Diversity and chromosomal evolution in the genus Ancistrus Kner , 1854 ( Loricariidae : Ancistrini ) from three hydrographic basins of Mato Grosso State , Brazil

Cytogenetic analyses were carried out in 117 specimens of seven species of the genus Ancistrus from three hydrographic in Mato Grosso State: Paraguay, Araguaia-Tocantins and Amazon basins. Conventional cytogenetic techniques were used to obtain mitotic chromosomes. C-banding was performed to detect heterochromatic regions and silver nitrate staining was used to identify nucleolar organizer regions (Ag-NORs). The counted and paired chromosomes revealed diploid numbers ranging from 2n = 40 to 2n = 54 with karyotype formulae varying from FN = 80 to FN = 86. Single marks in distinct chromosomes identified the nucleolar organizer regions. The constitutive heterochromatin was scarce in the diploid number from 2n = 50 to 2n = 54 and conspicuous blocks were observed in a single species with 2n = 40 chromosomes. These data corroborate the hypotheses of reduction of diploid number in species with derived features such as presence of sex chromosomes and polymorphisms, besides allowing inferences about the evolutionary mechanisms and the ancestor karyotype that favored the diversification of this important genus in the tribe Ancistrini.


Introduction
The genus Ancistrus comprises small fish, up to 15 cm in total length, characterized by a large and compressed body covered with dermal plates, including some species with several long coetaneous tentacles (Burgess, 1989).Amongst Loricariidae, Ancistrus ranks second in the number of species (59 described ones), right after Hypostomus (Ferraris, 2007).The species of this genus are regarded as more derived than the other 26 genera reported in the tribe Ancistrini, a sistergroup of Pterygoplichthini (Armbruster, 1997(Armbruster, , 2004)).
Chromosomal rearrangements, such as fusions, pericentric and paracentric inversions, heterochromatin accumulation and/or translocations have played a major role in the karyotype evolution and diversification of some freshwater fish groups from Neotropical region (Giuliano-Caetano, 1998;Artoni & Bertollo, 1999;Kavalco et al., 2005).According to Alves et al. (2003) and de Oliveira (2006), centric fusions are the main chromosomal rearrangement within Ancistrus, in which the reduction of diploid number seems to be an evolutionary trend.Nonetheless, other rearrangements, such as pericentric inversions and heterochromatin accumulation are also important for the chromosomal diversification and karyotype evolution of loricarids.
In the present work, cytogenetic analyses were carried out in different populations of seven Ancistrus species from three hydrographic basins in the state of Mato Grosso: Paraguay, Araguaia-Tocantins and Amazon.

Material and Methods
Cytogenetic studies were performed in 117 specimens from seven species of Ancistrus (Table 1) from three hydrographic basins in Mato Grosso State: Paraguay, Araguaia-Tocantins and Amazon basins.Voucher specimens were deposited in the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade Estadual de Maringá, Paraná State, Brasil and identified by Dr. Claudio Zawadzki and coworkers (NUP 6827), and in the fish collection of the Museu de Ciências e Tecnologia da PUC-RS, Porto Alegre (PUC/RS), identified by Alexandre Cardoso and Tiago Carvalho (MCP 41969, 41973, 41974, 41976, 41977, 41978).Many of these are undescribed species and wait for a proper taxonomic description.The localities were georeferenced, approximately, according to I3geo map from Ministério do Meio Ambiente (MMA).
The technique for obtaining of mitotic chromosomes and conventional analysis followed Bertollo et al. (1978); Cbanding was performed to identify heterochromatic regions according to Sumner (1972); and the nucleolar organizer regions (Ag-NORs) were identified by silver nitrate staining (Ag-NORs) (Howell & Black, 1980).

Results
Different diploid numbers were observed among the analyzed species: 2n = 40 to 2n = 54 with karyotype formulae ranging from FN (Fundamental Number) =80 to FN = 86.The distinct diploid numbers, karyotype formulae and Ag-NOR locations are shown in Table 2.
Besides the variation shown in Table 2 and Fig. 2, the species from localities a to f presented small amount of heterochromatin as revealed by faint and reduced C-bands at interstitial and pericentromeric regions of some chromosomes.More evident heterochromatic blocks were detected only at the NOR-bearing pair in some metaphases.No cytogenetic differences were observed between males and females in these species based on conventional and C-banding analyses.An intraindividual polymorphism was observed in relation to NOR size, once larger and minor Ag-NOR marks were observed between homologues although all species presented single NORs with interstitial and terminal sites (Fig. 2, box).In those species with higher diploid numbers, 2n = 50 to 2n = 54, a larger number of small acrocentric chromosomes was identified, hindering a defined analysis.The constitutive heterochromatin is scarce, with small and usually faint blocks after C-banding (Fig. 3 a-f).
The specimens of Ancistrus sp.13 from g site were highly differentiated from the others, with conspicuous heterochromatin blocks and reduced diploid number, 2n = 40 (Fig. 1g and g').In some individuals, the long arms of a large subtelocentric chromosome were nearly entirely heterochromatic while a small heterochromatin segment was observed in the homologue.
Chromosomal and heterochromatic heteromorphisms, including XX/XY and ZZ/ZW sex chromosome systems, were identified in four out of the five Ancistrus species with cytogenetic studies for the Pantanal region (Mariotto et al., 2004;Mariotto & Miyazawa, 2006).
According to Gold et al. (1990), a small amount of heterochromatin is a basal trait for teleosteans, including Siluriformes fishes.This statement supports the hypothesis that the karyotypes of Ancistrus from Paraguay basin presenting more heterochromatin segments and sex chromosome systems would be more derived, such as reported in Ancistrus cf.dubius (Mariotto et al., 2004;Mariotto & Miyazawa, 2006), a species in which heterochromatinization of one chromosome in the heteromorphic pair seems to have played a major role in the fixation of sex chromosome systems.According Artoni & Bertollo (1999) and Almeida-Toledo et al. (2001), the heterochromatin accumulation and changes in its distribution on chromosomes might favor the chromosomal heteromorphism and are usually associated with the origin of sex chromosomes in fish.However, in some Ancistrus species, the origin of sex chromosomes seems to be unrelated to constitutive heterochromatin, as observed in species from Amazon basin (de Oliveira et al., 2008).
Chromosomal rearrangements, including fusions, pericentric inversions, transpositions, and heterochromatin accumulation, were probably responsible in determining the derived karyotypes, sex chromosomes systems and polymorphisms observed in species from Cuiabá depression.The chromosomal fusions and pericentric inversions are supported by the identification of fundamental numbers (FN) from 80 to 86 in the analyzed species, especially when species with striking differences in the diploid number are considered, such as Ancistrus claro (2n = 54) and Ancistrus sp. 13 (2n = 40).
The fixation of chromosomal changes in small and low vagile populations (Oliveira et al., 1988), as typical of most Loricariidae, is once again reported in species of the genus Ancistrus.The basal diploid number of 2n = 54 chromosomes proposed for Loricariidae (Artoni & Bertollo, 2001) seems to corroborate the inferences about the putative ancestor karyotype of 2n = 54 chromosomes in the tribe Ancistrini, as observed in Ancistrus claro (present study) and in the genus Lasiancistrus (Mariotto, 2009), besides species from Hypoptopomatinae and Pterygoplichthini, phylogenetically related to Ancistrini.Armbruster (1997Armbruster ( , 2004) ) places Ancistrus as the most phylogenetically derived genus in the subfamily.The chromosomal data obtained in the present work confirms this hypothesis once this genus is characterized by divergent karyotypes, regarding either chromosomal structure or diploid number, leading to higher karyotype diversity than other Ancistrini representatives.
Since species bearing multiple sex chromosomes (de Oliveira et al., 2008), as reported in Amazon basin, and simple sex chromosome systems have already been identified in Ancistrus (Mariotto et al., 2004;Mariotto & Miyazawa, 2006;de Oliveira et al., 2006de Oliveira et al., , 2007;;Mariotto, 2009) according to each locality, a fixed pattern of sex heterogamety cannot be defined, thereby confirming the recurrence and the homoplasic character of sex chromosomes in the tribe Ancistrini.
The nucleolar organizer regions (NORs) in Ancistrus are highly conserved, comprising single marks in all species analyzed so far (Mariotto et al., 2004;Mariotto & Miyazawa, 2006;de Oliveira et al., 2008;Mariotto et al., 2009 and present study) without any case of multiple NORs in the genus.The few cases of multiple NOR systems were reported in other genera and species of the tribe Ancistrini like Peckoltia sp. 1 and Peckoltia sp. 2 (Souza, 2003) and Hemiancistrus spilomma (de Oliveira et al., 2006).
The present results show the remarkable plasticity in the genome organization of chromosomes in Ancistrus species from 2n = 40 to 2n = 54, that ultimately reflects their morphological plasticity, with representatives inhabiting fastflowing streams (plateau regions) up to flooded areas (plain regions), thus showing their adaptability to new niches.This evidence suggests speciation events once 13 cytotypes have already been reported in this group, which probably refer to new undescribed species.

Table 2 .
Karyotype data of analyzed Ancistrus species in increasing diploid number order.