Herpetology Notes, volume 11: 395-403 (2018) (published online on 09 May 2018) The reproductive biology of Dendropsophus minutus (Amphibia: Anura) in South of Brazil Peterson T. Leivas1,*, Tiago B. Mayer1 and Luis F. Fávaro1 Abstract. This study investigated the reproductive biology of Dendropsophus minutus (Peters, 1972), in a temperate area of Mixed Ombrophilous Mountainous Forest (Araucaria Forest) of southern Brazil, based on the histological analyses. A total of 89 adult males, four adult females and eight juvenile were collected during monthly sampling in the State of Paraná, Brazil. In the laboratory, sex determination and macroscopic determination of the gonad development stage of each specimen were carried out. Subsequently, the gonads were removed and their mass was determined to obtain an Individual Gonadosomatic Index (GSI) and processed according to the histological routine. Based on the quantity and the type of germinative cells, males had their testicles classified in four stage (immature, initial maturation, advanced maturation and mature) and females had their ovaries only in mature stage. Although we observed males in advanced stages of maturation throughout the year, they increased in frequency during winter and spring. The maturation curve for male showed that reproductive activity was more intense from August to January, when the mature females were recorded. We showed that male reproductive activity is affected by local abiotic factors that stimulate male sexual activity from the end of winter to the beginning of summer, period in which the temperature and rain are elevated. Key words: Atlantic rainforest, breeding phenology, histological analyses, reproduction, spermatogenesis Introduction in which individuals are most active, characterizing a seasonality in reproductive activity (Wells, 2007). Anuran amphibians present one of the most complex Besides abiotic factors, other biological interactions in behavioural repertories and the highest diversity of terrestrial and/or aquatic environments influence the reproductive mode among vertebrates (Haddad and reproductive cycle (Toledo et al., 2006; Leivas et al., Prado, 2005; Wells, 2007). Such complexity is due 2012). to the fact that species reproductive strategies are The understanding on species reproductive biology as determined by the combination of morphological, well as the influence of abiotic factors on reproductive physiological, behavioural and environmental factors activity has special interest in Hylidae because of the (Pombal and Haddad, 2005; Wells, 2007; Haddad et al., large distribution, high richness of species, diversity 2013). Anurans present two general temporal patterns of reproductive modes and behavioural repertoire. The of reproduction, the explosive which lasts for a few species of this family can present regional variations days and the prolonged which may last for weeks or regarding gonad development and reproductive period months (Wells, 2007; Haddad et al., 2013). Regardless (Wells, 2007; Haddad et al., 2013). Among the species of temporal pattern, abiotic factors (eg.: rainfall and of family Hylidae, Dendropsophus minutus (Peters, temperature) might interfere in the reproductive cycle 1972) is a small-sized species measuring 20–25 mm of amphibians (Oliveira et al., 2002; Hiert et al., 2012; (snout-ventral length) with widespread distribution Haddad et al., 2013) and determine the season of year from the lowlands up to 2.000 m in altitude, occurring from the lowlands east of the Andes from Colombia, Venezuela, and Trinidad southward through Ecuador, Peru and Brazil to Bolivia, Uruguay, until Argentina 1 Universidade Federal do Paraná (UFPR), Centro Politécnico, (Frost, 2017). Pós Graduação em Zoologia. End. Av. Coronel Francisco Heráclito dos Santos, 210 - Jardim das Americas, Curitiba In Brazil D. minutus occurs in different climatic - PR, CEP: 81531-970 - Caixa postal 19020. regions and biomes and the reproductive activity is * Corresponding author. E-mail: [email protected] influenced by regional climatic factors (ex. precipitation 396 Peterson T. Leivas et al. and temperature) (VanSluys and Rocha, 1998; Oliveira based on the classification proposed by Costa et al. et al., 2007; Santos et al., 2012). In subtropical climate (1998a and b), which considers the cell types present there is no studies on the reproductive biology of the in the parenchyma and their respective quantities in species and this knowledge is essential for understanding both sexes. This analysis enabled us to confirm, or how regional abiotic factors influence the reproductive to correct if necessary, the results of the macroscopic biology of frogs as widely distributed as D. minutus. analysis. After histological analyses, we classified the Considering the influence of climate and other abiotic testicular development as follows: Immature (A); Initial factors in reproductive process of frog species, this maturation (B1); advanced maturation (B2) and Mature study aims characterize the reproduction of D. minutus, (C). including the reproductive period based on histological The characterization of the reproductive period was analysis of the gonads, sex ratio, proportion juveniles based on the analyses of the Individual Gonadosomatic and adults, and the length at first maturity for males, and Index (GSI) from the maturation curve and on the monthly its relationship with environmental factors in subtropical distribution of the stages of gonadal development, which climate. were determined from the histological analyses of the gonads. Juvenile specimens that could not be sexed were Material and Methods excluded from the analysis. The GSI was measured by gonad mass related to total individual mass, represented The study was conducted in natural area with anthropic by the formula: GSI = (GM/BM) x 100, where (GM) interference in Campina Grande do Sul, state of Paraná, corresponds to gonad mass and (BM) to individual body Brazil (25º17’09”S, 49º00’05”W). The vegetation is mass (Costa et al., 1998a). Mixed Ombrophilous Mountainous Forest (Araucaria Length at first maturity (L50) was calculated by Forest) (IBGE, 2012) and the climate is temperate Cfb Fr=1–(℮–aSVL b) (Fr= relative frequency of adult following Koppen’s classification (Alvares et al., 2013). m individuals; ℮= base of the Napierian logarithm; a and b The mean annual temperature during the study (January are coefficients estimated by the least squares method, to December 2012) was 19.08 ± 3.00°C. During the transforming the variables involved; and SVL midpoint colder months the mean was 16.56 ± 1.79°C and, during of the length classes), according to Fávaro et al. (2003) the warmest months, was 21.60 ± 1.44°C. The average and Oliveira and Fávaro (2011). This analysis was annual precipitation was 1,483.8 mm/year with a performed only for males, since the number of females monthly mean of 123.7 ± 69.99 mm. The mean relative captured and the absence of immature females did not humidity was 80.73 ± 3.87%. Frosts occurred regularly allow this analysis. in winter. The sampling sites, at an approximate altitude The Spearman correlation was used to test the of 918 m, comprehend two permanent ponds with correlation between mean monthly temperature (°C), aquatic vegetation. mean monthly relative air humidity (%) and accumulated Monthly samplings were conducted from January to monthly precipitation (mm) with the frequency of December 2012, during the night, with sampling effort monthly incidence of each stage and individual GSI of three hours (two persons), using visual encounter (only for male). The stages of maturation in this based on auditory search (Crump and Scott, 1994). analyses were: Immature (A), Initial maturation (B1) After the capture, specimens were transported to the and Advances maturation + Mature (B2+C), grouped laboratory, where they were sacrificed according to the for possessing spermatozoids in the seminiferous loculi. current legislation at the Conselho Federal de Biologia Statistical significance was set a priori to p<0.05. In this (CFBIO - Resolution 308). Voucher specimens were study, the seasons of the year were defined as: Summer deposited at the Museu de História Natural do Capão da (January to March), Fall (April to June), Winter (July to Imbuia, Curitiba, state of Paraná, Brazil. September) and Spring (October to December). For each specimen we measured snout-ventral length (SVL) and body mass (BM). We then made a ventral Results incision in the carcass to determine the macroscopic stage of gonad development. Gonads were removed A total of 101 individuals of D. minutus was collected, and weighed (GM), and part of the gonadal tissue 89 adult males, four juvenile males and four adult was subjected to histological analyses. We used the females. For four juvenile specimens the sex was not precision of 0.01 mm in our length measurements determined. The SVL in adult males varied between and 0.001 g for mass. The microscopic analysis was 16.00 and 24.06 mm and the BM varied between 0.104 The reproductive biology of Dendropsophus minutus in South of Brazil 397 Table 1. Histological characteristics used to determine the stages of gonadal development in Dendropsophus minutus in climate temperate (Cfb) inTable south 1: Histological of Brazil. characteristics used to determine the stages of gonadal development in Dendropsophus minutus in climate temperate (Cfb) in south of Brazil. Characteristics Stage Males Females Immature Seminiferous loculi of reduced sizes, partially open, (A) predominance