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Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

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.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2021 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

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 Willi Hennig Society 2021.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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, tRNA Ile , tRNA Leu , and tRNA Val ) 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 Willi Hennig Society 2020.)

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., (© The Willi Hennig Society 2020.)

We report recent findings of Isthmohyla pictipes (Cope, 1875) in the Cordillera de Talamanca, Costa Rica, roughly two decades after it was last registered. We provide notes on microhabitat use, color variation, external morphology of adults and larvae, and geographic variation, and discuss some taxonomic characters employed to differentiate I. pictipes from I. tica (Starrett, 1966) and I. xanthosticta (Duellman, 1968). We also report fluorescence on the ventral surfaces of I. pictipes. Our findings are expected to shed light on the taxonomy of this species and should be useful in further population assessments and conservation plans.

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., Competing Interests: The authors declare no competing interest.

The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi, P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution., Competing Interests: The authors have declared that no competing interests exist.

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 Willi Hennig Society 2018.)

The genus Scinax currently includes more than 120 species, recovered in two major clades, the S. catharinae and the S. ruber clades. The latter comprises 75 species, most of which remain unassigned to any species groups, while 12 are included in the S. rostratus and S. uruguayus groups. In this paper we present a taxonomic review of the two species currently included in the S. uruguayus group, discussing some putative phenotypic synapomorphies of this group. Although S. pinima and S. uruguayus have been considered as distinct species, this has been based on scant evidence, and several authors doubted of their distinctiveness. Our study of available specimens of S. pinima and S. uruguayus corroborates that both are valid and diagnosable species based on phenotypic evidence. Furthermore, our results show that S. pinima previously known only from its type locality, has a much widespread distribution than previously thought (including the Brazilian states of Paraná, Santa Catarina, and Rio Grande do Sul), which, added to the biological information presented here allows to suggest the removal of this species from the "Data Deficient" IUCN Red List category to "Least Concern". Also, we describe a new species formerly reported as S. aff. pinima and S. uruguayus from NE Argentina and some localities from the Brazilian State of Rio Grande do Sul. All species are diagnosed and characterized using adult and larval morphology, osteology, vocalizations, cytogenetics, and natural history., Competing Interests: The authors have declared that no competing interests exist.

Peptides from skin secretions of amphibians are considered important components of their immune system and also play a relevant role in their defense mechanism against predators. Herein, by using mass spectrometry (MS), we characterize the sequence of 13 peptides from the gland secretion of the hylid tree frog, Boana punctata . Using in situ matrix-assisted laser desorption ionization imaging MS of a transverse section of the skin tissue, we show that some peptides are stored as longer molecules that are cleaved after being secreted, whereas others do not undergo any modification. Sequence comparison with peptides from other Boana species and analysis of the three-dimensional theoretical structure indicate that this cleavage depends on both the presence of a specific sequence motif and the secondary structure. The fact that peptides undergo a rapid cleavage upon secretion suggests that stored and secreted peptides may have distinct roles for anuran survival, including defense against pathogens and predators., Competing Interests: The authors declare no competing financial interest.

The hylid tribe Cophomantini is a diverse clade of Neotropical treefrogs composed of the genera Aplastodiscus, Boana, Bokermannohyla, Hyloscirtus, and Myersiohyla. The phylogenetic relationships of Cophomantini have been comprehensively reviewed in the literature, providing a suitable framework for the study of chromosome evolution. Employing different banding techniques, we studied the chromosomes of 25 species of Boana and 3 of Hyloscirtus; thus providing, for the first time, data for Hyloscirtus and for 15 species of Boana. Most species showed karyotypes with 2n = 2x = 24 chromosomes; some species of the B. albopunctata group have 2n = 2x = 22, and H. alytolylax has 2n = 2x = 20. Karyotypes are all bi-armed in most species presented, with the exception of H. larinopygion (FN = 46) and H. alytolylax (FN = 38), with karyotypes that have a single pair of small telocentric chromosomes. In most species of Boana, NORs are observed in a single pair of chromosomes, mostly in the small chromosomes, although in some species of the B. albopunctata, B. pulchella, and B. semilineata groups, this marker occurs on the larger pairs 8, 1, and 7, respectively. In Hyloscirtus, NOR position differs in the three studied species: H. alytolylax (4p), H. palmeri (4q), and H. larinopygion (1p). Heterochromatin is a variable marker that could provide valuable evidence, but it would be necesserary to understand the molecular composition of the C-bands that are observed in different species in order to test its putative homology. In H. alytolylax, a centromeric DAPI+ band was observed on one homologue of chromosome pair 2. The band was present in males but absent in females, providing evidence for an XX/XY sex determining system in this species. We review and discuss the importance of the different chromosome markers (NOR position, C-bands, and DAPI/CMA3 patterns) for their impact on the taxonomy and karyotype evolution in Cophomantini.

Anuran vocal sacs are elastic chambers that recycle exhaled air during vocalizations and are present in males of most species of frogs. Most knowledge of the diversity of vocal sacs relates to external morphology; detailed information on internal anatomy is available for few groups of frogs. Frogs of the family Hylodidae, which is endemic to the Atlantic Forest of Brazil and adjacent Argentina and Paraguay, have three patterns of vocal sac morphology-that is, single, subgular; paired, lateral; and absent. The submandibular musculature and structure of the vocal sac mucosa (the internal wall of the vocal sac) of exemplar species of this family and relatives were studied. In contrast to previous accounts, we found that all species of Crossodactylus and Hylodes possess paired, lateral vocal sacs, with the internal mucosa of each sac being separate from the contralateral one. Unlike all other frogs for which data are available, the mucosa of the vocal sacs in these genera is not supported externally by the mm. intermandibularis and interhyoideus. Rather, the vocal sac mucosa projects through the musculature and is free in the submandibular lymphatic sac. The presence of paired, lateral vocal sacs, the internal separation of the sac mucosae, and their projection through the m. interhyoideus are synapomorphies of the family. Furthermore, the specific configuration of the m. interhyoideus allows asymmetric inflation of paired vocal sacs, a feature only reported in species of these diurnal, stream-dwelling frogs., (© 2017 Wiley Periodicals, Inc.)

We studied the foot musculature of the fossorial family Odontophrynidae (composed of the genera Macrogenioglottus, Odontophrynus, and Proceratophrys) plus several outgroup taxa from Hyloidea and other burrowing taxa from across Anura. We found novel character-states supporting the monophyly of Odontophrynus, Proceratophrys, and Odontophrynus + Macrogenioglottus. The character-states observed in O. cultripes support conflicting phylogenetic positions within Odontophrynus. A comparison of some novel character-states with a diverse sample of burrowing taxa suggests that some modifications of the foot musculature might be involved in digging.

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.

Most anurans possess a tympanic middle ear (TME) that transmits sound waves to the inner ear; however, numerous species lack some or all TME components. To understand the evolution of these structures, we undertook a comprehensive assessment of their occurrence across anurans and performed ancestral character state reconstructions. Our analysis indicates that the TME was completely lost at least 38 independent times in Anura. The inferred evolutionary history of the TME is exceptionally complex in true toads (Bufonidae), where it was lost in the most recent common ancestor, preceding a radiation of >150 earless species. Following that initial loss, independent regains of some or all TME structures were inferred within two minor clades and in a radiation of >400 species. The reappearance of the TME in the latter clade was followed by at least 10 losses of the entire TME. The many losses and gains of the TME in anurans is unparalleled among tetrapods. Our results show that anurans, and especially bufonid toads, are an excellent model to study the behavioural correlates of earlessness, extratympanic sound pathways, and the genetic and developmental mechanisms that underlie the morphogenesis of TME structures.

We describe a new species of the Scinax catharinae group from coastal forest in the State of São Paulo, Brazil. We also describe the karyotype and the advertisement call of the new species, which is composed by a multipulsed note. The chromosome morphology of the new species shows the nucleolar organizer region (NOR) on Pair 6, a putative synapomorphy previously inferred for the S. catharinae clade. We report, for the first time, the presence of mental glands in a species of the genus Scinax. Additionally, we provide a brief discussion about the occurrence of glandular tissue on the mental and inguinal regions of the species of the S. catharinae clade.

This study describes the spermatozoa of 10 of the 15 species of the Neotropical frog genus Pleurodema through transmission electron microscopy. The diversity of oviposition modes coupled with a recent phylogenetic hypothesis of Pleurodema makes it an interesting group for the study of ultrastructural sperm evolution in relation to fertilization environment and egg-clutch structure. We found that Pleurodema has an unusual variability in sperm morphology. The more variable structures were the acrosomal complex, the midpiece, and the tail. The acrosomal complex has all the structures commonly reported in hyloid frogs but with different degree of development of the subacrosomal cone. Regarding the midpiece, the variability is given by the presence or absence of the mitochondrial collar. Finally, the tail is the most variable structure, ranging from single (only axoneme) to more complex (presence of paraxonemal rod, cytoplasmic sheath, and undulating membrane), with the absence of the typical axial fiber present in hyloid frogs, also shared with some other genera of Leiuperinae. J. Morphol. 277:957-977, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

A new species of treefrog of the genus Osteocephalus is described from the Rio Abacaxis, a southern tributary of the Amazonas in the state of Amazonas, Brazil. This member of the O. buckleyi group is characterized by green dorsal colouration with irregular blotches of various shades of brown, light venter with tan spots and bold dark markings on the posterior surfaces of the thighs. It can be distinguished from its closest relative, O. helenae from the same general area, by the lack of an axillary membrane, a few indistinct tubercles on the proximal segment of Finger IV and single ulnar tubercles.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (© 2015 Anatomical Society.)

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., (© The Willi Hennig Society 2015.)

A new species of the Scinax ruber Clade is described from Municipality of Barra do Garças, State of Mato Grosso, Brazil. It is diagnosed by its size (SVL 29.4-35.4 mm in males); dorsum with a background that varies from light and dark gray to dark brown, with round and irregular dark blotches; hidden surfaces of thigh and shank light or dark brown, with lighter, large and irregular blotches (yellow in life); and nuptial pad that covers the medial margin of Metacarpal I, and obscures the posterior portion and the outer margin of the inner metacarpal tubercle. It is most similar with Scinax rupestris, from which it differs by having larger adult males; less developed nuptial pad; and hidden surfaces of thigh and light or dark brown, with lighter, large and irregular blotches (yellow in life). The new species occurs near the headwaters of permanent or temporary streams, where males call from vegetation or rocks at the margins of small streams.

We describe a new treefrog species of Dendropsophus collected on rocky outcrops in the Brazilian Atlantic Forest. Ecologically, the new species can be distinguished from all known congeners by having a larval phase associated with rainwater accumulated in bromeliad phytotelms instead of temporary or lentic water bodies. Phylogenetic analysis based on molecular data confirms that the new species is a member of Dendropsophus; our analysis does not assign it to any recognized species group in the genus. Morphologically, based on comparison with the 96 known congeners, the new species is diagnosed by its small size, framed dorsal color pattern, and short webbing between toes IV-V. The advertisement call is composed of a moderate-pitched two-note call (~5 kHz). The territorial call contains more notes and pulses than the advertisement call. Field observations suggest that this new bromeligenous species uses a variety of bromeliad species to breed in, and may be both territorial and exhibit male parental care.

We describe the external morphology and oral cavity of the tadpoles of Bokermannohyla caramaschii and B. diamantina respectively from the states of Espírito Santo and Bahia, Brazil. Larvae of both species are distinguished from each other by external characters such as body shape, labial tooth-row formula, number of marginal papillae, coloration and internal oral anatomy features. Some of the character states of the tadpoles of B. caramaschii and B. diamantina that are shared with all other described tadpoles of the Bokermannohyla circumdata group, such as the absence/reduction of small flaps with accessory labial teeth laterally in the oral disc, and the absence/reduction of submarginal papillae, may represent morphological synapomorphies of this species group, or at least of some internal clade. The general pattern of brownish coloration with longitudinal stripes on the caudal muscle is also common to most species of the group. We did not find character states of the oral cavity that are exclusively shared by species of the B. circumdata group, or by other groups of Bokermannohyla.

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).

We describe the tadpole of Sphaenorhynchus caramaschii. It differs from tadpoles of other species of Sphaenorhynchus in having a short spiracle, submarginal papillae, and alternating short and large marginal papillae in the oral disc. Some larval characteristics, like morphology and position of the nostrils, length of the spiracle, and size of the marginal papillae on the oral disc are discussed for tadpoles of other species of Sphaenorhynchus.

The German naturalist Carl Hermann Conrad Burmeister (1807-1892) had a prolific scientific career, spanning multiple taxa from diverse insect groups and trilobites to temnospondyls, birds, and extant and fossil mammals (see Berg, 1895). His contributions to anuran taxonomy are concentrated in two books, "Erläuterungen zur Fauna Brasiliensis…" (Burmeister, 1856) and "Reise durch die La Plata-Staaten…" (Burmeister, 1861). The latter is an account of his travels in Argentina and Uruguay from 1857-1860 and includes descriptions of three new species of frogs: Leiuperus nebulosus, Cystignathus mystacinus, and Hyla leucotaenia. While the first two names currently designate valid species, with the combinations Pleurodema nebulosum and Leptodactylus mystacinus respectively, the last name has had a more complex taxonomic history. It involves confusions involving a homonym, its consideration as a junior synonym of Hypsiboas pulchellus (Duméril & Bibron, 1841)-a widely distributed species in eastern Argentina, southeastern Brazil, and Uruguay (Frost, 2014)-and its actual identity corresponding to another widespread species in the same geographic area, with which it has never before been associated: Scinax squalirostris (A. Lutz, 1925). All these issues are discussed in this paper.

We describe Dendropsophus ozzyi sp. nov., a new species of treefrog, tentatively included in the Dendropsophus microcephalus Group and most notably diagnosed by the presence of pointed fingers and an advertisement call with a very high dominant frequency. The new species is known from three localities in the Brazilian Amazon forest, two on western State of Pará and one (the type locality) in eastern State of Amazonas (03°56'50"S and 58°26'36"W, 45 m a.s.l.).

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., (© The Willi Hennig Society 2012.)

Many anuran species are characterized by sexually dimorphic skin glands. These glands often are concentrated on specific areas, such as the mental region, flanks, or the nuptial pads. We studied the histology and histochemistry of mental and lateral glands in Hypsiboas punctatus, and compared them to skin from other body regions. We describe four types of dermal glands, two types of mucous and two types of serous glands. The mucous glands are formed by a single layered epithelium. The mucocytes surrounding a central lumen are filled with polyhedral granules. Ordinary mucous glands are small sized glands with cubical epithelium, mucoid content, and small granules. Specialized mucous glands are characterized by a larger size, a columnar epithelium, a proteinaceous content and larger granules. Both types of serous glands are syncytial and share some structural features including size, shape, and morphology of secretory granules. However, ordinary and specialized serous glands differ in their histochemical properties, size and appearance of secretory granules, and glandular outlets. The specialized type of mucous glands in H. punctatus resembles most SDSGs described in anurans, whereas the presence of specialized serous glands that are sexually dimorphic is less common. Both specialized glands occur only in mental and lateral regions of males, whereas ordinary mucous and ordinary serous glands occur in males and females., (Copyright © 2012 Wiley Periodicals, Inc.)

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 tRNA Val ; 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., (© The Willi Hennig Society 2012.)

Many protein functions can be traced to linear sequence motifs of less than five residues, which are often found within intrinsically disordered domains. In spite of their prevalence, their role in protein evolution is only beginning to be understood. The study of papillomaviruses has provided many insights on the evolution of protein structure and function. We have chosen the papillomavirus E7 oncoprotein as a model system for the evolution of functional linear motifs. The multiple functions of E7 proteins from paradigmatic papillomavirus types can be explained to a large extent in terms of five linear motifs within the intrinsically disordered N-terminal domain and two linear motifs within the globular homodimeric C-terminal domain. We examined the motif inventory of E7 proteins from over 200 known papillomavirus types and found that the motifs reported for paradigmatic papillomavirus types are absent from many uncharacterized E7 proteins. Several motif pairs occur more often than expected, suggesting that linear motifs may evolve and function in a cooperative manner. The E7 linear motifs have appeared or disappeared multiple times during papillomavirus evolution, confirming the evolutionary plasticity of short functional sequences. Four of the motifs appeared several times during papillomavirus evolution, providing direct evidence for convergent evolution. Interestingly, the evolution pattern of a motif is independent of its location in a globular or disordered domain. The correlation between the presence of some motifs and virus host specificity and tissue tropism suggests that linear motifs play a role in the adaptive evolution of papillomaviruses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)