CHROMOSOME BANDING PATTERNS OF THE GUDGEON, GOBIO GOBIO (ACTINOPTERYGII, CYPRINIDAE)

The Gobioninae, along with other seven subfamilies, belongs to the family Cyprinidae (cf. Howes 1991). Hosoya (1986) considered them to be monophyletic, based on sensory canal patterns, morphology of supraoccipial and frontal, and modification of the anterior vertebrae. Three species; G. gobio, G. kessleri, and G. albipinnatus, occurring in Poland, have been traditionally assigned to the genus Gobio. According to Banarescu (1992) G. gobio from Europe, Siberia, and Central Asia represents Gobio sensu stricto. Naseka (1966) divided the genus Gobio sensu Banarescu into two genera Gobio and Romanogobio, and the latter genus containing two other species: R. kessleri and R. albipinnatus. Among Gobioninae only Gobio has a wide distribution range, extending throughout most of Europe, the Black Sea watershed in northern Anatolia, several landlocked lakes in Central Anatolia, some rivers in Central Asia, and most of Siberia in the watershed of the Arctic Ocean as far east as the Lena and Kolyma rivers (Banarescu 1999). Many geographical populations of this fish, classified as subspecies or “nations” have been described within its geographical range. According to Banarescu (1999) five European subspecies are now recognised. The majority of the morphological characters of the Gobio species and specifically the gudgeon, G. gobio, such as sexual dimorphism, age, growth, feeding habits, longevity, and some aspects of reproduction biology have been already described (see a review of Banarescu 1999). G. gobio, considered one of the most divergent fish species in Europe, with high level of interpopulational variability, has not been studied in relation to its banding chromosomal patterns. However, some cytogenetic features as C-banding, nucleolar organizer regions (NORs) and location of GC-rich DNA sites on chromosomes have been useful in cyprinid taxonomy (Buth et al. 1991, Boroƒ 2001). Some papers (Raicu et al. 1973, Sofradzija and Berberoviç 1975, Hafez et al. 1978, Vujoseviç et al. 1983, Vasil’ev 1985, Klinkhard et al. 1995) described this species as possessing, diploid number of 2n = 50 chromosomes, which is the most frequent among all other cyprinid lineages (Buth et al. 1991, Ráb and Collares-Pereira 1995). ACTA ICHTHYOLOGICA ET PISCATORIA (2005) 35 (2): 119–123


INTRODUCTION
The Gobioninae, along with other seven subfamilies, belongs to the family Cyprinidae (cf. Howes 1991). Hosoya (1986) considered them to be monophyletic, based on sensory canal patterns, morphology of supraoccipial and frontal, and modification of the anterior vertebrae. Three species; G. gobio, G. kessleri, and G. albipinnatus, occurring in Poland, have been traditionally assigned to the genus Gobio. According to Banarescu (1992) G. gobio from Europe, Siberia, and Central Asia represents Gobio sensu stricto. Naseka (1966) divided the genus Gobio sensu Banarescu into two genera Gobio and Romanogobio, and the latter genus containing two other species: R. kessleri and R. albipinnatus. Among Gobioninae only Gobio has a wide distribution range, extending throughout most of Europe, the Black Sea watershed in northern Anatolia, several landlocked lakes in Central Anatolia, some rivers in Central Asia, and most of Siberia in the watershed of the Arctic Ocean as far east as the Lena and Kolyma rivers (Banarescu 1999). Many geographical populations of this fish, classified as subspecies or "nations" have been described within its geo-graphical range. According to Banarescu (1999) five European subspecies are now recognised.
The majority of the morphological characters of the Gobio species and specifically the gudgeon, G. gobio, such as sexual dimorphism, age, growth, feeding habits, longevity, and some aspects of reproduction biology have been already described (see a review of Banarescu 1999). G. gobio, considered one of the most divergent fish species in Europe, with high level of interpopulational variability, has not been studied in relation to its banding chromosomal patterns. However, some cytogenetic features as C-banding, nucleolar organizer regions (NORs) and location of GC-rich DNA sites on chromosomes have been useful in cyprinid taxonomy (Buth et al. 1991, Boroƒ 2001. Some papers (Raicu et al. 1973, Sofradzija and Berberoviç 1975, Hafez et al. 1978, Vujoseviç et al. 1983, Vasil'ev 1985, Klinkhard et al. 1995 described this species as possessing, diploid number of 2n = 50 chromosomes, which is the most frequent among all other cyprinid lineages (Buth et al. 1991, Ráb andCollares-Pereira 1995). In the presently reported study we provide, for the first time, a new data on the location of Ag-stained NORs, AT-rich DNA regions (stained with DAPI), and GC-rich DNA regions on the chromosomes of the gudgeon G. gobio from the upper part of the Odra River in Poland.

MATERIALS AND METHODS
Fifteen specimens (six males and nine females) of gudgeon from the Odra River, near Legnica, (Poland) were studied. Mitotic chromosome preparations were obtained from the head kidney by standard air-drying technique (Ráb and Roth 1988). Conventional 5% Giemsa staining, C-banding (Haff and Schmid 1984), NOR sites by silver nitrate staining (Howell and Black 1980) and chromomycine A 3 staining (Sola et al. 1992) were applied. The distribution of A-T pairs on chromosomes was revealed by DAPI staining (Sola et al. 1992). The chromosomes were classified according to Levan et al. (1964).

RESULTS
All individuals of G. gobio were characterized by the same 2n = 50 number of chromosomes. The karyotypes consisted of 11 pairs of metacentric chromosomes, 13 pairs of submeta-subtelocentric, and one pair of acrocentric chromosomes, NF = 98 (Fig. 1). Ag-NOR sites on one submeta-subtelocentric chromosome pair No. 14 were found (Fig. 2a). Two cytotypes with size polymorphism in the NOR-bearing pair were observed (Fig. 2b). Chromosome sites with G-C-rich regions were shown on one submeta-subtelocentric pair ( Fig. 2c). C-positive blocks of heterochromatin mainly in the centromeric regions in some chromosomes pairs were detected (Fig. 1b). DAPI staining revealed A-T pair rich regions on "q" arm of one submeta-subtelocentric chromosome (Fig. 2d).

DISCUSSION
The chromosome diploid number of G. gobio 2n = 50 and diploid arm number (NF) ranging from 88 to 98 have been reported by other authors (Raicu et al. 1973, Sofradzija and Berberoviç 1975, Hafez et al. 1978, Vujoseviç et al. 1983). The same number of chromosomes was obtained in the presently reported study, and this feature is stable for the species of the genus Gobio, except Gobio uranoscopus from Slovakia with 2n = 52 chromosomes (Ráb and Collares--Pereira 1995). Diploid arm number NF = 98, determined in the present paper, and chromosome formulae of the gudgeon 2n = 50; (11 m + 13 sm-st + 1a) (Fig. 1) is similar with the results of the specimens from other populations previously described.
The majority of the European species, as well as most of other cyprinid fish species of North America, possessed single NOR-bearing chromosome pair (only about 30% species had multiple NOR sites) (Buth et al. 1991, Klinkhard et al. 1995. In cyprinids, NORs are located on all type of the chromosome (from metacentric to acrocentric) (Foresti et al. 1981, Galetti et al. 1985, Galetti jr. et al. 1984, Moreira-Filho et al. 1984, Takai and Ojima 1986, but in European species NORs are mainly located on sm/a chromosomes (Ráb and Collares--Pereira 1995). Classical rearrangements (inversion and translocation) are the most causes of interspecific NOR chro-Chromosome banding patterns of gudgeon 121 mosome differences (Gold and Amemiya 1986, Amemiya andGold 1988). Silver nitrate staining, which was done for the first time in this species, shown one sm-st NOR bearing chromosome pair (Fig. 2a). The size polymorphism of NORbearing chromosomes (pair No. 14) was observed in two individuals, and they have been described as two cytotypes "ss" and "sl" (Fig. 2b). The NORs enlargement that is an effect of amplification is common among different fish species including cyprinids. Size differences between homologous NORs have been found in other fish species (Sanchez et al. 1990, Jankun et al. 2003 and amphibian (Schmid 1982). Amplification or deletion of NOR sites could be an effect of the crossing-over disorders caused by wrong meiotic conjugation between repetitive nucleotide sequences of homologous chromosomes (Schmid and de Almeida 1988). These kinds of chromatin rearrangements lead to inactivation of NOR site (Foresti et al. 1981, Moreira-Filho et al. 1984, Takai and Ojima 1986. Distribution of heterochromatin in cyprinids generally is limited to centromeric regions and NOR sites (for review see: Klinkhardt et al. 1995, Ráb andCollares-Pereira 1995), but also some species with different type of heterochromatin location were found (Boroƒ 2001). C-positive blocks of heterochromatin in the centromeric regions of some investigated chromosomes in gudgeon are presented in this study (Fig. 1b).
In several cyprinid species there is non specific DAPI heterochromatin (Mayer et al. 1986, Schmid andGuttenbach 1988). In all investigated specimens, one small signal on a "q" arm of sm-st large chromosome after DAPI staining were found (Fig. 2d) that could be an effect of low accumulation of A-T pairs in gudgeon genome.
Using classical cytogenetic methods presented in this some species specific chromosome markers were found. However, they seem to be not sufficient for more detailed differentiation and identification of the gudgeon chromosomes. So, the next step using the molecular cytogenetic techniques (Fluorescence In Situ Hybridization-FISH) to enable identifying and description of some specific sequences on chromosomes are required.