Cladistics

Cladistics 23 (2007) 455463 10.1111/j.1096-0031.2007.00154.x

Phylogenetic relationships of Pseudis and Lysapsus (Anura, Hylidae, Hylinae) inferred from mitochondrial and nuclear gene sequences
O. Aguiar-Jr1,, M. Bacci Jr2, A. P. Lima3, D. C. Rossa-Feres4, C. F. B. Haddad5 and S. M. Recco-Pimentel1*
Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-863 Campinas, Sa Paulo, Brazil; 2Centro de Estudos de Insetos Sociais (CEIS), Universidade Estadual Paulista (UNESP), CP 199, 13056-900 Rio Claro, Sa Paulo, o o nia (INPA), 69011-970 Manaus, AM, Brazil; Brazil; 3Coordenadoria de Pesquisas em Ecologia, Instituto Nacional de Pesquisas da Amazo 4 Departamento de Zoologia e Botanica, Instituto de Biociencias, Letras e Ciencias Exatas, Universidade Estadual Paulista (UNESP), 15054-000 Sa Jose do Rio Preto, Sa Paulo, Brazil; 5Departamento de Zoologia, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), CP 199, o o 13056-900 Rio Claro, Sa Paulo, Brazil o Accepted 29 January 2007
1

Abstract The previous uncertain placement of Lysapsus and Pseudis within the neobatrachians was recently resolved by molecular and morphological studies, which supported them as members of the Hylinae subfamily. Their inter- and intrageneric relationships, however, have long been under debate and no studies shed light on these questions. Aiming to elucidate such questions, this paper used 3.2 kb comprising the mitochondrial genes 12S, tRNA valine, 16S and cytochrome b, and the nuclear exon 1 coding for rhodopsin, to all representatives of both genera (except to two subspecies of Pseudis paradoxa). The results identied three major clades: the clade 1 was composed by Lysapsus species and subspecies; clade 2 included the subspecies of the Pseudis paradoxa (Pseudis paradoxa paradoxa, P. paradoxa platensis and P. paradoxa occidentalis), P. fusca, P. bolbodactyla and P. tocantins, and clade 3 was composed by Pseudis southern Brazil species (Pseudis cardosoi and P. minuta). As closely related taxa we found Pseudis minuta + P. cardosoi; P. tocantins + P. fusca, and the subspecies within each genus. Evidence that Pseudis is not monophyletic with respect to Lysapsus was found and we suggest Lysapsus to be a junior synonym of Pseudis. Based on pair-wise comparison among gene sequences, we also suggest that the subspecies of Pseudis paradoxa and Lysapsus limellum must be considered as full species. The Willi Hennig Society 2007.

Frogs of the genera Pseudis and Lysapsus are aquatic and widespread in southern, south-eastern and midwestern Brazil, Uruguay, Argentina, Colombia and Venezuela (Busin et al., 2001). According to Frost (2006), Pseudis contains six species (P. paradoxa, P. bolbodactyla, P. cardosoi, P. fusca, P. tocantins and P. minuta) and Lysapsus has three species (L. limellum, L. caraya and L. laevis). Pseudis paradoxa has several

*Corresponding author: E-mail address: shirlei@unicamp.br Present address: Universidade Federal de Sao Paulo (UNIFESP), Campus Baixada Santista, Avenida Ana Costa, 95, 11060-001, Santos, Sao Paulo, Brazil, odaguiar@gmail.com The Willi Hennig Society 2007

subspecies, including the P. p. platensis, P. p. occidentalis, P. p. caribensis and P. p. nicefori (Gallardo, 1961; Cochran and Goin, 1970). Similarly, Lysapsus limellum encompasses the subspecies L. l. limellum and L. l. bolivianus (Gallardo, 1961). The validity of both genera and their placement among neobatrachians have long been debated (Boulenger, 1882; Noble, 1931; Parker, 1935; Savage and Carvalho, 1953; Burger, 1954; Duellman, 2001; Darst and Cannatella, 2004; Faivovich et al., 2005). Boulenger (1882) proposed the synonymization of these genera and recognized four species: P. paradoxa, P. minuta, P. limellum and P. mantidactyla. Noble (1931) suggested that Pseudis should be placed in its own subfamily, Pseudinae, in the family Bufonidae. This proposal was rejected by Parker

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(1935), who considered Pseudis to be a member of the family Hylidae based on the cartilaginous phalanx of Pseudinae, particularly as this phalanx was possibly homologous with the intercalary elements of hylids. Savage and Carvalho (1953), who resurrected the genus Lysapsus, argued that these genera could be considered distantly related when certain morphological and habitat traits were compared or, conversely, could be closely related based on the cartilaginous phalanx. These authors suggested that the cartilaginous phalanges were analogous to the cartilaginous intercalary elements present in Hylidae, Microhylidae and Rhacophoridae and took this as evidence that this character state did not indicate a direct relationship with the Hylidae. In addition, this trait dierentiated both genera from the Leptodactylidae, contrary to the proposition by Davis (1936), who claimed a relationship between leptodactylids and the Pseudinae. Based on these interpretations of the evidence, Savage and Carvalho (1953) proposed a separate family (Pseudidae) for these genera. Subspecies of P. paradoxa were described by Gallardo (1961) and Cochran and Goin (1970) based on morphometric characters and patterns of coloration. Without providing any specic evidence, Gallardo (1961) proposed that P. p. paradoxa, P. p. caribensis and P. p. occidentalis of northern and western South America were more closely related, and P. p. fucus and P. p. bolbodactylus of south-eastern Brazil formed another group, with P. p. platensis providing the connecting link according to this author. Two subspecies of Pseudis were subsequently raised to full species status (P. bolbodactyla and P. fusca) by Caramaschi and Cruz (1998). According to these authors, the absence or the vestigial presence of a carpal tubercle characterized and distinguished these subspecies from the remaining subspecies of P. paradoxa. Based on 23 morphological characters and chromosome number, Duellman (2001) suggested that Lysapsus and Pseudis were the sister group of the Hylinae, and again classied them as the subfamily Pseudinae in the family Hylidae. Based on an analysis of larval and adult morphological characteristics from 81 anuran species, Hass (2003) provided a cladogram showing that the Hylinae were paraphyletic with respect to the Pseudinae. According to this relationship, both of the Pseudinae genera would be nested within the Hylinae, with the Phyllomedusinae as the sister-group. Recent studies based on molecular and morphological data have clearly demonstrated that Lysapsus and Pseudis are members of the subfamily Hylinae (Barg, 2003; Darst and Cannatella, 2004; Faivovich et al., 2005; Frost et al., 2006). Whereas the allocation of these genera among the neobatrachians (Hylidae, Hylinae) has been claried by the studies mentioned above, the inter- and intrageneric relationships of Lysapsus and Pseudis remain unknown as all of the studies so far have used only a small number

of specimens from each of these genera (Hay et al., 1995; Ruvinsky and Maxson, 1996; Darst and Cannatella, 2004; Faivovich et al., 2005; Frost et al., 2006). In this study, we performed a molecular phylogenetic analysis of all species and subspecies of Lysapsus and all species of Pseudis, including three of the ve subspecies of P. paradoxa, to assess the relationships within each genus and to test their monophyly with respect to each other. We used sequences from one nuclear (exon 1 of the protein coding gene rhodopsin) and four mitochondrial (12S, tRNA-Val, 16S and cytochrome b) genes. In addition, the GenBank sequences for Lysapsus and Pseudis reported by Faivovich et al. (2005) provided additional specimens for our analysis. The results of the analysis were compared with those reported in the literature for various character systems (molecular, spermatological and morphological data).

Materials and methods Table 1 lists the sampled terminals and their collection data. At least three specimens from each taxon were sequenced for the cytochrome b, rRNA 12S, tRNA Val and 16S genes (except for Pseudis paradoxa platensis and Lysapsus laevis to which just two specimens were used) and one specimen for the gene corresponding to exon 1 of rhodopsin. For some species, data from dierent populations were analyzed. In addition, the same gene sequences available in GenBank for specimens of P. minuta, P. p. platensis, L. l. limellum and L. laevis from dierent collection localities, were also included. As outgroups, sequences from Scarthyla goinorum, Sphaenorhynchus lacteus and Scinax catharinae, sistergroups of the Lysapsus and Pseudis (Faivovich et al., 2005), were obtained from GenBank. Sequences from P. p. occidentalis (cytochrome b, 12S, tRNA-Val and 16S) were kindly provided by Dr Julian Faivovich. Extraction and quantication of genomic DNA Fragments of liver, muscle and heart were obtained from organs preserved in 95% ethanol or from frozen organs stored at )70 C. Whole genomic DNA was extracted using a Genomic Prep Cells and Tissues DNA isolation kit (GE Health Care, Buckinghamshire, UK) and was quantied by electrophoresis in 0.8% agarose gels using k DNA (Invitrogen, Carlsbad, CA) as a molecular size marker. DNA amplication and sequencing The target sequences were amplied using pureTaq Ready-to-Go PCR beads (GE Health Care). Each reaction contained up to 1.5 lL of genomic DNA, 2 lL of each primer solution (6 pmol mL) (see Table 2)

O. Aguiar et al. / Cladistics 23 (2007) 455463 Table 1 Localities and collection numbers for the taxa sampled in this work Species Pseudis minuta Localities Eldorado do Sul (RS) Eldorado do Sul (RS) Eldorado do Sul (RS) Entre Rios (Argentina) Tainhas (RS) Sao Francisco de Paula (RS)* Porto Nacional (TO)

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Collection numbers CAUPF 1947 CAUPF 1948 2 ZUEC 11584 4 MACN 37786 ZUEC 11601 ZUEC 11771 CAUPF 1525 CAUPF 1526 ZUEC 13227 ZUEC 13230 3 MNRJ 35457 MNRJ 34041 MNRJ 34044 MNRJ 34038 ZUEC 13235 ZUEC 13236 MNRJ 35459 MNRJ 33859 ZUEC 12829 ZUEC 12830 5 FN 189 FN 013 MFN 014 ZUEC 12828 ZUEC 13062 MACN 38642 MNRJ 34079 MNRJ 34071 MNRJ 34076 MNRJ 34060 MNRJ 34067 MNRJ 33954 MNRJ 33972 MNRJ 33944 MACN 38645 MNRJ 33883 MNRJ 33878 ZUEC 13201 ZUEC 13204 6 BC 204.16 FN 011 FN 012 7 AMCC 101720 8 USNM 152136 9 MCP 3734 10 QUCL 2340
1

Pseudis cardosoi Pseudis cardosoi Pseudis tocantins

Pseudis bolbodactyla

Quirinopolis (GO)

Pseudis fusca

Coronel Murta (MG)

Pseudis paradoxa paradoxa

Pseudis paradoxa platensis

Bacabal (MA) Bacabal (MA) Bacabal (MA) Macapa (AP) Pacaraima (RR) Pacaraima (RR) Nova Itapirema (SP) Nova Itapirema (SP) Corrientes (Argentina) Nossa Senhora do Livramento (MT)

Pseudis paradoxa occidentalis Lysapsus limellum limellum

Lysapsus limellum limellum Lysapsus limellum bolivianus

Corumba (MS) Guajara-Mirim (RO) Guajara-Mirim (RO) Guajara-Mirim (RO) Corrientes (Argentina) Santarem (PA) Santa Teresinha (MT)

Lysapsus limellum bolivianus Lysapsus caraya

Lysapsus laevis

Sphaenorhynchus lacteus Scinax catharinae Scarthyla goinorum

Pacaraima (RR) Pacaraima (RR) Guyana Madre de Dios (Peru) Pro-Mata (RS) Amazonas (Brazil)

Abbreviations: 1Amphibian collection of the Universidade de Passo Fundo, Rio Grande do Sul;2 Museu Nacional, Rio de Janeiro,3 Museu de Historia Natural Prof Dr Adao Jose Cardoso, Universidade Estadual de Campinas (UNICAMP), Campinas, Sao Paulo, 4Museo Argentino de Ciencias Naturales Bernardino Rivadavia, 5Field Numbers of Ulysses Caramaschi (to be accessioned in MNRJ collection), 6Laboratory register 7 8 number (to be accessioned in MNRJ collection), Ambrose Monell Cryo Collection, National Museum of Natural History, Smithsonian Institution, Washington, DC, 9Museu de Ciencias e Tecnologia da Ponticia Universidade Catolica do Rio Grande do Sul, Brazil, 10Queens University Laboratory Collection. *Specimens collected into the Centro de Pesquisas e ConservacaoPro-Mata, in the municipality of Pro-Mata. All specimens were collected under a permit issued by the Instituto Brazileiro do Ambiente e Recursos Naturais Renovaveis (IBAMAprocess 02001008875 0111, collection license number 083 04 and genetic material access authorization number 022 2005).

and MilliQ water to complete a nal volume of 25 lL. The PCRs were done in an MJ PTC 100 thermocycler. For all of the reactions, a long period of initial denaturation (94 C, 10 min) was followed by 39 cycles

of amplication and extension (94 C, 30 s; 5055 C, 1 min; 72 C, 2 min). The PCR-amplied products were cleaned with a GFX PCR DNA and Gel Band purication kit (GE Healthcare), and labeled with uorescent-dye

458 Table 2 Primers used in this work Primer MVZ 15 H15149(H) MVZ 59 MVZ 50 12SL13 16STitus I 16SL2A 16SH10 16SAR 16SBR Rhod 1A Rhod 1C Sequence

O. Aguiar et al. / Cladistics 23 (2007) 455463

Reference Moritz et al. (1992) Kocher et al. (1989) Graybeal (1997) Graybeal (1997) Feller and Hedges (1998) Titus and Larson (1996) Hedges (1994) Hedges (1994) Palumbi et al. (1991) Palumbi et al. (1991) Bossuyt and Milinkovitch (2000) Bossuyt and Milinkovitch (2000)

5-GAACTAATGGCCCACACWWTACGNAA-3 5-AAACTGCAGCCCCTCAGAAATGATATTTGTCCTCA-3 5-ATAGCACTGAAAAYGCTDAGATG-3 5-TYTCGGTGTAAGYGRAKGCTT-3 5-TTAGAAGAGGCAAGTCGTAACATGGTA-3 5-GGTGGCTGCTTTTAGGCC-3 5-CCAAACGAGCCTAGTGATAGCTGGTT-3 5-TGATTACGCTACCTTTGCACGGT-3 5-CGCCTGTTTATCAAAAACAT-3 5-CCGGTCTGAACTCAGATCAGGT-3 5-ACCATGAACGGAACAGAAGGYCC-3 5-CCAAGGGTAGCGAAGAARCCTTC-3

terminators (ABI Prism Big Dye Terminators v. 3.0 kit; Applied Biosystems, Perkin Elmer, Foster City, CA). The labeled PCR products were precipitated with isopropanol using an adaptation of the manufacturers protocol and then sequenced in an ABI 377 automatic sequencer. All of the samples were sequenced in both directions. Six pairs of primers were used to amplify the mitochondrial (rRNA 12S, tRNA valine, rRNA 16S and a fragment of cytochrome b) and nuclear (a fragment of exon 1 in rhodopsin) genes (see Table 2), and yielded a total data set of approximately 3.2 kb. Sequence editing and alignment The forward and reverse sequences were edited using the chromatograms provided by the sequencer in conjunction with the BioEdit Sequence Alignment Editor (Hall, 1999). Consensus sequences were generated from the alignment of opposite direction sequences and multiple sequence alignment with ClustalW v. 1.7 (Thompson et al., 1994), following the program default parameters, was subsequently used to align the consensus sequences. We have also tested an alternative alignment in which all regions of the gene fragments that could not be clearly aligned among all taxa were excluded, a process that has been used when RNA gene sequences are included (see Vences et al., 2003). Positions with gaps were also excluded. Phylogenetic analysis The data set was analyzed by the Maximum Parsimony (MP) criterion using PAUP* 4.0b10 (Swoord, 2002) with all characters equally weighted and gaps treated as missing data. To obtain the phylogenetic trees, aligned sequences were submitted to a heuristic search with 100 replicates, TBR swapping algorithm, random sequence addition, and collapse and multrees options. The same program and heuristic search settings were used to implement the bootstrap analysis (Felsenstein, 1985) with 1000 pseudo-replicates. In

addition, the parsimony ratchet algorithm (Nixon, 1999) was also used to search for the most parsimonious trees in the combined data set. This was done using the program PRAP (Muller, 2004), which writes commands into a command le to be executed by PAUP*. The following parameters were set in PRAP: 1000 ratchet replicates, 10 random addition cycles, weight 2, % weighted 25. The program Tree Graph 1.0 rc3 (Mul ler and Muller, 2004) was utilized to draw the tree. Decay values (Bremer, 1994) were also calculated with PRAP (Muller, 2004). To measure character congruence between partitions, the partition homogeneity test (ILD) (Farris et al., 1995) was applied to the matrix. Pair-wise distance analysis The percentage of similarity among the sequences of the subspecies from both genera was assessed using the pair-wise alignment option in BioEdit applicative. Pairwise comparisons of the sequences were done using the concatenated gene data and the cytochrome b, which was the most variable gene sequence and have been used to infer relationships among deep divergences and also at the species level (Goebel et al., 1999).

Results The P-value obtained with the ILD was 0.15, which indicated that the data sets were not incongruent. The combined data provide a matrix with 3234 characters, being 865 parsimony-informative characters using PAUP. Six most parsimonious trees with a length of 2887 were found. The trees presented a Consistency Index 0.56, a Homoplasy Index 0.43 and a Retention Index 0.85. The tree topology is shown in Fig. 1. Interindividual dierences (haplotypes) were observed in some species in both genera during sequence editing. All of the haplotypes were included in the nal matrix.

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Sphaenorhynchus lacteus Scinax catharinae Scarthyla goinorum

100/97 78/2 100/24 57/4 100/23 87/05 61/1 98/5

Lysapsus laevis (AR) Lysapsus laevis (RR) Lysapsus laevis (RR) Lysapsus limellum limellum (MS) Lysapsus limellum limellum (AR) Lysapsus limellum limellum (MS) Lysapsus limellum limellum (MT) Lysapsus limellum limellum (MT) Lysapsus limellum limellum (MT) Lysapsus limellum bolivianus (PA)
97/7

Clade 1

Lysapsus limellum bolivianus (PA) Lysapsus limellum bolivianus (RO) Lysapsus limellum bolivianus (RO) Lysapsus limellum bolivianus (RO) Lysapsus caraya (MT) Lysapsus caraya (MT)

100/28 100/66 91/3 100/11 100/39 100/27 51/3 91/5 100/18 100/14
100/11

79/2

Lysapsus caraya (MT) Pseudis paradoxa platensis (AR) Pseudis paradoxa occidentalis (AR) Pseudis paradoxa platensis (SP) Pseudis paradoxa platensis (SP) Pseudis paradoxa paradoxa (AP) Pseudis paradoxa paradoxa (RR) Pseudis paradoxa paradoxa (RR) Pseudis paradoxa paradoxa (MA) Pseudis paradoxa paradoxa (MA) Pseudis bolbodactyla (GO) Pseudis bolbodactyla (GO)

99/14

99/17

100/34 100/13 100/43

Clade 2

100/27 100/28 85/2 100/95 100/28

Pseudis bolbodactyla (GO)

Pseudis fusca
Pseudis fusca (MG) Pseudis fusca (MG) Pseudis tocantins (TO) Pseudis tocantins (TO) Pseudis tocantins (TO) Pseudis minuta (AR) Pseudis minuta (RS) Pseudis minuta (RS) Pseudis minuta (RS) Pseudis cardosoi (Tainhas, RS) Pseudis cardosoi (Pr-Mata, RS) Pseudis cardosoi (Tainhas, RS) Pseudis cardosoi (Pr-Mata, RS)

100/81 69/1 69/2 90/4 95/5 90/3 100/11 87/2

100/113

Clade 3

1
Fig. 1. Strict consensus of the six most parsimonious tree obtained from 865 parsimony-informative characters from cytochrome b, 12S, 16S, Valine tRNA and rhodopsin exon 1 markers from 43 Lysapus or Pseudis specimens. Numbers on left represent the bootstrap support values and those on right indicate the Bremer supports. Note the low support for the relationship between the clades 1 and 3 and for the placement of Lysapsus laevis. Dierent branch lengths are not proportional to the amount of change. Abbreviations are as follows: Brazilian localities: AP Amapa State; GO Goias State; MA Maranhao State; MG Minas Gerais State; MS Mato Grosso do Sul State; MT Mato Grosso State; PA Para State; RR Roraima State; RS Rio Grande do Sul State; SP Sao Paulo State; AR Argentina.

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The alignment excluding the ambiguous sections recovered an identical tree topology and approximately the same support values (bootstrap and Bremer). This indicated that the excluded alignment uncertainties had no inuence on the topology obtained with the complete data set. Only the topology obtained with the complete data set is shown here. The recovered clades and the relationships among them

Bremer support Groups with the lowest bootstrap frequencies were also found to have the lowest Bremer support. Lysapus laevis grouped with the remaining Lysapsus (D 4, Fig. 1 and the relationships between P. minuta and P. cardosoi were also conrmed by the Bremer support value of D 113 (Fig. 1). Pair-wise distance comparisons

Three major clades were recovered in our analysis. Clade 1 contained the subspecies of Lysapsus limellum (L. l. limellum and L. limellum bolivianus) and L. caraya and L. laevis, clade 2 included the subspecies of the Pseudis paradoxa complex (P. p. paradoxa, P. paradoxa platensis and P. paradoxa occidentalis) and P. fusca, P. bolbodactyla and P. tocantins, and clade 3 consisted exclusively of southern Brazilian species of Pseudis (P. cardosoi and P. minuta). The relationships among the three clades were well resolved with bootstrap value of 100%. The relationships of clades 1 and 2 was low (51%) (Fig. 1). The bootstrap support for the allocation of L. laevis with its congeneric species in clade 1 was also low (57%). Separate partition analysis (data not shown) was not helpful in clarifying these relationships. In contrast, the internal clade relationships received high bootstrap and Bremer support (Fig. 1). In clade 1, L. laevis appeared as the sister taxon of the remaining congeneric species, although the support for this was weak, and L. caraya was the sister taxon of the L. limellum subspecies, which were monophyletic with respect to each other (Fig. 1). A consecutive sister group relationship was found among the specimens of L. l. limellum collected in the Brazilian states of Mato Grosso, Mato Grosso do Sul and Argentina. Paraphyly was observed among the specimens of L. l. bolivianus from the two localities sampled (Fig. 1). Clade 2 shows Pseudis tocantins + P. fusca, with Pseudis bolbodactyla + subspecies of P. paradoxa as their sister group (Fig. 1). Pseudis bolbodactyla appeared as the sister taxon of this complex. The specimen of P. p. platensis from Argentina had P. p. occidentalis and the Brazilian specimens of P. p. platensis as consecutive sister groups. The specimens of P. p. paradoxa from the Brazilian states of Amapa, Roraima and Maranhao were recovered in a trichotomy within this clade (Fig. 1). In clade 3, P. minuta and P. cardosoi were strongly supported as sister taxa in all topologies (bootstrap 100%) (Fig. 1). The specimen of P. minuta from Argentina was the sister taxon of the southern Brazilian specimens, whereas paraphyly was observed among P. cardosoi specimens from Tainhas and ProMata in the Brazilian state of Rio Grande do Sul (Fig. 1).

Using the concatenated data set, the similarity between the sequences of P. p. paradoxa and P. p. platensis was 95%, which was the same as that obtained for the sequences of two full species (P. minuta and P. cardosoi). The P. p. occidentalis sequence showed 93% identity with that of P. p. paradoxa, 96% with that of P. p. platensis and 92% with the full species P. bolbodactyla. The cytochrome b comparisons showed very similar results. The similarity between the two subspecies was 92%, being lower than that observed between P. minuta and P. cardosoi (95%). Concerning P. p. occidentalis, its sequence presented 91% of identity with that of P. p. paradoxa, 97% with P. p. platensis and 90% with P. bolbodactyla. In the genus Lysapsus, pair-wise distance comparisons revealed 95% identity among the sequences of L. limellum subspecies, with practically the same values being obtained when each of them was compared with the sequence of Lysapsus caraya (95% and 94% when compared with L. l. bolivianus and L. l. limellum, respectively). The same is true to the comparisons using the cytochrome b fragment (92% of similarity between the subspecies and the same value when compared with L. caraya).

Discussion The results of this study provide a better understanding of the phylogenetic relationships among the species of Pseudis and Lysapsus as almost all of the nominal taxa described so far were represented. The close relationship between P. minuta and P. cardosoi, supported by high bootstrap (100%) and Bremer support (D 113) values, agreed with the great morphological similarity that caused Braun and Braun (1980) to consider them as a single species. The divergence between these two taxa involved dierentiation in morphology, in DNA sequences and also in karyotypical traits (see Busin et al., 2001). Our results corroborated the hypothesis proposed by Busin et al. (2001) that the karyotypes of P. minuta (2n 24) and P. cardosoi (Kwet, 2000) (denominated as Pseudis a. minuta in their study) (2n 28) had a common ancestry. Busin et al. (2001) found chromosomal evidence that the

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karyotype with 28 chromosomes was a derived condition. As the remaining Lysapsus and Pseudis also have a diploid number of 24 chromosomes (Barrio and Rubel, 1970; Busin, 2005; Busin et al., 2006), it is more parsimonious to consider that the 2n 28 karyotype may be an autapomorphy of P. cardosoi. This hypothesis is corroborated by the presence of 24-chromosome karyotypes in the current well-known sister clades of Pseudis and Lysapsus (Scinax and Sphaenorhynchus) in the tribe Dendropsophini (Faivovich et al., 2005), representatives of which were used here as outgroups. Barg (2003) also concluded that P. cardosoi and P. minuta were closely related, as indicated by four morphological synapomorphies, with both species appearing as a sister clade of the remaining species and subspecies of this genus. Another character state shared by P. cardosoi and P. minuta is the presence of a double vocal sac. In contrast, Pseudis tocantins, P. bolbodactyla, P. fusca (Caramaschi and Cruz, 1998), subspecies of P. paradoxa (Gallardo, 1961), all Lysapsus (Klappenbach, 1985), and Scarthyla goinorum (Duell man and de Sa, 1988), the sister taxon of the Lysapsus and Pseudis, have a single vocal sac, indicating that the double vocal sac is a putative morphological synapomorphy of P. cardosoi and P. minuta. The placement of P. paradoxa (and its subspecies), P. bolbodactyla and P. tocantins in the same clade, separated from P. minuta, agrees with the sperm ultrastructural characteristics described by Garda et al. (2004). According to these authors, the rst three species have granular material under the subacrosomal cone, whereas the fourth species has a multilaminar structure in the same region. However, until the state of this character is checked in an outgroup (e.g., Scarthyla goinorum), it cannot be used to reinforce the molecular data. Barg (2003) also described the relationships among Pseudis bolbodactyla and the P. paradoxa subspecies in this clade. In this case, P. p. caribensis (not included in the present analysis) appeared as a sister taxon of P. bolbodactyla, with the subspecies P. p. occidentalis, P. p. platensis and P. p. paradoxa being consecutive sister taxa. Our results showed that P. bolbodactyla was a sister taxon to the clade containing the P. paradoxa subspecies and corroborated its status of full species, as proposed by Caramaschi and Cruz (1998). The present paper is the rst to include P. tocantins and P. fusca in a phylogenetic analysis. Their strongly supported close relationship has not been reported in previous studies. The status of full species attributed to P. fusca by Caramaschi and Cruz (1998), which removed it from being a subspecies of P. paradoxa, was corroborated by the nding that these taxa were not even sister groups in the topology obtained here. Our ndings agree with the cytogenetic work of Busin (2005) who showed that P. bolbodactyla, P. fusca and P. tocantins

have C-banding patterns that dierentiate them from P. paradoxa subspecies. Although the Pseudis paradoxa subspecies appeared as closely related taxa, their subspecic status was not supported when the pair-wise distances were compared. The nding that the percentage of identity between their sequences was the same or lower than that between the sequences of two full species (P. cardosoi and P. minuta) indicated that there is no molecular reason to sustain their subspecic status. This observation, in addition to the morphological dierences noted by Caramaschi and Cruz (personal communication) allow us to propose that the P. paradoxa subspecies actually represent two full species (Pseudis paradoxa and Pseudis platensis). This conclusion is also valid for the subspecies P. p. occidentalis, for which practically the same percentage of identity was found when its sequence was compared with that of P. p. paradoxa and of a full species (P. bolbodactyla). Likewise, the percentage of identity of P. p. occidentalis with P. p. platensis was very similar to that found in a comparison between the full taxa P. minuta and P. cardosoi. The close relationship between P. p. occidentalis and P. p. platensis was not observed by Barg (2003), who considered them as a consecutive sister group to P. minuta and P. bolbodactyla + P. p. caribensis. The proximity between L. l. limellum and L. l. bolivianus observed here was also reported in the morphological study by Barg (2003). The samples of each L. limellum subspecies formed highly monophyletic groups as the pair-wise distance analysis showed that these subspecies were no closer to each other than they were to the full species L. caraya. This nding, together with some morphological (Caramaschi, personal communication) and slight karyotypical dierences (Busin et al., 2006), indicate that these subspecies should be raised to full species status, with the names Lysapsus limellum and Lysapsus bolivianus. Gallardo (1964) considered L. caraya as a subspecies of L. limellum. However, Klappenbach (1985) pointed out that Gallardo was conservative in considering L. caraya as a subspecies and that this taxon should be a valid species. In the present study, the sister group position of L. caraya in relation to both subspecies of L. limellum corroborated the conclusion of Klappenbach (1985) that this taxon is a valid species, and also agreed with the cytogenetic analysis of Busin et al. (2006) who reported dierences in the chromosomal morphology, C-banding pattern and NOR location between this species and the L. limellum subspecies. The present study is the rst to include Lysapsus laevis in a broader analysis of related taxa. In the report by Faivovich et al. (2005), in which only four taxa of this group were studied, this species appeared as a sister taxon of L. limellum, with a considerable Bremer support. The almost complete absence of a subacrosomal

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cone in sperm cells of L. laevis was considered as an apomorphy (Garda et al., 2004) and later suggested by Faivovich et al. (2005) as a possible synapomorphy of the genus. Our results did not indicate a strongly supported relationship between this taxon and its congeneric species in clade 1 (bootstrap 57%, D 4), hence providing no strong support for Lysapsus as a monophyletic genus. Some topologies obtained in an analysis of each gene partition (data not shown) recovered L. laevis as a fourth clade in a polytomy with the remaining three clades, or as being more related to the genus Pseudis. The results described here are the rst to indicate, in a quantitative phylogenetic framework, that Lysapsus and Pseudis may not be monophyletic. The monophyly of the subfamily Pseudinae has never been doubted, although some synapomorphies were found in morphological work by Duellman (2001) and in morphological and molecular work by Faivovich et al. (2005). However, the monophyly of each genus has always been an open question as the limits between Lysapsus and Pseudis (diagnostic characters), reviewed by Savage and Carvalho (1953) and Klappenbach (1985), are not clear and there have been no character states supporting the monophyly of either genus. As shown here, Pseudis (clades 2 and 3) was paraphyletic with respect to Lysapsus as clade 2, containing some species of Pseudis was more related to Lysapsus, albeit in a very weakly supported sister group relationship, than to clade 3, which contained the remaining congeneric species. Additional evidence for this relationship was provided by the nding that ve extra steps were required to hold the Pseudis members together in a monophyletic assemblage in the PAUP analysis, thus rendering the hypothesis of a monophyletic Pseudis less parsimonious. As noted by Faivovich et al. (2005), Savage and Carvalho (1953) implicitly assumed the paraphyly of Pseudis, with the suggestion that Lysapsus might have arisen from Pseudis. Taken together, our results lead us to conclude that, from a molecular perspective, Pseudis is not a monophyletic genus with respect to Lysapus. The monophyly or paraphyly of Lysapsus depends on the placement of Lysapsus laevis. When grouped within clade 1, the genus was monophyletic (Fig. 1), but when grouped with clades 2 or 3 in the topologies obtained from separate partitions (not shown), it was paraphyletic. Neither situation was highly supported and this question therefore remains open. However, in addition to the supposed spermatological synapomorphy (near absence of the subacrosomal cone) (Garda et al., 2004; Faivovich et al., 2005), a larger amount of heterochromatin has been found in the karyotypes of Lysapsus representatives than in Pseudis (Busin, 2005; Busin et al., 2006). If such a condition is an apomorphy within the Lysapsus and Pseudis, as has been suggested for

many anuran groups (King, 1991), then it provides an additional putative synapomorphy in support of the Lysapsus monophyly. In conclusion, our data corroborated some previous hypotheses of the relationships among species in the genera Lysapsus and Pseudis within the pseudines and helped to redene others. Our nding that Pseudis was not a monophyletic group with respect to Lysapsus suggests the need to synonymize these genera in a single genus (Pseudis).

Acknowledgments The authors thank Wanderley G. Martins, Lucina B. Lourenco, Danyelle C. Marini, Giovana G. Vinha and specially Joaquim M. Junior for helpful methodological discussions and valuable technical assistance. We also thank Ulisses Caramaschi for providing tissues of Lysapsus laevis and Pseudis paradoxa paradoxa (Rora ima and Amapa state populations) and Carlos Alberto Cruz and Ulisses Caramaschi for the species and subspecies identications. We are also grateful to Dr Julian Faivovich, who generously provided the P. p. occidentalis 12S, tRNA Val, 16S and cytochrome b sequences and critically reading this manuscript, and Monica Barg who kindly allowed us to cite her PhD thesis. O. Aguiar-Junior was supported by a FAPESP fellowship (grant no. 02 12133-0) and this work was supported by grants from FAPESP to S.M. ReccoPimentel (grant no. 02 12139-9) and C.F.B. Haddad (grant no. 01 13341-3). C.F.B. Haddad was also supported by CNPq.

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