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Chromosome analysis in Pseudopaludicola (Anura, Leiuperidae), with description of sex chromosomes XX/XY in P. saltica
2010; BioMed Central; Volume: 147; Issue: 2 Linguagem: Inglês
10.1111/j.1601-5223.2009.02153.x
ISSN1601-5223
AutoresThiago C. Duarte, Ana Cristina Prado Veiga‐Menoncello, Janaína F. R. Lima, Christine Strüssmann, Maria Lúcia Del-Grande, Ariovaldo A. Giaretta, Emiliane G. Pereira, Denise de Cerqueira Rossa‐Feres, Shirlei Maria Recco‐Pimentel,
Tópico(s)Plant Virus Research Studies
ResumoHereditasVolume 147, Issue 2 p. 43-52 Open Access Chromosome analysis in Pseudopaludicola (Anura, Leiuperidae), with description of sex chromosomes XX/XY in P. saltica Thiago C. Duarte, Thiago C. Duarte Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorAna Cristina P. Veiga-Menoncello, Ana Cristina P. Veiga-Menoncello Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorJanaína F. R. Lima, Janaína F. R. Lima Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorChristine Strüssmann, Christine Strüssmann Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorMaria L. Del-Grande, Maria L. Del-Grande Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorAriovaldo A. Giaretta, Ariovaldo A. Giaretta Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorEmiliane G. Pereira, Emiliane G. Pereira Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorDenise C. Rossa-Feres, Denise C. Rossa-Feres Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorShirlei M. Recco-Pimentel, Shirlei M. Recco-Pimentel Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this author Thiago C. Duarte, Thiago C. Duarte Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorAna Cristina P. Veiga-Menoncello, Ana Cristina P. Veiga-Menoncello Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorJanaína F. R. Lima, Janaína F. R. Lima Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorChristine Strüssmann, Christine Strüssmann Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorMaria L. Del-Grande, Maria L. Del-Grande Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorAriovaldo A. Giaretta, Ariovaldo A. Giaretta Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorEmiliane G. Pereira, Emiliane G. Pereira Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorDenise C. Rossa-Feres, Denise C. Rossa-Feres Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this authorShirlei M. Recco-Pimentel, Shirlei M. Recco-Pimentel Departamento de Anatomia, Biologia Celular, e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, BrazilSearch for more papers by this author First published: 04 May 2010 https://doi.org/10.1111/j.1601-5223.2009.02153.xCitations: 2 Shirlei M. Recco-Pimentel, Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, BR–13083-863 Campinas, São Paulo, Brazil. E-mail: shirlei@unicamp.br AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Taxonomic changes have frequently occurred in the anuran genus Pseudopaludicola as a consequence of high morphological similarity among its species. The present work reports karyotypic analysis of three Pseudopaludicola species sampled in their type locality and four Pseudopaludicola populations from distinct localities, aiming at contributing to the systematics of this genus. Chromosomes were stained with Giemsa or submitted to the silver staining (Ag-NOR) and C-banding techniques. The karyotype was 2n=22 in P. mineira, Pseudopaludicola sp. and two populations of P. saltica. The chromosome pair 8 was heteromorphic in P. saltica, characterizing a XX/XY sex-determination system with telocentric X and submetacentric Y. Highly similar karyotypes with 2n=18 chromosomes were observed in P. canga, P. aff. canga from Barreirinhas, State of Maranhão, Uberlândia, State of Minas Gerais and Icém, State São Paulo. The high similarity among the karyotypes 2n=18 suggested that the populations of P. aff. canga belong to the group 'pusilla', the same group of P. canga. The data demonstrated also that P. aff. canga from Barreirinhas (northeast region) is cytogenetically identical to P. canga with regarding the NOR site position in pair 3 and the presence of a heterochromatic block in the pair 2, whereas P. aff. canga from Uberlândia and Icém (southeast) had the NOR in the pair 9. Moreover, the cytogenetic data discriminated P. mineira and Pseudopaludicola sp. from the previously analyzed species with 22 chromosomes, and suggested that Pseudopaludicola sp. is an undescribed species. Sexual heteromorphic chromosomes are firstly reported in Pseudopaludicola and the data indicated the need of an extensive taxonomic review in this genus. The genus Pseudopaludicola Miranda Ribeiro, 1926, currently in the family Leiuperidae (Grant et al. 2006), consists of 12 species (Frost 2009). This genus is widely distributed throughout South America, from northern Colombia to Argentina. In Brazil, nine Pseudopaludicola species have been recognized (Lobo 1992; Frost 2009). These species are smaller than 20 mm, morphologically similar, and some of them exist in sympatry (Lobo 1992). The Pseudopaludicola genus has recently gone through several taxonomic changes, mainly based on molecular analyses. Frost et al. (2006) allocated Pseudopaludicola in the family Leptodactylidae, together with the genera Pleurodema, Edalorhina, Physalaemus, Paratelmatobius, Scythrophrys, Adenomera, Lithodytes, Leptodactylus and Vanzolinius. However, Grant et al. (2006) proposed a new arrangement for the family Leptodactylidae, presently consisting of four genera, Hydrolaetare, Leptodactylus, Paratelmatobius and Scythrophrys. The remaining genera, including Pseudopaludicola, were allocated in the family Leiuperidae. The species in the genus Pseudopaludicola are currently separated into the groups 'pusilla' and 'falcipes', according to their morphological traits (Lynch 1989). Their behavioral, bioacoustic and morphological characteristics have been intensively studied for taxonomic purpose (Haddad and Cardoso 1987; Lynch 1971, 1989; Lobo 1992, 1994, 1995, 1996). However, several aspects of intra and intergeneric phylogenetic relatedness and validation of new species remain unclear. According to several reports, a broad revision in this genus is still needed for the better understanding of its systematics (Lynch 1989; Lobo 1996). Cytogenetic analyses in the genus Pseudopaludicola are scarce in the literature and, thus far, the karyotypes described for P. falcipes and P. ameghini (sensuCope 1887), in the group 'falcipes', were analyzed only by conventional staining with Giemsa (Saez and Brum 1960; Brum and Saez 1968; Beçak 1968; Batistic et al. 1969; Batistic 1970). These techniques revealed uncommon variation in the number of chromosomes, from 2n=16 to 2n=22, in morphologically similar populations identified as P. falcipes (Beçak 1968; Batistic et al. 1969; Batistic 1970). However, a recent cytogenetic study on P. falcipes (2n=22) from the type locality revealed that the previously described chromosome number variation (2n=16 to 2n=22) attributed to the species P. falcipes (Beçak 1968; Batistic 1970), actually represents distinct species (Fávero et al. unpubl.). These karyotypes are related to diverse species, such as P. mystacalis (2n=16), P. ameghini (sensuCope 1887) and P. ternetzi (2n=20). Cytogenetic studies have been proven helpful as a tool to assist in the characterization and systematics of cryptic species, such as the ones existing in the Pseudopaludicola genus. The objective of the present work was to analyze the karyotypes of several Brazilian Pseudopaludicola species and populations, aiming at contributing to better understand the systematics of this genus. Cytogenetic data are described for the species P. mineira and P. saltica ('falcipes' group) and P. canga ('pusilla' group) from their type localities, and four populations, some of which are suspected of being new species. MATERIAL AND METHODS The analyzed specimens were sampled in eight Brazilian locations (Table 1). The voucher specimens were deposited in the Coleção Zoológica at the Federal University of Mato Grosso (UFMT), Mato Grosso State, Brazil, in Museu de Zoologia "Prof. Adão José Cardoso" (ZUEC) at the State University of Campinas (UNICAMP) and in Museu de Zoologia of the State University of São Paulo, São José do Rio Preto (DZSJRP), in São Paulo State, Brazil. A map of sampling locations in which the studied specimens were surveyed is shown in Fig. 1. Table 1. Number of specimens of the analyzed Pseudopaludicola species and their respective sampling localities in Brazil. (ZUEC = Zoology Museum of the State University of Campinas, SP, Brazil; DZSJRP = Zoology Museum of the State University of São Paulo, São José do Rio Preto, SP, Brazil; UFMT = Zoology Collection of the Federal University of Mato Grosso, Brazil). Species Locality Number of specimens Accession number P. mineira Serra do Cipó, Minas Gerais (type locality) (19°14′S; 43°33′W) 8 ♂; 1♀ ZUEC 14319 – 14323, 14326, 14329 e 14330 Pseudopaludicola sp. Andaraí, Bahia (12°48′S; 41°19′W) 10 ♂; 3 ♀ ZUEC 14255, 14256, 14257, 14260, 14261, 14264 – 14271 P. saltica Chapada dos Guimarães, Mato Grosso (type locality) (15°27′S; 55° 44′W) 13 ♂; 1♀ ZUEC 14227, 14228, 14232, 14234, 14236, 14237, 14240, 14242 – 14247 UFMT 8542 P. saltica Uberlândia, Minas Gerais (18°55′S; 48°16′W) 8 ♂; 2♀ ZUEC 14291, 14292, 14295, 14296, 14301, 14302, 14304, 14305, 14307, 14310 P. canga Serra dos Carajás, Pará (type locality) (6°03′S; 50°28′W) 3 ♂; 12♀ ZUEC 14343, 14353, 14363, 14364, 14366, 14369 – 14374, 14376, 14378, 14379, 14380 Pseudopaludicola aff. canga Barreirinhas, Maranhão (2°44′S; 42°49′W) 2♂ ZUEC 13860 e 13867 Pseudopaludicola aff. canga Uberlândia, Minas Gerais (18°55′S; 48°16′W) 8 ♂; 3♀ ZUEC 14181, 14185, 14186, 14187, 14209, 14212, 14214, 14216, 14217, 14222 e 14223 Pseudopaludicola aff. canga Icém, São Paulo (20°20′S; 49°H′W) 5 ♂; 1♀ DZSJRP 8727, 8728, 8747, 8749, 8750, 8752 Figure 1Open in figure viewerPowerPoint Map of sampling locations in which the studied specimens of Pseudopaludicola species were surveyed in Brazil, as listed in Table 1. Mitotic metaphases were obtained from cell suspensions of intestinal epithelium and testis of animals previously treated with 2% colchicine solution for five h, according to King and Rofe (1976) and Schmid (1978), with few modifications. Chromosomes were stained with Giemsa 10% or submitted to the (Ag-NOR) silver impregnation (Howell and Black 1980) and C-banding (Sumner 1972) techniques, with modification: samples were pre-treated with 50% acetic acid, according to Siqueira et al. (2008). The samples were examined under a photomicroscope Olympus BX60, and images were captured using QCapture™ 2.81.0 and Image Pro-plus™ 4.5. The chromosomes were measured and classified according to Green and Sessions (1991), as shown in Table 2. Table 2. Morphometric analysis of the chromosomes in the studied Pseudopaludicola. Classification according to Green and Sessions (1991): M=metacentric; SM=submetacentric; T=telocentric; †chromosome X; ♦ chromosome Y. Number of chromosomes 1 2 3 4 5 6 7 8 8′ 9 10 11 P. mineira (Serra do Cipó, Minas Gerais state) Relative size 15.66 13.47 11.29 10.33 9.20 8.49 7.92 6.71 - 6.11 5.72 5.03 Arm ratio 1.16 1.19 1.85 1.81 1.14 1.15 1.71 1.34 - 1.16 1.17 1.18 Classification M M SM SM M M SM M - M M M Pseudopaludicola sp. (Andaraí, Bahia state) Relative size 14.71 12.53 11.06 10.28 8.90 8.07 7.45 6.91 5.79 5.35 5.01 3.93 Arm ratio 1.15 1.37 1.99 1.87 1.19 1.31 1.39 2.31 1.85 1.25 1.25 1.93 Classification M M SM SM M M M SM SM M M SM P. saltica (Chapada dos Guimarães, Mato Grosso state) Relative size 14.65 12.50 11.80 10.90 8.23 7.77 7.33 5.72 6.10 5.37 4.95 4.64 Arm ratio 1.19 1.43 2.46 2.47 1.24 1.94 1.09 8.30 2.27 1.16 1.21 1.17 Classification M M SM SM M SM M T† SM ♦ M M M P. saltica (Uberlândia, Minas Gerais state) Relative size 14.75 12.60 11.60 10.80 9.23 8.31 7.67 5.68 6.26 4.94 4.87 4.53 Arm ratio 1.18 1.46 2.59 2.60 1.20 1.87 1.12 8.98 2.13 1.18 1.19 1.21 Classification M M SM SM M SM M T† SM ♦ M M M P. canga (Serra dos Carajás, Pará state) Relative size 14.89 12.31 11.49 10.55 9.68 8.88 7.46 6.64 - 6.57 - - Arm ratio 1.15 1.31 2.41 2.01 1.31 1.19 10.0 9.30 - 1.72 - - Classification M M SM SM M M T T - SM - - Pseudopaludicola aff. canga (Barreirinhas, Maranhão state) Relative size 14.73 13.13 12.16 9.63 9.15 8.61 7.56 6.86 - 5.97 - - Arm ratio 1.10 1.26 2.38 1.84 1.34 1.25 10.0 11.0 - 2.02 - - Classification M M SM SM M M T T - SM - - Pseudopaludicola aff. canga (Uberlândia, Minas Gerais state) Relative size 14.85 13.05 10.37 10.54 9.52 9.23 8.17 6.56 - 7.30 - - Arm ratio 1.27 1.39 2.59 2.08 1.28 1.41 13.6 10.72 - 2.01 - - Classification M M SM SM M M T T - SM - - Pseudopaludicola aff. canga (Icém, São Paulo state) Relative size 15.29 12.17 10.98 10.25 9.51 8.83 8.42 6.65 - 6.87 - - Arm ratio 1.18 1.47 2.09 2.68 1.19 1.39 14.8 12.0 - 1.87 - - Classification M M SM SM M M T T - SM - - RESULTS The karyotypes of the Pseudopaludicola specimens studied in the present work consisted of 2n=22 or 2n=18 chromosomes. Karyotypes 2n=22 The species P. mineira and P. saltica, and the population of Pseudopaludicola sp. showed 2n=22 chromosomes. The karyotypes of these species were observed to differ only in the morphology of the pairs 6, 7, 8 and 11 (Table 2, 3, Fig. 2). Table 3. Summary of differences observed among the karyotypes of Pseudopaludicola species with 2n=22 chromosomes. M = metacentric; SM = submetacentric; T = telocentric. Chromosome pairs Species 6 7 8 11 NOR in pair 8 C-band P. mineira M/M SM/SM M/M M/M telomeric 1, 2, 7, 8, 9, 10 Pseudopaludicola sp. M/M M/M SM/SM SM/SM sub-telomeric 1, 8, 9 SM/SM M/M T/SM M/M telomeric 1, 2, 3, 4, 6, 8 P. saltica SM/SM M/M T/T M/M telomeric 1, 2, 3, 4, 6, 8 Figure 2A–DOpen in figure viewerPowerPoint Karyotypes with 2n=22 chromosomes stained with Giemsa. (A) P. mineira, (B) Pseudopaludicola sp., (C) male specimen of P. saltica from Chapada dos Guimarães, (D) male specimen of P. saltica from Uberlândia. In the standout: (B) homomorphic pair 8 of Pseudopaludicola sp.; (C, D) the sex chromosomes XX of P. saltica. Bar = 5 µm. In most populations, the nucleolar organizing region (NOR) was detected in the telomeric region of the long arm of the pair 8 (Fig. 3). However, in Pseudopaludicola sp. the NOR was interstitially located near the telomere of the long arm of the pair 8 (Fig. 3B). With the exception of P. mineira and of two Pseudopaludicola sp. specimens (ZUEC 14255 and 14266), the NOR was heteromorphic in all of the analyzed individuals (Fig. 3B–D). The NOR heteromorphism comprised a difference in size among the homologous chromosomes of the pair 8, especially in the population of Pseudopaludicola sp. (Fig. 2B), and were identified as 8 and 8'. Figure 3A–DOpen in figure viewerPowerPoint Localization of the NOR site in the species with karyotype 2n=22, by the silver impregnation technique. (A) P. mineira, (B) Pseudopaludicola. sp. (C) P. saltica from Chapada dos Guimarães, (D) P. saltica from Uberlândia. In the outstand: (B) heteromorphic pair 8; (C, D) homomorphic pair 8. Bar = 5 µm. The C-banding technique revealed heterochromatic blocks in the centromeric region of all chromosomes. In Pseudopaludicola mineira, pericentromeric bands were observed in the long and short arms of the pair 1, in the short arm of pairs 2 and 7, and in the long arm of the pairs 9 and 10 (Fig. 4A). Interstitial blocks were present in both arms of pair 8 (Fig. 4A), and the one in the long arm was associated to the secondary constriction and coincident to the NOR (Fig. 3A). In Pseudopaludicola sp., heterochromatic interstitial blocks were observed in the short and long arms of the pair 1 and in the long arms of the pairs 8 and 9 (Fig. 4B). In the pair 8, this block was adjacent to a NOR positive secondary constriction. Figure 4A–DOpen in figure viewerPowerPoint Karyotypes with 2n=22 chromosomes submitted to the C-banding technique. (A) P. mineira (B) Pseudopaludicola sp. (C) male of P. saltica from Chapada dos Guimarães, (D) male of P. saltica from Uberlândia. In standout: (B) heteromorphic pair 8; (C, D) sex chromosomes XX of P. saltica. Bar = 5 µm. The karyotypes were identical in the two analyzed populations of P. saltica, from Chapada dos Guimarães, Mato Grosso, Brazil (type locality) and from Uberlândia, Minas Gerais, Brazil (Table 2, 3, Fig. 2C–D). They consisted of metacentric (pairs 1, 2, 5, 7, 9, 10 and 11) and submetacentric chromosomes (pairs 3, 4 and 6). In males, the NOR-bearing pair 8 was heteromorphic (XY) with telocentric and submetacentric homologous (Fig. 2C–D, 3C–D). In females, both chromosomes in the pair 8 were telocentric (XX) (Fig. 2C–D, 3D). In P. saltica, besides the centromeric heterochromatic blocks, positive C-bands were detected in the interstitial regions of the long arms of the pairs 1, 2, 3, 4 and 6, and in the short arms of the pairs 1 and 2 (Fig. 4C–D). A heterochromatic block coincident to the NOR was observed in the long arm of the pair 8, which in females showed a size heteromorphism (Fig. 4C–D). However, the Ag-NOR reaction was weaker and bluish compared to the intensely stained magenta heterochromatic blocks. The homologous of the pair 8 were polymorphic in the only analyzed P. saltica female from the type locality, due to the size heteromorphism of the secondary constriction, positive to C-banding, localized in the long arm coincident with the NOR (Fig. 5A). In males, the relative size of the submetacentric chromosome Y was a little larger than the telocentric X chromosome (Fig. 5B, Table 2). However, in the specimen UFMT 8542, the X chromosome was larger than the Y, with increased staining in the NOR site and in the heterochromatin block (Fig. 5C). Figure 5A–COpen in figure viewerPowerPoint Sex chromosomes of P. saltica from the type locality submitted to the Giemsa conventional staining, C-banding and Ag-NOR. (A) chromosomes X of the female voucher specimen ZUEC 14234, (B) chromosomes XY of the male voucher specimen ZUEC 14172 (C), chromosomes XY of the specimen UFMT 8542. Bar = 5 µm. Aiming at verifying the meiotic behavior of the sex chromosomes in P. saltica, they were analyzed at diakinesis in testis cells stained with Giemsa (Fig. 6A) and submitted to the C-banding technique (Fig. 6B). On the diakinesis, eleven bivalents were observed, which followed the expected behavior of homologous in this meiotic phase. Figure 6A–BOpen in figure viewerPowerPoint Diakinesis in the meiotic prophase I of germinative cells of male P. saltica from the Chapada dos Guimarães. Chromosomes were stained with Giemsa (A) and submitted to the C-banding technique (B). Arrows indicate the pair of sex chromosomes. Bar = 5 µm. Karyotypes with 2n=18 The karyotypes of P. canga from Serra dos Carajás and P. aff. canga from Barreirinhas, Uberlândia and Icém consisted of 2n=18, with no differences in the chromosome classification. The pairs 1, 2, 5 and 6 were metacentric, the pairs 3, 4 and 9 submetacentric and the pairs 7 and 8 telocentric (Table 2, Fig. 7). In all analyzed populations, the long arm of one of the homologous of the pair 3 had a slight size heteromorphism, which did not alter the classification of this pair (Table 2). Figure 7A–DOpen in figure viewerPowerPoint Karyotypes with 2n=18 chromosomes stained with Giemsa. (A) P. canga, (B) P. aff. canga - Barreirinhas, (C) P. aff. canga - Uberlândia, (D) P. aff. canga - Icém. Bar = 5 µm. The NOR site was localized in the pericentromeric region of the short arm of the pair 3 both in P. canga and P. aff. canga - Barreirinhas (Fig. 8A–B), near the telomere of the long arm of the pair 9 in P. aff. canga - Uberlândia (Fig. 8C) and at the telomeric region of the long arm of the pair 9 in P. aff. canga - Icém (Fig. 8D). Figure 8A–DOpen in figure viewerPowerPoint Localization of the NOR site in the species with karyotype 2n=18, by silver impregnation. (A) P. canga, (B) P. aff. canga - Barreirinhas, (C) P. aff. canga - Uberlândia (D) P. aff. canga - Icém. Bar = 5 µm. The C-banding pattern was similar among the four Pseudopaludicola populations and comprised few non-centromeric heterochromatic blocks. In all analyzed populations, a C-band was detected in the short arm of the pair 1. Only P. canga and P. aff. canga - Barreirinhas had the heterochromatic block in the pericentromeric region of the long arm of the pair 2 (Fig. 9). Figure 9A–DOpen in figure viewerPowerPoint Karyotypes with 2n=18 chromosomes submitted to the C-banding technique. (A) P. canga, (B) P. aff. canga - Barreirinhas, (C) P. aff. canga - Uberlândia, (D) P. aff. canga - Icém. In the outstand: the chromosome pair 2 of another P. canga metaphase, clearly showing the positive C-band in the pericentromeric region of the long arm. Bar = 5 µm. DISCUSSION The data presented herein revealed cytogenetic characteristics previously unknown in the Pseudopaludicola genus, which are of fundamental importance for the systematics of this anuran group and for the understanding of its evolutionary biology. Karyotype polymorphisms were suitable to separate the analyzed Pseudopaludicola species and populations into the 'falcipes' and 'pusilla' groups. The karyotypes (2n=22) were conservative between the species P. mineira and P. saltica, sampled in their type localities. However, the P. saltica from Uberlândia, and the population of Pseudopaludicola sp. showed morphological polymorphisms in the chromosomes 7, 8 and 11, which distinguished P. mineira from Pseudopaludicola sp. Moreover, these two species were differentiated by the NOR site in the pair 8, which was terminal in P. mineira and subterminal in Pseudopaludicola sp., and by polymorphisms in the heterochromatin distribution as well. Therefore, the karyotypic analysis indicated that Pseudopaludicola sp. is a distinct taxon, possibly a new, undescribed species. The fact that the P. saltica specimens from Uberlândia did not differ from the specimens sampled in the type locality (Chapada do Guimarães) confirmed that they are the same species. In these populations, the morphological differentiation of the pair 8 between males and females is an evidence of XX/XY sex-determination system, which was previously unknown in the genus Pseudopaludicola. The differentiation of the pair 8 might be directly involved in speciation within Pseudopaludicola, since the NOR is also localized in this same chromosome pair in other species (2n=22) of the genus, as for instance P. mineira and Pseudopaludicola sp., herein described, and P. falcipes from the type locality (Porto Alegre, Rio Grande do Sul state, Brazil) and Pseudopaludicola sp. from Poconé, Mato Grosso state, Brazil (Fávero et al. unpubl.). The morphology of the pairs 3 and 8 of these two last populations, however, was significantly different compared with the ones 2n=22 described herein. In all of these analyzed species, the pair 8 bears the NOR in the telomeric region and has a heterochromatic block associated to this region, while in P. falcipes the NOR is localized in the long arm of the subtelocentric chromosome 8, near the centromere. The data suggested that modifications in the nucleolar organizing region and, possibly, in the heterochromatin, can be related to changes in the structure of this chromosome pair and have contributed to the karyotypic differentiation of these species. Batistic (1970) observed a 2n=22 karyotype in meiotic meta-phases of a population identified as P. falcipes, from Feira de Santana, Bahia state, Brazil. As the study was restricted to meiotic metaphases and limited to the number of chromosomes detection, additional comparisons with this population were not possible. Pseudopaludicola canga is the first species within the group 'pusilla' to have its karyotype studied. The P. canga and the three populations of P. aff. canga– Barreirinhas, Uberlândia and Icém have 2n=18 karyotypes. The two former ones could be distinguished from the others only by the NOR site and by the presence of a C-band in chromosome 2. Considering this high karyotypic similarity, it is conceivable that these Pseudopaludicola populations are closely related to P. canga and possibly belong to the the same group 'pusilla'. This hypothesis could be tested in a phylogenetic study comprising species of the groups 'falcipes' and 'pusilla'. According to Giaretta and Kokubum (2003), P. canga is known only from its type locality, Serra dos Carajás, Pará, Brazil. However, P. canga and P. aff. canga - Barreirinhas were not distinguished in the cytogenetic analysis. Hence, the possibility that they belong to the same species cannot be discarded, which would expand the known P. canga geographic distribution. This hypothesis must be tested in additional studies on their morphology and bioacoustics, since they have been considered distinct taxa (G. A. Vasconcelos pers. comm.). Furthermore, no differences were detected in the karyotypes of P. aff. canga - Uberlândia and P. aff. canga - Icém, which also suggests that these two populations belong to the same taxon. The taxonomic identification of the population P. aff. canga (2n=18) from Icém (São Paulo state, Brazil) was based on morphological similarities with P. falcipes (2n=22). However, the chromosome analysis showed that this population is indeed closely related to the P. canga species. Correspondingly, the karyotype 2n=18 described by Beçak (1968) and Batistic (1970) for specimens from the region of São José do Rio Preto (São Paulo state) and identified as P. falcipes, is similar to the P. canga (2n=18) karyotype. Therefore, our data suggest that these populations are actually related to P. canga, despite having morphological similarities to P. falcipes and morphologically different of P. canga (D. C. Rossa-Feres unpubl.). In addition to the Pseudopaludicola karyotype 2n=18 in Icém, two other distinct karyotypes are found in sympatry, consisting of 2n=16 and 2n=20. These specimens are cytogenetically related, respectively, to P. mystacalis (2n=16) and to P. ameghini (sensuCope 1887) and P. ternetzi, both with 2n=20 (Fávero et al. unpubl.). The cytogenetic studies have indicated that the high morphological similarity among Pseudopaludicola species has frequently misled taxonomic identifications, and reinforces the need of an extensive review in the genus. The cytogenetic data presented herein suggested that the karyotypes 2n=18 chromosomes, probably belong to the group 'pusilla' and are evolutionary conservative, since they have rather similar chromosome morphology and heterochromatin pattern. However, they are variable in relation to the NOR-bearing pair and this characteristic clearly separated the 2n=18 karyotypes in two groups: one with the NOR site in the pair 3 (P. canga and P. aff. canga - Barreirinhas) and the other with the NOR in the pair 9 (P. aff. canga from Uberlândia and Icém). Chromosome number variation among species in the same genus, as demonstrated in Pseudopaludicola, is quite rare in the order Anura. Various chromosomic rearrangement mechanisms, involving centric fusion and/or fission events, were used to explain the origin of numerically different karyotypes, as well as possibl
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