Acta Herpetologica 16(1): 11-25, 2021 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-8955 Morphometric differentiation and sexual dimorphism in Limnomedusa macroglossa (Duméril & Bibron, 1841) (Anura: Alsodidae) from Uruguay Valeria de Olivera-López1,*, Arley Camargo2, Raúl Maneyro1 1 Laboratorio de Sistemática e Historia Natural de Vertebrados, Instituto de Ecología y Ciencias Ambientales, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay 2 Centro Universitario Regional Noreste, Sede Rivera, Universidad de la República, Ituzaingó 667, Rivera 40000, Uruguay * Corresponding author. E-mail address: vdeolivera@fcien.edu.uy Submitted on: 2020, 18th June; revised on: 2021, 9th March; accepted on: 2021, 21st March Editor: Raoul Manenti Abstract. Intersexual morphological differences within a species occur in many traits, including body size and shape. Many processes that cause geographic variability in morphology have been proposed: population structure, pheno- typic plasticity (environmental effects on development), and natural and/or sexual selection. Several hypotheses can explain patterns of sexual dimorphism in anurans, including natural or intra/inter-sexual selection, and differences in life history strategies between sexes. Limnomedusa macroglossa is considered a habitat specialist restricted to rocky outcrops in Brazil, Argentina, Paraguay, and Uruguay. We evaluated the extent of sexual (size and shape) dimorphism in L. macroglossa from Uruguay based on morphometrics and secondary sexual characteristics, while taking into account geographic variation. Sexual dimorphism in body size of adults was found, but multivariate analyses did not demonstrate the existence of significant differences in shape. There were also significant differences in body size and hind leg measurements among six hydrographic basins as a result from the phenotypic plasticity correlated with local temperature, representing a clinal variation along the latitudinal gradient of Uruguay. The sexual dimorphism found in body size is probably the consequence of higher growth rates and/or late sexual maturity in females, which favors larger body size for accommodating larger ovaries, and thus, higher reproductive output. Keywords. Sexual dimorphism, clinal variation, morphometrics, Limnomedusa macroglossa, Uruguay. INTRODUCTION Morphology is one of the main components of the phenotype that can be studied through qualitative as well as quantitative characteristics. In particular, morphol- ogy can be assessed via morphometrics to quantitatively describe, analyze and interpret morphological variation within and between species (Kaliontzopoulou, 2011; Rohlf, 1990). Morphological quantitative traits are usually polygenically inherited and show considerable plasticity in relation to environmental factors (Babik and Rafin- ski, 2000). Furthermore, plasticity can lead to geographic variability in morphology. In that sense, many processes have been proposed, such as: biogeographical barriers that partially isolate populations, effects of environmental parameters (precipitations and temperature) on growth rates, and action of sexual selection resulting in sexual dimorphism (Schäuble, 2004). Body size is a strongly plastic morphological trait (Green, 2015) fundamental in physiological and ecological contexts. Traditionally, snout-vent length (SVL) has been used as the gold stand- ard to measure body size in frogs (Kupfer, 2007). Among anurans, analyses of intraspecific geographical variability in morphology have often revealed extensive variation in body size (Schäuble, 2004). Due to the limited dispersal ability and high philopatry in frogs, it is common to find 12 V. de Olivera-López, A. Camargo, R. Maneyro intraspecific differences in morphology among geograph- ically separated populations, particularly in body size, caused by genetic divergence among isolated populations (Baraquet et al., 2012; Castellano et al., 2000). In addition to geographic distance, landscape features could account for spatial morphological variation. For instance, hydro- graphic basins could act as physical barriers promoting isolation and spatial structuring among populations as a result of changes in altitude, slope and landscape features among basins. Moreover, climate and food availability may also vary geographically, leading to differences in the ability to grow, resulting in morphological variation (Lovich and Gibbons, 1992; Hartmann, 2016). Another source of intraspecific variation could be sexual dimorphism; the occurrence of morphological differences between individuals of different sex within a species, may affect several traits like body size, shape and sometimes, secondary sexual characteristics (Wells, 2007). Several factors can influence sexual dimorphism including female reproductive strategy, sexual selection, and competition for resources (Fathinia et al., 2012). Sexual dimorphism may have important consequences for animal ecology, and is a key aspect for understand the evolution of life history traits (Kupfer, 2007). In par- ticular, sexual size dimorphism (SSD) is defined as the difference in body length or mass of sexually mature males and females (Fairbairn, 1997; Kupfer, 2007; Nali et al., 2014). Several evolutionary processes have been proposed to explain patterns of sexual dimorphism in anurans. On one hand, the usually biased SSD in favor of females (Shine, 1979) is hypothesized as the result of a fecundity advantage driven by natural selection: bigger females can harbor more eggs, and then produce larger clutches (Arak, 1988; Wells, 2007). Whereas in males, natural selection operates against of bigger body sizes, because higher vulnerability of prolonged breeders to predators increase their cost of reproduction in terms of survival at small body sizes (Camargo et al., 2008). On the other hand, some authors argue that sexual dimor- phism is a consequence of sexual selection. In this sense, Darwin envisioned that sexual selection depends on the struggle between males to access females, and recognized two mechanisms: intrasexual selection, through compe- tition between members of the same sex (usually males) for access to mates, where large males defeat small ones in aggressive encounters and displace them from territo- ries; and intersexual (epigamic) selection, where mem- bers of one sex (usually females) choose members of the opposite sex, by comparing traits of potential mates and select those that are more attractive (Darwin, 1871; Shine, 1979; Woolbright, 1983; Arak, 1988; Lovich and Gibbons, 1992). However, some authors proposed that sexual dimorphism is a function of differences in life history strategies between the sexes, as well as the result of a variety of selective forces. In this sense, SSD can be explained in terms of disparate age structure between sexes in reproductive populations; that is, females were larger because they were older than the males, which mature earlier at smaller size. In fact, Monnet and Cherry (2002) found that age differences between breeding males and females appear to have a major influence on the extent of dimorphism. Female anuran fecundity appears to be correlated with body size in all anuran species in which this phenomenon has been investigated, and, as anurans display indeterminate growth (Halliday and Ver- rell, 1986), this could be expected to lead to faster growth rates and delayed reproduction in females (Monnet and Cherry, 2002). Limnomedusa Fitzinger 1843, is the most basal genus within the family Alsodidae (Frost et al., 2006; Pyron and Wiens, 2011). The only species of the genus, “rapids frog” Limnomedusa macroglossa (Duméril and Bibron, 1841), is a generalist insect predator of medium to large size, with shades of brown-and-gray and conspicuous glands in the back, and an immaculate white belly (Maneyro and Car- reira, 2012). As secondary sexual characteristics, males present a single vocal sac and dark nuptial pads on their fingers. It is a habitat specialist, with a restricted distribu- tion in rocky outcrops of basaltic origin and superficial soils, with or without vegetation (Maneyro and Carreira, 2012). Regarding its geographic variation in Uruguay, lar- val dispersion appears to be connecting separate major basins via watercourses, although it is also likely that adults disperse between habitat patches by land. As a cor- ollary, an isolation pattern by distance was established, which maintains population stability and genetic diversity in northern populations (Fernández, 2016). Recently, de Olivera et al. (2018) found a correla- tion between body size and ovarian mass in populations of Limnomedusa macroglossa from Uruguay, suggesting a fecundity advantage for larger females since they can accommodate larger ovaries. Moreover, they also report- ed a prolonged pattern of reproduction for this species, which is usually associated with higher levels of intra/ inter-sexual selection (Wells, 2007). Further, in popula- tions from Rio Grande do Sul state, SSD has been found, where females attain larger SVL than males, and they also classified the pattern of reproduction as prolonged, although highly seasonal (Kaefer et al., 2009). Its geographic distribution includes the south of Bra- zil (from Paraná to Rio Grande do Sul), the northeast of Argentina (Misiones and Entre Ríos), the southeast of Paraguay (Alto Paraná), and almost the entire Uruguayan territory (Frost, 2020). However, despite being a relatively 13Sexual dimorphism and clinal variation in Limnomedusa macroglossa common species, geographical variation in morphology has not been investigated in L. macroglossa overall dis- tribution. This circumstance is relevant since most of the distribution range occurs in Uruguay, and thorough eval- uation of the morphological variation across such distri- bution is necessary given its latitudinal, environmental gradient. Lastly, in reference to its conservation status, is categorized nationally and globally as Least Concern according to the IUCN criteria (Silvano et al., 2004; Maneyro et al., 2019). The aim of this work was to evaluate the occurrence of sexual (size and shape) dimorphism in Limnomedusa macroglossa based on morphometrics and secondary sex- ual characteristics across populations from Uruguay. We hypothesized that: (1) Sexual dimorphism and minimum size at sexual maturity (MSSM) are important life history traits due of their value in reproductive output of a species. Besides, most anuran females have larger body sizes than males (female biased SSD) and, thus, females usually reach sex- ual maturity at larger sizes. (2) Isolation pattern by distance triggered by hydro- graphic basins favor geographical differences in morphol- ogy. From which the following predictions emerge: (1.1) we expect that L. macroglossa present sexual size dimorphism with females larger than males, and in fact, with females reaching MSSM at bigger sizes than males. (2.1) Finally, hydrographic basins, due to environ- mental differences, will favor a greater morphological dif- ferentiation in L. macroglossa between than within basins. MATERIALS AND METHODS Field data collection We hand-captured 180 individuals of Limnomedusa mac- roglossa between January 2012 and March 2015, of which 102 were juveniles, 34 mature females and 44 mature males. The indi- viduals were collected along a latitudinal gradient of six hydro- graphic basins from Uruguay (based on Achkar et al., 2013): Río Uruguay (7 females and 8 males), Río Santa Lucía (3 females and 4 males), Océano Atlántico (2 males), Laguna Merín (3 females and 1 male), Río de la Plata (6 females and 6 males), and Río Negro (11 females and 14 males) (Fig. 1) (see Appendices 1, 2 and 3). Latitude and longitude location data of these individuals were obtained from a GPS (Garmin, eTrex 20). In addition, 13 individuals not georeferenced (4 females and 9 males) were also used for SSD and SMA analyses. Lastly, juvenile individuals were used in another investigation (Fernández, 2016). All collected individuals were euthanized using topic lido- caine and intraperitoneal injection of sodium pentobarbital (0.5 ml of a 0.2 g/ml solution), fixed with 10% formaline, and preserved in 70% ethanol, following the experimental protocol “Euthanasia method for amphibians and reptiles in the field” approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Science, University of the Republic. Indi- viduals were euthanized with the purpose of being genetically studied by Fernández (2016), therefore in this work, those indi- viduals were reused. All the specimens are stored in the Verte- brate Zoology Collection (ZVC-B) of the Faculty of Sciences, University of the Republic. We measured eleven morphometric variables using a digi- tal calliper (0.01 mm precision) by a single observer for consist- ency (Grenat et al., 2012): snout-vent length (SVL), mandibular width (MW), head length (HL), inter-orbital distance (IOD), eye diameter (ED), inter-narial distance (IND), eye–nostril distance (END), arm length (ARML), tibia length (TiL), tar- sus length (TaL) and metatarsus length (MtL). We followed the methodology of Duellman (1970) to obtain the measurements of SVL, IOD, ED, IND, TiL, and MtL, as well as Napoli (2005) for END, and Greene and Funk (2009) for ARML. Finally, we measured TaL as the straight length of the tarsus, MW as the straight line between oral commissures, and HL as the straight line distance from the posterior edge of the skull to the tip of the snout. All individuals were measured twice to ensure accu- racy and all measurements were taken on the right side of the body (Fig. 2). For each individual, sex and maturation status (juvenile/ adult) were determined by gonadal analysis. Additionally, males were considered mature by the presence of nuptial pads in their fingers. Finally, to infer MSSM on each sex, we pooled all indi- viduals from all basins and register the size of the adult male/ female with the lowest SVL. Fig. 1. Maps of South America and Uruguay showing basins where Limnomedusa macroglossa was sampled for analyses of geographic variation in sexual dimorphism and morphometric differentiation. Names of sampling basins are as follows: a = Río Uruguay, b = Río Negro, c = Laguna Merín, d = Océano Atlántico, e = Río de la Plata and f = Río Santa Lucía. Black triangles are males and red circles are females (based on Achkar et al., 2013). 14 V. de Olivera-López, A. Camargo, R. Maneyro Data analysis Using the morphometric variables, we tested for sexual dimorphism and quantified morphometric variation through univariate and multivariate analyses while taking into account geographic distribution. To remove allometric effects of body size in the sexual dimorphism analyzes we applied the transformation proposed by Lleonart et al. (2000), which scales all individuals to same size and adjust their shape to that they would have in the new size. For all the variables we tested the normality (Lilliefors’ test) and homogeneity of the variance (Levene’s test) of raw and transformed data. A priori, the raw data did not reject the hypotheses of normality neither homogeneity of variances. Although, with the transformed data, there were rejected. No outlier individuals were found in the analyzed sample. We performed a t-test to evaluate for a significant differ- ence in SVL between males and females. Sexes were also com- pared through one-way perMANOVA using Euclidean simi- larity index. As differences in body size between sexes are not always related to SVL and can involve body parts used in vari- ous behavioral contexts (Kupfer, 2007), we conducted multivari- ate analyses. Differences in shape between males and females were examined through a Principal Component Analysis (PCA) using the Variance-Covariance matrix, and a Hierarchical Clus- ter with Unweighted Pair-Group Average algorithm and Euclid- ean similarity index with 9999 pseudoreplicates. SVL, MtL and TiL variables were log-transformed to esti- mate standardized mayor axis (SMA) regression slopes. This method estimates the line of best fit (slope) when both vari- ables are measured with error (Falster et al., 2006; Warton et al., 2006). We estimated the SMA relationship between SVL and MtL/TiL. We tested for significant allometry assuming the null hypothesis that the slope was equal to 1 (isometry), performed slope comparisons between sexes, tested for shifts along the common SMA slope and in elevation of slope between sexes using Wald test, with 1000 iterations and critical P-value to 0.05. To evaluate morphometric variation, we analyzed differenc- es among basins by one-way perMANOVA (using Bonferroni correction for P-values) and PCA, based on raw measured vari- ables, because our goal was also to evaluate the effect of body size, and we box plot SVL, TiL and MtL variables according to basins. In addition, we calculated the average leg length (= TiL + TaL + MtL) among individuals belonging to each basin. Finally, we tested for significant differences in the leg length and SVL among basins through t-test. The latitude vs. SVL relation- ship was evaluated through regression analysis (using Reduced Major Axis algorithm). In these analyses we used 35 mature males and 30 mature females because coordinate data were not available for all individuals. We used the freely available online programs PAST 3.21 (Hammer et al., 2001), GNUMERIC 1.12 (The Gnome Project, 2018), SMATR 2.0 (Falster et al., 2006) and QGIS 18.24 (QGIS Development Team, 2016) for all statistical analyses performed in this work. RESULTS Sexual dimorphism In total, we examined 180 specimens of which 102 were juveniles, 34 mature females and 44 mature males. We found dark nuptial pads in the first, second, and some- times, the third fingers of all mature males (Fig. 3). We found that females longer than 49.82 mm and males longer than 41.29 mm in SVL were sexually mature (i.e., nuptial pads in males and fully-developed oocytes in females). Taking this into account, we set the MSSM in females at 49.82 ± 0.01 mm and in males at 41.29 ± 0.01 mm. Significant differences in mature body size were found between sexes. Mature females had an average SVL (56.99 ± 4.27 mm) significantly higher than that of mature males (49.69 ± 4.73 mm) (t = 7.04, P < 0.001). Fig. 2. Morphometric measurements used for the analysis of sexual dimorphism in Limnomedusa macroglossa (Anura: Alsodidae) from Uruguay: SVL = snout–vent length; MW = mandibular width; HL = head length; IOD = inter-orbital distance; ED = eye diameter; IND = inter-narial distance; END = eye–nostril distance; ARML = arm length; TiL = tibia length; TaL = tarsus length and MtL = metatarsus length. Fig. 3. Male displaying dark nuptial pads above fingers of the fore- leg (ZVC-B 23281). 15Sexual dimorphism and clinal variation in Limnomedusa macroglossa Furthermore, significant differences were found in means of all other variables, with females reaching larger meas- urements than males (Table 1). When all the morpho- metric variables were introduced in a nonparametric per- MANOVA test, the comparison of sexes was not signifi- cant (F = 0.349, P = 0.865). The PCA of size-adjusted measurements showed a total of ten components, with 69.09 % of the vari- ance comprised by the first two components, with PC1 accounting for 54.14% and PC2 14.95% of the total varia- tion. The bi-dimensional projection of the first two com- ponents exhibited a substantial overlap of sexes (Fig. 4A). The loadings indicate that PC1 has a strong positive cor- relation with TiL (0.61) and MtL (0.58), and the lowest with ED, IND, END and IOD (Fig. 4B), whereas PC2 is moderately correlated with TaL (0.35) and ARML (0.33), while MtL stood out with a very strong negative correla- tion of - 0.76 (Fig. 4C). The dendrogram obtained, with hierarchical cluster- ing, was a good representation of the data matrix given the obtained coefficient of cophenetic correlation (CCC = 0.74) and showed a topology of females and males in congruence with PCA and perMANOVA analyses, determining the absence of morphometric, sexual shape dimorphism. However, the low bootstrap percentages (<50%) do not indicate high support for most of the sim- ilarity relationships. Morphometric differentiation Since multivariate analyses performed previously did not reveal significant differences between sexes, we pooled males and females within each basin in subse- quent analyses. When all the morphometric variables were analyzed through perMANOVA test, significant dif- ferences were found among the six hydrographic basins evaluated in this work (F = 2.553, P < 0.05). The pairwise comparisons showed significant differences between Río Negro and Río de la Plata basins (Table 2). The PCA of original measured variables (including SVL variable), showed a total of eleven components, with 96.4% of the variance comprised by the first two compo- nents, with PC1 accounting for 93.96% and PC2 2.47% of the total variation. The bi-dimensional projection of PC1 vs. PC2 showed the convex polygons grouping indi- Table 1. Descriptive statistics of each morphological variables measured in Limnomedusa macroglossa (Anura: Alsodidae) from Uruguay. Morphological differences between sexes were tested for each variable through t test. Sex: ♂ = male, ♀ = female; n: sample size; Min: mini- mum value; Max: maximum value; x: mean; SE: standard error; SD: standard deviation; Vc: variance coefficient. Variables for which signifi- cant differences were obtained are in bold. All measurements are shown in millimeters. Sex n Min. Max. x SE SD Vc. t test P value SVL ♂ 44 41.29 60.92 49.69 0.71 4.73 9.51 7.04 <0.001 ♀ 34 49.82 64.25 56.98 0.73 4.27 7.5 <0.001 MW ♂ 44 16.61 24.28 19.66 0.29 1.94 9.87 6.56 <0.001 ♀ 34 19.7 24.47 22.32 0.26 1.54 6.92 <0.001 HL ♂ 44 14.47 20.98 17.13 0.23 1.53 8.96 6.78 <0.001 ♀ 34 16.95 22.02 19.46 0.25 1.46 7.51 <0.001 IOD ♂ 44 7.05 9.82 8.38 0.12 0.79 9.44 5.71 <0.001 ♀ 34 8.2 10.93 9.4 0.13 0.77 8.15 <0.001 ED ♂ 44 4.17 7.2 5.45 0.1 0.69 12.59 4.27 <0.001 ♀ 34 5.07 7 6.05 0.09 0.51 8.51 <0.001 IND ♂ 44 3.26 5.48 4.22 0.08 0.56 13.22 5.34 <0.001 ♀ 34 3.69 5.76 4.85 0.08 0.46 9.48 <0.001 NED ♂ 44 4.01 6.33 5.02 0.08 0.56 11.16 7.32 <0.001 ♀ 34 4.92 7.05 5.88 0.08 0.45 7.73 <0.001 ARML ♂ 44 9.93 15.62 12.46 0.19 1.25 10.06 6.1 <0.001 ♀ 34 11.85 16.73 14.2 0.21 1.25 8.78 <0.001 TiL ♂ 44 24.7 37.72 30.53 0.49 3.26 10.68 6.43 <0.001 ♀ 34 30.21 39.86 35.02 0.47 2.76 7.89 <0.001 TaL ♂ 44 13.57 18.72 16.05 0.22 1.49 9.29 6.93 <0.001 ♀ 34 15.66 20.65 18.34 0.24 1.39 7.57 <0.001 MtL ♂ 44 21.82 31.93 26.46 0.41 2.72 10.28 5.59 <0.001 ♀ 34 25.1 32.85 29.63 0.37 2.15 7.24 <0.001 16 V. de Olivera-López, A. Camargo, R. Maneyro viduals from different basins with an elevated degree of overlap (Fig. 5A). According to Greene and Funk (2009), in PCA of morphological data, the first axis (PC1) is usu- ally associated with size, and the remaining axes describe orthogonal axes of variation in shape. Indeed, we found that, the first axis has a strong positive correlation with body size and a moderate correlation with a few hind leg measurements: SVL (0.69), TiL (0.45) and MtL (0.33). Meanwhile, head measurements (IOD, ED, IND and END) showed the weakest correlation (Fig. 5B). The sec- ond axis (shape axis) has a strong positive correlation with MtL (0.56), and a moderate correlation with MW (0.32) and TiL (0.31), while SVL stood out with a strong negative correlation -0.67 (Fig. 5C). Given the considerable contribution of SVL, TiL and MtL variables in size and shape axis of PCA, we did a box plot according to the hydrographic basins in order to show the differences between them. Significant differ- ences were found between hind leg length of individuals from Río de la Plata and Río Negro basins (t = 3.533, P < 0.001), being those of Río de la Plata basin the longest hind leg (82.12 ± 2.21 mm, n = 12), while those of Río Negro basin were the shortest legs (72.55 ± 1.55 mm, n = 25) (Fig. 6A,B), reaching a difference of 11,65%. Regard- ing SVL, we found a similar pattern, reaching higher val- ues in Río de la Plata basin (56.31 ± 1.57 mm, n = 12) and lower ones in Río Negro basin (50.12 ± 1.06 mm, n = 25; t = 3.302, P < 0.002; Fig. 7A), reaching a difference Fig. 4. (A) Scatter plot for the first two principal components obtained from a principal component analysis of eleven morphological vari- ables measured in Limnomedusa macroglossa (Anura: Alsodidae) from Uruguay, including convex polygons grouping individuals according to their sex. Red circles represent females and black crosses are males. Coefficients of association of each morphometric variable with the first principal component (PC1) (B) and with the second principal component (PC2) (C). SVL = snout-vent length, MW = mandibular width; HL = head length; IOD = inter-orbital distance; ED = eye diameter; IND = inter-narial distance; END = eye–nostril distance; ARML = arm length; TiL = tibia length; TaL = tarsus length and MtL = metatarsus length. 17Sexual dimorphism and clinal variation in Limnomedusa macroglossa Table 2. One-way perMANOVA results for morphometric data of Limnomedusa macroglossa from Uruguay taking into account the six hydrographic basins evaluated in this work: Río Uruguay, Río Santa Lucía, Océano Atlántico, Laguna Merín, Río de la Plata and Río Negro. Bonferroni corrected P values are displayed. Basins for which significant differences were obtained are in bold. Río Santa Lucía Río Negro Laguna Merín Río de la Plata Océano Atlántico Río Uruguay 1 0.462 1 1 1 Río Santa Lucía 1 1 1 1 Río Negro 1 0.024 1 Laguna Merín 1 1 Río de la Plata 1 Fig. 5. (A) Scatter plot for the first two principal components obtained from a principal component analysis of eleven morphological vari- ables measured in Limnomedusa macroglossa (Anura: Alsodidae) taking into account hydrographic basins from Uruguay, including convex polygons grouping individuals according to basins. Fill triangles represent males and circles are females. Basins: blue = Río Uruguay; sky blue = Río Santa Lucía; green = Océano Atlántico; violet = Laguna Merín; orange = Río de la Plata; black = Río Negro. Coefficients of asso- ciation of each morphometric variable with the first principal component (PC1) (B) and with the second principal component (PC2) (C) taking into account hydrographic basins from Uruguay. SVL = snout-vent length, MW = mandibular width; HL = head length; IOD = inter- orbital distance; ED = eye diameter; IND = inter-narial distance; END = eye–nostril distance; ARML = arm length; TiL = tibia length; TaL = tarsus length and MtL = metatarsus length. 18 V. de Olivera-López, A. Camargo, R. Maneyro of 11% between groups. Additionally, a significant corre- lation between latitude and SVL was found (r = 0.60, F = 4.35, P < 0.001; Fig. 7B). Allometric regressions We performed a SMA analysis with the variables that showed the highest correlations with PC1 and PC2 in PC analyzes. We found a significant SMA relationship between SVL and MtL in females [b=0.98, 95% confi- dence interval (CI) = 0.78-1.23] and males [b=1.09, 95% CI = 0.94-1.25]. It was also significant between SVL and TiL in females [b=1.06, 95% CI = 0.88-1.27] and males [b=1.13, 95% CI = 1.02-1.25] and between SVL and MW in females [b=0.93, 95% CI = 0.75-1.16] and males [b=1.03, 95% CI = 0.90-1.18]. In males, the SVL vs. TiL relationship showed a significant positive allometry (b=1.128). On the other hand, in all the other cases, there were no significant differences from isometry (Table 3). In all cases, the test for common slope across sexes indicated that there are no significant differences in com- mon slope between males and females. When testing for shifts along the common slope, we found significant shifts in all relationships with higher values in females (SVL-MtL relationship: W = 42.952, P < 0.01; SVL-TiL relationship: W = 47.729, p<0.01). The test for shift in elevation was only significant in the SVL vs. MtL rela- tionship in favor of males (W = 4.411, P < 0.05), but the difference in elevation was rather small and close to our resolution limit (0.01 mm). Fig. 7. (A) Box plot of body size (SVL) of Limnomedusa macroglossa (Anura: Alsodidae) according to hydrographic basins from Uruguay, considering males and females grouped. (B) Latitude-SVL relationship for L. macroglossa. The line represents the regression model. Red circles represent individuals of Río de la Plata basin and black circles are individuals of Río Negro basin. The line inside de boxes represents the median. SVL measurements are in millimeters. Fig. 6. Box plots of TiL (A) and MtL (B) variables of Limnomedusa macroglossa (Anura: Alsodidae) according to hydrographic basins from Uruguay, considering males and females grouped. TiL = tibia length and MtL = metatarsus length. The line inside de boxes represents the median. All measurements are in millimeters. 19Sexual dimorphism and clinal variation in Limnomedusa macroglossa DISCUSSION In this study we determined the minimum size at sexual maturity (MSSM) and described morphomet- ric and intersexual differences in Limnomedusa mac- roglossa. We showed that females and males differ in MSSM, presence of dark nuptial pads in males (a sexu- ally dimorphic characteristic) and body size, while no differences were found in body shape. Nuptial pads can be observed during the breeding season in response to increases in circulating levels of androgens, but later regress during the non-breeding period, although with- out resembling to a female-like morphology (Wells, 2007). Some authors argued that well-developed nup- tial pads are associated with breeding in water to pre- vent the female´s escape during amplexus (Duellman and Trueb, 1986). However, according to Savage (1961), nuptial pads also allow the male to hold the female while defending her against rival males. The MSSM is the size at which an individual has all the morphological and physiological conditions to begin to breed, and along with sexual dimorphism, are impor- tant life history traits to understand population changes through time. Life history theory explains the variation in MSSM between sexes through natural selection mecha- nisms, mainly related with adult mortality rates (Tolosa et al., 2014). We found that females of Limnomedusa mac- roglossa reach sexual maturity around 49.82 mm, while males reach it at a smaller size of about 41.29 mm. This difference between the sexes can be explained by sexual selection: selection for mating effort in males to defend territories, in detriment of larger males due to the high energetic expenditures and risks of mortality, and paren- tal effort in females to produce more eggs to maximize their reproductive output, which favors females with a larger size; both processes have been pointed out as potential explanations for sexual maturation at different ages (Howard, 1981). There was sexual dimorphism in size in Limnome- dusa macroglossa with females being larger than males, as it occurs in more than 90% of anurans species (Shine, 1979). These results agree with those found in a popula- tion of L. macroglossa in southern Brazil based on SVL only (Kaefer et al., 2009). Taking into account the main hypotheses regarding the causes of sexual dimorphism in anurans, natural and/or sexual selection might adequate- ly explain the differences in body sizes between females and males found in this work. Given the available data until date (Kaefer et al., 2009; de Olivera et al., 2018) and our results, it seems that the preference for larger females evolved because they produce more oocytes per clutch (Bionda et al., 2011) or bigger eggs (the fecundity advantage hypothesis), whereas in males, natural selection works against of bigger body sizes due to the existence of possible differential predation, since the long reproduc- tive period exposes and makes them more vulnerable to predators (Camargo et al., 2008). Furthermore, intra/inter- sexual selection could be playing an important role in the differentiation between males and females, through male- male competition and/or female choice (Darwin, 1871; Shine, 1979; Woolbright, 1983; Arak, 1988). Although, in our field work, we did not observe such behaviors, we cannot rule out their existence, since it has been reported that its a species with a prolonged reproduction pattern (Kaefer et al., 2009; de Olivera et al., 2018) which is usual- ly associated with more territorial males, choosy females, and overall higher levels of sexual selection (Wells, 2007). Finally, the age structure in the reproductive populations may also be operating between sexes (Halliday and Ver- rell, 1986; Monnet and Cherry, 2002). Thus, the sexual dimorphism in size found in L. macroglossa could be the result of distinct, possibly opposing, selective forces that trade-off differently in each sex. In addition to size, anurans exhibit other forms of sexual dimorphism, including: the proportions and mus- cular development of the forelimbs (related with clasping behavior), skin color, texture and glands (visual, tactile and chemical cues for sex recognition), fangs and tusks (related with combat), abdominal and laryngeal muscles, and lung capacities (calling behavior) (Wells, 2007; Bell and Zamudio, 2012) and head morphology (feeding strat- egies) (Khoshnamvand et al., 2018). No differences were found in shape between sexes, but significant differences were found among basins. Some variables related with the hind legs showed the highest contributions to overall shape differentiation. A functional interpretation of the differentiation in the hindlimb length found in L. macroglossa could be that Table 3. Standarized major axis (SMA) regression results and test of isometry for Limnomedusa macroglossa. Variables used in analyses were: SVL = snout–vent length; TiL = tibia length and MtL = meta- tarsus length. Abbreviations: a = intercept. Significant regressions are in bold. Variables SMA regression Test of isometry a r² p F p log MtL vs. log SVL Females -0.2471 0.575 < 0.01 0.034 0.855 Males -0.4193 0.800 < 0.01 1.422 0.240 log TiL vs. log SVL Females -0.3117 0.733 < 0.01 0.370 0.548 Males -0.4295 0.891 < 0.01 5.620 0.02 20 V. de Olivera-López, A. Camargo, R. Maneyro leg proportions may influence locomotor performance. Several experimental studies have shown how longer hindlimbs may improved locomotor performance (Ori- zaola and Laurila, 2009; Drakulic et al., 2016; Zamora- Camacho, 2018; Zamora-Camacho and Aragón, 2019), as well as jumping distance increases as the individ- ual grows larger (Zug, 1978). Meanwhile, other stud- ies revealed that locomotor performance is negatively affected at larger sizes (Moreno-Rueda et al., 2020), rela- tively large differences (>10%) in leg length can affect the jumping efficiency (Emerson, 1978; Babik and Rafinski, 2000). Differences in jumping ability could be occurring in L. macroglossa because our results showed differences greater than 10% in body size and leg length in individu- als from Río de la Plata basin compared to those from the Río Negro basin. Alternatively, differences in the hindlimb length may be the result of unequal growth and developmental rate during the larval and juvenile stages (Emerson et al., 1988; Babik and Rafinski, 2000). Because amphibians are ectotherms and depend on water, they show pheno- typic responses to changes in environmental factors. In this sense, some phenotypic plasticity can be attributed to environmental factors such as the duration of the lar- val period and its relation to size as a function of tem- perature (Vences et al., 2002). A general Temperature- Size rule for ectotherms states that higher temperatures increase developmental rates, at the cost of smaller size (Drakulic et al., 2016) and conversely, at low tempera- tures develop more slowly, so they metamorphose at larg- er body sizes (Harkey and Semlitsch, 1988). Moreover, some studies replace the idea of temperature and relate body size to latitude, predicting that body size within species increases with latitude (Lindsey, 1966; Schäuble, 2004). In this study we found that individuals which had the longest legs were from Río de la Plata basin, which correlates with the colder climate in the studied distribu- tion (InUMet, 2020). On the other hand, the individuals which had shortest legs were found in Río Negro basin, where the temperature is significantly higher (InUMet, 2020). So, we can expect that differences in environmen- tal temperature during the larval period may have been responsible for the variation in the hindlimbs length in L. macroglossa. This trend has already been reported in other studies (Atkinson, 1994, 1995; Angilletta et al., 2004). Furthermore, our results are consistent with the intraspecific version of Bergmann’s rule. It relates to geo- graphic variation in the body sizes of animals (Blackburn et al., 1999) which has been briefly stated by Mayr as: The smaller-sized geographic races of a species are found in the warmer parts of the range, the larger-sized races in the cooler districts (Ray, 1960). In this study we report a clinal variation in the rela- tive leg length and body size of Limnomedusa macroglos- sa along a latitudinal gradient in Uruguay. The body size dimorphism likely reflects differences in growth rates of males and females. In organisms with indetermi- nate growth, body size is a result of a trade-off between growth and reproduction (Camargo et al., 2008). There- fore, females of Limnomedusa macroglossa appear to delay sexual maturity, while maintaining the same body shape and proportions as the males, reaching larger sizes at maturity, based on the combined evidence of distinct MSSM and the body size shift along the common iso- metric slopes of males and females. This difference in size could be adaptive for females, since a larger body size would increase the volume of the abdominal cavity, being able to accommodate larger ovaries (de Olivera et al., 2018) and consequently, increasing their reproduc- tive output [the so called fecundity advantage hypoth- esis (Darwin, 1871)]. Therefore, sexual dimorphism in L. macroglossa could be determined by differential growth rate between the sexes, since the growth rates are usually asymptotic after maturation and sexes generally mature at different ages (sexual bimaturity) (Monnet and Cherry, 2002; Kupfer, 2007; Wells, 2007), or it may be the result of difference in the age distributions of males and females (Howard, 1981). Therefore, the sexual dimorphism found in body size is probably the consequence of high- er growth rates and/or late sexual maturity in females of Limnomedusa macroglossa, which favors a larger body size and larger ovaries, and consequently, higher repro- ductive output. CONCLUSIONS In conclusion, our data on MSSM and SSD of Lim- nomedusa macroglossa from Uruguay may contribute to the knowledge of the life history traits of this species. Our results show that females attained sexual matu- rity at larger sizes than males with a marked female biased sexual size dimorphism. These traits are driven by a trade- off between natural and sexual selection on each sex: parental effort in females does favor larger sizes to maxi- mize their reproductive output, because bigger females can accommodate more eggs in their abdominal cavity. Meanwhile, mating effort in males does not favor large sizes due to energetic expenditures and risk of mortal- ity during the long breading season, because bigger males invest most of their energy in search and calling behavior and have high mortality rates due to predation risk. We also report a clinal variation in the relative leg length and body size of Limnomedusa macroglossa along 21Sexual dimorphism and clinal variation in Limnomedusa macroglossa a latitudinal gradient in Uruguay. Individuals with long- est legs and bigger body sizes were from Río de la Pla- ta basin, meanwhile individuals with shortest legs and smaller body sizes were those found in Río Negro basin. These differences could be explained by phenotypic plas- ticity in age and size at metamorphosis when separate populations are exposed to different environmental con- ditions (Ruthsatz et al., 2018). Studies demonstrated a plastic response of metamorphic traits in anuran larvae to changes in environmental conditions such as tempera- ture. With increasing temperature time to metamorpho- sis may be reduced and metamorphosis occurs at smaller body sizes (Vences et al., 2002). Then, this may be occur- ring in L. macroglossa, since Río de la Plata basin is cor- related with the colder climate in the studied distribution, meanwhile Río Negro basin is correlated with a warmer one. All the evidence gathered in this work and its inter- pretations show that sexual dimorphism found in body size is likely the consequence of higher growth rates and/or late sexual maturity in females of Limnomedusa macroglossa, which favors a larger body size and bigger ovaries, and consequently, higher reproductive output. Examination of adult females and males, already in pro- gress, will soon allow a more in depth understanding of L. macroglossa reproductive biology in Uruguay. ACKNOWLEDGEMENTS We thank Ernesto Elgue and Claudia Fernández for his help in fieldwork and Pablo Toriño for drawings and statistical support. This work was supported by Comisión Sectorial de Investigación Científica under Grant CSIC I+D 2012 from University of the Republic. All animals have been captured, handled and euthanized in accord- ance with relevant guidelines in full compliance with spe- cific permits released by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Sciences, University of the Republic; the Honorary Committee for Animal Experimentation (CHEA) regulated in the National Law of Animal Experimentation N°18611 (Oct. 2, 2009); the Ordinance on the Use of Animals in Experi- mentation, Teaching and University research (C.D.C. Res. Nº11 Dec. 21,1999, University of the Republic) and the corresponding scientific collection permit issued by the Ministry of Livestock, Agriculture and Fisheries of Uru- guay (MGAP) (Res. 126/14). We thank two anonymous reviewers and the associ- ate editor that provided comments and suggestions for improving an earlier version of the manuscript. REFERENCES Achkar, M., Domínguez, A., Pesce, F. (2013): Cuencas hidrográficas del Uruguay: situación y perspectivas ambientales y territoriales. Redes Amigos de la Tierra, Montevideo. Angilletta, M. Jr., Steury, T., Sears, M. (2004)  : Tempera- ture, Growth Rate, and Body Size in Ectotherms  : Fitting Pieces of a Life-History Puzzle. Integr. Comp. Biol. 44: 498-509. Arak, A. (1988): Sexual size dimorphism in body size: a model and a test. Evol. 42: 820-825. Atkinson, D. (1994): Temperature and Organism Size – A Biological Law for Ectotherms? Adv. Ecol. Res. 25: 1-58. Atkinson, D. (1995): Effects of Temperature on the size of Aquatic Ectotherms: Exceptions to the General Rule. J. Therm. Biol. 20: 61-74. Babik, W., Rafinski, J. (2000). Morphometric differentia- tion of the moor frog (Rana arvalis Nilss.) in Central Europe. J. Zool. Syst. Evol. Res. 38: 239-247. Baraquet, M., Grenat, P.R., Salas, N.E., Martino, A.L. (2012): Variación morfométrica y geográfica entre poblaciones de Hypsiboas cordobae (Anura: Hylidae) en Argentina. Cuad. Invest. UNED. 4: 147-155. Bell, R.C., Zamudio, K.R. (2012): Sexual dichromatism in frogs: natural selection, sexual selection and unex- pected diversity. Proc. R. Soc. B. 279: 1-7. Bionda, C., Lajmanovich, R., Salas, N., Martino, A., Di Tada, I. (2011): Reproductive ecology of the common south american toad Rhinella arenarum (Anura: Bufo- nidae): Reproductive effort, clutch size, fecundity, and mate selection. J. Herpetol. 45: 261-264. Blackburn, T., Gaston, K., Loder, N. (1999): Geographic gradients in body size: A Clarification of Bergmann’s rule. Div. Distrib. 5: 165-174. Camargo, A., Sarroca, M., Maneyro, R. (2008): Repro- ductive effort and the egg number vs. size trade-off in Physalaemus frogs (Anura: Leiuperidae). Acta Oecol. 34: 163-171. Castellano, S., Giacoma, C., Dujsebayeva, T. (2000): Mor- phometric and advertisement call geographic varia- tion in polyploid green toads. Biol. J. Linnean Soc. 70: 341-360. Darwin, C.R. (1871): The descent of man and selection in relation to sex. John Murray, London. de Olivera, V., Camargo, A., Maneyro, R. (2018): Biología reproductiva de Limnomedusa macroglossa (Duméril & Bibron, 1841) (Anura: Alsodidae) en Uruguay. In: Book of abstracts V Congreso Uruguayo de Zoología, pp. 78. Sociedad Zoológica del Uruguay Eds, Monte- video. 22 V. de Olivera-López, A. Camargo, R. Maneyro Drakulic, S., Feldhaar, H., Lisicic, D., Mioc, M., Cizelj, I., Seiler, M., Spatz, T., Rodel, M-O. (2016): Population- specific effects of developmental temperature on body condition and jumping performance of a widespread European frog. Ecol. Evol. 6: 3115-3128. Duellman, W.E. (1970): The Hylid frogs of Middle Amer- ica. Museum of Natural History, University of Kansas. Duellman, W. E., Trueb. L. (1986): Biology of amphib- ians. McGraw-Hill, New York. Emerson, S.B. (1978): Allometry and jumping in frogs: helping the twain to meet. Evol. 32: 551-564. Emerson, S.B., Travis, J., Blouin, M. (1988): Evaluating a hypothesis about heterochrony: larval life-history traits and juvenile hind-limb morphology in Hyla cru- cifer. Evol. 42: 68-78. Fairbairn, D.J. (1997): Allometry for sexual size dimor- phism: Pattern and process in the coevolution of body size in males and females. Annu. Rev. Ecol. Syst. 28: 659-687. Falster, D.S., Warton, D.I., Wright, I.J. (2006): User’s guide to SMATR: Standardised Major Axis Tests and Rou- tines. Available from: http://bio.mq.edu.au/research/ groups/comparative/SMATR/SMATR_users_guide.pdf Fathinia, B., Rastegar-Pouyani, N., Darvishnia, H., Moham- adi, H., Faizi, H. (2012): Sexual size dimorphism in Rana (Pelophylax) ridibunda ridibunda Pallas, 1771 from a population in Darre-Shahr Township, Ilam Prov- ince, western Iran. Amphib. Reptile Conserv. 5: 92-97. Fernández, C. (2016.): Diversidad filogeográfica a múlti- ple escala en poblaciones de Limnomedusa macroglos- sa (Anura, Alsodidae) de Uruguay.  Master’s Thesis. Universidad de la República, Facultad de Ciencias, Montevideo. Only available online at: https://www. colibri.udelar.edu.uy/jspui/handle/20.500.12008/8438 Frost, D.R. (2020): Amphibian Species of the World 6.0, an Online Reference. [Internet]. New York: American Museum of Natural History. Available from: https:// amphibiansoftheworld.amnh.org/index.php Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C. (2006): The amphibian tree of life. Bull. Am. Mus. Nat. Hist. 297: 1-370. Green, D.M. (2015): Implications of female body-size variation for the reproductive ecology of an anuran amphibian. Ethol. Ecol. Evol. 27: 173-184. Greene, A.E., Funk, W.C. (2009): Sexual selection on morphology in an explosive breeding amphibian, the Columbia spotted frog (Rana luteiventris). J. Herpetol. 43: 244-251. Grenat, P.R., Salas, N.E., Martino, A.L. (2012): Intra- and inter-specific morphometric variation between Odon- tophrynus populations (Anura: Cycloramphidae) of central Argentina. Int. J. Trop. Biol. 60: 1589-1601. Gudynas, E., Gehrau, A. (1981): Notas sobre la dis- tribuición y ecología de Limnomedusa macroglossa (Dumeril & Bibron, 1841) en Uruguay (Anura, Lepto- dactylidae). Iheringia Ser. Zool. 60: 81-99. Halliday, T.R., Verrell, P.A. (1986): Review: Sexual selec- tion and body size in amphibians. Herpetol. J. 1: 86-92. Hammer, Ø., Harper, D.A.T., Ryan, P.D. (2001): PAST: Paleontological statistics software package for educa- tion and data analysis. Palaeontol. Electron. 4: 1-9. Harkey, G.A., Semlitsch, R.D. (1988): Effects of tempera- ture on growth, development, and color polymor- phism in the ornate chorus frog Pseudacris ornata. Copeia 1988: 1001-1007. Hartmann, D. (2016): Global Physical Climatology. Else- vier Science. University of Washington, Washington. Howard, R.D. (1981): Sexual dimorphism in bullfrogs. Ecology 62: 303-310. InUMet. (2020): Climatología del Verano (dic-ene-feb) en Uruguay 1981-2010. Instituto Uruguayo de Mete- orología. Only available online at: https://www.inu- met.gub.uy/sites/default/files/2020-12/CLIMATOLO- GIA%20DEL%20VERANO_LQ_0.pdf Kaefer, I.L., Both, C., Zanini Cechin, S. (2009): Breeding biology of the rapids frog Limnomedusa macroglossa (Anura: Cycloramphidae) in southern Brazil. J. Nat. Hist. 43: 1195-1206. Kaliontzopoulou, A. (2011): Geometric morphometrics in herpetology: modern tools for enhancing the study of morphological variation in amphibians and reptiles. Basic Appl. Herpetol. 25: 5-32. Khoshnamvand, H., Malekian, M., Keivany, Y. (2018): Morphological distinction and sexual dimorphism in divergent clades of Neurergus kaiseri (Amphibia: Sala- mandridae). Basic Appl. Herpetol. 32: 5-17. Kupfer, A. (2007): Sexual size dimorphism in amphib- ians: an overview. In: Sex, size and gender roles: evo- lutionary studies of sexual size dimorphism, pp. 50 – 59. Fairbairn, D.J., Blanckenhorn, W., Székely, T. Eds. Oxford University Press, Oxford. Lindsey, C.C. (1966): Body sizes of poikilotherm verte- brates at different latitudes. Evol. 20: 456-465. Lleonart, J., Salat, J., Torres, G.J. (2000): Removing allo- metric effects of body size in morphological analisys. J. Theor. Biol. 205: 85-93. Lovich, J., Gibbons, J. (1992): A review of Techniques for Quantifying Sexual Size Dimorphism. Growth Dev. Aging. 56: 269-281. 23Sexual dimorphism and clinal variation in Limnomedusa macroglossa Maneyro, R., Carreira, S. (2012): Guía de Anfibios del Uruguay. Ediciones de la fuga, Montevideo. Maneyro, R., Langone, J., Carreira, S. (2019): Anfibios. In: Libro Rojo de los Anfibios y Reptiles del Uruguay. Biología y conservación de los Anfibios y Reptiles en peligro de extinción a nivel nacional, pp. 17-106. Car- reira, S., Maneyro, R. Eds. Montevideo. Monnet, J-M., Cherry, M.I. (2002): Sexual size dimor- phism in anurans. Proc. R. Soc. Lond. B. 269: 2301- 2307. Moreno-Rueda, G., Requena-Blanco, A., Zamora-Cama- cho, F., Comas, M., Pascual, G. (2020): Morphological determinants of jumping performance in the Iberian green frog. Curr. Zool. 66: 417-424. Nali, R.C., Zamudio, K.R., Haddad, C.F.B., Prado, C.P.A. (2014): Size-dependent selective mechanisms on males and females and the evolution of sexual size dimorphism in frogs. Am. Nat. 184: 1-14. Napoli, M.F. (2005): A new species allied to Hyla cir- cumdata (anura: hylidae) from Serra da Mantiqueira, southeastern Brazil. Herpetologica. 61: 63-69. Orizaola, G., Laurila, A. (2009): Microgeographic varia- tion in the effects of larval temperature environment on juvenile morphology and locomotion in the pool frog. J. Zool. 277: 267-274. Pyron, R.A., Wiens, J.J. (2011): A large-scale phylogeny of amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecil- ians. Mol. Phylogenet. Evol. 61: 543-583. QGIS Development Team. (2016): QGIS Quantum Geo- graphic Information System. Open Source Geospatial Foundation Project. Available from: https://issues.qgis. org/projects/qgis/wiki/QGIS_Citation_Repository Ray, C. (1960): The Application of Bergmann’s and Allen’s Rules to the Poikilotherms. J. Morphol. 106: 85-108. Rohlf, F.J. (1990): Morphometrics. Annu. Rev. Ecol. Syst. 21: 299-316. Ruthsatz, K., Peck, M., Dausmann, K., Sabatina, N., Glos, J. (2018): Patterns of temperature induced develop- mental plasticity in anuran larvae. J. Therm. Biol. 74: 123-132. Savage, R.M. (1961): The ecology and life history of the common frog. Sir Isaac Pitman and Sons, London. Schäuble, C.S. (2004): Variation in body size and sexual dimorphism across geographical and environmental space in the frogs Limnodynastes tasmaniensis and L. Peronii. Biol. J. Linn. Soc. Lond. 82: 39-56. Shine, A. (1979): Sexual selection and sexual dimorphism in the Amphibia. Copeia 1979: 297-306. The Gnome Project. (2018): The Gnumeric Spreadsheet: Free, Fast, Accurate --- pick any three. Available from: http://www.gnumeric.org/ Silvano, D., Garcia, P., Kwet, A., Segalla, M.V., Lan- gone, J., Baldo, D. (2004): Limnomedusa macroglossa. The IUCN Red List of Threatened Species. Avail- able from: dx.doi.org/10.2305/IUCN.UK.2004.RLTS. T57176A11580028.en Tolosa, Y., Molina-Zuluaga, C., Restrepo, A., Daza, J. (2014): Sexual maturity and sexual dimorphism in a population of the rocket-frog Colostethus aff. frater- danieli (Anura: Dendrobatidae) on the northeastern Cordillera Central of Colombia. Actual. Biol. 37: 287- 294. Vences, M., Puente, M., Nieto, S., Vieites, D.R. (2002): Phenotypic plasticity of anuran larvae: environmental variables influence body shape and oral morphology in Rana temporaria tadpoles. J. Zool. 257: 155-162. Warton, D.I., Wright, I.J., Falster, D.S., Westoby, M. (2006): Bivariate line-fitting methods for allometry. Biol. Rev. 81: 259-291. Wells, K.D. (2007): The ecology and behaviour of amphibians. University of Chicago Press, Chicago. Woolbright, L.L. (1983): Sexual selection and size dimor- phism in anuran Amphibia. Am. Nat. 121: 110-119. Zamora-Camacho, F. (2018): Locomotor performance in a running toad: roles of morphology, sex and agrosys- tem versus natural habitat. Biol.  J. Linn.  Soc. 123: 411-421. Zamora-Camacho, F., Aragón, P. (2019): Failed preda- tor attacks have detrimental effects on antipredatory capabilities through developmental plasticity in Pelo- bates cultripes toads. Funct. Ecol. 33: 846-854. Zug, G.R. (1978): Anuran locomotion-structure and function, 2: jumping performance of semiaquatic, ter- restrial, and arboreal frogs. Smithsonian Institution Press, Washington. APPENDIX 1 Six hydrographic basins of Uruguay and its geographic location (based on Achkar et al., 2013). Basins Latitude (S) Longitude (W) Río Uruguay 30°5’10’’-33°54’59’’ 55°48’45’’-58°26’17’’ Río Santa Lucía 33°42’1’’-34°50’24’’ 54°59’24’’-57°07’11’’ Océano Atlántico 33°39’56’’-34°58’26’’ 53°22’13’’-55°10’8’’ Laguna Merín 31°54’18’’-34°24’51’’ 53°02’27’’-55°22’10’’ Río de la Plata 33°52’17’’-34°58’26’’ 54°55’14’’-58°24’47’’ Río Negro 30°49’59’’-33°57’37’’ 54°9’42’’-58°25’7’’ 24 V. de Olivera-López, A. Camargo, R. Maneyro APPENDIX 2 Thirty four mature females (♀) of Limnomedusa macroglossa used in the analyses and their respective morphometric measurements, basin and latitude/longitude from Uruguay. ZVC-B: vertebrate collection of the Faculty of Sciences, University of the Republic. SVL: snout–vent length; MW: mandibular width; HL: head length; IOD: inter-orbital distance; ED: eye diameter; IND: inter-narial distance; END: eye-nostril distance; ARML: arm length; TiL: tibia length; TaL: tarsus length; MtL: metatarsus length. ACRONYM (Adult ♀) SVL MW HL IOD ED IND END ARML TiL TaL MtL Basin Latitude Longitude 12 60.95 23.98 20.47 10.75 7 5.76 5.88 14.2 37.59 18.73 29.81 Río Uruguay -30.1166667 -57.05 24 53.06 21.3 18.27 8.39 5.49 4.77 5.76 13.05 32.81 17.59 30.48 Río Santa Lucía -34.0666667 -56.8833333 127 58.88 23.21 20.53 9.22 5.95 5.06 6.09 14.32 36.41 19.58 30 Río Uruguay -30.75 -56.3333333 132 56.15 21.17 18.02 8.66 6 4.76 5.75 14.34 32.44 17.4 28.55 Río Uruguay -30.75 -56.3333333 133 54.11 20.13 17.73 8.62 5.36 4.29 5.91 12.99 35.53 17.54 32.12 - - - 151 61.97 22.3 20.19 8.93 6.79 5.09 6.36 14.05 36.41 19.83 31.83 Río Negro -32.9166667 -54.9333333 153 56.55 24.12 20.36 10.22 6.62 5.15 5.77 15.14 36.56 19.36 31.83 Laguna Merín -34.2166667 -54.9333333 310 58.85 23.17 20.38 9.49 6.67 5.14 5.91 13.36 34.05 17.71 30.38 Laguna Merín -33.45 -54.5333333 317 55.34 20.79 18.85 9.01 5.83 4.35 6.03 13.09 33.2 18.38 27.91 Río de la Plata -34.5333333 -55.4 495 55.97 22.51 19.37 9.79 6.01 5.34 6.1 14.08 36.71 19.3 30.35 - - - 651 64.25 23.89 21.04 9.01 6.56 5.68 6.35 16.36 37.26 20.03 32.14 Río Santa Lucía -34.5833333 -56.4833333 691 62.54 23.65 21.93 9.78 5.99 4.82 6.66 15.12 37.23 19.27 32.74 Río Santa Lucía -34.3 -55.25 813 61.95 24.23 22.02 9.98 6.23 5.45 7.05 15.07 39.86 20.57 31.31 - - - 826 55.4 21.27 18.16 8.59 6.15 4.98 5.05 12.29 32.81 16.98 28.63 - - - 829 58.22 24.47 20.64 10.6 6.48 4.82 5.92 15.55 36.68 18.22 30.05 Laguna Merín -34.05 -54.7833333 996 59.34 24.34 21.63 10.68 6.72 5.15 6.3 14.11 38.53 18.96 32.66 Río Negro -32.9166667 -54.9333333 1189 50.7 21.48 19.06 9.36 6.24 4.29 5.43 13.72 32.48 16.63 27.55 Río de la Plata -34.55 -55.4 1247 58.21 23.69 18.62 9.3 6.44 4.85 5.93 13.24 34.01 17.62 30.09 Río Negro -31.55 -55.65 1324 62.79 23.58 21.14 10.05 5.96 5.12 6.57 16.46 37.97 20.08 32.85 Río de la Plata -34.8333333 -55.2666667 1414 51.05 19.83 17.55 8.23 5.96 4.54 5.6 13.26 31.57 16.03 27.47 Río Negro -32.5 -55.3166667 1511 53.55 22.15 19.26 8.99 5.44 4.95 6.18 13.66 33.94 16.96 28.9 Río Uruguay -30.95 -57.5333333 1523 54.17 22.39 19.45 9.14 6.29 5.14 6.07 14.3 34.84 18.28 29.76 Río Uruguay -30.9333333 -57.5 23088 51.71 21.63 18.21 9.03 5.44 4.39 5.56 13.14 33.35 18 27.64 Río Negro -31.0825 -55.8555556 23106 57.63 22.3 18.44 9.15 5.95 4.48 5.61 13.8 34.05 17.88 28.54 Río Negro -31.0466667 -55.8477778 23343 63.96 23.13 21.56 10.47 6.38 5.09 5.96 16.09 37.67 19.99 30.24 Río Uruguay -31.34177 -56.66407 23586 52.41 19.76 17.69 8.38 5.58 4.35 5.45 13.23 30.85 16.87 26.24 Río Negro -31.24676 -55.95104 23594 62.97 23.51 20.73 10.93 6.22 5.05 6.05 16.73 38.83 20.27 30.86 Río de la Plata -34.63791 -55.24744 23597 59.13 24.32 19.78 9.62 5.53 4.97 6.18 16.01 38.56 19.73 32.41 Río de la Plata -34.63791 -55.24744 23598 61.58 24.06 20.24 10.03 6.26 4.83 5.61 15.66 39.35 20.65 31.31 Río de la Plata -34.63791 -55.24744 23601 57.31 21.11 20.28 9.98 6.98 5.54 5.91 14.67 34.53 19.02 27.79 Río Uruguay -31.15043 -56.29138 23608 51.35 19.98 17.87 8.53 5.27 4.2 5.51 13.75 31.87 16.84 27.77 Río Negro -31.09436 -55.96907 23609 53.88 21 17.86 9.19 5.64 4.65 5.38 13.48 31.98 17.19 26.15 Río Negro -31.13739 -56.04582 23610 49.82 20.95 17.37 9.41 5.07 4.27 5.1 12.68 30.21 16.54 25.1 Río Negro -31.16724 -55.87382 23611 51.82 19.7 16.95 8.2 5.33 3.69 4.92 11.85 30.39 15.66 26.12 Río Negro -31.09436 -55.96907 25Sexual dimorphism and clinal variation in Limnomedusa macroglossa APPENDIX 3 Forty four mature males (♂) of Limnomedusa macroglossa used in the analyses and their respective morphometric measurements, basin and latitude/longitude form Uruguay. ZVC-B: vertebrate collection of the Faculty of Sciences, University of the Republic. SVL: snout–vent length; MW: mandibular width; HL: head length; IOD: inter-orbital distance; ED: eye diameter; IND: inter-narial distance; END: eye- nostril distance; ARML: arm length; TiL: tibia length; TaL: tarsus length; MtL: metatarsus length. ACRONYM (Adult ♂) SVL MW HL IOD ED IND END ARML TiL TaL MtL Basin Latitude Longitude 90 46.51 17.73 16.14 7.78 4.83 3.92 4.41 11.49 28.95 14.71 23.95 - - - 140 46.71 17.01 15.67 7.22 4.17 3.39 4.38 11.56 28.44 15.38 25.62 Río Uruguay -30.2833333 -57.1833333 329 57.13 22.88 20.3 9.41 6.11 5.22 5.57 13.81 35.57 17.97 31.82 Río Santa Lucía -34.0666667 -56.8833333 357 52.61 21.27 17.88 8.58 6.31 4.96 4.98 13.78 31.25 16.25 29.04 Río Uruguay -33.4666667 -58.4 549 51.73 19.68 18.28 7.85 5.04 3.7 5.52 13.38 32.51 17.24 26.8 Océano Atlántico -34.8166667 -54.9166667 588 42.8 17.04 15.5 7.35 4.68 4.23 4.93 11.34 26.87 15.47 23.72 - - - 908 44.37 19 16.85 7.36 5.35 3.86 4.54 11.68 26.7 13.91 23.7 Río Santa Lucía -34.5833333 -56.4833333 1121 50.87 19.84 17.73 9.15 5.65 4.28 5.3 12.6 31.87 14.64 27.81 - - - 1156 53.16 24.28 20.98 9.15 6.57 5.14 5.6 13.37 35.33 18.7 31.68 Océano Atlántico -34.7333333 -54.9833333 1195 58.65 23.76 19.77 9.82 6.07 5.48 6.33 15.62 37.72 17.12 31.93 Río Uruguay -33.85 -57.7333333 1245 43.22 16.99 14.73 7.99 5.56 4.25 5.07 11.38 27.78 14.87 24.29 Laguna Merín -33.1 -54.7 1342 45.21 18.21 16.38 7.88 4.88 4.01 5.1 11.89 29.07 15.27 26.89 Río Uruguay -30.9333333 -57.5 2120 60.92 22.81 19.61 9.82 7.2 5.24 5.86 13.81 36.88 18.72 30.62 Río de la Plata -34.8666667 -56.3666667 2124 56.47 22.36 18.85 9.45 6.55 5.14 5.58 13.17 33.2 18.02 30.36 Río de la Plata -34.8666667 -56.3666667 2853 44.19 17.68 14.71 7.48 5.03 3.47 4.04 10.51 24.7 13.57 21.96 Río Negro -31.1166667 -55.9833333 3013 47.1 18.95 16.45 7.9 5.27 3.84 4.11 12.36 28.96 14.88 24.44 Río Uruguay -31.8166667 -56.4166667 3444 52.17 20.43 17.24 9 6.06 4.66 5.19 12.08 31.68 16.82 27.14 - - - 3456 57.54 22.25 18.64 9.25 6.38 4.66 5.49 13.94 34.61 17.21 29.78 Río de la Plata -34.3333333 -57 4902 49.53 20.28 17.77 8.62 5.22 4.42 5.21 13.91 32.35 17.93 27.04 - - - 10254 51.5 21.19 17.31 8.98 5.5 4.29 5.55 12.59 32.01 16.81 28.36 - - - 10278 54.01 21.24 18.81 9.02 5.61 5.09 6.08 13.8 33.59 16.78 27.94 - - - 10279 53.21 20.67 18.29 9.78 5.9 5.05 5.99 13.94 34.98 18.38 28.82 - - - 10845 55.79 21.99 17.15 8.96 5.71 4.39 4.97 13.66 34.23 17.17 30.09 - - - 23105 45.68 18.31 16.37 7.78 5 3.56 4.18 10.8 27.58 15.04 25.23 Río Negro -31.0466667 -55.8477778 23341 46.66 18.33 15.81 7.43 5.27 4.06 4.52 10.82 26.64 13.76 23.12 Río Uruguay -31.60659 -56.43186 23358 48.55 18.87 15.91 7.86 5.54 3.81 4.64 12.15 29.37 15.63 25.83 Río de la Plata -34.471741 -55.529168 23583 49 18.48 16.49 8.44 5.11 4.07 5.08 12.6 29.33 15.42 27.6 Río Negro -31.18738 -55.9483 23587 52.19 19.95 18.14 8.46 5.89 3.93 4.99 13.95 32.14 17.52 27.27 Río Negro -31.23137 -56.09116 23588 49.49 19.95 16.74 8.94 5.38 4.16 5.03 12.56 29.73 16.2 27.03 Río Negro -31.3044 -56.05855 23589 48.9 19.61 17.14 8.4 5.69 3.83 4.64 12.12 29.64 15.61 25.81 Río Negro -31.30467 -56.05854 23590 41.29 17.28 14.47 7.43 4.26 3.26 4.16 11.2 25.46 14.29 22.07 Río Negro -31.32815 -56.17757 23591 50.04 18.75 17.04 8.95 4.64 4.11 5.11 11.92 28.91 15.88 25.02 Río Negro -31.69442 -56.12402 23592 47.05 17.24 15.46 7.54 5.48 3.57 4.01 10.48 28.59 14.85 25.16 Río Santa Lucía -34.28159 -55.27949 23593 52.11 20.85 17.79 9.52 6.91 4.52 4.83 13.22 33.38 17.23 28.57 Río Santa Lucía -34.28159 -55.27949 23595 48.21 20.16 16.79 8.05 4.98 4.21 5.17 12.63 30.95 16.81 25.31 Río de la Plata -34.63791 -55.24744 23596 51.53 19.58 17.17 8.37 4.94 4.25 5.19 12.33 31.21 16.74 25.11 Río de la Plata -34.63791 -55.24744 23599 52.37 20 17.48 8.41 5.65 4.56 5.41 13.77 32.47 17.47 26.21 Río Uruguay -30.67911 -56.51333 23600 55.14 21.15 19.55 9.06 6.29 4.45 5.37 14.37 33.82 18.33 27.39 Río Uruguay -30.67911 -56.51333 23602 45 19.81 17.11 8.35 4.91 3.84 4.94 10.97 28.12 14.49 24.95 Río Negro -31.19047 -55.90129 23603 48.79 18.62 16.56 8.13 5 3.75 5.12 12.3 29.26 15.55 26.65 Río Negro -31.19068 -55.90163 23604 48.13 19.05 16.68 8.06 4.94 3.96 5.08 11.45 29.42 15.87 25.33 Río Negro -31.19068 -55.90163 23605 44.46 17.86 16.14 7.79 4.96 3.74 4.52 11.45 26.73 13.78 22.39 Río Negro -31.18738 -55.9483 23606 43.54 16.61 15.16 7.1 4.72 3.63 4.31 11.53 25.93 14.24 22.67 Río Negro -31.18738 -55.9483 23607 43.11 17.04 14.79 7.05 4.66 3.81 4.6 9.93 25.46 13.86 21.82 Río Negro -31.13739 -56.04582