Acta Herpetologica 18(1): 11-22, 2023 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-11995 Age estimation and body size of the Parsley Frog, Pelodytes caucasicus Boulenger, 1896 from Lake Borçka Karagöl, Turkey Cantekin Dursun*, Serkan Gül, Nurhayat Özdemir Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, Rize, Turkey *Corresponding author. Email: cantekin.dursun@erdogan.edu.tr Submitted on: 2021, 31st August; revised on: 2022, 8th April; accepted on: 2022, 13th November Editor: Raoul Manenti Abstract. In this study, we described age structure, body size, body mass and the relationships among these param- eters for a population of P. caucasicus from Lake Borçka Karagöl, Artvin, Turkey. The mean SVL with standard error was 45.87 mm ± 0.55 (range: 39.98-50.28 mm) and the mean weight with standard error 8.81 g ± 0.39 (range: 6.10-11.47 g) in females whereas 48.16 mm ± 0.45 (range: 43.64-54.78 mm) and 11.32 ± 0.25 (range: 9.56-14.80 g) in males, respectively. We found a significant male-biased difference reflecting sexual dimorphism and statisti- cally significant positive relationships between these variables. According to the results, the age ranged between 2-5 years in females and 2-6 years in males. The mean age distributions significantly differed between the sexes (females: 3.28 years ± 0.19; males: 3.94 years ± 0.20). The mean ages and maximal ages were found identical to the previously reported results from Turkey, but the mean ages were higher than in Georgian populations. Von Bertalanffy growth models demonstrated similar curves, and the growth rate was faster up to 3 years in both sexes. To conclude, this study was the first to determine age structure and growth patterns in Borçka Karagöl population and weight data for P. caucasicus was presented for the first time in the literature. Keywords. Amphibia, skeletochronology, weight, Von Bertalanffy, growth rate. INTRODUCTION The determination of individual age is essential for studies like demographic and life history, including devel- opmental biology and population dynamics of a species. This provides the researchers basic ecological data related to population structure such as sexual maturity time, the age structure, growth rate, and life span. Moreover, this is a parameter essential to infer the life history traits of a species and to compare it to other species (de Buffrénil et al., 2021; Ma et al., 2022). In this context, skeletochro- nology is an effective and reliable method for estimating the growth and age of many amphibian species as the growth of amphibians is not independent of environmen- tal conditions (Guarino et al., 2019; Üzüm et al., 2020). In addition, the method can be also applied to animals such as mammals (Nacarino-Meneses et al., 2016), lizards (Beşer et al., 2019), turtles (Guarino et al., 2020), and even fossils (de Buffrénil et al., 2021). Skeletochronology calculates the lines involving the formation of calcium carbonate, called “annual rings” or “growth markers” in bone tissues (Castanet, 1994; Ma et al., 2022). For this, the diaphyseal region of their long bones, which show weaker vascularity, provides the best result for calculat- ing the age of individuals (Castanet et al., 1993). Rozen- blut and Ogielska (2005) showed that lines of arrested growth (LAGs) are most complete in the middle part of the phalangeal diaphysis in European water frogs and thus pointed out that the middle part of the long bone is optimal for age studies. Moreover, the skeletochronol- ogy helps to calculate the lifespan of the population in amphibians, explain the sexual size dimorphism, and 12 Cantekin Dursun, Serkan Gül, Nurhayat Özdemir reveal the differences between the sexes in terms of age and size. Also, the knowledge based on the skeletochro- nological studies tends to show population dynamics (Peng et al., 2021). Pelodytes caucasicus Boulenger, 1896, the Caucasian parsley frog, is a native species of the Caucasus fauna. The species is distributed throughout northwest Azer- baijan, Georgia (southwest and South Ossetia), Russia (Krasnodar district), and Turkey (Blacksea region) (Zaza- nashvili et al., 2012; Litvinchuk and Kidov, 2018; Çiçek et al., 2019). The species is considered as near threatened because of natural and anthropogenic pressures (Anan- jeva et al., 2009; Iskanderov, 2009; Kaya et al. 2009), so it is recommended that public campaigns should be conducted to raise the awareness for this endemic relict species in the border of Georgia and Turkey (Tarkhnish- vili and Kaya, 2009). From this aspect, it is important to reveal the population dynamics of P. caucasicus in a new population based on skeletochronology. Although the age structure of P. caucasicus was reported by two studies in Georgia (Gokhelasvili and Tarkhnisvili, 1994; Chubinish- vili et al., 1995), there is only single study in the border of Turkey (Erişmiş et al., 2009). Given the importance of skeletochronological information to better understand the population dynamics, ecological and evolutional pro- cesses, we aimed to present the age structure of the Bor- çka Karagöl population for the first time and compare the results with the previous studies. We also provided weight data for the first time in this species, as well as assessed the relationship between this trait and SVL for age and growth rate. MATERIAL AND METHODS Fieldwork We sampled 50 specimens (18 ♀♀, 32 ♂♂) during the breeding season (2013) from Lake Borçka Karagöl territory, in the vicinity of Artvin, Turkey (Fig. 1). The collected frogs were anesthetized with MS222. Snout‐ vent length (SVL) was measured at the nearest 0.01 mm using a digital calliper and the weight was noted using the nearest 0.01 g an electronic balance for each sample. To count growth lines, the longest toe of the left hindlimb was clipped and preserved in 70% ethanol. After the sam- pling procedure, all frogs were released at the site of cap- ture. Sex determination was possible due to the presence of secondary sexual characters, such as nuptial pads on the forearm, and presence of vocal sacs in males. The standard skeletochronology procedure (Castanet and Smirina, 1990) was applied to calculate the number of the lines of arrested growth (LAGs). After removing soft tissues, the phalanges were washed in tap water, then decalcified in 5% nitric acid for 2 hours. Lastly, the sam- ples were washed in distilled water overnight to ensure the removal of nitric acid. The 18 μm cross-sections were obtained from the diaphyseal part of each phalanx using a cryostat (Thermo Shandon Cryotome, Germany) and stained with Ehrlich Haematoxylin approximately for 15 minutes. The stained cross-sections were treated with glycerol to control under a light microscope. The images of selected sections were acquired using Olympus BX51 microscope with an integrated camera to estimate the precise age of each specimen. The number of LAGs was counted by two researchers (S. Gül and N. Özdemir). The distance between two adjacent LAGs is a well-known indicator demonstrating individual growth in a given year (Kleinenberg and Smirina, 1969; Kurnaz et al. 2018). Following previous studies (Özdemir et al., 2012; Üzüm et al., 2014), the endosteal resorption was evaluated by comparing the diameters of eroded marrow cavities with the diameters of non-eroded marrow cavities in sections from the youngest specimens. Statistical analyses To summarize data and present basic features of the measurements we calculated descriptive statistics using the psych package (Revelle, 2019). The normality assump- tion of the variables was checked using the Kolmogorov- Smirnov test. Since the variables followed a normal dis- tribution (P > 0.05), we used parametric tests in down- stream analyses. The homogeneity of variances was com- pared using Levene’s Test in the car (Fox and Weisberg, 2019) package. We utilized the student t-test to compare the mean differences of the variables between males and Fig. 1. Lake Borçka Karagöl and surrounding region. 13Age structure and body size of Pelodytes caucasicus females. Thereafter, Pearson’s product-moment correla- tion test was used to estimate the relationships among SVL, weight, and age. The relationship of SVL and weight was analysed using a linear regression model. Additionally, we used an ANCOVA test to explore the patterns of SVL and weight between sexes, with age as the covariate. Thereafter, post- hoc tests were applied using the emmeans package (Lenth, 2021) under Bonferroni correction (estimated marginal means: aka least-square means or adjusted means). We estimated growth curve models under the typi- cal Von Bertalanffy’s equation modified by Beverton and Holt (1957): Lt=L∞{1-exp[-k(t-t0)]} where Lt is the expected or average length at the time (or age) t, L∞ is the asymptotic average length, k is the so-called Bro- dy growth rate coefficient and t0 is a modelling artifact that is said to represent the time or age when the aver- age length was zero. To estimate parameter values and run the analyses FSA (Ogle et al., 2021), FSAdata (Ogle, 2019), FSAsim (Ogle, 2020) and nlstools (Baty et al., 2015) packages were used following the guide “fishR Vignette” prepared by Derek Ogle (2013). To visualize growth curve, we also added a hypothetical individual to the dataset by the reference of Erişmiş et al. (2009) under the presented parameters: SVL0 at metamorphosis is fixed to mean 20.15 and t0 (age at metamorphosis) is 0.3 year. Statistical analyses were carried out using the stats package. Data visualization was performed using ggplot2 (Wickham, 2016), ggpubr (Kassambara, 2020), and ggally (Schloerke et al., 2021) packages. All analyses were run in R Programming Language (R Core Team, 2020). RESULTS We successfully aged 50 individuals using skele- tochronology technique. Ages ranged between 2-5 years in females, and 2-6 years in males. Descriptive statistics of both sexes and age groups are presented in Table 1. The variances were found homogenous for SVL (F1,48 = 0.2926, P = 0.5911), weight (F1,48 = 1.5006, P = 0.2266) and age (F1,48 = 1.9470, P = 0.1693). According to the Student t-test results, statistically significant differences were found between sexes in all variables (Fig. 2). Males were signifi- cantly heavier (t = 5.6509, df = 48, P < 0.001) and larger (t = 3.1545, df = 48, P < 0.01) than females. The mean age was also found male-biased (t = 2.2053, df = 48, P < 0.05). A significantly positive correlation was found between SVL and age, SVL and weight for both sexes, but the correlation between weight and age was significant only in males. The correlation coefficients and p-values are presented in Fig. 3. The constructed linear regression model following the Table 1. The summary table of descriptive statistics based on the dataset. Variable Females Males N Mean ± SE Min Max N Mean ± SE Min Max All Specimens SVL (mm) 18 45.87 ± 0.55 39.98 50.28 32 48.16 ± 0.45 43.64 54.78 Weight (g) 18 8.81 ± 0.39 6.10 11.47 32 11.32 ± 0.25 9.56 14.80 Age (years) 18 3.28 ± 0.19 2.00 5.00 32 3.94 ± 0.20 2.00 6.00 2 Years Specimens SVL (mm) 3 43.54 ± 1.83 39.98 46.09 3 44.58 ± 0.49 43.64 45.26 Weight (g) 3 7.22 ± 0.55 6.18 8.05 3 10.38 ± 0.22 10.11 10.81 3 Years Specimens SVL (mm) 8 46.07 ± 0.71 43.42 49.26 9 47.18 ± 0.59 44.27 49.72 Weight (g) 8 9.55 ± 0.62 6.10 11.47 9 11.12 ± 0.37 10.05 13.28 4 Years Specimens SVL (mm) 6 46.02 ± 0.51 44.20 48.00 9 47.81 ± 0.60 45.08 51.12 Weight (g) 6 8.44 ± 0.53 6.70 10.05 9 10.72 ± 0.41 9.56 13.63 5 Years Specimens SVL (mm) 1 50.28 50.28 50.28 9 49.37 ± 0.47 47.13 51.95 Weight (g) 1 9.97 9.97 9.97 9 11.68 ± 0.38 10.48 13.90 6 Years Specimens SVL (mm) - - - - 2 54.08 ± 0.71 53.37 54.78 Weight (g) - - - - 2 14.65 ± 0.15 14.50 14.80 14 Cantekin Dursun, Serkan Gül, Nurhayat Özdemir correlations also presented significant equations between SVL and weight (males: F1,30 = 42.54, R2 = 0.59, P < 0.001; females: F1,16 = 11.23, R2 = 0.41, P < 0.01). According to the ANCOVA results, the effect of age on the intersexual differences was found significant in SVL (F1,47 = 39.8215, P < 0.001) and weight (F1,47 = 8.6144, P < 0.01). Post-hoc test indicated that the mean SVL score, with age as a covariate, was significantly dif- ferent between sexes (males: 47.8 ± 0.33; females: 46.6 ± 0.44; P < 0.05). Additionally, a statistically significant dif- ference was found in terms of the weight between both sex with the age covariate (males: 11.2 ± 0.25; females: 9.06 ± 0.34; P < 0.001). Growth curves estimated by Von Bertalanffy’s model fit adequately described the relationship between age and SVL, and the curves indicated similar shapes in both sex- es (Fig. 4). The final models were found statistically sig- nificant for all parameters (P < 0.01). According to the constructed models, the estimated asymptotic SVL was not higher than maximum recorded SVL values (Males: 54.78 mm; Females: 50.28 mm). The growth parameters were presented in Table 2. DISCUSSION The sexual size dimorphism (SSD) is an observ- able characteristic in animals, and it is known that the direction of size dimorphism in most of the amphib- ians is female biased (Kupfer, 2007). Shine (1979) noted that females are larger than males in 61% of urodeles and 90% of anurans. The female biased SSD is generally explained by the fecundity advantage hypothesis (Shine, 1989; Andersson, 1994) when the selection is supporting large females due to the larger energy storage capacity for reproduction and more offspring production. How- ever, a male biased SSD is relevant to the dominance in contests of strength, the extent of endurance, mate choice and higher sperm competition success in animal king- dom (Hudson and Fu, 2013). The male-biased SSD cor- responding to 10% amphibian species is especially associ- ated with territoriality behaviours and male-male compe- titions (Nali et al., 2014). The family Pelodytidae is including five different spe- cies from the single genus Pelodytes distributing in West- ern Europe especially in the Iberian Peninsula, Cauca- sia, and north-eastern Turkey (Amphibiaweb, 2022). The previous studies have reported the female biased sexual size dimorphism in Pelodtytes species inhabiting in Ibe- ria (Talavera, 1990; Escoriza, 2017). For instance, Este- ban et al. (2004) noted the larger average body length in females (43.31 mm) than males (36.32) mm in a north- ern Spain population of Pelodytes punctatus species. Diaz-Rodrigez et al. (2017) have described the presence of four valid species inhabiting along Western Europe by integrating molecular and morphological data. Regard- Fig. 2. The boxplots are representing the differences between the sexes (A: SVL, B: weight, C: age). The associated p-values were shown on the relevant plots. 15Age structure and body size of Pelodytes caucasicus ing their morphological measurements, all the species (P. punctatus, P. hespericus, P. ibericus and P. atlanticus) were characterized by larger average body length in females. However, our results revealed that unlike Iberian species, P. caucasicus males have a larger and heavier body than females. Our results are also consistent with the findings presented in previous studies. For example, Erişmiş et al. (2009) have comprehensively assessed the age struc- ture and growth in Pelodytes caucasicus from Uzungöl, Turkey and. The mean SVL of adult males with SD were reported as 47.16 ± 2.87 mm (n = 44; range 41.48- 52.58 mm), and significantly smaller in females (45.79 ± 2.29 mm; n = 31; range 40.28-50.62 mm). Arıkan et al. (2007) noted the range of SVL in sexually mature males between 45.06-52.08 mm, and 46.70-49.62 mm in mature females in Uzungöl population. Yildirimhan et al. (2009) also recorded the mean SVL 50.6 ± 3 mm (range: 43-57 mm) in the population of Çaykara, Trabzon including 47 males, 7 females. Erişmiş et al. (2009) emphasized that the bigger size of older males in their study may deviate the results linked to SSD, but the size differences could also be derived from the biotic or abiotic selective pres- sures in poikilotherm animals. They validated their find- ings based on the former studies conducted in climati- cally different regions in Caucasia (Gokhelashvili and Tarkhnishvili, 1994; Chubinishvili et al., 1995) and they suggested male biased SSD is the species characteristic of P. caucasicus. The common SSD pattern in anura is gen- erally female biased (Monnet and Cherry, 2002). Recent- ly, Pincheria-Donoso et al. (2021) have investigated the SSD of amphibian species in global scale. As a result, they observed 90.8% female biased SSD 7.5% male-biased SSD Fig. 3. The matrix is demonstrating the correlation coefficients, scatterplots, and density plots of binary variables. Corr values indicate the correlation coefficients (r). The significance level of correlation coefficients was represented with asterisk (*, P < 0.05; **, P < 0.01; ***, P < 0.001). 16 Cantekin Dursun, Serkan Gül, Nurhayat Özdemir and 1.7% monomorphic in anurans. However, Han and Fu (2013) determined male biased SDD in 19 different anuran families distributing in six distinct continents and along tropical and temperate habitats. Moreover, con- strained habitats and microhabitats can affect the female size and fecundity, and it can be resulted with more simi- lar body size of both sexes as observed in Hylid frogs (Silva et al., 2020). From this aspect, it can be suggested that the directional difference of SSD between the Ibe- rian species and Caucasian P. caucasicus may cause due to different ecological preferences in response to their habitats. The Iberian Peninsula which has complex oro- graphic structure is surrounded by Atlantic Ocean and Mediterranean Sea. The Mediterranean coast and sur- rounding areas show dry and warm/hot summers, and mild and wet winters. The Atlantic coasts are character- ized by oceanic type of climate, milder but humid winters and cooler/wetter summers, without large temperature variations. Lastly, the inner areas which is represented by continental climate type have hot and dry summers, Fig. 4. Graphical visualization of Von Bertalanffy’s growth curves and parameter optimization histograms. Dotted lines are representing 95% confidence intervals of fitted curves (A: Females; B: Males). Table 2. The constructed final models of Von Bertalanffy’s growth curves and associated parameters Estimated parameters L∞ CI K CI t0 Females 47.32 45.93-49.77 0.98 0.48-1.47 -0.56 Males 50.38 49.16-52.49 0.73 0.58-0.91 -0.70 17Age structure and body size of Pelodytes caucasicus and cold and humid winters with large-scale precipita- tion, but also semi-arid areas extremely low precipitation and very hot temperatures especially in summer season (Carvalho et al., 2021) which are corresponding to the distribution of Iberian Pelodytes taxa (see Diaz Rodrigez et al., 2017). However, Caucasus Ecoregion has quietly high mean annual rainfall in the southwestern part over 2000 mm in the coastal area of the Black Sea. The mean annual temperature in the South Caucasus part of the Black Sea coast around 15 centigrade degree (Zazanash- vili, 2009). Besides, the distribution of P. caucasicus spe- cies is restricted to the humid subtropics in Caucasia and Turkey with Colchic vegetation type (Tarkhnishvili, 1996; Iskanderov, 2009; Beşir and Gül, 2019). Gül (2014) also noted that the species prefers wet and warm microhabi- tats in Turkey. Additionally, the amount of precipitation is known as one of the most important factors construct- ing the distribution of P. caucasicus (Lukina and Koneva, 1996; Litvinchuk and Kidov, 2018). Pincheria-Donoso et al. (2021) proposed that the underlying impact of geo- graphical variation in climatic pressures can shape large- scale patterns of SSD in synergy with natural and sexual selection such as intensification of fecundity selection to shorten breeding season in anurans. They also implied that the different selection forms can shaped by macro- ecological factors climate, geographical gradients and temperature seasonality which are triggering the evolu- tion of life-history traits associated with fecundity. There- fore, we suggested that the ecological preferences of P. caucasicus are potential reason causing male-biased SSD comparing to the representatives of Iberian Peninsula. Radojićić et al. (2002) also revealed similar SSD pattern in the Bombina species. They noted that Bombina bom- bina showed male-biased SSD while the larger body size of females was observed in B. variegata subspecies, and the differences were associated with reproductive behav- iours and possible ecological differences. This pattern can also be observed in Pelobates syriacus (Bülbül et al., 2020 and references therein), Rana arvalis populations (Glandt and Jehle, 2008) and Rana nigrovittata which has simi- lar ecological needs with P. caucasicus such as streams in shaded forest environments (Khonsue et al., 2000). On the other hand, the male-male competition was reported in Pelodytes species (Pargana, 2003; Marquez et al., 2004 and unreported mating ball of P. caucasicus observed by S. Gül). Therefore, it should be also taken into considera- tion that sexual selection which maximize the fitness in reproductive traits may be an alternative force to describe the SSD pattern in our study as reported in different anu- ran species e.g., Paa spinosa (Gen-Yu et al., 2010) and Hypsiboas atlanticus (Camurugi and Juncá, 2013). SSD can be driven by life-history traits, so the accuracy of the age determination is critical to estimate these characteris- tics. The post-metamorphic terrestrial growth is a major part of total growth (over 90%) in amphibians (Werner, 1986) and the variation in terrestrial growth rates and age at maturity is playing an important role in the inter- sexual adult size variability (Marangoni et al., 2012). Fur- thermore, the age determination of amphibians yields crucial information on the demographic features such as size at sexual maturity growth, longevity, and growth rate (Duellman and Trueb, 1994; Otero et al., 2017; Baraquet et al., 2021). In this study, we found that the age is ranged between 2-6 years. The mean age is 3.28 years in females and 3.94 years in males. Our constructed Von Berta- lanffy’s growth curves adequately fitted age/SVL rela- tionship. The models demonstrated similar curve shape for both sexes, but the growth coefficient was higher in females. Previously, Gokhelasvili and Tarkhnisvili (1994) stud- ied the age distribution of the reproductive population of P. caucasicus in Borjomi canyon during two consecutive years (1992-1993). According to their results, the mean ages were 2.96 and 2.74 for males, 2.74 and 3 for females, respectively. They also reported the dominancy of young specimens in the reproductive portion of the populations and a very-high annual mortality rate index (0.78-0.83). The males reached maximum of 6 years, and 4 years in females, which is in accordance with what Gokhelasvili and Tarkhnisvili (1994) reported. In the following year, Chubinishvili et al. (1995) studied certain aspects of the population ecology of P. caucasicus. They reported sexual maturity between 2-3 years. Contrary to their findings, the mean age is found approximately one year above the one measured in the Borçka Karagöl population. Erişmiş et al. (2009) reported the mean age of males and females 3.61 ± 0.9 years and 3.03 ± 0.7 years, respec- tively and the age structure difference is statistically sig- nificant (P < 0.05). The oldest male was 5 years while the female was 4 years. The sexual maturity is reached at two years of age. We also obtained approximate val- ues from the mean SVL and age (see table 1). The differ- ences observed in SVL and age between sexes were also statistically significant. Given the identical results for Uzungöl and Borçka Karagöl populations, we can assume that there is a separation between Georgian and Turkish populations related to age structure. Erişmiş et al (2009) also calculated a survival rate at 0.78 in males and 0.76 in females, and these figures can be taken as a reference to explain the high mortality rate noted by Gokhelas- vili and Tarkhnisvili (1994). Based on the literature, the maximum age reported in the genus Pelodytes is 10 years for females and 8 years for males in Pelodytes puncta- tus species (Esteban et al., 2004). Additionally, the mean 18 Cantekin Dursun, Serkan Gül, Nurhayat Özdemir age (Burgos: 1.71 years ± 1.41 years, Valencia: 3.82 years ± 1.22 years) and mean SVL (Burgos: 34.36 mm ± 2.22 mm, Valencia: 36.41 mm ± 2.50 mm) of males in the study of Esteban et al. (2002) were lower than our mean values. The authors noted that males were aged between 1-6 years old, and the age structure was highly skewed corresponding 50% of the sample of males being 1 year old. This situation can cause uncertainties when age/SVL relationships in the genus Pelodtyes. In this study, we also did not age any individual in 1 year old from Lake Karagöl population. However, it is known that the low average age and high proportion of younger individuals might be associated with rapid decrease of local popula- tion of the species such as Rana porosa (Misawa et al., 2002) and Esteban et al. (2002) pointed that this pattern is more relevant to conservation status due to anthropo- genic pressures in the Burgos population. According to the growth curves estimation of Erişmiş et al. (2009), asymptotic SVL and growth coefficient were very similar between the sexes (Males: SVLmax: 53.42 mm ± 1.01 mm; K = 0.42 ± 0.03; Females: SVLmax = 52.04 mm ± 0.75 mm; K = 0.38 ± 0.01). In both sexes, the estimated asymptotic SVL was slightly higher than the maximum SVL record and the age-specific growth rate under the 3 years reported faster than higher ages. On the contrary, the estimated SVLmax values were lower, but the estimated K values were higher in our constructed growth model. From this aspect, we think that the more bell-shaped curve of our models is likely due to lack of the data from one-year individuals, and this may affect parameter fitting. The second reason is likely the pre- ferred formula equation differences. The common point of the models is a remarkable decrease of growth rate after 3 years following sexual maturity in both sexes. Data on body mass is limited in amphibians, but Santini et al. (2018) emphasized that weight data can be used to assess ecological and evolutionary processes such as dispersal distance, reproduction, population abun- dance and energy intake and it was also highly correlated with SVL in various anuran families (Bufonidae, Hylidae, Myobatrachidae, Ranidae). Moreover, they said that rapid body mass rise is associated with SVL rise for terrestrial and semi-aquatic species because allometric relationships between length and mass vary in amphibian species based relevant to the different habitat preferences. Lastly, they suggested the geometric similarity hypothesis (Hill, 1950) is fitting these assumptions better because if two organ- isms are geometrically similar their linear dimensions can be made equal by multiplying those of one of them by a constant. In this study, we contributed to the literature by first recorded weight data of P. caucasicus species. In the genus Pelodytes the available weight data was presented by Esteban et al. (2002) for male P. punctatus specimens (Burgos: 2.36 g ± 0.28 g, Valencia: 3.71 g ± 0.58 g). Con- trary to the mean weight in our male specimens, Span- ish populations have lower values in parallel with mean SVL and age differences. The linear regression model and ANCOVA results also indicated the allometric relation- ship between SVL and weight, and the weight differed both between sexes and among age groups. Habitat pref- erence of P. caucasicus are terrestrial and freshwater sys- tems (Kaya et al., 2009), thus our findings are suggesting the geometric similarity hypothesis and habitat preference effect proposed by Santini et al. (2018) to describe the relationship between SVL and weight. In addition, Xiong et al. (2020) noted that age is affecting the intersexual dif- ference on the weight for the Shangcheng Stout Salaman- der Pachyhynobius shangchengensis. From this aspect, the synchronized rise of SVL with aging may contribute to the intersexual difference associated with weight in P. cauca- sicus species. Camurugi and Juncá (2013) said that male- biased sexual size dimorphism corresponding to length and weight is unusual pattern and among frogs and even if it is generally described by male-male competition, the reasons are not fully understood yet. They also described the male-biased SSD as synapomorphyc characteristic in Hypsiboas punctatus species group. Monnet and Cherry (2002) indicated that Bufo achalensis, Rana cascadae, R. nigrovittata species showed male-biased SSD because males were older sex as we determined that the mean age of P. caucasicus males. This pattern is causing due to delayed maturity which is determinant factor when larg- er body is contributing to mating success and males are not accelerating the growth to breed earlier than females. Tarkhnishvili (1993) said that the lower fecundity, smaller eggs, the volume of clutch and smaller body are the differ- ences in spawning mood associated with female body size, and they are causing to rapid maturation for the species including Pelodytes caucasicus as a strategy to shorten the period between generations and to increase the number of adult individuals. Therefore, we assume that the male biases SSD observed in weight data may also occur in response to the life-history and reproductive traits. To sum up, we determined age structure and growth patterns in Borçka Karagöl population. Future studies can comprehensively investigate the relationship between these variables and ecological conditions, life-history traits, and reproductive characteristics. ACKNOWLEDGEMENTS The study was done with the permission of Recep Tayyip Erdogan University Local Ethics Committee for 19Age structure and body size of Pelodytes caucasicus Animal experiments (2018/1). We also thank Dr. Tuğba Ergül Kalaycı for assistance during the laboratory experi- ments. REFERENCES Afsar, M., Afsar, B., Ayaz, D., Çiçek, K., Tok, C. (2018): Borçka-Karagöl Tabiat Parkı (Artvin/Türkiye) ve Çevresinin Kurbağaları. Jist, 8: 49-55. AmphibiaWeb (2022): Information on amphibian biology and conservation. [web application]. Berkeley, Cali- fornia: AmphibiaWeb. Available: https://amphibiaweb. org/. [Accessed on 22 April 2022]. Ananjeva, N.B., Tuniyev, B.S., Orlov, N.L. (2009): Reas- sessment of the IUCN Red List for Amphibians and Reptiles of the Caucasus. In: Status and Protection 24 of Globally Threatened Species in the Caucasus, pp. 131-136. Zazanashvili, N., Mallon, D., Eds, CEPF, WWF. Contour Ltd, Tbilisi. Andersson M (1994): Sexual Selection. Princeton Univer- sity Press, Princeton. Arıkan, H., Tosunoğlu, M., Atatür, M.K., Göçmen, B. (2007): Some Comments of the Breeding Biology of Pelodytes caucasicus Boulenger, 1896 (Anura: Pelodyt- idae) from Uzungöl, Northeast Anatolia. Turk. J. Zool. 31: 53-64. Baran, İ., Tosunoğlu, M., Kaya, U., Kumlutaş, Y. (1997): Çamlıhemşin Rize civarının Herpetofaunası hakkında. Doğa Türk Zooloji Dergisi. 21: 409-416. Baraquet, M., Pollo, F.E., Otero, M.A., Grenat, P.R., Salas, N.E., Martino, A.L. (2021): Body size, age and growth in males populations of Boana pulchella (Anura, Hyli- dae). An. Acad. Bras. Cienc. 93(suppl 4): e20200991. Baty, F., Ritz, C., Charles, S., Brutsche, M., Flandrois, J.P., Delignette-Muller, M.L. (2015): A Toolbox for Non- linear Regression in R: The Package nlstools. J. Stat. Softw. 66: 1-21. Beşer, N., Ilgaz, Ç., Kumlutaş, Y., Avcı, A., Candan, K., Üzüm, N. (2019): Age structure and body size of a critically endangered species, Acanthodactylus har- ranensis (Squamata: Lacertidae) and its demography. Anim. Biol. 69: 421-431. Beşir, İ.H., Gül, S. (2019): Mapping of distribution of amphibian species in Rize Province, Turkey. Herpetol. Notes 12: 45-51. Beverton, R.J.H., Holt, S.J. (1957): On the dynamics of exploited fish populations. Fisheries Investigations, United Kingdom Ministry of Agriculture and Fisher- ies. Bülbül, U., Koç, H., Odabaş, Y., Eroğlu, A. Y., Kurnaz, M., & Kutrup, B. (2020). Life history traits in a population of Pelobates syriacus (Boettger, 1889) from Turkey. Russ. J. Herpetol. 27: 195-200. Camurugi, F., Juncá, F. (2013): Reproductive biology of Hypsiboas atlanticus (Anura: Hylidae). Herpetol. Notes 6: 489-495. Carvalho, D., Cardoso Pereira, S., Rocha, A. (2021): Future surface temperature changes for the Iberian Peninsula according to EURO-CORDEX climate pro- jections. Clim. Dyn. 56: 123-138. Castanet, J., Smirina, E. (1990): Introduction to the skel- etochronological method in amphibians and reptiles. Ann. Sci. Nat. Zool. 11: 191-196. Castanet, J., Francillon-Vieillot, H., Meunier, F.J., de Ricqlès, A. (1993): Bone and individual aging. Bone 7: 245-283. Caucasus Nature Fund (2020): https://www.caucasus- naturefund.org/ecoregion/caucasus-hotspot. [Accessed on 31 Aug 2021]. Chubinishvili, T., Gokhelashvili, R.K., Tarkhnishvili, D.N. (1995): Population ecology of the Caucasian parsley frog (Pelodytes caucasicus Boulenger) in the Borjomi Canyon. Russ J. Herpetol. 2: 79-86. Çiçek, K., Yakın, B.Y., Afsar, M., Ayaz, D., Tok, C.V. (2019): Some records of Caucasian Parsley Frog and Caucasian Salamander from Eastern Blacksea Region, Turkey. Acta Biologica Turcica 32: 37-41. de Buffrénil, V, Quilhac, A, Castanet, J. (2021): Cyclical growth and skeletochronology. In: Vertebrate skeletal histology and paleohistology, pp. 626-644. de Buffré- nil, V., de Ricqlès, A.J., Zylberberg, L., Padian, K., Eds, CRC Press, Boca Raton. Diaz-Rodriguez, J., Gehara, M., Marquez, R., Vences, M., Goncalves, H., Sequeira, F., Solano, I.M, Tejedo, M. (2017): Integration of molecular, bioacoustical and morphological data reveals two new cryptic species of Pelodytes (Anura, Pelodytidae) from the Iberian Pen- insula. Zootaxa. 4243: 1-41. Duellman, W. E., Trueb, L. (1994): Biology of amphib- ians. Johns Hopkins University Press, Baltimore, Lon- don. Erişmiş, U.G., Arıkan, H., Konuk, M., Guarino, F.M. (2010): Age structure and growth in caucasian parsley frog Pelodytes caucasicus (Boulenger, 1896) from Tur- key. Russ. J. Herpetol. 16: 19-26. Escoriza, D. (2017): Sapillo moteado septentrional-Pelo- dytes punctatus (Daudin, 1802). In: Enciclopedia Vir- tual de los Vertebrados Españoles. López, P., Martín, J., Martínez-Solano, I., Eds, Museo Nacional de Cien- cias Naturales, Madrid. Esteban, M., Sánchez-Herráiz, M.J., Barbadillo, L.J., Cas- tanet, J. (2004): Age structure and growth in an iso- lated population of Pelodytes punctatus in northern Spain. J. Nat. Hist. 38: 2789-2801. 20 Cantekin Dursun, Serkan Gül, Nurhayat Özdemir Esteban, M., Sánchez-Herráiz, M.J., Barbadillo, L.J., Cas- tanet, J., Márquez, R. (2002): Effects of age, size and temperature on the advertisement calls in two Spanish populations of Pelodytes punctatus. Amphibia-Reptilia 23: 249-258. Fox, J., Weisberg, S. (2019): An R Companion to Applied Regression. Third Edition. Sage, Thousand Oaks, CA. Gen-Yu, B., Zheng, R.Q., Zhang, Y., Liu, C.T. (2010): Geo- graphic variation in body size and sexual size dimor- phism in the giant spiny frog Paa spinosa (David, 1875) (Anura: Ranoidae). J. Nat. Hist. 44: 1729-1741. Glandt, D., Jehle, R. (2008): Geographical variation of sexual size dimorphism in the moor frog (Rana arva- lis) in East Europe. Z. Feldherpetol. 13: 113-120. Gokhelashvili, R.K., Tarkhnishvili, D.N. (1994): Age structure of six Georgian anuran populations and its dynamics during two consecutive years (Anura). Her- petozoa 7: 11-18. Guarino, F.M., Crottini, A., Mezzasalma, M., Randrianiri- na, J.E., Andreone, F. (2019): A skeletochronological estimate of age and growth in a large riparian frog from Madagascar (Anura: Mantellidae: Mantidacty- lus). Herpetozoa 32: 39-44. Guarino, F.M., Nocera, F.D., Pollaro, F., Galiero, G., Iac- carino, D., Iovino, D., Mezzasalma, M., Petraccioli, A., Odierna, G., Maio, N. (2020): Skeletochronology, age at maturity and cause of mortality of loggerhead sea turtles Caretta caretta stranded along the beaches of Campania (south-western Italy, western Mediterrane- an Sea). Herpetozoa 33: 39-51. Gül, S. (2014): Habitat preferences of endemic Caucasian parsley frog (Pelodytes caucasicus) Boulenger, 1896 and Caucasian salamander (Mertensiella caucasica) (Waga, 1876) based on bioclimatic data of Fırtına Val- ley (Rize, Northeastern Anatolia). J. Anatol. Wildl. Sci. 5: 24-29. Gül, S., Kumlutaş, Y., Ilgaz, Ç. (2018): Potential distri- bution under different climatic scenarios of climate change of the vulnerable Caucasian salamander (Mertensiella caucasica): A case study of the Caucasus Hotspot. Biologia 73: 175-184. Han, X., Fu, J. (2013). Does life history shape sexual size dimorphism in anurans? A comparative analysis. BMC Evol. Biol. 13: 27. Hill, A.V. (1950): The dimensions of animals and their muscular dynamics. Sci. Prog. 38: 209-230 Hudson, C.M., Fu, J. (2013): Male-Biased Sexual Size Dimorphism, Resource Defense Polygyny, and Multi- ple Paternity in the Emei Moustache Toad (Leptobra- chium boringii). PLoS One. 8: e67502 Iskanderov, T. (2009): Current status of the Caucasus toad (Bufo verrucosissimus Pall., 1814) and Caucasus pars- ley frog (Pelodytes caucasicus Boul., 1896) in Azerbai- jan. In: Status and Protection of Globally Threatened Species in the Caucasus, pp. 151-156. Zazanashvili, N., Mallon, D., Eds, CEPF, WWF. Contour Ltd, Tbi- lisi. Kassambara, A. (2020): ggpubr: ‘ggplot2’ Based Publica- tion Ready Plots. R package version 0.4.0. https:// CRAN.R-project.org/package=ggpubr Kaya, U., Tuniyev, B., Tuniyev, S., Kuzmin, S., Tarkhnish- vili, D., Papenfuss, T., Sparreboom, M., Ugurtas, I., Anderson, S., Eken, G., Kiliç, T., Gem E. (2009): Pelo- dytes caucasicus. In: The IUCN Red List of Threatened Species 2009. Kleinenberg, S.E., Smirina, E.M. (1969): Method of deter- mining the age of amphibians. Zool Zh. 48: 1090- 1094. Kupfer, A. (2007): Sexual size dimorphism in amphibians: an overview. In: Sex, size and gender roles: evolution- ary studies of sexual size dimorphism, pp. 50-59. Fair- bairn D., Blanckenhorn, W., Szekely, T., Eds, Oxford University Press, Cary, NC, USA. Kurnaz, M., Bülbül, U., Eroğlu, A. İ., Uzun, F., Koc, H., Kutrup, B. (2018): Age and growth of the Artvin Liz- ard, Darevskia derjugini (NIKOLSKY, 1898), in Tur- key. Herpetozoa 30: 147-158. Length, R.V. (2021): emmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1.5.4. https://CRAN.R-project.org/package=emmeans Litvinchuk, S.N., Kidov, A.A. (2018): Distribution and conservation status of the Caucasian Parsley Frog, Pel- odytes caucasicus (Amphibia: Anura). Nat. Conserv. Res. 3: 51-60. Lukina, G.P., Koneva, V.A. (1996): Notes on the biology of the Caucasian parsley frog (Pelodytes caucasicus) in Krasnodar Region. Advances in Amphibian Research in the Former Soviet Union 1: 109-116. Ma, M., Luo, S., Tang, X., Chen, Q. (2022): Age structure and growth pattern of a high-altitude lizard population based on age determination by skeletochronology. J. Exp. Zool. A: Ecol. Integr. Physiol. 337: 491-500. Marangoni, F., Barrasso, D.A., Cajade, R., Agostini, G. (2012): Body size, age and growth pattern of Physalae- mus fernandezae (Anura: Leiuperidae) of Argentina. North-West. J. Zool. 8: 63-71. Márquez, R., Pargana, J. M., Crespo, E. G. (2001). Acous- tic competition in male Pelodytes ibericus (Anura: Pelodytidae): Interactive playback tests. Copeia. 2001: 1142-1150. Misawa, Y., Khonsue, W., Matsui, M. (2002): Age deter- mination of Daruma pond frog, Rana porosa brevip- oda from Japan towards its conservation (Amphibia: Anura). Amphibia-Reptilia 23: 259-268. 21Age structure and body size of Pelodytes caucasicus Mittermeier, R.A., Robles, G.P., Hoffmann, M., Pilgrim, J., Brooks, T., Mittermeier, C.G., Lamoreux, J., da Fonseca, G.A.B. (2004): Hotspots revisited: Earth’s biologically richest and most endangered ecoregions. CEMEX, Mexico City. Nacarino-Meneses, C., Jordana, X., Koehler, M. (2016): First approach to bone histology and skeletochronol- ogy of Equus hemionus. Comptes. Rendus. Palevol. 15: 267-277. Nali, R.C., Zamudio, K.R., Haddad, C.F., Prado, C.P. (2014): Size-dependent selective mechanisms on males and females and the evolution of sexual size dimorphism in frogs. Am. Nat. 184: 727-740. Naseka, A.M. (2010): Zoogeographical freshwater divi- sions of the Caucasus as a part of the West Asian transitional region. Proceedings of the Zoological Institute RAS 314: 469-492. Ogle, D. (2020): FSAsim: Simulate Data for Fisheries Stock Assessment Methods. http://derekogle.com/ fishr, https://github.com/droglenc/FSAsim Ogle, D.H. (2013): fishR Vignette-von Bertalanffy growth models. Ashland, WI: Northland College, 54. Ogle, D.H. (2019): FSAdata: Fisheries Stock Analysis, Datasets. R package version 0.3.8. Ogle, D.H., Wheeler, P., Dinno, A. (2021): FSA: Fisher- ies Stock Analysis. R package version 0.8.32. https:// github.com/droglenc/FSA Otero, M., Baraquet, M., Pollo, F., Grenat, P., Salas, N., Martino, A. (2017): Sexual size dimorphism in rela- tion to age and growth in Hypsiboas cordobae (Anura: Hylidae) from Córdoba, Argentina. Herpetol. Con- serv. Biol. 12: 141-148. Özdemir, N., Altunisik, A., Ergül, T., Gül, S., Tosunoğlu, M., Cadeddu, G., Giacoma, C. (2012): Variation in body size and age structure among three Turkish populations of the tree frog Hyla arborea. Amphibia- Reptilia 33: 25-35. Pargana, J.M., Márquez, R., Reques, R., Sánchez-Herráiz, M.J., Tejedo, M., Crespo, E.G. (2003): The mating call of Pelodytes ibericus (Anura, Pelodytidae). Herpetol. J. 13: 199-204. Peng, Z., Zhang, L., Lu, X. (2021): Global gaps in age data based on skeletochronology for amphibians. Integr. Zool. 17: 752-763. Pincheira‐Donoso, D., Harvey, L.P., Grattarola, F., Jara, M., Cotter, S.C., Tregenza, T., Hodgson, D.J. (2021): The multiple origins of sexual size dimorphism in global amphibians. Glob. Ecol. Biogeogr. 30: 443-458. Radojićić, J.M., Cvetković, D., Tomović, L.J., Dzukić, G., Kalezić, M.L. (2002). Sexual dimorphism in fire‐bel- lied toads Bombina spp. from the central Balkans. Folia Zool. 51: 129-140. R Core Team (2020): R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project. org/ Revelle, W. (2019): psych: Procedures for Personality and Psychological Research. Northwestern University, Evanston, Illinois, USA. Rozenblut, B., Ogielska, M. (2005): Development and growth of long bones in European water frogs (Amphibia: Anura: Ranidae), with remarks on age determination. J. Morphol. 265: 304-317. Santini, L., Benítez-López, A., Ficetola, G.F., Huijbregts, M.A.J. (2018): Length–mass allometries in amphib- ians. Integr. Zool. 13: 36-45. Schloerke, B., Cook, D., Larmarange, J., Briatte, F., Mar- bach, M., Thoen, E., Elberg, A., Crowley, J. (2021): GGally: Extension to ‘ggplot2’. R package version 2.1.0. https://CRAN.R-project.org/package=GGally Silva, N. R., Berneck, B. V., da Silva, H. R., Haddad, C. F., Zamudio, K. R., Mott, T., Nali, R.C., Prado, C. P. (2020): Egg-laying site, fecundity and degree of sexual size dimorphism in frogs. Biol. J. Linn. Soc. 131: 600- 610. Shine, R. (1979). Sexual selection and sexual dimorphism in the Amphibia. Copeia 2: 297-306. Shine, R. (1989): Ecological causes for the evolution of sexual dimorphism: a review of the evidence. Q. Rev. Biol. 64: 419-461. Talavera, R.R. (1990): Evolución de pelo batidos y peloditidos (amphibia, anura): morfología y desarrollo del sistema esquelético. Unpublished doctoral disser- tation. Universidad Complutense de Madrid, Madrid. Tarkhnishvili, D. (1993): Anurans of Borjomi Canyon: clutch parameters and guild structure. Alytes. 11: 140- 154. Tarkhnishvili, D. (1996): The distribution and ecology of the amphibians of Georgia and the Caucasus: a bio- geographical analysis. Z. Feldherpetol. 3: 167-196. Tarkhnishvili, D., Kaya, U. (2009): Status and Conserva- tion of the Caucasian Salamander (Mertensiella cauca- sica). In: Status and protection of globally threatened species in the Caucasus, pp. 157-164. Zazanashvili, N., Mallon, D., Eds, CEPF, WWF, Tbilisi, Georgia. Üzüm, N., Ilgaz, Ç., Kumlutaş, Y., Gümüş, Ç., Avcı, A. (2014): The body size, age structure, and growth of Bosc’s fringe-toed lizard, Acanthodactylus boskianus (Daudin, 1802). Turk. J. Zool. 38: 383-388. Üzüm, N., Avcı, A., Özcan, Ç.G., Olgun, K. (2020): Com- parison of the age structure, body size, and some growth parameters of two insular populations of Lyciasalamandra fazilae from southwestern Turkey. Russ. J. Herpetol. 27: 19-25. 22 Cantekin Dursun, Serkan Gül, Nurhayat Özdemir Werner, E. (1986): Amphibian metamorphosis: growth rate, predation risk, and the optimal size at transfor- mation. Am. Nat. 128: 319-341. Wickham, H. (2016): ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag, New York. Williams, L., Zazanashvili, N., Sanadiradze, G., Kandau- rov, A. (2006): An Ecoregional Conservation Plan for the Caucasus. Contour Ltd, Tbilisi. Xiong, J., Gou, j., Huang, Y., Zhang, B., Ren, H., Tao, P. (2020): Age and Body Size of the Shangcheng Stout Salamander Pachyhynobius shangchengensis (Caudata: Hynobiidae) from Southeastern China. Asian Herpe- tol. Res. 11: 219-224. Yildirimhan, H.S., Bursey, C.R., Goldberg, S.R. (2009): Helminth parasites of the Caucasian parsley frog, Pel- odytes caucasicus, from Turkey. Comp. Parasitol. 76: 247-257. Zazanashvili, N. (2009): The Caucasus hotspot. In: Status and protection of globally threatened species in the Caucasus. Zazanashvili, N., Mallon, D., Eds, CEPF, WWF, Tbilisi, Georgia. Zazanashvili, N., Garforth, M., Jungius, H., Gamkrelidze, T., Montalvo, C. (2012): Ecoregion conservation plan for the Caucasus, revised and updated edition. Cauca- sus Biodiversity Council, Tbilisi. Threats of the emerging pathogen Batrachochytrium salamandrivorans (Bsal) to Italian wild salamander populations Lorenzo Dondero1, Giorgia Allaria1, Giacomo Rosa1, Andrea Costa1, Gentile Francesco Ficetola2, Roberto Cogoni3, Elena Grasselli1, Sebastiano Salvidio1,* Age estimation and body size of the Parsley Frog, Pelodytes caucasicus Boulenger, 1896 from Lake Borçka Karagöl, Turkey Cantekin Dursun*, Serkan Gül, Nurhayat Özdemir Patterns of acoustic phenology in an anuran assemblage of the Yungas Andean forests of Argentina Martín Boullhesen1,2,*, Marcos Vaira1, Rubén Marcos Barquez2, Mauricio Sebastián Akmentins1 Diet and trophic niche overlap of four syntopic species of Physalaemus (Anura: Leptodactylidae) in southern Brazil Renata K. Farina1, Camila F. Moser2, Stefano Scali3, Mateus de Oliveira4, Patrícia Witt5, Alexandro Marques Tozetti1,* Screening of Ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of San Domenico e dei Serpari (Cocullo, AQ, Italy) Daniele Marini1,2, Ernesto Filippi3,*, Gianpaolo Montinaro4, Francesco C. Origgi5,6 Assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community Carter Dollen1,2, Tracy J. Coleman1,2, Travis R. Robbins1,2,* Revisiting the polyploidy in the genus Odontophrynus (Anura: Odontophrynidae) André Luis de Souza, Mayara Aparecida das Neves Micalichen, Roger Alves da Rocha, Rafael Bueno Noleto*