Herpetology Notes, volume 11: 395-403 (2018) (published online on 09 May 2018)

The reproductive biology of minutus (Amphibia: Anura) in South of

Peterson T. Leivas1,*, Tiago B. Mayer1 and Luis F. Fávaro1

Abstract. This study investigated the reproductive biology of (Peters, 1972), in a temperate area of Mixed Ombrophilous Mountainous (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 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 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 , , and Trinidad southward through , and Brazil to , , until 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 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 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 of spermatogonia.

Seminiferous loculi increased in size compared to Initial maturation previous stage, predominance of spermatogonia and (B1) spermatocytes. Seminiferous loculi increased in size compared to Advanced maturation previous stage, with few spermatogonia, (B2) predominance of spermatids and few spermatozoids. Seminiferous loculi of bigger size, compared to Predominance of ovarian Mature previous stages, containing large quantities of follicles full of vitelline (C) spermatozoids. granules.

and 0.977 g. The SVL in adult females varied between (Fig. 2B) where the frequency of mature and nearly Table 2 –Spearman correlation test (p) and of the probability associated to the null hypothesis (p) between 21.00 and 24.25the mm frequency and ofthe male BM individuals varied ofbetween Dendropsophus 0.451 minutus mature in maturation males stages coincides and the environmental with months with the highest and 1.145 g. Formean malemonthly juvenile temperature SVL (ºC), monthly varied accumulated between precipitationGSI values. (mm) and mean relative humidity (%) in climate temperate (Cfb) in south of Brazil. * = significant values (p ” 0,05). 12.70 and 18.70 mm and the BM varied between 0.090 The correlation analysis between the monthly mean and 0.392 g. All four females collected had their ovaries GSI and the abiotic variables did not indicate significant Stages Males classified as Mature (C) (Table 1 and Fig. 1). The results values. The frequency of males with advanced maturing/ Temperature Precipitation Humidity for the male maturationTable curves 1: Histological revealed characteristics low variation used to determine mature the stages testis of gonadal (B2+C) development was negatively in correlated with ȡ p ȡ p ȡ p on monthly GSI, withDendropsophus greatest variation minutus inwas climate observed temperate (Cfb)relative in south humidityof Brazil. whereas the incidence of males stage A 0,5463 0,0660 0,7257 0,0075* 0,3545 0,2581 from August to January coinciding with the period with A was positively correlated with precipitation. There B1 0,4649 0,1277 Characteristics 0,1816 0,5721 -0,3705 0,2357 highest reproductiveB2 e C activity.Stage 0,0878 Conversely, 0,7859 theMales -0,3093smallest 0,3279were not -0,6678 detected 0,0176* significant Females values of the stages of variation in GSI was observedImmature from February Seminiferous to May, loculi ofmaturation reduced sizes, with partially temperature open, (Table 2). where the reproductive(A) activity was the lowestpredominance (Fig. 2of spermatogonia.Although males were predominant throughout Seminiferous loculi increased in size compared to A and B). Initial maturation the study period, significant differences in sexual previous stage, predominance of spermatogonia and (B1) The analysis of the percentage distributionspermatocytes. of monthly proportion occurred in January, February July, August, testicular development (Fig. 3) showed that, from September, October, November and December. Females Seminiferous loculi increased in size compared to Advanced maturation August to December, there was an abundanceprevious of males stage, with fewwere spermatogonia, observed in low frequency in February, October, (B2) in advanced stages of maturation in additionpredominance to three of spermatidsNovember and few andspermatozoids. December. Seasonal analyses of the Seminiferous loculi of bigger size, compared to Predominance of ovarian mature males (65% ofMature the total males captured). This sexual proportion demonstrated significant differences previous stages, containing large quantities of follicles full of vitelline (C) result supports our findings for the maturationspermatozoids. curve between all the seasons (Tablegranules. 3).

Table 2. Spearman correlation test (p) and of the probability associated to the null hypothesis (p) between the frequency of male individuals of DendropsophusTable 2 minutus–Spearman in correlation maturation test (stagesp) and ofand the probabilitythe environmental associated tomean the null monthly hypothesis temperature (p)1 between (ºC), monthly the frequency of male individuals of Dendropsophus minutus in maturation stages and the environmental accumulated precipitationmean (mm) monthly and temperaturemean relative (ºC), humiditymonthly accumulated (%) in climate precipitation temperate (mm) and(Cfb) mean in relative south humidityof Brazil. * = significant values (p ≤ 0,05). (%) in climate temperate (Cfb) in south of Brazil. * = significant values (p ” 0,05).

Stages Males Temperature Precipitation Humidity ȡ p ȡ p ȡ p A 0,5463 0,0660 0,7257 0,0075* 0,3545 0,2581 B1 0,4649 0,1277 0,1816 0,5721 -0,3705 0,2357 B2 e C 0,0878 0,7859 -0,3093 0,3279 -0,6678 0,0176*

1 398 Peterson T. Leivas et al.

Figure 1. Histological sections of testicles and ovaries of Dendropsophus minutus. A – Immature testicle (A) with predominance of spermatogonia (SG); B – Testicle in Initial maturation (B1) with predominance of spermatocytes (SC); C and D – Testicles in Advanced maturation (B2), demonstrating spermatogonia (SG), spermatocytes (SC), spermatids (ST) and spermatozoids (SZ); E – Mature testicle (C) with predominance of spermatozoids (SZ); F – Mature ovary (C) with mature ovarian follicles, full of vitelline granules. Coloration hematoxylin-eosin.

The ontogenetic analysis characterizes the Significant differences in the number of juveniles and predominance of adult individuals in all the study adults were verified from July to December. Seasonally, period, except in June. Juveniles occurred in January, significant differences were found in the summer, the February, June and December, in reduced numbers. winter and the spring (Table 4). The reproductive biology of Dendropsophus minutus in South of Brazil 399

number of females obtained and to the absence of immature females in the samples. However, the smallest female captured measured 21.00 mm in SVL.

Discussion In this study, we characterized the reproduction of males as prolonged seasonal based on the presence of reproductively active males (i.e. testis with sperm) throughout the year. The peak reproductive activity of mature males lay between the end of winter and beginning of summer, after which we observed the presence of immature males. Male vocalization is intensified during the reproductive season. Cunha et al. (2010) found that the most intense period of vocalization in D. minutus is between spring and summer, which corroborates our findings for male peak reproductive activity. However, males do not vocalize only for calling females but also by different behavioural conditions such as territorial defence (Pombal, 1997; Prado et al., 2005). Thus, the GSI and Figure 2. Values of Individual Gonadosomatic Index (GSI) histological data values to make a reliable parameter (A) and maturation curve (B) for males of Dendropsophus to determine the potential reproductive period of the minutus in climate temperate (Cfb) in south of Brazil species. The small variation in male GSI during the analysed period is explained by the high frequency of testicles in advanced maturation throughout the year. Of all The SVL of the first sexual maturation for males was mature females captured in the study, most part were estimated at 14.85 mm and the SVL in which all males obtained during the largest average monthly values GSI are adults was of 22.97 mm (Fig. 4). The first maturation for males, suggesting an increased reproductive activity length curve for females was not made due to the low in the spring and in the summer (Oliveira et al., 2007;

Figure 3. Monthly distribution frequency (in %) of the stages Figure 4. First maturation length estimated for males of of testicular development of Dendropsophus minutus. A Dendropsophus minutus in climate temperate (Cfb) in south (immature), B1 (initial maturation), B2 (advanced maturation) of Brazil. and C (mature) in climate temperate (Cfb) in south of Brazil. Number above the bars = absolute frequency of males. 400 Peterson T. Leivas et al.

Table 3. Number ofTable males 3: andNumber females of males as well and asfemales the monthly as well asand the seasonal monthly sexand ratioseasonal test sex (χ 2 ratio) of Dendropsophus test (Ȥ 2) of minutus in climate temperate (Cfb) in Dendropsophussouth of Brazil. minutus * = significant in climate values.temperate Seasons: (Cfb) in Summersouth of Brazil. (Sum), * =Fall significant (Fal), Winner values. (Win)Seasons: and Springer (Spr). Summer (Sum), Fall (Fal), Winner (Win) and Springer (Spr).

Months Females Males x2 Seasons Females Males Ȥ2 Jan 12 0 9 9,00* Feb 12 1 10 7,36* Sum 1 21 18,18* Mar/12 0 2 2,00 Apr 12 0 2 2,00 May/12 0 1 1,00 Fal 0 4 4,00* Jun 12 0 1 1,00 Jul 12 0 7 7,00* Aug 12 0 10 10,00* Win 0 31 31,00* Sep 12 0 14 14,00* Oct/12 1 21 18,18* Nov 12 1 8 5,44* Spr 3 34 28,90* Dec/12 1 8 5,44* Table 3: Number of males and females as well as the monthly and seasonal sex ratio test (Ȥ 2) of Dendropsophus minutus in climate temperate (Cfb) in south of Brazil. * = significant values. Seasons: Summer (Sum), Fall (Fal), Winner (Win) and Springer (Spr). Table 4 – Monthly and seasonal absolute frequency of juveniles and adults of Dendropsophus minutus in 2 2 climateMonths temperate Females (Cfb) in Malessouth of Brazilx analyzedSeasons by the Chi-Square Females test Males(x 2). * = significantȤ values. Wells, 2007; WogelSeasons:Jan and12 SummerPombal, 0 (Sum), 2007). Fall 9 (Fal), Winner 9,00* (Win) andpredation Springer (Spr). pressure and availability of food resources

Feb 12 1 10 7,36*2 Sum 1 21 18,18*2 Our observationsMonths of mature Juveniles females Adults together Ȥ with Seasonscan also Juveniles interfere Adults with the reproductiveȤ process (Prado Mar/12 0 2 2,00 the high male GSIJan values 12 during 1 the period 8 of 5,44*highest and Uetanabaro, 2000). Food resources are essential Apr 12 0 2 2,00 reproductive activityFeb 12can be related 4 to the 9 physiological 1,92 Sumin reproductive 5 activity 19 of 8,16* since the energy is May/12 0 1 1,00 Fal 0 4 4,00* induction of sexualMar/12 maturation 0 through 2abiotic factors, 0,00 critical to reproduction (Wells, 2007). Jun 12 0 1 1,00 which adds to previousApr 12 literature 0 showing 2 the 0,00 effects While analysing the histology of our samples, we Jul 12 0 7 7,00* of other environmentalMay/12 factors 0 on the reproduction 1 0,00 of Falfound sperm 1 in the 3 testis 1,00 of the majority of males Aug 12 0 10 10,00* Win 0 31 31,00* amphibians (Wells,Jun 2007; 12 Ferreira 1 and Mehanna, 0 1,00 2012, throughout the year. However, we could not characterize Sep 12 0 14 14,00* Leivas et al. 2012).Jul However, 12 factors 0 not measured 7 7,00* in this D. minutes reproduction as continuous because we could Oct/12 1 21 18,18* study as morphologicalAug 12 and physiological 0 10 components, 10,00* Winonly collect 0 three 31 mature 31,00* males. Moreover, the high Nov 12 1 8 5,44* Spr 3 34 28,90* Sep 12 0 14 14,00* Dec/12 1 8 5,44* Oct/12 0 22 22,00* Nov 12 0 8 8,00* Spr 2 38 34,38* Table 4. Monthly andDec/12 seasonal absolute 2 frequency 8 of juveniles 3,60 and adults of Dendropsophus minutus in climate temperate (Cfb) in south of Brazil analyzedTable 4 – Monthlyby the Chi-Square and seasonal test absolute (x 2). *frequency = significant of juveniles values. and Seasons: adults of Summer Dendropsophus (Sum), minutus Fall (Fal), in Winner (Win) 2 and Springer (Spr).climate temperate (Cfb) in south of Brazil analyzed by the Chi-Square test (x ). * = significant values. Seasons: Summer (Sum), Fall (Fal), Winner (Win) and Springer (Spr).

Months Juveniles Adults Ȥ2 Seasons Juveniles Adults Ȥ2 Jan 12 1 8 5,44* Feb 12 4 9 1,92 Sum 5 19 8,16* Mar/12 0 2 0,00 Apr 12 0 2 0,00 May/12 0 1 0,00 Fal 1 3 1,00 Jun 12 1 0 1,00 2 Jul 12 0 7 7,00* Aug 12 0 10 10,00* Win 0 31 31,00* Sep 12 0 14 14,00* Oct/12 0 22 22,00* Nov 12 0 8 8,00* Spr 2 38 34,38* Dec/12 2 8 3,60

2 The reproductive biology of Dendropsophus minutus in South of Brazil 401 frequency of males in advanced stages of maturation in precipitation values is explained by the fact that after our sample did not allow us to pinpoint the reproductive metamorphosis immature specimens are registered period of this species. Nonetheless, we could still infer during summer months (December to February), period the relative differences in temporal reproductive activity with higher pluviometric rates in the studied area. that was derived from the maturation curve. The difference in the sexual ratio, with predominance Even though there are temporal differences in the of males over females in the studied population, might reproductive activity of the species, in this study the be related to factors like the non-location of females for reproduction of males were characterized as prolonged, their not calling, different spatial occupation compared to corroborated with the described by Oliveira et al., males (Wogel and Pombal, 2007; Thomé and Brasileiro, (2007) for Minas Gerais state, a region characterized as 2007) and a natural higher proportion of males in the with Cwa climate (humid subtropical with hot summer) studied area what appears to be a standard for hylids (Alvares et al., 2013). However, it differs from the (Eggert and Guyétant, 2003; Wells 2007). described by Santos e Oliveira (2007) and Santos et al. The determination of the length of the first maturation (2012) for the state of São Paulo, in which the climate provides important data regarding the biology of the was characterized as Aw (tropical with dry winter) species. Our results indicate that the L50 of D. minutus (Alvares et al., 2013). Therefore, we can consider that corresponded to 61% of the maximum SVL obtained in the type of climate influences the characterization of the the studied period, close to the value found for Lysapsus reproductive process. The tropical climate, evidenced limellus, whose L50 value corresponded to 69% of the within the studied area of the State of São Paulo, favours maximum SVL (Prado and Uetanabaro, 2000). These a continuous reproduction because it presents higher differences first maturation size can be influenced by temperatures throughout the year. The subtropical abiotic factors, such as temperature, oxygen availability climate, evidenced within the studied area of the State and hydroperiod (Young et al., 2004), which mainly of Minas Gerais and also in the location of the present affect the amount of water in breeding points (Barreto study, favours a prolonged reproduction for the species. and Andrade, 1995). However, biotic factors such as This is because lower temperatures are achieved during competition for females and predation pressure, can also the winter, whereas in the spring and summer higher interfere with the reproductive cycle and anticipate the environmental temperatures occur. sexual maturation of a species (Hinshaw and Sullivan, The negative relation between potentially reproductive 1990; Haddad and Bastos, 1997), allowing individuals males (B2 and C) and humidity, is due to the fact that start reproducing earlier and participate in more during less humid seasons of the year (fall and winter) reproductive events (Wells, 2007; Leivas et al., 2012). the reproductive activity was decreased, and individuals In conclusion, we showed that local abiotic factors presented low values of GSI, compared to the period affect the reproductive cycle of D. minutus in tropical of spring and summer. Furthermore, the absolute or subtropical climates. In climate tropical this specie frequency of males in advanced maturation was reduced has reproductive activity continued and in subtropical during the fall (April to June), while mature males were climate has reproductive activity it is prolonged. In the absent. In a study conducted with D. minutus in the state present study, the presence of males with advanced of Mato Grosso, a decreased reproductive activity was and mature maturation are found in periods with lower observed between the months of May and September, relative humidity and males intensify their reproductive period which corresponds to the lowest temperatures activity in a period comprising the end of winter and and values of precipitation, and a total absence of beginning of summer. spermatozoids was registered in August (Ferreira and Frogs with great geographic distribution may have Mehanna, 2012). different reproductive characteristics throughout The positive relation between immature males and occurrence by local climatic factors. Abiotic factors precipitation is due to the juveniles born from the also influence the population structure and indirectly first spawns, which occurred in the months of August/ determine the increase juveniles in the population September, corresponding to the end of the winter (rainfall period in this study). This allows the conclusion and beginning of spring. According to Eterovik and that D. minutus reaches reproductive success through Sazima (2004), tadpoles have larval development of reproductive strategies, which are under the influence approximately three months. Therefore, the positive of local abiotic factors. relation between juvenile individuals and an increase in 402 Peterson T. Leivas et al.

Acknowledgments. Peterson T. Leivas and Tiago Burda received Brazil. Bioscience 55(3): 207-217. a masters stipend from the Coordenação de Aperfeiçoamento de Hiert, C., Roper, J.J., Moura, M. O. (2012): Constant breeding and Pessoal de Nível Superior - CAPES. The study was conducted on low survival rates in the subtropical Striped Frog in southern the environmental permit n° 10277-1 of ICMbio. Brazil. Journal of Zoology 288: 151-158. Hinshaw, S.H., Sullivan, B.K. (1990): Predation on versicolor References and Pseudacris crucifer during reproduction. Journal of Herpetology 24: 196-197. Alvares, C.A., Stape, J.L., Sentelhas, P.C., Gonçalvez, J.L., Huang, W.S., Lin, J.Y., Yu, J.Y.L. (1997): Male reproductive cycle Sparovek, G. (2013). Koppen´s climate classification map for of the toad Bufo melanostictus in Taiwan. Zoological Science Brazil. Meteorologische Zeitschrift 22(6): 711-728. 14(3): 497-503. Barreto, L., Andrade, G.V. (1995): Aspects of the reproductive IBGE – Instituto Brasileiro de Geografia e Estatística (2012): biology of Physalaemus cuvieri (Anura: Leptodactylidae) in Manual Técnico da Vegetação Brasileira, 2nd Edition. Rio de northeastern Brazil. Amphibia- Reptilia 16(1): 67-76. Janeiro, BR, Instituto Brasileiro de Geografia e Estatística. Costa, C.L.S., Lima, S.L., Andrade, D.R., Agostinho, C.A. (1998a): Leivas, PT., Moura, M.O., Favaro, L.F. (2012): The Reproductive Caracterização morfológica dos estádios de desenvolvimento do Biology of the Invasive Lithobates catesbeianus (Amphibia: aparelho reprodutor masculino de rã-touro, Rana catesbeiana, Anura). Journal of Herpetology 46(2): 153-161. no Sistema Anfigranja de Criação Intensiva. Revista Brasileira Oliveira, E.C., Fávaro L.F. (2011). Reproductive biology of the de Zootecnia 27(4): 642-650. flatfish Etropus crossotus (Pleuronectiformes: Paralichthyidae) Costa, C.L.S, Lima, S.L., Andrade, D.R., Agostinho, C.A. (1998b): in the Paranaguá Estuarine Complex, Paraná State, subtropical Caracterização morfológica dos estádios de desenvolvimento do region of Brazil. Neotropical Ichthyology 9: 795-805. aparelho reprodutor masculino de rã-touro, Rana catesbeiana, Oliveira, E.F., Feio, R.N., Matta, S.L.P. (2007): Aspectos no Sistema Anfigranja de Criação Intensiva. Revista Brasileira reprodutivos de Dendropsophus minutus (Peters, 1872) no de Zootecnia 27(4): 651-657. município de Viçosa, Minas Gerais. Revista Ceres 54(313): Crump, M.L., Scott Jr., N.J. (1994): Visual encounter surveys. 230-238. In: Measuring and monitoring biological diversity: standard Oliveira, C., Zanetoni, C., Zieri, R. (2002): Morphological methods for amphibians, p. 84-92. Heyer, W.R., Donnelly, observations on the testes of Physalaemus cuvieri (Amphibia, M.A., Mcdiarmid, R.W., Foster M.S., Eds., Washington, USA, Anura). Revista Chilena de Anatomia 20: 263-268. Smithsonian Institution Press. Pombal, J.P. Jr (1997): Distribuição espacial e temporal de anuros Cunha, A.K., Oliveira, I.S., Hartmann, M.T. (2010): Anurofauna (Amphibia) em uma poça permanente na Serra de Paranapiacaba, da Colônia Castelhanos, na Área de Proteção Ambiental de Sudeste do Brasil. Revista Brasileira de Biologia 57: 583-594. Guaratuba, Serra do Mar paranaense, Brasil. Biotemas 23(2): Pombal, J.P. Jr., Haddad, C.F.B. (2005): Estratégia e modos 123-134. reprodutivos de anuros (Amphibia) em uma poça permanente Eggert, C., Guyétant, R. (2003): Reproductive behaviour of spade na Sera de Paranapiacaba, Sudeste do Brasil. Papeis Avulsos de foot toads (Pelobates fuscus): Daily sex ratios and males’ tactics, Zoologia 45(15): 201-213. ages, and physical condition. Canadian Journal of Zoology 81: Prado, C.P.A., Uetanabaro, M. (2000): Reproductive biology 46-51. of Lysapsus limellus, Cope, 1862 (Anura, Pseudidae) in the Eterovik, P.C., Sazima, I. (2004): Anfíbios da Serra do Cipó - Minas Pantanal, Brazil. Zoocriaderos 3(1): 25-30. Gerais - Brasil. Belo Horizonte, BR, Editora PUC Minas. Prado, C.P.A., Uetanabaro, M., Haddad, C.F.B. (2005): Breeding Fávaro, L.F., Lopes S.C.G., Spach, H.L. (2003): Reprodução activity patterns, reproductive modes, and use by anurans do peixe-rei, Atherinella brasiliensis (Quoy and Gaimard) (Amphibia) in a seasonal environment in the Pantanal, Brazil. (Atheriniformes, Atherinidae), em uma planície de maré Amphibia Reptilia 26(2): 211-221. adjacente à gamboa do Baguaçu, Baía de Paranaguá, Paraná, Santos, L.R.S., Oliveira, C. (2007): Morfometria testicular durante Brasil. Revista Brasileira de Zoologia 20: 501-506. o ciclo reprodutivo de Dendropsophus minutus (Peters) (Anura, Ferreira, A., Mehanna, M. (2012): Seasonal testicular changes in Hylidae). Revista Brasileira de Zoologia 24(1): 64-70. Dendropsophus minutus Peters, 1872 (Anura, Hylidae). Biocell Santos, L.R.S., Franco-Belussi, L., Oliveira, C. (2012): Germ 36(2): 57-62. Cell Dynamics during the Annual Reproductive Cycle of Frost, D. R. (2017): species of the world: an online Dendropsophus minutus (Anura: Hylidae). Zoological Science reference. Available at: http://amphibiaweb.org. Accessed on 10 28: 840-844. January 2017. Thomé, M.T.C., Brasileiro, C.A. (2007): Dimorfismo sexual, uso Haddad, C.F.B., Bastos, R.P. (1997): Predation on the toad Bufo do ambiente e abundância sazonal de Elachistocleis cf. ovalis crucifer during reproduction (Anura; Bufonidae). Amphibia and (Anura: Microhylidae) em um remanescente de Cerrado no Reptilia 18: 295-298. Estado de São Paulo, Sudeste do Brasil. Biota Neotropica 7(1): Haddad, C.F.B., Toledo, L.F., Prado, C.P.A., Loebmann, D., 27-33. Gasparini, J.L., Sazima, I. (2013): Guia dos anfíbios da Mata Toledo, L.F., Ribeiro, R.S., Haddad, C.F.B. (2006): Anurans as Atlântica - Diversidade e Biologia. São Paulo, BR, Anolis prey: an exploratory analysis and size relationships between Books. predators and their prey. Journal of Zoology 271: 170-177. Haddad, C.F.B., Prado, C.P.A. (2005): Reproductive Modes in VanSluys, M., Rocha, C.F.D. (1998): Feeding habits and Frogs and Their Unexpected Diversity in the Atlantic Forest of microhabitat utilization by two syntopic Brazilian Amazonian The reproductive biology of Dendropsophus minutus in South of Brazil 403

frogs (Hyla minuta and Pseudopaludicula sp. (gr. falcipes). Revista Brasileira de Biologia 58(4): 559-562. Wells, K.D. (2007): The Ecology and Behavior of Amphibians. Chicago and London, USA, The University of Chicago Press. Wogel, H., Pombal, J.P. Jr (2007): Comportamento reprodutivo e seleção sexual em Dendropsophus bipunctatus (Spix, 1824) (Anura, Hylidae). Papeis Avulsos de Zoologia 47(13): 165-174. Young, B.E., Stuart, S.N., Chanson, J.S., Cox, N.A., Boucher, T.M. (2004): Disappearing jewels: The status of NewWorld amphibians. Virginea, USA, NatureServe.

Accepted by Anamarija Zagar