Cytotaxonomy, heterochromatic polymorphism and natural triploidy of a species of Astyanax (Pisces, Characidae) endemic to the Iguaçu river basin

HUMAN AND ANIMAL HEALTH

Cytotaxonomy, heterochromatic polymorphism and natural triploidy of a species of Astyanax (Pisces, Characidae) endemic to the Iguaçu river basin

Daniel Luis Zanella KantekI, ^*; Rafael Bueno Noleto^I; Alberto Sérgio Fenocchio^II; Marta Margarete Cestari^I

^ILaboratório de Citogenética Animal; Universidade Federal do Paraná; [e-mail]; 81531-990; Curitiba - PR - Brasil

^IILaboratório de Citogenética; Universidad Nacional de Misiones; 3300 Posadas - Misiones - Argentina

ABSTRACT

Cytogenetic analysis with Astyanax sp. D revealed a karyotype of 2n=50 with 2M+26SM+6ST+16A, besides a triploid specimen showing 2n=75 chromosomes (3M+39SM+9ST+24A). C-banding strongly stained the terminal regions of several SM-ST-A chromossomes. Two pairs of acrocentric chromosomes presented interstitial heterochromatin, this state being polymorphic and occuring due to possible paracentric inversions. The results obtained with the AluI restriction enzyme and A₃ chromomycin were similar to the C-banding. Relationships were proposed between Astyanax sp. D and A. scabripinnis, as well as considerations for a possible origin of the triploid specimen (2n=3x=75). When comparing the present results with cytogenetic features of other endemic Astyanax species in the Iguaçu river (A. sp. B and C), a clear differentiation was observed between them, indicating cytogenetics as an important cytotaxonomic tool.

Key words: Cytogenetics; triploidy; paracentric inversion

RESUMO

Análises citogenéticas em Astyanax sp. D evidenciaram 2n=50 cromossomos e um cariótipo com 2M+26SM+6ST+16A. O bandamento C destacou as regiões teloméricas de diversos cromossomos SM-ST-A. Dois pares de cromossomos acrocêntricos possuem heterocromatina intersticial, sendo este estado polimórfico decorrente de prováveis inversões paracêntricas. O resultados obtidos com a enzima de restrição AluI e a Cromomicina A₃ foram semelhantes aos do bandamento C. São propostas relações de parentesco entre Astyanax sp. D e Astyanax scabripinnis, bem como considerações sobre a possível origem do exemplar triplóide (2n=3x=75). Ao comparar os resultados deste trabalho com outras espécies de Astyanax do Rio Iguaçu, estas espécies se tornam claramente distinguíveis, evidenciando a citogenética como uma importante ferramenta taxonômica.

INTRODUCTION

The genus Astyanax has received special attention due to its biological and cytogenetic characteristics, with cases where different populations of the same species demonstrate cytogenetic and morphological differences. This genus shows a broad karyotypic variability, being observed from 2n=36 in A. schubarti (Morelli et al.,1983; Daniel-Silva and Almeida-Toledo, 2001) to 2n=50 in other species of the genus (Souza and Moreira-Filho, 1995). Variations in the diploid number have been detected within single Astyanax species, as is the case of A. fasciatus (Morelli et al., 1983; Justi, 1993) and A. scabripinnis (Moreira-Filho and Bertollo, 1991; Souza and Moreira-Filho, 1995; Néo et al., 2000; Alves and Martins-Santos, 2002).

According to Sampaio (1988), the Iguaçu river has at least six Astyanax species. These species received the denominations of Astyanax sp.A, B, C, D, E, F. Althugh they lack a valid scientific name, these species are considered as real taxonomic entities in the literature concerning the Iguaçu river (Severi and Cordeiro 1994; Agostinho and Gomes 1997; Kantek et al., 2003; Ingenito et al., 2004). Garutti and Britski (2000) described the ocurrence of Astyanax altiparanae in the river. The objective of this study was to cytogenetically characterize Astyanax sp.D from the first plateau of the Iguaçu river (Paraná, Brasil), comparing with other taxa and reporting one more case of natural triploidy.

MATERIAL AND METHODS

Seven Astyanax sp. D specimens (5 males and 2 females) from the first plateau of the Iguaçu river, in the township of Piraquara (PR), were studied. The mitotic metaphases were obtained by the indirect method described by Fenocchio et al. (1991). C-banding was performed as described by Sumner (1972), while chromomycin A₃ (CMA₃) and the restriction endonuclease AluI assays were done according to Schmid (1980) and Mezzanote et al. (1983), respectively.

RESULTS

The studied fish had a modal diploid number of 50 chromosomes, comprising 2 metacentric (M), 26 submetacentric (SM), 6 subtelocentric (ST) and 16 acrocentric (A) chromosomes, adding up to the fundamental number (FN) of 84 (Fig. 1). Heteromorphic sex chromosomes were not detected. The constitutive heterochromatin was detected (Fig. 2A, 3A) on weak little centromeric C⁺ marks in some chromosomes and on large interstitial and terminal blocks located on the long arms of all ST chromosomes, on several A chromosomes and some SM chromosomes. Pairs 18 and 19 showed interindividual C-banding variations in the following situations: (1) both homologues with C⁺ interstitial blocks; (2) one of the chromosomes with the C⁺ terminal block and its homologue with an interstitial mark; (3) both homologues with C⁺ terminal blocks (Fig. 2B). It was possible to observe a size heteromorphism of the heterochromatic blocks between homologues (Fig. 2A-pair 21), as well as heterochromatic marks that were located at a short distance above the telomere (Fig. 2A-pair 5). The number of C-bands was also variable. Figure 2A shows only seven A chromosomes bearing such regions, while on Figure 3A it is possible to visualize ten A chromosomes with C-bands. The AluI⁺ pattern was identical to the C-banding distribution (Fig. 3A,^B).

A triploid specimen was also identified, with 2n=3x=75 chromosomes: 3M+39SM+9ST+24A (Fig. 4A). Its karyotype showed the same features observed for the diploid specimens. C-banding, AluI and CMA₃ data showed that triads 18 and 19 were formed by two identical chromosomes plus a third distinct chromosome (Fig. 4B, 5A,^B).

DISCUSSION

The diploid number of 2n=50 for Astyanax sp. D did not differ from those of most species of this genus, including A. scabripinnis (Moreira-Filho and Bertollo 1991; Maistro et al., 1998; Souza et al., 1996), Astyanax sp.C (Kantek et al., 2003), Astyanax sp.B (Fazoli et al., 2003) and A. altiparanae (Cenci and Margarido, 2003; Kantek et al., 2004). The C-banding pattern could be considered equilocal on the chromosomes. A possible explanation for this pattern would be based on the model proposed by Schweizer and Loidl (1987), which disserted on the orientation of the chromosomes during the meiotic interphase (Rabl orientation), allowing the transference of heterochromatin between equidistant positions of non-homologue chromosomes. The similarity between the C⁺ blocks, also demonstrated by the AluI and CMA₃ techniques, supported this hypothesis.

When comparing patterns of Astyanax. sp.D, some A. scabripinnis populations (Mantovani et al., 2000; Maistro et al., 2001) and A. janeiroensis (Carvalho et al., 2002) showed a similar C-banding pattern. These species presented large terminal heterochromatic blocks located on the long arm of the ST and A chromosomes. The pattern obtained with AluI was also similar for Astyanax sp. D and A. scabripinnis from the Cascatinha river (Maistro et al., 2001). However, other populations differed in C-band distribution (Maistro et al., 1998; Moreira-Filho and Bertolo, 1991), the same occurring with Astyanax sp. B. (Fazoli et al., 2003), Astyanax. sp.C (Kantek et al., 2003) and A. altiparanae (Cenci and Margarido, 2003; Kantek et al., 2004) from the Iguaçu river, which had less heterochromatin than Astyanax. sp. D.

The C-band polymorphism on the 18^th and 19^th chromosome pairs of Astyanax sp.D seemed to be related to paracentric inversions. Such polymorphism was not detected in other Astyanax species thus far. A. janeiroensis (Carvalho et al., 2002) also showed interstitial C⁺ blocks, but without a polymorphic condition.

CMA₃ staining showed that C⁺ regions of Astyanax sp. D were GC-rich, as occured in A. scabripinnis (Souza et al., 1996; Daniel-Silva, 1996). The differences in the amount of heterochromatin between homologues were also registered for A. scabripinnis (Mantovani et al., 2000; Maistro et al., 2000).

The identification key for Astyanax species elaborated by Eigenmann (1921) identified Astyanax sp. D as A. scabripinnis paranae. However, Sampaio (1988), through morphological measurements, considered these two fish forms as distinct species. The current diagnosis for A. scabripinnis (Eigenmann, 1921) was considered to be little specific as it joined many forms of Astyanax that inhabited headwaters, making it not informative for the taxonomy of this group. The high diversity recognized for this taxon would be a reflex of this flaw on its taxonomy. Many populations identified before as A. scabripinnis subspecies could now be considered new species. Future analysis would result in the recognition of other taxa (Bertaco and Malabarba, 2001).

Astyanax, as well as most fishes, exhibited external fertilization, with the oocyte finishing the second mitotic division in the external medium (water), resulting in a non-disjunction of the sister chromatids (II meiosis) in adverse environment conditions (low temperatures, for example), producing one oocyte with 2n chromosomes (Fauaz et al., 1994).

Electrophoretic analysis of hybrids from the crossing of two carp species demonstrated that triploid individuals possesed two maternal alleles and only paternal allele (Tsukomo and Rigolino, 1993), supporting the hypothesis that the natural triploids came through a diploid egg x haploid sperm fertilization.

The identification of two identical chromosomes and one differentiated on the 18^th and 19^th triads of the triploid specimen (Figs. 4, 5) corroborated this hypothesis, which had been suggested for other fish species (Morelli et al., 1983; Almeida-Toledo et al., 1985; Giuliano-Caetano and Bertollo, 1990; Maistro et al., 1994). However, the alternative hypothesis, haploid egg x diploid sperm fertilization, could not be ruled out in the origin of the triploidy.

These results supported the proximity of the A. sp. D and A. scabripinnis groups. However presently, it would not be possible to differentiate between equal or distinct biological units. When confronting Astyanax sp. D with Astyanax sp. C (Kantek et al., 2003), Astyanax sp. B (Fazoli et al., 2003) and Astyanax altiparanae (Cenci and Margarido, 2003; Kantek et al., 2004) from the Iguaçu river, these species were clearly distinguishable by their cytogenetic features, showing the probable lack of gene flow between them as well as the importance of karyotypic data for the taxonomy of this fish group.

ACKNOWLEDGMENTS

We thank Vinicius Abilhoa from the Museu de Historia Natural Capão da Imbuia (Curitiba-PR) for the sample identification and Gil Caveira for translation of this paper. Financial support was provided by CNPq (National Council for Scientific and Technologic Development).

Received: March 11, 2005;

Revised: October 03, 2005;

Accepted: August 31, 2006.

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*

Author for corresponce

Publication Dates

• Publication in this collection
  30 May 2007
• Date of issue
  Jan 2007

History

• Accepted
  31 Aug 2006
• Reviewed
  03 Oct 2005
• Received
  11 Mar 2005