Karyotype description of five species of Trichomycterus ( Teleostei : Siluriformes : Trichomycteridae )

Trichomycteridae is a family of small catfish which are widely distributed throughout Southern Central America and South America. The present study showed that the cis-Andean species Trichomycterus florensis, Trichomycterus sp. aff. Trichomycterus itatiyae, Trichomycterus reinhardti, Trichomycterus davisi and Trichomycterus auroguttatus had 2n = 54 chromosomes (42 metacentric, 10 submetacentric and 2 subtelocentric), with T. reinhardti, T. auroguttatus and T. sp. aff. T. itatiyae exhibiting only one chromosome pair with silver-stained nucleolus organizer regions (NORs). The cytogenetic data suggest the existence of at least two groups of species in the cis-Andean representatives of the genus Trichomycterus. In the first group the first metacentric pair is considerably larger than the second metacentric pair and the NORs occur in the pericentromeric position of the short arm of a large submetacentric pair while in the second group the first and second metacentric pairs are about the same size and larger than the other metacentric pairs and the NORs are located in the pericentromeric position of the long arm of a large metacentric pair. The relative conservatism of the karyotype of the cis-Andean Trichomycterus species contrasts with the wide diversification observed in the trans-Andean species, reinforcing the hypothesis that the genus is not monophyletic.


Introduction
Trichomycteridae is a family of small-sized catfish which are widely distributed throughout Southern Central America and South America (Wosiacki, 2002).This family has 171 described species, distributed between 39 genera and eight subfamilies: Trichogeninae, Copionodontinae, Sarcoglanidinae, Glanapteryginae, Tridentinae, Vandellinae, Stegophilinae, and Trichomycterinae (de Pinna and Wosiacki, 2003).According to de Pinna (1998), the Trichomycteridae is a monophyletic group because it exhibits seven synapomorphies, most of which are concentrated in the opercular suspensory arch, a highly modified structure that enables fish to anchor on either the substrate or the tissue of their hosts.The subfamily Trichomycterinae is a non-monophyletic assemblage that currently includes eight genera and about 100 species (Wosiacki, 2002).However, according to Mário C.C. de Pinna (personal communication) a large number of new species are yet to be described in this genus.
Only a few cytogenetic studies of the family Trichomycteridae have been conducted, in spite of the large number of species and the wide distribution of this family (Table 1).The diploid chromosome number ranges from 2n = 50 in a Trichomycterus species to 2n = 64 in Vandellia cirrhosa (Table 1), although most species of this family have 2n = 54 chromosomes (Table 1).
In this paper we describe the karyotypes of five Trichomycterus species collected from different Brazilian hydrographic basins and discuss the evolution of chromosomes in the family Trichomycteridae.

Materials and Methods
Five Trichomycterus species were analyzed, the species, collection site and number (male, female and unsexed) of specimens collected being shown in Table 2.The fish were identified and kept in the fish collection of Laboratório de Biologia de Peixes (LBP), Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil.
Chromosome preparations and staining techniques, including silver-staining for the nucleolus organizer regions (NORs), were carried out according to Foresti et al. (1993).Chromosome morphology was determined on the basis of arm ratio as proposed by Levan et al. (1964) and chromosomes were classified as metacentric (M), submetacentric (SM) or subtelocentric (ST).
Another common characteristic of the T. reinhardti and T. auroguttatus sampled was the presence of NORs in the pericentromeric position of the short arm of a large submetacentric pair (Figures 1c and 2b, respectively), this characteristic having also been described in T. spegazzini (Gonzo et al., 2000).
In T. sp.aff.T. itatiyae (Figure 1b) and T. davisi (Figure 2a) the first and the second metacentric pairs were about the same size and larger than the other metacentric pairs.This characteristic was also observed in the karyotypes of one T. paolence cytotype (Torres, 1998) and also in T. davisi, Trichomycterus stawiarski and Trichomycterus sp.(Borin and Martins-Santos, 1999).
We also identified NORs in the second metacentric pair of T. sp.aff.T. itatiyae in the pericentromeric position 46 Sato et al.   of the long arm (Figure 1b), the same characteristic having been observed in Trichogenes longipinnis (Lima and Galetti Jr., 1990).Torres et al. (1998) found NORs in the pericentromeric position of the long arm of a large submetacentric pair of two different samples of T. paolence and a third specimen showed NORs in the pericentromeric position of the long arm of a large metacentric pair.
Even though silver-staining did not produce good results for the T. davisi metaphasic chromosomes the presence of a secondary constriction in the short arm of the second metacentric pair (Figure 2a) suggests that this is the NOR-bearing pair in this species, NORs in the same chromosome pair having also been reported by Borin and Martins-Santos (1999) in T. davisi, T. stawiarski and Trichomycterus sp.
We also found differences between the sizes of the two last submetacentric pairs and the other submetacentric chromosomes in T. auroguttatus from the Rio Doce basin and the Rio Paraíba do Sul basin.In the T. auroguttatus specimens from the Rio Doce basin the last two submetacentric pairs were smaller than the other submetacentric pairs, while in the Rio Paraíba do Sul T. auroguttatus specimens all the submetacentric pairs had almost the same size.
The data discussed above suggest the existence of at least two groups of species in the cis-Andean species of the genus Trichomycterus that exhibit the same diploid number as well as a different karyotype structure.In the first group the first metacentric pair is considerably larger than the second metacentric pair and the species presented silverstaining NORs in the pericentromeric position on the short arm of a large submetacentric pair; in the second group the first and second metacentric pairs were about the same size and larger than the other metacentric pairs and the NORs were located in the pericentromeric position on the long arm of a large metacentric pair.
Our data corroborates the morphological studies conducted by Wosiacki (2002) who suggested that T. davisi and T. stawiarski belong to a natural group, although Wosiacki's (2002) study does not support our cytogenetic analyses indicating that T. reinhardti and T. auroguttatus belong to a natural group.New morphological and cytogenetic studies involving a wider sample of Trichomycterus are needed so that the karyotypic units of this group can be better defined.
Although many groups of Neotropical fish (e.g. the Callichthyidae and Loricariidae), exhibit high chromosome diversity at both the interspecific and intraspecific level,  several groups (e.g. the Anostomidae and Curimatidae) have a very conservative karyotypic macrostructure as has been shown by Oliveira et al. (1988), who suggested that these karyotypic characteristics could be associated with the type of population structure of each group because species with small populations isolated in headwaters usually have different karyotypes while species belonging to groups with large populations inhabiting large river systems usually have very little karyotypic diversity.
The cytogenetic study of Trichomycterus species has shown that in spite of the fact that many species of this genus are usually found isolated in the headwaters of small rivers most species, such as those cytogenetically analyzed by us, exhibit the same diploid number and karyotypic macrostructure (Table 1).In all cis-Andean Trichomycterus species studied 2n = 54 chromosomes were found (Table 1) and different karyotypes have not been found even in widely distributed species such as T. auroguttatus (Table 1) and T. spegazzini (Gonzo et al., 2000).On the other hand, all trans-Andean Trichomycterus species studied have shown different diploid numbers, i.e. 2n = 56 for Trichomycterus areolatus, 2n = 52 for Trichomycterus chiltoni, 2n = 58 and 2n = 62 for Trichomycterus laucaensis, and 2n = 50 for Trichomycterus sp.(Table 1).The phylogenetic study conducted by Wosiacki (2002) showed that the trans-Andean species T. areolatus and T. chiltoni belong to a new unnamed clade, composed of these species and Bullockia maldonadoi (2n = 60) and Hatcheria macraei (2n = 52) (Arratia and Campos, 1997).Thus, at least some of the karyotypic variability presently assigned to the genus Trichomycterus is probably a karyotypic characteristic of Wosiacki's (2002) unnamed clade.
The most primitive species of Trichomycteridae karyotyped to date, Trichogenes longipinnis (Lima and Galetti Jr., 1990), exhibits 2n = 54 chromosomes and is found in coastal streams in Southeastern Brazil.Since this same diploid number is found in all cis-Andean Trichomycterus species it is reasonable to suppose that these species are exhibiting the primitive diploid number, although, at present, the reason for the conservation of this diploid number cannot be explained.Future studies of other Trichomycteridae species will be very important to elucidate this and other questions related to chromosome evolution in this group.
The family Trichomycteridae is a sister-group of the family Nematogenyidae (de Pinna, 1998) of which only one species, Nematogenys inermis, exhibits 2n = 94 chromosomes (Arratia and Veloso, 1980).In a review study of chromosome number in Siluriformes, Oliveira and Gosztonyi (2000) suggested that the putative primitive diploid number of this order could be 2n = 56.The information currently available suggests that the diploid number increased considerably in Nematogenyidae but diminished in Trichomycteridae, although further cytogenetic and phylo-genetic data are needed to allow a better understanding of chromosome evolution in these families.

Figure 2 -
Figure 2 -Karyotypes of: a) Trichomycterus davisi; b) Trichomycterus auroguttatus from the Rio Doce basin, the NOR-bearing chromosome pairs being shown in the insets; c) Trichomycterus auroguttatus from the Rio Paraíba do Sul basin.

Table 1 -
Cytogenetic data for members of the family Trichomycteridae.