Research Article |
Corresponding author: Vladimir Margarido ( vladimir.margarido@unioeste.br ) Academic editor: Inna Kuznetsova
© 2015 Leonardo Paiz, Lucas Baumgärtner, Weferson Júnio da Graça, Vladimir Margarido.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Paiz LM, Baumgärtner L, da Graça WJ, Margarido VP (2015) Basic cytogenetics and physical mapping of ribosomal genes in four Astyanax species (Characiformes, Characidae) collected in Middle Paraná River, Iguassu National Park: considerations on taxonomy and systematics of the genus. Comparative Cytogenetics 9(1): 54-65. https://doi.org/10.3897/CompCytogen.v9i1.9002
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Karyotypes and chromosomal characteristics of both minor and major rDNAs in four fish species known popularly as “lambaris”, namely Astyanax abramis (Jenyns, 1842), Astyanax asuncionensis Géry, 1972, Astyanax correntinus (Holmberg, 1891) and Astyanax sp. collected from downstream of the Iguassu Falls (Middle Paraná River basin), preservation area of the Iguassu National Park, were analyzed by conventional and molecular protocols. A. abramis had diploid chromosome number 2n=50 (4m+30sm+8st+8a) and single AgNORs (pair 22), A. asuncionensis had 2n=50 (8m+24sm+6st+12a) and single AgNORs (pair 20), Astyanax sp. had 2n=50 (4m+26sm+8st+12a) and single AgNORs (pair 25), and A. correntinus had 2n=36 (12m+16sm+2st+6a) and multiple AgNORs (pairs 12, 15, 16, 17). FISH with 18S rDNA showed a single site for A. abramis, A. asuncionensis and Astyanax sp. and multiple for A. correntinus (14 sites). FISH with 5S rDNA showed single 5S-bearing loci chromosome pair only for A. asuncionensis and multiple for A. abramis (four sites), A. correntinus (five sites) and Astyanax sp. (four sites). Distinct distribution patterns of heterochromatin were observed for karyotypes of all species, with the exception of the first acrocentric chromosome pair characterized by centromeric, interstitial-proximal and telomeric blocks of heterochromatin on the long arm, which may represent homeology between karyotypes of A. abramis and A. asuncionensis. Our study showed species-specific characteristics which can serve in diagnosis and differentiation between A. abramis and A. asuncionensis, considered cryptic species, as well as strengthening the occurrence of a species of Astyanax not yet described taxonomically. In addition, the data obtained from first cytogenetic studies in A. correntinus suggest a high similarity with A. schubarti Britski, 1964, suggesting that these species may belong to the same morphological group and that can be phylogenetically related.
Fish cytogenetics, chromosome banding, rDNA-FISH, karyotype differentiation, rDNA sites multiplication
Characiformes are considered one of the most diversified groups in the world freshwater ichthyofauna, comprising 18 families with 270 genera and more than 1,700 species (
Among the families that comprise Characiformes, four are in the African continent (Alestidae, Citharinidae, Distichodontidae and Hepsetidae) and 14 are in Neotropical regions (Acestrorhynchidae, Anostomidae, Characidae, Chilodontidae, Crenuchidae, Ctenolucidae, Curimatidae, Cynodontidae, Erythrinidae, Gasteropelecidae, Hemiodontidae, Lebiasinidae, Parodontidae and Prochilodontidae) (
Astyanax Baird & Girard, 1854 known popularly as “lambaris”, includes around 140 valid species and probably many not yet discovered and/or awaiting formal description (
Ichthyofaunal researches in river systems of southern Brazil were carried out particularly in systems that comprise the Upper Paraná River basin (
Astyanax comprises interesting species for cytogenetic studies, with different evolutionary models that show from maintenance of a preserved chromosomal condition to derived karyotype characteristics, used as important tools in the differentiation and identification of species (
The physical mapping of genes 5S rDNA and 18S rDNA has also been used to characterize different populations in the species of A. scabripinnis complex (
The aim of the present study was to characterize using the conventional and molecular cytogenetic techniques, the karyotypes and chromosomal characteristics of rDNA in the species A. abramis, A. asuncionensis, Astyanax sp. and A. correntinus, collected downstream from the Iguassu River Falls (middle Parana River), to contribute to the taxonomy of one of the major component of Neotropical Characidae fish diversity.
The specimens analyzed were deposited in Coleção Ictiológica do Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura - (NUP), Universidade Estadual de Maringá: nine specimens of A. abramis (four males and five females, NUP 14581), 25 specimens of A. asuncionensis (13 males and 12 females, NUP 14584), 25 specimens of A. correntinus (11 males and 14 females, NUP 14582) and one specimen of Astyanax sp. (female, NUP 14583), in the Iguassu River, in the stretch with around 25 km between downstream of the Iguassu Falls and its mouth on the Paraná River, Middle Parana River basin, located in the preservation area of the Iguassu National Park (25°38'18.72"S; 54°28'4.74"W).
All the specimens were anesthetized and sacrificed by an overdose of clove oil (
The AgNORs were revealed by silver impregnation according to
The physical mapping of 5S rDNA and 18S rDNA loci was carried out by fluorescence in situ hybridization (FISH) according to
The metaphases were photographed using a BX 61 epifluorescence microscope, coupled with Olympus DP 71 digital camera with the Olympus DP Controller software 3.2.1.276. The chromosomes were classified and organized in accordance with
The 2n was 50 chromosomes (4m+30sm+8st+8a, FN=92) for males and females (Fig.
Karyotypes arranged from Giemsa-stained chromosomes of: a A. abramis c A. asuncionensis e A. correntinus g A. sp.; and from C-banded chromosomes of: b A. abramis d A. asuncionensis f A. correntinus h Astyanax sp. The AgNORs bearing chromosomes are framed. Bar = 10 µm.
The 2n was 50 chromosomes (8m+24sm+6st+12a, FN=88) for males and females (Fig.
The 2n was 36 chromosomes (4m+26sm+8st+12a, FN=66) for males and females (Fig.
The 2n was 50 chromosomes (12m+16sm+2st+6a, FN=88) (Fig.
Table
Summary of the cytogenetical data for the Astyanax species analyzed in the present study.
Species | A. abramis | A. asuncionensis | A. correntinus | Astyanax sp. |
---|---|---|---|---|
2n | 50 | 50 | 36 | 50 |
Karyotype formula | 4m+30sm+8st+8a | 8m+24sm+6st+12a | 4m+26sm+8st+12a | 12m+16sm+2st+6a |
AgNORs | Single: - pair 22, a, tel, p arm |
Single: - pair 20, a, tel, p arm |
Multiple: - pair 12, sm, tel, p arm - pair 15, st, tel, q arm - pair 16, a, tel, q arm - pair 17, a, bitel |
Single: - pair 25, a, tel, p arm |
Heterochromatin
(C-banding) |
Centromeric, pericentromeric and telomeric | Centromeric, pericentromeric and telomeric | Centromeric and telomeric | Pericentromeric and telomeric |
18S rDNA | Single: - pair 22, a, tel, p arm |
Single: - pair 20, a, tel, p arm |
Multiple: - pair 4, m, tel, q arm - pair 9, sm, tel, q arm - pair 12, sm, tel, p arm - pair 15, st, tel, q arm - pair 16, a, tel/bitel - pair 17, a, tel/bitel - pair 18, a, tel, p arm |
Single: - pair 25, a, tel, p arm |
5S rDNA | Single: - pair 7, sm, cent |
Single: - pair 9, sm, cent |
Multiple: - pair 2, m, cent - pair 4, m, cent - pair 12, sm, cent |
Multiple: - pair 4, sm, cent - pair 21, a, tel |
Although the present study revealed the same diploid chromosome number (2n= 50) for A. abramis, A. asuncionensis and Astyanax sp., with karyotypes dominated by bi-armed chromosomes, karyotypes differed among these three species, and can be used as a species-specific cytogenetic profile (Fig.
The number and position of NORs (Ag-impregnation and 18S rDNA-FISH), i.e. NOR phenotypes, observed in karyotypes of Astyanax species under study were conserved for the three species with 2n=50, with presence of a single site always located on the p arm in terminal position of a chromosome pair in A. abramis, A. asuncionensis and Astyanax sp. (Fig.
As to the 5S rDNA-FISH, simple sites were observed in karyotype of A. asuncionensis located in centromeric position (Fig.
With regards to the distribution pattern of heterochromatin, although low amount in A. abramis, A. asuncionensis and Astyanax sp., it was found mainly in centromeric and interstitial-proximal position, in addition to NORs associated (Fig.
The present study shows species-specific cytogenetic markers which can serve in diagnosis and differentiation between A. abramis and A. asuncionensis, considered cryptic species (deep body; presence of a well defined, black, horizontal humeral spot; absence of maxillary tooth; and the presence of circuli in posterior field of scales), as well as strengthening the occurrence of a species of Astyanax not yet described taxonomically (elongated body; absence of a well defined, black, horizontal humeral spot; presence of one maxillary tooth; and the absence of circuli in posterior field of scales). In addition, the data obtained from first cytogenetic studies in A. correntinus suggest a high similarity with A. schubarti, suggesting that these species may belong to the same morphological group (deep body; absence of a well defined, black, horizontal humeral spot; presence of one maxillary tooth; and the absence of circuli in posterior field of scales; broad silvery lateral band) and that can be phylogenetically related. Further studies, including another species of the “clade Astyanax” and molecular analyses of mitochondrial genes sequences, may confirm these hypotheses.
The authors are grateful to Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for authorizing the capture of the fishes (License number: SISBIO 10522-1). The authors thank to Universidade Estadual do Oeste do Paraná (UNIOESTE), Parque Nacional do Iguaçu, Macuco Safari, Grupo de Pesquisa em Tecnologia de Produção e Conservação de Recursos Pesqueiros e Hídricos (GETECH) and to the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (NUPÉLIA) for logistical support. This study was financed by CAPES (Coordenadoria de Aperfeiçoamento de Ensino Superior), Fundação Araucária (Fundação Araucária de Apoio e Desenvolvimento Científico e Tecnológico do Estado do Paraná) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).