A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions

Biochemical Systematics and Ecology 34 (2006) 457e466 www.elsevier.com/locate/biochemsyseco

A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions Natan Medeiros Maciel a,*, Carlos Alberto Schwartz a, Guarino Rinaldi Colli b, Mariana Souza Castro a,c, Wagner Fontes c, Elisabeth N. Ferroni Schwartz a a

Laborato´rio de Toxinologia, Departamento de Cieˆncias Fisiolo´gicas, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, Campus Darcy Ribeiro, Brası´lia DF 70910-900, Brazil b Departamento de Zoologia, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, Campus Darcy Ribeiro, Brası´lia DF 70910-900, Brazil c Centro Brasileiro de Servic¸os e Pesquisas em Proteı´nas, Laborato´rio de Bioquı´mica e Quı´mica de Proteı´nas, Departamento de Biologia Celular, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, Campus Darcy Ribeiro, Brası´lia DF 70910-900, Brazil Received 20 April 2005; accepted 9 January 2006

Abstract This research tested the utility of two classes of skin secretion compounds to the phylogeny of the Bufo crucifer group. Skin secretions from specimens of nine populations of B. crucifer group were obtained and submitted to qualitative analysis. We observed a clear difference in the composition of the skin secretion molecules obtained from the species of Bufo studied. Fiftynine molecules, 16 indolealkylamines and 43 proteins, were used as characters, and 39 of these were parsimonious informative. The tree topology of the skin secretion combined data showed areas of congruence and conflict when compared to an mtDNA phylogeny of the B. crucifer group. We used the Templeton test to evaluate the heterogeneity between the skin secretion and mtDNA data. Although not recommended, we performed a combined analysis with the two partitions. The skin secretion characters from the species of Bufo studied have phylogenetic signal. These data are indicative, at least as a preliminary study, of the phylogenetic relationships among the B. crucifer group taxa. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Bufo; Bufo crucifer group; Toad; Skin secretion; Proteins; Indolealkylamines; Alternative character to infer phylogeny

1. Introduction The genus Bufo Laurent, 1768 is cosmopolitan and encompasses more than 250 species, arranged in approximately 40 phenetical groups (Frost, 2004). One of these, the Bufo crucifer group, occurs in Atlantic Rain Forest and adjacent * Corresponding author. Fax: þ55 61 3274 1251. E-mail address: [email protected] (N.M. Maciel). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2006.01.005

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areas from the state of Ceara´ to Rio Grande do Sul in Brazil and possibly in northeastern Argentina (Baldissera et al., 2004). The group is presumably most closely related to the Bufo marinus group (Blair, 1972; Low, 1972; Maxson, 1984; Baldissera et al., 1999), from which it differs by having relatively lower cranial crests, smoother dorsal skin, and smaller parotoid glands (Duellman and Schulte, 1992). Wied-Neuwied (1821) described the epithet crucifer as a triple cross, formed by a dark dorsal pattern that follows the vertebral line. This character is extremely polymorphic and has contributed to some taxonomic confusion in the group. In the past, different forms in the B. crucifer group were regarded as species or subspecies (Lutz, 1934; Cochran, 1955) but were placed under the synonymy of B. crucifer by Cochran (1955). Cei (1980) redescribed B. crucifer using a specimen from S~ao Paulo state (Brazil), and some recent works suggest the existence of more than one species in the group (Haddad and Sazima, 1992; Izecksohn and Carvalho-e-Silva, 2001). Recently, Baldissera et al. (2004) revised the B. crucifer group on the basis of external morphological and morphometric characters and recognized five species: B. crucifer Wied-Neuwied, 1821 (ranges from Ceara´ to southern Espı´rito Santo and northeastern Minas Gerais); Bufo ornatus Spix, 1824 (occurs from southern Espı´rito Santo, through Rio de Janeiro and S~ao Paulo to northern Parana´ and possibly in northeastern Argentina, in the provinces of Misiones and Corrientes); Bufo henseli A. Lutz, 1934 (southern Santa Catarina to the coast of Rio Grande do Sul); Bufo abei Baldissera, Caramaschi, and Haddad, 2004 (Parana´ through Santa Catarina to northern Rio Grande do Sul); and Bufo pombali Baldissera, Caramaschi, and Haddad, 2004 (occurs in transitional areas between the Atlantic Rain Forest and ‘‘cerrados’’ of Minas Gerais). Additionally, Baldissera (2001) proposed a phylogeny of the group based on 12S and 16S rRNA mitochondrial gene sequences. Several data sets have been used in phylogenetic analysis of amphibians, including DNA or RNA sequence (Liu et al., 2000; Chek et al., 2001; Read et al., 2001; Pramuk et al., 2001; Dawood et al., 2002; Wilkinson et al., 2002; Symula et al., 2003; Moriarty and Cannatella, 2004; Hillis and Wilcox, 2005), hormones (Conlon et al., 2000), morphology (Ford, 1993; Mendelson et al., 2000; Faivovich, 2002; Haas, 2003), protein sequences (Alrubaian et al., 2002), skin secretions (Maciel et al., 2003), and sperm ultrastructure (Garda et al., 2002). These different data sets have been used in isolation or in combined analyses (e.g. Pramuk, 2002). The granular glands of amphibian skin produce a great variety of substances responsible for the noxious or poisonous character of this tissue. These substances have been grouped according to their chemical structure as biogenic amines, steroids, alkaloids, peptides, and proteins (Daly et al., 1987; Clarke, 1997; Erspamer, 1994). They act as passive defense mechanisms against predators and microorganisms, and they have evolved independently in different groups of amphibians as a result of their interaction with the environment (Toledo and Jared, 1995). Three groups of aromatic amines are found in amphibian skin (Erspamer, 1971): indolealkylamines, imidazolealkylamines, and hydroxyphenylalkylamines. Owing to their variety, abundance, and widespread distribution, indolealkylamines are by far the most important and characteristic biogenic amines of amphibian skin (Erspamer, 1994). According to Cei et al. (1972), the skin of Bufo provides a spectacular representation of indolealkylamines. Since the occurrence of a given indole derivative implies the occurrence of the enzyme systems catalyzing its biosynthesis and several of these enzyme systems are likely highly specific, these substances might trace species evolution and provide information about phylogenetic relationships. Several works describe differences in indolealkylamine composition from species of Bufo (e.g. Cei et al., 1972), and they have been used in phylogenetic analyses of species of Bufo (Ceriotti et al., 1989; Maciel et al., 2003). Herein we evaluate the utility of indolealkylamines and proteins from skin secretions in a phylogenetic analysis of species in the B. crucifer group, and conduct partitioned and combined analyses of the different forms (as recognized by Baldissera et al., 2004), using data from skin secretions and gene sequences. 2. Materials and methods Specimens from nine populations of the B. crucifer group were collected in different regions in eastern Brazil (Table 1). Specimens of B. marinus, Bufo granulosus, and Bufo gr. margaritifer were also collected and used as outgroups in phylogenetic analyses (see below). The surveys were done under Centro de Conservac¸~ao e Manejo de Anfı´bios e Re´pteis/Instituto Brasileiro do Meio Ambiente e Recursos Naturais Renova´veis (RAN/IBAMA) license numbers 12/2001 and 054/02. Voucher specimens were deposited in the Colec¸~ao Herpetolo´gica da Universidade de Brası´lia (CHUNB), Brası´lia, DF, Brazil e B. marinus e Bele´m do Para´ (PA): CHUNB 35659; B. gr. margaritifer e Balsas (MA): CHUNB 35660; B. granulosus e Porto de Galinhas (PE): CHUNB 35661, 35662; Bufo cf. crucifer e Manhumirim (MG): CHUNB 35661e35669; B. crucifer e Itacare´ (BA): CHUNB 35670e35672; B. pombali e Nova Lima (MG):

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Table 1 Specimens used in this study, city and state in Brazil where the specimens were collected, and number of specimens used Species

City/State of Brazil

Number of specimens

B. B. B. B. B. B. B. B. B. B. B. B.

Bele´m do Para´ e PA Porto de Galinhas e PE Balsas e MA Itacare´ e BA Manhumirim e MG Nova Lima e MG Mogi das Cruzes e SP Ubatuba e SP Itanhae´m e SP Teleˆmaco Borba e PR Santo Amaro da Imperatriz e SC Torres e RS

3 2 1 18 18 6 4 4 6 5 6 3

marinus granulosus gr. margaritifer crucifer cf. crucifer pombali ornatus ornatus ornatus ornatus abei henseli

CHUNB 35673, 35674; B. ornatus e Mogi das Cruzes (SP): CHUNB 35675e35677; Ubatuba (SP): CHUNB 35678e 35679; Itanhae´m (SP): CHUNB 35680e35682; Teleˆmaco Borba (PR): CHUNB 35683e35686; B. abei e Santo Amaro da Imperatriz (SC): CHUNB 35687e35690; B. henseli e Torres (RS): CHUNB 35691.

2.1. Indolealkylamines Parotoid secretions were obtained by gland compression, resuspended in deionized water, lyophilized, and frozen at
20  C. For thin layer chromatography, dried secretion was prepared in methanol to the final concentration of 17 mg/ml. Indolealkylamine standards were prepared using commercial products (serotonin, 5-HT from Sigma Chemical Company), extracts from seeds of Anadenanthera macrocarpa (bufotenin, BTN; Stromberg, 1954), parotoid secretions of Bufo ictericus (dehydrobufotenin, DHB; Slotta and Neisser, 1937), or methylation of BTN (bufotenidin, BTD; Pereira et al., 1963). The identification of bufothionin (DHB-S) was done as described by Deulofeu and Duprat (1944). To analyze indolealkylamines from skin secretions, we used thin layer chromatography (TLC). Ascendant unidimensional chromatography was developed using silica-gel H plates 20  20 cm (Sigma Chemical Company), as described previously (Maciel et al., 2003). We used a mixture of ethyl acetate, isopropylic acid, and ammonium hydroxide (25%) as eluent in the proportion (v:v:v) 90:70:40. The plates were sprayed with o-phthalaldehyde reagent (OPT), according to Narasimhacheri and Plaut (1971), and the visualization of the spots was done under UV light (375 nm). The qualitative indolealkylamines analysis was done by the respective retention factor, determined by the distance (cm) moved by the amine divided by the distance (cm) moved by the solvent front.

2.2. Proteins Skin secretions were obtained according to Mor et al. (1991), with modifications. Animals were killed by the destruction of the nervous system and, afterwards, the skin was carefully removed, with the exception of that covering the parotoid gland. Skins were weighed, washed with distilled water, chopped, and extracted three times with 20 volumes (w/v) of 10% acetic acid at 4  C. Skin secretions from specimens in each population were combined in the analysis, except for specimens collected in Ubatuba and Mogi das Cruzes, SP, Brazil. To analyze proteins from skin secretions, we used vertical block polyacrylamide gel electrophoresis. The concentration gel was 12% following Maniatis et al. (1989). Lyophilized skin secretions (250 mg from each population) were first precipitated using 50% trichloroacetic acid. Molecular mass markers were also used (Sigma Chemical Company). Protein bands were stained with silver (protocol provided by Dr. P.M. Larsen and Dr. S. Fey, Center for Proteome Analysis, Odense, Denmark) and analyzed with Image Master 3.1 (Elite SoftwareAmersham Pharmacia Biotech).

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2.3. Phylogenetic analysis Partitioned (data not shown) and combined parsimony analyses of skin secretion data sets (indolealkylamines and proteins) were performed to test their utility in constructing a phylogeny of the B. crucifer group. We also performed a combined analysis with the skin secretion and mtDNA data sets (obtained from Baldissera, 2001). We coded skin secretion characters as absent (0) or present (1). The Templeton test, implemented in PAUP* 4.0 b10 (Swofford, 2000), was used to assess the heterogeneity between the two data sets. To perform this analysis we constructed a matrix with the five following taxa of each partition: B. marinus, B. crucifer, B. ornatus, B. abei, and B. henseli. Baldissera (2001) used only one outgroup (B. marinus) and did not evaluate B. pombali in his analysis. Baldissera (2001) evaluated more than one population of each species in the B. crucifer group, as we also did with B. ornatus. To construct the matrix of each data partition (mtDNA and skin secretions), we chose one population of each species of Baldissera’s work and one population of ours. Phylogenetic analysis was conducted using MacClade 4.0 (Maddison and Maddison, 1992) and PAUP* 4.0 b10 (Swofford, 2000). We sought the shortest trees using an exhaustive search. Support for individual clades was evaluated using non-parametric bootstrapping (Felsenstein, 1985), with 10,000 pseudoreplicates per analysis with 10 random addition sequences per pseudoreplicate. Bootstrap values higher than 70% are considered significant (Hillis and Bull, 1993). If more than one most parsimonious tree were recovered, we performed a strict consensus analysis. 3. Results Populations of the B. crucifer group and outgroups differed in the composition of indolealkylamines (Table 2a). The retention factors of standard amines were 0.09 (BTD), 0.29 (DHB), 0.39 (DHB-S), 0.56 (5-HT), and 0.745 (BTN). Furthermore, 11 unidentified indolealkylamines were observed with the following retention factors: 0.016, 0.083, 0.109, 0.145, 0.358, 0.477, 0.49, 0.60, 0.645, 0.762, and 0.78. All indolealkylamines, conspicuous or traces, identified or not, were considered in the phylogenetic analysis. The composition of skin secretion proteins differed among populations of the B. crucifer group (Table 2b). The molecular mass of 43 proteins extended from 15 to 90 kDa. In the qualitative analysis, all stained bands were considered. The combined matrix contained 59 characters, from which 19 were ambiguous: six indolealkylamine characters e BTN, DHB-S, and 0.358, 0.49, 0.60 and 0.645 (Rfs), and 13 protein characters e 18, 19, 20, 23, 24, 25, 29, 32, 43, 44, 52, 53, and 55. The parsimony analysis made by exhaustive search resulted in four trees with a treelength of 98. The resultant cladogram from the strict consensus analysis (treelength of 107) is shown in Fig. 1. From all 59 characters, 39 were parsimony informative, 19 were uninformative, and one was constant. Each one of the trees presented CI ¼ 0.5918 and RI ¼ 0.4937. The strict consensus analysis presented CI ¼ 0.5421 and RI ¼ 0.3797. The CI, excluding uninformative characters, was 0.4432. The Templeton test was implemented using the most parsimonious tree of each data set (mtDNA and skin secretion) (Fig. 2a and b). The analyses of mtDNA and skin secretion data sets showed, respectively, values of p ¼ 0.0005 and p ¼ 0.0009. These results suggest the existence of different evolutionary processes for each data set. Hence, according to this test, combining the data sets was not recommended. However, the combined analysis was done. The analysis of parsimony by exhaustive search resulted in one tree with a treelength of 154 (Fig. 3). 4. Discussion The topology of the strict consensus tree (indolealkylamines and proteins combined data) shows some differences when compared to the strict consensus tree of the protein data. According to combined data, the clade formed by B. abei þ B. pombali þ B. henseli is unresolved. Meanwhile, considering the protein data set separately, B. abei is more closely related to B. pombali than to B. henseli. B. ornatus is the sister species of the clade formed by B. pombali, B. abei and B. henseli and these taxa are sister taxa of B. crucifer. Populations of Manhumirim (MG) and Itacare´ (BA) are closely related. The relationships between B. abei þ B. pombali þ B. henseli would be resolved in the phylogeny using protein data but unresolved by combined data. B. marinus would be the basal outgroup. The ingroup was supported in 43.5% (bootstrap).

Table 2 Character states of (a) 16 indolealkylamines among nine specimens and (b) 43 proteins among seven specimens of the B. crucifer group and three other species of Bufo used as outgroups Taxa

Characters 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

1 1 0 1

1 1 0 1

1 0 1 0

0 1 1 1

0 1 0 1

0 1 0 1

0 0 0 1

0 0 0 1

1 0 0 1

0 0 0 0

0 0 1 0

0 0 0 0

0 0 1 0

0 0 0 0

0 0 1 0

1

1

0

1

1

1

1

1

1

0

0

0

0

0

0

1 1 1 1 1 1

1 1 1 1 1 1

1 0 0 0 0 0

1 1 1 1 1 1

1 0 0 0 0 0

1 1 0 0 1 0

1 1 1 0 1 1

1 1 1 1 1 1

1 1 0 0 1 0

1 1 1 0 1 1

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

1 0 1 0 0 0

0 0 0 0 0 0

1

1

0

1

0

0

1

1

0

1

0

0

0

0

0

a

Taxa

Characters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

(b) 43 Proteins among seven Bufo marinus 1 Bufo granulosus 1 Bufo gr. margaritifer 0 Bufo crucifer 1 Itacare´ e BA Bufo cf. crucifer 1 Manhumirim e MG Bufo pombali 1 Nova Lima e MG Bufo ornatus 0 Itanhae´m e SP Bufo ornatus 1 Teleˆmaco Borba e PR Bufo abei Santo Amaro 1 da Imperatriz e SC Bufo henseli 1 Torres e RS

specimens 1 0 1 0 1 0 1 1 0 0 0 1 1 0 0 0

0 0 0 1

1 1 0 0

0 0 1 1

0 1 1 0

0 0 0 0

0 0 0 0

1 0 0 1

0 0 0 0

0 0 0 0

0 1 0 0

0 1 0 1

0 0 1 0

0 1 0 0

1 0 0 1

0 0 0 0

0 0 1 0

0 0 0 0

0 1 0 0

1 0 0 0

0 1 1 0

0 0 0 0

0 0 0 1

1 1 0 1

0 0 0 0

0 0 1 0

0 0 0 1

0 1 0 1

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 1

0 1 1 1

0 1 0 0

0 0 0 0

0 1 1 0

0 0 0 0

0 0 0 0

0 0 1 1

1 0 0 0 0 1 1 0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

1

1

0

0

0

1

1

0

0 1 0 0 0 1 0 1 0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

1

0

0

1

1

0

0

0

0

1 0 0 0 1 0 1 0 0

1

0

0

1

0

0

0

0

0

1

0

0

0

0

0

1

1

0

1

0

0

0

0

1

0

0

0

0

0

0

1

0

0

0 0 0 0 0 0 0 0 1

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

1

1

0

0

0

0

0

0

1

0

0

0

0

1

0

0

0

0

0 1 0 0 0 1 0 1 0

0

0

1

0

0

1

0

0

0

0

0

1

0

0

0

0

1

0

0

0

1

1

0

0

1

0

0

1

1

0

0

0

0

0 1 0 0 0 0 0 1 0

0

0

1

0

0

1

0

0

0

0

0

1

0

0

0

0

1

0

0

1

1

0

1

0

0

1

0

0

1

0

0

0

0

461

The characters were coded as absent (0) or present (1). a Character list: 1 (5-HT), 2 (DHB), 3 (*0.78), 4 (*0.358), 5 (DHB-S), 6 (*0.49), 7 (*0.645), 8 (BTN), 9 (*0.60), 10 (*0.477), 11 (*0.145), 12 (*0.762), 13 (BTD), 14 (*0.109), 15 (*0.083) and 16 (*0.016). *Indolealkylamines not known.

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(a) 16 Indolealkylamines among nine specimens Bufo marinus 1 Bufo granulosus 1 Bufo gr. margaritifer 0 Bufo crucifer 1 Itacare´ e BA Bufo cf. crucifer 1 Manhumirim e MG Bufo pombali Nova Lima e MG 1 Bufo ornatus Ubatuba e SP 1 Bufo ornatus Itanhae´m e SP 1 Bufo ornatus Mogi das Cruzes e SP 1 Bufo ornatus Teleˆmaco Borba e PR 1 Bufo abei Santo Amaro 1 da Imperatriz e SC Bufo henseli Torres e RS 1

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B. marinus Belém do Pará-PA

B. granulosus Porto de Galinhas-PE

B. gr. margaritifer Balsas-MA

B. cf. crucifer Manhumirim-MG 50 B. crucifer Itacaré-BA 43.5 64

B. ornatus Telêmaco Borba-PR

B. ornatus Itanhaém-SP

B. pombali Nova Lima-MG

B. abei Santo Amaro da Imperatriz-SC

B. henseli Torres-RS Fig. 1. Strict consensus of four trees of 59 skin secretion characters (treelength ¼ 107, CI ¼ 0.5421 and RI ¼ 0.3797). Numbers above nodes indicate bootstrap values obtained from 10,000 pseudoreplicates. Localities are listed after the taxa.

Combining different data sets is controversial (Bull et al., 1993; De Queiroz et al., 1995). However, combined analysis increased the number of characters and resolved, for the most part, the relationships among the taxa studied, in spite of two polytomies. The bootstrap value was satisfactory only for the clade formed by B. abei þ B. pombali þ B. henseli (83.9%). The clade formed by populations of B. ornatus was supported in 64%. The topologies of the mtDNA data and the combined skin secretion data differ in the position of B. henseli. In the phylogeny obtained using mtDNA data B. henseli is the sister group of B. abei þ B. ornatus þ B. crucifer. In the phylogeny generated by skin secretion data, B. henseli is more closely related to B. abei. It appears more plausible, in biogeographic terms, to think that B. henseli would be more closely related to B. abei. However, this kind of inference is speculative, and further studies are needed. The mtDNA phylogenetic analysis showed CI ¼ 0.9516 and RI ¼ 0.8333. These values are higher than those obtained in the skin secretion analysis (CI ¼ 0.8400 and RI ¼ 0.5789). According to the mtDNA data, the clade B. crucifer þ B. ornatus þ B. abei showed a bootstrap value of 100% and B. ornatus þ B. abei of 78%. The skin secretion data analysis supported the clade B. ornatus þ B. abei þ B. henseli (63%). The bootstrap value for B. abei þ B. henseli was 99%. The source of heterogeneity between the data sets may be related to the use of populations from different localities in each analysis. Because we obtained low bootstrap values, the skin secretion phylogenetic hypothesis did not support the monophyly of the B. crucifer group, contrary to Baldissera (2001). Excluding the position of B. henseli, the skin secretion phylogenetic hypothesis corroborates the acoustic and morphology analyses made by Baldissera (2001) and Baldissera et al. (2004). The relationship of B. pombali with other members of the B. crucifer group has not been resolved (Baldissera, 2001). According to this author, evidence from external morphology and geographic distribution supports a close relationship between B. crucifer and B. ornatus. However, skin secretion characters indicate that B. pombali forms a clade with B. abei and B. henseli.

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B. marinus Manaus-AM

a B. henseli Santa Maria-RS

B. crucifer Itabuna-BA 100 B. ornatus Ubatuba-SP 78

B. abei Blumenau-SC B. marinus Belém do Pará-PA

b B. crucifer Itacaré-BA

B. ornatus Itanhaém-SP 63 B. abei Santo Amaro da Imperatriz-SC 99

B. henseli Torres-RS Fig. 2. (a) Tree based on mtDNA (Baldissera, 2001). Treelength ¼ 62, CI ¼ 0.9516, and RI ¼ 0.8333. Numbers above nodes indicate bootstrap values obtained from 10,000 pseudoreplicates. Localities are given after the taxa. (b) Tree based on skin secretions. Treelength ¼ 50, CI ¼ 0.8400, and RI ¼ 0.5789.

Tests such as Templeton are statistically elegant but relatively unrefined (Kitching et al., 1998). Thus, results from these tests are difficult to interpret. The combined analysis used 1182 characters (mtDNA e 1123 characters and skin secretion e 59 characters), of which 52 were phylogenetically informative and 1071 were constant. This analysis presented CI ¼ 0.7468, RI ¼ 0.4730 and CI ¼ 0.5761 (excluding non-informative characters). The bootstrap analysis supported B. abei þ B. pombali in 73% and B. crucifer þ B. ornatus in 55%. All these taxa together with B. henseli are supported in 51%. The topology of the combined analysis is similar to the topologies generated by mtDNA and skin secretion data partitions. The cladogram presented B. henseli as basal in the mtDNA data analysis, although this relationship is weakly supported (51%). Furthermore, the taxa B. granulosus, B. gr. margaritifer, and B. pombali were not sampled in the mtDNA partition (missing data), possibly influencing the results. The skin secretion partition also showed a higher number of parsimony informative characters (39) compared to the mtDNA data (13). 5. Conclusion The utilization of different types of data sets is fundamental for constructing supported phylogenies. The indolealkylamines and proteins of granular glands of species of B. crucifer group indicate their relationships. Skin secretions

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B. marinus

B. granulosus

B. gr. margaritifer

B. henseli

B. crucifer

51 55

B. ornatus

B. pombali 73 B. abei Fig. 3. Trees of 1182 combined characters (mtDNA and skin secretion) (treelength ¼ 154, CI ¼ 0.7468, and RI ¼ 0.4730).

may be good characters for inferring phylogenetic relationships. Knowledge of skin secretion evolution, however, is deficient. These molecules may show phylogenetic signal and, at least in a preliminary way, indicate relationships. Additional work is necessary to understand the relationships among Bufo in South America. New species are still being described (Ve´lez-R and Ruiz-C, 2002; Stevaux, 2002; Pramuk and Kadivar, 2003; Maneyro et al., 2004). Morphological (Faivovich, 2002; Ford and Cannatella, 1993; Pramuk, 2002) and larval (Haas, 2003) characters have been used to infer anuran phylogeny. Together with morphological characters, other types of characters may be useful in elucidating relationships of the B. crucifer group and the other species of Bufo in South America. Acknowledgements The authors are indebted to R.H. Matsushita, B.A. Duar, A. Sebben, A.Q. Teixeira Jr., O.R. Pires Jr., R. Morales, P. Fattorelli, R.A. Machado, P. Colombo, F. Vilella, P.C. Eterovick, and F. Nunes for their assistance in collecting specimens. R.D. Teixeira, H.C. Wiederhecker, and G.H.C. Vieira helped with the cladistic analysis. G.H.C. Vieira, M.G. Zatz, and L. Tavares-Bastos helped with figures depicting phylogenetic trees. R.A. Brand~ao, C.F.B. Haddad, and F. Baldissera Jr. helped in the identification of Bufo species. The authors also thank P.M. Larsen and S.J. Fey (Center for Proteome Analysis, Odense, Denmark) for the staining gel method. Many others were generous in sharing their time or scientific knowledge. Their help and suggestions improved this work. We thank J.P. Caldwell by the helpful comments and suggestions in the last version of the manuscript and for the English improvements. The surveys were done under RAN/IBAMA license. This work was supported by Programa de Po´s-graduac¸~ao em Biologia Animal, Universidade de Brası´lia, and Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) (Fellowship to N.M. Maciel). References Alrubaian, J., Danielson, P., Walker, D., Dores, R.M., 2002. Cladistic analysis of anuran POMC sequences. Peptides 23, 443e452. Baldissera Jr., F.A., 2001. Taxonomia e Filogenia do Grupo de Bufo crucifer Wied-Neuwied, 1821 (Anura, Bufonidae). Tese. Universidade Estadual Paulista, Rio Claro, S~ao Paulo, Brasil. Baldissera Jr., F.A., Caramaschi, U., Haddad, C.F.B., 2004. Review of the Bufo crucifer species group, with descriptions of two new related species (Amphibia, Anura, Bufonidae). Arq. Mus. Nac. Rio J. 62, 255e282.

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