Austrian biologist Heinz Tunner, who used electrophoresis of protein markers to distinguish different frog forms, was able to explain these results by means of the hypothesis of clonal inheritance (Tunner, 1974). Tunner and other researchers (e.g., Borkin et al., 1987) were able to demonstrate that one of the parental genomes is eliminated from the cell prior to meiosis, while the other is duplicated and transmitted to the gametes clonally, without recombination. A schematic explanation of the results described by Berger, as well as certain other phenomena arising from crosses within the Rana esculenta complex, is given in Table 1 (in the table, the genomes are denoted by letters: L for the pool frog and R for the marsh frog). The genotype LL corresponds to the pool frog, RR to the marsh frog, and LR to their hybrid, the edible frog. One of the genomes of the hybrid frogs (shown in bold) is transmitted clonally, without recombination with the other genome. The other, non-clonal genome is utilised throughout the life of the hybrid individual and is eliminated prior to meiosis. Table 1. Schemes of various types of reproduction in the Rana esculenta complex. The genome of the pool frog is designated L, and that of the marsh frog R; the genome eliminated during gametogenesis is shown with a strikethrough. A. Formation of a hybrid by crossing the parental species B. Reproduction of the hybrid when crossed with R. lessonae Parents Gametes Offspring R. lessonae R. ridibunda LL x RR L R LR R. esculenta R. esculenta R. lessonae LR x LL L R L LR R. esculenta C. Reproduction of the hybrid when crossed with R. ridibunda D. Hybridolysis — segregation of the parental species when hybrids are crossed Parents Gametes Offspring R. esculenta R. ridibunda LR x RR R L R LR R. esculenta R. esculenta R. esculenta LR x LR R L L R LL R. lessonae As can be seen when comparing variants B and C in Table 1, when co-existing with one of the parental species, hybrid frogs produce gametes of the other species (complementary gametes), which enables them to reproduce hybrids in the next generation as well. This rule does not hold in all cases, but those hybrid forms that produce gametes of the same parental species with which they interbreed will disappear from the population as a result of such crossings. Conversely, hybrid forms that produce gametes complementary to the parental species will reproduce successfully and will be favoured by selection. When hybrid frogs producing gametes of the same parental species are crossed with one another, that species may segregate in the offspring (see Table 1, D). This phenomenon has been termed hybridolysis (from Latin hybrida — a hybrid, and Greek lysis — decomposition, dissolution). Individuals arising as a result of hybridolysis usually exhibit reduced viability, which may be a consequence of changes accumulating in the clonal genome as it is transmitted from generation to generation without recombination. A whole series of terms has been proposed to denote the mode of reproduction registered in green frogs. An analogous phenomenon was first described in Mexican topminnows of the genus Poeciliopsis (Schultz, 1969) under the name "hybridogenesis" (from hybrida and Greek genesis — origin). This name is not very apt, since the essence of the phenomenon consists not so much in the origin of hybrids as in their reproduction at the expense of one of the parental species — a kind of "gametic (genomic) parasitism" (Günther, 1979). Since the genome of one of the parental forms is used only temporarily, L.Ya. Borkin and I.I. Darevsky (1980) proposed calling it "creditogenesis" (from Latin creditum — a loan). Various authors also use the terms "kleptogenesis" (from Latin klepto — I steal; Dubois, Günther, 1982), as well as "hemiclonal" (from Latin hemi- — half- and Greek clon — offspring, branch) and "meroclonal" (from Greek meros — part, portion) inheritance. In our view, the optimal term is meroclonal inheritance, as it is the most general in character. It is not contradicted either by the inheritance of one or two genomes in triploid hybrids, or by the recorded cases of recombination between genomes (see below). Some of these terms are compatible with one another: for example, one may speak of merohemiclonal inheritance in creditogenesis. In connection with the foregoing, a natural question arises: should the species name R. esculenta be used to designate hybrid frogs? In favour of granting edible frogs independent status is the fact that hybrids do not arise anew with each crossing of parental species, but are stably reproduced through successive generations, transmitting the clonal genome and acquiring new characteristics in the course of evolution. Hybrids have their own range, which extends beyond the intersection of the ranges of the parental species. Moreover, it is known that hybridisation of the parental species in nature occurs fairly frequently, but the products of such hybridisation usually have reduced viability and are often sterile, and therefore lose out to hybrids reproducing by hybridogenesis (Plötner, 2005). A significant consideration is that hybrids can form pure populations in which they exist without interbreeding with the parental species. Incidentally, R. esculenta was described by Carl Linnaeus considerably earlier than later researchers described its parental species R. lessonae and R. ridibunda. In this case, as in many others, the richness of life's manifestations does not fit into the Procrustean bed of unambiguous rules and unified categories. The edible frog is neither an ordinary hybrid nor a true species. To assign a definite taxonomic status to such hybrid forms, the taxonomic category of klepton was proposed (Dubois, Günther, 1982). In that case, the name of the hybrid frogs widespread in the Kharkiv region should be written as Rana klepton esculenta (abbreviated R. kl. esculenta). Although in this article hybrid forms are simply designated as R. esculenta, it should be borne in mind that this name is applied to a special form rather than an ordinary species. Diversity of population systems of green frogs Despite the ecological specificity of R. lessonae, R. ridibunda, and R. esculenta, all of them are capable of inhabiting the same biotopes, being associated with the banks of various freshwater bodies. During the breeding season they may form joint breeding groups comprising representatives of different forms. Within these groups, not only do the said forms reproduce, but hybridisation also occurs. Since the term "population" does not apply to groups including representatives of different species, the aggregations of green frogs inhabiting certain biotopes and jointly participating in reproduction are customarily referred to as "population systems." Following Berger's discovery, a whole series of population system types for green frogs was described, differing both in their composition and in the character of the gametes produced by the hybrid forms. The most general of the proposed classifications of population systems of the Rana esculenta complex (Uzzell, Berger, 1975; Lada, 1995) includes the following 7 types: L-type — ordinary populations of R. lessonae, the pool frog; R-type — ordinary populations of R. ridibunda, the marsh frog; E-type — so-called "pure" populations of the hybrid form, R. esculenta; L-R-type — population systems consisting of marsh and pool frogs reproducing without the formation of hybrids; L-E-type — population systems of R. lessonae and hybrids (see the second case in Table 1); R-E-type — population systems analogous to the preceding, in which hybrids coexist with R. ridibunda; R-E-L-type — mixed systems comprising both parental species and their hybrids. For a considerable part of the range (Western Europe), "pure" population systems of the E-type include a significant proportion of triploid hybrids. In population reproduction, triploids may replace the parental species, producing gametes that complement the gametes of diploid hybrids. Such systems are recommended to be classified as the E-t-type and not considered "pure," recognising only diploid Rana esculenta as a species or klepton (Plötner, 2005). It should be noted that the classification given above does not describe the full diversity of population systems of green frogs. These systems may differ from one another in several additional parameters, a detailed discussion of which lies beyond the scope of this article. Among these are: — the quantitative ratio of the various forms; — the sex composition of each form, primarily the hybrids, which is linked to the fact that the clonal genomes may be either male or female; — the presence or absence of individuals with recombined genomes; — the presence or absence of hybrids differing in ploidy (number of genomes) and their composition; — the character of the gametes (number and composition of genomes) produced by the various hybrid forms; — the composition of the parental forms, each of which may be represented by different genomes or even cryptic species (Plötner, 2005; Borkin et al., 2004). It must be noted that in recent years the number of described taxa — both species and hybrid forms — of European green frogs has substantially increased. According to the latest review (Plötner, 2005), the R. ridibunda group also includes R. bedriagae Camerano, 1882, R. terentievi Mezhzherin, 1992, R. cretensis Beerli, Hotz, Tunner, Heppich & Uzzell, 1994, and R. epeirotica Schneider, Sofianidou & Kyriakopoulou-Sklavounou, 1984. The R. lessonae group includes R. bergeri Günther, 1985, R. shqiperica Hotz, Uzzell, Günther, Tunner & Heppich, 1987, R. perezi Seoane, 1885, and R. saharica Boulenger in Hartert, 1913. Not associated with either of the "principal" species but belonging to the same complex are R. cerigensis Beerli, Hotz, Tunner, Heppich & Uzzell, 1994 and R. caralitana Arikan, 1988, as well as certain other forms currently under investigation but not yet formally named. In addition to R. esculenta, the kleptones include R. hispanica Bonaparte, 1839 (the product of crossing R. bergeri and R. ridibunda) and R. grafi Crochet, Dubois, Ohler & Tunner, 1995 (a hybrid of R. perezi and R. ridibunda). It may be anticipated that further study of this remarkable group of animals will lead to an even greater increase in the number of forms included in it. In studying the population systems of green frogs, it should be borne in mind that they are not bounded by rigid limits. This group of animals is characterised by the formation of metapopulations — aggregations of many permanent or temporary breeding groups, between which exchange of individuals takes place. Individual breeding groups may differ from one another in composition, and through migration may exert an influence on other groups within the overall metapopulation. From what has been said it becomes clear why the study of green frogs attracts more attention in European herpetology than any other problem. Methods for studying the hybridogenetic complex of frogs For the diagnosis of frog forms, a whole complex of methods has been employed by various researchers. The three principal forms are sufficiently well distinguished from one another morphologically (Table 2). Unfortunately, the existence of individual variability, the presence of triploids and tetraploids, and of partially recombinant forms renders morphological discrimination of the entire complex impossible. For recognition of the three principal forms of the Rana esculenta complex under field conditions, the acoustic criterion proves useful — different frog forms differ in their vocalisations. In addition, R. lessonae, R. ridibunda, and R. esculenta differ (Table 2) in habitat preferences, behaviour, colouration (Lada, 1995; Broska, 1982; Ananeva et al., 1998), and even in odour (our data). For most of these parameters, Rana esculenta exhibits values intermediate between R. lessonae and R. ridibunda. A number of works (e.g., Plenet et al., 2000; Negovetic et al., 2001) have recorded substantial ecophysiological differences among the named forms of green frogs. The ecological differences among members of the Rana esculenta complex lead to a certain partitioning of their ecological niches and alleviate interspecific competition. The niches of tadpoles are partitioned to a considerably lesser degree. Reproduction within shared population systems acts as a factor impeding deeper niche partitioning between R. esculenta and its parental species (Holenweg Peter, Reyer, 2002). The data presented in Table 2 allow fairly reliable identification of the parental species and diploid hybrids under field conditions, but are not suitable for identifying polyploid hybrids. Study of the size of erythrocytes and eggs can provide information about the number of chromosomal sets (cells of tri- and tetraploids are larger), but does not allow determination of the origin of the genomes. Study of karyotypes by classical methods (e.g., Manilo, 2005) likewise provides only general information about the chromosomal complement (enabling the detection of polyploid and aneuploid cells), but does not provide reliable methods for identifying the species affiliation of the chromosomes under study. The repeatedly published data on morphological differences between the chromosomes of R. lessonae and R. ridibunda are contradictory (e.g., Suryadnaya, 2003 and Suryadna, 2005) and do not provide a reliable basis for determining the genotype of the individuals studied. Identification of individual genomes is possible only with the aid of differential chromosome staining technologies (e.g., Miura et al., 1997). Table 2. Comparison of certain characteristics of the three principal forms in the Rana esculenta complex (Lada, 1995 et al.) Character Pool frog (R. lessonae Camerano, 1882) Edible frog (R. esculenta Linnaeus, 1758) Marsh frog (R. ridibunda Pallas, 1771) Size and shape of the inner metatarsal tubercle and length of the first toe [IMG_1] Relative length of the shin Colouration of the resonators in males White or yellowish-white Light grey Dark grey to black General body colouration Usually green, sometimes bright light green; nuptial colouration present; yellow colour on the hind limbs Intermediate between the parental species; yellow colour on the hind limbs From green to almost black Odour Weak odour Strong odour Pungent, distinctive odour Male advertisement call (after the introductory note) Chirping Intermediate in character between the parental species Rolling, resembling laughter Behaviour when threatened Swim some distance underwater and resurface Intermediate between the parental species Lie concealed on the bottom Behaviour of males towards competitors Highly aggressive Aggressive Relatively neutral Hibernation On land Together with the parental species In water Preferred habitats Small waterbodies in the forest zone; lives on land outside the breeding period Various, except the extreme types characteristic of the parental species Large waterbodies in open landscapes Significant progress in the study of merohemiclonal inheritance and the diversity of green frogs was linked to the study of protein markers by gel electrophoresis. This method also allows recognition of certain recombinant individuals (in which recombination has affected the markers under consideration), and — owing to the gene dosage effect — enables the identification of triploids among hybrids (Günther, 1975). A convenient and powerful method for studying the hybridogenetic complex proved to be flow DNA cytometry, which allows determination of the amount of DNA per cell (Borkin et al., 1987). This method is based on measurement of the UV fluorescence of DNA in individual cells (erythrocytes or spermatozoa). The measurement error in such a study is considerably smaller than the difference in genome size between the parental species, which makes it possible not only to reliably determine the number and species affiliation of the genomes of adult individuals, but also to establish which genomes the spermatozoa carry. Since the genome weight of R. ridibunda is approximately 8 pg (picograms), and that of R. lessonae approximately 7 pg, it is clear, for example, that an individual with a genome weight of 22 pg is a triploid LLR. Recombinant individuals with an altered amount of DNA in the genome can be identified by this method with a considerably lower degree of confidence. In recent years, identification of individual genotypes of green frogs by characteristic microsatellite DNA sequences has become widespread (e.g., Christiansen et al., 2005). Probably, in the future this method will become the primary one used for studying both green frogs and many other groups of organisms. Interest in the hybridogenetic complex of European green frogs, which flared up some forty years ago, has not abated to this day. Various aspects of this problem are investigated by a number of European universities, museums, and laboratories. To date, three international conferences devoted to the problems of studying the Rana esculenta complex have already been held. The two principal centres for the study of green frogs situated in the territory of the former USSR are located in St. Petersburg and Kyiv. At the ZIN and CIN RAS, the method of flow DNA cytometry is used primarily. At the I.I. Schmalhausen Institute of Zoology of the NAS of Ukraine and the Zoological Museum of the NMNH of the NAS of Ukraine, electrophoresis of protein markers, karyoanalysis, and immunological studies are also conducted. More recently, field, morphometric, and cytogenetic research has been initiated at Kharkiv University by the authors of the present work. As G.A. Lada (1995) aptly remarked, "in the problem of Central European green frogs, questions of morphology, ecology, ethology, systematics, cytology, biochemistry, genetics, palaeontology, and evolutionary theory are all tied together in one tight knot"; possibly developmental biology and biogeography should also be added to this list. Study of green frogs in Kharkiv region The study of green frogs inhabiting the Kharkiv region began as early as the mid-nineteenth century (Chernai, 1852). Even earlier, Professor I.A. Krynicki of Kharkiv University (1837) described from the vicinity of Pyatigorsk the species Rana dentex Krynicki, 1837, now placed in synonymy with R. ridibunda. One stage in the history of the study of frogs is associated with the activities of A.M. Nikolsky, the founder of the school of herpetology in Russia and the USSR. Most of his scientific career was spent in Kharkiv, including at Kharkiv University. Encountering the existence of transitional forms among different forms of green frogs, Nikolsky regarded them as subspecies of a single species, R. esculenta. It should be noted, however, that even during his Kharkiv period, A.M. Nikolsky studied mainly material from a considerable part of the territory of the former Russian Empire, rather than local specimens. Following the discovery of the hybrid nature of R. esculenta, many researchers were drawn to study green frogs. The Kyiv herpetologist S.V. Tarashchuk (1985) proposed a multiplicative index intended for the discrimination of different forms of green frogs. This index was applied by V.I. Vedmedera (1984), a staff member of Kharkiv University, to describe the diversity of frogs from the Kharkiv region even before the publication of information about this index in print. The study of frogs from this region by biochemical methods was initiated by staff members of the Zoological Institute of the Russian Academy of Sciences in 1989, with the participation of A.M. Rudik. In 1995 and 1996, the Tambov herpetologist G.A. Lada visited the Kharkiv region and described at Iskov Pond in the vicinity of the biological field station of Kharkiv University in Haidary a large "pure" population of diploid R. esculenta (Lada, 1998). The Kharkiv region is situated on the boundary of the range of one of the parental species, R. lessonae. The range of R. esculenta broadly coincides with that of R. lessonae (R. ridibunda is distributed far more widely), but in some places extends beyond it. R. lessonae enters the Kharkiv region only in its north-west (Krasnokutsky and Bohodukhivsky districts) and south-west (Zachepylivsky district, the Russky Orchik tract). It is possible that local populations of R. lessonae also exist in other northern districts of the region, but their existence has not been reliably demonstrated at present. At the same time, the range of R. esculenta extends beyond these limits in a south-easterly direction, reaching as far as the Izium district of the Kharkiv region, and extends further downstream along the Siverskyi Donets. In this part of the range, hybrid frogs exist mainly within population systems of the R-E-type. According to preliminary data based on DNA cytometry and field observations, six variants of population systems belonging to the R-, R-E-, and R-E-L-types have so far been registered in the Kharkiv region: I. Individuals of both sexes with genotype RR (R-type); II. Individuals of both sexes with genotypes RR and LR, as well as solitary immature individuals LL, probably segregating as a result of hybridolysis (R-E-type); III. Individuals of both sexes with genotypes RR, LRR, LR, LLR, as well as solitary immature individuals LL and LLRR (R-E-type with triploids); IV. Individuals of both sexes with genotypes LR, as well as solitary adult individuals RR (the so-called "pure" E-type, which in all probability is connected by transitional forms with the R-E-type); V. Males LR and solitary adult individuals RR (this system may be referred to as the Em-type); VI. Individuals of both sexes with genotypes RR, LR, and LL (R-E-L-type). Polyploid hybrids are fairly widespread in Western Europe. In the territory of the former Soviet Union, despite systematic searches, by 2001 only a few isolated individuals had been recorded. In 2002, in the course of joint work by staff members of the Zoological Institute of the RAS and the Institute of Cytology of the RAS (St. Petersburg), together with Kharkiv University, a significant proportion of triploid hybrids was registered in samples from the Kharkiv region (Borkin et al., 2004). At present, the number of representatives of the Rana esculenta complex from the Kharkiv region examined by cytometry has reached 813 individuals from 48 sites (Borkin et al., 2005). The composition of the gametes produced by males of the most widespread forms of green frogs has been determined. A preliminary assessment of the distribution of different forms of green frogs and types of their population systems across the studied territory has been obtained (Borkin et al., 2004; Borkin et al., 2005). The results of investigations in the Kharkiv region indicate the systematic occurrence of polyploids in the population systems of green frogs inhabiting waterbodies located in the Siverskyi Donets basin. In some of these systems, the proportion of triploids amounts to approximately 40% of the total number of hybrid frogs. The zone of triploid distribution that has been discovered is separated from similar regions by a considerable gap: the nearest areas where mass finds of polyploid individuals were previously made are located approximately 1,000 km away (in Poland) and approximately 1,500 km away (in western Hungary). Of special interest is the recent discovery of triploid hybrid frogs in waterbodies of the Siverskyi Donets floodplain in the Rostov oblast (Borkin et al., 2006). The proportion of triploids in this area is even higher than in the Kharkiv region. Presumably, the phenomena registered in the Kharkiv and Rostov regions are closely linked and pertain to a single centre of green frog diversity situated in the Siverskyi Donets basin. The Rana esculenta complex in the vicinity of the KhNU Biological Field Station and on the territory of the Homilsha Forests National Nature Park To characterise the complexity of the task involved in studying the diversity of green frogs, the composition of the most thoroughly studied metapopulation from the Kharkiv region may be considered (Table 3). It is situated in the Zmiiivsky district, in the vicinity of the KhNU Biological Field Station and on the territory of the Homilsha Forests National Nature Park (the floodplain of the Siverskyi Donets and the Homilsha river, temporary waterbodies and ponds in the Koryakiv and Iskov ravines near the village of Haidary, and in the Dobrytsky ravine near the village of Velyka Homilsha). On this territory, local population systems of green frogs, connected by migration routes, are situated. To date, staff members of the ZIN and CIN RAS have determined the genotypes of more than 350 individuals from this metapopulation. The composition of spermatozoa has been determined for 38 diploid hybrid males, as well as 3 triploids LRR (Borkin et al., 2005). In addition, a substantial volume of data on the distribution and abundance of different forms, identified on the basis of morphological and bioacoustic criteria, has been accumulated. Among diploid males of R. esculenta, three types of gamete formation were registered (Borkin et al., 2005), represented in approximately equal numbers of individuals. These are males producing exclusively spermatozoa with the R genome, males producing exclusively spermatozoa with the L genome, and males producing a mixture of spermatozoa with both the R and L genomes. Of greatest interest are the individuals of the last group, capable of simultaneously producing gametes of two different species. The cytogenetic mechanism providing for this type of gametogenesis remains enigmatic. One probable explanation might be the mosaic nature of such individuals, if they consist of a mixture of cells with different genotypes. Evidence that such mosaicism is possible may be provided by the recently discovered mixoploid green frogs from Zhytomyr oblast (Manilo, 2005). It is possible that male green frogs may also produce diploid spermatozoa; however, it is impossible to distinguish them from other diploid cells that may be present in the sample under study by means of flow cytometry (S.N. Litvinchuk, personal communication). Table 3. Composition of the metapopulation of green frogs in the vicinity of the KhNU Biological Field Station and on the territory of the Homilsha Forests National Nature Park, and the gametes produced by the various forms ("+++ " — more than 10% of individuals of the corresponding category; "++" — from 1 to 10%; "+" — 1% or less; "?" — data differ for different local population systems) RR LR LLR LRR LL LLRR Immature +++ +++ + +++ + + Females +++ ++ (?) + ++ (?) Possibly do not survive to reproductive age Males +++ +++ no data ++ (?) Gametes of males R L R L, R no data R Gametes of females no data Within the said metapopulation, individual, particularly interesting local population systems may be distinguished. One of them inhabits Iskov Pond, situated near the village of Haidary. As already mentioned, G.A. Lada (1998) studied this pond in 1995 and 1996 and found there a large "pure" population of R. esculenta, noting that effective population reproduction was taking place in it. In 2001, Iskov Pond was drained, but by 2002 it had already been refilled. In 2003, R. esculenta individuals of both sexes were observed in it. However, in 2005, during the breeding season at Iskov Pond, only male R. esculenta were recorded, while among females it was only possible to find 2 R. ridibunda individuals. These data, together with the results of studying other population systems resembling Iskov Pond, made it possible to advance the hypothesis that "pure" population systems of R. esculenta, consisting only of males, can exist owing to female R. ridibunda immigrants arriving from other habitats (Korshunov, 2005). An analogous explanation has been proposed in the literature to account for the existence in western Ukraine of pure R. esculenta populations consisting entirely of females (Mezhzherin et al., 2005). According to our hypothesis, the overwhelming majority of crossings at Iskov Pond (and in other population systems of the Em-type) corresponds to the scheme shown in Table 4. It may be supposed that previously the hybrid frogs inhabiting Iskov Pond were transmitting different types of genomes clonally. When the stable population structure was disrupted as a result of the draining and subsequent refilling of the pond, and as a result of mixing with frogs from other population systems, only one clonal line survived from the various clonal lines — that with the male clonal genome of R. lessonae. Males transmitting the genome LY, in any crossings with females producing R-gametes, produce the same type of males (and in crossings with hybrid females producing L-gametes, hybridolysis R. lessonae arise, which do not survive to maturity). The population system of Iskov Pond has found itself in an evolutionary dead end. Even if other male frogs enter it, due to the surplus of LYRX males, they have very slim chances of fertilising the available females. According to the hypothesis being expounded, the effective population size of the green frog population system at Iskov Pond is critically low, and its reproduction depends on migrant individuals. The result of this should be the degradation of this population system, which is to be expected in the coming years. Table 4. Hypothetical mechanism of reproduction in "Em-type" populations, using Iskov Pond in the village of Haidary as an example. The subscript y corresponds to the genome containing the male (heterogametic) sex chromosome, and X the female (homogametic) one. Parents Gametes Offspring ♀ R. ridibunda ♂ R. esculenta RXRX x LYRX RX LY RX LYRX ♂ R. esculenta Dynamics and stability of different types of population systems The hypothesis reflected in Table 4 illustrates that different types of population systems of green frogs may differ in their stability. This means that such systems should be studied not only in statics but also in dynamics. Various transformations of population systems have been registered by several authors (e.g., Holenweg Peter, 2001; Vorburger, Reyer, 2003; Mezhzherin et al., 2005), and methods of mathematical modelling of the processes occurring in them have also been used (Hellriegel, Reyer, 2000; Reyer et al., 2004, and others). In our view, the described processes may be regarded as parts of a general sequence of possible transformations of population systems. To study their diversity, we propose a general scheme of changes in population systems of the Rana esculenta complex (Fig. 1). In our view, such a scheme may prove useful for modelling the dynamics of the described processes and for studying them under field conditions. [IMG_2] Fig. 1. Possible sequence of transformations of different types of population systems of the Rana esculenta complex Before proceeding to a detailed examination of the proposed scheme, it must be noted that it does not account for three circumstances that are significant for the analysis of the dynamics of population systems of green frogs in each specific case. First, these are the differences between R. lessonae and R. ridibunda, whose relationships with the hybrid forms are not entirely symmetrical. For example, owing to the preference of male frogs for larger females, crossing of ♀ R. ridibunda × ♂ R. lessonae is far more probable than ♀ R. lessonae × ♂ R. ridibunda. An analogous selectivity is manifested in crossings in which R. esculenta participates (Abt, Reyer, 1993). Such and other manifestations of sexual selection may exert a substantial influence on the composition of frog population systems (Som et al., 2000). Second, these are the differences between population systems with respect to the sex composition of the forms within them and the sex affiliation of the clonal genomes. Thus, a female clonal genome may be transmitted to both females and males of the hybrid forms, whereas a male one — only to males. Third, the proposed scheme does not account for the specifics of the habitats, which correspond to the requirements of the three forms within the Rana esculenta complex to varying degrees and thereby influence their ratios in population systems. As the starting point of the evolution of population systems, a population of one of the parental species should be considered, into which representatives of the other species or merohemiclonal hybrids immigrate (Fig. 1, stage 1). If the immigrating individuals of the other species cannot form merohemiclonal hybrids with the host species but occupy their own niche, the population system will transition to the L-R-type (Fig. 1, stage 2). Immigration of the other parental species that does form merohemiclonal hybrids with the host species is equivalent to immigration of the hybrids themselves. The hybrids may be represented by different lines, differing in the clonal genomes they transmit. These clonal genomes may differ in their species affiliation, sex, and genetic characteristics. Only those hybrid lines that produce complementary gametes (for example, gametes of R. lessonae when immigrating into a population of R. ridibunda; see variants B and C in Table 1) will be able to reproduce successfully in the parental species population. Lines producing gametes similar to the parental species dominating in the population system will simply dissolve through crossings. Thus, populations of the parental species dominating in a given region act as a filter, retaining one of the hybrid forms. In the joint inhabitation of the numerically dominant parental species and a hybrid producing complementary gametes, the proportion of hybrid individuals will increase (Fig. 1, stage 3; Vorburger, Reyer, 2003, and other works). The reason for this is that the offspring from crossing parental species individuals with hybrids will be entirely hybrid. As the proportion of hybrids increases, the number of individuals of the other parental species segregating as a result of hybridolysis will grow. If the products of hybridolysis prove to be viable and fertile, the population system will transform into the R-E-L-type (Fig. 1, stage 4). If hybridolysis individuals perish, the increase in the proportion of hybrids will lead to an increase in the genetic load in the population. Nevertheless, as parental species individuals continue to interbreed with hybrids, the proportion of the latter will increase further (Fig. 1, stage 5). As field observations show, in many cases a certain stable proportion of hybrids is reached in natural population systems (Fig. 1, stage 6). This is probably connected with various manifestations of hybrid dysgenesis: elevated mortality of hybrids, developmental disorders, reduced fertility, and increased proportion of aneuploid gametes from which nonviable offspring develop (Christiansen et al., 2005, and others). In that case, the equilibrium ratio between hybrids and parental individuals will reflect their relative viability (Holenweg Peter, 2001; Reyer et al., 2004). As already noted, when hybrids immigrate into a parental species population, selection should favour lines producing gametes complementary to the parental species. However, selection of hybrid lines is frequency-dependent, and as the proportion of hybrids of one line increases, favourable opportunities will open up for other hybrid forms. Thus, in a population dominated by hybrids producing L gametes (Fig. 1, stage 7), selection may favour the spread of diploid and triploid individuals producing R gametes (Table 3). The equilibrium proportion of representatives of these lines in the population will be determined, among other things, by the level of viability and the specifics of gametogenesis of hybrid individuals that have received the clonal L genome from one parent and the clonal R genome from the other. Thus, one of the outcomes of the evolution of a population system may be a transition to a system with several cytogenetic forms of hybrids (Fig. 1, stage 8). However, if for some reason (the absence of the necessary hybrid lines or their nonviability) this does not occur, hybrids complementary to the original parental species may displace it completely or almost completely. The result may be a population system whose reproduction depends on the influx of migrants and whose effective size has been reduced to a critical level (Fig. 1, stage 9). Selection in such population systems will favour the spread of hybrid lines producing other types of gametes. If the population system develops a variant combination of different frog forms differing in genotype and in the gametes they produce, which ensures effective reproduction, it will persist; if not — it will degrade. After some time, the vacated habitat will be colonised by migrants from other local populations. Iskov Pond is probably at this stage of evolution of the frog population system. Moreover, the clonally transmitted female genome was probably lost in it as a result of the temporary disruption and restoration of the pond (Table 4). "Pure" population systems of the edible frog may be an example of a stable type of reproduction that has developed following the displacement of the parental species by hybrids. As examples of Western European population systems show, a stable variant may be a combination of di- and triploid hybrids. Frequency-dependent selection may establish the optimal ratio of such forms in common population systems. According to the concept of reticulate evolution advanced by L.Ya. Borkin and I.I. Darevsky (1980), the next stage in the evolution of "pure" population systems may be their transition to parthenogenetic reproduction. It must be noted that diploid hybrids producing two types of gametes simultaneously (see Table 3) may be the only hybrid form capable of forming populations consisting of uniform individuals. However, in such populations (assuming equal production of L and R gametes), half the offspring will consist of hybridolysis individuals, which should die at one stage or another of ontogeny before reaching sexual maturity. As is clear from the foregoing reasoning, the evolution of any given frog population system depends on the properties of the hybrids comprising it. Many researchers point out that clonal transmission of a genome should promote the accumulation of mutations within it. The model referred to as "Muller's ratchet" is invoked, in particular, to describe this process (Plötner, 2005; Hedrick, 2003; a ratchet is a gear with hooked teeth that can rotate in only one direction). In accordance with this analogy, it may be expected that in the clonal genome, accumulation of mutant alleles will occur without their removal through recombination. It is precisely the rotation of such a "ratchet" that leads to the nonviability of individuals segregating as a result of hybridolysis. Hybrid frogs arising from crossing different clonal lines receive different clonal genomes, for which coincidence of lethal mutations is improbable. The fact that such individuals are often viable (Vorburger, 2001) confirms the hypothesis that changes in the clonal genome are predominantly deleterious. However, in our view, alternative hypotheses should also be considered. In merohemiclonal hybrids, unlike the overwhelming majority of other organisms, the genomes perform different functions. The clonal genome ensures its own transmission from generation to generation and the elimination of non-clonal genomes. The non-clonal genome performs the usual role, aside from the fact that it functions jointly with the clonal genome and is excluded from transmission to subsequent generations. Changes (both adaptive in terms of the functions performed, and stochastic) in the non-clonal genome are lost, while those in the clonal genome accumulate and determine its specific evolution. As noted above, the clonal lines of R. esculenta reproduce far more effectively than hybrids arising from crossing of parental forms (Plötner, 2005). This circumstance is probably a consequence of the adaptation of the clonal genome to merohemiclonal reproduction. Competition between different hybrid lines may lead to an increase in the proportion of clonal genomes that more successfully ensure their own reproduction. Such clonal genomic evolution belongs to a theoretically novel, practically unstudied category of evolutionary change. The most important conclusion from the foregoing reasoning is that the diversity of types of frog population systems reflects a continuously ongoing evolutionary search within them. The study of these phenomena requires a combination of field and cytogenetic research, as well as the use of mathematical modelling. Prospects for further study of the Rana esculenta complex in Kharkiv region There is no doubt that the centre of green frog diversity discovered in the Kharkiv region requires further study. Among the most important tasks requiring resolution, the following should be mentioned: — determining the boundaries of the distribution of the various frog forms and types of their population systems in the Siverskyi Donets basin; — describing the ecological, ethological, morphological, and other differences among individuals belonging to different cytogenetic forms (differing in the number, composition, and origin of their genomes and the composition of the gametes they produce); — recording the composition of population systems in different types of natural habitats and establishing the system of their reproduction (mate choice, composition of parental gametes, and composition of offspring); — elucidating the specifics of meiosis and gametogenesis in triploid individuals; — establishing the cytological mechanisms enabling certain hybrid frog individuals to simultaneously produce gametes of both parental species; — developing mathematical models of population reproduction in green frogs and verifying the predictions derived from these models against natural population systems. For solving complex cytogenetic problems, karyoanalysis in squash preparations of macerated tissues according to the modified method of V.V. Klymenko (Klymenko, 2001) appears promising. Probably, with the aid of this method it will be possible to determine the karyotype of oocytes, to detect diploid spermatocytes, and to study the topography of cells with different karyotypes in frog tissues — something that is practically impossible by other means. The authors express sincere gratitude to their colleagues in the study of green frogs in the Kharkiv region: L.Ya. Borkin, M.V. Vladimirova, G.A. Lada, S.N. Litvinchuk, V.V. Manilo, S.Yu. Morozov-Leonov, Yu.M. Rozanov, T.S. Fomenko, and A.V. Shabanova, and also thank Professors L.A. Atramentova, G.N. Zholtkevich, and V.V. Klymenko for valuable consultations. References Ananeva N.B., Borkin L.Ya., Darevsky I.S., Orlov N.L. Amphibians and Reptiles. 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A. Formation of a hybrid when crossing parent species
B. Reproduction of a hybrid when crossing it with R. lessonae
Parents
Gametes
Descendants
R. lessonae
R. ridibunda
LL
x
RR
L
R
LR
R. esculenta
R. esculenta
R. lessonae
LR
x
LL
L
R
L
LR
R. esculenta
C. Reproduction of a hybrid when crossing it with R. ridibunda
D. Hybridization - segregation of the parent species when crossing hybrids
Parents
Gametes
Descendants
R. esculenta
R. ridibunda
LR
x
RR
R
L
R
LR
R. esculenta
R. esculenta
R. esculenta
LR
x
LR
R
L
L
R
LL
R. lessonae