Research Article |
Corresponding author: Daniel Pacheco Bruschi ( danielpachecobruschi@gmail.com ) Academic editor: Teresa Capriglione
© 2019 Michelle Louise Zattera, Luana Lima, Iraine Duarte, Deborah Yasmin de Sousa, Olívia Gabriela dos Santos Araújo, Thiago Gazoni, Tamí Mott, Shirlei Maria Recco-Pimentel, Daniel Pacheco Bruschi.
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Citation:
Zattera ML, Lima L, Duarte I, Sousa DY, Araújo OGS, Gazoni T, Mott T, Recco-Pimentel SM, Bruschi DP (2019) Chromosome spreading of the (TTAGGG)n repeats in the Pipa carvalhoi Miranda-Ribeiro, 1937 (Pipidae, Anura) karyotype. Comparative Cytogenetics 13(3): 297-309. https://doi.org/10.3897/CompCytogen.v13i3.35524
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Pipidae is a clade of Anura that diverged relatively early from other frogs in the phylogeny of the group. Pipids have a unique combination of morphological features, some of which appear to represent a mix of adaptations to aquatic life and plesiomorphic characters of Anura. The present study describes the karyotype of Pipa carvalhoi Miranda-Ribeiro, 1937, including morphology, heterochromatin distribution, and location of the NOR site. The diploid number of P. carvalhoi is 2n=20, including three metacentric pairs (1, 4, 8), two submetacentric (2 and 7), three subtelocentric (3, 5, 6), and two telocentric pairs (9 and 10). C-banding detected centromeric blocks of heterochromatin in all chromosome pairs and the NOR detected in chromosome pair 9, as confirmed by FISH using the rDNA 28S probe. The telomeric probes indicated the presence of interstitial telomeric sequences (ITSs), primarily in the centromeric region of the chromosomes, frequently associated with heterochromatin, suggesting that these repeats are a significant component of this region. The findings of the present study provide important insights for the understanding of the mechanisms of chromosomal evolution in the genus Pipa, and the diversification of the Pipidae as a whole.
Pipidae, chromosome banding, interstitial telomeric sequences, rearrangements, chromosome evolution
Chromosome studies provide important insights into the diversification of karyotypes and represent an effective approach for the identification of homologies among species (
Pipids are a clade of anurans that diverged relatively early from other frogs in the phylogeny of the group (
However, based on molecular phylogenetic inferences and presumed ancestral diploid numbers, some authors have distinguished a fifth lineage, Silurana, which includes all the species derived from an ancestor with 2n = 20 (
Pipa is the only non-African representative of the Pipidae, and evidences from a number of different sources indicates that this South American lineage is derived from an ancestor closely related to the extant members of the genus Hymenochirus. Pipidae was widely distributed in Gondwana and after its splintering, those lineages had distributions associated with the Afro-Tropical (Hymenochirus, Pseudhymenochirus and Xenopus) and Neotropical Regions (Pipa). The historical isolation resulted in the diversification of the ancestral lineage of the genus Pipa, which is found in South America, as far north as Panama (
The genus currently contains seven species: P. arrabali Izecksohn, 1976, P. aspera Mueller, 1924, P. carvalhoi Miranda-Ribeiro, 1937, P. myersi Trueb, 1984, P. parva Ruthven & Gaige, 1923, P. pipa (Linnaeus, 1758), and P. snethlageae Muller, 1914 (
As no data whatsoever are available for the other five Pipa species, further studies will be essential for the understanding of the genomic rearrangements that have occurred during the adaptive radiation of this lineage in South America. Here, we describe the karyotype of P. carvalhoi, including the position of the NORs and the distribution pattern of the heterochromatin. We also documented the intrachromosomal spread of the telomeric (TTAGGG)n motifs and discuss these findings in the context of the phylogenetic scenario of the family Pipidae.
We analyzed three specimens of Pipa carvalhoi collected in Buerarema (three male), Bahia state, Brazil, and three from Buíque (two male + one juvenile), Pernambuco state, Brazil. The collection of specimens was authorized by SISBIO/Instituto Chico Mendes de Conservação da Biodiversidade through protocol number 55481-1. The specimens were deposited in the “Célio Fernando Baptista Haddad” Amphibian Collection (CFBH), on the Rio Claro campus of São Paulo State University (UNESP) and in Natural History Museum in Universidade Federal de Alagoas (
The chromosomal preparations were obtained from intestinal and testicular cells treated with 2% colchicine for 4 hours, using techniques modified from
Loci of 28S rDNA were detected fluorescence in situ hybridization (FISH). We used the 28S fragment isolated by
The diploid number of the P. carvalhoi karyotype was 2n = 20 chromosomes (Fig.
The C-banding technique detected centromeric blocks of heterochromatin in all chromosome pairs. Interstitial heterochromatin blocks were also detected in the long arms of pair 5 (Fig.
Karyotype of P. carvalhoi. a Prepared by conventional Giemsa staining b C-banding and c DAPI staining. The arrow indicates the NOR site.
Under conventional Giemsa staining, a secondary constriction was observed in the subterminal regions of the homologs of pair 9, which coincides with the NOR site (in both populations), detected by the Ag-NOR method and confirmed by FISH using the rDNA 28S probe (Fig.
Fluorescence in situ hybridization in P. carvalhoi karyotype. a Hybridized with the 28S rDNA probe b the NOR-bearing chromosome highlighted by DAPI-staining, the Ag-NOR method, and FISH with 28S rDNA c In situ hybridization with the telomeric probe in the karyotype of P. carvalhoi from Pernambuco, Brazil. The arrows in c indicate the interstitial telomeric sequences (ITSs) and the constriction in chromosome 8 are indicates by asterisk.
The chromosomal evolution of the pipids appears to have involved complex rearrangements, including recurrent polyploidization events and associated shifts in the diploid number (Table
Detailed cytogenetic data available for species of the Pipidae family. NOR: Nucleolar Organizer Region; M= metacentric; SM= submetacentric; ST=subtelocentric; T=telocentric; p= short arm; q=long arm; (–) no data.
Species | Ploidy level | Karyotype formula | NOR site | Reference |
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Xenopus tropicalis group | ||||
X. tropicalis | 2n = 20 | 2 M + 14 SM + 4 A | 5q |
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X. epitropicalis | 4n = 40 | 4M + 28 SM+ 8 A | 5q |
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X. new tetraploid 1 | 4n = 40 | 4M + 28 SM+ 8 A | 5q |
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X. new tetraploid 2 | 4n = 40 | 4M + 28 SM+ 8 A | 5q |
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Xenopus laevis group | ||||
X. borealis | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 4p |
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X. clivii | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 4p |
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X. fraseri | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 6q |
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X. gilli | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 12p |
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X. laevis laevis | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 12p |
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X. laevis bunyoniensis | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | – |
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X. laevis petersi | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | – |
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X. laevis poweri | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | – |
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X. laevis sudanensis | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | – |
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X. laevis victorianus | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | – |
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X. largeni | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | – |
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X. muelleri | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 4p |
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X. pygmaeus | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 6q |
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X. sp. nov. VI | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 4p |
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X. sp. nov. IX | 4n = 36 | 6 M+ 14 SM+ 2 ST + 14 T | 12p |
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X. amieti | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | 5q |
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X. andrei | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | 18q |
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X. boumbaensis | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | 6p+ 4p |
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X. itombwensis | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | – |
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X. lenduensis | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | – |
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X. vestitus | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | 12p |
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X. wittei | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | 12p |
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X. sp. nov. X | 8n = 72 | 12 M + 28 SM + 4 ST + 28 T | 18q |
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X. longipes | 12n = 108 | 18 M + 42 SM + 6 ST + 42 T | 7p |
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X. ruwenzoriensis | 12n = 108 | 18 M + 42 SM + 6 ST + 42 T | 11q |
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X. cf. boumbaensis | 12n = 108 | 18 M + 42 SM + 6 ST + 42 T | 7p |
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X. sp. nov. VIIIa | 12n = 108 | 18 M + 42 SM + 6 ST + 42 T | 7p |
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Genus Pseudhymenochirus | ||||
P. merlini | 2n = 20 | 8 M + 4 SM + 6 ST + 2 T | 10q |
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Genus Hymenochirus | ||||
H. boettgeri | 2n = 20 | 14 M + 2 SM + 4 ST | 4p |
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Genus Pipa | ||||
P. carvalhoi | 2n = 20 | 6 M+ 4 SM+6 ST + 4 T | 9q | Present study |
8 M + 8 SM + 4 T† |
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P. pipa | 2n = 22 | 8 M + 14 A | – |
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6M + 2ST + 14A |
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P. parva | 2n = 30 | 30 T | – | Morescalchi 1981 |
Subsequently,
The diploid number (2n = 20) recorded here in P. carvalhoi also corresponds to a retention of the plesiomorphic condition of the pipids, and an overview of all the known karyotypes of pipid species indicates that the morphology of pairs 1, 2, 3, and 4 is highly conserved, as it is in the outgroup, Rhinophrynus dorsalis (
The comparison of the karyotypes of P. carvalhoi and P. pipa (
The pericentromeric heterochromatin block in the homologs of pair 3 of P. carvalhoi could be a common feature of pipid karyotypes. Interestingly, this heterochromatin block, is also present in Xenopus (= Silurana) tropicalis (Tymowska & Fischberg, 1982), Hymenochirus boettgeri, and Pseudhymenochirus merlini karyotype (
We detected interstitial telomeric sequences (ITSs) in the centromeric/pericentromeric region of the metacentric and submetacentric chromosomes of the P. carvalhoi karyotype. Based in
Despite being an unusual feature of vertebrate genomes, we found ITS sites in euchromatic regions (in pair 9, for example), as found in some other anuran species.
Adopting the parsimony criterion, we rejected the hypothesis that the ITSs detected in the P. carvalhoi karyotype are remnants of centric (Robertsonian) fusions, given that P. carvalhoi has the plesiomorphic pipid diploid number. However, for some chromosomes (pairs 6, 8 and 9) theses ITS to confirm occurrence of the intrachromosomal rearrangements during evolution of this karyotype. Already, for others ITS signals, our data support the conclusion that the presence of the intrachromosomal telomeric motif (TTAGGG)n represents a component of the repetitive DNA sequences spread throughout these chromosomes. Furthermore, the ITSs found in the P. carvalhoi chromosomes coincide with the heterochromatic blocks detected by C-banding in chromosomes 1, 2, 4, 5 and 7. The role of telomeric repeats as repetitive motifs of part of the satellite DNA has already been described in a number of rodent genera, with a unique signal being found in the pericentromeric heterochromatin together with Msat-160 or in telomeric probes, in experiments with co-located het-ITSs and the Msat-160 satellite DNA (
Overall, then, the results of the present study indicate that P. carvalhoi has a karyotype of 2n = 20 chromosomes, supporting that this chromosome formula represents the pleiomorphic condition of the pipids, with interspecific chromosomal homologies indicating a highly conserved karyotype configuration. The presence of ITSs in some chromosomes may have originated independently during the chromosomal evolution of this species which in others pairs correspond to evidences of the pericentromeric inversions occurred during Pipidae karyotype diversification. The findings of the present study provide important insights into the mechanisms of chromosomal evolution in the genus Pipa and the diversification of the family Pipidae as a whole.
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 MLZ, LL, ID and DY, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/PQ/309904/2015-3). We thank the Multi-User Confocal Microscopy Center of the Federal University of Paraná for the capture of the images included in this study.