Anuran Assemblage Changes Along Small-Scale Phytophysiognomies in Natural Brazilian Grasslands

Home , Leptodactylus latinasus, Physalaemus gracilis, Pseudis minuta, Scinax granulatus, Scinax squalirostris

bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Anuran assemblage changes along small-scale phytophysiognomies in natural Brazilian grasslands

Diego Anderson Dalmolin1*, Volnei Mathies Filho2, Alexandro Marques Tozetti3

1 Laboratório de Metacomunidades, Departamento de Ecologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

2 Fundação Universidade Federal do Rio Grande, Rio Grande, Rio Grande do Sul, Brasil

3Laboratório de Ecologia de Vertebrados Terrestres, Universidade do Vale do Rio dos Sinos, Avenida Unisinos 950, 93022-000 São Leopoldo, Rio Grande do Sul, Brazil.

* Corresponding author: Email: [email protected]

1 Abstract 2 We studied the species composition of frogs in two phytophysiognomies (grassland and 3 forest) of a Ramsar site in southern Brazil. We aimed to assess the distribution of 4 species on a small spatial scale and dissimilarities in community composition between 5 grassland and forest habitats. The sampling of individuals was carried out through 6 pitfall traps and active search in the areas around the traps. We evaluated the existence 7 of these differences by using permutational multivariate analysis of variance and 8 multivariate dispersion. We found 13 species belonging to six families. Leptodactylidae 9 and Hylidae were the most representative families. The compositional dissimilarity was 10 higher between the sampling sites from different phytophysiognomies than within the 11 same phytophysiognomy, suggesting that forest and grassland drive anuran species 12 composition differently. Also, the difference in anuran species composition between the 13 sampling sites within the forest was considerably high. Based on our results, we could 14 assume that the phytophysiognomies evaluated here offer quite different colonization 15 opportunities for anurans, especially those related to microhabitat characteristics, such 16 as microclimate variables.

21 Keywords 22 Dissimilarity; anuran; south Brazil; Taim; forest; grasslands

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

29 Introduction 30

31 The composition of species in communities is a result of a complex interaction between 32 organisms and environmental characteristics. The presence of a singular species in an 33 assemblage depends on intrinsic (such as the individual's physiological needs for 34 homeostatic maintenance) and extrinsic processes (such as competition, predation and 35 other biotic interactions). Combined, both of them determine the possibility of a given 36 species to colonize a habitat, as well their long-term maintenance in there (Schluter & 37 Ricklefs 1993; Ximenez et al. 2014). It is assumed that heterogeneous habitats comprise 38 a richer biota (Semlitsch et al. 2015) since they offer many different microhabitats, 39 which precisely favors the establishment of species without high niche overlap 40 (Barrows & Allen 2010; Oliveira et al. 2013)

41 The concept of habitat heterogeneity is related to much confusion, being frequently used 42 as a synonym of habitat complexity. The process of describing and evaluating the 43 habitat structure is one of the challenges for carrying out ecological studies (Stein & 44 Kreft 2015). In structural terms, environments can be described by (i) their complexity - 45 vertical development of vegetation - and (ii) their heterogeneity - horizontal variation in 46 descriptors such as density, dominance and frequency of plant species that compose 47 them (August 1983). In a review on the topic, Tews et al. (2004) defend the idea that the 48 concept of habitat heterogeneity includes both vertical and horizontal structure of plant 49 community - a concept adopted in the present study. The possibility of assessing the 50 effect of habitat heterogeneity on the structuring of their communities comes up against 51 the huge number of areas never sampled in the Neotropical region (Garcia & 52 Vinciprova 2003). This fact also has implications for the establishment of conservation 53 strategies for potentially threatened species - as is the case with many Brazilian anurans 54 (Silvano & Segalla 2005).

55 The variety of available habitats and the high levels of species endemism throughout the 56 Brazilian biomes makes it an adequate location to assess the role of environmental 57 heterogeneity in the activity patterns and community structure of anurans (Silvano & 58 Segalla 2005). However, most studies on anuran ecology in Brazil are concentrated in 59 forest environments, especially in the Cerrado and the Atlantic Forest biomes (da Silva 60 et al. 2011a; Iop et al. 2012; Prado & Rossa-Feres, 2014; Saccol et al., 2017). The bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

61 Pampa grassland formations and the coastal portion of southern Brazil are home to 62 anuran species that are common in open habitats (Núñez et al. 2004, Vasconcelos et al. 63 2019; Dalmolin et al. 2019). Although the landscape of these environments consists 64 predominantly of open areas (Machado et al. 2012), some forest patches can also be 65 observed (such as swamp forests and dry or sandy forests).

66 Studies that describe changes in amphibian assemblages over structural changes in 67 vegetation cover can contribute to propose scenarios of landscape changes (Da Silva et 68 al. 2011a). The process of forests expanding over grasslands under climate change or 69 even converting forested areas into human use would reflect changes in the amphibian 70 composition (Manenti et al. 2013; Oliveira & Rocha 2015). In this sense, our work 71 aimed to: (i) verify the distribution of anuran species on a small spatial scale; (ii) 72 describe the association between the patterns of dissimilarity in anuran composition 73 between communities of different phytophysiognomies (forest and grassland).

74 The study design on a small spatial scale is interesting to assess adjacent habitats whose 75 probability of colonization is theoretically the same for all species in the region 76 (Ximenez et al. 2014). In addition, the habitat elements linked to the micro-habitat scale 77 are important descriptors of the spatial partition of species (Freitas et al. 2002, Oliveira 78 & Rocha 2015) in particular because they deal with transition zones in a refined way 79 (Bersier et al. 2007). The detection of differences in anuran assemblages between 80 habitats provides a strong indication of the segregation process of these species along a 81 vegetation gradient (Da Silva et al. 2011a; Prado & Rossa-Feres 2014). We expected to 82 find greater differences in composition in places belonging to different 83 phytophysiognomies than between areas belonging to the same phytophysiognomies, 84 since the environmental and microenvironmental conditions must be similar in these 85 areas, thus reducing their composition dissimilarity (Da Silva et al. 2011).

87 Material and Methods 88

89 Study site 90 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

91 The study was carried out at the Estação Ecológica do Taim (ESEC Taim; S 92 32°32'16.6" W 052°32'16.7"), located in the municipality of Rio Grande, Brazil’s 93 extreme south (Figure 1). ESEC Taim encompasses ecosystems of great relevance and, 94 since 2017, was recognized as a Ramsar site (Ramsar Wetland of International 95 Importance; Ramsar, 2018). The landscape consists of wetlands and grasslands in 96 association with remnants of “restinga” and flooded forests, which are dominated by 97 cork trees (Erythrina crista-galli) and fig trees (Ficus organensis; Waechter & 98 Jarenkow 1998).

99 The climate of the region is humid sub-temperate, with an annual average temperature 100 of 18.1º C and average temperature of the coldest month of 12.7° C. The annual rainfall 101 is 1162 mm, with periods of drought in the spring and a higher incidence of rain in the 102 winter (Maluf 2000). The maximum temperature varied between 9.1° C and 39.3° C 103 during the study period (May 2011 to April 2012), with the hottest months being 104 February and March 2012; the minimum temperature varied between 0.9 ° C and 28.1 ° 105 C, with the coldest months being July and August 2011. Meteorological data were 106 obtained from the database provided by the Meteorological Station of the Universidade 107 Federal do Rio Grande (FURG), located in the municipality of Rio Grande.

108

109 Figure 1: Geographic location of the study area (Estação Ecológica do Taim) in the 110 state of Rio Grande do Sul, Brazil.

111

112 Anuran sampling 113

114 Sampling was carried out in the two most remarkable phytophysiognomies of ESEC 115 Taim: (a) sites of grassland dominance and (b) sites of forest dominance (Figure 2). We 116 were able to distinguish the boundary between these two environments precisely even 117 by a visual inspection. This was possible because the forests are distributed in patches 118 merged along the grassland with well-defined boundaries.

119

120 Figure 2: Phytophysiognomies of ESESC Taim: (a) sites of grassland dominance and 121 (b) sites of forest dominance. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

122 We selected two sampling areas in sites of grassland dominance and two in sites of 123 forest dominance. Sampling areas were chosen based on vegetation integrity, area with 124 continuous habitat and presence of potential anuran breeding sites. In addition, we 125 considered our accessibility to these areas since many regions of ESEC Taim are 126 permanently flooded, making the displacement of researchers and the installation of 127 pitfall traps not possible. Sampling areas are at least about 800 m from each other. Field 128 campaigns were carried out every two weeks between May 2011 and April 2012. Each 129 campaign comprised four days when all sites were simultaneously sampled. Frogs were 130 sampled by the combination of three methods: (i) pitfall traps with drift fence; (ii) 131 auditory surveys; (iii) visual search.

132

133 Pitfall traps with drift fence (passive sampling) 134

135 We installed sets of buckets arranged in pitfall lines in all the areas of grassland 136 dominance and forest dominance. We installed two lines of buckets in each area, 137 resulting in eight lines and 32 buckets. Pitfalls were arranged following the description 138 of similar studies (e.g. Cechin & Martins 2000). Each pitfall was formed by four 100-l 139 buckets buried about 13.3 m from each other and connected by a mosquito net (60 cm in 140 height) that served as a guide fence. The guide fence was buried 10 cm into the ground 141 to avoid individuals to transpose the fence (Cechin & Martins 2000). All buckets had 142 holes at the bottom to prevent them from being flooded by rain.

143 We grouped the records obtained from each pair of lines of the same sampling point and 144 consider it to be an assemblage of species. Pitfalls remained open during four 145 consecutive days in intervals of 15 days. During the samplings, we carried out daily 146 inspections in the buckets. We identified every captured animal, which was later 147 released about 20 meters from the trap. The collection was carried out with the 148 authorization number 27755-1 from SISBIO.

149

150 Visual search and auditory surveys (active sampling) 151 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

152 Visual search and auditory surveys (Heyer et al. 1994) were performed during the night, 153 starting one hour after sunset. Active sampling was conducted in the vicinity of the 154 pitfall traps. For standardization of sampling effort, we conducted a visual search in an 155 area of about 1 ha around each pitfall line. Based on our previous experience in auditory 156 surveys, we were able to perform a safe species identification of calling males in a 157 maximum area of 5 ha. This was the covered area for calling surveys at each pitfall line. 158 Active sampling was made along four sampling nights and repeated monthly. Sampling 159 started each night in a different environment (grassland or forest), alternately to avoid 160 the same physiognomy to be always sampled at the same time. We applied two hours 161 per person of active sampling.

162

163 Phytophysiognomy evaluation 164

165 To better characterize the phytophysiognomies of the sampling areas, the vegetation 166 structure was measured through plots (following Tozetti & Martins 2008). We 167 characterized the vegetation structure in the vicinity of the areas where pitfall traps were 168 installed. We delimited two plots of 3 m x 10 m, which were distributed perpendicularly 169 in each of the pitfall lines, one of which was placed at the beginning and the other at the 170 end of the line. The following variables of the vegetation were measured in each plot:

171

172 Table 1: Vegetation descriptors measured in each transect.

173

174 Statistical Analysis 175 176 We evaluated differences in the anuran composition in the areas within and between 177 habitat types by using permutational multivariate analysis of variance (PERMANOVA) 178 and multivariate dispersion (BETADISPER). We considered the presence and absence 179 of each specie in each transect. We used the Jaccard index as a dissimilarity measure 180 and performed 9999 permutations. PERMANOVA and BETADISPER were run using 181 the “adonis” and “betadisper” functions in the R package vegan. 182 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

183 Results 184 185 We recorded 13 species of six families in both phytophysiognomies (Figure 3; Table 2). 186 Leptodactylidae and Hylidae were the most representative, with six and four species 187 respectively. The hylid Hypsiboas pulchellus occurred exclusively in grasslands and 188 Scinax squalirostris occurred exclusively in forests. The number of recorded species 189 varied during the sampling period, with the largest number of species recorded in May 190 (12 species), September and November (11 species) in both phytophysiognomies. 191 192 193 Figure 3: Anuran species found in two phytophysiognomy types of Estação Ecológica 194 do Taim, southern Brazil, between May 2011 and April 2012. (a) Elachistocleis bicolor; 195 (b) Hypsiboas pulchellus; (c) Leptodactylus gracilis; (d) Leptodactylus latinasus; (e) 196 Leptodactylus latrans; (f) Odontophrynus maisuma; (g) Physalaemus biligonigerus; (h) 197 Physalaemus gracilis; (i) Pseudis minuta; (j) Pseudopaludicola falcipes; (k) Rhinella 198 dorbignyi; (l) Scinax granulatus; (m) Scinax fuscovarius. 199 200 Table 2: Anuran species found in two phytophysiognomy types of Estação Ecológica 201 do Taim, southern Brazil, between May 2011 and April 2012. Black squares represent 202 the occurrence of species in the relative month. 203 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

204 We found that the patterns of compositional dissimilarity (β diversity) were higher 205 between the sampling sites from different phytophysiognomies than within the same 206 phytophysiognomy (F = 4.72; p=0.05; Figure 4). Also, the difference in composition 207 between the sampling sites in forest areas was considerably high. 208

209 Figure 4: Dissimilarity in anuran composition in two different phytophysiognomies of 210 Reserva Ecológica do Taim, southern Brazil. 211

212 Discussion 213 214 Our results showed differences in species composition between grassland and forest 215 habitats. They pointed out that differences in vegetation cover are capable to drive 216 significant changes in anuran species composition. We also highlight that we surveyed 217 adjacent (spatially closely related) sites, which reveals that vegetation has a powerful 218 effect on defining the composition of anuran assemblages. We may assume that the 219 phytophysiognomies evaluated here offer quite different colonization opportunities to 220 anurans, with drastic changes in anuran assemblages across a small geographical area. 221 An obvious relationship between phytophysiognomies and colonization opportunities is 222 related to microhabitat characteristics generated by shading, wind reduction, humidity 223 maintenance, and reduction of dial range of air temperature. Microenvironmental 224 conditions (especially those related to the microclimate) vary substantially between 225 forests and grassland (D’Odorico et al. 2013) and, in our sampling site, mainly because 226 the transition between them is relatively abrupt. Based on these differences, the 227 heterogeneity affects a set of microhabitat proprieties, which could enable the 228 maintenance of some species. The effects of substrate characteristics (e.g., litter deep, 229 moisture retention), and dial variation in air temperature are well-known as determining 230 factors for structuring anuran assemblages (Da Silva et al. 2011b; Van Dyke et al., 231 2017). Based on this, we point out that the relatively great difference in anuran species 232 composition between sampling sites in forest is a result of the greater heterogeneity of 233 this habitat in relation to grassland. It makes sense since forest sites would vary in both 234 vertical and horizontal components of vegetation cover whereas grasslands vary mostly 235 across the horizontal component (Dalmolin et al. 2019; Dalmolin et al. 2020). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

236 In general, it is expected that forest environments will have a greater variety of 237 microhabitats (Afonso & Eterovick 2007; Prado & Rossa-Feres 2014; Van Dyke et al. 238 2017). As a result, the occurrence of a greater number of species with different 239 functional traits and evolutionary histories is enhanced (Dalmolin et al. 2019). This 240 possibility opens a precedent for new studies with the specific objective of evaluating 241 the way species use these habitats, as well as their preferences in terms of microhabitats.

242 We must highlight the fact that Scinax squalirostris was recorded only in forests. This 243 result is different from what was expected since this species is associated with low 244 vegetation such as grass and shrubs (Ximenez et al. 2014; Maneyro et al. 2017; Dias et 245 al. 2019). Another aspect to be taken into account is that hylids are less likely to be 246 caught in pitfall traps because of their climbing habits (Oliveira et al. 2013; Dosso et al. 247 2019). This could have caused some sampling bias and, consequently, affected the 248 observed patterns (which was not our case, since we used different, complementary 249 sampling methods). In addition, some studies indicate that even specific sampling 250 methods such as calling surveys are prone to the observer’s effect on the pattern of 251 results (Dosso et al. 2019).

252 The fact that we have associated different sampling methods favored the record of 253 species that make wide use of each habitat. Estimations based exclusively on auditory 254 surveys, for example, prioritize the record of habitat use related to reproductive activity 255 (Santos et al. 2016, Moser et al. 2019) limiting the projections regarding habitat 256 requirements (Oliveira et al. 2016; Santos et al. 2016). This is not the case in our study, 257 as previous work carried out in the same region reported the occurrence of nine to 258 fifteen species (Ximenez et al. 2014; Ximenez & Tozetti 2014; Dalmolin et al. 2019; 259 Dalmolin et al. 2020). Thus, our samplings can be considered effective for the 260 representativeness of anuran species in Brazil’s extreme south.

261 The results herein obtained draw attention to the role of heterogeneity and 262 environmental complexity on anuran composition. They also warn about the imminent 263 risk of changing landscapes due to the invasion of forest species such as Eucalyptus and 264 Pinus species. Although the sampled communities do not include any endangered 265 species, it is worth mentioning that the study area is a Ramsar site, which is of particular 266 concern when dealing with an area of enormous ecological relevance and importance 267 for the maintenance of many ecosystem functions. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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379 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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381 Tables 382

Table 1: Vegetation descriptors measured in each transect. Descriptor Levels Categories: exposed (without any type of Types of land cover cover); cover by grassy vegetation; presence of shrubs; presence of trees Vegetation cover Percentual Vegetation height Centimeters 383

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403 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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Table 2: Anuran species found in two phytophysiognomy types of Estação Ecológica do Taim, southern Brazil, between May 2011 and April 2012. Black squares represent the occurrence of species in the relative month. Sampling Method Months with registers Species Family Pitf Audito Visu M J J A S O N D J F M A all ry al Bufonidae Rhinella dorbignyi X X X Cycloramph Odontophrynus X X X idae maisuma Hypsiboas X X X pulchellus

Hylidae Pseudis minuta X X X Scinax granulatus X X X Scinax squalirostris X X X Physalaemus X biligonigerus Physalaemus X X X gracilis Pseudopaludicola X X Leptodactyli falcipes dae Leptodactylus X X gracilis Leptodactylus cf. X X X latrans Leptodactylus X X X latinasus Microhylida Elachistocleis X X e bicolor 406

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410 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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412 Figures 413

414 415 Figure 1: Geographic location of the study area (Estação Ecológica do Taim) in the 416 state of Rio Grande do Sul, Brazil.

417 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

418 419 Figure 2: Phytophysiognomies of ESESC Taim: (a) sites of grassland dominance and 420 (b) sites of forest dominance.

421 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

422 423 Figure 3: Anuran species found in two phytophysiognomy types of Estação Ecológica 424 do Taim, southern Brazil, between May 2011 and April 2012. (a) Elachistocleis bicolor; 425 (b) Hypsiboas pulchellus; (c) Leptodactylus gracilis; (d) Leptodactylus latinasus; (e) 426 Leptodactylus latrans; (f) Odontophrynus maisuma; (g) Physalaemus biligonigerus; (h) 427 Physalaemus gracilis; (i) Pseudis minuta; (j) Pseudopaludicola falcipes; (k) Rhinella 428 dorbignyi; (l) Scinax granulatus; (m) Scinax fuscovarius. 429 430 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.31.229310; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

431 432 Figure 4: Dissimilarity in anuran composition in two different phytophysiognomies of 433 Reserva Ecológica do Taim, southern Brazil.