Cytogenetical analyses in three fish species of the genus Pimelodus ( Siluriformes : Pimelodidae ) from rio São Francisco : considerations about the karyotypical evolution in the genus

Karyotypes and other chromosomal markers were investigated in three species of the catfish genus Pimelodus, namely P. fur, P. maculatus and Pimelodus sp., from municipality of Três Marias, Minas Gerais, Brazil, using differential staining techniques (C-banding, Silver nitrate and CMA 3 staining). The diploid chromosome number was 2n = 56 in P. maculatus and Pimelodus sp., while in P. fur 2n = 54. The karyotype of P. fur consisted in 32M + 8SM + 6ST + 8A with fundamental number (NF) of 100, that of P. maculatus 32M + 12SM + 12A with NF = 112, and that of Pimelodus sp. had 32M + 12Sm + 6ST + 6A with NF = 106.The nucleolar organizer regions (NORs) in all three species were invariably detected in telomeres of longer arm of the 20 chromosome pair. These sites were also positive after CMA 3 and C-banding. No heteromorphic sex chromosomes were detected and C-banding pattern was species specific. Inferences about the karyotype differentiation in Pimelodus and putative chromosomal rearrangements are hypohesized.


Introduction
The rio São Francisco basin is the fourth most important Brazilian basin, comprising nearly 7,4% of national territory, which includes North, Northeastern and Center-Western regions (Paiva, 1982).According to Britski et al. (1988), 133 fish species were described in the São Francisco and 57 of them occur at Três Marias, including: endemic catfish species Pimelodus fur and Pimelodus sp., and P. maculatus, also found in the rio Paraná basin.These three species are representatives of the family Pimelodidae and share several morphological similarities, being identified especially by their color patterns.
The family Pimelodidae is one of the most important speciose groups within Siluriformes that includes 31 recognized genera and 83 valid species (Lundberg & Littmanm, 2003).Pimelodus, with 24 species (Lundberg & Littmanm, 2003) is one of most cytogenetically studied genus of Pimelodidae.It is widespread throughout South American rivers, comprising endemic species, such as P. ortomanni (rio Iguaçu basin, Southern Brazil), as well as widely distributed ones, like P. maculatus.
The genus Pimelodus is known by the presence of the diploid number 2n = 56 associated with great variation in karyotype composition (Swarça et al., 2000).Only one case of structural polymorphism was detected in this genus (Dias & Foresti, 1993).

O F S
In the present work, cytogenetic analyses were carried out in three species of Pimelodus from rio São Francisco, in order to provide useful information for both chromosomal characterization of these species and identification of putative chromosomal rearrangements involved in the evolution of the group.
Single Ag-NORs located at terminal position of the long arms of 20 th chromosome pair was observed in all three species (Fig. 1 boxes).An NOR-size heteromorphism between homologous in P. fur. was also observed C-banding pattern was peculiar for each species.The NOR sites were C-band positive.P. fur and Pimelodus sp. (Fig. 2a  and 2c) had C-band positive blocks distributed over pericentromeric and terminal regions of most of chromosomes.In Pimelodus sp., C-band positive segments were also observed in both terminal regions of a pair of M chromosomes.P. maculatus had the lowest amount of C-band positive segments, located in pericentromeric regions (Fig. 2b). CMA

Discussion
The present work reports on the diploid number lower than 2n = 56 among catfishes of the genus Pimelodus.The descrease in 2n in P. fur is likely due to the chromosomal rearrangement of fusion or translocation type.On the other hand, a diploid number higher than 2n = 56 was reported for P. blochii where equals 2n = 58 (Della-Rosa et al., 1980).Oliveira & Gosztonyi (2000) suggested that 2n = 56 chro- New observation is the absence of A chromosomes in the karyotype of P. maculatus.Specimens under study as yet, the lowest number of A chromosomes was one pair (Pimelodus sp.from Iguaçu river, Souza et al., 2004a), whereas the highest was six pairs (P.fur, P. maculatus and Pimelodus sp., Toledo & Ferrari, 1976).The most frequent pattern in the genus is the presence of three pairs of Achromosomes (Vissoto et al., 1999;Swarça et al., 2001;Borin & Martins-Santos, 2002) and the lack observed in P. maculatus could be a result of
Based on the available cytogenetic data for the family Pimelodidae, and Siluriformes as well, we hypothesis that pericentric inversions appear to be an important, if not the most common rearrangement karyotype differentiation in the family keeping the diploid number but diversifying the karyotype morphology.Dias & Foresti (1993) have also analyzed P. maculatus and Pimelodus sp. from rio São Francisco.They reported the same karyotype composition for both species (40 M/SM + 16 ST/A), besides the occurrence of a structural polymorphism.Their results however do not agree with our data, differences between both reports can be attributed to fact that chromosomes were assigned into particular categories on the basis of differently condensated chromosomes.
Ag-NOR analyses confirmed that the species under study retain the pattern observed in the genus, i.e., a predominance of single Ag-NORs at terminal position of longer arm of ST chromosomes (Dias & Foresti, 1993;Vissoto et al., 1999;Borin & Martins-Santos, 2002).On the other hand, the occurrence of Ag-NORs at chromosomal short arms appears to be relatively rare (Borin & Martins-Santos, 2002;Souza et al., 2003).
The NOR sites of the three species were C-positive and thus heterochromatic, as also reported in another population of P. maculatus (Swarça et al., 2001a).A size heteromorphism of these sites between homologues was frequently observed in P. fur after C-banding, CMA 3 and Ag-nitrate staining.Such size heteromorphism has been frequently reported in Pimelodidae (Gil et al., 1998;Vissoto et al., 1999;Swarça et al., 1999, Souza et al., 2004a, b).Because of their redundancy, the rDNA regions are likely more susceptible to unequal crossing-over or chromosomal rearrangements, leading to size differences between NOR-bearing homologous, particularly if these sites are associated to heterochromatic regions (Moreira-Filho et al.,1984).
Correspondence between CMA 3 and Ag-NOR sites were observed in several pimelodid species, such as Pirinampus pirinampu (Swarça et al., 1999), Zungaro zungaro (Swarça et al., 2001b), Pimelodus maculatus (Swarça et al.,2001a;Borin & Martins-Santos, 2002), P. abscontidus and P. ornatus (Borin & Martins-Santos, 2002), and P. argentus and P. mysteriosus (Souza et al., 2003).In the present study, the same correspondence between CMA 3 and Ag-NOR positive sites was observed.Besides, other fluorescent signals were detected in C-band positive chromatin of Pimelodus sp.documenting presence of heterochromatin of GC-type This character shows that this species presents a distinctive heterochromatin classes then other two species.A similar situation was reported in P. maculatus from rio Paraguay (Souza et al., 2003).
Species specific C-banding pattern permitted the determination of each species: P. fur and Pimelodus sp. had small Cband positive sites at the centromeric and terminal regions of some chromosomes, similarly to the pattern observed in this group (Vissoto et al., 1999;Borin & Martins-Santos, 2002;Souza et al., 2003) while P. maculatus had less C-band positive segments.
Nevertheless, a pair of M chromosomes bearing C-band positive segments at both telomeres were observed in Pimelodus sp.only Similar C-banding pattern was reported in other species of the genus Pimelodus (Pimelodus sp. and P. heraldoi -Souza et al., 2004a;P. maculatus, P. ornatus and P. absconditus -Borin & Martins-Santos, 2002) and some pimelodids and heptapterids (Pimelodella sp.-Vasconcelos & Martins-Santos, 2000;Rhamdia sp. -Garcia et al., 2003;Iheringichthys labrosus -Carvalho et al., 2004).This situation is frequently reported in these catfishes, indicating that the presence of this chromosome pair could be a shared condition and a cytotaxonomic marker for both families.
The present study shows, for the first time, two peculiar situations for representatives of the genus Pimelodus: the occurrence of a lower diploid number in P. fur and the absence of A chromosomes in P. maculatus.These findings might be related to intrinsic features of São Francisco River basin, which could favor the endemism of fish species in this basin.The present results contribute to the evolutionary and cytogenetic understanding of the phylogenetic relationships within this genus and the family Pimelodidae.
3 staining revealed positive fluorescent signals cor-responding to Ag-positive signals in all three species.Moreover, other fluorescent signals corresponding with some other C-band positive segments were also observed in Pimelodus sp.(Figs.2d, 2e, and 2f).

Fig. 2 .
Fig. 2. Somatic chromosome metaphases of P. fur (a, d), P. maculatus (b, e) and Pimelodus sp.(c, f) submitted to: C banding (a, b, c) and staining with CMA 3 (d, e, f).The major arrows indicate the NOR-bearing pairs.NOR association (b, f) and NOR size heteromorphism (a) are exemplified.The arrows indicate positive chromosome regions corresponding with heterochromatin sites after treatment with fluorochrome and the arrowheads indicate a pair of metacentric chromosomes with both telomeres heterochromatic.