Bapx1 is required for jaw joint development in amphibians.

The acquisition of a movable jaw and a jaw joint are key events in gnathostome evolution. Jaws are derived from the neural crest derived pharyngeal skeleton and the transition from jawless to jawed vertebrates consists of major morphological changes, which must have a genetic foundation. Recent studies on the effects of bapx1 knockdown in fish and chicken indicate that bapx1 has acquired such a role in primary jaw joint development during vertebrate evolution, but evidence from amphibians is missing so far. In the present study, we use Ambystoma mexicanum, Bombina orientalis, and Xenopus laevis to investigate the effects of bapx1 knockdown on the development of these three different amphibians. Using morpholinos we downregulated the expression of bapx1 and obtain morphants with altered mandibular arch morphology. In the absence of bapx1 Meckels cartilage and the palatoquadrate jaw joint initially develop separately but during further development the joint cavity between both fills with chondrocytes. This results in the fusion of both cartilages and the loss of the jaw joint. Despite this the jaw itself remains usable for feeding and breathing. We show that bapx1 plays a role in jaw joint maintenance during development and that the morphants morphology possibly mirrors the morphology of the jawless ancestors of the gnathostomes.

Heterochronies in the cranial development of Asian tree frogs (Amphibia: Anura: Rhacophoridae) with different life histories.

The development of bony skull was studied in four species of Asian tree frogs (Rhacophoridae) with different life histories: biphasic development with free larval stage and direct development. In biphasic rhacophorids the sequence of the appearance of cranial bones generally followed the generalized pattern of craniogenesis, which was described for most studied anurans. In contrast, direct-developing species displayed some heterochronies in the formation of skull bones, namely, the accelerated formation of the anlagen of jaw and suspensorium bones. The obtained results support that the embryonization in amphibians is regularly accompanied by a heterochronic repatterning of craniogenesis, rather similar in different phyletic groups.

Ecology of the bromeligenous frog Phyllodytes luteolus (Anura, Hylidae) from three restinga remnants across Brazil's coast.

In this study, we analyzed diet, sexual dimorphism and bromeliad use in three populations of the hylid frog Phyllodytes luteolus from restinga habitats along the Brazilian coast. We found 13 arthropods categories in 161 stomachs. Ants and termites were the dominant prey items. The similar trophic niche across populations suggests this species has a conservative diet. We found sexual dimorphism regarding body size and jaw width. We recordedP. luteolus in five bromeliad species, but predominantly inAechmeablanchetiana (35.6% of individuals recorded). We recorded solitary individuals in 44% of occupied bromeliads, and never found two males sharing the same bromeliad. The data is suggestive that populations ofP. luteolus has a conservative diet independent of area, with ants and termites the being most relevant prey items. The sexual dimorphism in jaw and the solitary males may suggest that this species have territorial behavior.

Multiple genetic lineages challenge the monospecific status of the West African endemic frog family Odontobatrachidae.

BACKGROUND: Correct species identification is crucial in different fields of biology, and in conservation. The endemic West African frog family Odontobatrachidae currently contains a single described species, Odontobatrachus natator. From western Guinea to western Côte d'Ivoire it inhabits forests around waterfalls or cascades. Based on more than 130 specimens from 78 localities, covering the entire distribution, we investigated the molecular diversity of these frogs. RESULTS: Our analyses included mitochondrial and nuclear genes, with a concatenated alignment of 3527 base pairs. We detected high level of genetic differentiation with five distinct lineages or operational taxonomic units (OTUs). These OTUs were also identified by two different species delimitation approaches, Generalized Mixed Yule Coalescent (GMYC) and cluster algorithm. All OTUs occur in parapatry in the Upper Guinean forests. One OTU, assigned to the "true" Odontobatrachus natator, covers the largest distribution, ranging from the border region of western Sierra Leone-Guinea to eastern Liberia. Two OTUs are restricted to western Guinea (Fouta Djallon and foothills), while two others occur in eastern Guinea and the border region of Guinea-Liberia-Côte d'Ivoire. The OTU representing O. natator consists of two divergent subclades: one restricted to the Freetown Peninsula (Sierra Leone) and the other covering all populations further inland. Environmental niche models indicated that the restricted Freetown Peninsula population is separated by unsuitable habitat from remaining populations. CONCLUSION: Geographic isolation of OTUs and molecular differences comparable to species level differentiation in other frog families indicate that O. natator contains cryptic species diversity. Respective distribution patterns most probably resulted from repeated changes of forest cover (contraction and expansion) over evolutionary timescales. The survival within forest refugia that have persisted through multiple drier periods and subsequent dispersal during wetter times may best explain the observed geographic distributions of OTUs. According to the IUCN Red List range criteria each OTU should be classified as "Endangered." If the Freetown Peninsula "natator" population is recognized as a distinct species it would warrant recognition as "Critically Endangered." The identification of cryptic lineages highlights the urgent need to protect these frogs, all of which are endemic to small areas within the Upper Guinean biodiversity hotspot.

Mitochondrial phylogeny of an Asian tree frog genus Theloderma (Anura: Rhacophoridae).

We assessed phylogenetic and systematic relationships among 17 out of 23 species of Theloderma and all three species of Nyctixalus from 2412bp sequences of the mitochondrial DNA genes of 12S rRNA, tRNA(val), and 16S rRNA using maximum likelihood and Bayesian inference methods. With the exception of T. moloch, Theloderma and Nyctixalus are confirmed to form a clade, in which each genus also forms a clade. Theloderma moloch is phylogenetically outside these clades and closer to samples from Chiromantis, Feihyla, Gracixalus, Kurixalus, Philautus, Polypedates, Raorchestes, and Rhacophorus. Within Theloderma, T. horridum and T. stellatum form the sister taxon to a clade comprising the remaining species. The basal split within the latter clade groups T. asperum, T. licin, T. petilum, and T. ryabovi as the sister to a clade comprising T. bicolor, T. chuyangsinense, T. corticale, T. gordoni, T. laeve, T. lateriticum, T. nebulosum, T. rhododiscus, and T. truongsonense. Our phylogenetic results indicate homoplastic evolution of four morphological characters: small vs. large body size, presence of vomerine teeth, presence of a vocal opening in males, and interdigital webbing on hands. The common ancestor of Theloderma and Nyctixalus is inferred to have arisen in the area including the current Sunda region.

Light shines through the spindrift--phylogeny of African torrent frogs (Amphibia, Anura, Petropedetidae).

Torrent frogs of the genus Petropedetes Reichenow, 1874 as currently understood have a disjunct distribution with species endemic to West, Central or East Africa. We herein present a phylogenetic analysis including all but one of the currently described 12 species of the genus. Maximum Likelihood and Bayesian analyses of combined nuclear (rag1, SIA, BDNF) and mitochondrial (16S, 12S, cytb) genes of more than 3500 base pairs, revealed clades corresponding to the three sub-Saharan regions. Molecular results are confirmed by morphological differences. Surprisingly, the three geographic clades do not form a monophyletic group with respect to closely related families Pyxicephalidae and Conrauidae and therefore require taxonomic changes. We resurrect Arthroleptides Nieden, 1911 for the East African taxa. The Central African taxa remain in the genus Petropedetes. The West African members are placed in the new genus Odontobatrachus gen. nov. The taxonomic position of the new genus remains incertae sedis as it was not assigned to any of the four families included in our analyses. Potential new species have been detected within all three major clades, pointing to a still not fully clarified diversity within African torrent frogs.

Feeding habits of the leaf litter frog Haddadus binotatus (Anura, Craugastoridae) from two Atlantic Forest areas in southeastern Brazil.

Haddadus binotatus is an endemic anuran of the Brazilian Atlantic Forest and currently, there is no information about the diet of this species. We analyzed the diet of two populations of this anuran in two states in southeast Brazil. Samplings were carried out in 2004 in the state of Rio de Janeiro and in 2009 and 2010 in the state of Espírito Santo. Haddadus binotatus presented a rich diet composition, preying 19 prey types. Orthoptera, Coleoptera, and Blattodea were the most important preys in the Rio de Janeiro population, and Orthoptera, Araneae and Hemiptera were the most important in the Espírito Santo population. The diet composition differed numerically between the two localities, but not in terms of volume, which can reflect local differences in the prey availability in the two habitats. The jaw width limited the size of prey, which is expected for predators who swallow the preys without chewing. The proportion of individuals with empty stomachs was higher in the Rio de Janeiro population (39.2%) than in the Espírito Santo population (17.9%), suggesting that the former could be in a lower energy balance. The females of the species were larger than the males, which may result from the production of larger eggs.

Polyploidy and epigenetic events in the evolution of Anura.

This article reviews the polyploidy events that have long been demonstrated to play a role in the evolution of Anura, while also discussing the importance of epigenetic control of gene expression and diversity. Findings on Brazilian autopolyploid anurans, mainly of the genus Odontophrynus, obtained in previous studies on their cytogenetics, chromatin ultrastructure, and molecular gene regulation are discussed here. Our data on genome duplication and on epigenetic events were analyzed here regarding phylogenetic trees, including the classic 2R model for vertebrate evolution and the growing evidence of similar epigenetic mechanisms in animal and allopolyploid plants. We propose that polyploidy and epigenetic events led to rapid Anura diversity and speciation. Also, recent advances in molecular studies in other organisms led us to revisit some controversial models of evolution.

Description of two new species of the genus Megophrys (Amphibia: Anura: Megophryidae) from Heishiding Nature Reserve, Fengkai, Guangdong, China, based on molecular and morphological data.

Two new species, Megophrys acuta sp. nov. and Megophrys obesa sp. nov., are described based on a series of specimens collected from Heishiding Nature Reserve, Fengkai County, Guangdong Province, China. They can be distinguished from other known congeners occurred in southern and eastern China by morphological characters and molecular divergence in the mitochondrial 16S rRNA gene. M. acuta is characterized by small and slender body with adult females measuring 28.1-33.6 mm and adult males measuring 27.1-33.0 mm in snout-vent length; snout pointed, strongly protruding well beyond margin of lower jaw; canthus rostralis well developed and sharp; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the pupil of eye. M. obesa is characterized by stout and slightly small body with adult females measuring 37.5-41.2 mm, adult male measuring 35.6 mm in snout-vent length; snout round in dorsal view; canthus rostralis developed; hindlimbs short, the heels not meeting, tibio-tarsal articulation reaching forward the posterior margin of eye. The discovery of these two new species further confirms that the diversity of this genus has been significantly underestimated. At present the genus Megophrys contains 56 species of which 35 species are distributed in China.

Two new species of Kalophrynus Tschudi, 1838 (Anura: Microhylidae) from the Annamite mountains in southern Vietnam.

We describe two new mountain-dwelling microhylid species of the genus Kalophrynus from the southern part of the Annamite Mountains in Vietnam. The two new species differ from all known congeners in morphological characters and mtDNA; phylogenetically, they form a sister clade to the large-bodied K. interlineatus (1009 bp, 16S rRNA gene, mtDNA). Both species share the following characteristics: snout pointed in dorsal and lateral views, slightly sloping in profile; tympanum distinct, smaller than eye in diameter; toe webbing moderate; outer metatarsal tubercle present; light dorsolateral line absent. Kalophrynus cryptophonus sp. nov. from Loc Bao, Lam Dong Province is a small-sized species distinguishing from its congeners by a combination of: SVL 27.9-30.4 mm in males, 23.4 mm in female; canthus rostralis indistinct; males with large sharp conical spines on the skin covering mandible margins and finely asperous nuptial pads on the dorsal surface of fingers I-III; dark ocelli in the inguinal region usually present, small, without a light border; anterior palatal dermal ridge short, restricted to medial part of palate. Kalophrynus honbaensis sp. nov. from Hon Ba, Khanh Hoa Province is a medium-sized Kalophrynus, distinguishing from its congeners by a combination of: SVL 26.7-36.8 mm in males; canthus rostralis distinct; males without distinguishable spines on the mandible margins nor the nuptial pads; dark ocelli in the inguinal region present, large, without a distinct light border, anterior palatal dermal ridge developed, parallel to posterior one. Kalophrynus cryptophonus sp. nov. reproduces in hollow bamboo stems; we describe larval morphology and bioacoustics of this species in relation to phytotelm breeding. A review of the distribution of the genus Kalophrynus in Indochina is provided.

The tadpoles of nine Cameroonian Leptodactylodon species (Amphibia, Anura, Arthroleptidae).

We describe and compare the tadpoles of nine Leptodactylodon species from Cameroon. The tadpoles of Leptodactylodon bicolor, L. mertensi, L. ovatus, L. perreti and L. ventrimarmoratus are herein reinvestigated, partly based on larger series than previously available. In addition we present first descriptions for the tadpoles of L. boulengeri, L. erythrogaster, L. ornatus, and L. cf. polyacanthus. The morphology of these exotrophic, lotic and neustonic tadpoles is discussed in comparison with other stream-dwelling tadpoles. Based on the assumed biology of these tadpoles, living in interstices of gravel or debris, the functioning of several special morphological features, in particular the funnel-mouth of Leptodactylodon tadpoles, are interpreted.

Three new species of Proceratophrys Miranda-Ribeiro 1920 from Brazilian Cerrado (Anura, Odontophrynidae).

Based on the analyses of specimens collected at several areas in the Cerrado domain from Central Brazil and others housed in scientific collections and on specimens collected at the type-locality, herein we describe three new species belonging to the P. cristiceps group: Proceratophrys bagnoi sp. nov., from UHE Serra da Mesa power plant (13°49'47.5"S, 48°19'17"W; 570 m a.s.l.; WGS84 datum), Municipality of Minaçu, State of Goiás; Proceratophrys branti sp. nov., from Jalapão, Municipality of Mateiros (05o15'25"S, 48o12'00"W; 109 m a.s.l.; WGS84 datum), State of Tocantins; and Proceratophrys dibernardoi sp. nov., Municipality of Mineiros (17o33'52"S, 52o33'20"W; 803 m a.s.l.; WGS84 datum), State of Goiás. The diversity of Proceratophrys in Brazilian Cerrado is still underscored and several species will be described in the following years.

A new species of Crossodactylodes (Anura: Leptodactylidae) from Minas Gerais, Brazil: first record of genus within the Espinhaço Mountain Range.

The genus Crossodactylodes comprises three species of Atlantic Rainforest endemic frogs strictly dependent on bromeliads where they spend their entire life cycle. The current geographic distribution of the genus covers highland areas of Atlantic Rainforest in the States of Rio de Janeiro and Espírito Santo, Southeastern Brazil. We describe a new species of the genus from Parque Estadual do Pico do Itambé, at Santo Antônio do Itambé municipality, State of Minas Gerais, Southeastern Brazil. Crossodactylodes itambe sp. nov. is characterized by the following combination of traits: male SVL 16.2 ± 1.3 (14.0-17.6 mm, n = 10), female SVL 16.2 ± 1.0 (13.5-18.0 mm, n = 15); snout short, rounded in dorsal view, sloping in lateral view; absence of vocal sac and vocal slits in males; absence of vomerine teeth; males with upper arms and forearms hypertrophied; cloacal flap prominent, simple; dorsal skin coarsely granular. The new species inhabits rupicolous bromeliads in open areas of rocky fields, and is recorded in altitudes between 1836 and 2062 m above sea level. This record extends the genus distribution for about 325 km northwest from where it was known. Crossodactylodes sp. nov. is the only species of the genus that occurs in open field habitats (campos rupestres), in very high altitudes of a non-costal mountain range (the Espinhaço Range). 

Erythrocyte nuclear size as a better diagnostic character than cell size in the identification of live cryptic polyploid species.

It is well documented in anurans the cryptic condition of many species complexes involving polyploids. In these complexes the character that clearly differentiates them is the number of chromosome complements. The blood cells of amphibians conserve their nucleus, and so the erythrocyte size is correlated with the DNA content. We analyzed two cryptic-polyploid complexes occurring in the center of Argentina: Odontophrynus cordobae (2n)/O. americanus (4n) and Pleurodema kriegi (4n)/P. cordobae (8n). Our aim was evaluate the efficiency in the utilization of nuclear area with respect to cellular area of the erythrocytes to define the limits values for the identification of cryptic-polyploid species. We studied 110 individuals of Pleurodema and 116 individuals of Odontoprhynus. For each individual, we measured the cellular and nuclear length (L) and width (A) of 40 erythrocytes (Area = L*A*π/4) and boundary values were calculated using distribution curves. In both complexes studied, the erythrometric parameters showed significant differences between related species. Moreover, in both complexes the nuclear area was more efficient for identifying the species (Pleurodema: 34.39 µm2 (probability = 99.96%) and Odontophrynus: 24.02 µm2 (99.075%)) than the cell area (Pleurodema: 273.08 µm2 (97.55%) y Odontophrynus: 197.69 µm2 (97.94%)). Greater efficiency found using nuclear area is novel and significant because most studies use only the cell area to differentiate polyploid complexes.

The genus Odontophrynus (Anura: Odontophrynidae): a larval perspective.

The genus Odontophrynus consists of 11 species of medium-sized frogs distributed across south and east South America. This study examines and describes the chondrocrania and oral cavities of O. americanus, O. maisuma, O. carvalhoi, and O. cultripes, and review current knowledge about the larval external morphology of the genus. Twenty-one tadpoles were cleared and double-stained for chondrocranium description and five tadpoles were dissected for analysis in a scanning electron microscope. The presence of a tectum parientale may be considered here as a putative synapomorphy of the genus. The O. americanus and O. cultripes species groups were partially differentiated by the length of the processus pseudopterigoideus, shape of divergence of the hypobranchial plates, number of postnarial papillae, and number of projections of the lateral ridge papillae. The larvae of O. occidentalis species group, in turn, differed from others by presenting a greater total length.

Morphological and acoustic characterization of Proceratophrys goyana (Lissamphibia: Anura: Odontophrynidae), with the description of a sympatric and related new species.

Proceratophrys goyana was until recently the only species of the genus described from central Brazil. In this paper we characterize the adult morphology and advertisement call of this species, based on data collected at its type-locality (Chapada dos Veadeiros, Goiás, Brazil). These new data allowed us to recognize a new species of Proceratophrys, sympatric to P. goyana, which is described herein. Proceratophrys goyana is mainly characterized by the male SVL = 38.8-46.5 mm; a well-developed and continuous pair of dorsal sagittal ridges; upper eyelids triangular; developed frontoparietal crests, delimiting a shallow depression between them; overall color pattern browned, with the symmetrical dorsal ridges bordered laterally by dark brown undulations. Proceratophrys rotundipalpebra sp. nov. is characterized by the male SVL = 30.4-39.3 mm; the pair of symmetrical dorsal ridges well-developed anteriorly and somewhat interrupted in the sacral region; upper eyelids short and rounded; frontoparietal crests not well-developed; overall color pattern stained by 3-4 tonalities of gray, without a clear background color. The advertisement calls of both species are emitted in a multi-note pattern, each note pulsed, the first and last notes differing from each other and from those amidst the call in temporal features. The description of this new species is another example of the underestimated diversity of Proceratophrys in the Cerrado of central Brazil. 

A new species of the genus Gracixalus (Amphibia: Anura: Rhacophoridae) from Southern Guangxi, China.

A new species of the genus Gracixalus is described from Nonggang National Nature Reserve, based on eleven specimens from evergreen karst forests in Sino-Vietnamese border region of southern China. The new species, Gracixalus nonggan-gensis sp. nov., is medium sized species of Gracixalus that can be distinguished from its congeners by a combination of the following characters: SVL ranging from 29.9-35.3 mm in males and 33.6-38.0 mm in females; vomerine teeth absent; distinct tympanum as wide as the disc of finger III; the lower part of the tympanum with many small tubercles; tibiotarsal articulation reaching the tip of the snout; dorsum smooth yellowish-olive in life with a wide, dark-green irregular mark; throat, chest and belly white with light grey-blue tint and brown marbling; broad, dark olive, transverse stripes on limbs; finger webbing absent, toes one-third webbed; male with internal subgular vocal sac. Based on a 16S ribosomal RNA mitochondrial gene fragment, G. nonggangensis sp. nov. forms a monophyletic group embedded within the genus Gracixalus.

Two new species of Proceratophrys Miranda-Ribeiro, 1920 (Anura; Odontophrynidae) from the Atlantic forest, with taxonomic remarks on the genus.

We describe two new species of Proceratophrys allied to the P. appendiculata species complex by the presence of single and long palpebral appendages and a triangular rostral appendage. Proceratophrys izecksohni sp. nov. is characterized by having small to medium size (SVL 32.1-54.2 mm in males), elongated hindlimbs (thigh length plus tibia length corresponding to more than 90% of snout-vent length), a broad head (head width corresponding to 55% of the snout-vent length), and by the light brown gular region and a cream colored ventral surface with scattered brown dots. Proceratophrys belzebul sp. nov. is characterized by its medium size (SVL 40.5-51.3 mm in males), by the absence of contact between the nasals bones and between the nasals and frontoparietals, by a very reduced iliac projection, by having frontoparietal bones very depressed and broad rostrally, by the smooth surface of the squamosal and nasal, by shallow, inconspicuous ventral pits on the maxillae, and by the females presenting the gular region dark brown. The two new species were previously confused with P. appendiculata for which we provide a new diagnosis. A molecular analysis based on mitochondrial and nuclear genes recovers a monophyletic Proceratophrys with high support, and the two new species in a clade with P. appendiculata and P. tupinamba. The data also reinforce the idea that the species groups presently admitted to the genus are not monophyletic.

Larval chondrocranium morphology of five species of Proceratophrys Miranda-Ribeiro (Amphibia; Anura; Odontophrynidae).

The chondrocranium and hyobranchial morphology are described for the tadpoles of five species of Proceratophrys Miranda-Ribeiro. The chondrocranium of P. appendiculata, P boiei, P laticeps and P. tupinamba are very similar and can be distinguished from that of P. cristiceps by: 1) less developed processus muscularis; 2) thinner palatoquadrate; 3) broadest ethmoidal region; 4) longer cornua trabeculae; and 5) morphology f the suprarostral cartilages. A morphological variation among Proceratophrys boiei is described. It reveals the existence of at least three distinct evolutionary lineages under this name. A brief comparison with other cycloramphid species is also provided.

[Intra- and inter-specific morphometric variation between Odontophrynus populations (Anura: Cycloramphidae) of central Argentina]. / Variación morfométrica intra e interespecífica entre poblaciones de Odontophrynus (Anura: Cycloramphidae) del área central de Argentina.

Intra- and inter-specific morphometric variation between Odontophrynus populations (Anura: Cycloramphidae) of central Argentina. Morphometric analyses are particularly important, and for many years they have supported evolutionary and ecological phenomena, and have been useful for the classification of new species, mainly to the lowest taxonomic levels. In order to assess the degree of sexual dimorphism, the intra-specific morphometric variation and the inter-specific morphological differences, we performed morphometric analyses of two morphologically cryptic species, Odontophrynus cordobae (diploid) and O. americanus (tetraploid). For this, we measured 15 morphometric variables on 211 individuals from 18 localities of Córdoba province. We found sexual dimorphism in six and three parameters in O. cordobae and O. americanus, respectively. Diploid and tetraploid males significantly differed in six morphometric variables. Discriminant Function Analysis (DFA) including all populations correctly classified a 76.37% of individuals within their respective species. DFA based on four groups (1- allopatric O. cordobae, 2-syntopic O. cordobae; 3- allopatric O. americanus; 4- syntopic O. americanus) accurately classified a 73.45% of individuals within their respective groups. Allopatric individuals of each species and, allopatric and syntopic individuals within each species were clearly segregated. Syntopic individuals of O. cordobae and O. americanus showed the lowest percentages of correct classification. Morphometric differences between sympatric diploids and tetraploids were not greater than those observed in allopatry. These findings deviate from the expected results under the hypothesis of character displacement, and they suggest that external morphological characters would not have a major influence on the recognition and choice of conspecific males by females.