Mitochondrial Genes Reveal Cryptic Diversity in Plant-Breeding Frogs from Madagascar (Anura, Mantellidae, Guibemantis)

Molecular Phylogenetics and Evolution 44 (2007) 1121–1129 www.elsevier.com/locate/ympev

Mitochondrial genes reveal cryptic diversity in plant-breeding frogs from Madagascar (Anura, Mantellidae, Guibemantis)

Richard M. Lehtinen a,*, Ronald A. Nussbaum b, Christina M. Richards c, , David C. Cannatella d, Miguel Vences e

a Biology Department, 931 College Mall, The College of Wooster, Wooster, OH 44691, USA b University of Michigan Museum of Zoology, Division of Amphibians and Reptiles, 1109 Geddes Avenue, Ann Arbor, MI 48109-1079, USA c Wayne State University, Department of Biological Sciences, Detroit, MI 48202, USA d Section of Integrative Biology and Texas Memorial Museum, University of Texas, 24th and Speedway, Austin, TX 78712, USA e Zoological Institute, Technical University of Braunschweig, Spielmannstr. 8, 38106 Braunschweig, Germany

Received 10 October 2006; revised 21 May 2007; accepted 23 May 2007 Available online 9 June 2007

Abstract

One group of mantellid frogs from Madagascar (subgenus Pandanusicola of Guibemantis) includes species that complete larval development in the water-filled leaf axils of rainforest plants. This group consists of six described species: G. albolineatus, G. bicalcaratus, G. flavobrunneus, G. liber, G. pulcher, and G. punctatus. We sequenced the 12S and 16S mitochondrial rRNA genes (1.8 kb) from multiple specimens (35 total) of all six species to assess phylogenetic relationships within this group. All reconstructions strongly supported G. liber as part of the Pandanusicola clade, even though this species does not breed in plant leaf axils. This result confirms a striking reversal of reproductive specialization. However, all analyses also indicated that specimens assigned to G. liber include genetically distinct allopatric forms that do not form a monophyletic group. Most other taxa that were adequately sampled (G. bicalcaratus, G. flavobrunneus, and G. pulcher) likewise consist of several genetically distinct lineages that do not form monophyletic groups. These results suggest that many of the recognized species in this group are complexes of cryptic species. Ó 2007 Elsevier Inc. All rights reserved.

Keywords: Amphibia; Pandanusicola; Phylogeny; Phytotelmata

1. Introduction rather striking in appearance and are clearly different from other known forms. Others are minimally morphologically Madagascar contains a disproportionately large portion differentiated from one another and have been detected of Earth’s biological diversity and nearly all Malagasy only by careful combination of morphological compari- frogs (all except one introduced species) are endemic to sons, bio-acoustic analysis, and molecular genetic tools. the island. Considering described species only, the amphib- Many of these frogs have now been subject to molecular ian fauna of Madagascar now includes over 200 frog spe- phylogenetic analysis (e.g., Andreone et al., 2005; Glaw cies in five families (nearly 4% of the world’s total; Frost and Vences, 2006; Vences and Glaw, 2001; Vences et al., et al., 2006; Glaw and Vences, 2003). Since the early 2003). Based on the phylogenies obtained, it became clear 1990s, herpetologists have uncovered dozens of new frog that various genera as previously understood were non- taxa from Madagascar and this rapid rate of description monophyletic (Richards et al., 2000; Vences et al., 2003). shows no sign of slowing. Many of these new species are Consequently, the classification of various groups has been recently revised, especially in the endemic Malagasy family

* Mantellidae (e.g., Glaw and Vences, 2006). Because the Corresponding author. Fax: +1 330 263 2378. E-mail address: [email protected] (R.M. Lehtinen). Malagasy poison frogs of the genus Mantella were deeply Deceased. nested within the genus Mantidactylus as formerly recog-

1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.05.020 1122 R.M. Lehtinen et al. / Molecular Phylogenetics and Evolution 44 (2007) 1121–1129 nized (e.g., Richards et al., 2000), the latter has now been To address this problem, we use mitochondrial sequence partitioned into the eight genera Blommersia, Boehmantis, data from these previous studies and add sequences for 30 Gephyromantis, Guibemantis, Mantidactylus, Spinomantis, additional specimens attributed to this problematic group Wakea, and the recently discovered Tsingymantis. Within (including G. punctatus, which was not included earlier). the Mantellidae, however, one problematic group has By using a more comprehensive sampling of the available received little attention thus far: the plant-breeding frogs taxa, we herein reconstruct the evolutionary history of this currently classified in the subgenus Pandanusicola in the group. genus Guibemantis (Mantellidae; Glaw and Vences, 2006). Most of these species lay their eggs on the leaves 2. Materials and methods of Pandanus plants (or less commonly other phytotelmata), and the tadpoles develop in the tiny water bodies 2.1. DNA sequences/laboratory methods in the leaf axils of these plants (Lehtinen, 2002). Parental care in the form of amphisexual egg attendance is con- A subset of the 12S and 16S mitochondrial rRNA firmed for two species in this group (Lehtinen, 2003), sequences used in this study was taken from Richards and the tadpoles are specialized in a number of ways et al. (2000) and Lehtinen et al. (2004). New sequences were for life in a small, desiccation-prone, and food-poor envi- added to these using standard laboratory protocols. DNA ronment (Lehtinen, 2004). was extracted from frozen tissue samples using Qiagen There are six species currently assigned to the subgenus DNeasyÒ tissue kits. The 12S and 16S rRNA genes were Pandanusicola. These include G. albolineatus (Blommers- amplified via the polymerase chain reaction with the prim- Schlo¨sser and Blanc, 1991), G. bicalcaratus (Boettger, ers reported in Richards et al. (2000). PCR products were 1913), G. flavobrunneus (Blommers-Schlo¨sser, 1979), G. purified using Qiagen MinEluteä PCR column purification liber (Peracca, 1893), G. pulcher (Boulenger, 1882), and kits. Purified PCR products were sequenced using the G. punctatus (Blommers-Schlo¨sser, 1979). Many of these amplification primers on an automated sequencer (ABI species (as currently understood) show substantial varia- 3100) at the MCIC at the Ohio Agricultural Research tion in coloration patterns; this is particularly notable in and Development Center. A subset of the sequencing was G. liber. Guibemantis liber was tentatively placed with these done using similar protocols in the Cannatella Lab at the species in the phenetic pulcher group by Blommers-Schlo¨s- University of Texas. ser (1979) and Blommers-Schlo¨sser and Blanc (1991), but Sequence chromatograms were directly imported into was placed by later authors (e.g., Andreone, 2003; Glaw Codon Code Aligner 1.5.1 where bases called using Phred and Vences, 1994) into the subgenus Guibemantis (the software and contigs were assembled. Multiple sequence depressiceps group of Blommers-Schlo¨sser and Blanc, alignment was performed using ClustalX 1.81 (Thompson 1991). Members of the subgenus Guibemantis (currently et al., 1997). Alternative alignments were explored by sys- composed of four species), as far as is known, lay their eggs tematically varying gap opening and gap extension penal- on vegetation overhanging ponds, lack parental care and ties in varying ratios. Alternative alignments were have unspecialized, typical pond-type tadpoles. compared visually for positional homology. Hypervariable Previous analysis of mitochondrial genes indicated that regions of the sequences (corresponding to secondary Pandanusicola and Guibemantis are sister clades (Richards structure) were excluded from analysis. The best alignment et al., 2000). Consequently, both subgenera were included was found by setting the gap creation/extension penalties in one genus (Guibemantis) in a recent comprehensive clas- to 10/5. Novel sequences were submitted to GenBank sification of the mantellids (Glaw and Vences, 2006). How- (Table 1). ever, the analysis by Richards et al. (2000) included only a single member of Pandanusicola and Guibemantis (G. 2.2. Phylogenetic analysis flavobrunneus and G. kathrinae, respectively). Lehtinen et al. (2004) extended this analysis by including all known To estimate of the phylogenetic relationships of the spe- members of Pandanusicola (except G. punctatus, for which cies in the subgenus Pandanusicola, we used maximum par- tissues were unavailable) and two additional species of simony, maximum likelihood and Bayesian analyses. Guibemantis (G. liber and G. tornieri). These results sug- Partition homogeneity was examined with the ILD test in gested that G. liber may be properly placed in Pandanusico- PAUP* version 4b10 (Swofford, 1998), using a heuristic la, rather than Guibemantis. Based on their tree, Lehtinen search strategy and 1000 replicates. et al. (2004) also suggested that plant-breeding evolved The maximum parsimony (MP) and maximum likeli- only once in this group, but there may have been a reversal hood (ML) analyses were implemented in PAUP* 4.0b10 back to the ancestral reproductive mode (pond-breeding) using heuristic searches and 100 random addition sequence in G. liber. Even with this work, however, only a single replicates. The tree-bisection-reconnection algorithm was specimen from each putative species was used, leaving open used for branch swapping with the steepest descent option the question of whether cryptic diversity within the recog- off. In the MP analyses, clade support was assessed using nized species was obscuring the real evolutionary history 2000 bootstrap replicates and characters were unordered of this group. and unweighted. In the ML analysis, we used the R.M. Lehtinen et al. / Molecular Phylogenetics and Evolution 44 (2007) 1121–1129 1123

Table 1 Collection localities of specimens used in this study and GenBank accession numbers of sequenced gene fragments (12S and 16S, respectively) Species Subgenus Locality Voucher GenBank Accession Numbers Guibemantis cf. albolineatus Pandanusicola Andasibe [A] ZSM 250/2002 AY454354, AY454376 Guibemantis cf. bicalcaratus Pandanusicola Marojejy [B] ZCMV 2044 EF468005, EF472505 Guibemantis bicalcaratus Pandanusicola Manongarivo Reserve [C] UMMZ 212592 EF468006, EF472506 Guibemantis bicalcaratus Pandanusicola Besariaka [D] ZCMV 932 EF468023, EF472522 Guibemantis bicalcaratus Pandanusicola Marojejy Reserve, Manantenina River [B] UMMZ 212597 EF468007, EF472507 Guibemantis bicalcaratus Pandanusicola Sainte Luce [E] FGZC 2588 EF468008, EF472508 Guibemantis (?) bicalcaratus Pandanusicola? Maharira [F] ZMA 20444 EF468024, EF472523 Guibemantis (?) bicalcaratus Pandanusicola? Vohiparara [F] ZSM 745/2003 EF468025, EF472524 Guibemantis bicalcaratus Pandanusicola Sainte Luce [E] UMMZ 197485 AY454356, AY454379 Guibemantis cf. depressiceps Guibemantis Andohahela FGZC 2434 EF468009, EF488668 Guibemantis flavobrunneus Pandanusicola Manombo [G] ZMA 20119 EF468010, EF472509 Guibemantis flavobrunneus Pandanusicola Mangoro River area, precise locality unknown FGZC 2655 EF468011, EF472510 Guibemantis flavobrunneus Pandanusicola Ambohitantely [H] FMNH 259923 AY454367, AY454392 Guibemantis flavobrunneus Pandanusicola Andringitra, Iatara River [I] UMMZ 212929 EF468012, EF472511 Guibemantis flavobrunneus Pandanusicola Cap Est [J] UMMZ 212922 EF468013, EF472512 Guibemantis flavobrunneus Pandanusicola Ankaratra Mountains [O] UMMZ 212919 EF468014, EF472513 Guibemantis kathrinae Guibemantis Manantantely forest UMMZ 198114 AF261242, AF261260 Guibemantis liber Pandanusicola Montagne d’Ambre [L] ZMA 19644 EF468029, EF472528 Guibemantis liber Pandanusicola Vevembe [K] ZMA 20117 EF468028, EF472527 Guibemantis liber Pandanusicola Montagne d’Ambre [L] UMMZ 213064 EF468015, EF472514 Guibemantis liber Pandanusicola Manongarivo Reserve, Ambalafary [C] UMMZ 213068 EF468016, EF472515 Guibemantis liber Pandanusicola Tsaratanana, Befosa [M] UMMZ 213101 EF468017, EF472516 Guibemantis cf. liber Pandanusicola Vevembe [K] ZMA 20116 EF468027, EF472526 Guibemantis cf. liber Pandanusicola Ankavanana River [N] UMMZ 213093 AY454364, AY454387 Guibemantis cf. liber Pandanusicola Marojejy Reserve, Manantenina River [B] UMMZ 213070 EF468018, EF472517 Guibemantis cf. liber Pandanusicola Andringitra, Sahavaby River [I] UMMZ 213314 AY454368, AY454393 Guibemantis pulcher Pandanusicola Samalaotra [F] ZCMV 2002.277 EF468035, EF472534 Guibemantis pulcher Pandanusicola Maharira [F] ZCMV 265 EF468034, EF472533 Guibemantis pulcher Pandanusicola Besariaka [D] ZMA 20317 EF468030, EF472529 Guibemantis pulcher Pandanusicola Ranomafana, Kidonavo bridge [F] ZMA 20242 EF468032, EF472531 Guibemantis pulcher Pandanusicola Vohiparara bridge [F] ZMA 19415 EF468033, EF472532 Guibemantis cf. pulcher Pandanusicola Sainte Luce [E] RML 262 EF468019, EF472518 Guibemantis cf. pulcher Pandanusicola Sainte Luce [E] RML 132 EF468020, EF472519 Guibemantis pulcher Pandanusicola Andasibe [A] ZSM 58/2002 EF468031, EF472530 Guibemantis punctatus Pandanusicola Sainte Luce [E] RML 290 EF468021, EF472520 Guibemantis punctatus Pandanusicola Sainte Luce [E] RML 265 EF468022, EF472521 Guibemantis tornieri Guibemantis Zahamena Reserve UMMZ 213353 AY454369 Letters correspond to locations in Fig. 1. Collection abbreviations are as follows: FGZC, Frank Glaw Zoological Collection; FMNH, Field Museum of Natural History; RML, uncatalogued tissue samples of R. Lehtinen at UMMZ; UMMZ, University of Michigan Museum of Zoology; ZCMV, Zoological collection of Miguel Vences; ZMA, Zoological Museum Amsterdam; ZSM, Zoologische Staatssammlung Mu¨nchen.

GTR + I + G substitution model, as determined by hierar- simultaneously with three heated chains and one cold chical likelihood ratio tests in Modeltest version 3.7 chain, as per the program defaults. The run was conducted (Posada and Crandall, 1998). Empirical base frequencies for 1.4 million generations, with the first 400,000 being dis- were: freq A = 0.3499; freq C = 0.2110; freq G = 0.1716; carded as burn-in. This produced 1 million post burn-in freq T = 0.2675. The proportion of invariable sites (I) generations. The analysis was terminated when stationarity was 0.2733 and the gamma distribution shape parameter was reached (the standard deviation of split frequencies (G) was 0.6913. Because of computational limitations, we was less than 0.01). Majority rule consensus trees were used used only 100 bootstrap replicates to assess clade support to display the results of all analyses. For the Bayesian anal- in the ML analyses. For both MP and ML analyses, nodes ysis, nodes with posterior probability values over 95% were with 70% bootstrap support were regarded as sufficiently deemed well supported. resolved (Hillis and Bull, 1993). Sequences from three of the four members of the subge- The Bayesian analysis was conducted in MrBayes 3.1.1 nus Guibemantis (G. cf. depressiceps, G. kathrinae, and G. (Huelsenbeck and Ronquist, 2001) using the substitution tornieri) were used as outgroups in the ML and MP analy- model indicated by Modeltest (above). Priors were set ses. Since MrBayes allows only a single outgroup, G.cf. using the default setting in MrBayes, and the model param- depressiceps was arbitrarily chosen as the outgroup for eters were estimated in the analysis. Two independent anal- the Bayesian analysis. Guibemantis is the sister taxon to yses with different random starting trees were run Pandanusicola (Richards et al., 2000; Lehtinen et al., 2004). 1124 R.M. Lehtinen et al. / Molecular Phylogenetics and Evolution 44 (2007) 1121–1129

3. Results external morphology come mostly from the northern part of the island (Manongarivo, Tsaratanana, Montagne After excluding 219 hypervariable or ambiguous charac- d’Ambre) but also include one specimen from the south ters, 1748 characters were available for analysis. The results (Vevembe; Fig. 2). Two other specimens attributed to of the ILD test were significant (p = 0.03), so we conducted G. liber (clade 3) are genetically very divergent from partitioned analyses of the 12S and 16S genes separately as the G. liber in clade 2 (average pairwise sequence diver- well as a total evidence analysis of the concatenated data- gence >9.0%; Table 3). These occur in a relatively small set. The total evidence parsimony reconstruction (1748 area of northeastern Madagascar (Marojejy and the unordered characters, 514 parsimony informative) pro- Ankavana River area; Fig. 2). Lastly, one morphologi- duced two equally parsimonious trees of 1945 steps cally similar lineage from Vevembe and Andringitra in (CI = 0.453; RI = 0.657). The likelihood and Bayesian south and central eastern Madagascar is also very diver- analyses of these data generally produced similar topolo- gent (clade 6 in Figs. 1 and 2) and, at least in the Bayes- gies, and for simplicity we present and discuss the consen- ian analysis, is the sister taxon to all other members of sus tree from the Bayesian analysis. The separate analyses Pandanusicola (Table 2). of the 12S and 16S genes also yielded very similar topolo- Similar genetic differentiation and non-monophyly is gies, differing primarily in the level of support at basal also seen in G. pulcher (Fig. 1). There is strong support nodes. Table 2 summarizes support for major branches for the monophyly of the highland populations of G. pul- recovered from our phylogenetic analyses. cher (clade 1 in Figs. 1 and 2, Table 2); however, a low- In all reconstructions, G. liber is strongly supported as land form from extreme southeastern Madagascar is being nested deep within a clade containing only mem- only distantly related to highland forms (average pairwise bers of Pandanusicola (Fig. 1) and does not group with sequence divergence >8.0%; Table 3) and groups in yet members of the outgroup (subgenus Guibemantis). How- another strongly divergent clade (clade 4, Figs. 1 and 2, ever, while sequences of typical G. liber (i.e., as defined Table 2). by Blommers-Schlo¨sser (1979) and subsequent authors Specimens attributed to G. flavobrunneus are found in who referred to non-phytotelm-breeding populations) three different portions of our tree (Fig. 1). Two specimens strongly group together (clade 2 in Fig. 1), other speci- (one from the Ankaratra Mountains and one from the mens attributed to this species by morphological similar- Mangoro River region) are the sister taxon to the highland ity are strongly genetically differentiated and occur in forms of G. pulcher (clade 1). Two other specimens attrib- relatively distant portions of the tree. Specimens that uted to G. flavobrunneus (from the area in and around we attributed to typical G. liber from clade 2 based on Andringitra) are the sister taxon to the lowland G. pulcher

Table 2 Summary of partitioned and total evidence phylogenetic analyses Branch supported MP (total) ML (total) B (total) MP (12S) ML (12S) B (12S) MP (16S) ML (16S) B (16S) 1. G. (?) bicalcaratus from Maharira and + (96) + (86) + (1.00) + (97) + (90) + (1.00) + (74) + (73) + (0.78) Vohiparara 2. Pandanusicola + G. liber + (100) + (91) + (1.00) + (100) + (89) + (1.00) + (75) + (63) + (0.80) 3. G. cf. liber from Andringitra, Besariaka + (100) + (100) + (1.00) + (100) + (99) + (1.00) + (100) + (100) + (1.00) and Vevembe 4. Pandanusicola excluding G. cf. liber (À)(À) + (1.00) (À)(À) À (À)(À)(À) from Andringitra, Besariaka and Vevembe 5. G. liber in clade 2 of Fig. 1 + (100) + (100) + (1.00) (À)(À) + (0.89) + (100) + (99) + (0.95) 6. G. liber in clade 2 of Fig. 1 + (À)(À) + (0.89) (À)(À) + (0.92) (À)(À) + (0.57) G. punctatus and G. bicalcaratus from Sainte Luce 7. G. bicalcaratus from Sainte Luce + (À)(À) + (0.62) (À)(À)(À)(À)(À) + (0.97) G. punctatus 8. G. pulcher in clade 1 of Fig. 1 + (100) + (98) + (1.00) + (92) + (76) + (1.00) + (100) + (97) + (1.00) 9. G. pulcher in clade 1 of Fig. 1 + + (100) + (97) + (1.00) + (75) + (78) + (1.00) + (97) + (84) + (1.00) G. ﬂavobrunneus from Ankaratra and Mangoro River region 10. G. cf. pulcher from Sainte Luce + + (73) + (68) + (1.00) + (89) + (88) + (1.00) + (93) + (94) + (1.00) G. ﬂavobrunneus from Andrigitra and Manombo (= clade 4 of Fig. 1) 11. G. cf. liber in clade 3 of Fig. 1 + (100) + (100) + (1.00) + (70) + (85) + (1.00) + (100) + (100) + (1.00) 12. G. bicalcaratus in clade 5 of Fig. 1 + (54) + (59) + (1.00) + (68) (À) + (1.00) + (67) + (54) + (0.99) ML, maximum likelihood; MP, maximum parsimony; B, Bayesian; +, topology was supported; À, topology not supported; (À), topology not resolved. Numbers in parentheses are bootstrap support values (MP and ML) and Bayesian posterior probabilities (only given if >50%). Node designations follow Fig. 1. R.M. Lehtinen et al. / Molecular Phylogenetics and Evolution 44 (2007) 1121–1129 1125

Fig. 1. Fifty percent majority rule consensus phylogram of Bayesian analysis of concatenated 12S and 16S mitochondrial genes (1.8 kb). Posterior probabilities are given to indicate the level of support for each node. Collection localities are given after each taxon name with letters that refer to those used in Fig. 2 and Table 1. Numbers refer to clades as used in the text. All ingroup taxa are currently in the subgenus Pandanusicola and all outgroup taxa are in the subgenus Guibemantis. in clade 4 (Fig. 1). The last specimen attributed to G. mens from Maharira and Vohiparara group outside subge- ﬂavobrunneus was collected in the central highlands nus Pandanusicola altogether (average pairwise sequence (Ambohitantely) and is grouped with a specimen attributed divergence 16%; Table 3). to G. bicalcaratus from Manongarivo (northwestern The single specimen attributed to G. albolineatus from Madagascar). Andasibe groups with clade 4 and the clade comprising Specimens attributed to G. bicalcaratus occur in three G. bicalcaratus (Manongarivo) and G. ﬂavobrunneus portions of the tree, including the specimen mentioned (Ambohitantely). There is modest support for a sister- above. Four G. bicalcaratus specimens from northern and group relationship between G. punctatus + G. bicalcaratus central Madagascar group together in clade 5. Two other from Sainte Luce and the G. liber in clade 2, primarily from G. bicalcaratus specimens (both from Sainte Luce in coastal the Bayesian analyses (Fig. 1; Table 2). Several other nodes southeastern Madagascar) are the sister taxon to G. punct- are supported in the Bayesian analyses that are not atus although this topology is not well supported (Fig. 1; resolved in the parsimony and likelihood reconstructions, Table 2). Lastly, in all analyses the G. bicalcaratus speci- which tended to be more conservative (see Table 2). 1126 R.M. Lehtinen et al. / Molecular Phylogenetics and Evolution 44 (2007) 1121–1129

voucher specimens, it is beyond doubt that the taxa in clade 2(Fig. 1) represent typical non-phytotelm-breeding G. liber (sensu Blommers-Schlo¨sser, 1975, 1979; Blommers-Schlo¨s- ser and Blanc, 1991; Glaw and Vences, 1994). A larger DNA barcoding survey of Malagasy frogs based on a short fragment of the 16S rRNA gene (Vences et al., 2005a) also provided evidence that populations from central eastern Madagascar (Mandraka, Andasibe) studied by Blom- mers-Schlo¨sser (1975, 1979) have haplotypes belonging to this clade (Genbank Accession Numbers AY684187, AY848079–AY848082). Therefore, based on the nesting of clade 2 within a group of phytotelm-breeders (e.g., clades 1, 5 and G. bicalcaratus and G. punctatus from Sainte Luce; Fig. 1), we can strongly confirm that non-phytotelm- breeding in G. liber constitutes a reversal from specialized reproduction in leaf axils. Also remarkable is clade 6; these frogs are morphologically similar to G. liber but have a greenish color reminis- Fig. 2. Map of Madagascar with collection localities indicated. Letters cent of G. pulcher. We have previously collected refer to specific place names indicated in Table 1. Clade numbers refer to individuals of this form from other localities in the south- those presented in the phylogram in Fig. 1. east. It has a very different advertisement call and clearly represents a separate species. Field observations indicate Generally, the most nested portions of the tree are well that this species breeds in swamps rather than in leaf axils, resolved in most reconstructions, but many of the more although this inference is so far based only on calling basal nodes in our tree are not well supported (bootstrap behavior, as eggs or tadpoles have not yet been identified values less than 70%; posterior probabilities less than (M. Vences, personal observation). If confirmed (and if this 95%; Table 2). species is the sister taxon to other Pandanusicola as our Bayesian tree indicates; Fig. 2) this would suggest that 4. Discussion pond-breeding is the ancestral reproductive mode in this group. A recent analysis of the molecular phylogenetic relation- As with G. liber, our tree indicates that G. bicalcaratus, ships within the subgenus Guibemantis placed G. liber as G. flavobrunneus, and G. pulcher are also non-monophyletic the sister taxon to this clade (Vences and Glaw, 2005). (Fig. 1). This again suggests that very probably unde- However, this analysis did not include any members of scribed, cryptic species masquerade under these names. Pandanusicola, and the authors suggested that the place- For example, specimens assigned to G. pulcher occur in ment of G. liber needs to be confirmed. Our analysis (the three distinct parts of the tree with high support. The G. first to include multiple members of both subgenera Guibe- pulcher specimens from Sainte Luce (a coastal site in south- mantis and Pandanusicola) confirms that G. liber is part of eastern Madagascar) are morphologically and acoustically Pandanusicola and not subgenus Guibemantis, as reflected distinct from G. pulcher from highland areas (R.M.L., in the most recent classification (Glaw and Vences, 2006). unpublished data), are genetically divergent and likely rep- Natural history studies indicate that G. liber is a pond- resent an undescribed species. Similarly, specimens cur- breeding species, with normal pond-type tadpoles, a rently assigned to G. bicalcaratus and G. flavobrunneus relatively large clutch size, and no parental care appear in a number of areas of the tree and may represent (Blommers-Schlo¨sser, 1975; Vejarano et al., 2006). As far cryptic lineages deserving species status. as it is known, all other members of Pandanusicola lay their Interestingly, the G. bicalcaratus from Maharira and relatively small clutches of eggs in plant-held water bodies Vohiparara group outside Pandanusicola altogether. A (phytotelmata), have specialized tadpoles for surviving in comprehensive phylogenetic analysis of over 1500 mantel- these small pools (Blommers-Schlo¨sser, 1979; Glaw and lid sequences for a fragment of the 16S rRNA gene (Vences Vences, 1994; Lehtinen, 2004), and at least some have et al., 2005b, unpublished data) indicate that this popula- parental care (Lehtinen, 2003). Using only a single repre- tion may belong to the genus Spinomantis. Any labeling sentative of each putative species in Pandanusicola (includ- error can be excluded since multiple 16S rRNA sequences ing G. liber), Lehtinen et al. (2004) suggested that these of this population are available (Genbank Accession Num- traits in G. liber may represent a reversal to the ancestral bers AY848381–AY848386). The population from Mahar- reproductive mode (see also Lehtinen and Nussbaum, ira consists of small frogs living in Pandanus, the males 2003). In the present study, specimens assigned to G. liber bearing femoral glands typical for Pandanusicola. They occur in three distinct portions of the tree with strong are rather common in Ranomafana National Park (Mahar- support. However, from field observations linked to the ira being just one locality within the park where the species Table 3

Pairwise sequence diﬀerences among sampled taxa in the subgenus Pandanusicola (corrected for gaps and ambiguities) 1121–1129 (2007) 44 Evolution and Phylogenetics Molecular / al. et Lehtinen R.M.

albo bic bic bic bic bic bic bic bic flav flav flav flav flav flav liber liber liber liber liber liber liber liber pulch pulch pulch pulch pulch pulch pulch pulch pulch punct punct 250 444 745 592 597 932 044 588 485 923 119 929 922 919 655 064 117 644 068 101 070 093 116 262 132 265 277 242 415 317 58 314 290 265 albo 250 — 0.1612 0.1622 0.1020 0.0909 0.0928 0.0931 0.0877 0.0811 0.0790 0.0821 0.0933 0.0938 0.0880 0.1174 0.0837 0.0756 0.0831 0.0821 0.0877 0.097 0.0837 0.0895 0.0815 0.0810 0.0724 0.0724 0.0763 0.0746 0.0747 0.0767 0.0875 0.0883 0.0889 bic 444 0.1612 — 0.1728 0.1741 0.1862 0.1603 0.1609 0.1544 0.1644 0.1739 0.1661 0.1674 0.1678 0.1810 0.1690 0.1310 0.1641 0.1590 0.1642 0.1755 0.1670 0.1817 0.1586 0.1595 0.1490 0.1492 0.1656 0.1609 0.1517 0.1567 0.1604 0.1607 0.1603 bic 745 0.1622 0.0000 — 0.1748 0.1752 0.1627 0.1608 0.1632 0.1589 0.1649 0.1580 0.1618 0.1702 0.1695 0.1863 0.1684 0.1549 0.1549 0.1613 0.1666 0.1713 0.1669 0.1633 0.1628 0.1641 0.1504 0.1506 0.1510 0.1504 0.1571 0.1510 0.1628 0.1643 0.1627 bic 592 0.1020 0.1728 0.1748 — 0.1143 0.1396 0.1056 0.1081 0.1030 0.0897 0.1172 0.0899 0.1165 0.1261 0.1214 0.1025 0.0963 0.1141 0.0977 0.0950 0.1247 0.1082 0.1133 0.1010 0.1005 0.1002 0.1021 0.1232 0.1239 0.1033 0.1043 0.1079 0.1056 0.1059 bic 597 0.0909 0.1741 0.1752 0.1143 — 0.0871 0.0729 0.0895 0.0834 0.0901 0.0912 0.1110 0.0774 0.0931 0.1327 0.0878 0.0811 0.0828 0.0877 0.0914 0.1058 0.0880 0.0989 0.0920 0.0927 0.0836 0.0836 0.0831 0.0819 0.0813 0.0858 0.0947 0.0949 0.0943 bic 932 0.0928 0.1862 0.1627 0.1396 0.0871 — 0.0875 0.1167 0.1050 0.1017 0.0845 0.0901 0.1157 0.0957 0.1597 0.0883 0.0759 0.0858 0.0888 0.0943 0.1169 0.0980 0.1581 0.0826 0.0816 0.0841 0.0848 0.0956 0.0948 0.0910 0.1133 0.1056 0.0994 0.0995 bic 044 0.0931 0.1603 0.1608 0.1056 0.0729 0.0875 — 0.0756 0.0748 0.0844 0.0965 0.1038 0.0807 0.0934 0.1233 0.0829 0.0773 0.0848 0.0810 0.0841 0.0933 0.0822 0.0933 0.0789 0.0795 0.0822 0.0831 0.0936 0.0923 0.0821 0.0838 0.0860 0.0862 0.0868 bic 588 0.0877 0.1609 0.1632 0.1081 0.0895 0.1167 0.0756 — 0.0093 0.0822 0.0995 0.0981 0.0926 0.0863 0.1113 0.0742 0.0728 0.0743 0.0715 0.0763 0.0937 0.0745 0.0936 0.0801 0.0808 0.0694 0.0702 0.0767 0.0747 0.0742 0.0808 0.0852 0.0721 0.0727 bic 485 0.0811 0.1544 0.1589 0.1030 0.0834 0.1050 0.0748 0.0093 — 0.0779 0.0919 0.0938 0.0894 0.0820 0.1069 0.0697 0.0673 0.0689 0.0687 0.0725 0.0905 0.0714 0.0832 0.0771 0.0777 0.0637 0.0638 0.0685 0.0678 0.0675 0.0691 0.0804 0.0689 0.0695 flav 923 0.0790 0.1644 0.1649 0.0897 0.0901 0.1017 0.0844 0.0822 0.0779 — 0.0821 0.0885 0.0941 0.0913 0.1212 0.0789 0.0736 0.0780 0.0725 0.0764 0.0953 0.0850 0.1007 0.0761 0.0749 0.0758 0.0765 0.0759 0.0743 0.0785 0.0768 0.0934 0.0833 0.0816 flav 119 0.0821 0.1739 0.1580 0.1172 0.0912 0.0845 0.0965 0.0995 0.0919 0.0821 — 0.0299 0.1219 0.0852 0.1444 0.0778 0.0618 0.0729 0.0769 0.0816 0.1082 0.0836 0.1286 0.0385 0.0375 0.0563 0.0569 0.0776 0.0788 0.0758 0.0789 0.0915 0.0981 0.0974 flav 929 0.0933 0.1661 0.1618 0.0899 0.1110 0.0901 0.1038 0.0981 0.0938 0.0885 0.0299 — 0.1198 0.0856 0.1020 0.1012 0.0974 0.0612 0.0957 0.098 0.1111 0.0964 0.1110 0.0862 0.0881 0.0952 0.0965 0.0694 0.0656 0.0974 0.0970 0.1036 0.0981 0.0990 flav 922 0.0938 0.1674 0.1702 0.1165 0.0774 0.1157 0.0807 0.0926 0.0894 0.0941 0.1219 0.1198 — 0.1095 0.1269 0.0943 0.0800 0.1004 0.0903 0.0951 0.1022 0.0906 0.1141 0.0966 0.0974 0.0921 0.0922 0.1125 0.1114 0.0944 0.0950 0.1084 0.0978 0.0973 flav 919 0.0880 0.1678 0.1695 0.1261 0.0931 0.0957 0.0934 0.0863 0.0820 0.0913 0.0852 0.0856 0.1095 — 0.0757 0.0723 0.0638 0.0703 0.0753 0.0801 0.1056 0.0814 0.0850 0.0934 0.0925 0.0494 0.0493 0.0514 0.0504 0.0481 0.0518 0.0901 0.0852 0.0836 flav 655 0.1174 0.1810 0.1863 0.1214 0.1327 0.1597 0.1233 0.1113 0.1069 0.1212 0.1444 0.1020 0.1269 0.0757 — 0.1168 0.0719 0.1316 0.1130 0.1128 0.1314 0.1095 0.1198 0.1184 0.1193 0.0823 0.0830 0.1108 0.1097 0.0886 0.0879 0.1238 0.1146 0.1135 liber 064 0.0837 0.1690 0.1684 0.1025 0.0878 0.0883 0.0829 0.0742 0.0697 0.0789 0.0778 0.1012 0.0943 0.0723 0.1168 — 0.0064 0.0092 0.0256 0.0274 0.0945 0.0728 0.0916 0.0817 0.0831 0.0662 0.0662 0.0625 0.0625 0.0675 0.0691 0.0869 0.0714 0.0714 liber 117 0.0756 0.1310 0.1549 0.0963 0.0811 0.0759 0.0773 0.0728 0.0673 0.0736 0.0618 0.0974 0.0800 0.0638 0.0719 0.0064 — 0.0095 0.0278 0.0338 0.0916 0.0710 0.0807 0.0798 0.0814 0.0607 0.0615 0.0572 0.0569 0.0637 0.0635 0.0782 0.0659 0.0644 liber 644 0.0831 0.1641 0.1549 0.1141 0.0828 0.0858 0.0848 0.0743 0.0689 0.0780 0.0729 0.0612 0.1004 0.0703 0.1316 0.0092 0.0095 — 0.0271 0.0312 0.0997 0.0671 0.1143 0.0724 0.0723 0.0624 0.0632 0.0623 0.0618 0.0622 0.0635 0.0768 0.0627 0.0628 liber 068 0.0821 0.1590 0.1613 0.0977 0.0877 0.0888 0.0810 0.0715 0.0687 0.0725 0.0769 0.0957 0.0903 0.0753 0.1130 0.0256 0.0278 0.0271 — 0.0206 0.0902 0.0745 0.0887 0.0816 0.0822 0.0666 0.0674 0.0619 0.0612 0.0657 0.0683 0.0828 0.0682 0.0682 liber 101 0.0877 0.1642 0.1666 0.0950 0.0914 0.0943 0.0841 0.0763 0.0725 0.0764 0.0816 0.098 0.0951 0.0801 0.1128 0.0274 0.0338 0.0312 0.0206 — 0.0928 0.0753 0.0905 0.0852 0.0865 0.0669 0.0683 0.0642 0.0643 0.0671 0.0685 0.0870 0.0714 0.0714 liber 070 0.097 0.1755 0.1713 0.1247 0.1058 0.1169 0.0933 0.0937 0.0905 0.0953 0.1082 0.1111 0.1022 0.1056 0.1314 0.0945 0.0916 0.0997 0.0902 0.0928 — 0.0577 0.1078 0.1071 0.1075 0.0773 0.0788 0.0992 0.1005 0.0884 0.0906 0.1032 0.1018 0.1013 liber 093 0.0837 0.1670 0.1669 0.1082 0.0880 0.0980 0.0822 0.0745 0.0714 0.0850 0.0836 0.0964 0.0906 0.0814 0.1095 0.0728 0.0710 0.0671 0.0745 0.0753 0.0577 — 0.0920 0.0892 0.0899 0.0682 0.0682 0.0733 0.0717 0.0721 0.0746 0.0854 0.0832 0.0832 liber 116 0.0895 0.1817 0.1633 0.1133 0.0989 0.1581 0.0933 0.0936 0.0832 0.1007 0.1286 0.1110 0.1141 0.0850 0.1198 0.0916 0.0807 0.1143 0.0887 0.0905 0.1078 0.0920 — 0.0961 0.0958 0.0809 0.0824 0.1179 0.0975 0.1001 0.1232 0.0249 0.0915 0.0915 pulch 262 0.0815 0.1586 0.1628 0.1010 0.0920 0.0826 0.0789 0.0801 0.0771 0.0761 0.0385 0.0862 0.0966 0.0934 0.1184 0.0817 0.0798 0.0724 0.0816 0.0852 0.1071 0.0892 0.0961 — 0.0034 0.0781 0.0795 0.0808 0.0802 0.0782 0.0782 0.0917 0.0922 0.0923 pulch 132 0.0810 0.1595 0.1641 0.1005 0.0927 0.0816 0.0795 0.0808 0.0777 0.0749 0.0375 0.0881 0.0974 0.0925 0.1193 0.0831 0.0814 0.0723 0.0822 0.0865 0.1075 0.0899 0.0958 0.0034 — 0.0793 0.0807 0.0809 0.0793 0.0789 0.0789 0.0914 0.0911 0.0912 pulch 265 0.0724 0.1490 0.1504 0.1002 0.0836 0.0841 0.0822 0.0694 0.0637 0.0758 0.0563 0.0952 0.0921 0.0494 0.0823 0.0662 0.0607 0.0624 0.0666 0.0669 0.0773 0.0682 0.0809 0.0781 0.0793 — 0.0011 0.0011 0.0000 0.0127 0.008 0.0813 0.0688 0.0689 pulch 277 0.0724 0.1492 0.1506 0.1021 0.0836 0.0848 0.0831 0.0702 0.0638 0.0765 0.0569 0.0965 0.0922 0.0493 0.0830 0.0662 0.0615 0.0632 0.0674 0.0683 0.0788 0.0682 0.0824 0.0795 0.0807 0.0011 — 0.0023 0.0011 0.0133 0.0092 0.0820 0.0696 0.0697 pulch 242 0.0763 0.1656 0.1510 0.1232 0.0831 0.0956 0.0936 0.0767 0.0685 0.0759 0.0776 0.0694 0.1125 0.0514 0.1108 0.0625 0.0572 0.0623 0.0619 0.0642 0.0992 0.0733 0.1179 0.0808 0.0809 0.0011 0.0023 — 0.0031 0.0152 0.0162 0.0829 0.0717 0.0700 pulch 415 0.0746 0.1609 0.1504 0.1239 0.0819 0.0948 0.0923 0.0747 0.0678 0.0743 0.0788 0.0656 0.1114 0.0504 0.1097 0.0625 0.0569 0.0618 0.0612 0.0643 0.1005 0.0717 0.0975 0.0802 0.0793 0.0000 0.0011 0.0031 — 0.0157 0.0133 0.0831 0.0710 0.0693 pulch 317 0.0747 0.1517 0.1571 0.1033 0.0813 0.0910 0.0821 0.0742 0.0675 0.0785 0.0758 0.0974 0.0944 0.0481 0.0886 0.0675 0.0637 0.0622 0.0657 0.0671 0.0884 0.0721 0.1001 0.0782 0.0789 0.0127 0.0133 0.0152 0.0157 — 0.0068 0.0803 0.0740 0.0727 pulch 58 0.0767 0.1567 0.1510 0.1043 0.0858 0.1133 0.0838 0.0808 0.0691 0.0768 0.0789 0.0970 0.0950 0.0518 0.0879 0.0691 0.0635 0.0635 0.0683 0.0685 0.0906 0.0746 0.1232 0.0782 0.0789 0.008 0.0092 0.0162 0.0133 0.0068 — 0.0815 0.0729 0.0717 pulch 314 0.0875 0.1604 0.1628 0.1079 0.0947 0.1056 0.0860 0.0852 0.0804 0.0934 0.0915 0.1036 0.1084 0.0901 0.1238 0.0869 0.0782 0.0768 0.0828 0.0870 0.1032 0.0854 0.0249 0.0917 0.0914 0.0813 0.0820 0.0829 0.0831 0.0803 0.0815 — 0.0922 0.0928 punct 290 0.0883 0.1607 0.1643 0.1056 0.0949 0.0994 0.0862 0.0721 0.0689 0.0833 0.0981 0.0981 0.0978 0.0852 0.1146 0.0714 0.0659 0.0627 0.0682 0.0714 0.1018 0.0832 0.0915 0.0922 0.0911 0.0688 0.0696 0.0717 0.0710 0.0740 0.0729 0.0922 — 0.0017 punct 265 0.0889 0.1603 0.1627 0.1059 0.0943 0.0995 0.0868 0.0727 0.0695 0.0816 0.0974 0.0990 0.0973 0.0836 0.1135 0.0714 0.0644 0.0628 0.0682 0.0714 0.1013 0.0832 0.0915 0.0923 0.0912 0.0689 0.0697 0.0700 0.0693 0.0727 0.0717 0.0928 0.0017 — 1127 1128 R.M. Lehtinen et al. / Molecular Phylogenetics and Evolution 44 (2007) 1121–1129 occurs). It is unknown if these frogs use Pandanus plants additional geographic sampling and detailed morphologi- for breeding or just as shelter; more fieldwork is necessary cal and acoustic studies (Vences et al., 2005a). There is a to understand the reproductive mode of this species, which possibility that we amplified non-functional nuclear copies has acquired phytotelm-dwelling habits and possibly phy- (Numts) of these mitochondrial genes (Sorenson and Flei- totelm-breeding independently from Pandanusicola. scher, 1996). However, these transposed sequences are Many of the clades revealed in these analyses are apparently less common than initially thought and most concordant with geography (Fig. 2), suggesting that they often involve the mitochondrial control region and the diagnose population lineages rather than representing cytochrome b gene (Pereira and Baker, 2004). incomplete lineage sorting or introgression of haplotypes. Our data support two major conclusions. First, even For example, all of the central highland forms assigned though it has a different mode of reproduction, G. liber is to G. pulcher group together in clade 1 (Figs. 1 and 2), part of the Pandanusicola lineage, confirming recent taxon- and clade 4 represents a number of related forms from omy and strongly suggesting a reversal of its reproductive the southeastern part of the island. Similarly, clade 3 mode. Second, most of the described species in Pandanusi- (Fig. 1) tightly clusters geographically in the northeast cola consist of two or more genetically well differentiated, (Fig. 2). Other clades, however, are less geographically but morphologically cryptic, allopatric lineages that may concordant. For example, four of five haplotypes deserve recognition at the species level. assigned to G. liber in clade 2 are from northwestern Madagascar, while the remaining specimen is geographi- Acknowledgments cally distant, in the southeast (Figs. 1 and 2), confirming that this taxon (the typical G. liber) occurs over a wide Thanks to D. Lightle, J. Baker (COW), and B. Caudle range in Madagascar with slight to moderate regional (UT) for help with laboratory work, and Frank Glaw differentiation. (Zoologische Staatssammlung Mu¨nchen) for samples The 12S and 16S mitochondrial genes have been used and discussion. We also thank Y. Zhang and T. Meulia repeatedly in molecular systematic studies of amphibians. at the Molecular and Cellular Imaging Center at the Ohio Hertwig et al. (2004) asserted that these genes may not be Agricultural Research and Development Center and D. well suited for assessing deep splitting events. While the Fraga and W. Morgan at the College of Wooster for younger portions of our tree were well resolved, the more assistance with molecular protocols. Fieldwork in Mada- basal portions lack robust support from at least some gascar was made possible through collaboration accords methods of analysis (Table 1). This may be due to rapid with the De´partement de Biologie Animale, Universite´ diversification in this lineage or the decreased ability of d’Antananarivo and the Association Nationale pour la the 12S and 16S genes to discern deep divergences. Better Gestion des Aires Protegeés (ANGAP). We are grateful taxon sampling and additional data, using nuclear genes to the Malagasy authorities for issuing research and ex- or other characters, will likely help to resolve the deepest port permits. This project was funded through grants to nodes in this group. As others have suggested, multiple R.M.L. from the William H. Wilson Fund and Sopho- sources of data are likely to provide the most robust esti- more Research program at the College of Wooster. We mates of evolutionary history (Rubinoff and Holland, would like to dedicate this paper to the memory of Chris 2005). Richards, who passed away while this manuscript was in For intra-familial and intra-generic assessment, how- press. ever, these genes appear to function well and have even been suggested as bar codes for amphibians (Vences References et al., 2005b). However, other recent studies have revealed surprisingly high estimates of intra-specific variation in Andreone, F., 2003. Mantellidae: Mantidactylus. In: Goodman, S.M., mitochondrial genes in amphibians from both temperate Benstead, J.P. (Eds.), The Natural History of Madagascar. The and tropical areas (e.g., Austin et al., 2002; Camargo University of Chicago Press, Chicago, pp. 910–913. Andreone, F., Vences, M., Vieites, D.R., Glaw, F., Meyer, A., 2005. et al., 2006; Lougheed et al., 1999). For example, Vences Recurrent ecological adaptations revealed through a molecular anal- et al. (2005a) found high levels of intra-population nucleo- ysis of the secretive cophyline frogs of Madagascar. Molecular tide diversity (up to 14%) in some mantellids. However, the Phylogenetics and Evolution 34, 315–322. cytochrome oxidase I gene used there is known to evolve Austin, J.D., Lougheed, S.C., Neidrauer, L., Chek, A.A., Boag, P.T., faster than the rRNA genes used in the present study. 2002. Cryptic lineages in a small frog: the post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Molecular Phy- While mitochondrial genes may be particularly well suited logenetics and Evolution 25, 316–329. to delimiting species, especially when concordance with Blommers-Schlo¨sser, R.M.A., 1975. A unique case of mating behaviour in other data sets is found (Wiens and Penkrot, 2002), many a Malagasy tree frog, Gephyromantis liber (Peracca, 1893), with believe that strong mitochondrial sequence divergence observations on the larval development (Amphibia, Ranidae). Beau- should not be used as the sole determinant for species diag- fortia 23, 15–25. Blommers-Schlo¨sser, R.M.A., 1979. Biosystematics of the Malagasy frogs. nosis (e.g., Funk and Omland, 2003). Nevertheless, mito- I. Mantellinae (Ranidae). Beaufortia 29, 1–77. chondrial divergences can be an excellent starting point Blommers-Schlo¨sser, R.M.A., Blanc, C.P., 1991. Amphibiens (premie`re to delimit candidate species that may then be the focus of partie). Faune de Madagascar 75, 176–192 (363). R.M. Lehtinen et al. / Molecular Phylogenetics and Evolution 44 (2007) 1121–1129 1129

Camargo, A., De Sa, R.O., Heyer, W.R., 2006. Phylogenetic analyses of diversification using a dart-poison frog (Epipedobates femoralis). mtDNA sequences reveal three cryptic lineages in the widespread Proceedings of the Royal Society of London Series B—Biological Neotropical frog Leptodactylus fuscus (Schneider, 1799) (Anura, Sciences 266, 1829–1835. Leptodactylidae). Biological Journal of the Linnean Society 87, 325– Pereira, S.L., Baker, A.J., 2004. Low number of mitochondrial pseudo- 341. genes in the chicken (Gallus gallus) nuclear genome: implications for Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Haddad, molecular inference of population history and phylogenetics. BMC C.F.B., De Sa, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Evolutionary Biology 4, 17–24. Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., substitution. Bioinformatics 14, 817–818. 2006. The amphibian tree of life. Bulletin of the American Museum of Richards, C.M., Nussbaum, R.A., Raxworthy, C.J., 2000. Phylogenetic Natural History 297, 370 pp. relationships within the Madagascan boophids and mantellids as Funk, D.J., Omland, K.E., 2003. Species-level paraphyly and polyphyly: elucidated by mitochondrial ribosomal genes. African Journal of frequency, causes, and consequences, with insights from animal Herpetology 49, 23–32. mitochondrial DNA. Annual Review of Ecology Evolution and Rubinoff, D., Holland, B.S., 2005. Between two extremes: mitochondrial Systematics 34, 397–423. DNA is neither panacea nor the nemesis of phylogenetic and Glaw, F., Vences, M., 1994. A Fieldguide to the Amphibians and Reptiles taxonomic inference. Systematic Biology 54, 952–961. of Madagascar. Vences and Glaw Verlag, Cologne. Sorenson, M.D., Fleischer, R.C., 1996. Multiple independent transposi- Glaw, F., Vences, M., 2003. Introduction to Amphibians. In: Goodman, tions of mitochondrial DNA control region sequences to the nucleus. S.M., Benstead, J.P. (Eds.), The Natural History of Madagascar. The Proceedings of the National Academy of Sciences USA 93, 15239– University of Chicago Press, Chicago, pp. 883–898. 15243. Glaw, F., Vences, M., 2006. Phylogeny and genus-level classification of Swofford, D.L., 1998. PAUP*. Phylogenetic Analysis using Parsimony mantellid frogs (Amphibia, Anura). Organisms Diversity & Evolution (*and Other Methods). Sinauer Associates, Sunderland, MA. 6, 236–253. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, Hertwig, S., de Sa, R.O., Haas, A., 2004. Phylogenetic signal and the D.G., 1997. The ClustalX windows interface: flexible strategies for utility of 12S and 16S mtDNA in frog phylogeny. Journal of multiple sequence alignment aided by quality analysis tools. Nucleic Zoological Systematics and Evolutionary Research 42, 2–18. Acids Research 24, 4876–4882. Hillis, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a Vejarano, S., Thomas, M., Vences, M., 2006. Comparative larval method for assessing confidence in phylogenetic analysis. Systematic morphology in Madagascan frogs of the genus Guibemantis (Anura: Biology 42, 182–192. Mantellidae). Zootaxa 1329, 39–57. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of Vences, M., Glaw, F., 2001. Systematic review and molecular phylogenetic phylogeny. Bioinformatics 17, 754–755. relationships of the direct developing Malagasy anurans of the Lehtinen, R.M., 2002. The use of screw pines (Pandanus spp.) by Mantidactylus asper group (Amphibia, Mantellidae). Alytes (Paris) amphibians and reptiles in Madagascar. Herpetological Bulletin 82, 19, 107–139. 20–25. Vences, M., Glaw, F., 2005. A new species of Mantidactylus from the east Lehtinen, R.M., 2003. Parental care and reproduction in two species of coast of Madagascar and its molecular phylogenetic relationships Mantidactylus (Anura: Mantellidae). Journal of Herpetology 37, 766– within the subgenus Guibemantis. Herpetological Journal 15, 37–44. 768. Vences, M., Raxworthy, C.J., Nussbaum, R.A., Glaw, F., 2003. A revision Lehtinen, R.M., 2004. Tests for competition, cannibalism, and priority of the Scaphiophryne marmorata complex of marbled toads from effects in two phytotelm-dwelling tadpoles from Madagascar. Herpe- Madagascar, including the description of a new species. Herpetological tologica 60, 1–13. Journal 13, 69–79. Lehtinen, R.M., Nussbaum, R.A., 2003. Parental care: a phylogenetic Vences, M., Thomas, M., Bonett, R.M., Vieites, D.R., 2005a. Deciphering perspective. In: Jamieson, B.G.M. (Ed.), Reproductive Biology and amphibian diversity through DNA barcoding: chances and challenges. Phylogeny of Anura. Science Publishers, Inc., Enfield, NH, pp. 343–387. Philosophical Transactions of the Royal Society B—Biological Sci- Lehtinen, R.M., Richards, C.M., Nussbaum, R.A., 2004. Origin of a ences 360, 1859–1868. complex reproductive trait: phytotelm breeding in Mantelline frogs. In: Vences, M., Thomas, M., Van der Meijden, A., Chiari, Y., Vieites, D.R., Lehtinen, R.M. (Ed.), Ecology and Evolution of Phytotelm-Breeding 2005b. Comparative performance of the 16S rRNA gene in DNA Anurans. Museum of Zoology, University of Michigan, Ann Arbor, barcoding of amphibians. Frontiers in Zoology 2, 1–12. pp. 45–54. Wiens, J.J., Penkrot, T.A., 2002. Delimiting species using DNA and Lougheed, S.C., Gascon, C., Jones, D.A., Bogart, J.P., Boag, P.T., 1999. morphological variation and discordant species limits in spiny lizards Ridges and rivers: a test of competing hypotheses of Amazonian (Sceloporus). Systematic Biology 51, 69–91.