Cytogenetic study of the giant otter Pteronura brasiliensis Zimmermann 1780 ( Carnivora , Mustelidae , Lutrinae )

The giant otter, Pteronura brasiliensis Zimmermann 1780 (Carnivora, Mustelidae, Lutrinae), was widely distributed in South America but stable populations are now only found in the Pantanal and Amazon regions and the species is classified as endangered. There is only one recognized species of giant otter, although two subspecies of doubtful value have also been cited in the literature. We present the first karyotype of four captive P. brasiliensis specimens, all of which posses 2n = 38 chromosomes as 14M+8SM+6ST+8A and one pair of sexual chromosomes. An heteromorphic secondary constriction, associated with the nucleolar organizer region (NOR), was seen on the long arms of chromosome pair 17. The C-banding technique revealed heterochromatin in the centromeric region of all the chromosomes and the NOR was C-banding positive. The giant otter presented the same diploid number as most mustelids, although its karyotype is quite species-specific.

There are five Amazonian aquatic mammals, the Amazonian manatee (Trichechus inunguis), the pink dolphin (Inia geoffrensis), the tucuxi (Sotalia fluviatilis), the Neotropical otter (Lontra longicaudis) and the giant otter (Pteronura brasiliensis).The World Conservation Union (IUCN) classifies the Amazonian manatee and the pink dolphin as vulnerable and the tucuxi and the neotropical otter as belonging to the "data deficient" category, while the giant otter is classified as an endangered species (IUCN, 2006).
Riverine habitats are highly vulnerable to anthropogenic activities, this being especially true in the Amazon basin, which is suffering rapid development leading to degradation and loss of essential habitats for giant otters (Borobia and Rosas, 1991;Rosas, 1994;Carter and Rosas, 1997).
The giant otter, Pteronura brasiliensis Zimmermann 1780 (Carnivora, Mustelidae, Lutrinae), is the largest of all otters and is endemic to South America, with historical records of sightings from north to south-central South America.However, currently stable populations are limited to the Amazon basin and upper Pantanal in the Paraná-Paraguay river basin (Carter and Rosas, 1997).Two P. brasiliensis subspecies have been cited in the literature but are of doubtful value (Duplaix, 1980).
Karyotypic studies have been carried out to analyze intra-and interspecific and intra-and interpopulation variations as well as to identify cryptic species, mutagenic effects and to solve taxonomic problems (Guerra, 1988;Macgregor, 1993).Nie et al. (2002) used chromosomal rearrangements, a rare type of genomic change, to investigate some systematic questions relating to the genome phylogeny of the domestic cat, red panda and five mustelid species.However, while cytogenetic studies have previously been carried out on other mustelid species (Freitas et al., 1975;Kurose et al., 2000;Graphodatsky et al., 2002) there have been no published studies on the chromosomal constitution of P. brasiliensis.
In this paper we describe for the first time the karyotype of P. brasiliensis, providing valuable data to verify the validity of the alleged subspecies in future studies using specimens from the Pantanal region.
We carried out chromosomal analyses on two male and two female giant otters from the Brazilian Amazon basin held in captivity at the Aquatic Mammals Laboratory of the National Institute for Amazonian Research (Instituto Nacional de Pesquisas da Amazônia, INPA), Manaus, Amazonas, Brazil (Figure 1).The giant otters were sedated with 1.93 ± 0.57 mg kg -1 of Zoletil ® (Virbac, Brazil) and blood samples collected from the femoral vein using an heparinized syringe which was maintained in a vertical position for 30 min after collection to allow the lymphocytes to separate.After separation, the lymphocyte layer was added to Complete Medium for Karyotyping (Cultilab, Campinas, SP) and the cultures incubated for 72 h at 37 °C.One hour before the end of incubation, 0.3 mL of colchicine at 0.0125% (w/v) was added.The cells were harvested by centrifugation at 1000 rpm for 10 min and 10 mL of hypotonic 0.075 M KCl solution at 37 °C was progressively added in 2 mL aliquots at 10 min intervals, after which the cells were fixed using four changes of Carnoy fluid (methanol/acetic acid 3:1 v/v).The methodology was modified from the protocol described by Verma and Babu (1995).Constitutive heterochromatin was detected using Cbanding (Sumner, 1972) and the nucleolar organizer regions (NORs) by silver nitrate staining (Ag-NORs) as described by Howell and Black (1980).The chromosomes were identified by the arm ratio criteria (Levan et al., 1964) where the metacentric (M), submetacentric (SM) and subtelocentric (ST) were considered as bi-armed chromosomes and the acrocentric (A) as one-armed chromosomes.In each group, the chromosomes were arranged in order of decreasing size.
The four giant otters examined showed a diploid number of 38 chromosomes, of which 36 were autosomes (14 metacentric + 8 submetacentric + 6 subtelocentric + 8 acrocentric) and two were sex chromosomes (Figure 2a and  b).The fundamental number of autosomes (FNa) was 64.The C-banding technique identified positive centromeric heterochromatin in all the chromosomes, with chromosomes 8, 10 and 13 also showing telomeric heterochromatin (Figure 2c).Chromosome pair 17 showed a nucleolar region which was C-banding positive (Figure 2c) and cor-responded to a secondary constriction in the proximal region of the long arms of this chromosome (Figure 2d).
The diploid chromosome number in Mustelidae ranges from 30 in Mustela vison (Graphodatsky et al., 2000) to 44 in Mustela altaica (Graphodatsky et al., 1976), Mustela ermina orientalis and Mustela mustela anakuma (Obara, 1991).However, in more than 60% of mustelids the diploid number is 2n = 38 (Table 1).Although mustelid karyotypes are conserved in regard to diploid number they present considerable differences in chromosome structure, indicating that several rearrangements occurred during the evolution of this group (Couturier and Dutrillaux, 1986).Sex chromosomes also differ in morphology (M, SM, ST, A) and size (large, medium, small), but the Y chromosome is always the smallest.
According to Graphodatsky et al. (2002), the differentiation mustelid karyotypes from the common ancestor probably occurred by means of several chromosomal rearrangements (e.g.centric fusions and fissions, addition of heterochromatin, pericentric inversions) which caused the large karyotype variability observed in the Mustelidae family.Comparative analysis of the chromosomal segments in mustelids and out-group species revealed 18 putative conserved autosomal segments, which are probably ancestral chromosomes or chromosome arms (Graphodatsky et al., 2002).Franco-de-Sá, et al.Our results show that the P. brasiliensis karyotype is very similar to the karyotype of the Japanese weasel, Mustela itatsi (Kurose et al., 2000), since both species present the same karyotypic formulae, a secondary constriction on the long arm of chromosome pair 17 and a submetacentric X chromosome.However, it is clear that the P. brasiliensis and M. itatsi karyotypes are species-specific since, among other factors, the Y chromosome is submetacentric in P. brasiliensis and acrocentric in M. itatsi.
The mustelids are also very diverse in regard to the C-banding pattern, with some presenting several heterochromatic short arms while others only show heterochromatic centromeric regions (Freitas et al., 1975;Kurose et al., 2000).We found that P. brasiliensis showed constitutive heterochromatin blocks at the centromeric region of all chromosomes and on the telomeric region of some of chro-mosomes.This pattern is totally different from that of the neotropical otter Lontra longicaudis which belongs to the same subfamily (Lutrinae) (Freitas et al., 1975).
The karyotype and chromosome constitution of the giant otter described in this paper could provide useful information to clarify the situation regarding the existence of the P. brasiliensis subspecies mentioned in the literature (Duplaix, 1980), if similar chromosomal studies are carried out with giant otters from the Pantanal region. 1094

Figure 1 -
Figure 1 -Amazonian giant otter (Pteronura brasiliensis) held in captivity at the Aquatic Mammals Laboratory of the Brazilian National Institute for Amazonian Research (Instituto Nacional de Pesquisas da Amazônia, INPA), Manaus, Amazonas (AM), Brazil.

Table 1 -
List of the chromosome number of 22 mustelid species.