Research Article |
Corresponding author: Filipe Schitini Salgado ( filipesalgado126@hotmail.com ) Corresponding author: Marina Souza Cunha ( marina.souza.cunha@gmail.com ) Academic editor: Artem Lisachov
© 2021 Filipe Schitini Salgado, Marina Souza Cunha, Silvana Melo, Jorge Abdala Dergam.
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:
Salgado FS, Cunha MS, Melo S, Dergam JA (2021) Cytogenetic analysis of Hypomasticus copelandii and H. steindachneri: relevance of cytotaxonomic markers in the Anostomidae family (Characiformes). Comparative Cytogenetics 15(1): 65-76. https://doi.org/10.3897/compcytogen.v15.i1.61957
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Recent phylogenetic hypotheses within Anostomidae, based on morphological and molecular data, resulted in the description of new genera (Megaleporinus Ramirez, Birindelli et Galetti, 2017) and the synonymization of others, such as the reallocation of Leporinus copelandii Steindachner, 1875 and Leporinus steindachneri Eigenmann, 1907 to Hypomasticus Borodin, 1929. Despite high levels of conservatism of the chromosomal macrostructure in this family, species groups have been corroborated using banding patterns and the presence of different sex chromosome systems. Due to the absence of cytogenetic studies in H. copelandii (Steindachner, 1875) and H. steindachneri (Eigenmann, 1907), the goal of this study was to characterize their karyotypes and investigate the presence/absence of sex chromosome systems using different repetitive DNA probes. Cytogenetic techniques included: Giemsa staining, Ag-NOR banding and FISH using 18S and 5S rDNA probes, as well as microsatellite probes (CA)15 and (GA)15. Both species had 2n = 54, absence of heteromorphic sex chromosomes, one chromosome pair bearing Ag-NOR, 18S and 5S rDNA regions. The (CA)15 and (GA)15 probes marked mainly the subtelomeric regions of all chromosomes and were useful as species-specific chromosomal markers. Our results underline that chromosomal macrostructure is congruent with higher systematic arrangements in Anostomidae, while microsatellite probes are informative about autapomorphic differences between species.
Anastomid, coastal basins, cytogenetics, endemic species, fluorescence in situ hybridization, freshwater fishes, repetitive sequences
Within the order Characiformes, the family Anostomidae encompasses around 150 valid species distributed throughout South America (
Recently, phylogenetic hypotheses based on morphological and molecular data have suggested the creation of the new genus Megaleporinus Ramirez, Birindelli et Galetti, 2017 (
Cytogenetic studies in this group have revealed a conserved karyotype macrostructure of 2n = 54 and fundamental number (NF) = 108 (Table
Cytogenetic data available on the Anostomidae species regarding their chromosome number (2n), karyotype description, presence or absence of sex-chromosome systems, number of chromosomes marked by the Ag-NOR banding technique, and also 18S and 5S rDNA probes.
Species | 2n | Karyotype | Sex-System | Ag-NOR | 18S | 5S | References |
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Abramites hypselonotus | 54 | – | no | – | 2 | – |
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A. solaria | 54 | – | no | 2 | – | – |
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Anostomus ternetzi | 54 | – | no | 2 | – | – |
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Hypomasticus copelandii | 54 | 28m+26sm | no | 2 | 2 | 2 | Present Study |
H. steindachneri | 54 | 30m+24sm | no | 2 | 2 | 2 | Present Study |
Laemolyta taeniata | 54 | 28m+26sm | no | 2 | 2 | 2 † |
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Leporellus vittatus | 54 | 28m+26sm | no | 2 | 2 | 2–4 † |
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Leporinus agassizi | 54 | 28m+26sm | no | 2 | 2 | 2 |
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L. amblyrhyncus | 54 | – | no | 2 | – | – |
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L. fasciatus | 54 | 28m+26sm | no | 2 | 2 | 2 |
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L. friderici | 54 | 28m+26sm/32m+22sm | no | 2 | 2 | 2–4 |
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L. lacustris | 54 | 30m+24sm | no | 2 | 2 | – |
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L. multimaculatus | 54 | 26m+28sm | ZZ/ZW | 2 | – | – |
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L. octofasciatus | 54 | – | no | 2 | – | – |
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L. piau | 54 | – | no | 2 | – | – |
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L. striatus | 54 | – | no | 2 | 2 | – |
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L. taeniatus | 54 | – | no | 2 | – | – |
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Megaleporinus conirostris ‡ | 54 | – | ZZ/ZW | 2 | – | – |
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M. elongatus ‡ | 54 | Z1Z1Z2Z2/Z1W1Z2W2 | 2 | 2 | 4 |
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M. macrocephalus ‡ | 54 | – | ZZ/ZW | – | 2 | – |
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M. obtusidens ‡ | 54 | 26m+28sm/ 28m+26sm | ZZ/ZW | 2 | 2 | 2–4 |
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M. reinhardti ‡ | 54 | 28m+26sm | ZZ/ZW | – | 2 | 2 |
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M. trifasciatus ‡ | 54 | 26m+28sm | ZZ/ZW | 2–3 | 6 § | 2 † |
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Pseudanos trimaculatus | 54 | – | no | 2 | – | – |
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Rhytiodus microlepis | 54 | 28m+26sm | no | 2 | 4 § | 2 |
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Schizodon altoparanae | 54 | – | no | 2 | – | 4 |
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S. borellii | 54 | – | no | 2 | 2 | 4 |
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S. fasciatus | 54 | 28m+26sm | no | 2 | 22 § | 2 † |
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S. intermedius | 54 | – | no | 2 | – | – |
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S. isognathus | 54 | – | no | 2 | 2 | 4 |
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S. knerii | 54 | – | no | 2 | – | 4 |
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S. nasutus | 54 | – | no | 2 | – | 4 |
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S. vittatus | 54 | – | no | 2 | – | 4 |
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Hypomasticus copelandii was collected from Glória (Paraíba do Sul River Basin), Itabapoana (Itabapoana River Basin), Matipó (Doce River Basin) and Mucuri (Mucuri River Basin) rivers, covering its full range of distribution in southeastern Brazil. Hypomasticus steindachneri was collected from Tiririca Lake (Doce River Basin) (Table
Locales and sample size of Hypomasticus copelandii and Hypomasticus steindachneri from southeastern Brazil.
Species | Voucher | Locality | GPS coordinates | Sample size |
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Male/Female | ||||
Hypomasticus copelandii | MZUFV4500 MZUFV 4504 | Glória River, Paraíba do Sul River Basin | 21°05'21"S, 42°20'30"W | 01/02 |
MZUFV4503 MZUFV 4504 | Itabapoana River, Itabapoana River Basin | 20°59'26"S, 41°42'56"W | 02/02 | |
MZUFV4502 | Matipó River, Doce River Basin | 20°06'59"S, 42°24'14"W | 04/04 | |
MZUFV4354 | Mucuri River, Mucuri River Basin | 17°42'21"S, 40°45'42"W | 0/1 | |
Hypomasticus steindachneri | MZUFV3596 MZUFV3607 MZUFV3635 MZUFV4658 | Tiririca Lake, Doce River Basin | 19°18'51"S, 42°24'13"W | 4/4 |
The specimens were anesthetized with clove oil 300 mg.L-1 (
Digital images were captured in a BX53F Olympus microscope equipped with DP73 and MX10 Olympus camera for classical and molecular techniques respectively, both using the CellSens imaging software. Chromosomes were measured with the Image-Pro Plus software and classified according to their size and arm ratios as metacentric (m) or submetacentric (sm) (
Our results showed 2n = 54 in all H. copelandii populations, karyotype of 28m + 26sm and NF = 108, no heteromorphic sex chromosomes were detected, and Ag-NOR was located at the terminal region of chromosome pair 4 (Fig.
Giemsa-stained karyotypes of Hypomasticus copelandii and Hypomasticus steindachneri. Ag-NORs are shown in the boxes. Scale bar: 10 μm.
The microsatellite (CA)15 was detected in both arms of all chromosomes in H. copelandii, whereas microsatellite (GA)15 showed the same pattern with the exception of submetacentric pair 18 that showed signals in the interstitial region of the short arm (Fig.
The conserved Anostomidae karyotype macrostructure is observed in both H. copelandii and H. steindachneri, i.e. 2n = 54 and NF = 108, with some differences in the karyotypic formula regarding the number of metacentric and submetacentric chromosomes (Table
Phylogenetic tree of the Anostomidae family adapted from
Although Ag-NOR number is conserved for most anastomid species with only two markings (Table
In Anostomidae, 5S rDNA variation is restricted to two or four markings and, interestingly, with intraspecific variation among populations in a few species (Table
Microsatellite (CA)15 and (GA)15 probes marked the terminal region of both arms in most of the chromosomes in both species, a pattern that is observed in the autosomes of species with sex chromosome systems, whereas the heteromorphic sex chromosomes have specific accumulation patterns of distinct repetitive DNA classes (
The authors thank “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)”, “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),” and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” for the financial support. The authors would also like to thank Raul Silveira from VERT Ambiental for field support in the Paraíba do Sul and Itabapoana rivers.