﻿Karyotype of Sabanejewiabulgarica (Drensky, 1928) (Teleostei, Cobitidae) from the Danube Delta, Romania

﻿Abstract The karyotype of the freshwater fish Sabanejewiabulgarica (Drensky, 1928), from the Danube Delta, was studied by conventional Giemsa staining and the C-banding technique. The diploid chromosome number was 2n = 50. The karyotype contained 2 pairs of metacentric (the first one was much larger than the second one), 6 pairs of submetacentric and 17 pairs of subtelocentric to acrocentric chromosomes. Pericentromeric blocks of heterochromatin were revealed in most of the chromosome pairs. The karyotype phenotype of S.bulgarica was the same as found for S.balcanica from Northern Carpathian Mountains.


Introduction
Freshwater fishes of the genus Sabanejewia Vladykov, 1929 are small (max 15 cm TL), have a benthic lifestyle in river habitats and can be distinguished from all other genera of Cobitidae by a specific sexual dimorphism (males with lateral body swellings) (Nalbant 1994;Kottelat and Freyhof 2007;Šlechtová et al. 2008). Their distribution spans from northern Italy to the Aral Sea basin including tributaries of the Black Sea, Caspian Sea, Baltic Sea, Aral Sea and Mediterranean Sea (Kottelat and Freyhof 2007). The highest taxonomic diversity was long time assumed to be in the Danube basin, but phylogenetic studies (Perdices and Doadrio 2001;Šlechtová et al. 2008;Vasil'eva et al. 2022) have shown that several phenotypes within this area are very closely related (Sabanejewia balcanica (Karaman, 1922) species complex), so that the exact species composition is still not known.
Sabanejewia bulgarica (Drensky, 1928) was originally described as a species of the genus Cobitis Linnaeus, 1758 occurring in the lowest Danube basin including the Danube River itself. Junior synonyms are C. albicoloris Chichkoff, 1932and C. taenia tesselatus Pietschman, 1937(Kottelat 2012. For some time, most authors included S. bulgarica in the polytypic Balkan golden loach complex, with Sabanejewia balcanica, as a subspecies (Nalbant 1957;Bănărescu 1964;Bănărescu et al. 1972). Vasil'eva andVasil'ev (1988) analysed the geographical variations of characters among a number of local forms of golden loach throughout its range they showed that bulgarica is quite distinct from all other populations and deserves species rank (Nalbant 1994;Kottelat 1997Kottelat , 2012. The main differences include the overall colour of body, character of Gambetta´s zone, pigmentation and character of spots on the end of the caudal peduncle and basis of caudal fin as well as the habitat, as it is the only deep-water riverine form of Sabanejewia. Recently, Križek et al. (2020) based on several lines of evidence and analysis of individuals from type localities of both S. balcanica and S. bulgarica sensu Bănărescu et al. (1972) have claimed that Danubian golden loaches are genetically closer to S. bulgarica and S. balcanica itself is to be restricted to the drainage of the Vardar River.
Chromosomes of the loaches of the genus Sabanejewia remain poorly studied. Ráb et al. (1991) summarized three previous studies dealing with S. larvata (De Filippi, 1859), S. caspia (Eichwald, 1838) and S. kubanica Vasil'eva et Vasil 'ev, 1988. Vasil'ev and Vasil'eva (1994, 2019 reported karyotypes of S. caspia and S. aurata (De Filippi, 1863) from other locations (Kizylach Bay of the Caspian Sea, Kura River in Tbilisi and southern basin of the Bug River of the Black Sea, respectively). The karyotype of S. baltica Witkowski, 1994 from the Bug River in Poland was reported by Boroń (2000). All these studies document invariable 2n = 50 and remarkably small karyotype variability among the loaches of this genus. To contribute to the cytotaxonomy of Sabanejewia, the present report describes the karyotype and C-banding pattern of S. bulgarica from Danube Delta, Romania. Additionally, the comparison of karyotypes of all karyologically studied species of Sabanejewia was conducted.

Materials and methods
The five Sabanejewia bulgarica specimens examined (all females) were collected in the Saint George Branch, Danube Delta (July 1997). The loach individuals arrived at the laboratory (Danube Delta Institute, Tulcea) in very bad condition and died subsequently. One female displayed the colour pattern and high body as described in original description by Drensky (1928), while four other individuals displayed other colour patterns (Fig. 1).
Standard procedure for chromosome preparation followed Ráb and Roth (1988) and C-banding technique followed Haaf and Schmid (1984). Chromosomes were clas-sified according to the system of Levan et al. (1964). The analysed individuals are deposited as vouchers in fish collection of Laboratory of Fish Genetics, IAPG, AS CR, Liběchov (Accs. code. SB 7/97).

Results and discussion
The diploid chromosome number was determined as 2n = 50 in all five individuals. The detailed karyotype analysis was carried out on a single specimen with morphological characters corresponding to the original description (Fig. 1, top individual). The karyotype comprised 2 pairs of metacentric (m), 6 pairs of submetacentric (sm) and 17 pairs of subtelocentric (st) to acrocentric (a) chromosomes (Fig. 2). C-banding procedure reveals conspicuous pericentromeric blocks of heterochromatin in most of the chromosome pairs with very prominent blocks in the largest m chromosome and the first sm element.
Our results enable us to compare the karyotype of Sabanejewia bulgarica with karyotypes of other species of this genus analysed so far (Table 1). It is evident that the karyotype of S. bulgarica shows some apparent similarities with those of congeneric species: i) the same diploid chromosome number 2n = 50, ii) the low number of m chromosomes (two pairs), iii) the numbers of sm chromosomes (six pairs) and st to a chromosomes (17 pairs), iv) likely C-banding pattern correspond to those of S. balcanica (Ráb et al. 1991). The comparison also demonstrates some differences in karyotype structure, which, however, need explanation. Lodi andMarchioni (1980) andVasil'ev (1985) distinguished the categories of st and a chromosome while Vasil'eva and Vasil'ev (1988), Ráb et al. (1991) and Vasil'ev and Vasil'eva (1994, 2019 did not recognize these morphological types of chromosomes as different ones but combined them together. This is especially due to the presence of small acrocentric-like chromosomes with their centromere position ranging gradually from subterminal to nearly terminal which makes difficult the precise assignment of these chromosomes to particular morphological types. The possible interspecific differences in proportion of such st and a chromosomes are in addition masked by the effect of chromosome arms contraction during mitosis due to the effect of timing and dose  of colchicine treatment. Thus, it is difficult to interpret the karyotype descriptions of different authors.
In conclusion, the karyotype of S. bulgarica from the Danube Delta is very similar to karyotypes of other Sabanejewia species in respect of the chromosome number and morphological types of chromosomes, and is nearly identical with karyotype of S. balcanica from the Northern Carpathian range in Slovakia. Further detailed cytotaxonomic as well as other genetic surveys of populations of Sabanejewia throughout its distribution will answer problems of systematics of Danubian golden loaches.