A unique diploid-tetraploid unisexual-bisexual fish complex (Pisces, Cobitidae)

Doklady Biological Sciences, Vol. 404, 2005, pp. 364–366. Translated from Doklady Akademii Nauk, Vol. 404, No. 4, 2005, pp. 559–561. Original Russian Text Copyright © 2005 by Vasil’ev, Lebedeva, Vasil’eva, Levenkova, Ryskov.

GENERAL BIOLOGY

A Unique Diploid-Tetraploid Unisexual-Bisexual Fish Complex (Pisces, Cobitidae) V. P. Vasil’ev, E. B. Lebedeva, E. D. Vasil’eva, E. S. Levenkova, and Corresponding Member of the RAS A. P. Ryskov Received March 29, 2005

The discovery of vertebrate clonal forms offered new prospects in evolutionary genetic, cytological, and ecological research, which can be divided into four major blocks: clonal form origin, cytogenetic mechanisms of clonal inheritance, clonal diversity and its sources, the ecological problems of the coexistence of clonal and bisexual forms. The important results of these studies were the following: clonal forms proved to have hybrid origin, the relationships between hybridization, unisexual reproduction, and polyploidy have been understood in the evolutionary respect. As a result, a concept of reticular speciation in vertebrates have been developed [1, 2] and the origin of even-polyploid species with restored bisexuality proved to be the final stage of this process [3, 4]. By now, about 80 forms were identified among fish, amphibia, and reptiles, which are reproduced by parthenogenesis, gynogenesis, and hybridogenesis. Unlike parthenogenesis that occurs in some groups of reptiles [5], natural gynogenesis involves males whose sperms stimulate egg development, although there is no true fertilization. Therefore, fish and amphibian gynogenetic forms inhabit the same areas as one or two closely related bisexual species from whose hybridization these forms have originated. In rare cases, the reproduction of clonal forms involves males of a third, less closely related, bisexual species. This way of clonal form reproduction leads to the development of unisexual–bisexual (clonal–bisexual) complexes, such as those indentified in the genera Poecilia, Poeciliopsis (Poeciliidae), Fundulus, Menidia (Atherinidae), Phoxinus (Cyprinidae), and Cobitis (Cobitidae) [2, 5, 6]. These complexes are diploid–diploid, diploid–diploid–triploid, and, in rare cases, diploid–triploid–tetraploid. In this study, we describe fish complexes from the genus Cobitis, in

Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 119071 Russia Institute of Gene Biology, Russian Academy of Sciences, ul. Vavilova 34/5, Moscow, 117334 Russia Research Zoological Museum, Moscow State University, ul. Bol’shaya Nikitskaya 6, Moscow, 125009 Russia

which the unisexual form is represented exclusively by tetraploid females. The material for this study was collected in the Don River basin in 2004 (Table 1). The level of ploidy was determined by calculating the number of chromosomes and measuring erythrocyte square (Table 2). In triploid forms (3n), the chromosome numbers depend on their origin and comprise 74 (in all water bodies of Russia and Ukraine studied) or 73, 74, and 75 (in many waters of Eastern and Western Europe) [8, 9]. The following forms of the genus Cobitis inhabit waters of the Don basin: diploid bisexual species C. melanoleuca (the chromosome number 2n = 50, the chromosome arm number NF = 78) and C. rossomeridionalis (2n = 49, NF = 86 in males and 2n = 50, NF = 86 in females), as well as a unisexual female form (which was visually indistinguishable from the latter) with a chromosome number of 98 and NF = 161–163 (Fig. 1) (97–99 chromosomes, taking into account the accuracy of counting the chromosomes). Table 2 shows data obtained by erythrocyte area measuring in Cobitis from the Don River basin and in triploid individuals from the Moskva River for comparison. These data testify to the fact that individuals from the Don River basin with a chromosome number of 97–99 and erythrocyte square of 145.9 ± 5.47 are tetraploid. The ratio of tetraploids represented only by female and bisexual C. rossomeridionalis and C. melanoleuca was the following: 8 : 1: 5 in the Sosna River; 8 : 4 : 8 in the Don River; 17 : 7 : 145 in the Lesnoi Voronezh River. The first report on the clonal–bisexual complex of Cobitis was published in 1981 [10]. It was found in the Moskva River near Zvenigorod and shown to include five forms: two diploid bisexual species (C. taenia and C. melanoleuca), a triploid form, and two tetraploid forms [11]. The triploid form is of hybrid origin, and its genome consists of a haploid karyotype of C. taenia and a diploid karyotype of an unknown species, as we previously suggested [4]. However, today, we assume that this may be a trihybrid form, i.e., it carries haploid karyotypes of three different species. DNA fingerprinting with a large set of microsatellite probes showed a high homogeneity (monoclonality) of this triploid form [12]. The tetraploid forms are also of hybrid origin: (1) a triploid form × C. taenia and (2) a triploid form ×

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A UNIQUE DIPLOID-TETRAPLOID UNISEXUAL-BISEXUAL FISH COMPLEX

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Table 1. The composition of the unisexual–bisexual complex of Cobitis in waters of Russia and Ukraine Composition of complexes

Locality

Bisexual diploid species

Zapadnaya Dvina River Dniester River Dnieper River basin Upper Dnieper River Bol’shaya Desna River Lower Dnieper River Yuzhnyi Bug River basin Savranka River Kodyma River Volga River basin Moskva River Sura River Oka River Sea of Azov basin Obitochnaya River Don River Basin Sosna River Don River (Eletz town) Lesnoy Voronezh River

Unisexual forms

C. taenia C. rossomeridionalis

Triploid Triploid

C. taenia C. taenia C. rossomeridionalis

Triploid Triploid Triploid

C. taenia C. rossomeridionalis

Triploid Triploid

C. melanoleuca, C. taenia C. melanoleuca, C. taenia C. melanoleuca, C. taenia

Triploid and tetraploid* Triploid and tetraploid* Triploid and tetraploid*

C. rossomeridionalis

Triploid and tetraploid*

C. rossomeridionalis, C. melanoleuca C. rossomeridionalis, C. melanoleuca C. rossomeridionalis, C. melanoleuca

Tetraploid Tetraploid Tetraploid

*Our data.

Table 2. The size of erythrocytes from diploid, triploid, and tetraploid forms of Cobitis Species/form C. rossomeridionalis Tetraploid form Triploid form

Number of erythrocytes measured

Length, µm Don River basin 13.3 ± 0.52 18.5 ± 0.29 Moskva River basin 16.4 ± 0.69

32 7 241

C. melanoleuca. The latest studies showed that diploid–polyploid complexes frequently occur in the Volga River basin and in the rivers of the Black Sea, Baltic Sea, and North Sea basins (Table 1) [7–9, 13]. The proportion of tetraploids in the Moskva River is small (about 10%); a significant part of them appeared de novo in each progeny [14]. Among other unisexual– bisexual complexes of species from the genus Cobitis, no tetraploids were found (Table 1) or their frequency was extremely low (less than in the Moskva River), which suggests their sporadic origin and the lack of evolutionary independence. All these complexes are in essence diploid–triploid or diploid–diploid–triploid. Thus, the diploid–tetraploid complexes that we have identified in the Don River basin represent a unique phenomenon. Note that these complexes were not DOKLADY BIOLOGICAL SCIENCES

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Width, µm

Area, µm2

8.7 ± 0.39 10.1 ± 0.42

90.3 ± 5.89 145.9 ± 5.47

9.9 ± 0.49

127.9 ± 9.16

found in other groups of vertebrates (fish or amphibian) in which clonal–bisexual complexes exist. Regarding the origin of discovered tetraploid forms, only preliminary conclusions can be made now. Since the tetraploid form lives together with C. rossomeridionalis and C. melanoleuca, it might have appeared due to the hybridization of a triploid form with one of these species. The gynogenetic triploid form produces unreduced triploid eggs (3n = 74, NF = 120). When the latter are fertilized by haploid sperms, various karyotypes may appear. Crosses between a triploid form and C. melanoleuca (in the haploid karyotype, n = 25, NF = 39) should yield tetraploids with 4n = 99, NF = 159. The hybridization of the triploid form with C. rossomeridionalis (two types of sperms: n = 24, NF = 43 and n = 25, NF = 43) yields two tetraploid lines: 4n = 98, NF = 163 and 4n = 99, NF = 163. Since the number of

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complexes, reticular speciation, and clonal and bisexual forms of vertebrates in general. ACKNOWLEDGMENTS This study was supported by the programs of the Russian Academy of Sciences “Fundamental Principles of Biological Resource Managements” and “Gene Pool Dynamics,” Russian Foundation for Basic Research (project no. 05-04-48229), and the Program for Supporting Leading Scientific Schools (project no. NSh1995.203.4). (a)

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(b) Fig. 1. Metaphases of (a, b) two individuals of the tetraploid form of the Cobitis: 4n = 98.

chromosomal arms in the tetraploid form is 161–163, it appeared most likely due to hybridization between the triploid form and C. rossomeridionalis. Note that the triploid form itself has been superseded by the tetraploid form with time. Probably, the tetraploid form appeared due to the hybridization between the triploid form and some other species that is now absent in the Don River basin. We are studying the diploid–tetraploid complexes from the Don River basin at the level of the nuclear genome (by DNA fingerprinting) and at the level of the mitochondrial genome (by sequencing the cytochrome b gene). This approach is of great interest and provides new information on the evolution of clonal–bisexual

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