Vocal repertoire of Microhyla nilphamariensis from Delhi and comparison with closely related M. ornata populations from the western coast of India and Sri Lanka.

Advertisement calls in frogs have evolved to be species-specific signals of recognition and are therefore considered an essential component of integrative taxonomic approaches to identify species and delineate their distribution range. The species rich genus Microhyla is a particularly challenging group for species identification, discovery and conservation management due to the small size, conserved morphology and wide distribution of its members, necessitating the need for a thorough description of their vocalization. In this study, we provide quantitative description of the vocal behaviour of Microhyla nilphamariensis, a widely distributed south Asian species, from Delhi, India, based on call recordings of 18 individuals and assessment of 21 call properties. Based on the properties measured acrossed 360 calls, we find that a typical advertisement call of M. nilphamariensis lasts for 393.5 ±  57.5 ms, has 17 pulses on average and produce pulses at rate of 39 pulses/s. The overall call dominant frequency was found to be 2.8 KHz and the call spectrum consisted of two dominant frequency peaks centered at 1.6 KHz and 3.6 KHz, ranging between 1.5-4.1 KHz. Apart from its typical advertisement call, our study also reveals the presence of three 'rare' call types, previously unreported in this species. We describe variability in call properties and discuss their relation to body size and temperature. We found that overall dominant frequency 1 (spectral property) was found to be correlated with body size, while first pulse period (temporal property) was found to be correlated with temperature. Further, we compare the vocal repertoire of M. nilphamariensis with that of the congener Microhyla ornata from the western coast of India and Sri Lanka and also compare the call properties of these two populations of M. ornata to investigate intra-specific call variation. We find statistically significant differentiation in their acoustic repertoire in both cases. Based on 18 call properties (out of 20), individuals of each locality clearly segregate on PCA factor plane forming separate groups. Discriminant function analysis (DFA) using PCA factors shows 100% classification success with individuals of each locality getting classified to a discrete group. This confirms significant acoustic differentiation between these species as well as between geographically distant conspecifics. The data generated in this study will be useful for comparative bioacoustic analysis of Microhyla species and can be utilized to monitor populations and devise conservation management plan for threatened species in this group.

Prey capture kinematics of horned frogs (Anura: Ceratophryidae).

Horned frogs, members of the Ceratophryidae family, encompass a group of anurans varying in size and behavior, yet unified by morphological and behavioral traits enabling them to adopt a megalophagous diet (i.e., large prey feeding). Although the group has been the focus of numerous studies, our understanding of its feeding behavior remains limited. In this study, we characterize the feeding mechanism in five species representing the three extant genera of ceratophryid anurans, both in terrestrial and aquatic environments. We also explore the ability of Chacophrys pierottii to adjust feeding behavior based on prey type. Our findings show that all species are capable of wide mouth opening, displaying an asymmetric feeding cycle. While tongue usage is the primary method for capturing prey on land, ceratophryids may use their forelimbs to manipulate prey into their mouths, exhibiting different behavioral patterns. C. pierottii shows modulation of its feeding kinematics and is also capable of some modulation of its feeding in response to prey properties.

A new species of the Scinax cruentomma group (Anura: Hylidae) from the Ucayali River basin of Loreto, Peru.

We describe a new species of the Scinax cruentomma species group, with a red streak in the iris and a weakly bilobate vocal sac. It is known from oligotrophic soils in the sedimentary basin of the Ucayali River near Jenaro Herrera (province of Requena, Peru) and Ro Blanco (buffer zone of the Matses Indigenous territory and reserve). The new species can be distinguished from the other species of the S. cruentomma group by its small snout-vent length, body and iris color patterns, weakly bilobate vocal sac, myological characters, and the number of notes and pulses of the advertisement call. It is morphologically most similar to S. strussmannae, from which the advertisement call, nostril, canthus rostralis, and loreal region can distinguish it.

The interglenoid tubercle of the atlas is ancestral to lissamphibians.

Lissamphibians, represented today by frogs, salamanders, and caecilians, diverged deep in the tetrapod tree of life. Extensive morphological adaptations to disparate lifestyles have made linking extant lissamphibians to one another and to their extinct relatives difficult and controversial. However, the discovery of a feature on the atlas of the frog Xenopus laevis, may add to the small set of osteological traits that unite lissamphibians. In this study, we combine our observations of atlas development in X. laevis with a deep examination of atlantal interglenoid tubercle (TI) occurrence in fossil taxa. The TI is shown herein to occur transiently on the ossifying atlas of roughly one-third of X. laevis tadpoles but is absent in adults of this species. In ancestral character state estimations (ACSE), within the evolutionary context of lissamphibians as dissorophoid temnospondyls, this feature is found to be ancestrally shared among lissamphibians, its presence is uncertain in stem batrachians, and then the TI is lost in extant caecilians and frogs. However, our data suggests apparent TI loss around the origin of frogs may be explained by its ontogenetically transient nature. The only nonamphibian tetrapods with a TI are "microsaurs," and this similarity is interpreted as one of many convergences that resulted from convergent evolutionary processes that occurred in the evolution of "microsaurs" and lissamphibians. The TI is thus interpreted to be ancestral to lissamphibians as it is found to be present in some form throughout each extant lissamphibian clade's history.

Distinguishing fanged frogs (Limnonectes) species (Amphibia: Anura: Dicroglossidae), from Thailand using high resolution melting analysis.

Morphologically, species of fanged frogs (Limnonectes) are exceedingly similar, making it difficult to distinguish them within the complex. In Thailand, it has been difficult to distinguish between the sympatric species L. bannaensis and L. taylori, particularly among tadpoles, adolescents, and adult females. A precise identification contributes to a greater understanding of biodiversity, particularly for assessing distributions and population dynamics. Therefore, a novel approach is required. The objective of this study was to develop a high resolution melting analysis (HRM) for the rapid and accurate identification of six species of Limnonectes of the L. kuhlii complex found in Thailand, particularly the two sympatric fanged frogs. Here, HRM assays using 16S rRNA mitochondrial primers were designed and developed. There was as much as a 25.3% variation in the nucleotide sequence of the fragment amplified by HRM16S primers among the six species of Limnonectes. Prior to conducting an in vitro HRM, the DNA sequences were used in a simulation HRM, uMELT Quartz, to predict the melting curve for each species of Limnonectes. There were discrepancies between the predicted melting curves of each species generated by the programme. Consequently, in vitro HRM tests were conducted. The obtained melting curve and Tm values were consistent with those predicted, albeit with a slightly different Tm value and a more distinct melting curve. All evaluated species of Limnonectes could be easily distinguished from one another by comparing the melting curve shapes. The HRM assay was then used to confirm the species of 18 Limnonectes samples in comparison to the reference samples (confidence interval > 90%). In addition, the results of HRM were consistent with those of experts who used morphological analysis to identify species. The HRM was found to be useful, and therefore the method would also contribute to future ecological and systematic studies on the target species.

First description of buccopharyngeal anatomy in Pelodryadinae larvae: Morphological comparison and systematic implications (Anura: Hylidae: Pelodryadinae: Litoria rubella and Ranoidea caerulea).

Pelodryadinae, the Australian tree frogs, is a monophyletic group endemic to the Australo-Papuan region. Although we have a relatively good knowledge about tadpoles' phenotypic diversity in terms of external morphology, information about internal anatomy is rare for the subfamily; for instance, their buccopharyngeal cavity is completely unknown. Herein I describe for the first time the buccopharyngeal anatomy of two pelodryadins: Litoria rubella and Ranoidea caerulea. I compare my results with available evidence from Phyllomedusidae, that is, the sister clade to Pelodryadinae, and briefly comment on buccopharyngeal cavity within Hylidae. Both species can be readily distinguished based on lateral ridge, postnarial, buccal roof arena, infralabial papillae, and lingual papillae. Variation between the two species may suggest a large diversity within Pelodryadinae. Pelodryadinae and Phyllomedusinae present similar buccopharyngeal morphologies, although Agalychnis callidryas has a unique morphology and putative apomorphic transformations can be observed in Pithecopus + Phyllomedusa, Ranoidea, and Phasmahyla.

Vascularization inside the epidermis of Neotropical anurans (Nobleobatrachia).

Anuran skin is a dynamic organ involved in essential functions that strongly correlate with specific morphological traits. Particularly, gas exchange has been associated with epidermal modifications, such as reduced cell layers and increased vascularization. Here, we describe the epidermal morphology and its association with capillary networks in the dorsal skin of 103 Neotropical anurans (Nobleobatrachia) from different ecomorphs and habitats. Additionally, we examined the lateral and ventral skin for a subset of these species. We report intraepidermal capillaries in (i) dorsal skin of Lepidobatrachus laevis and Lepidobatrachus llanensis (burrowing and semi-aquatic Chacoan species), Hyloscirtus colymba and Hyloscirtus palmeri (arboreal species from humid forests), and Alsodes neuquensis and 15 Telmatobius spp. (aquatic and semi-aquatic species from cold environments); (ii) lateral skin of Boana benitezi and H. colymba (arboreal species from humid forests), and (iii) ventral skin of B. benitezi, H. colymba, Atelognathus patagonicus (aquatic species from cold environments), and four Chacoan species, Chacophrys pierottii, Ceratophrys cranwelli (burrowing/terrestrial species), and Lepidobatrachus asper and L. llanensis (burrowing/semi-aquatic species). Also, verrucae hydrophilicae were observed exclusively in the ventral skin of Leptodactylus fuscus, Leptodactylus laticeps (terrestrial and Chacoan species), and B. benitezi. Regardless of the skin region, the capillaries always penetrate the epidermis from the dermis, while epidermal cell layers are flattened. Our findings support previous hypotheses stating that the environment where species occur influences skin changes related to cutaneous respiration (intraepidermal capillaries in different body regions) and water absorption (intraepidermal capillaries associated with verrucae hydrophilicae within ventral skin). Also, phylogeny might influence the development of these structures, as revealed by the presence of intraepidermal capillaries in almost all analyzed species of Telmatobius. Finally, the co-occurrence of verrucae hydrophilicae in the ventral skin of hylids from humid forests, and leptodactylids from the subhumid Chacoan region suggest an independent origin.

Ecology, sexual dimorphism, and jumping evolution in anurans.

Sexual dimorphism (SD) is a common feature of animals, and selection for sexually dimorphic traits may affect both functional morphological traits and organismal performance. Trait evolution through natural selection can also vary across environments. However, whether the evolution of organismal performance is distinct between the sexes is rarely tested in a phylogenetic comparative context. Anurans commonly exhibit sexual size dimorphism, which may affect jumping performance given the effects of body size on locomotion. They also live in a wide variety of microhabitats. Yet the relationships among dimorphism, performance, and ecology remain underexamined in anurans. Here, we explore relationships between microhabitat use, body size, and jumping performance in males and females to determine the drivers of dimorphic patterns in jumping performance. Using methods for predicting jumping performance through anatomical measurements, we describe how fecundity selection and natural selection associated with body size and microhabitat have likely shaped female jumping performance. We found that the magnitude of sexual size dimorphism (where females are about 14% larger than males) was much lower than dimorphism in muscle volume, where females had 42% more muscle than males (after accounting for body size). Despite these sometimes-large averages, phylogenetic t-tests failed to show the statistical significance of SD for any variable, indicating sexually dimorphic species tend to be closely related. While SD of jumping performance did not vary among microhabitats, we found female jumping velocity and energy differed across microhabitats. Overall, our findings indicate that differences in sex-specific reproductive roles, size, jumping-related morphology, and performance are all important determinants in how selection has led to the incredible ecophenotypic diversity of anurans.

Locomotor, ecological and phylogenetic drivers of skeletal proportions in frogs.

Frogs exhibit complex anatomical features of the pelvis, limbs and spine, long assumed to represent specialisations for jumping. Yet frogs employ a wide range of locomotor modes, with several taxa featuring primary locomotor modes other than jumping. Using a combination of techniques (CT imaging and 3D visualization, morphometrics, phylogenetic mapping), this study aims to determine the link between skeletal anatomy and locomotor style, habitat type and phylogenetic history, shedding new light on how functional demands impact morphology. Body and limb measurements for 164 taxa from all the recognised anuran families are extracted from digitally segmented CT scans of whole frog skeletons and analysed using various statistical techniques. We find that the expansion of the sacral diapophyses is the most important variable for predicting locomotor mode, which was more closely correlated with frog morphology than either habitat type or phylogenetic relationships. Predictive analyses suggest that skeletal morphology is a useful indicator of jumping but less so for other locomotor modes, suggesting that there is a wide range of anatomical solutions to performing locomotor styles such as swimming, burrowing or walking.

Frog hatchlings use early environmental cues to produce an anticipatory resource-use phenotype.

Developmental plasticity can occur at any life stage, but plasticity that acts early in development may give individuals a competitive edge later in life. Here, we asked if early (pre-feeding) exposure to a nutrient-rich resource impacts hatchling morphology in Mexican spadefoot toad tadpoles, Spea multiplicata. A distinctive carnivore morph can be induced when tadpoles eat live fairy shrimp. We investigated whether cues from shrimp--detected before individuals are capable of feeding--alter hatchling morphology such that individuals could potentially take advantage of this nutritious resource once they begin feeding. We found that hatchlings with early developmental exposure to shrimp were larger and had larger jaw muscles--traits that, at later stages, increase a tadpole's competitive ability for shrimp. These results suggest that early developmental stages can assess and respond to environmental cues by producing resource-use phenotypes appropriate for future conditions. Such anticipatory plasticity may be an important but understudied form of developmental plasticity.

Triassic stem caecilian supports dissorophoid origin of living amphibians.

Living amphibians (Lissamphibia) include frogs and salamanders (Batrachia) and the limbless worm-like caecilians (Gymnophiona). The estimated Palaeozoic era gymnophionan-batrachian molecular divergence1 suggests a major gap in the record of crown lissamphibians prior to their earliest fossil occurrences in the Triassic period2-6. Recent studies find a monophyletic Batrachia within dissorophoid temnospondyls7-10, but the absence of pre-Jurassic period caecilian fossils11,12 has made their relationships to batrachians and affinities to Palaeozoic tetrapods controversial1,8,13,14. Here we report the geologically oldest stem caecilian-a crown lissamphibian from the Late Triassic epoch of Arizona, USA-extending the caecilian record by around 35 million years. These fossils illuminate the tempo and mode of early caecilian morphological and functional evolution, demonstrating a delayed acquisition of musculoskeletal features associated with fossoriality in living caecilians, including the dual jaw closure mechanism15,16, reduced orbits17 and the tentacular organ18. The provenance of these fossils suggests a Pangaean equatorial origin for caecilians, implying that living caecilian biogeography reflects conserved aspects of caecilian function and physiology19, in combination with vicariance patterns driven by plate tectonics20. These fossils reveal a combination of features that is unique to caecilians alongside features that are shared with batrachian and dissorophoid temnospondyls, providing new and compelling evidence supporting a single origin of living amphibians within dissorophoid temnospondyls.

Diversity of cortical bone morphology in anuran amphibians.

The cortical bones of mammals, birds, and reptiles are composed of a complex of woven bone and lamellar bone (fibrolamellar bone) organized into a variety of different patterns; however, it remains unclear whether amphibians possess similar structures. Importantly, to understand the evolutionary process of limb bones in tetrapods, it is necessary to compare the bone structure of amphibians (aquatic to terrestrial) with that of amniotes (mostly terrestrial). Therefore, this study compared the cortical bones in the long bones of several frog species before and after metamorphosis. Using micro-computed tomography (CT), we found that the cortical bones in the fibrolamellar bone of Xenopus tropicalis (Pipoidea superfamily) and Lithobates catesbeianus (Ranoidea superfamily) froglets are dense, whereas those of Ceratophrys cranwelli (Hyloidea superfamily) are porous. To clarify whether these features are common to their superfamily or sister group, four other frog species were examined. Histochemical analyses revealed porous cortical bones in C. ornata and Lepidobatrachus laevis (belonging to the same family, Ceratophryidae, as C. cranwelli). However, the cortical bones of Dryophytes japonicus (Hylidae, a sister group of Ceratophryidae in the Hyloidea superfamily), Microhyla okinavensis (Microhylidae, independent of the Hyloidea superfamily), and Pleurodeles waltl, a newt as an outgroup of anurans, are dense with no observed cavities. Our findings demonstrate that at least three members of the Ceratophryidae family have porous cortical bones similar to those of reptiles, birds, and mammals, suggesting that the process of fibrolamellar bone formation arose evolutionarily in amphibians and is conserved in the common ancestor of amniotes.

Taxonomic reassessment and nomenclatural status of Niceforonia dolops and Hylodes philippi (Amphibia: Anura: Strabomantidae).

The taxonomic and nomenclatural status of Hylodes philippi and H. verrucosus have been in state of flux. Some problems regarding their identity were noticed in 1971, which were partly solved with the finding of the purportedly lost type of H. verrucosus in 2006 and the morphological revisions of both H. philippi and H. verrucous in 2008 and 2012, upon which they were considered conspecific and closely related to the Eleutherodactylus dolops group; H. verrucosus became a junior synonym of H. philippi, and the latter, a nomen dubium within Hypodactylus (later, Niceforonia). The reexamination of the type series of Eleutherodactylus dolops Lynch & Duellman, 1980 and the types of Hylodes verrucosus Jimnez de la Espada, 1875 and H. philippi Jimnez de la Espada, 1875, suggests that they are conspecific. Accordingly, Eleutherodactylus dolops is herein regarded as junior synonym of H. philippi and a new combination, Niceforonia philippi (Jimnez de la Espada, 1875), and a new diagnosis are granted. This species is known from a few scattered localities along the montane forest of the Amazonian versant of the Andes from Orellana and Napo provinces in northern Ecuador to Sibundoy in southwestern Colombia, and little to nothing is known about its natural history and population trends.

Revision of Litoria rothii (Anura: Pelodryadidae) from northern Australia.

Litoria rothii is a widespread pelodryadid frog with a charismatic laughing advertisement call, distributed across the Australian Monsoon Tropics and southern New Guinea. Given its large distribution spanning well-known biogeographic barriers, variation in male advertisement calls and the prevalence of unresolved species complexes in the Australian frog fauna, we examine the genetic, morphological and acoustic diversity in the species from across its range. Our analyses reveal the presence of a previously unrecognised species in western parts of the range of L. rothii sensu lato, which we describe herein as a new species. Litoria ridibunda sp. nov. is distinguished from L. rothii on the basis of paraphyly of nuclear gene trees with L. everetti from Indonesia, colour patterns on the posterior thigh and male advertisement calls. Compared to L. rothii, the new species has a less contrasting pattern on the posterior thigh and a male advertisement call with a greater number of notes per call and a greater call duration. In particular, the magnitude of call differences between the species is highest where the ranges of the two species are in proximity in north-western Queensland. Our study further emphasises the undiagnosed diversity that remains in Australian frogs, even in relatively large, charismatic, frequently encountered species that often share human dwellings.

Lessons on transparency from the glassfrog.

Transparency in glassfrogs has potential implications for human blood clotting.

Glassfrogs conceal blood in their liver to maintain transparency.

Transparency in animals is a complex form of camouflage involving mechanisms that reduce light scattering and absorption throughout the organism. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells (RBCs) that strongly attenuate light. Here, we document how glassfrogs overcome this challenge by concealing these cells from view. Using photoacoustic imaging to track RBCs in vivo, we show that resting glassfrogs increase transparency two- to threefold by removing ~89% of their RBCs from circulation and packing them within their liver. Vertebrate transparency thus requires both see-through tissues and active mechanisms that "clear" respiratory pigments from these tissues. Furthermore, glassfrogs' ability to regulate the location, density, and packing of RBCs without clotting offers insight in metabolic, hemodynamic, and blood-clot research.

Ocular lens morphology is influenced by ecology and metamorphosis in frogs and toads.

The shape and relative size of an ocular lens affect the focal length of the eye, with consequences for visual acuity and sensitivity. Lenses are typically spherical in aquatic animals with camera-type eyes and axially flattened in terrestrial species to facilitate vision in optical media with different refractive indices. Frogs and toads (Amphibia: Anura) are ecologically diverse, with many species shifting from aquatic to terrestrial ecologies during metamorphosis. We quantified lens shape and relative size using 179 micro X-ray computed tomography scans of 126 biphasic anuran species and tested for correlations with life stage, environmental transitions, adult habits and adult activity patterns. Across broad phylogenetic diversity, tadpole lenses are more spherical than those of adults. Biphasic species with aquatic larvae and terrestrial adults typically undergo ontogenetic changes in lens shape, whereas species that remain aquatic as adults tend to retain more spherical lenses after metamorphosis. Further, adult lens shape is influenced by adult habit; notably, fossorial adults tend to retain spherical lenses following metamorphosis. Finally, lens size relative to eye size is smaller in aquatic and semiaquatic species than other adult ecologies. Our study demonstrates how ecology shapes visual systems, and the power of non-invasive imaging of museum specimens for studying sensory evolution.

Six new species of Pristimantis (Anura: Strabomantidae) from Llanganates National Park and Sangay National Park in Amazonian cloud forests of Ecuador.

We describe six new species of rainfrogs of the genus Pristimantis (Strabomantidae) from Amazonian cloud forests in Ecuador. We also present a phylogeny showing the relationships of the new species. The phylogeny is based on mitochondrial genes 16S rRNA (16S), 12 rRNA (12S), NADH-ubiquinone oxidoreductase chain 1 (ND1) and the nuclear gene recombination-activating 1 (RAG1). We also describe the osteology of two of the new species using high-resolution x-ray computed tomography. The new species belong to two clades. The first clade is sister to the subgenus Huicundomantis and includes P. tamia sp. nov., P. miktos, and P. mallii. Pristimantis tamia sp. nov. is morphologically similar to P. miktos, P. mallii, P. martiae, and P. incomptus, but differs from them by lacking vocal slits and tympanic membrane and by having light greenish blue iris. Based in our results we expand the subgenus Huicundomantis to include the P. miktos species group. The second clade is remarkable by being highly divergent and consisting exclusively of new species: P. anaiae sp. nov., P. glendae sp. nov., P. kunam sp. nov., P. resistencia sp. nov., and P. venegasi sp. nov. The new species resemble P. roni, P. yanezi, P. llanganati, P. katoptroides, P. verecundus, and P. mutabilis but can be distinguished from them by lacking vocal slits and tympanic membrane and by having large dark round areas with thin clear borders in the sacral region. All six new species occur in the eastern slopes of the Ecuadorian Andes and are known from a single locality in Llanganates or Sangay National Park. We recommend assigning all of them to the Data Deficient (DD) Red List category. Based in our high-resolution x-ray tomographies, we report the presence of structures that appear to be intercalary elements. This would be the first report of such structures in Terrarana.

A spectacular new species of Hyloscirtus (Anura: Hylidae) from the Cordillera de Los Llanganates in the eastern Andes of Ecuador.

We have discovered a spectacular new species of frog in the genus Hyloscirtus, belonging to the H. larinopygion species group. The adult female is characterized by a mostly black body with large bright red spots on the dorsal and ventral surface, extremities, and toe pads. The adult male is unknown. Small juveniles are characterized by a yellow body with variable black markings on the flanks; while one larger juvenile displayed irregular orange or yellow marks on a black background color, with light orange or yellow toe pads. Additional distinctive external morphological features such as cloacal ornamentation are described, and some osteological details are imaged and analyzed. The performed phylogeny places the new species as the sister to a clade consisting of ten taxa, all of which are part of the H. larinopygion group. We use genetic distances to fit the new species into a published time-calibrated phylogeny of this group; our analysis based on the published chronology suggests that the divergence of the new species from its known congeners pre-dates the Quaternary period. The new species is currently only known only from Cerro Mayordomo, in Fundación EcoMinga´s Machay Reserve, at 2,900 m in the eastern Andes of Tungurahua province, Ecuador, near the southern edge of Los Llanganates National Park, but its real distribution may be larger.