Water environment and social isolation affect association preference rather than kinship in toad tadpoles (Bufo formosus)

Water environment and social isolation affect association preference rather than kinship in toad tadpoles (Bufo formosus) | 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 Water environment and social isolation affect association preference rather than kinship in toad tadpoles (Bufo formosus) Kazuko Hase This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7930339/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 Kin-biased association is widespread among animal societies. Animals capable of kin recognition often rely on phenotype matching based on odor cues. Because individuals must learn the scent of their relatives, kin discrimination frequently depends on early social experience and environmental conditions. This study investigated how water environment and social experience influence association preferences in Bufo formosus (eastern Japanese common toad) tadpoles. Using a 2×2 factorial design, subject tadpoles were reared either in tap or pond water and under group or solitary conditions. Binary-choice tests were then conducted to examine whether tadpoles preferred siblings or non-siblings. No significant kin-biased association was detected in any treatment, suggesting that B. formosus tadpoles do not employ phenotype-matching-based kin recognition. However, both social experience and water type significantly affected general social tendencies: group-reared tadpoles interacted more with conspecifics than solitary-reared ones, and those raised in tap water spent more time near siblings than those reared in pond water. These findings indicate that social aggregation in B. formosus is shaped by social experience and environmental context rather than kinship. Aggregation behavior Bufo formosus Kin recognition Phenotype-matching Social experience Toad tadpole Figures Figure 1 Figure 2 Figure 3 Significance Statement Kin-biased association does not play a central role in Bufo formosus social aggregation. Tadpoles did not show significant preference for siblings over non-siblings in any condition, indicating no phenotype-matching-based kin recognition. However, social experience and aquatic environment significantly affected social associations. Group-reared tadpoles were more active and spent more time near conspecifics than isolated individuals. Pond-water conditions appeared to reduce interest in conspecifics. Tadpoles reared in tap water spent more time near siblings than those reared in pond water. INTRODUCTION Social animals often show association preferences. During group formation, recognizing suitable partners is essential. Because individuals rely on cooperation, they must discriminate among conspecifics. Inclusive fitness theory (Hamilton 1964a, b) predicts that related individuals are more likely to cooperate than unrelated ones, reducing the risk of harm among kin even under intense competition. Assessing kin recognition ability thus offers key insights into the evolution of social grouping (Breed 2014). Kin recognition occurs across diverse animal taxa and typically involves three steps: production of a kin label, its perception, and kin-biased behavior (Reeve 1989; Pfennig 1997; Starks 2004). The first step requires a template for assessing relatedness, often established through early social learning (Holmes and Sherman 1982; Blaustein 1983; Mateo 2017). For example, in ground squirrels, recognition relies on both genetic relatedness and learned odor cues from the natal nest (Holmes and Sherman 1982; Mateo 2002). Kin labels can be acquired through prior association, resulting in an “odor of kin” used to evaluate unfamiliar conspecifics. This process, known as phenotype matching, includes two forms: simple phenotype matching , in which individuals learn the labels from familiar conspecifics and generalize them to others with shared traits, and self-referent phenotype matching , in which individuals use their own phenotype as a template for kin recognition (Holmes 1986; Hauber et al. 2000, 2001; Mateo and Johnston 2000; Mateo 2010, 2017). The learning process is particularly crucial in phenotype-matching-based kin recognition, with kin templates typically shaped through interactions with conspecifics during the early life stage. In many animal species, individuals benefit from grouping with conspecifics through predator avoidance (e.g., fish school, Eggers 1978; Godin and Morgan 1985), thermal control (e.g., see lion, Liwanag et al. 2014), and enhanced foraging efficiency (e.g., in bird, Camphuysen and Garthe 2004; in spider, Rypstra 1989). Amphibian larvae (tadpoles) also form schools similar to fish and can often recognize their siblings (Wells 2010). Because individuals hatch together within the same egg mass, they naturally learn the odor of relatives through early association. Thus, tadpoles which exhibit kin recognition ability typically discriminate kin from non-kin via phenotype matching and form kin-biased associations. The adaptive significance of kin discrimination in tadpoles remains unclear, but intraspecific competition likely plays a major role. From the perspective of inclusive fitness, competition among relatives should be reduced. In Spea multiplicata and S. bombifrons , species with both cannibalistic and omnivorous morphs, cannibalistic individuals discriminate kin more strongly than omnivorous ones (Pfennig 1990, 1999; Pfennig et al. 1993). In Rana ornativentris , to reduce kin competition, large-sized tadpoles avoid smaller siblings but approach smaller non-siblings for feeding (Hase and Kutsukake 2019). Fouilloux et al. (2022) noted that cannibalism in Dendrobates tinctorius tadpoles reflects a balance between relatedness and size, with individuals trading off their own fitness for that of their relatives, as shown through experimental and theoretical approaches. Furthermore, several kin discrimination manners are plastically determined rather than only innate. Kin-biased associations observed in tadpoles reared with siblings disappear when reared with a mixture of siblings and non-siblings group (Waldman 1981; O’Hara and Blaustein 1982; Blaustein and O’Hara 1986). In R. ornativentris , tadpoles raised with non-siblings no longer distinguish between siblings and non-siblings, indicating that rearing environment shapes recognition of relatives and preferred conspecifics (Hase and Kutsukake 2022). This plasticity is likely common among animals with phenotype-matching-based kin recognition, where social association depends on rearing conditions (Hepper 1991; Breed 2014). The Japanese eastern common toad ( Bufo formosus ) occurs in northeastern Japan and exhibits explosive breeding in early spring following hibernation. Breeding typically occurs less than a week from mid-February to mid-March, and tadpoles complete metamorphosis within roughly six weeks (Hase and Shimada 2014; Matsui and Maeda 2018). As in other Bufoideae species, the tadpoles are black, toxic, and form cohesive aggregations. Such grouping likely provides both predator dilution(Watt et al. 1997) and aposematic benefits through their dark coloration (Stynoski and Porras-Brenes 2022). Kin recognition based on phenotype matching has been documented in several Bufo species [in B. americunus (Waldman 1982, 1986); in B. bufo (Kiseleva 1989) ; in B. melanostictus (Saidapur and Girish 2000; Eluvathingal et al. 2009); in B. scaber (Gramapurohit et al. 2006)], where individuals learn odour cues from prior association with siblings. If B. formosus shows similar kin-biased associations, learning is likely essential. However, no studies have examined association preferences in the absence of prior social experience—that is, after rearing in social isolation. Environmental factors, such as water quality, have also received little attention. Despite numerous studies, the processes by which tadpoles acquire kin templates and develop aggregation behavior remain unresolved. In this study, I conducted behavioral experiments on B. formosus tadpoles to examine how aquatic environment and social experience influence their association preferences. If tadpoles recognize kin through odor cues, water from natural ponds rich in microorganisms may enhance odor-based learning compared to tap water. To test this, larvae were reared in either tap water or pond water. In addition, to assess the effect of social experience, tadpoles were raised either in groups or in solitary. Kin recognition by solitary-reared individuals would indicate self-referent phenotype matching . Using a 2×2 factorial design combining two water types and two social conditions, I tested whether B. formosus tadpoles discriminate kin through self-referent or simple phenotype matching , and whether microbe-rich environments facilitate odor-based learning. MATERIAL AND METHODS Animals In April 2021, four egg strings (about 30 cm) were collected from four separated puddles on the grounds of Botanical Gardens, Graduate School of Science, the University of Tokyo, Tochigi Prefecture, Japan. Each egg-derived larvae were treated as a different sibling cohort in the rearing experiment. In the laboratory, after embryo hatching (developmental stage 22, Gosner 1960), the larvae were transferred to rearing containers. The four rearing treatments were divided according to whether the water environment was tap water or pond water, and the social environment in which the larvae were reared, group (N = 20) or solitary (N = 1): tap water and group (hereinafter, Tg); tap water and solitary (Ts); pond water and group (Pg); pond water and solitary (Ps). Exact container sizes and social conditions, as well as the amount of environmental waters, are shown in Fig. 1 a. Tap water was dechlorinated water. Pond water was transported from Ichijiro Pond at the campus of the University of Tokyo, and the water characteristics are the same as in the previous study (Hase 2022). All individuals were reared for two weeks in a large incubator at 18°C, 12:12-h dark: light cycle with a sufficient supply of fish food pellets containing vegetable stock as the main component (PLECO; Kyorin, Hyōgo, Japan). And Individuals reaching the 30–35 developmental stage (Gosner 1960) were used in behavioral experiment. Behavioral experiment To examine kin identification in juveniles, the four types of choice tests were designed according to previous studies. The differences in the four tests reflect the differences in the four rearing treatment types. A common scheme was used to verify kin recognition ability and the influence of the rearing conditioning in B. formosus tadpole. Diagram of the arenas where the binary-choice tests were conducted is shown in Fig. 2 b. The arena was divided into four equal sections, and 10 stimulus individuals, 10 siblings or 10 non-siblings were placed. The time spent by the subject tadpole was measured to determine which effective stimuli side they stayed in longer. The subject individuals, placed quietly in the center of the arena, were captured by a camera (HCV720-M; Panasonic, Osaka, Japan). Each trial was conducted with 5 minutes of acceleration followed by 30 minutes, then the process was repeated with left and right turns to eliminate bias, for a total of 60 minutes. All subjects were tracked by UMAtracker (Yamanaka and Takeuchi 2018), and the time spent in the stimulus zones at both ends was measured in 1-second intervals. During the test, water was used under conditions similar to those under which the subjects were grown. For example, when individuals grown in pond water were tested, the stimulus individuals were also grown in pond water, and the arena was filled with pond water (Fig. 1 b). Four types of subjects were used in accordance with four different rearing conditions. All stimuli (siblings and non-siblings) were unfamiliar, i.e., stimuli tadpoles had been raised separately in a container from subjects. Raw data of the four types of choice test were shown in supplementary table S1 . Data analysis Statistical analyses were conducted using R version 4.3.1(R Core Team 2023). To access whether tadpoles discriminate siblings from non-siblings by phenotype-matching, I compared the time spent on each effective stimulus side (out of 3600s), sibling and non-sibling, using a paired t-test. Differences were considered significant at p < 0.05. If a statistically significant difference is obtained in both groups and solitary social treatments (Tg and Ts or Pg and Ps), then B. formosus has a self-referent kin recognition system. If the superiority is significant only in group treatments (Tg or Pg, or both), then B. formosus possess a simple phenotype-matching. Next, to examine the effects of social experience (group or solitary) and the water environment (tap or pond), I run a generalized linear mixed model (GLMM) with a binomial error distribution. To put it in concrete terms, among the four binary-choice tests (Tg, Ts, Pg, and Ps), whether there were differences in the time spent on the effective stimuli sides were examined by GLMM and post hoc test. GLMM was performed twice, depending on whether the stimuli were siblings or non-siblings. That is, a GLMM was performed with time spent on the sibling side (out of 3600s) as the response variable, and a separate GLMM was performed with time spent on the non-sibling side (out of 3600s) as the response variable. In both GLMM, social experimence, water type, and those of two-way interaction were treated as explanatory variables. The type of origin of egg strings of subject tadpoles was treated as a random effect. When the two-way interactions were significant, I conducted post hoc tests using Tukey-adjusted pairwise comparisons. RESULTS The results of the binary-choice tests are shown in Table 1 and Fig. 2 . In all tests, no statistical significance was obtained between the time spent on sibling and non-sibling effective sides ( p > 0.05). This means that tadpoles of B. formosus do not use any phenotype-matching kin discrimination system in the context of the association preferences. Table 1 Summary of the binary-choice test between unfamiliar siblings and unfamiliar non-siblings. Rearing treatments for subject tadpoles. Water, type of water. Sociality, social treatments. Sample size, number of trials (subject tadpoles). Spent time on sibling, non-sibling sides, spent not on either side, i.e., near the center (out of 3600s). P value, paired two-tailed t test Rearing treatments Sample size Spent time on sibling side (mean ± SD) Spent time on non-sibling side (mean ± SD) Spent not on either side (mean ± SD) P value Water Sociality Abbreviation Tap Group Tg 22 1419.0 ± 702.2s 1306.0 ± 608.3s 875.0 ± 352.3s 0.680 Tap Solitary Ts 21 1213.3 ± 511.7s 1184.6 ± 706.6s 1202.1 ± 696.9s 0.899 Pond Group Pg 25 1393.3 ± 518.2s 1384.4 ± 568.3s 822.2 ± 364.6s 0.966 Pond Solitary Ps 22 1075.2 ± 576.9s 1192.8 ± 692.2s 1332 ±695.8s 0.619 GLMM analyses indicated a significant effect of rearing treatment on association preference, and Table 2 summarizes this effect. Social experience, water environment, and their interaction showed significant effects on the time spent on the sibling side (Fig. 3 a). In the post hoc test, differences in time spent on the sibling side were observed among all conditioning cohorts (Tg, Ts, Pg, and Ps; Table 2 ). Tadpoles reared in groups spent more time on the sibling side than those reared solitarily, and those raised in tap water spent more time on the sibling side than those raised in pond water (Tg > Pg > Ts > Ps). In contrast, similarity, social experience, water environment, and their interaction affected the time spent on the non-sibling side (Fig. 3 b). Although no difference in time spent was found between the Ts and Ps conditions, all other pairwise comparisons showed significant differences (Table 2 ). Tadpoles reared in groups and pond water spent more time on the non-sibling side than those reared in groups and tap water (Tg < Pg), whereas no significant difference was detected between solitary tadpoles reared in tap and pond water (Ts = Ps). The differences in time spent on the sibling and non-sibling sides corresponded to variations in the time spent near the center of the arena (Table 1 ). GLMM analyses indicated a significant effect of rearing treatment on association preference, and Table 2 summarizes this effect. Social experience, water environment, and their interaction showed significant effects on the time spent on the sibling side (Fig. 3 a). In the post hoc test, differences in time spent on the sibling side were observed among all conditioning cohorts (Tg, Ts, Pg, and Ps; Table 2 ). Tadpoles reared in groups spent more time on the sibling side than those reared solitarily, and those raised in tap water spent more time on the sibling side than those raised in pond water (Tg > Pg > Ts > Ps). In contrast, similarity, social experience, water environment, and their interaction affected the time spent on the non-sibling side (Fig. 3 b). Although no difference in time spent was found between the Ts and Ps conditions, all other pairwise comparisons showed significant differences (Table 2 ). Tadpoles reared in groups and pond water spent more time on the non-sibling side than those reared in groups and tap water (Tg < Pg), whereas no significant difference was detected between solitary tadpoles reared in tap and pond water (Ts = Ps). The differences in time spent on the sibling and non-sibling sides corresponded to variations in the time spent near the center of the arena (Table 1 ). Table 2 Results of analyses by the general linear mixed model (GLMM) and Post hoc tests. Rearing treatments are explanatory fixed variables in GLMM. Water : Socility means interaction between water types (tap or pond) and Sociality treatments (solitary or group). The abbreviations used for comparisons in the Post hoc test correspond with Table 1 . Bold text denotes statistically significant values. Time allocation for Analysis Variable / Comparison Estimate ± SE z/z ratio P Sibling GLMM Water type 0.027 ± 0.010 2.666 0.0077 Sociality –0.396 ± 0.010 –38.251 < .0001 Water : Socility 0.145 ± 0.015 9.758 Ts 0.251 ± 0.010 23.632 Ps 0.396 ± 0.010 38.251 Pg 0.027 ± 0.010 2.666 0.0384 Tg > Ps 0.423 ± 0.011 39.662 < .0001 Ts < Pg –0.225 ± 0.010 –21.703 Ps 0.171 ± 0.011 15.623 < .0001 Non-sibling GLMM Water type –0.0813 ± 0.010 –8.045 < .0001 Sociality –0.232 ± 0.010 –22.695 < .0001 Water : Socility 0.093 ± 0.015 6.252 Ts 0.139 ± 0.011 12.923 Ps 0.232 ± 0.010 22.695 < .0001 Tg < Pg –0.081 ± 0.010 –8.045 Ps 0.150 ± 0.011 14.195 < .0001 Ts < Pg –0.220 ± 0.010 –21.184 < .0001 Ts < Ps 0.011 ± 0.011 1.047 0.722 DISCUSSION Despite expectations, the results of the behavioral tests indicated that B. formosus tadpoles do not exhibit phenotype-matching-based kin recognition. This occurred even though B. formosus tadpoles display social aggregation, as do several related species that possess the kin recognition abilities (Waldman 2005). Differences in the spent time of stimuli sides were observed in the results of the choice tests among the four rearing treatments. However, kin-biased association did not appear to play an essential role in the tadpoles’ grouping behavior. The reasons for this can be discussed from two perspectives. First, in the grouping behavior of toad tadpoles, the number of conspecifics may be more important than kin-biased association. The primary function of tadpole aggregation is likely predator avoidance. It has been proposed that the distinctive black coloration of these tadpoles serves as a warning signal to predators, which is enhanced by cohesive aggregation (Wells 2010; Stynoski and Porras-Brenes 2022). In this context, kin-biased association with siblings is not essential for effective aggregation. Some species may therefore achieve cohesive social aggregation without kin discrimination. Watt et al. (1997) showed that tadpoles are less likely to be predated upon when in larger groups. Additionally, a binary-choice test in Miyako toads ( B. gargarizans miyakonis ) indicated that group size is a critical factor (Hase 2023). In the present study, the same number of tadpoles (10 vs. 10) was used in the binary-choice tests, and no significant differences were observed (Table 1 , Fig. 2 ). This suggests that, in black-colored toad tadpoles, group size may be more important than kinship in aggregation behavior. Second, a mechanistic issue in kin discrimination of tadpoles, primarily related to their genetic background (Green et al. 2015; Scott et al. 2022). A theoretical study suggests that phenotype-matching–based kin recognition systems allow individuals to distinguish between full siblings, half siblings, and nonrelatives by referring to their own matching traits (Lacy and Sherman 1983). Previous studies using B. americanus demonstrated the ability to discriminate unfamiliar siblings from familiar non-siblings (Waldman 1986). The experimental design of Waldman (1986) did not differ fundamentally from the present study, although it is possible that the siblings and non-siblings in previous studies had greater genetic divergences than those used in this study. This background pattern may be common across species in which phenotype-matching-based kin recognition is present in some tadpoles but absent in others. For example, B. boreas did not exhibit phenotype matching.(O’Hara and Blaustein 1982) The ‘kin labels’ of B. formosus and B. boreas tadpoles may not differ enough to allow sibling–non-sibling distinction. However, the absence of observed kin discrimination in B. formosus does not necessarily indicate a lack of kin recognition ability. They may instead use learned 'kin labels' after metamorphosis and during maturity, for example in the context of inbreeding avoidance (Waldman 2005). B. formosus tadpoles varied the time spent on both stimulus sides (sibling and non-sibling) depending on social treatment and water environment (Fig. 3 ). In both water types, social treatment influenced the time spent on stimulus sides. Tadpoles reared in isolation (belonging to Ts and Ps) spent more time near the center, indicating reduced interaction with siblings or non-siblings. Compared to group-reared individuals, solitary-reared tadpoles appeared less interested in conspecifics. This result suggests that, regardless of kin recognition, sociality in tadpoles requires learning. Solitary conditioning—used here to test self-referent phenotype matching—is equivalent to social isolation and can affect nervous system development, as shown in mice (Koike et al. 2009; Lander et al. 2017). This may manifest as reduced interest in conspecifics. In contrast, group-reared subjects frequently moved between sibling and non-sibling sides, as if evaluating which side to choose (Table 2 ; Fig. 2 ). These observations suggest that B. formosus tadpoles develop grouping behavior only after being raised in a group. Regarding water environments, we predicted that tadpoles raised in pond water, which contains more microorganisms, would exhibit stronger odor-based differences between siblings and non-siblings than those raised in tap water. However, this prediction was not supported. No kin-biased association was observed under pond-water conditioning, regardless of social treatment. In fact, pond water appeared to reduce interest in siblings: tadpoles spent less time on sibling sides (Tg > Pg; Ts > Ps; Fig. 2 ). Pond water may interfere with recognition mechanisms. A previous study using the same pond water reported higher mortality in B. japonicus tadpoles when reared in sibling groups (Hase 2022). If the pond environment contains lethal pathogens, it may not be advantageous to crowd with siblings, whose odors are familiar. Consistent with this, subject tadpoles belonging to Pg spent less time with siblings and more time with non-siblings compared to Tg (Fig. 3 ). For tadpoles in Pg, when pathogens are present, avoiding siblings may be adaptive—a form of “social distancing” (Sandel et al. 2020; Stockmaier et al. 2021). In this study, no kin discrimination was detected in Bufo formosus tadpoles. Although no kin-biased association preference was observed in the binary-choice tests with equal stimulus group sizes (10 vs. 10), the results suggest that tadpoles develop sociality through their rearing environment. Whether the failure to acquire kin recognition ability and/or sociality during the larval stage affects individual fitness remains to be investigated. Declarations Competing Interests The author declares that there are no competing interests. Ethical Approval All tadpoles were treated carefully to minimise physical stress, in compliance with the ethical guidelines of the institute. The protocol was reviewed and accepted by the Ethics Committee for Animal Research of the Graduate University for Advanced Studies (no. SKD2018AR002). Funding This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) Research Fellowship (No. 19K16226). Author Contribution Kazuko Hase designed the study, conducted the experiments, analyzed the data, and wrote the manuscript. Acknowledgement I thank the staff of the Botanical Gardens, Graduate School of Science, the University of Tokyo, for their cooperation during field surveys and sampling. I am deeply grateful to Emeritus Prof. M. Shimada of the University of Tokyo for providing the large incubator used in the rearing experiments. This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) Research Fellowship (No. 19K16226). Data Availability All datasets generated during and/or analyzed during the current study are available in the supplementary material. References Blaustein AR (1983) Kin Recognition Mechanisms: Phenotypic Matching or Recognition Alleles? Am Nat 121:749–754. https://doi.org/10.1086/284101 Blaustein AR, O’Hara RK (1986) An investigation of kin recognition in Red-legged frog (Rana aurora) tadpoles. 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R Foundation for Statistical Computing Reeve HK (1989) The Evolution of Conspecific Acceptance Thresholds. https://doi.org/101086/284926 133:407–435. https://doi.org/10.1086/284926 Rypstra AL (1989) Foraging success of solitary and aggregated spiders: insights into flock formation. Anim Behav 37:274–281 Saidapur SK, Girish S (2000) The ontogeny of kin recognition in tadpoles of the toad Bufo melanostictus (Anura; Bufonidae). J Biosci 25:267–273. https://doi.org/10.1007/BF02703935 Sandel AA, Rushmore J, Negrey JD, et al (2020) Social Network Predicts Exposure to Respiratory Infection in a Wild Chimpanzee Group. Ecohealth 17:437–448. https://doi.org/10.1007/S10393-020-01507-7/FIGURES/4 Scott TW, Grafen A, West SA (2022) Multiple social encounters can eliminate Crozier’s paradox and stabilise genetic kin recognition. Nature Communications 2022 13:1 13:1–19. https://doi.org/10.1038/s41467-022-31545-4 Starks PT (2004) Recognition systems: from components to conservation. Ann Zool Fenn 41:689–690 Stockmaier S, Stroeymeyt N, Shattuck EC, et al (2021) Infectious diseases and social distancing in nature. Science (1979) 371:. https://doi.org/10.1126/SCIENCE.ABC8881/ASSET/7B75219B-DB20-4BF7-9F56-CF1A2C3E20F9/ASSETS/GRAPHIC/371_ABC8881_F5.JPEG Stynoski JL, Porras-Brenes K (2022) Meta-analysis of tadpole taste tests: consumption of anuran prey across development and predator strategies. Oecologia 199:845–857. https://doi.org/10.1007/s00442-022-05221-9 Waldman B (1981) Sibling Recognition in Toad Tadpoles: The Role of Experience. Z Tierpsychol 56:341–358. https://doi.org/10.1111/J.1439-0310.1981.TB01306.X Waldman B (1986) Preference for unfamiliar siblings over familiar non-siblings in American toad (Bufo americanus) tadpoles. Anim Behav 34:48–53. https://doi.org/10.1016/0003-3472(86)90005-9 Waldman B (1982) Sibling association among schooling toad tadpoles: field evidence and implications. Anim Behav 30:700–713. https://doi.org/10.1016/S0003-3472(82)80141-3 Waldman B (2005) Kin recognition in amphibians. In: Hepper PG (ed) Kin Recognition. Cambridge University Press, Cambrige, pp 162–219 Watt PJ, Nottingham SF, Young S (1997) Toad tadpole aggregation behaviour: evidence for a predator avoidance function. Anim Behav 54:865–872. https://doi.org/10.1006/ANBE.1996.0512 Wells KD (2010) The Ecology and Behavior of Amphibian Larvae. In: The Ecology and Behavior of Amphibians. University of Chicago press, pp 557–598 Yamanaka O, Takeuchi R (2018) UMATracker: An intuitive image-based tracking platform. Journal of Experimental Biology 221:. https://doi.org/10.1242/JEB.182469 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1.xlsx Table S1. Raw data of the binary choice tests 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7930339","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":537350085,"identity":"ae5babe3-3dbe-4c81-bd8a-214163219237","order_by":0,"name":"Kazuko Hase","email":"data:image/png;base64,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","orcid":"","institution":"Tohoku University","correspondingAuthor":true,"prefix":"","firstName":"Kazuko","middleName":"","lastName":"Hase","suffix":""}],"badges":[],"createdAt":"2025-10-23 08:54:17","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7930339/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7930339/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96247768,"identity":"4c219dbb-bf5f-41f6-8bed-f906c4cdd45c","added_by":"auto","created_at":"2025-11-19 07:27:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":220122,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of the four rearing treatments (\u003cstrong\u003ea\u003c/strong\u003e) and the binary-choice test arena (\u003cstrong\u003eb\u003c/strong\u003e). Tap water was dechlorinated, and pond water was filtered through a 100-mesh polyethylene net. Water temperature was maintained at 18 ℃. Group- and solitary-reared containers were supplied with 1.5 L and 0.2 L of tap or pond water, respectively, which was changed every two days. For the binary-choice tests, the tank was filled with 1.5 L of water, and it was replaced between each trial. Subjects could perceive visual and chemical cues from stimuli across polyethylene nets (strings: 0.1 mm diameter; mesh: 13–14 threads/cm²). Dashed and solid line areas indicate quarters of the arena corresponding to the effective “Non-sibling stimuli side” and “Sibling stimuli side,” respectively.\u003c/p\u003e","description":"","filename":"Fig.1arena.png","url":"https://assets-eu.researchsquare.com/files/rs-7930339/v1/d06e60505609d6b522f626b7.png"},{"id":96087115,"identity":"28fb5ac0-f5dc-45ca-ad5f-df640629f019","added_by":"auto","created_at":"2025-11-17 12:39:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":201082,"visible":true,"origin":"","legend":"\u003cp\u003eTime allocation in four types of binary-choice tests. The y-axis shows time spent on the effective sibling stimulus side (+) and the effective non-sibling stimulus side (−). Dark-filled and light-dashed bars represent the mean ± standard deviation for the sibling and non-sibling sides, respectively. Rearing treatments for the subject tadpoles are tap water with group rearing (Tg), tap water with solitary rearing (Ts), pond water with group rearing (Pg), and pond water with solitary rearing (Ps), shown from left to right on the x-axis. Numbers in parentheses indicate the number of trials. NS indicates not significant (paired two-tailed t-test).\u003c/p\u003e","description":"","filename":"Fig.2result20250827.png","url":"https://assets-eu.researchsquare.com/files/rs-7930339/v1/c5562b21501ed839c3c1df4f.png"},{"id":96087117,"identity":"cf5dde20-8830-489b-b04e-f39c5469d8d1","added_by":"auto","created_at":"2025-11-17 12:39:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":855428,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction plots for the mean time spent on the effective sibling stimulus side (\u003cstrong\u003ea\u003c/strong\u003e) and the effective non-sibling stimulus side (\u003cstrong\u003eb\u003c/strong\u003e) by water types, tap or pond water, in relation to social treatment status.\u003c/p\u003e","description":"","filename":"Fig.3interaction0828.png","url":"https://assets-eu.researchsquare.com/files/rs-7930339/v1/dba12aa3d2dd6e2e57afc84a.png"},{"id":96737843,"identity":"724c2260-d90e-4529-8593-50f9405f0248","added_by":"auto","created_at":"2025-11-25 14:39:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1770100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7930339/v1/9ddc98c7-58ff-4cb1-9973-69137610f385.pdf"},{"id":96087118,"identity":"91b6e700-a881-4235-b32e-eaef4e2dacb7","added_by":"auto","created_at":"2025-11-17 12:39:19","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e. Raw data of the binary choice tests\u003c/p\u003e","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7930339/v1/ee2f3991a83ed3520d3428fc.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Water environment and social isolation affect association preference rather than kinship in toad tadpoles (Bufo formosus)","fulltext":[{"header":"Significance Statement","content":"\u003cp\u003eKin-biased association does not play a central role in \u003cem\u003eBufo formosus\u003c/em\u003e social aggregation. Tadpoles did not show significant preference for siblings over non-siblings in any condition, indicating no phenotype-matching-based kin recognition. However, social experience and aquatic environment significantly affected social associations. Group-reared tadpoles were more active and spent more time near conspecifics than isolated individuals. Pond-water conditions appeared to reduce interest in conspecifics. Tadpoles reared in tap water spent more time near siblings than those reared in pond water.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eSocial animals often show association preferences. During group formation, recognizing suitable partners is essential. Because individuals rely on cooperation, they must discriminate among conspecifics. Inclusive fitness theory (Hamilton 1964a, b) predicts that related individuals are more likely to cooperate than unrelated ones, reducing the risk of harm among kin even under intense competition. Assessing kin recognition ability thus offers key insights into the evolution of social grouping (Breed 2014).\u003c/p\u003e\u003cp\u003eKin recognition occurs across diverse animal taxa and typically involves three steps: production of a kin label, its perception, and kin-biased behavior (Reeve 1989; Pfennig 1997; Starks 2004). The first step requires a template for assessing relatedness, often established through early social learning (Holmes and Sherman 1982; Blaustein 1983; Mateo 2017). For example, in ground squirrels, recognition relies on both genetic relatedness and learned odor cues from the natal nest (Holmes and Sherman 1982; Mateo 2002). Kin labels can be acquired through prior association, resulting in an \u0026ldquo;odor of kin\u0026rdquo; used to evaluate unfamiliar conspecifics. This process, known as phenotype matching, includes two forms: \u003cem\u003esimple phenotype matching\u003c/em\u003e, in which individuals learn the labels from familiar conspecifics and generalize them to others with shared traits, and \u003cem\u003eself-referent phenotype matching\u003c/em\u003e, in which individuals use their own phenotype as a template for kin recognition (Holmes 1986; Hauber et al. 2000, 2001; Mateo and Johnston 2000; Mateo 2010, 2017). The learning process is particularly crucial in phenotype-matching-based kin recognition, with kin templates typically shaped through interactions with conspecifics during the early life stage.\u003c/p\u003e\u003cp\u003eIn many animal species, individuals benefit from grouping with conspecifics through predator avoidance (e.g., fish school, Eggers 1978; Godin and Morgan 1985), thermal control (e.g., see lion, Liwanag et al. 2014), and enhanced foraging efficiency (e.g., in bird, Camphuysen and Garthe 2004; in spider, Rypstra 1989). Amphibian larvae (tadpoles) also form schools similar to fish and can often recognize their siblings (Wells 2010). Because individuals hatch together within the same egg mass, they naturally learn the odor of relatives through early association. Thus, tadpoles which exhibit kin recognition ability typically discriminate kin from non-kin via phenotype matching and form kin-biased associations. The adaptive significance of kin discrimination in tadpoles remains unclear, but intraspecific competition likely plays a major role. From the perspective of inclusive fitness, competition among relatives should be reduced. In \u003cem\u003eSpea multiplicata\u003c/em\u003e and \u003cem\u003eS. bombifrons\u003c/em\u003e, species with both cannibalistic and omnivorous morphs, cannibalistic individuals discriminate kin more strongly than omnivorous ones (Pfennig 1990, 1999; Pfennig et al. 1993). In \u003cem\u003eRana ornativentris\u003c/em\u003e, to reduce kin competition, large-sized tadpoles avoid smaller siblings but approach smaller non-siblings for feeding (Hase and Kutsukake 2019). Fouilloux et al. (2022) noted that cannibalism in \u003cem\u003eDendrobates tinctorius\u003c/em\u003e tadpoles reflects a balance between relatedness and size, with individuals trading off their own fitness for that of their relatives, as shown through experimental and theoretical approaches. Furthermore, several kin discrimination manners are plastically determined rather than only innate. Kin-biased associations observed in tadpoles reared with siblings disappear when reared with a mixture of siblings and non-siblings group (Waldman 1981; O\u0026rsquo;Hara and Blaustein 1982; Blaustein and O\u0026rsquo;Hara 1986). In \u003cem\u003eR. ornativentris\u003c/em\u003e, tadpoles raised with non-siblings no longer distinguish between siblings and non-siblings, indicating that rearing environment shapes recognition of relatives and preferred conspecifics (Hase and Kutsukake 2022). This plasticity is likely common among animals with phenotype-matching-based kin recognition, where social association depends on rearing conditions (Hepper 1991; Breed 2014).\u003c/p\u003e\u003cp\u003eThe Japanese eastern common toad (\u003cem\u003eBufo formosus\u003c/em\u003e) occurs in northeastern Japan and exhibits explosive breeding in early spring following hibernation. Breeding typically occurs less than a week from mid-February to mid-March, and tadpoles complete metamorphosis within roughly six weeks (Hase and Shimada 2014; Matsui and Maeda 2018). As in other Bufoideae species, the tadpoles are black, toxic, and form cohesive aggregations. Such grouping likely provides both predator dilution(Watt et al. 1997) and aposematic benefits through their dark coloration (Stynoski and Porras-Brenes 2022). Kin recognition based on phenotype matching has been documented in several \u003cem\u003eBufo\u003c/em\u003e species [in \u003cem\u003eB. americunus\u003c/em\u003e (Waldman 1982, 1986); in \u003cem\u003eB. bufo\u003c/em\u003e(Kiseleva 1989) ; in \u003cem\u003eB. melanostictus\u003c/em\u003e (Saidapur and Girish 2000; Eluvathingal et al. 2009); in \u003cem\u003eB. scaber\u003c/em\u003e (Gramapurohit et al. 2006)], where individuals learn odour cues from prior association with siblings. If \u003cem\u003eB. formosus\u003c/em\u003e shows similar kin-biased associations, learning is likely essential. However, no studies have examined association preferences in the absence of prior social experience\u0026mdash;that is, after rearing in social isolation. Environmental factors, such as water quality, have also received little attention. Despite numerous studies, the processes by which tadpoles acquire kin templates and develop aggregation behavior remain unresolved.\u003c/p\u003e\u003cp\u003eIn this study, I conducted behavioral experiments on \u003cem\u003eB. formosus\u003c/em\u003e tadpoles to examine how aquatic environment and social experience influence their association preferences. If tadpoles recognize kin through odor cues, water from natural ponds rich in microorganisms may enhance odor-based learning compared to tap water. To test this, larvae were reared in either tap water or pond water. In addition, to assess the effect of social experience, tadpoles were raised either in groups or in solitary. Kin recognition by solitary-reared individuals would indicate \u003cem\u003eself-referent phenotype matching\u003c/em\u003e. Using a 2\u0026times;2 factorial design combining two water types and two social conditions, I tested whether \u003cem\u003eB. formosus\u003c/em\u003e tadpoles discriminate kin through \u003cem\u003eself-referent\u003c/em\u003e or \u003cem\u003esimple phenotype matching\u003c/em\u003e, and whether microbe-rich environments facilitate odor-based learning.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eIn April 2021, four egg strings (about 30 cm) were collected from four separated puddles on the grounds of Botanical Gardens, Graduate School of Science, the University of Tokyo, Tochigi Prefecture, Japan. Each egg-derived larvae were treated as a different sibling cohort in the rearing experiment. In the laboratory, after embryo hatching (developmental stage 22, Gosner 1960), the larvae were transferred to rearing containers. The four rearing treatments were divided according to whether the water environment was tap water or pond water, and the social environment in which the larvae were reared, group (N\u0026thinsp;=\u0026thinsp;20) or solitary (N\u0026thinsp;=\u0026thinsp;1): tap water and group (hereinafter, Tg); tap water and solitary (Ts); pond water and group (Pg); pond water and solitary (Ps). Exact container sizes and social conditions, as well as the amount of environmental waters, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. Tap water was dechlorinated water. Pond water was transported from Ichijiro Pond at the campus of the University of Tokyo, and the water characteristics are the same as in the previous study (Hase 2022). All individuals were reared for two weeks in a large incubator at 18\u0026deg;C, 12:12-h dark: light cycle with a sufficient supply of fish food pellets containing vegetable stock as the main component (PLECO; Kyorin, Hyōgo, Japan). And Individuals reaching the 30\u0026ndash;35 developmental stage (Gosner 1960) were used in behavioral experiment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBehavioral experiment\u003c/h3\u003e\n\u003cp\u003eTo examine kin identification in juveniles, the four types of choice tests were designed according to previous studies. The differences in the four tests reflect the differences in the four rearing treatment types. A common scheme was used to verify kin recognition ability and the influence of the rearing conditioning in \u003cem\u003eB. formosus\u003c/em\u003e tadpole. Diagram of the arenas where the binary-choice tests were conducted is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe arena was divided into four equal sections, and 10 stimulus individuals, 10 siblings or 10 non-siblings were placed. The time spent by the subject tadpole was measured to determine which effective stimuli side they stayed in longer. The subject individuals, placed quietly in the center of the arena, were captured by a camera (HCV720-M; Panasonic, Osaka, Japan). Each trial was conducted with 5 minutes of acceleration followed by 30 minutes, then the process was repeated with left and right turns to eliminate bias, for a total of 60 minutes. All subjects were tracked by UMAtracker (Yamanaka and Takeuchi 2018), and the time spent in the stimulus zones at both ends was measured in 1-second intervals. During the test, water was used under conditions similar to those under which the subjects were grown. For example, when individuals grown in pond water were tested, the stimulus individuals were also grown in pond water, and the arena was filled with pond water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Four types of subjects were used in accordance with four different rearing conditions. All stimuli (siblings and non-siblings) were unfamiliar, i.e., stimuli tadpoles had been raised separately in a container from subjects. Raw data of the four types of choice test were shown in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were conducted using R version 4.3.1(R Core Team 2023). To access whether tadpoles discriminate siblings from non-siblings by phenotype-matching, I compared the time spent on each effective stimulus side (out of 3600s), sibling and non-sibling, using a paired t-test. Differences were considered significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. If a statistically significant difference is obtained in both groups and solitary social treatments (Tg and Ts or Pg and Ps), then \u003cem\u003eB. formosus\u003c/em\u003e has a self-referent kin recognition system. If the superiority is significant only in group treatments (Tg or Pg, or both), then \u003cem\u003eB. formosus\u003c/em\u003e possess a simple phenotype-matching.\u003c/p\u003e\u003cp\u003eNext, to examine the effects of social experience (group or solitary) and the water environment (tap or pond), I run a generalized linear mixed model (GLMM) with a binomial error distribution. To put it in concrete terms, among the four binary-choice tests (Tg, Ts, Pg, and Ps), whether there were differences in the time spent on the effective stimuli sides were examined by GLMM and post hoc test. GLMM was performed twice, depending on whether the stimuli were siblings or non-siblings. That is, a GLMM was performed with time spent on the sibling side (out of 3600s) as the response variable, and a separate GLMM was performed with time spent on the non-sibling side (out of 3600s) as the response variable. In both GLMM, social experimence, water type, and those of two-way interaction were treated as explanatory variables. The type of origin of egg strings of subject tadpoles was treated as a random effect. When the two-way interactions were significant, I conducted post hoc tests using Tukey-adjusted pairwise comparisons.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe results of the binary-choice tests are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In all tests, no statistical significance was obtained between the time spent on sibling and non-sibling effective sides (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This means that tadpoles of \u003cem\u003eB. formosus\u003c/em\u003e do not use any phenotype-matching kin discrimination system in the context of the association preferences.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of the binary-choice test between unfamiliar siblings and unfamiliar non-siblings. Rearing treatments for subject tadpoles. Water, type of water. Sociality, social treatments. Sample size, number of trials (subject tadpoles). Spent time on sibling, non-sibling sides, spent not on either side, i.e., near the center (out of 3600s). P value, paired two-tailed t test\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eRearing treatments\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSample size\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpent time on sibling side (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpent time on non-sibling side (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpent not on either side (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWater\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSociality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAbbreviation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTap\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1419.0\u0026thinsp;\u0026plusmn;\u0026thinsp;702.2s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1306.0\u0026thinsp;\u0026plusmn;\u0026thinsp;608.3s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e875.0\u0026thinsp;\u0026plusmn;\u0026thinsp;352.3s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.680\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTap\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolitary\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1213.3\u0026thinsp;\u0026plusmn;\u0026thinsp;511.7s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1184.6\u0026thinsp;\u0026plusmn;\u0026thinsp;706.6s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1202.1\u0026thinsp;\u0026plusmn;\u0026thinsp;696.9s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.899\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePond\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1393.3\u0026thinsp;\u0026plusmn;\u0026thinsp;518.2s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1384.4\u0026thinsp;\u0026plusmn;\u0026thinsp;568.3s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e822.2\u0026thinsp;\u0026plusmn;\u0026thinsp;364.6s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.966\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePond\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolitary\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1075.2\u0026thinsp;\u0026plusmn;\u0026thinsp;576.9s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1192.8\u0026thinsp;\u0026plusmn;\u0026thinsp;692.2s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1332 \u0026plusmn;695.8s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.619\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eGLMM analyses indicated a significant effect of rearing treatment on association preference, and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes this effect. Social experience, water environment, and their interaction showed significant effects on the time spent on the sibling side (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the post hoc test, differences in time spent on the sibling side were observed among all conditioning cohorts (Tg, Ts, Pg, and Ps; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Tadpoles reared in groups spent more time on the sibling side than those reared solitarily, and those raised in tap water spent more time on the sibling side than those raised in pond water (Tg\u0026thinsp;\u0026gt;\u0026thinsp;Pg\u0026thinsp;\u0026gt;\u0026thinsp;Ts\u0026thinsp;\u0026gt;\u0026thinsp;Ps). In contrast, similarity, social experience, water environment, and their interaction affected the time spent on the non-sibling side (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Although no difference in time spent was found between the Ts and Ps conditions, all other pairwise comparisons showed significant differences (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Tadpoles reared in groups and pond water spent more time on the non-sibling side than those reared in groups and tap water (Tg\u0026thinsp;\u0026lt;\u0026thinsp;Pg), whereas no significant difference was detected between solitary tadpoles reared in tap and pond water (Ts\u0026thinsp;=\u0026thinsp;Ps). The differences in time spent on the sibling and non-sibling sides corresponded to variations in the time spent near the center of the arena (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). GLMM analyses indicated a significant effect of rearing treatment on association preference, and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes this effect. Social experience, water environment, and their interaction showed significant effects on the time spent on the sibling side (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the post hoc test, differences in time spent on the sibling side were observed among all conditioning cohorts (Tg, Ts, Pg, and Ps; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Tadpoles reared in groups spent more time on the sibling side than those reared solitarily, and those raised in tap water spent more time on the sibling side than those raised in pond water (Tg\u0026thinsp;\u0026gt;\u0026thinsp;Pg\u0026thinsp;\u0026gt;\u0026thinsp;Ts\u0026thinsp;\u0026gt;\u0026thinsp;Ps). In contrast, similarity, social experience, water environment, and their interaction affected the time spent on the non-sibling side (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Although no difference in time spent was found between the Ts and Ps conditions, all other pairwise comparisons showed significant differences (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Tadpoles reared in groups and pond water spent more time on the non-sibling side than those reared in groups and tap water (Tg\u0026thinsp;\u0026lt;\u0026thinsp;Pg), whereas no significant difference was detected between solitary tadpoles reared in tap and pond water (Ts\u0026thinsp;=\u0026thinsp;Ps). The differences in time spent on the sibling and non-sibling sides corresponded to variations in the time spent near the center of the arena (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of analyses by the general linear mixed model (GLMM) and Post hoc tests. Rearing treatments are explanatory fixed variables in GLMM. Water : Socility means interaction between water types (tap or pond) and Sociality treatments (solitary or group). The abbreviations used for comparisons in the Post hoc test correspond with Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Bold text denotes statistically significant values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime allocation for\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAnalysis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVariable / Comparison\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEstimate\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ez/z ratio\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSibling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGLMM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWater type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.0077\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSociality\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;0.396\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;38.251\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWater : Socility\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.145\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePost hoc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTg\u0026thinsp;\u0026gt;\u0026thinsp;Ts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.251\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e23.632\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePg\u0026thinsp;\u0026gt;\u0026thinsp;Ps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.396\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e38.251\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTg\u0026thinsp;\u0026gt;\u0026thinsp;Pg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.0384\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTg\u0026thinsp;\u0026gt;\u0026thinsp;Ps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.423\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e39.662\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTs\u0026thinsp;\u0026lt;\u0026thinsp;Pg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;21.703\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTs\u0026thinsp;\u0026gt;\u0026thinsp;Ps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.171\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e15.623\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNon-sibling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGLMM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWater type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;0.0813\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;8.045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSociality\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;0.232\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;22.695\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWater : Socility\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.093\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.252\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePost hoc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTg\u0026thinsp;\u0026gt;\u0026thinsp;Ts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.139\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePg\u0026thinsp;\u0026gt;\u0026thinsp;Ps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.232\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.695\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTg\u0026thinsp;\u0026lt;\u0026thinsp;Pg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;0.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;8.045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTg\u0026thinsp;\u0026gt;\u0026thinsp;Ps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.150\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.195\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTs\u0026thinsp;\u0026lt;\u0026thinsp;Pg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;0.220\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;21.184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTs\u0026thinsp;\u0026lt;\u0026thinsp;Ps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.011\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.722\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDespite expectations, the results of the behavioral tests indicated that \u003cem\u003eB. formosus\u003c/em\u003e tadpoles do not exhibit phenotype-matching-based kin recognition. This occurred even though \u003cem\u003eB. formosus\u003c/em\u003e tadpoles display social aggregation, as do several related species that possess the kin recognition abilities (Waldman 2005). Differences in the spent time of stimuli sides were observed in the results of the choice tests among the four rearing treatments. However, kin-biased association did not appear to play an essential role in the tadpoles\u0026rsquo; grouping behavior. The reasons for this can be discussed from two perspectives.\u003c/p\u003e\u003cp\u003eFirst, in the grouping behavior of toad tadpoles, the number of conspecifics may be more important than kin-biased association. The primary function of tadpole aggregation is likely predator avoidance. It has been proposed that the distinctive black coloration of these tadpoles serves as a warning signal to predators, which is enhanced by cohesive aggregation (Wells 2010; Stynoski and Porras-Brenes 2022). In this context, kin-biased association with siblings is not essential for effective aggregation. Some species may therefore achieve cohesive social aggregation without kin discrimination. Watt et al. (1997) showed that tadpoles are less likely to be predated upon when in larger groups. Additionally, a binary-choice test in Miyako toads (\u003cem\u003eB. gargarizans miyakonis\u003c/em\u003e) indicated that group size is a critical factor (Hase 2023). In the present study, the same number of tadpoles (10 vs. 10) was used in the binary-choice tests, and no significant differences were observed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that, in black-colored toad tadpoles, group size may be more important than kinship in aggregation behavior.\u003c/p\u003e\u003cp\u003eSecond, a mechanistic issue in kin discrimination of tadpoles, primarily related to their genetic background (Green et al. 2015; Scott et al. 2022). A theoretical study suggests that phenotype-matching\u0026ndash;based kin recognition systems allow individuals to distinguish between full siblings, half siblings, and nonrelatives by referring to their own matching traits (Lacy and Sherman 1983). Previous studies using \u003cem\u003eB. americanus\u003c/em\u003e demonstrated the ability to discriminate unfamiliar siblings from familiar non-siblings (Waldman 1986). The experimental design of Waldman (1986) did not differ fundamentally from the present study, although it is possible that the siblings and non-siblings in previous studies had greater genetic divergences than those used in this study. This background pattern may be common across species in which phenotype-matching-based kin recognition is present in some tadpoles but absent in others. For example, \u003cem\u003eB. boreas\u003c/em\u003e did not exhibit phenotype matching.(O\u0026rsquo;Hara and Blaustein 1982) The \u0026lsquo;kin labels\u0026rsquo; of \u003cem\u003eB. formosus\u003c/em\u003e and \u003cem\u003eB. boreas\u003c/em\u003e tadpoles may not differ enough to allow sibling\u0026ndash;non-sibling distinction. However, the absence of observed kin discrimination in \u003cem\u003eB. formosus\u003c/em\u003e does not necessarily indicate a lack of kin recognition ability. They may instead use learned 'kin labels' after metamorphosis and during maturity, for example in the context of inbreeding avoidance (Waldman 2005).\u003c/p\u003e\u003cp\u003e\u003cem\u003eB. formosus\u003c/em\u003e tadpoles varied the time spent on both stimulus sides (sibling and non-sibling) depending on social treatment and water environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In both water types, social treatment influenced the time spent on stimulus sides. Tadpoles reared in isolation (belonging to Ts and Ps) spent more time near the center, indicating reduced interaction with siblings or non-siblings. Compared to group-reared individuals, solitary-reared tadpoles appeared less interested in conspecifics. This result suggests that, regardless of kin recognition, sociality in tadpoles requires learning. Solitary conditioning\u0026mdash;used here to test self-referent phenotype matching\u0026mdash;is equivalent to social isolation and can affect nervous system development, as shown in mice (Koike et al. 2009; Lander et al. 2017). This may manifest as reduced interest in conspecifics. In contrast, group-reared subjects frequently moved between sibling and non-sibling sides, as if evaluating which side to choose (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These observations suggest that \u003cem\u003eB. formosus\u003c/em\u003e tadpoles develop grouping behavior only after being raised in a group.\u003c/p\u003e\u003cp\u003eRegarding water environments, we predicted that tadpoles raised in pond water, which contains more microorganisms, would exhibit stronger odor-based differences between siblings and non-siblings than those raised in tap water. However, this prediction was not supported. No kin-biased association was observed under pond-water conditioning, regardless of social treatment. In fact, pond water appeared to reduce interest in siblings: tadpoles spent less time on sibling sides (Tg\u0026thinsp;\u0026gt;\u0026thinsp;Pg; Ts\u0026thinsp;\u0026gt;\u0026thinsp;Ps; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Pond water may interfere with recognition mechanisms. A previous study using the same pond water reported higher mortality in \u003cem\u003eB. japonicus\u003c/em\u003e tadpoles when reared in sibling groups (Hase 2022). If the pond environment contains lethal pathogens, it may not be advantageous to crowd with siblings, whose odors are familiar. Consistent with this, subject tadpoles belonging to Pg spent less time with siblings and more time with non-siblings compared to Tg (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For tadpoles in Pg, when pathogens are present, avoiding siblings may be adaptive\u0026mdash;a form of \u0026ldquo;social distancing\u0026rdquo; (Sandel et al. 2020; Stockmaier et al. 2021).\u003c/p\u003e\u003cp\u003eIn this study, no kin discrimination was detected in \u003cem\u003eBufo formosus\u003c/em\u003e tadpoles. Although no kin-biased association preference was observed in the binary-choice tests with equal stimulus group sizes (10 vs. 10), the results suggest that tadpoles develop sociality through their rearing environment. Whether the failure to acquire kin recognition ability and/or sociality during the larval stage affects individual fitness remains to be investigated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe author declares that there are no competing interests.\u003c/p\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003cp\u003e All tadpoles were treated carefully to minimise physical stress, in compliance with the ethical guidelines of the institute. The protocol was reviewed and accepted by the Ethics Committee for Animal Research of the Graduate University for Advanced Studies (no. SKD2018AR002).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) Research Fellowship (No. 19K16226).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eKazuko Hase designed the study, conducted the experiments, analyzed the data, and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eI thank the staff of the Botanical Gardens, Graduate School of Science, the University of Tokyo, for their cooperation during field surveys and sampling. I am deeply grateful to Emeritus Prof. M. Shimada of the University of Tokyo for providing the large incubator used in the rearing experiments. This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) Research Fellowship (No. 19K16226).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll datasets generated during and/or analyzed during the current study are available in the supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBlaustein AR (1983) Kin Recognition Mechanisms: Phenotypic Matching or Recognition Alleles? Am Nat 121:749\u0026ndash;754. https://doi.org/10.1086/284101\u003c/li\u003e\n\u003cli\u003eBlaustein AR, O\u0026rsquo;Hara RK (1986) An investigation of kin recognition in Red-legged frog (Rana aurora) tadpoles. J Zool 209:347\u0026ndash;353. https://doi.org/10.1111/J.1469-7998.1986.TB03598.X\u003c/li\u003e\n\u003cli\u003eBreed MD (2014) Kin and nestmate recognition: The influence of W. D. Hamilton on 50years of research. 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Journal of Experimental Biology 221:. https://doi.org/10.1242/JEB.182469\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Aggregation behavior, Bufo formosus, Kin recognition, Phenotype-matching, Social experience, Toad tadpole","lastPublishedDoi":"10.21203/rs.3.rs-7930339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7930339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKin-biased association is widespread among animal societies. Animals capable of kin recognition often rely on phenotype matching based on odor cues. Because individuals must learn the scent of their relatives, kin discrimination frequently depends on early social experience and environmental conditions. This study investigated how water environment and social experience influence association preferences in \u003cem\u003eBufo formosus\u003c/em\u003e (eastern Japanese common toad) tadpoles. Using a 2\u0026times;2 factorial design, subject tadpoles were reared either in tap or pond water and under group or solitary conditions. Binary-choice tests were then conducted to examine whether tadpoles preferred siblings or non-siblings. No significant kin-biased association was detected in any treatment, suggesting that \u003cem\u003eB. formosus\u003c/em\u003e tadpoles do not employ phenotype-matching-based kin recognition. However, both social experience and water type significantly affected general social tendencies: group-reared tadpoles interacted more with conspecifics than solitary-reared ones, and those raised in tap water spent more time near siblings than those reared in pond water. These findings indicate that social aggregation in \u003cem\u003eB. formosus\u003c/em\u003e is shaped by social experience and environmental context rather than kinship.\u003c/p\u003e","manuscriptTitle":"Water environment and social isolation affect association preference rather than kinship in toad tadpoles (Bufo formosus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 12:39:15","doi":"10.21203/rs.3.rs-7930339/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":"6e86cfd4-10d0-4099-b191-0b73efc9d68e","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T09:51:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 12:39:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7930339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7930339","identity":"rs-7930339","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}