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Using a natural marker, we documented breeding site attendance patterns by males and females of the Trinidad Leaf Frog, Phyllomedusa trinitatis. We followed attendance at a cluster of three isolated ponds over 53 and 56 consecutive nights in 2016 and 2019 respectively. Most females attended only once, but for those that attended more than once we calculated an inter-nesting interval (mean 27.6 days, N = 7). Males showed high pond fidelity, but some did attend at two of the ponds, always with a strong preference for one of them. Males showed three attendance patterns. A few attended on multiple consecutive nights (maximum, 19 nights); more were sporadic (one attended seven times over 46 nights with gaps of 15 and 19 days in the sequence); some attended only once (2016: 12, 2019: 15), but most were found to be present on multiple nights (2016: 38, 2019: 32). Our analysis suggested that these latter frogs were either newly recruited individuals or had been predated during the study. Our data show that rainfall has some influence on attendance. We found no relationship between male body condition and attendance pattern. In addition, there was no evidence that a particular male attendance pattern was optimal for breeding success.

Stable individual variation in ventral spotting patterns in Phyllomedusa trinitatis (Anura: Phyllomedusidae) and other Phyllomedusa species: a minimally invasive method for recognizing individuals. We describe a distinctive pattern of individually variable white ‘islands’ and dots on the ventral surfaces of the throat and forearm regions of male and female Phyllomedusa trinitatis. Crucially, we report from captive-reared individuals that these patterns are ontogenetically stable. We have used these patterns to recognize individual frogs in populations of 60 and more. Examination of the ventral surfaces of other Phyllomedusa species in museum specimens and from published accounts suggests that use of these patterns provides a generally useful, minimally invasive recognition method in this genus. We fnd that species previously classed as Phyllomedusa but now regarded as belonging to different genera lack these patterns.

An acoustic and morphometric evaluation of the geographic distribution of Phyllomedusa burmeisteri (Anura: Phyllomedusidae), with comments on P. bahiana. A recent study, based on phylogenetic and phylogeographic multilocus approaches, detected two evolutionary units (BUR and BUR-RJ) within the range of P. burmeisteri. BUR has a wide distribution, whereas BUR-RJ is restricted to a small area coincident with the Serra do Mar mountain range in the Brazilian state of Rio de Janeiro. We evaluate if acoustic and morphometric data support these two major clades within P. burmeisteri that were proposed using molecular evidence. We also provide for the frst time detailed morphometric data for adult males of P. burmeisteri (including topotypes) and the holotype of P. bahiana, and we revisit the comparative acoustic diagnosis between P. burmeisteri and P. bahiana. We were unable to distinguish BUR and BUR-RJ evolutionary units based on morphometric, acoustic or any other feature of external morphology or coloration. Given the high levels of similarity in morphometric and acoustic traits between P. burmeisteri and P. bahiana, these features appear not to be informative in the differentiation of the two sister species.

Graphical abstract Highlights • We identified six cryptic lineages under Pithecopus rohdei from Atlantic Forest. • A integrative model-based species delimitation approach was used. • The potential new species are allopatric. • Putative species distribution corresponds to reported forest refuges. • Plio-Pleistocene climatic changes probably influenced lineages diversification. Abstract Even though Brazil is the world leader in amphibian diversity, a significant part of its richness remains unknown or hidden under cryptic taxa. Here, we used model-based species delimitation in an integrative taxonomic approach, by gathering molecular and morphometric data to assess cryptic taxa within the monkey frogs Pithecopus rohdei , from the Atlantic Forest, and P. megacephalus , from campos rupestres ecosystem. We sampled one mitochondrial, five nuclear loci, and 18 morphometric variables. Using species-delimitation methods with genetic and morphometric data, we recovered five divergent lineages within P. rohdei and no cryptic lineages were recovered for P. megacephalus. Morphometric data show differentiation only for one of the candidate species revealed by the delimitation approaches, suggesting that individuals from Doce River basin constitute a putative species for formal taxonomic description. The time-calibrated mtDNA tree shows that P. rohdei complex lineages began to diverge in late Miocene. However, dates from the multilocus species tree are more recent, occurring in Pleistocene, and suggesting their persistence in refuges of forest and sky islands within the Atlantic Forest biome. The existence of cryptic taxa within P. rohdei is, therefore, relevant for planning conservation strategies for this species complex in the Atlantic Forest biodiversity hotspot. [ABSTRACT FROM AUTHOR]

Abstract Skin secretions are known as a highly-complex mixture of abundant and diverse bioactive molecules and its study has attracted increasing attention over recent years. Phylloseptin is a unique family of antimicrobial peptides which have been only isolated from frogs of the Phyllomedusinae subfamily. Here, three novel peptide precursors were successfully cloned from a cDNA library, which was constructed from the skin secretion of Phyllomedusa burmeisteri , as pair of primers (one nested universal primer and a designed degenerate sense primer) were employed for "shotgun" cloning. The encoded mature peptides were validated by MS/MS sequencing, and subsequently termed as Phylloseptin-PBa1, -PBa2 and -PBa3. Phylloseptin-PBa1 and -PBa2 were demonstrated to possess potent antimicrobial activities against Gram-positive bacteria and yeast, as well as broad-spectrum anticancer activities, while they possess varying haemolytic activity at the effective concentration. In contrast, Phylloseptin-PBa3 was found to exhibit a strong haemolytic activity even though it was only found to possess a weak antimicrobial activity and inconspicuous anticancer activity. Highlights • Three full-length cDNAs were cloned from the skin secretion-derived cDNA library. • Encoded novel phylloseptins were validated by RP-HPLC and MS/MS sequencing. • Novel phylloseptins showed broad-spectrum antimicrobial and anticancer activities. • No obvious hemolytic activity showed at the detected minimal inhibitor concentrations. [ABSTRACT FROM AUTHOR]

Abstract Clinal variation and cryptic species complexes are challenges to taxa targeted conservation. These issues are particularly problematic for the estimation of species geographic distributions, generally resulting in erroneous estimations due to unrecognized species. However, integrative taxonomy could help to solve these problems, revealing geographic and genealogical limits of species when they are not clear through morphological comparisons solely. Here we evaluated the efficiency of integrative taxonomy in identifying a new population of leaf frog from a species complex. This example was chosen because correct species identification can have an impact in the conservation assessment of a threatened species. The new population belongs to Pithecopus centralis , extending its distribution by approximately 400 km to the east of its known distribution. This identification was confirmed by the consensus between genetic, morphometric and bioacoustic evidence, even with some level of uncertainty when using each piece of evidence. We reassessed the conservation status of the species following recommendations from the International Union for Conservation of Nature (IUCN), after having checked and cleaned the data used for the species categorization in the Brazilian Red List of Threatened Fauna. In spite of the distribution extension reported here, our results demonstrate that the status of the species should be revised in the national Red List, reinforcing the importance of a rigorous taxonomic approach to improve Red Lists assessments. [ABSTRACT FROM AUTHOR]

Protected areas ( PAs) are essential for biodiversity conservation, but their coverage is considered inefficient for the preservation of all species. Many species are subdivided into evolutionarily significant units ( ESUs) and the effectiveness of PAs in protecting them needs to be investigated. We evaluated the usefulness of the Brazilian PAs network in protecting ESUs of the critically endangered Pithecopus ayeaye through ongoing climate change. This species occurs in a threatened mountaintop ecosystem known as campos rupestres. We used multilocus DNA sequences to delimit geographic clusters, which were further validated as ESUs with a coalescent approach. Ecological niche modeling was used to estimate spatial changes in ESUs' potential distributions, and a gap analysis was carried out to evaluate the effectiveness of the Brazilian PAs network to protect P. ayeaye in the face of climate changes. We tested the niche overlap between ESUs to gain insights for potential management alternatives for the species. Pithecopus ayeaye contains at least three ESUs isolated in distinct mountain regions, and one of them is not protected by any PA. There are no climatic niche differences between the units, and only 4% of the suitable potential area of the species is protected in present and future projections. The current PAs are not effective in preserving the intraspecific diversity of P. ayeaye in its present and future range distributions. The genetic structure of P. ayeaye could represent a typical pattern in campos rupestres endemics, which should be considered for evaluating its conservation status. [ABSTRACT FROM AUTHOR]

Some nocturnal animals are brightly marked, yet the adaptive significance of their colourful patterns in low light is poorly understood, and this phenomenon is particularly prevalent in amphibians. One way to resolve this seeming paradox is to determine whether these colour patterns serve as visual cues that evolve through sexual and/or natural selection. Accordingly, we focused on Central American phyllomedusines, a clade of leaf-frogs well known for variable and colourful markings. We first tested the null hypothesis that phylogeny explains colour pattern evolution and then tested for overdispersion in sympatry. Phylogenetic analysis confirms that colour pattern variation is not correlated with lineage diversification and tests of overdispersion show that colour patterns of closely related taxa diverge in sympatry. Combined, these evolutionary and community-based lines of evidence support the hypothesis that flank colour patterns probably function in species recognition. They are thus analogous to widespread visual displays among diurnal vertebrates, suggesting that the richness of similar sensory interactions among animals after dark might be severely under-appreciated. [ABSTRACT FROM AUTHOR]

Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer . These 15 novel antimicrobial peptides of 20–32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD– and the sequence motifs –VALGAVSK– or –GKAAL(N/G/S) (V/A)V– in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77 μM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3–fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11 μM). CZS-1 reached 100% haemolysis at 120.87 μM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use. Biological significance Through the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named “Cruzioseptins” and contains cationic amphipathic peptides of 20–32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical application. [ABSTRACT FROM AUTHOR]

The dermaseptin antimicrobial peptide family contains members of 27-34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 51-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested. [ABSTRACT FROM AUTHOR]

We provide a detailed description of the reproduction of the tree frogAgalychnis granulosa, including its advertisement call.Agalychnis granulosabreeds in ponds and the backwaters of streams within primary forests. The breeding season started at the beginning of the wet season and ended two months before the beginning of the dry season. Males called at night in the vegetation around the ponds at a maximum distance of 5 m from the water. The call was composed of a single note with three to seven pulses and a dominant frequency ranging from 1490 to 2101 Hz. Oviposition sites included tree trunks, attached leaves, fallen leaves and bromeliad roots, and ranged in height from 0 to 300 cm above the water. Egg clutches were composed of approximately 45 eggs encapsulated by a transparent gelatinous matrix. Empty egg capsules (4–7 per clutch) were recorded in 36% of the clutches. Great plasticity was observed in the reproductive strategies ofA. granulosa, probably reflecting the varying competition and predation at the different life stages of the species. [ABSTRACT FROM PUBLISHER]

Introduction In Europe body purification and natural balance restoring rituals are becoming increasingly popular, but an introduction of Amazonian shamanic rituals in urban Europe can result in unexpected adverse events. Case report A 44-year-old woman attended a Kambô or Sapo ritual in Slovenia where dried skin secretion from a giant leaf frog ( Phyllomedusa bicolor ) was applied to five freshly burned wounds at her shoulder. Afterwards, she drank 6 litres of water and gradually developed nausea and vomiting, confusion, lethargy, muscle weakness, spasms and cramps, seizure, decreased consciousness level and short-term memory loss. The initial laboratory tests showed profound plasma hypoosmolality (251 mOsm/kg) proportional to hyponatremia (116 mmol/L) combined with inappropriately elevated urine osmolality (523 mOsm/kg) and high urine sodium concentration (87 mmol/L) indicating a syndrome of inappropriate antidiuretic hormone secretion. The patient was treated with 0.9% sodium chloride and a restriction of water intake. Plasma osmolality and hyponatremia improved one day after venom exposure, but the symptoms disappeared as late as the third day. Conclusion In patients presenting with neurological symptoms and a line of small body burns Phyllomedusa bicolor venom exposure should be suspected. Acute symptomatic hyponatremia after Phyllomedusa bicolor venom exposure is the result of inappropriate antidiuretic hormone secretion that can be exacerbated by excessive water intake. [ABSTRACT FROM AUTHOR]