Cytogenetic analysis of five Hypostomus species (Siluriformes, Loricariidae)

In this work, we analyzed the karyotypes of five Hypostomus species. Hypostomus cf. heraldoi, from the Mogi-Guaçu River, had 2n = 72 chromosomes, with a nucleolar organizer region (NOR) in one chromosomal pair. Hypostomus regani, from the Mogi-Guaçu River had 2n = 72 chromosomes with NORs in two chromosomal pairs. Hypostomus sp., from the Mogi-Guaçu River basin, had 2n = 68 chromosomes, with NORs in two chromosomal pairs. Hypostomus aff. agna, from Cavalo Stream, had 2n = 74 chromosomes with NORs in two chromosomal pairs. Hypostomus cf. topavae, from Carrapato Stream, had 2n = 80 chromosomes, with NORs in two chromosomal pairs. Hypostomus species showed marked diversity in the karyotypic formula, which suggested the occurrence of several Robertsonian rearrangements and pericentric inversions during the evolutionary history of this genus. This hypothesis was supported by the occurrence of a large number of uniarmed chromosomes and multiple NORs in a terminal position in most species and may be a derived condition in the Loricariidae.


Introduction
Siluriformes is an extremely large fish order with a wide distribution throughout tropical regions (Ferraris, 2007). The number of known species in this region is about 3,100, but may be considerably higher (Reis et al., 2003;Nelson, 2006;Ferraris, 2007). The largest family within the Siluriformes is the Loricariidae, with approximately 700 species distributed in eight subfamilies (Reis et al., 2006;Ferraris, 2007;Chiachio et al., 2008).
Loricariids occur in several habitats, from lagoons and swamps to rapids in sloping streams or rivers with rocky bottoms, at altitudes up to 3000 m. In large water channels, these fish are usually found on rocky bottoms facing into strong water currents (Garavello and Garavello, 2004) or along the margins where the current is moderate (Burgess, 1989). According to Suzuki et al. (2000), these fish exhibit a large diversity of adaptive strategies, with many species showing nest defense, parental care of eggs, brooder larvae behavior, and mouths adapted for feeding on algae and detritus.
Although the Loricariidae is one of the largest fish families in the world, the number of cytogenetically studied species is still very low. There is marked inter-specific diversification in the diploid number, which ranges from 2n = 36 chromosomes in Rineloricaria latirostris (Giuliano-Caetano L, Doctoral thesis, Universidade Federal de São Carlos, 1998) to 2n = 84 in Hypostomus sp. (Cereali et al., 2008). Cytogenetically, the Hypostominae is the best studied group within the Loricariidae, but it is also the most complex, with the diploid number varying from 2n = 38 in Ancistrus sp. (Alves et al., 2003) to 2n = 84 in Hypostomus sp. (Cereali et al., 2008). A very interesting feature in the Hypostominae (particularly within Hypostomini) is the inverse relationship between the diploid number and the number of chromosomes with two arms, which suggests the occurrence of several events of centric fusion/fission (Robertsonian rearrangements) during the evolution of this group (Artoni and Bertollo, 2001).
The Hypostomini consists of a single genus, Hypostomus, whose representatives have a relatively small, stout body, without a depressed caudal peduncle and adipose fin (Armbruster, 2004). This genus, which contains 125 valid species (Zawadzki et al., 2008a;Carvalho et al., 2010) and is distributed from Central America to southern South America (Ferraris, 2007), has the greatest karyotypic diversity within the family Bertollo, 1996, 2001;Artoni et al., 1998). According to Artoni and Bertollo (1996), these fish exhibit non-conservative characteristics in diploid number, karyotypic macrostructure and chromo-somal banding (Artoni and Bertollo, 1996). Currently, most of the cytogenetic data on Hypostomus relate to the diploid number, karyotypic formulas and location of the NOR (Rubert et al., 2008). The diploid number ranges from 2n = 52 in Hypostomus emarginatus (Artoni and Bertollo, 2001) to 2n = 84 in Hypostomus sp. (Cereali et al., 2008) ( Table 2). Some species have distinct karyotypic formulas and their chromosomal variation is accompanied by an increase in the number of subtelo/acrocentric chromosomes (Table 2). According to Artoni and Bertollo (1996), chromosomal rearrangements, such as centric fission and pericentric inversions, play an important role in the karyotype evolution of these fish. Sex chromosomes have been found in some Hypostominae, such as Hypostomus sp., with ZZ/ZW (Artoni et al., 1998) and Ancistrus sp. 1, with XX/X0 (Alves et al., 2006). Hypostomus has single or multiple NORs in the terminal portion of the chromosomes, as observed for other species of this genus (Table 2), with the number of silver-stained chromosomes varying from one (Artoni and Bertollo, 1996;Cereali et al., 2008) to three (Artoni and Bertollo, 1996;Alves et al., 2006) pairs.
Several Hypostomus species are morphologically very similar (Schubart, 1964;Schaefer, 1987;Reis et al., 1990;Muller and Weber, 1992;Mazzoni et al., 1994;Weber and Montoya-Burgos, 2002;Oyakawa et al., 2005;Zawadzki et al., 2008a,c), which makes their identification difficult. In addition, several new species await formal description. Cytogenetic studies have been very useful taxonomically since several fish groups identified only on the basis of morphological studies have been further characterized as a cluster of two or more isolated genetic units.
To improve our knowledge of the diversity and species relationships in Hypostomus, in this study we undertook a cytogenetic analysis of five species in this genus. We provide information on the karyotypic organization of these species and discuss some aspects of karyotypic evolution in this group of fish.

Material and Methods
Specimens of five species of Hypostomus were collected in streams and rivers from the upper Paraná River basin and Atlantic coastal Rivers ( Figure 1, Table 1). The specimens were collected under a license from Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). After the cytogenetic procedures, the fish were fixed in 10% formaldehyde and preserved in 70% ethanol for future taxonomic studies. Voucher specimens were deposited in the ichthyological collection of the Laboratório de Biologia e Genética de Peixes (LBP) of the Departamento de Morfologia do Instituto de Biociências, Universidade Estadual Paulista "Júlio de Mesquita Filho", campus of Botucatu, São Paulo state.
Chromosomal preparations were obtained using the air drying technique (Foresti et al., 1981) and nucleolar organizer regions (NORs) were detected by the silver impregnation technique of Howell and Black (1980). Chromosomal morphology was established based on the proportions of the arms, as proposed by Levan et al. (1964), and the chromosomal nomenclature commonly applied to fish (a -acrocentric, m -metacentric, sm -submetacentric and st -subtelocentric) was used. Martinez et al. 563

Results and Discussion
The five species analyzed (Table 2) showed diploid numbers ranging from 2n = 68 chromosomes in Hypostomus sp. to 2n = 80 in Hypostomus cf. topavae. All of the species, except for Hypostomus regani, were analyzed karyotypically for the first time. There were no sexlinked chromosomal differences in any of the species.
Hypostomus regani, from the Mogi-Guaçu River, had 2n = 72 chromosomes, with a karyotypic formula of 6 m, 6 sm, 32 st and 28 a ( Figure 2B, Table 2), which partially confirmed the results of Artoni and Bertollo (1996) and Alves et al. (2006), who observed the same diploid number as found here but different karyotypic formulas (Table 2). Hypstomus regani is one of the most widely-distributed species throughout the Paraná-Paraguay River basin. Based on alloenzymatic data, Zawadzki et al. (2008b) identified genetically-structured populations of H. regani from the Manso Reservoir (Paraguay River basin), Itaipu Reservoir (lower portion of the upper Paraná River basin) and Corumbá Reservoir (upper portion of the upper Paraná River basin). These findings indicate that differences in the karyotypic formulas of H. regani populations are not uncommon.
Hypostomus cf. topavae, from Carrapato Stream, had 2n = 80 chromosomes, consisting of 6 m, 8 sm, 42 st and 24 564 Cytogenetic analysis of five Hypostomus species (Siluriformes, Loricariidae)  a ( Figure 3B, Table 2). This diploid number was also found in Hypostomus sp. E (Artoni and Bertollo, 1996) (Table 2), although H. cf. topavae can be differentiated from its congeners by its karyotypic organization. Based on cytogenetic studies, Artoni and Bertollo (2001) found that in the Hypostominae higher chromosomal numbers are associated with a greater number of uniarmed chromosomes, whereas low diploid numbers are associated with a higher number of biarmed chromosomes. Similarly, the high diploid numbers in Hypostomus are associated with a high number of uniarmed chromosomes (Table 2). Alves et al. (2003Alves et al. ( , 2005Alves et al. ( , 2006 suggested that the diploid number 2n = 54 and the presence of many biarmed chromosomes are primitive characteristics of the Loricariidae. Their conclusion was based mainly on the wide occurrence of this diploid number and karyotypic formulas in basal loricariid taxa, such as members of the subfamilies Neoplecostominae and Hypoptopomatinae. The available data (Table 2) therefore corroborate and reinforce the hypothesis of Artoni and Bertollo (1996) that centric fissions and pericentric inversions have had an important role in the evolution of this fish group.
Our results showed that Hypostomus species have single or multiple NORs in the terminal position of their chromosomes, as observed in other species of this genus (Table 2). In H. cf. heraldoi, NORs occurred on the long arm of an acrocentric chromosomal pair (pair 20) (Figure 2A, Table 2); in H. regani, NORs occurred on the short arms of two subtelocentric chromosomal pairs (pairs 15 and 16) ( Figure 2B, Table 2); in Hypostomus sp., from the Mogi-Guaçu River, NORs occurred on the short arms of two subtelocentric chromosomal pairs (pairs 7 and 14) ( Figure 2C, Table 2); in H. aff. agna, NORs occurred on the long arms of two chromosomal pairs, one submetacentric (pair 5) and one subtelocentric (pair 13) ( Figure 3A, Table 2); finally, in H. cf. topavae, NORs occurred on two chromosomal pairs: on the short arms of a subtelocentric (pair 11) and on the long arms of an acrocentric pair (pair 30) ( Figure 3B, Table 2). These finding highlight the extensive diversity in NOR phenotype among the Loricariidae. Oliveira and Gosztonyi (2000) stated that in the Siluriformes the basal NOR condition was probably a single NOR at a terminal position on the chromosome. Artoni and Bertollo (1996) proposed that NORs located terminally on the long arm of a single metacentric chromosomal pair represented the primitive condition in the Hypostominae. Based on these hypotheses, species with multiple NORs 566 Cytogenetic analysis of five Hypostomus species (Siluriformes, Loricariidae) would be derived in a monophyletic group. Since most Hypostomus species studied here had multiple NORs, we suggest that these NORs either originated independently among Hypostomus species or originated only once in a monophyletic Hypostomus group. The hypothesis that the five Hypostomus species analyzed here represent a derived cytogenetic condition is coherent with the available biogeographic data for Hypostomus. Based on geological and molecular data, Montoya-Burgos (2003) estimated that the origin of the main clade of Hypostomus was on the former Amazon River basin. According to this author, the vicariant and dispersal events from Amazonian areas to Paraná-Paraguayan areas occurred about 10-12 million years ago. Thus, if the cytogenetic hypotheses are congruent with the biological evolution of Hypostomus, then Amazon basin species should have chromosomal numbers close to 2n = 54 and a single NOR in the terminal position. However, although Hypostomus species have been described from the Amazon River basin (Weber, 2003;Zawadzki et al., 2008a) only H. emarginatus has been karyotyped. If we consider that some authors consider H. emarginatus as pertaining to the genus Squaliforma (Weber 3003;Ferraris 2007;Eschmeyer, 2011), then, to date, no nominal Hypostomus species from the Amazon River basin have been karyotyped. Clearly, an adequate understanding of the karyotypic evolutionary history of Hypostomus requires detailed cytogenetic studies of Amazonian species.