Chromosome Banding in Amphibia. XXXI. the Neotropical Anuran Families Centrolenidae and Allophrynidae
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Original Article Cytogenet Genome Res 2014;142:268–285 Accepted: February 6, 2014 DOI: 10.1159/000362216 by J. Smith Published online: April 25, 2014 Chromosome Banding in Amphibia. XXXI. The Neotropical Anuran Families Centrolenidae and Allophrynidae a a a b Michael Schmid Claus Steinlein Wolfgang Feichtinger James P. Bogart a b Department of Human Genetics, University of Würzburg, Würzburg , Germany; Department of Integrative Biology, University of Guelph, Guelph , Ont., Canada Key Words During the pre-banding era of cytogenetics, very few Allophrynidae · Anura · Centrolenidae · Chromosome studies included tropical glass frogs (family Centroleni- evolution · Sex chromosomes dae). These were confined to 4 species (Cochranella gra- nulosa, Espadarana prosoblepon, Hyalinobatrachium fleischmanni, and Sachatamia albomaculata) and were Abstract carried out using classical squash techniques and uni- The mitotic chromosomes of 11 species from the anuran formly stained chromosomes in the diakinesis stage of families Centrolenidae and Allophrynidae were analyzed by male meiosis ( table 1 ). The early studies did demonstrate means of conventional staining, banding techniques, and in the existence of a strikingly low chromosome number situ hybridization. The amount, location, and fluorochrome (n = 10, 2n = 20) in centrolenids. A single further study affinities of constitutive heterochromatin, the number and on somatic tissues of Vitreorana eurygnatha revealed that positions of nucleolus organizer regions, and the patterns of all 10 chromosomes have a biarmed (metacentric or sub- telomeric DNA sequences were determined for most of the metacentric) morphology ( table 1 ). No cytogenetic stud- species. The karyotypes were found to be highly conserved ies have previously been published for the sister taxon of with a low diploid chromosome number of 2n = 20 and mor- the Centrolenidae, the family Allophrynidae. phologically similar chromosomes. The sister group relation- Banding analyses were only applied to 2 males and 2 ship between the Centrolenidae and Allophrynidae (un- females of the Venezuelan centrolenid species Vitreorana ranked taxon Allocentroleniae) is clearly corroborated by the antisthenesi which revealed XY♂ /XX♀ sex chromosomes cytogenetic data. The existence of heteromorphic XY♂ /XX♀ in an initial stage of morphological differentiation and sex chromosomes in an initial stage of morphological differ- unusually large amounts of brightly labeled, AT-rich con- entiation was confirmed in Vitreorana antisthenesi . The ge- stitutive heterochromatin in the centromeric and peri- nome sizes of 4 centrolenid species were determined using centromeric regions of all autosomes and in the X chro- flow cytometry. For completeness and for comparative pur- mosome [Schmid et al., 1989]. poses, all previously published cytogenetic data on centro- A new phylogenetic taxonomy, based on molecu- lenids are included. © 2014 S. Karger AG, Basel lar, morphological, anatomical, behavioral, and biogeo- © 2014 S. Karger AG, Basel Michael Schmid 1424–8581/14/1424–0268$39.50/0 Department of Human Genetics, University of Würzburg Biozentrum, Am Hubland E-Mail [email protected] DE–97074 Würzburg (Germany) www.karger.com/cgr E-Mail m.schmid @ biozentrum.uni-wuerzburg.de graphical data, has been proposed for the Centrolenidae et al., 2009; Castroviejo-Fisher et al., 2012; Caramaschi et [Guayasamin et al., 2008, 2009]. These studies recognized al., 2013]. Allophrynids live in forests and flooded forests. the evolutionary proximity of the families Centrolenidae They are arboreal or semi-arboreal and nocturnal. and Allophrynidae and combined both into the unranked The present study examines the mitotic chromosomes taxon Allocentroleniae. The family Centrolenidae in- of 10 species in the family Centrolenidae and 1 species in cludes the subfamilies Centroleninae and Hyalinoba- the family Allophrynidae and provides results for several trachinae and comprises 11 genera: Centrolene (34 spe- banding techniques and fluorescence in situ hybridiza- cies), Chimerella (1 species), Cochranella (24 species), tion (FISH) experiments that were applied to the chro- Espadarana (3 species), Nymphargus (30 species), Ruly- mosomes of 8 species of Centrolenidae. The amount, rana (8 species), Sachatamia (3 species), Teratohyla (4 chromosomal location and composition of the constitu- species), Vitreorana (8 species), Celsiella (2 species), and tive heterochromatin, the number and location of nucle- Hyalinobatrachium (27 species). The genus Ikakogi (1 olus organizer regions (NORs) as well as the patterns of species) was described by Guayasamin et al. [2009] and, repetitive telomeric DNA sequences were determined. because it could not be assigned with confidence to either The existence of heteromorphic XY♂ /XX♀ sex chromo- subfamily, was included as incertae sedis in the Centro- somes in an initial stage of morphological differentiation lenidae. The family Allophrynidae contains the single ge- was confirmed for V. antisthenesi . The genome sizes of 4 nus Allophryne (3 species). Two of these species were only centrolenid species were determined using DNA flow cy- recently described [Castroviejo-Fisher et al., 2012; Cara- tometry. For completeness and for comparative purpos- maschi et al., 2013]. es, all previously published cytogenetic data on centrole- Glass frogs are distributed throughout the Neotropics, nids are included and re-evaluated. from Mexico to Bolivia, including the Caribbean island of Tobago, with highest species diversity in the northern Andes (Colombia and Ecuador), and with a large hiatus Materials and Methods in the lower Amazon basin. An isolated group of species occurs in southeastern Brazil and northeastern Argenti- Animals na [Duellman, 1977; Cannatella and Duellman, 1982; Individuals representing 11 species belonging to the genera Co- Guayasamin et al., 2009]. They live mainly in cloud and chranella (C. granulosa), Espadarana (E. prosoblepon), Hyalinoba- trachium (H. colymbiphyllum, H. duranti, H. fleischmanni, H. fra- rain forests and are nocturnal and arboreal. Females de- gile, H. orientale, H. valerioi), Vitreorana (V. antisthenesi, V. eury- posit egg clutches on the underside of leaves, or on rocks, gnatha) (family Centrolenidae), and Allophryne (A. ruthveni) above running water. After hatching, the tadpoles fall into (family Allophrynidae) were collected during various expeditions the water to complete their development. Some species, to Costa Rica, Venezuela, Brazil and the island of Tobago ( table 1 ). however, are also known to occasionally breed in ponds. The chromosomes of the Venezuelan species were prepared in a temporary cytogenetic laboratory in the field station Estación Bio- As a further diagnostic character, the skin and flesh of the lógica de Rancho Grande located in the Henri Pittier National ventral surfaces are partially or completely transparent Park, Aragua State. The Costa Rican specimens were prepared in owing to the scarcity of pigments. This allows the viscera a temporary cytogenetic laboratory in the Departamento de Bio- to be observed through the abdominal wall [Duellman logía, University of Costa Rica, San José. Tissue obtained for chro- and Burrowes, 1989; Ruiz-Carranza and Lynch, 1991; mosomes was transferred to 1.8-ml plastic tubes (Nunc), stored at 4 ° C or –20 ° C and transported to the laboratory in Würzburg Duellman and Trueb, 1994; Cisneros-Heredia and McDi- (Germany), either packed in dry ice or at room temperature. Frogs armid, 2007; Kubicki, 2007; Guayasamin et al., 2009]. As collected in Tobago were carried alive to Würzburg where their pointed out by Guayasamin et al. [2009], glass frogs rep- chromosomes were prepared. Chromosomes of the specimens resent one of the most interesting neotropical anuran sampled in Brazil were prepared in the field. All procedures with groups because of their morphological and ecological the living animals strictly conformed to the guidelines established by the Animal Care Committees of the respective countries. characteristics, as well as their phylogenetic and biogeo- graphic complexity. Chromosome Preparations The 3 described species of the family Allophrynidae oc- Mitotic chromosomes were prepared directly from the bone cur from the Guiana region of South America (Venezuela, marrow, intestines or the cornea of the eye after in vivo colchicine Guyana, Surinam, French Guiana, northern central Bra- treatment. Meiotic chromosomes were obtained from testes. De- tailed techniques used for squash preparations (cornea) and cell zil) to central Brazil, northeastern Amazonian Peru and suspensions (bone marrow, intestine, testes), as well as the hypo- the Atlantic rain forest of eastern Brazil [Langone and Se- tonic treatment and fixation of the cells have been previously de- galla, 1997; Caldwell and Hoogmoed, 1998; Guayasamin scribed [Schmid et al., 2010]. Chromosome Banding in Amphibia. Cytogenet Genome Res 2014;142:268–285 269 XXXI. Centrolenidae and Allophrynidae DOI: 10.1159/000362216 Table 1. Species from the families Centrolenidae and Allophrynidae cytogenetically examined in the present study and in previous re- ports Speciesa Country Speci- Localityc Geographical 2n/FNe mensb coordinatesd Family Centrolenidae, subfamily Centroleninae Cochranella granulosa* Present study Costa Rica 1♂ Carara National Park, Puntarenas Province 09°44′N/84°37′W 20/40 1♂ Río Sucio, Braulio Carrillo National Park, Limón Province 10°09′N/83°58′W 20/40 Duellman, 1967 Nicaragua 2♂ Finca Tepeyac, 10.5 km N,