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 (Albert and Crampton 2005).

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, 1962 and Stegostenopos Triques, 1997) is accepted as monophyletic (Albert and Campos-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, 2002, Lacerda and Maistro 2007, Milhomem et al. 2007, 2008, Silva et al. 2009, Nagamachi et al. 2010).

In Rhamphichthyoidea, the available chromosome information comes from only six species (Table 2): Hypopomus artedi Kaup, 1856 with diploid number (2n) = 38, Fundamental Number (FN) = 70 and Karyotypic Formula (KF) = 32m/sm+6st/a; Hypopygus lepturus Hoedman, 1962with 2n = 50, FN = 86 and KF = 36m/sm+10st+4a; Brachyhypopomus brevirostris Steindachner, 1868, with 2n = 36, FN = 42 and KF = 6m/sm+30st/a (Almeida-Toledo et al. 2000); Brachyhypopomus pinnicaudatus Hopkins, 1991, with 2n = 41 in males and 42 in females (X₁X₂Y sex system) and FN = 42, with all acrocentric chromosomes except the Y (Almeida-Toledo 1978); Steatogenys elegans Steindachner, 1880, with 2n = 50 (ZZ/ZW sex system), FN = 62 and KF = 12m/sm+38st/a; Steatogenys duidae La Monte, 1929, with 2n = 50, FN = 100 and KF=50m/sm (Cardoso et al. 2011); and Rhamphichthys hahni Meinken, 1937, with 2n = 50, FN = 94 and FK = 44m/sm+6st/a (Mendes et al. 2012).

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.

Fishes were collected using a bioamplification device that detects electric fields and translate them into sounds (Crampton et al. 2007). We analyzed 13 animals (seven males and six females) of Rhamphichthys marmoratus Castelnau, 1855, collected from rivers in the Reserva de Desenvolvimento Sustentável Mamirauá (Mamirauá Sustainable Development Reserve, RSDM), Amazonas state, Brazil (03°07'32.5"S, 064°46'47.3"W). The sample was deposited in the museum of the RSDM (IDSMIctio000735 and IDSMIctio000750). The two individuals of Rhamphichthys prope rostratus Linnaeus, 1766, one male and one female, came from the Parú River, Pará state, Brazil (01°31'13.39"S, 52°38'49.00"W). This sample was deposited in the Museu Paraense Emílio Goeldi (MPEG 18347). Figure 1 shows the collection sites.

Metaphase chromosomes were obtained according to the method described by Bertollo et al. (1978) and analyzed by Giemsa staining, C-banding (Sumner 1972), Ag-NOR staining (Howell and Black 1980), CMA₃ banding (Schweizer 1980) and DAPI banding (Pieczarka et al. 2006). Fluorescent In Situ Hybridization (FISH) was performed using 18S rDNA probes from Prochilodus argenteus Spix et Agassiz, 1829 (Hatanaka and Galetti Jr 2004). Microscopic images were obtained using a Zeiss Axiophot 2 microscope and a Zeiss Axiocam Mrm controlled by the Zeiss Axiovision software. Metaphase organization was performed following the method of Levan et al. (1964).

Both Rhamphichthys marmoratus and Rhamphichthys prope rostratus had 2n = 50, but differed in their KFs, with Rhamphichthys marmoratus having 44m/sm+6a and Rhamphichthys 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 situation 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 Rhamphichthys prope rostratus and Rhamphichthys marmoratus is AT-rich (i.e., DAPI banding-positive), which is consistent with other species of Gymnotiformes (Milhomem et al. 2007, 2008, Silva et al. 2008, Silva et al. 2009). The CH blocks found in pairs 4 and 12 of Rhamphichthys marmoratus and in pairs 3, 4 and 9 of Rhamphichthys 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). Mendeset 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 CMA₃ as previously observed on other species (Pendáset 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 Rhamphichthys 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 CMA₃ banding can be explained by a variation in the amount of GC-rich sequences interspersed among the ribosomal genes in this region. In Rhamphichthys hanni (Mendes et al. 2012), the results of the Ag-NOR staining and 18S rDNA probe hybridization were very similar to our findings in Rhamphichthys 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.