Karyotypic variation in the long-whiskered catfish Pimelodusblochii Valenciennes, 1840 (Siluriformes, Pimelodidae) from the lower Tapajós, Amazonas and Trombetas Rivers

Abstract The genus Pimelodus LaCépède, 1803 comprises 35 formally recognized species distributed along the major neotropical river basins. Despite conservatism in diploid number with 2n=56, an intense variation of chromosomal morphology (karyotypic formula) has been documented in Pimelodus species. In the present study, we analyzed karyotypes of 20 specimens, identified as Pimelodusblochii Valenciennes, 1840 and collected from the lower courses of the Tapajós, Amazonas and Trombetas Rivers. The karyotypes were characterized by Giemsa conventional staining, C-banding, silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The karyotypes showed 2n=56 chromosomes in fish from the Tapajós River. In contrast, fish from the Amazonas and Trombetas Rivers had 2n=58. The nucleolus organizing regions were labeled on the short arm of an acrocentric chromosome as demonstrated by silver staining and FISH. Signals for 18S and 5S rDNA were co-localized on one chromosome pair. Our results demonstrate karyotypic divergence between Tapajós and Amazonas-Trombetas populations of P.blochii, interpreted as supporting the existence of a species complex in this taxon.

Diploid chromosome numbers of eleven previously investigated Pimelodus species showed variation from 54 to 58 with eleven distinct karyotype formula (see Table 2 in the Discussion section). Additionally, karyotype variation with B chromosomes had been reported in P. ortmanni and Pimelodus sp. (Borin and Santos 2004).
So far, two distinct karyotypes were reported for two Pimelodus blochii populations; 2n=56 for the Araguaia River, and 2n=58 for the Amazon River ). Although the chromosome numbers of P. blochii were declared in three meetings and published in abstracts by Swarça et al. (2007), no karyotype image of the species is available in any peer-reviewed literature. Therefore, the taxon has been considered as poorly described in the scientific literature. Rocha (2006) suggested that the name P. blochii is arbitrarily assigned to many different long-whiskered catfish in the Brazilian part of the Amazon Basin. Based on morphometric and molecular data, a large collection of P. blochii specimens was examined by Rocha (2006). It was demonstrated that the Brazilian specimens are distinct from P. blochii topotypes from Suriname and possibly represent a species complex with six undescribed taxa.
In the present paper, we investigate the karyotype of Pimelodus blochii from the lower portions of the Tapajós, Amazonas and Trombetas Rivers in order to evaluate their chromosomal features and contribute the debate on the species taxonomy. The karyotypes were characterized by conventional Giemsa staining, C-banding, silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) technique with 5S and 18S rDNA probes.

Samples and collection sites
Twenty (20) specimens were collected from four localities in the Tapajós, Amazonas and Trombetas Rivers ( Table 1). The fish were captured by local fishermen using hooks and gillnets. The specimens were transferred to plastic tanks (50 L capacity) filled with water from the collection site and aerated with an aquarium pump. After cytogenetic procedures, the specimens were photographed, fixed in 10% formalin for 48 h, washed with running water and preserved with 70% ethanol. The voucher specimens were deposited in the Fish Collection of the Water Science and Technology Institute at Federal University of Western Pará, Brazil. External morphology and coloration features are shown in Fig. 1. The experimental procedures were approved by the Ethical Committee of Animal Research at Federal University of Western Pará (CEUA/UFOPA) under Protocol N. 10001/2015.

Chromosome preparation
Intra-abdominal colchicine (0.0125%) injection was performed at 0.01 ml/g (Bertollo et al. 1978) to stop cell division. The exposed fish were placed in an aerated tank for 40 min and euthanized with water that contains a lethal concentration of clove oil. The posterior kidney tissue was removed and minced in 6 ml of hypotonic KCl solution (0.075 M, 5.6 g/L). The cell suspension was incubated at 37 °C for 20 min and then fixed with fresh methanol-acetic acid (3:1 v/v) solution; the fixative was changed three times.  A specimen from the Tapajós River (ITB-14, SL=150 mm; W=55 g) B specimen from the lower Amazonas River at Santarém (STXVI-2, SL=145 mm; W=52 g) C specimen from the lower Amazonas River at Almeirim (ALC-2, SL=110 mm; W=9 g) D specimen from the Trombetas River (PO-22, SL=108 mm, W=33 g).
Post-hybridization washes were performed with 15% formamide at 42 °C for 10 min, three washes in 0.1×SSC at 60 °C for 5 min, and 0.5% Tween20 at room temperature for 5 min. For signal detection, slides were placed in NFDM buffer (20 ml of 20×SSC, pH 7.0 + 5 g of powdered skim milk + 80 ml of distilled water) for 15 min, followed by two washes in 5% Tween20 for 5 min at room temperature.

Microscopy and karyotype analysis
At least 30 metaphases were counted to determine the diploid chromosome number. The best spread metaphase plates were photographed with a CCD camera (Moticam 10 MP) coupled to a Zeiss Axioskop40 microscope for conventional/banding images, and a Nikon Eclipse CI for FISH images. The contrast and brightness were adjusted with ADOBE PHOTOSHOP CS3. The chromosomes were arranged as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (t) following Levan et al. (1964).
The 5S rDNA probe showed distinct localizations among the samples. Co-localization of 5S and 18S rDNA to a single chromosome pair was detected in the Trombetas and Amazonas Rivers populations (Almeirim population); this syntenic pattern also occurred in the Santarém population but on just one homologous chromosome. The Tapajós River specimens' karyotypes showed a distinct position for 18S and 5S rDNA (Figs 4, 5).

Discussion
The karyotype macrostructure of Pimelodus blochii from the Tapajós, Amazonas and Trombetas Rivers are compatible with a previous report ) ( Table 2). The diploid chromosome number was found to be 58 (30m/sm+28a) in the lower Amazonas population; this is compatible with the Solimões River population (Della-Rosa et al. 1980). The specimens from the Trombetas River conserved 2n=58 but shifted the  karyotypic formula to 26m/sm+32a; this could be a result of pericentric inversions. On the other hand, Pimelodus blochii from the Tapajós River with 2n=56 (36m/sm+20a) showed a resemblance to the population from the Araguaia River (Barra do Garça, Mato Grosso State) (Farias et al. 2000).
Despite extensive conservatism in diploid number, variation in karyotypic formula has been frequently detected ). Pericentric inversions can explain such modifications of chromosomal morphology without alteration of the diploid number and have been previously demonstrated, such as in Pimelodus maculatus and Pimelodus sp. (Dias and Foresti 1993).
Our specimens collected from the Chicaia River, a tributary of the lower Amazonas (Almeirim population), have the typical pigmentation for the A variety, whereas the specimens from the Santarém population, collected at the confluence of the Amazonas and Tapajós Rivers, had the B variety pigmentation. Although both populations conserved the diploid number 2n=58 and karyotypic formula (30m/sm+28a), they diverged in their 18S and 5S rDNA locations (Fig. 5). The specimens from the Trombetas River also have the B variety pigmentation, but diverge in the karyotypic formula (26m/sm+32a) and 18S and 5S rDNA locations (Fig. 5). The most differentiated karyotype was observed in specimens from the Tapajós population; these had a clearly distinct diploid number, karyotypic formula and 18S and 5S location. In general, their coloration resembles the B variety, but their karyotypic distinctiveness leads us to suggest that these specimens may be a new, undescribed Pimelodus species. Ribeiro and Lucena (2006), Azpelicueta et al. (2008), and Lucinda et al. (2016) discussed three distinct patterns of pigmentation among species of Pimelodus. According to Lucinda et al. (2016), among the species formally described, a striped-pattern is shared by P. albicans, P. albofasciatus, and P. tetramerus. However, our results, as well as those of Ribeiro and Lucena (2006), suggest that the diversity of Pimelodus with blackish stripes along its flanks that inhabit the waters of the Amazonas River basin is greater than the diversity currently described. This includes undescribed species that have commonly been misidentified as P. blochii. P. blochii from the lower Amazonas River occurs as two morphotypes (A and B) distinguished through body pigmentation and characterized by a 2n=58 karyotype with minor variations. Additional studies of this group are needed in order to clarify the evolutionary dynamics of the 18S and 5S rRNA genes as well as to acquire morphological and molecular data to evaluate the taxonomy and phylogeny of Pimelodus species.

Conclusions
The populations of Pimelodus blochii from the lower courses of Amazonas, Tapajós and Trombetas rivers presented differentiated karyotypes based on variation in diploid number and chromosome morphology. The specimens collected from the Tapajós River, with 2n=56, are clearly distinguished from the others and may constitute a new, undescribed Pimelodus species.