Karyotypic studies on Hoplerythrinus unitaeniatus (Pisces, Erythrinidae) populations. A biodiversity analysis

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1 CARYOLOGIA Vol. 56, no. 3: , 2003 Karyotypic studies on Hoplerythrinus unitaeniatus (Pisces, Erythrinidae) populations. A biodiversity analysis DÉBORA DINIZ and LUIZ ANTONIO CARLOS BERTOLLO* Universidade Federal de São Carlos, Departamento de Genética e Evolução, Laboratório de Citogenética Animal, Via Washington Luiz, Km 235, Cx. Postal 676, São Carlos, SP, Brazil. Abstract - Three populations of Hoplerythrinus unitaeniatus from two distinct Brazilian hydrographic basins were cytogenetically analyzed. Karyotypical variations were detected among them. The populations from Upper Paraná basin presented 2n=48, FN=96 and karyotype formula 44M/SM+4ST whereas that from São Francisco basin presented 2n=52, FN=100 and 44M/SM+4ST+4A. Constitutive heterochromatin showed a same pattern for all populations. Multiple Ag-NORs were found, ranging from four to six, and located on interstitial regions of the chromosomes, as observed in 2n=48 populations, and also in telomeric regions, as in 2n=52 specimens. Fluorescence in situ hybridizations (FISH) with 18S rdna probe were in agreement with Ag-NORs, further revealing a size heteromorphism between NOR sites as well as additional regions than those detected by the AgNO 3 staining. 5S rrna genes were present on pericentromeric regions of M/SM chromosomes in the three populations, displaying numerical variations specific to each locality. The present study adds new data about the karyotypic diversity of H. unitaeniatus and corroborates the divergent chromosomal evolution observed within the family Erythrinidae. Key words: fish; Hoplerythrinus unitaeniatus; karyotypic structure; biodiversity. INTRODUCTION Erythrinidae is a relatively small family of Neotropical fish, comprising three genera: Erythrinus Scopoli, 1977, Hoplerythrinus Gill, 1895 and Hoplias Gill, 1903, all endemic to South and Central Americas (GODOY 1975). Hoplerythrinus is regarded as a monotypic genus, and represented by the species Hoplerythrinus unitaeniatus (GERY 1977). This species exhibits some adaptive mechanisms such as accessory breathing (AZEVEDO and GOMES 1943) and locomotion ability out of the water (FINK and FINK 1978). Due to these features, it presents a great resistance during occasional dry seasons. * Corresponding author: fax ; bertollo@power.ufscar.br Cytogenetic analyses in the Erythrinidae fish were intensified from the initial studies in the genus Hoplias (BERTOLLO 1978). This genus is now the most studied one, revealing great karyotypic divergences among populations of the H. malabaricus group, which includes distinct sex chromosomes systems (BERTOLLO et al. 1986, 1997 a, b, 1998, 2000; BORN and BERTOLLO 2000). Erythrinus is the less known genus, but preliminary data also evidence a karyotypic diversity in this group, and a multiple sex chromosome system (MOLINA and BERTOLLO 1993; SILVESTRO and MARGARIDO 2001). Hoplerythrinus was firstly surveyed by GIU- LIANO-CAETANO (1986), who carried out a comparative analysis among H. unitaeniatus populations from distinct Brazilian hydrographic basins, with evidence of an intra-specific diversity. A

2 304 DINIZ and BERTOLLO chromosome polymorphic condition due to structural rearrangements was described for H. unitaeniatus from the Negro river, Amazonas state (GIULIANO-CAETANO and BERTOLLO 1988), as well as the occurrence of natural triploidy in one specimen from this same population (GIU- LIANO-CAETANO and BERTOLLO 1990). These data, added to some others from distinct populations, were recently compiled (GIULIANO- CAETANO et al. 2001), in order to provide an overview of the karyotypic diversity in the species. In the present study, we analyzed three other H. unitaeniatus populations from the Upper Paraná and São Francisco basins (Brazil). New chromosomal data about the karyotypic divergence in this fish group and its probable biological meaning are included. MATERIAL AND METHODS Samples from three H. unitaeniatus populations belonging to distinct Brazilian hydrographic basins were analyzed. The specimens from the Mogi-Guaçú river (Guatapará, São Paulo state) were named A population, and those ones from the Paraná river (Bataguassú, Mato Grosso do Sul state) were named B population. Both of these hydrographic systems belong to the major Upper Paraná basin. The specimens from marginal lagoons of the Prata river, a tributary of the Paracatu river (Minas Gerais state) - São Francisco basin - were named C population (Fig. 1). Fig. 1 South America map showing the collection sites of H. unitaeniatus populations already analyzed. 1 = Manaus (AM), 2 = Aripuanã (MT), 3 = Porto Velho (RO), 4 = Miranda (MS), 5 = Corrientes (Argentina), 6 = Posadas (Argentina), 7 = Paramaribo (Surinam), 8 = Parque Estadual do Vale do Rio Doce (MG), 9 = Guatapará (SP), 10 = Bataguassu (MS), 11 = Prata river (MG). Sites 9-11 refer to the present study. Table 1 Karyotypic features of H. unitaeniatus populations (adapted from Giuliano-Caetano et al. (2001). (*) = acrocentric chromosomes of similar size; (**) = acrocentric chromosomes of different size; AM = Amazonas, MG = Minas Gerais, MS = Mato Grosso do Sul, MT = Mato Grosso, RO = Rondônia, SP = São Paulo Brazilian states; Ar = Argentina; Su = Surinam; F = female; M = male; FN = fundamental number; M = metacentric, SM = submetacentric, ST = subtelocentric, A = acrocentric chromosomes. Locality Sample 2n FN Karyotype Ref. Manaus-AM 48(a) 96 48M/SM 1 Manaus-AM 48(b) 94 46M/SM+2A(*) 1 Manaus-AM 48(c) 94 46M/SM+2A(**) 1 Manaus-AM 48(d) 95 47M/SM+1A 1 Aripuanã-MT M/SM 2 Porto Velho-RO 2F+3M M/SM 3 Miranda-MS 4F+5M M/SM 3 Corrientes-Ar 5F+2F M/SM 3 Posadas-Ar M/SM 4 Paramaribo-Su 1F M/SM+2A 3 Vale do Rio Doce-MG 1F+1M M/SM+6A 3 Guatapará-SP 11F+9M M/SM+4ST 5 Bataguassú-MS 13F+7M M/SM+4ST 5 Rio da Prata-MG 4F+1M M/SM+4ST+4A 5 References: 1 = GIULIANO-CAETANO and BERTOLLO (1988); 2 = GIULIANO-CAETANO (1986); 3 = GIULIANO-CAETANO et al. (2001); 4 = BERTOLLO, L.A.C. (unpublished); 5 = present study.

3 KARYOTYPIC STUDIES ON HOPLERYTHRINUS UNITAENIATUS 305 About 400 metaphases from A population (20 specimens: 9 males and 11 females), 400 metaphases from B population (20 specimens: 7 males and 13 females) and 200 metaphases from C population (5 specimens: 1 male and 4 females were analyzed. The mitotic chromosomes were obtained by air drying technique (BERTOLLO et al. 1978). The Ag-NORs (nucleolar organizer regions detected by silver nitrate staining) were analyzed according to HOWELL and BLACK (1980). The constitutive heterochromatin was detected by C-banding, as described by SUMNER (1972), with slight modifications. The chromosomes were classified as metacentric (M), submetacentric (SM), subtelocentric (ST) and acrocentric (A), accord- Fig. 2 Karyotypes of H. unitaeniatus from population A (Guatapará, SP), after conventional Giemsa staining (a) and C- banding (b).

4 306 DINIZ and BERTOLLO ing to their arm ratio (LEVAN et al. 1964). The fundamental number (FN=number of chromosomal arms) was calculated taking into account the M, SM and ST chromosomes bearing two arms, and the A chromosomes bearing only one arm. The chromosomes were ordered in the karyotype by decreasing size in each morphological category. Fluorescent in situ hybridization (FISH), using 18S rdna and 5S rdna probes, was performed following the procedure described by PINKEL et al. (1986), with slight modifications. The 18S rdna probe was obtained by PCR, from the fish Prochilodus affinis, using the primers NS1 (5 -GTAGTCATATGCTTGTCTC-3 ) and NS8 (5 -TCCGCAGGTTCACCTACGGA-3 ) (WHITE Fig. 3 Karyotypes of H. unitaeniatus from population B (Bataguassu, MS), after conventional Giemsa staining (a) and C-banding (b).

5 KARYOTYPIC STUDIES ON HOPLERYTHRINUS UNITAENIATUS ). Another probe, composed by the recombinant plasmid p-bsiiks bearing the 5S rdna gene, obtained from the fish Leporinus elongatus (MARTINS and GALET- TI 1999, 2000), was also used in FISH experiments. The probes were targeted with byotinilated adenine (BdATP) by nick translation, according to manufacturer s instructions (BIONICK TM Labelling System, Gibco BRL). RESULTS The specimens of H. unitaeniatus from populations A and B presented similar karyotypic formulae, with 44 M/SM + 4 ST chromosomes (2n=48 and FN=96). The specimens from population C presented 44 M/SM + 4ST + 4A chro- Fig. 4 Karyotypes of H. unitaeniatus from population C (Prata river, MG), after conventional Giemsa staining (a) and C-banding (b). In (a) the first chromosome of the acrocentric group shows a large secondary constriction on the short arm.

6 308 DINIZ and BERTOLLO Fig. 5 Metaphases of H. unitaeniatus from populations A (a), B (b, c), and C (d) after FISH with 18S rdna probe. The arrows indicate the ribosomal sites. Below, on the square, chromosomes showing the Ag-NORs and FISH sites from the populations A (e - f), population B (g h i) and population C (j - k).

7 KARYOTYPIC STUDIES ON HOPLERYTHRINUS UNITAENIATUS 309 mosomes (2n=52 and FN=100). Males and females did not evidenced any chromosomal differentiation among sexes (Figs. 2-4). All populations showed a similar pattern of constitutive heterochromatin distribution, along on the pericentromeric region of most of chromosomes, besides on the telomeric and interstitial regions of some others. The C-banding pattern was also informative enough to improve chromosomal pairing (Figs. 2b-4b). Multiple Ag-NORs were observed in the populations A, B and C. In A and B it was verified a variation ranging from two to four Ag-NORs, located on two M/SM chromosomal pairs at interstitial region. From four to six Ag-NORs were observed in the population C (usually four), located on the short arm of one ST plus one acrocentric chromosome pair, besides an occasional fourth M/SM chromosome bearing an interstitial NOR (Fig. 5). FISH with 18S rdna probe confirmed the results obtained for Ag-NORs, but evidenced some other nucleolar organizing regions not detected by AgNO 3 staining (Fig. 5). Thus, four chromosomes were labelled in the populations A and B, all in agreement with Ag-NORs. However, in the population B, two additional signals were occasionally detected on the telomeric region of two non homologous chromosomes. In the population C, six chromosomes were also positively signed corresponding to the Ag-NORs sites. Fig. 6 Metaphases of H. unitaeniatus from population A (a), B (b) and C (c, d) after FISH with 5S rdna probe. The arrows indicate the ribosomal sites.

8 310 DINIZ and BERTOLLO The 5S rdna sites were located on the pericentromeric regions of M/SM chromosomes in the three populations, but displaying numerical variations. Three signals were observed in the population A, four in the population B, and four to five in the population C (Fig. 6). DISCUSSION Cytogenetical studies carried out on H. unitaeniatus have demonstrated karyotypical diversity among some populations. In the present work, populations showing karyotypical divergences in relation to others previously analyzed (GIULIANO-CAETANO et al. 2001) were identified. Although the populations A and B presented 2n=48 and FN=96, similar to that found among the populations from Manaus (a), Aripuanã, Porto Velho, Miranda, Corrientes and Posadas, they karyotypic formulas were slightly different, i.e., 44M/SM+4ST, whereas the previously cited ones presented 48M/SM (Table 1). Besides, ST chromosomes were not reported for other H. unitaeniatus until the moment. In addition, the specimens from population C presented numerical and structural differences (44M/SM+4ST+4A; FN=100) if compared to the other populations. Even the population from Parque Estadual do Rio Doce, which shows the same diploid number, differs from population C regarding the chromosomal types present in the karyotype (46M/SM+6A; FN=98) (Table 1). Although some influence of the chromosomal measurements and classification methods concerning the karyotypic formulas can not be ruled out, the chromosome divergence among populations of H. unitaeniatus is a real feature. A larger amount of constitutive heterochromatin is observed in H. unitaeniatus than in other Erythrinidae species (Figs. 2b-4b). Its distribution on the chromosomes permits a better identification of several homologous pairs; however, it was not a differential character among the populations, which show a similar C-banding pattern. NORs in fish karyotypes can occur on a single or in several chromosome pairs (FORESTI et al. 1981; GALETTI 1998). Regarding this character, the Erythrinidae fish present some peculiarities, as bitelomeric NORs in several Hoplias malabaricus populations (BERTOLLO 1996). The species of this family, including H. unitaeniatus, are characterized by multiple NORs, involving up to 10 chromosomes, as in Hoplias malabaricus. A probable exception of this scenario is represented by the H. lacerdae group, which usually shows a single Ag- NOR pair (BERTOLLO 1996). In addition, a NORbearing X chromosome was also observed in a H. malabaricus population with an XX / XY sex chromosome system (BORN and BERTOLLO 2000), representing an uncommon feature among fish species (REED and PHILLIPIS 1997; ARTONI et al. 2001; ARTONI and BERTOLLO 2002). Telomeric NORs is the predominant situation in the Erythrinidae (BERTOLLO 1996), although they can also be located on interstitial regions of the chromosomes, as usually occur in Hoplerythrinus genus (BERTOLLO 1996; GIULIANO-CAETANO et al. 2001; present study). In general, the NORs bearing chromosomes are of M/SM type in the Erythrinidae, excepting Erythrinus erythrinus where they are predominantly acrocentric (BERTOLLO 1996). Both situations are found among the H. unitaeniatus populations. Thus, in this species, the NORs are located on the M/SM chromosomes and also on the ST/A ones, both at interstitial and telomeric positions (Fig. 5). FISH with 18S rdna probe, besides to confirm the Ag-NORs locations, also showed size differences among rdna sites as well as non homologous sites (Fig. 5). Unequal exchanges, regional duplications/deletions and transpositions are some events that can explain such occurrences. These events appear to be facilitated in redundant DNA classes, as the rdna, leading to a discrepancy in the number of ribosomal cistrons throughout the genome (RITOSSA et al. 1986; SMITH 1976; RUIZ 1982). Indeed, size variation between rdna cistrons has been detected as a widespread feature among fish species, including those with divergent as well as conserved karyotypes (MANTOVANI et al. 2000; WASKO and GALETTI 2000). The 5S rdna occurs on the pericentromeric region of M/SM chromosomes (Fig. 6), showing numerical variation among the populations (3-4 sites in the populations A-B and 4-5 sites in the population C). The small size showed by these sites is a factor that impairs its analysis in H. unitaeniatus, and probably related to the variation in the number of the signals detected on the chromosomes. The interstitial location of the 5S rdna sites is seen in several fish, mammal and amphibian species (VITELLI et al. 1982; LUCCHI- NI et al. 1993; MELLINK et al. 1996; FREDERIKSEN et al. 1997; MARTINS and GALETTI 2001), and

9 KARYOTYPIC STUDIES ON HOPLERYTHRINUS UNITAENIATUS 311 might not be a random event, presenting some advantages in the genomic organization of the vertebrates (MARTINS and GALETTI 2001). 45S (18S + 5.8S + 28S) and 5S rdnas can assume a syntenic distribution on chromosomes of some fishes (PENDÁS et al. 1994; MÓRAN et al. 1996; HATANAKA 2001). Otherwise, they can be addressed to distinct chromosomal pairs, which seems to be the usual situation in these animals (MARTINS and GALETTI 1999, 2000, 2001), and agrees with the results of the present study. Summarizing, the populations A and B, now analyzed, differ from the population C for several karyotypical features. Thus, besides the chromosomal number, these populations present a well differentiated karyotype structure, including acrocentric chromosomes that are exclusive to the population C. Furthermore, the number and location of Ag-NORs are also differentiated among both population groups, what was confirmed by FISH using 18S rdna probe, revealing that the differences are not just related to the activity of ribosomal cistrons. Besides, the 5S rdna sites are also distinct among them, occurring at an increased number in the population C. The available data (Table 1) show that H. unitaeniatus is a karyotypic diversified species. Indeed, chromosomal rearrangements have occurred during the evolution of this fish group, leading to distinct karyotypic structures, with an increase/decrease of the chromosome number. Populations with 2n=48 chromosomes are the most frequent ones and widely distributed (Table 1; Fig. 1). On the other hand, populations with 2n=52 chromosomes have a restrict distribution, by far observed in few localities. One of such localities is the Parque Estadual do Vale do Rio Doce, which comprises several isolated lakes, belonging to the Eastern basin, and the other is the Prata river, belonging to the São Francisco river basin. Although nowadays independent, possible communications might have occurred between these two hydrographic basins in the past. According to BIZERRIL (1994), the Paraná and the São Francisco river systems have contributed on the fish fauna composition from Eastern Brazil. In fact, it is interesting to point out the occurrence of the rarest H. unitaeniatus karyotype, 2n=52, with a similar karyotypic structure, in the Eastern and São Francisco river basins. Accordingly, another specimen from the Eastern basin (São João river, Casimiro de Abreu, Rio de Janeiro state), also showed a similar karyotype (GIULIANO-CAETANO 1986). These data agree with the previous proposition (BIZERRIL 1994), about the ichthyofauna relationships concerning these basins. The distribution of populations with 2n=48 chromosomes is quite widespread (Table 1, Fig.1), suggesting a putative ancestor condition in this group (GIULIANO-CAETANO et al. 2001). However, reduced chromosomal numbers seem to indicate a derived condition amongst Erythrinidae, as in some populations of Hoplias (BERTOLLO et al. 2000) and Erythrinus (BERTOLLO et al. in prep.). It is possible that a similar condition is also present in Hoplerythrinus, where the karyotypes with 2n=52 chromosomes, bearing a higher number of Ag-NORs, 18S and 5S rdna sites, may represent a more ancestral condition within this group. Actually, karyotypes with 2n=52 chromosomes are also detected in the genus Erythrinus, demonstrating their distribution amongst Erythrinidae. Nevertheless, the correct pathway of the chromosomal evolution in this fish group still remains an open question. The population of H. unitaeniatus from Manaus region (AM) showed a chromosomal polymorphism (Table 1), probably due to pericentric inversions, altering the morphology of some pairs in the karyotype, but conserving the diploid number 2n=48 (GIULIANO-CAETANO and BERTOLLO 1988). In the population from the Prata river (São Francisco basin), a karyotypic variation was also verified, with individuals bearing 2n=50 and 2n=51 chromosomes (data not showed). However, these variations are followed by modifications on the morphology of several chromosomes, as well by the occurrence of non homologous chromosomes, which impair their interpretation. Thus, these data are going to be submitted to further analyses, which are necessary to confirm the results and to understand such events. The present study improves the data on the karyotypic divergence in H. unitaeniatus, and the analyses of its biodiversity. It reinforces the proposition that Hoplerythrinus may not correspond to a monotypic genus (GIULIANO-CAE- TANO et al. 2001), and, if so, corresponding to a species complex, as also supposed for the Hoplias malabaricus group (BERTOLLO et al. 2000) and Erythrinus erythrinus (BERTOLLO et al. in prep.). Thus, it is evident the need of a taxonomic revision in the Erythrinidae family on the whole.

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