Research Article |
Corresponding author: Daniel Pacheco Bruschi ( danielpachecobruschi@gmail.com ) Academic editor: Larissa Kupriyanova
© 2019 Joana Moura Gama, Camilla Borges Gazolla, Deborah Yasmin de Souza, Shirlei Maria Recco-Pimentel, Daniel Pacheco Bruschi.
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:
Gama JM, Gazolla CB, de Souza DY, Recco-Pimentel SM, Bruschi DP (2019) Recurrent variation in the active NOR sites in the monkey frogs of the genus Pithecopus Cope, 1866 (Phyllomedusidae, Anura). Comparative Cytogenetics 13(4): 325-338. https://doi.org/10.3897/CompCytogen.v13i4.37687
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Treefrogs of the genus Pithecopus Cope, 1866 exhibit expressive chromosomal homogeneity which contrasts with a high variation frequency of the nucleolus organizer region (NOR) related to the group. Currently, the genus contains eleven species and no chromosomal data are available on P. palliatus Peters, 1873, P. ayeaye Lutz, 1966 and P. megacephalus Miranda-Ribeiro, 1926. Here, we describe the karyotypes of these three species based on Giemsa staining, C-banding, silver impregnation and in situ hybridization (FISH). We were also analyze the evolutionary dynamic of the NOR-bearing chromosome in species of genus under a phylogenetic view. The results indicate that P. palliatus, P. ayeaye, and P. megacephalus have similar karyotypes, which are typical of the genus Pithecopus. In P. palliatus the NOR was detected in the pericentromeric region of pair 9p whereas in P. ayeaye and P. megacephalus we report cases of the multiple NOR sites in karyotypes. In P. ayeaye the NOR was detected in the pericentromeric region of pair 9p in both homologues and additional sites was detected in pairs 3q, 4p, and 8q, all confirmed by FISH experiments. Already in P. megacephalus the NOR sites were detected in pericentromeric region homologues of pair 8q and additionally in one chromosome of pair 13q. A comparative overview of all the Pithecopus karyotypes analyzed up to now indicates the recurrence of the NOR-bearing chromosome pairs and the position of the NORs sites on these chromosomes. We hypothesized that this feature is a result of a polymorphic condition present in the common ancestor of Pithecopus. In such case, the lineages derived from polymorphic ancestor have reached fixation independently after divergence of lineages, resulting in a high level of homoplasy observed in this marker. Our findings help to fill the gaps in the understanding of the karyotype of the genus Pithecopus and reinforce the role of the evolutionary dynamics of the rDNA genes in karyotype diversification in this group.
chromosomal evolution, Pithecopus
No published chromosomal data are available on P. palliatus, P. ayeaye, and P. megacephalus. Pithecopus palliatus is a member of the lowland clade (
Pithecopus ayeaye is endemic to high altitudes in southeastern Brazil. This species is listed as critically endangered (CR) by the International Union for Conservation of Nature, IUCN (
Pithecopus megacephalus occurs at high elevations (above 800 m a.s.l.) in the campo rupestre systems of the Southern Espinhaço Mountain Range (
Here, we advance our understanding of the cytogenetics of the genus Pithecopus and compile the karyotype data available on the genus to discuss its chromosomal features from a phylogenetic perspective.
We analyzed populations of P. ayeaye, P. megacephalus and P. palliatus sampled in Brazilian localities (Table
Details of the Pithecopus species and specimens sampled for the cytogenetic analyses presented in this study.
Species | Number of specimens | Locality/State1 | Geographic coordinates | ZUEC 2 number |
---|---|---|---|---|
P. ayeaye | 03 ♂ | Brumadinho/MG | 20°29'S, 44°19'W | 16403–16405 |
P. megacephalus | 03 ♂ | Santana do Riacho/MG | 19°10'S, 43°42'W | In the accept |
P. palliatus | 12 ♂ + 3 ♀ | Boca do Acre/AM | 8°44'S, 67°23'W | 17037–17051 |
Metaphase cells were obtained from the intestines and testes of animals previously treated with 2% colchicine (Sigma – Aldrich; 0.02 ml per 1 g of body weight), following procedures modified from
The nucleolus organizer regions (NOR) were revealed by the silver nitrate impregnation technique (Ag-NOR) following
We analyzed twenty metaphase plates per individual for each of the applied methods. The metaphases were photographed under an Olympus microscope and analyzed using the Image Pro-Plus software, version 4 (Media Cybernetics, Bethesda, MD, USA). The chromosomes were ranked and classified according to the scheme of
All three species analyzed here had a diploid number of 26 chromosomes. The chromosomal complement of all three species (Figs
The heterochromatin revealed by the C-banding was arranged in centromeric blocks in the karyotypes of all three species studied here (Figs
Karyotype of P. ayeaye prepared by conventional Giemsa staining (A) C- banding (B) Ag-NOR (D) DAPI staining after C-banding (C). Chromosomes submitted to Mytramicim (MM) (E) and FISH experiments with a nucleolar 28S rDNA probe (F). The arrow indicates indicates secondary constrictions; the arrowheads indicate multiple NOR site.
In all the karyotypes, the secondary constrictions revealed by conventional Giemsa staining coincided with the NOR sites detected by the Ag-NOR method. In P. ayeaye the NORs were detected in the pericentromeric region of the short arm of the both homologs of pair 9 (Fig.
Karyotype of P. palliatus prepared by conventional Giemsa staining (A) C-banding (B) and Ag-NOR method (C). Secondary constrictions observed coincided with the Ag-NOR sites (C).
The analysis of the chromosomes of the three Pithecopus species, presented here, reinforces the conclusion that the macrostructure of the karyotypes of the members of this genus (diploid number and chromosome morphology) is highly conserved (
Like the other species of the genus Pithecopus (
The extreme chromosomal conservation observed in the Pithecopus species contrasts with its considerable inter- and intrapopulation variation in the chromosomal pairs that carry the 28S rDNA gene clusters. In the present study, two new cases of multiple NOR sites were recorded in the genus Pithecopus, in the karyotypes of P. ayeaye and P. megacephalus. However, a comparative overview of all the Pithecopus karyotypes analyzed up to now indicates the recurrence of the NOR-bearing chromosome pairs, and the position of the NORs on these chromosomes, in particular in pairs 3, 4, 8, 9, 11, and 13. Multiple NORs are common in this genus, and have been recorded in practically all the species (Morando and Hernando 1997,
The interesting feature of the genus Pithecopus is that when the polymorphic condition is recorded in the different species, it to be located in the same chromosomes and NOR positions. Thus, it is difficult to recognize the phylogenetic signal of this marker for the application of a parsimonious evolutionary analysis. Here, we suggest two possible scenarios to explain this variation: (i) the NOR in pair 9q represents the plesiomorphic condition in Pithecopus, with subsequent rearrangements resulting in the repositioning of the NOR to pair 8 in P. azureus and in the ancestor of P. hypochondrialis + P. araguaius, with the NOR in pair 8q also representing an autapomorphy in P. megacephalus. Subsequent independent events of the loss or gain of rDNA would have resulted in the appearance of the rDNA sites in chromosomes 3, 4, 7, 11, and 13 in the species with the polymorphic condition. In this context, the NOR in pair 9q should be present in the most recent common ancestor (TMRC) of the Pithecopus genus (see Figure
While the first of these explanations depends on high rates of loss/gain of copies of the rDNA in the genomes of the species, the second hypothesis would depend on the recurrence of the same pairs as the NOR-bearing chromosomes in the different species in the genus Pithecopus (see Fig.
The active NOR-bearing chromosomes found in the karyotypes of the Pithecopus species and the broader phylogenetic context of the genus. Two possible scenarios to explain NOR variation are shown in inset (see details in discussion). The phylogenetic arrangement was reconstructed from
Assuming the ancestral polymorphism hypothesis, the total reproductive isolation of each evolutionary lineage would have resulted in the fixation of the principal active NOR sites in at least one pair of homologous chromosomes (the homozygous condition), which would permit the degeneration of the other sites, or at least the reduction or silencing of their expression. In P. nordestinus and P. ayaye, respectively, the position of the active NOR detected by Ag-NOR was confirmed by the FISH using 18S/28S rDNA probes (
One particularly illustrative example of this scenario is the variation in P. hypochondrialis found by
The results of the present study also indicate clearly a predominance of rDNA sites located in the pericentromeric and/or subterminal regions of the chromosomes (Fig.
Our findings help to fill the gaps in the knowledge of the karyotype variability of the genus Pithecopus and constitute a good example of the complex role of the rDNA genes in karyotype evolution. Ours results reveals that evolutionary dynamics of the NOR sites in genus and its potential as hotspot of chromosomal rearrangements, which implies that it may be a fundamental feature of chromosomal evolution in the genome of Pithecopus.
We thank the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP 2016/07717-6), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PROAP – Finance Code 001) for the scholarships provided to JGM, CBG and DYS. We thank the Multi-User Confocal Microscopy Center of the Federal University of Paraná for the capture of the images included in this study.