Evaluation of the taxonomic status of populations assigned to Phyllomedusa hypochondrialis (Anura, Hylidae, Phyllomedusinae) based on molecular, chromosomal, and morphological approach
© Bruschi et al.; licensee BioMed Central Ltd. 2013
Received: 25 January 2013
Accepted: 30 July 2013
Published: 12 August 2013
The taxonomic and phylogenetic relationships of the genus Phyllomedusa have been amply discussed. The marked morphological similarities among some species hamper the reliable identification of specimens and may often lead to their incorrect taxonomic classification on the sole basis of morphological traits. Phenotypic variation was observed among populations assigned to either P. azurea or P. hypochondrialis. In order to evaluate whether the variation observed in populations assigned to P. hypochondrialis is related to that in genotypes, a cytogenetic analysis was combined with phylogenetic inferences based on mitochondrial and nuclear sequences.
The inter- and intra-population variation in the external morphology observed among the specimens analyzed in the present study do not reflect the phylogenetic relationships among populations. A monophyletic clade was recovered, grouping all the specimens identified as P. hypochondrialis and specimens assigned P. azurea from Minas Gerais state. This clade is characterized by conserved chromosomal morphology and a common C-banding pattern. Extensive variation in the nucleolar organizing region (NOR) was observed among populations, with four distinct NOR positions being recognized in the karyotypes. Intra-population polymorphism of the additional rDNA clusters observed in specimens from Barreiras, Bahia state, also highlights the marked genomic instability of the rDNA in the genome of this group. Based on the topology obtained in the phylogenetic analyses, the re-evaluation of the taxonomic status of the specimens from the southernmost population known in Brazil is recommended.
The results of this study support the need for a thorough revision of the phenotypic features used to discriminate P. azurea and P. hypochondrialis. The phylogenetic data presented here also contribute to an extension of the geographic range of P. hypochondrialis, which is known to occur in the Amazon basin and neighboring areas of the Cerrado savanna, where it may be sympatric with P. azurea, within contact zones. The misidentification of specimens may have led to inconsistencies in the original definition of the geographic range of P. azurea. The variability observed in the NOR of P. hypochondrialis reinforces the conclusion that these sites represent hotspots of rearrangement. Intraspecific variation in the location of these sites is the result of constant rearrangements that are not detected by classical cytogenetic methods or are traits of an ancestral, polymorphic karyotype, which would not be phylogenetically informative for this group.
KeywordsPhyllomedusa Morphological variation Chromosome Phylogenetic inference
The taxonomic classification [1, 2] and phylogenetic relationships [3, 4] of the frogs of the genus Phyllomedusa have been subjected of extensive debate. Representatives of the genus are distributed throughout Central America and in South America east of the Andes, as far south as Argentina . The genus Phyllomedusa is currently composed of 30 species, of which 26 have been allocated to four species groups, based on morphological features – the P. burmeisteri (5 spp.), P. hypochondrialis (9 spp.), P. perinesos (4 spp.), and P. tarsius (8 spp.) species groups [4, 5]).
The marked morphological similarities of members of this genus hamper the reliable identification of species, often resulting in taxonomic inaccuracies, redefinition of species, and frequent description of new species. Based on morphological traits, Caramaschi  redefined the phenetic P. hypochondrialis species group, which currently consists of P. azurea, P. centralis, P. hypochondrialis, P. megacephala, P. nordestina, P. oreades, and P. rohdei. Morphological data ) and phylogenetic inferences  indicate that P. araguari is a synonym of P. oreades, while Baêta et al.  recognized P. itacolomi as a synonym of P. ayeaye. These recent studies are indicatives of the taxonomic instability that the genus is still subjected.
A recent molecular phylogenetic analysis by Faivovich et al.  revealed the presence of two subclades within the P. hypochondrialis species group. One of these clades included P. azurea, P. hypochondrialis, and P. nordestina, while the other is composed of the remaining four species. There are numerous reports of taxonomic errors involving P. azurea, P. hypochondrialis, and P. nordestina, which have barely distinguishable diagnostic characteristics . Phyllomedusa azurea is known to occur in open habitats of the Cerrado savannas, Pantanal wetlands, and Chaco scrub biomes, whereas P. hypochondrialis is distributed mainly in the Amazonian region and areas of Amazonian influence in the Pantanal  and P. nordestina is found in wet habitats amidst the Caatinga scrublands of the Brazilian Northeast. Recent records of P. azurea extend previously known distribution to a Cerrado-Amazon transitional zone in the state of Rondônia , and to open upland habitats in Santa Catarina, southern Brazil . However, the correct taxonomic classification of these populations is still unclear .
Chromosomal characteristics of members of the genus Phyllomedusa are relatively poorly known, although the karyotypes of a number of species have been described, including P. rohdei[12–14], P. camba, P. nordestina, P. hypochondrialis, P. distincta, and P. tetraploidea[16–18]. However, potentially informative chromosomal features have been observed, in particular, the marked variability in the number and position of the NORs in different populations [12, 13, 15, 16]. Morphological variation has also been observed in some populations assigned to P. hypochondrialis suggesting the need for the complementary application of different interpretative tools as a helpt to clear their taxonomic status.
Considering these fundamental problems, the present study focused on the morphological variation found in frog populations attributed to P. hypochondrialis, and examined whether this variation is interspecific or inter-populational. Cytogenetic and molecular approaches are also used in order to verify whether the observed phenotypic variation is related to genotype-level variation, based on the analysis of specimens obtained from Brazilian populations assigned to P. hypochondrialis and P. azurea from a number of distinct regions. It was also considered specimens that could not be safely identified up to the specific level, such as Phyllomedusa cf. hypochondrialis, Phyllomedusa sp. (aff. hypochondrialis).
The variation in external morphology observed among the specimens allowed the recognition of four morphotypes, based on the diagnostic traits used to distinguish P. azurea from P. hypochondrialis (Figure 1):
Morphotype 1 Narrow white stripe on the upper lip extending to the lower eyelid together with the presence of a discontinuous, wide green stripe along 2/3 to 3/4 of the length of the upper surface of the thighs. Specimens presenting these character conditions correspond to P. hypochondrialis, according to Caramaschi .
Morphotype 2 White stripe on the upper lip extending to the lower eyelid together with the presence of a wide green stripe along the full length of the upper surface of the thighs.
Morphotype 3 White stripe on the upper lip extending to the lower eyelid. Green stripe absent on the upper surface of the thighs.
Two of the other morphological characteristics used by Caramaschi  to distinguish P. azurea and P. hypochondrialis vary considerably both within and between populations, and cannot be used reliably to identify the species: the size of the adhesive discs relative to the eardrum (discs larger than the eardrum in P. hypochondrialis and smaller in P. azurea) and the white stripe on the upper lip, which is visible dorsally in P. hypochondrialis, but not in P. azurea.
Distribution of the four recognized morphotypes in each study population
Laranjal do Jari/AP
Chapada dos Guimarães/MT
All the populations tentatively assigned to P. hypochondrialis [P. hypochondrialis, Phyllomedusa sp. (aff. hypochondrialis), Phyllomedusa cf. hypochondrialis)] formed a monophyletic clade together with the GenBank sequences of specimens from the Guyanas (96% posterior probability for Bayesian Inference-Figure 2). By contrast, specimens tentatively attributed to P. azurea from Uberlândia (L16), state of Minas Gerais – in the Cerrado biome– were paraph yletic in relation to the P. azurea haplotypes from Argentina, Bolivia, and Paraguay. In fact, the specimens from Uberlândia grouped with the P. hypochondrialis clade.
In the BI topology, the specimen from Alta Floresta, Mato Grosso (L8), formed a subclade with the population from Belterra, in Pará (L7), with aposterior probability of 99%. A second subclade was composed of populations from Chapada dos Guimarães and Santa Teresinha, both in Mato Grosso (posterior probability 99%). The third subclade included haplotypes from Maranhão (L11-L13), Tocantins (L14), Bahia (L15) and Minas Gerais (L16), with a posterior probability of 95%. Finally, the specimens from Amapá (L1) and some localities in the state of Pará (L2-L6) grouped with the GenBank sequences of one specimen from Suriname (the type locality of P. hypochondrialis) and two from French Guiana and Guyana.
These results also provide useful insights into the taxonomic status of a population recently discovered in Água Doce, in the Brazilian state of Santa Catarina by Lucas et al. , which is morphologically similar to P. azurea. However, the haplotypes of the specimens from this population were paraphyletic in relation to the other haplotypes of the P. azurea clade, and were closely related to the second major clade in the P. hypochondrialis group (P. rohdei, P. ayeaye, P. centralis, P. megacephala and P. oreades) inferred in phylogenetic reconstruction.
Description of the karyotypes
Nucleolar organization region (NOR)
In specimens from Maranhão (L11, L12 and L13, respectively, from São Luí s, Bacabeira, Urbano Santos) and from Porto Nacional, in Tocantins (L14), NORs were located in the interstitial region of the short arm of pair 7, coinciding with the secondary constrictions observed by Giemsa staining (Figure 4). Intra-population variation was also observed in specimens from Barreiras (L15), in which the NOR was located in the interstitial region of the short arm of pair 7 in all individuals. Additional NOR was observed in subterminal region of one homologue of pair 4 (specimen ZUEC 17072), in the subterminal region of the short arm in one homologue of pair 3 (specimens ZUEC 17082 and 17083), and in the pericentromeric region of the long arm of one homologue of pair 3 (specimens ZUEC 17071 and 17078) (Figure 4E). In all these cases, the regions were identified as secondary constrictions by Giemsa staining. In specimens from Uberlândia (L16), NORs were detected in the pericentromeric region of the short arm of pair 4, coinciding with secondary constrictions (Figure 4F).
Additionally, C-bands were observed in the pericentromeric region of the short arm and interstitially on the long arm of pair 1 (Figure 6E) and in the subterminal region of the long arm of pair 2 in specimens from Barreiras (L15). A pericentromeric block was also observed in the short arm of pair 4 and a subterminal block of heterochomatin in a single homologue of pair 1 (morph 1b) in specimens from Uberlândia (L16), not detected in morpho 1a (Figure 6F). Morph 1b was observed in all the metaphases analyzed, irrespective of the sex of the specimen.
The phylogenetic analyses of the genus Phyllomedusa presented here support emphatically the monophyletic status of the P. burmeisteri, P. tarsius, P. perinesos and P. hypochondrialis species groups, further reinforcing the topology obtained by Faivovich et al.  However, a number of questions remain with regard to the group-level classification of certain species. The analyses cluster all the Brazilian specimens examined (L1-16) in the same clade, together with sequences from Suriname, French Guiana, and Guyana. The Brazilian populations (L1-16) analyzed here were all identified as P. hypochondrialis, based on the fact that the type locality of P. hypochondrialis was identified as “Suriname” in the original description (see reference ). The inter- and intra-population variation in external morphology observed among the specimens of this clade does not reflect the differences in the phylogenetic relationships among populations.
Morphotype 1 corresponds to the set of characteristics used to describe the species P. hypochondrialis, while morphotype 4 corresponds to the description of P. azurea. Morphotype 2 corresponds to a mixture of the diagnostic traits of the two species, while morphotype 3 combines characteristics that do not correspond to any formal species description. Overall, then, the results of the present study indicate that diagnostic traits employed by Caramaschi  for the differentiation of P. hypochondrialis and P. azurea are in fact combined in varying proportions in the populations examined. In this case, it is not possible to identify any specific morphological pattern associated with the geographic distribution of the populations. The high frequency of intermediate morphotypes in individuals of these two species (morphotype 2) re-emphasizes the difficulties in morphologically distinguishing P. azurea from P. hypochondrialis. The morphological variation observed in the present study supports the need for a careful re-analysis of the phenotypic features used to discriminate P. azurea and P. hypochondrialis.
Chromosomal morphology and C-banding patterns are conserved within the P. hypochondrialis clade, indicating the presence of homologies among the different karyotypes. While there is some variation in the morphology of pair 7 (ST/SM) in population L11-15 in comparison with the other populations (L1-10, L16), it is possible to infer homologies between these karyotypes, which can be explained by the presence of NORs in submetacentric pair 7, the increase in the arm ratio, and the centromeric position of this pair. Other common features in this clade include the distribution of heterochromatin in pairs 7 (pericentromeric) and 9 (subterminal), which appears to be a diagnostic feature of P. hypochondrialis.
The topology obtained from the phylogenetic analysis indicated the presence of subclades within P. hypochondrialis, related to variations in the position of the rDNA cluster in the genome. The subclade formed by specimens from Belterra (L7) and Alta Floresta (L8) is related to the presence of NORs in the short arm of pair 8. Despite the presence of a paracentric inversion involving NOR segments in pair 8 (morph 8b) in the single specimen from Alta Floresta (L8), the 8a morph in this karyotype appears to be homologous with pair 8 in the specimens from Belterra (L9). The NOR position detected by the Ag-NOR method and the heterochromatic block detected by C-banding and DAPI staining support this conclusion. This type of rearrangement has been reported in other anurans, such as Agalychnis and Scythrophrys. Nevertheless, examination of additional specimens from the populations analyzed here would be necessary for a more conclusive understanding of NOR dynamics in these animals.
Two P. hypochondrialis subclades presented NORs in a pericentromeric position on long arm of the pair 8. One subclade included specimens from Mato Grosso – Chapada dos Guimarães (L10) and Santa Terezinha (L9) – while the ot her encompassed the populations from Amapá (L1) and Pará (L2-L6), together with Suriname, French Guiana, and Guyana. Despite this similarity, the shared trait is not phylogenetically informative for the diagnosis of the two groups.
The subclade composed of populations from Maranhão (L11-L13), Tocantins (L14), Bahia (L15), and Minas Gerais (L16) presents a complex and potentially interesting pattern of NOR variation. Within this subclade, some populations had NOR fixed in pair 7 (L11-L14), while others had NOR in pair 4 (L16). In the population from Bahia (L15), NOR was observed primarily in pair 7 (all specimens), but there was also a polymorphism in the additional rDNA clusters, which were distributed in distinct patterns in different specimens. Additional NOR has been recorded in other anurans (e.g., [19, 21–23]).
The distribution (number and position) of rDNA clusters in the genome has been used as a chromosomal marker in cytogenetic studies of a range of taxonomic groups, and in some cases, it has been useful for the discrimination of species [21, 24, 25] and the interpretation of phylogenic relationships [26–28]. However, the usefulness of this characteristic for the diagnosis of phylogenetic relationships must be assessed carefully, given that the variation in the location of NORs is not necessarily a reliable indicator of the distinction between taxa, and in some cases must be interpreted as variation between populations.
The extensive NOR variation within species and between populations observed in the present study impedes a reliable interpretation of the evolution of this characteristic in the study group. A similar pattern of NOR variation has been observed in other Phyllomedusa species, such as P. rohdei[12, 13], P. camba, P. ayeaye (Bruschi - personal observation; ), P. burmeisteri, P. tarsius, P. tetraploidea[16, 29], P. distincta[16, 29]. This suggests recurrent variation in this characteristic during the course of the evolutionary history of this group. The recurrent variation in the position of the NORs may reflect either the rapid rate of evolution of this character in this genus or a polymorphic ancestral karyotype.
Studies of a number of different taxonomic groups indicate that NOR sites represent unstable regions of the genome and therefore are important “hotspots” of rearrangement [30–36]. These clusters have a number of features in common with other rearrangement hotspot regions, such as the presence of tandem repeats , and play an important role in non-homologous recombination . The association of a transposon with clusters of rDNA is thought to contribute to the instability of the genome in these regions [38, 39].
The results of the present study reconfirm Caramaschi’s  revalidation of P. azurea as a taxon distinct from P. hypochondrialis, and reinforce the conclusions of Faivovich et al. . These results also confirm the need for a more precise morphological definition of each species, given that some P. hypochondrialis populations presented characteristics thought to be diagnostic of P. azurea. This was case of specimens from Uberlândia, Minas Gerais state, tentat ively attributed to P. azurea- in the Cerrado biome, where the species typically occurs [2, 40]) – but in Bayesian inference was recovered in P. hypochondrialis clade, suggesting a misidentification. The inclusion of the population from Água Doce (Santa Catarina) - identified by Lucas et al. (2011) as P. azurea – in ours analysis reinforced the difficulties of differentiating these taxa based on external morphology. In fact, the morphological variation observed in the specimens from Água Doce indica tes the possible presence of a species complex in P. azurea. In the present study, this population was included in the second major clade of the P. hypochondrialis group, and was paraphyletic in relation to the P. azurea haplotypes from Paraguay, Bolivia, and Argentina. The topology obtained here indicates that the identification of the Água.
Doce population must be reevaluated, preferably through the integrated analysis of chromosomal, morphological, and bioacoustic data. Interestingly, Água Doce is located within the Pampas biome, which is characterized by extensive grassland habitats , also characteristic of the ecosystems inhabited by other species included in the second subclade of the P. hypochondrialis group, such as P. megacephala, P. ayeaye, P. centralis, and P. oreades, which are also found in plateau and upland areas. The only exception is P. rohdei, found in the Brazilian Atlantic Forest.
The P. hypochondrialis samples analyzed in the present study confirmed the considerable morphological variation found among populations, and reinforced the need for more systematic phenotypic studies for the definition of reliable diagnostic features for the identification of P. hypochondrialis and P. azurea. The results of the present phylogenetic analysis also contributed to the extension of the known geographic distribution of P. hypochondrialis, previously known only from the Amazon region, to areas of open Cerrado savanna (such as Uberlândia/Minas Gerais State), including areas of possible sympatry with P. azurea. Additionally the misidentification of P. azurea may have led to mistakes in this species’ range limits.
The analysis of chromosomal markers allowed the identification of homologies and contributed to a better understanding of chromosomal evolution in this genus. Interestingly, the observed NOR variability in P. hypochondrialis reinforces the suggestion that NOR sites are hotspots of rearrangement and that the intraspecific variation in the location of these sites is either the result of processes that are not detected by classical cytogenetic methods or the remnants of a polymorphic ancestral karyotype.
Species, code in the map, collecting localities of the samples examined in morphological, phylogenetic and cytogenetic analysis
Laranjal do Jari/AP
16194; 16196; 16198; 16550
19916; 19929; 19938; 19940; 19944;
19916; 19929; 19938;
19940; 19944; 19945
19917; 19920; 19923–19925; 19927;
19917; 19930; 19932; 19942
19917; 19920; 19923–19925;
19930; 19932; 19935–19937; 19941;
19927; 19930; 19932; 19935-
19918, 19922; 19926; 19928; 19929
19918, 19922; 19926;
19914; 19945; 19919; 19921; 19933
19914; 19915; 19921
19914; 19945; 19919;
Phyllomedusa cf. hypochondrialis
Phyllomedusa cf. hypochondrialis
Chapada dos Guimarães/MT
15884; 158 86;
Phyllomedusa cf. hypochondrialis
Phyllmedusa sp. (aff. hypochondrialis)
16208; 16209; 16211;
Phyllomedusa sp. (aff. hypochondrialis)
16221; 16225; 16226; 16237;
16224; 16228; 16229;
Phyllomedusa sp. (aff.hypochondrialis)
13662; 13663; 13665
Phyllomedusa sp. (aff. hypochondrialis)
13486; 13491; 13492
13486 - 13495; 13506-13508
Phyllomedusa sp. (aff. hypochondrialis)
15882; 15889–15894; 13474
The two principal morphological characters identified by Caramaschi  for the diagnosis of P. azurea and P. hypochondrialis were evaluated in the present study: (1) the presence of a narrow white stripe on the upper lip and (2) the presence/absence and configuration of the green stripe on the upper surface of the thighs. All specimens collected were analyzed and photographed in a Zeiss stereomicroscope. Cytogenetic and molecular analyses of all specimens were conducted to assess the reliability of the morphology-based diagnosis.
Isolation, amplification, and sequencing of DNA
Genomic DNA was extracted from liver or muscle tissues and stored at −70°C in the tissue bank at the Department of Structural and Structural Biology of the Campinas State University (Unicamp) in Campinas, São Paulo, Brazil, using t he TNES method as applied by Bruschi et al. . The mitochondrial tRNA-Val, 12S and 16S ribosomal genes were amplified using the primers MVZ 59 (L), MVZ 50 (H), 12L13, Titus I (H), Hedges16L2a, Hedges16H10, 16Sar-L and 16Sbr-H (for primer sequences, see reference ). The nuclear gene RAG-1 was amplified using the primers RAG-1R and RAG-1F . The amplified PCR products were purified with a GFX PCR and Gel Band DNA Purification kit (GE Healthcare, England) and used directly as templates for sequencing in an automatic ABI/Prism DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator kit (Applyed Biosystems, Foster City, CA, USA), as recommended by the manufacturer. The DNA sequences were sequenced bi-directionally, edited in Bioedit version 7.0.1 (http://www.mbio.ncsu.edu/BioEdit/ bioedit.html), and aligned using Clustal W.
Analysis of molecular data
The phylogenetic relationships among the populations were inferred from the concatenated matrix of the mitochondrial DNA 12S, tRNAval, and 16S rDNA sequences and nuclear Rag-1 gene (totalized 2920pb). To evaluate this approach, we selected 39 specimens analyzed by morphology and cytogenetic methods, representing at least one specimens to each morphotype observed within population. The data matrix was complemented with 70 Phyllomedusa sequences available in GenBank (Additional file 1). The species used as outgroups were Hylomantis hulli and Phasmahyla guttata, which were chosen based on the topology reported by Faivovich et al. . The sequence was aligned using the Clustal W program . The initial alignments were checked by eye and adjusted wherever necessary. Phylogenetic trees were constructed using Bayesian inference, based on the Markov chain Monte Carlo (MCMC) analysis, in MrBayes 3.1.2  with two independent runs, each with four chains and sampling every 1000 generations for 6 million generations. The adequate burn-in (the first 25% trees excluded) was determined by examining a plot of the likelihood scores of the heated chain for convergence and stationary. The most appropriate evolutionary model selected by Modeltest 3.7 for the BI analysis was the GTR+R+I model .
Based in morphotype variation, we selected 110 specimens (complete description to specimens used are describes in Table 2) to submitted cytogenetic methods, representing each one of the morphotypes found in the screened populations. Metaphase cells were obtained from intestines and testes of animals previously treated with 2% colchicine, following procedures modified from King and Rofe  and Schmid . Prior to the removal of the intestine and testes, the animals were anesthetized profoundly. Cell suspensions were dripped onto clean plates and stored at −20°C. The chromosomes were stained with 10% Giemsa, silver stained by the Ag–NOR method , and C-banded . In two populations (L7 and L8), the C-banded chromosomes were also stained with DAPI (500 μg/mL), after Giemsa distaini ng with ethanol, to better characterize the heterochromatin. 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 measured and the centromere index (CI), relative length (RL), and centromere ratio (CR) were estimated. The chromosomes were ranked and classified according to the scheme of Green and Sessions .
Nucleolar organizer region
We thank the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP; grants 2010/11300-7 and 2010/17464-1), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; PROAP), and Sérgio Siqueira for helping collect frog specimens in Santa Terezinha, Mato Grosso and Janaina Reis Lima collect frog specimens in Laranjal do Jari, Amapá. We also thank Luciana Bolsoni Lourenço, Cíntia Pelegrineti Targueta de Azevedo Brito, and Ana Cristina Prado Veiga-Menoncello for discussions and/or information provided.
- Cruz CAG: Sobre as relações intergenéricas de Phyllomedusinae da Floresta Atlântica (Amphibia, Anura, Hylidae). Rev Bras Biol. 1982, 50: 709-726.Google Scholar
- Caramaschi U: Redefinição do grupo de Phyllomedusa hypochondrialis, com redescrição de P. megacephala (Miranda-Ribeiro, 1926), revalidação de P. azurea Cope, 1826 e descrição de uma nova espécie (Amphibia, Anura, Hylidae). Arq Mus Nac. 2006, 64: 159-179.Google Scholar
- Faivovich J, Haddad CFB, Garcia PCA, Frost DR, Campbell JA, Wheeler WC: A systematics review of the frog family Hylidae, with special reference to the Hylinae, a phylogenetic analysis and taxonomic revision. Bul Am Nat Hist. 2005, 294: 1-240. 10.1206/0003-0090(2005)294[0001:SROTFF]2.0.CO;2.View ArticleGoogle Scholar
- Faivovich J, Haddad CFB, Baêta D, Jungfer KH, Álvares GFRA, Brandão RA, Sheil C, Barrientos LS, Barrio-Amós CL, Cruz CAG, Wheeler WC: The phylogenetic relationships of the charismatic poster frogs, Phyllomedusinae (Anura, Hylidae). Cladistics. 2010, 25: 1-35.Google Scholar
- Frost DR: Amphibian Species of the World: An online reference. http://research.amnh.org/vz/herpetology/amphibia/,
- Brandão RA, Álvares GFR: Remarks on “a new Phyllomedusa Wagler (Anura: Hylidae) with reticulated pattern on flanks from Southeastern Brazil”. Zootaxa. 2044, 2009: 61-64.Google Scholar
- Giaretta AA, Filho JCO, Kokubum MN: A new Phyllomedusa Wagler (Anura, Hylidae) with reticulated pattern on flanks from Southeastern Brazil. Zootaxa. 2007, 1614: 31-41.Google Scholar
- Baêta D, Caramaschi U, Cruz CAG, Pombal JP: Phyllomedusa itacolomi Caramaschi, Cruz & Feio, 2006, a junior synonym of Phyllomedusa ayeaye (B. Lutz, 1966) (Hylidae, Phyllomedusinae). Zootaxa. 2226, 2226: 58-65.Google Scholar
- Caramaschi U, Cruz CAG, Feio RN: A new species of Phyllomedusa Wagler, 1830 from the state of Minas Gerais, Brazil (Amphibia Anura, Hylidae). Bol Mus Nac. 2007, 524: 1-8.Google Scholar
- Calderon LA, Messias MR, Serrano RP, Zaqueo KD, Souza ES, Nienow SS, Cardozo-Filho JL, Diniz-Souza R, Delaix-Zaqueo K, Stabeli RG: Amphibia, Anura, Hylidae, Phyllomedusinae, Phyllomedusa azurea: Distribution extension and geographic distribution map. Check List. 2009, 5 (2): 317-319.Google Scholar
- Lucas EM, Fortes VB, Garcia PCA: Amphibia, Anura, Hylidae, Phyllomedusa azurea Cope, 1862: Distribution extension to southern Brazil. Check List. 2010, 6 (1): 164-166.Google Scholar
- Paiva CR, Nascimento J, Silva APZ, Bernarde OS, Ananias F: Karyotypes and Ag-NORs in Phyllomedusa camba De La Riva, 1999 and P. rohdei Mertens, 1926 (Anura, Hylidae, Phyllomedusinae): cytotaxonomic considerations. Ital J Zool. 2010, 77: 116-121. 10.1080/11250000903187585.View ArticleGoogle Scholar
- Barth A, Solé M, Costa MA: Chromosome polymorphism in Phyllomedusa rohdei populations (Anura, Hylidae). J Herpetol. 2009, 43: 676-679. 10.1670/08-210.1.View ArticleGoogle Scholar
- Bruschi DP, Busin CS, Siqueira S, Recco-Pimentel SM: Cytogenetic analysis of two species in the Phyllomedusa hypochondrialis group (Anura, Hylidae). Hereditas. 2012, 149: 34-40. 10.1111/j.1601-5223.2010.02236.x.View ArticlePubMedGoogle Scholar
- Morando M, Hernando A: Localización cromosómica de genes ribosomales activos em Phyllomedusa hypochondrialis y P. sauvagii (Anura, Hylidae). Cuad Herpetol. 1997, 11: 31-36.Google Scholar
- Kasahara S, Campos JRC, Catroli GF, Haddad CFB: Cytogenetics of Phyllomedusa distincta (2n=2x=26), Phyllomedusa tetraploida (2n=4x=52) and their triploid hybrids (2n=3x=39) [abstract]. Chrom Res. 2007, 5: s10-Google Scholar
- Beçak ML, Denaro L, Beçak W: Polyploidy and mechanisms of karyotypic diversification in Amphibia. Cytogenetics. 1970, 9: 225-238. 10.1159/000130093.View ArticlePubMedGoogle Scholar
- Haddad CF, Pombal-Jr JP, Batistic RF: Natural hybridization between diploid and tetraploid species of leaf-frogs, genus Phyllomedusa (Amphibia). J Herpetol. 1994, 28: 425-430. 10.2307/1564953.View ArticleGoogle Scholar
- Schmid M, Feichtinger W, Weimer R, Mais C, Bolaños F, León P: Chromosome banding in Amphibia XXI. Inversion polymorphism and multiple nucleolus organizer regions in Agalychnis callidryas (Anura, Hylidae). Cytogenet Cell Genet. 1995, 69: 18-26. 10.1159/000133929.View ArticlePubMedGoogle Scholar
- Lourenço LB, Garcia PC, Recco-Pimentel SM: Cytogenetics of a new species of the Paratelmatobius cardosoi group (Anura: Leptodactylidae) with the description of an apparent case of pericentric inversion. Amphibia-Reptilia. 2003, 24 (1): 47-55. 10.1163/156853803763806939.View ArticleGoogle Scholar
- Busin CS, Lima AP, Strussmann C, Siqueira-Jr S, Recco-Pimentel SM: Chromosomal differentiation of populations of Lysapsus limellus limellus and L. l. bolivianus, and of Lysapsus caraya (Hylinae, Hylidae). Micron. 2006, 37: 355-362. 10.1016/j.micron.2005.11.009.View ArticlePubMedGoogle Scholar
- Silva APZ, Haddad CFB, Kasahara S: Nucleolus organizer in Physalaemus cuvieri (Anura, Leptodactylidae), with evidence of a unique case of Ag-NOR variability. Hereditas. 1999, 131: 135-141.View ArticlePubMedGoogle Scholar
- Targueta CP, Rivera M, Souza MB, Recco-Pimentel SM, Lourenço LB: Cytogenetics contributions for the study of the Amazonian Engystomops (Anura: Leiuperidae) assessed in the light of phylogenetic relationships. Mol Phyl Evol. 2010, 54: 709-725. 10.1016/j.ympev.2009.10.018.View ArticleGoogle Scholar
- Cazaux B, Catalan J, Veyrunes F, Douzery EJP, Britton-Davidian J: Are ribosomal DNA clusters rearrangement hotspots? A case in the genus Mus (Rodentia, Muridae). BMC Evol Biol. 2011, 11: 124-10.1186/1471-2148-11-124.PubMed CentralView ArticlePubMedGoogle Scholar
- Britton-Davidian J, Cazaux B, Catalan J: Chromosomal dynamics of nucleolar organizer regions (NORs) in the house mouse: micro-evolutionary insights. Heredity. 2012, 108: 68-74. 10.1038/hdy.2011.105.PubMed CentralView ArticlePubMedGoogle Scholar
- Medeiros LR, Rossa-Feres DC, Recco-Pimentel SM: Chromosomal differentiation of Hyla nana and Hyla sanborni (Anura, Hylidae) with a description of NOR polymorphism in H. nana. J Hered. 2003, 94: 149-154. 10.1093/jhered/esg019.View ArticlePubMedGoogle Scholar
- Siqueira S, Aguiar O, Pansonato A, Giaretta AA, Strüssmann C, Martins I, Recco-Pimentel SM: The karyotype of three Brazilian Terrarana frogs (Amphibia, Anura) with evidence of a new Barycholos species. Genet Mol Biol. 2009, 32 (3): 470-476. 10.1590/S1415-47572009005000044.PubMed CentralView ArticlePubMedGoogle Scholar
- Andreone F, Aprea G, Vences M, Odierna G: A new frog of the genus Mantidactylus from the rainforests of North-Eastern Madagascar, and its karyological affinities. Amphibia-Reptilia. 2003, 24: 285-303. 10.1163/156853803322440763.View ArticleGoogle Scholar
- Nguyen TT, Aniskin VM, Gerbault-Seureau M, Planton H, Renard JP, Nguyen BX, Hassanin A, Volobouev VT: Phylogenetic position of the saola (Pseudoryx nghetinhensis) inferred from cytogenetic analysis of eleven species of Bovidae. Cytogenet Genome Res. 2008, 122: 41-54. 10.1159/000151315.View ArticlePubMedGoogle Scholar
- Nguyen P, Sahara K, Yoshio A, Marec F: Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera). Genetica. 2010, 138: 343-354. 10.1007/s10709-009-9424-5.View ArticlePubMedGoogle Scholar
- Batistic RF: Aspectos citogenéticos da evolução em Phyllomedusa (Anura-Amphibia). PhD thesis. 1989, Ribeirão Preto, São Paulo: Faculdade de Medicina, USP,Google Scholar
- Wiley JE, Little ML, Romano MA, Blount DA, Cline CR: Polymorphism in the location of the 18S and 28S rDNA genes in chromosomes of the diploid-tetraploid tree frogs Hyla chrysoscelis and Hyla versicolor. Chromosoma. 1989, 97: 481-487. 10.1007/BF00295033.View ArticleGoogle Scholar
- Kaiser H, Mais C, Bolaños F, Steinlein C, Feichtinger W, Schmid M: Chromosomal investigation of three Costa Rica frogs from the 30-chromosome radiation of Hyla with description of a unique geographic variation in nucleolus organizer regions. Genetica. 1996, 98: 95-102. 10.1007/BF00120223.View ArticleGoogle Scholar
- Datson PM, Murray BG: Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism?. Chrom Res. 2006, 14: 845-857. 10.1007/s10577-006-1092-z.View ArticlePubMedGoogle Scholar
- Cabrero J, Camacho JPM: Location and expression of ribosomal RNA genes in grasshoppers: abundance of silent and cryptic loci. Chrom Res. 2008, 16: 595-607. 10.1007/s10577-008-1214-x.View ArticlePubMedGoogle Scholar
- Huang J, Ma L, Fei S, Li L: 45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp. Plos One. 2008, 3: e2167-10.1371/journal.pone.0002167.PubMed CentralView ArticlePubMedGoogle Scholar
- Stankiewicz P, Lupski JR: Genome architecture, rearrangements and genomic disorders. Trends Genet. 2002, 18: 74-81. 10.1016/S0168-9525(02)02592-1.View ArticlePubMedGoogle Scholar
- Schubert I, Wobus U: In situ hybridization confirms jumping nucleolus organizing regions in Allium. Chromosoma. 1985, 92: 143-148. 10.1007/BF00328466.View ArticleGoogle Scholar
- Raskina Q, Barber JC, Nevo E, Belyayev A: Repetitive DNA and chromosomal rearrangements: speciation-related events in plant genomes. Cytogent Genome Res. 2008, 120: 351-10.1159/000121084.View ArticleGoogle Scholar
- Prado VHM, Borges RE, Silva FR, Tognolo TT, Rossa-Feres DC: Amphibia, Anura, Hylidae, Phyllomedusa azurea: Distribution extension. Check List. 2008, 4 (1): 55-56.View ArticleGoogle Scholar
- Bencke GA, Mauricio GN, Develey PF, Goerck JM: Áreas Importantes Para a Conservação das Aves no Brasil: Parte 1 – Estados do Domínio da Mata Atlântica. 2006, São Paulo: SAVE BrasilGoogle Scholar
- Goebel AM, Donnelly JM, Atz ME: PCR primers and amplification methods for 12S ribosomal DNA, the control region, cytochrome oxidase I, and cytochrome b in bufonids and other frogs, and an overview of PCR primers which have amplified DNA in amphibians successfully. Mol Phylogenet Evol. 1999, 11: 163-199. 10.1006/mpev.1998.0538.View ArticlePubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680. 10.1093/nar/22.22.4673.PubMed CentralView ArticlePubMedGoogle Scholar
- Roquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003, 19: 1572-1574. 10.1093/bioinformatics/btg180.View ArticleGoogle Scholar
- Posada D, Crandall KA: Model Test: testing the model of DNA substitution. Bioinformatics. 1998, 14: 817-818. 10.1093/bioinformatics/14.9.817.View ArticlePubMedGoogle Scholar
- King M, Rofe R: Karyotypic variation in the Australian gekko Phyllodactylus marmoratus (Gray) (Gekkonidae: Reptilia). Chromosoma. 1976, 54: 75-87. 10.1007/BF00331835.View ArticlePubMedGoogle Scholar
- Schmid M: Chromosome banding in Amphibia. I. Constitutive heterochromatin and nucleolus organizer regions in Bufo and Hyla. Chromosoma. 1978, 66: 361-368. 10.1007/BF00328536.View ArticleGoogle Scholar
- Howell WM, Black DA: Controlled silver staining of nucleolar organizer regions with a protective colloidal developer: a −1 step method. Experientia. 1980, 36: 1014-1015. 10.1007/BF01953855.View ArticlePubMedGoogle Scholar
- Sumner AT: A simple technique for demostrating centromeric heterochromatin. Exp Cell Res. 1972, 83: 438-442.View ArticleGoogle Scholar
- Green DM, Sessions SK: Nomenclature for chromosomes. Amphibian cytogenetics and evolution. 1991, San Diego: Academic Press, 431-432.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.