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Corresponding author: Lidia Nogueira ( lidia.nogueira@yahoo.com.br ) Academic editor: Nikolay Tzankov
© 2015 Lidia Nogueira, Fabilene Paim, Débora Diniz, Mirco Sole, Paulo Roberto Affonso, Sérgio Siqueira, Iracilda Sampaio.
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:
Nogueira L, Paim F, Diniz D, Sole M, Affonso P, Siqueira S, Sampaio I (2015) Cytogenetic analysis of Scinax auratus and Scinax eurydice (Anura, Hylidae) with emphasis on cytotaxonomy. Comparative Cytogenetics 9(2): 227-236. https://doi.org/10.3897/CompCytogen.v9i2.4593
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Scinax Wagler, 1830 is a species-rich genus of amphibians with relatively few detailed chromosomal reports. In this work, cytogenetic analyses of Scinax auratus (Wied-Neuwied, 1821) and Scinax eurydice (Bokermann, 1968) were carried out based on conventional (Giemsa staining, Ag-NOR and C-banding) and cytomolecular (base-specific fluorochrome staining and fluorescence in situ hybridization – FISH of ribosomal probes) techniques. Both species shared the same karyotype, location of active nucleolar organizer regions on pair 11 and GC-rich heterochromatin, as reported for most species in S. ruber clade. Interpopulation chromosomal variation was observed in S. eurydice, indicating the occurrence of cryptic species. The mapping of 18S ribosomal genes by FISH is reported for the first time in both species.
Amphibians, chromosomes, FISH, heterochromatin
Classic and cytomolecular chromosomal studies have been efficient to infer intra and interspecific relationships in anurans, besides supporting the validation of new and cryptic species (
The genus Scinax Wagler, 1830 encompasses 114 species (
S. auratus (Wied-Neuwied, 1821) inhabits rocky areas in Atlantic forest and forest borders in northeastern Brazil (
S. euridyce (Bokermann, 1968) is also widespread in Brazil with records in five states of northeastern and southeastern Brazil (Bahia, Espírito Santo, Minas Gerais, Rio de Janeiro and São Paulo) (
In the present work, we provide new chromosomal data for both S. auratus and S. eurydice in order to respond the following questions: (1) Are the NORs observed in 3q of S. alter also present in S. auratus? (2) Is the polymorphism of NORs previously reported in S. eurydice from southeastern Brazil shared by populations from Bahia? (3) Are there chromosomal differences among geographically distant populations? (4) Can the mapping of 18S rDNA by FISH reveal additional non-active NORs previously undetected by silver nitrate staining?
Five specimens of S. auratus and S. euridyce were collected for cytogenetic analyses in Jequié, state of Bahia, northeastern Brazil (13°51'4"S, 40°4'52"W) (Table
Analyzed species, number of individuals (N), sex (J = juveniles of undentified sex) and collection site.
Species | Voucher | N | Locality |
---|---|---|---|
S. auratus | MZUESC11051 (♀), MZUESC11052 (♀), MZUESC11053 (♀), MZUESC11054 (♀), MZUESC11055 (♀) | 5 | Jequié - BA |
S. eurydice | MZUESC11047 (♂), MZUESC11049 (J), MZUESC11005 (J), MZUESC11006 (♂), MZUESC11007 (♂) | 5 | Jequié - BA |
The slides were stained with Giemsa at 10% in phosphate buffer (pH 6.8) for about 10 minutes and air dried. For karyotyping, the chromosomes were classified according to centromere position into: m (metacentric), sm (submetacentric) and st (subtelocentric) following the nomenclature suggested by
Fluorescence in situ hybridization using 18S rDNA probes was carried out according to
The best metaphase spreads were photographed using an Olympus BX51 epifluorescence microscope equipped with digital image capture system (ImagePro Plus – Media Cybernetics) and processed in the software Adobe Photoshop CS 8.0.1.
S. auratus and S. eurydice presented 2n = 24 and FN = 48 besides sharing the same chromosomal formula: 16 metacentric (pairs 1, 2, 7, 8, 9, 10, 11 and 12) and eight submetacentric (pairs 3, 4, 5 and 6) chromosomes (Table
Karyotypes of S. auratus (a, c, e) and S. eurydice (b, d, f) after Giemsa-staining (a, b), C-banding (c, d) and base-specific fluorochrome staining (e, f). The NOR-bearing chromosomes after silver nitrate staining and FISH with 18S rDNA probes of each species are shown in boxes. Bar = 10 µm.
Chromosomal measurements of studied species: relative length (RL), centromeric index (CI) and classification (CT) according to
Species | Chromosomal Pairs | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
S. auratus | RL | 16.35 ± 0.09 | 13.66 ± 0.07 | 11.40 ± 0.03 | 10.34 ± 0.55 | 9.22 ± 0.04 | 8.73 ± 0.01 | 6.76 ± 0.26 | 6.30 ± 0.01 | 5.48 ± 0.50 | 5.30 ± 0.01 | 5.21 ± 0.01 | 5.11 ± 0.01 |
CI | 0.49 ± 0.01 | 0.42 ± 0.01 | 0.34 ± 0.01 | 0.36 ± 0.01 | 0.34 ± 0.01 | 0.31 ± 0.01 | 0.45 ± 0.01 | 0.48 ± 0.01 | 0.41 ± 0.01 | 0.38 ± 0.01 | 0.48 ± 0.01 | 0.47 ± 0.01 | |
CP | M | M | SM | SM | SM | SM | M | M | M | M | M | M | |
S. eurydice | RL | 14.35 ± 0.09 | 11.52 ± 0.15 | 10.82 ± 0.43 | 9.56 ± 0.55 | 9.01 ± 0.37 | 7.81 ± 0.21 | 6.61 ± 0.21 | 6.45 ± 0.09 | 5.79 ± 0.60 | 5.71 ± 0.09 | 5.56 ± 0.96 | 4.53 ± 0.15 |
CI | 0.48 ± 0.01 | 0.42 ± 0.01 | 0.27 ± 0.01 | 0.32 ± 0.01 | 0.32 ± 0.01 | 0.3 ± 0.01 | 0.41 ± 0.01 | 0.37 ± 0.01 | 0.43 ± 0.01 | 0.48 ± 0.01 | 0.45 ± 0.02 | 0.47 ± 0.01 | |
CP | M | M | SM | SM | SM | SM | M | M | M | M | M | M |
Silver nitrate staining revealed active nucleolus organizer regions (Ag-NORs) at interstitial region of 11q (Fig.
Heterochromatin was distributed over centromeric regions of all chromosomes in S. auratus while telomeric C-bands were observed in most chromosomes of S. eurydice along with telomeric heterochromatic blocks at centromeric regions of pairs 5 and 8 (Fig.
FISH with 18S rDNA probes confirmed the single NOR-bearing pair visualized by silver nitrate staining in the analyzed species (Fig.
The karyotypes of S. auratus and S. eurydice followed the pattern proposed for Scinax (2n = 24 and FN = 48). Similarly, the karyotype formulae agree with those reported for species within S. ruber clade (
Based on morphological traits and vocalization, S. auratus seems to be closely related to Scinax alter, S. cretatus, S. crospedospilus, S. cuspidatus, S. imbegue, S. juncae and S. tymbamirim (
The NORs were associated with CMA3+ signals in both analyzed species, indicating the presence of GC-rich repetitive DNA interspersed with ribosomal genes, as commonly observed in anurans (
The specimens of S. eurydice from the state of São Paulo, southeastern Brazil (
Other cases of NOR polymorphism have been previously reported in anurans such as Hyla nana (Boulenger, 1889) (
The presence of heterozygous NORs (Ag+/Ag-) in S. eurydice might be related to sex, since this heteromorphic pattern was observed only in males. For instance, females and males of Gastrotheca riobambae (Fowler, 1913) were characterized by two and single NOR marks, respectively, mapped on X chromosomes (
Nonetheless, experimental evidence has shown that individuals of salamanders Plethodon cinereus (Green, 1818) and Xenopus laevis (Daudin, 1802) bearing heterozygous NORs (Ag+/Ag-), independently on sex, are viable but their fertility is reduced since crosses between heterozygous specimens will produce unviable tadpoles bearing homozygous NORs (
The interpopulation variation of NOR and C-banding pattern among populations of S. eurydice, associated with slight differences in vocalization between samples from northeastern and southeastern Brazil (
In conclusion, the detailed cytogenetic characterization of S. auratus and S. eurydice showed that S. auratus shares some chromosomal traits with most of species in S. ruber clade, but diverges from the putatively closely related S. alter. The results in S. eurydice from Bahia revealed differences in chromosomal banding when compared to populations of southeastern Brazil, indicating the presence of cryptic species that should be systematically revised. Therefore, the chromosomal analyses in Scinax are potentially useful to both taxonomy and systematics of this group of anurans.
We thank Amanda Santiago, Rodrigo Araújo and Euvaldo Júnior for the collection and organization of specimens; Financial support to this work was provided by CAPES and CNPq. The specimens were collected with the permission of ICMBio (license number 28684-1).