03_Tsyba-1.indd UDC 597.4/.5:575.1 REGULAR INTERGENERIC HYBRIDIZATION OF LEUCISCINE CYPRINIDS (CYPRINIDAE, LEUCISCINAE) IN THE DNIPRO RIVER AFFLUENTS A. A. Tsyba*, M. Ghazali, S. V. Kokodiy, S. V. Mezhzherin Schmalhausen Institute of Zoology NAS of Ukraine, vul. B. Khmelnytskogo, 15, Kyiv, 10030 Ukraine *Corresponding authour E-mail: tsyba1974@gmail.com A. A. Tsyba (https://orcid.org/0000-0001-5838-0948) M. Ghazali (https://orcid.org/0000-0001-9195-0914) S. V. Kokodiy (https://orcid.org/0000-0002-0651-6935) S. V. Mezhzherin (https://orcid.org/0000-0003-2905-5235) Regular Intergeneric Hybridization of Leuciscine Cyprinids (Cyprinidae, Leuciscinae) in the Dnipro Affl uants. Tsyba, A. A., Ghazali, M., Kokodiy, S. V., Mezhzherin, S. V. — Th e large-scale hybridization of fi shes of the subfamily Leuciscinae in the subordinate systems of the Dnipro River basin is presented by the data on two pairs of species, roach Rutilus rutilus × bream Abramis brama, and bleak Alburnus alburnus × rudd Scardinius erythrophthalmus. Th e hybridization and occurrence of F1 hybrids are confi rmed with allozyme spectra and morphological characters on series of samples. A complete morphometric analysis of the characteristics of bleak and rudd hybrids was performed. Th e paper discusses the intergeneric hybridization in nature, which is a unique phenomenon characteristic only of some groups of cyprinids. Th e most likely reason is the overestimation of the taxonomic status of European cyprinids, which is confi rmed by the insignifi cant level of intergeneric genetic divergence. K e y w o r d s : Cyprinidae, bream, roach, bleak, rudd, hybridization, allozymes, morphometry. Th e issues of natural hybridization are attracting a lot of attention. Interest is caused not only by the wide presence of this natural phenomenon that was previously thought to be exceptional, but also by its consequences, oft en in the form of irreversible evolutionary changes (Arnold, 1992; Barton, 2001; Abbott et al., 2013). Th e intensity of hybridization phenomena has increased in recent decades (Brennan et al., 2015) because of climate changes, destabilization of ecosystems, destruction of historically formed landscapes, and invasions. Cases of interspecifi c hybridization of freshwater and anadromous fi shes are especially numerous. Th is phenomenon began to be studied in detail in the middle of the 20th century (Hubbs, 1955; Schwartz, 1972) using morphological characters to identify hybrids, and gene markers are used since the 1970s. Hence, the fact of hybridization was unambiguously proved and the parental species were reliably established. At present, there are hundreds of hybridizing pairs of species, and hybridization mainly occurs in freshwater and anadromous fi sh of the Holarctic region (Scribner et al., 2000). Zoodiversity, 55(4): 295–306, 2021 DOI 10.15407/zoo2021.04.295 296 A. A. Tsyba, M. Ghazali, S. V. Kokodiy, S. V. Mezhzherin Depending on parent species and the degree of their genetic diff erentiation, hybridization can lead to diff erent results. Crosses between genetically close vicarious species are accompanied by genetic recombination and introgression of genetic fragments of one species into the genome of another, and with the formation of hybrid zones of various widths (Arnold, 1992). Th e random hybridization is characteristic of sympatric genetically distant species (Mayr, 1963). In this case, allodiploid off spring are formed. Typically, the proportion of such hybrids is negligible, produced in random recurring interspecifi c crosses. Th e allodiploid hybrids can even numerically dominate over individuals of parental species in small water bodies for a while, as in the case with the hybridization of Carassius carassius and Carassius auratus (Mezhzherin et al., 2012). Allodiploid hybrids can be divided into two groups depending on the modes of gametogenesis. In some cases, meiosis is replaced by ameiosis (mitosis), which ultimately leads to the formation of diploid-polyploid hybrid complexes that self-reproduce by sperm-dependent parthenogenesis (Vasiliev, 1985). Alternatively, various kinds of meiotic anomalies are observed, leading to a signifi cant loss of fertility. Th e large cyprinid family comprising about 367 genera and more than 3,000 living species (Nelson et al., 2016) is of particular interest. Intergeneric hybridization is oft en observed for its extratropical representatives (Schwartz, 1972; Scribner et al., 2000). Hybrids identifi ed by external characteristics as crosses of the roach Rutilus rutilus (Linnaeus, 1758) and the bream Abramis brama (Linnaeus, 1758) (Berg, 1912; Beling, 1928), bleak Alburnus alburnus (Linnaeus, 1758) and rudd (Scardinius erythrophthalmus (Linnaeus, 1758) (Berg, 1912; Velykokhatko, 1929), were noted in the Dnipro River system in the fi rst quarter of the 20th century. Since then, the hybridization of these species in this river system was not reported. In order to resolve the problem, it is necessary to prove the fact of natural hybridization at the level of gene markers. In addition, it remains unclear how widespread this phenomenon is in the modern, anthropogenically transformed Dnipro River system. Material and methods Th e materials of the present work are samples and single specimens of cyprinids with morphological characters either within the standard range or clearly deviating. A series of 16 supposed hybrids of A. brama and roach R. rutilus was collected in June 1991 on the mouth of the Sozh River (left tributary of the Dnipro) in a fl oodplain lake (51.975 N, 30.868 E). A total of 51 putative hybrids of the bleak and rudd were caught in a small lake-type reservoir on the Snitka River (50.106 N, 29.976 E), located in the watershed of the upper reaches of Unava (basin of Irpin River, right tributary of the Dnipro River) and Stugna Rivers (right tributary of the Dnipro) in May–June 2016–2019. Fishes were caught with a sport fi shing gear. Alozyme analysis according to the standard method (Peacock, Buntig, 1965) was carried out on supposed hybrids and the following leuciscine species: bream, Abramis brama, blue bream, Abramis ballerus (Linnaeus, 1758), silver bream, Blicca bjoerkna (Linnaeus, 1758), common roach, R. rutilus, common rudd, S. erythrophthalmus, common bleak, A. alburnus, ide, Leuciscus idus (Linnaeus, 1758), chub, Squalius cepha lus (Linnaeus, 1758), asp, Aspius aspius (Linnaeus, 1758), as well as a representative of the subfamily Acheilognathinae Bleeker, 1863, the Amur bitterling, Rhodeus sericeus (Pallas, 1776). Multilocus analysis was performed using polyacrylamide gel electrophoresis and running tris-borate- EDTA buff er, pH 8.5, for a number of water-soluble and structural muscle proteins. Th e variability of the following enzymes and proteins encoded by the corresponding loci was analysed: aspartate aminotransferase (Aat-1, -2), glycerol-3-phosphate dehydrogenase (G3pdh-1), malate dehydrogenase (Mdh-1A, B, Mdh-2A), superoxide dismutase (Sod-1), lactate dehydrogenase (Ldh-A, -B), umbelliferyl esterase (Es-D), structural muscle proteins stained for total protein (Pt-1, -2, -3, -4) and albumin (Alb). A total of eight enzymes and proteins encoded by 15 loci were analysed. Morphometric analysis was performed on a series of 30 hybrids of rudd and bleak, according to the standard scheme of body measurements for cyprinids (Pravdin, 1966). For this purpose, 22 body measurements (H — body depth, iH — body thickness, aD — antidorsal length, pD — postdorsal length, aV — anteventral length, aA — antianal length, lD — dorsal fi n length, hD — dorsal fi n height, lA — anal fi n length, hA — anal fi n height, lP — pectoral fi n length, lV — pelvic fi n length, PV — distance between pectoral and pelvic fi n, pl — caudal peduncle length, h — caudal peduncle depth, C1 — upper lobe of the caudal fi n length, C2 — lower lobe of the caudal fi n length, c — head length, hc — head height, r — snout length, o — eye diameter, io — іnterorbital distance standard length) were used. All plastic characters are given relative to l - standart body length, except for the plastic characters of head, they are given relative to head length. And also fi ve meristic characters (A — the number of rays in the anal, D — dorsal, P — pectoral, and V — pelvic fi ns, and ll — scales in the lateral line) were used. Group characterizing was performed with the principal component analysis (PCA) on correlation matrix. In total, we used 12 specimens of A. alburnus, 4 specimens of S. erythrophthalmus and 26 specimens of their hybrid for which all traits were available. We used function PCA of package FactoMineR, v. 1.42 (Le et al., 2008); visualization was done with package factoextra, v. 1.0.5 (Kassambara, Mundt, 2017) in statistical system R, v. 3.6.3 (R Core Team, 2020). 297Regular Intergeneric Hybridization of Leuciscine Cyprinids in the Dnipro Affl uants Results S e l e c t i o n o f g e n e m a r k e r s a n d a s s e s s m e n t o f g e n e t i c d i v e r g e n c e . All studied species of cyprinids had a unique set of allelic variants of enzymes (allozymes) and structural proteins (table 1). Moreover, the degree of interspecies diff erences varied signifi cantly. Minor fi xed diff erences in alleles aff ecting two, less oft en three loci, took place in most pairwise interspecifi c comparisons. Minimal diff erences were found when T a b l e 1 . Allele pools of enzyme and protein loci in cyprinid species Loci Allele Abr. bra. Abr. bal. Bl. bjor. Alb. alb. Rut. rut. Sc. eryth. L. idus Sq. ceph. As. asp. Rh. ser. Aat-1 99 1 100 1 1 1 1 1 1 1 110 1 1 Aat-2 -90 1 -95 1 1 -98 1 1 -100 1 1 1 1 0,8 -110 0,2 Ldh-B 90 1 92 1 100 1 1 1 1 1 1 1 1 Mdh-1A 90 0,05 100 0,95 1 1 1 1 1 1 1 1 1 Mdh-1B 90 1 100 1 1 1 1 1 1 1 1 1 Mdh-2A 99 1 1 100 1 1 1 1 1 1 1 1 Sod-1 80 1 90 0,1 100 1 1 0,9 1 1 1 1 110 1 1 G3pdh 85 1 90 1 100 1 1 1 1 1 1 1 1 Pt-2 80 0,4 98 0,7 99 1 100 1 1 0,6 1 1 0,3 1 1 120 1 Pt-3 96 1 97 1 98 99 1 1 100 1 1 1 1 102 1 1 Pt-4 -100 1 1 1 1 1 1 1 100 1 1 1 Loci: Alb, Es-D, Ldh-A, Pt-1 under used conditions of electrophoresis were monomorphic. 298 A. A. Tsyba, M. Ghazali, S. V. Kokodiy, S. V. Mezhzherin comparing R. rutilus and A. alburnus. In this case, fi xation of alternative alleles aff ected only one Ldh-B locus, which in roach has an allele that distinguishes it from the rest of the studied cyprinids. In this case, fi xation of alternative alleles aff ected only one locus. In 4–5 loci, diff erences were observed between representatives of the Abramis group and other European cyprinid species. Signifi cant diff erentiation aff ecting 7–8 loci, largely expected, was observed by comparing the gene pool of R. sericeus with other species. All these features of genetic relationships between the studied species are clear on a phenogram based on genetic distances (fi g. 1). Th ree or four clusters (groups of species) can be distinguished on diff erent hierarchical levels of genetic diff erences. H y b r i d i z a t i o n o f A . a l b u r n u s × S . e r y t h r o p h t h a l m u s . Analysis of enzymatic and protein spectra in 30 putative hybrids collected in an artifi cial reservoir on the Snitka River confi rmed their status as hybrids of A. alburnus × S. erythrophthalmus for all of Fig. 1. Phenogram of genetic distances (Nei’s D) between the studied species of cyprinids, UPGMA algorithm on the basis of 15 biochemical loci. Fig. 2. Electrophoregram of muscle structure proteins spectra of A. alburnus (1), S. erythrophthalmus (2), and their hybrids A. alburnus × S. erythrophthalmus (3). 299Regular Intergeneric Hybridization of Leuciscine Cyprinids in the Dnipro Affl uants them. Th e electrophoretic spectrum of the products of the Pt-3 locus, presented in hybrids by two fractions (fi g. 2), was found to be diagnostic and the most convenient in practice, which indicates its monomeric nature. Th eoretically, hybridization of the above fi sh with R. rutilus, which is a common species in the Snitka River here, is quite possible in this reservoir. However, the absence of heterozygotes in hybrids at the Ldh-B locus does not confi rm such an assumption. A four-year study conducted on this reservoir showed a stable presence of hybrids in bleak schools, where their proportion in diff erent samples varied from 5 to 25  % of caught fi shes. Th is means that the hybridization between bleak and rudd is a common phenomenon, which repeats annually. Th e analysis of muscle spectra of samples from seven populations of the bleak from the rivers of the Kaniv reservoir, the Desna and Stugna Rivers showed the absence of hybrids. Th is indicates that hybridization between these species is possible primarily in “confi ned” conditions of reservoirs with a limited number of suitable spawning grounds. Hybrids of bleak and rudd are more similar in size and externally to bleak, although they reliably diff er from the latter in the reddish colour of the caudal and anal fi ns (fi g. 3). For these hybrids, the almost red colour of the caudal fi n is especially characteristic, which is never dark grey at the edge as in bleak. Th e body is elongated, laterally compressed. Th e upper mouth is small. Th e back is dark, while the sides are silvery. Ovaries fi lled with eggs were found in 16 of 51 individuals. Th e number of eggs ranged from 31 to 435, with an average of 130. Such a low individual fecundity means a limited fertility of the hybrids. Fig. 3. Standard hybrid A. alburnus × S. erythrophthalmus from Snitka River. T a b l e 2 . Statistical parameters of meristic characters and body indices in two parent species and hybrids Characters A. alburnus (n = 17) A. alburnus × S. erythrophthalmus (n = 30) S. erythrophthalmus (n = 4) M SE Min Max M SE Min Max M SE Min Max ll 48 0.75 43 55 43.6 0.28 40 47 39.8 0.48 39 41 A 18.3 0.29 15 20 14.3 0.12 13 16 11.5 0.29 11 12 H/l, % 22.4 0.38 19.4 24.9 24.8 0.29 22.0 28.4 29.1 0.61 28 30.9 aA/l, % 64.0 0.33 62.0 66.8 66.7 0.47 56.0 70.1 70.1 1.32 66.9 73.2 lA/l, % 20.0 0.18 18.3 21.3 17.6 0.20 15.2 19.6 15.4 0.12 15.3 15.8 Io/c, % 26.3 0.46 22.2 29.4 30.9 0.30 28.3 34.9 37.1 0.4 36.1 38 L e g e n d : ll — number of scales in the lateral line; A — number of branched rays in the anal fi n; l — body length to the end of the scale cover; H – body depth; aA — anteanal distance; lA — anal fi n length; Io — infraorbital distance; c — head length; SE — standard error. 300 A. A. Tsyba, M. Ghazali, S. V. Kokodiy, S. V. Mezhzherin In a number of body proportions and some meristic characters, the hybrids signifi cantly diff er from the two parental species (table 2). Th e parameters in question are the relative height of the body, measurements associated with the anal fi n, infraorbital distance, as well as the number of scales in the lateral line and branched rays in the anal fi n. By all these features, hybrids have intermediate values compared to the parental species. An important diagnostic feature of cyprinids is the formula of the pharyngeal teeth, which is 2.5–5.2 in bleak and 3.5– 5.3 in rudd. Hybrids showed a high variability in that parameter: there were 1–2 teeth on the outer row, and at least 4–6 teeth on the inner row. Th e formula of the meristic traits of hybrids and parental species, for comparison, is presented in table 3. First two PCs explain 42 % of total variance (table 4). Multivariate statistical analysis based on body indices and meristic parameters shows a clear separation of parental species and hybrids in the morphological space of the fi rst two components (fi g. 4). Th e most variation is associated with io, H, iH, hc, aA (larger in A. alburnus) and A, lA, ll (larger in S. erythrophthalmus) (table 5). Distribution of hybrids, although it is of an intermediate values, is somewhat biased towards bleak (fi g. 5). Greater similarity T a b l e 3 . Th e formula of meristic traits in hybrids, bleak, rudd Charac- ters Hybrids Bleak* Rudd* D III (8)9(10) III (6–7)8–9 III (7) 8 (9–10) A III (13)14–15(16) III 17–20 III 10–12 P I (12)13–14(15 I (11–12) 13–16 I 13–15 (16) V I 8 I (7) 8 (9) II (7) 8 l.l. 40–47 45–48 (37) 38–42 (43) d.f. 2.5–5.2, 2.4–5.2, 3.5–5.3, 2.5–5.1, 1.5–5.1, 2.5–6.2, 1.4–5.2 2.5–5.2 3.5–5.3, 3.5–5.2 *Movchan, 2011 Fig. 4. Results of the principal component analysis of the A. alburnus (squares), S. erythrophthalmus (triangles) and their hybrid (crosses): Top — position of the specimens in the space of fi rst two principal components; Bottom — loadings of the traits. 301Regular Intergeneric Hybridization of Leuciscine Cyprinids in the Dnipro Affl uants with bleak than rudd was also noted in off spring obtained under artifi cial conditions (Nikolyukin, 1952). A high degree of morphometric diff erences between hybrids is also confi rmed by the results of discriminant analysis. Th e level of identifi cation of hybrids and parental individuals by 22 indices and fi ve meristic characters is 100 %. H y b r i d i z a t i o n b e t w e e n A b r a m i s b r a m a × R . r u t i l u s . Hybridization between the bream and the roach was observed in fl oodplain of the Sozh River. Th ey were established by the presence of fi xed heterozygous states in the putative hybrids at the loci Aat-1, Ldh-B, Pt-3, and Pt-4. In the heterozygote, the product of the Pt-4 locus, which had a spectrum of fi ve fractions, was a tetramer (fi g. 6). A similar set of diagnostic loci in relation to roach is also characteristic of the silver bream, which also looks very similar to the bream. Th e single fi xed allele at the Pt-3 locus is the same in roach and silver bream. Th erefore, fi nding the bream and roach hybrids is proved by the presence of a constant heterozygote Pt-3100/102. Some of the found hybrids of roach and bream from the Sozh River sharply diff ered in size. In addition, they were collected in diff erent years. Th at, together with the fi nds of similar hybrids in the fl oodplain water bodies of the Desna River, proves that the hybridization of roach and bream in the Dnipro basin from the Desna to Sozh Rivers has been a fairly common and recurring phenomenon in recent decades. Discussion F a c t o r s a n d b i o l o g i c a l c o n s e q u e n c e s o f h y b r i d i z a t i o n . Natural hybridization of the roach and bream has been repeatedly reported (Witkowski & Blachuta 1980; Pervozvanskiy & Zelinskiy, 1981; Blachuta, Witkowski, 1984; Economidis et al., 1989; Fahy et al., 1988; Pitts et al., 1997; Slynko, 2000; Kodukhova & Karabanov, 2013; Hayden et al., 2014; Kuparinen et al., 2014; Konopiński & Amirowicz, 2018; Wood & Jordan, 1987) and confi rmed by the analyses of gene markers (Golubtsov et al., 1990; Slynko, 2000; Pitts et al., 1997; Konopiński & Amirowicz, 2018). In the middle of the 19th century, Abramis leuckarii Heckel et Kner, 1858 was even described from hybrid T a b l e 4 . Description of principal components Components Eigenvalue % of variance Cumulative % of variance PC1 8.536 30.5 30.5 PC2 3.121 11.1 41.6 T a b l e 5 . Correlation of principal components and variables Characters PC1 PC2 ll –0.744 –0.260 D –0.223 –0.068 A –0.851 –0.295 P –0.021 0.419 V 0.081 –0.110 L –0.081 –0.258 H 0.877 –0.167 H 0.609 –0.085 iH 0.808 –0.267 aD 0.298 –0.454 pD –0.343 0.268 aV 0.505 –0.139 aA 0.700 –0.135 PV 0.204 0.031 pl –0.003 0.453 lD 0.697 0.044 hD 0.652 0.556 lA –0.768 –0.103 hA 0.401 0.719 lP –0.411 0.566 lV 0.408 0.556 lC1 –0.584 0.250 lC2 –0.504 0.344 c 0.297 –0.157 hc 0.744 0.187 r 0.404 –0.234 o –0.395 0.546 io 0.925 0.132 302 A. A. Tsyba, M. Ghazali, S. V. Kokodiy, S. V. Mezhzherin individuals of the bream and roach. Numerous cases of hybridization gave reason to believe that hybridization between these species, although it occurs randomly, is an obligatory event during cohabitation, and individuals of A. brama × R. rutilus are perhaps the most common interspecifi c freshwater fi sh hybrids in Europe. Studies at the Rybinsk Reservoir have shown that at unstable spring fl ood, the ratio of hybrids can be 1.5  % (Kodukhova & Karabanov, 2013). Hybrids reach sexual maturity, are fertile, and their gametogenesis occurs by semi-cloning, abnormal meiosis and ameiosis, judging by the size of gametes (Slynko, 2000). Under artifi cial conditions, backcrosses can be obtained from hybrids (Wood & Jordan, 1987; Slynko, 2000; Peittse et al., 2005). However, eff ective reproduction of hybrids in nature is questionable. Triploid hybrid associates of the bream and roach, which should form in backcrosses and copulations of diploid hybrid gametes with haploid gametes of parental species, have not been found in natural conditions. In the Dnipro basin, hybridization between the bream and roach was noted by L. S. Berg (1912) and, somewhat later, by D. E. Beling (1928). Information about hybrids of these two species for the Dnipro basin has not been presented later. A series of 67 hybrids caught in the Hirskyi Tikych River (basin of the Southern Bug River) in 1990 is kept in the Department of Zoology of the National Museum of Natural History, National Academy of Sciences of Ukraine (Movchan et al., 2003). Interestingly, the hybrids in the Sozh River were mostly caught under fl ooded willow shrubs. In years with low fl oods, coastal shrubs were not inundated, and hybrids were not Fig. 5. A putative recurrent hybrid A. alburnus × S. erythrophthalmus from Snitka River. Fig. 6. Electrophoregram of muscle structure proteins spectra of A. brama (1); R. rutilus (2), and their hybrids A. brama × R. rutilus (3). 303Regular Intergeneric Hybridization of Leuciscine Cyprinids in the Dnipro Affl uants found. It seems obvious that the physically defective hybrids of bream and roach keep to poorly accessible microhabitats, occupying small riverside reservoirs, by which they are clearly inferior to the standards of parental species. Hybrids of bleak and rudd were obviously known back in the 19th century. It is thought that their presence in the aquatic systems of Germany has served as the basis for erroneous descriptions of the species Alburnus rosenhaueri Jäskel, 1866 and Scardiniopsis alburniformis Beneske, 1884 (Berinkey, 1960). In the fi rst decades of the 20th century, Berg (1912) pointed out the hybridization between the bleak and rudd, and F. D. Velykokhatko (1929) mentioned such hybrids in the middle reaches of the Ros River. In the 1970–1980s, hybrids of A. alburnus with species of the genera Abramis, Rutilus, Squalius, but never with S. erythrophthalmus, were oft en observed in European waters (Wheeler, 1978; Witkowski & Blachuta, 1980; Kammerad & Wuestemann, 1989). On the Iberian Peninsula, bleak is an invasive species that easily hybridizes with endemic representatives of the genus Squalius (Vinyoles et al., 2007), displacing them. Nevertheless, until now the fact of hybridization between bleak and rudd in nature has not been confi rmed at the level of gene makers, which, due to the intermediate nature of the morphology of hybrids, raises the question of the correctness of their determination purely by external features. Th erefore, our results, proving the high frequency of hybridization of rudd and bleak at the level of gene markers, are of interest. Th e studied cases of hybridization of the roach and bream, bleak and rudd are obviously the most common cases of interspecifi c hybridization within the European cyprinid subfamily Leuciscinae. Th is tendency is mainly determined by the following two ecological factors.  Firstly, they are the most numerous and widespread species of these subfamilies in Europe. Secondly, the spawning grounds and time of these fi sh overlap strongly. At the same time, intergeneric hybridization is in no way associated with the genetic proximity or remoteness of the crossing species and in this respect is completely random. I n t e r g e n e r i c h y b r i d i z a t i o n a s a p h e n o m e n o n o f t h e c y p r i n i d f a m i l y . Intergeneric hybridization is extremely rare in nature, but it is common in the cyprinid family. Th e ratio of intergeneric hybrids within Cyprinidae is 84 % of the total number of recorded cases of natural hybridization in this family (Scribner et al., 2000). Most of them belong to the European-West Siberian subfamily Leuciscinae and, to a lesser extent, to small cyprinids of North America, genetically close to this subfamily. In the other 10 families of freshwater fi shes, for which the natural hybridization was established, only one case of intergeneric hybridization was revealed within Catostomidae (Scribner et al., 2000). Th e ease and effi ciency of intergeneric hybridization in cyprinids is also confi rmed by artifi cial crosses (Nikolyukin, 1952), and the resulting hybrids oft en reach sexual maturity. What is the reason for this strange phenomenon? Obviously, the more distant are the species are, the more reliable is the reproductive isolation and less viable and fertile are their hybrids. Usually, intergeneric genetic diff erences are so great that hybrids can only be obtained in vitro, and they are usually sterile. Th e ease and prevalence of intergeneric hybridization in European cyprinids, fi rst of all, means that the generic status in this group is overestimated, which is confi rmed by both the taxonomic structure of the Leuciscinae subfamily and the insignifi cant level of genetic diff erentiation of species in these taxonomic units. Th e latter circumstance has long attracted the attention of researchers and even became the reason for raising the question of a sharp ambiguity in the rates of molecular evolution in diff erent taxonomic groups (Avise, 1977). In the system of European cyprinids, monotypization at the genus level is expressed like in no other group of Holarctic fi shes. In fact, all morphologically reliably isolated species of these European subfamilies are considered as a separate genus, and most of the recently recognized species are vicarious forms confi ned to diff erent basins (Kottelat & Freyhof, 2007; Perea et al., 2010). Th e level 304 A. A. Tsyba, M. Ghazali, S. V. Kokodiy, S. V. Mezhzherin of genetic distances between genera of the subfamily Leuciscinae is drastically low for vertebrates and other freshwater fi shes and is in sharp contrast to the diff erentiation of the East Asian and Trans-Palaearctic genera of this family (Hänfl ing, 2000; Briolaya et al., 1998; Perea et al., 2010). According to genetic distances (Hänfl ing, 2000) and the molecular clock recalculated on this basis, the age of generic radiation in modern Leuciscinae should be estimated at a level from 1.0 to 2.4 million years and dated to the Pleistocene. Th e divergence of the East Asian genus Rhodeus from Europe occurred, apparently, about 4 million years ago. Th e fi rst case corresponds to the period of formation of species, and the second one — to the genera of modern mammals (Mezhzherin, 1997). However, according to palaeontological data (Kovalchuk, 2017), the radiation of European cyprinid genera occurred in the late Miocene about 10–11 Ma. Th is contradiction between molecular and paleontological data can be explained by presenting the evolution of cyprinids not as a result of one-time radiation, but as a series of successive speciations that occurred every 2–4 million years. As a result, a number of species were formed with a similar set of morphological characters each time. 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