Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Medeiros, L. R.
Right arrow Articles by Recco-Pimentel, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Medeiros, L. R.
Right arrow Articles by Recco-Pimentel, S. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Journal of Heredity 2003:94(2)
© 2003 The American Genetic Association 94:149-154

Chromosomal Differentiation of Hyla nana and Hyla sanborni (Anura, Hylidae) With a Description of NOR Polymorphism in H. nana

L. R. Medeiros, D. C. Rossa-Feres, and S. M. Recco-Pimentel

From the Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), 13084-971 Campinas, São Paulo, Brazil (Medeiros and Recco-Pimentel), and Departamento de Zoologia e Botânica, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista (UNESP), 15054-000 São José do Rio Preto, São Paulo, Brazil (Rossa-Feres).

Address correspondence to Shirlei M. Recco-Pimentel at the address above, or e-mail: shirlei{at}unicamp.br.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Specimens of Hyla nana and Hyla sanborni from a syntopic population were studied cytogenetically. These species are morphologically very similar and are frequently misidentified, confused with each other. Both species had a diploid chromosome number, 2n = 30. However, the karyotypes of H. nana and H. sanborni differed considerably from each other in the number of submetacentric and telocentric chromosomes. The two species also differed in their primary NOR-bearing chromosomes (metacentric pair 13 in H. nana and telocentric pair 12 in H. sanborni). Additional nucleolus organizer regions (NORs) were detected by Ag-NOR staining and FISH in chromosome pairs 1, 5, 6, 12, and 14 in seven specimens of H. nana. Thus, a total of six patterns of NOR were identified. These differences in karyotype and in NOR location allowed the unambiguous identification of syntopic individuals of the two species. However, the chromosomal morphology of both species differed from that reported for populations from other geographic regions, suggesting that a systematic reevaluation of this group of Hyla may be necessary.

Hyla nana Boulenger, 1889, and Hyla. sanborni Schmidt, 1944, are morphologically (Langone 1994; Langone and Basso 1987) and ecologically (Del-Grande 1995; Rossa-Feres 1997) very similar, and museum specimens of both species are frequently confused with each other. Live specimens of H. nana and H. sanborni can be distinguished from each other by the frequency of their dominant mating call (3.0–4.0 kHz and 4.6–6.6 kHz, respectively); male snout-vent length (20.22 ± 1.03 mm, n = 211, and 16.70 ± 1.51 mm, n = 109); coloration of the vocal sac (yellow or transparent); seasonal occurrence (September/October to March and December/January to March); and the height of the call site above ground (24.3 ± 14 cm, n = 600, and 47.8 ± 18 cm, n = 109; Rossa-Feres 1997; Rossa-Feres and Jim 2001).

Barrio (1967) considered H. sanborni to be a subspecies of H. nana, but this placement was not accepted by others (Braun and Braun 1976; Cardoso 1981). Fitzgerald and Scott (1984) and Basso et al. (1985) recognized H. sanborni as a full species, basing their conclusion on geographic distribution, morphology, and mating call differences. However, the relationships of these two species with other members of the genus Hyla are still unclear. Frost (1985) included both species in the H. nana group, but they have also been included in the Hyla minuta group (Cei, 1987) and in the Hyla microcephala group (Langone and Basso, 1987).

There are contradictory karyotype data for different populations of both species (Bogart 1973; Rabello 1970; Skuk and Langone 1992), although these analyses have shown only the standard karyotype of H. nana and H. sanborni. In this report we provide a more complete cytogenetic analysis of both species, based on specimens from a syntopic population, in an attempt to distinguish between these two species.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Forty-three specimens of H. nana (40 males and 3 females) and 27 specimens of H. sanborni (all males) were studied. All specimens were collected at Nova Itapirema (21°11'S–49°42'W), in the State of São Paulo, southeastern Brazil, from February to May 1998 and October 1998 to May 1999.

Specimens were identified according to Rossa-Feres (1997) and Rossa-Feres and Jim (2001) and were deposited in the ZUEC (Museu de História Natural "Prof. Adão José Cardoso," Universidade Estadual de Campinas, Campinas, SP, Brazil) and in the DZSJRP (Departamento de Zoologia, Universidade Estadual Paulista, São José do Rio Preto, SP, Brazil) collections, under the accession numbers ZUEC 11405–11412, 11416–11418, 11647–11677 (H. nana), ZUEC 11678–11692 (H. sanborni), DZSJRP 1111 (H. nana), and DZSJRP 990–1001 (H. sanborni).

Chromosomal preparations were obtained from a suspension of intestinal or testicular cells. Slides were stained with 10% Giemsa solution or processed for C-banding (King 1980), Ag-NOR staining (Howell and Black 1980), and fluorescent in situ hybridization (FISH) with rDNA probes (Viégas-Péquignot 1992). The probes used consisted of a recombinant plasmid HM123 or a mixture of the two recombinants HM123 and HM456 containing fragments of Xenopus laevis rDNA (Meunier-Rotival et al. 1979), which were biotin-labeled by a nick translation reaction according to the manufacturer's (GIBCO) protocol. To investigate the additional NOR detected only by FISH in one specimen, a subclone pBS28 (Lourenço LB et al., in preparation) containing only the coding region of the 28S RNA of HM123 was also used in FISH experiments. The nomenclature of Green and Sessions (1991) was used to describe the chromosome morphology. The centromere position was determined by chromosomal measurements in 31 metaphases of 18 specimens of H. nana and 12 metaphases of 7 specimens of H. sanborni.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
H. nana and H. sanborni had a diploid chromosome complement of 2n = 30, but the fundamental number (FN) was 52 and 50, respectively. In H. nana there were six pairs of metacentric chromosomes (3, 8, 9, 10, 11, and 13), five submetacentric pairs (1, 2, 4, 5, and 7), and four telocentric pairs (6, 12, 14, and 15; Figure 1A). In some Giemsa-stained mitotic metaphases, a secondary constriction was detected on the short arm of pair 1 and on the long arms of pair 5 (Figure 1A,B). The karyotype of H. sanborni consisted of six pairs of metacentric chromosomes (2, 8, 9, 10, 11, and 14), four submetacentric pairs (1, 3, 4, and 6), and five telocentric pairs (5, 7, 12, 13, and 15; Figure 1C), and a secondary constriction was not seen.



View larger version (54K):
[in this window]
[in a new window]
  		Figure 1.. Karyotypes of H. nana (A) and H. sanborni (C) after Giemsa staining. (B) shows the secondary constrictions of chromosome pairs 1 and 5 of H. nana. Karyotypes of H. nana (D) and H. sanborni (E) after C-banding (Bar = 5 mm)

 
Both species had the same C-banding pattern, with only the centromeric region of all chromosomes staining strongly (Figure 1D,E). In all specimens of H. nana the NOR was located on the long arm of pair 13 (Figures 2 and 3). Seven of the 43 specimens examined showed additional NOR-bearing pairs, representing five distinct patterns (Figures 2 and 3). NORs were observed in pairs 1, 5, 12, and 14 in the heterozygous condition. The NOR was located close to the telomere on the long arms of chromosomes 5 and 12 and on the short arms of pair 1, and interstitially on the long arm of chromosome 14 (Figure 3B–F). The NORs in chromosomes 1, 5, and 13 coincided with secondary constrictions in Giemsa-stained metaphases.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2.. Ag-NOR patterns in the metaphase chromosomes of H. nana (A–F) and H. sanborni (G). Karyotype with the most common staining only in chromosome pair 13 (A); NOR in pairs 1 and 13 (B); in pairs 1, 13, and 14 (C); in pairs 1, 12, and 13 (D); in pairs 12 and 13 (E); and in pairs 5 and 13, as well as pair 6, which showed no silver staining (F). The arrows indicate the NOR regions. Metaphase chromosomes of H. sanborni with staining only in chromosome pair 12 (G). (Bar = 5 mm)

 


View larger version (19K):
[in this window]
[in a new window]
  		Figure 3.. Metaphases of H. nana (A–F) and H. sanborni (G) after in situ hybridization. Karyotype with the most common staining only in chromosome pair 13 (A); NOR in pairs 1 and 13 (B); in pairs 1, 13, and 14 (C); in pairs 1, 12, and 13 (D); in pairs 12 and 13 (E); and in pairs 5 and 13, as well as pair 6, which showed staining by FISH (F). FISH of H. sanborni with staining only in pair 12 (G). (Bar = 5 mm)

 
FISH experiments in the seven specimens of H. nana with different NOR patterns, revealed the same regions detected by the silver-staining method (Figure 3A–E). Additionally, in one specimen of H. nana a heterozygous condition not detected by the Ag-NOR technique was identified in pair 6 (Figure 3F). FISH using the pBS28 probe also detected the additional NOR in pair 6. In all specimens of H. sanborni, silver staining and FISH detected the NOR in the telomeric region of telocentric pair 12 (Figures 2G and 3G).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
H. nana and H. sanborni belong to the 30-chromosome Hyla, a diverse group of neotropical frogs. Although very similar in external morphology, these species are easily distinguished by the number of telocentric chromosomes (four in H. nana and five in H. sanborni). Species of Hyla with 2n = 30 have a variable number of telocentrics, from none in H. minuta to five in Hyla labialis (Bogart 1973). As reported by Bogart (1973), pericentric inversions may have shifted the position of the centromeres in many 30-chromosome Hyla from a telocentric to a more metacentric position.

The karyotypes of H. nana and H. sanborni studied here differ from those of populations from other regions. The karyotype of H. nana was first described by Rabello (1970), who classified pairs 5 and 9 as acrocentrics (= telocentrics); in the karyotype studied here these chromosomes were submetacentric and metacentric, respectively. Indeed, the two karyotypes were very similar and had the same number of telocentrics. Rabello (1970) did not state where the specimens were collected.

The H. nana karyotype was also described by Bogart (1973), who studied a population from northern Argentina. Surprisingly, this karyotype had five telocentrics as found here for H. sanborni, and, if the position of pairs 13 and 14 is reversed, these karyotypes become identical.

In contrast, the karyotype of H. sanborni from a population at Ponta Grossa, Paraná State, Brazil, had four telocentrics instead of five (Skuk and Langone 1992). In addition, pairs 7 and 13 were metacentric, and pairs 9 and 14 were acrocentric, whereas in the present study these pairs are telocentric and metacentric, respectively. A single exchange in the position of the largest telocentric chromosome (9) to position 6 in the karyotype of H. sanborni from Paraná made this karyotype identical to that of H. nana from Nova Itapirema. Given that Skuk and Langone (1992) measured only two metaphases in H. sanborni and that these medium sized chromosomes are similar in length, an inversion in their position in the karyotype is probable. Thus, with only one inversion in the position of two chromosome pairs in H. nana and H. sanborni from Argentina and Paraná (Skuk and Langone 1992), the karyotypes become identical to those of H. sanborni and H. nana from Nova Itapirema. This discrepancy can be explained by misidentification, because these species are morphologically very similar, but may also reflect small interpopulational variations in their karyotypes.

NOR analyses reveals considerable differences between the H. nana and H. sanborni karyotypes. In H. nana the primary NOR-bearing chromosome was the telocentric pair 12, whereas in H. sanborni the NOR was observed on metacentric pair 13. However, 14% of the specimens of H. nana showed additional NORs. This type of intrapopulational variation has been described in other anurans, such as Hyla chrysoscelis and Hyla versicolor (Wiley et al. 1989), Bufo terrestris (Foote et al. 1991), Agalychnis callidryas (Schmid et al. 1995), and Physalaemus petersi (Lourenço et al. 1998). Multiple NOR-bearing chromosome pairs have been considered a derived state in the Anura (King et al. 1990) and have been found in species of several families (Lourenço et al. 1998, 2000; Schmid 1978; Schmid et al. 1995; Wiley et al. 1989). Possible mechanisms involved in NOR dispersion in anuran genomes include inversions and translocations involving chromosomal segments containing NORs, transpositions by mobile genetic elements, amplifications of "orphan" rDNA cistrons, and reinsertion error during extrachromosomal amplification of ribosomal cistrons (Foote et al. 1991; Kaiser et al. 1996; King et al. 1990; Lourenço et al. 1998, 2000; Schmid et al. 1995; Wiley et al. 1989).

FISH experiments showed labeling in one of the homologs of pair 6 of H. nana that was not detected by silver staining. Similar findings have been reported for H. chrysoscelis and H. versicolor (Wiley et al. 1989). Given that the results obtained by FISH are independent of NOR activity, it is possible that this labeling detected some homologous rDNA sequences in chromosome 6. In any case, multiple NOR-bearing chromosomes were observed only in H. nana.

This comparative cytogenetic analysis of H. nana and H. sanborni indicates that the two species can be easily distinguished from each other by their chromosome morphology, especially by the number of telocentric pairs and NOR location. These data, together with the differences among populations from other regions (see Bogart 1973; Rabello 1970; Skuk and Langone 1992), suggest that there may be more than two species under the names H. nana and H. sanborni.

A cytogenetic study of populations from different regions, including the type locality, together with morphological and ecological data, could help to establish the taxonomic status of these species and their different populations.


   Acknowledgments
 
The authors thank M. Menin for help with the field work, K. A. Carvalho for technical assistance, L. B. Lourenço for assistance with the FISH experiments and for valuable discussion, and F. Langeani for useful comments on evolution and speciation. This work was supported by the Brazilian agency CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Proc. 00/11031-4).


   Footnotes
 
Corresponding Editor: William S. Modi Back

Received April 3, 2002
Accepted November 14, 2002


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

    Barrio A, 1967. Sobre la validez de Hyla sanborni y H. uruguaya (Anura. Hylidae). Physis. 76:521-524.

    Basso NJ, Peri SI, Di Tada IE, 1985. Revalidación de Hyla sanborni Schmidt: 1944 (Anura: Hylidae). Cuad Herpet. 3:1-11.

    Bogart JP, 1973. Evolution of anuran karyotypes. In: Evolutionary biology of anurans (Vial JL, ed). Columbia: University of Missouri Press; 337–349.

    Braun PC, Braun CAS, 1976. Contribuição ao estudo da fauna anfibiológica da região metropolitana (Grande Porto Alegre), Rio Grande do Sul, Brasil. Comum. Mus. Ci. PUCRGS. 10:1-16.

    Cardoso AJ, 1981. Organização espacial e temporal na reprodução e vida larvária de uma comunidade de hilídeos no sudeste do Brasil (Amphibia. Anura) (Master's thesis). Campinas, Brazil: Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP); 106 pp.

    Cei JM, 1987. Additional notes to "Amphibians of Argentina": an update. 1980–1986. Monit Zool Ital. 21:209-272.

    Del-Grande ML, 1995. Estudo comparado da biologia de Hyla nana e de Hyla sanborni (Amphibia, Anura, Hylidae) em Corumbataí, Estado de São Paulo (master's thesis). Rio Claro, Brazil: Instituto de Biociências, Universidade Estadual Paulista; 70 pp.

    Fitzgerald LA, Scott NJ, 1984. Taxonomic status of Hyla sanborni and Hyla nana (Hylidae, Anura). Program and Abstracts 32nd Annual Meeting. Herpetol. League, 27th Annual Meeting SSAR, 64th Annual Meeting ASIH (July 28–August 3, 1984, Norman, OK); 113 pp.

    Foote DL, Wiley JE, Little ML, Meyne J, 1991. Ribosomal RNA gene site polymorphism in Bufo terrestris. Cytogenet Cell Genet. 57:196-199.[ISI][Medline]

    Frost DR, 1985. Amphibian species of the world. A taxonomic and geographical reference. Lawrence, KS: Allen Press and the Association of Systematic Collections.

    Green DM, Sessions SK, 1991. Nomenclature for chromosomes. In: Amphibian cytogenetics and evolution (Green DM and Sessions SK, eds). San Diego: Academic Press; 431–432.

    Howell WM, Black DA, 1980. Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia. 36:1014-1015.[CrossRef][ISI][Medline]

    Kaiser H, Mais C, Bolanos F, Steinlein C, Feichtinger W, Schmid M, 1996. Chromosomal investigation of three Costa Rica frogs from the 30-chromosome radiation of Hyla with the description of a unique geographic variation in nucleolus organizer regions. Genetica. 98:95-102.[CrossRef]

    King M, 1980. C-banding studies in Australian hylid frogs: secondary constriction structure and the concept of euchromatin transformation. Chromosoma (Berl). 80:191-207.[CrossRef]

    King M, Contreras N, Honeycutt RL, 1990. Variation within and between nucleolar organizer region in Australian hylid frogs (Anura) shown by 18S + 28S in situ hybridization. Genetica. 80:17-29.[CrossRef][ISI][Medline]

    Langone JA, 1994. Ranas y sapos del Uruguay (reconocimiento y aspectos biologicos). Mus Damaso Antonio Larrañaga. 5:9-45.

    Langone JA, Basso NG, 1987. Distribucion geografica y sinonima de Hyla nana Boulenger, 1889 y Hyla sanborni Schmidt, 1944 (Anura, Hylidae) y observaciones sobre formas afines. Comun Zool Mus Hist Nat Montev. 11:1-17.

    Lourenço LB, Garcia PCA, Recco-Pimentel SM, 2000. Cytogenetics of two species of Paratelmatobius (Anura, Leptodactylidae) with phylogenetic comments. Hereditas. 133:201-209.[CrossRef][ISI][Medline]

    Lourenço LB, Recco-Pimentel SM, Cardoso AJ, 1998. Polymorphism of the nucleolus organizer regions (NORs) in Physalaemus petersi (Amphibia, Anura, Leptodactylidae) detected by silver staining and fluorescence in situ hybridization. Chromosome Res. 6:621-628.[CrossRef][ISI][Medline]

    Meunier-Rotival M, Cortadas J, Macaya G, 1979. Isolation and organization of calf ribosomal DNA. Nucleic Acids Res. 6:2109-2123.[Abstract/Free Full Text]

    Rabello MN, 1970. Chromosomal studies in Brazilian anurans. Caryologia. 1:45-59.

    Rossa-Feres D de C, 1997. Ecologia de uma comunidade de anfíbios da região noroeste do Estado de São Paulo: microhabitat, sazonalidade, dieta e nicho multidimensional (PhD dissertation). Rio Claro, Brazil: Instituto de Biociências, Universidade Estadual Paulista (UNESP), 181 pp.

    Rossa-Feres D de C, Jim J, 2001. Similaridade do sítio de vocalização em uma comunidade de anfíbios anuros na região noroeste do Estado de São Paulo, Brasil. Rev Bras Zool. 18:439-454.

    Schmid M, 1978. Chromosome banding in Amphibia. I. Constitutive heterochromatin and nucleolus organizer regions in Bufo and Hyla. Chromosoma. 66:361-388.

    Schmid M, Feichtinger W, Weimer R, Mais C, Bolaños F, Leon P, 1995. Chromosome banding in Amphibia. XXI. Inversion polymorphism and nucleolus organizer regions in Agalychnis callidryas (Anura, Hylidae). Cytogenet Cell Genet. 69:18-26.[ISI][Medline]

    Skuk G, Langone JA, 1992. Los cromosomas de cuatro especies del género Hyla (Anura: Hylidae) com número diploide de 2n = 30. Acta Zool Lilloana. 41:165-171.

    Viegas-Péquignot E, 1992. In situ hybridization to chromosomes with biotinlated probes. In: In situ hybridization: a practical approach (Willernson D, ed). Oxford: Oxford University Press and IRL Press, 137–158.

    Wiley JE, Little ML, Romano MA, Blount DA, Cline GR, 1989. Polymorphism in the location of the 18S and 28S rDNA genes on the chromosomes of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor. Chromosoma. 97:481-487.[CrossRef]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Medeiros, L. R.
Right arrow Articles by Recco-Pimentel, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Medeiros, L. R.
Right arrow Articles by Recco-Pimentel, S. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?