Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Multiple origins of green coloration in frogs mediated by a novel biliverdin-binding serpin.

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.

Structure and evolution of the sexually dimorphic integumentary swelling on the hands of dendrobatid poison frogs and their relatives (Amphibia: Anura: Dendrobatoidea).

The hands of adult males of many dendrobatid poison frogs and their relatives possess swelling formed by glandular tissue hypothesized to secrete courtship pheromones delivered to the female during cephalic amplexus. Variation in the occurrence and external morphology of the swelling of finger IV has provided important evidence for dendrobatoid systematics for decades, but its underlying structure has not been investigated. We undertook a detailed comparative analysis of the integument of the hand, including both external morphology and histology, of 36 species representing the diversity of dendrobatoid frogs and several close relatives. The swelling is caused by four densely packed, hypertrophic, morpho-histochemical types of specialized mucous glands (SMGs). We observed type I SMGs on fingers II-V and the wrist, including areas that are not swollen, types II and IV exclusively on finger IV, and type III on finger IV and the wrist. Type I SMGs occur either in isolation or together with types II, III, or IV; types II, III, and IV never occur together or without type I. We delimited 15 characters to account for the variation in external morphology and the occurrence of SMGs. Our data suggest that type I SMGs are a new synapomorphy for Dendrobatoidea and that type II SMGs originated in either the most recent common ancestor (MRCA) of Dendrobatidae or independently in the MRCAs of Aromobatidae and Colostethinae, respectively, while types III and IV are restricted to Anomaloglossus. The discovery of these SMGs adds a new dimension to studies of poison frog reproductive biology, which have investigated acoustic, visual, and tactile cues in courtship, mating, and parental care across the diversity of Dendrobatoidea for decades but have almost entirely overlooked the possible role of chemical cues.

A closer look at pupil diversity and evolution in frogs and toads.

The eyes of frogs and toads (Anura) are among their most fascinating features. Although several pupil shapes have been described, the diversity, evolution, and functional role of the pupil in anurans have received little attention. Studying photographs of more than 3200 species, we surveyed pupil diversity, described their morphological variation, tested correlation with adult habits and diel activity, and discuss major evolutionary patterns considering iris anatomy and visual ecology. Our results indicate that the pupil in anurans is a highly plastic structure, with seven main pupil shapes that evolved at least 116 times during the history of the group. We found no significant correlation between pupil shape, adult habits, and diel activity, with the exception of the circular pupil and aquatic habits. The vertical pupil arose at least in the most-recent common ancestor of Anura + Caudata, and this morphology is present in most early-diverging anuran clades. Subsequently, a horizontal pupil, a very uncommon shape in vertebrates, evolved in most neobatrachian frogs. This shape evolved into most other known pupil shapes, but it persisted in a large number of species with diverse life histories, habits, and diel activity patterns, demonstrating a remarkable functional and ecological versatility.

Phylogenetic relationships of the Boana pulchella Group (Anura: Hylidae).

In this paper we present a phylogenetic analysis of the treefrogs of the Boana pulchella Group with the goals of (1) providing a rigorous test of its monophyly; (2) providing a test of relationships supported in previous studies; and (3) exploring the relationships of the several species not included in previous analyses. The analyses included>300 specimens of 37 of the 38 species currently included in the group, plus 36 outgroups, exemplars of the diversity of Boana and the other genera of the hylid tribe Cophomantini. The dataset included eight mitochondrial genes (12S, 16S, CytB, COI, ND1, tRNAIle, tRNALeu, and tRNAVal) and five nuclear genes (RHO, TYR, RAG-1, CXCR4, SIAH1). The phylogenetic analyses recover the monophyly of the B. pulchella Group with lower support than previous studies, as a result of the inclusion of the B. claresignata Group, which is recovered as its sister taxon. Within the B. pulchella Group, the inclusion of almost all species of the group had little impact on previous notions of its phylogeny, except for the rejection of the hypothesized B. polytaenia Clade (B. goiana and B. phaeopleura are nested in the clade here called the B. prasina Clade), which is redefined. Phylogenetic support is strong for five major clades, which collectively include all but three of the species sampled: the B. balzani Clade (B. aguilari, B. balzani, B. gladiator, B. melanopleura, B. palaestes), the redefined B. polytaenia Clade (B. botumirim, B. buriti, B. cipoensis, B. jaguariaivensis, B. leptolineata, B. polytaenia, B. stenocephala, and two undescribed species), the B. prasina Clade (B. bischoffi, B. caingua, B. cordobae, B. goiana, B. guentheri, B. marginata, B. phaeopleura, B. prasina, B. pulchella, and one undescribed species), the B. riojana Clade (B. callipleura, B. marianitae, B. riojana), and the B. semiguttata Clade (B. caipora, B. curupi, B. joaquini, B. poaju, B. semiguttata, B. stellae, and two undescribed species). The monophyly of the B. prasina + B. riojana Clades, and that of the B. polytaenia + B. semiguttata Clades are well-supported. The relationships among these two clades, the B. balzani Clade, B. ericae + B. freicanecae, and B. cambui (representing the deepest phylogenetic splits within the B. pulchella Group) are recovered with weak support. We discuss the phenotypic evidence supporting the monophyly of the B. pulchella Group, and the taxonomy of several species, identifying three new synonyms of Boana polytaenia, one new synonym of Boana goiana, and one new synonym of B. riojana.

Evolution of the strikingly diverse submandibular muscles in Anura.

The most ventral muscles of the head (the mm. submentalis, intermandibularis, and interhyoideus) provide support to the gular region and lift the buccal floor during ventilation and feeding. These muscles show limited variation in most gnathostomes, but in Anura they exhibit a surprising diversity. The few studies that have explored this character system highlighted its potential as a source of phylogenetic information. In this paper we explored the diversity of this character system studying specimens of 567 anuran species and reviewing published data to cover a total of 1321 species, belonging to 53 of the 54 currently recognized anuran families, as well as caudates and caecilians. We defined 27 discrete characters including the number of muscle bellies, supplementary layers, hypertrophy and diversity of elastic fibres, and pigmentation, among others, and optimized them on a comprehensive phylogenetic hypothesis. We recognized 223 unambiguously optimized synapomorphies for numerous clades on different scales, including three for Anura and many for suprafamiliar clades with poor phenotypic support. Finally, we discussed the evolution of this highly diverse character system, including homology, development, and its functional role in vocalization and feeding. Interestingly, the striking levels of variation in some structures contrast with the amount of phylogenetic inertia, allowing us to recognize several general patterns. Supplementary elements of the m. intermandibularis evolved first as broad layers occuring in more than half of extant anuran species and then concentrated forming discreet bellies in several clades. The anterior portion of the gular region is not sexually dimorphic, and is likely related to ventilation and tongue protraction. Conversely, the diversity of the m. interhyoideus is strongly linked to vocal sacs, which are present only in adult males, suggesting the presence of two independent modules.

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.

Naturally occurring fluorescence in frogs.

Fluorescence, the absorption of short-wavelength electromagnetic radiation reemitted at longer wavelengths, has been suggested to play several biological roles in metazoans. This phenomenon is uncommon in tetrapods, being restricted mostly to parrots and marine turtles. We report fluorescence in amphibians, in the tree frog Hypsiboas punctatus, showing that fluorescence in living frogs is produced by a combination of lymph and glandular emission, with pigmentary cell filtering in the skin. The chemical origin of fluorescence was traced to a class of fluorescent compounds derived from dihydroisoquinolinone, here named hyloins. We show that fluorescence contributes 18-29% of the total emerging light under twilight and nocturnal scenarios, largely enhancing brightness of the individuals and matching the sensitivity of night vision in amphibians. These results introduce an unprecedented source of pigmentation in amphibians and highlight the potential relevance of fluorescence in visual perception in terrestrial environments.

A total evidence analysis of the phylogeny of hatchet-faced treefrogs (Anura: Hylidae: Sphaenorhynchus).

The Neotropical hylid genus Sphaenorhynchus includes 15 species of small, greenish treefrogs widespread in the Amazon and Orinoco basins, and in the Atlantic Forest of Brazil. Although some studies have addressed the phylogenetic relationships of the genus with other hylids using a few exemplar species, its internal relationships remain poorly understood. In order to test its monophyly and the relationships among its species, we performed a total evidence phylogenetic analysis of sequences of three mitochondrial and three nuclear genes, and 193 phenotypic characters from all species of Sphaenorhynchus. Our results support the monophyly of Sphaenorhynchus with molecular and phenotypic evidence, with S. pauloalvini as the earliest diverging taxon, followed by S. carneus, as the sister taxon of all remaining species of the genus. We recognize three species groups in Sphaenorhynchus (the S. lacteus, S. planicola and S. platycephalus groups), to facilitate its taxonomic study; only three species (S. carneus, S. pauloalvini and S. prasinus) remain unassigned to any group. Sequence data were not available for only two species (S. bromelicola and S. palustris) for which we scored phenotypic data; wildcard behaviour was detected only in S. bromelicola nested inside the S. platycephalus group. On the basis of the resulting phylogenetic hypothesis, we discuss the evolution of oviposition site and a number of phenotypic characters that could be associated with heterochronic events in the evolutionary history of this group.

The Green Clade grows: A phylogenetic analysis of Aplastodiscus (Anura; Hylidae).

Green tree frogs of the genus Aplastodiscus occur in the Atlantic Forest and Cerrado biomes of South America. The genus comprises 15 medium-sized species placed in three species groups diagnosed mainly by cloacal morphology. A phylogenetic analysis was conducted to: (1) test the monophyly of these species groups; (2) explore the phylogenetic relationships among putative species; and (3) investigate species boundaries. The dataset included eight mitochondrial and nuclear gene fragments for up to 6642 bp per specimen. The results strongly support the monophyly of Aplastodiscus and of the A. albofrenatus and A. perviridis groups. Aplastodiscus sibilatus is the sister taxon of all other species of Aplastodiscus, making the A. albosignatus Group non-monophyletic as currently defined. At least six unnamed species are recognized for Aplastodiscus, increasing the diversity of the genus by 40%. A fourth species group, the A. sibilatus Group is recognized. Aplastodiscus musicus is transferred from the A. albofrenatus Group to the A. albosignatus Group, and A. callipygius is considered a junior synonym of A. albosignatus. Characters related to external cloacal morphology reveal an interesting evolutionary pattern of parallelisms and reversions, suggesting an undocumented level of complexity. We analyze, in light of our phylogenetic results, the evolution of reproductive biology and chromosome morphology in Aplastodiscus.

Odorous secretions in anurans: morphological and functional assessment of serous glands as a source of volatile compounds in the skin of the treefrog Hypsiboas pulchellus (Amphibia: Anura: Hylidae).

Serous (granular or venom) glands occur in the skin of almost all species of adult amphibians, and are thought to be the source of a great diversity of chemical compounds. Despite recent advances in their chemistry, odorous volatile substances are compounds that have received less attention, and until now no study has attempted to associate histological data with the presence of these molecules in amphibians, or in any other vertebrate. Given the recent identification of 40 different volatile compounds from the skin secretions of H. pulchellus (a treefrog species that releases a strong odour when handled), we examined the structure, ultrastructure, histochemistry, and distribution of skin glands of this species. Histological analysis from six body regions reveals the presence of two types of glands that differ in their distribution. Mucous glands are homogeneously distributed, whereas serous glands are more numerous in the scapular region. Ultrastructural results indicate that electron-translucent vesicles observed within granules of serous glands are similar to those found in volatile-producing glands from insects and also with lipid vesicles from different organisms. Association among lipids and volatiles is also evidenced from chemical results, which indicate that at least some of the volatile components in H. pulchellus probably originate within the metabolism of fatty acids or the mevalonate pathway. As odorous secretions are often considered to be secreted under stress situations, the release of glandular content was assessed after pharmacological treatments, epinephrine administrated in vivo and on skin explants, and through surface electrical stimulation. Serous glands responded to all treatments, generally through an obvious contraction of myoepithelial cells that surround their secretory portion. No response was observed in mucous glands. Considering these morpho-functional results, along with previous identification of volatiles from H. pulchellus and H. riojanus after electrical stimulation, we suggest that the electron-translucent inclusions found within the granules of serous glands likely are the store sites of volatile compounds and/or their precursors. Histochemical and glandular distribution analyses in five other species of frogs of the hylid tribe Cophomantini, revealed a high lipid content in all the species, whereas a heterogeneous distribution of serous glands is only observed in species of the H. pulchellus group. The distribution pattern of serous glands in members of this species group, and the odorous volatile secretions are probably related to defensive functions.

Phylogenetic relationships of toads of the Rhinella granulosa group (Anura: Bufonidae): a molecular perspective with comments on hybridization and introgression.

The Rhinella granulosa group consists of 13 species of toads distributed throughout open areas of South America and Panama. In this paper we perform a phylogenetic analysis considering all but one species of the group, employing five nuclear and four mitochondrial genes, for up to 7910 bp per specimen. Separate phylogenetic analyses under direct optimization (DO) of nuclear and mitochondrial sequences recovered the R. granulosa group as monophyletic and revealed topological incongruence that can be explained mainly by multiple events of hybridization and introgression, both mitochondrial and nuclear. The DO combined analysis, after the exclusion of putatively introgressed or heterozygous genomes, resulted in a phylogenetic hypothesis for the R. granulosa group in which most of the species are recovered as monophyletic, but with interspecific relationships poorly supported. The optimization of morphological (adult and larval), chromosomal, and behavioural characters resulted in 12 putative phenotypic synapomorphies for this species group and some other synapomorphies for internal clades. Our results indicate the need for additional population genetic studies on R. dorbignyi and R. fernandezae to corroborate the taxonomic status of both taxa. Finally, we discuss biological and genetic characteristics of Bufonidae, as possible explanations for the common occurrence of hybridization and introgression observed in some lineages of this family.

Frog volatile compounds: application of in vivo SPME for the characterization of the odorous secretions from two species of Hypsiboas treefrogs.

A novel in vivo design was used in combination with solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) to characterize the volatile compounds from the skin secretion of two species of tree frogs. Conventional SPME-GC/MS also was used for the analysis of volatiles present in skin samples and for the analysis of volatiles present in the diet and terraria. In total, 40 and 37 compounds were identified in the secretion of Hypsiboas pulchellus and H. riojanus, respectively, of which, 35 were common to both species. Aliphatic aldehydes, a low molecular weight alkadiene, an aromatic alcohol, and other aromatics, ketones, a methoxy pyrazine, sulfur containing compounds, and hemiterpenes are reported here for the first time in anurans. Most of the aliphatic compounds seem to be biosynthesized by the frogs following different metabolic pathways, whereas aromatics and monoterpenes are most likely sequestered from environmental sources. The characteristic smell of the secretion of H. pulchellus described by herpetologists as skunk-like or herbaceous is explained by a complex blend of different odoriferous components. The possible role of the volatiles found in H. pulchellus and H. riojanus is discussed in the context of previous hypotheses about the biological function of volatile secretions in frogs (e.g., sex pheromones, defense secretions against predators, mosquito repellents).

Does counting species count as taxonomy? On misrepresenting systematics, yet again.

Recent commentary by Costello and collaborators on the current state of the global taxonomic enterprise attempts to demonstrate that taxonomy is not in decline as feared by taxonomists, but rather is increasing by virtue of the rate at which new species are formally named. Having supported their views with data that clearly indicate as much, Costello et al. make recommendations to increase the rate of new species descriptions even more. However, their views appear to rely on the perception of species as static and numerically if not historically equivalent entities whose value lie in their roles as "metrics". As such, their one-dimensional portrayal of the discipline, as concerned solely with the creation of new species names, fails to take into account both the conceptual and epistemological foundations of systematics. We refute the end-user view that taxonomy is on the rise simply because more new species are being described compared with earlier decades, and that, by implication, taxonomic practice is a formality whose pace can be streamlined without considerable resources, intellectual or otherwise. Rather, we defend the opposite viewpoint that professional taxonomy is in decline relative to the immediacy of the extinction crisis, and that this decline threatens not just the empirical science of phylogenetic systematics, but also the foundations of comparative biology on which other fields rely. The allocation of space in top-ranked journals to propagate views such as those of Costello et al. lends superficial credence to the unsupportive mindset of many of those in charge of the institutional fate of taxonomy. We emphasize that taxonomy and the description of new species are dependent upon, and only make sense in light of, empirically based classifications that reflect evolutionary history; homology assessments are at the centre of these endeavours, such that the biological sciences cannot afford to have professional taxonomists sacrifice the comparative and historical depth of their hypotheses in order to accelerate new species descriptions.

Phylogenetic relationships of a Patagonian frog radiation, the Alsodes + Eupsophus clade (Anura: Alsodidae), with comments on the supposed paraphyly of Eupsophus.

The frog clade composed of the alsodid genera Alsodes + Eupsophus is the most species-rich of the Patagonian endemic frog clades, including nearly 31 of the slightly more than 50 species of that region. The biology of this group of frogs is poorly known, its taxonomy quite complex (particularly Alsodes), and its diversity in chromosome number striking when compared with other frogs (collectively, there are species having 2n = 22, 2n = 26, 2n = 28, 2n = 30 or 2n = 34). We present a phylogenetic analysis of this Patagonian frog clade based on mitochondrial and nuclear gene sequences. We sequenced five mitochondrial genes (cytochrome b, cytochrome oxidase I, 12S, 16S, NADH dehydrogenase subunit 1) with three intervening tRNAs, and fragments of three nuclear genes (seven in absentia homolog 1, rhodopsin exon 1, RAG-1), for a maximum of 6510 bp for multiple specimens from 26 of the 31 species. We recovered Eupsophus as polyphyletic, with E. antartandicus, E. sylvaticus, and E. taeniatus in Batrachylidae, in accordance with most previous hypotheses. Based on this result, we transfer E. antartandicus and E. taeniatus back to Batrachyla, and E. sylvaticus to Hylorina (resurrected from the synonymy of Eupsophus), remediating the paraphyly of Eupsophus. Our results strongly corroborate the monophyly of Alsodes + Eupsophus (sensu stricto), the individual monophyly of these genera, and the monophyly of the species groups of Eupsophus. They also show the non-monophyly of all non-monotypic species groups of Alsodes proposed in the past. Our results expose several taxonomic problems particularly in Alsodes, and to a lesser extent in Eupsophus. This phylogenetic context suggests a rich evolutionary history of karyotypic diversification in the clade, in part corroborating previous hypotheses. In Alsodes, we predict three independent transformations of chromosome number from the plesiomorphic 2n = 26. All these, strikingly, involve increments or reductions of pairs of haploid chromosomes. Finally, the phylogenetic pattern recovered for Alsodes and Eupsophus suggests a trans-Andean origin and diversification of the group, with multiple, independent ingressions over cis-Andean regions.

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.

Structure and function of cationic hylin bioactive peptides from the tree frog Boana pulchella in interaction with lipid membranes.

Amphibians have a great diversity of bioactive peptides in their skin. The cDNA prepro-peptide sequencing allowed the identification of five novel mature peptides expressed in the skin of Boana pulchella, four with similar sequences to hylin peptides having a cationic amphipathic-helical structure. Whole mature peptides and some of their fragments were chemically-synthesized and tested against Gram-positive and Gram-negative bacterial strains. The mature peptide hylin-Pul3 was the most active, with a MIC= 14 µM against Staphylococcus aureus. Circular dichroism assays indicated that peptides are mostly unstructured in buffer solutions. Still, adding large unilamellar vesicles composed of dimyristoyl phosphatidylcholine and dimyristoylphosphatidylglycerol increased the α-helix content of novel hylins. These results demonstrate the strong influence of the environment on peptide conformation and highlight its significance while addressing the pharmacology of peptides and their biological function in frogs.

A phylogenetic analysis of Pleurodema (Anura: Leptodactylidae: Leiuperinae) based on mitochondrial and nuclear gene sequences, with comments on the evolution of anuran foam nests.

Species of the genus Pleurodema are relatively small, plump frogs that mostly occur in strong-seasonal and dry environments. The genus currently comprises 14 species distributed from Panama to southern Patagonia. Here we present a phylogenetic analysis of Pleurodema, including all described species and several outgroups. Our goals include testing its monophyly and the monophyly of the species groups that were historically proposed, and studying the evolution of some character systems, particularly macroglands and egg-clutch structure; this last point also provided the chance for a discussion of foam nest evolution in anurans. Our dataset includes portions of the mitochondrial genes cytochromeb, 12S, 16S, and the intervening tRNAVal ; the nuclear gene sequences include portions of rhodopsin exon 1 and seven in absentia homolog I. Our results support a clade composed of Pleurodema and including the monotypic SomuncuriaLynch, 1978 nested within it. The latter genus is therefore considered a junior synonym of Pleurodema and its sole species is added to this genus. Furthermore, our results indicate the non-monophyly of several species groups proposed previously. We recognize four clades in Pleurodema: the P. bibroni clade (P. bibroni, P. cordobae and P. kriegi), the P. thaul clade (P. bufoninum, P. marmoratum, P. somuncurensis and P. thaul), the P. brachyops clade (P. alium, P. borellii, P. brachyops, P. cinereum, P. diplolister and P. tucumanum) and the P. nebulosum clade (P. guayapae and P. nebulosum). Our results further indicate the need for a taxonomic reassessment of P. borellii and P. cinereum (as did previous studies), P. guayapae and P. nebulosum, and the three species in the P. bibroni clade. Pleurodema shows a striking pattern of variation in presence/absence of lumbar glands. Our results indicate multiple losses or independent gains of this character associated with defensive displays. The reproductive modes of Pleurodema include four different egg-clutch structures. The optimization of these indicates that there are at least two independent transformations from the plesiomorphic mode of foam nests to egg-clutch structures involving gelatinous masses of different sorts (ovoid plates, masses, or strings). We hypothesize that these independent transformations could involve changes at the behavioural (the loss of foam beating behaviour by the parent) and/or structural level (transformations involving the pars convoluta dilata, the section of the oviduct where the foam-making substance is secreted). Finally, our study of foam nest evolution in Pleurodema is extended to the other groups of anurans where foam-nesting occurs, on the basis of available data and recent phylogenetic hypotheses. In the different hyloid groups where it occurs, foam-nesting evolved from clutches laid in water. However, in all ranoids in which foam-nesting occurs, it evolved from terrestrial clutches, with eggs laid hanging in vegetation, or, if the clutches are laid on a restricted volume of water, involving endotrophic development. © The Willi Hennig Society 2012.

A new reproductive mode in anurans: Natural history of Bokermannohyla astartea (Anura: Hylidae) with the description of its tadpole and vocal repertoire.

Anurans have the greatest diversity of reproductive modes among tetrapod vertebrates, with at least 41 being currently recognized. We describe a new reproductive mode for anurans, as exhibited by the Paranapiacaba Treefrog, Bokermannohyla astartea, an endemic and poorly known species of the Brazilian Atlantic Forest belonging to the B. circumdata group. We also describe other aspects of its reproductive biology, that are relevant to understanding the new reproductive mode, such as courtship behavior, spawning, and tadpoles. Additionally, we redescribe its advertisement call and extend its vocal repertoire by describing three additional call types: courtship, amplectant, and presumed territorial. The new reproductive mode exhibited by B. astartea consists of: (1) deposition of aquatic eggs in leaf-tanks of terrestrial or epiphytic bromeliads located on or over the banks of temporary or permanent streams; (2) exotrophic tadpoles remain in the leaf-tanks during initial stages of development (until Gosner stage 26), after which they presumably jump or are transported to streams after heavy rains that flood their bromeliad tanks; and (3) tadpole development completes in streams. The tadpoles of B. astartea are similar to those of other species of the B. circumdata group, although with differences in the spiracle, eyes, and oral disc. The vocal repertoire of B. astartea exhibits previously unreported acoustic complexity for the genus. Bokermannohyla astartea is the only bromeligenous species known to date among the 187 known species within the tribe Cophomantini. We further discuss evolutionary hypotheses for the origin of this novel reproductive mode.