The karyotype of Greek cobitid fish Cobitis strumicae Karaman, 1955, from Lake Volvi, Greece, a representative of one of its two major intraspecific phylogenetic lineages, was analysed by means of sequential Giemsa-staining, C-banding, silver-staining, CMA₃ fluorescence banding and also by in situ hybridization (FISH) with rDNA probe. The diploid chromosome number was 2n = 50, karyotype composed of 10 pairs of metacentric to submetacentric and 15 pairs of subtelocentric to acrocentric chromosomes. The nucleolus organizer regions (NORs) as revealed by Ag- and CMA₃ staining and FISH were situated in the telomeric region of the fourth submetacentric chromosome pair. The chromosomes contained very low content of C-positive heterochromatin. No heteromorphic sex chromosomes were detected. This first karyotype report for any species of lineage Bicanestrinia Băcescu, 1962 shows a simple karyotype dominated by acrocentric chromosomes and possessing single NOR-bearing chromosome pair. Cytotaxonomic implications of this finding for the taxonomy of the genus Cobitis Linnaeus, 1758 are further discussed.

chromosome banding, NOR phenotype, FISH, rDNA, cytotaxonomy of Cobitis loaches

The genus Cobitis Linnaeus, 1758 attracted the interest of evolutionary biologists by producing several gynogenetic female-only lineages after hybridisation of species (Bohlen and Ráb 2001). As reasons for the asexual reproduction in these hybrids differences in the karyotype and chromosome structure between the parental species have been proposed. Indeed, within Cobitis a large variability of karyotypes and chromosomal markers have been observed (Janko et al. 2007). On the other hand, species of Cobitis are morphologically highly similar and difficult to identify on the basis of morphologic characters. They have a pronounced sexual dimorphism with males being smaller than females and developing an ossified plate-like structure on the dorsal side of the pectoral fins, called ‘lamina circularis’. The widespread presence of hybrid lineages further complicates the systematics and taxonomy of Cobitis loaches, therefore genetic methods are applied in identification of species. Chromosome studies have shown that most species have a diploid chromosome number of 2n = 50, but highly diversified karyotypes (reviewed in Ráb and Slavík 1996, Arai 2011). This genetic marker therefore appears to be one of the key parameter in the genetic and taxonomic studies of Cobitis loaches, e.g. Ráb and Slavík (1996), Boroň and Danilkiewicz (1998), Vasil’eva and Vasil’ev (1998), Ráb et al. (2007), and serves as one of the determination tools to identify genome composition in hybridogenous clonal asexual biotypes (Janko et al. 2007, Majtánová et al. 2016).

Recent phylogenetic studies (Buj et al. 2014, Ludwig et al. 2001, Perdices and Doadrio 2001, Perdices et al. 2016) demonstrated that the European representatives of Cobitis include five major lineages, namely the ‘Siberian lineage’, represented by a single species C. melanoleuca Nichols, 1925, Băcescu's (1962) subgenera Acanestrinia (now often referred to as ‘Adriatic lineage’), Iberocobitis, Bicanestrinia, and Cobitis s. str. The subgenus Bicanestrinia is morphologically well characterized by having two laminae circulares on the pectoral fins of males. Species of Bicanestrinia occur in the Middle East (Turkey, Iran, Syria) and southeast Europe (Bulgaria, Greece) (Bohlen et al. 2006). Up to now, only one species of Bicanestrinia, C. linea (Heckel, 1847), has ever been analysed in a cytogenetic study, therefore little is known about cytogenetic similarities and differences between Bicanestrinia and Cobitis s. str. One of the European species of Bicanestrinia, C. strumicae Karaman, 1955, has long been known from rivers draining into the Aegean Sea, such as Struma, Maritza and the lakes adjacent to the Struma basin such as Volvi and Koronia in Greece. However, it has recently been found in the Danube basin, where it is genetically involved in asexual hybrid forms (Choleva et al. 2008). Since further studies on this example of a sperm-dependent hybrid switch of the sexual hosts require a proper identification of the genetic material of C. strumicae, the cytogenetic analysis of Struma spiny loach will complete identification tool box of hybrid biotypes of the genus Cobitis.

This study reports on the karyotype and other chromosomal characteristics of Greek cobitid fish C. strumicae from population inhabiting Lake Volvi, Greece, analysed by means of sequential Giemsa-staining, C-banding, silver-staining, CMA₃ fluorescence banding and by in situ hybridization (FISH) with 28S rDNA.

Ten males and two females were collected at the outlet of a thermal spring into Lake Volvi, Greece, by dip net and transferred alive to the laboratory. The examined specimens are deposited as voucher samples in the collection of the Laboratory of Fish Genetics, IAPG, CAS, Liběchov, under Accession Code CoS/97. Valid Animal Use Protocol was in force during study in IAPG (No. CZ 02386). Standard procedures for chromosome preparation followed Ráb and Roth (1988). Silver (Ag-) staining and Chromomycin A₃ (CMA₃) fluorescence banding, for detection of NORs, followed Howell and Black (1980) and Sola et al. (1992), respectively. The sequence of stainings followed protocol of Rábová et al. (2016). Fluorescence in situ hybridization (FISH) with a mouse rDNA biotinylated probe (clone I-19, a 4.2-kb EcoRI-SalI fragment containing most of the 28S rDNA-coding region) to detect chromosomal sites of rDNA, i.e. sites of NORs, followed the procedure of Reed and Phillips (1995) and Ozouf-Costaz et al. (1996). Briefly, a mouse rDNA clone, was biotin-labelled by nick translation (Oncor, Inc). Chromosomes were pretreated by incubating the slides in 2X SSC (pH 7.0) at 37 °C for 30 min, dehydrated in a 4 °C ethanol series, and air-dried. Chromosomal DNA was denatured by incubating the slides in a filtered 70% formamide/2X SSC solution (pH 7.0) at 70 °C for 2 min, followed by dehydration in 4 °C ethanol series. Labelled probe was diluted to 16.6 ng/µl in hybridization solution (Hybrisol VII, Oncor; 50% formamide), denatured by incubation at 70 °C for 5 min and placed immediately on ice until applied to slides. Hybridization was performed using 20–25 µl (~ 250 ng) of probe mixture/slide and incubated overnight in a 37 °C humidity chamber. After hybridization, slides were washed in a 50% formamide/2X SSC solution (pH 7.0) at 37 °C for 15 min, followed by an 8 min wash in 2X SSC (37 °C, pH 7.0). Slides were washed at room temperature for 2 min each in the following series: 4X SSC; 4X SSC + triton X; and a 1:1 mix of 4X SSC and PN buffer (0.1 M NaHP0, 0.1 M NaH₂P0, 5% NP-40 detergent, pH 8.0). Fluorescein-isothiocyanate (FITC)-conjugated avidin was used to detect hybridization signal. Chromosomes were counterstained with propidium iodide (0.375 µg/ml) in antifade (10 mg/ml p-phenylenediamine dihydrochloride (DAPI) in PBS/90% glycerol, pH 8.0) and chromosomes viewed under epifluorescence.

At least 25 Giemsa-stained or banded metaphases plates per individual were examined, most of them sequentially. Chromosomes were classified according to Levan et al. (1964), metacentric to submetacentric and subtelocentric to acrocentric chromosomes, respectively were grouped together in Fig. 1.

Figure 1.

Karyotypes of a male (a) and female (b) of C. strumicae from Lake Volvi arranged from sequentially Giemsa-stained (upper row) and C-banded (lower row) chromosomes; sequentially Ag-stained chromosome pair with positive signal is framed in (a) and (b); metaphase cells of C. strumicae after CMA₃ staining (c) and FISH with rDNA probe (d) chromosomes bearing CMA₃ and FISH signals are framed; m – metacentric, sm – submetacentric, st – subtelocentric and a – acrocentric chromosomes. Scale bar: 10 μm.

This study was supported by the project EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE and RVO: 67985904 (E. H., P.R., Z.M.,), Czech Science Foundation 17-09807S (L. Ch.) and (partly) by a Research network of the French Ministère de l´Education Nationale (C.O.- C.). The authors thank P.S. Economidis, T.T. Nalbant for valuable help, advices and stimulating discussions about diversity of Bicanestrinia loaches and M. Rábová, IAPG, Liběchov, for laboratory assistance. This study is the part of a series Cytogenetics of bisexual species and their asexual hybrid clones in European spined loaches, genus Cobitis (Cobitidae, Cypriniformes).