And H. Semilineatus Species Groups

Short Communication

Patterns of ribosomal DNA distribution in hylid frogs from the Hypsiboas faber and H. semilineatus species groups

Rosana dos Reis Abrante Nunes and Valéria Fagundes Departamento de Ciências Biológicas, Centro de Ciências Humanas e Naturais, Universidade Federal do Espírito Santo, Vitória, ES, Brazil.

Abstract We performed a comparative analysis of the nucleolus organizer region (NOR) distribution in the karyotypes of hylid frogs from two different taxonomic groups, Hypsiboas faber and H. semilineatus. Silver nitrate staining of NORs (Ag-NORs) and fluorescence in situ hybridization (FISH) with a rDNA probe were used to investigate the chromosomal location of rDNA loci in two species. The karyotype of H. semilineatus and the Ag-NORs distribution of the four species are presented herein for the first time. After conventional staining, the four species presented very similar karyotypes with 2n = 24, but Ag-NORs analyses revealed species-specific characteristics. H. albomarginatus, H. faber and H. semilineatus had one pair of interstitial Ag-NORs in the short arm of pairs 2, 11, and in the long arm of pair 7, respectively. H. pardalis presented telomeric NORs in the long arm of pair 11. Ag-NORs were heteromorphic in three of the species (H. pardalis, H. semilineatus and H. albomarginatus) and FISH confirmed the differential activity of rDNA genes in H. semilineatus. In the present study, 2n = 24 karyotypes could be distinguished by their Ag-NORs distribution. Our results further the knowledge about the cytogenetics of hylids from Brazil. Key words: hylids, Hypsiboas, karyotypes, FISH, Ag-NORs, Hypsiboas semilineatus, H. faber. Received: February 18, 2008; Accepted: June 17, 2008.

Until recently, the family Hylidae was believed to Anuran amphibians are frequently considered as a contain 42 genera arranged into four subfamilies: Pelodry- karyologically conservative group. In the family Hylidae, a adinae, Hemiphractinae, Hylinae and Phyllomedusinae. basic karyotype with 2n = 24, morphologically similar Nevertheless the many taxonomic and systematic questions chromosomes and no differentiated sex chromosomes has led Faivovich et al. (2005) to review hylids systematics, been described. About 25% of the known anuran species evaluate the monophyly of the named taxa and examine the had their karyotype described until the 90s (Kuramoto, basis of the existing taxonomy. They performed a phylo- 1990; King, 1990), but the studies were mainly based on genetic analysis using mostly DNA sequence data in order conventional staining analyses providing insufficient data to test the monophyly of the Hylidae. They were able to de- to characterize species of hylids. More recent studies, termine the constituent taxa of Hylidae, with special atten- which included chromosome banding (mainly C-banding tion to the genera and species of the subfamily Hylinae. and BrdU incorporation) and localization of the nucleolus According to Faivovich et al. (2005), the family Hylidae in- organizer regions (NORs), revealed some minor differ- cludes 877 species, divided into three subfamilies: Hylinae, ences among species, which became important tools in Pelodryadinae, and Phyllomedusinae. These authors pro- comparative chromosome analyses (Baldissera et al., 1993; posed taxonomic changes for some Hylinae, in which the Schmid et al., 1995; Silva et al., 2000; Kasahara et al., species previously included in the genus Hyla were rear- 2003; Ananias et al., 2004; Raber et al., 2004). ranged into 16 genera. According to this revision, Hyla Interindividual variation and size heteromorphisms albomarginata, Hyla semilineata, Hyla pardalis and Hyla of the NORs detected by silver nitrate staining (Ag-NORs) faber must be referred to as Hypsiboas albomarginatus frequently allow the karyological distinction of hylids. Ac- Spix, 1824, Hypsiboas semilineatus Spix, 1824, Hypsiboas cording to Schmid (1978; 1982), the Ag-NORs frequently pardalis Spix, 1824 and Hypsiboas faber Wied- occur at the same chromosomal location in the karyotypes Neuwied,1821, respectively. of species of the same group or in groups of related species, reinforcing the importance of extensive karyological analy- Send correspondence to Valéria Fagundes. Laboratório de Gené- ses to characterize the karyotypes of the group. tica Animal, Departamento de Ciências Biológicas, Centro de Ciên- cias Humanas e Naturais, Universidade Federal do Espírito Santo, In this work, we aimed to compare the karyotypes and Av. Marechal Campos 1468, Maruípe, 29040-90 Vitória, ES, Brazil. Ag-NORs patterns of four hylid species from the Atlantic E-mail: [email protected] or [email protected]. Forest of southeastern Brazil. The species studied belong to Nunes and Fagundes 983 two groups: the H. semilineatus group (H. semilineatus) per. Ten metaphases per species had their chromosomes and the H. faber group (H. albomarginatus, H. faber and H. measured (in millimeters) to determine the chromosome pardalis). We also intended to verify if the Ag-NORs pat- relative length (RL) and the centromeric ratio (CR), i.e. the terns were consistent with the monophyletic clades identi- proportion between the short and the long arms. We fied by molecular and morphological traits. We expected adopted the nomenclature for chromosome morphology each species to present a distinctive Ag-NORs distribution proposed by Green and Sessions (1991), which is based on which would be useful to distinguish individual species or the centromeric ratio. Fluorescence in situ hybridization species groups of hylids. (FISH) was performed with the biotin-labeled probe HM123, which contains fragments of the 18S and 28S Our sample consisted of 13 specimens of species in- rDNA of Xenopus leavis (Meunier-Rotival et al., 1979). H. cluded in the hylid species groups H. semilineatus (H. semilineatus and H. pardalis metaphases were hybridized semilineatus) and H. faber (H. albomarginatus, H. faber with the probe following the protocol described by Viégas and H. pardalis). The animals were collected in four locali- – Péquignot (1992). ties of the state of Espírito Santo, Brazil (Table 1). They were treated with a solution of 0.1% colchicine (1 mL/100 g All specimens exhibited 2n = 24 and FN = 48 (Figure of body weight) during 4-6 h prior to the sacrifice. Mitotic 1a-d), but comparative analyses revealed some differences chromosome preparations were obtained from the intestine in the relative length (RL) and chromosome morphologies by the squash technique described by Bogart (1973a). among the karyotypes of the four species, even though they Briefly, the intestine was immersed for 30 min in distilled were all exclusively composed of biarmed chromosomes water and then fixed in an ethanol/acid acetic solution. The (Figure 1). No sex chromosomes were observed in any of intestine was fragmented with a scalpel onto a glass slide the karyotypes. The chromosomes varied gradually in size, with a few drops of the fixative and then squashed with a with pair 1 corresponding to 18-19% and pair 12 corre- glass coverslip. The slide was immersed in liquid nitrogen sponding to 2-3% of the genome. Pair 6 of Hypsiboas faber for about one minute and the coverslip was removed. The was slightly smaller than the same chromosome pair in the slide was then immersed in 90% ethanol for about one min- other species (Table 2). ute and air-dried. For conventional staining, the chromo- The karyotype of H. semilineatus (HSE) is described some preparations were hydrolyzed in HCl 1N at 60 °C for herein for the first time. It is composed of five pairs of 10 min and stained with a 3% Giemsa solution for 10 min. metacentric chromosomes (pairs 1, 2, 10, 11 and 12), three Ag-NORs staining followed Howell and Black (1980). pairs of submetacentrics (pairs 4, 5 and 8) and four pairs of Metaphases of each species were analyzed to determine the subtelocentrics (pairs 3, 6, 7 and 9). diploid number (2n) and the number of chromosome arms Hypsiboas albomarginatus (HAL) presented six pairs (fundamental number, FN). The metaphases were photo- of metacentric chromosomes (pairs 1, 2, 3, 10, 11 and 12), graphed and copies were made in Kodak photographic pa- five pairs of submetacentrics (pairs 4, 5, 7, 8 and 9) and one

Table 1 - Analyzed specimens of Hypsiboas from the state of Espírito Santo, Brazil.

Species group Species Animal Sex1 Municipality Number of cells analyzed2 CS Ag-NOR Total H. faber H. albomarginatus LGA 221 M Anchieta 13 9 22 LGA 586 M Cariacica 10 8 18 LGA 587 M Cariacica 14 6 20 H. faber LGA 202 M Pedra Azul 14 7 21 LGA 1178 M Pedra Azul 10 4 14 H. pardalis LGA 209 M Cariacica 22 6 28 LGA 208 M Cariacica 15 6 21 LGA 287 M Cariacica 12 5 17 LGA 289 M Cariacica 16 10 26 H. semilineatus H. semilineatus LGA 132 M Santa Teresa 15 7 22 LGA 133 M Santa Teresa 27 12 39 LGA 145 ND Santa Teresa 19 9 28 LGA 148 ND Santa Teresa 10 5 15 Total 13 197 94 291

1M = Male; ND = not determined; 2CS – number of cells analyzed after conventional staining; Ag-NORs – number of cells analyzed after Ag-NOR staining. 984 rDNA distribution in hylid frogs

Table 2 - Relative chromosome length (RL), centromeric ratio (CR) and morphology (CP) of the mitotic chromosomes of Hypsiboas albomarginatus (HAL), H. semilineatus (HSE), H. pardalis (HPA) and H. faber (HFA).

Pair HSE HAL HPA HFA 1 RL 19.27 17.88 16.67 18.23 CR 1.14 0.93 1.01 0.94 CPmmmm 2 RL 15.03 13.41 13.25 14.81 CR 1.25 1.56 1.25 0.82 CPmmmm 3 RL 11.18 13.41 11.97 12.54 CR 4.20 1.54 1.80 2.17 CP st m sm sm 4 RL 9.63 9.20 11.54 10.83 CR 2.65 2.25 3.50 5.17 CP sm sm st st 5 RL 9.25 7.98 10.04 9.12 CR 2.22 2.90 2.57 1.96 CP sm sm sm sm 6 RL 9.25 7.15 8.12 6.27 CR 6.00 4.10 4.50 3.75 CP st st st st 7 RL 7.32 6.07 5.34 5.70 CR 3.75 2.17 2.79 3.50 CP st sm sm st 8 RL 5.39 5.75 5.13 5.70 CR 2.00 2.00 2.25 2.33 CP sm sm sm sm 9 RL 5.01 5.11 5.13 5.70 CR 3.00 1.75 1.17 0.83 CP st sm m m 10 RL 3.28 5.11 5.13 4.56 CR 1.38 1.42 2.00 2.50 CP m m sm sm Figure 1 - a-d) Karyotypes with 2n = 24, FN = 48 after conventional staining: a) Hypsiboas albomarginatus,b)H. semilineatus,c)H. pardalis and 11 RL 3.08 5.11 4.27 3.42 d) H. faber; e-g) Pair 2 in H. albomarginatus: e) secondary constriction CR 1.33 1.00 1.33 3.50 (arrow), f) homomorphic Ag-NORs at the short arms, g) heteromorphic CP m m m st Ag-NORs with an extra NOR in one homologue (arrow); h-i) Pair 11 of H. pardalis: h) homomorphic Ag-NORs, terminal in the short arms, i) homo- 12 RL 2.31 3.83 3.42 3.13 morphic FISH signals with rDNA probes (arrows); j-l) Pair 7 in H. CR 1.00 1.00 1.00 1.00 semilineatus: j) secondary constriction (arrow), k) heteromorphic CPmmmm Ag-NORs, l) homomorphic FISH signals with rDNA probes (arrows); m) Pair 11 of H. faber with interstitial heteromorphic Ag-NORs in the long m = metacentric; sm = submetacentric and st = subtelocentric. arms (arrow). subtelocentrics (pairs 4 and 6). The karyotype of Hyla pair of subtelocentrics (pair 6). The karyotypes of H. pardalis from southeastern Brazil previously described by albomarginatus from Espírito Santo were similar to those Bogart (1973b) is similar to the one that we observed of found in specimens from São Paulo identified as Hyla Hypsiboas pardalis from Espírito Santo. albomarginata by Beçak (1968). H. faber (HFA) presented four pairs of metacentric H. pardalis (HPA) presented five pairs of metacentric chromosomes (pairs 1, 2, 9, and 12), four pairs of submeta- chromosomes (pairs 1, 2, 9, 11 and 12), five pairs of centrics (pairs 3, 5, 8 and 10) and four pairs of subtelo- submetacentrics (pairs 3, 5, 7, 8 and 10) and two pairs of centrics (pairs 4, 6, 7 and 11). The karyotype of Hyla faber Nunes and Fagundes 985 from São Paulo described by Beçak (1968) is similar to that morphic Ag-NORs and in 69% of them the size differences described herein for Hypsiboas faber. could be attributed to tandem duplications or triplications A comparative analysis between the four species affecting one of both rDNA clusters. karyotypes revealed some conserved chromosome pairs We observed Ag-NORs heteromorphisms in three of (pairs 1, 2, 5, 6, 8, and 12), contrasting with some variable the four species investigated (H. semilineatus, H. ones (pairs 3, 4, 7, 9, 10 and 11). Some more suggestive dif- albomarginatus and H. faber). The difference in the size of ferences observed between homologues are more likely due the Ag-NORs by silver staining of H. semilineatus was not to variations from the squashing technique than to real confirmed by FISH with the rDNA probe, suggesting that heteromorphisms. Pair 3 varied from subtelocentric (HSE) the Ag-NORs heteromorphism is caused by differential to submetacentric (HPA and HFA) and metacentric (HAL). gene activity (and consequent differential accumulation of Pair 4 was submetacentric (HSE and HAL) or subtelo- ribonucleoproteins). The duplicated Ag-NORs sites in pair centric (HPA and HFA). Pair 7 was submetacentric (HAL 2ofH. albomarginatus were also not tested by FISH. We and HPA) or subtelocentric (HSE and HFA). Pair 9 was were unable to determine if the Ag-NORs size hetero- subtelocentric (HSE), submetacentric (HAL) or metacen- morphism observed in H. faber was due to variation in the tric (HPA and HFA). Pair 10 was metacentric (HSE and number of ribosomal genes or to their differential activity, HAL) or submetacentric (HPA and HFA) and pair 11 was since FISH results were not obtained. metacentric (HSE, HAL and HPA) or subtelocentric We observed duplicated Ag-NOR in H. (HFA). albomarginatus. Duplicated Ag-NORs were previously re- Although a single chromosome pair presented an ported by Schmid et al. (1995) in the frog Agalychnis Ag-NOR in all metaphases from all the specimens, it was callidryas. Possible mechanisms responsible for the disper- not the same pair in all species. Pairs 2 and 7 are the sion of NOR sites in anuran genomes have been discussed NORs-bearing chromosomes in H. albomarginatus and H. by some authors (Wiley et al., 1989, King 1990, Foote et semilineatus, respectively, and present a secondary con- al., 1991, Schmid et al. 1995, Kaiser et al., 1996). They striction, not always observed on both homologues (Figure suggested that NORs transposition could have occurred by 1e and 1j), which coincided with the location of rDNA gene the movement of mobile genetic elements closely linked to clusters. NOR cistrons, amplifications of “orphan” rDNA cistrons, In H. albomarginatus, the Ag-NORs were intersti- reinsertion errors during extrachromosomal amplification tially located at the short arm of pair 2 (Figure 1e-f). In all of ribosomal cistrons, or chromosomal rearrangements metaphases both homologues had positive Ag-NORs sig- such as translocations and inversions involving the seg- nals. One specimen (LGA 221) exhibited an additional in- ments containing NORs. Oliveira et al. (1996) proposed terstitial Ag-NOR, proximal to the short arm of one that pericentric inversions could split NOR cistrons result- homologue of pair 2 (Figure 1g). ing in two Ag-NORs with about half of the regular size. H. pardalis presented one pair of telomeric Ag-NORs Considering the similar sizes of each of the duplicate in the short arms of pair 11 (Figure 1h). There was an exact Ag-NORs in H. albomarginatus, it is unlikely that peri- correspondence in the position and size of the NORs after centric inversions involving the rDNA region occurred in FISH and silver staining (Figure 1i). this species. We are thus inclined to attribute the duplica- All four specimens of H. semilineatus presented one tion to an intrachromosomal duplication (likely due to mo- interstitial Ag-NOR in the long arm of pair 7 (Figure 1j-k). bile elements). A conspicuous difference in the size of the Ag-NORs be- However, it is important to emphasize that the Ag- tween homologues was observed in all specimens. Despite NOR technique detected heterochromatic regions in some of the heteromorphism in the Ag-NORs, the size of signals species (Lourenço et al., 1998). Such regions were easily of the rDNA probes observed after FISH were similar on distinguished from the NORs sites by the intensity of silver both homologues (Figure 1l). impregnation, which resulted in black dots in true NORs Both specimens of H. faber presented interstitial Ag- and in brown sections in heterochromatic regions (Lou- NORs in the long arms of pair 11, which showed renço et al., 1998). We did not observe brownish staining in heteromorphism after silver-nitrate staining (Figure 1m). the extra signal, which allows us to discard that it is a sil- Nucleolus organizer regions have been considered ver-stained heterochromatic region. Moreover, the speci- important markers for the study of chromosome evolution men with the heteromorphic duplicated Ag-NOR was in amphibians (Lourenço et al., 1998). Although intra- collected in a locality different from that where the animals specific variation of NORs location is rare (Schmid, 1978), with non-duplicated Ag-NORs were found. New data on interindividual heteromorphisms of Ag-NORs size were more individuals collected in the same locality would help widely observed in anurans. In an extensive study, Schmid, to verify a possible regional variation. (1982) used silver staining and GC-specific fluorochromes Although the NORs location may be used to charac- to analyze the NORs of 260 individuals from 23 genera of terize species of amphibians, our analysis revealed that anurans. In this study, 67% of the animals presented hetero- NORs are more useful to discriminate groups of species 986 rDNA distribution in hylid frogs rather than species with similar 2n = 24 karyotypes. Anuran ing the rDNA cluster from a telomeric to an interstitial posi- species frequently revealed only a single NOR-bearing tion. chromosome pair in diploid karyotypes (Schmid, 1982; H. albomarginatus (present paper), also included in Mahony and Robinson, 1986; Anderson, 1991). Schmid H. faber group, presented NORs in a different chromosome (1978; 1982) observed that the NORs usually occur at the pair (pair 2). Although we only analyzed conventionally same chromosomal location in the karyotypes of the spe- stained chromosomes we do not think that it is possible to cies of the same group or in groups of related species. Ex- misidentify pairs 2 and 11, and we believe that distinct ceptions to this rule indicate that rearrangements may have chromosome pairs are NOR-bearing in each of these two contributed to the species evolution (Schmid, 1978). clades. We plotted our data on Ag-NORs location on Faivo- In the H. pulchellus group, a sister group of H. faber, vich et al. (2005) phylogenetic tree and we observed that the species H. semiguttatus and H. joaquini (Ananias et al., each monophyletic clade had the ribosomal cistron located 2004) grouped in a monophyletic clade and shared the pres- in a specific chromosome pair (Figure 2). ence of a telomeric Ag-NOR in pair 1. H. prasinus and H. H. pardalis (present study), H. faber (present study) pulchellus (Ananias et al., 2004) form another clade in the and H. crepitans (Gruber et al., 2006), which belong to a H. pulchellus group and shared the presence of Ag-NORs monophyletic clade and are included in the H. faber group, in pair 12. H. guentheri, H. bischoffi (Raber et al., 2004) shared the Ag-NORs at the pair 11. Differences of the rela- and H. marginatus (Ananias et al., 2004) are arranged in a tive size of chromosome 11, as specified for H. pardalis third monophyletic clade species group and present Ag- and H. faber in Table 2, are not rare. The variation in the NORs in pair 10, at the telomeric position of the long arm of NORs location (terminal in the short arm of a metacentric H. guentheri and H. bischoffi, and at the pericentromeric re- pair in H. pardalis, interstitial in the long arm of a subtelo- gion in H. marginatus. These differences in NORs location centric pair in H. faber and interstitial in the long arm of a can be explained by pericentric inversion. submetacentric pair in H. crepitans) can be explained by a Schmid (1978, 1982) stated that the NORs were re- single pericentric inversion in these chromosomes, chang- currently observed at the same chromosomal location in the karyotypes of species from the same groups and that chromosome translocations could be frequent in the evolution of the group. Our data corroborate this assumption and sug- gest that: (1) inversions also contributed to chromosome changes and NORs transpositions; (2) Ag-NORs present a pattern in hylid frogs species groups (as observed in the H. faber group) or inside clades of species groups (as in the H. pulchellus group). These data may be useful in the cyto- taxonomic and evolutionary studies of hylids.

Acknowledgments This work was supported by the Conselho Nacional Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, RRAN Masters scholarship). We thank the Instituto Brasi- leiro do Meio Ambiente e dos Recursos Naturais Renová- veis for the license to collect (IBAMA n. 02009.00-1492/00, license 021/2000 DIFAS), and Pedro Luis Peloso and João Luís Gasparini for collecting and identifying some specimens. We are also grateful to Dr. S. Recco-Pimentel for providing the conditions in the labora- tory to carry out the FISH experiments.

References Ananias F, Garcia PCA and Recco-Pimentel SM (2004) Con- served karyotype in the Hyla pulchella species group (Anura, Hylidae). Hereditas 140:42-48. Figure 2 - Partial view of the maximum parsimony consensus tree pre- Anderson K (1991) Chromosome evolution in Holarctic Hyla sented by Faivovich et al. (2005), modified with the inclusion of our treefrogs. In: Green DM and Sessions SK (eds) Amphibian Ag-NORs data. Wherever NOR data are missing, this is due to the absence Cytogenetics and Evolution. Academic Press, San Diego, pp of data in the literature. 299-328. Nunes and Fagundes 987

Baldissera FA, Oliveira PSL and Kasahara S (1993) Cytogenetics Lourenço LB, Recco-Pimentel SM and Cardoso AJ (1998) Poly- of four Brazilian Hyla species (Amphibia, Anura) and de- morphism of the nucleolus organizer regions (NORs) in scription of a case with a supernumerary chromosome. Rev Physalemus petersi (Amphibia, Anura, Leptodactylidae) de- Bras Genet 16:335-345. tected by silver staining and fluorescence in situ hybridiza- Beçak ML (1968) Chromosomal analysis of eighteen species of tion. Chromosome Res 6:621-628. Anura. Caryologia 21:191-208. Mahony MJ and Robinson ES (1986) Nucleolar organiser region Bogart JP (1973a) Method for obtaining chromosomes. Caldasia (NOR) location in karyotypes of Australian ground frogs 11:29-40. (family Myobatrachidae). Genetica 68:119-127. Meunier-Rotival M, Cortadas J, Macaya G and Bernardi G (1979) Bogart JP (1973b) Evolution of anuran karyotypes. In: Vial JL Isolation and organization of calf ribosomal DNA. Nucleic (ed) Evolutionary Biology of the Anurans. University of Acids Res 6:2109-2123. Missouri Press, Columbia, pp 337-349. Oliveira C, Foresti F and Tabata YA (1996) Pericentric inversion Faivovich J, Haddad CFB, Garcia PCA, Frost DR, Campbell JA involving a NOR bearing chromosome of rainbow trout and Wheeler WC (2005) Systematic review of the frog fam- (Oncorhynchus mykiss): Electron microscopy studies of the ily Hylidae, with special reference to Hylinae: Phylogenetic synaptonemal complex. Caryologia 49:335-342. analysis and taxonomic revision. Bull Am Mus Nat Hist Raber SC, Carvalho KA, Garcia PCA, Vinciprova G and Recco- 294:1-240. Pimentel SM (2004) Chromosomal caracterization of Hyla Foote DL, Wiley JE, Little ML and Meyne J (1991) Ribosomal bischoffi and Hyla guentheri (Anura, Hylidae). RNA gene site polymorphism in Bufo terrestris. Cytogenet Phylomedusa 3:43-49. Cell Genet 57:196-199. Schmid M (1978) Chromosome banding in Amphibia. I. Constitu- Green DM and Sessions SK (1991) Nomenclature for chromo- tive heterochromatin and nucleolus organizer regions in somes. In: Green DM and Sessions SK (eds) Amphibian Bufo and Hyla. Chromosoma 66:361-388. Cytogenetics and Evolution. Academic Press, San Diego, pp Schmid M (1982) Chromosome banding in Amphibia VII: Analy- 431-432. sis of the structure and variability of NORs in Anura. Chro- Gruber SL, Haddad CFB and Kasahara S (2006) Chromosome mosoma 87:327-344. banding in three species of Hypsiboas (Hylidae, Hylinae), Schmid M, Feichtinger W and Weimer R (1995) Chromosome with special reference to a new case of B-chromosome in banding in Amphibia XXI. Inversion polymorphism and nu- anuran frogs and to the reduction of the diploid numbers of cleolus organizer regions in Agalychnis callidryas (Anura, 2n = 24 to 2n = 22 in the genus. Genetica 130:281-29. Hylidae). Cytogenet Cell Genet 69:18-26. Howell WM and Black DA (1980) Controlled silver-staining of Silva APZ, Baldissera Jr FA, Haddad CFB and Kasahara S (2000) nucleolus organizer regions with a protective colloidal de- Karyotypes and nucleolus organizer regions of the genus veloper: A 1- step method. Experientia 36:1014-1015. Physalaemus (Anura, Leptodactylidae). Iheringia Ser Zool 88:159-164. Kaiser H, Mais C, Bolanos F, Steinlein C, Feichtinger W and Viegas-Péquignot E (1992) In situ hybridization to chromosomes Schmid M (1996) Chromosomal investigation of three Costa with biotinylated probes. In: Willerson D (ed) In situ Hy- Rica frogs from the 30-chromosome radiation of Hyla with bridization: A Practical Approach. Oxford University Press the description of a unique geographic variation in nucleolus and IRL Press, Oxford, pp 137-158. organizer regions. Genetica 98:95-102. Wiley JE, Little ML, Romano MA, Blount DA and Cline GR Kasahara S, Silva APZ, Gruber SL and Haddad CFB (2003) Com- (1989) Polymorphism in the location of the 18S and 28S parative cytogenetic analysis on four tree frog species rRNA genes on the chromosomes of the diploid-tetraploid (Anura, Hylidae, Hylinae) from Brazil. Cytogenet Gen Res tree frogs Hyla chrysoscelis and Hyla versicolor. 103:155-162. Chromosoma 97:481-487. King M (1990) Amphibian. In: John B (ed) Animal Cytogenetics. Associate Editor: Fábio de Melo Sene Gebruder Borntraegger, Berlin and Stuttgart, pp 1-241. License information: This is an open-access article distributed under the terms of the Kuramoto M (1990) A list of chromosome number of anuran am- Creative Commons Attribution License, which permits unrestricted use, distribution, and phibians. Bull Fukoka Univ Educ 39:83-127. reproduction in any medium, provided the original work is properly cited.