Triploid forms’ karyotypes of spined loaches from the genus Cobitis (Actinopterygii: Cypriniformes: Cobitidae) of the upper Dnieper and Western Dvina rivers: Analysis of the triploids’ origin

Triploid forms’ karyotypes of spined loaches from the genus Cobitis (Actinopterygii: Cypriniformes: Cobitidae) of the upper Dnieper and Western Dvina rivers: Analysis of the triploids’ origin. Acta Abstract Unisexual forms of lower vertebrates (fish, amphibians, and reptiles) reproduced by parthenogenesis, gynogenesis, or hybridogenesis are represented by diploids, triploids, or tetraploids, whose origin is associated with interspecific hybridization. Among fish species, the highest variability of unisexual polyploids was found in the genus Cobitis . The structure of their genomes and putative parental species holds great interest for the investigation in association with questions about possible evolutionary success. In particular, it serves to elucidate the possible high colonization properties of a few polyploid forms, in contrast to the local history of rather numer ous hybrid forms with a limited distribution. Therefore, the aim of this study was to describe the karyotype structure of two newly discovered triploid forms of the genus karyotype submeta-, 22 subtelo-acrocentric loaches per Dnieper River, triploid 23 meta-, 26 submeta-, subtelo-ac rocentric n hybridization Cobitis species Cobitis triploid of complexes, diploid C. elongatoides


Introduction
About 90 unisexual forms reproducing by parthenogenesis, gynogenesis, or hybridogenesis are known among fish, amphibians, and reptiles at present. These forms can be diploid, triploid, and tetraploid; however triploid unisexual forms are much more widespread, while tetraploids are extremely rare. The genesis of unisexual forms is associated with interspecific hybridization, while triploid and tetraploid forms can have not only dihybrid, but even trihybrid origin (see Vasil'ev et al. 1989Vasil'ev et al. , 2011Vrijenhoek et al. 1989).
The first clonal-bisexual (diploid-polyploid) complex in the genus Cobitis was noted in 1981 in the Volga River basin (Vasil′ev and Vasil′eva 1982;Vasil′ev et al. 1989). Further studies revealed the wide distribution of diploid-polyploid loach complexes in the Volga River basin, as well as in the rivers of the Black, Baltic, and North seas (Vasil′ev 1990;Boroń 1992Boroń , 1995Boroń , 1999Boroń , 2003Ráb and Slavik 1996;Vasil′eva and Vasil′ev 1998;Boroń and Danilkiewicz 1998;Bohlen and Ráb 2001;Bohlen et al. 2002;Vasil′ev et al. 2005Vasil′ev et al. , 2007Vasil′ev et al. , 2011Janko et al. 2007Janko et al. , 2012Choleva et al. 2008;Buj et al. 2008;Mezhzherin et al. 2022). The structure of these complexes varies, but their obligatory elements are at least one all-female form and one diploid bisexual species (host), which is a sperm donor for gynogenetic reproduction. The most common complexes include a diploid and/or triploid unisexual form and one diploid bisexual species (Vasil′ev et al. 2011). The high variability of unisexual forms in Cobitis observed in ploidy levels and in karyotype structure is the result of different combinations of haploid sets from several diploid bisexual species distributed in European waters. According to various genetic studies, the presumed parental species for the unisexual forms are as follows: Cobitis taenia Linnaeus. 1758, distributed in the North Atlantic basin from France and Great Britain to Russia; Cobitis melanoleuca Nichols, 1925 widespread from the Volga River system to the waters of the Western Pacific in the Far East and China; the Black Sea species Cobitis tanaitica Băcescu et Mayer, 1969, C. taurica Vasil′eva, Vasil′ev, Janko, Ráb et Rábová, 2005, and C. pontica Vasil′eva et Vasil′ev, 2006Cobitis elongatoides Băcescu et Mayer, 1969, common in the Danube basin;C. strumicae Karaman, 1955, recorded in the basins of the Aegean and southwestern Black Seas (Vasil′ev et al. 1989(Vasil′ev et al. , 2011Ráb et al. 2000;Bohlen et al. 2002;Boroń 2003;Janko et al. 2005aJanko et al. , 2007Janko et al. , 2018Choleva et al. 2008). It is assumed that their ranges fluctuated during the Quaternary (Janko et al. 2005a;Culling et al. 2006) and overlapped in Central Europe, the Lower Danube basin, and southern Ukraine (Janko et al. 2007). Hybrid unisexual forms that arose in the mentioned hybrid zones achieved outstanding evolutionary successes and colonized most of the European continent (Janko et al. 2018). However, the observed diversity of unisexual genotypes suggests a local history of bisexual-unisexual complexes (Bohlen and Ráb 2001;Vasil′ev et al. 2011). That is why further work on the discovery of new complexes, the study of their origin and distribution in our opinion holds great interest for the investigation.
In this study, we describe the karyotypes of two triploid forms found in the Western Dvina and Upper Dnieper rivers, respectively, and analyze their origin and putative parental species.  (Fig. 1). In total, 182 spined loach individuals were studied karyologically from the first locality, and 91 individuals from the second. The voucher specimens were deposited in the collection of the Zoological Museum of Moscow State University under the numbers P-21434, P-21800, P-21801, P-21805, P-23738, P-24010. All individuals were injected with about 0.05 mL colchicine solution (0.3%-0.4%). After 4 h cells of anterior part of kidney were used for chromosomal slides prepared according to Vasil′ev (1978). The study was performed using anesthesia of fishes with a solution of MS-222. Metaphase chromosomes stained in 4% Giemsa solution (pH 6.8) were counted with PC software Quick Photo Micro. From 3 to 5 metaphase plates were analyzed for each studied fish. The chromosomes were classified according to Levan et al. (1964). Photos of metaphase chromosomes were taken with a Leica DFC 295 digital camera. For a comparative analysis, we used previously published data on loaches collected in the Lower Dnieper and Dnieper estuary (Vasil′ev 1990).

Results
Among the karyotyped spined loaches of the Western Dvina River, 44 (63.8%) were identified as triploid females in the sample collected in 2003 and 77 (68.1%)-in the sample collected in 2005. These triploids had 74 chromosomes, including 13 meta-, 39 submeta-and 22 subtelo-and acrocentrics (Table 1, Fig. 2). Other fishes were karyologically identified as C. taenia. The karyotype of this species was described earlier (Vasil′ev and Vasil′eva 1982;Vasil′ev et al. 1989) and is shown in Table 2 Fig. 3). Other spined loaches found in this location belonged to C. taenia.

Discussion
Triploid form in the Western Dvina basin. According to the number of chromosomes ( Fig. 2A), the karyotype of this triploid form includes two haploid sets of 50-chromosome species and one haploid set of 48-chromosome species. Cobitis taenia Linnaeus, 1758 is the only 48-chromosome species among the karyotyped European spined loaches (Table 2). After removing the haploid set of C. taenia with 5 meta-(m), 9 submeta-(sm) and 10 subtelo-and acrocentric (sta) chromosomes ( Fig. 2B) from the triploid karyotypes, the remaining chromosomes of the triploid form of the Western Dvina (8 m + 30 sm + 12 sta) correspond fairly well to the sum of  the haploid set of C. tanaitica females (4 m + 15 sm + 6 sta) and the male of the same species (4 m + 15 sm + 6 sta) (Fig. 2C, D). Differences in the ratios of submeta-/ subtelocentrics may be associated with a difference in the classification associated with various degrees of spiralization of chromosomes in the analyzed triploids or diploid species. Other karyotyped 50-chromosome species are unsuitable as putative parental species: they have a higher number of metacentrics and differ in sm/sta ratio (Table 2). Accordingly, the proposed scheme of the origin of this triploid form should include the following stages: 1) hybridization of a female C. tanaitica and a male C. taenia; 2) hybridization between a hybrid C. tanaitica × C. taenia and a male C. tanaitica. This scheme is due to the unique karyotype of C. tanaitica, characterized by an evolutionarily fixed Y-autosomal translocation (Vasil′eva and Vasil′ev 1998), leading to differences in the number of chromosomes in males and females (Table 2). It is also confirmed by our preliminary results that demonstrated a close relation between the triploids of the Western Dvina and C. tanaitica from the Don River based on the variability of the cytochrome b gene (Vasil′ev et al. 2005).
In this line, it is necessary to discuss the differences observed in the structure of the karyotype of C. tanaitica in our studies and in publications of other authors (Bohlen et al. 2002;Janko et al. 2007). Previously, we studied the karyotype of C. tanaitica (the species was originally described from the Don River basin) based on materials, collected from 7 populations of the Black Sea and Sea of Azov basins: Don River at Rogozhkino (47°10.367′N, 039°20.  A Species identification is provided by Bohlen et al. 2002 andJanko et al. 2007 (see comments in the text); m = meta-, sm = submeta-, sta = subteloand acrocentric chromosomes.
Dnieper estuary (46°32.333′N, 032°3.967′E) (Vasil′eva and Vasil′ev 1998). A total of 50 individuals were studied, all of them had a characteristic karyotype with 2n = 50 with 8 metacentrics in females and 2n = 49 with 9 metacentrics (an unpaired large chromosome is noticeably prominent) in males (Vasil′ev 1990, fig. 1a-b;Vasil′ev 1995, fig. 1). All studied specimens, as well as non-karyotyped fishes from the same samples and fishes from the south Bug system (Savranka River) were morphologically identified and described as a new species Cobitis rossomeridionalis Vasil′eva et Vasil′ev, 1998, a junior synonym of C. tanaitica. This species differed from C. taenia sympatrically found in the Kalka and Obitochnaya rivers, Dnieper estuary, and Dniester River at Mayaki both in the karyotype structure (36 individuals were karyotyped) and morphological features (Vasil′eva and Vasil′ev 1998). Bohlen et al. (2002) presented the karyotype of C. tanaitica, which they isolated from the chromosome set of the tetraploid specimen (4n = 100) from the Danube River in Germany. They proposed that the genome of this tetraploid includes 3 haploid sets of C. elongatoides and 1 set of C. tanaitica. Janko et al. (2007) indicated that they karyotyped or examined by flow cytometry (?) 8 specimens of C. tanaitica from the Odra River, 4 specimens from the Danube River, 6 specimens from the Lake Sinoe, and 9 specimens from the Don River (Janko et al. 2007, Table 1). They stated that a biotype corresponding to C. tanaitica (2n = 50, 10 m + 24 sm + 8 st + 8 a) was found in both sexes in the lower Danube River, upper Odra River, and in the Don River (p. 395); this karyotype was presented by the authors in Fig.  1C. However, the presence of diploid bisexual C. tanaitica specimens has never been confirmed in the Odra basin despite the fine-scale intensive researches (Janko et al. 2012;Kotusz et al. 2014). The karyotypes proposed by Bohlen et al. (2002) and Janko et al. (2007) are similar in structure, but differ significantly from the karyotype described by us from the confirmed range of C. tanaitica not only in the absence of sex differences, but also in the higher number of metacentric (Table 2) and acrocentric chromosomes: 8 acrocentrics versus 4-6 in males and females from the Sea of Azov basin. Janko et al. (2003Janko et al. ( , 2007 have never confirmed that the studied fish belong to C. tanaitica according to the accepted morphological diagnostic characters (Vasil′eva and Vasil′ev 1998;Vasil′eva 2004;Kottelat and Freyhof 2007). Most likely, Janko et al. (2007) studied the karyotype of some other spined loach species, found only in the lower reaches of the Danube and in Lake Sinoe, a lagoon in northern Dobrudja, Romania, in the Danube Delta. Indeed, Bohlen and Ráb (2001) noted that only specimens from Sinoe Lake were karyotyped. It should also be noted that during two years of our field work in the Danube Delta and in the adjacent territories of Ukraine (2010-2011) we did not find a single specimen of C. tanaitica. The spined loaches in our samples from the Danube system were mainly represented by triploids (3n = 75), a small number of tetraploids (4n = 100), and diploid males and females with 2n = 50, which were identified as C. elongatoides according to our preliminary DNA studies (unpublished). Therefore, in association with the foregoing, in this paper we designate the diploid loach species found in the lake Sinoe, in the Odra and the lower reaches of the Danube with 2n = 50, 10 m, 22-26 sm, 14-18 sta as Cobitis sp.
Among the described triploid karyotypes, the fishes of the Western Dvina are most similar to the fishes of the Oława (Odra River system) and Bug (Vistula River system) rivers. They also have 3n = 74 with a low number of metacentric and a high number of submetacentric chromosomes (Table 3). It has been suggested that the Table 3. The karyotype structure of previously studied triploid forms, their putative parental diploid species of Cobitis (as identified in the cited publication) and diploid species coexisting with triploids. A Identified as Cobitis sp., but not the same as suggested in the triploid genome; AA identified as C. danubialis Nalbant, 1993. m = meta-, sm = submeta-, sta = subtelo-and acrocentric chromosomes.
genome of triploids of the Vistula basin includes a haploid set of C. taenia and two haploid sets of an unknown species with 2n = 50, 12 m + 24 sm + 14 sta (Boroń 1999). Taking into account the different degree of chromosome spiralization, we can assume the already mentioned Cobitis sp. as the second parental species for this triploid. The same dihybrid origin can be assumed for the triploids of the Odra River. Boroń and Kotusz (2000) revealed in the Odra River a diploid spined loach with 2n = 49 (19 m + 18 sm + 12 sta) and primarily identified it as a separate undescribed species, parental to sympatric triploids. However, according to the described karyotype structure, this diploid form should be considered as a hybrid of C. taenia and C. elongatoides (see Table 2), as Boroń (2003) later concluded; both species are known from the Odra system (Boroń 2003). Thus, the triploid form of the Western Dvina differs from other triploids found in the Baltic Sea basin (Odra, Vistula) by the karyotype structure and the supposed origin; this form also differs from other triploids revealed in various European river systems (Table 3). Judging by the present-day distribution of its parental species, C. taenia and C. tanaitica, the triploid form did not originate from the Baltic Sea basin; its origin, of course, should be associated with the Black Sea basin and dated to the Late Pleistocene according to Culling et al. (2006).
It is well known that the recent freshwater fish fauna of the Baltic Sea is one of the youngest. The Baltic Sea depression was covered with ice during the last glacial advance in the Pleistocene and was filled with fresh water at the end of the Quaternary after the retreat of the ice sheet, starting about 13 thousand years ago (Reid and Orlova 2002). Berg (1949) associated the genesis of southern fish forms in the Baltic zoogeographic province with their northward dispersal after the retreat of the ice. However, Reid and Orlova (2002) concluded that much of the current biological diversity in the Baltic Sea reflects relatively recent colonization of non-native species, intentionally or unintentionally introduced by human activities. In our case, an important transport artery connecting the Western Dvina with the Dnieper basin was the Berezinskaya water system (Berezina Canal)-a former artificial waterway that connected the Berezina River (tributary of the Dnieper) with the Western Dvina on the territory of the Vitebsk region of (the present day) Belarus, built within 1797-1805. The total length of this system was 169 km, it had 14 locks and connecting and bypass canals with a length of about 24 km. The system was opened to traffic in 1805; timber rafting continued until the early 1950s; but at present, the canals are impassable due to blockages (Snytko et al. 2016).
As follows from the current distribution of polyploid forms of spined loaches in the river systems of Europe, after penetrating the Baltic Sea basin (about 13 000-200 years ago), the analyzed triploid form disappeared from its natural range. Probably, it was replaced by another triploid form, which is now widespread in the upper Dnieper basin.
Triploid form in the upper Dnieper. The karyotype structure of triploid females in the upper Dnieper basin indicates its trihybrid origin (Fig. 3A). A similar karyotype structure was found in triploid forms from the Lower Dnieper and Volga systems (Table 3). Since their genomes consist of 74 chromosomes, one of the parental species must have 24 chromosomes in the haploid set. According to the first hypothesis of the origin of studied triploids, C. taenia can be such a parental species. After the removal of its haploid set, the remaining chromosomes of triploids (18 m + 16-17 sm + 15-16 sta) suggest that the other parental species was a diploid spined loach with a high number of metacentrics and a low number of uni-armed chromosomes; the only corresponding karyotyped species is C. elongatoides (Fig.  3B, Table 2). The rest of the chromosomes (4-7 m + 4-8 sm + 13-15 sta) are similar to haploid sets of spined loach species common in the Caucasus (Eastern Black Sea region and Caspian basin) (Vasil′ev 1995;Vasil′eva et al. 2021): Cobitis saniae Eagderi, Jouladeh-Roudbar, Jalili, Sayyadzadeh et Esmaeili, 2017, Cobitis satunini Gladkov, 1935, Cobitis derzhavini Vasil′eva, Solovyeva, Levin et Vasil′ev, 2020, and populations requiring identification. It should be noted that our long-term studies in the Caucasus showed the absence of polyploid loaches in this area (Vasil′eva et al. 2021). C. taenia, C. elongatoides and unknown species Cobitis sp. were previously assumed as parental species for the triploid form, widespread in the Volga River basin (Vasil′ev et al. 2011). C. taenia was confirmed as its maternal species by our preliminary study of the variability of the cytochrome b gene (Vasil′ev et al. 2005). At the same time, triploids from the Lower Dnieper basin showed pronounced divergence from both C. taenia and triploids of the Volga system and were closer to C. tanaitica and triploids from the Western Dvina basin (Vasil′ev et al. 2005).
According to the second hypothesis of the origin of triploids from the Dnieper, the parental diploid with 24 chromosomes in the haploid set may be a male of C. tanaitica. After removing its haploid set (Fig. 3C), the remaining genome of triploids should include 19 m, 14 sm, 17 sta. And after further removal of the haploid set of C. elongatoides (Fig. 3B), the resulting set with 4 m + 6 sm + 15 sta (Fig. 3D) will be similar to haploid sets of spined loaches from the Caucasus, as in the previous hypothesis. Based on our preliminary mtDNA data (Vasil′ev et al. 2005), we suggest that the most probable origin of the Dnieper triploids is via hybridization of C. elongatoides, C. tanaitica, and Cobitis sp., with the first species as the maternal form. Anyway, this triploid form undoubtedly arose in the water bodies of the northwestern part of the Black Sea basin, where C. elongatoides, currently found in the Danube basin, could meet the Caucasian Cobitis sp., as well as C. tanaitica (or C. taenia), living in northeastern Europe. The strong competition and deep northward dispersion observed for this triploid form may explain the absence of the putative maternal species in diploid-triploid complex, where the role of the host species belongs to C. taenia.

Conclusions
The triploid form of spined loaches of the Western Dvina River most likely arose as a result of the hybridization of Cobitis tanaitica and C. taenia. Since the range of C. tanaitica, whose karyotype is characterized by an evolutionarily fixed Y-autosomal translocation, locally distributed in the rivers of the northern coast of the Black Sea, hybridization probably happened in the Dnieper River system, where both parental species occur. The triploid form that arose here, unique for the Baltic Sea basin, probably colonized the Western Dvina through the artificial Berezinskaya water system (Berezina Canal), but at the same time it was forced out of its area of origin. According to the karyotype structure, the triploid form, common both in the upper and lower reaches of the Dnieper, has a trihybrid origin, with probable hybridization of C. elongatoides, C. tanaitica and an unknown species Cobitis sp. Both studied triploid forms are parts of unisexual-bisexual complexes, in which their putative diploid maternal species (C. tanaitica and C. elongatoides) are absent, and the role of the host species involved in reproduction belongs to C. taenia. This phenomenon can be explained by the strong ecological competition of the unisexual form and its maternal species, as well as the deep northward dispersion observed for the triploid forms of the Western Dvina and upper Dnieper basins, where maternal species cannot exist due to their more southerly origin.