Accepted Manuscript
Short communication
Brazilian marsupial frogs are diphyletic (Anura: Hemiphractidae: Gastrotheca)
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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Short Communication
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 frog 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 species in these
20 clades inhabit humid forests at low to mid-elevations, and their life histories are
21 characterized by lacking free-living tadpoles (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 amphibians (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 genus Gastrotheca. Similar to few other amphibian
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., Cryptobatrachus, 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 taxonomy 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 Flectonotus 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
212 References 213 Blackburn, D.C., Wake, D.B., 2011. Class Amphibia Gray, 1825. In: Zhang, Z.-Q. (Ed.)
214 Animal biodiversity: an outline of higher-level classification and survey of
215 taxonomic richness. Zootaxa 3148: 39–55.
216 Canedo, C., Haddad, C.F.B., 2012. Phylogenetic relationships within anuran clade
217 Terrarana, with emphasis on the placement of Brazilian Atlantic rainforest frogs
218 genus Ischnocnema (Anura: Brachycephalidae). Mol. Phylogenet. Evol. 65: 610–
219 620.
220 Caramaschi, U., Rodriguez, M.T., 2007. Taxonomic status of the species of Gastrotheca
221 Fitzinger, 1843 (Amphiba, Anura, Amphignathodontidae) of the Atlantic Rain Forest
222 of eastern Brazil, with description of a new species. Bol. Mus. Nac. Rio de Janeiro,
223 Zoologia 525: 1–19.
224 Darst, C.R., Cannatella, D.C., 2004. Novel relationships among hyloid frogs inferred
225 from 12S and 16S mitochondrial DNA sequences. Mol. Phylogenet. Evol. 31: 462–
226 475.
227 Duellman, W.E., 1974. A systematic review of the marsupial frogs (Hylidae:
228 Gastrotheca) of the Andes of Ecuador. Occ. Pap. Mus. Nat. Hist. Univ. Kansas 22:
229 1–27.
230 Duellman, W.E., 1984. Taxonomy of Brazilian hylid frogs of the genus Gastrotheca. J.
231 Herpetol. 18: 302–312.
232 Duellman, W.E., 1989. Alternative life-history styles in anuran amphibians: Evolutionary
233 and ecological implications. In Alternative life-history styles of animals, ed. M. N.
234 Bruton, 101–126. Dordrecht: Kluwer Academic.
235 Duellman, W.E., Catenazzi, A., Blackburn, D.C., 2011a. A new species of marsupial frog 236 (Anura: Hemiphractidae: Gastrotheca) from the Andes of southern Peru. Zootaxa
237 3095: 1–14.
238 Duellman, W.E., Fritts, T.H., 1972. A taxonomic review of the southern Andean
239 marsupial frogs (Hylidae: Gastrotheca). Occ. Pap. Mus. Nat. Hist. Univ. Kansas 9:
240 1–37.
241 Duellman, W.E., Hillis, D.M., 1987. Marsupial frogs (Anura: Hylidae: Gastrotheca) of
242 the Ecuadorian Andes: Resolution of taxonomic problems and phylogenetic
243 relationships. Herpetologica 43: 141–173.
244 Duellman, W.E., Jungfer, K.-H., Blackburn, D.C., 2011b. The phylogenetic relationship
245 of geographically separated “Flectonotus” (Anura: Hemiphractidae), as revealed by
246 molecular, behavioral, and morphological data. Phyllomedusa 10: 15–29.
247 Duellman, W.E., Lehr, E. Rodríguez, D., von May, R., 2004. Two new species of
248 marsupial frogs (Anura: Hylidae: Gastrotheca) from the Cordillera Oriental in central
249 Peru. Sci. Pap. Nat. Hist. Mus. Univ. Kansas 32: 1–10.
250 Duellman, W.E., Maness, S.J., 1980. The reproductive behavior of some hylid marsupial
251 frogs. J. Herpetology 14: 213–222.
252 Duellman, W.E., Maxson, L.R., Jesiolowski, C.A., 1988. Evolution of marsupial frogs
253 (Hylidae: Hemiphractinae): Immunological evidence. Copeia 1988: 527–543.
254 Faivovich, J., Haddad, C.F.B., Garcia, P.C.A., Frost, D.R., Campbell, J.A., Wheeler,
255 W.C., 2005. Systematic review of the frog family Hylidae, with special reference to
256 Hylinae: phylogenetic analysis and taxonomic revision. Bull. Amer. Mus. Nat. Hist.
257 294: 1–240.
258 Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Haddad, C.F.B., de Sá, R.O., 259 Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A.,
260 Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, D.M., Wheeler,
261 W.C. 2006., The amphibian tree of life. Bull. Amer. Mus. Nat. Hist. 297: 1–370.
262 Guayasamin, J.M., Castroviejo-Fisher, S., Ayarzagüena, J., Trueb, L., Vilà, C., 2008.
263 Phylogenetic relationships of glassfrogs (Centrolenidae) based on mitochondrial and
264 nuclear genes. Mol. Phylogenet. Evol. 48: 574–595.
265 Hedges, S.B., Duellman, W.E., Heinicke, M.P., 2008. New World direct-developing
266 frogs (Anura: Terrarana): molecular phylogeny, classification, biogeography, and
267 conservation. Zootaxa 1737: 1–182.
268 Heinicke, M.P., Duellman, W.E., Trueb, L., Means, D.B., MacCulloch, R.D., Hedges,
269 S.B., 2009. A new frog family (Anura: Terrarana) from South America and an
270 expanded direct-developing clade revealed by molecular phylogeny. Zootaxa 2211:
271 1–35.
272 Izecksohn, E., Carvalho-e-Silva, S. P., Peixoto, O.L., 2009. Sobre Gastrotheca fissipes
273 (Boulenger, 1888), com a descrição de uma nova espécie (Amphibia, Anura,
274 Amphignathodontidae). Arq. Mus. Nac. Rio de Janeiro 67: 81–91.
275 Lehr, E., Fritzsch, G., Müller, A., 2005. Analysis of Andes frogs (Phrynopus,
276 Leptodactylidae, Anura) phylogeny based on 12S and 16S mitochondrial rDNA
277 sequences. Zool. Scripta 34: 593–603.
278 Lynch, J.D., 2008. A taxonomic revision of frogs of the genus Cryptobatrachus (Anura:
279 Hemiphractidae). Zootaxa 1883: 28–68.
280 Katoh, K., Kuma, K.-I., Toh, H., Miyata, T., 2005. MAFFT version 5: improvement in
281 accuracy of multiple sequence alignment. Nucleic Acids Res. 33: 511–518. 282 Mendelson, J.R., III, Da Silva, H.R., Maglia, A.M., 2000. Phylogenetic relationships
283 among marsupial frog genera (Anura: Hylidae: Hemiphractinae) based on evidence
284 from morphology and natural history. Zool. J. Linn. Soc. 128: 125–148.
285 Nylander, J.A.A., Wilgenbusch, J.C., Warren, D.L., Swofford, D.L., 2008. AWTY (are
286 we there yet?): A system for graphical exploration of MCMC convergence in
287 Bayesian phylogenetics. Bioinformatics 24: 581–583.
288 Posada, D., 2008. jModelTest: Phylogenetic model averaging. Mol. Biol. Evol. 25: 1253–
289 1256.
290 Pyron, A.R., Wiens, J.J., 2011. A large-scale phylogeny of Amphibia including over
291 2,800 species, and a revised classification of extant frogs, salamanders, and
292 caecilians. Mol. Phylogenet. Evol. 61: 543–583.
293 Sukumaran, J., Holder, M.T., 2010. DendroPy: a Python library for phylogenetic
294 computing. Bioinformatics 26: 1569–1571.
295 Teixeira, M., Jr., Amaro, R.C., Recoder, E.S., de Sena, M.A., Rodrigues, M.T., 2012a. A
296 relict new species of Oreobates (Anura, Strabomantidae) from the seasonally dry
297 tropical forests of Minas Gerais, Brazil, and its implication to the biogeography of
298 the genus and that of South American dry forests. Zootaxa 3158: 37–52.
299 Teixeira, M., Jr., dal Vechio, F., Recoder, R.S., Carnaval, A.C., Strangas, M.,
300 Damasceno, R.P., de Sena, M.A., Rodrigues, M.T., 2012b. Two new species of
301 marsupial tree-frogs genus Gastrotheca Fitzinger, 1843 (Anura, Hemiphractidae)
302 from the Brazilian Atlantic Forest. Zootaxa 3437: 1–23.
303 Wassersug, R.J., Duellman, W.E., 1984. Oral structures and their development in egg-
304 brooding hylid frog embryos and larvae: Evolutionary and ecological implications. J. 305 Morphol. 182: 1–37.
306 Wiens, J.J., 2007. Global patterns of diversification and species richness in amphibians.
307 Am. Nat. 170: S86–S106.
308 Wiens, J.J., Fetzner, Jr., J.W., Parkinson, C.L., Reeder, T.W., 2005. Hylid frog phylogeny
309 and sampling strategies for speciose clades. Syst. Biol. 54: 719–748.
310 Wiens, J.J., Kuczynski, C.A., Duellman, W.E., Reeder, T.W., 2007. Loss and re-
311 evolution of complex life cycles in marsupial frogs: does ancestral trait
312 reconstruction mislead? Evolution 61: 1886–1899. 313 Fig. 1. Maximum likelihood phylogeny depicting relationships among marsupial
314 frogs and their allies (family Hemiphractidae). Samples with an asterisk were
315 sequenced for this study. Nodes with high support are indicated by black circles and
316 support values are indicated on other selected nodes. Species are color-coded by life
317 history mode. See Materials and methods for collection acronyms.
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 Gastrotheca pseustes 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 Stefania scalae S. evansi S. coxi S. ginesi Fritziana ssilis Fr. cf. ssilis Fr. ohausi Hemiphractus 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