Karyotype description of possible new species of the Hypostomus ancistroides complex ( Teleostei : Loricariidae ) and other Hypostominae

Cytogenetic analyses were performed in four species of the Hypostominae subfamily, three from Hypostomus (Hypostomini) genus and Rhinelepis aspera (Rhinelepini). Three populations of Hypostomus ancistroides were analyzed, which had 2n=68 chromosomes, but presented different karyotype formulas. Hypostomus regani and H. strigaticeps, both from Ivaí river, showed 2n=72 chromosomes with two distinct cytotypes. In turn, R. aspera of the upper Paraná river basin presented 2n=54 chromosome. Multiple Nucleolar Organizer Regions (NORs) have been evidenced by silver nitrate staining in species of Hypostomus and single NOR in R. aspera. The observed variation in the chromosome number and the marked variability in karyotype formulas and NORs reveal a certain amount of karyotype variation in the genus Hypostomus suggesting the probable existence of cryptic species with independent chromosome traits. Therefore, our data can be of great value in discriminating species and understanding their chromosomal evolution.

Cytogenetically, the subfamily Hypostominae is the most well studied of the family Loricariidae, but it is also the most complex comprising a variation of the diploid number from 2n=38 chromosomes in Ancistrus sp.(ALVES et al., 2005a) to 2n=84 chromosomes in Hypostomus sp.(CEREALI et al., 2008).A very interesting feature in Hypostominae, particularly among Hypostomus, is the inverse relationship between the diploid number and the number of chromosomes with two arms, suggesting the occurrence of many events of centric fusion/fission during the development of the group (ARTONI; BERTOLLO, 2001).
According to Artoni and Bertollo (1996) these fish exhibit not conservative characteristics on the diploid number, karyotypic macrostructure and chromosome banding (ARTONI; BERTOLLO, 1996).Currently, the majority of cytogenetic data on Hypostomus is related to the diploid number, karyotype formula and location of NOR (RUBERT et al., 2008).
The Hypostominae, showing high adaptive performance, inhabit several freshwater environments from headwaters (ALVES et al., 2005a) to major hydrographic basins (JEREP et al., 2007).This distribution pattern could lead to distinct groups without apparent geographic barriers, which makes the cytogenetic and molecular studies useful tools for identification of cryptic species (BICKFFORD et al., 2007;IRWIN, 2002).The Karyotype description of Hypostomus ancistroides from different localities showing different karyotype constitutions seems to reinforce such a view.In this study, three species of the genus Hypostomus, H. ancistroides (Ihering), H. regani (Ihering) and H. strigaticeps (Regan), and Rhinelepis aspera Spix & Agassiz are karyotyped aiming to enlarge the knowledge on patterns of diversity and karyotype evolution in catfishes of the upper Paraná river basin.

Material and methods
Three species of the genus Hypostomus and one of the Rhinelepis were collected (Table 1 and Figure 1).The specimens were collected under license from the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA), protocol number 11360-1.They were anesthetized with benzocaine (5%) and then sacrificed for subsequent cytogenetic analysis.The collected specimens were fixed in 10% formalin and are stored in 70% alcohol for further taxonomic studies.Metaphasic preparations of chromosomes were performed through the technique of air drying (FORESTI et al., 1981) and the detection of the nucleolus organizer regions (NOR) by the Silver Impregnation technique of Howel and Black (1980).Chromosome morphology was established on the basis of arm ratio as proposed by Levan et al. (1964), and the chromosome nomenclature commonly attributed to fish as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a) was used.For the description of the chromosomes bearing NORs the karyotypes were sequentially assembled for all species.

Results and discussion
Cases of sex chromosomes were found in the literature for some Hypostominae, as ZZ/ZW in Hypostomus sp.(ARTONI et al., 1998) and XX/X0 in Ancistrus sp. 1 (ALVES et al., 2006), which was not observed in this study, showing that chromosomal changes related to gender/sex should be a variable character in the group.
Specimens of Hypostomus ancistroides analyzed showed 2n=68 chromosomes, however, variations in the karyotype formula were observed.
Differences in karyotype formulas have also been described for other populations of H. ancistroides by Michele et al. (1977), Artoni and Bertollo (1996) and Alves et al. (2006) (Table 2).These differences in the karyotype formula between submetacentric and subtelocentric may be involved in the karyotype assembly since chromosomes are small, with size not more than 5μm, but herein the variation that occurs between metacentric and acrocentric chromosomes, does not allow assembly errors, ensuring the karyotypic formula as unique to each species.
These results suggest that the group known as H. ancistroides form a complex of species not so far diagnosed by morphological characteristics.The impregnation by silver nitrate revealed multiple NORs in the three populations of Hypostomus ancistroides analyzed, differing only in the type of chromosome pairs where they were found (Figure 2).In the population of Dourados stream (Figure 2a), the stained regions were observed in the terminal portion of short arms of pair nine and only one chromosome of pair 10, while for the population of Maringá stream (Figure 2b) the markings were in the terminal regions of short arms of two pairs to chromosomes 11 and 15 and only in a single chromosome of pair five.The population of Ximbaúva stream (Figure 2c) showed three markings in the terminal regions of short arms of only single chromosomes to pairs tree (metacentric), five and nine (submetacentric).
Hypostomus ancistroides is a widely distributed species throughout the upper Paraná river basin, inhabiting streams and creeks, specially under marginal vegetation (CASATTI, 2005).The fact of its preference for streams, the sedentary habits of most Hypostomus species and its wide distribution in this basin, make the many populations of H. ancistroides prone to show some degree of genetic divergence to other conspecific populations.
The maintenance of this variation could be tentatively explained by the 'dilution gene flow cascate', a model for non migratory species (Figure 3), based in the works of Irwin (2002) and Bickfford et al. (2007).In this model, the conspecific populations inhabiting their extremity ranges could present some karyotypic differences despite being morphologically similar.In such a scenario one population with fixed chromosome alterations (population a = Pa), has immediate contact with a subsequent population (b = Pb), and this has contact with another subjacent population (c = Px), and so on.Only a few individuals could maintain a gene flow between adjacent populations, however in the extreme edges of the species range we will probably detect differences among Pa and Px.The gene flow should occur in both directions when free of barriers.Such a kind of differentiation is very common in fishes (DE AGUIAR et al., 2009;CAPISTANO et al., 2008;NIRCHIO;OLIVEIRA, 2006;PANSONATO-ALVES et al., 2010).However, sometimes, the genetic variation does not origin immediate phenotypic differentiation in the external morphology of an organism.Herein, chromosome alterations as pericentric inversions support the reported karyotype structure due to the significant alterations that occurred in the metacentric and acrocentric chromosomes.Thus, we highlight to the possibility of two cryptic species when comparing the populations of H. ancistroides from the Pirapó river basin and the Ivaí river basin.Hypostomus regani presented a diploid number of 72 chromosomes, showing 12m, 14sm, 26st and 20a (Figure 4a, Table 2), as found by Artoni and Bertollo (1996) and Alves et al. (2006) (Table 2), showing different shapes for chromosomes.Hypostomus regani also had multiple NORs, with stained regions found in the short arms of the subtelocentric chromosome pairs 12 and 19 (Figure 4a).Despite species of
Rhinelepis aspera had a diploid number of 54 chromosomes, showing 24m, 18sm and 12st (Figure 4c, Table 2).Rhinelepis aspera was the only species to exhibit single NOR, located on the short arm of the first metacentric chromosome (Figure 4c).
This diploid number was also found in populations analyzed by Artoni and Bertollo (2001) of R. aspera, exhibiting a distinct karyotype formula (Table 2).Zawadzki et al. (2005) et al., 2005b) all the studied populations have 2n=54 chromosomes.In representatives of the subfamily Hypoptopomatinae as Corumbataia cuestae and Hisonotus depressicauda (FERREIRA et al., 2005) it was also found the same diploid number.The karyotype morphology of these species show a higher number of metacentric/submetacentric chromosomes, corroborating the hypothesis of Artoni and Bertollo (2001) that the inverse relationship between the diploid number and the number of chromosomes with two arms suggests the occurrence of many events of centric fusion/fission in the developments of the group.Thus, it is proposed that the higher number of chromosomes to most of the species of Hypostomus should be a derived condition, and Hypostomus seems to be one of the most derivative taxon in karyotypic terms within Loricariidae.
The diploid number in Hypostomus varies from 2n=52 in H. emarginatus (ARTONI; BERTOLO, 2001) to 84 in Hypostomus sp. 3 (CEREALI et al., 2008).Some studied species have distinct karyotype formulas and the chromosome variation is accompanied by an increase in the number of subtelo/acrocentric chromosome types.According to Artoni and Bertollo (1996), chromosomal rearrangements as centric fission and pericentric inversions play an important role in karyotype evolution of this group of fish.
The karyotyped species of the tribes of Hypostominae show the same relationship proposed by Artoni and Bertollo (2001)    This relatively conserved karyotype number of 52 and 54 among the representatives of different Loricariidae subfamilies corroborates the hypothesis that these karyotype numbers should be a basal condition for Loricariidae.Therefore, in the light of the results herein obtained the specimens of these three species of Hypostomus presented a more derived karyotype structure within Loricariidae.

Conclusion
NORs on Loricariidae present a great phenotypic diversity.However, single NOR in terminal position on the chromosome presents itself as a basal character, which is usually found in Hypostominae and Hypoptopomatinae (ARTONI; BERTOLLO, 1996;OLIVEIRA;GOSZTONYI, 2000).In sum, the high amount of variation in karyotype and NOR structure draw a karyotypically fluctuating evolutive condition for this group.Additionally, many species of Hypostomus are taxonomically undefined and there is still a lack of characters enabling nominate them as distinct species.
, H. regani is one of the most widely-distributed species throughout Paraná-Paraguay river basin.Through alloenzymatic data, Zawadzki et al. (2008b) found 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).Therefore, differences in karyotype formulas between different populations of H. regani are somewhat expected, as well.

Table 1 .
Species, collecting sites and sex of analyzed specimens.