South American Journal of Herpetology, 4(2), 2009, 164-172 © 2009 Brazilian Society of Herpetology
MORPHOLOGICAL AND KARYOTYPIC CONTRIBUTIONS FOR A BETTER TAXONOMIC DEFINITION OF THE FROG ISCHNOCNEMA RAMAGII (BOULENGER, 1888) (ANURA, BRACHYCEPHALIDAE)
Marcelo F. Napoli1,4, Fernando Ananias2, Patricia M. Fonseca1, and Ana Paula Z. Silva3
1 Museu de Zoologia, Departamento de Zoologia, Instituto de Biologia, Universidade Federal da Bahia, Rua Barão de Jeremoabo, Ondina, 40170‑115, Salvador, BA, Brasil. E‑mail: [email protected], [email protected] 2 Curso de Ciências Biológicas, Universidade São Francisco (USF), Avenida São Francisco de Assis, 218, Jardim São José, 12916‑900, Bragança Paulista, SP, Brasil. E‑mail: [email protected] 3 Laboratório Especial de Ecologia e Evolução, Instituto Butantan, Avenida Vital Brasil, 1500, 05503‑900, São Paulo, SP, Brasil. E‑mail: [email protected] 4 Corresponding author.
Abstract. In this study we describe the morphological variation of Ischnocnema ramagii from a population sample within the Municipality of Salvador, Bahia State, Brazil, and characterize two extremely distinct morphotypes within this taxon (striped vs. non-striped dorsum); we describe its karyotype, and compare the karyotypic structure of the distinct morphotypes recognized herein. Specimens with striped dorsa were less common (13‑18%) than those with non-striped dorsa (81‑86%), the latter group comprising a mix of discrete color pattern states, whose frequencies were not different among juveniles, males, and females. The PCAs on morphometric data resulted in a strong degree of superposition between striped and non-striped dorsa, for both genders. We encountered a diploid number of 30 chromosomes for both males and females, with chromosome pairs 1 to 15 being telocentric. The karyotypic comparison between specimens with striped and non-striped dorsa did not show any noticeable difference. Nevertheless the karyotype of I. ramagii from Salvador, Bahia, differed from the karyotype described for I. paulodutrai from Ilhéus, Bahia, which showed a submetacentric chromosome pair. This result supports the presence of two full “sibling” species in Bahia State, one from the south (I. paulodutrai) and another from the north (I. ramagii), validating the identity of I. paulodutrai as a full species, and refuting the recognition of Salvador samples as I. paulodutrai. The high number of telocentric chromosomes suggests a closer relationship between I. ramagii and “Eleutherodactylinae” species from Central America and the northern region of Brazil.
Keywords. Ischnocnema paulodutrai, Intra-population variation, Karyotype, Polymorphism, Size and shape variation.
Introduction I. paulodutrai; (3) possible morphological polymor- phism within populations; and (4) complex variation The “Eleutherodactylinae” frog Ischnocnema ra‑ of advertisement calls within and among geographic magii (Boulenger, 1888) (sensu Heinicke et al., 2007) populations. The current poor knowledge of local inhabits Tropical Atlantic forests of northeastern Bra- and geographic variation of both species prevents an zil, from Paraíba State to Bahia State (Frost, 2008). accurate assignment of a specimen to a given taxa, The species I. ramagii was described as Hylodes which is usually done by taking into account the geo- ramagii, based on a single specimen collected by graphic distance between the area of collection and G. A. Ramage in the Municipality of Igarassu, State the respective type-localities (I. paulodutrai, Ilhéus, of Pernambuco, northeastern Brazil. In the last few southern Bahia State, 14°47’S, 39°02’W; I. rama‑ years, similar morphotypes have been registered from gii, Igarassu, Pernambuco State, 07°50’S, 34°54’W; the State of Bahia, Brazil, usually being recognized ca. 890 km distance from each other). Because of as I. ramagii (e.g., Munduruca, 2005; Juncá, 2006; the above statements, we did not follow Napoli and Bastazini et al., 2007) or I. paulodutrai (Bokermann, Brandão (2003) in allocating specimens from Salva- 1975) (e.g., Napoli and Brandão, 2003). Other closely dor to I. paulodutrai. related morphotypes have been captured within the In this study, we make a first assessment to im- Tropical Atlantic Forest and Caatinga biomes by us prove the morphologic and cytogenetic knowledge of and by other researchers during surveys of anurans, I. ramagii, using population samples from a patch of but probably belong to species not yet described Atlantic Forest remnant located in the campus of the (pers. obs.). Universidade Federal da Bahia (UFBA), at a neigh- Concerning the correct identification of specimens borhood named Ondina, in the Municipality of Sal- of Ischnocnema ramagii and I. paulodutrai, four ma- vador, Bahia State, Brazil. We decided to limit our jor problems can be postulated: (1) the short original analysis to this local population for three reasons: description of I. ramagii; (2) the poor diagnosis of (1) only one type of advertisement call is found in Napoli, M. F. et al. 165 this population, which led us to assume that only one collections (acronyms in parenthesis): Comissão Ex- species occurs at the locality; (2) this local population ecutiva do Plano de Cultivo da Lavoura Cacaueira, contains two extremely distinct morphotypes, distin- Municipality of Ilhéus, Bahia State (CEPLAC); Cé- guished by the presence or not of longitudinal brown lio Haddad Collection (CFBH), deposited in the De- stripes on the dorsum, which makes their descriptions partamento de Zoologia, Instituto de Biociências, very important for an accurate definition of species Universidade Estadual Paulista, Municipality of Rio limits; (3) there is no doubt about the conspecific- Claro, São Paulo State; Museu de Zoologia, Univer- ity of these morphotypes, as we obtained 13 frogs of sidade Federal da Bahia, Municipality of Salvador, both (UFBA 2914; 54% striped dorsum; 46% non- Bahia State (UFBA). striped dorsum) within a single clutch of large-yolked We analyzed 151 specimens of Ischnocnema ra‑ terrestrial eggs; and (4) many specimens are available magii (89 males, 22 females, and 40 juveniles) from from this locality (n = 151). a patch of Atlantic Forest remnant located in the cam- The goals of the present study are to (1) charac- pus of the Universidade Federal da Bahia, at a neigh- terize the morphological variation of Ischnocnema borhood named Ondina, in the Municipality of Salva- ramagii from a population sample within the Munici- dor, Bahia State, Brazil (12°59’S; 38°27’W). pality of Salvador, Bahia State, Brazil; (2) describe We developed outline drawings as a standard the karyotype; (3) assess the level of karyotypic poly- series for color patterns of dorsum, interocular bar, morphisms in the population; and (4) compare the post-orbital region, and sacral surface, recorded for karyotype of I. ramagii with the karyotype of I. pau‑ each specimen examined. When a new pattern was lodutrai from Ilhéus, Bahia State, by Siqueira et al. encountered, it was added to the standards. We fol- (2008). lowed Heyer (1984) as several aspects of the dorsal pattern are inherited independently in some species of Terrarana (Goin, 1950, 1960). The purpose of Material and Methods this procedure was to analyze (1) variation within this population and (2) differences among juveniles, Specimens used for descriptions or examined for males, and females. Drawings were made using a comparisons are deposited in the following Brazilian Leica MZ6 stereomicroscope with a drawing tube.
Figure 1. Dorsal views of Ischnocnema ramagii from the Municipality of Salvador, Bahia State, Brazil. A, adult female with striped dor- sum, UFBA 3158, SVL 25.0 mm; B, adult female with non-striped dorsum, UFBA 2271, SVL 25.5 mm. Photographs by Rafael O. Abreu. 166 Morphology and Karyotype of Ischnocnema ramagii
Chi-square analyses were performed to test whether measurements followed Heyer et al. (1990): UAR patterns observed differed in occurrence among juve- (upper arm), FAR (forearm), HAL (hand length). The niles, males, and females. The α-level of significance remaining measurements are LF1 (length of finger I) (P ≤ 0.05) was Bonferroni corrected (Bland, 2004). and LF2 (length of finger II), both as the straight line We measured 20 morphometric characters from 89 distance between the lower and upper borders of the adult males and 22 adult females of I. ramagii, which finger. We measured SVL, HL, HW, ITD, THL, TL, were recorded in millimeters. Fifteen measurements FL, UAR, FAR, and HAL with a vernier caliper, and followed Napoli (2005): SVL (snout-vent length), HL used an ocular micrometer in an SZ Olympus stereo- (head length), HW (head width), ED (eye diameter), microscope for the remaining variables. UEW (upper eyelid width), IOD (interorbital dis- We used the principal component analysis (PCA) tance), IND (internarial distance), END (eye-nostril to study multivariate patterns of shape and size varia- distance), NSD (nostril to tip of snout distance), TD tion. The first principal component (PC1) typically (tympanum diameter), THL (thigh length), TL (tibia expresses “size” variation when it has loadings of the length), FL (foot length), 3FD (third finger disk di- same magnitude and sign for all growth-dependent ameter), and 4TD (fourth toe disk diameter). Three variables (Humphries et al., 1981). The remaining
Figure 2. Standards for color patterns of (A) dorsum, (B) interocular bar, (C) post-orbital region, and (D) sacral surface. Additional stan- dards not figured are: A1, B1, C1, and D1, which represent immaculate surfaces. Patterns D2 and D3 may be comprised by only one or two transverse stripes. Napoli, M. F. et al. 167 axes are uncorrelated with PC1 (orthogonal) and are 09657, 09662‑09663; striped males, CFBH 09644, usually considered as “shape” variation. We obtained 09665‑09669; non-striped males, 09651‑09652, eigenvectors and associated eigenvalues from a vari- 09655, 09658‑096661), and transported them alive to ance-covariance matrix, and the loadings were the the cytogenetics laboratory of the São Francisco Uni- correlations between the original variables and the versity (São Paulo State, Brazil). Karyotypes were scores. The scores were projected onto reduced space prepared by two different procedures: (1) intestinal using the axes that account for the largest amounts of epithelium – animals pretreated with 1% colchicine variation among the sample groups. solution for at least 4 h (Schmid, 1978) and (2) cyto- For cytogenetic analysis, we captured 19 speci- logical preparations of bone marrow and liver – ani- mens of Ischnocnema ramagii (striped female, CFBH mals previously submitted to Phytohemagglutinin‑P 09670; non-striped females, CFBH 09653‑09654, (Cultilab) and 0.1% colchicine treatments, in the
Table 1. Occurrence of pattern states (Fig. 2) among juveniles, males, and females of Ischnocnema ramagii from Ondina, Municipality of Salvador, Bahia State, Brazil. Values in parentheses are percentages.
General dorsal pattern Interocular bar Post-orbital maculae Sacral marks Sample A1 A2 A3 A4 B1 B2 B3 C1 C2 C3 C4 D1 D2 D3 D4 11 23 6 1 18 10 1 19 7 2 3 7 13 6 Juveniles (27.5) (57.5) (15.0) (3.4) (62.0) (34.4) (3.4) (65.5) (24.1) (6.8) (10.3) (24.1) (44.8) (20.6) 6 12 46 14 45 3 5 41 15 5 9 17 35 5 Males (7.6) (15.3) (58.9) (17.9) (93.7) (6.2) (7.5) (62.1) (22.7) (7.5) (13.6) (25.7) (53.0) (7.5) 4 9 4 9 4 8 4 1 1 3 7 2 Females (23.5) (52.9) (23.5) (69.2) (30.7) (61.5) (30.7) (7.6) (7.6) (23.0) (53.8) (15.3)
Table 2. Standardized coefficients and factor loadings (r) from Table 3. Standardized coefficients and factor loadings (r) from principal component analysis (PCA) for 20 morphometric char- principal component analysis (PCA) for 20 morphometric char- acters of Ischnocnema ramagii adult males from the Municipal- acters of Ischnocnema ramagii adult females from the Munici- ity of Salvador, State of Bahia, Brazil. NS, not significant; * pality of Salvador, State of Bahia, Brazil. NS, not significant; * P ≤ 0.05; ** P ≤ 0.01; ** P ≤ 0.001; Cum. Prop., cumulative P ≤ 0.05; ** P ≤ 0.01; ** P ≤ 0.001; Cum. Prop., cumulative proportion of eigenvalues in percentage. proportion of eigenvalues in percentage.
PC1 PC2 PC3 (r) PC1 (r) PC2 (r) PC3 PC1 PC2 PC3 (r) PC1 (r) PC2 (r) PC3 SVL ‑0.50 0.74 ‑0.31 ‑0.90*** 0.42*** ‑0.09NS SVL ‑0.50 0.74 ‑0.31 ‑0.91*** ‑0.40NS 0.11NS HL ‑0.23 0.00 0.34 ‑0.88*** 0.01NS 0.21NS HL ‑0.23 0.00 0.34 ‑0.91*** ‑0.25NS ‑0.05NS HW ‑0.18 0.26 0.49 ‑0.78*** 0.37*** 0.35*** HW ‑0.18 0.26 0.49 ‑0.91*** ‑0.21NS ‑0.11NS ED ‑0.04 0.05 0.10 ‑0.57*** 0.23* 0.21NS ED ‑0.04 0.05 0.10 ‑0.49* ‑0.32NS ‑0.23NS END ‑0.07 ‑0.02 0.20 ‑0.73*** ‑0.07NS 0.31** END ‑0.07 ‑0.02 0.20 ‑0.73*** ‑0.10NS ‑0.27NS TD ‑0.03 0.06 0.05 ‑0.48*** 0.31** 0.14NS TD ‑0.03 0.06 0.05 ‑0.64*** ‑0.38NS ‑0.05NS UEW ‑0.03 0.06 0.16 ‑0.41*** 0.23* 0.29** UEW ‑0.03 0.06 0.16 ‑0.49*** ‑0.57** ‑0.33NS IOD ‑0.04 ‑0.04 0.06 ‑0.55*** ‑0.16NS 0.11NS IOD ‑0.04 ‑0.04 0.06 ‑0.61*** ‑0.23NS ‑0.49* IND ‑0.04 0.00 0.05 ‑0.74*** 0.01NS 0.13NS IND ‑0.04 0.00 0.05 ‑0.83*** ‑0.37NS ‑0.07NS NSD ‑0.02 0.00 0.04 ‑0.56*** 0.05NS 0.14NS NSD ‑0.02 0.00 0.04 ‑0.78*** ‑0.36NS ‑0.10NS UAR ‑0.24 ‑0.09 0.20 ‑0.86*** ‑0.11NS 0.12NS UAR ‑0.24 ‑0.09 0.20 ‑0.86*** 0.01NS ‑0.42* FAR ‑0.12 0.01 0.13 ‑0.79*** 0.04NS 0.13NS FAR ‑0.12 0.01 0.13 ‑0.90*** ‑0.19NS 0.10NS HAL ‑0.16 ‑0.04 ‑0.01 ‑0.91*** ‑0.08NS ‑0.01NS HAL ‑0.16 ‑0.04 ‑0.01 ‑0.87*** 0.21NS 0.01NS THL ‑0.30 ‑0.11 0.35 ‑0.94*** ‑0.12NS 0.18NS THL ‑0.30 ‑0.11 0.35 ‑0.94*** 0.22** ‑0.01NS TL ‑0.37 ‑0.42 0.11 ‑0.92*** ‑0.33** 0.05NS TL ‑0.37 ‑0.42 0.11 ‑0.81*** 0.52NS 0.02NS FL ‑0.52 ‑0.35 ‑0.44 ‑0.96*** ‑0.20NS ‑0.13NS FL ‑0.52 ‑0.35 ‑0.44 ‑0.94*** 0.31NS 0.08NS 3FD ‑0.02 0.02 0.03 ‑0.50*** 0.12NS 0.11NS 3FD ‑0.02 0.02 0.03 ‑0.21NS ‑0.33NS ‑0.17NS 4TD ‑0.02 0.02 0.05 ‑0.52*** 0.14NS 0.16NS 4TD ‑0.02 0.02 0.05 ‑0.34NS ‑0.31NS ‑0.22NS LF1 ‑0.10 ‑0.12 ‑0.16 ‑0.79*** ‑0.29** ‑0.19NS LF1 ‑0.10 ‑0.12 ‑0.16 ‑0.72*** 0.41NS ‑0.10NS LF2 ‑0.10 ‑0.13 ‑0.11 ‑0.79*** ‑0.32** ‑0.14NS LF2 ‑0.10 ‑0.13 ‑0.11 ‑0.77*** 0.31NS ‑0.13NS Eigenvalues 4.21 0.42 0.11 — — — Eigenvalues 5.73 0.81 0.17 — — — Cum. prop. Cum. prop. 80.37 88.49 90.59 — — — 78.51 89.72 92.17 — — — (%) (%) 168 Morphology and Karyotype of Ischnocnema ramagii proportion of 0.1 ml/10 g animal weight, during 48 h (Fig. 2; Table 1), whose frequencies were not differ- and 6 h, respectively (Baldissera et al., 1993; Silva ent among juveniles, males, and females (after Bon- et al., 2000). We stained the slides with 10% Giemsa ferroni correction significant at P < 0.01: X2 = 0.46, solution for chromosome morphological studies, and DF = 2, P = 0.79; X2 = 1.45, DF = 2, P = 0.48; performed the nucleolus organizer regions (NOR) X2 = 0.11, DF = 2, P = 0.94; X2 = 0.46, DF = 2, by silver nitrate staining (Ag‑NORs) (Howell and P = 0.79; respectively). Black, 1980). We identified chromosomes according Principal component analyses showed a strong de- to Green and Sessions (1991). gree of overlap between specimens of I. ramagii with striped and non-striped dorsa, separately for males and females (Fig. 3; Tables 2 and 3). Results We encountered a diploid number of 30 chromo- somes in mitotic cells for both males and females. We found two clearly distinct color patterns with- Chromosome pairs 1 to 15 were telocentric. A slight in the examined population of Ischnocnema ramagii; and continuous size decrease was observed in chro- a morphotype with striped dorsum (Fig. 1A) and an- mosome length from pairs 1‑5 and 6‑15 (Fig. 4). other with non-striped dorsum (Fig. 1B). Specimens Stained chromosomes presented Ag‑NOR at the dis- with non-striped dorsa were more common (males: tal region of chromosome 7, which coincided with n = 79, 86.81%; females: n = 18, 81.8%) than striped the site of the secondary constriction (Fig. 5). The specimens (males: n = 12, 13.18%; females: n = 4, karyotypic comparison between I. ramagii speci- 18.1%); these two morphotypes exhibit a mixture of mens with striped and non-striped dorsa (dorsal discrete pattern states of general dorsal patterns, in- staining polymorphism) did not show any noticeable terocular bar, post-orbital maculae, and sacral marks difference.
Figure 3. Projection of individual scores resulted from the principal component analysis (PCA) for 20 morphometric characters from adult males (A, B) and adult females (C, D) of Ischnocnema ramagii in the space of the first with the second axes. Standardized coefficients and loadings are presented in tables 2 and 3, respectively. Napoli, M. F. et al. 169
Discussion karyotype of I. paulodutrai lacks the submetacentric chromosome pair present in I. ramagii from Salvador. The two morphotypes and the color patterns de- This result supports the presence of two full “sibling” scribed herein for Ischnocnema ramagii from Salva- species in Bahia State: one from the south (I. pau‑ dor (northern Bahia) are also observed within popu- lodutrai) and another from the north (I. ramagii). It lation samples from southern Bahia, Municipality of also validates the identity of I. paulodutrai as a full Ilhéus (type-locality of I. paulodutrai). Comparisons species, and refutes the recognition of Salvador sam- of specimens of I. paulodutrai with I. ramagii from ples as I. paulodutrai by Napoli and Brandão (2003). Salvador did not reveal any clear differences in exter- Chromosome numbers 2n = 28 and 2n = 30, with a nal morphology. Furthermore, I. ramagii from Salva- high number of telocentric pairs, have been described dor showed the same diploid number (2n = 30) and for species of the Central American genera Euhyas similar chromosome morphology as I. paulodutrai Fitzinger, 1843, Pelorius Hedges, 1989, and Syrrho‑ from Ilhéus (Siqueira et al., 2008). Nevertheless, the phus Cope, 1878 (see Campos and Kasahara, 2006 for
Figure 4. Karyotypes of Ischnocnema ramagii (A) with striped dorsum and (B) with non-striped dorsum after Giemsa staining. The second- ary constrictions are indicated by arrows in pair 7, corresponding to homomorphic state of nucleolar organizer regions (NOR). 170 Morphology and Karyotype of Ischnocnema ramagii a synthesis), currently placed in the genus Eleuthero‑ to 2n = 36, and also a range of the fundamental num- dactylus Duméril and Bibron, 1841 and Pristimantis ber of chromosome arms, from FN = 32 to FN = 58, Jiménez de la Espada, 1870 (Frost, 2008), and also even among species sharing the same diploid number. for the Brazilian species Pristimantis dundeei (Heyer In Brazil, seven “Eleutherodactylinae” species have and Muñoz, 1999), P. aff. dundeei, and I. paulodutrai been karyotyped: Haddadus binotatus (Spix, 1824), (Siqueira et al., 2008). I. guentheri (Steindachner, 1864), I. holti (Cochran, According to King (1990), Kuramoto (1990), and 1948), I. juipoca (Sazima and Cardoso, 1978), I. lac‑ Campos and Kasahara (2006), nearly 100 species of tea (Miranda-Ribeiro, 1923), I. parva (Girard, 1853), Eleutherodactylus (many of them currently assigned and I. paulodutrai. With the exception of I. pau‑ to other genera; see Heinicke et al., 2007 and Hedges lodutrai all species cytogenetically analyzed showed et al., 2008 for taxonomic arrangement) have, to some 2n = 20 or 2n = 22 chromosomes (Beçak, 1968; Beçak extent, been chromosomally examined. Contrary to and Beçak, 1974; de Lucca et al., 1974; de Lucca and the conservative karyotypic pattern of most anuran Jim, 1974; Siqueira et al., 2004; Siqueira et al., 2008; genera, Eleutherodactylus and allied genera show a Campos et al., 2008). The number of telocentric chro- high variation in diploid number, ranging from 2n = 18 mosomes is highest among those species possessing the largest chromosome numbers, which suggest that karyotype evolution may have occurred through cen- tric fusions and/or centric fissions. Sexual chromo- somes were described for Eleutherodactylus maussi Stejneger, 1904 (currently Strabomantis biporcatus Peters, 1863; Schmid et al. 1992, 2002b); E. euphro‑ nides Schwartz, 1967 and E. shrevei Schwartz, 1967 (both currently allocated in the genus Pristimantis; Schmid et al. 2002a), and for E. riveroi (= Pristiman‑ tis, Schmid et al. 2003). The high number of telocentric chromosomes suggests a closer relationship between I. ramagii and “Eleutherodactylinae” species from Central Amer- ica and northern Brazil. Molecular studies obtained by Heinicke et al. (2007) and Hedges et al. (2008) revealed three large radiations of species with un- expected geographic isolations: a South American clade, a Caribbean clade, and a Middle American clade. According to Heinicke et al. (2007), the great diversity of species of the South American clade is as- sociated to the Andes uplift, a relatively recent event. Mountain building and associated climatic changes resulted in repeating patterns of habitat isolation, which, in turn, probably resulted in genetic isolation and speciation in these amphibians. The use of other banding techniques, as well as an increase in the number of specimens and population samples of Ischnocnema and allied genera are neces- sary for the understanding of the chromosome evolu- tion in this group of frogs.
Resumo
No presente estudo descrevemos a variação Figure 5. Chromosomes of Ischnocnema ramagii treated with the Ag‑NOR method: (A) with striped dorsum and (B) with non-striped morfológica de Ischnocnema ramagii a partir de dorsum. Note the chromosome pair 7 bearing de Ag‑NOR site. uma população presente no Município de Salvador, Napoli, M. F. et al. 171
Estado da Bahia, Brasil, e caracterizamos dois Literature Cited morfótipos extremamente distintos neste táxon (dorso listrado vs. não-listrado); descrevemos seu Baldissera Jr., F. A., P. L. S. Oliveira, and S. Kasahara. 1993. cariótipo e comparamos a estrutura cariotípica dos Cytogenetics of four Brazilian Hyla species (Amphibia- Anura) and description of a case with a supernumerary distintos morfótipos aqui reconhecidos. Espécimes chromosome. Revista Brasileira de Genética, 16:335‑345. com dorso listrado foram menos comuns (13‑18%) Bastazini, C. V., J. V. Munduruca, P. L. B. Rocha, and M. do que aqueles com dorso não-listrado (81‑86%), F. Napoli. 2007. Which environmental variables better os últimos compostos por uma mistura de padrões explain changes in anuran community composition? A case study in the Restinga of Mata de São João, Bahia, Brazil. de colorido, cujas freqüências não foram diferentes Herpetologica, 63:459‑471. entre juvenis, machos e fêmeas. Os PCAs sobre Beçak, M. L. 1968. Chromosomal analysis of eighteen species of os dados morfométricos resultaram em alto grau Anura. Caryologia, 21:191‑208. de sobreposição entre espécimes de dorso listrado Beçak, M. L. and W. Beçak. 1974. Diploidization in Eleutherodactylus (Leptodactylidae-Amphibia). Experientia, e não-listrado, para ambos os sexos. Encontramos 30:624‑625. o número diplóide de 30 cromossomos para Bland, M. 2004. Multiple significance tests and the Bonferroni machos e fêmeas, com pares de cromossomos correction. Available from
Howell, W. M. and D. A. Black. 1980. Controlled silverstaining Schmid, M., W. Feichtinger, C. Steinlein, A. Rupprecht, T. of nucleolus organizer regions with a protective colloidal Haaf, and H. Kaiser. 2002a. Chromosome banding in developer: a 1‑step method. Experientia, 36:1014‑1015. Amphibia XXIII. Giant W sex chromosomes and extremely Humphries J. M., F. L. Bookstein, B. Chernoff, G. R. Smith, R. small genomes in Eleutherodactylus euphronides and L. Elder, and S. G. Poss, 1981. Multivariate discrimination Eleutherodactylus shrevei (Anura, Leptodactylidae). by shape in relation to size. Systematic Zoology, 30:291‑308. Cytogenetic and Genome Research, 97:81‑94. Juncá, F. A. 2006. Diversidade e uso de hábitat por anfíbios Schmid, M., W. Feichtinger, C. Steinlein, T. Haaf, M. Schartl, anuros em duas localidades de Mata Atlântica, no norte do R. Visbal García, J. Manzanilla Pupo, and A. Fernández estado da Bahia. Biota Neotropica, 6:1‑17. Badillo. 2002b. Chromosome banding in Amphibia XXVI. King, M. 1990. Amphibia; volume 4, Chordata 2, pp. 1‑241. In: Coexistence of homomorphic XY sex chromosomes and John, B. (Ed.), Animal cytogenetics. Gebriider Borntraeger, a derived Y‑autosome translocation in Eleutherodactylus Berlin/Stuttgart. maussi (Anura, Leptodactylidae). Cytogenetic and Genome Kuramoto, M. 1990. A list of chromosome numbers of anuran Research, 99:330‑343. amphibians. Bulletin of Fukuoka University of Education. Schmid, M., W. Feichtinger, C. Steinlein, R. Visbal Garcia Part III. Mathematics, Natural Sciences and Technology, and A. Fernández Badillo. 2003. Chromosome banding in 39:83‑127. Amphibia XXVIII. Homomorphic XY sex chromosomes Munduruca, J. F. V. 2005. Gradientes ambientais e composição and a derived Y autosome translocation in Eleutherodactylus da comunidade de anuros da Reserva Camurujipe, Mata de riveroi (Anura, Leptodactylidae). Cytogenetic and Genome São João, Bahia, Brasil. Master dissertation, Postgraduate Research, 101:62‑73. Program in Ecology and Biological Monitoring, Universidade Silva, A. P. Z., C. F. B. Haddad, and S. Kasahara, S. 2000. Federal da Bahia, Salvador, Bahia, Brazil, 100 pp. Chromosomal studies on five species of the genus Napoli, M. F. 2005. A new species allied to Hyla circumdata Leptodactylus Fitzinger, 1826 (Amphibia, Anura) using (Anura: Hylidae) from Serra da Mantiqueira, southeastern differential staining. Cytobios, 103:25‑38. Brazil. Herpetologica, 61:63‑69. Siqueira, S., F. Ananias, and S. M. Recco-Pimentel. 2004. Napoli, M. F. and F. Brandão. 2003. Eleutherodactylus Cytogenetic analysis of three species of Eleutherodactylus paulodutrai (Paulo’s Robber Frog): Geographic distribution. (Anura, Leptodactylidae) from southeastern Brazil. Genetics Herpetological Review, 34:161. and Molecular Biology, 27:363‑372. Schmid, M. 1978. Chromosome banding in Amphibia I. Siqueira, S., O. Aguiar-Jr, C. Strüssmann, M. L. Del-Grande, Constitutive heterochromatin and nucleolus organizers and S. M. Recco-Pimentel. 2008. Chromosomal analysis of regions in Bufo and Hyla. Chromosoma, 66:361‑88. three Brazilian “eleutherodactyline” frogs (Anura: Terrarana), Schmid, M., C. Steinlein, and W. Feichtinger. 1992. Chromosome with suggestion of a new species. Zootaxa, 1860:51‑59. banding in Amphibia XVII. First demonstration of multiple sex chromosome in amphibian: Eleutherodactylus maussi Submitted 03 February 2009 (Anura, Leptodactylidae). Chromosoma, 1001:284‑292. Accepted 23 April 2009
Appendix 1
Specimens Examined
Ischnocnema paulodutrai – BRASIL: Bahia: Ilhéus, type-locality (CEPLAC 347, 458, 466, 473‑476, 533, 541‑548, 708‑709, 711‑712, 714‑717, 7119, 1534, 1545‑1547, 1597‑1599, 1606, 1612‑1622, 1619, 1620‑1621, 1622, 1715‑1717; UFBA 819‑823).
Ischnocnema ramagii – BRASIL: Bahia: Salvador (CFBH 9644, 9651‑9655, 9657‑9663, 9665‑9670; UFBA 809‑818, 824‑842, 844, 846‑847, 2158‑2168, 2170‑2238, 2268‑2284, 2914‑2915, 3150, 3152, 3158‑3160, 5575). Pernambuco: Igarassu, type-locality (UFBA 7511, 7669‑7677). Sergipe: Itaporanga d’Ajuda (UFBA 6809‑6815).