Amphibians on the hotspot: Molecular biology and conservation in the South American Atlantic Rainforest

Amphibians are the focus of a recent debate and public attention owing to the global decline in their populations worldwide. Amphibians are one of the most threatened and poorly known groups of vertebrates in several geographic areas, even though they play a central role in their own ecosystems. At different levels, amphibians make their contribution to proper ecosystem functioning. They act as regulators of the food web and nutrient cycling, and they also provide several valuable ecosystem services, e.g., as a food source and as animal models for lab research. In this sense, it seems clear that the maintenance of amphibian diversity should be one of the major goals for the several countries where their population decline is observed. However, we are still struggling with the very first step of this process, i.e., the correct identification of the amphibian species diversity. Over the past few decades, research on molecular identification of amphibians using DNA barcoding has encountered some difficulties related to high variability in the mitochondrial genome of amphibians, and a research gap is noticeable in the literature. We herein evaluated both COI and 16S rRNA mitochondrial genes for the molecular identification of frogs and tadpoles in a large fragment of the South American Atlantic Rainforest in Rio de Janeiro, Brazil. Our results suggest that both COI and 16S rRNA are informative markers for the molecular identification of the amphibian specimens with all specimens unambiguously identified at the species level. We also made publicly available 12 new sequences of Atlantic Rainforest amphibian species for the first time, and we discussed some conservation issues related to amphibians within the Atlantic Rainforest domains in the state of Rio de Janeiro, Brazil.The authors benefited from grants provided to CFDR (304791/2010-5; 470265/2010- 8 and 302974/2015-6) from Conselho Nacional do Desenvolvimento Cientifico e Tecnologico (CNPq) and through "Cientistas do Nosso Estado" Program from FAPERJ to CFDR (process No. E-26/ 102.765.2012 E-26/202.920.2015). We would like to thank C. Haddad (Universidade Estadual Paulista) and M. L. Lyra (Universidade Estadual Paulista) for some of the specimens and the primers used in the present manuscript and L. A. Fusinatto, who kindly revised the manuscript offering helpful suggestions. FP was supported by the Portuguese Foundation for Science and Technology (FCT) [IF/01356/2012] and Northern Regional Operational Programme (NORTE2020) through the European Regional Development Fund (ERDF) [MARINFO NORTE-01-0145-FEDER- 000031]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Differential success in sampling of Atlantic Forest amphibians among different periods of the day

In general, anurans tend to be nocturnal, though diurnal activity is characteristic of some groups. Studies show that frog activity may be inferred based on the number of individuals collected at different periods of the day, during large-scale field surveys. We investigated the best period of the day to conduct amphibian sampling in nine Atlantic Rainforest areas in southeastern Brazil, based on intensive field surveys. At each locality we employed similar sampling effort during diurnal, crepuscular and nocturnal searches (totaling 704.5 sampling hours). We pooled data from all localities for each period and estimated the proportion of frogs of each species active at each period based on the total number of individuals and on the number of species found during all surveys for that period. We recorded a total of 817 individual frogs from 69 species. Species richness was highest at night (median = 12 species), intermediate at dusk (median = 8), and lowest during the day (median = 4). The percentage of the total number of individual frogs found (pooled species) was highest during the night (ca. 53%) and lowest during the day (ca. 14%). Analyzing each species separately, the number of individuals recorded was consistently higher at dusk and night for most species. Our study evidences a trend for nocturnal activity for most Atlantic Rainforest frogs, with few species having primarily diurnal habits. Those results may favor future studies and conservation efforts for amphibian species

Differential success in sampling of Atlantic Forest amphibians among different periods of the day

In general, anurans tend to be nocturnal, though diurnal activity is characteristic of some groups. Studies show that frog activity may be inferred based on the number of individuals collected at different periods of the day, during large-scale field surveys. We investigated the best period of the day to conduct amphibian sampling in nine Atlantic Rainforest areas in southeastern Brazil, based on intensive field surveys. At each locality we employed similar sampling effort during diurnal, crepuscular and nocturnal searches (totaling 704.5 sampling hours). We pooled data from all localities for each period and estimated the proportion of frogs of each species active at each period based on the total number of individuals and on the number of species found during all surveys for that period. We recorded a total of 817 individual frogs from 69 species. Species richness was highest at night (median = 12 species), intermediate at dusk (median = 8), and lowest during the day (median = 4). The percentage of the total number of individual frogs found (pooled species) was highest during the night (ca. 53%) and lowest during the day (ca. 14%). Analyzing each species separately, the number of individuals recorded was consistently higher at dusk and night for most species. Our study evidences a trend for nocturnal activity for most Atlantic Rainforest frogs, with few species having primarily diurnal habits. Those results may favor future studies and conservation efforts for amphibian species

Anurofauna of an Atlantic Rainforest fragment and its surroundings in Northern Rio de Janeiro State, Brazil

We carried out a study on the anurofaunal community from an Atlantic Forest fragment (Monte Verde mountains) and the surrounding area in Cambuci municipality, Rio de Janeiro State, Brazil, which constitutes one of the largest fragments remaining in the largely deforested landscape of the northern portion of the State. We combined three sampling methods: plot sampling, transects and pit-fall traps. We recorded twenty species of amphibians, of which only eleven were found within the forest fragment (and five of these also occurred in the surrounding matrix). Two of the species recorded in the present study (Crossodactylus sp. and Ischnocnema cf. parva) may represent undescribed taxa. Our records expand the distribution range of one species (Scinax trapicheiroi) to the north, and fill a geographic distribution gap for another one (Ischnocnema oea). The estimated overall density of frogs living in the leaf litter of the fragment (based on results of plot sampling) was 3.1 individuals/100 m², with Haddadus binotatus being the most abundant species (2.4 individuals/100 m²). Comparisons of our data with those of other studies suggest that anuran communities in forest fragments ca. 1,000 ha or smaller may be severely limited in their richness, and often include a large proportion of species tolerant to open areas, such as many hylids. Our results show the importance of increasing knowledge about the anurofaunal community of the northern portion of the State of Rio de Janeiro and preserve the forest remnants that still exist in the region

Anurofauna of an Atlantic Rainforest fragment and its surroundings in Northern Rio de Janeiro State, Brazil

We carried out a study on the anurofaunal community from an Atlantic Forest fragment (Monte Verde mountains) and the surrounding area in Cambuci municipality, Rio de Janeiro State, Brazil, which constitutes one of the largest fragments remaining in the largely deforested landscape of the northern portion of the State. We combined three sampling methods: plot sampling, transects and pit-fall traps. We recorded twenty species of amphibians, of which only eleven were found within the forest fragment (and five of these also occurred in the surrounding matrix). Two of the species recorded in the present study (Crossodactylus sp. and Ischnocnema cf. parva) may represent undescribed taxa. Our records expand the distribution range of one species (Scinax trapicheiroi) to the north, and fill a geographic distribution gap for another one (Ischnocnema oea). The estimated overall density of frogs living in the leaf litter of the fragment (based on results of plot sampling) was 3.1 individuals/100 m², with Haddadus binotatus being the most abundant species (2.4 individuals/100 m²). Comparisons of our data with those of other studies suggest that anuran communities in forest fragments ca. 1,000 ha or smaller may be severely limited in their richness, and often include a large proportion of species tolerant to open areas, such as many hylids. Our results show the importance of increasing knowledge about the anurofaunal community of the northern portion of the State of Rio de Janeiro and preserve the forest remnants that still exist in the region