Expanding the knowledge about the occurrence of anurans in the highest amphibian diversity area of Atlantic Forest :

The vegetation cover in the Atlantic Forest has been converted to human use or degraded by human activities, which declined the vegetation to 16% of its original extent. Although several protected areas have been created in this Neotropical biome over the past decades, our knowledge of the amphibian species within these areas is still far from adequate. Here, we present lists of anuran species from three different areas of Parque Estadual da Serra do Mar: núcleos Curucutu, Santa Virgínia and São Sebastião. To survey anuran species, we visited six sampling units (two ponds, two streams and two trails inside forest fragments) in each locality. Samplings were accomplished from December 2014 to February 2015, December 2015 to February 2016 and December 2016 to February 2017, totalizing 27 days of field samplings in each sampling unit. We recorded 34 anuran species in the Núcleo Curucutu, 44 species in the Núcleo São Sebastião and 42 species in the Núcleo Santa Virgínia, totalizing 65 species belonging to 12 families (number of species in parentheses): Bufonidae (5), Brachycephalidae (6), Centrolenidae (1), Craugastoridae (1), Cycloramphidae (2), Hemiphractidae (1), Hylidae (29), Hylodidae (3), Leptodactylidae (12), Microhylidae (2), Odontophrynidae (2), and Phyllomedusidae (1). We hope that these lists can be useful for future studies as well as helping in the management and conservation planning of these protected areas.


Introduction
The Atlantic Forest, a global hotspot for biodiversity conservation (Mittermeier et al. 2004), originally covered 1.3 million km 2 , of which between 84% and 89% have been converted to human use or degraded by human activities (Ribeiro et al. 2009).This biome harbors one of the highest diversity of amphibians in the world, hosting approximately 600 species of amphibians (Haddad et al. 2013).Although several protected areas have been created in this Neotropical biome over the past three decades, our knowledge of the amphibian species within these areas is still far from adequate.For example, the Parque Estadual da Serra do Mar (PESM) in the state of São Paulo, Brazil, is the largest protected area of the entire Atlantic Forest biome but some administrative areas such as Núcleo São Sebastião still do not have an amphibian survey published.This is worrisome because PESM harbors the highest amphibian diversity of the Atlantic Forest biome (Loyola et al. 2014).Here, we surveyed amphibian species of Núcleo Curucutu, Núcleo Santa Virgínia and Núcleo São Sebastião and present the first list of amphibian species for Núcleo São Sebastião.Natural history knowledge and species composition are the basic dataset for ecology, systematics, biogeography, and conservation biology (e.g., Collen et al. 2008, Da Silva et al. 2012, 2014); therefore, we hope that our lists can be useful for future studies as well as helping in the management and conservation planning of these protected areas.

Survey techniques
In each of the three localities we sampled amphibian species in two ponds, two streams and two trails inside forest fragments (Table 1, Figure 2) from December 2014 to February 2015, December 2015 to February 2016 and December 2016 to February 2017 totalizing 27 days of field samplings in each sampling unit.We selected this period because is the time of the year when most of annual rainfall occurs and most anuran species are active.We sought to choose sampling units with similar size area and physiognomic characteristics in the three núcleos (Figure 2).We carried out the surveys on streams and trails inside forest fragments considering always the same extension of 100 m in all núcleos.Furthermore, we selected trails in which there were no water bodies along the extension sampled.We used three methods to record the presence of anurans in each locality: (i) surveys at breeding sites (Scott & Woodward 1994) -we recorded calling males from 19.00 hours to 24.00 hours; (ii) survey of larvae with dipnetting -we used a long and wire hand net (3 mm 2 mesh size) along the margins of ponds and streams, sampling the available microhabitats from 12.00 hours to 18.00 hours; and (iii) visual encounter (Crump & Scott Jr. 1994) -we walked slowly for 30 minutes in trails inside the forest fragment, streams and around ponds looking at microhabitats for individuals hidden under trunks, bromeliads, stones, branches, and leaf litter.All collected specimens (Table 2) were anesthetized and killed, fixed in 10% formaldehyde and stored in 70% ethanol (adults) or 10% formaldehyde (tadpoles).We also collected tissue samples that were fixed in 100% ethanol and maintained in -20ºC in freezers (Table 2)

Data Analysis
To assess the sampling efficiency of surveys separately for each locality, we used species accumulation curve (Gotelli & Colwell 2001) generated from the data of incidence of anurans during the inventory period.The total number of species recorded each day was considered as a sample, totaling 27 samples.Considering the diverse richness estimators available, we chose to use the Jackknife algorithm first order based on its performance when compared to other estimators (Magurran 2004, Walther & Moore 2005, Hortal et al. 2006).All analyzes were performed in R v 3.2.2(R Core Team 2015) with 1,000 randomizations using vegan (Oksanen et al. 2016
According to the threatened species categories used in IUCN (2016), Bokermannohyla izecksohni is classified as critically endangered, Physalaemus atlanticus is classified as vulnerable, Adenomera ajurauna, Megaelosia cf.bocainensis, Paratelmatobius cardosoi and P. bokermanni are classified as data deficient and other anuran species are classified as least concern (Table 2).On the other hand, considering threatened species categories used in the Brazilian (ICMBio 2015) and state of São Paulo (Garcia et 2).It is important to recognize that IUCN trend populations it is not consensual among researchers and it may contain imprecisions.For example, the populations of widespread species with recognized tolerance to anthropogenic habitats such as T. mesophaeus probably may not be declining (Carvalho-e-Silva & Garcia 2004).We registered 1282 individuals being that Ololygon perpusillus and Boana albopunctata were the most abundant species at Núcleo Curucutu (Figure 8B), Ischnocnema parva and Dendropsophus microps were the most abundant species at Núcleo Santa Virgínia (Figure 8C) and Adenomera marmorata and Ololygon littoralis were the most abundant species at Núcleo São Sebastião (Figure 8D).Most anuran species were recorded more than once during the sampling period; however, Thoropa taophora, B. izecksohni, O. argyreornata, Ololygon cf.obtriangulata, T. mesophaeus, Megaelosia cf.bocainensis and R. hoogmoedi were recorded on only one occasion based on a single individual (Figure 8A).Except for Megaelosia cf.bocainensis whose natural history and population trend are unknown, Thoropa taophora, B. izecksohni, O. argyreornata, Ololygon cf.obtriangulata, T. mesophaeus and R. hoogmoedi are common species that reproduce during one or few days along the year (i.e.explosive breeding).Therefore, the low abundance recorded for these species probably is due to sampling effort.Information about species richness and composition are the basic dataset for ecology, systematics, biogeography and conservation biology (e.g., Collen et al. 2008, Da Silva et al. 2012, 2014) as well as they are relevant to subsidize management plans and conservation strategies.Therefore, we hope that these lists can be useful for future studies as well as helping in the management and conservation planning of these protected areas.

Figure 1 .
Figure 1.Map of the state of São Paulo, Brazil, highlighting the area of the Parque Estadual da Serra do Mar and the location of Núcleo Curucutu, Núcleo São Sebastião and Núcleo Santa Virginia.
) and BiodiversityR(Kindt & Coe 2005) packages.Taxonomic nomenclature followsFrost (2017).The species conservation status was obtained from The International Union for Conservation of Nature Red List of Threatened Species (IUCN 2016), Brazil Red Book of Threatened Species of Fauna (ICMBio 2015) and Faunal Species of São Paulo state Threatened with Extinction(Garcia et al. 2009).

Figure 2 .
Figure 2. Photographies of ponds (a-b), streams (c-d) and trails inside forest fragments (e-f) used as sample units in the survey of anurans in the núcleos Curucutu, São Sebastião and Santa Virgínia of the Parque Estadual da Serra do Mar, state of São Paulo, southeastern Brazil.

Figure 7 .
Figure 7. Species accumulation curve (black line) and Jackknife algorithm first order (gray line) representing the anuran species richness observed and estimated, respectively, based on 18 samples from December 2014 to February 2017 at the Parque Estadual da Serra do Mar, Núcleo Curucutu, Núcleo São Sebastião and Núcleo Santa Virgínia, state of São Paulo, southeastern Brazil.The dots show the mean generated by 1000 randomizations and the vertical bars indicate the standard deviation.

Table 1 .
Geographical coordinates of sample units used in the survey of anurans in Núcleo Curucutu, Núcleo São Sebastião and Núcleo Santa Virgínia of the Parque Estadual da Serra do Mar, state of São Paulo, southeastern Brazil.Geographical coordinates in degrees, minutes and seconds, based on the WGS84 datum.

Table 2 .
(Garcia et al. 2009osition and abundance from Núcleo Curucutu (PECU), Núcleo São Sebastião (PESB) and Núcleo Santa Virgínia (PESV) of the Parque Estadual da Serra do Mar, state of São Paulo, southeastern Brazil.STATUS IUCN = threatened species categories used in the IUCN Red List Data (IUCN 2016); STATUS BRAZIL = categories of threatened species used in the Brazil Red Book of Threatened Species of Fauna (ICMBio 2015); STATUS SÃO PAULO = categories of threatened species used in the São Paulo Red Book of Threatened Species of Fauna(Garcia et al. 2009): DD (Data Deficient), LC (Least Concern) and CE (Critically Endangered).POP.TREND = current population trends used in IUCN Red List Data: increasing, decreasing, stable or unknown.* = species not evaluated or not cited.# Cryptic species: in this case, abundance was defined as only one species in the fieldwork.