Research Article |
Corresponding author: Fábio Porto-Foresti ( fp.foresti@unesp.br ) Academic editor: Inna Kuznetsova
© 2020 Caio Augusto Gomes Goes, Sandro Natal Daniel, Lucas Henrique Piva, George Shigueki Yasui, Roberto Ferreira Artoni, Diogo Teruo Hashimoto, Fausto Foresti, Fábio Porto-Foresti.
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:
Goes GAG, Daniel SN, Piva LH, Yasui GS, Artoni RF, Hashimoto DT, Foresti F, Porto-Foresti F (2020) Cytogenetic markers as a tool for characterization of hybrids of Astyanax Baird & Girard, 1854 and Hyphessobrycon Eigenmann, 1907. Comparative Cytogenetics 14(2): 231-242. https://doi.org/10.3897/CompCytogen.v14i2.49513
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Astyanax Baird et Girard, 1854, is one of the largest genera in the family Characidae and comprises 177 valid species. This genus has been the focus of cytogenetic studies primarily owing to the presence of B chromosomes and high karyotypic diversity among different populations. The intense genetic variability in Astyanax is one of the factors responsible for the occurrence of species complexes, which are groups (1) with certain difficulties in establishing common genetic pools or (2) belonging to different cryptic species. To evaluate cytogenetic marker inheritance and the possibility of the identification of these hybrids, this study aimed to describe cytogenetic hybrids from three strains of species of the genera Astyanax and Hyphessobrycon Eigenmann, 1908. A. lacustris Lütken, 1875, A. schubarti Britski, 1964, A. fasciatus Cuvier, 1819, and H. anisitsi Eigenmann, 1907 were used to generate three hybrid lineages. The diploid number, heterochromatin sites, and ribosomal genes (18S and 5S rDNA) of the parental strains and the hybrids were analyzed. The results indicated that the three hybrid lineages had cytogenetic markers of both parents, presenting Mendelian inheritance. However, differences in distribution of heterochromatic blocks were observed between the hybrids and the parent strains. Our results allowed the identification of the hybrid strains based on the cytogenetic markers applied, reinforcing the efficiency of cytogenetic markers as tools for identification and indicating that such events may increase the karyotypic diversity in the genera Astyanax and Hyphessobrycon.
neotropical fishes, B chromosomes, chromosome polymorphism, repetitive DNAs, species complex
Interspecific hybridization is the union of distinct genetic pools, the progenies of which are usually individuals posessing intermediate taxonomic characteristics of both parental species (
Astyanax Baird et Girard, 1854, belonging to the family Characidae, is one of the most species-rich genus and currently comprises 177 valid species (
There has been a report of interspecific hybridization among Astyanax species in the nature (
The parent strains used in this study were obtained from the Instituto Chico Mendes de Conservação da Biodiversidade (CEPTA –
The hybrids were identified and deposited in the Laboratório de Genética de Peixes, Bauru, São Paulo, Brazil, under the accession numbers LGP8291–LGP8382. Fifty-nine animals were anesthetized using 1% benzocaine. Mitosis stimulation was performed using the method described by
5S (two different bands: 255 and 525 bp) and 18S (one band: 600 pb) rDNA probes were obtained using polymerase chain reaction with the primers 5S A (5'-TCAACCAACCACAAAGACATTGGCAC-3') and 5S B (5'-TAGACTTCTGGGTGGCCAAAGGAATCA-3') for the 5S gene (
All parent strains displayed stable diploid chromosome numbers; A. lacustris displayed 2n = 50 (6m+12sm+14st+18a) chromosomes; A. fasciatus, 2n = 48 (10m+12sm+12st+14a); A. schubarti, 2n = 36 (10m+10sm+10st+6a); and H. anisitsi, 2n = 50 (10m+2sm+20st+18a) (Fig.
Karyotypes of the parental individuals analyzed: Astyanax lacustris (3m+6sm+7st+9a), Hyphessobrycon anisitsi (5m+1sm+10st+9a), A. fasciatus (5m+6sm+6st+7a), and A. schubarti (5m+5sm+5st+3a). Scale bar: 5 µm.
Karyotypes of three hybrids of species of the genus Astyanax: A. lacustris × Hyphessobrycon anisitsi (8m+7sm+17st+18a), A. lacustris × A. fasciatus (8m+7sm+17st+18a), and A. lacustris × A. schubarti (8m+11sm+12st+12a). Scale bar: 5 µm.
The results of C-positive heterochromatin revealed some interesting features. Astyanax lacustris and A. schubarti hybrids showed regular heterochromatic blocks inherited from both parent strains. The terminal heterochromatic blocks in subtelocentric/acrocentric chromosomes of A. fasciatus and the typical location of As51 satellite DNA were not detected in the hybrids (Figure
Heterochromatic markers obtained by C-banding on metaphase plates of Astyanax lacustris (a), A. fasciatus (b), and A. schubarti (c), and Hyphessobrycon anisitsi (d) and hybrids A. lacustris × A. fasciatus (e), A. lacustris × A. schubarti (f), and A. lacustris × H. anisitsi (g, h) after C-banding. The arrows indicate heterochromatic markers. In h, a metaphase with 51 chromosomes, the chromosome being completely heterochromatic, can be observed. Scale bar: 5µm.
The ribosomal sites showed Mendelian inheritance, as revealed in Figure
Fluorescence in situ hybridization with the probes DNAr 5S (green) and 18S (red). The results are labeled as: Astyanax lacustris (a, d, g), A. schubarti (b), hybrid A. lacustris × A. schubarti (c), A. fasciatus (e), hybrid A. lacustris × A. fasciatus (f), Hyphessobrycon anisitsi (h), and hybrid A. lacustris × H. anisitsi (i). Arrows and arrowheads indicate chromosomes bearing 18S and 5S rDNA clusters: arrows, A. lacustris; arrowheads, other species in the cross. Scale bar: 5µm.
The genus Astyanax is rich in chromosomal polymorphisms (
Hybridization between different fish species can generate individuals that diverge from simple diploids with equal parental contribution (
The C-banding patterns revealed interesting features, as conspicuous heterochromatic blocks did not appear to be regularly inherited in some cases, indicating some degree of chromatin remodeling, similar to that in plant and mammal hybrids (
Fertile hybrids have been described for different Neotropical fish species such as hybrids of the catfishes “cachapinta” and “pintachara,” Pseudoplatystoma corruscans (Spix et Agassiz, 1829) and P. reticulatum Eigenmann et Eigenmann, 1889, (
The cytogenetic markers applied to the hybrid strains analyzed in this study were efficient in terms of identification based on the known karyotype of the parent strains, which differentiates the hybrids involving species of the genus Astyanax from other hybrids of Neotropical fish, which keeps its cytotypes conserved (
The authors thank the Brazilian funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: Processo n° 2015/12902-4), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing financial support. We would like to thank Editage (www.editage.com) for English language editing. This work was supported by the Fundação de Amparo à Pesquisa (FAPESP) under Grant (no. 2015/12902-4)