Research Article |
Corresponding author: Marco Antônio A. Peixoto ( marco.peixotom@gmail.com ) Academic editor: Natalia Golub
© 2016 Marco Antônio A. Peixoto, Marina P.C. Oliveira, Renato N. Feio, Jorge A. 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:
Peixoto MAA, Oliveira MPC, Feio RN, Dergam JA (2016) Karyological study of Ololygon tripui (Lourenço, Nascimento and Pires, 2009), (Anura, Hylidae) with comments on chromosomal traits among populations. Comparative Cytogenetics 10(4): 505-516. https://doi.org/10.3897/CompCytogen.v10i4.9176
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To increase the number of cytogenetic characters used in Ololygon tripui systematics, we applied some cytogenetic techniques such as Giemsa, C- and NOR-banding, and fluorescence
Cytotaxonomy, population cytogenetics, Ag-NOR, heterochromatic blocks, 18S rDNA, FISH, microsatellite DNA probes
The genus Ololygon Fitzinger, 1843 belongs to Hylidae and currently includes 46 species (
In the genus Ololygon, the NOR is observed on chromosome pair 6, with exception of O. canastrensis (Cardozo and Haddad, 1982), where NORs occur on chromosome pair 6 and 11 (
Either as dispersed elements or repetitive elements organized in tandem, the repetitive DNA represents a large portion of the eukaryotic genome, and includes satellite DNA, microsatellites, minisatellites, telomeric sequences, multigene family, and transposable elements (
Relatively a few systematic data are available for the taxonomically complex and diverse genus Ololygon. Thus, cytogenetic data are potentially informative for understanding the phylogenetic relationships of the species within the genus (
As a total, 32 specimens of O. tripui were collected from four populations from the state of Minas Gerais, Brazil: Estação Ecológica do Tripuí, Ouro Preto municipality (Tripuí – Type locality), Parque Estadual Serra do Brigadeiro, Ervália municipality (PESB), RPPN Mata do Sossego, Simonésia municipality (Sossego), and Usina da Fumaça, Muriaé municipality (Fumaça) (Table
Mitotic chromosomes were obtained from gut epithelial cells according to
Sample sizes (N) per gender, sample locality and voucher identification of Ololygon tripui populations.
Population | Sample locality | N | Gender | Voucher (MZUFV) |
---|---|---|---|---|
PESB | Parque Estadual Serra do Brigadeiro, Ervália – MG (20°51'52"S, 042°31'17"W) | 9 | Male | 9865, 9870, 9871, 9872, 10294, 10299-301, 11421 |
3 | Female | 11511, 12103-104 | ||
Sossego | RPPN Mata do Sossego, Simonésia – MG (20°04'22"S, 042°04'12"W) | 6 | Male | 11441-443, 12571, 12575-576 |
2 | Female | 12573, 12577 | ||
Fumaça | Usina da Fumaça, Muriaé – MG (21°00'58"S, 042°26'36"W) | 6 | Male | 11444-449 |
3 | Female | 11438-440 | ||
Tripuí | Estação Ecológica do Tripuí, Ouro Preto – MG (20°23'22"S, 043°32'20"W) | 3 | Male | 12447-449 |
To identify heterochromatic regions, the C-banding technique followed
All the individuals analyzed had a diploid complement of 24 chromosomes. All chromosomes were biarmed and their FN was 48 (Suppl. material
a–c. Karyotype of Ololygon tripui from the Fumaça locality. a Giemsa and Ag-NOR staining of male chromosomes b Giemsa and Ag-NOR staining of female chromosomes c C-banding and 18S rDNA markers on chromosome pair number 6. Bar = 10 µm.
a–c. Karyotype of Ololygon tripui from PESB locality. a Giemsa and Ag-NOR staining of male chromosomes b Giemsa and Ag-NOR staining of female chromosomes c 18S rDNA sites on chromosome pair number 6. Bar = 10 µm.
a–c. Karyotype of Ololygon tripui from the Sossego locality. a Giemsa and Ag-NOR staining of male chromosomes b Giemsa and Ag-NOR staining of female chromosomes c 18S rDNA markers on chromosome pair number 6. Bar = 10 µm.
Karyotype of Ololygon tripui from the Tripuí locality. Giemsa, Ag-NOR staining, and 18S rDNA markers on multiple chromosomes of a male specimen. Bar = 10 µm.
Comparative morphology and measurements of chromosome pairs in Ololygon tripui populations. m = metacentric, sm = submetacentric, st = subtelocentric, CR = centromeric ratio, CT = chromosome type.
Population | Gender | Chromosome pairs | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |||
PESB | Female | CR | 2.41 | 2.53 | 2 | 3.38 | 2.46 | 3.25 | 4.55 | 2.4 | 1.25 | 1.11 | 1.23 | 1.19 |
CT | sm | sm | sm | st | sm | st | st | sm | m | m | m | m | ||
Male | CR | 2.11 | 2.32 | 2.3 | 3.57 | 1.99 | 3.4 | 3.19 | 2.31 | 1.1 | 1.1 | 1.1 | 1.17 | |
CT | sm | sm | sm | st | sm | st | st | sm | m | m | m | m | ||
Sossego | Female | CR | 2.38 | 2.28 | 2.41 | 3.91 | 2.04 | 3.96 | 3.63 | 2.17 | 1.49 | 1.35 | 1.15 | 1.26 |
CT | sm | sm | sm | st | sm | st | st | sm | m | m | m | m | ||
Male | CR | 2.17 | 2.18 | 2.09 | 3.49 | 2.1 | 3.45 | 3.53 | 2.05 | 1.23 | 1.32 | 1.19 | 1.14 | |
CT | sm | sm | sm | st | sm | st | st | sm | m | m | m | m | ||
Fumaça | Female | CR | 2.58 | 2.62 | 1.97 | 3.68 | 2.56 | 3.66 | 3.53 | 2.13 | 1.45 | 1.33 | 1.28 | 1.16 |
CT | sm | sm | sm | st | sm | st | st | sm | m | m | m | m | ||
Male | CR | 2.3 | 2.42 | 1.8 | 3.46 | 2.06 | 3.57 | 3.95 | 2.31 | 1.31 | 1.33 | 1.2 | 1.28 | |
CT | sm | sm | sm | st | sm | st | st | sm | m | m | m | m | ||
Tripuí | Male | CR | 2.39 | 2.44 | 2.51 | 3.76 | 2.31 | 3.67 | 3.67 | 1.81 | 1.35 | 1.19 | 1.15 | 1.14 |
CT | sm | sm | sm | st | sm | st | st | sm | m | m | m | m |
Heterochromatic blocks were detected in the centromeric and pericentromeric regions of chromosomes, only in the specimens from the Fumaça locality. Heterochromatic patterns were similar in both sexes (Fig.
FISH showed 18S rDNA sites in chromosome pair 6 in males and females from the Fumaça, PESB, and Sossego populations. Multiple tags for 18S rDNA FISH were also observed in a male of the Tripuí population, with markings restricted to one homolog in chromosome pair 3 and 4, and on both homologs of chromosome pair 6. The microsatellites repetitive DNA probe (GA)15 accumulated in the terminal region of all chromosomes (Fig.
a–d. Male mitotic chromosomes of Ololygon tripui from different populations, labeled with the (GA)10 repetitive DNA probe. a Fumaça bPESBc Sossego d Tripuí. Bar = 10 µm.
The diploid number of 2n = 24 and FN = 48 observed in O. tripui was similar to those reported for all studied species of the genus and considered as a highly conserved character, which is shared with other genera within Hylidae (i.e. Scinax, Xenohyla Izecksohn, 1998, and Lysapsus Cope, 1862) (
The polymorphic presence of more than one pair of Ag-NOR cistrons in the Tripuí population of O. tripui have also been reported in other species of this genus (
Markings obtained with 18S rDNA FISH revealed some chromosomal aspects that were not evident using the Ag-NOR protocol. Although high correlation was observed in the Fumaça and Sossego populations between the FISH and NOR banding patterns, in the PESB population this correlation was partial: only one homolog showed Ag-NOR banding, whereas both homologs bore 18S rDNA sites. Notably, in the Tripuí population, 18S rDNA sites were detected in one homolog of chromosome pair 3 and 4 and in both homologs of chromosome pair 6; Ag-NOR-banding was evident on one homolog of chromosome pair 3, and on the chromosome pair 6. Unlike Ag-NORs, FISH exposure of rDNA regions occurs regardless of previous interphase activation of this region, allowing to detect actual levels of polymorphisms between populations or species (
Partial correlation between results obtained by Ag-NOR and FISH techniques can be explained by the action of different mechanisms, such as: i) a limitation of the technique in detecting a rDNA sequences present in low copy number; ii) the mobility of rDNA sequences spread by transposable elements; iii) a real polymorphic condition; iv) the occurrence of chromosome rearrangements such as translocations; v) the physiological amplification of rDNA cistrons (
Chromosome mapping using microsatellite sequences in hylids is scarce, however available data suggest that this kind of sequences accumulate in the vicinity of centromeres and telomeres (
Our results indicate that despite O. tripui populations were conservative in most of their cytogenetic characters such as diploid and fundamental number, Ag-NOR, repetitive DNA, and 18S rDNA patterns in relation to Ololygon sp., O. arduoa, O. belloni, and O. cosenzai, the former species differs in its chromosome morphology and in the repetitive DNA pattern, that probably occurs due to independent evolution of the species, thus corroborating its taxonomic status.
We concluded that although repetitive DNA patterns of variation are largely unknown in anurans, the cytogenetic mapping of different repetitive DNA sequences provided reliable chromosomal markers, revealing species-specific differences, when compared with other species of the genus. Our study showed initial insights on the use of repetitive DNA probes to discuss evolutionary issues.
We are grateful to friends from the Museu de Zoologia João Moojen (MZUFV) and the Laboratório de Sistemática Molecular (BEAGLE) for their assistance in the data collection, especially to J.V. Lacerda, M. Moura, C. Coelho, P. Santos, B. Assis, D. Santana, A. Zaidan, A.P. Silva, and N. Travenzoli. We also thanks to K. L. Souza, by laboratory assistance. This work is a contribution to the project “Biogeografia e Conservação da anurofauna no Complexo Serrano da Mantiqueira, Sudeste do Brasil”, supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Project #068437-2014/06). The authors are grateful to the Coordenação de Aperfeiçoamento de Nível Superior (CAPES), Fundação do Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and the Universidade Federal de Viçosa for their financial support. The Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA), and the Instituto Estadual de Florestas issued the collecting permits (IBAMA / ICMBIO #20672, IEF #040/11, and IEF #041/11). Financial support was given to RNF through a scientific productivity fellowship by CNPq.
Data on chromosomal and fundamental number, chromosomal formulae, C and NOR banding, 18S, and sample locality to all species from the genus Ololygon with some karyotypic study
Data type: molecular data