Corresponding author: Sanae Kasahara ( email@example.com )
Academic editor: Tereza Capriglione
© 2017 Simone Lilian Gruber, Gabriela Isabela Gomes de Oliveira, Ana Paula Zampieri Silva, Hideki Narimatsu, Célio Fernando Baptista Haddad, Sanae Kasahara.
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: Gruber SL, de Oliveira GIG, Silva APZ, Narimatsu H, Haddad CFB, Kasahara S (2017) Comparative analysis based on replication banding reveals the mechanism responsible for the difference in the karyotype constitution of treefrogs Ololygon and Scinax (Arboranae, Hylidae, Scinaxinae). Comparative Cytogenetics 11(2): 267-283. https://doi.org/10.3897/CompCytogen.v11i2.11254
According to the recent taxonomic and phylogenetic revision of the family Hylidae, species of the former Scinax catharinae (Boulenger, 1888) clade were included in the resurrected genus Ololygon Fitzinger, 1843, while species of the Scinax ruber (Laurenti, 1768) clade were mostly included in the genus Scinax Wagler, 1830, and two were allocated to the newly created genus Julianus
Anura, Hylidae, 5-bromodeoxiuridine, comparative cytogenetics, NOR, karyotype evolution
The family Hylidae was recently revised by
Over the years, the hylid frogs of the former Scinax genus have been subjected to several taxonomic and phylogenetic reviews (
The Ololygon and Scinax species are distributed across the Americas from Mexico to Argentina and Uruguay, and the islands of Tobago, Trinidad, and Saint Lucia (
Early researches on the cytogenetics of the former Scinax species determined the chromosome number by observing mitotic or meiotic cells. All of the species exhibited a diploid number of 2n = 24 bi-armed chromosomes, corresponding to the fundamental number of FN = 48 chromosome arms (revisions in
In the present work our efforts were the use of replication banding after 5-bromodeoxiuridine treatment, an useful approach to identify homeologous chromosomes (
Replication banding was fundamental to provide evidence of the structural rearrangement responsible for the difference in the karyotypes between Ololygon and Scinax species, thereby contributing for clarify the phylogenetic relationships between these two genera.
Five species currently included in the genus Ololygon, O. albicans (Bokermann, 1967), O. argyreornata (Miranda-Ribeiro, 1926), O. hiemalis (Haddad & Pombal, 1987), O. littoralis (Pombal & Gordo, 1991), and O. obtriangulata (Lutz, 1973), and six species of genus Scinax, S. caldarum (Lutz, 1968), S. crospedospilus (Lutz, 1925), S. eurydice (Bokermann, 1968), S. fuscovarius (Lutz, 1925), S. hayii (Barbour, 1909), and S. similis (Cochran, 1952) were collected from Brazilian locations (Table
Analyzed species, voucher number (CFBH), sample size, sex, and collection locality.
|Species||Voucher Number (CFBH)||Sample size||Sex||Collection locality|
|Ololygon albicans||101783||1||Male||Petrópolis, RJ
|Ololygon argyreornata||172893, 172903||2||Males||Ilha do Cardoso, SP
|Ololygon hiemalis||285912, 362312, 362332||3||Males||Mogi Guaçu, SP
|Ololygon littoralis||406312||1||Male||Bertioga, SP
|Ololygon obtriangulata||CFBHT*203292||1||Female||Biritiba Mirim, SP
|Scinax caldarum||225521||1||Male||Poços de Caldas, MG
|Scinax crospedospilus||362011, 362021||2||Males||Mogi das Cruzes, SP 23°31'29"S; 46°11'14"W|
|Scinax eurydice||167363||1||Male||Serra do Japi, Jundiaí, SP
|Scinax fuscovarius||224152||1||Female||Biritiba Mirim, SP
|Scinax hayii||242162||1||Male||Biritiba Mirim, SP
|285882||1||Male||Mogi das Cruzes, SP
|Scinax similis||59333, 59323||2||Females||Três Marias, MG
The sample includes species whose chromosomes are described for the first time in this paper: Scinax caldarum and S. crospedospilus; specimens belonging to species already described in
The animals were identified by Dr. Célio F. B. Haddad and the vouchers were fixed in formalin (10%), preserved in 70% ethanol and deposited in the amphibian collection CFBH of the Departamento de Zoologia, Instituto de Biociências, UNESP, Rio Claro, SP, Brazil. Identification of the specimen CFBHT 20329 was confirmed by COI sequencing from muscle sample collected soon after the animal euthanasia and preserved in 70% ethanol. Direct cytological preparations were obtained from bone marrow, liver, and also the testes of male samples (
Mitotic and meiotic preparations were analyzed after Giemsa staining. Mitotic chromosomes were also submitted to the techniques of silver impregnation (Ag-NOR) (
Chromosomes were analyzed under standard and UV light. The best mitotic and meiotic cells were photographed using an Olympus BX51 microscope and digital capture system DP71. Copies of the material were obtained digitally. Karyograms were assembled according to the morphology of the chromosomes in decreasing order of size (Table
Relative length (RL), centromeric index (CI), and nomenclature for centromeric position (CP) on mitotic chromosomes of Ololygon and Scinax, according to
All investigated specimens presented 24 bi-armed chromosomes. In Ololygon species, chromosome pairs 1 and 2 were submetacentric with a slight size difference between them, while in the species of Scinax genus chromosome pairs 1 and 2 were metacentric with great size difference. For illustration, the Giemsa-stained karyotypes of one of the species of Ololygon (O. hiemalis) from a population not studied before and of the two species of Scinax (S. caldarum and S. crospedospilus) karyotyped for the first time are presented in Figure
Replication banding patterns for the chromosomes of O. hiemalis, S. crospedospilus, S. eurydice, S. fuscovarius, and for two specimens of S. similis are presented in Figure
Nucleolus organizer regions were located on pair 6 for the Ololygon species and in pair 11 for the Scinax species. Figure
Chromosome preparations of O. hiemalis, O. littoralis, O. obtriangulata, S. crospedospilus, and S. hayii were submitted to hybridization with a telomeric probe and only the terminal regions of all chromosomes in these species were labeled (Figure
Testis preparations were obtained from male specimens of O. argyreornata, O. hiemalis, O. littoralis, S. caldarum, S. crospedospilus, S. eurydice, and S. hayii. In all of them, metaphase I cells presented 12 bivalents, whereas metaphase II cells presented 12 chromosomes, as shown in Figure
Giemsa stained karyotypes of Ololygon and Scinax species. a O. hiemalis, male b S. caldarum, male c S. crospedospilus, male d S. similis, female, with heteromorphic chromosomes pairs 3 and 4. Secondary constrictions (arrowhead) are visible on chromosome 6 in a and on chromosomes 11 in c. Bar = 10 μm.
Replication banding in Ololygon and Scinax species. a O. hiemalis b S. crospedospilus c S. eurydice d S. fuscovarius e–f S. similis. Note heteromorphic chromosome pairs 3 and 4 in inset. Bar = 10 μm.
a Comparison of replication bands of the chromosomes of: Ol = O. littoralis; Oh = O. hiemalis (two distinct metaphases of the same specimen); Sc = S. crospedospilus; Se = S. eurydice; Sf = S. fuscovarius; Ss = S. similis. b Ideogram of chromosomes 1 and 2 of Ololygon and Scinax evidencing loss of segment in the short arms of both chromosomes in Ololygon. Bar = 10 μm.
Nucleolus organizer regions in Ololygon obtriangulata (a, b, c) and Scinax fuscovarius (d, e, f) identified by a, d silver impregnation b, e CMA3 staining, and c, f FISH with rDNA probe.
FISH with telomeric probes in Ololygon and Scinax species. a O. hiemalis b O. littoralis c O. obtriangulata d S. crospedospilus e S. hayii. Bar = 10 μm.
C-banding of Ololygon and Scinax species. a O. albicans b O. argyreornata c O. littoralis d S. crospedospilus e S. fuscovarius f S. hayii. Bar = 10 μm.
Cromomycin A3 staining in Ololygon and Scinax species. a O. littoralis b S. crospedospilus c S. hayii. NOR-bearing chromosomes in b (arrow). Bar = 10 μm.
Although Ololygon and Scinax species share the same diploid and fundamental numbers, two karyotypic constitutions were unequivocally distinguished by visual inspection of standard stained chromosomes as well as comparative measurements. One of these karyotypes is characteristic for all five species, currently recognized as belonging to the genus Ololygon (
The difference between the two karyotypes, which is related to the morphology and size of chromosome pairs 1 and 2, had already been indicated in one of the first cytogenetic studies performed in the 1970’s by
Although without experimental evidence,
The probable direction of chromosome evolution, in other words, the loss or gain of repetitive segments in chromosome pairs 1 and 2, is suggested in light of the cytogenetic data of Ololygon and Scinax species, and the phylogeny of
Obtaining replication bands along chromosomes was helpful to clarify the heteromorphism in pairs 3 and 4 in one of the two specimens of the sample of Scinax similis described in
Based on C-banding data of species of the former Scinax clades,
The FISH technique using telomeric probes tested in the chromosome preparations of Ololygon and Scinax of our sample, which could potentially clarify the occurrence of structural rearrangement during the chromosome evolution, as demonstrated by
Our cytogenetic data confirm the loss of repetitive sequence in the short arms of chromosomes 1 and 2 as the mechanism responsible for the difference in the karyotypic constitutions of Ololygon and Scinax. Nevertheless, relationships in Scinaxcinae species are not easy to interpret. Despite the taxonomic difficulties, increasing the number of analyzed species and using more cytological markers based on molecular cytogenetics methodologies will eventually help to clarify phylogenetic questions within Ololygon, Scinax, and related genera.
This work was supported by grants #2013/50741-7, #2014/50342-8, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors thank to Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for providing the collection permits. The authors are grateful to Daniela Moraes Leme and Glaucilene Catrolli for help during chromosome preparation and analysis, to Dr. Juliana Zina for collecting the specimen CFBH16736 in Serra do Japí, Jundiaí, SP, and to Mariana Lyra for sequencing COI gene from tissue sample of one specimen.