Chromosomes of Pelophylax ridibundus Pallas, 1771 in different population systems of southern Ukraine

This paper provides the comparative morphological analysis of P. ridibundus chromosomes in different population systems of southern Ukraine. A certain variability and heteromorphism of individual homologs and of whole chromosome pairs was established. The chromosomes of P. ridibundus in populations consisting of a single species (Crimea, Zalisne, Arabat Spit) are relatively stable in quantitative and qualitative terms. Karyotypes are more variable in the lower reaches of the Dnipro River and Kharkiv Region, where the hybridogenic taxon P. esculentus lives together with P. ridibundus. It is assumed that such a relative constancy of chromosomes of southern Ukrainian P. ridibundus in pure RR-systems indicates both a high potential of their resistance to factors and an intraspecific genetic balance. This study is especially important in terms of conservation and enhancement of genetic and biological diversity, which mainly ensures the sustainability of ecosystems functioning.


Introduction
The South of Ukraine is characterized by a unique biological and genetic diversity of both typical widespread species and rare ones, which can preserve their unique relict nature with their own mechanisms of evolution and speciation [1].What is interesting here is the hybridogenic speciation as a special source of increasing biological diversity, and this requires a new strategy for its assessment, protection and preservation.Pelophylax ridibundus (Pallas, 1771) is involved in all these processes.It is the most common typical species of amphibians of freshwater reservoirs of southern Ukraine and one of the parent species of the hybridogenic complex of green frogs of the genus Pelophylax.The vast majority of "pure" RR populations of P. ridibundus are distributed in the southern and southeastern parts of Ukraine.In other regions, it is often found in a complex with other species, namely P. lessonae and the hybrid taxon P. esculentus.
It is known that P. ridibundus is considered to be a complex of cryptic species.According to the analysis of mitochondrial and nuclear DNA, it was established that this species, in particular in the south of Ukraine and Crimea, is represented by two genetically differentiated forms -"western" (= Central European -P.ridibundus) and "eastern" (= Anatolian P. cf. bedriagae) [2,3].On the basis of our morphological studies and the latest literature data, it has been preliminarily proven that P. cf. bedriagae lives in Crimea, while P. ridibundus lives in the lower reaches of the Dnieper and further to the north and west.We also assume that P. cf. bedriagae, might inhabit southeastern Ukraine, connected with the small rivers of the Azov region.Almost all known studies in this area are based on molecular and genetic data.It is quite important at this stage to establish more "visible" diagnostic traits, in particular at the level of ecology, morphology, and chromosomal indicators.The latter are important given the fact that amphibians are characterized by stable chromosomal conservatism, in which the same number of chromosomes is characteristic not only for species, but also for genera and families.Since it is the karyotype that ensures the stability of the existence of the species, therefore it is important to investigate how karyologically stable this species is, especially in the conditions of southern Ukraine.The importance of such features is essential from the point of view of protection, preservation, and increase of biological diversity, which is based on complex invisible genetic mechanisms, which can have great potential for understanding the role of hybridization as a source of speciation.

Research aim and objectives
The aim of the study was to identify specific characteristics at the level of karyotypes and to conduct a comparative morphological analysis of P. ridibundus chromosomes from different populations of southern Ukraine, including the Crimea.

Material and methods
The study was carried out in accordance with all modern requirements for the handling of experimental objects.Eight individuals of P. ridibundus were used in the study.For comparison of karyotypes, typical populations of P. ridibundus were selected (figure 1): Kherson Region, Henichesk District, Arabat Spit, recreation center "Valok" -1 specimen (point 1); Kherson region, Hola Prystan District, near Stara Zburyivka village -2 specimens (point 3); Crimea, Bakhchysarai District, village Zalisne (the material was collected before 2013) -3 specimens (point 4) and a more distant point where the relevant population systems are widespread -Kharkiv Region, Izium District, near Snizhkivka village -2 specimens (point 2).
A total of 49 metaphase plates were analyzed, 20 of which were selected for morphological analysis.The preparation methods of karyological preparations, video recording, analysis and karyotyping of chromosomes, peculiarities of homologues selection and their typing are described in detail in our previous works.

Results
The standard karyotype of P. ridibundus is symmetrical and represented by two-armed chromosomes.It is characterized by the diploid number of 2n = 26, and the number of arms of N.F.= 52.The karyotype includes 5 pairs of large and 8 pairs of small chromosomes.The 1 st is a large metacentric, the 2 nd , 3 rd and 4 th pairs of chromosomes are submetacentric, the 5th pair is the smallest metacentric in the group of large chromosomes.The 6 th and 7 th pairs are also metacentrics among small chromosomes.The 8 th and 9 th pairs are represented by subtelocentric chromosomes (on some plates they can be submetacentric), especially the 9 th pair, which is characterized by a certain variability, since there is a secondary band on its long arm.The 10 th , 11 th , 12 th , and 13 th pairs are submetacentrics.The formula of the P. ridibundus karyotype is 4 meta-(m) + 7 submeta-(sm) + 2 subtelocentric-(st), 2n = 26, N.F.= 52.
First of all, it should be noted that all the analyzed metaphase plates and karyograms correspond to the standard karyotypes of P. ridibundus, but at the same time there is a certain variability and heteromorphism of individual homologs and of whole chromosome pairs.Particular attention should be paid to the study and clarification of the standard types of the last 4 pairs of chromosomes.Regarding the 10 th pair, it can be said that, in contrast to the indicated submetacentric type, in the vast majority it is represented by subtelocentric homologs.All analyzed plates show secondary bands on the long arm of the 9 th chromosome pair, which is not always characteristic of other closely related species, such as the hybridogenic P. esculentus.The chromosomes of P. ridibundus from Crimea and the Arabat Spit are relatively more stable in terms of quantity and quality (figure 2).Homologs are easily selected, even with total coloring.The chromosomes are more variable in the karyotypes of P. ridibundus from the lower reaches of the Dnipro and Kharkiv Region (figure 3).Pairs often include chromosomes, which are different in size, but identical in morphology.This may be typical for the first pair of chromosomes, which was repeatedly emphasized in our previous studies.Heteromorphism of individual pairs and homologs is also found among the group of small chromosomes, especially the 6th, and the last three (11 th , 12 th , 13 th ).Incomplete sets are often found, which is typical for all analyzed populations.More often homologs or even whole pairs are lost among the small chromosomes (figure 4).Among the large ones, homologs of the 5th pair can be lost occasionally (figure 5).

Discussion
First of all, the prevalence of population systems of green frogs in Ukraine should be mentioned.There are pure and mixed populations of parental species -RR, LL, RL, hemiclonal mixed -RE, LE, REL, and hybridogenic EE-type systems.In southern Ukraine, the most common are populations of P. ridibundus (RR) and mixed semi-clonal systems -RE and REL with different ploidy, abundance, and specific sexual structure.In different parts of their range, they have corresponding percentages, genetic structure, and other specific population characteristics [4].
The main result of the conducted comparative morphological analysis is the relative stability of P. ridibundus chromosomes in RR-populations (Crimea, Arabat Spit).Perhaps in such populations P. ridibundus has a more homogeneous genetic structure, which is manifested in the relative uniformity of chromosomal parameters, while incomplete sets are still present.
Populations in which individuals with mosaic, i.e. with di-, tri-, and more, and aneuploid cell lines [5] are found mostly majority in P. esculentus and in all mixed population systems.One of the reasons for this may be a violation of the process of genome elimination or other mechanisms that arise during multidirectional hybridogenic speciation.According to our data, such karyotypes are also present in RR populations.As noted, P. ridibundus from southern Ukraine has an ambiguous systematic status, in particular in the Crimea there are two genetically differentiated forms [3,6] that can make their own adjustments of gamete production, introgression, genome composition, which can be manifested at the chromosomal level.It is interesting that alleles of the "western" form are less common than the "eastern" form in the south, moreover, it is believed that the territory of Crimea was inhabited by P. ridibundus of the Anatolian lineage, directly from the Caucasus.Probably, the extensive delta systems, which were formed in the regressive stages of the Black Sea basin during the Holocene, Pleistocene, to the southeast and west of the Crimea, contributed to this.The karyotypes of P. ridibundus in the lower reaches of the Dnipro River and Kharkiv Region are much more variable.In this part of Ukraine P. ridibundus lives together with hybridogenic taxon P. esculentus.The RE-type population system has a very diverse genetic structure, with the presence of polyploids, a large diversity of parental genomes that are produced, transmitted, and inherited through complex genetic mechanisms [7][8][9][10].Therefore, all these factors can be manifested in the variability of homologs, the qualitative and quantitative characteristics of chromosomes.Of course, we allow for the possibility that our results are related to geographical variability, different nature spiralization and condensation of the chromosomes and their individual parts, which can lead to heteromorphism of homologs.

Conclusion
The results of the study suggest that such a relative constancy of chromosomes of the southern Ukrainian P. ridibundus in pure RR-systems indicates a high potential of their resistance to factors and their intraspecific genetic balance.At the same time, we allow for the possibility of introgression between the "eastern" and "western" forms.P. ridibundus in mixed RE-systems has a certain genetic and ecological interaction in the process of hybridogenic reproduction that directly affects the morphology of the karyotype, which is manifested in the variability of homologs, their qualitative and quantitative characteristics.The presented materials are only preliminary and require confirmation with the use of comparative statistical methods of the relevant chromosomal indicators.It is also necessary to investigate the composition of the genome, the genetic structure, to what extent molecular variability is comparable with chromosomal variability, and how it is reflected in different populations of green frogs.This is especially important from the point of view of preserving and increasing the genetic and biological diversity, which mainly ensures the sustainability of ecosystems functioning.

Figure 1 .
Figure 1.Populations of P. ridibundus used for analysis (the points are described in the text).