Karyotypic differentiation through chromosome fusion and number reduction in Imparfinis hollandi ( Ostariophysi , Heptapteridae )

The Neotropical Heptapteridae fish Imparfinis hollandi, endemic to the Iguaçu River Basin (Brazil), was cytogenetically analyzed and the diploid chromosome number of 2n = 42 chromosomes was determined (22m + 10sm + 10st), the lowest diploid number in this genus and family. Like other Heptapteridae species, only one NOR-bearing chromosome pair was detected by silver nitrate staining. Dark heterochromatic blocks were visualized in only three chromosome pairs, and chromomycin A3 + bands were coincident with Ag-NORs. Although no intercalary (TTAGGG)n sequence was observed through FISH with a telomere probe, an asymmetric karyotype showing four large chromosome pairs with diploid chromosome number reduction suggests that tandem chromosome fusions probably occurred during the karyotypic differentiation of Imparfinis hollandi.

Comprising 1,548 valid species grouped into 15 families, Siluriformes is the most diversified and widely distributed Ostariophysi fish order, and its members are commonly known as catfishes (Reis et al., 2003) and occur in all continents.The genus Imparfinis belongs to Heptapteridae, an endemic family of the Neotropics that includes 26 genera of small to medium-sized fishes with 186 valid species (Bockmann and Guazzelli, 2003).Imparfinis hollandi was previously identified as Pariolius hollandi (Julio et al., 1997) or Heptaterus hollandi (Eschmeyer, 1998), thus reflecting the systematic difficulties of this group.Recent studies in Heptapteridae have been performed in order to discover the family phylogeny and to provide major rearrangements in the classification of the group, as well as to recognize the correct name of some taxa, since the species-level taxonomy is poorly developed in their genera (Pinna, 1998;Bockmann and Guazzelli, 2003).
Due to the rare occurrence of Imparfinis hollandi, only three individuals (2 males and 1 female) were collected from the Salto Osório reservoir of the Iguaçu River, in Quedas do Iguaçu (Paraná State, Brazil).Mitotic chromosomes were obtained from kidney cells.Ag-NOR (Nucleolar Organizer Regions) sites were detected using silver nitrate staining.Chromomycin A 3 staining, using distamycin A as a counterstaining, was performed to investigate GC-rich isochores.C-banding was produced with barium hydroxide.The classification of chromosomes in metacentric (m), submetacentric (sm), and subtelocentric (st) followed the arm ratio criterion, and the chromosomes were organized into types and in a decreasing order of size in the karyotype.All these methods were carried out according to procedures found in Margarido and Galetti (2000).A telomere repeat probe was generated by PCR according to IJdo et al. (1991) using primers (TTAGGG) 5 and (CCCTAA) 5 .The probe was marked with 14-dATP biotin by nick translation following the manufacturer's instructions (Bionick Labelling System -Invitrogen).Chromosomes were denatured in 0.05 N NaOH/2xSSC for 3 min.After overnight hybridization at 37 °C, the hybridization signal was detected using conjugated avidin-fluorescein (FITC) and biotinylated anti-avidin antibody.Chromosomes were counterstained with propidium iodide (50 μg/mL) and analyzed with an Olympus BX50 epifluorescence microscope.Chromosome images were captured with the use of the CoolSNAP-Pro software (Media Cybernetic).
The diploid chromosome number determined for Imparfinis hollandi (2n = 42; 22m + 10sm + 10 st) (Figure 1a) is different from the diploid number of all the other Imparfinis species studied until now (predominantly 2n = 58; Vissoto et al., 1997;Vissoto et al., 2001;Stolf et al., 2004), and is the lowest diploid number in the Heptapteridae family.Until the present study, the lowest diploid chromosome number described for a heptapterid fish was 2n = 46 chromosomes in the genus Pimelodella (Vissoto et al., 1999;Dias and Giuliano-Caetano, 2002).Even though Pimelodella shows 2n = 46 chromosomes, the karyotype of this species is symmetric.This stands in contrast with Imparfinis hollandi which shows an asymmetric karyotype with four outstandingly large chromosomes pairs (pairs 1m, 2m, 12sm, and 17st) that correspond to almost twice the size of the other chromosomes in the complement, a characteristic not shared by any studied heptapterid.This diploid chromosome number reduction suggests that tandem chromosome fusions may have occurred during the karyotypic differentiation of this species.Despite the fact that C-banding showed a reduced amount of heterochromatin (Figure 1b), like in other Heptapteridae (Abucarma and Martins-Santos, 2001;Swarça et al., 2003;Stolf et al., 2004), these results corroborate the idea of chromosome fusions.Although dark heterochromatic blocks were observed in only three chromosome pairs (numbers 8, 12, and 18), slightly differential longitudinal staining can be observed in the four larger chromosome pairs by both Giemsa staining and C-banding, in accordance with the suggestion of their origin though the fusion of ancestral minor chromosomes.
A prerequisite for fusion should either be elimination or inactivation of telomeres (Slijepcevic, 1998), since telomeres are specialized structures at chromosome ends required for maintaining chromosome stability and integrity (Zakian, 1997).The absence of intercalary (TTAGGG) n sites in the four larger chromosome pairs of Imparfinis hollandi indicates the elimination of telomeres during fusion of ancestral minor chromosomes (Figure 2).Another explanation is the high stringency used in the FISH technique since the hybridization solution consisted of 50% formamide.Abuín et al. (1996) reported the verification (presence/absence) of interstitial telomeric sequences by varying the formamide concentration (low/ high, respectively).On the contrary, Meyne et al. (1990) verified the occurrence of interstitial telomeric sites from a number of vertebrate species, suggesting fusion without telomere loss.In fish, chromosome fusion has been reported in processes involved in the differentiation of multiple sex chromosome systems mainly through centric fusion of acrocentric chromosomes, such as in Eigenmannia sp.(Almeida-Toledo et al., 2000a), Brachyhypopomus pinnicaudatus (Almeida-Toledo et al., 2000b), Erythrinus eyrthrinus (Bertollo et al., 2004), Gymnotus pantanal (cited as Gymnotus sp., Silva and Margarido, 2005), and Harttia carvalhoi (Centofante et al., 2006), although fusion between metacentric and submetacentric chromosomes had already been well documented in Hoplias malabaricus (Bertollo et al., 1997).
In conclusion, silver and CMA 3 + staining were neither good tools to detect rearrangements in karyotype structure nor good cytogenetic markers, since only pair 18 was evidenced by both techniques (Figure 1a).A single chromosome pair bearing either NORs or CMA 3 + bands is a common feature shared by several Heptapteridae (Swarça et al., 2003;Stolf et al., 2004).Furthermore, the data presented in this study confirm that karyotypic evolution in Heptapteridae is more divergent than conservative and that more studies, especially morphological and cytogenetic analyses, would be of great value in order to better understand the family systematics and evolution and the chromosomal mechanisms involved in the speciation process.