Phylogenetic resolution and systematics of the Asian tree frogs,Rhacophorus (Rhacophoridae,Amphibia)

Li, J.T., Li, Y., Murphy, R.W., Rao, D.‐Q. & Zhang, Y.‐P. (2012). Phylogenetic resolution and systematics of the Asian tree frogs, Rhacophorus (Rhacophoridae, Amphibia). —Zoologica Scripta, 41, 557–570. The treefrog genus Rhacophorus, a large genus with 80 species, has a wide range, occurring eastward from India to China, Japan, South‐east Asia, the Greater Sunda Islands and the Philippines. The phylogenetic relationships and taxonomic recognition of many species are very controversial. To stabilize the taxonomy, the phylogenetic relationships among about 52 species are investigated from 96 samples using mtDNA sequence data. Matrilineal relationships based on maximum likelihood and Bayesian inference methods resolve three well‐supported lineages (A, B and C), although the phylogenetic relationships among three lineages remain ambiguous. Analyses support recognition of two previously assigned subgenera, Leptomantis and Rhacophorus, and these correspond to lineages A and B, respectively. Given that we have three strongly supported lineages, that these lineages are morphologically distinct, and the constrained geographic distributions of these groups, we recognize each lineage as a taxon. Subgenus Leptomantis includes species mainly from Malaysia, Indonesia and the Philippines. Subgenus Rhacophorus contains a mix of species occurring in India, Indochina and southern China. Lineage C accommodates species distributed mostly in East Asia, including Japan and China. Based on genetic and morphological data from type localities, the taxonomic recognition of some species needs to be reconsidered. Rhacophorus pingbianensis and Polypedates spinus are considered as junior synonyms of Rhacophorus duboisi. Specimens of Rhacophorus rhodopus from Vietnam and Hainan, China likely represent an undescribed, cryptic species.     

Evolution of reproduction in the Rhacophoridae (Amphibia, Anura)

Rhacophorid treefrogs have different reproductive modes: some go through a tadpole stage and some have direct development, and the adults of some species produce foam nests. Philautus is the only genus characterized by direct development. The production of foam nests has been reported in the genera Polypedates, Rhacophorus, Chiromantis and Chirixalus. Recent molecular studies did not provide a robust hypothesis concerning the origin of these reproductive modes in the Rhacophoridae. In order to better understand the evolution of these reproductive modes, we tried to clarify relationships within this group, using DNA sequencing. Our data set consists in a large number of new sequences (1676 base pairs corresponding to threee genes) for five outgroup ranoids and 48 Rhacophoridae, including 16 undescribed species from Sri Lanka and southern India, and all homologous data available in Genbank. After the inclusion of Philautus from India, our data show that the separation of Philautus into clades does not coincide with their geographic distribution. Our data point to the existence of a clade, including the genera Rhacophorus, Polypedates, Chiromantis and Chirixalus, which confirms the results of Wilkinson et al. (2002) and suggests that the ability to produce foam nests has emerged only once in the Rhacophoridae, as already stated by these authors.     

A subordinal classification of frogs (Amphibia: Anura)

Two new anuran suborders, based on two states of the trigeminofacial ganglion character complex are proposed. A subsidiary character is the presence or absence of free ribs in extant taxa. These new suborders are more clades (sister groups) than sequential levels of organization. Discoglossoidei, retaining separate trigeminal and facial ganglia and free ribs, encompasses only Leiopelmatidae and Discoglossidae, although by definition it would include the common ancestor of both lineages. Ranoidei have the trigeminal and facial ganglia fused and extant taxa lack free ribs. This group includes all other frogs.
Only the superfamiliesPelobatoidea and Pipoidea are reallocated by the new arrangement. The former are now regarded as representing the ranoidean stem group. Both laival and adult morphology show that pipoids are highly derived rather than primitive frogs, and their trigeminofacial systems show that they are ranoideans rather than discoglossoideans. They presumably are ultimately derived from pelobatoids, but the known taxa are too specialized for direct derivation and there must have been an intermediate group with pipoid tadpoles but without extreme specializations for either fossorial or aquatic life.     

Patterns of reproductive‐mode evolution in Old World tree frogs (Anura,Rhacophoridae)

The Old World tree frogs (Anura: Rhacophoridae), with 387 species, display a remarkable diversity of reproductive modes – aquatic breeding, terrestrial gel nesting, terrestrial foam nesting and terrestrial direct development. The evolution of these modes has until now remained poorly studied in the context of recent phylogenies for the clade. Here, we use newly obtained DNA sequences from three nuclear and two mitochondrial gene fragments, together with previously published sequence data, to generate a well‐resolved phylogeny from which we determine major patterns of reproductive‐mode evolution. We show that basal rhacophorids have fully aquatic eggs and larvae. Bayesian ancestral‐state reconstructions suggest that terrestrial gel‐encapsulated eggs, with early stages of larval development completed within the egg outside of water, are an intermediate stage in the evolution of terrestrial direct development and foam nesting. The ancestral forms of almost all currently recognized genera (except the fully aquatic basal forms) have a high likelihood of being terrestrial gel nesters. Direct development and foam nesting each appear to have evolved at least twice within Rhacophoridae, suggesting that reproductive modes are labile and may arise multiple times independently. Evolution from a fully aquatic reproductive mode to more terrestrial modes (direct development and foam nesting) occurs through intermediate gel nesting ancestral forms. This suggests that gel nesting is not only a possible transitional state for the evolution of terrestriality, but also that it is a versatile reproductive mode that may give rise to other terrestrial reproductive modes. Evolution of foam nesting may have enabled rhacophorids to lay a larger number of eggs in more open and drier habitats, where protection from desiccation is important. Terrestrial direct development allows frogs to lay eggs independent of bodies of water, in a diversity of humid habitats, and may represent a key innovation that facilitated the evolution of nearly half of all known rhacophorid species.     

A New Species of Rhacophorus(Amphibia: Anura: Rhacophoridae)from Vietnam

We describe a new species of Rhacophorus based on two adult specimens collected from Hoa Binh Province, northern Vietnam. Rhacophorus hoabinhensis sp. nov. is distinguishable from its congeners on the basis of a combination of the following morphological characters: size small(SVL 31.1–32.5 mm in males); head slightly longer than wide; vomerine teeth absent; snout short(SNL/SVL 0.16); dorsal skin smooth; forearm and tarsus with dermal fringes; dermal appendage at vent present; webbing formula on fingers Ⅰ12/3-12/3 Ⅱ1-2 Ⅲ1-1 Ⅳ and on toes Ⅰ3/4-1 Ⅱ1/2-1Ⅲ1/2-1 Ⅳ1-1/2Ⅳ; dorsal surface grey yellow with brown spots; lower jaw region dark grey, throat, chest and belly cream; anterior and posterior thighs, as well as ventral surface of tibia orange. The interspecific uncorrected genetic distances(16 S rRNA gene) between the new species from Hoa Binh and other analyzed congeners varied from 9.8% to 17.4%. In the phylogenetic analyses, the new species revealed to be a representative of Rhacophorus and was nested within the R. hoanglienensis-orlovi species group.     

Genetic differentiation of the Iberian tree frogs Hyla arborea molleri and Hyla meridionalis (Amphibia: Anura)

Genetic differentiation of Hyla arborea molleri and H. meridionalis was studied through allozyme electrophoresis and through essays of artificial hybridization. Samples were taken at several localities in Portugal, where these species occur either in sympatry or allopatry, and at one locality in France. Of the 21 presumptive loci analyzed, 6 were monoallelic, while 9 loci were discriminative between both species. A relatively high level of genetic differentiation was found between the two species (NEI's D = 0.725), contrasting with their morphological similarity. Remarkably low levels of genetic variability were found within species, with greater differentiation found among samples of H. meridionalis than of H. a. molleri. Although the capacity for interspecific hybridization was low, hybrid embryos resulted from both reciprocal crosses, but with much greater success in the cross ♂H. a. molleri x δH. meridionalis. Hybrids showed greater similarity with the male parent, and showed heterozygote allozymic phenotypes for discriminative loci. Results are compared with those of similar studies on the same and different species of Hyla, and differentiation of H. a. molleri and H. meridionalis is discussed in relation to their taxonomic status and origin.     

A new species of Gracixalus (Amphibia: Anura: Rhacophoridae) from northern Vietnam

We describe a new species of small tree frog from northern Vietnam based on morphological differences and molecular divergence. Gracixalus waza sp. nov. is distinguishable from its congeners and other small rhacophorid species on the basis of a combination of the following characters: (1) size small (snout-vent length of males 27.1–32.9 mm, of females 37.6 mm); (2) head as wide as or wider than long; (3) vomerine teeth absent; (4) snout rounded and long (16–18 % of the snout-vent length); (5) spines on upper eyelid absent; (6) tibiotarsal projection absent; (7) dorsal skin smooth; (8) dermal fringes on forearm and tarsus absent; (9) dorsal surface of head and body greyish-green to moss-green with dark brown pattern forming an inverse Y marking; and (10) throat and chest with dark marbling. Our molecular data showed that the new species is nested in the same group with Gracixalus jinxiuensis sensu lato.     

The ecomorphology of Bornean tree frogs (family Rhacophoridae)

Ecomorphological studies generally assume a causal relationship between morphology and ecology mediated by performance. That assumption was tested in a community of Bornean tree frogs of the family Rhacophoridae. Biomechanical models allow a priori predictions about expected intra- and interspecific correlations among toepad area, hind-limb length, body mass, sticking ability, jumping distance, microhabitat, vertical height and substrate. Both intra- and interspecifically, size and shape variables showed patterns of association with ecology predicted on the basis of maintaining relative as well as absolute levels of performance. Unexpectedly, the form-function relationship between toepad area and sticking ability differed intra- and interspecifically. These findings indicate the need for more empirical work examining morphology-performance relationships and their use in ecology.     

Evolution of parental care in dart poison frogs (Amphibia: Anura: Dendrobatidae)

Dendrobatid frogs perform a unique mode of parental care, ranging from egg attendance and tadpole transport to tadpole feeding. It is hypothesized that a behaviour in which the egg attending parent remains with the cluch is the most primitive condition. In more advanced forms or parental care, the male is able to attend several clutches of eggs. Tadpole attendance and feeding, finally, started as deceit; the male induced the female to lay eggs into a bromeliad leaf axil already occupied by a larva. Costs and benefits of the different modes of parental care and possible alternative reproductive tactics are discussed.     

Phylogeny and genus-level classification of mantellid frogs (Amphibia, Anura)

We propose a novel classification of frogs in the family Mantellidae, based on published phylogenetic information and on a new analysis of molecular data. Our molecular tree for 53 mantellid species is based on 2419 base pairs of the mitochondrial 12S rRNA, 16S rRNA, tRNAVal and cytochrome b genes, and of the nuclear rhodopsin gene. Because the genus Mantidactylus Boulenger sensu lato is confirmed to be paraphyletic with respect to Mantella Boulenger, and is highly diverse in morphology and reproductive biology, we propose to partition Mantidactylus into seven genera by elevating four subgenera to genus rank (Blommersia Dubois, Guibemantis Dubois, Spinomantis Dubois, and Gephyromantis Methuen) and creating two new genera (Boehmantis gen. n. and Wakea gen. n.). In addition, we create the new subgenera Boophis (Sahona) subgen. n., Gephyromantis (Duboimantis) subgen. n., G. (Vatomantis) subgen. n., and Mantidactylus (Maitsomantis) subgen. n. The following species are transferred to Spinomantis, based on their phylogenetic relationships: S. elegans (Guibé) comb. n. (formerly in Mantidactylus subgenus Guibemantis); S. bertini (Guibé) comb. n. and S. guibei (Blommers-Schlösser) comb. n. (both formerly in Mantidactylus subgenus Blommersia); S. microtis (Guibé) comb. n. (formerly in Boophis Tschudi). Within Boophis, the new B. mandraka species group and B. albipunctatus species group are established. Boophis rhodoscelis (Boulenger) is transferred to the B. microtympanum group. The following five species are revalidated: Mantidactylus bellyi Mocquard and M. bourgati Guibé (not junior synonyms of M. curtus (Boulenger)); M. cowanii (Boulenger) (not syn. M. lugubris (Duméril)); M. delormei Angel (not syn. M. brevipalmatus Ahl); Mantella ebenaui (Boettger) (not syn. M. betsileo (Grandidier)). The new classification accounts for recent progress in the understanding of the phylogeny and natural history of these frogs, but it is still tentative for a number of species. Future modifications may be necessary, especially as concerns species now included in Gephyromantis and Spinomantis.Full article published online at: http://www.senckenberg.de/odes/06-11.htm     

Resurrection of the Genus Leptomantis,with Description of a New Genus to the Family Rhacophoridae(Amphibia:Anura)

Genus Rhacophorus Kuhl and Van Hasselt, 1 822 is one of the most diverse genera of the family Rhacophoridae, and its taxonomy of genus Rhacophorus faces major challenges because of rapidly described new species and complex interspecies relations. In this study, we investigate the generic taxonomy within the genus Rhacophorus based on 1 972 bp of mitochondrial genes(12S rRNA, tRNA-val and 16S rRNA), containing 102 sequences from 58 species. The results reveal three well-supported and highly diverged matrilines that correspond with morphological characteristics and geographic distribution. Accordingly, we consider these three lineages as distinct genera: Rhacophorus sensu stricto, resurrected genus Leptomantis Peters, 1867, and the genus Zhangixalus gen. nov.     

The larval hyobranchial apparatus of discoglossoid frogs: its structure and bearing on the systematics of the Anura (Amphibia: Anura)

The morphology of the larval hyobranchial apparatus of discoglossoid frog species representing the genera Ascaphus, Alytes, Bombina, and Discoglossus is described and the resulting characters were analysed cladistically. Seven species representing seven major lineages of frogs were included in the cladistic analysis of characters. Several changes in the terminology of the musculature are introduced, and a new interpretation of the subarcualis-muscle system is presented. The phylogenetic analysis suggest that the hyobranchial apparatus was substantially altered in the lineages leading to and within the Pipanura. This notably involved fusion, reduction and loss of skeletal structures and muscles, and splitting of certain muscles into muscle groups. The result confirm previous hypotheses based on the study of adults: discoglossoid species retain the most numlerous plesiomorphic characters among extant ianurans. The larval hyobranchial apparatus is in many features structrually similar to that of urodeles. Many of their character states were most likely present in the most recent common ancestor of all living forgs. The cladistic analysis of 31 characters of ithe larval hyobranchial apparatus supports major clades: Anura, Bombinanura, Pipanura, and Pelobatoidea + Neobatrachia. The cladiostic analysis and interpretation of larval characters is in part compatible with phylogenetic hypotheses based on characters of adults and rRNA sequences, but is in conflict with the Mesobatrachia and Archaeobatrachia concepts of other authors.     

Female-offspring communication in a Taiwanese tree frog, Chirixalus eiffingeri (Anura: Rhacophoridae)

We assessed the roles of visual and olfactory cues in female-tadpole communication inChirixalus eiffingeri . The mean cumulative time that at least one tadpole was active or begged for food was significantly longer when a female C. eiffingeri was present than when a plastic frog was introduced and when no frog was present. Tadpoles did not respond visually to a female frog physically separated from them by transparent Plexiglas. However, tadpoles were more active in water conditioned by female frogs than in unconditioned water. Tadpole activity was further elevated by water conditioned by a female frog and tadpoles. Tadpoles were more active in water conditioned by male frogs than in unconditioned water, but water conditioned by a male frog and tadpoles did not further elevate tadpole activity. Thus, water conditioned by adults of either sex contains substances that increase tadpole activity, but only females show a synergistic effect with conditioning by tadpoles. Copyright 2002 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour     

Muscle development in the abdominal region of Larval Hylidae (Amphibia: Anura)

Larval muscle development in the abdominal region of five species of hylid frogs (Scinax nasicum, S. fuscovarium, Hyla andina, Phyllomedusa boliviana, Gastrotheca gracilis) was studied using differential staining techniques. These five species represent three major hylid subfamilies. The development of the main abdominal muscles, the rectus abdominis, the two lateral muscles (obliquus externus and transversus), and the lateral pectoralis abdominalis is described. The number of myotomes of the rectus abdominis varies between five and six, and the abdominal muscles associated with the rectus abdominis (obliquus externus, pectoralis abdominalis, and rectus cervicis) vary interspecifically in time of appearance and configuration. The presence of gaps in the configuration of the rectus abdominis has been related to the lotic habits of the larvae. However, our observations indicate the presence of such gaps in larvae that inhabit lentic environments as well. These results suggest that the presence of these gaps is unrelated to larval habitat. There are relatively small differences in muscle morphology among these closely related species, which apparently cannot be explained by morphological adaptations related to their ecology. In the species studied, the number of elements that form the abdominal musculature in larvae is equal to that observed in adults. Likewise, the general morphology of the muscles is ontogenetically conserved. This suggests that both the axial skeleton and musculature are more ontogenetically conserved in relation to the substantial changes that are observed in the skull and head muscles of developing anurans. J. Morphol. 241:275–282, 1999. © 1999 Wiley‐Liss, Inc.     

Phylogenetic relationships among Rhacophorinae (Rhacophoridae, Anura, Amphibia), with an emphasis on the Chinese species

The partial nucleotide sequence of mitochondrial 12S and 16S rRNA genes was determined for 23 Chinese species of Rhacophoridae (Amphibia: Anura), representing four of the eight recognized genera. Using Buergeriinae as the outgroup, phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference) were performed in combination with already published mitochondrial 12S and 16S sequences of Rhacophorinae frogs. In all cases, Philautus romeri Smith, 1953 is recovered as the sister taxon to all other Rhacophorinae, although the support values are weak. Chirixalus doriae Boulenger, 1893 is closer to Chiromantis [ Chiromantis rufescens (Günther, 1868) and Chiromantis xerampelina Peters, 1854] than to Chirixalus vittatus (Boulenger, 1887). The clade { Philautus odontotarsus Ye & Fei, 1993, [ Philautus idiootocus (Kuramoto & Wang, 1987), Kurixalus eiffingeri (Boettger, 1895)]} is recovered with strong support. The monophyly of Theloderma and Rhacophorus rhodopus Liu & Hu, 1959 is not supported. It is suggested that Philautus albopunctatus Liu & Hu, 1962 should be placed into the synonymy of Theloderma asperum (Boulenger, 1886), and that Philautus rhododiscus Liu & Hu, 1962 should be assigned to Theloderma , so as to correct the paraphyly. Additionally, the monophyly of ' Aquixalus ' is not supported, and this requires further examination. Results also indicate that the Rhacophorus leucomystax (Gravenhorst, 1829)/ Rhacophorus megacephalus (Hallowell, 1861) complex needs further revision. Studies employing broader sampling and more molecular markers will be needed to resolve the deep relationships within the subfamily Rhacophorinae.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 153 , 733–749.     

Phylogeny of frogs as inferred from primarily larval characters (Amphibia:Anura)★

This paper presents larval evidence and evaluates its contribution to the discussion of frog phylogeny;136 larval characters,6 reproductive biology characters, and 14 adult morphology characters were scored for 81 frog and 4 caudate species.More than 90% of the data matrix entries represent original data derived from personal direct examination of specimens.Some larval characters are described for the rest time and many others have not been assessed for specic taxa or in a broad phylogenetic context before.Ho‐ moplasy appears common in this and other amphibian morphological data sets.The data supported and conrmed various well‐ known clades, among others the Anura, Bufonidae, Ceratophryinae, Discoglossidae, Dendrobatidae, Hyperoliidae, Microhylidae, South American microhylids, Phyllomedusinae, Pseudinae, Pipoidea, Pipidae, and Scoptanura.The Ascaphidae was sister group to all other anurans and the Pipoidea was placed more basally than in some previous analyses.The Eurasian pelobatids formed a clade, whereas Spea and Pelodytes did not group robustly with them.Pelobatoid frogs emerged as a paraphyletic “transitional” assemblage including Heleophryne. The resolution of basal neobatrachian splits remained labile, although some subclades within the Neobatrachia were robustly supported. The “Hylidae” was paraphyletic, and hyline species were paraphyletic with respect to the Pseudinae.Hemisus clearly was in a clade with the Hyperoliidae and is proposed to be included in that family.Scaphiophryne was conrmed as basal taxon within the Microhylidae.Compared to the larval stages of the most recent common ancestor of anurans, members of the Scoptanura (microhylids except scaphiophrynines)have accumulated the highest number of apomorphic character states in anuran evolution.     

Timing of development of the middle ear of Anura (Amphibia)

Summary The opercularis system and tympanum-stapes complex of the anuran middle ear develop at different times relative to metamorphosis. In early larvae, the fenestra ovalis is represented by a large lateral opening in the otic capsule filled with connective tissue. At later larval stages, but well before metamorphosis, a cartilaginous operculum begins to form at the posterior margin of the fenestra ovalis, and proceeds to expand to fill all except the anterior part of the fenestra. The opercularis muscle forms along with the levator scapulae superior muscle at the anteromedial edge of the developing suprascapular cartilage of the shoulder girdle. The muscle fibers extend anteroventrally towards the operculum and otic capsule, and, just before emergence of the forelimbs, that portion that will form the opercularis muscle inserts on the lateral surface of the operculum. At this stage, when the metamorphosing frogs first show terrestrial habits, the opercularis system is complete and presumably functional. Timing of development of the tympanum-stapes complex is more variable. The stapes begins as a cartilaginous condensation in the anterior part of the fenestra ovalis, and develops laterally to eventually contact the epidermis and dermis that together will form the tympanum. Meanwhile a middle ear cavity and tympanic annulus form to complete the complex. In several species, especially those that metamorphose at a smaller body size, the tympanum-stapes complex is quite incomplete by the end of metamorphosis, and in Hyla crucifer it takes about 60 days to fully develop. The presence of a complete opercularis system by the start of terrestrial activity is consistent with an hypothesized seismic function of the system. The independent timing of development of the opercularis system and tympanum-stapes complex does not support functional hypotheses linking the opercularis system with modulation of responsiveness of the tympanum-stapes complex to aerial sound. Newly metamorphosed frogs with poorly developed tympanum-stapes complexes are presumably either insensitive to aerial sound or employ alternate mechanisms for transmission of sound energy to the inner ear, possibly involving the opercularis system.     

Target-capture phylogenomics provide insights on gene and species tree discordances in Old World treefrogs (Anura: Rhacophoridae)

Genome-scale data have greatly facilitated the resolution of recalcitrant nodes that Sanger-based datasets have been unable to resolve. However, phylogenomic studies continue to use traditional methods such as bootstrapping to estimate branch support; and high bootstrap values are still interpreted as providing strong support for the correct topology. Furthermore, relatively little attention has been given to assessing discordances between gene and species trees, and the underlying processes that produce phylogenetic conflict. We generated novel genomic datasets to characterize and determine the causes of discordance in Old World treefrogs (Family: Rhacophoridae)—a group that is fraught with conflicting and poorly supported topologies among major clades. Additionally, a suite of data filtering strategies and analytical methods were applied to assess their impact on phylogenetic inference. We showed that incomplete lineage sorting was detected at all nodes that exhibited high levels of discordance. Those nodes were also associated with extremely short internal branches. We also clearly demonstrate that bootstrap values do not reflect uncertainty or confidence for the correct topology and, hence, should not be used as a measure of branch support in phylogenomic datasets. Overall, we showed that phylogenetic discordances in Old World treefrogs resulted from incomplete lineage sorting and that species tree inference can be improved using a multi-faceted, total-evidence approach, which uses the most amount of data and considers results from different analytical methods and datasets.