Cytotaxonomy of Parodon nasus and Parodon tortuosus (Pisces, Characiformes). A case of synonymy confirmed by cytogenetic analyses

Morphological and cytogenetical studies were carried out on the freshwater fish Parodon nasus and Parodon tortuosus in order to evaluate a putative synonymy. The diploid chromosome number observed in both species was 2n = 54 (48M/SM and 6ST) with no differences between the sexes. Despite slight differences in the pattern of heterochromatin distribution and the number of cusps in symphysean teeth, the location of nucleolar organizer regions (NORs) and 5S rRNA genes (both species-specific features) were similar in both species. The remarkable similarity observed between these allopatric species supports recent taxonomical reviews indicating that P. tortuosus is a synonym for P. nasus.


Introduction
Over the last three decades, cytogenetic studies in Neotropical fishes have widely contributed to the taxonomy of Neotropical fishes.It has been pointed out that karyotypical analyses provides useful information for evolutionary and phylogenetic studies and aids in the identification of controversial species (Bertollo et al., 1986), and, indeed, such techniques have been successfully applied to characterize phenotypically similar cryptic species.Cytogenetical analyses of some fish species (e.g. from the genera Leporinus (Galetti et al., 1981a, b) and Brycon (Margarido and Galetti, 1996)) have agreed with their taxonomic status but cytogenetical studies of species such as Astyanax scabripinnis (Moreira-Filho and Bertollo, 1991), Hoplias malabaricus (Bertollo et al., 2000), Eigenmannia virescens (Almeida-Toledo et al., 2002) and Hoplerythrinus unitaeniatus (Diniz and Bertollo, 2003) have diverged from taxonomic classifications, suggesting that these groups should be taxonomically reviewed.
The Parodontidae, a relatively small freshwater fish family within the order Characiformes, comprises fish commonly known in Brazilian Portuguese as 'canivetes' or 'charutos' ('penknives' or 'cheroots') (Pavanelli, 2003) which are widely distributed throughout nearly all South America.Three genera are reported for this family: Parodon Valenciennes, 1850;Apareiodon Eigenmann, 1916;andSaccodon Kner, 1964. Since 1849, 13 Apareiodon, 18 Parodon and 3 Saccodon species have been described for the Parodontidae, although after the detailed review of Pavanelli (2003) this number was reduced to a total of 21 species.Parodontid fish present distinct, nonoverlapping, reproductive periods (Barbieri et al., 1983) and a pale gray streamlined body (Nomura, 1979), the coloration being basically composed of stripes, bars and blotches providing an obliterative effect in their rocky and turbulent freshwater habitats (Sazima, 1980).The pectoral and pelvic fins of members of this family are well developed and adapted to stabilizing the fish on the rocky bottoms of the rivers and streams where these fish are usually found grazing on algae (Sazima, 1980).
Dentary structure is an important feature of the family Parodontidae and is helpful in the identification of the three genera (Garavello, 1977), small lateral teeth being present in the genus Parodon but absent in Apareiodon and Saccodon.In the Parodontidae the pre-maxilla is surrounded by a series of tiny teeth with rounded cusps, the number of cusps of pre-maxilla teeth being a reliable character for the diagnosis of Parodon and Apareiodon species but not for Saccodon species, which are characterized by extensive teeth polymorphism (Garavello, 1977).
The aim of the work described in the present paper was to better characterize P. tortuosus and P. nasus using both cytogenetic analysis and a comparative analysis of the number of teeth cusps in these two taxonomic units.(1980,1984,1985), Jesus and Moreira-Filho (1999) females) of P. nasus and 22 specimens (8 males and 14 females) of P. tortuosus using the methodology described by Bertollo et al. (1978).Silver nitrate (Ag) staining was used to detect the nucleolar organizer regions (Ag-NORs) according to the method of Howell and Black (1980).Fluorescent in situ hybridization (FISH) with 18S rDNA probes obtained from Prochilodus argenteus (Hatanaka and Galetti, 2004) was performed according to the protocol of Pinkel et al. (1986).This same procedure was employed for FISH with a 5SDNA probe obtained from Leporinus elongatus (Martins and Galetti, 1999).Differentiation of GC-rich chromosome regions was carried out by Schmid's method (Schmid, 1980) followed by staining with the GCspecific fluorochrome Chromomycin A 3 , and the constitutive heterochromatin being analyzed by the C-banding technique (Sumner, 1972).The chromosome types were classified according their arm ratio, as proposed by Levan et al. (1964).The teeth cusps analyses were carried out on 23 specimens of P. tortuosus and 33 specimens of P. nasus according to the methodology described by Moreira-Filho and Garavello (1994).
This paper presents for the first time a cytogenetical analyses of P. nasus, the diploid number obtained by us being 2n = 54 (48 M/SM + 6ST) for both sexes (Figure 1 6,17,25,. A single Ag-NOR pair was found in both P. nasus and P. tortuosus, located at the terminal position on the long arm of the 25 th chromosome pair (Figure 1).Four GC-rich signals were observed for both species, two on the short arms and two on the long arms (coincident with the NOR sites) of chromosome pair 25 (Figure 1).A single NOR pair on the long arms of chromosome pair 25 was confirmed by FISH with the 18S rDNA probe (Figure 3 a-b).However, while 5S rDNA FISH showed regions in a single pair on P. tortuosus chromosomes, this probe showed regions on three P. nasus chromosomes (Figure 3 c-d).

Discussion
The results obtained for diploid number (2n = 54), karyotype formulae, NORs, GC-rich regions, C-banding and 5S rDNA location were quite similar in P. tortuosus and P. nasus.Our P. tortuosus data supports that previously published by Moreira-Filho et al. (1984, 1985), Jesus and Moreira-Filho (2000a), Vicente et al. (2001) and Centofante et al. (2002).A diploid number of 54 has been reported for all Parodontidae species (Table 1), with the exception of A. affinis females from Upper Paraná which present a female heterogamety characterized by a multiple ZW 1 W 2 sex chromosome system.While Apareiodon species present extensive karyotypical similarity (Moreira-Filho et al., 1985), a diversification of chromosomal morphology is found within the genus Parodon, with a ZZ/ZW sex chromosome system distinguishing P. hilarii and Parodon sp. from the other species of this genus (Centofante et al., 2002).
A single NOR-bearing chromosomal pair has been found in Parodontidae fish (Table 1).The location of the NOR seems to be more conserved within Apareiodon, except that A. piracicabae shows intra-and inter-individual polymorphism related to the occurrence of double NORs in  each chromosome of a subtelocentric pair (Moreira-Filho et al., 1984).The NORs of P. hilarii, P. pongoensis (also known as Parodon sp.) and P. tortuosus have been considered to be species-specific markers located on different chromosomes (Jesus and Moreira-Filho, 2000a), and our results show that the NORs of both P. tortuosus and P. nasus are located on the same chromosomal pair, reinforcing the idea of synonymy between these two species.
Chromomycin A 3 staining showed that the NORs of P. tortuosus and P. nasus were GC-rich, a result identical to those published for other Parodontidae species (Jesus, 1996).The distribution of constitutive heterochromatin in Parodon and Apareiodon chromosomes occurs preferentially at the centromeric and, sometimes, the telomeric regions (Jesus and Moreira-Filho, 2000b).Our C-banding results were quite similar for P. tortuosus and P. nasus, with slight differences found in only a few chromosomes.
713  These slight differences have also been observed in P. tortuosus by other authors (Jesus and Moreira-Filho, 2000a;Vicente, 2001;Centofante et al., 2002) and also in P. hilarii (Jesus, 1996;Vicente, 2001), and are probably related to technical artifacts and/or interpopulation differences.These observations lead us to conclude that in the family Parodontidae the pattern of constitutive heterochromatin cannot be considered a useful marker for species diagnosis, contrasting with the situation in the genera Leporinus (Galetti et al., 1991a) and Brycon (Margarido and Galetti, 1996) where the C-banding pattern is species-specific.Vicente et al. (2001) detected a major and a minor 5S rDNA cluster on distinct chromosomal pairs of P. hilarii, P. pongoensis and P. tortuosus providing a species-specific marker.The occurrence of 5S rRNA genes in clusters involving more than a single chromosomal pair has been reported in several fish species, including those from the genera Schizodon (Martins and Galetti, 2000), Leporinus (Martins and Galetti, 1999) and Brycon (Wasko and Galetti, 2001).In these species, as in Parodon, besides the presence of two clusters there are also differences related to the location of 18S and 5S rDNA, which seems to represent a common situation in fish (Martinez et al., 1996).According to Martins and Galetti (1999), this distinctive location of ribosomal genes is required for the efficient maintenance of the conserved sequence displayed in tandem arrays.
We found that in P. nasus and P. tortuosus only the 5S rDNA major cluster was well-defined and on the same chromosome (syntenic) as the NORs, similar to the situation in Salmo salar described by Pendás et al. (1994).Since P. tortuosus is the only species within the genus Parodon to present such a feature, our results concerning the location of the 5S rDNA major clusters reinforces the similarity between P. nasus and P. tortuosus.In P. nasus, however, a third chromosome also contained the 5S rDNA major cluster, probably representing a homologue from a second 5S rDNA-bearing pair (a minor cluster) absent from the P. tortuosus specimens analyzed by us.The small size of these clusters (i.e.only a few copies of the 5S rRNA genes) in P. nasus may have been why this homologue was not detected by FISH, as it was reported for the genus Brycon (Wasko and Galetti, 2001).Our morphological analyses based on the number of symphysean teeth cusps showed a variation of 16-19 for P. tortuosus and 17-21 for P. nasus.According to Jesus and Moreira-Filho (2000a) such an overlap in the number of cusps is uncommon among Parodon species, although a symphysean teeth analyses of some Parodontidae species has shown a similar number of cusps in distinct species such as Apareiodon ibitiensis and Apareiodon sp. which were found to have 8-11 cusps (Jesus, 1996).
Taken together, our results demonstrate a high degree of similarity between P. tortuosus from the Passa-Cinco river and P. nasus from the Cuiabá river, supporting the taxonomical review carried out by Pavanelli (2003) who states that P. tortuosus should be considered a synonymy for P. nasus.

Figure 1 -
Figure 1 -Karyotypes of: a. Parodon tortuosus and b.Parodon nasus with conventional Giemsa staining.The nucleolar organizer regions after silver nitrate (Ag-NOR) and D/CMA 3 staining are showed in the boxes.

Figure 4 -
Figure 4 -Symphysean teeth of: a. Parodon tortuosus and b.Parodon nasus, with the variation of the number of cusps for each species.

Figure 5 -
Figure 5 -Histogram showing the distribution of frequency of cusps by teeth in P. tortuosus (grey) and in P. nasus (white).

Table 1 -
of P. tortuosus were collected in the Passa-Cinco river (Upper Paraná river basin) and P. nasus in the Cuiabá river (Paraguay river basin).Mitotic chromosomes were obtained from 20 specimens (10 males and 10 An overview of karyotypical data in Parodontidae species.