Meleshko et al. (2014) The study of three HPS Pelophylax esculentus complex

The study of three hemiclonal population systems Pelophylax esculentus complex from the Seversko-Donetskiy center of green frogs’ diversity O. V. Meleshko, O. V. Korshunov, D. A. Shabanov V.N. Karazin Kharkiv National University meleshko.e.v@gmail.com The structure of hemiclonal population systems (HPS) Pelophylax esculentus complex from the Iskov pond (Gaydary village, Zmiyiv District), the Lower Dobritskiy pond (the region of Gomolsha river floodplain, Dobritskiy yar, the territory of the National Park “Gomolshanski lisy”) and from the pond near the village Zhovtneve (Vovchanskiy District, Kharkiv Region) was studied. All studied HPS were defined as REt-type. We observed the triploid hybrids number increasing in the HPS from the Iskov pond. The large part of triploid hybrids was found in the HPS of the Lower Dobritskiy pond. It was observed that all triploid hybrids from the pond near the village Zhovtneve were females. There was significant bond between the diploid erythrocytes size and sex of the frogs. It seems that triploids in this HPS are originating in other way than compare to other HPS. There were two groups of females differs in body size which possibly corresponds to different ontogenetic strategies of green frog females. The males have a normal size distribution. Key words : hemiclonal population systems, Seversko-Donetskiy center of green frogs diversity, Pelophylax esculentus complex, species composition, triploid hybrids, size distribution. Introduction There are three main forms of European green frogs ( Pelophylax esculentus complex): the pool frog, Pelophylax lessonae (Camerano, 1882), the lake frog, Pelophylax ridibundus (Pallas, 1771), and the edible frog, Pelophylax esculentus (Linnaeus, 1758). In 1964, L. Berger discovered the green frogs’ phenomenon of interspecific hybridization (Berger, 1964) and found that P. esculentus is interspecies hybrid. The main distinctive feature of green frogs is a hemiclonal inheritance comprising the clonally transfer of one of the parental genomes to gametes, i.e. without recombination. In the case of crossing between hybrids frogs producing gametes of the same parental species, offspring are usually unviable. This phenomenon is called hybridolysis (Plötner, 2005). Apart from that, different forms of the green frogs from Pelophylax esculentus complex are able to live together and breed in the same area despite of their ecological specificity. This property determines the existence of various types hemiclonal population systems (HPS), which are differs in the presence and dominance of the one or another form, as well as the nature of the gametes produced by hybrid forms (Shabanov et al., 2009; Shabanov, Litvinchuk, 2010). There are several basic types of HPS (Uzzell, Berger, 1975): L-type — ordinary populations of P. lessonae , the pool frog; R-type — ordinary populations of P. ridibundus , the lake frog; E-type — so called «pure» populations of hybrids, P. esculentus ; LR-type — population systems of P. lessonae and P. ridibundus ; breeding produces no hybrids; LE-type — population systems of P. lessonae and P. esculentus ; RE-type — population systems of P. ridibundus and P. esculentus ; LER-type — mixed systems, comprising both parental species and their hybrids. It is common to indicate the presence of triploids in HPS by adding the letter “t” to mark the population system. For example, REt-type systems compose of P. ridibundus , as well as di- and triploid P. esculentus ; Et-type systems consist of diploid and triploid hybrids only. The features of Seversko-Donetskiy center of green frogs’ diversity. The region of the HPS’ high diversity is associated with the Seversky Donets river basin within Kharkiv and Donetsk Regions. Moreover, polyploid hybrids are often found in this region (Borkin et al., 2004), called Seversko-Donetskiy center of green frogs’ diversity (Shabanov et al., 2009). The population systems of R, RE, REt and Et-types with P. esculentus of both sexes have been found in the Seversky Donets river valley. Coexisting di-and triploid hybrids are typical for Seversky Donets floodplain and adjacent habitats (Korshunov, 2010). Seversko-Donetskiy center of green frogs’ diversity is defined by the following: (i) P. ridibundus and various forms of P. esculentus inhabit the center; mature P. lessonae are absent; individual representatives of the parental species have been recorded only at the stage of fingerlings (froglets before the first hibernation). Subsequently all genomes of the P. lessonae are transmitted via diploid and triploid hybrids. (ii) Diploids of the both sexes are numerous among P. esculentus in the diversity center, as well as a significant number of triploids of the both sexes: LRR (more numerous) and LLR, and also single immature tetraploids LLRR were found. (iii) Both sexes’ specimens, producing either P. lessonae gametes or P. ridibundus gametes, or both P. lessonae and P. ridibundus gametes were observed among diploid P. esculentus . (iv) Genetic diversity of P. lessonae genomes transmitted via hybrid frogs in this center is not lower than that of F1 hybrids of those regions inhabited by both parental species (Mezhzherin et al., 2010). (v) The recombinant specimens have been registered among green frogs from this center i.e. P. ridibundus genome has fragments of the P. lessonae genome (Mezhzherin et al., 2005). The history of previous researches The most long lasting studies of the HPS structure was undertaken at the Iskov pond (hereinafter – IpHPS), situated near the biological station of V.N. Karazin Kharkiv National University (Gaidary village, Zmiyiv District, Kharkiv Region). Initially the E-type system was described there (Lada, 1995). This HPS is represented by almost exclusively diploid specimens of the P. esculentus of both sexes. However, because the pond was hauled down, significant change of the HPS structure occurs. After the pond restoring, spawning areas almost without females have been observed for a long time. Tadpoles haven’t been recorded in the pond since 2004 till 2011, when tadpoles and then fingerlings were observed in large quantities. Further observations have led us to conclusion that the frog reproduction in the pond is restoring. Suspected mechanisms of the HPS reproduction, considering females coming from other spawning habitats as a source of new clonal genomes, have been described (Meleshko et al., 2011). This allows us to assume that the dynamics of the IpHPS structure is of considerable interest to study. HPS of Lower Dobritskiy Pond (hereinafter – DpHPS, near the Gomolsha river valley, the territory of National Park “Gomolshanski lisy”) was a subject of primary interest. The single specimen of the hybrid tetraploid frog was recorded close to the habitat of this HPS (Borkin et al., 2004). In the top of that, high proportion of triploid hybrids was recorded for this HPS. The HPS of the Zhovtneve (hereinafter – ZhpHPS, Vovchanskiy District, Kharkiv Region) is located far away from the two latter. The high proportion of triploid hybrids is typical for the green frogs from this HPS. In all HPS which was in focus of our studies, according to the data of Yu. Rozanov and S. Litvinchuk, RL genotypes were recorded. In addition to that, RRL triploid form was recorded from the IpHPS, and RL – in the DpHPS and ZhpHPS. Their numbers had not been estimated earlier, because separate specimens of particular interest were collected instead of the representative samples. The genotypes and ploidy level of specimens were determined by DNA flow cytometry. The details of the technique have been published previously (Borkin et al., 2001). The study of these three HPS can provide valuable information for understanding the reproduction processes within Pelophylax esculentus complex in the Seversko-Donetskiy center of green frogs’ diversity. Materials and methods The research was undertaken in June-July, 2012-2013. The mature green frog individuals were collected from Iskov Pond (Gaydary village, Zmiyiv District), Lower Dobritskiy Pond (near the Gomolsha river valley, Dobritskiy tract, territory of the National Park “Gomolshanski lisy”) and from the pond in the Zhovtneve village (Vovchanskiy District, Kharkiv Region). Collection sites and the information about the samples are composed in Table. 1. Table 1. The studied samples of green frogs

Field work was carried out using standard techniques. Animals were caught at night with a light tube or in the daytime manually or with a hand net. Captured frogs were placed in wet canvas or mesh bags or in plastic boxes. In some cases, during the temporary handling of the animals in the laboratory, they were placed in cans or plastic bottles, which lay on its side without water, once a day frogs were washed with running water.

Determination of the species and sex was carried out visually based on complex of the morphological characters (Korshunov, 2010; Lada, 1995). It is known that visual determination of green frogs based on morphological features does not allow making a precise assumption on their species status. In particular, the triploid hybrid individuals are harder to distinguish visually from individuals of the parental species due to the effect of gene dosage. However, determination of ploidy level by measuring the average length of erythrocytes, the results of which are regularly confirmed by karyological research, significantly reduces the probability errors in the determination.

All specimens from the ZhpHPS aliquot, 99 individuals from the DpHPS aliquot and 183 individuals from the IpHPS aliquot were analysed cytologically, i.e. erythrocytes size was determined. The air dried blood smears of each specimen were made according to the standard technique: a drop of blood obtained from a frog finger was coated on a slide glass and smeared with a thin layer of the second slide edge (Bondarieva et al., 2012). Smears were dried up and photographed under high magnification (13 of a light microscope using a USB-eyepieces camera. The object-micrometer was photographed under the same conditions. Major axis length measurement of 15-20 erythrocytes was performed on photos using the PDF XChange Viewer software. All measurements are given in µ. Sex of adults, determined by dissection, and the age composition (juveniles or adults) of samples are composed in Table 1. 

Results and discussion

The composition of the samples studied is represented in Tables 2, 3. Only data for specimens with cytologically determined ploidy level are given in Table 2, Table 3 includes all sample data. The diploid specimens of the P. esculentus dominated in number green frogs taxocenes of the IpHPS (Table 2). It should be noted that we have registered a significant (p <0.02) increasing in the proportion of triploid P. esculentus in 2013 in comparison with 2012. 

Table 2. Determination of ploidy of individuals from studied HPS

Previously it was assumed that the IpHPS crisis was associated with the loss of all clonal genomes except the male P. lessonae genome (Shabanov et al., 2006). The resumption of frog reproduction in the pond during the past years could potentially indicate that the HPS got new clonal genomes. We can only assume what kind of genomes was brought in the pond and by which specimens. It is most likely that the observed change of triploid hybrids proportion in 2013 is also a consequence of the ongoing transformations caused by penetrating of new clonal genomes into HPS. We assume that HPS state is premature in this case, and now it is going to the populational equilibrium.

Table 3. Sex composition of the studied HPS

The DpHPS samples were primarily composed of diploid *P. esculentus*. Many triploid hybrids were also found, consistent with previous research (Borkin et al., 2004). The proportion of triploids did not significantly differ between samples collected in 2012 and 2013. Notably, in 2012, only male frogs were found in this HPS sample, while in 2013, females constituted about 40% of the sample (Table 3). This difference is likely due to the timing of sample collection rather than a change in the HPS composition: in 2012, frogs were caught during spawning, capturing males in their breeding areas, whereas in 2013, collections occurred after the spawning period. The ZhpHPS study yielded particularly interesting results. Diploids of the edible frog also predominated here. *P. ridibundus* made up 7% of the total frog population in the 2012 sample and 20% in the 2013 sample (Table 2), though this difference in sample composition was not statistically significant. Triploid *P. esculentus* accounted for approximately 30% of the studied samples and consisted exclusively of females (group I, Fig. 1). In contrast, triploid hybrids in the two previously described HPS included both sexes. This suggests a different reproductive mechanism for triploids in the ZhpHPS. Another significant finding from the ZhpHPS was the difference in red blood cell size between diploid males (group II, Fig. 1) and diploid females (group III, Fig. 1); diploid males had significantly larger erythrocytes. It was once hypothesized that males might include both diploid and triploid individuals, but karyological analysis disproved this (O. Mikhailova, personal communication). We observed that females in the 2013 samples were considerably smaller than those from 2012, likely because the 2012 collections were made during spawning, while the 2013 collections were made afterward. Body size distribution was examined across all studied HPS. The distribution patterns were similar for all HPS, so the samples were analyzed together in Figure 2. The size distribution for females showed two peaks, indicating the presence of both relatively small and larger individuals. This observation supports the hypothesis (Usova, 2010) of two distinct growth strategies in female green frogs: early maturation (rapid growth, high fertility, shorter lifespan) and slow growth (lower growth rate, longer lifespan). Consequently, all studied HPS were classified as REt-type, each with its own specific population structure. DpHPS was characterized by a high percentage of triploid hybrids. In IpHPS, which had previously faced critical conditions, reproduction was observed to be restored. All triploid individuals in ZhpHPS were female. The size distribution pattern was consistent across all three HPS, with females separating into relatively small and relatively large size groups.