Cytogenetic study of heptapterids (Teleostei, Siluriformes) with particular respect to the Nemuroglanis subclade

Abstract The catfish family Heptapteridae (order Siluriformes) is endemic to the Neotropics and is one of the most common of the fish families in small bodies of water. Although over 200 species have been identified in this family, very few have been characterized cytogenetically. Here, we analyze the chromosome genomes of four species of Heptapteridae: Cetopsorhamdia iheringi (Schubart & Gomes, 1959), 2n = 58, comprising 28 metacentric (m) + 26 submetacentric (sm) + 4 subtelomeric (st) chromosomes; Pimelodella vittata (Lütken, 1874), 2n = 46, comprising 16m + 22sm + 8st; Rhamdia prope quelen (Quoy & Gaimard, 1824), 2n = 58 comprising 26m + 16sm + 14st + 2 acrocentric; and Rhamdiopsis prope microcephala (Lütken, 1874), 2n = 56, comprising 12m + 30sm + 14st. The nucleolus organizer regions (NORs) were located in a single chromosome pair in all species. The two species that belonged to the subclade Nemuroglanis, Cetopsorhamdia iheringi and Rhamdia prope quelen, had a diploid chromosome number of 58 and an interstitial NOR adjacent to a C+ block located on one of the larger chromosome pairs in the complement. Our results from conventional cytogenetic techniques in combination with FISH using 18S and 5S rDNA probes corroborated the taxonomical hypothesis for the formation of the Nemuroglanis subclade.


Introduction
In recent years, various classification changes have led to the current taxonomic status of the catfish family Heptapteridae. Lundberg et al. (1991a, b) suggested the division of the family Pimelodidae into the subfamilies Pimelodinae, Pseudopimelodinae, and Rhamdiinae. Subsequently, on the basis of phylogenetic studies in the Siluriformes, Pinna (1998) elevated the subfamily Rhamdiinae to the level of a family, Rhamdiidae. Bockmann and Guazzelli (2003) later established the family Heptapteridae instead of Rhamdiidae; this family includes 24 genera and 189 valid species (Ferraris 2007) of small fish, commonly known as "bagres" or "mandis". These fish are characterized by a long adipose fin, three pairs of barbels, an elongated body, and a grayish body color. They are endemic to the Neotropics and have a wide distribution in the water courses of Central and South America, with many species distributed in areas of ichthyological endemism. They live on the bottom of small and medium rivers, at low to medium depths, and are usually solitary with nocturnal habits (Bockmann and Guazelli 2003).
The diploid chromosome number in the Heptapteridae varies from 2n = 42 in Imparfinis hollandi (Haseman, 1911) (Margarido and Moreira-Filho 2008) to 2n = 58 in many other species. The latter chromosome number is the most frequent and is also considered a plesiomorphic character (Swarça et al. 2007, Borba et al. 2011. The karyotypes of heptapterid species comprise mainly metacentric and submetacentric chromosomes (see below) suggesting that pericentric inversions were more frequent than centric fissions in the evolution of the family. Nucleolus organizer regions (NORs) are usually present on one chromosome pair, and may be terminal or interstitial. These data suggest that extensive chromosomal rearrangements were involved in speciation within this group (Swarca et al. 2007). The reduction in diploid number may have been produced by successive chromosome fusions with deletions and inversions, such as those responsible for NOR position variation among species. B chromosomes are present in some species and are considered to be of recent origin, and without phylogenetic implications (Borba et al. 2011).
The presence of an interstitial NOR, which is usually located on the largest chromosome pair of the complement and adjacent to a C + block, and the predominance of 2n = 58, are all cytogenetic characters strongly associated with the Nemuroglanis subclade (Kantek et al. 2009).
As there have been relatively few cytogenetic studies in the Heptapteridae, and because of the need to obtain further data to substantiate proposals on the cytotaxonomy of the family (Borba et al. 2011), the present study was undertaken to provide the first analysis, to our knowledge, of the karyotype of Pimelodella vittata (Lütken, 1874). We also used various cytological methods to analyze three other heptapterid species and compared the new data with those previously published to examine the cytotaxonomy of this family.
Mitotic metaphase preparations were made as described by Bertollo et al. (1978). Chromosome morphologies were assigned using the arm size ratio criteria proposed by Levan et al. (1964). Heterochromatin was identified by C-banding (Sumner 1972) and NORs were detected by silver nitrate staining (Howell and Black 1980). Metaphase preparations analyzed after conventional staining (Giemsa) were also subjected to C-banding, allowing the assemblage of sequential karyotypes.
The 18S and 5S rDNA sites on the chromosomes were located by the fluorescence in situ hybridization (FISH) technique (Pinkel et al. 1986), with a stringency of 77%, using probes obtained from Prochilodus argenteus (Agassis, 1829) (Hatanaka and Galetti Jr 2004) and Leporinus elongatus (Valenciennes, 1850) , respectively. The two probes were labeled with 14-dATP-biotin through nick translation in accordance with the manufacturer's instructions (Bionick Labelling System, Invitrogen). Chromosomes were counterstained with DAPI (0.2 mg/ml) and analyzed using an Olympus BX50 epifluorescence microscope. Image-Pro Plus software (Media Cybernetics) was used for image capture.
Silver staining showed that the NOR was located interstitially on the short arm of pair 1, and formed a secondary constriction (Fig. 1a box). Constitutive heterochromatin was present in the pericentromeric regions of several chromosome pairs (Fig. 1b) in addition to visible C + blocks in the NOR-bearing pair (Fig. 1a, b).
FISH with the 18S rDNA probe confirmed that the NOR was located interstitially on the short arm of pair 1 (Fig. 4a). FISH using the 5S ribosomal probe revealed the existence of a large number of these sequences on the NOR-bearing chromosomes, covering a large part of the chromosomes above and below the 18S ribosomal sites. There was synteny between the 18S and 5S rDNAs (Fig. 4b, c).

Pimelodella vittata
All cells from P. vittata specimens had 2n = 46 and a karyotypic formula of 16m, 22sm and 8st chromosomes (Fig. 1c), with no evidence of heteromorphic sex chromosomes.
Silver staining located the NORs to the terminal region of the short arm of pair 17, where they formed a secondary constriction (Fig. 1c box). It was possible to see weak C + bands close to the centromeres in some chromosomes (Fig. 1d).
FISH using the 18S rDNA probe confirmed the NOR location (Fig. 4d). Only one 5S rDNA locus was present in P. vittata in the terminal region of a submetacentric/subtelocentric chromosome pair (Fig. 4e). The 18S and 5S rDNA loci were not on the same pair of chromosomes (Fig. 4e, f).
Silver staining indicated the NOR was located in the terminal region of chromosome pair 10, where it formed a secondary constriction (Fig. 2a box). The chromosomes did not show any heterochromatic segments (Fig. 2b).
FISH using the 18S rDNA probe hybridized to the same region as the Ag-NOR (Fig. 5a, c). Only one 5S rDNA locus was identified; this was located at an interstitial position on a submetacentric chromosome pair (Fig. 5b, d).

Rhamdiopsis prope microcephala
Cells from all specimens had 2n = 56 and a karyotypic formula of 12m, 30sm and 14st chromosomes (Fig. 2c), with no evidence of heteromorphic sex chromosomes.     quelen (a, b, c, d) and Rhamdiopsis prope microcephala (e, f) subjected to fluorescence in situ hybridization with an 18S rDNA probe (a, c, e) and 5S rDNA (b, d, f). Metaphases c and d belong to the triploid specimen. Bar =10 µm. Silver staining indicated the NOR was located in an interstitial region of chromosome pair 16, where it formed a secondary constriction (Fig. 2c box). Constitutive heterochromatin was present in the pericentromeric regions of several chromosome pairs (Fig. 2d).
FISH using the 18S rDNA probe hybridized to the same region as the Ag-NOR (Fig. 5e). Two 5S rDNA loci were identified at a terminal position on a submetacentric/subtelocentric chromosome pair (Fig. 5f).
The C-banding and Ag-NOR patterns of Rhamdia and Pimelodella species (Swarça et al. 2007, Borba et al. 2011, Gouveia et al. 2012) are distinctly different from most taxa of the Nemuroglanis subclade that have been analyzed. The existence of cytogenetic characteristics that separate the recognized groups of Heptapteridae was initially proposed by Fenocchio et al. (2003). Thus, for example, the interstitial C + band pattern is a more common feature of species of the Nemuroglanis subclade, such as C. iheringi and R. prope microcephala (Fig. 1b, 2d, respectively). Other species of the family Heptapteridae that do not belong to this subclade, such as P. vittata (Fig. 1d) and R. prope quelen (Fig. 2b), have different patterns of heterochromatin distribution (Swarça et al. 2007. However, as the majority of heptapterid species have not been studied cytogenetically studied, then it is difficult to elaborate broader proposals.
Another cytogenetic characteristic that may be diagnostic of the Nemuroglanis subclade is the synteny between 18S and 5S rDNA. Up until now, only Imparfinis schubarti (Gomes, 1956) (Kantek et al. 2009) and C. iheringi (present study) have been found to show this characteristic. Other genera in the Heptapteridae that do not belong to the Nemuroglanis subclade, such as Pimelodella and Rhamdia (Garcia et al. 2003, do not show this synteny. However, as R. prope microcephala did not have this characteristic, then the association between 5S and 18S rDNA might be a synapomorphy, shared by the group of species in the Nemuroglanis subclade that have a 2n = 58 karyotype and possess interstitial NORs on the largest chromosome pair of the complement. The 5S ribosomal gene consists of multiple copies of a highly conserved 150 base pair sequence, separated by highly variable non-transcribed spacers (Williams and Strobeck 1985). These variable sequences, which were caused by insertions/deletions, mini-repetitions and pseudogenes, are useful for evolutionary studies and serve as population markers for many organisms, including plants (Zanke et al. 1995), mammals (Suzuki et al. 1994) and fishes (Martins et al. 2002). Variations in these spacers have also been detected in some neotropical fishes, such as Leporinus  and Brycon (Wasko et al. 2001). A comparison of the outcome of analysis of I. schubarti (Kantek et al. 2009) and C. iheringi indicates that despite the relative evolutionary proximity of the species (both belong to the Nemuroglanis clade), and the likely localization of these sequences to homeologous chromosomes, there is nevertheless considerable differences in the signals obtained with the 5S rDNA probe. The large 5S rDNA blocks on C. iheringi chromosomes presumably originated through duplication of the 5S rDNA of an ancestral species close to these taxa. Other species of heptapterids considered more basal in the family, such as species belonging to the genera Rhamdia and Pimelodella, have only small 5S rDNA signals; this suggests that the presence of the large 5S rDNA block in C. iheringi is an apomorphic character. Based on the supposed homogeneity among 5S rDNA repeats, several studies have proposed that 5S rDNA is subject to concerted evolution (Arnheim 1983), where duplicated gene family members evolve as a single unit that undergoes a high degree of homogenization (as a unit in concert) (Pinhal et al. 2011). Prior to this study, variability in the number and location of 5S ribosomal genes has been reported among Siluriformes (Kavalko et al. 2004) except for Rhamdia . The analyses here confirm the variability observed by other authors, and also the conservation of 5S rDNA in Rhamdia.
Until now, only the genera Imparfinis, Cetopsorhamdia, Heptapterus, Phenacorhamdia, Rhamdiopsis, Pimelodella, Rhamdia, and Taunayia had been cytogenetically analyzed; these represent only eight of the 24 genera in the family Heptapteridae (Yano and Margarido 2012). The first five belong to the subclade Nemuroglanis. More studies involving this family may assist in the elucidation of cytotaxonomy and chromosome evolution in this family.