Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae)

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18 mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate.     

Morphology and development of additional bony elements in the genus Brachycephalus (Anura: Brachycephalidae)

We describe the extra bony elements, plates, and osteoderms present in species of the genus Brachycephalus. Samples of eight species of Brachycephalus, including seven populations of Brachycephalus ephippium, were examined. The large additional elements associated with the skull (parotic plate) and vertebrae (vertebral and paravertebral plates) all comprise intramembranous bone, similar to that of the frontoparietal or nasal bones of the skull of most of frogs. Additionally, in the dermis of one unnamed species, we discovered and described true osteoderms. We discuss the morphological nature and diversity of theses elements and their importance as evidence of phylogenetic relationship within Brachycephalus. In summary, three distinct conditions of extra bony elements occur in the genus Brachycephalus: (1) bony plates may be present or absent in species of the genus; (2) a few, small bony plates may be developed and these may be represented by (a) paravertebral plates small and restricted to the distal ends of the transverse processes of the presacral IV, (b) parotic plates small and not covering the tops of the squamosals, and (c) ornamented spinal plates on all vertebrae; and (3) well‐developed bony plates may be present as (a) paravertebral plates forming a ‘bone‐shield’ on the dorsal surface of the trunk, ornamented, and visible through the integument, (b) parotic plates covering the tops of the squamosals, and (c) spinal plates associated with all vertebrae, and ornamented on vertebrate I–VI. Although the phenomenon of miniaturization may be associated with the appearance of new elements in at least some of the species in the genus, the traditional rule may not be universally applicable. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 752–767.     

The mitochondrial genome of Brachycephalus brunneus (Anura: Brachycephalidae), with comments on the phylogenetic position of Brachycephalidae

The mitochondrial genome of Brachycephalus brunneus was determined by next-generation sequencing of mitochondrial DNA. Without its control region, it has a total length of 15,485 bp, consisting of 37 genes: 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. Except for eight tRNAs and the nd6 gene, all other mitochondrial genes are encoded on the heavy strand. ATG and ATC act mainly as the initial codon in 10 protein-coding genes, whereas nd2 and cox1 use ATT and nad3 uses ATA. Gene order is generally consistent with that observed in closely-related families. The cloverleaf structures for trnS1 and trnC lacked the DHU-stem and DHU-loop, respectively. Phylogenetic analyses of mitogenomes of closely-related families indicate that Brachycephalidae is more closely-related to Craugastoridae than to Eleutherodactylidae. This is the first sequenced mitochondrial genome for the entire Brachycephalidae and can provide the basis for the development of mitochondrial markers for other members of the family, including many species that are critically endangered.     

Ultrastructure variation in the spermatozoa of Pseudopaludicola frogs (Amphibia,Anura, Leptodactylidae), with brief comments on its phylogenetic relevance

The taxonomic history of the small frogs of the genus Pseudopaludicola from South America has been controversial. Phylogenetic inferences based on molecular data have identified four Pseudopaludicola clades, correlating with the known variation in karyotypes (2n = 22, 20, 18, and 16). In this study, the ultrastructure of the spermatozoa was analyzed in 12 species of the Pseudopaludicola, with the aim of describing their morphology and identifying characters that may contribute to a better understanding of the phylogenetic relationships. The spermatozoa presented marked differences in tail structures. The tails of the spermatozoa of the species with 2n = 22 chromosomes (Pseudopaludicola sp. 1 [P. pusilla group], Pseudopaludicola falcipes, P. mineira, and Pseudopaludicola saltica), as well as Pseudopaludicola ameghini and Pseudopaludicola ternetzi (2n=20), have juxta‐axonemal fibers, undulating membranes and axial fibers. In contrast, in the species with 2n = 18 (P. facureae, P. giarettai, Pseudopaludicola canga, P. atragula, and Pseudopaludicola sp. 2) and 2n = 16 (Pseudopaludicola mystacalis), there are no evident axial or juxta‐axonemal fibers, but a paraxonemal rod with a thick undulating membrane, which is shorter than that found among Pseudopaludicola species. The ultrastructural morphological differences observed in the spermatozoa of these species may be phylogenetically informative, given that they coincide with the consensus phylogeny of the group and appear to represent a progressive simplification of the spermatozoon. J. Morphol. 276:1495–1504, 2015. © 2015 Wiley Periodicals, Inc.     

Chondrocranial, hyobranchial and internal oral morphology in larvae of the basal bufonid genus Melanophryniscus (Amphibia: Anura)

Melanophryniscus is a genus of small toads inhabiting the southern portion of South America. This genus is considered basal within the family Bufonidae. Data on larval chondrocranial morphology do not exist for the genus and larval internal oral anatomy has only been described for a single species. Here, we describe chondrocranial and internal oral morphology in Melanophryniscus montevidensis , M. orejasmirandai and M. sanmartini . Chondrocranial morphology is similar among the species examined. Comparisons with other bufonids and with outgroup taxa suggest that the following chondrocranial characters may represent synapomorphies for the Bufonidae: free (or absent) ceratobranchial IV, a reduced or absent larval crista parotica, the lack of a larval otic process, and late development of thin, poorly chondrified orbital cartilages. The presence of an elongated processus anterior dorsalis of the suprarostral alae and the absence of a chondrified commissura quadratoorbitalis appear to be unique in Melanophryniscus among bufonids. Internal oral anatomy is conserved in Melanophryniscus , and among bufonids in general.     

A new genus and three new species of miniaturized microhylid frogs from Indochina(Amphibia: Anura:Microhylidae: Asterophryinae)

We report on the discovery of a new genus of microhylid subfamily Asterophryinae from northern and eastern Indochina, containing three new species.Vietnamophryne Gen. nov. are secretive miniaturized frogs(SVL21 mm) with a mostly semi-fossorial lifestyle. To assess phylogenetic relationships, we studied 12 S rRNA – 16 S rRNA mt DNA fragments with a final alignment of 2 591 bp for 53 microhylid species. Morphological and osteological characters were analyzed using micro-CT scanning and used to describe the new genus. Results of phylogenetic analyses assigned the new genus into the mainly Australasian subfamily Asterophryinae as a sister taxon to the genus Siamophryne from southern Indochina. The three specimens collected from Gia Lai Province in central Vietnam, Cao Bang Province in northern Vietnam, and Chiang Rai Province in northern Thailand proved to be separate species, different both in morphology and genetics(genetic divergence 3.1%≤P≤5.1%). Our work provides further evidence for the "out of Indo-Eurasia"scenario for Asterophryinae, indicating that the initial cladogenesis and differentiation of this group of frogs occurred in the Indochina Peninsula. To date, each of the three new species of Vietnamophryne Gen. nov. is known only from a single specimen; thus,their distribution, life history, and conservation status require further study.     

Cranial ontogeny in Philautus silus (Anura: Ranidae: Rhacophorinae) reveals few similarities with other direct-developing anurans

Direct development has evolved in rhacophorine frogs independently from other anuran lineages, thereby offering an opportunity to assess features associated with this derived life history. Using a developmental series of the direct-developing Philautus silus (Ranidae: Rhacophorinae) from Sri Lanka, we examine features of cranial morphology that are part of a suite of adaptations that facilitate feeding in free-living tadpoles, but have been changed or lost in other direct-developing lineages. Larval-specific upper jaw cartilages, which are absent from many non-rhacophorine direct-developing species (such as Eleutherodactylus coqui), develop in embryos of P. silus. Similarly, lower jaw cartilages initially assume a larval morphology, which is subsequently remodeled into the adult jaw configuration before hatching. However, the cartilaginous jaw suspension and hyobranchial skeleton never assume a typical larval morphology. The palatoquadrate, which suspends the lower jaw, lacks the posterior connections to the braincase found in many metamorphosing species. Unlike in metamorphosing species, bone formation in P. silus begins before hatching. However, the sequence of bone formation resembles that of metamorphosing anurans more than that of other direct developers. In particular, P. silus does not exhibit precocious ossification of the lower jaw, which is characteristic of some frogs and caecilians that lack a free-living tadpole. These data reveal some similarities between Philautus and other direct-developing anurans. However, the departure of Philautus embryos from the generalized tadpole skeletal morphology is less pronounced than that observed in other direct-developing taxa.     

Dissimilarities in auditory tuning in midwife toads of the genus Alytes (Amphibia: Anura)

The auditory sensitivity in three species of the anuran genus Alytes (Alytidae) was examined to determine patterns of intra‐ and interspecific variation, relating these measurements to behavioural preferences measured in previous studies and to the adaptive and evolutionary significance of this sensory function. The audiograms obtained with multi‐unit recordings in the torus semicircularis of 13 Alytes cisternasii, 10 Alytes obstetricans, and eight Alytes dickhilleni show two regions of enhanced sensitivity, between approximately 100–500 and 1200–2400 Hz, with minimum thresholds at approximately 40 and 45 dB SPL, respectively. The mean and range of the high‐frequency region differed among species, although the sensitivity, measured as minimum thresholds, was similar. The region of high‐frequency sensitivity was centred at approximately the frequency of the advertisement call in A. cisternasii but, in A. obstetricans and A. dickhilleni, was centred at frequencies higher than the conspecific calls. These results contrast with preferences for lower frequencies exhibited by Alytes in female phonotactic and in male evoked vocal responses. Such loose relationships between signals and receivers suggest that the divergence of the sound communication system in Alytes has implied environmental and phylogenetic factors in addition to sexual selection processes.     

Development and variation of the anuran webbed feet (Amphibia, Anura)

Webbed feet evolved convergently in most groups of aquatic tetrapods. However, extensive webbing is not always limited to an aquatic life style. In Anurans, hind limbs display great variation, including absence, of interdigital membranes, which is explained by differential growth rates of digital and interdigital tissues during early limb development. In order to explore web diversification in anurans, this paper presents analyses of: (1) hind limb early development and its relationship to the expression of interdigital membranes; (2) intraordinal variation of interdigital membranes in adult feet; and (3) intraordinal variation of metatarsal and digit lengths, including comments on metatarsal development. Study of limb development is carried out in larval series of 12 anuran species. Analysis of intraordinal variation comprises a sample of adults of 111 species. We recognize two configurations in the autopodium bud: (1) paddle-like shape with digits differentiated within the confines of interdigital tissues, and (2) pointed autopodium with digits differentiated beyond interdigital tissues. These early differences are conserved in adult morphology, in which allometry and isometry of digit IV (and metatarsal IV) with respect to other digits (and metatarsals) result in asymmetrical and paddle-like autopodium, respectively. The paddle-like autopodium is restricted to fossil and extant pipids and the hylids Pseudis and Lysapsus , whereas the asymmetrical one is present in most anurans. Both configurations seem to represent an early divergence of the autopodium shape. The paddle-like configuration observed in hylids appears as a reversion to an ancient condition that results from a conserved program of limb development.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 39–58.     

Variation of apomorphic characters in stream-dwelling tadpoles of the bufonid genus Ansonia (Amphibia: Anura)

Six larval forms of the bufonid genus Ansonia from Borneo share the following synapomorphies: cup-like, ventral oral disc; an expanded post-dental portion of the lower lip, which has a papillate margin; upper keratinized jaw sheath divided; body markedly flattened ventrally; eyes set relatively far back on the body. All of these tadpoles live on the bottom in strong currents, except for larval Ansonia leptopus , which lives in drifts of dead leaves that accumulate in eddies within streams. These larval forms differ among themselves in body shape, development of inframarginal papillae on the lower lip, size of the gap between the keratinized parts of the upper jaw sheath, width of the post-dental portion of the lower lip, relative lengths of upper and lower rows of labial teeth, and arrangement of the gut coils. One form has an abdominal sucker. Changes in these characters are not correlated; the derived condition in one character is not always associated with the derived state in another. Consequently, these tadpoles cannot be arranged in a simple morphocline from least to most derived, again with the exception of A. leptopus , which is the least modified in all respects. Although tadpoles of Ansonia resemble those of the neotropical bufonid genus Atelopus in general specialization for benthic life in flowing water, they differ from that group in body form and details of the oral disc.     

The mating calls of 12 species and sub-species of the genus Xenopus (Amphibia : Anura)

The principal acoustic characteristics of the mating calls of 12 species and sub-species of the genus Xenopus were determined: X. laevis laevis, X. laevis petersi, X. laevis victorianus, X. gilli, X. muelleri, X. borealis, X. clivii, X. fraseri, X. ruwenzoriensis, X. wittei, X. vestitus and X. tropicalis . These calls are very specific, especially among species whose hybrids are particularly viable in the laboratory. Twin species have not been discovered. Most of the species emit calls with high harmonic frequencies of 16 kHz; these frequencies reach 80 and 150 kHz in X. I. laevis and X. ruwenzoriensis , the ultra-sound level. This was a previously unknown phenomenon in the Batrachians.     

蛙科精子形态结构研究进展

本文对已有的蛙科精子形态结构的研究进行总结,归纳了蛙科精子形态结构及量度的特征和类型,以及蛙科精子结构与其它两栖类精子结构的异同,探讨蛙科精子形态结构研究中存在和亟待解决的问题。     

Interspecific patterns for egg and clutch sizes of African Bufonidae (Amphibia: Anura)

Little is known about reproductive trade-offs in African amphibians, but such data, particularly in the form of quantitative measurements, are a key for investigating life history evolution. Here we compile and analyze known data on African bufonids from published material and new data from preserved museum specimens, to investigate interspecific patterns of egg and clutch sizes variation. Our data is a composite of mixed sources, including ova data from dissected females and laid clutches from observations in the field. Our study shows that, as body size increases, clutch size increases but egg size decreases, and when correcting for body size, egg size is inversely correlated with clutch size. These parameter interactions however, are different for different reproductive modes. In free-swimming larval developing species, the same trends are recovered, but for lecithotrophic viviparous species no significant correlations could be recovered for clutch size and body size nor for the trade-off between clutch size and egg size, and egg size is positively related to body size. The egg size of Nimbaphrynoides occidentalis (Angel, 1943) is a clear outlier, which may be due to its matrotrophic viviparous reproduction. In addition, we observed no statistical difference between ova data collected from dissections and laid clutch data from field observations, which suggests that such a mixed dataset has utility in comparative analyses.     

Discerning evolutionary processes in patterns of tamarin (genus Saguinus) craniofacial variation

Quantitative genetic theory specifies evolutionary expectations for morphological diversification by genetic drift in a monophyletic clade. If genetic drift is responsible for the evolutionary morphological diversification of a clade, patterns of within- and between-taxon morphological variance/covariance should be proportional. We tested for proportionality of within- and between-species craniofacial morphological variation in 12 species of tamarins (genus Saguinus). We found that within- and between-taxon morphological variations across the entire genus were not proportional, and hence not likely to be due to genetic drift alone. The primary deviation from proportionality is that size and size-related shape in the cranium is more variable relative to other aspects of cranial morphology than expected under genetic drift, suggesting differential size selection between the two major clades, the small-bodied and large-bodied tamarins. Within each of these major clades, most of the interspecific variation is consistent with the pattern expected under genetic drift, although specific contrasts may indicate the involvement of differential selection. Morphological distances among taxa do not correspond very closely to the phylogeny derived from mtDNA. In particular, S. oedipus and S. geoffroyi are very distinct morphologically from the rest of the tamarins, although they are phylogenetically the sister clade to a clade containing S. midas and S. bicolor. Morphological similarity is not a good guide to phylogenetic affinity in the tamarins, especially with regard to deeper nodes in the phylogenetic tree.