Cytogenetic data on Ancistrus sp. (Siluriformes, Loricariidae) of the Paraguay River basin (MS) sheds light on intrageneric karyotype diversification

Abstract Ancistrus Kner, 1854 is a diverse catfish genus, currently comprising 66 valid species, but karyotype data were recorded for 33 species, although only ten have their taxonomic status defined. Considerable karyotype diversity has been found within this genus, with 2n varying from 34 to 54 and structural variability including heteromorphic sex chromosomes. In many cases, uncertainty on the taxonomic status of the study populations hampers reliable interpretation of the complex chromosomal evolutionary history of the group. This study aims to present the first karyotype data for a population of the Ancistrus sp. collected in Criminoso stream (tributary of the Paraguay River Basin, Mato Grosso do Sul, Brazil) in which a combination of different chromosomal markers was used and results integrated in broad discussion on karyotype evolution in the genus. The specimens presented 2n=42 with 18m+16sm+8st and a single NOR revealed by silver nitrate and fluorescence in situ hybridization (FISH) with 18S rDNA probe, located in pair No. 10. Clusters of 5S rDNA were located in the pericentromeric region of three chromosomes: pair No. 1 (metacentric) and one of the homologues of the nucleolar pair No. 10. Heterogeneity in the molecular composition of the heterochromatin was confirmed by the association of C-banding and fluorochrome CMA3/DAPI-staining. Exploring the differential composition of constitutive heterochromatin in Ancistrus may provide an important perspective to understand genome organization and evolution within this group. Our data reinforce the chromosomal diversity present in Ancistrus genus and we discuss the potential sources these variation. The karyotype structure of Ancistrus sp. “Criminoso stream” appears to be consistent with the existence of a new candidate species.


Introduction
Ancistrus Kner, 1854 is the most species-rich genus of the tribe Ancistrini (Hypostominae), currently consisting of 66 valid species (Bifi et al. 2009, Froese andPauly 2016). This genus is widespread in the Neotropical region, from Panama to Rio de La Plata in Argentina, although the greatest diversity of Ancistrus species is found in the basin of the Amazon River (Fisch-Muller 2003).
Up until now, the karyotypes of 33 Ancistrus species have been described, even though most of these species have yet to be formally identified (e.g. de Oliveira et al. 2009, Mariotto et al. 2011, 2013, Favarato et al. 2016. The karyotype data available for this genus indicate considerable chromosomal diversity, with diploid numbers ranging from 2n=34 chromosomes in Ancistrus cuiabae ) to 2n=54 in Ancistrus claro (Mariotto et al. 2011). In addition to this numerical diversity, there is considerable variation in chromosome structure, including differences among populations that suggest the existence of species complexes, as observed in Ancistrus prope dubius (Mariotto and Miyazawa 2006). Another remarkable feature of Ancistrus is the occurrence of heteromorphic sex chromosomes in some species, including simple (Mariotto et al. 2004, Alves et al. 2006, Mariotto and Miyazawa 2006 and multiple systems (de Oliveira et al. 2007(de Oliveira et al. , 2008 which has also contributed to karyotype diversification within the genus.
The chromosomal mapping of the two classes of rDNA (45S and 5S genes) has contributed to the understanding of the organization and evolutionary dynamics of these genes in fish genomes. The 5S rDNA sites are commonly located in interstitial or proximal positions and separated from the 45S rDNA genes, as observed in many groups, but in Loricariidae little is known about the distribution and number of 5S ribosomal genes, being more studied for some species of Hypostomus Lacépède, 1803 (Kavalco et al. 2004, 2005, Mendes-Neto et al. 2011, Traldi et al. 2012, Bueno et al. 2014) and some Ancistrus species (Mariotto et al. 2011, Konerat et al. 2015, Favarato et al. 2016. These studies indicate that the distribution of 5S rDNA genes varies considerably and may occur on one or more chromosome pairs, with or without synteny among the 18S rDNA sites. The complex taxonomic scenario that has been noted in the Ancistrus genus also contributes to the difficulty in understanding the karyotype evolution of this group. In the present study, we provide a detailed description of the karyotype of Ancistrus sp. "Criminoso stream" based on specimens collected in the basin of the Paraguay River (Brazil), using classical and molecular cytogenetic techniques. We provide physical chromosome maps of the 18S and 5S rDNA clusters, and of the heterochromatin, highlighting the GC-and AT-rich composition using base-specific fluorochromes. We also compiled the cytogenetic data available for the genus Ancistrus to provide a more systematic overview of the karyotypic variation in this group.

Material and methods
Authorization for the collection of specimens was granted by the Brazilian Environment Ministry through its Biodiversity Information and Authorization System (SIS-BIO), under the license number 36575-1. The protocols used in this study were submitted and reviewed by the Ethics Committee on the use of animals (CEUA) of Universidade Estadual de Maringá under the case number 013/2009. Cytogenetic analyses were conducted on 13 specimens (5 females, 6 males and 2 of undetermined sex) of Ancistrus sp. collected from the Criminoso stream (18°29.333'S, 54°45.233'W), a small tributary of the Taquari River, near the town of Coxim in the basin of the upper Paraguay River, in Mato Grosso do Sul state, Brazil. The specimens were identified as Ancistrus sp. "Criminoso stream" (NUP 12018) and deposited in the ichthyological collection of the Limnology, Ichthyology and Aquaculture Research Center (Nupélia) at Maringá State University, Paraná, Brazil.
Chromosome preparations were obtained from kidney cells following the technique described by Bertollo et al. (1978). The nucleolus organizer regions (NOR) were detected by impregnation with silver nitrate (Ag-NO 3 ), as described by Howell and Black (1980). Double staining was carried out with chromomycin A 3 (CMA 3 ) and DAPI, according to Schweizer (1976). The constitutive heterochromatin was identified by the C-banding technique as described in Sumner (1972) and stained with propidium iodide according to the method of Lui et al. (2012). The physical mapping of the 18S and 5S rDNA sequences was carried out by Fluorescence in situ Hybridization (FISH) according to Pinkel et al. (1986). The 18S probe was obtained from Prochilodus argenteus Spix et Agassiz, 1829 (Hatanaka and Galetti Jr. 2004), and labelled with the Nick Translation Biotin kit and 5S rDNA probes from Leporinus elongatus Valenciennes, 1850 (Martins and Galetti Jr. 1999) labelled with the Nick Translation Digoxigenin kit. The hybridization signals were detected using avidin-FITC (fluorescein isothiocyanate) for the 18S rDNA probe and anti-digoxigenin-rhodamine for the 5S rDNA probe. The chromosomes were counterstained with DAPI. The metaphases were photographed using an epifluorescence microscope and optimized for best contrast and brightness with Adobe Photoshop CS6 software.
The chromosomes were identified based on the modified arm ratio (AR) criteria of Levan et al. (1964), and classified as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a). The fundamental number (FN) was established considering the meta-submetacentric and subtelocentric chromosomes to have two arms and the acrocentric chromosomes, only one.

Results
The specimens of Ancistrus sp. "Criminoso stream" had a diploid number of 2n=42 with 18m+16sm+8st and a FN of 84, in both sexes (Figure 1a). The Ag-NOR sites were found in the terminal position of the long arm of a submetacentric pair (No. 10), which presented a clear size heteromorphism (Figure 1a, in box), also confirmed by 18S rDNA-FISH (Figure 1b, in box). The 5S rDNA sites were detected in the pericentromeric regions of the first pair of metacentric chromosomes and in one of the homologues of the nucleolar pair (Figure 1b). and 18S rDNA (pair No. 10) were also positive for C-bands. Some of the chromosomes also presented interstitial heterochromatic blocks (pairs Nos. 2, 3, 6, 9 and 11). Chromomycin A 3 staining revealed several GC-rich regions besides the Ag-NORs sites ( Figure  2b), which coincided with most of the heterochromatic blocks in the telomeric regions ( Figure 2a). The DAPI fluorochrome produced a brighter signal in the centromeric regions of chromosome pairs Nos. 1, 5, 10, 11, 12 and 13 (Figure 2c). It should be noted that these DAPI + blocks in pairs Nos. 1 and 10 are interspersed with the 5S rDNA sites (Figures 1b, 2c). These heterochromatin blocks were observed in all specimens analyzed, irrespective of the sex. Figure 3 highlights the pairs 1 and 10 with all banding.

Discussion
The karyotypes described for the genus Ancistrus have been obtained from species found in the basins of the Paraguay and Amazon Rivers, with extensive variability observed in the genus, whose diploid numbers vary of 34, 38, 40, 42, 44, 46, 48, 50, 52 and 54 chromosomes (de Oliveira et al. 2009, Mariotto et al. 2011, 2013, Favarato et al. 2016. The available descriptions of karyotypes for unidentified Ancistrus species emphasize the potential diversity of the genus in these basins. The 2n=42 karyotype as observed in Ancistrus sp. "Criminoso stream" has also been recorded in other Ancistrus species albeit with distinct karyotype formulae (Table 1), indicates the presence of another species, yet to be identified, in the Pantanal region. Additional taxonomic studies are clearly needed to determine the full diversity of the genus in the region.  (green), positive for C-band and CMA 3 ; pericentromeric 5S rDNA sites (red) were shown in only one of the homologues of the pair 10, whose sequence was coincident with heterochromatic blocks (C-band) and DAPI in this pair. The karyotype data available for Ancistrus indicate a relation between diploid number and chromosome types; species with a 2n=44 or more have a larger number of acrocentric chromosomes, while species with smaller diploid numbers (2n=34 to 42) have few or no acrocentric chromosomes (de Oliveira et al. 2009, Medeiros et al. 2016. Artoni and Bertollo (2001) proposed that a karyotype of 2n=54 with a predominance of metasubmetacentric pairs was the primitive condition for the family Loricariidae, which was thus far been found in Ancistrus sp. 01, Ancistrus sp. 03 (Mariotto et al. 2013) and Ancistrus claro (Mariotto et al. 2011) species, with karyotype formula of 14m+8sm+8st+24a. These species have a proportion karyotype of 22 meta-submetacentric (40.7%) and 32 st-acrocentric chromosomes (59.3%). From this starting point, there has been an extensive reduction in the diploid number in the genus Ancistrus (54 to 34) due to possible chromosomal fusions, which can be deduced from the inverse relationship between the number of acrocentric and meta-submetacentric chromosomes. This can be seen in the species with reduced diploid numbers (40, 38, 34), which show little or no acrocentric chromosomes (Mariotto et al. 2004, 2009, de Oliveira et al. 2009, Favarato et al. 2016, Medeiros et al. 2016). However, other rearrangements such as inversions and/or translocations also occurred during the evolution of the karyotype, resulting in the considerable diversity of the fundamental number observed in this genus Chromosome banding in Ancistrus sp. "Criminoso stream" revealed a single nucleolar pair (Ag-NOR), a character shared with all other species of the genus analyzed to date, except Ancistrus sp. (Reis et al. 2012). This arrangement has been confirmed by FISH of the 18S rDNA in some species. A single NOR in an interstitial location has been considered the plesiomorphic condition in Loricariidae (Artoni and Bertollo 2001, Alves et al. 2003, de Oliveira et al. 2009, Mendes-Neto et al. 2011. However, in Ancistrus, the NOR has been observed in a terminal position for most species but, there is also a significant number of species with interstitial NORs (Medeiros et al. 2016). The structural diversity of NORs in Ancistrus related to the different types and sizes of chromosomes may be associated with extensive structural rearrangements (fusions, translocations and/or inversions) that occurred during the karyotype diversification of the genus (de Oliveira et al. 2009, Mariotto et al. 2011. A clear example of these rearrangements was provided by Mariotto et al. (2009) in Ancistrus cuiabae, which has a pericentric inversion in the nucleolar pair (Ag-NOR) supported by C-banding and 18S rDNA-FISH.
Assuming that the primitive Loricariidae karyotype was composed of 2n=54 chromosomes, with synteny between the 18S and 5S rDNA sites as in Ancistrus claro, Mariotto et al. (2011) suggested that this layout may represent the basal condition for the genus. This proposal was supported for the species A. dolichopterus, Ancistrus prope dolichopterus, A. ranunculus and A. maximus analyzed by Favarato et al. (2016). Thus, Ancistrus sp. "Criminoso stream" presented a derived condition related to synteny break between the rDNA 18S and 5S sites (pairs 10 and 1, respectively), but the presence of an interstitial 5S rDNA site in one homologues of nucleolar (pair No. 10), may be a remnant of the primitive condition in this genus.
The heterogeneity in the molecular composition of the heterochromatin of Ancistrus sp. "Criminoso stream" was demonstrated by the combination of C-banding, CMA 3 and DAPI-staining, representing a valuable approach in comparative cytogenetics. DAPI bands were clearly related with the pericentromeric heterochromatin observed by Cbanding in most chromosomes and coincided with the 5S rDNA sites (pair No. 1 and one homologue of pair No. 10). The GC-rich heterochromatin associated with (or interspersed between) ribosomal genes, as observed, is common in most fish chromosomes; however, the presence of terminal CMA 3 + /C-band + blocks in most chromosomes is not a feature frequent in fishes karyotype, included Ancistrus species. In addition, to date there is no karyotypic study in Ancistrus demonstrating coincidence of heterochromatin blocks in several chromosomes with GC/AT rich content, as obtained in the species under study. So, it could be a good chromosomal mark to characterize the karyotype of this putative new species. All Ancistrus species cytogenetically described to date exhibit profiles of low constitutive heterochromatin content, with varied distribution form being found in interstitial and pericentromeric regions to occupying large portions of the long or short chromosome arms (e.g. de Oliveira et al. 2009). In some cases, the accumulation of heterochromatin could be related to the origin of heteromorphic sex chromosomes, as found in Ancistrus prope dubius (Mariotto et al. 2004, Mariotto andMiyazawa 2006)

and
A. taunayi (Konerat et al. 2015), being also associated with paracentric inversions in the latter. Particularly in Ancistrus sp. "Criminoso stream" we hypothesized that unique constitutive heterochromatin pattern observed in their karyotype sheds light on intrageneric karyotype diversification, however such studies are needed in other species of the genus.

Conclusion
Our results further reinforce the considerable variation in karyotype macrostructure within the genus through the description of a new karyotype formula and unique constitutive heterochromatin pattern observed in the population of the Ancistrus sp. "Criminoso stream", contributing with information about the complex chromosomal evolution history of the catfish genus.
This result also appears to be consistent with the existence of a new candidate species. Notwithstanding, our results also emphasize the need for an integrated approach to the understanding of the taxonomic status of this population, based on morphological, ecological, and molecular data. Ultimately, ecological and behavioral traits other than reproductive strategies may be contributing to the mechanisms of isolation that underpin the chromosomal diversification in Ancistrus.