Different approaches to understanding methodological adequacy in ecophysiological studies on small ectotherms.

Body temperature (Tb) variation and environmental temperature gradients are more intense in small individuals because their body size allows for a more intimate relationship between Tb and the environment. To contribute to a methodological consensus on the ecophysiology of small ectotherms, we aimed to investigate whether different approaches and methodological techniques affect the measurement of critical temperatures in a small lizard (Coleodactylus meridionalis, Sphaerodactylidae) from the Atlantic Forest of southern Bahia, Brazil, and subsequently its vulnerability assessment. We measured two metrics of thermal physiology: critical thermal minimum (CTmin) and critical thermal maximum (CTmax). In total, four types of temperature measurements (protocols) were defined. In the first protocol, we estimated CTmax/CTmin without heating/cooling rate by directly measuring the lizard's midbody temperature. In the other three protocols, we used a ramping assay with a heating/cooling rate to estimate CTmax/CTmin in the chamber (height: 11.3 cm), substrate, and Tb of the lizard, respectively. In total 116 individuals of Coleodactylus meridionalis were collected, of which 177 CTmax and 131 CTmin were performed. C. meridionalis showed a mean CTmax of 41 °C and a mean CTmin of 8.9 °C when considering the Tb protocol, which is intermediate compared to the other protocols. The substrate temperature protocol was the closest to Tb, and for this, the best method for the small lizards using an infrared thermometer.

Effects of temperature on growth, development, and survival of amphibian larvae: macroecological and evolutionary patterns.

Temperature affects the rate of biochemical and physiological processes in amphibians, influencing metamorphic traits. Temperature patterns, as those observed in latitudinal and altitudinal clines, may impose different challenges on amphibians depending on how species are geographically distributed. Moreover, species' response to environmental temperatures may also be phylogenetically constrained. Here, we explore the effects of acclimation to higher temperatures on tadpole survival, development, and growth, using a meta-analytical approach. We also evaluate whether the latitude and climatic variables at each collection site can explain differences in species' response to increasing temperature and whether these responses are phylogenetically conserved. Our results show that species that develop at relatively higher temperatures reach metamorphosis faster. Furthermore, absolute latitude at each collection site may partially explain heterogeneity in larval growth rate. Phylogenetic signal of traits in response to temperature indicates a non-random process in which related species resemble each other less than expected under Brownian motion evolution (BM) in all traits, except survival. The integration of studies in a meta-analytic framework allowed us to explore macroecological and macroevolutionary patterns and provided a better understanding of the effects of climate change on amphibians.

Tracking the little ones: use of fluorescent powder to follow a leaf litter lizard (Coleodactylus meridionalis, Squamata: Sphaerodactylidae) in the Atlantic Forest of southern Bahia, Brazil.

The ecology of movement is an expanding area, marked by the diversity of analytical methods and protocols, which enables this integrative reading. We investigated movement ecology aspects of Coleodactylus meridionalis in southern Bahia, northeastern Brazil, using fluorescent powder with mineral oil to track individuals. We monitored 69 individuals of C. meridionalis that walked an average distance of 148 cm in 2h. We identified this movement as foraging due to the orientation of the step sequence and microenvironments used. We find no significant differences between walking distance and weight. However, we found a decrease in activity over the follow-up period. Most of the lizard's movements were directed north, while south, east, and west were followed equally. The individuals stayed predominantly on the ground (leaf litter), but it was possible to observe the use of other surfaces, such as trunks and burrows on the ground. Therefore, we studied the movement in three dimensions (ground height, distance traveled, and orientation of steps). We observed the lizard's foraging, one of the most common and least investigated movements in small lizards like C. meridionalis. This involves not only the species' activity schedule but other intrinsic and extrinsic factors that shape the movement decisions of individuals.

Roundup Original DI® and thermal stress affect survival, morphology and thermal tolerance in tadpoles of Boana faber (Hylidae, Anura).

In amphibians, stressful environments can lead to accelerated metamorphosis at the expense of total length, resulting in the occurrence of morphological abnormalities. Many studies have linked the occurrence of these phenomena to the pollution of habitats by pesticides and thermal stress. Here, we assessed how exposure to Roundup Original DI® and higher constant temperatures affect the survival of Boana faber tadpoles and estimate the CL5096hs for the population. In addition, we evaluated how exposure to Roundup affects larval growth, morphology and thermal tolerance. Our findings suggest that even at sublethal doses, Roundup Original DI® may affect the survival of Boana faber larvae. There also appears to be an additive effect between Roundup and temperature increase on larval survival, however, we need to further explore this point to determine a pattern, proving to be a promising issue to be investigated in the future. We observed effects of chronic exposure to the herbicide formulation on the morphology and growth of the tadpoles, resulting in a reduction in total length and differences in the shape of the larvae. Although we did not recover any direct effects of herbicide exposure on CTMax, we did observe an upward trend in CTMax for tadpoles exposed to Roundup. Understanding how anthropogenic changes affect anuran persistence is fundamental for the management and conservation of the species and can be considered an initial step toward the formulation of legislations that regulate the use of herbicides.

DNA barcode reveals candidate species of Scinax and Ololygon (Anura: Hylidae) in Atlantic Forest.

Molecular species delimitation methods are efficient tools to identify species, including the discovery of new taxa and cryptic organisms, thus being useful to biodiversity studies. In the present work, 16S mitochondrial sequences and cytochrome oxidase I (COI) were used to evaluate the richness of species in the genus Scinax and Ololygon from a biodiversity hotspot in Atlantic Forest. A total of 109 specimens formally belonging to eight species of Scinax and three species of Ololygon were collected in 13 localities along the state of Bahia (northeastern Brazil) and one site in Espírito Santo (southeastern Brazil). Of the Scinax species collected in this study, three were morphologically differentiated from other described species and identified as putative new species (Scinax sp.1, Scinax sp.2 and Scinax sp.3). The species delimitations were inferred using three different methods: ABGD, PTP and mPTP which allowed recognizing 11 Scinax species and five Ololygon species. Scinax sp. 1, Scinax sp. 2 and Scinax sp. 3, have been confirmed as new putative species and Ololygon argyreornata possibly contains cryptic species. We suggest additional studies, including morphological and bioacoustic data to validate these new putative species.

Quaternary climatic fluctuations influence the demographic history of two species of sky-island endemic amphibians in the Neotropics.

We evaluated the role of Quaternary climatic fluctuations on the demographic history and population structure of amphibian species endemic to the 'campo rupestre' in the Neotropics, evaluating their distributional shifts, demographic changes, and lineage formation from the end of Pleistocene to present. We chose two anurans endemic to the high-elevation 'campo rupestre' in the Espinhaço Range (ER) in northeastern and southeastern Brazil (Bokermannohyla alvarengai and Bokermannohyla oxente), as models to test the role of Quaternary climatic fluctuations over their distribution range in this region. We collected tissue samples throughout their distribution range and used statistical phylogeography to examine processes of divergence and population demography. We generated spatial-temporal reconstructions using Bayesian inference in a coalescent framework in combination with hind-cast projections of species distribution models (SDMs). We also used the results and literature information to test alternative diversification scenarios via approximate Bayesian computation (ABC). Our results show that Quaternary climatic fluctuations influenced the geographic ranges of both species showing population expansion during the last glacial maximum (LGM) and range contraction during interglacial periods, as inferred from selected ABC models and from past projections of SDMs. We recovered Pleistocene diversification for both species occuring in distinctly unique periods for each taxon. An older and range-restricted lineage was recovered in a geographically isolated geological massif, deserving conservation and further taxonomic study. The diversification and distribution of these amphibian species endemic to the Neotropical 'campo rupestre' were influenced by Quaternary climatic fluctuations. The expansion of cold adapted species restricted to higher elevations during glacial periods and their concomitant retraction during interglacial periods may have been crucial for producing patterns of species richness and endemism along elevation gradients in tropical and subtropical domains. Such processes may influence the evolution of the biota distributed in heterogeneous landscapes with varied topography.

The phylogeny of Dendropsophini (Anura: Hylidae: Hylinae).

The relationships of the hyline tribe Dendropsophini remain poorly studied, with most published analyses dealing with few of the species groups of Dendropsophus. In order to test the monophyly of Dendropsophini, its genera, and the species groups currently recognized in Dendropsophus, we performed a total evidence phylogenetic analysis. The molecular dataset included sequences of three mitochondrial and five nuclear genes from 210 terminals, including 12 outgroup species, the two species of Xenohyla, and 93 of the 108 recognized species of Dendropsophus. The phenomic dataset includes 46 terminals, one per species (34 Dendropsophus, one Xenohyla, and 11 outgroup species). Our results corroborate the monophyly of Dendropsophini and the reciprocal monophyly of Dendropsophus and Xenohyla. Some species groups of Dendropsophus are paraphyletic (the D. microcephalus, D. minimus, and D. parviceps groups, and the D. rubicundulus clade). On the basis of our results, we recognize nine species groups; for three of them (D. leucophyllatus, D. microcephalus, and D. parviceps groups) we recognize some nominal clades to highlight specific morphology or relationships and facilitate species taxonomy. We further discuss the evolution of oviposition site selection, where our results show multiple instances of independent evolution of terrestrial egg clutches during the evolutionary history of Dendropsophus.

The phylogeny of the Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini).

The South American and West Indian Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini) include 85 species. These are notably diverse in morphology (e.g. disparate levels of cranial hyperossification) and life history (e.g. different reproductive modes, chemical defences), have a wide distribution, and occupy habitats from the tropical rainforests to semiarid scrubland. In this paper, we present a phylogenetic analysis of this hylid tribe based on sequence fragments of up to five mitochondrial (12S, 16S, ND1, COI, Cytb) and six nuclear genes (POMC, RAG-1, RHOD, SIAH, TNS3, TYR). We included most of its species (> 96%), in addition to a number of new species. Our results indicate: (i) the paraphyly of Trachycephalus with respect to Aparasphenodon venezolanus; (ii) the nonmonophyly of Aparasphenodon, with Argenteohyla siemersi, Corythomantis galeata and Nyctimantis rugiceps nested within it, and Ap. venezolanus nested within Trachycephalus; (iii) the polyphyly of Corythomantis; (iv) the nonmonophyly of the recognized species groups of Phyllodytes; and (v) a pervasive low support for the deep relationships among the major clades of Lophyohylini, including C. greeningi and the monotypic genera Itapotihyla and Phytotriades. To remedy the nonmonophyly of Aparasphenodon, Corythomantis, and Trachycephalus, we redefined Nyctimantis to include Aparasphenodon (with the exception of Ap. venezolanus, which we transferred to Trachycephalus), Argenteohyla, and C. galeata. Additionally, our results indicate the need for taxonomic work in the following clades: (i) Trachycephalus dibernardoi and Tr. imitatrix; (ii) Tr. atlas, Tr. mambaiensis and Tr. nigromaculatus; and (iii) Phyllodytes. On the basis of our phylogenetic results, we analyzed the evolution of skull hyperossification and reproductive biology, with emphasis on the multiple independent origins of phytotelm breeding, in the context of Anura. We also analyzed the inter-related aspects of chemical defences, venom delivery, phragmotic behaviour, co-ossification, and prevention of evaporative water loss.

Cryptic diversity and ancient diversification in the northern Atlantic Forest Pristimantis (Amphibia, Anura, Craugastoridae).

The Neotropical region is known both for its megadiverse fauna and for the significant deficiency of our knowledge on species limits. The Amazon and Atlantic Forest are the two most diverse and large rainforests in South America, and they harbor many groups of sister species and sister genera. The frog genus Pristimantis is the most speciose genus of terrestrial vertebrates with 546 species, but only three of them occur in the Atlantic forest. Herein, we investigate the diversification history and phylogenetic relationship among the Atlantic Forest Pristimantis lineages in a spatial-temporal framework, using mitochondrial and nuclear genes. Our results reveal a remarkable hidden diversity, with nine highly structure lineages that may correspond to undescribed species, with many cases of sympatry among these divergent lineages. Atlantic forest Pristimantis form a monophyletic group that started to diversify over 40 million years ago. This ancient group shows diversification events that remount the early bursts of the Atlantic Forest diversification history, as well as lineage diversification likely resulting from recent Pleistocene climatic fluctuations. Future work must concentrate in comparing these lineages under an integrative framework including morphology, advertisement calls and other ecology traits to confidently delimit species of Pristimantis in the Atlantic Forest.

Searching for Networks: Ecological Connectivity for Amphibians Under Climate Change.

Ecological connectivity depends on key elements within the landscape, which can support ecological fluxes, species richness and long-term viability of a biological community. Landscape planning requires clear aims and quantitative approaches to identify which key elements can reinforce the spatial coherence of protected areas design. We aim to explore the probability of the ecological connectivity of forest remnants and amphibian species distributions for current and future climate scenarios across the Central Corridor of the Brazilian Atlantic Forest. Integrating amphibian conservation, climate change and ecological corridors, we design a landscape ranking based on graph and circuit theories. To identify the sensitivity of connected areas to climate-dependent changes, we use the Model for Interdisciplinary Research on Climate by means of simulations for 2080-2100, representing a moderated emission scenario within an optimistic context. Our findings indicate that more than 70% of forest connectivity loss by climate change may drastically reduce amphibian dispersal in this region. We show that high amphibian turnover rates tend to be greater in the north-eastern edges of the corridor across ensembles of forecasts. Our spatial analysis reveals a general pattern of low-conductance areas in landscape surface, yet with some well-connected patches suggesting potential ecological corridors. Atlantic Forest reserves are expected to be less effective in a near future. For improved conservation outcomes, we recommend some landscape paths with low resistance values across space and time. We highlight the importance of maintaining forest remnants in the southern Bahia region by drafting a blueprint for functional biodiversity corridors.

Genetic analysis reveals candidate species in the Scinax catharinae clade (Amphibia: Anura) from Central Brazil.

Scinax (Anura: Hylidae) is a species-rich genus of amphibians (113 spp.), divided into five species groups by morphological features. Cladistic analyses however revealed only two monophyletic clades in these groups: Scinax catharinae and Scinax ruber. Most species from the S. catharinae clade are found in Atlantic rainforest, except for Scinax canastrensis,S. centralis, S. luizotavioi, S. machadoi,S. pombali and S. skaios. In the present work, specimens of Scinax collected in Chapada dos Guimarães, central Brazil, were morphologically compatible with species from theS. catharinae group. On the other hand, genetic analysis based on mitochondrial (16S and 12S) and nuclear (rhodopsin) sequences revealed a nucleotide divergence of 6 to 20% between Scinax sp. and other congeners from the Brazilian savannah (Cerrado). Accordingly, Bayesian inference placed Scinax sp. in the S. catharinae clade with high support values. Hence, these findings strongly indicate the presence of a new species in the S. catharinae clade from the southwestern portion of the Brazilian savannah. To be properly validated as a novel species, detailed comparative morphological and bioacustic studies with other taxa from Brazil such asS. canastrensis, S. centralis, S. luizotavioi, S. machadoi, S. pombali and S. skaios are required.

Cytogenetic studies in six species of Scinax (Anura, Hylidae) clade Scinax ruber from northern and northeastern Brazil.

Scinax species are still underrepresented in cytogenetic studies, mainly with respect to populations from northeastern and northern Brazil. In this study, we provide new chromosomal information on Scinax boesemani, S. camposseabrai, S. garbei, S. pachycrus, S. trilineatus and S. x-signatus, all belonging to clade S. ruber. They were collected at two locations in the Caatinga biome (northeastern Brazil) and at one in the Amazon (northern Brazil) biomes. Chromosomes were analyzed by conventional staining, C-banding, Ag-NOR staining, and fluorochrome staining. All species shared a modal diploid value of 2n = 24 and fundamental arm number (FN) of 48. Moreover, both chromosomal size and morphology were similar to other species in this Scinaxclade. C-banding revealed centromeric heterochromatin in all species, along with terminal species-specific C-bands in some species. Active nucleolar organizer regions (Ag-NORs) were identified at 11q in most species, except for S. boesemani and S. garbei (Ag-NORs at interstitial region of 8q). Differing from most anurans, GC-rich regions were not restricted to NORs, but also coincident with some centromeric and terminal C-bands. These data contribute to the cytotaxonomy of Scinax by providing chromosomal markers and demonstrating the occurrence of microstructural rearrangements and inversions on chromosomal evolution of Scinax.

Diversity patterns, research trends and mismatches of the investigative efforts to amphibian conservation in Brazil.

The number of papers on biology of amphibians has increased in the recent years. A detailed overview of the publications on amphibians can be very useful in assessing the status of our knowledge about this taxonomic group. Due to the large number of articles published, we aimed to assess the scientific contribution of herpetological researches carried out between the years 2001 and 2010 on Brazilian amphibians, considering the diversity patterns, the threats and the research topics that have been published most often. We applied scientometric attention indexes in the reviewed studies from seven scientific databases. To examine the relationship between the numbers of species recorded locally and regionally at different spatial scales, we used an additive partitioning of diversity in three hierarchical levels (i.e., states, geographic regions and biomes). We evaluated 892 articles and 914 species, which showed that 65 % of the total diversity of Brazilian amphibian species was represented by the beta diversity among the biomes. We identified many differences in the allocation of research efforts for taxonomic groups, threats categories, geographic regions, and research topics, highlighting the main research trends conducted and the priority themes for investigation of further papers on Brazilian amphibians.

Reproductive biology of a bromeligenous frog endemic to the Atlantic Forest: Aparasphenodon arapapa Pimenta, Napoli and Haddad, 2009 (Anura: Hylidae).

The genus Aparasphenodon is restricted to South America and comprises five poorly studied species which present a straight relationship with bromeliads. Herein we present the reproductive biology of the species Aparasphenodon arapapa. Our observations indicate that A. arapapa is a prolonged breeder, reproducing throughout the year using bromeliads as a calling and breeding site. The tadpoles complete their development inside those plants. Males, females and juveniles may also use the bromeliads as a shelter. We also describe the courtship behavior and the parental care of the species with an evidence of temporary fidelity between male and female and propose a new reproductive mode.

Influence of conspecifics and road noise on the properties of the advertisement call of Phyllodytes luteolus (Anura, Hylidae).

Acoustic communication in animals can be affected by multiple biotic (intra and interspecific) and abiotic (e.g., wind and rain) natural noises. In addition, human beings produce additional novel sources of noise, which can reduce or inhibit the reception of acoustic signals by conspecifics, leading to behavioral changes. In this study, we investigated whether sound of conspecifics and road noise additively affect the acoustic parameters of the advertisement call of males of a Yellow Heart-tongued Frog (Phyllodytes luteolus). We hypothesized that males that vocalize in choruses (males calling nearby) and in areas close to highways (anthropic noise) will increase their temporal and spectral acoustic parameters, respectively, to avoid acoustic signal masking. We recorded the vocalizations of 38 males in environments close (N = 18) to and distant (N = 20) from highways in different social contexts (many or few individuals calling nearby). Contrary to our expectation, the results indicated that males calling in areas close to highways had lower dominant frequency calls than those from natural areas (far from highways), and that the density of males in the chorus had no influence on the acoustic parameters. Furthermore, we found a positive relationship between body size and intensity, indicating that larger individuals can emit calls that can reach greater distances. The advertisement call of Phyllodytes luteolus has a high dominant frequency, with little overlap with the frequency of anthropic noises (roads), which may explain its presence and reproductive success of this species in bromeliads from urbanized areas.

Phenotypic variation of Leptodactylus cupreus Caramaschi, São-Pedro and Feio, 2008 (Anura, Leptodactylidae).

This study describes for the first time the female of Leptodactylus cupreus and provides new information concerning its geographical distribution, male's morphology and bioacustics. Leptodactylus cupreus, a poorly known species from the Brazilian Atlantic Forest, was originally allocated in the L. mystaceus complex of the L. fuscus species group. Based on morphological observations, we infer that L. cupreus should be in fact related to L. mystacinus, a species that, although assigned to the L. fuscus species group, is not assigned to the L. mystaceus complex. Therefore, we comment the phylogenetic relationships concerning L. cupreus, L. mystaceus and L. mystacinus.

Captive breeding, embryonic and larval development of Ranitomeyavariabilis (Zimmermann & Zimmermann, 1988), (Anura, Dendrobatidae).

A solid basis to address the conservation challenges of amphibians requires an increased knowledge on their natural history and biology. Recent data on reproductive modes in amphibians suggest that they are much more complex and variable than previously thought but understudied. However, detailed information on the reproductive history is especially important to fill the current knowledge gaps. Following recent taxonomic changes in Ranitomeyavariabilis, information about captive-breeding management, image-based measurements of total length and surface area of the silhouette for individuals from embryonic to metamorphic development, and detailed larval staging for captive-bred specimens are provided from French Guiana. The development of R.variabilis from the stage eight (Gosner 1960) through metamorphosis took 79 to 91 days (n = 6) with a survival rate of 46%. The developmental stages largely matched those of the generalized staging system of Gosner (1960), with differences in the stages when labia and teeth differentiation and atrophy of the oral apparatus occurred. Compared with other studies the total length of R.variabilis tadpoles was greater at given stages than those of R.variabilis from a Peruvian population and those of the sister species, R.amazonica. Other studies concerning growth curves based on surface area data revealed that R.variabilis tadpoles at peak size were larger than those of R.amazonica, R.imitator, R.reticulata, R.sirensis, and R.vanzolinii, but smaller than R.benedicta. Our results represent the first embryonic and larval staging for R.variabilis, and detailed information is provided on their initial life phases. These data may facilitate the identification of R.variabilis tadpoles in the wild, as well helping to clarify the biogeographical distribution and taxonomic arrangement of the species. In addition, knowledge is added to the captive-breeding methodology for the species.

An elusive giant: A new species of Vitreorana Guayasamin et al., 2009 (Anura: Centrolenidae) from the northern Atlantic Forest with an osteological description and comments on integumentary spicules.

We describe a new species of Vitreorana from the Atlantic Forest of southern Bahia state, in north-eastern Brazil. Vitreorana assuh sp. nov. is by far the largest species of the genus (snout-to-vent length [SVL] = 30.9 mm and 34.1 mm in one male and female, respectively; maximum SVL recorded for all other species = 28.0 mm). The new species is morphologically most similar to V. franciscana, from the Cerrado biome in Brazil. Besides its large size, the new species has, among other diagnostic characters, the presence of vomerine teeth and the occurrence of enameled iridophores in the pericardium, hepatic and urinary bladder peritonea. Furthermore, we describe and compare the complete mineralized skeleton of the new species and V. uranoscopa, and describe and discuss the variation of integumentary spicules in all species of the genus and their relevance to Vitreorana systematics.

Following the footsteps of Burmeister's leaf frog (Phyllomedusa burmeisteri) in the Atlantic forest of Brazil.

Amphibians are organisms which mainly have a biphasic life cycle. When at the larval stage, their habitat is generally aquatic, and when adults, they become terrestrial. Pond-breeding amphibians are sensitive to some disturbances in their environment which lead to the decline of a population. The interactions between the species and their environment are performed through movement. Movement ecology combines and explains the movement data of organisms with biotic and abiotic factors and because of this, knowing the movement of these creatures is of great ecological importance. We used the spool-and-line methodology in individuals of the treefrog Phyllomedusa burmeisteri, at the Reserva Ecologica Michelin, located in the southern region of Bahia in Brazil to study their movement patterns in different environments inside and outside of the forest. We monitored 19 P. burmeisteri individuals that presented a mean total movement distance of 2160.76 cm (S.D. 1152.42). We found no significant difference in the trajectories of individuals in forested and open areas, as well as a positive relationship between the distances individuals moved and their weight only in forested areas. no relationship between distance and weight of these individuals. We observed that individuals followed non-linear paths and the number of steps from one place to another varied among individuals. Our movement ecology data allows us to answer questions about short-term movement patterns of Phyllomedusa burmeisteri. This is the first step to start understanding the spatial cognition of treefrogs from the Atlantic Forest and to fill gaps about life habits of these frogs. Information on the movement patterns of a species, as well as its home range can help to create conservation strategies, regarding the creation or delimitation of protected areas, for example.

A new species of Scinax Wagler (Hylidae: Scinaxini) from the tropical forests of Northeastern Brazil.

We describe a new species of the Scinax ruber clade from Northeastern Brazil that occurs in widely separated geographic areas in the Atlantic Forest of southern Bahia state and the Highland Humid Forest of Serra de Baturité, northeast Ceará state. Scinax tropicalia sp. nov. (holotype coordinates: -14.795694°, -39.172645°) is diagnosed from all 75 currently recognize species of the S. ruber clade by bioacoustical and morphological adult traits, such as duration (0.11-0.31 s) and dominant frequency (1.59-1.85 kHz) of the advertisement call, snout shape rounded, nearly rounded, or semi-circular in dorsal view and rounded to slightly protruding in profile, bilobate vocal sac, absence of pectoral glands and spicule-shaped papillary epidermal projections on nuptial pads, and color pattern on the dorsum of body and hidden surfaces of hindlimbs.