Karyotypic similarities between two species of Rhamphichthys (Rhamphichthyidae, Gymnotiformes) from the Amazon basin

Abstract The family Rhamphichthyidae includes three genera: Rhamphichthys Müller et Troschel, 1846, Gymnorhamphichthys M. M. Ellis, 1912 and Iracema Triques, 1996. From this family, only the species Rhamphichthys hanni Meinken, 1937 has had its karyotype described. Here, we describe the karyotypes of two additional Rhamphichthys species: Rhamphichthys marmoratus Castelnau, 1855 from the Reserva de Desenvolvimento Sustentável Mamirauá, Amazonas state and Rhamphichthys prope rostratus Linnaeus, 1766 from Pará state, both in Brazil. Our karyotypic analyses demonstrated that the diploid number is conserved for the genus (2n = 50), but the karyotypic formulas (KFs) differed between Rhamphichthys marmoratus (44m/sm+6a) and Rhamphichthys prope rostratus (42m/sm+8a). In both species, the constitutive heterochromatin (CH) was located in the centromeric region of most chromosomes. Large heterochromatic blocks were found on the long arms of pairs 4 and 14 in Rhamphichthys marmoratus and on chromosomes 3, 4 and 19 in Rhamphichthys prope rostratus, which also has a heteromorphism in chromosome pair 1. The CH was DAPI positive, indicating that it is rich in AT base pairs. The Nucleolus Organizer Region (NOR) showed staining at a single location in both species: the long arm of pair 1 in Rhamphichthys marmoratus and the long arm of pair 12 in Rhamphichthys prope rostratus, where it showed a size heteromorphism. CMA3 staining coincided with that of Ag-NOR, indicating that the ribosomal genes contain interspaced GC-rich sequences. FISH with an 18S rDNA probe confirmed that there is only one NOR site in each species. These results can be used as potential cytogenetic markers for fish populations, and comparative analysis of the karyotypes of Hypopygus Hoedman, 1962, Rhamphichthys and Steatogenys Boulenger, 1898 suggests that the first two genera diverged later that the third.


Introduction
The family Rhamphichthyidae comprises three genera: Rhamphichthys Müller et Troschel, 1846, with eight described species, Gymnorhamphichthys Ellis, 1912, with six species, and Iracema Triques, 1996, with only one species (Ferraris 2003, Lundberg 2005, Triques 2005, Carvalho et al. 2011 (Table 1). These numbers are likely to be an underestimate, since the number of species described in Gymnotiformes has increased over the last 15 years .
The species of Rhamphichthys have a long and narrow body, a long tubular snout, no teeth in the jaw, and an anal fin with more than 300 rays. They are slow swimmers and spend most of their time at the bottoms of rivers (Mago-Leccia 1994, Ferraris 2003, Triques 2005. Among the Gymnotiformes, Rhamphichthys has the largest diversity and abundance in the Amazon basin, and the species Rhamphichthys rostratus Linnaeus, 1766 has the largest geographic distribution when compared with the other species of this genus (Ferraris 2003). All Rhamphichthys species generate electrical pulses that are used to communicate and identify mating partners and other species. This trait allows them to be nocturnal and live in rivers with dark waters (Kawasaki et al. 1996, Crampton 1998, Nanjappa et al. 2000, Gouvêa et al. 2002. The phylogeny of the Gymnotiformes proposed by Albert (2001) was based on morphophysiological, behavioral and DNA sequence analyses by Alves-Gomes et al. (1995). In it, the families Rhamphichthyidae and Hypopomidae form a monophyletic group (Rhamphichthyoidea) that is separated from the clade that includes the families Sternopygidae and Apteronotidae. Among the Rhamphichthyoidea, the tribe Steatogenini (Steatogenys Boulenger, 1898, Hypopygus Hoedman, 1962and Stegostenopos Triques, 1997 is accepted as monophyletic (Albert andCampos-da-Paz 1998, Crampton et al. 2007), but there is some debate as to whether this tribe belongs to the Rhamphichthyidae (Alves- Gomes et al. 1995) or the Hypopomidae (Albert 2001).
Relatively few cytogenetic studies have been performed in Gymnotiformes. According to Oliveira et al. (2009), only 48 species of this order have had their karyotypes described. The genera Gymnotus Linnaeus, 1758 and Eigenmannia Jordan et Evermann, 1896 have the most available information on their karyotypic diversity (Almeida-Toledo et al. 2001, Lacerda and Maistro 2007, Milhomem et al. 2007, Silva et al. 2009, Nagamachi et al. 2010. In Rhamphichthyoidea, the available chromosome information comes from only six species (Table 2) In the present work, we studied the karyotypes of two species of Rhamphichthys from the Amazon region in an effort to better define the boundaries between the species, and compared our findings with those from the single previously described species of Rhamphichthys to better understand the phylogenetic relationships in this genus.

Material and methods
Fishes were collected using a bioamplification device that detects electric fields and translate them into sounds ). We analyzed 13 animals (seven males and six females) of Rhamphichthys marmoratus Castelnau, 1855, collected from  Ferraris 2003 andAlbert and.

Rhamphichthys marmoratus
All samples of R. marmoratus (Fig. 2) had 2n = 50 and a karyotypic formula (KF) consisting of 44 metacentric/submetacentric (m/sm) and 6 acrocentric chromosomes (Fig.  2a), with no evidence of any sex-determination chromosome system. Ag-NOR staining showed that the NOR is located in the interstitial region of the long arm of pair 1, in a secondary constriction (Fig. 2b, box). Constitutive heterochromatin (CH) was found in the centromeric regions of all chromosomes (Fig. 2c). Pair 4 was notable for a large heterochromatic block running from the proximal region across most of the long arm, while pair 14 had a CH block covering most of its short arm. CH was also found in the distal region of the long arm of pair 1 (Fig. 2c). DAPI fluorochrome banding coincided with positive C-banding in all centromeres, and was especially strong in pairs 4 (Fig. 3a). The CMA 3 fluorochrome banding localized to the same region as the NOR, suggesting that this region is GC-rich (Fig. 3b). FISH with 18S rDNA probes confirmed that the NOR is located in the interstitial region of the long arm of pair 1 (Fig. 3c).

Rhamphichthys prope rostratus
R. prope rostratus (Fig. 4a) had 2n = 50 and a KF of 42m/sm+8a, with no evidence of a sex-determination system (Fig. 4b). Ag-NOR staining was noted in the interstitial region of the long arm of pair 12 (Fig. 4b, box). CH was found in the pericentromeric regions of most chromosomes, and large CH blocks were found in the proximal regions of the long arm of pairs 3, 4 and 9. Pair 1 had a heteromorphism in both males and females, probably because of a heterochromatin block, as did pair 12 (Fig. 4c). DAPI banding was positive in the CH regions, suggesting that these regions are ATrich (Fig. 5a). CMA 3 banding showed size differences between the homologs, suggesting the presence of a size difference in this GC-rich region (Fig. 5b). Finally, FISH against the 18S rDNA hybridized to the same region that was positive for Ag-NOR staining (Fig. 5c).

Discussion
Both Rhamphichthys marmoratus and Rhamphichthys prope rostratus had 2n = 50, but differed in their KFs, with R. marmoratus having 44m/sm+6a and R. prope rostratus having 42m/sm+8a. Previously, Rhamphichthys hanni was described as having 2n = 50, but 20m+24sm+6a (Mendes et al. 2012). These differences can be explained by chromosome rearrangements that have altered the chromosome morphology but not the diploid number (e.g., pericentric inversions). These rearrangements can be sufficient to act as a post-mating reproductive barrier (King 1993). A more refined analysis, such as the use of chromosome painting, will be necessary for the precise determination of the rearrangements that differentiate the karyotypes of these three species. In a similar situ-  ation in Gymnotiformes, Nagamachi et al. (2010) demonstrated that two cytotypes of Gymnotus carapo Linnaeus, 1758 (2n = 42 and 2n = 40) differed not just by the fusion event suggested by the conventional analysis, but also by many rearrangements. The CH in R. prope rostratus and R. marmoratus is AT-rich (i.e., DAPI bandingpositive), which is consistent with other species of Gymnotiformes (Milhomem et al. 2007, Silva et al. 2008, Silva et al. 2009). The CH blocks found in pairs 4 and 12 of R. marmoratus and in pairs 3, 4 and 9 of R. prope rostratus can be used as cytogenetic markers for these species, as suggested for other Neotropical fish species (Almeida-Toledo 1998, Silva et al. 2008. Mendes et al. (2012) found only three submetacentric pairs with heterochromatin blocks in Rhamphichthys hanni. This is an important trait and can be used along with other characteristics to differentiate populations of these species, since there is some debate regarding their interspecific boundaries.
The NOR was found on a secondary constriction and stained positive with CMA 3 as previously observed on other species (Pendás et al. 1993, Fernandes et al. 2005, Milhomem et al. 2007, Silva et al. 2008, De Souza et al. 2009). Each of the species studied herein had a single NOR, but R. prope rostratus had a size heteromorphism in this region. The 18S rDNA probe hybridized to a similar-sized segment in both homologs, suggesting that the size difference is not likely to be the result of an in-tandem duplication of the ribosomal genes (Martins-Santos and Tavares 1996), as described in Eigenmannia sp.1 by Almeida-Toledo et al. (1996). Instead, the heteromorphism found by CMA 3 banding can be explained by a variation in the amount of GC-rich sequences interspersed among the ribosomal genes in this region. In R. hanni (Mendes et al. 2012), the results of the Ag-NOR staining and 18S rDNA probe hybridization were very similar to our findings in R. rostratus.
The phylogeny proposed by Albert (2001) places the families Rhamphichthyidae and Hypopomidae into a monophyletic group (Rhamphichthyoidea) that is only distantly related to the clade that joins the families Sternopygidae and Apteronotidae. The monophyly of Rhamphichthyoidea was supported by the synapomorphic characteristics described by Triques (2005).
However Alves-Gomes et al. (1995) suggested that Hypopomidae is not monophyletic, in that the genera Hypopygus and Steatogenys are more closely related to Rhamphichthyidae. The cytogenetic data described herein, as well as the recent work of Cardoso et al. (2011), seem to support the latter phylogenetic arrangement, since all the Rhamphichthys karyotypes described to date have 2n = 50. Among the Hypopomidae, Hypopygus and Steatogenys have 2n = 50, but all of the other genera have lower diploid numbers (2n = 26 to 42, Table 2). However, while the Rhamphichthys have karyotypes with KFs similar to those of Hypopygus and Steatogenys (42-44 bi-armed and 6-8 mono-armed chromosomes) the KFs diverge considerably into Steatogenys, ranging from all bi-armed chromosomes (Steatogenys duidae) to mostly mono-armed chromosomes (Steatogenys elegans). Conversely, the karyotype of Hypopygus has a KF similar to those of Rhamphichthys. These differences seem to indicate that the genera Hypopygus and Steatogenys split from Rhamphichthys at an earlier date than the Rhamphichthys species split from one another, which is consistent with the phylogeny of Alves-Gomes et al. (1995). The chromosome similarity between Hypopygus and Rhamphichthys suggests that these genera separated more recently than Steatogenys, or that chromosome evolution proceeded more quickly in the latter genus, with a buildup of autoapomorphies.
The available cytogenetic information on Gymnotiformes may be sparse (of eight species of this genus, only three have had their karyotypes analyzed), but the existing data show an important variability in this group. More cytogenetic investigations on the family Rhamphichthyidae are warranted, as they will help us better understand the chromosomal evolution of these fishes for use in other fields of science, and assist us in defining the boundaries of the Rhamphichthys species.