Accepted Manuscript

Short communication

Brazilian are diphyletic (Anura: : )

David C. Blackburn, William E. Duellman

PII: S1055-7903(13)00179-6 DOI: http://dx.doi.org/10.1016/j.ympev.2013.04.021 Reference: YMPEV 4580

To appear in: Molecular Phylogenetics and Evolution

Received Date: 7 January 2013 Revised Date: 2 April 2013 Accepted Date: 22 April 2013

Please cite this article as: Blackburn, D.C., Duellman, W.E., Brazilian marsupial frogs are diphyletic (Anura: Hemiphractidae: Gastrotheca), Molecular Phylogenetics and Evolution (2013), doi: http://dx.doi.org/10.1016/ j.ympev.2013.04.021

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2

3 Brazilian marsupial frogs are diphyletic (Anura: Hemiphractidae: Gastrotheca)

4

5 David C. Blackburna,*, William E. Duellmanb

6 a Department of Vertebrate Zoology & Anthropology, California Academy of Sciences, 55

7 Music Concourse Drive, San Francisco, CA 94118, USA

8 b Biodiversity Institute, University of Kansas, 1345 Jayhawk Boulevard, Lawrence, KS

9 66045, USA

10 * Corresponding author. E-mail address: [email protected] (D.C. Blackburn)

11 12 Abstract

13 Molecular phylogenetic analyses based on expanded taxonomic and geographic sampling

14 support the monophyly of the marsupial genera (family Hemiphractidae), resolve six

15 geographically circumscribed lineages within Gastrotheca, and, for the first time, reveal

16 that two divergent lineages of Gastrotheca inhabit the Atlantic Coastal Forests of Brazil.

17 Within Gastrotheca, the earliest diverging clade is confined to northeastern Brazil,

18 whereas the three subsequent diverging lineages are restricted to northern Venezuela (G.

19 walkeri), southeastern Brazil, and northwestern South America. All in these

20 clades inhabit humid forests at low to mid-elevations, and their life histories are

21 characterized by lacking free-living (i.e., direct development). Two derived

22 clades inhabit the Andes, and both contain species with either direct development or

23 tadpoles. One Andean clade of Gastrotheca ranges in the high Andes from Colombia to

24 extreme northern Peru, whereas the other clade inhabits high elevations in the Andes of

25 southern Ecuador, Peru, and Bolivia, and lower elevations in the Andes of northwestern

26 Argentina. The presence of two non-sister lineages on each side of the Amazon Basin

27 suggests that vicariance across this central region played an important role in

28 diversification within Gastrotheca.

29 30 1. Introduction

31 Marsupial frogs and their allies (family Hemiphractidae) are restricted to South America.

32 Species occur throughout the northern and central Andes, on the Guyana Shield, in the

33 upper Amazon Basin, and also in the Atlantic Coastal Forests of Brazil. Studies of these

34 frogs have played an important role in understanding the evolution of life history

35 diversity in (Duellman and Maness 1980; Wassersug and Duellman 1984;

36 Wiens et al. 2007). Yet, phylogenetic analyses have not addressed the biogeography of

37 hemiphractid frogs, especially the Gastrotheca. Similar to few other

38 genera, Gastrotheca spans both sides of the Amazonian Basin, being found in western

39 South America and the Andes as well as the Atlantic Coastal Forests, but not within the

40 basin itself. However, no other genus of frogs has the same pattern as Gastrotheca; taken

41 as a group, genera of holoadenine strabomantids have a similar pattern (Hedges et al.

42 2008) and strabomantids of the genus Oreobates have discontinuous distributions in dry

43 forest south of the Amazon Basin (Teixeira et al. 2012a). The exclusion of most Brazilian

44 Gastrotheca from previous analyses precluded addressing a major biogeographic

45 question in this distinctive genus.

46 Our study has two objectives. First, we increase species-level taxon sampling

47 across the Hemiphractidae, but especially within Gastrotheca. We expand previous

48 datasets by providing novel sequence data for fifteen additional species of Gastrotheca,

49 including six undescribed species and several only recently described (Duellman et al.

50 2004, 2011a). Second, we specifically address whether species of Gastrotheca from the

51 Atlantic Coastal Forests form a single clade. Previous analyses included only one 52 Brazilian species; here we include four additional species to test the monophyly of

53 Gastrotheca from this region.

54

55 2. Materials and methods

56 Our sampling expands that of Wiens et al. (2007) by including fifteen additional species

57 (Appendix 1), several of which are undescribed species for which descriptions are in

58 preparation (L. Coloma and S. Carvajal, unpubl. data). Early molecular phylogenetic

59 work did not recover Hemiphractidae as monophyletic (e.g., Darst and Cannatella 2004;

60 Faivovich et al. 2005; Frost et al. 2006) but monophyly was supported by subsequent

61 analyses (Wiens 2007; Guayasamin et al. 2008; Heinicke et al. 2009; Duellman et al.

62 2011b; Pyron and Wiens 2011; for discussion, see Blackburn and Wake 2011). We did

63 not include species from previous studies in our analyses if DNA sequence data were

64 available for only one gene region (e.g., , Gastrotheca ovifera). Previous

65 analyses included data for a specimen identified as Gastrotheca fissipes from Guarapari

66 in Espírito Santo, Brazil (Faivovich et al. 2005; Wiens et al. 2007). We follow the more

67 recent of Izecksohn et al. (2009) who described populations in this region as a

68 new species, G. megacephala, and restricted G. fissipes to localities farther north in the

69 state of Pernambuco. Collection codes for specimens sequenced in this study are as

70 follows: CFBH – Célio F.B. Haddad collection, Rio Clara, Brazil; CHP – Círculo

71 Herpetológico de Panamá, Smithsonian Tropical Research Institute, Panama; CORBIDI –

72 Centro de Ornitologia y Biodiversidad, Lima, Peru; CTMZ – tissue collection at Museu

73 de Zoologia, Universidade de São Paulo (MZUSP), Brazil; MNCN – Museo Nacional de

74 Ciencias Naturales, Madrid, Spain; MNRJ – Museu Nacional Rio de Janeiro, Brazil; 75 MUSM – Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima,

76 Peru; MVZ – Museum of Vertebrate Zoology, University of California, Berkeley; QCAZ

77 – Museo de Zoologia, Pontífica Universidad Católica de Ecuador, Quito.

78 The four sampled genes were two mitochondrial genes (16S ribosomal RNA;

79 NADH dehydrogenase subunit 1, ND1) and two nuclear genes (proopiomelanocortin,

80 POMC; recombination activating gene 1, RAG-1). These gene regions are the same as

81 those sampled in Wiens et al. (2007), except we did not include the mitochondrial 12S

82 ribosomal RNA gene because of difficulties in amplification using polymerase chain

83 reaction (PCR). Several additional primers were designed for amplification of POMC and

84 RAG1 (Table 1). Protocols used for both PCR amplification and direct-sequencing were

85 standard and similar to that of Duellman et al. (2011b) with some minor changes to

86 annealing temperatures for PCR as needed. DNA sequences generated for this study are

87 deposited in GenBank (KC844921–845002).

88 Phylogenetic analyses were performed using maximum likelihood and Bayesian

89 methods in GARLI-Partition v.097 (Zwickl 2006) and MrBayes v.3.1.2, respectively, and

90 partitioning nucleotide positions by gene. A multiple alignment for each gene was

91 generated using MAFFT v. (Katoh et al. 2005) with minor adjustments made by eye; the

92 alignment used for analysis is deposited in Dryad

93 (http://dx.doi.org/10.5061/dryad.9dd42). The best-fit model of sequence evolution for

94 each partition was selected as that with the lowest Akaike Information Criterion (AIC)

95 score using jModelTest v.0.1.1 (Posada 2008); the GTR+I+Γ model was the preferred

96 model for 16S, ND1, and RAG-1, whereas GTR+Γ was preferred for POMC. For the

97 maximum likelihood (ML) estimate of the phylogeny, we used the ML phylogeny with 98 the lowest -ln likelihood score from 100 search replications; each search was terminated

99 after 10 x 105 generations after the last topological improvement. Support was estimated

100 using 100 nonparametric bootstrap pseudoreplicates in GARLI using similar search

101 specifications but with one search per bootstrap replicate. Bayesian phylogenetic analyses

102 were conducted using MrBayes v.3.1.2 using four runs of four MCMC chains run for 20

103 million generations, sampled every 2000 generations, and using a temperature of 0.2 and

104 default priors; likelihoods reached a plateau by one million generations. Following

105 assessment of convergence by examination of split frequencies among runs in AWTY

106 (Nylander et al. 2008), we conservatively discarded the first 10 million generations from

107 each run. Split support for both maximum likelihood and Bayesian analyses was

108 calculated using SumTrees in DendroPy (Sukumaran and Holder 2010).

109

110 3. Results

111 Maximum likelihood and Bayesian analyses resulted in similar estimates of phylogenetic

112 relationships with many nodes resolved with high support (≥ 70% bootstrap support; ≥

113 0.95 posterior probability). Each genus of marsupial frog is determined to be

114 monophyletic with high support (Fig. 1), and many relationships are similar to those

115 resolved by Wiens et al. (2007). Within Gastrotheca, there are six lineages, each of

116 which has a distinctive biogeography. Gastrotheca from the Atlantic Coastal Forests of

117 Brazil comprise two distinct lineages that do not together form a clade.

118 The earliest diverging lineage of Gastrotheca is represented by G. megacephala

119 of Brazil, followed by a clade consisting of G. walkeri from northern Venezuela and a

120 clade of six species from northwestern South America (Fig. 1). The remaining species of 121 Gastrotheca are resolved as members of two clades, one of which is restricted to

122 southeastern Brazil (four species). The other clade contains the majority of Gastrotheca

123 diversity and is partitioned into a clade from the central Andes and another from the

124 northern Andes; Gastrotheca galeata is shown to be the basal member of this large

125 Andean clade.

126

127 4. Discussion

128 The expanded taxonomic and geographic sampling of our analyses reveals that the

129 Gastrotheca found in the Atlantic Coastal Forests of Brazil consists of two divergent

130 lineages that are not sister taxa. This supports previous suggestions that Brazilian species

131 of Gastrotheca may not be closely related (Duellman 1984; Caramaschi and Rodrigues

132 2007). A similar pattern of non-monophyly has been found in other anuran taxa

133 containing species endemic to the Atlantic Coastal Forests (Hedges et al. 2008; Canedo

134 and Haddad 2012). Recently, Teixeira et al. (2012b) described two new species of

135 Gastrotheca from the Atlantic Coastal Forests of Brazil. These authors suggested non-

136 monophyly of Brazilian Gastrotheca by finding a closer relationship of G. pulchra

137 (eastern Brazil) to the Andean G. cf. marsupiata than to the species that they described

138 from northeastern Brazil. The analysis by Teixeira et al. (2012b) lacked the more

139 extensive sampling of Gastrotheca of our study, including species from southeastern

140 Brazil. Because the mitochondrial gene ND1 is the only locus sampled in both studies,

141 comparisons are somewhat difficult. However, pairwise divergences indicate that our

142 data for G. megacephala are more similar (< 2%) to that presented by Teixeira et al.

143 (2012b) for G. prasina, G. recava, and G. fissipes (from Pernambuco) than to our data for 144 G. albolineata, G. ernestoi, G. fulvorufa, or G. microdiscus (≥ 2.5%). Unfortunately,

145 ND1 data are not presented for G. pulchra by Teixeira et al. (2012b), we cannot further

146 evaluate the relationships of G. pulchra to other Brazilian species. If G. megacephala, G.

147 prasina, G. recava, and G. fissipes form a clade exclusive of the other Brazilian

148 Gastrotheca (as suggested by Teixeira et al. 2012b), then the biogeography of these two

149 clades is interesting because there is relatively little geographic overlap between them.

150 However, if G. pulchra is in the clade found mostly in southeastern Brazil, then these two

151 clades would have more extensive overlap because G. pulchra occurs throughout most of

152 the region inhabited by G. fissipes, G. megacephala, and G. recava (Teixeira et al.

153 2012b).

154 Because available data for Gastrotheca ovifera is limited only to the

155 mitochondrial 16S gene, we chose not to include it in our analyses. Separate preliminary

156 analyses suggest that this species is a member of the large clade containing Gastrotheca

157 species from the Central and Northern Andes and southeastern Brazil but its position

158 within this large clade remains unresolved (data not shown). This, however, suggests

159 biogeographic complexity because another Venezuelan species, G. walkeri, is not a

160 member of this larger clade (Fig. 1). Another species occurring in both Venezuela and

161 neighboring Columbia, G. nicefori, is clearly nested within the Northern Andean clade,

162 further indicating that the marsupial frog fauna in the northernmost Andes represents a

163 mixture of lineages.

164 Similar to previous analyses, we recovered each of the five hemiphractid genera

165 as monophyletic (Fig. 1), although relationships among these genera do not receive

166 strong support. The sixth genus, Crytobatrachus, was not included because of the 167 absence of tissues; however, the morphology of species in the genus suggests that it is

168 monophyletic (Lynch 2008). Within Gastrotheca, there is strong support for six major

169 geographically circumscribed lineages, one of which is represented by a single species,

170 G. walkeri, from northern Venezuela (another presumably related species, G. williamsoni

171 was not included). Our results suggest that is the most basal lineage within

172 Hemiphractidae as found in other studies (Wiens et al. 2007; Duellman et al. 2011b;

173 Pyron and Wiens 2011). Recently, Duellman et al. (2011b) revealed that species

174 previously assigned to Flectonotus represent two highly divergent lineages restricted to

175 either to northern South America or the Atlantic Coastal Forests of Brazil; these are now

176 recognized as Flectonotus and Fritziana, respectively.

177 Similar to Wiens et al. (2007), we find G. megacephala (their G. fissipes) and a

178 clade in northwestern South America as successively branching lineages at the base of

179 Gastrotheca. Within the larger Andean clade, there are two large subclades restricted to

180 the central and northern Andes, respectively. In part because of recent descriptions of

181 new species, as well as new genetic data for previously described species (e.g., Duellman

182 et al. 2011a), we have uncovered support for a clade of eight direct-developing species

183 within the central Andes clade. Two Andean species complexes contain several presently

184 unnamed species (Fig. 1); these complexes contain species that are distinct genetically

185 but so far not readily distinguishable morphologically. Both of the large clades found in

186 the central or northern Andes contain a diversity of species with either direct

187 development or free-living and feeding tadpoles (Fig. 1). Similar to the study of Wiens et

188 al. (2007), a complicated pattern of life history evolution is revealed by mapping these

189 two life history states on the phylogeny. 190

191 5. Conclusion

192 An expanded phylogeny of marsupial frogs of the genus Gastrotheca based on

193 two mitochondrial and two nuclear genes reveals that the genus Gastrotheca is

194 monophyletic and contains six lineages that are geographically distinct. The greatest

195 diversity of Gastrotheca is in the Andean region, and the genus does not occur in the

196 Amazon Basin, except for G. longipes at the westernmost periphery. Our study reveals

197 that Gastrotheca in the Atlantic Coastal Forests of Brazil belong to two clades, one of

198 which is the sister to all other species of Gastrotheca. The two species-rich clades found

199 in the northern and central Andes both contain species with either tadpoles or direct

200 development and suggest a complicated pattern of life history evolution in these montane

201 clades.

202

203 Acknowledgments

204 Tissues of Brazilian taxa were provided by C.F.B. Haddad, J.M. Pombal, Jr., and

205 H. Zaher. Sequences of unnamed Ecuadorian taxa are from S. Carvajal and L.A. Coloma.

206 Duellman was supported by NSF DEB-1118879. We thank A.B. Sellas (Center for

207 Comparative Genomics, California Academy of Sciences) for her assistance with

208 laboratory work necessary for the completion of this project and J. Wiens (SUNY–Stony

209 Brook) for making the matrix of Wiens et al. (2007) available to us. During portions of

210 this work, DCB was supported in part by NSF DEB-0334928 to L. Trueb.

211

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Gastrotheca_Table1.doc

Table 1. Primers developed in this study for polymerase chain reaction amplification of nuclear-coding genes.

Gene Primer name Primer sequence 5’ to 3’ (indicated by arrow) proopiomelanocortin (POMC) POMC-Hemi-f TGTGACTCTTCCTCAGCGTC  recombination activation gene 1 (RAG-1) RAG1-Hemi-f1 CTTCCRGGATATCATCCHTT  RAG1-Hemi-f2 TGATGAATCHGACCATGA AA  RAG1-Hemi-r1 AAGTATTCCTCCAAYTTCCAACA  RAG1-Hemi-r2 GCTTKGCAGAGACTCCCTTA 

Figure QCAZ 42862 G. pseustes * G. lateonota QCAZ 45113 * G. sp. A QCAZ 22635 * G. sp. B QCAZ 21105 * Feeding tadpoles G. sp. C QCAZ 47299 G. peruana * G. stictopleura Non-feeding tadpoles G. psychrophila G. marsupiata MVZ 269022 Direct development G. marsupiata * G. griswoldi Central Andes G. lauzuricae MNCN 9529 G. christiani * > 0.95 posterior probablity, > 70% bootstrap support G. chrysosticta G. pachachacae MUSM 284492 G. rebeccae CORBIDI 8006 * G. ochoai * 0.1 substitions per site G. excubitor G. testudinea QCAZ 16444 G. atympana * G. nebulanastes MUSM 27943 G. antoniiochoai * G. zeugocystis G. litonedis G. litonedis QCAZ 42721 G. litonedis QCAZ 42726 * 0.93 G. sp. D QCAZ 42725 * 60% G. orophylax * G. plumbea G. monticola G. sp. E QCAZ 21213 Northern Andes G. trachyceps * G. argenteovirens 0.95 G. ruizi 53% G. aureomaculata G. dunni G. nicefori 1.00 G. riobambae 65% G. galeata G. microdiscus CFBH T3069 G. microdiscus CFBH T3068 * G. microdiscus CFBH T1250 * G. fulvorufa CTMZ 7467 * SE Brazil G. ernestoi MNRJ 64000 * 0.97 G. ernestoi MNRJ 57129 * 66% G. albolineata MNRJ 54401* G. dendronastes * G. cornuta G. longipes NW S. America G. helenae G. weinlandii G. guentheri G. walkeri N. Venezuela G. megacephala CFBH T377 * G. megacephala NE Brazil scalae S. evansi S. coxi S. ginesi Fritziana ssilis Fr. cf. ssilis Fr. ohausi fasciatus CHP 6670 H. fasciatus CHP 6397 * H. scutatus * H. bubalus H. proboscideus Flectonotus tzgeraldi Fl. pygmaeus Highlights

Brazilian marsupial frogs are diphyletic (Anura: Hemiphractidae: Gastrotheca) David C. Blackburn, William E. Duellman

 An expanded phylogeny of marsupial frogs and allies is presented.  Six geographically circumscribed lineages are recovered, including two widespread Andean clades.  Gastrotheca from coastal Brazil belong to two divergent clades that are not sister taxa. graphical_abstract.tif