Anuran fauna of the Parque Estadual Carlos Botelho - Núcleo Sete Barras, southeastern Brazil: species composition, use of breeding sites, and seasonal patterns of breeding activity

: The goal of this work was to study the species composition, the use of breeding sites, and the seasonal patterns of breeding activity of the anuran fauna from Parque Estadual Carlos Botelho - Núcleo Sete Barras, state of São Paulo, southeastern Brazil. Fieldwork was carried out from September 2005 to October 2007 through two main methods: active visual search inside a 10 ha- permanent plot and aural and visual search in seven previously selected aquatic breeding habitats. Species richness was related to the sampling effort by means of species accumulation curve and through non-parametric estimators. Thirty-three species distributed in 12 families were recorded, from which 69% are endemic to the Atlantic Forest. Trachycephalus mesophaeus represents a new record for the park. Cycloramphus lutzorum is included as Data Deficient in the IUCN list. The species accumulation curve did not stabilize, showing some tendency to rise. The use of breeding sites by 20 species was compared using cluster analysis, which revealed two major groups: the first with five species (two habitat generalists and three stream specialists) and the second composed by the other species (with different reproductive modes associated with flooded environments). The breeding period of most species was associated to the rainy season (October to February), and only Scinax hayii showed continuous breeding activity during the entire period of study.


Introduction
The Atlantic Rainforest is home to 528 species of anurans, of which about 80% are endemic (Vancine et al. 2018). This astonishing diversity is commonly attributed to the highly rugged terrain and the existence of geographical barriers that isolate populations and create high levels of speciation and endemism; the stratified physiognomy of the vegetation, with a great diversity of epiphytes, together with high levels of rainfall and humidity, result in intense partitioning of habitats and microhabitats by anurans and the evolution of specialized reproductive modes (see Haddad & Prado 2005 and references herein).
The greatest diversity in São Paulo state was recorded in the Parque Estadual Carlos Botelho, with about 85 anuran species, which is perhaps due to the abundance of water bodies and the excellent state of conservation of this conservation area (e.g., Guix et al. 1994, 2000, Forlani et al. 2010. Spatial distribution and reproductive success of amphibians are strongly dependent on the physical characteristics of reproductive environments, varying in space and time. Environments with similar abiotic factors harbor different species compositions, which indicates that there is more than one factor influencing the choice of reproductive sites, such as habitat heterogeneity and degree of preservation of terrestrial habitats around aquatic breeding sites (Wells 1977, Gascon 1991, Rudolf & Rödel 2005, which affect assemblage diversity, local abundance of some species and interspecific interactions (Underwood 2000).
Rainfall distribution and air temperature variation along the year strongly influence anuran breeding activity (Wells 2007). A higher number of species is able to reproduce throughout the year in aseasonal (Hero 1990, Gascon 1991 than in seasonal environments, where the calling and breeding activity of most species are associated with the rainy season (Aichinger 1987, Bertoluci 1998, Bertoluci & Rodrigues 2002b, Canelas & Bertoluci 2007, Narvaes et al. 2009).
The present study aimed to investigate the composition of the anuran fauna of the Núcleo Sete Barras of the Parque Estadual Carlos Botelho, São Paulo state, southeastern Brazil, the use of breeding sites by the species and their seasonal patterns of breeding activity.

Study site
The Parque Estadual Carlos Botelho (PECB) (24º00' -24º15' S, 47º45' -48º10' W) is part of an ecological complex of the Atlantic Rainforest in the southern part of the State of São Paulo, encompassing an area of approximately 38.000 ha, with altitudes that very between 30 to 1600 m above sea level, covered by Dense Ombrophylous Forest (Ferraz & Varjabedian 1999) (Figure 1). According to Veloso et al. (1991), the vegetation at PECB may perform three different physiognomic and floristic compositions: montane rainforest, submontane and low lands. The Núcleo Sete Barras (NSB) comprises two forest formations (sub-montane and low lands) and is located in the municipality of Sete Barras, Vale do Ribeira de Iguape, in a contact zone between two geomorphological formations, Guapiara Plateau (low lands) and Serra de Paranapiacaba (sub-montane) (24º11'37'' S, 47º55'11'' W) (Ferraz & Varjabedian 1999).
The climate is Cfa of Köppen, mesothermal, subtropical, hot and humid, with no dry season (Setzer 1946). In the study period, the mean annual temperature was 21.8˚ and the annual accumulated precipitation was 1,582 mm ).

Data sampling and collection
The anuran fauna sampling was carried out using two main methods: active visual (Crump & Scott 1994) in a 2500 m transect inside a permanent plot of 10 ha (more detail in Bertoluci et al. 2007a); and male vocal activity monitoring in seven different previously selected breeding habitats (Table 1). In the plot area, we carried out two visits a month between September 2005 and October 2006, always two researchers (20h/person), totalizing a sampling effort of 240h/person. The breeding habitats were visited for two consecutive nights per month between September 2005 and October 2007, totaling 52 visits in the period. In addition, we recorded all individuals found by chance in the sampling areas. All selected reproductive habitats were located along the main track called "Trilha das Figueiras", which is located along the main road that connects the municipalities of São Miguel Arcanjo and Sete Barras (Figure 1).

Data analysis
The species richness registered in relation to the sampling effort was assessed by the species accumulation curve (sensu Colwell et al. 2004), calculated through the exact method (Kindt 2004) using the function "specaccum". To estimate the richness we used the function "specpool" and "poolaccum" and the non-parametric estimators Bootstrap and Jacknife I (Santos 2003) with 10,000 randomizations with sample reposition, "vegan" package (Oksanen et al. 2011), developed for R environment (R Development Core Team 2012). A matrix with monthly occurrence was used for all species registered in the reproductive habitats and chance encounters.
To evaluate the similarity in the use of the breeding sites, we considered only species that show reproductive activity in the selected aquatic sites between September 2006 and October 2007. Based on the breeding sites use, species were classified as habitat generalists and specialists. Dissimilarity between species pairs was also calculated by the complement of the Jaccard index (DJ = 1-J), and this triangular matrix was submitted to a cluster analysis by the UPGMA method (e.g., Sneath & Sokal 1973), which calculates the coefficient of cofenetic correlation (r), indicating the degree of representativeness of the similarity matrix in the dendrogram, so that values of r ≥ 0.8 allow to consider that the dendrogram adequately represents the dissimilarity matrix (Rohlf 2000). For this analysis we use the "hclust" function, applying the "average" method, also from the "vegan" package (Oksanen et al. 2011), developed for the R-environment (R Development Core Team 2012).

Use of breeding sites
Twenty species were recorded in the seven breeding sites we monitored (Table 3). The number of species in the sites varied from four to 12. Site L1 showed the highest number of species (12), followed by Site L2 (11). The lowest number of species was found in Site R2 (4), the only temporary site studied. Fritziana fissilis, Dendrophryniscus brevipollicatus and Physalaemus spiniger were recorded in the surroundings of water bodies. Cluster analysis identified two major groups: (I) formed by Rhinella ornata and R. icterica (Ia) and Cycloramphus lutzorum, Vitreorana uranoscopa and Bokermannohyla hylax (Ib); (II) formed by the four species of Dendropsophus (IIa) and other species (IIb) ( Figure 5). Table 4 summarizes the annual breeding patterns of the 20 anuran species whose males vocalized in the seven aquatic sites showed in Table 3. Classes shown here are the highest classes of the number of calling males recorded each month considering all monitoring sessions and all sites. Evidences of actual reproduction (amplectant pairs, oviposition behavior or egg clusters) are indicated. Anurans called in almost every month of the two-years period, with higher number of active species (more than 10) being recorded in the rainy seasons (October to February) of both years. Only Scinax hayii displayed a continuous breeding pattern, with males calling in 22 of the 24 samples.

Species composition
The species composition at the NSB is typical of Neotropical sites, with high representativeness of Hylidae (Duellman 1999). In addition, 69% of the species are endemic to the Atlantic Rainforest; the high degree of endemism is also typical of this biome (Vancine et al. 2018), however the geomorphological characteristics of NSB, with altitudes close to the sea level, rising in a gradient that reaches 1,000 m in altitude, influenced the composition of the anuran fauna, which is composed by species typical of the low Atlantic Rainforest (Rhinella hoogmoedi, Ololygon argyreornata and O. littoralis) (Pombal & Gordo 1991, Caramaschi & Pombal 2006) alongside of species restricted to the hillside forest (e.g., Ischnocnema aff. guentheri) ).
The estimated richness values theoretically indicate the possibility of increasing at least three species in the sampling survey, which can be observed when considering the high ß diversity of anuran fauna found in PECB in its different phytophysiognomies or even in an altitudinal gradient (e.g. Guix et al. 1994, Guix et al. 2000, Moraes et al. 2007, Forlani et al. 2010).
The anuran fauna of different portions of PECB was described in four other studies (Guix et al., 1994;Guix et al., 2000;Bertoluci et al., 2007;Forlani et al., 2010). The compilation of these lists by Forlani et al. (2010) resulted in a richness of 85 species for the entire park. We add here one species to that list, Trachycephalus mesophaeus, maybe due to our higher sampling effort compared to those of the studies cited above, raising the overall richness of PECB to 86 species. This richness corresponds to 37% of the anuran known for the state of São Paulo (Rossa-Feres et al. 2011). As expected the composition of the anuran fauna of NSB is similar to those of other localities of southeastern Brazil covered by the Dense Ombrophilous Forest of São Paulo state ), like the Estação Biológica de Boracéia (Heyer et al., 1990;Bertoluci et al., 2005), the Parque Estadual Intervales (Bertoluci, 1998), and the Reserva Biológica do Alto da Serra de Paranapiacaba (Verdade et al. 2009). The different physiognomies of the Atlantic Forest, the large altitudinal gradient (Guix et al. 1994(Guix et al. , 2000 and the good state of conservation of PECB areas, and habitat heterogeneity (e.g., Silva et al. 2011 and references therein) are also relevant factors in the interpretation of this high richness.
Although none of the species recorded in the present study were included in the Brazilian red list of endangered fauna, Cycloramphus lutzorum is considered Data Deficient with decreasing populations by IUCN (2019); major threats to this frog are residential and commercial development, agriculture and aquaculture, and biological resource use.

Breeding site utilization
Species that used the monitored reproductive habitats did not distribute homogeneously among sites. Rhinella ornata, R. icterica and Ololygon littoralis were classified as habitat generalists (they used all habitat types); the pattern of habitat use of these species has already been observed in other taxocenoses (e.g., Izecksohn & Carvalho-e-Silva 2001, Bertoluci & Rodrigues 2002c, Narvaes et al. 2009  metamorphosis (Heyer 1985a(Heyer , 1990. Cycloramphus species lay their eggs on rocks and in wet rock crevices, with semi-terrestrial tadpoles living at the water-land interface, microhabitats commonly found on the banks of rocky streams (Haddad & Prado 2005), as sampled in PECB.
Bokermannohyla hylax is a typical species of dense ombrophilous forest streams (Heyer 1985b;Bertoluci 2002;Bertoluci et al. 2003). Males vocalize from the vegetation on the edge of forest streams, tadpoles seek shelter under rocks, dead leaves or burrow into the mud (Bertoluci et al. 2003). The presence of this species in Site L1 (classified as a pond) is probably due to the interruption of the stream course by the road slope, altering the original structure of the habitat. Changes caused by anthropogenic disturbances modify the local structure of the forest, form new microhabitats and make different resources available, which influences community structure (Connell & Slatyer 1977, Pickett & White 1985, Tocher et al. 2001). Species of the genus Rhinella were considered habitat generalists, showing the primitive reproductive mode for the group, which favors the exploration of different habitat types (Haddad & Prado 2005). Subgroup Ib of the dendrogram includes only species with specialized reproductive modes associated with streams (Heyer et al. 1990, Bertoluci et al. 2004,  Haddad & Prado 2005). Group II includes only species that breed in lentic water bodies, all of the family Hylidae, except Leptodactylus latrans (Leptodactylidae). In subgroup IIa are Dendropsophus species, typical of lagoons (e.g., Bertoluci & Rodrigues 2002c, Narvaes et al. 2009) and with generalized reproductive mode (e.g., Haddad & Prado 2005). In subgroup IIb there are two smaller branches; the former includes all species of Scinaxinae (Scinax and Ololygon), as well as Leptodactylus latrans and Phyllomedusa distincta, pond-associated species. Among the species of Scinax all have generalized reproductive mode except Ololygon rizibilis, which produces an aquatic foam nest (Haddad & Prado 2005). Leptodactylus latrans builds floating foam nests in permanent or semipermanent environments (Vaz-Ferreira & Gerhau 1975) and P. distincta lays eggs on leaves hanging over water in permanent aquatic environments (Woehl & Woehl 2000). The most intimate grouping between O. rizibilis and L. latrans may be due to the use of semi-permanent environments, suitable for their specialized reproductive mode. The second branch is formed by all Boana species, among which only Boana faber has a specialized reproductive mode (eggs in nests dug in mud in the banks of lentic water bodies; Martins & Haddad 1988), despite being habitat generalist.
The high degree of overlap in the use of reproductive habitats corroborates the well-established idea that suitable water bodies for reproduction behave as limiting factors for anurans (e.g., Bertoluci & Rodrigues 2002c). However habitat heterogeneity permits anurans use specific microhabitats as calling, oviposition and developmental sites with low or no overlap, influencing anuran richness and the composition of the assemblages (e.g., Bertoluci & Rodrigues 2002c, Vasconcelos & Rossa-Feres 2008, Vasconcelos et al. 2009, Figueiredo et al. 2019).

Seasonal patterns of breeding activity
The breeding activity of anurans at PECB-NSB was closely related to the rainy season. This general pattern is commonly found in seasonal tropical areas of both Amazonia (Toft & Duellman 1979, Aichinger, 1987, Hero, 1990, Gascon 1991, Duellman, 1995 and southeastern Brazil (Bertoluci 1998, Eterovick & Sazima 2000, Bertoluci & Rodrigues 2002b, Canelas & Bertoluci 2007, Narvaes et al. 2009     (1) 1-2, (2) 3-5, (3) 6-10, (4) 11-20, (5) 21-50, and (6) more than 50; V, abundance of calling males not estimated; * evidence of actual reproduction (amplexes or egg clusters); + presence of individuals without breeding activity.   Bertoluci 1998), also in São Paulo state. These differences seems to be related to the different climate regimes of these localities. A high proportion of continuous breeders is characteristic of aseasonal environments (e.g. Crump 1974). We conclude claiming attention to the importance of preserving all types of habitats and microhabitats in the entire altitudinal gradient of PECB as a strategy for the conservation of frogs of southeastern Brazil, one of the richest regions of the world.

Supplementary Material
The following online material is available for this article: Appendix I -Voucher material.