Presence of an alien turtle accelerates hatching of common frog (Rana temporaria) tadpoles

13 The presence of a predator affects prey populations either by direct pr edation or by modifying 14 various parts of their life history. We investigated whether the hatching time, developmental 15 stage, and body size at hatching of common frog (Rana temporaria) embryos would alter in the 16 presence of a red -eared slider ( Trachemys scripta elegans) as a predator. The presence of a 17 predator affected all factors examined. We found that in the absence of the slider, the embryos 18 hatched in 12 days, while hatching was accelerated by two days in slider treatment. At the same 19 time, the embr yos hatched smaller and at a lower stage of development with the slider than 20 without it. Our study extends the range of predators studied, including the effect on different 21 phases of development of potential amphibian prey. 22 23 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 2

24 The impacts of invasive species on native communities are still difficult to generalise due to the 25 limited number of species and environments researched (Griesemer et al. 2018; Ramírez 26 Albores et al. 2019; Rolim et al. 2015; Tricarico et al. 2016). However, inappropriate responses 27 of individuals to invasive predators can strongly affect native populations (Mooney and Cleland 28 2001). In amphibians, predation can account for a significant proportion of the total mortality 29 of all their developmental stages (Gunzburger and Travis 2005; Chivers et al. 2001; Laurila et 30 al. 2002; Nyström et al. 1997) . The ability to detect, recognise, and respond to potential 31 predators is, therefore, an important part of antipredatory behaviour (Bennett et al. 2013; Polo‐32 Cavia and Gomez‐Mestre 2014), and native populations can have especially serious problems 33 facing the presence of new alien predators (Gomez-Mestre and Díaz-Paniagua 2011; Nunes et 34 al. 2019; Polo -Cavia et al. 2010) . In general, embryonic and early larval stages are the most 35 vulnerable to predation (Laurila et al. 2002; Wells 2007) , and the ability to respond to the 36 presence of a predator can therefore significantly increase the fitness of an individual and thus 37 the viability of the entire population (Vonesh and Bolker 2005; Warkentin 1995). 38 Whether intentionally or unintentionally introduced, the recent wide occurrence of the 39 red-eared slider ( Trachemys scripta elegans ) in Europe (GISD 2021) presents a new 40 opportunity to investigate the responses of naive native amphibian populations to a new 41 predator. Although red -eared slider (hereafter referred to as slider) is not reproductively 42 successful throughout Europe (Cadi et al. 2004; Ficetola et al. 2009; Mikátová and Šandera 43 2015; Standfuss et al. 2016), even the mere presence of adults may pose a certain risk to native 44 species. In previous studies, we found that the presence of the sliders affect several life history 45 parameters of common frog (Rana temporaria) tadpoles, such as movement activity, trajectory 46 of movement (Berec et al. 2016), time to metamorphosis, or size at metamorphosis (Vodrážková 47 et al. 2020). Although sliders are usually still hibernating at the time of common frog breeding 48 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 3 (Gibbons et al. 1990; Speybroeck et al. 2016) , which eliminates the risk of direct predation, 49 kairomones released by sliders into the aquatic environment provide amphibians with 50 information about their presence. Since the slider is an opportunistic predator and can consume 51 frog eggs (Ernst and Lovich 2009), some response of common frog embryos is to be expected. 52 For frog embryos, there are two basic strategies for avoiding predation or significantly 53 reducing its effects: the development of egg unpalatability and hatching plasticity (Wells 2007). 54 The unpalatability of eggs is a passive strategy in which the embryo relies on the predator's 55 inability or unwillingness to consume eggs, which imposes costs on its host even if the host 56 never comes in contact with the predator; environmentally cued hatching is characterised by an 57 embryo’s active capability to alter the time of hatching according to the conditions it encounters 58 during embryonic development. Hatchi ng plasticity has been documented many times in 59 amphibian embryos, and predator presence has been shown to trigger early hatching from eggs 60 incubated in both air and water (Chivers et al. 2001; Warkentin 2011). In terrestrially laid eggs, 61 hatching can be stimulated by vibrational cues during the direct physical attacks of predators, 62 such as snakes (Jung et al. 2019; Warkentin 1995) , frogs (Vonesh and Bolker 2005), katydids 63 (Poo and Bickford 2014), wasps (Warkentin 2000), or egg-eating fly larvae (Vonesh and Bolker 64 2005). In aquatic environments, these responses are induced mainly by chemical cues from 65 predators (kairomones) or by chemical cues that are released from injured prey during predation 66 events (Dodson 1988; Laurila et al. 2002; Nicieza 1999; 2000; Petranka et al. 1987; Smith and 67 Fortune 2009; Tollrian 1994). 68 This study aimed to shift our previous focus (Berec et al. 2016; Vodrážková et al. 2020; 69 in review) to a different developmental stage, namely, embryos in eggs. We investigated 70 whether the presen ce of a slider can alter the hatching time of common frog embryos. We 71 hypothesised that the presence of a slider would accelerate the hatching time, so the ontogenetic 72 stage and body size at hatching were also measured. The uniqueness of this study lies in the use 73 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 4 of a stage-nonspecific predator, which is virtually absent in the literature. At the same time, it 74 is an alien predator from a taxonomic group to which the prey has no common history. 75 76

77 Five freshly laid clutches of common fro gs were collected in a pool between Holubov and 78 Vrábče, South Bohemia, the Czech Republic (48.9078633N, 14.3485608E), on 2 April 2021. 79 Collection locality was monitored daily to collect egg clutches laid during the night before. 80 Neither the slider nor any other species of turtle occurs at the collection locality, so the eggs 81 and their parents are naive prey relative to the turtles. The experiment was performed in six 82 glass tanks – three replications with the sliders and three repl ications of control . Glass tanks 83 (size: 1 00 × 55 × 50 cm) filled with 20 cm of aged tap water were equipped with a Claro 84 300 filter pump (300 L.h −1) and rinsed three times a week. The room temperature was set at 85 15 °C and the datalogger (Dostman LOG200 PDF) recorded a mean air temperature of 14.8 ± 86 0.4 °C (± S.D.; measured at hourly intervals) during the experiment . Fluorescent tubes (2 x 87 36 W) with a light regime of 12 h/12 h were used. During the dark phase of the day, the glass 88 tanks were illuminated with red light to allow permanent monitoring of egg hatching. 89 Three adult sliders (carapace length: 18 cm, 20 cm, and 21 cm) were used as predators. 90 The slider was placed in each of three glass tanks three days to release kairomones into the 91 water before the experiment was initiated and fed three times a week with ReptoMin Tetra turtle 92 gammarus. To prevent physical but not chemical contact between the slider and frog eggs, a 93 glass barrier was placed inside each glass tank with a 6 cm gap at both ends so that water could 94 flow freely throughout the tank. On the other side of this barrier, five perforated opaque boxes 95 (20 × 14 cm) with holes 1 mm in diameter were glued to the bottom of the glass tanks to contain 96 the eggs (Fig. 1). 97 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 5 98 Figure 1. 99 Diagram of the glass tank showing the position of the slider (if present) and the boxes for clutch 100 samples. These were placed randomly in the boxes in each glass tank (see Materials and 101 methods). Three replications with the sliders and three replications without them (control) were 102 used. Slider drawing by Jakub Berec. 103 104 Six samples of approximately 150 eggs each were taken from the collected clutch and 105 randomly placed in six boxes, one in each glass tank. This procedure was repeated for all five 106 clutches, so that there were five boxes in each tank with sample from each clutch. Each glass 107 tank was continuously monitored using a camera (Niceboy Stream Pro). Hatched tadpoles were 108 counted every 24 h. Hatching was defined as the moment at which the whole hatchling had left 109 the protective jelly of the eggs . To maintain a good processing of the camera recording s (the 110 large number of hatched tadpoles in a small box makes it difficult to count them ), hatched 111 tadpoles were transferred every six hours to a depot tank. At the time when half of the eggs in 112 each box had hatched, two tadpoles were taken from the group of tadpoles hatched in the last 113 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 6 six hours. These tadpoles were photographed under a stereomicroscope (Olympus SZX 7) and 114 measured (to the nearest 0.01 mm) using QuickPHOTO MICRO 3.2 software. Their 115 developmental phase was determined according to Gosner (1960). 116 The experiment involved a four factor design (slider: presence/absence, glass tank: 1-3 117 with slider, 4-6 controls, box: five in each glass tank, and clutch: six samples). The slider was 118 used as a fixed factor as both levels of this factor (presence/absence) were tested. All other three 119 factors (glass tank, clutch, and box) were random (Allen 2017) with the glass tank factor nested 120 in the slider presence/absence factor. According the experimental design, linear mixed model 121 was used for analysis (Quinn and Keough 2002). Three analyses were performed – for hatching 122 time, developmental stage, and the size at hatching. Adjusted R -squared was used as measure 123 of variability explained in the statistical model. Effect sizes were evaluated by partial eta -124 squared (Richardson 2011). Given the number of eggs, statistical significance was assessed at 125 the 99.9% level (Steel et al. 2013). All calculations were done in Tibco Statistica (TIBCO 126 2017). 127 128

129 All three models for life history parameters measured were statistically significant (hatching 130 time: F=688.7, p<<0.001; adjusted R2=0.749; developmental stage: F=27.1, p<<0.001; adjusted 131 R2=0.852; size at hatching: F=23.6 , p<<0.001; adjusted R 2=0.833). For all these parameters, 132 the presence of the slider was the only significant or far most important factor in each mo del 133 (Supplementary file: partial eta-squared in Tables 1-3). 134 We found a significant difference in hatching time between the presence and absence of 135 the slider (F(1,4)=915.0; p<< 0.001). In the absence of the slider, embryos hatched in 136 12 ± 0.6 days (mean ± S.D.). The presence of the sli der accelerated hatching by two days 137 (10 ± 0.6 days) (Fig. 2). Hatching time differed significantly also among glass tanks 138 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 7 (F(1,4)=9.5; p<<0.001), boxes (F(1,4)=7.6; p<< 0.001) and clutches (F(1,4)=44.3; p<< 0.001), 139 but the effect sizes of these three factors were negligible in comparison to the effect of slider 140 presence (Supplementary file: partial eta-squared in Table 1). 141 Similarly, significant differences were found between the developmental stage 142 (F(1,4)=4608.0; p<0.01) and size (F(1,4)=75.1; p< 0.001) of freshly hatched embryos in the 143 presence of the slider and without it. In the presence of a slider, embryos hatched at 144 developmental stage 20 ± 1.5 (mean ± S.D.) with an average size of 5.92 ± 1.460 mm, while in 145 the control, freshly hatched embryos had developed to stage 23 ± 1.0, with an average size of 146 10.77 ± 1.042 mm (Fig. 2). Neither developmental stage nor hatching size was significantly 147 affected by glass tank, clutch and boxes (Supplementary file: Tables 2 and 3). 148 149 Figure 2. 150 Histogram of A hatching time, B Gosner (developmental) stage, and C size at hatching of the 151 embryos of common frogs in the presence of red-eared slider and control. 152 153

154 Hatching Time (days) 8 9 10 11 12 13 0 20 40 60 80 Number of Individuals (%) 0 20 40 60 80 100 Developmental Stage 18 19 20 21 22 23 24 Size at Hatching (mm) 12 Control Red-eared Slider A B C Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 8 Developmental plasticity is an adaptive response of anuran embryos and larvae to the risk of 155 predation (Altig and McDiarmid 1999; Benard 2004; Warkentin 2011) . Here, we present 156 evidence for the developmental plasticity of common frog embryos in the presenc e of a red -157 eared slider and, in addition to a previous study (Vodrážková et al. 2020), provide a 158 comprehensive insight of the influence of this alien predator on the early phases of the common 159 frog life cycle. We have previously shown (Vodrážková et al. 2020) that, in the slider presence, 160 tadpoles of common frogs are able to modify the duration of larval development. In the present 161 study, we confirmed a similar response in common frog embryos, which hatched earlier in the 162 presence of a slider. At the same time, the embryos were smaller and less developed when 163 exposed to the chemical signals of a predator. We also found the effect of glass tank, clutch and 164 box on hatching time, which was nevertheless negligible in comparison with t he effect of 165 predator presence. 166 In the presence of stage -specific predators, amphibians can modify the duration of the 167 relevant developmental stage (Chivers et al. 2001; Ireland et al. 2007; Mitchell et al. 2017). In 168 anuran embryos, specifically, the presence of egg predators has mostly been shown to induce 169 early hatching of embryos (Chivers et al. 2001; Johnson et al. 2003; Laurila et al. 2001; Segev 170 et al. 2015; Warkentin 1995; 2000) , while tadpole predators induce delayed hatching (Laurila 171 et al. 2002; Mitchell et al. 2017; Schalk et al. 2002; Sih and Moore 1993), thus increasing their 172 chance of survival by escaping possibl e attacks. However, the slider is not a stage -specific 173 predator, as it is capable of consuming both amphibian eggs and larvae (Brown et al. 1995; 174 Ernst and Lovich 2009; Chen 2006); thus, the allocation of risk between developmental stages 175 of the frog may b e more complex in this case (Warkentin 2011). Studies examining predator 176 effects on the developmental rates of both eggs and larvae are rare because few predators 177 consume both eggs and larvae simultaneously. Muraro et al. (2021) used a stage -nonspecific 178 predator ( Procambarus clarkii ) and found, in concordance with our results, a reduction in 179 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 9 hatching time in Rana latastei embryos. However, they did not study larval development. 180 Ireland et al. (2007) solved the problem of predator stage specificity by simult aneously 181 exposing frog eggs to stage-specific predators of eggs (leech: Nephelopsis obscura) and larvae 182 (dragonfly: Aeshna canadensis nymphs), which resulted in no change in hatching time, whereas 183 tests with separately acting predators produced the expected response of a reduction in hatching 184 time in the egg predator treatment and an increase in hatching time in the larval predator 185 treatment. This study on embryos and a previous study on tadpoles (Vodrážková et al. 2020) 186 jointly clarify that the embryos/tadpoles of the common frog responded to the presence of a 187 predator by shortening the stage of development during which the embryo/tadpole would be 188 exposed to the predator. It would be interesting to analyse how common frog tadpoles react to 189 the presence of a slider if the entire development from eggs to metamorphosis was taking place 190 with this predator present. 191 However, some studies have shown that frog embryos, including the common frog, do 192 not always respond specifically to stage -specific predators by short ening hatching time 193 (Capellán and Nicieza 2010; Laurila et al. 2001; Laurila et al. 2002; Saglio and Mandrillon 194 2006; Schalk et al. 2002; Touchon et al. 2006; Touchon and Wojdak 2014) . The published 195 differences in embryo responses may correspond to differe nt signal intensities of the presence 196 of a specific predator, and thus, the responses to indirect waterborne cues might be weaker than 197 those to the direct, mechanical cues of a predator attack (Warkentin 2011). An evident response 198 to water-borne cues of sl iders may be related to a markedly stronger signal of a much larger -199 sized predator in our experiment compared to commonly tested invertebrate predators. The 200 ability to scale predator danger and adjust hatching time accordingly has been found, for 201 example, in embryos of southern leopard frogs ( Lithobates sphenocephalus) (Johnson et al. 202 2003). Moreover, a possible absence of a change in hatching time does not necessarily imply a 203 complete lack of response to the presence of a predator. It may be manifested by other types of 204 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 10 responses, such as changes in the body shape of tadpoles (Laurila et al. 2001; Mandrillon and 205 Saglio 2007; Saglio and Mandrillon 2006; Touchon and Wojdak 2014) or their behaviour 206 (Saglio and Mandrillon 2006; Touchon and Wojdak 2014). 207 Native and naive prey may fail to detect the novel predator adequately as a dangerous 208 threat, resulting in no (Cox and Lima 2006; Sih et al. 2010) or inefficient antipredator responses 209 to counter the predator’s attack strategies (Sih et al. 2010; Strauss et al. 2006). However, when 210 responses in hatching time in naive prey are detected, they are often explained by the presence 211 of syntopic, taxonomically related predators (Melotto et al. 2021; Muraro et al. 2021; Sih et al. 212 2010), although the time since invasion ma y also play an important role (Gomez-Mestre and 213 Díaz-Paniagua 2011; Nunes et al. 2013) . Our results suggested that a common evolutionary 214 history is not necessary for a detectable response. Such a result has already been published for 215 tadpole development time (Stav et al. 2007; Vodrážková et al. 2020), but as far as we know, it 216 has not yet been published for hatching time in frog embryos. An explanation for embryo 217 response to an alien slider may be in the ability of embryos to detect a kind of general "smell 218 of fear" that is elicited by most predators, regardless of taxonomic classification (Sih et al. 219 2010). 220 Finding a general tendency in the phenotypic plasticity responses of prey across a broad 221 range of animal predators (different taxa and feeding spectra), environmental and experimental 222 conditions is a challenge even in anurans themselves ( Relyea et al. 2018 ). However, in frogs, 223 the earlier hatching time was generally associated with smaller size at hatching (Capellán and 224 Nicieza 2007 ; Chivers et al. 2001 ; Ireland et al. 2007 ; Laurila et al. 2002) and lower 225 developmental stage (Capellán and Nicieza 2007 ; Chivers et al. 2001 ; Ireland et al. 2007 ; 226 Laurila et al. 2002 ; Muraro 2021), and our results confirm this relationship. In some cases, 227 earlier hatched tadpo les performed higher growth rate and reached the size of later ha tched 228 tadpoles at metamorphosis (Capellán and Nicieza 2007 ). However, if tadpoles are unable to 229 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 11 compensate for their smaller size at hatching, this can impose significant costs in later 230 developmental phases. These costs have been demonstrated in increased mortality during the 231 larval stage (Smith 1987; Warkentin 1995; but see Vonesh and Bolker (2005) where early 232 hatchlings survived better), reduced size at metamorphosis (Vonesh and Bolker 2005; 233 Vodrážková et al. 2020), lower post -metamorphic survival (Altwegg and Reyer 2003; Berven 234 1990), change of behaviour (Buckley et al. 2005, Capellán and Nicieza 2007), delayed maturity 235 (Smith 1987) and lower reproductive success (Smith 1987). 236 Our work added a slider as an additional predator inducing changes in the embryonic 237 developmental rate in Ranidae. Since the impact of earlier embryo hatching (lower body size 238 and lower stage of development) on fitness has been confirmed in several frog species (Laurila 239 et al. 2002; Touchon et al. 2013; Vonesh and Bolker 2005; Warkentin 1995) , the same impact 240 can be expected for the common frog. T he existence of defensive responses in slider -exposed 241 embryos may reduce the threat that poses the spreading of this inva sive species in Europe. On 242 the other hand, the reduced size at hatching and developmental stage of common frog hatchlings 243 represents additional risks of negative fitness impacts, and at the very least, the presence of 244 sliders in non-native areas should receive increased attention. 245 246 Funding/Acknowledgements/Competing interests 247 M.V. acknowledges GAJU 045/2019/Z for financial support. The authors have no support to 248 report. The authors have declared that no competing interests exist. 249 All methods were carried ou t in accordance with relevant guidelines and regulations. All 250 experimental protocols were approved by the Czech Ministry of Agriculture, Department of 251 Animal Welfare according to article No. 15, section 2 of the act registered under number 252 9103/2009-17210. 253 254 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 12

255 Allen M. (2017) The sage encyclopedia of communication research methods (Vols. 1-4). SAGE 256 Publications, Inc., (Thousand Oaks, CA): 1-2064. 257 Altig R, McDiarmid RW (1999) Tadpoles: The biology of anuran larvae. University of Chicago 258 Press, (Chicago): 1-454. 259 Altwegg, R., & Reyer, H. U. (2003). Patterns of natural selection on size at metamorphosis in 260 water frogs. Evolution, 57, 872 -882. https://doi.org/10.1111/j.0014-261 3820.2003.tb00298.x 262 Benard MF (2004) Predator -induced phenotypic plasticity in organisms with complex life 263 histories. Annual Review of Ecology, Evolution, and Systematics 35: 651 -673. 264 https://doi.org/10.1146/annurev.ecolsys.35.021004.112426. 265 Bennett AM, Pereira D, Murray DL (2013) Investment into defensive traits by anuran prey 266 (Lithobates pipiens) is mediated by the starvation-predation risk trade-off. PLoS One 8: 267 e82344. https://doi.org/10.1371/journal.pone.0082344. 268 Berec M, Klapka V, Zemek R (2016) Effect of an alien turtle predator on movement activity 269 of European brown frog tadpoles. Italian Journal of Zoology 83: 68 -76. 270 https://doi.org/10.1080/11250003.2016.1139195. 271 Berven, K. A. (1990). Factors affecting population fluctuations in larval and adult stages of the 272 wood frog (Rana sylvatica). Ecology, 71, 1599-1608. https://doi.org/10.2307/1938295 273 Brown HA, Bury RB, Darda DM, Diller L, Peterson C, Storm R (1995) Reptiles of Washington 274 and Oregon. Seattle Audubon Society, (Seattle, WA): 1-176. 275 Buckley, C. R., Michael, S. F., & Irschick, D. J. (2005). Early hatching decreases jump ing 276 performance in a direct-developing frog, Eleutherodactylus coqui. Functional ecology, 277 67-72. 278 Cadi A, V D, Prévot‐Julliard AC, Joly P, Pieau C, Girondot M (2004) Successful reproduction 279 of the introduced slider turtle ( Trachemys scripta elegans ) in the South of France. 280 Aquatic Conservation: Marine and Freshwater Ecosvstems 14: 237 -246. 281 https://doi.org/10.1002/aqc.607. 282 Capellán E, Nicieza AG (2007) Trade-offs across life stages: does predator–induced hatching 283 plasticity reduce anuran post -metamorphic pe rformance? Evolutionary Ecology 21: 284 445-458. https://doi.org/10.1007/s10682-006-9133-9. 285 Capellán E, Nicieza AG (2010) Constrained plasticity in switching across life stages: pre -and 286 post-switch predators elicit early hatching. Evolutionary Ecology 24: 49 -57. 287 https://doi.org/10.1007/s10682-008-9289-6. 288 Chen T-H (2006) Distribution and status of the introduced red-eared slider (Trachemys scripta 289 elegans) in Taiwan. In: Koike F, Cloud M, Kawamichi M, De Poorter M, Iwatsuki K 290 (Eds) Assessment and control of bio logical invasion risks. Shoukadoh Book Sellers 291 (Kyoto, Japan and IUCN, Gland, Switzerland): 187-195. 292 Chivers DP, Kiesecker JM, Marco A, Devito J, Anderson MT, Blaustein AR (2001) Predator‐293 induced life history changes in amphibians: egg predation induces h atching. Oikos 92: 294 135-142. https://doi.org/10.1034/j.1600-0706.2001.920116.x. 295 Cox JG, Lima SL (2006) Naiveté and an aquatic –terrestrial dichotomy in the effects of 296 introduced predators. Trends in Ecology & Evolution 21: 674 -680. 297 https://doi.org/10.1016/j.tree.2006.07.011. 298 Dodson S (1988) The ecological role of chemical stimuli for the zooplankton: Predator‐299 avoidance behavior in Daphnia. Limnology and Oceanography 33: 1431 -1439. 300 https://doi.org/10.4319/lo.1988.33.6part2.1431. 301 Ernst CH, Lovich JE (2009) Turtles of the United States and Canada. JHU Press, (Baltimore): 302 1-840. 303 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 13 Ficetola GF, Thuiller W, Padoa‐Schioppa E (2009) From introduction to the establishment of 304 alien species: bioclimatic differences between presence and reproduction localities in 305 the sli der turtle. Diversity and Distributions 15: 108 -116. 306 https://doi.org/10.1111/j.1472-4642.2008.00516.x. 307 Gibbons J, Greene J, Congdon J (1990) Temporal and spatial movement patterns of sliders and 308 other turtles. In: Gibbons J (Ed) Life history and ecology of the slider turtle. Smithsonian 309 Institution Press (Washington, D.C.): 201-215. 310 Global Invasive Species Database. http://www.issg.org/database [accessed 14/10/2021] 311 Gomez-Mestre I, Díaz-Paniagua C (2011) Invasive predatory crayfish do not trigger inducible 312 defences in tadpoles. Proceedings of the Royal Society B: Biological Sciences 278: 313 3364-3370. https://doi.org/10.1098/rspb.2010.2762. 314 Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on 315 identification. Herpetologica 16: 183-190. 316 Griesemer J, Jeschke J, Heger T (2018) Mapping theoretical and evidential landscapes in 317 ecological science: Levin's virtue trade -off and the hierarchy -of-hypotheses approach. 318 In: Jeschke J, Heger T (Eds) Invasion biology: Hypotheses and evide nce. CABI 319 (Boston, MA): 23-29. 320 Gunzburger MS, Travis J (2005) Critical literature review of the evidence for unpalatability of 321 amphibian eggs and larvae. Journal of Herpetology 39: 547 -571. 322 https://doi.org/10.1670/1-05A.1. 323 Ireland D, Wirsing A, Murray D (2007) Phenotypically plastic responses of green frog embryos 324 to conflicting predation risk. Oecologia 152: 162 -168. https://doi.org/10.1007/s00442-325 006-0637-3. 326 Johnson JB, Saenz D, Adams CK, Conner RN (2003) The influence of predator threat on the 327 timing of a life-history switch point: predator-induced hatching in the southern leopard 328 frog ( Rana sphenocephala ). Canadian Journal of Zoology 81: 1608 -1613. 329 https://doi.org/10.1139/z03-148. 330 Jung J, Kim SJ, Pérez Arias SM, McDaniel JG, Warkentin KM (2019) How do red -eyed 331 treefrog embryos sense motion in predator attacks? Assessing the role of vestibular 332 mechanoreception. Journal of Experimental Biology 222: jeb206052. 333 https://doi.org/10.1242/jeb.206052. 334 Laurila A, Crochet P -A, Merilä J (2001) Predation -induced effects on hatchling morphology 335 in the common frog ( Rana temporaria ). Canadian Journal of Zoology 79: 926 -930. 336 https://doi.org/10.1139/z01-045. 337 Laurila A, Pakkasmaa S, Crochet P-A, Merilä J (2002) Predator-induced plasticity in early life 338 history and morphology in two anuran amphibians. Oecologia 132: 524 -530. 339 https://doi.org/10.1007/s00442-002-0984-7. 340 Mandrillon A-L, Saglio P (2007) Herbicide exposure affects the chemical recognition of a non 341 native predator in common toad tadpoles ( Bufo bufo ). Chemoecology 17: 31 -36. 342 https://doi.org/10.1007/s00049-006-0354-8. 343 Melotto A, Ficetola GF, Alari E, Romagnoli S, Manenti R (2021) Visual recognition and 344 coevolutionary history drive responses of amphibians to an invasive predator. 345 Behavioral Ecology 32: 1352-1362. https://doi.org/10.1093/beheco/arab101. 346 Mikátová B, Šandera M (2015) První rozmnožování volně žijící želvy nádherné ( Trachemys 347 scripta) na území České republiky. Herpeta 1: 5-6. 348 Mitchell MD, Bairos -Novak KR, Ferrari MC (2017) Mechanisms underlying the control of 349 responses to predator odours in aquatic prey. Journal of Experimental Biology 220: 350 1937-1946. https://doi.org/10.1242/jeb.135137. 351 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 14 Mooney HA, Cleland EE (2001) The evolutionary impact of invasive species. Proceedings of 352 the National Academy of Sciences 98: 5446 -5451. 353 https://doi.org/10.1073/pnas.091093398. 354 Moore RD, Newton B, Sih A (1996) Delayed hatching as a response of streamside salamander 355 eggs to chemical cues from predatory sunfish. Oikos: 331 -335. 356 https://doi.org/10.2307/3546073. 357 Muraro M, Romagnoli S, Barzaghi B, Falaschi M, Manenti R, Ficetola GF (2021) Invasive 358 predators induce plastic and adaptive responses during embryo deve lopment in a 359 threatened frog. NeoBiota 70: 69. https://doi.org/10.3897/neobiota.70.65454. 360 Nicieza AG (1999) Context‐dependent aggregation in Common Frog Rana temporaria 361 tadpoles: influence of developmental stage, predation risk and social environment. 362 Functional Ecology 13: 852-858. https://doi.org/10.1046/j.1365-2435.1999.00375.x. 363 Nicieza AG (2000) Interacting effects of predation risk and food availability on larval anuran 364 behaviour and development. Oecologia 123: 497 -505. 365 https://doi.org/10.1007/s004420000343. 366 Nunes AL, Fill JM, Davies SJ, Louw M, Rebelo AD, Thorp CJ, Vimercati G, Measey J (2019) 367 A global meta-analysis of the ecological impacts of alien species on native amphibians. 368 Proceedings of the Royal Society B 286: 20182528. 369 https://doi.org/10.1098/rspb.2018.2528. 370 Nunes AL, Richter -Boix A, Laurila A, Rebelo R (2013) Do anuran larvae respond 371 behaviourally to chemical cues from an invasive crayfish predator? A community-wide 372 study. Oecologia 171: 115-127. https://doi.org/10.1007/s00442-012-2389-6. 373 Nyström P, Axelsson E, Sidenmark J, Brönmark C (1997) Crayfish predation on amphibian 374 eggs and larvae. Amphibia -Reptilia 18: 217 -228. 375 https://doi.org/10.1163/156853897X00107. 376 Petranka JW, Kats LB, Sih A (1987) Predator -prey interactions among fish and larval 377 amphibians: use of chemical cues to detect predatory fish. Animal Behaviour 35: 420 -378 425. https://doi.org/10.1016/S0003-3472(87)80266-X. 379 Polo-Cavia N, Gonzalo A, López P, Martín J (2010) Predator recognition of native but not 380 invasive turtle predators by n aïve anuran tadpoles. Animal Behaviour 80: 461 -466. 381 https://doi.org/10.1016/j.anbehav.2010.06.004. 382 Polo‐Cavia N, Gomez‐Mestre I (2014) Learned recognition of introduced predators determines 383 survival of tadpole prey. Functional Ecology 28: 432-439. https://doi.org/10.1111/1365-384 2435.12175. 385 Poo S, Bickford DP (2014) Hatching plasticity in a Southea st Asian tree frog. Behavioral 386 Ecology and Sociobiology 68: 1733-1740. https://doi.org/10.1007/s00265-014-1781-0. 387 Quinn G P, Keough MJ (2002) Experimental Design a nd Data Analysis for Biologists. 388 Cambridge University Press (Cambridge): 1-537. 389 Ramírez Albores JE, Badano EI, Flores Rivas JD, Flores Flores JL, Yáñez Espinosa L (2019) 390 Scientific literature on invasive alien species in a megadiverse country: advances and 391 challenges in Mexico. https://doi.org/10.3897/neobiota.48.36201. 392 Relyea, RA, Stephens, PR, Barrow, LN, Blaustein, AR, Bradley, PW, Buck, JC, Chang, A, 393 Collins, JP, Crother, B, Earl, J, Gervasi, SS (2018) Phylogenetic patterns of trait and 394 trait plasticity evolution: Insights from amphibian embryos. Evolution 72: 663-678. 395 https://doi.org/10.1111/evo.13428. 396 Richardson JTE (2011) Eta squared and partial eta squared as measures of effect size in 397 educational research. Educational Research Review 6: 135 -147. 398 https://doi.org/10.1016/j.edurev.2010.12.001. 399 Rolim RG, de Ferreira PMA, Schneider AA, Overbeck GE (2015) How much do we know 400 about distribution and ecology of naturalized and invasive alien plant species? A case 401 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 15 study from subtropical southern Brazil. Biological Invasions 17: 1497 -1518. 402 https://doi.org/10.1007/s10530-014-0811-1. 403 Saglio P, Mandrillon A -L (2006) Embryonic experience to predation risk affects tadpoles of 404 the common frog ( Rana temporaria ). Archiv fur Hydrobiologie: 505 -523. 405 10.1127/0003-9136/2006/0166-0505. 406 Segev O, Rodríguez A, Hauswaldt S, Hugemann K, Vences M (2015) Flatworms ( Schmidtea 407 nova) prey upon embryos of the common frog ( Rana temporaria) and induce minor 408 developmental acceleration. Amphibia -Reptilia 36: 155 -163. 10.1163/15685381 -409 00002992. 410 Schalk G, Forbes MR, Weatherhead PJ (2002) Development al plasticity and growth rates of 411 green frog (Rana clamitans) embryos and tadpoles in relation to a leech ( Macrobdella 412 decora) predator. Copeia 2002: 445 -449. https://doi.org/10.1643/0045-413 8511(2002)002[0445:DPAGRO]2.0.CO;2. 414 Sih A, Bolnick DI, Luttbeg B, Orrock JL, Peacor SD, Pintor LM, Preisser E, Rehage JS, Vonesh 415 JR (2010) Predator –prey naïveté, antipredator behavior, and the ecology of predator 416 invasions. Oikos 119: 610-621. https://doi.org/10.1111/j.1600-0706.2009.18039.x. 417 Sih A, Moore RD (1993) Delayed hatching of salamander eggs in response to enhanced larval 418 predation risk. The American Naturalist 142: 947-960. 419 Smith, D. C. (1987). Adult recruitment in chorus frogs: effects of size and date at 420 metamorphosis. Ecology, 68, 344-350. https://doi.org/10.2307/1939265 421 Smith GR, Fortune DT (2009) Hatching plasticity of wood frog ( Rana sylvatica ) eggs in 422 response to mosquitofish ( Gambusia affinis ) cues. Herpetological Conservation and 423 Biology 4: 43-47. 424 Speybroeck J, Beukema W, Bok B, Van Der Voort J (2016) Field guide to the amphibians and 425 reptiles of Britain and Europe. Bloomsbury Publishing, (London, UK): 1-434. 426 Standfuss B, Lipovšek G, Fritz U, Vamberger M (2016) Threat or fiction: is the pond slider 427 (Trachemys scripta) really invasive in Central Europe? A case study from Slovenia. 428 Conservation Genetics 17: 557-563. https://doi.org/10.1007/s10592-015-0805-2. 429 Stav G, Kotler BP, Blaustein L (2007) Direct and indirect effects of dragonfly (Anax imperator) 430 nymphs on green toad ( Bufo viridis ) tadpoles. Hydrobi ologia 579: 85 -93. 431 https://doi.org/10.1007/s10750-006-0388-5. 432 Steel, E. A., M. C. Kennedy, P. G. Cunningham, and J. S. Stanovick. 2013. Applied statistics 433 in ecology: common pitfalls an d simple solutions. Ecosphere 4 :115. 434 http://dx.doi.org/10.1890/ES13-00160.1. 435 Strauss SY, Lau JA, Carroll SP (2006) Evolutionary responses of natives to introduced species: 436 what do introductions tell us about natural communities? Ecology Letters 9: 357 -374. 437 https://doi.org/10.1111/j.1461-0248.2005.00874.x. 438 TIBCO. 2017, Tibco Statistica™ Quick Reference . 439 https://docs.tibco.com/pub/stat/13.3.0/doc/pdf/TIB_stat_13.3_quick_ref.pdf [accessed 440 6/2/2022] 441 Tollrian R (1994) Fish-kairomone induced morphological changes in Daphnia lumholtzi (Sars). 442 Archiv fur Hydrobiologie: 69 -75. https://doi.org/10.1127/archiv-443 hydrobiol/130/1994/69. 444 Touchon J, Gomez-Mestre I, Warkentin K (2006) Hatching plasticity in two temperate anurans: 445 responses to a pathogen and predation cues. Canadian Journal of Zoology 84: 556-563. 446 https://doi.org/10.1139/z06-058. 447 Touchon JC, McCoy MW, Vonesh JR, Warkentin KM (2013) Effects of plastic hatching timing 448 carry over through metamorphosis in red‐eyed treefrogs. Ecology 94: 850 -860. 449 https://doi.org/10.1890/12-0194.1. 450 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751 16 Touchon JC, Wojdak JM (2014) Plastic hatching timing by red-eyed treefrog embryos interacts 451 with larval predator identity and sublethal predation to affect prey morphology but not 452 performance. PLoS One 9: e100623. https://doi.org/10.1371/journal.pone.0100623. 453 Tricarico E, J unqueira AO, Dudgeon D (2016) Alien species in aquatic environments: a 454 selective comparison of coastal and inland waters in tropical and temperate latitudes. 455 Aquatic Conservation: Marine and Freshwater Ecosystems 26: 872 -891. 456 https://doi.org/10.1002/aqc.2711. 457 Vodrážková M, Šetlíková I, Berec M (2020) Chemical cues of an invasive turtle reduce 458 development time and size at metamorphosis in the common frog. Scientific Reports 459 10: 1-6. https://doi.org/10.1038/s41598-020-64899-0. 460 Vodrážková M, Šetlíková I, Berec M (in review) Different time patterns of the presence of red-461 eared slider influence the ontogeny dynamics of common frog tadpoles. Scientific 462 Reports: x-x. 463 Vonesh JR, Bolker BM (2005) Compensatory larval responses shift trade‐offs associated with 464 predator‐induced hatching plasticity. Ecology 86: 1580 -1591. 465 https://doi.org/10.1890/04-0535. 466 Warkentin KM (1995) Adaptive plasticity in hatching age: a response to predation risk trade -467 offs. Proceedings of the National Academy of Sciences 92: 3507 -3510. 468 https://doi.org/10.1073/pnas.92.8.3507. 469 Warkentin KM (2000) Wasp predation and wasp -induced hatching of red -eyed treefrog eggs. 470 Animal Behaviour 60: 503-510. https://doi.org/10.1006/anbe.2000.1508. 471 Warkentin KM (2011) Plasticity of hatching in amphibians: evol ution, trade-offs, cues and 472 mechanisms. Integrative and Comparative Biology 51: 111 -127. 473 https://doi.org/10.1093/icb/icr046. 474 Wells KD (2007) The Ecology and Behavior of Amphibians. The University of Chicago Press, 475 (Chicago): 1-114. 476 Author-formatted, not peer-reviewed document posted on 25/02/2022. DOI:  https://doi.org/10.3897/arphapreprints.e82751