Impacts of isolated or mixed Roundup® Original DI and Boral® 500 SC herbicides on the survival and metamorphosis of Melanophryniscus admirabilis tadpoles.

The bufonid species Melanophryniscus admirabilis is restricted to a single location in the southern Atlantic Forest, Brazil. Although the site of occurrence of M. admirabilis is covered with native forest and it is not directly exposed to pesticides application, the area is surrounded by agricultural activity. Our objectives were to evaluate possible alterations in morphological parameters (body mass, snout-vent length, and body index), metamorphosis (time to reach Gosner stages 42, 46 and to complete metamorphosis), and survival of M. admirabilis exposed to isolated Roundup® Original DI (R1: 234 and R2: 2340 µg.L-1 of glyphosate) and Boral® 500 SC, (B1: 130 and B2: 980 µg.L-1 of sulfentrazone) or mixed (R1+B1, R2+B1, R1+B2, R2+B2). Spawns of M. admirabilis were collected in natural lakes in the municipality of Arvorezinha and taken to laboratory cultivation. After the tadpoles acquired free swimming, the animals were acclimated for five days and fed ad libitum. The aquariums were contaminated with herbicides on the sixth day of cultivation, and the animals stayed in these aquariums for four days. Afterwards, the tadpoles were transferred to aquariums with clean water and monitored until metamorphosis (Gosner stage 46), when they were weighed, measured (snout-cloacal length) and cryoeuthanized. We observed no alterations in morphological parameters; however, survival was reduced in exposed groups (mortality index: 71 % in R2 and 29-64 % in mixed groups), suggesting energy allocation for metamorphosis at the expense of survival. Boral did not alter metamorphosis time. Roundup isolated and mixed with Boral altered the timing of Gosner stages 42 and 46 and reduced metamorphosis time, suggesting endocrine disruption. Thus, monitoring the presence and limiting the use of these pesticides in the area where M. admirabilis occurs can be crucial for conservation strategies.

Anthropic Impact on the Critically Endangered Melanophryniscus admirabilis (Admirable Redbelly Toad): Evidence from the Presence of Multiresistant Enterobacteriaceae.

Melanophryniscus admirabilis is a microendemic and critically endangered toad, known from a single population. This microendemic species inhabits a small fragment of the Atlantic Forest in South Brazil, an area significantly impacted by hydroelectric power plant projects, livestock farming, agricultural activities, biopiracy, and tourism. Given the exclusive and limited population of M. admirabilis, preserving and conserving this species is of utmost importance in Brazil. Research on this species primarily concentrates on its biology, ecology, and ecotoxicology. Currently, there is no knowledge about antimicrobial resistance (AMR) bacteria present in wild M. admirabilis, despite the potential for studying them to provide valuable insights into environmental pollution. To this end, Enterobacteriaceae species (n = 82) obtained from 15 wild M. admirabilis toads were subjected to the standard Kirby-Bauer disk diffusion method to test their AMR. The results showed that Enterobacteriaceae species had the highest antibiotic resistance to IPM (45.1%), CIP (39%), NIT (32.5%), AMP (31.3%), TET (18.3%), and FOX (17%). Of the tested species, 18 (21.9%) species tested were susceptible, 40 (48.8%) were resistant to 1 or 2 different antibiotic classes, and 24 (29.3%) were classified as multidrug-resistant. Overall, our findings suggest that the incidence of AMR in Enterobacteriaceae isolated from wild M. admirabilis is high, indicating environmental stress caused by anthropic pollution in their habit.

First Report of Culturable Skin Bacteria in Melanophryniscus admirabilis (Admirable Redbelly Toad).

Melanophryniscus admirabilis is a small toad, critically endangered with a microendemic distribution in the Atlantic Forest in southern Brazil. The amphibian skin microbiome is considered one of the first lines of defense against pathogenic infections, such as Batrachochytrium dendrobatidis (Bd). The knowledge of skin amphibian microbiomes is important to numerous fields, including species conservation, detection, and quantification of environmental changes and stressors. In the present study, we investigated, for the first time, cultivable bacteria in the skin of wild M. admirabilis, and detected Bd fungus by nested polymerase chain reaction (PCR) technique. Skin swab samples were collected from 15 wild M. admirabilis, and the isolation of bacteria was performed by means of different culture strategies. A total of 62 bacterial isolates being Bacillus (n = 22; 34.48%), Citrobacter (n = 10; 16.13%), and Serratia (n = 12; 19.35%) were more frequently isolated genera. Interestingly, all skin samples tested were Bd negative. Some bacterial genera identified in our study might be acting in a synergic relationship and protecting them against the Bd fungus. In addition, these bacteria may play an essential role in maintaining this species in an environment modulated by anthropic actions. This first report of skin cultivable bacteria from M. admirabilis natural population improves our knowledge of skin amphibian microbiomes, contributing to a better understanding of their ecology and how this species has survived in an environment modulated by anthropic action.

More than meets no eyes: taxonomic status of a Liotyphlops (Serpentes: Anomalepididae) blindsnake from the atlantic rainforest

Liotyphlops Peters, 1881 is an anomalepidid blindsnake genus that encompasses 12 species, widely distributed in Central and South America. In this study, we evaluate the taxonomic status of Liotyphlops sousai Santos & Reis, 2018, a species described based on a single specimen from the Atlantic Rainforest of southern Brazil, and after analyzing evidences based upon molecular systematics, external morphology and osteology, we propose its synonymy with Liotyphlops beui (Amaral, 1924), a common and widely distributed Anomalepidid species. We also provide comments and recommendations on the taxonomy of Liotyphlops, assessing the risks associated with describing a new species based on small type series.

The Oral Bacterial Community in Melanophryniscus admirabilis (Admirable Red-Belly Toads): Implications for Conservation.

Melanophryniscus admirabilis (admirable red-belly toad) is a microendemic and critically endangered species found exclusively along 700 m of the Forqueta River, in a fragment of the Atlantic Forest of southern Brazil. One of the greatest concerns regarding the conservation of this species is the extensive use of pesticides in areas surrounding their natural habitat. In recent years, the adaptation and persistence of animal species in human-impacted environments have been associated with microbiota. Therefore, the present study aimed to characterize the oral bacterial community of wild M. admirabilis and to address the question of how this community might contribute to this toad's adaptation in the anthropogenic environment as well as its general metabolic capabilities. A total of 11 oral samples collected from wild M. admirabilis were characterized and analyzed via high-throughput sequencing. Fragments of the 16S rRNA variable region 4 (V4) were amplified, and sequencing was conducted using an Ion Personal Genome Machine (PGM) System with 316 chips. A total of 181,350 sequences were obtained, resulting in 16 phyla, 34 classes, 39 orders, and 77 families. Proteobacteria dominated (53%) the oral microbiota of toads, followed by Firmicutes (18%), Bacteroidetes (17%), and Actinobacteria (5%). No significant differences in microbial community profile from among the samples were reported, which suggests that the low dietary diversity observed in this population may directly influence the bacterial composition. Inferences of microbiome function were performed using PICRUSt2 software. Important pathways (e.g., xenobiotic degradation pathways for pesticides and aromatic phenolic compounds) were detected, which suggests that the bacterial communities may serve important roles in M. admirabilis health and survival in the anthropogenic environment. Overall, our results have important implications for the conservation and management of this microendemic and critically endangered species.

Melanophryniscus admirabilis tadpoles' responses to sulfentrazone and glyphosate-based herbicides: an approach on metabolism and antioxidant defenses.

Melanophryniscus admirabilis is a frog endemic to the southern Atlantic Forest (Brazil), with restricted distribution and considered as critically endangered. The aim of this study was to evaluate possible alterations in biomarkers of metabolism (glycogen, proteins, and uric acid) and oxidative balance (superoxide dismutase, catalase, glutathione S-transferase, and lipoperoxidation) of tadpoles of Melanophryniscus admirabilis exposed to commercial herbicide formulations containing sulfentrazone (Boral® 500 SC: 130 and 980 µg a.i./L) and glyphosate (Roundup® Original: 234 and 2340 µg a.i./L). Mortality was not observed in any of the groups studied. Our results show that a 96-h exposure to the herbicides decreased glycogen levels, indicating increased energy demand for xenobiotic metabolism. Protein levels increased in the Boral group but decreased in the higher concentration of Roundup, and uric acid levels did not change significantly between the experimental groups. Lipoperoxidation decreased in the Boral group and in the higher concentration of Roundup. Decreased levels of superoxide dismutase in both treatments and of catalase in the lowest concentration of the herbicides were observed. Glutathione S-transferase activity increased in the Roundup group; this enzyme seems to be crucial in the metabolization of the herbicides and in the survival of the tadpoles. Our results suggest that M. admirabilis has a high antioxidant capacity, which guaranteed the survival of tadpoles. Nevertheless, exposure to pesticides could impose a serious risk to this species, especially considering its restricted distribution, habitat specificity, and high physiological demand to metabolize xenobiotics.

Advertisement call of two threatened red-bellied-toads, Melanophryniscus cambaraensis and M. macrogranulosus (Anura: Bufonidae), from the Atlantic Rainforest, southern Brazil.

In anuran amphibians, acoustic signals are fundamental mechanisms of mate recognition and mate choice, which makes frog calls a fundamental tool for anuran taxonomy. In this work, we describe the advertisement call of two species for the genus Melanophryniscus, M. cambaraensis and M. macrogranulosus and use the descriptions to try to solve a taxonomic problem between them. We collected data after heavy rains in three different sample sites in Rio Grande do Sul, Brazil, between 2012 and 2013. The advertisement call of both species is composed of two segments. It always begins with part A (about 0.44-6 seconds) composed of single modulated pulses separated by long time intervals. It is followed by part B, a long train of unmodulated pulses with short time intervals, lasting from 9 to 32.2 seconds. Principal Component Analysis (PCA) indicated some variation between temporal parameters of the two species, but Multivariate Analysis of Variance showed no significant differences. Within-individual Coefficient of Variation (CV) showed only two static parameters: pulse rate and peak frequency, both in the part B of the call. Despite intra-male variation in some acoustic parameters, it is not possible to differentiate between M. cambaraensis and M. macrogranulosus species only using bioacoustics.

Species limits in the Tawny-crowned Pygmy-tyrant Euscarthmus meloryphus complex (Aves: Passeriformes: Tyrannidae).

Euscarthmus pygmy-tyrants include two species of tiny passerines that inhabit semi-open landscapes in South America. One of these, Euscarthmus meloryphus is a taxonomic complex comprising three recognized subspecies for which previously proposed splits and lumps were not founded on proper taxonomic analysis. We assessed morphology (morphometrics and plumage), distribution models, and vocalizations of a substantial dataset with the aim of evaluating species limits within the E. meloryphus complex. Our results support the recognition of two species, the widespread E. meloryphus from east-central South America, and E. fulviceps from Ecuador and Peru, which are clearly diagnosable on morphological and vocal characters. We consider that E. meloryphus paulus from Colombia and Venezuela is a synonym of the nominotypical taxon based on the absence of diagnostic vocal and morphological characters. The allopatric distribution of the two populations of E. meloryphus may be related to the savannah connections during the LGM in South America. Further, we show that Lepturus ruficeps Swainson, 1838 is a synonym of E. rufomarginatus instead of E. meloryphus as it has long been treated.

Anthropogenic substrate-borne vibrations impact anuran calling.

Anthropogenic disturbance is a major cause of the biodiversity crisis. Nevertheless, the role of anthropogenic substrate vibrations in disrupting animal behavior is poorly understood. Amphibians comprise the terrestrial vertebrates most sensitive to vibrations, and since communication is crucial to their survival and reproduction, they are a suitable model for investigating this timely subject. Playback tests were used to assess the effects of substrate vibrations produced by two sources of anthropogenic activity- road traffic and wind turbines- on the calling activity of a naïve population of terrestrial toads. In their natural habitat, a buried tactile sound transducer was used to emit simulated traffic and wind turbine vibrations, and changes in the toads' acoustic responses were analyzed by measuring parameters important for reproductive success: call rate, call duration and dominant frequency. Our results showed a significant call rate reduction by males of Alytes obstetricans in response to both seismic sources, whereas other parameters remained stable. Since females of several species prefer males with higher call rates, our results suggest that anthropogenically derived substrate-borne vibrations could reduce individual reproductive success. Our study demonstrates a clear negative effect of anthropogenic vibrations on anuran communication, and the urgent need for further investigation in this area.

Atlas of Brazilian snakes: verified point-locality maps to mitigate the wallacean shortfall in a megadiverse snake fauna

Accurate and detailed species distribution maps are fundamental for documenting and interpreting biological diversity. For snakes, an ecologically diverse group of reptiles, syntheses and detailed data on distribution patterns remain scarce. We present the first comprehensive collection of detailed, voucher-based, point-locality, range maps for all described and documented Brazilian snakes, with the major aim of mitigating the Wallacean shortfall and as a contribution towards a better understanding of this rich, threatened, and poorly studied megadiverse fauna. We recorded a total of 412 snake species in Brazil on the basis of an extensive and verified point-locality database of 163,498 entries and 75,681 unique records (available here as Online Supporting Information). Our results reveal previously undocumented patterns of distribution, sampling effort, richness, and endemism levels, resulting in a more objective view of snake diversity in the Neotropics. Apart from these achievements, we understand that the most relevant and enduring contribution of the present atlas is to stimulate researchers to publish corrections, additions, and new discoveries.

Atlas of Brazilian snakes: verified point-locality maps to mitigate the wallacean shortfall in a megadiverse snake fauna

Accurate and detailed species distribution maps are fundamental for documenting and interpreting biological diversity. For snakes, an ecologically diverse group of reptiles, syntheses and detailed data on distribution patterns remain scarce. We present the first comprehensive collection of detailed, voucher-based, point-locality, range maps for all described and documented Brazilian snakes, with the major aim of mitigating the Wallacean shortfall and as a contribution towards a better understanding of this rich, threatened, and poorly studied megadiverse fauna. We recorded a total of 412 snake species in Brazil on the basis of an extensive and verified point-locality database of 163,498 entries and 75,681 unique records (available here as Online Supporting Information). Our results reveal previously undocumented patterns of distribution, sampling effort, richness, and endemism levels, resulting in a more objective view of snake diversity in the Neotropics. Apart from these achievements, we understand that the most relevant and enduring contribution of the present atlas is to stimulate researchers to publish corrections, additions, and new discoveries.

Correction: Effects of traffic noise on the calling behavior of two Neotropical hylid frogs.

[This corrects the article DOI: 10.1371/journal.pone.0183342.].

Are the unken reflex and the aposematic colouration of Red-Bellied Toads efficient against bird predation?

Aposematic signals as well as body behaviours may be important anti-predator defences. Species of the genus Melanophryniscus are characterised by having toxic lipophilic alkaloids in the skin and for presenting a red ventral colouration, which can be observed when they perform the behaviour called the unken reflex. Both the reflex behaviour and the colouration pattern are described as defence mechanisms. However, there are currently no studies testing their effectiveness against predators. This study aimed to test experimentally if both ventral conspicuous colouration and the unken reflex in Melanophryniscus cambaraensis function as aposematic signals against visually oriented predators (birds). We simulated the species studied using three different clay toad models as follows: (a) in a normal position with green coloured bodies, (b) in the unken reflex position with green coloured body and extremities and (c) in the unken reflex position with a green body and red extremities. Models were distributed on a known M. cambaraensis breeding site and in the adjacent forest. More than half of the attacks on the models were from birds; however, there was no preference for any model type. Thus, just the presence of the red colour associated with the motionless unken reflex position does not seem to prevent attacks from potential predators. It is possible that the effective aposematic signal in Melanophryniscus is achieved through the unken reflex movement together with the subsequent exhibition of the warning colouration and the secretion of toxins.

Reptile diversity of Sinos River Basin / Diversidade de répteis da Bacia Hidrográfica do Rio dos Sinos

Abstract This work aimed to catalog the species of reptiles of the Sinos River Basin based on records from scientific collections and data collected in the field. We recorded 65 species, including 46 snakes, nine lizards, five turtles, four amphisbaenians and one caiman. Snakes composed most of the recorded specimens (91.3%), and the three most representative are venomous and of medical importance. The most urban region of the basin (Lowland) has the highest number of records. This fact may be a reflection of the high human population density in this region, which would have favored the encounter of specimens and their sending to scientific collections and research centers. It is worth highlighting that most species with few specimens in the collections are also rarely observed in the wild, such as Clelia hussani and Urostrophus vautieri. This observation makes it feasible that these populations are small or that they are declining.

Resumo Este trabalho teve como objetivo catalogar as espécies de répteis da Bacia Hidrográfica do Rio Sinos com base em registros de coleções científicas e dados coletados em campo. Registramos 65 espécies, incluindo 46 serpentes, nove lagartos, cinco tartarugas, quatro anfisbenas e um jacaré. As serpentes compuseram a maioria dos espécimes registrados (91,3%), e as três espécies mais representativas são peçonhentas e de importância médica. A região mais urbana da bacia (planície) possui o maior número de registros. Este fato pode ser um reflexo da alta densidade populacional humana nesta região, o que teria favorecido o encontro de espécimes e seu envio para coleções científicas e centros de pesquisa. Vale ressaltar que a maioria das espécies com poucos exemplares nas coleções também são raramente observadas na natureza, como Clelia hussani e Urostrophus vautieri. Esta observação torna viável que essas populações sejam pequenas ou que estejam em declínio.

Effects of traffic noise on the calling behavior of two Neotropical hylid frogs.

Anthropogenic disturbance has been pointed to as one of the major causes of the world´s biodiversity crisis. Among them, noise pollution is a potential underestimated threat, projected to increase in the next decades accompanying urban expansion. Rising levels of noise pollution may result in negative impacts on species highly dependent on acoustic communication. Amphibians have long served as model organisms for investigating animal acoustic communication because their reproduction depends on transmitting and receiving acoustic signals. A few studies have investigated the effects of anthropogenic noise on anurans, but there is still limited knowledge on how it affects them. In this study, we test the effect of two intensities of traffic noise on calling males of two Neotropical treefrogs species. We expect to record more changes in call parameters, to avoid masking effect, at higher intensity noise treatments, and in the species with higher call/noise frequency overlap. We performed a set of field playback experiments exposing male frogs to road noise at two different intensities (65dB and 75dB). Focal species are Boana bischoffi (high call/noise frequency overlap) and B. leptolineata (low call/noise frequency overlap). Both species changed acoustic parameters during or after the exposure to traffic noise. Advertisement call rate of B. bischoffi decreased during road noise, and dominant frequency decreased over time. Call length of B. leptolineata increased or decreased, depending on the order of noise intensity. We also observed spatial displacement in both species, which moved away from the noise source. Our results provide evidence that traffic noise affects anuran calling behavior, and noise intensity is an important factor affecting how species respond.

Climate change and the distribution of neotropical red-bellied toads (Melanophryniscus, Anura, Amphibia): how to prioritize species and populations?

We used species distribution modeling to investigate the potential effects of climate change on 24 species of Neotropical anurans of the genus Melanophryniscus. These toads are small, have limited mobility, and a high percentage are endangered or present restricted geographical distributions. We looked at the changes in the size of suitable climatic regions and in the numbers of known occurrence sites within the distribution limits of all species. We used the MaxEnt algorithm to project current and future suitable climatic areas (a consensus of IPCC scenarios A2a and B2a for 2020 and 2080) for each species. 40% of the species may lose over 50% of their potential distribution area by 2080, whereas 28% of species may lose less than 10%. Four species had over 40% of the currently known occurrence sites outside the predicted 2080 areas. The effect of climate change (decrease in climatic suitable areas) did not differ according to the present distribution area, major habitat type or phylogenetic group of the studied species. We used the estimated decrease in specific suitable climatic range to set a conservation priority rank for Melanophryniscus species. Four species were set to high conservation priority: M. montevidensis, (100% of its original suitable range and all known occurrence points potentially lost by 2080), M. sp.2, M. cambaraensis, and M. tumifrons. Three species (M. spectabilis, M. stelzneri, and M. sp.3) were set between high to intermediate priority (more than 60% decrease in area predicted by 2080); nine species were ranked as intermediate priority, while eight species were ranked as low conservation priority. We suggest that monitoring and conservation actions should be focused primarily on those species and populations that are likely to lose the largest area of suitable climate and the largest number of known populations in the short-term.

Community structure of helminth parasites of the "Cururu" toad, Rhinella icterica (Anura: Bufonidae) from southern Brazil.

Sixty specimens of the "cururu" toad, Rhinella icterica (Spix 1824) (Bufonidae), were collected in Campo Belo do Sul, State of Santa Catarina, Brazil, between May 2009 and January 2011, and were examined for the presence of helminth parasites. Nine species of adult helminths were found: Catadiscus cohni, Rudolphitrema rudolphii, Cylindrotaenia sp., Rhabdias fuelleborni, Strongyloides sp., Cosmocerca rara, Cosmocerca brasiliensis, Aplectana elenae, and Oxyascaris sp., in addition to an unidentified adult nematode species. Females of cosmocercid nematodes, proteocephalan plerocercoid, and acanthocephalan cystacanth were found but not identified for lack absolute of taxonomic characters. The sex of the anurans had no influence on prevalence, abundance, and richness of helminth species. Length and body mass of hosts did not influence the prevalence and richness of helminths, while the abundance of R. fuelleborni was significantly correlated with both parameters.

A new microendemic species of Tropidurus (Squamata: Tropiduridae) from southern Brazil and revalidation of Tropidurus catalanensis Gudynas & Skuk, 1983.

The South American and cis-andean lizard genus Tropidurus has a complex taxonomic history. Most species were recently described and previous revisions included few specimens from the southern part of the continent. Tropidurus torquatus has the broadest geographic distribution in the genus and several morphological and ecological differences were described within its distribution. We analyzed the geographic variation in external morphological characters of Tropidurus torquatus, including large number of samples from southern Brazil. Tropidurus catalanensis is revalidated and Tropidurus imbituba sp. nov., with a restrict distribution in the southern coast of Brazil, is described. The new species is distinguished from Tropidurus catalanensis by the number of dorsal scales. It can be distinguished from T. catalanensis and T. torquatus by a distinct orange ventral coloration in adults, which can reach the lateral portion of the body in adult males. Our analysis also suggests that at least two other undescribed species could be recognized under the name Tropidurus torquatus in southeastern and central Brazil.