Oviposition strategies of amphibians in ephemeral streams: a multi-species perspective

Oviposition strategies of amphibians in ephemeral streams: a multi-species perspective | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oviposition strategies of amphibians in ephemeral streams: a multi-species perspective Dogeun Oh, Jaehun Kang, Uhram Song, JeongYoon Ahn, Changku Kang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4019240/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Many amphibians are selective about where to lay eggs, as this greatly affects their offpsring’s survival. Theoretically, species sharing the same habitat are expected to experience similar selective pressures, leading to the prediction that they might share similar oviposition preferences. However, this hypothesis has not yet been tested. In this study, through an extensive field survey, we examined the oviposition site selection of four amphibian species (Rana uenoi, Hynobius quelpaertensis, Bombina orientalis, and Dryophytes japonicus) that reproduce in the same natural ephemeral stream. Our focus was on understanding how various abiotic and biotic factors influence their reproductive choices. We primarily found that drought avoidance is a universal selective pressure affecting all species, with a tendency to avoid laying eggs in smaller pools prone to drought. Species-specific responses to leaf litter and canopy coverage were observed, but none of the species' oviposition choices correlated with the quantity of stones in pools. The study also explored biotic influences, revealing species-specific trends in the selection of pools with conspecific and heterospecific juveniles, predators, and mosquito larvae, indicating a complex ecological interplay. These findings highlight the complexity of amphibian reproductive strategies, where decisions are not driven solely by a single factor such as the avoidance of predators or competitors but also by intricate assessments of multiple factors. The study highlights that amphibian oviposition in ephemeral streams is influenced by a diverse interplay of biotic and abiotic factors, essential for understanding their reproductive strategies in dynamic environments. ephemeral pool anuran reproduction predator avoidance conspecific avoidance multiple factors Figures Figure 1 Figure 2 Figure 3 Significance statement Where and when eggs are deposited greatly impacts offspring survival in egg-laying species, particularly in fluctuating environments. Despite extensive research on egg-laying behaviors, mainly focusing on single species, there is a knowledge gap regarding the behavior of multiple species sharing the same habitat. We hypothesized species sharing the same habitat might exhibit analogous egg-laying choices due to shared selective pressures. A thorough field study on four amphibian species in an ephemeral stream reveals a shared trend of avoiding drought among all species. Additionally, various biotic and abiotic factors influenced egg-laying decisions differently across species. Surprisingly, factors detrimental to juvenile survival, such as predators or competitors, were not consistently avoided in natural systems. Introduction Animals have evolved diverse parental care strategies to increase the survival of their progeny (Clutton-Brock 1991). For example, many species show nesting, guarding, or brooding behaviours to directly protect and care for their young (Royle et al. 2012). Parental care varies widely between taxa: many birds and mammals have evolved the costly parental feeding while other vertebrates and invertebrates often have simpler forms of parental behaviours that do not involve direct feeding of their offspring (Balshine 2012; Trumbo 2012). These simpler forms include the lack of post-laying care. In such animals, the choice of where and when to lay eggs has profound consequences on the reproductive success, especially when the eggs and/or juvenile stages are immobile or confined to local regions (Doak et al. 2006; Refsnider and Janzen 2010). Thus, natural selection favours the ability of parents to prefer oviposition sites that increase the chance of offspring survival, their performance, or even parental survival (Brodin et al. 2006; Hirayama and Kasuya 2013; Dimitrie and Benard 2023). There has been ample evidence of the selective oviposition in wide array of taxa including insects, reptiles, amphibians, and fish (Schwarzkopf and Brooks 1987; Spieler and Linsenmair 1997; Mokany and Shine 2003a). Oviposition site selection is more likely to evolve under conditions when (i) potential egg-depositing sites are temporally and spatially heterogeneous in terms of suitability for offspring survival, (ii) juveniles are unable to disperse from the egg-spawning spots, and (iii) parents are able to evaluate suitable sites by comparison of alternatives (Resetarits Jr 1996). Amphibians in highly ephemeral environments present an excellent platform for studying oviposition site selection because the habitat structure often meets all of the above conditions: (i) potential oviposition sites are locally scattered, allowing parents to compare the suitability of multiple pools, (ii) the characteristics (i.e. suitability as a egg-laying site) of pools are often both spatially and temporally varying, and (iii) juveniles are restricted to the pools where mothers laid eggs unless disturbed (e.g. heavy rains) (Buxton and Sperry 2017). Indeed, amphibians in ephemeral streams have been extensively studied for their selective oviposition (Spieler and Linsenmair 1997; Murphy 2003; Baek et al. 2021; Gould et al. 2021). Both biotic and abiotic factors have been demonstrated to affect female choice of amphibians. Mothers often choose oviposion sites based on the degree of risk from predators, conspecifics or heterospecifics. Predators are ubiquitous problems for eggs/juveniles and yield negative consequences for offspring survival not only through direct predation but also through non-consumptive effects (Preisser et al. 2005; Buxton and Sperry 2017; Buxton et al. 2017). Naturally, many studies have revealed the presence of predator avoidance (Buxton and Sperry 2017; Resetarits et al. 2021). The presence of conspecifics or heterospecifics of related taxa may function as predators (through cannibalism) or putative competitors but may also give a cue about site quality (Dillon and Fiaño 2000; Rudolf and Rödel 2005; Gould et al. 2020). The risk of cannibalism also affects female choice, and mothers may avoid the sites with higher risk of cannibalism (Iwai et al. 2007); however, reducing the risk of cannibalism does not appear to be universal, with some notable exceptions (Poelman and Dicke 2007; Rojas 2014). Food availability, such as leaf litter, could also affect female choice especially when egg-laying sites are small and isolated (McDiarmid and Altig 1999; Stoler and Relyea 2021; Magee-Christian and Earl 2022). When it comes into abiotic factors, temperature and water are demonstrated as main selective agents because the avoidance of thermal damage or desiccation is critical for juvenile survival and development (Spieler and Linsenmair 1997; Goldberg et al. 2006). To date, studies on female oviposition preference have mostly been conducted on a single species, and studies that examined the selective oviposition in multi-species level are surprisingly scarce (Miaud 1995; Ortiz-Ross et al. 2020). However, various species of amphibians co-occur in the same habitat and one can predict that species that co-occur in the same habitat would be under similar selective pressures for oviposition or affect each other interactively. In this study, we investigated the oviposition choice of multiple amphibian species that reproduce in the same ephemeral stream to (i) identify biotic and abiotic factors that correlate with female oviposition choice for each species and (ii) investigate that species that occur in the same habitat (thus likely to be under similar selective pressures) show similar oviposition decisions. We formulated certain predictions regarding assemblage-wide or species-specific responses to several abiotic and biotic factors. About assemblage-wide responses, we firstly predict that natural selection may shape female behaviours to avoid the pools with high risk of dessication (Spieler and Linsenmair 1997; Goldberg et al. 2006). Second, females would selectively lay eggs in pools with sufficient organic matters or potential food materials for tadpoles (Kern et al. 2013; Magee-Christian and Earl 2022). Third, predation (or cannibalism) risk might also affect female choice. This includes (i) the avoidance of pools that are already occupied by aquatic predators and (ii) laying eggs in pools with potential refuges (Resetarits Jr and Wilbur 1989). In contrast, there are certain factors that we predict species-specific or circumstantial responses. First, in regards to the presence of con-/heterospecific juveniles, we had two competing predictions. Females might avoid to lay eggs in pools with con-/heterospecific juveniles to avoid the risk of cannibalism. Alternatively, the presence of other juveniles may be an indicator of pool quality or decrease individual risk of predation through dilution (Rudolf and Rödel 2005). In that case, females may selectively lay eggs in pools with other juveniles. Second, canopy coverage may also affect female choice in two different ways. Pools with less canopy coverage should be exposed to the sunlight directly which can cause faster evaporation of water. In that case, we predict the females to avoid laying eggs in pools under low canopy covergae. However, under the direct sunlight, the water temperature can be maintained higher than those under high canopy coverage which can boost development, a favourable condition in ephemeral streams (Oh et al. 2021). In the latter scenario, we predict females to selectively lay eggs in pools under higher canopy coverage. Materials And Methods Study species and environments We conducted this study within Bangcheon stream in Jeju Island, South Korea (N33.4722, E126.5432). Jeju Island, a volcanic island, is largely covered by basalt layers that quickly drain surface water underground. As a consequence, in most streams, including our study site, water flows only during rain. In these ephemeral streams, oviposition sites were available in the form of pools of varying sizes. Along the stream, the pools were patchily distributed, with distances varying from less than a metre between each pool and its nearest neighbour (Fig 1A). Each pool remained isolated until rainfall occurred. Heavy rains usually swept most, if not all, organisms in the pools. Rains did not alter the location of each pool, although some pools temporarily merged into larger ones just after heavy rains. There were no fish in our study sites. We chose a specific study site occupying approximately 730 m 2 area. This area spanned a longitudinal distance of approximately 120 metres along the stream and contained 321 different pools when estimated just after rain when all available pools can be identified. In the study site, three frogs and one salamander species mainly occurred and reproduced during our study period from 8th March to 24th August, 2021: Rana uenoi , Hynobius quelpaertensis , Bombina orientalis , and Dryphytes japonicus . While Kaloula borealis also laid some eggs in our study site, their numbers were only a few and we did not include K. borealis in our analysis. The timing of reproduction largely overlapped between R. ueonoi and H. quelpaertensis , and between B. orientalis and D. japonicus (Fig. 1B). For R. uenoi , although reproduction started before our study period, we only analysed the eggs laid during our study period. Field surveys to study oviposition preference in relation to abiotic and biotic factors To monitor the oviposition choices of females, we visited all available pools within our study site between 0800 and 1300 on every non-rainy day of our study period. We refrained from visiting the stream on rainy days for the safety of the experimenters. Each pool was marked with a unique number on a rock surface adjacent to it, using non-toxic silicone. We recorded several biotic and abiotic characteristics of each pool daily (see Table 1): (i) whether water was present or absent (Dry; a binary variable), (ii) the amount of leaf litter (Leaves; an ordinal variable), (iii) the quantity of stones as potential refuges (Stones; ordinal), (iv) the turbidity of the pool water (Turbidity; ordinal), (v) the presence of aquatic predators (Predators; binary), and (vi) the presence of mosquito larvae (MosqLarvae; binary). Variables concerning the presence of amphibian juveniles were coded for each species as follows: (vii) the presence of newly spawned eggs (NewEggs; binary), (viii) the presence of conspecific eggs that were not newly spawned (ConEggs; binary), (ix) the presence of conspecific tadpoles (ConTadpoles; binary), (x) the presence of heterospecific eggs that were not newly spawned (HeteroEggs; binary), and (xi) the presence of heterospecific tadpoles (HeteroTadpoles; binary). Although we initially started by counting the number of con-/heterospecific eggs and tadpoles, we decided to use these variables in binary form due to the heterogeneity of errors among pools; pools with larger size, more turbid water, or a higher number of stones were more likely to have inaccuracies in their counts. Leaves, Stones, and Turbidity (all ordinal variables) were subjectively but consistently assessed and rated by a single experimenter (DO) throughout the survey. The criteria can be found in Table 1. Mosquito larvae have primarily been described as competitors, but also as predators or prey of tadpoles (Blaustein and Margalit 1994, 1996; Mokany and Shine 2003b). While we were unsure of the ecological relationship between our studied species and mosquito larvae, we nonetheless analysed correlational patterns between their presence and female oviposition choice. Invertebrates other than mosquito larvae, such as mayfly or Chironomidae larvae, were also occasionally present, but their occurrence was very low (less than 0.3% of pools) and thus were not included in our analysis. Occasionally, a pool was transiently divided into multiple pools (under consecutive non-rainy days), or two pools merged together (this rarely happened just after rain). When a pool divided, we recorded the characteristics of each separate pool and used the averaged value for analysis, retaining the original pool ID for these transiently separated pools. When pools merged, we noted the characteristics of the merged pool and applied them to both original pools. During the breeding seasons of R. uenoi and H. quelpaertensis , from March to May, all 321 pools were surveyed. However, for the breeding seasons of B. orientalis and D. japonicus , spanning May to August, we limited the survey to 192 pools in the lower parts of our study site, where more oviposition activities were observed. This reduced scale was necessary to complete the surveys in a timely manner, as the increased oviposition activities resulted in longer times required to survey each pool. Egg clutches from different females were clearly distinguishable for R. uenoi and H. quelpaertensis , in which eggs were either densely grouped together or found in a sac. However, distinguishing eggs from different mothers in the same pool posed a challenge for B. orientalis and D. japonicus . This difficulty arose because not only was the number of eggs highly variable, but also the boundaries between different egg clutches were not clearly defined. Therefore, instead of counting the number of egg clutches, we employed a binary variable to denote whether newly spawned eggs were found in each pool each day. This approach, however, resulted in an inability to detect instances where more than one female laid eggs in the same pool on the same day. In addition to the pool characteristics that we surveyed daily, we also measured (i) pool volume (PoolVolume) and (ii) canopy coverage (CanopyCover). Unlike the aforementioned variables, these two characteristics were measured only once during our study period. While PoolVolume could change daily (decreasing gradually due to evaporation but increasing during rain), all pools were expected to be simultaneously affected by these changes. Therefore, we measured PoolVolume only once and used this as a size index for each pool. PoolVolume was estimated just after a rainy day when all pools were fully filled with water. The three-dimensional shape of each pool was highly irregular; thus, we crudely estimated PoolVolume by multiplying the surface area by the deepest depth of each pool. The surface area was measured by aerial photography using a drone (MAVIC PRO, DJI, Guangdong, China), with a ruler placed next to each pool for area calibration. CanopyCover was estimated using a mobile phone camera (Galaxy Note 8, Samsung, Seoul, South Korea). This measurement was taken on a summer day when foliage was dense. We positioned the camera just above the suspected centroid of the water surface, facing upwards, and took a photograph. The percentage of plant cover in the image was then calculated, using this percentage as an index of CanopyCover with ImageJ 1.80 (National Institute of Health, Maryland, USA). The camera’s angle of view was 77°, as specified by the manufacturer. Abiotic factor analysis We considered Dry, Leaves, Stones, Turbidity, PoolVolume, and CanopyCover as abiotic factors that could potentially affect female oviposition choice. All analyses were conducted separately for each species. Firstly, we calculated a representative index for each variable for each pool, generating a pool-level dataset. For Dry, we calculated the proportion of the days that each pool found to be dry during the breeding season of each species. For Leaves, Stones, and Turbidity, we first examined whether the intra-pool variation of each variable across the study period was smaller than the inter-pool variation. For this, we employed the intraclass correlation coefficient (ICC) test in the ‘irr’ package in R, using a one-way consistency model (Koo and Li 2016). Within-pool variability was lower than among-pool variability for all three variables (all P < 0.001, estimated ICC ranged from 0.39 to 0.77). Then, for each pool, we averaged each variable across the breeding season and used these averaged values to characterise each pool. PoolVolume was log-transformed to reduce distributional skewness. After all these processes, we had one representative value for each abiotic factor for each pool. We excluded transiently appearing pools in the abiotic factor analysis (i.e., when either a pool was divided into multiple pools during consecutive non-rainy days due to the exposure of bottom surfaces, or multiple pools were connected to each other just after rain) because averaging across the entire breeding season was impossible for those temporary pools. As an index of each pool's oviposition frequency, we employed two different methods depending on the species. For R. uenoi and H. quelpaertensis , we used a binary variable to indicate whether eggs were found in each pool at least once during the breeding season. This approach was chosen because (i) the total number of egg clutches per pool was generally one or a few, and (ii) a small fraction of pools (14% for R. uenoi and 7% for H. quelpaertensis ) were used for oviposition. Conversely, for B. orientalis and D. japonicus , a higher number of pools were used for oviposition (56% and 36%, respectively), with each pool often being used multiple times on different dates. Therefore, we counted the number of days new eggs were found in each pool throughout the breeding season and used this count variable for our analysis. We fitted generalised linear models (GLZs) to examine the effect of abiotic variables on female oviposition choice. Firstly, we examined the presence of multicollinearity among predictors and found that Leaves and Turbidity were highly correlated ( Pearson’s r > 0.78 for all species data). To resolve the multicollinearity issue, we removed the Turbidity variable from further analysis, leaving only five abiotic factors (Dry, Leaves, Stones, Pool Volume, and Canopy Cover). For R. uenoi and H. quelpaertensis , we fitted binomial GLZs using the binary response of whether each pool was used for oviposition at least once and the five abiotic factors as predictors. For B. orientalis and D. japonicus , we used the number of days new eggs were found during the breeding season for each pool as a response variable and the abiotic factors as predictors. Overdispersion was detected when we fitted Poisson GLZs, so we instead fitted negative binomial GLZs (Lindén and Mäntyniemi 2011). Visual inspections of predictors and our response variables suggested putative polynomial relationships for Leaves and PoolVolume for B. orientalis and D. japonicus ; thus, we additionally included quadratic terms of Leaves and PoolVolume for these two species. We did not include interaction terms in our predictors because (i) we did not specifically predict interactive effects among the predictors, and (ii) with five main predictors, including unplanned interaction terms leads to the addition of 10 extra predictors (more when polynomial terms are simultaneously considered) for each species, which makes interpretations challenging in our multi-species datasets. To estimate the parameters of variables and predict their effects, we applied multimodel inference and performed model averaging using the ‘MuMIn’ package (Grueber et al. 2011). We averaged the parameters of models that appeared within the 0.95 cumulative sum of AIC weights using conditional averaging. Biotic factor analysis We considered Predators, MosqLarvae, ConEggs, ConTadpoles, HeteroEggs, HeteroTadpoles as biotic factors that could potentially affect female oviposition site choice. For the main results, we did not distinguish between eggs and tadpoles, thus using the merged categories of ConEggs/Tadpoles (where either or both conspecific eggs or tadpoles were present) and HeteroEggs/Tadpoles as variables, in order to keep the main results from being overly complex. However, we nonetheless performed additional analyses, considering eggs and tadpoles separately, and included these results in the Supporting Information, which are also discussed. Unlike abiotic factors, which did not vary much throughout the breeding season within each pool, biotic conditions changed frequently; thus, generating and analysing averaged pool-level data was inappropriate. The most ecologically relevant approach may be analysing each day separately, as the female's decision was made by comparing the conditions of currently available pools. However, this was statistically challenging in our data because only a limited number of pools were found with new eggs on most days, making GLZ analysis for each day impossible due to the excessive number of pools with no new eggs each day. Instead, we pooled the data across our survey period for each species, generated frequency tables, and examined whether the frequency of eggs laid in pools with specific biotic conditions deviated from what would be expected if females randomly laid eggs without considering the biotic condition. Pooling the data across all survey dates was necessary due to the low number of new egg clutches found in each individual survey (often fewer than three), which precluded us from estimating the expected frequencies for each specific date. In the biotic factor analysis, we were able to include temporary pools in the analysis, but their numbers were minor (less than 1% of the total pools). Firstly, we excluded the pools that had never been used during the study period from the analysis. We considered that these pools were not used for oviposition, regardless of the biotic conditions, likely because the abiotic conditions were not favourable to frogs. For example, pools that were substantially smaller than other pools were never used for oviposition in all four species. Thus, we compared the biotic conditions among the pools that had been used for oviposition at least once for each species. Then, for each biotic factor, we calculated the frequency of available pools and the pools containing a specific biotic factor. For instance, to investigate the potential influence of the presence of predators on female oviposition choice, we determined the frequency of available pools and the pools containing predators during each survey. Specifically, we used the biotic conditions from the day before newly spawned eggs were found because female oviposition mostly, if not always, occurred during the night. Therefore, the conditions experienced by females were not those in which the new eggs were found, but rather the conditions of the previous day. These frequencies were subsequently pooled across all survey dates. Utilising this information, we estimated the expected frequencies of pools with and without egg clutches if female frogs randomly laid their eggs irrespective of the presence of predators. Subsequently, we constructed a contingency table using the frequencies (expected and observed frequencies of pools with egg clutches in both predator-free and predator-present pools) and conducted a chi-square test. We performed the above procedures for all biotic factors. Results Correlational patterns between abiotic factors and female oviposition choice The results are summarised in Table 2 (showing significance and trends), Fig. 2, and Table S1 (detailed statistics). In all four species, PoolVolume and Dry consistently affected the oviposition choice of females. Specifically, all species tended to avoid laying eggs in pools where the risk of desiccation was higher. Also, all species were more likely to lay eggs in larger pools over smaller ones, except for B. orientalis , which laid more eggs in intermediate-sized pools compared to smaller or larger ones. In contrast, we found no evidence that Stones affected female oviposition choice in any of the species. The effect of Leaves and CanopyCover varied among species. H. quelpaertensis and B. orientalis females laid eggs more frequently in pools with more leaf litter and in pools with intermediate-amount of leaf litter, respectively. In contrast, Leaves did not affect the oviposition choice of R. uenoi and D. japonicus . In terms of CanopyCover, D. japonicus females were more likely to lay eggs in pools under higher canopy coverage, while other species did not show such patterns. Correlational patterns between biotic factors and female oviposition choice The correlational patterns between biotic factors and female oviposition preferences were species-specific (see Table 2 for the summary, Fig. 3 for plots, and Table S2 for detailed statistics). The results of the effect of Predators and MosqLarvae were not evident for R. uenoi and H. quelpaertensis , as both predators and mosquito larvae were scarcely present during their breeding seasons. In both R. uenoi and H. quelpaertensis , the observed frequencies of pools with newly spawned eggs significantly deviated from the frequencies expected if females were laying eggs irrespective of the presence of ConEggs/Tadpoles. When we separately analysed the presence of ConEggs and ConTadpoles, we found that the avoidance of ConTadpoles, not ConEggs, drove the observed patterns (see Fig. S1). While pools with HeteroEggs/Tadpoles tended to be avoided in the main results, separate analyses did not reveal the same patterns (Fig. S2), suggesting that the avoidance of heterospecifics is not as apparent as the avoidance of conspecifics in R. uenoi . In H. quelpaertensis , pools with ConEggs/Tadpoles were avoided, which was mainly driven by the avoidance of ConEggs (Fig. 3; Fig. S1), but pools with HeteroEggs/Tadpoles were not avoided. In B. orientalis , we did not find any patterns related to either ConEggs/Tadpoles or HeteroEggs/Tadpoles in the main analysis (Table 2; Fig. 3). However, separate analyses for eggs and tadpoles revealed that female B. orientalis laid eggs in pools with HeteroEggs already present more frequently than would be expected by random chance, but avoided laying eggs in pools with HeteroTadpoles (Fig. S2). In contrast, in D. japonicus , the observed frequencies of pools with newly spawned eggs significantly deviated from the frequencies expected by random chance for both ConEggs/Tadpoles and HeteroEggs/Tadpoles. Female D. japonicus tended to lay eggs in pools containing both ConEggs/Tadpoles and HeteroEggs/Tadpoles (Table 2; Fig. 2). Separate analyses showed that D. japonicus laid more eggs in pools with both ConEggs, ConTadpoles, HeteroTadpoles but not HeteroEggs (Fig. S1,2). The observed frequencies of pools with newly spawned eggs did not significantly deviate from the frequencies expected by random chance for Predators and MosqLarvae, except in D. japonicus ; they laid eggs in pools with Predators and MosqLarvae more frequently than would be expected by random chance (Table 2; Fig. 3). Discussion Our results firstly reveal that the risk of drought is an important factor in oviposition site choice, shared by all four species; all species selectively laid eggs in pools where the risk of pool desiccation was lower. This is reflected in the patterns where all species avoided laying eggs in small-sized pools and in those that had experienced more frequent drought conditions. These results firmly demonstrate that desiccaion is a universal selective pressure that shared by all studied species. The results also align well with accumulated evidence that many animals evolved to lay eggs in places with lower risk of desiccation in ephemeral environments (Rudolf and Rödel 2005; Goldberg et al. 2006; Kern et al. 2013; Baek et al. 2021). The results that B. orientalis laid more eggs in intermediate-sized pools corrobrates a previous study, even though larger pools are less likely to experience drought (Baek et al. 2021). One potential reason is that the water temperature in large pools remains cooler even in summer, thus delaying the development time of juveniles (Álvarez and Nicieza 2002). Ephemeral streams are not a favourable environment for tadpoles due to the high risk of desiccation, flooding, scarcity of food sources, and limited space for movement (Gould et al. 2022). Thus, faster development time is often preferred in ephemeral streams (Richter‐Boix et al. 2011; Oh et al. 2021). In this context, the avoidance of larger pools can be adaptive because juvenile development is more rapid in smaller-sized pools where the water temperature can easily warm during the daytime (see Fig. S3 showing that large pools were cooler than small pools in our study site). However, it is still uncertain why the other species do not exhibit similar large-pool avoidance behaviours. The breeding seasons of R. uenoi and H. quelpaertensis are before summer, thus they may be adapted to lower temperatures. Also, D. japonicus lays a substantially larger number eggs at once than B. orientalis ; thus, D. japonicus may require more space for their progeny, which could also lead to the absence of large-pool avoidance. For the rest of the abiotic factors, we found that oviposition choice was either species-specific or not present at all. Firstly, the amount of leaf litter was correlated with oviposition choice in two species: H. quelpaertensis and B. orientalis . Leaf litter is one of the main food sources for tadpoles (McDiarmid and Altig 1999), so it is not surprising that these two species avoided pools with less leaf litter. The reason for the avoidance of pools with a large amount of leaf litter in B. orientalis may be that those with the highest level of leaf litter at our study site were almost filled with it, thus being highly turbid, leaving less space for tadpole movement and probably having a low amount of oxygen. In contrast, during the breeding season of H. quelpaertensis (early spring when new leaves had not yet proliferated), most of the leaf litter was the remnants of the previous year, so no pools were fully filled with leaf litter. This phenological difference may affect the differences between the two species. The reasons why the other two species, R. uenoi and D. japonicus , did not show any preference for leaf litter is puzzling. Ecological and behavioural differences may be a potential explanation; R. uenoi and D. japonicus lay substantially more eggs in a single pool than the other two species (several hundred versus 10-100 eggs). Therefore, R. uenoi and D. japonicus may be better equiped to allleviate the costs of cannibalism and utilise alternative food sources (e.g., through cannibalism) compared to the other two species. This may explain the species-specific responses to the amount of leaf litter. Both canopy coverage and the presence of stones were hardly correlated with oviposition choice, except for D. japonicus , which preferentially laid eggs in pools with open canopies. We had two competing predictions for canopy coverage, and D. japonicus females were more likely to lay eggs in pools that were at a higher risk of desiccation but warmer. As D. japonicus selectively lay eggs in larger-sized pools, this may alleviate the risk of desiccation. The lack of correlation between the amount of stone and oviposition preference suggests that the presence of potential refuges is not a strong driver of oviposition site selection. In our study sites, predation pressures from the predators that refuges could benefit (e.g., terrestrial predators) may not be strong. In terms of biotic factors, R. uenoi and H. quelpaertensis laid eggs in pools without conspecific juveniles more frequently than would be expected by chance. This corroborates our prediction, as well as other studies, that mothers would lay eggs where the risk of cannibalism is low (Spieler and Linsenmair 1997; Iwai et al. 2007). The evidence for avoiding pools with heterospecifics is equivocal; a weak trend was found in R. uenoi but not in H. quelpaertensis . The total number of egg clutches found at our study sites was not very high for both R. uenoi and H. quelpaertensis throughout our study period (68 for R. uenoi and 30 for H. quelpaertensis ). Thus, we consider that either or both (i) statistical power was low for these two species to reveal the true patterns, especially when we separately analysed eggs and tadpoles, and/or (ii) the encounter rates between these two species were too low to drive avoidance behaviours. Indeed, visual inspection of the patterns suggests that the oviposition patterns seem to deviate from what would be expected by random chance (see Fig. S1,2), but the absence of statistical support for this difference suggests that statistical power was low. Thus, our evidence for female avoidance of heterospecific juveniles in these two species remains inconclusive. Unexpectedly, B. orientalis did not show any avoidance of pools with conspecific juveniles. This contrasts with a previous finding that showed conspecific avoidance (Baek et al. 2021). The discrepancy between Baek et al.’s study (2021) and ours may be due to the sampling interval; Baek et al. sampled twice per week, while we conducted the survey on a daily basis. Thus, it is possible that, in Baek et al.’s study, those that laid eggs in pools with others’ juveniles were quickly cannibalised, so that an intermittent survey would not reveal the true oviposition patterns. Indeed, in their own study, most of the newly spawned eggs were cannibalised under the presence of conspecific tadpoles within 24 hours. Considering that (i) egg cannibalism commonly occurs in this population (Baek et al. 2021; Oh et al. 2021), and (ii) there were always some available pools without conspecifics throughout the seasons, it is unclear why B. orientalis did not show any avoidance of others’ juveniles. D. japonicus even preferred to lay eggs in pools with others’ eggs/juveniles present. Possibly, during the breeding seasons of R. uenoi and H. quelpaertensis , pools were mostly empty, leaving a variety of options for females on where to lay eggs after avoiding the filled ones. However, during the breeding seasons of B. orientalis and D. japonicus , a greater number of pools (that were probably preferred) were already filled on many days, thus the costs of choosing empty and likely less-preferred pools may not outweigh the benefits of choosing filled but more-preferred pools. Addiionally, the preference to oviposit in pools that already occuppeid might have adaptive advantage in ephemeral pools because it can supply additional nutrient via cannibalism or decrease predatory risk through dilution effects (Doody et al. 2009; Buxton and Sperry 2017; Gould et al. 2021). While our initial prediction was the avoidance of both predators and mosquito larvae, the presence of these organisms did not correlate with the oviposition choice in B. orientalis but did correlate in D. japonicus in an unexpected manner; D. japonicus laid more eggs in pools with predators or mosquito larvae already present than would be expected by chance. One potential reason behind this observed pattern may be that the pool preference of predators (or mosquitoes) matches that of frogs. Indeed, in our data, there were positive correlations between the number of days new eggs were found and the number of days predators were present for each pool (Fig. S4). The same correlations were found with mosquito larvae (Fig. S4). Therefore, there may be conflicting pressures in the wild; while avoiding organisms with any negative interactions may benefit individuals, the choice is not simple in reality because the competitors or predators can share similar preferences for certain abiotic conditions (Giao and Godoy 2007), such as those with a low risk of desiccation. The oviposition patterns in D. japonicus could be observed if their preference for abiotic factors overrides predator/competitor avoidance. This constrasts a previous finding that the risk of predators was prioritised over desiccation risk in the pantless treefrog which lay both aquatic and arboreal eggs (Touchon and Worley 2015). Whether D. japonicus and B. orientalis indeed show preference for, or an avertion to, pools with predators or mosquito larvae requires experimental work in manipulative environments. However, our results imply that such preference/avoidance may conflict with abiotic condition preference if predators or competitors share similar preferences. Will the observed selective oviposition have fitness consequences? Although our data cannot confirm this due to the inability to track individuals, most of the eggs and juveniles were flushed out during occasional heavy rain before they become froglets. We observed only a few individuals that became froglets at our study site, but even it was unclear whether they had grown in the same pool or been flushed down from upstream. Therefore, we consider that the oviposition choice of females may have limited consequences only during the eggs and early juvenile stages before subsequent heavy rain events. Nevertheless, natural selection would favour an aversion to pools with a higher risk of desiccation, as this could lead to the complete death of the progeny at any time. After accounting for avoiding desiccation, the impact of other abiotic/biotic factors may become not straightforward because pools with a low risk of desiccation are fewer, making the choice multifaceted. This assumes that female amphibians prioritise the risk of desiccation over other factors when selecting an egg-laying site, which remains to be tested. Ephemeral environments are complex systems with many biotic and abiotic factors interacting. In these circumstances, females may not decide where to lay eggs based on individual factors but rather use a holistic evaluation of the overall adequacy of the sites (Stahlschmidt and Adamo 2013; Gould et al. 2021). Although our study is observational and therefore does not reveal the causality of the relationship, the important insights that can be drawn from our study include (i) avoiding drought appears to be a universal driver shared by all amphibian species in the amphibian assemblage of ephemeral streams, (ii) both abiotic and biotic factors correlate with the oviposition choice of females, showing species-specific patterns, and (iii) factors that apparently have negative consequences for juveniles, such as predators/competitors, are not always avoided. This could be because predators or competitors share similar pool preferences; thus, avoiding these organisms could lead to laying eggs in less profitable pools from other perspectives (Marsh and Borrell 2001; Gould et al. 2021). We consider that employing cost-benefit approaches, which account for the interplay of multiple factors, would advance our understanding of female oviposition behaviour in natural systems. Declarations Author contributions CK, DO, and US designed the study. DO and JL conducted field studies. DO, J-Y A, CK performed data analyses. DO and CK wrote the first version of the mansucript. All authors contributed in revising the manuscript. Acknowledgments We appreciate Sangeun Gwak, Seokhyun Lee for their invaluable assistant during the field survey. This study was supported by National Research Foundation of Korea (NRF-2019R1C1C1002466, RS-2023-00239493) and Creative-Pioneering Researchers Program through Seoul National University. Competing interests We declare no conflict of interest. Ethics approval All field studies were approved by Mokpo National University Institutional Animal Care and Use Committee (MNU-IACUC-2021-002). We adhered to the guidelines for the treatment of animals in behavioural research and teaching (Buchanan et al. 2012). Data availability Data available from Figshare repository (https://doi.org/10.6084/m9.figshare.25009685.v2) (Oh et al. 2024) Code availability The code used for analyses and figures in this manuscript are available from Figshare repository (https://doi.org/10.6084/m9.figshare.25009685) (Oh et al. 2024) Consent to participate Not applicable Consent for publication Not applicable References Álvarez D, Nicieza AG (2002) Effects of temperature and food quality on anuran larval growth and metamorphosis. Funct Ecol 16:640–648. https://doi.org/10.1046/j.1365-2435.2002.00658.x Baek S-Y, Lee M-H, Kim Y-S, et al (2021) Selective oviposition by oriental fire-bellied toads in temporally fluctuating environments. Curr Herpetol 40:120–128. https://doi.org/10.5358/hsj.40.120 Balshine S (2012) Patterns of parental care in vertebrates. Evol Parent care 62–80 Blaustein L, Margalit J (1994) Mosquito larvae (Culiseta longiareolata) prey upon and compete with toad tadpoles (Bufo viridis). 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Ichthyol Herpetol 109:785–790. https://doi.org/10.1643/h2020090 Touchon JC, Worley JL (2015) Oviposition site choice under conflicting risks demonstrates that aquatic predators drive terrestrial egg-laying. Proc R Soc B Biol Sci 282:20150376. https://doi.org/10.1098/rspb.2015.0376 Trumbo ST (2012) Patterns of parental care in invertebrates. Evol Parent care 81–100 Tables Table 1. Variables measured for each pool during the field survey. Variable Explanation Measured interval Measured scale Criteria used Dry Whether each pool had water or not Daily Presence/absence (binary) Leaves The amount of leaf litter Daily 0 – 4 (ordinal) 0: No leaf litter 1: A small amount of leaf litter covering less than 5 % of pool bottom 2: Leaf litter covering more than 5% but less than 1/3 of the pool bottom 3: Leaf litter covering about half of the pool bottom 4: Pool bottom fully covered with leaf litter Stones The amount of stones Daily 0 – 3 (ordinal) 0: No stones 1: A few small stones that covered less than 1/3 of the pool bottom 2: Stones covering about half of the pool bottom 3: Stones fully covering the pool bottom Turbidity The degree of water turbidity Daily 0 – 3 (ordinal) 0: Fully transparent with no signs of turbidity 1: Low turbidity; slight murkiness detectable, but underwater visibility remains clear 2: High turbidity; underwater visibility is reduced but still possible at close range 3: Severe turbidity; underwater visibility is virtually impossible NewEggs Whether new eggs, oviposited the previous night, were found Daily Presence/absence (binary) ConEggs The presence of conspecific eggs Daily Presence/absence (binary) ConTadpoles The presence of conspecific tadpoles Daily Presence/absence (binary) HeteroEggs The presence of heterospecific eggs Daily Presence/absence (binary) HeteroTadpoles The presence of heterospecific tadpoles Daily Presence/absence (binary) Predators The presence of aquatic insect predators Daily Presence/absence (binary) Cybister chinensis Laccotrphes japonensis Notonecta triguttata Odonate larvae MosqLarvae The presence of mosquito larvae Daily Presence/absence (binary) PoolVolume Pool volume Once Continuous CanopyCover Canopy coverage Once Percentage Table 2. Summary of the results showing the factors that were correlated with oviposition preferences. ‘+’ and ‘–’ symbol indicates positive and negative correlation, respectively. ‘ ∩’ symbols show quadratic effects, indicating that oviposition was more frequent in the mid-range values. Variable type Variables Rana uenoi Hynobius quelpaertensis Bombina orientalis Dryophytes japonicus Abiotic factors PoolVolume + + ∩ + Dry – – – – Leaves 0 + ∩ – CanopyCover 0 0 0 - Stones 0 0 0 0 Biotic factors ConEggs and ConTadpoles – – 0 + HeteroEggs and HeteroTadpoles – 0 0 + Predators N.A. N.A. 0 + MosqLarvae N.A. N.A. 0 + Supplementary Files SI.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4019240","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290410542,"identity":"2fc35b9d-13b2-4f2c-ba15-f040b5ef27b6","order_by":0,"name":"Dogeun Oh","email":"","orcid":"","institution":"Mokpo National University","correspondingAuthor":false,"prefix":"","firstName":"Dogeun","middleName":"","lastName":"Oh","suffix":""},{"id":290410543,"identity":"15823c49-2e42-461b-a8a6-21c9693e45cc","order_by":1,"name":"Jaehun Kang","email":"","orcid":"","institution":"Jeju National University","correspondingAuthor":false,"prefix":"","firstName":"Jaehun","middleName":"","lastName":"Kang","suffix":""},{"id":290410544,"identity":"c71ebced-d063-41b8-8e8e-2aeeb2676654","order_by":2,"name":"Uhram Song","email":"","orcid":"","institution":"Jeju National University","correspondingAuthor":false,"prefix":"","firstName":"Uhram","middleName":"","lastName":"Song","suffix":""},{"id":290410545,"identity":"0675fa1e-3789-42a3-8c08-488a464f7782","order_by":3,"name":"JeongYoon Ahn","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"JeongYoon","middleName":"","lastName":"Ahn","suffix":""},{"id":290410546,"identity":"f1d5787b-6607-4d36-8115-6f67fae6671d","order_by":4,"name":"Changku Kang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie3RMQrCMBSA4VcCurR0TanYK6QU6lAPYxezdHER3YRCvYIgeAxxrATqklkCOujirCDSbsbinOommH9IQshHAgHQ6X4x/BpyIHI0Tu899DFB5GvSwh8Re5leznfOAns530+rDQN7nqNgorrkWPSCjmAhPvLxweIMMB+gmCsIwYPQxVfWB5EMD0bGAASg7UxJ6KMmniSjShKvmSShc5UPI4IWYElCJIlVBItk7AKngS8S5FoZNX0ep76K2Au6dsoi8leCnm9VFnW7O8YcFXmFzHoy65+Ua6MJyCNlPbVPjSd1Op3uP3sClXdQlG1SRsAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3707-4989","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Changku","middleName":"","lastName":"Kang","suffix":""}],"badges":[],"createdAt":"2024-03-06 04:11:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4019240/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4019240/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54781613,"identity":"e32c0e7c-e9ad-4efb-bcba-c7d10e390465","added_by":"auto","created_at":"2024-04-16 16:59:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3314794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) Photos displaying the habitat structure of our field site under no rain (left) and rainy conditions (right). (B) The frequency distribution of the number of new eggs found for each species throughout our study period. Image ©: Dogeun Oh\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4019240/v1/04f9630e8ce1c431ea44db14.png"},{"id":54781327,"identity":"6248d717-e378-4ca0-9a00-5e752f8123a0","added_by":"auto","created_at":"2024-04-16 16:51:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":426445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScatterplots showing the relationship between each abiotic factor and oviposition choice for each species. Lines represent the predicted values estimated from GLZs, and the shaded areas indicate the 95% confidence intervals.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4019240/v1/70dd1319e60dd1d94767abde.png"},{"id":54781326,"identity":"babf43a0-6207-47b6-b2bc-d3d02eef12f6","added_by":"auto","created_at":"2024-04-16 16:51:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2662744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe observed frequencies of pools with new eggs, both with and without each biotic factor, are compared to the expected frequencies in scenarios where females lay their eggs randomly, irrespective of the presence of the biotic factor.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4019240/v1/31d16cff6c5cd61cb21cb1ce.png"},{"id":56875085,"identity":"844ef550-d1d1-43ac-91de-4f3e82983eac","added_by":"auto","created_at":"2024-05-21 14:47:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9056978,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4019240/v1/61171727-5616-48bf-836b-5953a47f639b.pdf"},{"id":54781328,"identity":"32349c4d-e441-4af4-be38-08734fe1e781","added_by":"auto","created_at":"2024-04-16 16:51:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5022233,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-4019240/v1/439e7e611f7bbb1d4ef9ac15.docx"}],"financialInterests":"","formattedTitle":"Oviposition strategies of amphibians in ephemeral streams: a multi-species perspective","fulltext":[{"header":"Significance statement","content":"\u003cp\u003eWhere and when eggs are deposited greatly impacts offspring survival in egg-laying species, particularly in fluctuating environments. Despite extensive research on egg-laying behaviors, mainly focusing on single species, there is a knowledge gap regarding the behavior of multiple species sharing the same habitat. We hypothesized species sharing the same habitat might exhibit analogous egg-laying choices due to shared selective pressures. A thorough field study on four amphibian species in an ephemeral stream reveals a shared trend of avoiding drought among all species. Additionally, various biotic and abiotic factors influenced egg-laying decisions differently across species. Surprisingly, factors detrimental to juvenile survival, such as predators or competitors, were not consistently avoided in natural systems.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Animals have evolved diverse parental care strategies to increase the survival of their progeny\u0026nbsp;(Clutton-Brock 1991). For example, many species show nesting, guarding, or brooding behaviours to directly protect and care for their young\u0026nbsp;(Royle et al. 2012). Parental care varies widely between taxa: many birds and mammals have evolved the costly parental feeding while other vertebrates and invertebrates often have simpler forms of parental behaviours that do not involve direct feeding of their offspring\u0026nbsp;(Balshine 2012; Trumbo 2012). These simpler forms include the lack of post-laying care. In such animals, the choice of where and when to lay eggs has profound consequences on the reproductive success, especially when the eggs and/or juvenile stages are immobile or confined to local regions\u0026nbsp;(Doak et al. 2006; Refsnider and Janzen 2010). Thus, natural selection favours the ability of parents to prefer oviposition sites that increase the chance of offspring survival, their performance, or even parental survival\u0026nbsp;(Brodin et al. 2006; Hirayama and Kasuya 2013; Dimitrie and Benard 2023). There has been ample evidence of the selective oviposition in wide array of taxa including insects, reptiles, amphibians, and fish\u0026nbsp;(Schwarzkopf and Brooks 1987; Spieler and Linsenmair 1997; Mokany and Shine 2003a).\u003c/p\u003e\n\u003cp\u003eOviposition site selection is more likely to evolve under conditions when (i) potential egg-depositing sites are temporally and spatially heterogeneous in terms of suitability for offspring survival, (ii) juveniles are unable to disperse from the egg-spawning spots, and (iii) parents are able to evaluate suitable sites by comparison of alternatives\u0026nbsp;(Resetarits Jr 1996). Amphibians in highly ephemeral environments present an excellent platform for studying oviposition site selection because the habitat structure often meets all of the above conditions: (i) potential oviposition sites are locally scattered, allowing parents to compare the suitability of multiple pools, (ii) the characteristics (i.e. suitability as a egg-laying site) of pools are often both spatially and temporally varying, and (iii) juveniles are restricted to the pools where mothers laid eggs unless disturbed (e.g. heavy rains)\u0026nbsp;(Buxton and Sperry 2017). Indeed, amphibians in ephemeral streams have been extensively studied for their selective oviposition\u0026nbsp;(Spieler and Linsenmair 1997; Murphy 2003; Baek et al. 2021; Gould et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth biotic and abiotic factors have been demonstrated to affect female choice of amphibians. Mothers often choose oviposion sites based on the degree of risk from predators, conspecifics or heterospecifics. Predators are ubiquitous problems for eggs/juveniles and yield negative consequences for offspring survival not only through direct predation but also through non-consumptive effects\u0026nbsp;(Preisser et al. 2005; Buxton and Sperry 2017; Buxton et al. 2017). Naturally, many studies have revealed the presence of predator avoidance\u0026nbsp;(Buxton and Sperry 2017; Resetarits et al. 2021). The presence of conspecifics or heterospecifics of related taxa may function as predators (through cannibalism) or putative competitors but may also give a cue about site quality\u0026nbsp;(Dillon and Fia\u0026ntilde;o 2000; Rudolf and R\u0026ouml;del 2005; Gould et al. 2020). The risk of cannibalism also affects female choice, and mothers may avoid the sites with higher risk of cannibalism\u0026nbsp;(Iwai et al. 2007); however, reducing the risk of cannibalism does not appear to be universal, with some notable exceptions\u0026nbsp;(Poelman and Dicke 2007; Rojas 2014). Food availability, such as leaf litter, could also affect female choice especially when egg-laying sites are small and isolated\u0026nbsp;(McDiarmid and Altig 1999; Stoler and Relyea 2021; Magee-Christian and Earl 2022). When it comes into abiotic factors, temperature and water are demonstrated as main selective agents because the avoidance of thermal damage or desiccation is critical for juvenile survival and development\u0026nbsp;(Spieler and Linsenmair 1997; Goldberg et al. 2006).\u003c/p\u003e\n\u003cp\u003eTo date, studies on female oviposition preference have mostly been conducted on a single species, and studies that examined the selective oviposition in multi-species level are surprisingly scarce\u0026nbsp;(Miaud 1995; Ortiz-Ross et al. 2020). However, various species of amphibians co-occur in the same habitat and one can predict that species that co-occur in the same habitat would be under similar selective pressures for oviposition or affect each other interactively. In this study, we investigated the oviposition choice of multiple amphibian species that reproduce in the same ephemeral stream to (i) identify biotic and abiotic factors that correlate with female oviposition choice for each species and (ii) investigate that species that occur in the same habitat (thus likely to be under similar selective pressures) show similar oviposition decisions.\u003c/p\u003e\n\u003cp\u003eWe formulated certain predictions regarding assemblage-wide or species-specific responses to several abiotic and biotic factors. About assemblage-wide responses, we firstly predict that natural selection may shape female behaviours to avoid the pools with high risk of dessication (Spieler and Linsenmair 1997; Goldberg et al. 2006). Second, females would selectively lay eggs in pools with sufficient organic matters or potential food materials for tadpoles (Kern et al. 2013; Magee-Christian and Earl 2022). Third, predation (or cannibalism) risk might also affect female choice. This includes (i) the avoidance of pools that are already occupied by aquatic predators and (ii) laying eggs in pools with potential refuges (Resetarits Jr and Wilbur 1989). In contrast, there are certain factors that we predict species-specific or circumstantial responses. First, in regards to the presence of con-/heterospecific juveniles, we had two competing predictions. Females might avoid to lay eggs in pools with con-/heterospecific juveniles to avoid the risk of cannibalism. Alternatively, the presence of other juveniles may be an indicator of pool quality or decrease individual risk of predation through dilution (Rudolf and R\u0026ouml;del 2005). In that case, females may selectively lay eggs in pools with other juveniles. Second, canopy coverage may also affect female choice in two different ways. Pools with less canopy coverage should be exposed to the sunlight directly which can cause faster evaporation of water. In that case, we predict the females to avoid laying eggs in pools under low canopy covergae. However, under the direct sunlight, the water temperature can be maintained higher than those under high canopy coverage which can boost development, a favourable condition in ephemeral streams (Oh et al. 2021). In the latter scenario, we predict females to selectively lay eggs in pools under higher canopy coverage.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cem\u003eStudy species and environments\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We conducted this study within Bangcheon stream in Jeju Island, South Korea (N33.4722, E126.5432). Jeju Island, a volcanic island, is largely covered by basalt layers that quickly drain surface water underground. As a consequence, in most streams, including our study site, water flows only during rain. In these ephemeral streams, oviposition sites were available in the form of pools of varying sizes. Along the stream, the pools were patchily distributed, with distances varying from less than a metre between each pool and its nearest neighbour (Fig 1A). Each pool remained isolated until rainfall occurred. Heavy rains usually swept most, if not all, organisms in the pools. Rains did not alter the location of each pool, although some pools temporarily merged into larger ones just after heavy rains. There were no fish in our study sites.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe chose a specific study site occupying approximately 730 m\u003csup\u003e2\u003c/sup\u003e area. This area spanned a longitudinal distance of approximately 120 metres along the stream and contained 321 different pools when estimated just after rain when all available pools can be identified. In the study site, three frogs and one salamander species mainly occurred and reproduced during our study period from 8th March to 24th August, 2021: \u003cem\u003eRana uenoi\u003c/em\u003e, \u003cem\u003eHynobius quelpaertensis\u003c/em\u003e, \u003cem\u003eBombina orientalis\u003c/em\u003e, and \u003cem\u003eDryphytes japonicus\u003c/em\u003e. While \u003cem\u003eKaloula borealis\u003c/em\u003e also laid some eggs in our study site, their numbers were only a few and we did not include \u003cem\u003eK. borealis\u003c/em\u003e in our analysis. The timing of reproduction largely overlapped between \u003cem\u003eR. ueonoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, and between \u003cem\u003eB. orientalis\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e (Fig. 1B). For \u003cem\u003eR. uenoi\u003c/em\u003e, although reproduction started before our study period, we only analysed the eggs laid during our study period.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eField surveys to study oviposition preference in relation to abiotic and biotic factors\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo monitor the oviposition choices of females, we visited all available pools within our study site between 0800 and 1300 on every non-rainy day of our study period. We refrained from visiting the stream on rainy days for the safety of the experimenters. Each pool was marked with a unique number on a rock surface adjacent to it, using non-toxic silicone.\u003c/p\u003e\n\u003cp\u003eWe recorded several biotic and abiotic characteristics of each pool daily (see Table 1): (i) whether water was present or absent (Dry; a binary variable), (ii) the amount of leaf litter (Leaves; an ordinal variable), (iii) the quantity of stones as potential refuges (Stones; ordinal), (iv) the turbidity of the pool water (Turbidity; ordinal), (v) the presence of aquatic predators (Predators; binary), and (vi) the presence of mosquito larvae (MosqLarvae; binary). Variables concerning the presence of amphibian juveniles were coded for each species as follows: (vii) the presence of newly spawned eggs (NewEggs; binary), (viii) the presence of conspecific eggs that were not newly spawned (ConEggs; binary), (ix) the presence of conspecific tadpoles (ConTadpoles; binary), (x) the presence of heterospecific eggs that were not newly spawned (HeteroEggs; binary), and (xi) the presence of heterospecific tadpoles (HeteroTadpoles; binary).\u003c/p\u003e\n\u003cp\u003eAlthough we initially started by counting the number of con-/heterospecific eggs and tadpoles, we decided to use these variables in binary form due to the heterogeneity of errors among pools; pools with larger size, more turbid water, or a higher number of stones were more likely to have inaccuracies in their counts. Leaves, Stones, and Turbidity (all ordinal variables) were subjectively but consistently assessed and rated by a single experimenter (DO) throughout the survey. The criteria can be found in Table 1. Mosquito larvae have primarily been described as competitors, but also as predators or prey of tadpoles\u0026nbsp;(Blaustein and Margalit 1994, 1996; Mokany and Shine 2003b). While we were unsure of the ecological relationship between our studied species and mosquito larvae, we nonetheless analysed correlational patterns between their presence and female oviposition choice. Invertebrates other than mosquito larvae, such as mayfly or Chironomidae larvae, were also occasionally present, but their occurrence was very low (less than 0.3% of pools) and thus were not included in our analysis.\u003c/p\u003e\n\u003cp\u003eOccasionally, a pool was transiently divided into multiple pools (under consecutive non-rainy days), or two pools merged together (this rarely happened just after rain). When a pool divided, we recorded the characteristics of each separate pool and used the averaged value for analysis, retaining the original pool ID for these transiently separated pools. When pools merged, we noted the characteristics of the merged pool and applied them to both original pools. During the breeding seasons of \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, from March to May, all 321 pools were surveyed. However, for the breeding seasons of \u003cem\u003eB. orientalis\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e, spanning May to August, we limited the survey to 192 pools in the lower parts of our study site, where more oviposition activities were observed. This reduced scale was necessary to complete the surveys in a timely manner, as the increased oviposition activities resulted in longer times required to survey each pool.\u003c/p\u003e\n\u003cp\u003eEgg clutches from different females were clearly distinguishable for \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, in which eggs were either densely grouped together or found in a sac. However, distinguishing eggs from different mothers in the same pool posed a challenge for \u003cem\u003eB. orientalis\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e. This difficulty arose because not only was the number of eggs highly variable, but also the boundaries between different egg clutches were not clearly defined. Therefore, instead of counting the number of egg clutches, we employed a binary variable to denote whether newly spawned eggs were found in each pool each day. This approach, however, resulted in an inability to detect instances where more than one female laid eggs in the same pool on the same day.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In addition to the pool characteristics that we surveyed daily, we also measured (i) pool volume (PoolVolume) and (ii) canopy coverage (CanopyCover). Unlike the aforementioned variables, these two characteristics were measured only once during our study period. While PoolVolume could change daily (decreasing gradually due to evaporation but increasing during rain), all pools were expected to be simultaneously affected by these changes. Therefore, we measured PoolVolume only once and used this as a size index for each pool. PoolVolume was estimated just after a rainy day when all pools were fully filled with water. The three-dimensional shape of each pool was highly irregular; thus, we crudely estimated PoolVolume by multiplying the surface area by the deepest depth of each pool. The surface area was measured by aerial photography using a drone (MAVIC PRO, DJI, Guangdong, China), with a ruler placed next to each pool for area calibration. CanopyCover was estimated using a mobile phone camera (Galaxy Note 8, Samsung, Seoul, South Korea). This measurement was taken on a summer day when foliage was dense. We positioned the camera just above the suspected centroid of the water surface, facing upwards, and took a photograph. The percentage of plant cover in the image was then calculated, using this percentage as an index of CanopyCover with ImageJ 1.80 (National Institute of Health, Maryland, USA). The camera\u0026rsquo;s angle of view was 77\u0026deg;, as specified by the manufacturer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Abiotic factor analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We considered Dry, Leaves, Stones, Turbidity, PoolVolume, and CanopyCover as abiotic factors that could potentially affect female oviposition choice. All analyses were conducted separately for each species. Firstly, we calculated a representative index for each variable for each pool, generating a pool-level dataset. For Dry, we calculated the proportion of the days that each pool found to be dry during the breeding season of each species. For Leaves, Stones, and Turbidity, we first examined whether the intra-pool variation of each variable across the study period was smaller than the inter-pool variation. For this, we employed the intraclass correlation coefficient (ICC) test in the \u0026lsquo;irr\u0026rsquo; package in R, using a one-way consistency model\u0026nbsp;(Koo and Li 2016). Within-pool variability was lower than among-pool variability for all three variables (all P \u0026lt; 0.001, estimated ICC ranged from 0.39 to 0.77). Then, for each pool, we averaged each variable across the breeding season and used these averaged values to characterise each pool. PoolVolume was log-transformed to reduce distributional skewness. After all these processes, we had one representative value for each abiotic factor for each pool. We excluded transiently appearing pools in the abiotic factor analysis (i.e., when either a pool was divided into multiple pools during consecutive non-rainy days due to the exposure of bottom surfaces, or multiple pools were connected to each other just after rain) because averaging across the entire breeding season was impossible for those temporary pools.\u003c/p\u003e\n\u003cp\u003eAs an index of each pool\u0026apos;s oviposition frequency, we employed two different methods depending on the species. For \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, we used a binary variable to indicate whether eggs were found in each pool at least once during the breeding season. This approach was chosen because (i) the total number of egg clutches per pool was generally one or a few, and (ii) a small fraction of pools (14% for \u003cem\u003eR. uenoi\u003c/em\u003e and 7% for \u003cem\u003eH. quelpaertensis\u003c/em\u003e) were used for oviposition. Conversely, for \u003cem\u003eB. orientalis\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e, a higher number of pools were used for oviposition (56% and 36%, respectively), with each pool often being used multiple times on different dates. Therefore, we counted the number of days new eggs were found in each pool throughout the breeding season and used this count variable for our analysis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We fitted generalised linear models (GLZs) to examine the effect of abiotic variables on female oviposition choice. Firstly, we examined the presence of multicollinearity among predictors and found that Leaves and Turbidity were highly correlated (\u003cem\u003ePearson\u0026rsquo;s r\u003c/em\u003e \u0026gt; 0.78 for all species data). To resolve the multicollinearity issue, we removed the Turbidity variable from further analysis, leaving only five abiotic factors (Dry, Leaves, Stones, Pool Volume, and Canopy Cover). For \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, we fitted binomial GLZs using the binary response of whether each pool was used for oviposition at least once and the five abiotic factors as predictors. For \u003cem\u003eB. orientalis\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e, we used the number of days new eggs were found during the breeding season for each pool as a response variable and the abiotic factors as predictors. Overdispersion was detected when we fitted Poisson GLZs, so we instead fitted negative binomial GLZs\u0026nbsp;(Lind\u0026eacute;n and M\u0026auml;ntyniemi 2011). Visual inspections of predictors and our response variables suggested putative polynomial relationships for Leaves and PoolVolume for \u003cem\u003eB. orientalis\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e; thus, we additionally included quadratic terms of Leaves and PoolVolume for these two species. We did not include interaction terms in our predictors because (i) we did not specifically predict interactive effects among the predictors, and (ii) with five main predictors, including unplanned interaction terms leads to the addition of 10 extra predictors (more when polynomial terms are simultaneously considered) for each species, which makes interpretations challenging in our multi-species datasets.\u003c/p\u003e\n\u003cp\u003eTo estimate the parameters of variables and predict their effects, we applied multimodel inference and performed model averaging using the \u0026lsquo;MuMIn\u0026rsquo; package\u0026nbsp;(Grueber et al. 2011). We averaged the parameters of models that appeared within the 0.95 cumulative sum of AIC weights using conditional averaging.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Biotic factor analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We considered Predators, MosqLarvae, ConEggs, ConTadpoles, HeteroEggs, HeteroTadpoles as biotic factors that could potentially affect female oviposition site choice. For the main results, we did not distinguish between eggs and tadpoles, thus using the merged categories of ConEggs/Tadpoles (where either or both conspecific eggs or tadpoles were present) and HeteroEggs/Tadpoles as variables, in order to keep the main results from being overly complex. However, we nonetheless performed additional analyses, considering eggs and tadpoles separately, and included these results in the Supporting Information, which are also discussed.\u003c/p\u003e\n\u003cp\u003eUnlike abiotic factors, which did not vary much throughout the breeding season within each pool, biotic conditions changed frequently; thus, generating and analysing averaged pool-level data was inappropriate. The most ecologically relevant approach may be analysing each day separately, as the female\u0026apos;s decision was made by comparing the conditions of currently available pools. However, this was statistically challenging in our data because only a limited number of pools were found with new eggs on most days, making GLZ analysis for each day impossible due to the excessive number of pools with no new eggs each day. Instead, we pooled the data across our survey period for each species, generated frequency tables, and examined whether the frequency of eggs laid in pools with specific biotic conditions deviated from what would be expected if females randomly laid eggs without considering the biotic condition. Pooling the data across all survey dates was necessary due to the low number of new egg clutches found in each individual survey (often fewer than three), which precluded us from estimating the expected frequencies for each specific date. In the biotic factor analysis, we were able to include temporary pools in the analysis, but their numbers were minor (less than 1% of the total pools).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Firstly, we excluded the pools that had never been used during the study period from the analysis. We considered that these pools were not used for oviposition, regardless of the biotic conditions, likely because the abiotic conditions were not favourable to frogs. For example, pools that were substantially smaller than other pools were never used for oviposition in all four species. Thus, we compared the biotic conditions among the pools that had been used for oviposition at least once for each species.\u003c/p\u003e\n\u003cp\u003eThen, for each biotic factor, we calculated the frequency of available pools and the pools containing a specific biotic factor. For instance, to investigate the potential influence of the presence of predators on female oviposition choice, we determined the frequency of available pools and the pools containing predators during each survey. Specifically, we used the biotic conditions from the day before newly spawned eggs were found because female oviposition mostly, if not always, occurred during the night. Therefore, the conditions experienced by females were not those in which the new eggs were found, but rather the conditions of the previous day. These frequencies were subsequently pooled across all survey dates. Utilising this information, we estimated the expected frequencies of pools with and without egg clutches if female frogs randomly laid their eggs irrespective of the presence of predators. Subsequently, we constructed a contingency table using the frequencies (expected and observed frequencies of pools with egg clutches in both predator-free and predator-present pools) and conducted a chi-square test. We performed the above procedures for all biotic factors.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eCorrelational patterns between abiotic factors and female oviposition choice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The results are summarised in Table 2 (showing significance and trends), Fig. 2, and Table S1 (detailed statistics). In all four species, PoolVolume and Dry consistently affected the oviposition choice of females. Specifically, all species tended to avoid laying eggs in pools where the risk of desiccation was higher. Also, all species were more likely to lay eggs in larger pools over smaller ones, except for \u003cem\u003eB. orientalis\u003c/em\u003e, which laid more eggs in intermediate-sized pools compared to smaller or larger ones. In contrast, we found no evidence that Stones affected female oviposition choice in any of the species. The effect of Leaves and CanopyCover varied among species. \u003cem\u003eH. quelpaertensis\u003c/em\u003e and \u003cem\u003eB. orientalis\u003c/em\u003e females laid eggs more frequently in pools with more leaf litter and in pools with intermediate-amount of leaf litter, respectively. In contrast, Leaves did not affect the oviposition choice of \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e. In terms of CanopyCover, \u003cem\u003eD. japonicus\u003c/em\u003e females were more likely to lay eggs in pools under higher canopy coverage, while other species did not show such patterns.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCorrelational patterns between biotic factors and female oviposition choice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe correlational patterns between biotic factors and female oviposition preferences were species-specific (see Table 2 for the summary, Fig. 3 for plots, and Table S2 for detailed statistics). The results of the effect of Predators and MosqLarvae were not evident for \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, as both predators and mosquito larvae were scarcely present during their breeding seasons.\u003c/p\u003e\n\u003cp\u003eIn both \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, the observed frequencies of pools with newly spawned eggs significantly deviated from the frequencies expected if females were laying eggs irrespective of the presence of ConEggs/Tadpoles. When we separately analysed the presence of ConEggs and ConTadpoles, we found that the avoidance of ConTadpoles, not ConEggs, drove the observed patterns (see Fig. S1). While pools with HeteroEggs/Tadpoles tended to be avoided in the main results, separate analyses did not reveal the same patterns (Fig. S2), suggesting that the avoidance of heterospecifics is not as apparent as the avoidance of conspecifics in \u003cem\u003eR. uenoi\u003c/em\u003e. In \u003cem\u003eH. quelpaertensis\u003c/em\u003e, pools with ConEggs/Tadpoles were avoided, which was mainly driven by the avoidance of ConEggs \u0026nbsp;(Fig. 3; Fig. S1), but pools with HeteroEggs/Tadpoles were not avoided.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eB. orientalis\u003c/em\u003e, we did not find any patterns related to either ConEggs/Tadpoles or HeteroEggs/Tadpoles in the main analysis (Table 2; Fig. 3). However, separate analyses for eggs and tadpoles revealed that female \u003cem\u003eB. orientalis\u003c/em\u003e laid eggs in pools with HeteroEggs already present more frequently than would be expected by random chance, but avoided laying eggs in pools with HeteroTadpoles (Fig. S2). In contrast, in \u003cem\u003eD. japonicus\u003c/em\u003e, the observed frequencies of pools with newly spawned eggs significantly deviated from the frequencies expected by random chance for both ConEggs/Tadpoles and HeteroEggs/Tadpoles. Female \u003cem\u003eD. japonicus\u003c/em\u003e tended to lay eggs in pools containing both ConEggs/Tadpoles and HeteroEggs/Tadpoles (Table 2; Fig. 2). Separate analyses showed that \u003cem\u003eD. japonicus\u003c/em\u003e laid more eggs in pools with both ConEggs, ConTadpoles, HeteroTadpoles but not HeteroEggs (Fig. S1,2).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The observed frequencies of pools with newly spawned eggs did not significantly deviate from the frequencies expected by random chance for Predators and MosqLarvae, except in \u003cem\u003eD. japonicus\u003c/em\u003e; they laid eggs in pools with Predators and MosqLarvae more frequently than would be expected by random chance (Table 2; Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Our results firstly reveal that the risk of drought is an important factor in oviposition site choice, shared by all four species; all species selectively laid eggs in pools where the risk of pool desiccation was lower. This is reflected in the patterns where all species avoided laying eggs in small-sized pools and in those that had experienced more frequent drought conditions. These results firmly demonstrate that desiccaion is a universal selective pressure that shared by all studied species. The results also align well with accumulated evidence that many animals evolved to lay eggs in places with lower risk of desiccation in ephemeral environments\u0026nbsp;(Rudolf and R\u0026ouml;del 2005; Goldberg et al. 2006; Kern et al. 2013; Baek et al. 2021).\u003c/p\u003e\n\u003cp\u003eThe results that \u003cem\u003eB. orientalis\u003c/em\u003e laid more eggs in intermediate-sized pools corrobrates a previous study, even though larger pools are less likely to experience drought\u0026nbsp;(Baek et al. 2021). One potential reason is that the water temperature in large pools remains cooler even in summer, thus delaying the development time of juveniles\u0026nbsp;(\u0026Aacute;lvarez and Nicieza 2002). Ephemeral streams are not a favourable environment for tadpoles due to the high risk of desiccation, flooding, scarcity of food sources, and limited space for movement\u0026nbsp;(Gould et al. 2022). Thus, faster development time is often preferred in ephemeral streams\u0026nbsp;(Richter‐Boix et al. 2011; Oh et al. 2021). In this context, the avoidance of larger pools can be adaptive because juvenile development is more rapid in smaller-sized pools where the water temperature can easily warm during the daytime (see Fig. S3 showing that large pools were cooler than small pools in our study site). However, it is still uncertain why the other species do not exhibit similar large-pool avoidance behaviours. The breeding seasons of \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e are before summer, thus they may be adapted to lower temperatures. Also, \u003cem\u003eD. japonicus\u003c/em\u003e lays a substantially larger number eggs at once than \u003cem\u003eB. orientalis\u003c/em\u003e; thus, \u003cem\u003eD. japonicus\u003c/em\u003e may require more space for their progeny, which could also lead to the absence of large-pool avoidance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;For the rest of the abiotic factors, we found that oviposition choice was either species-specific or not present at all. Firstly, the amount of leaf litter was correlated with oviposition choice in two species: \u003cem\u003eH. quelpaertensis\u003c/em\u003e and \u003cem\u003eB. orientalis\u003c/em\u003e. Leaf litter is one of the main food sources for tadpoles\u0026nbsp;(McDiarmid and Altig 1999), so it is not surprising that these two species avoided pools with less leaf litter. The reason for the avoidance of pools with a large amount of leaf litter in \u003cem\u003eB. orientalis\u003c/em\u003e may be that those with the highest level of leaf litter at our study site were almost filled with it, thus being highly turbid, leaving less space for tadpole movement and probably having a low amount of oxygen. In contrast, during the breeding season of \u003cem\u003eH. quelpaertensis\u003c/em\u003e (early spring when new leaves had not yet proliferated), most of the leaf litter was the remnants of the previous year, so no pools were fully filled with leaf litter. This phenological difference may affect the differences between the two species. The reasons why the other two species, \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e, did not show any preference for leaf litter is puzzling. Ecological and behavioural differences may be a potential explanation; \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e lay substantially more eggs in a single pool than the other two species (several hundred versus 10-100 eggs). Therefore, \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e may be better equiped to allleviate the costs of cannibalism and utilise alternative food sources (e.g., through cannibalism) compared to the other two species. This may explain the species-specific responses to the amount of leaf litter.\u003c/p\u003e\n\u003cp\u003eBoth canopy coverage and the presence of stones were hardly correlated with oviposition choice, except for \u003cem\u003eD. japonicus\u003c/em\u003e, which preferentially laid eggs in pools with open canopies. We had two competing predictions for canopy coverage, and \u003cem\u003eD. japonicus\u003c/em\u003e females were more likely to lay eggs in pools that were at a higher risk of desiccation but warmer. As \u003cem\u003eD. japonicus\u003c/em\u003e selectively lay eggs in larger-sized pools, this may alleviate the risk of desiccation. The lack of correlation between the amount of stone and oviposition preference suggests that the presence of potential refuges is not a strong driver of oviposition site selection. In our study sites, predation pressures from the predators that refuges could benefit (e.g., terrestrial predators) may not be strong.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In terms of biotic factors, \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e laid eggs in pools without conspecific juveniles more frequently than would be expected by chance. This corroborates our prediction, as well as other studies, that mothers would lay eggs where the risk of cannibalism is low\u0026nbsp;(Spieler and Linsenmair 1997; Iwai et al. 2007). The evidence for avoiding pools with heterospecifics is equivocal; a weak trend was found in \u003cem\u003eR. uenoi\u003c/em\u003e but not in \u003cem\u003eH. quelpaertensis\u003c/em\u003e. The total number of egg clutches found at our study sites was not very high for both \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e throughout our study period (68 for \u003cem\u003eR. uenoi\u003c/em\u003e and 30 for \u003cem\u003eH. quelpaertensis\u003c/em\u003e). Thus, we consider that either or both (i) statistical power was low for these two species to reveal the true patterns, especially when we separately analysed eggs and tadpoles, and/or (ii) the encounter rates between these two species were too low to drive avoidance behaviours. Indeed, visual inspection of the patterns suggests that the oviposition patterns seem to deviate from what would be expected by random chance (see Fig. S1,2), but the absence of statistical support for this difference suggests that statistical power was low. Thus, our evidence for female avoidance of heterospecific juveniles in these two species remains inconclusive.\u003c/p\u003e\n\u003cp\u003eUnexpectedly, \u003cem\u003eB. orientalis\u003c/em\u003e did not show any avoidance of pools with conspecific juveniles. This contrasts with a previous finding that showed conspecific avoidance\u0026nbsp;(Baek et al. 2021). The discrepancy between Baek et al.\u0026rsquo;s study (2021) and ours may be due to the sampling interval; Baek et al. sampled twice per week, while we conducted the survey on a daily basis. Thus, it is possible that, in Baek et al.\u0026rsquo;s study, those that laid eggs in pools with others\u0026rsquo; juveniles were quickly cannibalised, so that an intermittent survey would not reveal the true oviposition patterns. Indeed, in their own study, most of the newly spawned eggs were cannibalised under the presence of conspecific tadpoles within 24 hours. Considering that (i) egg cannibalism commonly occurs in this population\u0026nbsp;(Baek et al. 2021; Oh et al. 2021), and (ii) there were always some available pools without conspecifics throughout the seasons, it is unclear why \u003cem\u003eB. orientalis\u003c/em\u003e did not show any avoidance of others\u0026rsquo; juveniles. \u003cem\u003eD. japonicus\u003c/em\u003e even preferred to lay eggs in pools with others\u0026rsquo; eggs/juveniles present. Possibly, during the breeding seasons of \u003cem\u003eR. uenoi\u003c/em\u003e and \u003cem\u003eH. quelpaertensis\u003c/em\u003e, pools were mostly empty, leaving a variety of options for females on where to lay eggs after avoiding the filled ones. However, during the breeding seasons of \u003cem\u003eB. orientalis\u003c/em\u003e and \u003cem\u003eD. japonicus\u003c/em\u003e, a greater number of pools (that were probably preferred) were already filled on many days, thus the costs of choosing empty and likely less-preferred pools may not outweigh the benefits of choosing filled but more-preferred pools. Addiionally, the preference to oviposit in pools that already occuppeid might have adaptive advantage in ephemeral pools because it can supply additional nutrient via cannibalism or decrease predatory risk through dilution effects\u0026nbsp;(Doody et al. 2009; Buxton and Sperry 2017; Gould et al. 2021).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;While our initial prediction was the avoidance of both predators and mosquito larvae, the presence of these organisms did not correlate with the oviposition choice in \u003cem\u003eB. orientalis\u003c/em\u003e but did correlate in \u003cem\u003eD. japonicus\u003c/em\u003e in an unexpected manner; \u003cem\u003eD. japonicus\u003c/em\u003e laid more eggs in pools with predators or mosquito larvae already present than would be expected by chance. One potential reason behind this observed pattern may be that the pool preference of predators (or mosquitoes) matches that of frogs. Indeed, in our data, there were positive correlations between the number of days new eggs were found and the number of days predators were present for each pool (Fig. S4). The same correlations were found with mosquito larvae (Fig. S4). Therefore, there may be conflicting pressures in the wild; while avoiding organisms with any negative interactions may benefit individuals, the choice is not simple in reality because the competitors or predators can share similar preferences for certain abiotic conditions\u0026nbsp;(Giao and Godoy 2007), such as those with a low risk of desiccation. The oviposition patterns in \u003cem\u003eD. japonicus\u003c/em\u003e could be observed if their preference for abiotic factors overrides predator/competitor avoidance. This constrasts a previous finding that the risk of predators was prioritised over desiccation risk in the pantless treefrog which lay both aquatic and arboreal eggs\u0026nbsp;(Touchon and Worley 2015). Whether \u003cem\u003eD. japonicus\u003c/em\u003e and \u003cem\u003eB. orientalis\u003c/em\u003e indeed show preference for, or an avertion to, pools with predators or mosquito larvae requires experimental work in manipulative environments. However, our results imply that such preference/avoidance may conflict with abiotic condition preference if predators or competitors share similar preferences.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Will the observed selective oviposition have fitness consequences? Although our data cannot confirm this due to the inability to track individuals, most of the eggs and juveniles were flushed out during occasional heavy rain before they become froglets. We observed only a few individuals that became froglets at our study site, but even it was unclear whether they had grown in the same pool or been flushed down from upstream. Therefore, we consider that the oviposition choice of females may have limited consequences only during the eggs and early juvenile stages before subsequent heavy rain events. Nevertheless, natural selection would favour an aversion to pools with a higher risk of desiccation, as this could lead to the complete death of the progeny at any time. After accounting for avoiding desiccation, the impact of other abiotic/biotic factors may become not straightforward because pools with a low risk of desiccation are fewer, making the choice multifaceted. This assumes that female amphibians prioritise the risk of desiccation over other factors when selecting an egg-laying site, which remains to be tested.\u003c/p\u003e\n\u003cp\u003eEphemeral environments are complex systems with many biotic and abiotic factors interacting. In these circumstances, females may not decide where to lay eggs based on individual factors but rather use a holistic evaluation of the overall adequacy of the sites (Stahlschmidt and Adamo 2013; Gould et al. 2021). Although our study is observational and therefore does not reveal the causality of the relationship, the important insights that can be drawn from our study include (i) avoiding drought appears to be a universal driver shared by all amphibian species in the amphibian assemblage of ephemeral streams, (ii) both abiotic and biotic factors correlate with the oviposition choice of females, showing species-specific patterns, and (iii) factors that apparently have negative consequences for juveniles, such as predators/competitors, are not always avoided. This could be because predators or competitors share similar pool preferences; thus, avoiding these organisms could lead to laying eggs in less profitable pools from other perspectives (Marsh and Borrell 2001; Gould et al. 2021). We consider that employing cost-benefit approaches, which account for the interplay of multiple factors, would advance our understanding of female oviposition behaviour in natural systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;CK, DO, and US designed the study. DO and JL conducted field studies. DO, J-Y A, CK performed data analyses. DO and CK wrote the first version of the mansucript. All authors contributed in revising the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate Sangeun Gwak, Seokhyun Lee for their invaluable assistant during the field survey. This study was supported by National Research Foundation of Korea (NRF-2019R1C1C1002466,\u0026nbsp;RS-2023-00239493) and Creative-Pioneering Researchers Program through Seoul National University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll field studies were approved by Mokpo National University Institutional Animal Care and Use Committee (MNU-IACUC-2021-002). We adhered to the guidelines for the treatment of animals in behavioural research and teaching\u0026nbsp;(Buchanan et al. 2012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available from Figshare repository (https://doi.org/10.6084/m9.figshare.25009685.v2)\u0026nbsp;(Oh et al. 2024)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe code used for analyses and figures in this manuscript are available from Figshare repository (https://doi.org/10.6084/m9.figshare.25009685) (Oh et al. 2024)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026Aacute;lvarez D, Nicieza AG (2002) Effects of temperature and food quality on anuran larval growth and metamorphosis. 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Entomol Exp Appl 149:250\u0026ndash;255. https://doi.org/10.1111/eea.12124\u003c/li\u003e\n\u003cli\u003eIwai N, Kagaya T, Okochi I (2007) Choice of oviposition site by \u003cem\u003eRana japonica\u003c/em\u003e: Role of the developmental stage of conspecific eggs. Herpetologica 63:31\u0026ndash;34. https://doi.org/10.1655/0018-0831(2007)63[31:COOSBR]2.0.CO;2\u003c/li\u003e\n\u003cli\u003eKern MM, Nassar AA, Guzy JC, Dorcas ME (2013) Oviposition site selection by spotted salamanders (\u003cem\u003eAmbystoma maculatum\u003c/em\u003e) in an isolated wetland. J Herpetol 47:445\u0026ndash;449. https://doi.org/10.1670/11-179\u003c/li\u003e\n\u003cli\u003eKoo TK, Li MY (2016) A guideline of selecting and reporting intraclass correlation coefficients for reliability research. J Chiropr Med 15:155\u0026ndash;163. https://doi.org/10.1016/j.jcm.2016.02.012\u003c/li\u003e\n\u003cli\u003eLind\u0026eacute;n A, M\u0026auml;ntyniemi S (2011) Using the negative binomial distribution to model overdispersion in ecological count data. Ecology 92:1414\u0026ndash;1421. https://doi.org/10.1890/10-1831.1\u003c/li\u003e\n\u003cli\u003eMagee-Christian RE, Earl JE (2022) Effects of leaf litter species on Cope\u0026rsquo;s Gray Treefrog oviposition site selection. Ichthyol Herpetol 110:750\u0026ndash;758. https://doi.org/10.1643/h2021096\u003c/li\u003e\n\u003cli\u003eMarsh DM, Borrell BJ (2001) Flexible oviposition strategies in t\u0026uacute;ngara frogs and their implications for tadpole spatial distributions. Oikos 93:101\u0026ndash;109. https://doi.org/10.1034/j.1600-0706.2001.930111.x\u003c/li\u003e\n\u003cli\u003eMcDiarmid RW, Altig R (1999) Tadpoles: the biology of anuran larvae. University of Chicago Press, Chicago\u003c/li\u003e\n\u003cli\u003eMiaud C (1995) Oviposition site selection in three species of European newts (Salamandridae) genus Triturus. Amphibia-Reptilia 16:265\u0026ndash;272. https://doi.org/10.1163/156853895X00064\u003c/li\u003e\n\u003cli\u003eMokany A, Shine R (2003a) Oviposition site selection by mosquitoes is affected by cues from conspecific larvae and anuran tadpoles. Austral Ecol 28:33\u0026ndash;37. https://doi.org/10.1046/j.1442-9993.2003.01239.x\u003c/li\u003e\n\u003cli\u003eMokany A, Shine R (2003b) Competition between tadpoles and mosquito larvae. Oecologia 135:615\u0026ndash;620. https://doi.org/10.1007/s00442-003-1215-6\u003c/li\u003e\n\u003cli\u003eMurphy PJ (2003) Does reproductive site choice in a neotropical frog mirror variable risks facing offspring? Ecol Monogr 73:45\u0026ndash;67. https://doi.org/10.1890/0012-9615(2003)073[0045:DRSCIA]2.0.CO;2\u003c/li\u003e\n\u003cli\u003eOh D, Kang J, Song U, et al (2024) Data for the manuscript \u0026ldquo;Oviposition strategies of amphibians in ephemeral streams: a multi-species perspective\u0026rdquo;\u003c/li\u003e\n\u003cli\u003eOh D, Kim Y, Yoo S, Kang C (2021) Habitat ephemerality affects the evolution of contrasting growth strategies and cannibalism in anuran larvae. PeerJ 9:e12172. https://doi.org/10.7717/peerj.12172\u003c/li\u003e\n\u003cli\u003eOrtiz-Ross X, Thompson ME, Salicetti-Nelson E, et al (2020) Oviposition site selection in three glass frog species. Copeia 108:333\u0026ndash;340. https://doi.org/10.1643/CE-19-243\u003c/li\u003e\n\u003cli\u003ePoelman EH, Dicke M (2007) Offering offspring as food to cannibals: oviposition strategies of Amazonian poison frogs (\u003cem\u003eDendrobates ventrimaculatus\u003c/em\u003e). Evol Ecol 21:215\u0026ndash;227. https://doi.org/10.1007/s10682-006-9000-8\u003c/li\u003e\n\u003cli\u003ePreisser EL, Bolnick DI, Benard MF (2005) Scared to death? The effects of intimidation and consumption in predator\u0026ndash;prey interactions. Ecology 86:501\u0026ndash;509. https://doi.org/10.1890/04-0719\u003c/li\u003e\n\u003cli\u003eRefsnider JM, Janzen FJ (2010) Putting eggs in one basket: ecological and evolutionary hypotheses for variation in oviposition-site choice. Annu Rev Ecol Evol Syst 41:39\u0026ndash;57. https://doi.org/10.1146/annurev-ecolsys-102209-144712\u003c/li\u003e\n\u003cli\u003eResetarits Jr WJ (1996) Oviposition site choice and life history evolution. Am Zool 36:205\u0026ndash;215. https://doi.org/10.1093/icb/36.2.205\u003c/li\u003e\n\u003cli\u003eResetarits Jr WJ, Wilbur HM (1989) Choice of oviposition site by Hyla chrysoscelis: role of predators and competitors. Ecology 70:220\u0026ndash;228. https://doi.org/10.2307/1938428\u003c/li\u003e\n\u003cli\u003eResetarits WJ, Bohenek JR, Pintar MR (2021) Predator-specific responses and emergent multi-predator effects on oviposition site choice in grey treefrogs, \u003cem\u003eHyla chrysoscelis\u003c/em\u003e. Proc R Soc B Biol Sci 288:20210558. https://doi.org/10.1098/rspb.2021.0558\u003c/li\u003e\n\u003cli\u003eRichter‐Boix A, Tejedo M, Rezende EL (2011) Evolution and plasticity of anuran larval development in response to desiccation. A comparative analysis. Ecol Evol 1:15\u0026ndash;25. https://doi.org/10.1002/ece3.2\u003c/li\u003e\n\u003cli\u003eRojas B (2014) Strange parental decisions: Fathers of the dyeing poison frog deposit their tadpoles in pools occupied by large cannibals. Behav Ecol Sociobiol 68:551\u0026ndash;559. https://doi.org/10.1007/s00265-013-1670-y\u003c/li\u003e\n\u003cli\u003eRoyle NJ, Smiseth PT, K\u0026ouml;lliker M (2012) The evolution of parental care. Oxford University Press\u003c/li\u003e\n\u003cli\u003eRudolf VHW, R\u0026ouml;del M-O (2005) Oviposition site selection in a complex and variable environment: the role of habitat quality and conspecific cues. Oecologia 142:316\u0026ndash;325. https://doi.org/10.1007/s00442-004-1668-2\u003c/li\u003e\n\u003cli\u003eSchwarzkopf L, Brooks RJ (1987) Nest-site selection and offspring sex ratio in painted turtles, Chrysemys picta. Copeia 53\u0026ndash;61. https://doi.org/10.2307/1446037\u003c/li\u003e\n\u003cli\u003eSpieler M, Linsenmair KE (1997) Choice of optimal oviposition sites by \u003cem\u003eHoplobatrachus occipitalis\u003c/em\u003e (Anura: Ranidae) in an unpredictable and patchy environment. Oecologia 109:184\u0026ndash;199. https://doi.org/10.1007/s004420050073\u003c/li\u003e\n\u003cli\u003eStahlschmidt ZR, Adamo SA (2013) Warm and cozy: temperature and predation risk interactively affect oviposition site selection. Anim Behav 86:553\u0026ndash;558. https://doi.org/10.1016/j.anbehav.2013.06.009\u003c/li\u003e\n\u003cli\u003eStoler AB, Relyea RA (2021) Love it or leaf it: site selection of breeding treefrogs based on leaf litter subsidies. Ichthyol Herpetol 109:785\u0026ndash;790. https://doi.org/10.1643/h2020090\u003c/li\u003e\n\u003cli\u003eTouchon JC, Worley JL (2015) Oviposition site choice under conflicting risks demonstrates that aquatic predators drive terrestrial egg-laying. Proc R Soc B Biol Sci 282:20150376. https://doi.org/10.1098/rspb.2015.0376\u003c/li\u003e\n\u003cli\u003eTrumbo ST (2012) Patterns of parental care in invertebrates. Evol Parent care 81\u0026ndash;100\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Variables measured for each pool during the field survey.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003e\u003cstrong\u003eExplanation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasured interval\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasured scale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCriteria used\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eDry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eWhether each pool had water or not\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eLeaves\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe amount of leaf litter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003e0 \u0026ndash; 4 (ordinal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e0: No leaf litter\u003c/p\u003e\n \u003cp\u003e1: A small amount of leaf litter covering less than 5 % of pool bottom\u003c/p\u003e\n \u003cp\u003e2: Leaf litter covering more than 5% but less than 1/3 of the pool bottom\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3: Leaf litter covering about half of the pool bottom\u003c/p\u003e\n \u003cp\u003e4: Pool bottom fully covered with leaf litter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eStones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe amount of stones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003e0 \u0026ndash; 3 (ordinal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e0: No stones\u003c/p\u003e\n \u003cp\u003e1: A few small stones that covered less than 1/3 of the pool bottom\u003c/p\u003e\n \u003cp\u003e2: Stones covering about half of the pool bottom\u003c/p\u003e\n \u003cp\u003e3: Stones fully covering the pool bottom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eTurbidity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe degree of water turbidity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003e0 \u0026ndash; 3 (ordinal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e0: Fully transparent with no signs of turbidity\u003c/p\u003e\n \u003cp\u003e1: Low turbidity; slight murkiness detectable, but underwater visibility remains clear\u003c/p\u003e\n \u003cp\u003e2: High turbidity; underwater visibility is reduced but still possible at close range\u003c/p\u003e\n \u003cp\u003e3: Severe turbidity; underwater visibility is virtually impossible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eNewEggs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eWhether new eggs, oviposited the previous night, were found\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eConEggs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe presence of conspecific eggs\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eConTadpoles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe presence of conspecific tadpoles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eHeteroEggs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe presence of heterospecific eggs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eHeteroTadpoles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe presence of heterospecific tadpoles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003ePredators\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe presence of aquatic insect predators\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u003cem\u003eCybister chinensis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eLaccotrphes japonensis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eNotonecta triguttata\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eOdonate larvae\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eMosqLarvae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eThe presence of mosquito larvae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eDaily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePresence/absence (binary)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003ePoolVolume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003ePool volume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eOnce\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003eContinuous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.746913580246915%\"\u003e\n \u003cp\u003eCanopyCover\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.061728395061728%\"\u003e\n \u003cp\u003eCanopy coverage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.796296296296296%\"\u003e\n \u003cp\u003eOnce\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.444444444444443%\"\u003e\n \u003cp\u003ePercentage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.95061728395062%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:justify;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 2. Summary of the results showing the factors that were correlated with oviposition preferences.\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u0026lsquo;+\u0026rsquo; and \u0026lsquo;\u0026ndash;\u0026rsquo; symbol indicates positive and negative correlation, respectively. \u0026lsquo;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026cap;\u0026rsquo; symbols show quadratic effects, indicating that oviposition was more frequent in the mid-range values.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:63.55pt;border:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eVariable type\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:103.25pt;border:solid windowtext 1.0pt;border-left: none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eVariables\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.65pt;border:solid windowtext 1.0pt;border-left: none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eRana uenoi\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.8pt;border:solid windowtext 1.0pt;border-left: none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eHynobius quelpaertensis\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.7pt;border:solid windowtext 1.0pt;border-left: none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eBombina orientalis\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.85pt;border:solid windowtext 1.0pt;border-left: none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDryophytes japonicus\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" style=\"width:63.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eAbiotic factors\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:103.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003ePoolVolume\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e+\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e+\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B0F0;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026cap;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e+\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:103.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDry\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026ndash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026ndash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026ndash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026ndash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:103.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eLeaves\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e+\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B0F0;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026cap;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026ndash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:103.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eCanopyCover\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:103.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eStones\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;font-size:13px;font-family:\"Malgun Gothic\",sans-serif;line-height:200%;'\u003e\u003cspan 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conspecific avoidance, multiple factors","lastPublishedDoi":"10.21203/rs.3.rs-4019240/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4019240/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Many amphibians are selective about where to lay eggs, as this greatly affects their offpsring’s survival. Theoretically, species sharing the same habitat are expected to experience similar selective pressures, leading to the prediction that they might share similar oviposition preferences. However, this hypothesis has not yet been tested. In this study, through an extensive field survey, we examined the oviposition site selection of four amphibian species (Rana uenoi, Hynobius quelpaertensis, Bombina orientalis, and Dryophytes japonicus) that reproduce in the same natural ephemeral stream. Our focus was on understanding how various abiotic and biotic factors influence their reproductive choices. We primarily found that drought avoidance is a universal selective pressure affecting all species, with a tendency to avoid laying eggs in smaller pools prone to drought. Species-specific responses to leaf litter and canopy coverage were observed, but none of the species' oviposition choices correlated with the quantity of stones in pools. The study also explored biotic influences, revealing species-specific trends in the selection of pools with conspecific and heterospecific juveniles, predators, and mosquito larvae, indicating a complex ecological interplay. These findings highlight the complexity of amphibian reproductive strategies, where decisions are not driven solely by a single factor such as the avoidance of predators or competitors but also by intricate assessments of multiple factors. The study highlights that amphibian oviposition in ephemeral streams is influenced by a diverse interplay of biotic and abiotic factors, essential for understanding their reproductive strategies in dynamic environments.","manuscriptTitle":"Oviposition strategies of amphibians in ephemeral streams: a multi-species perspective","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-16 16:51:20","doi":"10.21203/rs.3.rs-4019240/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1374a7ba-d849-4df6-9988-304a4836f490","owner":[],"postedDate":"April 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-21T14:39:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-16 16:51:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4019240","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4019240","identity":"rs-4019240","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}