Karyotypic and morphological divergence between two cryptic species of Eigenmannia in the Amazon basin with a new occurrence of XX / XY sex chromosomes ( Gymnotiformes : Sternopygidae )

Eigenmannia species are widely distributed in the Neotropics, with eight valid species currently recognized. Populations of Eigenmannia from three locations in the eastern Amazon were investigated using cytogenetic and morphological techniques, revealing two taxa designated here as Eigenmannia sp. “A” and Eigenmannia sp. “B”. The species differ in three morphometric characters, two meristic characters, and one osteological character. Eigenmannia sp. “A” presents 2n = 34 (22 m/sm+12 st/a) and Eigenmannia sp. “B” presents 2n = 38 (14 m/sm+24st/a) and simple differentiated sex chromosomes of the type XX/ XY. In both species the Constitutive Heterochromatin (CH) rich in A-T bases is distributed in the centromeric region of all chromosomes. Eigenmannia sp. “B” also presents CH blocks in the interstitial region of chromosome pairs 8, 9 and X which are positively stained with CMA3, indicating G-C rich regions. The NOR is located on the short arm of chromosome pair 17 of Eigenmannia sp. “A” and on the short arm of pair 14 of Eigenmannia sp. “B”. FISH with rDNA probes hybridized to different-sized regions between homologs, suggesting heteromorphism. The differentiation of the X chromosome in Eigenmannia sp. “B” could be the result of amplification of repetitive DNA sequences.

Cytogenetic studies of Eigenmannia demonstrate a high level of karyotypic diversity (Table 1).Eigenmannia gr.virescens is the most studied group, presenting karyotypic variation which ranges from 2n = 28 in Eigenmannia sp. 1 (Almeida-Toledo et al., 1988), to 2n = 38 in Eigenmannia virescens (Almeida-Toledo et al., 2001, 2002;Silva et al., 2009).Previous results demonstrate the existence of differentiated sex chromosomes in the species of the E. gr.
Although the taxa described in Eigenmannia are considered valid, various authors suggest that the species in this genus represent a complex, which is difficult to resolve (Shibatta, 1993;Campos-da-Paz, 1997).This is corroborated by cytogenetic studies that demonstrate karyotypic variation among samples both within and between hydrological basins (Moysés et al., 2005).
We investigated three populations of Eigenmannia from streams of eastern Amazonia, using cytogenetic, morphometric, meristic and osteological analyses.The results reveal the existence of two cryptic species, and the description of a new occurrence of sex chromosomes of the simple XX/XY type for Eigenmannia in the Amazon basin.Brazil Almeida-Toledo et al. (1984, 1988, 2000) Eigenmannia sp.Morphometry, meristics and osteology.Measurements were made on the left hand side of each specimen using a digital calliper to a precision of 0.1 mm while viewed under a stereomicroscope.Morphometric analyses were based on Mago Leccia (1978), Triques (1996) and Crampton et al. (2004).Taxonomic parameters for Gymnotiformes were followed where measures of the body are described in proportion to the length as measured from the tip of the snout to the end of the anal fin (LEA -Length to the end of the anal fin) and measures of parts of the head described in proportion to the Head Length (HL).Meristic analyses included: number of rays of the pectoral fin, number of lateral line scales (counting from the first scale with a lateral line tube to the end of the anal fin) and the number of scales above the lateral line (counted at the highest point along the body, approximately in line with the distal portion of the longest ray of the anal fin).Specimens were cleared and stained following Taylor & Van Dyke (1985).The osteological nomenclature of the maxilla follows Lundberg & Mago-Leccia ( 1986) and de Santana & Crampton (2011).
Cytogenetic methods.Metaphase chromosomes were obtained following the protocol of Bertollo et al. (1978).Conventional analyses were performed, including staining with Giemsa (Merck), C-banding (Sumner, 1972), impregnation with silver nitrate (Ag-NOR) (Howell & Black, 1980), staining with the fluorochrome DAPI (Pieczarka et al., 2006), and staining with Chromomycin A 3 (CMA 3 ) (Schweizer, 1980).The gonads were removed and prepared as a smear with a 32x20mm glass slide and observed under a stereomicroscope to determine the sex of the individuals.
The morphology of the maxilla in Eigenmannia sp."A" presents a small antero-dorsal process which is about half the size of the anterior naris, and the descending process is large, approximately the same width as the anterior naris (Fig. 4a).In Eigenmannia sp."B", the antero-dorsal process is hypertrophied, being about the same size as the anterior naris, and the descending process is narrow (about half the width of the anterior naris) (Fig. 4b).
The two species present a yellow colouration when preserved, with three longitudinal stripes, one above the lateral line, one along the proximal portion of the pterygiophores of the anal fin and one along the base of the anal fin.They also present a dark band along the body localized between the lateral line and the stripe, which runs along the proximal portion of the pterygiophores of the anal fin.

Cytogenetic analyses.
Approximately 30 metaphase cells of each specimen were examined, revealing a difference in the karyotype of the two species.The species Eigenmannia sp."A" presents 2n = 34 chromosomes and a karyotypic formula (KF) of 22 m/sm and 12 st/a without the presence of differentiated sex chromosomes (Fig. 5).Eigenmannia sp."B" presents 2n = 38 and a KF of 14 m/sm and 24 st/a (Fig. 6).In males, a heteromorphic pair of acrocentric chromosomes was observed, with one of the homologous chromosomes distinctly larger than the other (Fig. 6b).This heteromorphism was not observed in the females, where the homologous chromosomes were both large (Fig. 6a).The Constitutive Heterochromatin (CH) is mainly distributed in the centromeric region of all chromosomes of both species (Figs.5b, 6c-d).In Eigenmannia sp."B" CH blocks were also observed in the interstitial regions of acrocentric chromosomes 8, 9 and 10.It was also possible to note a large CH block in the interstitial region of the X chromosome, which was absent in the Y (Figs. 6c-d).
The NOR was localized on the short arm of chromosome pair 17 in Eigenmannia sp."A" and on the short arm of chromosome pair 14 in Eigenmannia sp."B" (Figs.5-6 box), which was also found to present size heteromorphism between the homologous chromosomes of the two species.
DAPI fluorescence was found in the centromeric regions of all chromosome pairs of both species, consistent with the C-banding results (Figs. 7a, 8a-b).CMA 3 marked the short arm of chromosome pair 17 most intensely in Eigenmannia sp."A" and the short arm of chromosome pair 14 most intensely in Eigenmannia sp."B".Additionally, in Eigenmannia sp."B" it was possible to observe intense marking of chromosome pairs 8 and 9, coincident with the CH blocks (Figs.8c-d).FISH with 18S rDNA probes hybridized the region of the short arm of chromosome pair 17 in Eigenmannia sp."A", and the region of the short arm of chromosome pair 14 in Eigenmannia sp."B" (Figs.8e-f).

Discussion
Taxonomic considerations.The morphometric, meristic and osteological divergences among Eigenmannia sp."A" and Eigenmannia sp."B" indicate the occurrence of two distinct lineages, corroborated by the karyotypic differences.The karyotypic differences (2n = 34 vs. 2n = 38, XX/XY) are sufficient to act as a post-zygotic reproductive isolation mechanism (King, 1993).As such, we find that the two cryptic species currently occur in sympatry in the rio Quatipuru basin (specifically at igarapé do Açaiteuazinho), but that Eigenmannia sp."A" also occurs allopatrically in the rio Marapanim basin.
The species Eigenmannia sp."A" and Eigenmannia sp."B" are distinct from E. virescens, E. macrops, E. humboldtii, E. limbata and E. nigra by the presence of three longitudinal stripes (vs.uniform colouration without stripes).Additionally, they are distinct from E. humboldtii, E. limbata and E. nigra due to the colouration pattern of the anal fin, which is hyaline (vs.darkened along the distal margin in E. humboldtii and E. limbata or uniformly darkened in E. nigra).Eigenmannia sp."A" shares the presence of stripes along the body with E. trilineata, E. vicentespelaea and E. microstoma.It differs from E. trilineata and E. microstoma in the size of the antero-dorsal process of the maxilla, being equivalent to half the size of the posterior naris (vs.equivalent to the size of the posterior naris) and in the suborbital height, 22.3-29.9%HL (vs.32.5-46.6%;29.9-40.8%,respectively).It is distinguished from E. vicentespelaea by the number of pectoral fin rays, 15-16 vs. 17-19 and by the number of scales above the lateral line, 12-14 vs. 7-8.

Cytogenetic considerations. The analysis of Gymnotiformes
has revealed significant cytogenetic variation between populations (Milhomem et al., 2008;Nagamachi et al., 2010).The karyotypic diversity encountered in the genus Eigenmannia, reflects to some extent the population structure where the formation of small aggregations with low dispersion favours the fixation of chromosome rearrangements (Moysés et al., 2005;Silva et al., 2009).The karyotype 2n = 34 (22 m/sm+12st/a) described by Moysés et al. (2010) for Eigenmannia sp. in the rio São Francisco basin is different from that of Eigenmannia sp."A" (24 m/ sm+10st/a) based on KF.This difference in KF could be a result of a pericentric inversion event, which would also act as a post-zygotic isolation mechanism (King, 1993).
The CH encountered in the centromeric region of chromosomes of Eigenmannia sp."A" and Eigenmannia sp."B" is typical of Gymnotiformes (Silva et al., 2008;Milhomem et al., 2012a), as well as being observed in other vertebrate species (Sumner, 2003;Gomes et al., 2012).C bands are usually positively marked by DAPI as they are composed predominantly of A-T bases (Silva et al., 2009).However, in Eigenmannia sp."B" the CH blocks in the interstitial regions of chromosomes 8, 9 and X were marked strongly with CMA 3 (indicating G-C rich regions), demonstrating that this region presents a distinct composition compared to other CH classes present in the autosomes.
In the genus Eigenmannia, the NOR is simple, frequently located on the short arm of a subtelocentric/acrocentric chromosome (Table 1), as was observed in both Eigenmannia sp."A" and sp."B" (17p and 14p, respectively).However, the species Eigenmannia sp. 1 and Eigenmannia sp. 2 (Almeida-Toledo et al., 1984, 1988, 2000), presented the NOR on the long arm of a metacentric chromosome, pair 10 (Table 1).As such, in order to confirm that the simple NOR is a shared character among the different species it is important to know whether the rDNA sequence sites are always located on the same chromosome in the different species, as demonstrated for Gymnotus gr.carapo (Milhomem et al., 2013).
In both species the NOR is positively stained by CMA 3 , indicating that this region is interlaced with sequences rich in G-C bases (Nascimento et al., 2006;Silva et al., 2008;Milhomem et al., 2007Milhomem et al., , 2008Milhomem et al., , 2012aMilhomem et al., , 2012b)).FISH with rDNA probes hybridized to different-sized regions between homologs, suggests that heteromorphism in the size of the NOR could either be associated with differences in transcription activity of the ribosomal genes or may be the result of differences in the copy numbers of the ribosomal genes (Oliveira et al., 2009;Milhomem et al., 2013).Sex chromosomes in Eigenmannia.In fishes, sex chromosomes are not present in basal taxa and their origin within genera or families is probably convergent.Additional information suggests that the origin of sex chromosomes in Neotropical fishes is recent, with diversification in some taxa approximately 7-10 Ma (Charlesworth et al., 2005;Cioffi et al., 2011;Henning et al., 2011).
Different sex chromosome systems have previously been described in Eigenmannia, including both simple XX/ XY and ZZ/ZW systems and a multiple X 1 X 1 X 2 X 2 /X 1 X 2 Y system (Table 1), suggesting that these different systems do not have a common origin.The species Eigenmannia sp. 2 (Almeida-Toledo et al., 1984, 1988, 2000) and E. trilineata (2n = 31/32) present the multiple X 1 X 1 X 2 X 2 /X 1 X 2 Y system, where a centric fusion between two acrocentric chromosomes (pairs 6 and 11) in the male karyotype, resulted in a metacentric Y chromosome (neo-Y) in these species (Almeida-Toledo et al., 1984, 1988, 2000;Fernandes et al. 2010).
Karyotypes described for E. virescens (2n = 38) from the rio Mogi-Guaçu without sex chromosomes and karyotypes from the rio Tietê, with a simple XX/XY system (Table 1) (Almeida-Toledo et al., 2001), are similar to that described here for Eigenmannia sp."B".Karyotypes of Eigenmannia virescens from the rio São Francisco, middle rio Amazonas, Island of Marajó, Abaetetuba, Belém and Benevides, present the ZZ/ZW system (Almeida-Toledo et al., 2002;Silva et al., 2009).Silva et al. (2009) suggest that the mechanisms involved in the differentiation of the W chromosome are the result of a pericentric inversion event in the proximal region of the short arm of an acrocentric chromosome, followed by a heterochromatization event.These events would have occurred in a distinct manner in independent populations, derived from an ancestral karyotype without differentiated sex chromosomes (Henning et al., 2011).It is highly possible that the sex chromosomes differentiation also contributed to species differentiation/divergence and worked as a reproductive isolation mechanism.
The events that occur during differentiation of sex chromosomes are still not well described.During the differentiation of sex chromosomes, the suppression or partial restriction of recombination between the sex determining pair of chromosomes should occur.This phenomenon is associated with the accumulation of heterochromatin on the sex chromosomes (Ohno, 1967).The absence of recombination favours the accumulation of repetitive sequences of DNA, permitting the morphological differentiation of the sex chromosomes (Almeida-Toledo et al., 2001;Cioffi et al., 2012).Such events are expected to have been involved in the morphological differentiation of the X and Y chromosomes of Eigenmannia sp."B".
The investigation of the organization of repetitive sequences can provide evidence for the origin and evolution of sex chromosomes.Studies with chromosome painting, using sex chromosome specific probes and FISH with probes for repetitive DNA sequences, combined with phylogenetic analyses are fundamental to elucidate the mechanisms involved in the origin and differentiation of sex chromosomes.Eigenmannia represents a potential comparative model within the Gymnotiformes for such comparative analyses.The inclusion of diverse analytical methods using morphological, osteological and cytogenetic data can be extremely useful to clarify taxonomic problems in Neotropical fishes such as those encountered in Eigenmannia, and may well indicate further cryptic species.

Fig. 7 .
Fig. 7. (a) DAPI fluorescent banding coinciding with C banding.(b) CMA 3 fluorescent banding is coincident with the NOR region on short arm of pair 17 of Eigenmannia sp."A".(c) FISH with 18S rDNA probes hybridizing on the short arm of chromosome 17 (arrows).

Fig. 8 .
Fig. 8. DAPI fluorescent banding showing centromeric regions of both males (a) and females (b) of Eigenmannia sp."B".CMA 3 fluorescent banding hybridizing areas, which coincide with the NOR (Fig. 6) on chromosome 14 of males (c) and females (d), and strong signal on pairs 8, 9 and the X -inset boxes.FISH with 18S rDNA probes of Eigenmannia sp."B", hybridizing on the short arm of chromosome 14 (arrows) of males (e, f).

Table 1 .
Karyotypic studies in the genus Eigenmannia.

299 Material and Methods Samples.
Fifty three samples were collected from three localities in eastern Amazonia, Pará State, Brazil (Fig.1): two localities in the municipality of Capanema, rio Quatipuru basin and one in the municipality of São Francisco, rio Marapanim basin.All specimens were deposited in the ichthyology collection of the Museu Paraense Emílio Goeldi, Pará State, Brazil (MPEG) (Table2).Samples were collected using seine nets, and kept alive with portable aeration in thermally protected receptacles for transport to the laboratory.Sample collection was made under licence 020/2005 (ICMBio Registration: 207419).

Table 2 .
Localities of the samples of Eigenmannia in north-eastern Pará State.?= undetermined sex.