Cytogenetic analysis of Baryancistrus xanthellus ( Siluriformes : Loricariidae : Ancistrini ) , an ornamental fish endemic to the Xingu River , Brazil

Baryancistrus xanthellus is a species from the Ancistrini tribe known commonly as “amarelinho” or “golden nugget pleco”. It is one of the most popular and valued ornamental fishes due to its color pattern. Also, it is an endemic species from the Xingu River occurring from Volta Grande do Xingu, region where the Belo Monte Hydropower Dam is being built, to São Félix do Xingu. The current study aimed to cytogenetically characterize B. xanthellus. Results point to the maintenance of 2n=52, which is considered the most common condition for the tribe, and a single nucleolus organizer region (NOR). Mapping of the 18S rDNA confirmed the NOR sites, and the 5S rDNA was mapped in the interstitial position of a single chromosome pair. The 18S and 5S rDNA located in different pairs constitute an apomorphy in Loricariidae. Large blocks of heterochromatin are present in pairs 1 and 10 and in the regions equivalent to NOR and the 5S rDNA. Data obtained in this study corroborated with the currently accepted phylogenetic hypothesis for the Ancistrini and demonstrate evidence that the genus Baryancistrus occupies a basal position in the tribe.

Baryancistrus Rapp Py-Daniel, 1989 is allocated into the Ancistrini and has six described species (Rapp Py-Daniel et al., 2011) that are unique due to their exuberance and diversity of coloration and are, therefore, highly demanded in the fishkeeping market.The presence of yellow spots throughout its body and yellow markings on its dorsal and caudal fins characterize this species.Due to the presence of these spots, which vary in size and intensity, this species is commonly known as "amarelinho" or "golden nugget pleco".This species is rheophilic, and its distribution is strongly linked to the rapids of the Xingu River (Rapp Py-Daniel et al., 2011), which is target for several constructions to take advantage of its hydroelectric potential (Junk & Mello, 1990).Near the middle of its course, the Xingu River receives the Iriri River and posteriorly suffers an accentuated deflection, forming the region known as the Volta Grande do Xingu.According to Zuanon (1999), the most commonly found species in this part of the river are from the Loricariidae family.Also, according to a report developed by several specialists on Belo Monte dam environmental impacts (Painel de Especialistas -Análise Crítica do Estudo de Impacto Ambiental do Aproveitamento Hidrelétrico de Belo Monte, 2009), the situation of the rheophilic fish there is dire.
Thus, all studies involving these species are extremely important not only for acquiring basic knowledge about them but also to design conservation strategies, since their habitats are being seriously impacted.For the Baryancistrus genus, only B. aff.niveatus has cytogenetic data published (Souza et al., 2004).Therefore, the present study investigated the conventional and molecular karyotype macrostructure of one more specie of Baryancistrus, B. xanthellus, in order to increase the information on the genetic diversity of Ancistrini on Amazon region.
Mitotic induction was performed with the application of a yeast solution according to the protocol of Oliveira et al. (1988).Mitotic chromosomes were obtained from kidney cells through the air drying technique modified for fishes by Bertollo et al. (1978).For the characterization of the nucleolus organizer regions (NORs), an AgNO 3 stain was done according to Howell & Black (1980).The heterochromatic regions were identified according to the protocol of Sumner (1972).DNA extraction followed Sambrook et al. (1989).An amplification through PCR (polymerase chain reaction) was done for the development of 18S and 5S rDNA probes using the primers 18Sf (5'-CCG CTT TGG TGA CTC TTG AT-3') and 18Sr (5'-CCG AGG ACC TCA CTA AAC CA-3') (Gross et al., 2010) and primers 5Sa (5' TAC GCC CGA TCT CGT CCG ATC-3') and 5Sb (5'-CAGGCT GGT ATG GCC GTA AGC-3') (Martins & Galetti, 1999), respectively.The final volume of each reaction was of 25µl containing 200ng of genomic DNA, 10X buffer with 1.6mM of MgCl 2 , Taq DNA polimerase (5U/µl), dNTPs (1mM), primer pair (5mM) and Milli-Q water.The 18S rDNA probe obtained was isolated and labeled with digoxigenin-11-dUTP (Roche Applied Science) through the Nick Translation method and the signal detection was performed using anti-digoxigenin-rhodamine (Roche Applied Science).The 5S rDNA probe was labeled with biotin-16-dUTP (Roche Applied Science) using Nick translation and signal detection was performed using a conjugated avidin-fluorescein (FITC).
The chromosomes were analyzed in an epifluorescence Olympus BX51 microscope and the images were captured with a mounted Olympus DP71 camera through the Image-Pro MC 6.3 software.The karyotypes were organized with the aid of the Adobe Photoshop CS6 software, measured with the ImageJ software and classified according to Levan et al. (1964).

Results
Baryancistrus xanthellus presents a diploid number of 52 chromosomes, (16m+28sm+8st), the fundamental number (FN) was equal 104 for males and females, and no differentiated sexual chromosomes were observed (Fig. 2).Active NOR sites were located at the interstitial portion of the short arm of the fourth metacentric pair of all the individuals analyzed.Size heteromorphism of the NOR was observed between the homologues in some specimens (Fig. 2).
Constitutive heterochromatin was found in the centromeric region in the majority of the chromosomes, extending into the proximal region of both arms in some cases.Large blocks occupied the short arms completely on pair 1 and the long arms of pair 10.The NOR was C-band positive (Fig. 3a).
Mapping of the 18S rDNA confirmed the results obtained by silver staining.As in the Ag-NOR, size heteromorphism was also observed.The 5S rDNA pattern was in the pericentromeric region of metacentric pair 7, which presented a conspicuous heterochromatic block on all analyzed specimens (Fig. 3b).
Baryancistrus xanthellus presented 2n=52, which was already verified in species of the genera Peckoltia, Hemiancistrus, Hypancistrus, Scobinancistrus and Panaque (Artoni & Bertollo, 2001;Oliveira et al., 2006;Souza et al., 2009;Cardoso et al., 2013;Silva et al., 2014).There was a single NOR located in the interstitial region of the short arm, in the third pair in B. aff.niveatus (Souza et al., 2004) and the fourth pair in B. xanthellus.This character cannot be considered specific for each genus, since species from the same genus may present sites in different locations.In Ancistrus and Hemiancistrus, for example, the single sites can be found in the short or long arms, and many times even multiple NORs were found (Oliveira et al., 2006(Oliveira et al., , 2009)).Like in B. aff.niveatus, the NOR was C-band positive differently from species of the genera Peckoltia and Scobinancistrus that have heterochromatic blocks adjacent to the NOR (Souza et al., 2004;Souza et al., 2009;Cardoso et al., 2013).
In some specimens of B. xanthellus, the NOR was heteromorphic in regards to size when compared the homologues, which was also observed by Souza et al. (2004) in some specimens of B. aff.niveatus.This heteromorphism is very frequent in Neotropical fish and has been explained as a duplication of ribosomal genes or by a process of accumulation of these genes in one of the homologues through unequal crossing-over (Foresti et al., 1981;Almeida-Toledo et al., 2000;Swarça et al., 2001).This might be due to the presence of constitutive heterochromatin between the ribosomal genes, which might have promoted unequal exchanges between the chromatids (Sola et al., 1988) or by accumulating constitutive heterochromatin in an adjacent position to the NOR (Vicari et al., 2008).
In B. xanthellus, large blocks of heterochromatin were observed in the short arm of pair 1 and the long arm of pair 10, and conspicuous blocks were co-located with the 18S and 5S rDNA sites (pairs 4 and 7, respectively) of all specimens (Fig. 3a).A similar pattern of C-banding was observed in B. aff.niveatus where pairs 1 and 10 also presented one of the arms almost completely heterochromatic (Souza et al., 2004), which could be a pattern for the genus.However, B. aff.niveatus also presented large blocks on pairs 11 and 22. Ziemniczak et al. (2012) suggested that the absence of large and numerous blocks of heterochromatin seem to be a plesiomorphic character in Loricariidae.This characteristic may be proven by comparing the basal genera with the derived ones (Artoni &Bertollo, 2001;Oliveira, 2006;Oliveira et al., 2008;Mariotto et al., 2009;Traldi et al., 2012).Large heterochromatic blocks are found in two or more chromosomal pairs in the genera Scobinancistrus, Hypancistrus and mainly in Peckoltia (Souza et al., 2009;Cardoso et al., 2013;Silva et al., 2014).This characteristic seems to be common to the Ancistrini and corroborates the suggestion proposed by Ziemniczak et al. (2012).
For the Baryancistrus, this is the first record of the mapping of the rDNA 5S and 18S.The simultaneous hybridization of both probes (double-FISH) did not result in syntenic markings (Fig. 3b).This rDNA distribution in different chromosomal pairs constitutes an apomorphy in the Loricariidae (Ziemniczak, 2011).
In the Ancistrini so far, only two genera have data on 5S rDNA: Ancistrus (Mariotto et al., 2011;Favarato et al., 2016) and Hypancistrus (Silva et al., 2014).As it has been observed for the 18S rDNA, the 5S rDNA also presented variable forms among the different species of Ancistrini.In a study by Mariotto et al. (2011), in which the 18S and 5S rDNA probes were hybridized in seven species of Ancistrus, all species presented 18S rDNA markings in a single pair.However, only one species, Ancistrus sp.06, presented a single pair with 5S.This same species also presented synteny between the two ribosomal genes.The remaining species presented two or three pairs with the 5S rDNA.In regards to the position in the chromosome, 5S rDNA was found to be variable, occupying pericentromeric, interstitial, or terminal positions (Table 1).
The existence of multiple sites of the 5S rDNA in several species may be considered an important indication of the great karyotypic diversity present in Ancistrini and should correspond to an apomorphic condition in the group.Studies conducted so far in the group suggest that the localization of the 5S rDNA in a single chromosome pair is less frequent (Table 1), occurring in a few species of Ancistrus (Mariotto et al., 2011;Favarato et al., 2016) and in B. xanthellus.According to Martins & Galetti (1999), the localization of the ribosomal genes in different chromosomes may be advantageous if compared to the syntenic disposition because it might avoid unfavorable arrangements (Dover, 1986), since the occurrence of unequal crossing-over might be frequent in chromosomes with co-located ribosomal genes.
In B. xanthellus, it is possible to visualize the heterochromatin association with 18S and 5S rDNA.This is a recurrent characteristic in the evolutionary history of Neotropical fishes (Vicari et al., 2003).In some cases, the heterochromatic blocks might be adjacent to the nucleolar regions, while in other cases the markings may be overlapping or intercalated (Pendás et al., 1993a,b;Artoni & Bertollo, 2001).The presence of heterochromatin, which holds large quantities of satellite DNA and transposable elements (Dimitri et al., 2009), might facilitate transposition events, moving ribosomal genes to other regions of the genome (Moreira-Filho et al., 1984;Vicari et al., 2008;Gross et al., 2009Gross et al., , 2010)), promoting genetic duplication and unequal crossing-over, and still lead to size variation of the heterochromatin segments as well as in the number of rDNA cistrons (Sola et al., 1988;Vicari et al., 2003Vicari et al., , 2008)).
In general, B. xanthellus conserves the karyotipic macrostructure of the Ancistrini.The maintenance of 2n=52 with a few heterochromatic blocks, a single NOR, and single rDNA sites are evidences that the genus occupies a basal position in the tribe.Our results can help to better understand the chromosomal evolution in this remarkable fish group, but the continuity of cytogenetic studies for the Baryancistrus is indispensable for a better comprehension of the evolutionary trends.
A karyotypic diversity might result in great morphological diversity and color pattern in the species of Ancistrini endemic to the Xingu River.This species diversity represents an invaluable richness; therefore, it is important that there are efforts to understand the origin, evolution, behavior, ecology, and the subsequent preservation of such diversity, since many species are threatened by extinction due to the changes in their original habitats caused by the construction of hydropower dams.

Fig. 2 .
Fig. 2. Karyotype of Baryancistrus xanthellus in conventional staining.The square indicates the pair that bears the nucleolus organizer region (NOR).

Table 1 .
Survey of cytogenetic data of the species of Ancistrini.2n (diploid number), FN (fundamental number), NOR (nucleolar organizer region), 18S (position that the rDNA 18S occupies in the karyotype) and 5S (pairs that have a rDNA 5S marking).