Evolutionary cytogenetics of the Hoplias lacerdae , Miranda Ribeiro , 1908 group . A particular pathway concerning the other Erythrinidae fish

The taxonomy/systematics of the Erythrinidae fish is still imprecise, with several doubts on their relationships. Karyotypes and chromosomal characteristics of some species of the Hoplias lacerdae group (Erythrinidae), from different Brazilian hydrographic basins and pisciculture stations, were analyzed in the present study, using conventional Giemsa staining, C-banding, silver staining, Mithramycin and Distamycin/DAPI fluorochromes, and fluorescent in situ hybridization (FISH). A diploid chromosome number of 2n = 50 and karyotypes composed of metaand submetacentric chromosomes without sex-related differences were found. Only one active NOR (Nucleolar Organizer Region) site was found, which was identified by silver staining (Ag-NOR) and FISH, located on the chromosome pair 11, although additional 45S rDNA sites were also mapped on other chromosome pairs only by FISH. The Ag-NOR of the chromosome pair 11 was found to be GC-rich, appearing positive after Mithramycin staining. Mithramycin-positive/ DAPI-negative sites were also observed in the centromeric/pericentomeric regions of the chromosome pairs 4, 6, 15, and 19, which have also affinity to silver nitrate. However, these four sites were not detected by FISH with the rDNA probe, indicating to be only argentophilic GC-rich heterochromatic regions. Chromosome data show that the karyotype evolution in Hoplias lacerdae group is relatively conserved and follows a particular pathway concerning the other Erythrinidae fishes, such as Hoplias malabaricus, Hoplerythrinus unitaeniatus, and Erythrinus erythrinus, in which polytypic karyotypes are found. Thus, the H. lacerdae group shows chromosome features that are not closely related to those of the congeneric H. malabaricus group. These finds, together with genetic and morphologic data, are important tools to be considered in a major revision of the Erythrinidae family, as well as for conservation programs.


Introduction
Erythrinidae is a small freshwater fish family widely distributed throughout South America, with only three genera (Hoplias, Hoplerythrinus, and Erythrinus), with a low number of nominal species.However, the taxonomy/ systematics of this family is not well understood.Hoplias malabaricus (Bloch, 1794), and H. lacerdae Miranda Ribeiro, 1908, for example, belong to two distinct species groups, the H. malabaricus and the H. lacerdae groups, respectively, in which several species do not present a definitive taxonomy (Oyakawa, 1990(Oyakawa, , 2003)).
The taxonomic doubts are also supported by several cytogenetic data showing a great karyotypic diversity among different populations, indicating the occurrence of distinct hidden species.Indeed, H. malabaricus, Hoplerythrinus unitaeniatus (Agassiz, 1829), and Erythrinus erythrinus (Bloch and Schneider, 1801) have different karyotypes (cytotypes) easily characterized by the chromosome number and composition, as well as by the presence of distinct sex chromosome systems (Bertollo et al., 2000;Giuliano-Caetano et al., 2001;Bertollo et al., 2004).
Concerning the H. lacerdae group, chromosome data are only available for specimens from two Brazilian populations.Although these two populations were widely isolated, one of them from the Southeastern region and the other one from the North region, they showed similar karyotypes with 2 n = 50 meta-and submetacentric chromosomes (Bertollo et al., 1978;Bertollo and Moreira-Filho, 1983).The present study deals with chromosome data from other distinct populations, using conventional analyses, fluorochrome staining, banding methods, and fluorescent in situ hybridization with an rDNA probe, in order to improve the cytogenetic data of the H. lacerdae group.The obtained data was also used to verify if their general karyotypic macrostructure remains stable or, on the contrary, if a karyotypic diversity is also found among populations, as occur in other erythrinids.
C-bands were located in the centromeric/pericentromeric region of all the chromosome pairs and several of them were very conspicuous.An exception was the specimens from population C which did not show satisfactory results for C-banding.In addition, chromosome number 5 showed a telomeric C-band on the long arm (Figures 3a, 4a).
One Ag-NOR site was identified close to the centromere, on the long arms of the chromosome pair number 11, in the four populations (Figures 3b, 4b), which is Mithramycin-positive (Figure 3e).However, the centromeric/pericentromeric sites of the chromosome pairs 4, 6, 15, and 19 were also highlighted after silver staining (Figures 3b, 4b), showing a conspicuously bright fluorescence after Mythramycin staining (Figures 3c, 3e, 4c) DAPI (Schweizer, 1980) staining, in order to detect GC-and AT-rich sites on the chromosomes, respectively.Silver nitrate staining (Howell and Black, 1980), and C-banding (Sumner, 1972), were also used to analyze the active nucleolar organizer regions (Ag-NORs) and the C-positive heterochromatin, respectively.The rDNA sites were mapped on the chromosomes using the fluorescent in situ hybridization (FISH) with an rDNA probe, according to Pinkel et al. (1986).The probe consisted of a mixture of two recombinant plasmids (HM123 and HM456) containing rDNA fragments of Xenopus laevis (Meunier-Rotival et al., 1979) that comprise the totality of the 45S rDNA gene.
The diploid chromosome number was found for females and males; at least 30 metaphases per each specimen were analyzed.The classification of the chromosomes followed Levan et al. (1964).
No karyotypic differentiation was observed between the males and females from the populations A, B, and C (Figure 2).In addition, the male karyotype from population D did not suggest the occurrence of chromosome heterogamety.

Discussion
All analyzed specimens have 2n = 50 meta-and submetacentric chromosomes, showing the same macro karyotype structure.Some minor differences were observed in the centromere position of the chromosome pair number 20 in the populations A and C, and in the chromosome pair number 23 in the populations B and D. However, it is difficult to conclude if these features indicate real differentiations among the populations or if they would be resultant of technical problems, such as the correct pairing in the karyotypes or the degree of the chromosomal condensation.Similar karyotypes were also previously observed in the specimens from Usina do Limoeiro pisciculture station, São José do Rio Pardo, São Paulo State (Bertollo et al., 1978) and from the Aripuanã River -Aripuanã, Mato Grosso State (Bertollo and Moreira-Filho, 1983).This karyotype stability is also reinforced by the similar pattern of the heterochromatin distribution, which is shared by the specimens of the distinct populations.
It is generally accepted that the nuclear organizer regions are GC-rich regions in lower vertebrates (Schmid and Guttenbach, 1988), i.e., they are Mithramycin-or Chromomycin A 3 -positive sites, which is also true for specimens of the H. lacerdae group.Fluorescent in situ hybridization, carried out in the population B, mapped the 45S rRNA genes in the NOR site on the chromosome pair 11, in addition to some sites in other chromosomes.These data indicate the occurrence of multiple NORs, although only that on the 11 th pair was silver stained, i.e., an active NOR, which was also observed in the other analyzed specimens.Thus, all the additional 45S rDNA sites found in the population B appear to be under genetic inactivation.Until now, there is no available information on ribosomal gene mapping by FISH in the other populations.
On the other hand, the four argentophilic regions in the chromosome pairs 4, 6, 15, and 19 were Mithramycinpositive, a fact that, at first, could also indicate the occurrence of nucleolar organizer regions.However, ribosomal cistrons were not detected in these four chromosomes after FISH.Similar cases in which GC-rich sites were not correlated with NORs have also been observed in few other fish, such as in species of the genus Schizodon (Martins and Galetti Jr., 1998), Liposarcus and Leporinus (Artoni et al., 1999), Eigenmannia (Almeida-Toledo et al., 2000), and some Loricariidae (Kavalco et al., 2004).According to Sumner (1990), chromosome staining with silver nitrate may also highlight argentophilic heterochromatic sites, which can explain the present results.
The comparison between male and female karyotypes from populations A-C, as well as the analysis of the male karyotype from population D, indicate the absence of heteromorphic sex chromosomes.Bertollo et al. (1978), suggested an XX/XY sex chromosome system for Hoplias cf.lacerdae from Usina do Limoeiro pisciculture station, based on the comparative analysis between male and female karyotypes.Considering that it was the first study in this fish group using only conventional Giemsa stained chromosomes, the reevaluation of the proposed sex chromosome system would be necessary using more informative chromosomal analyses for its confirmation.Unfortunately, specimens from that locality are not more available hindering this confirmation.Thus, at this time, the Hoplias lacerdae group emerges as an Erythrinidae and H. brasiliensis (Agassiz, 1829) as valid species in the lacerdae group, which are found in the Ribeira de Iguape River (São Paulo and Paraná States), and in coastal basins (Bahia State), respectively.In addition, other species not yet formally described occur in several other South American hydrographic systems, such taxon where a heteromorphic sex chromosome system seems not to occur.
Despite the reduced number of species, several questions about the taxonomic state of the H. lacerdae group, as well as the whole Erythrinidae family, are still to be solved.Oyakawa (1990Oyakawa ( , 2003) ) recognizes H. lacerdae  as Amazon basin, upper Paraná and upper São Francisco basins, Uruguay basin, and South Brazil.Strictly, none of the specimens from natural populations with available chromosomal data (Figure 1) must correspond to H. lacerdae and H. brasiliensis.Indeed, besides the fish from pisciculture stations (samples 2, 4, and 5) which have unknown origin, those from natural environments (samples 1, 3, and 6) are not representative specimens from the hydrographic basins where H. lacerdae and H. brasiliensis occur.Thus, it is very likely that they correspond to undescribed species from the Eastern and Northern Brazilian regions (São Francisco River, and Aripuanã and Pitinga Rivers, respectively).
Our data demonstrate that the karyotype evolution in the H. lacerdae group is characterized by a macrostructural homogeneity, contrasting with other Erythrinidae species, such as H. malabaricus, Hoplerythrinus unitaeniatus, and Eryhtrinus erythrinus in which polytypic karyotypes are found.Indeed, many populations of the H. malabaricus group show distinct karyotypes (cytotypes) identified by their chromosome number and/ or structure.The same is valid for the nominal species H. unitaeniatus and E. erythrinus from different Brazilian rivers.In addition, several populations of H. malabaricus and E. erythrinus also differ in the occurrence of distinct sex chromosome systems (XX/XY, X 1 X 1 X 2 X 2 /X 1 X 2 Y, and XX/XY 1 Y 2 ), or in the absence of chromosome dif-f f ferentiation between sexes (Table 1).All these chromosomal features of H. malabaricus, H. unitaeniatus, and E. erythrinus are found among isolated populations in nature, or even among sympatric cytotypes without evidences of hybridization, showing that the chromosomal rearrangements are already fixed, suggesting the existence of several unnamed species (Bertollo et al., 2000;Diniz and Bertollo, 2003;Bertollo et al., 2004).Thus, it is remarkable that two very divergent karyotype pathways can be characterized within the genus Hoplias, i.e., H. malabaricus group, with a pronounced chromosome diversity and a smaller diploid number (2n = 39-42 chromosomes), and H. lacerdae group, with a relative chromosomal homogeneity and a higher diploid number (2 n = 50 chromosomes).In addition, in the H. lacerdae group only a single chromosome pair bearing active NORs (Ag-NORs) was found, in contrast to what occurs in the H. malabaricus group, where all the cytotypes showed multiple Ag-NORs (Bertollo, 1996), as well as in the other erythrinids (Table 1).Thus, H. malabaricus and H. lacerdae groups do not appear closely related if we consider their chromosomal features.As the taxonomy and systematics of the Erythrinidae fish are still imprecise showing several doubts about their relationships, the current data, together with other genetic and morphologic data, will be useful tools for the revision of this fish family, as well as for conservation programs.

Figure 2 .
Figure 2. Karyotype of the Hoplias lacerdae group from population a (São Francisco River) showing 2 n = 50 meta/ submetacentric chromosomes, with no differentiation between females and males.Idiograms of the chromosome pairs 20 and 23 showing slight interpopulational differences related to the centromere position: 20a and 23a refer to populations A and B; 20b and 23b refer to populations C and D. Bar = 5 µm.

Figure 3 .
Figure 3. Chromosomes of the Hoplias lacerdae group from populations A (a-b) and B (c-f) showing a) the preferential location of the C-positive heterochromatin on the centromeric/pericentromeric regions and the chromosome pair number 5 with a telomeric C-band (arrows); b) Ag-NORs on the chromosome pair number 11 (arrows) and the argentophilic centromeric/ pericentromeric regions on the chromosome pairs numbers 4, 6, 15, and 19 (arrowheads); c) conspicuous Mithramycinpositive sites and d) DA/DAPI-negative sites (arrows) related to the argentophilic regions showed in b), respectively; e) Mithramycin-positive NORs on the chromosome pair number 11 (arrows) and the conspicuous Mithramycin-positives sites on the chromosome pairs numbers 4, 6, 15, and 19; and f) FISH metaphase showing the 45S rDNA mapping on the Ag-NOR bearing chromosome pair number 11 (arrows), as well as on some other chromosome pairs (arrowheads). e

Figure 4 .
Figure 4. Representative idiograms of the Hoplias lacerdae group from populations A and B showing a) the distribution of the C-positive heterochromatin on the chromosomes; b) Ag-NOR/Mithramycin positive site/45S rDNA location on the chromosome pair number 11, and the argentophilic regions on the chromosome pairs numbers 4, 6, 15, and 19; c) and d) Mithramycin positive sites and DA/DAPI negative sites related to the argentophilic regions on the chromosome pairs numbers 4, 6, 15, and 19, respectively.