A biological invasion reduces rates of cannibalism by Japanese toad tadpoles

A biological invasion reduces rates of cannibalism by Japanese toad tadpoles | 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 Article A biological invasion reduces rates of cannibalism by Japanese toad tadpoles Michael R. Crossland, Richard Shine, Takashi Haramura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2558797/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jun, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract By imposing novel evolutionary pressures, biological invasions can favour rapid changes in intraspecific competitive mechanisms such as cannibalism. In their invasive range in Australia but not in their native range in South America, cane toads ( Rhinella marina ) exhibit high rates of cannibalism of embryos (eggs and hatchlings) by tadpoles. To explore the generality of such changes, we examined cannibalism in Japanese common toads ( Bufo japonicus ) on the islands of Honshu (native range) and Hokkaido (invasive range). Contrary to the Australian system, invasion has been accompanied by a reduction rather than increase in rates of cannibalism under standard laboratory conditions. That reduction in cannibalistic tendency by invasive-range toad tadpoles occurs despite an increase in the attractivity of invasive-range toad embryos as prey to other predatory amphibian larvae, including native-range conspecifics. Our data thus support the idea that biological invasions can generate rapid changes in rates of cannibalism, but also show that decreases as well as increases can occur. Future work could investigate the proximate cues and selective forces responsible for this rapid decrease in rates of cannibalism in invasive populations of Japanese common toads. Biological sciences/Evolution Biological sciences/Ecology Biological sciences/Ecology/Evolutionary ecology invasive species adaptive response anuran egg hatchling predation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Biological invasions offer a unique opportunity to examine rapid adaptive changes induced by exposure to new challenges (e.g. 1 ). A species extending its range into hitherto-unoccupied areas may encounter a suite of novel predators, prey, parasites, competitors and abiotic extremes 2 , 3 . In response to the opportunities and risks posed by those new interactions, invaders sometimes exhibit remarkably rapid evolved shifts in morphology, physiology and behaviour 4 . Some of the best examples of rapidly evolved changes in invasive species come from studies on a South American anuran (the cane toad, Rhinella marina ) that was introduced to Australia in 1935 in an ill-considered attempt to control insect pests 5 . Although the toad invasion of Australia has been in progress for less than a century, the animals have already expanded their range by thousands of kilometres 6 . In the process of that accelerating range expansion, cane toads have accumulated a wide range of heritable differences in traits that affect locomotor ability and resilience to novel abiotic challenges 7 – 10 . One of the most spectacular examples involves the evolution of cannibalism. In the cane toad’s native range in South America, larvae rarely consume conspecific eggs or hatchlings. In contrast, larvae from all Australian populations tested to date are voracious cannibals, actively searching out and consuming newly-laid eggs at the hatching stage 11 , 12 . This predation is species-specific; toad tadpoles ignore the eggs of other anuran species unless they are exposed to chemical cues from conspecific eggs 13 . The evolution of this intense cannibalism has been attributed to higher abundances of toads in the invasive range than in the native range, placing a selective premium on traits that maximise intraspecific competitive ability 12 . Consistent with that hypothesis, laboratory trials show that cannibalism enhances larval viability by reducing competitor abundance 14 . To assess the generality of this result, we need to examine other invasive species. The strongest comparisons are with other bufonid (toad) taxa, because of phylogenetic conservatism in life histories and in the morphology and ecology of eggs and larvae 15 . In this paper, we describe the results of experimental studies on rates of cannibalism by a toad species (Japanese common toad, Bufo japonicus ) that has been translocated from the island of Honshu (native range) to the northern island of Hokkaido (invasive range). The translocation occurred around 100 years ago, similar to the timescale of the invasion of Australia by cane toads 16 , 17 . Like Australia, Hokkaido had no native bufonid species prior to the invader’s arrival 16 , 17 . Those similarities between the Australian and Japanese invasive-toad systems stimulated us to design and conduct laboratory experiments to ask (1) if the invasion of Hokkaido has affected the intensity of cannibalism; and (2) if any such changes are due to evolved shifts in vulnerability of embryos versus predatory behaviour of larvae. Materials And Methods Study species and area Like most sympatric anuran species, Japanese common toads lay their eggs in lentic water-bodies and the tadpoles develop in the aquatic environment 18 – 20 . The shared use of waterbodies as breeding sites with other amphibian species on Honshu and Hokkaido makes the eggs and hatchlings of Japanese common toads vulnerable to predation by a variety of amphibian larvae 16 , 19 , 21 – 23 . Cannibalism by conspecifics is also possible because, although the breeding period of Japanese common toads is brief (e.g., up 3 weeks on Honshu: 21 , 22 , eggs hatch after 1 week 16 giving tadpoles of early clutches a short period of time to prey upon eggs and hatchlings of later-breeding conspecifics. Predation typically is high on the hatchling stage: that is, larvae that have hatched out from the egg capsule but have not yet attained locomotor capacity 16 , 23 . Although the eggs and hatchlings of Japanese common toads contain maternally-invested toxins 24 , 25 , amphibian taxa that are native to Honshu (and hence, have a long history of sympatry with the toads) readily consume invasive toad hatchlings and can tolerate those toxins (predatory frog tadpoles: Rana ornativentris , predatory salamander larvae: Hynobius nigrescens ; 16 , 17 . The situation is different in the invasive range on Hokkaido, where the hatchlings of invasive toads are toxic to amphibian species native to that island (which have no evolutionary history of exposure to bufonids). On Hokkaido, tadpoles of Rana pirica readily consume invasive toad hatchlings, but are almost always killed by the toxins 16 , 17 , 23 , 25 . Native Hokkaido salamander larvae ( Hynobius retardus ) also readily consume invasive toad hatchlings, with survival rates varying from 6 to 77% among populations 16 , 23 . Predation rates by vulnerable species on Hokkaido can be high. Kazila and Kishida 23 found that 90% of H. retardus larvae offered an invasive-range (Hokkaido) toad hatchling ate the hatchling, whereas 49% of R. pirica tadpoles consumed a hatchling. Overall aims and experimental design We investigated predatory interactions among tadpoles and embryos (eggs and hatchlings: 26 ) of “true toads” (family Bufonidae: B. japonicus comprising the two subspecies B. j. formosus and B. j. japonicus ) and “true frogs” (family Ranidae: Rana japonica , R. ornativentris , R. dybowski ). In our experiments, the two Bufo subspecies (respectively, the eastern Japanese common toad and western Japanese common toad: 27 ) did not differ significantly in their predatory responses or vulnerability to predation within the native range (see Results). Therefore, we combined data for these two subspecies, and refer to them collectively as B. japonicus for brevity. Three of our study species ( B. japonicus , R. japonica , R. ornativentris ) are native to the main islands of Honshu, Shikoku and Kyushu. Bufo japonicus (specifically, B. j. formosus ) has been introduced to Hokkaido as well as to Sadogashima Island and the Izu Islands 27 . Rana dybowski is native to Tsushima Island, located between Kyushu and the Korean Peninsula, and is not sympatric with B. japonicus (indeed, this island has no native Bufonid species). We assessed rates of predation by anuran tadpoles on anuran embryos for toad ( Bufo ) and frog ( Rana ) species within their native range (Honshu) to determine the role that tadpole phylogeny (Bufonidae vs. Ranidae) and embryo phylogeny (Bufonidae vs. Ranidae) play in the outcome of predator-prey interactions within the native range. We then tested whether the effect of tadpole phylogeny on predation varies between native populations of toad embryos versus invasive populations of toad embryos. We did this in two ways. First, we combined the data for all native-range (Honshu) tadpoles tested with invasive-range (Hokkaido) toad embryos and compared these data to predation by native-range (Honshu) tadpoles on native-range (Honshu) toad embryos. Secondly, we specifically compared the predatory responses of tadpoles of two species ( B. japonicus, R. ornativentris ) to native-range (Honshu) toad embryos versus invasive-range (Hokkaido) toad embryos. Finally, we assessed whether propensity for cannibalism varies with tadpole population invasion history by comparing cannibalism on invasive-range (Hokkaido) toad embryos by native-range (Honshu) toad tadpoles versus invasive-range (Hokkaido) toad tadpoles. Collection and husbandry of eggs and larvae We collected native-range eggs in the wild from Honshu (Tochigi Prefecture: B. japonicus , R. japonica , R. ornativentris ; Okayama Prefecture: B. japonicus , R. ornativentris ; Wakayama Prefecture: R. ornativentris ), Kyushu (Miyazaki Prefecture: B. japonicus ) and Tsushima Island (Nagasaki Prefecture: R. dybowski ). Invasive-range B. japonicus were collected from Hakodate, Sapporo and Tsukigata on Hokkaido. Eggs were transported to the laboratory (Seto Marine Research Station of Kyoto University, and Rakuno Gakuen University) where they hatched. Tadpoles were reared in 120 L tanks (66 x 86 x 34 cm) on a diet of algal pellets (Hikari Algal Wafers, Kyorin) fed ad libitum daily until used in experiments described below. Laboratory experiments We conducted experiments in the laboratory using 1000 ml plastic containers filled with 750 ml water. In each experiment, a single tadpole was randomly allocated to either a control container or an egg treatment container. Tadpoles in egg treatment containers were offered 5 or 10 anuran eggs. Tadpoles in control containers were fed cat food ad libitum daily. We recorded the number of embryos eaten by each tadpole every 24 h for 72 h (at which time eggs had developed through the hatchling stage and into free-swimming larvae (tadpoles), and were no longer vulnerable to predation). We recorded water temperature in each container daily. We also recorded tadpole mortality daily. Experiment 1. Is predation among native species on Honshu determined by tadpole phylogeny and/or embryo phylogeny? We tested one toad species and three frog species in a series of tadpole-embryo predation experiments (Table 1 ). Table 1 Toad (Bufo) and frog (Rana) species from native range populations (Honshu) tested in embryo (eggs and hatchlings) predation experiments. N = number of replicates. Tadpole Embryo N Bufo japonicus Bufo japonicus Rana japonica Rana ornativentris 25 20 25 Rana japonica Rana japonica Rana ornativentris 5 5 Rana ornativentris Bufo japonicus Rana japonica Rana ornativentris 15 10 10 Rana dybowski Bufo japonicus 10 Experiment 2. Does the effect of tadpole phylogeny on predation vary between native versus invasive toad embryo populations? We tested tadpoles of B. japonicus (N = 15), R. japonica (N = 6) and R. ornativentris (N = 10) with invasive-range (Hokkaido) toad embryos for comparison with native-range (Honshu) predation data collected in Experiment 1. Experiment 3. Does propensity for cannibalism by B. japonicus tadpoles vary with tadpole population invasion history? We investigated the effect of invasion history of tadpole populations on B. japonicus cannibalism by testing native-range (Honshu) B. japonicus tadpoles (N = 15) vs. invasive-range (Hokkaido) B. japonicus tadpoles (N = 30) with invasive-range (Hokkaido) B. japonicus embryos. Statistical analyses We analysed data in R 28 as a binomial response to treatment (embryo eaten, not eaten; tadpole ate embryos, did not eat embryos) using logistic regression 29 and quasi-binomial models to account for data over-dispersion (mixed effects models: package MASS:glmmPQL 30 followed by Anova (package car, 31 ). We conducted post-hoc multiple comparisons among treatments using Tukey tests adjusted with the Holm method (package multcomp, 32 ). We included container water temperature as a covariate in all models. Because temperature values are continuous data, we centred these data based on mean values for the dataset in question prior to analysis. In some instances there was zero predation in all replicates within a treatment, resulting in models failing to reach convergence. When this occurred, we assigned a single embryo to have been eaten (or a single tadpole to have eaten an embryo) in that treatment to obtain a conservative estimate of treatment effect 29 . For models that included an interaction term, we removed the interaction term when it was non-significant and re-ran the model to obtain final estimates. We retained all main effects in the final models, regardless of their statistical significance. Experiment 1. Is predation among native species on Honshu determined by tadpole phylogeny and/or embryo phylogeny? We analysed overall effects of phylogeny on predation using fixed effects of tadpole phylogeny (Bufonidae, Ranidae), embryo phylogeny (Bufonidae, Ranidae) and their interaction (tadpole phylogeny x embryo phylogeny). Time and container water temperature were included as fixed effect covariates. Container was included as a random effect to account for non-independence of observations of the same container over time. Following the above analysis, we conducted a multiple comparisons test to evaluate all combinations of the four tadpole/embryo treatments (i.e., toad tadpole/toad embryos, toad tadpole/frog embryos, frog tadpole/toad embryos, frog tadpole/frog embryos). This analysis used tadpole/egg treatment as a fixed effect, with time and container water temperature as covariates. Container was included as a random effect. Experiment 2. Does the effect of tadpole phylogeny on predation vary between native versus invasive toad embryo populations? We first combined the data for all native-range (Honshu) tadpoles tested with invasive-range (Hokkaido) toad embryos and compared these data to predation by native-range (Honshu) tadpoles on native-range (Honshu) toad embryos. For this analysis, we used embryo population (native-range (Honshu), invasive-range (Hokkaido)), tadpole phylogeny (Bufonidae, Ranidae) and their interaction (embryo population x tadpole phylogeny) as fixed main effects. Time and container water temperature were included as covariates. Container was included as a random effect. We then conducted a multiple comparison test to assess predation among the four tadpole/embryo combination treatments (i.e., native-range (Honshu) toad tadpole/native-range (Honshu) toad embryos; native-range (Honshu) toad tadpole/invasive-range (Hokkaido) toad embryos; native-range (Honshu) frog tadpole/native-range (Honshu) toad embryos; native-range (Honshu) frog tadpole/invasive-range (Hokkaido) toad embryos). In this analysis, we used tadpole phylogeny/embryo population treatment as a fixed effect, with time and container water temperature as covariates. Container was included as a random effect. For native-range (Honshu) tadpoles ( B. japonicus and R. ornativentris ), we conducted separate analyses to compare responses of these tadpoles to native-range (Honshu) toad embryos versus invasive-range (Hokkaido) toad embryos. These analyses used toad embryo population (native, invasive) as a fixed effect, with time and container water temperature as covariates. Container was included as a random effect. Experiment 3. Does propensity for cannibalism by toad tadpoles vary with tadpole population invasion history? We analysed the data from this experiment using the fixed effect of toad tadpole population (native, invasive), with time and container water temperature as covariates. Container was included as a random effect. Ethics approval All procedures were approved by Rakuno Gakuen University Animal Care Committee (permit #DH22D8). This study was carried out in compliance with the ARRIVE guidelines, and all methods were carried out in accordance with relevant guidelines and regulations. Results Comparison of two subspecies of toads from the native range (Honshu) We tested native-range B. j. formosus tadpoles versus native-range B. j. japonicus tadpoles as predators on embryos of three anuran taxa ( B. j. japonicus , R. japonica , R. ornativentris ). There was no significant effect of toad tadpole subspecies on rate of embryo consumption (logistic regression: B. j. japonicus embryos t = 0.000, df = 13, p = 1.000, R. japonica embryos t = -0.855, df = 22, p = 0.402, R. ornativentris embryos t = -1.929, df = 22, p = 0.067). There was similarly no difference in vulnerability to predation for native-range B. j. formosus embryos versus native-range B. j. japonicus embryos: none of these embryos were eaten by any native-range Bufo or Rana tadpoles. On this basis, we combined data for the two Bufo subspecies for subsequent analyses and refer to them as B. japonicus (toad; identified as native-range vs. invasive-range). Experiment 1. Is predation among native species on Honshu determined by tadpole phylogeny and/or embryo phylogeny? Both tadpole phylogeny and embryo phylogeny were significant predictors of rates of predation for native-range (Honshu) species (Table 2 , Fig. 1 ). Overall, frog tadpoles were more likely to eat embryos (of all kinds tested) than were toad tadpoles (Table 2 , Fig. 1 ), and frog embryos were more likely to be eaten by tadpoles (of all kinds tested) than were toad embryos (Table 2 , Fig. 1 ). The tadpole phylogeny x embryo phylogeny interaction was non-significant (Chi-square = 2.54, df = 1, p = 0.11). Time and water temperature were both significant covariates (Table 2 ). Table 2 ANOVA results for effect of tadpole phylogeny (Bufonidae, Ranidae) and embryo phylogeny (Bufonidae, Ranidae) on rates of embryo predation for native anuran species on Honshu. Analyses were conducted using mean water temperature = 18.0° C. Chi-square df P Tadpole phylogeny Time Water temperature 25.760 5.841 63.260 1 1 1 < 0.0001 0.0157 < 0.0001 Embryo phylogeny Time Water temperature 11.199 10.566 36.041 1 1 1 0.0008 0.0012 < 0.0001 Overall, predation varied significantly among the four native-range (Honshu) tadpole/embryo treatment groups (Table 3 , Fig. 2 ). Both time and container water temperature were significant covariates for predation (Table 3 ). Table 3 ANOVA results for effect of tadpole/embryo phylogeny treatment (i.e., Bufo tadpole/Bufo embryos, Bufo tadpole/Rana embryos, Rana tadpole/Bufo embryos, Rana tadpole/Rana embryos) on rates of embryo predation for native anuran species on Honshu. Analyses were conducted using mean water temperature = 18.0° C. Chi-square df P Tadpole/embryo phylogeny Time Water temperature 46.085 9.515 27.2266 3 1 1 < 0.0001 0.0020 < 0.0001 Specific comparison of the four native-range (Honshu) tadpole/embryo treatments showed that no toad or frog tadpoles ate any toad embryos, whereas predation rate by toad tadpoles on frog embryos was low (Fig. 2 : respectively A, B, C). The greatest predation rate was by frog tadpoles on frog embryos, with frog tadpoles eating more frog embryos than toad embryos (Table 4 and Fig. 2 : D vs. B). Predation by frog tadpoles on frog embryos was also more common than predation by toad tadpoles on either frog embryos (Table 4 and Fig. 2 : D vs. C) or toad embryos (Table 4 and Fig. 2 : D vs. A). Table 4 Multiple comparison results for the four tadpole/embryo treatments in Experiment 1 (native-range (Honshu) species). Letters in parentheses refer to tadpole/embryo treatment combinations identified in Fig. 2 . Analyses were conducted using mean water temperature = 18.0° C. Tadpole + Embryo Treatment Comparison z p Bufo tadpole + Rana embryos vs. Bufo tadpole + Bufo embryos; (Fig. 2 : C vs A) Rana tadpole + Bufo embryos vs. Bufo tadpole + Bufo embryos; (Fig. 2 : B vs A) Rana tadpole + Rana embryos vs. Bufo tadpole + Bufo embryos; (Fig. 2 : D vs A) Rana tadpole + Bufo embryos vs. Bufo tadpole + Rana embryos; (Fig. 2 : B vs C) Rana tadpole + Rana embryos vs. Bufo tadpole + Rana embryos; (Fig. 2 : D vs C) Rana tadpole + Rana embryos vs. Rana tadpole + Bufo embryos; (Fig. 2 : D vs B) 1.719 0.553 4.690 -0.944 5.624 3.768 0.2571 0.6905 < 0.0001 0.6905 < 0.0001 0.0007 Experiment 2. Does the effect of tadpole phylogeny on predation vary between native-range versus invasive-range toad embryo populations? Overall, embryo population (native-range (Honshu) toad vs. invasive-range (Hokkaido) toad) was a significant predictor for the rate of predation by native-range (Honshu) toad tadpoles (Table 5 , Fig. 3 ). Tadpole phylogeny (Bufonidae, Ranidae) was also a significant main effect (Table 5 ). The tadpole phylogeny x embryo phylogeny interaction was non-significant (Chi-square = 2.059, df = 1, p = 0.1513). Time was a significant covariate, but container water temperature was not (Table 5 ). Table 5 ANOVA results for effect of toad (Bufo japonicus) embryo population invasion history (native-range (Honshu), invasive-range (Hokkaido)) and tadpole phylogeny (Bufonidae, Ranidae) on embryo predation by native-range (Honshu) tadpoles. Analyses were conducted using mean water temperature = 20.0 ° C. Chi-square df P Embryo population Tadpole phylogeny Time Water temperature 93.666 5.187 54.763 1.709 1 1 1 1 < 0.0001 0.0228 < 0.0001 0.1911 Specific comparisons among tadpole/embryo treatments showed that tadpole phylogeny was not a significant predictor of predation by native-range (Honshu) tadpoles on native-range (Honshu) toad embryos (Table 6 and Fig. 3 : B vs. A) but was a significant predictor for predation by native-range (Honshu) tadpoles on invasive-range (Hokkaido) toad embryos (Table 6 and Fig. 3 : D vs. C). Native-range (Honshu) frog tadpoles ate more invasive-range (Hokkaido) toad embryos than did native-range (Honshu) toad tadpoles (Table 6 and Fig. 3 : D vs. C). Table 6 Multiple comparison results for predation by native-range (Honshu) tadpoles (Bufo, Rana) on toad (B. japonicus = Bufo) embryos (native-range (Honshu) vs. invasive-range (Hokkaido)). Letters in parentheses refer to tadpole/egg treatment combinations identified in Fig. 3 . Analyses were conducted using mean water temperature = 17.5° C. Tadpole + Embryo Treatment Comparison z p Bufo tadpole + Bufo Honshu embryos vs. Bufo tadpole + Bufo Hokkaido embryos; (Fig. 3 : A vs C) Rana tadpole + Bufo Hokkaido embryos vs. Bufo tadpole + Bufo Hokkaido embryos; (Fig. 3 : D vs C) Rana tadpole + Bufo Honshu embryos vs. Bufo tadpole + Bufo Hokkaido embryos; (Fig. 3 : B vs C) Rana tadpole + Bufo Hokkaido embryos vs. Bufo tadpole + Bufo Honshu embryos; (Fig. 3 : D vs A) Rana tadpole + Bufo Honshu embryos vs. Bufo tadpole + Bufo Honshu embryos; (Fig. 3 : B vs A) Rana tadpole + Bufo Honshu embryos vs. Rana tadpole + Bufo Hokkaido embryos; (Fig. 3 : B vs D) -6.145 2.733 -6.209 8.030 -0.113 -8.045 < 0.0001 0.0126 < 0.0001 < 0.0001 0.9097 < 0.0001 In addition to these phylogenetic effects, both native-range (Honshu) frog tadpoles and native-range (Honshu) toad tadpoles ate more invasive-range (Hokkaido) toad embryos than native-range (Honshu) toad embryos (Table 6 and Fig. 3 : respectively, B vs. D, A vs. C). Individual species comparisons showed that native-range (Honshu) toad tadpoles and frog tadpoles both ate more invasive toad embryos than native toad embryos (Table 7 , Figs. 4 and 5 ). Time was a significant covariate for predation by Honshu toad tadpoles, but not for Honshu frog tadpoles. Water temperature was not a significant covariate for either species (Table 7 ). Table 7 ANOVA results for effect of toad (Bufo japonicus) embryo population (native-range (Honshu), invasive-range (Hokkaido)) on predation by native-range (Honshu) toad (B. japonicus) tadpoles and native-range (Honshu) frog (R. ornativentris) tadpoles. B. japonicus analyses were conducted using mean water temperature = 19.0° C. R. ornativentris analyses were conducted using mean water temperature = 16.8° C. Chi-square df p B. japonicus tadpole B. japonicus embryo population Time Water temperature 20.288 17.189 1.626 1 1 1 < 0.0001 < 0.0001 0.2022 R. ornativentris tadpole B. japonicus embryo population Time Water temperature 10.619 3.360 0.3652 1 1 1 0.0011 0.0668 0.5456 Experiment 3. Does propensity for cannibalism by toad tadpoles vary with tadpole population invasion history? Native-range (Honshu) toad tadpoles ate more invasive-range (Hokkaido) toad embryos than did invasive-range (Hokkaido) toad tadpoles (Table 8 , Fig. 6 ). Time was a significant covariate, but water temperature was not. Table 8 ANOVA results for effect of toad (Bufo japonicus) tadpole population (native-range (Honshu), invasive-range (Hokkaido)) on predation on invasive-range Hokkaido toad (B. japonicus) embryos. Analyses were conducted using mean water temperature = 19.3 ° C. Treatment Chi-square df p Native-range vs. invasive-range tadpoles Time Water temperature 12.643 30.524 0.010 1 1 1 0.0004 < 0.0001 0.9193 Tadpole mortality We did not formally analyse tadpole mortality due to low mortality rates and an obvious lack of treatment effects. During our experiments, 5 toad tadpoles in control containers died, 1 Honshu toad tadpole offered frog embryos died without any evidence of eating embryos, and 2 Honshu toad tadpoles offered invasive-range (Hokkaido) toad embryos died after eating either 0 or 1 embryo. Discussion In our experiments, cannibalism was less common in invasive (Hokkaido) populations of toads than in native-range (Honshu) populations. That lower frequency was due to characteristics of the toad tadpoles not the toad embryos. Hokkaido (invasive-range) toad embryos were eaten more often, by frogs as well as by toads, than were native-range (Honshu) toad embryos. All else being equal, that palatability should increase rates of cannibalism in invasive (Hokkaido) populations. However, the larvae of invasive-range toads ate fewer conspecific embryos (from the invasive-range population) than did the larvae of native-range toads. That is, cannibalism has declined over the course of the toad’s invasion of Hokkaido because of shifts in the attributes of the cannibals (larvae) not the victims (embryos). Our data on the tadpoles and embryos of frogs provide a context for that shift in cannibalism rates within toads. Rates of predation (intra- and interspecific) overall were higher in frogs than in toads, and including frogs as both predators and prey allowed us to tease apart mechanisms underlying the reduced rates of cannibalism in invasive populations of toads. Thus, data from frogs showed that the greater vulnerability of toad embryos from invasive populations was not a species-specific effect, because frogs as well as toads consumed more invasive-range toad embryos than native-range toad embryos. By including the additional anuran species in our trials, we could show that invasive-population toads exhibit overall an overall increase in embryo vulnerability. Our results for predation by native-range (Honshu) tadpoles on invasive-range (Hokkaido) toad embryos concur with previous studies. We found that native-range tadpoles ( B. japonicus , R. japonica , R. ornativentris ) consumed embryos of invasive-range toads without ill effect. Similarly, Oyake et al. 16 and Okamiya et al. 17 found that Honshu species ( R. ornativentris tadpoles, H. nigrescens larvae) consume invasive-range (Hokkaido) toad hatchlings without dying. This ability to tolerate toad toxins is likely due to co-evolutionary adaptation on Honshu 16 , 17 . However, as far as we are aware, our study is the first to examine embryo cannibalism responses for either native-range or invasive-range Japanese common toads. Our experimental design does not identify the proximate cues involved in the decreased rates of cannibalism in invasive populations of Japanese toads. Detailed studies in Australian cane toads have shown that toxins released from eggs close to the time of hatching attract cannibalistic larvae, and also induce foraging responses by those larvae 13 , 33 , 34 . A similar situation is plausible with the Japanese toads, that also produce eggs containing toxins (as evidenced by high mortality rates of toad-naïve predators consuming those eggs or hatchlings: 16 , 17 , 23 – 25 ). The decrease in rates of cannibalism in invasive populations of Japanese toads thus might reflect a lower attraction to such cues by conspecific larvae and a decrease in toxin content, a change in toxin composition, or a decrease in rates of toxin release by embryos. Laboratory experiments exposing predators to scent cues rather than embryos could test those ideas (cf. 33 , 34 ), and direct measures of the types and amounts of toxins in embryos from each population also would be informative (cf. 34 , 35 ). Adult B. japonicus exhibit geographic variation in toxin (bufadienolide) composition within their native range 36 . Because the toxins in B. japonicus eggs and hatchlings are maternally-invested 24 , 25 , similar geographic variation presumably also occurs in the toxin composition of toad embryos. However, whether the toxin composition of B. japonicus in the invasive-range (Hokkaido) differs from that of native-range populations remains to be determined. Interestingly, the fact that native-range toad tadpoles readily consumed invasive-range toad embryos suggests that factors other than geographic variation in toxin composition may also be involved in causing lower rates of cannibalism by invasive-range toad tadpoles. The evolutionary forces responsible for the decline in rates of cannibalism in invasive Japanese toads also warrant further study. Most obviously, why has invasion been accompanied by a reduced rate of cannibalism in this system, whereas Australian cane toads exhibit the opposite pattern 11 , 12 ? Field studies are needed to clarify this paradox. The colder climate of Hokkaido (the invasive range) than Honshu (the native range) may affect temporal overlap in contact between conspecific embryos and larvae. For example, if breeding is restricted to a brief period in spring, and tadpoles metamorphose during the following summer, then there would be few opportunities for older larvae to consume newly-laid eggs and hatchlings. In contrast, a longer breeding season may generate within-year contact between embryos and larvae, increasing opportunities for cannibalism. Field studies could clarify that phenology in native versus invasive ranges. The importance of cannibalism also covaries with population density, and with the occurrence of other predators of embryos 12 , 37 . Thus, for example, the evolution of cannibalism in Australian cane toads has been attributed to higher densities in the invasive range than in the native range, coupled with a scarcity of alternative predators on embryos 12 . To examine this idea, we need field data on densities of toads in their native range and invasive range, and estimates of rates of egg mortality due to intraspecific versus interspecific predation. Field enclosures in natural waterbodies may provide a way to measure these variables 11 , 12 . The availability and nutritional value of alternative food sources for larvae also deserve attention. Although we found a decrease rather than increase in rates of cannibalism in invasive populations of Japanese toads, contrary to the situation with cane toads in Australia 11 , 12 , it is striking that in both cases a recent invasion (~ 100 years) has initiated a substantial shift in the importance of cannibalism. That similarity supports the idea that invasions impose rapid shifts on the selective forces involved in intraspecific competition generally, and cannibalism specifically. The proximate cues and adaptive significance of such shifts are amenable to studies in the laboratory and in the field, rendering cannibalism in toads an ideal study system in which to examine rapid evolutionary change. Declarations Acknowledgements We thank Koshiro Eto (Kitakyushu Museum of Natural History & Human History) for collecting samples on Tsushima Island. This work was supported by an internal grant from The Hakubi Center, Kyoto University, and the Environment Research and Technology Development Fund (4RF-1402) of the Ministry of the Environment, Japan. Data availability Data is available from Dryad Digital Repository https://datadryad.org/stash/share/Szhq4YDLypxYit13FGv-J6JQ6LAdtf3lCKsTBp3oZAk. References Prentis, P. J., Wilson, J. R., Dormontt, E. E., Richardson, D. M. & Lowe, A. J. Adaptive evolution in invasive species. Trends in plant science 13 , 288–294 (2008). Moran, E. V. & Alexander, J. M. Evolutionary responses to global change: lessons from invasive species. Ecology Letters 17 , 637–649 (2014). Borden, J. B. & Flory, S. L. Urban evolution of invasive species. Frontiers in Ecology and the Environment 19 , 184–191 (2021). Rollins, L. A., Richardson, M. F. & Shine, R. A genetic perspective on rapid evolution in cane toads ( Rhinella marina ). Molecular Ecology 24 , 2264–2276 (2015). Shine, R. Cane toad wars (Univ of California Press, 2018). Urban, M. C., Phillips, B. L., Skelly, D. K. & Shine, R. A toad more traveled: the heterogeneous invasion dynamics of cane toads in Australia. The American Naturalist 171 , E134–E148 (2008). Hudson, C. M., McCurry, M. R., Lundgren, P., McHenry, C. R. & Shine, R. Constructing an invasion machine: the rapid evolution of a dispersal-enhancing phenotype during the cane toad invasion of Australia. PloS one 11 , e0156950 (2016). Gruber, J., Brown, G., Whiting, M. J. & Shine, R. Is the behavioural divergence between range-core and range-edge populations of cane toads ( Rhinella marina ) due to evolutionary change or developmental plasticity?. Royal Society Open Science 4 , 170789 (2017). Kosmala, G., Christian, K., Brown, G. & Shine, R. Locomotor performance of cane toads differs between native-range and invasive populations. Royal Society Open Science 4 , 170517 (2017). Kosmala, G. K., Brown, G. P., Shine, R. & Christian, K. Skin resistance to water gain and loss has changed in cane toads ( Rhinella marina ) during their Australian invasion. Ecology and Evolution 10 , 13071–13079 (2020). DeVore, J. L., Crossland, M. R. & Shine, R. Trade-offs affect the adaptive value of plasticity: stronger cannibal-induced defenses incur greater costs in toad larvae. Ecological Monographs 91 , e01426 (2021a). DeVore, J. L., Crossland, M. R., Shine, R. & Ducatez, S. The evolution of targeted cannibalism and cannibal-induced defenses in invasive populations of cane toads. Proceedings of the National Academy of Sciences 118, e2100765118 (2021b). Crossland, M. R., Shine, R. & DeVore, J. L. Choosy cannibals: Targeted consumption of conspecific hatchlings by larval cane toads is triggered by species-specific defensive toxins. Ecology and Evolution 12 , e8655 (2022). Crossland, M. R., Hearnden, M. N., Pizzatto, L., Alford, R. A. & Shine, R. Why be a cannibal? The benefits to cane toad, Rhinella marina [= Bufo marinus ], tadpoles of consuming conspecific eggs. Animal Behaviour 82 , 775–782 (2011). Haas, A. Phylogeny of frogs as inferred from primarily larval characters (Amphibia: Anura). Cladistics 19, 23–89 (2003). Oyake, N., Sasaki, N., Yamaguchi, A., Fujita, H., Tagami, M., Ikeya, K., Takagi, M., Kobayashi, M., Abe, H. & Kishida, O. Comparison of susceptibility to a toxic alien toad ( Bufo japonicus formosus ) between predators in its native and invaded ranges. Freshwater Biology 65 , 240–252 (2020). Okamiya, H., Inoue, Y., Takai, K., Crossland, M. R. & Kishida, O. Native frogs ( Rana pirica ) do not respond adaptively to alien toads ( Bufo japonicus formosus ) 100 years after introduction. Ecological Research 36 , 1005–1014 (2021a). Zheng, X., Natuhara, Y., Li, J., Li, G., Du, Y., Jia, H., Dai, Z., Du, D., Zhong, S. & Qin, D. Effects of multiple stressors on amphibian oviposition: landscape and local determinants in central Japan. Ecological Indicators 128 , e107824 (2021). Haramura, T., Eto, K., Crossland, M. R., Nishikawa, K. & Shine, R. Competition between the tadpoles of Japanese toads versus frogs. Scientific Reports 12 , 1627 (2022). Tanaka, K. & Ishikawa, K. Developmental stages of lotic-breeding toad, Bufo torrenticola , with a comparison to lentic-breeding B. japonicus formosus (Amphibia: Anura: Bufonidae). Current Herpetology 41 , 8–23 (2022). Kusano, T., Maruyama, K. & Kanenko, S. Breeding site fidelity in the Japanese toad, Bufo japonicus formosus . Herpetological Journal 9 , 9–13 (1999). Kusano, T., Miura, T., Terui, S. & Maruyama, K. Factors affecting the breeding activity of the Japanese common toad, Bufo japonicus formosus (Amphibia: Bufonidae) with special reference to the lunar cycle. Current Herpetology 34 , 101–111 (2015). Kazila, E. & Kishida, O. Foraging traits of native predators determine their vulnerability to a toxic alien prey. Freshwater Biology 64 , 56–70 (2019). kamiya, H., Takai, K. & Kishida, O. The Japanese common toad, Bufo japonicus formosus , contains toxin in the egg stage. Current Herpetology 40 , 103–106 (2021b). Okamiya, H., Tagami, M., Crossland, M. & Kishida O. Are toxic effects of alien species affected by their prey? Evaluation by bioassay with captive-bred toad embryos and a vulnerable predator. Hydrobiologia 848 , 4445–4452 (2021c). Gosner, K. L. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16 , 183–190 (1960). Maeda, N. & Matsui, M. Frogs and toads of Japan. (Bun-ichi Sogo Shuppan, 1989)((in Japanese)). R Core Team R : A language and environment for statistical computing (R Foundation for Statistical Computing, 2021). Warton, D. I. & Hui, F. K. C. The arcsine is asinine: the analysis of proportions in ecology. Ecology 92 , 3–10 (2011). Venables, W. N. & Ripley, B. D. Modern Applied Statistics with S Fourth edition (Springer, 2002). Fox, J. & Weisberg, S. An R Companion to Applied Regression Third edition (Sage, Thousand Oaks CA, 2019). Hothorn, T., Bretz, F. & Westfall, P. Simultaneous inference in general parametric models. Biometrical Journal 50 , 346–363 (2008). Crossland, M. R., Haramura, T., Salim, A. A., Capon, R. J. & Shine, R. Exploiting intraspecific competitive mechanisms to control invasive cane toads ( Rhinella marina ). Proceedings of the Royal Society B 279, 3436–3442 (2012). Crossland, M. R., Salim, A. A., Capon, R. J. & Shine, R. Chemical cues that attract cannibalistic cane toad ( Rhinella marina ) larvae to vulnerable embryos. Scientific Reports 11 , 12527 (2021). Hayes, R. A., Crossland, M. R., Hagman, M., Capon, R. J. & Shine, R. Ontogenetic variation in the chemical defenses of cane toads ( Bufo marinus ): toxin profiles and effects on predators. Journal of chemical ecology 35 , 391–399 (2009). Inoue, T., Nakata, R., Savitzky, A. H., Yoshinaga, N., Mori, A. & Mori, N. Variation in bufadienolide composition of parotoid gland secretion from three taxa of Japanese toads. Journal of Chemical Ecology 46 , 997–1009 (2020). Richardson, M. L., Mitchell, R. F., Reagel, P. F. & Hanks, L. M. Causes and consequences of cannibalism in noncarnivorous insects. Annual review of entomology 55 , 39–53 (2010). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 13 Jun, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 07 Apr, 2023 Reviews received at journal 14 Mar, 2023 Reviewers agreed at journal 05 Mar, 2023 Reviewers invited by journal 02 Mar, 2023 Editor assigned by journal 22 Feb, 2023 Editor invited by journal 15 Feb, 2023 Submission checks completed at journal 15 Feb, 2023 First submitted to journal 07 Feb, 2023 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-2558797","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":176226098,"identity":"45123eea-8c53-4999-bf3e-c910f6eef77f","order_by":0,"name":"Michael R. Crossland","email":"","orcid":"","institution":"University of Sydney","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"R.","lastName":"Crossland","suffix":""},{"id":176226099,"identity":"67e86775-440c-42ac-bd16-bbe3fa24c440","order_by":1,"name":"Richard Shine","email":"","orcid":"","institution":"Macquarie University","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Shine","suffix":""},{"id":176226100,"identity":"0cfa2c62-b9f4-4a43-896d-be2919472d0d","order_by":2,"name":"Takashi Haramura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYFACHhiD+QADwwEMUWwa4JJsCSRr4TFA0oIH2DPwHvxcUbMtcXv7mW8ffpw5nMfPwPzwA4PMHTy28CVLnjl2O3HOmdzNM3tuHC6WbGAzlmDgeYZbi/wbA6Ci24kzGHI3M/B8OJy44QCDGVD8MD6/GP9s+AfUwv/mMeMfsBb2b4S0mEk2tgG1SOQwM/PcAGnhIWALUIFlY99t4xkSz4yZZc6kJ85s5imWSMDjF/YGHuObDd9uy87gT37M+OaYdWI/e/vGDx97cIcYFsAMxIk9B0jRAgY/SNcyCkbBKBgFwxYAAKk0VfL5QHMjAAAAAElFTkSuQmCC","orcid":"","institution":"Rakuno Gakuen University","correspondingAuthor":true,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Haramura","suffix":""}],"badges":[],"createdAt":"2023-02-07 06:59:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2558797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2558797/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-36743-8","type":"published","date":"2023-06-13T21:10:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":33057510,"identity":"e2dcfa8a-dfa8-4cdc-8929-bd86fe95ff6c","added_by":"auto","created_at":"2023-02-16 22:46:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":169545,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of tadpole phylogeny and embryo phylogeny on rates of predation among native-range (Honshu) amphibian species. Data plotted are number of embryos (eggs and hatchlings) consumed after 72 h.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558797/v1/51b6543ebc9ed892a2c71c70.jpg"},{"id":33056809,"identity":"ceef3e8c-48f9-4142-b908-2600306c4583","added_by":"auto","created_at":"2023-02-16 22:38:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169026,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple comparison of tadpole/embryo combinations for native-range (Honshu) species. A = toad (\u003cem\u003eBufo\u003c/em\u003e) tadpole vs. \u003cem\u003eBufo\u003c/em\u003e embryos, B = frog (\u003cem\u003eRana\u003c/em\u003e) tadpole vs. \u003cem\u003eBufo\u003c/em\u003e embryos, C = \u003cem\u003eBufo\u003c/em\u003e tadpole vs. \u003cem\u003eRana\u003c/em\u003eembryos, D = \u003cem\u003eRana\u003c/em\u003e tadpole vs. \u003cem\u003eRana\u003c/em\u003e embryos. Data plotted are number of embryos (eggs and hatchlings) consumed after 72 h.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558797/v1/53aecec6bbeb630b3ca5a847.jpg"},{"id":33057511,"identity":"fb48d780-c3bd-47a4-a8f6-5264cc898735","added_by":"auto","created_at":"2023-02-16 22:46:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":178742,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple comparison of tadpole/embryo combinations for native-range (Honshu) tadpoles eating native-range (Honshu) toad (\u003cem\u003eB. japonicus\u003c/em\u003e) embryos (A, B) vs. invasive-range (Hokkaido) toad (\u003cem\u003eB. japonicus\u003c/em\u003e) embryos (C, D). Data plotted are number of embryos (eggs and hatchlings) consumed after 72 h.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558797/v1/2df5f767e3c4ba0721a4ed91.jpg"},{"id":33056813,"identity":"046b6f78-39c3-411c-8694-c4d7dee7f85a","added_by":"auto","created_at":"2023-02-16 22:38:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":172092,"visible":true,"origin":"","legend":"\u003cp\u003ePredation by native-range (Honshu) toad (\u003cem\u003eB. japonicus\u003c/em\u003e) tadpoles on conspecific native-range (Honshu) toad embryos vs. conspecific invasive-range (Hokkaido) toad embryos. Data plotted are number of embryos (eggs or hatchlings) consumed at 24 h intervals.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558797/v1/1885ec74aa22e4307af49df8.jpg"},{"id":33056812,"identity":"cc04010c-59fa-4d7d-8eaa-0f0409f0aaae","added_by":"auto","created_at":"2023-02-16 22:38:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":171086,"visible":true,"origin":"","legend":"\u003cp\u003ePredation by native-range (Honshu) frog (\u003cem\u003eR. ornativentris\u003c/em\u003e) tadpoles on native-range (Honshu) toad (\u003cem\u003eB. japonicus\u003c/em\u003e) embryos vs. invasive-range (Hokkaido) toad (\u003cem\u003eB. japonicus\u003c/em\u003e) embryos. Data plotted are number of embryos (eggs or hatchlings) consumed at 24 h intervals.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558797/v1/4abed9f92662d5661c76f710.jpg"},{"id":33056814,"identity":"67f1e9b4-9332-4fc4-8e32-f0f0cc7a3b9f","added_by":"auto","created_at":"2023-02-16 22:38:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":171581,"visible":true,"origin":"","legend":"\u003cp\u003ePredation on invasive-range (Hokkaido) toad (\u003cem\u003eB. japonicus\u003c/em\u003e) embryos by conspecific native-range (Honshu) toad tadpoles vs. conspecific invasive-range (Hokkaido) toad tadpoles. Data plotted are number of embryos (eggs or hatchlings) consumed at 24 h intervals.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558797/v1/08c13923bc98076056823e4e.jpg"},{"id":44732389,"identity":"16d9b9eb-09fa-427e-b0eb-001c7c30c3a5","added_by":"auto","created_at":"2023-10-16 21:54:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":771008,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2558797/v1/1c861fdd-ba05-4c04-9b53-ce2acda02541.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A biological invasion reduces rates of cannibalism by Japanese toad tadpoles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiological invasions offer a unique opportunity to examine rapid adaptive changes induced by exposure to new challenges (e.g.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e). A species extending its range into hitherto-unoccupied areas may encounter a suite of novel predators, prey, parasites, competitors and abiotic extremes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In response to the opportunities and risks posed by those new interactions, invaders sometimes exhibit remarkably rapid evolved shifts in morphology, physiology and behaviour\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSome of the best examples of rapidly evolved changes in invasive species come from studies on a South American anuran (the cane toad, \u003cem\u003eRhinella marina\u003c/em\u003e) that was introduced to Australia in 1935 in an ill-considered attempt to control insect pests\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although the toad invasion of Australia has been in progress for less than a century, the animals have already expanded their range by thousands of kilometres\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In the process of that accelerating range expansion, cane toads have accumulated a wide range of heritable differences in traits that affect locomotor ability and resilience to novel abiotic challenges\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne of the most spectacular examples involves the evolution of cannibalism. In the cane toad\u0026rsquo;s native range in South America, larvae rarely consume conspecific eggs or hatchlings. In contrast, larvae from all Australian populations tested to date are voracious cannibals, actively searching out and consuming newly-laid eggs at the hatching stage\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This predation is species-specific; toad tadpoles ignore the eggs of other anuran species unless they are exposed to chemical cues from conspecific eggs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The evolution of this intense cannibalism has been attributed to higher abundances of toads in the invasive range than in the native range, placing a selective premium on traits that maximise intraspecific competitive ability\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Consistent with that hypothesis, laboratory trials show that cannibalism enhances larval viability by reducing competitor abundance\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo assess the generality of this result, we need to examine other invasive species. The strongest comparisons are with other bufonid (toad) taxa, because of phylogenetic conservatism in life histories and in the morphology and ecology of eggs and larvae\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In this paper, we describe the results of experimental studies on rates of cannibalism by a toad species (Japanese common toad, \u003cem\u003eBufo japonicus\u003c/em\u003e) that has been translocated from the island of Honshu (native range) to the northern island of Hokkaido (invasive range). The translocation occurred around 100 years ago, similar to the timescale of the invasion of Australia by cane toads\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Like Australia, Hokkaido had no native bufonid species prior to the invader\u0026rsquo;s arrival\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThose similarities between the Australian and Japanese invasive-toad systems stimulated us to design and conduct laboratory experiments to ask (1) if the invasion of Hokkaido has affected the intensity of cannibalism; and (2) if any such changes are due to evolved shifts in vulnerability of embryos \u003cem\u003eversus\u003c/em\u003e predatory behaviour of larvae.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy species and area\u003c/h2\u003e \u003cp\u003eLike most sympatric anuran species, Japanese common toads lay their eggs in lentic water-bodies and the tadpoles develop in the aquatic environment\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The shared use of waterbodies as breeding sites with other amphibian species on Honshu and Hokkaido makes the eggs and hatchlings of Japanese common toads vulnerable to predation by a variety of amphibian larvae\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Cannibalism by conspecifics is also possible because, although the breeding period of Japanese common toads is brief (e.g., up 3 weeks on Honshu:\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, eggs hatch after 1 week\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e giving tadpoles of early clutches a short period of time to prey upon eggs and hatchlings of later-breeding conspecifics. Predation typically is high on the hatchling stage: that is, larvae that have hatched out from the egg capsule but have not yet attained locomotor capacity\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Although the eggs and hatchlings of Japanese common toads contain maternally-invested toxins\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, amphibian taxa that are native to Honshu (and hence, have a long history of sympatry with the toads) readily consume invasive toad hatchlings and can tolerate those toxins (predatory frog tadpoles: \u003cem\u003eRana ornativentris\u003c/em\u003e, predatory salamander larvae: \u003cem\u003eHynobius nigrescens\u003c/em\u003e;\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe situation is different in the invasive range on Hokkaido, where the hatchlings of invasive toads are toxic to amphibian species native to that island (which have no evolutionary history of exposure to bufonids). On Hokkaido, tadpoles of \u003cem\u003eRana pirica\u003c/em\u003e readily consume invasive toad hatchlings, but are almost always killed by the toxins\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Native Hokkaido salamander larvae (\u003cem\u003eHynobius retardus\u003c/em\u003e) also readily consume invasive toad hatchlings, with survival rates varying from 6 to 77% among populations\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Predation rates by vulnerable species on Hokkaido can be high. Kazila and Kishida\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e found that 90% of \u003cem\u003eH. retardus\u003c/em\u003e larvae offered an invasive-range (Hokkaido) toad hatchling ate the hatchling, whereas 49% of \u003cem\u003eR. pirica\u003c/em\u003e tadpoles consumed a hatchling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eOverall aims and experimental design\u003c/h2\u003e \u003cp\u003eWe investigated predatory interactions among tadpoles and embryos (eggs and hatchlings:\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e) of \u0026ldquo;true toads\u0026rdquo; (family Bufonidae: \u003cem\u003eB. japonicus\u003c/em\u003e comprising the two subspecies \u003cem\u003eB. j. formosus\u003c/em\u003e and \u003cem\u003eB. j. japonicus\u003c/em\u003e) and \u0026ldquo;true frogs\u0026rdquo; (family Ranidae: \u003cem\u003eRana japonica\u003c/em\u003e, \u003cem\u003eR. ornativentris\u003c/em\u003e, \u003cem\u003eR. dybowski\u003c/em\u003e). In our experiments, the two \u003cem\u003eBufo\u003c/em\u003e subspecies (respectively, the eastern Japanese common toad and western Japanese common toad:\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e) did not differ significantly in their predatory responses or vulnerability to predation within the native range (see Results). Therefore, we combined data for these two subspecies, and refer to them collectively as \u003cem\u003eB. japonicus\u003c/em\u003e for brevity.\u003c/p\u003e \u003cp\u003eThree of our study species (\u003cem\u003eB. japonicus\u003c/em\u003e, \u003cem\u003eR. japonica\u003c/em\u003e, \u003cem\u003eR. ornativentris\u003c/em\u003e) are native to the main islands of Honshu, Shikoku and Kyushu. \u003cem\u003eBufo japonicus\u003c/em\u003e (specifically, \u003cem\u003eB. j. formosus\u003c/em\u003e) has been introduced to Hokkaido as well as to Sadogashima Island and the Izu Islands\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eRana dybowski\u003c/em\u003e is native to Tsushima Island, located between Kyushu and the Korean Peninsula, and is not sympatric with \u003cem\u003eB. japonicus\u003c/em\u003e (indeed, this island has no native Bufonid species).\u003c/p\u003e \u003cp\u003eWe assessed rates of predation by anuran tadpoles on anuran embryos for toad (\u003cem\u003eBufo\u003c/em\u003e) and frog (\u003cem\u003eRana\u003c/em\u003e) species within their native range (Honshu) to determine the role that tadpole phylogeny (Bufonidae vs. Ranidae) and embryo phylogeny (Bufonidae vs. Ranidae) play in the outcome of predator-prey interactions within the native range.\u003c/p\u003e \u003cp\u003eWe then tested whether the effect of tadpole phylogeny on predation varies between native populations of toad embryos \u003cem\u003eversus\u003c/em\u003e invasive populations of toad embryos. We did this in two ways. First, we combined the data for all native-range (Honshu) tadpoles tested with invasive-range (Hokkaido) toad embryos and compared these data to predation by native-range (Honshu) tadpoles on native-range (Honshu) toad embryos. Secondly, we specifically compared the predatory responses of tadpoles of two species (\u003cem\u003eB. japonicus, R. ornativentris\u003c/em\u003e) to native-range (Honshu) toad embryos \u003cem\u003eversus\u003c/em\u003e invasive-range (Hokkaido) toad embryos.\u003c/p\u003e \u003cp\u003eFinally, we assessed whether propensity for cannibalism varies with tadpole population invasion history by comparing cannibalism on invasive-range (Hokkaido) toad embryos by native-range (Honshu) toad tadpoles \u003cem\u003eversus\u003c/em\u003e invasive-range (Hokkaido) toad tadpoles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCollection and husbandry of eggs and larvae\u003c/h2\u003e \u003cp\u003eWe collected native-range eggs in the wild from Honshu (Tochigi Prefecture: \u003cem\u003eB. japonicus\u003c/em\u003e, \u003cem\u003eR. japonica\u003c/em\u003e, \u003cem\u003eR. ornativentris\u003c/em\u003e; Okayama Prefecture: \u003cem\u003eB. japonicus\u003c/em\u003e, \u003cem\u003eR. ornativentris\u003c/em\u003e; Wakayama Prefecture: \u003cem\u003eR. ornativentris\u003c/em\u003e), Kyushu (Miyazaki Prefecture: \u003cem\u003eB. japonicus\u003c/em\u003e) and Tsushima Island (Nagasaki Prefecture: \u003cem\u003eR. dybowski\u003c/em\u003e). Invasive-range \u003cem\u003eB. japonicus\u003c/em\u003e were collected from Hakodate, Sapporo and Tsukigata on Hokkaido. Eggs were transported to the laboratory (Seto Marine Research Station of Kyoto University, and Rakuno Gakuen University) where they hatched. Tadpoles were reared in 120 L tanks (66 x 86 x 34 cm) on a diet of algal pellets (Hikari Algal Wafers, Kyorin) fed \u003cem\u003ead libitum\u003c/em\u003e daily until used in experiments described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory experiments\u003c/h2\u003e \u003cp\u003eWe conducted experiments in the laboratory using 1000 ml plastic containers filled with 750 ml water. In each experiment, a single tadpole was randomly allocated to either a control container or an egg treatment container. Tadpoles in egg treatment containers were offered 5 or 10 anuran eggs. Tadpoles in control containers were fed cat food \u003cem\u003ead libitum\u003c/em\u003e daily.\u003c/p\u003e \u003cp\u003eWe recorded the number of embryos eaten by each tadpole every 24 h for 72 h (at which time eggs had developed through the hatchling stage and into free-swimming larvae (tadpoles), and were no longer vulnerable to predation). We recorded water temperature in each container daily. We also recorded tadpole mortality daily.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 1. Is predation among native species on Honshu determined by tadpole phylogeny and/or embryo phylogeny?\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe tested one toad species and three frog species in a series of tadpole-embryo predation experiments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eToad (Bufo) and frog (Rana) species from native range populations (Honshu) tested in embryo (eggs and hatchlings) predation experiments. N\u0026thinsp;=\u0026thinsp;number of replicates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTadpole\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEmbryo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBufo japonicus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBufo japonicus\u003c/p\u003e \u003cp\u003eRana japonica\u003c/p\u003e \u003cp\u003eRana ornativentris\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRana japonica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRana japonica\u003c/p\u003e \u003cp\u003eRana ornativentris\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRana ornativentris\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBufo japonicus\u003c/p\u003e \u003cp\u003eRana japonica\u003c/p\u003e \u003cp\u003eRana ornativentris\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRana dybowski\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBufo japonicus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\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 \u003cb\u003eExperiment 2. Does the effect of tadpole phylogeny on predation vary between native versus invasive toad embryo populations?\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe tested tadpoles of \u003cem\u003eB. japonicus\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;15), \u003cem\u003eR. japonica\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;6) and \u003cem\u003eR. ornativentris\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;10) with invasive-range (Hokkaido) toad embryos for comparison with native-range (Honshu) predation data collected in Experiment 1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 3. Does propensity for cannibalism by B. japonicus tadpoles vary with tadpole population invasion history?\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe investigated the effect of invasion history of tadpole populations on \u003cem\u003eB. japonicus\u003c/em\u003e cannibalism by testing native-range (Honshu) \u003cem\u003eB. japonicus\u003c/em\u003e tadpoles (N\u0026thinsp;=\u0026thinsp;15) vs. invasive-range (Hokkaido) \u003cem\u003eB. japonicus\u003c/em\u003e tadpoles (N\u0026thinsp;=\u0026thinsp;30) with invasive-range (Hokkaido) \u003cem\u003eB. japonicus\u003c/em\u003e embryos.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eWe analysed data in R\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e as a binomial response to treatment (embryo eaten, not eaten; tadpole ate embryos, did not eat embryos) using logistic regression\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and quasi-binomial models to account for data over-dispersion (mixed effects models: package MASS:glmmPQL\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e followed by Anova (package car,\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e). We conducted post-hoc multiple comparisons among treatments using Tukey tests adjusted with the Holm method (package multcomp,\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e). We included container water temperature as a covariate in all models. Because temperature values are continuous data, we centred these data based on mean values for the dataset in question prior to analysis.\u003c/p\u003e \u003cp\u003eIn some instances there was zero predation in all replicates within a treatment, resulting in models failing to reach convergence. When this occurred, we assigned a single embryo to have been eaten (or a single tadpole to have eaten an embryo) in that treatment to obtain a conservative estimate of treatment effect\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. For models that included an interaction term, we removed the interaction term when it was non-significant and re-ran the model to obtain final estimates. We retained all main effects in the final models, regardless of their statistical significance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 1. Is predation among native species on Honshu determined by tadpole phylogeny and/or embryo phylogeny?\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe analysed overall effects of phylogeny on predation using fixed effects of tadpole phylogeny (Bufonidae, Ranidae), embryo phylogeny (Bufonidae, Ranidae) and their interaction (tadpole phylogeny x embryo phylogeny). Time and container water temperature were included as fixed effect covariates. Container was included as a random effect to account for non-independence of observations of the same container over time.\u003c/p\u003e \u003cp\u003eFollowing the above analysis, we conducted a multiple comparisons test to evaluate all combinations of the four tadpole/embryo treatments (i.e., toad tadpole/toad embryos, toad tadpole/frog embryos, frog tadpole/toad embryos, frog tadpole/frog embryos). This analysis used tadpole/egg treatment as a fixed effect, with time and container water temperature as covariates. Container was included as a random effect.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 2. Does the effect of tadpole phylogeny on predation vary between native versus invasive toad embryo populations?\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe first combined the data for all native-range (Honshu) tadpoles tested with invasive-range (Hokkaido) toad embryos and compared these data to predation by native-range (Honshu) tadpoles on native-range (Honshu) toad embryos. For this analysis, we used embryo population (native-range (Honshu), invasive-range (Hokkaido)), tadpole phylogeny (Bufonidae, Ranidae) and their interaction (embryo population x tadpole phylogeny) as fixed main effects. Time and container water temperature were included as covariates. Container was included as a random effect.\u003c/p\u003e \u003cp\u003eWe then conducted a multiple comparison test to assess predation among the four tadpole/embryo combination treatments (i.e., native-range (Honshu) toad tadpole/native-range (Honshu) toad embryos; native-range (Honshu) toad tadpole/invasive-range (Hokkaido) toad embryos; native-range (Honshu) frog tadpole/native-range (Honshu) toad embryos; native-range (Honshu) frog tadpole/invasive-range (Hokkaido) toad embryos). In this analysis, we used tadpole phylogeny/embryo population treatment as a fixed effect, with time and container water temperature as covariates. Container was included as a random effect.\u003c/p\u003e \u003cp\u003eFor native-range (Honshu) tadpoles (\u003cem\u003eB. japonicus\u003c/em\u003e and \u003cem\u003eR. ornativentris\u003c/em\u003e), we conducted separate analyses to compare responses of these tadpoles to native-range (Honshu) toad embryos \u003cem\u003eversus\u003c/em\u003e invasive-range (Hokkaido) toad embryos. These analyses used toad embryo population (native, invasive) as a fixed effect, with time and container water temperature as covariates. Container was included as a random effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 3. Does propensity for cannibalism by toad tadpoles vary with tadpole population invasion history?\u003c/h2\u003e \u003cp\u003eWe analysed the data from this experiment using the fixed effect of toad tadpole population (native, invasive), with time and container water temperature as covariates. Container was included as a random effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e All procedures were approved by Rakuno Gakuen University Animal Care Committee (permit #DH22D8). This study was carried out in compliance with the ARRIVE guidelines, and all methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparison of two subspecies of toads from the native range (Honshu)\u003c/h2\u003e \u003cp\u003eWe tested native-range \u003cem\u003eB. j. formosus\u003c/em\u003e tadpoles \u003cem\u003eversus\u003c/em\u003e native-range \u003cem\u003eB. j. japonicus\u003c/em\u003e tadpoles as predators on embryos of three anuran taxa (\u003cem\u003eB. j. japonicus\u003c/em\u003e, \u003cem\u003eR. japonica\u003c/em\u003e, \u003cem\u003eR. ornativentris\u003c/em\u003e). There was no significant effect of toad tadpole subspecies on rate of embryo consumption (logistic regression: \u003cem\u003eB. j. japonicus\u003c/em\u003e embryos t\u0026thinsp;=\u0026thinsp;0.000, df\u0026thinsp;=\u0026thinsp;13, p\u0026thinsp;=\u0026thinsp;1.000, \u003cem\u003eR. japonica\u003c/em\u003e embryos t = -0.855, df\u0026thinsp;=\u0026thinsp;22, p\u0026thinsp;=\u0026thinsp;0.402, \u003cem\u003eR. ornativentris\u003c/em\u003e embryos t = -1.929, df\u0026thinsp;=\u0026thinsp;22, p\u0026thinsp;=\u0026thinsp;0.067). There was similarly no difference in vulnerability to predation for native-range \u003cem\u003eB. j. formosus\u003c/em\u003e embryos \u003cem\u003eversus\u003c/em\u003e native-range \u003cem\u003eB. j. japonicus\u003c/em\u003e embryos: none of these embryos were eaten by any native-range \u003cem\u003eBufo\u003c/em\u003e or \u003cem\u003eRana\u003c/em\u003e tadpoles. On this basis, we combined data for the two \u003cem\u003eBufo\u003c/em\u003e subspecies for subsequent analyses and refer to them as \u003cem\u003eB. japonicus\u003c/em\u003e (toad; identified as native-range vs. invasive-range).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 1. Is predation among native species on Honshu determined by tadpole phylogeny and/or embryo phylogeny?\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBoth tadpole phylogeny and embryo phylogeny were significant predictors of rates of predation for native-range (Honshu) species (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, frog tadpoles were more likely to eat embryos (of all kinds tested) than were toad tadpoles (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and frog embryos were more likely to be eaten by tadpoles (of all kinds tested) than were toad embryos (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The tadpole phylogeny x embryo phylogeny interaction was non-significant (Chi-square\u0026thinsp;=\u0026thinsp;2.54, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.11). Time and water temperature were both significant covariates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\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\u003eANOVA results for effect of tadpole phylogeny (Bufonidae, Ranidae) and embryo phylogeny (Bufonidae, Ranidae) on rates of embryo predation for native anuran species on Honshu. Analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;18.0\u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChi-square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\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\u003eTadpole phylogeny\u003c/p\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003eWater temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.760\u003c/p\u003e \u003cp\u003e5.841\u003c/p\u003e \u003cp\u003e63.260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.0157\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmbryo phylogeny\u003c/p\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003eWater temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.199\u003c/p\u003e \u003cp\u003e10.566\u003c/p\u003e \u003cp\u003e36.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0008\u003c/p\u003e \u003cp\u003e0.0012\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\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 \u003cp\u003eOverall, predation varied significantly among the four native-range (Honshu) tadpole/embryo treatment groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Both time and container water temperature were significant covariates for predation (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA results for effect of tadpole/embryo phylogeny treatment (i.e., Bufo tadpole/Bufo embryos, Bufo tadpole/Rana embryos, Rana tadpole/Bufo embryos, Rana tadpole/Rana embryos) on rates of embryo predation for native anuran species on Honshu. Analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;18.0\u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChi-square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\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\u003eTadpole/embryo phylogeny\u003c/p\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003eWater temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.085\u003c/p\u003e \u003cp\u003e9.515\u003c/p\u003e \u003cp\u003e27.2266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.0020\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\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 \u003cp\u003eSpecific comparison of the four native-range (Honshu) tadpole/embryo treatments showed that no toad or frog tadpoles ate any toad embryos, whereas predation rate by toad tadpoles on frog embryos was low (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: respectively A, B, C). The greatest predation rate was by frog tadpoles on frog embryos, with frog tadpoles eating more frog embryos than toad embryos (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: D vs. B). Predation by frog tadpoles on frog embryos was also more common than predation by toad tadpoles on either frog embryos (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: D vs. C) or toad embryos (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: D vs. A).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultiple comparison results for the four tadpole/embryo treatments in Experiment 1 (native-range (Honshu) species). Letters in parentheses refer to tadpole/embryo treatment combinations identified in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;18.0\u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTadpole\u0026thinsp;+\u0026thinsp;Embryo Treatment Comparison\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ez\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\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\u003eBufo tadpole\u0026thinsp;+\u0026thinsp;Rana embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: C vs A)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Bufo embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: B vs A)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Rana embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: D vs A)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Bufo embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Rana embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: B vs C)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Rana embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Rana embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: D vs C)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Rana embryos vs. Rana tadpole\u0026thinsp;+\u0026thinsp;Bufo embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: D vs B)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.719\u003c/p\u003e \u003cp\u003e0.553\u003c/p\u003e \u003cp\u003e4.690\u003c/p\u003e \u003cp\u003e-0.944\u003c/p\u003e \u003cp\u003e5.624\u003c/p\u003e \u003cp\u003e3.768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2571\u003c/p\u003e \u003cp\u003e0.6905\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.6905\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.0007\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 \u003cb\u003eExperiment 2. Does the effect of tadpole phylogeny on predation vary between native-range versus invasive-range toad embryo populations?\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOverall, embryo population (native-range (Honshu) toad vs. invasive-range (Hokkaido) toad) was a significant predictor for the rate of predation by native-range (Honshu) toad tadpoles (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Tadpole phylogeny (Bufonidae, Ranidae) was also a significant main effect (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The tadpole phylogeny x embryo phylogeny interaction was non-significant (Chi-square\u0026thinsp;=\u0026thinsp;2.059, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.1513). Time was a significant covariate, but container water temperature was not (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA results for effect of toad (Bufo japonicus) embryo population invasion history (native-range (Honshu), invasive-range (Hokkaido)) and tadpole phylogeny (Bufonidae, Ranidae) on embryo predation by native-range (Honshu) tadpoles. Analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;20.0 \u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChi-square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\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\u003eEmbryo population\u003c/p\u003e \u003cp\u003eTadpole phylogeny\u003c/p\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003eWater temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.666\u003c/p\u003e \u003cp\u003e5.187\u003c/p\u003e \u003cp\u003e54.763\u003c/p\u003e \u003cp\u003e1.709\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.0228\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.1911\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 \u003cp\u003eSpecific comparisons among tadpole/embryo treatments showed that tadpole phylogeny was not a significant predictor of predation by native-range (Honshu) tadpoles on native-range (Honshu) toad embryos (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: B vs. A) but was a significant predictor for predation by native-range (Honshu) tadpoles on invasive-range (Hokkaido) toad embryos (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: D vs. C). Native-range (Honshu) frog tadpoles ate more invasive-range (Hokkaido) toad embryos than did native-range (Honshu) toad tadpoles (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: D vs. C).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultiple comparison results for predation by native-range (Honshu) tadpoles (Bufo, Rana) on toad (B. japonicus\u0026thinsp;=\u0026thinsp;Bufo) embryos (native-range (Honshu) vs. invasive-range (Hokkaido)). Letters in parentheses refer to tadpole/egg treatment combinations identified in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;17.5\u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTadpole\u0026thinsp;+\u0026thinsp;Embryo Treatment Comparison\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ez\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\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\u003eBufo tadpole\u0026thinsp;+\u0026thinsp;Bufo Honshu embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo Hokkaido embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: A vs C)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Bufo Hokkaido embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo Hokkaido embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: D vs C)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Bufo Honshu embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo Hokkaido embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: B vs C)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Bufo Hokkaido embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo Honshu embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: D vs A)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Bufo Honshu embryos vs. Bufo tadpole\u0026thinsp;+\u0026thinsp;Bufo Honshu embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: B vs A)\u003c/p\u003e \u003cp\u003eRana tadpole\u0026thinsp;+\u0026thinsp;Bufo Honshu embryos vs. Rana tadpole\u0026thinsp;+\u0026thinsp;Bufo Hokkaido embryos; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: B vs D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.145\u003c/p\u003e \u003cp\u003e2.733\u003c/p\u003e \u003cp\u003e-6.209\u003c/p\u003e \u003cp\u003e8.030\u003c/p\u003e \u003cp\u003e-0.113\u003c/p\u003e \u003cp\u003e-8.045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.0126\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.9097\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\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\u003eIn addition to these phylogenetic effects, both native-range (Honshu) frog tadpoles and native-range (Honshu) toad tadpoles ate more invasive-range (Hokkaido) toad embryos than native-range (Honshu) toad embryos (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: respectively, B vs. D, A vs. C).\u003c/p\u003e \u003cp\u003eIndividual species comparisons showed that native-range (Honshu) toad tadpoles and frog tadpoles both ate more invasive toad embryos than native toad embryos (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Time was a significant covariate for predation by Honshu toad tadpoles, but not for Honshu frog tadpoles. Water temperature was not a significant covariate for either species (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA results for effect of toad (Bufo japonicus) embryo population (native-range (Honshu), invasive-range (Hokkaido)) on predation by native-range (Honshu) toad (B. japonicus) tadpoles and native-range (Honshu) frog (R. ornativentris) tadpoles. B. japonicus analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;19.0\u0026deg; C. R. ornativentris analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;16.8\u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChi-square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\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\u003eB. japonicus tadpole\u003c/p\u003e \u003cp\u003eB. japonicus embryo population\u003c/p\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003eWater temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.288\u003c/p\u003e \u003cp\u003e17.189\u003c/p\u003e \u003cp\u003e1.626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.2022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR. ornativentris tadpole\u003c/p\u003e \u003cp\u003eB. japonicus embryo population\u003c/p\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003eWater temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.619\u003c/p\u003e \u003cp\u003e3.360\u003c/p\u003e \u003cp\u003e0.3652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0011\u003c/p\u003e \u003cp\u003e0.0668\u003c/p\u003e \u003cp\u003e0.5456\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 \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 3. Does propensity for cannibalism by toad tadpoles vary with tadpole population invasion history?\u003c/h2\u003e \u003cp\u003eNative-range (Honshu) toad tadpoles ate more invasive-range (Hokkaido) toad embryos than did invasive-range (Hokkaido) toad tadpoles (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Time was a significant covariate, but water temperature was not.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA results for effect of toad (Bufo japonicus) tadpole population (native-range (Honshu), invasive-range (Hokkaido)) on predation on invasive-range Hokkaido toad (B. japonicus) embryos. Analyses were conducted using mean water temperature\u0026thinsp;=\u0026thinsp;19.3 \u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChi-square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\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\u003eNative-range vs. invasive-range tadpoles\u003c/p\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003eWater temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.643\u003c/p\u003e \u003cp\u003e30.524\u003c/p\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e0.9193\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 \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTadpole mortality\u003c/h2\u003e \u003cp\u003eWe did not formally analyse tadpole mortality due to low mortality rates and an obvious lack of treatment effects. During our experiments, 5 toad tadpoles in control containers died, 1 Honshu toad tadpole offered frog embryos died without any evidence of eating embryos, and 2 Honshu toad tadpoles offered invasive-range (Hokkaido) toad embryos died after eating either 0 or 1 embryo.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our experiments, cannibalism was less common in invasive (Hokkaido) populations of toads than in native-range (Honshu) populations. That lower frequency was due to characteristics of the toad tadpoles not the toad embryos. Hokkaido (invasive-range) toad embryos were eaten more often, by frogs as well as by toads, than were native-range (Honshu) toad embryos. All else being equal, that palatability should increase rates of cannibalism in invasive (Hokkaido) populations. However, the larvae of invasive-range toads ate fewer conspecific embryos (from the invasive-range population) than did the larvae of native-range toads. That is, cannibalism has declined over the course of the toad\u0026rsquo;s invasion of Hokkaido because of shifts in the attributes of the cannibals (larvae) not the victims (embryos).\u003c/p\u003e \u003cp\u003eOur data on the tadpoles and embryos of frogs provide a context for that shift in cannibalism rates within toads. Rates of predation (intra- and interspecific) overall were higher in frogs than in toads, and including frogs as both predators and prey allowed us to tease apart mechanisms underlying the reduced rates of cannibalism in invasive populations of toads. Thus, data from frogs showed that the greater vulnerability of toad embryos from invasive populations was not a species-specific effect, because frogs as well as toads consumed more invasive-range toad embryos than native-range toad embryos. By including the additional anuran species in our trials, we could show that invasive-population toads exhibit overall an overall increase in embryo vulnerability.\u003c/p\u003e \u003cp\u003eOur results for predation by native-range (Honshu) tadpoles on invasive-range (Hokkaido) toad embryos concur with previous studies. We found that native-range tadpoles (\u003cem\u003eB. japonicus\u003c/em\u003e, \u003cem\u003eR. japonica\u003c/em\u003e, \u003cem\u003eR. ornativentris\u003c/em\u003e) consumed embryos of invasive-range toads without ill effect. Similarly, Oyake et al.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and Okamiya et al.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e found that Honshu species (\u003cem\u003eR. ornativentris\u003c/em\u003e tadpoles, \u003cem\u003eH. nigrescens\u003c/em\u003e larvae) consume invasive-range (Hokkaido) toad hatchlings without dying. This ability to tolerate toad toxins is likely due to co-evolutionary adaptation on Honshu\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, as far as we are aware, our study is the first to examine embryo cannibalism responses for either native-range or invasive-range Japanese common toads.\u003c/p\u003e \u003cp\u003eOur experimental design does not identify the proximate cues involved in the decreased rates of cannibalism in invasive populations of Japanese toads. Detailed studies in Australian cane toads have shown that toxins released from eggs close to the time of hatching attract cannibalistic larvae, and also induce foraging responses by those larvae\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. A similar situation is plausible with the Japanese toads, that also produce eggs containing toxins (as evidenced by high mortality rates of toad-na\u0026iuml;ve predators consuming those eggs or hatchlings:\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e). The decrease in rates of cannibalism in invasive populations of Japanese toads thus might reflect a lower attraction to such cues by conspecific larvae and a decrease in toxin content, a change in toxin composition, or a decrease in rates of toxin release by embryos. Laboratory experiments exposing predators to scent cues rather than embryos could test those ideas (cf.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e), and direct measures of the types and amounts of toxins in embryos from each population also would be informative (cf.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e). Adult \u003cem\u003eB. japonicus\u003c/em\u003e exhibit geographic variation in toxin (bufadienolide) composition within their native range\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Because the toxins in \u003cem\u003eB. japonicus\u003c/em\u003e eggs and hatchlings are maternally-invested\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, similar geographic variation presumably also occurs in the toxin composition of toad embryos. However, whether the toxin composition of \u003cem\u003eB. japonicus\u003c/em\u003e in the invasive-range (Hokkaido) differs from that of native-range populations remains to be determined. Interestingly, the fact that native-range toad tadpoles readily consumed invasive-range toad embryos suggests that factors other than geographic variation in toxin composition may also be involved in causing lower rates of cannibalism by invasive-range toad tadpoles.\u003c/p\u003e \u003cp\u003eThe evolutionary forces responsible for the decline in rates of cannibalism in invasive Japanese toads also warrant further study. Most obviously, why has invasion been accompanied by a reduced rate of cannibalism in this system, whereas Australian cane toads exhibit the opposite pattern\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e? Field studies are needed to clarify this paradox. The colder climate of Hokkaido (the invasive range) than Honshu (the native range) may affect temporal overlap in contact between conspecific embryos and larvae. For example, if breeding is restricted to a brief period in spring, and tadpoles metamorphose during the following summer, then there would be few opportunities for older larvae to consume newly-laid eggs and hatchlings. In contrast, a longer breeding season may generate within-year contact between embryos and larvae, increasing opportunities for cannibalism. Field studies could clarify that phenology in native \u003cem\u003eversus\u003c/em\u003e invasive ranges.\u003c/p\u003e \u003cp\u003eThe importance of cannibalism also covaries with population density, and with the occurrence of other predators of embryos\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, for example, the evolution of cannibalism in Australian cane toads has been attributed to higher densities in the invasive range than in the native range, coupled with a scarcity of alternative predators on embryos\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. To examine this idea, we need field data on densities of toads in their native range and invasive range, and estimates of rates of egg mortality due to intraspecific \u003cem\u003eversus\u003c/em\u003e interspecific predation. Field enclosures in natural waterbodies may provide a way to measure these variables\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The availability and nutritional value of alternative food sources for larvae also deserve attention.\u003c/p\u003e \u003cp\u003eAlthough we found a decrease rather than increase in rates of cannibalism in invasive populations of Japanese toads, contrary to the situation with cane toads in Australia\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, it is striking that in both cases a recent invasion (~\u0026thinsp;100 years) has initiated a substantial shift in the importance of cannibalism. That similarity supports the idea that invasions impose rapid shifts on the selective forces involved in intraspecific competition generally, and cannibalism specifically. The proximate cues and adaptive significance of such shifts are amenable to studies in the laboratory and in the field, rendering cannibalism in toads an ideal study system in which to examine rapid evolutionary change.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Koshiro\u0026nbsp;Eto (Kitakyushu Museum of Natural History \u0026amp; Human History) for collecting samples on\u0026nbsp;Tsushima Island.\u0026nbsp;This work was supported by an internal grant from The Hakubi Center, Kyoto University, and the Environment Research and Technology Development Fund (4RF-1402) of the Ministry of the Environment, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available from Dryad Digital Repository https://datadryad.org/stash/share/Szhq4YDLypxYit13FGv-J6JQ6LAdtf3lCKsTBp3oZAk.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePrentis, P. J., Wilson, J. R., Dormontt, E. E., Richardson, D. M. \u0026amp; Lowe, A. J. Adaptive evolution in invasive species. \u003cem\u003eTrends in plant science\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 288\u0026ndash;294 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoran, E. V. \u0026amp; Alexander, J. M. Evolutionary responses to global change: lessons from invasive species. \u003cem\u003eEcology Letters\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 637\u0026ndash;649 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorden, J. B. \u0026amp; Flory, S. L. Urban evolution of invasive species. \u003cem\u003eFrontiers in Ecology and the Environment\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 184\u0026ndash;191 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRollins, L. A., Richardson, M. F. \u0026amp; Shine, R. A genetic perspective on rapid evolution in cane toads (\u003cem\u003eRhinella marina\u003c/em\u003e). \u003cem\u003eMolecular Ecology\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 2264\u0026ndash;2276 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShine, R. \u003cem\u003eCane toad wars\u003c/em\u003e (Univ of California Press, 2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrban, M. C., Phillips, B. L., Skelly, D. K. \u0026amp; Shine, R. A toad more traveled: the heterogeneous invasion dynamics of cane toads in Australia. \u003cem\u003eThe American Naturalist\u003c/em\u003e \u003cb\u003e171\u003c/b\u003e, E134\u0026ndash;E148 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHudson, C. M., McCurry, M. R., Lundgren, P., McHenry, C. R. \u0026amp; Shine, R. Constructing an invasion machine: the rapid evolution of a dispersal-enhancing phenotype during the cane toad invasion of Australia. \u003cem\u003ePloS one\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, e0156950 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGruber, J., Brown, G., Whiting, M. J. \u0026amp; Shine, R. Is the behavioural divergence between range-core and range-edge populations of cane toads (\u003cem\u003eRhinella marina\u003c/em\u003e) due to evolutionary change or developmental plasticity?. \u003cem\u003eRoyal Society Open Science\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 170789 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosmala, G., Christian, K., Brown, G. \u0026amp; Shine, R. Locomotor performance of cane toads differs between native-range and invasive populations. \u003cem\u003eRoyal Society Open Science\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 170517 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosmala, G. K., Brown, G. P., Shine, R. \u0026amp; Christian, K. Skin resistance to water gain and loss has changed in cane toads (\u003cem\u003eRhinella marina\u003c/em\u003e) during their Australian invasion. \u003cem\u003eEcology and Evolution\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 13071\u0026ndash;13079 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeVore, J. L., Crossland, M. R. \u0026amp; Shine, R. Trade-offs affect the adaptive value of plasticity: stronger cannibal-induced defenses incur greater costs in toad larvae. \u003cem\u003eEcological Monographs\u003c/em\u003e \u003cb\u003e91\u003c/b\u003e, e01426 (2021a).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeVore, J. L., Crossland, M. R., Shine, R. \u0026amp; Ducatez, S. The evolution of targeted cannibalism and cannibal-induced defenses in invasive populations of cane toads. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 118, e2100765118 (2021b).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrossland, M. R., Shine, R. \u0026amp; DeVore, J. L. Choosy cannibals: Targeted consumption of conspecific hatchlings by larval cane toads is triggered by species-specific defensive toxins. \u003cem\u003eEcology and Evolution\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, e8655 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrossland, M. R., Hearnden, M. N., Pizzatto, L., Alford, R. A. \u0026amp; Shine, R. Why be a cannibal? The benefits to cane toad, \u003cem\u003eRhinella marina\u003c/em\u003e [= \u003cem\u003eBufo marinus\u003c/em\u003e], tadpoles of consuming conspecific eggs. \u003cem\u003eAnimal Behaviour\u003c/em\u003e \u003cb\u003e82\u003c/b\u003e, 775\u0026ndash;782 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaas, A. Phylogeny of frogs as inferred from primarily larval characters (Amphibia: Anura). \u003cem\u003eCladistics\u003c/em\u003e 19, 23\u0026ndash;89 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyake, N., Sasaki, N., Yamaguchi, A., Fujita, H., Tagami, M., Ikeya, K., Takagi, M., Kobayashi, M., Abe, H. \u0026amp; Kishida, O. Comparison of susceptibility to a toxic alien toad (\u003cem\u003eBufo japonicus formosus\u003c/em\u003e) between predators in its native and invaded ranges. \u003cem\u003eFreshwater Biology\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e, 240\u0026ndash;252 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkamiya, H., Inoue, Y., Takai, K., Crossland, M. R. \u0026amp; Kishida, O. Native frogs (\u003cem\u003eRana pirica\u003c/em\u003e) do not respond adaptively to alien toads (\u003cem\u003eBufo japonicus formosus\u003c/em\u003e) 100 years after introduction. \u003cem\u003eEcological Research\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 1005\u0026ndash;1014 (2021a).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, X., Natuhara, Y., Li, J., Li, G., Du, Y., Jia, H., Dai, Z., Du, D., Zhong, S. \u0026amp; Qin, D. Effects of multiple stressors on amphibian oviposition: landscape and local determinants in central Japan. \u003cem\u003eEcological Indicators\u003c/em\u003e \u003cb\u003e128\u003c/b\u003e, e107824 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaramura, T., Eto, K., Crossland, M. R., Nishikawa, K. \u0026amp; Shine, R. Competition between the tadpoles of Japanese toads \u003cem\u003eversus\u003c/em\u003e frogs. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 1627 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, K. \u0026amp; Ishikawa, K. Developmental stages of lotic-breeding toad, \u003cem\u003eBufo torrenticola\u003c/em\u003e, with a comparison to lentic-breeding \u003cem\u003eB. japonicus formosus\u003c/em\u003e (Amphibia: Anura: Bufonidae). \u003cem\u003eCurrent Herpetology\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e, 8\u0026ndash;23 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKusano, T., Maruyama, K. \u0026amp; Kanenko, S. Breeding site fidelity in the Japanese toad, \u003cem\u003eBufo japonicus formosus\u003c/em\u003e. \u003cem\u003eHerpetological Journal\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 9\u0026ndash;13 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKusano, T., Miura, T., Terui, S. \u0026amp; Maruyama, K. Factors affecting the breeding activity of the Japanese common toad, \u003cem\u003eBufo japonicus formosus\u003c/em\u003e (Amphibia: Bufonidae) with special reference to the lunar cycle. \u003cem\u003eCurrent Herpetology\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e, 101\u0026ndash;111 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazila, E. \u0026amp; Kishida, O. Foraging traits of native predators determine their vulnerability to a toxic alien prey. \u003cem\u003eFreshwater Biology\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 56\u0026ndash;70 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ekamiya, H., Takai, K. \u0026amp; Kishida, O. The Japanese common toad, \u003cem\u003eBufo japonicus formosus\u003c/em\u003e, contains toxin in the egg stage. \u003cem\u003eCurrent Herpetology\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 103\u0026ndash;106 (2021b).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkamiya, H., Tagami, M., Crossland, M. \u0026amp; Kishida O. Are toxic effects of alien species affected by their prey? Evaluation by bioassay with captive-bred toad embryos and a vulnerable predator. \u003cem\u003eHydrobiologia\u003c/em\u003e \u003cb\u003e848\u003c/b\u003e, 4445\u0026ndash;4452 (2021c).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGosner, K. L. A simplified table for staging anuran embryos and larvae with notes on identification. \u003cem\u003eHerpetologica\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 183\u0026ndash;190 (1960).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaeda, N. \u0026amp; Matsui, M. \u003cem\u003eFrogs and toads of Japan.\u003c/em\u003e (Bun-ichi Sogo Shuppan, 1989)((\u0026lt;bvertical-align:super;\u0026gt;in\u0026lt;/bvertical-align:super;\u0026gt;\u0026lt;bvertical-align:super;\u0026gt; \u0026lt;/bvertical-align:super;\u0026gt;\u0026lt;bvertical-align:super;\u0026gt;Japanese\u0026lt;/bvertical-align:super;\u0026gt;)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team \u003cem\u003eR\u003c/em\u003e: \u003cem\u003eA language and environment for statistical computing\u003c/em\u003e (R Foundation for Statistical Computing, 2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarton, D. I. \u0026amp; Hui, F. K. C. The arcsine is asinine: the analysis of proportions in ecology. \u003cem\u003eEcology\u003c/em\u003e\u003cb\u003e92\u003c/b\u003e, 3\u0026ndash;10 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenables, W. N. \u0026amp; Ripley, B. D. \u003cem\u003eModern Applied Statistics with S\u003c/em\u003e Fourth edition (Springer, 2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFox, J. \u0026amp; Weisberg, S. \u003cem\u003eAn R Companion to Applied Regression\u003c/em\u003e Third edition (Sage, Thousand Oaks CA, 2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHothorn, T., Bretz, F. \u0026amp; Westfall, P. Simultaneous inference in general parametric models. \u003cem\u003eBiometrical Journal\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 346\u0026ndash;363 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrossland, M. R., Haramura, T., Salim, A. A., Capon, R. J. \u0026amp; Shine, R. Exploiting intraspecific competitive mechanisms to control invasive cane toads (\u003cem\u003eRhinella marina\u003c/em\u003e). \u003cem\u003eProceedings of the Royal Society B\u003c/em\u003e 279, 3436\u0026ndash;3442 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrossland, M. R., Salim, A. A., Capon, R. J. \u0026amp; Shine, R. Chemical cues that attract cannibalistic cane toad (\u003cem\u003eRhinella marina\u003c/em\u003e) larvae to vulnerable embryos. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 12527 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayes, R. A., Crossland, M. R., Hagman, M., Capon, R. J. \u0026amp; Shine, R. Ontogenetic variation in the chemical defenses of cane toads (\u003cem\u003eBufo marinus\u003c/em\u003e): toxin profiles and effects on predators. \u003cem\u003eJournal of chemical ecology\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 391\u0026ndash;399 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue, T., Nakata, R., Savitzky, A. H., Yoshinaga, N., Mori, A. \u0026amp; Mori, N. Variation in bufadienolide composition of parotoid gland secretion from three taxa of Japanese toads. \u003cem\u003eJournal of Chemical Ecology\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 997\u0026ndash;1009 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichardson, M. L., Mitchell, R. F., Reagel, P. F. \u0026amp; Hanks, L. M. Causes and consequences of cannibalism in noncarnivorous insects. \u003cem\u003eAnnual review of entomology\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e, 39\u0026ndash;53 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"invasive species, adaptive response, anuran, egg, hatchling, predation","lastPublishedDoi":"10.21203/rs.3.rs-2558797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2558797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBy imposing novel evolutionary pressures, biological invasions can favour rapid changes in intraspecific competitive mechanisms such as cannibalism. In their invasive range in Australia but not in their native range in South America, cane toads (\u003cem\u003eRhinella marina\u003c/em\u003e) exhibit high rates of cannibalism of embryos (eggs and hatchlings) by tadpoles. To explore the generality of such changes, we examined cannibalism in Japanese common toads (\u003cem\u003eBufo japonicus\u003c/em\u003e) on the islands of Honshu (native range) and Hokkaido (invasive range). Contrary to the Australian system, invasion has been accompanied by a reduction rather than increase in rates of cannibalism under standard laboratory conditions. That reduction in cannibalistic tendency by invasive-range toad tadpoles occurs despite an increase in the attractivity of invasive-range toad embryos as prey to other predatory amphibian larvae, including native-range conspecifics. Our data thus support the idea that biological invasions can generate rapid changes in rates of cannibalism, but also show that decreases as well as increases can occur. Future work could investigate the proximate cues and selective forces responsible for this rapid decrease in rates of cannibalism in invasive populations of Japanese common toads.\u003c/p\u003e","manuscriptTitle":"A biological invasion reduces rates of cannibalism by Japanese toad tadpoles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-16 22:38:00","doi":"10.21203/rs.3.rs-2558797/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-04-07T09:16:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-03-14T07:02:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9e3d2648-d7ab-4379-b00f-6a07672fb5c5","date":"2023-03-06T03:08:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-03-03T00:27:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-22T12:30:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-02-15T07:20:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-02-15T06:54:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-02-07T06:50:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67dd1083-bd9e-4788-bf9b-082c617a7316","owner":[],"postedDate":"February 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":19229063,"name":"Biological sciences/Evolution"},{"id":19229064,"name":"Biological sciences/Ecology"},{"id":19229065,"name":"Biological sciences/Ecology/Evolutionary ecology"}],"tags":[],"updatedAt":"2023-10-16T21:38:46+00:00","versionOfRecord":{"articleIdentity":"rs-2558797","link":"https://doi.org/10.1038/s41598-023-36743-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-06-13 21:10:34","publishedOnDateReadable":"June 13th, 2023"},"versionCreatedAt":"2023-02-16 22:38:00","video":"","vorDoi":"10.1038/s41598-023-36743-8","vorDoiUrl":"https://doi.org/10.1038/s41598-023-36743-8","workflowStages":[]},"version":"v1","identity":"rs-2558797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2558797","identity":"rs-2558797","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}