From the Cover: The evolution of targeted cannibalism and cannibal-induced defenses in invasive populations of cane toads

Biotic conflict can create evolutionary arms races, in which innovation in one group increases selective pressure on another, such that organisms must constantly adapt to maintain the same level of fitness. In some cases, this process is driven by conflict among members of the same species. Intraspecific conflict can be an especially important selective force in high-density invasive populations, which may favor the evolution of strategies for outcompeting or eliminating conspecifics. Cannibalism is one such strategy; by killing and consuming their intraspecific competitors, cannibals enhance their own performance. Cannibalistic behaviors may therefore be favored in invasive populations. Here, we show that cane toad tadpoles (Rhinella marina) from invasive Australian populations have evolved an increased propensity to cannibalize younger conspecifics as well as a unique adaptation to cannibalism—a strong attraction to vulnerable hatchlings—that is absent in the native range. In response, vulnerable conspecifics from invasive populations have evolved both stronger constitutive defenses and greater cannibal-induced plastic responses than their native range counterparts (i.e., rapid prefeeding development and inducible developmental acceleration). These inducible defenses are costly, incurring performance reductions during the subsequent life stage, explaining why plasticity is limited in native populations where hatchlings are not targeted by cannibalistic tadpoles. These results demonstrate the importance of intraspecific conflict in driving rapid evolution, highlight how plasticity can facilitate adaptation following shifts in selective pressure, and show that evolutionary processes can produce mechanisms that regulate invasive populations.

In our changing world, adaptations of invasive species to their introduced habitats provide key examples of how species can rapidly evolve in response to changes in both abiotic conditions and biotic interactions (1). For example, competitive abilities can be favored in introduced populations that have been freed from their natural predators and parasites and are therefore under relatively stronger selective pressure from competition (2, 3). The potential advantages of evolving increased competitive abilities are generally considered in the context of interspecific competition with native species, where such abilities could facilitate establishment and spread. However, a key attribute of successful invaders is that they become hyperabundant, at which point intraspecific competition can have stronger effects on fitness. This shift in selective pressure can instead favor mechanisms that reduce intraspecific conflict or enhance intraspecific competitive abilities; these mechanisms can differ from those favored by interspecific competition (4, 5). Whether and how strategies for alleviating competition with conspecifics evolve in invasive species remains a key question and could provide insights into the factors that eventually reduce invasiveness [as for invasive plants (5)] and/or regulate the populations of these species postinvasion (6).Cannibalism can reduce intraspecific competition, as cannibalized conspecific competitors are both eliminated and consumed. This widespread and ecologically important phenomenon not only affects wildlife populations via effects on recruitment (7), population stabilization (8), and community structure (9) but also promotes dispersal (10) and migration (11) and can facilitate invasion (12, 13). For predatory species, cannibalism may represent natural diet extension. However, cannibalistic behaviors in herbivorous or detritivorous organisms that lack predatory adaptations can instead be indicative of resource limitation (14), as cannibalism both provides food resources and reduces intraspecific competition. This can be an adaptive strategy in certain contexts [such as resource-limited or temporary environments (15, 16)]. For example, within temporary waterbodies, pond drying is a substantial mortality risk for larval amphibians, especially if competition reduces developmental rates, delaying metamorphosis. In these environments, some amphibians display remarkable adaptations that facilitate cannibalism and accelerate development [e.g., inducible carnivorous/cannibalistic morphs (17, 18)]. Across species, cannibalistic tendencies not only vary among individuals (19) but can evolve under selective pressures such as artificial selection (20) or resource limitation (14). However, although the evolution of cannibalistic behaviors has been documented in laboratory populations, understanding of the significance of this evolutionary process is limited by a scarcity of evidence from natural populations (14, 15).In Australia, cane toads are an abundant invasive species, achieving densities ∼10 times greater than those in their native South American range (21). This invasion has been facilitated by the toads’ high reproductive output and novel toxic defenses: as Australia lacks native toads, Australian predators and parasites are poorly adapted to toad toxins, and their ingestion is often lethal (22). As in their native range, Australian cane toads often breed in resource-limited, temporary waterbodies where their tadpoles graze on algae and detritus. Intraspecific competition reduces performance in these habitats (23, 24), but tadpoles lack adaptations for killing the conspecific tadpoles with which they compete (24). However, tadpoles can consume conspecific eggs that are laid in their pond, and the prefeeding hatchling stage, when the relatively immobile hatchlings have emerged from the protective egg capsule, is exceptionally vulnerable to cannibalism (25). In contrast, these hatchlings are relatively well defended against Australian predators by maternally invested toxins (e.g., bufadienolides) that provide protection throughout the aquatic stages. In Australia, cannibalism is the principal source of mortality in ponds where conspecific tadpoles are present, and cannibals often reduce the survival of newly laid clutches by >99% (25). This behavior improves cannibal performance by reducing competition and providing trophic resources (24) and may therefore be especially favored in these high-density, invasive populations (26). However, whether a high propensity to cannibalize conspecific hatchlings is characteristic of this species or has evolved within the invasive range is unknown. To determine whether cannibalism rates differ between native and invasive populations, we used 43 tadpole clutches and 22 hatchling clutches to conduct 514 cannibalism trials in which 10 hatchlings were exposed to a single tadpole over a 24-h period. Although tadpoles from both the native and invasive range cannibalized conspecific hatchlings, cannibalism rates were higher in invasive populations, such that the odds a hatchling would be cannibalized when exposed to an Australian tadpole were 2.55 times those in the native range (SE = 2.15 to 3.02, degrees of freedom [df] = 41, t = 5.50, P < 0.0001, Fig. 1B and SI Appendix, Table S1).Open in a separate windowFig. 1.Cane toad tadpoles from invasive Australian populations cannibalized conspecifics at a higher rate than did native range tadpoles and, unlike native range tadpoles, exhibited a strong attraction to conspecifics during the vulnerable hatchling stage. Adult toads were collected from across French Guiana and the extent of their current Australian distribution (A); dots indicate collection sites, and arrows indicate historic exportation and introduction sites. When their offspring were offered 10 conspecific hatchlings, Australian tadpoles consumed more hatchlings than did tadpoles from native range populations (B) (proportion cannibalized shown for a 100-mg tadpole, n = 43 clutches, P < 0.0001). In attraction trials, Australian tadpoles were also more strongly attracted to hatchlings than native range tadpoles (n = 31 clutches, P < 0.0001). In these trials, tadpoles from the native range did not differentiate between an empty control trap and a trap containing hatchlings (C), such that 46% of native range tadpoles selected the hatchlings trap (SE: 39 to 53%). However, tadpoles from invasive Australian populations were strongly attracted to conspecifics during this vulnerable period (D), with 88% of tadpoles from invasive populations selecting the trap that contained hatchlings (SE: 87 to 90%). Means ± SE.For species that lack adaptations for subduing and killing conspecifics, an ability to detect and locate vulnerable life stages can facilitate cannibalism (14). Because cane toad hatchlings are only vulnerable to cannibalism during the early, prefeeding stages (25), the ability to target conspecifics during this relatively brief period would enhance the ability of tadpoles to eliminate newly laid clutches. We determined whether tadpoles from native or invasive populations were attracted to the vulnerable hatchling stage using attraction trials, in which 2 traps (one control, one baited with 300 hatchlings) were placed in 90 L pools containing 50 tadpoles. We conducted 69 trials using 31 tadpole and 14 hatchling clutches. Within the native range, tadpoles were equally likely to enter the control trap as the trap containing hatchlings (odds ratio: 0.82, SE = 0.56 to 1.20, df = 12, t = −0.525, P = 0.61, Fig. 1C). However, Australian tadpoles were strongly attracted to conspecific hatchlings, such that a tadpole was 29.5 times as likely to enter a trap containing hatchlings as the paired control trap (SE = 24.7 to 35.1, df = 55, t = 19.26, P < 0.0001, Fig. 1D). Cannibalism therefore shifts from an opportunistic behavior in the native range to a targeted response in Australia, whereby tadpoles cease their normal foraging activities upon detecting hatchling cues in order to locate and consume conspecifics (SI Appendix, Fig. S1 and Table S2). In Australia, this behavior, which is not known for any other amphibians, is mediated by the detection of maternally invested toxins that are present in newly hatched conspecifics [e.g., bufadienolides; native amphibians are not targeted (27)]. These cues both attract tadpoles and induce feeding behaviors (27, 28). Attraction is only induced by the detection of conspecifics during this vulnerable prefeeding stage; Australian tadpoles are not attracted to conspecifics during the invulnerable tadpole stage (SI Appendix, Fig. S2 and Table S3). Remarkably, this adaptation has also facilitated control measures by allowing the targeted trapping of tadpoles from invaded waterbodies using toxin baits (27). These invasive populations have therefore evolved a behavior that facilitates the control of their own populations, not only because targeted cannibalism limits conspecific recruitment (25) but also because it increases tadpole susceptibility to the removal efforts of resource managers.Innovation in one group can increase selective pressure on another, creating an evolutionary arms race (29, 30) in which organisms must constantly adapt in order to maintain fitness. Although seldom considered in the context of cannibalism, conflict with conspecifics can drive this process [e.g., in cases of sexual or maternal–offspring conflict (31)]. Stronger defensive strategies during the vulnerable, prefeeding life stages may therefore emerge in response to increased cannibalistic behaviors in tadpoles from invasive populations. When faced with a novel challenge, phenotypic plasticity (i.e., the ability for a given genotype to produce alternative phenotypes based on environmental conditions) is a key mechanism through which individuals can rapidly respond (32, 33). Individuals that exhibit an adaptive plastic response when confronted with a novel threat may be more likely to survive, increasing the persistence of plastic individuals, and thereby favoring plastic genotypes [i.e., Baldwin effects (34)]. In contrast, maladaptive plastic responses should be rapidly eliminated by natural selection (35). This may result in the evolution of increased adaptive plasticity. Inducible developmental acceleration is a plastic response that has evolved in response to stage-specific threats in a wide variety of species; this defense reduces mortality risk by allowing an individual that perceives a threat to reduce the duration of the risky period (36–39). This kind of inducible defense has been documented in response to cannibalism risk in Australian cane toad hatchlings (25), but whether it is present in native populations or has emerged in response to increased cannibalism risk in the invasive range is unknown. We used 23 newly laid clutches to determine whether hatchlings from invasive populations exhibit stronger cannibal-induced defenses than those from native populations. We divided each clutch between cannibal exposed and control treatment tanks (presence versus absence of two caged conspecific tadpoles) and measured the duration of the vulnerable prefeeding period for each individual (for five individuals/tank in 189 cannibal exposed and 109 control tanks). Developmental plasticity in response to conspecific cues was relatively rare and inconsistent in native range clutches, such that only 1 of 10 clutches exhibited significant developmental acceleration. In contrast, most Australian clutches accelerated development (Fig. 2A). The regulation of prefeeding development has therefore been modified in invasive populations; on average, Australian populations exhibit significant, adaptive, cannibal-induced plasticity in prefeeding developmental rates (−2.34 ± 0.30 h, df = 163.0, t = −7.93, P < 0.0001), whereas native populations do not (−1.23 ± 0.64 h, df = 108.4, t = −1.93, P = 0.056; SI Appendix, Table S4). Although increased selective pressure from cannibalism could favor an inducible defense, the relative rarity of a response in the native range raises a question: why is this inducible defense not more common in native populations where opportunistic cannibalism still poses a risk? One possibility is that, where cannibalism risk is low, the fitness costs associated with this inducible defense outweigh the benefits.Open in a separate windowFig. 2.Relative to native range clutches, cane toad clutches from invasive populations developed more rapidly through the vulnerable prefeeding stages, exhibited stronger cannibal-induced plasticity in developmental rates, and performed more poorly following exposure to cannibal cues. Clutches from the invasive Australian range reached the invulnerable tadpole stage more quickly than did native range clutches in both control and cannibal-cue–exposed treatments (A) [n = 23 clutches, P = 0.0266 and 0.0209, respectively; clutch means, Australian data were adapted from DeVore et al. (25)]. Plasticity in developmental rates also differed between native and invasive populations (A); on average, Australian clutches accelerated prefeeding development in response to cannibal cues (n = 13 clutches, P < 0.0001), whereas clutches from the native range did not (n = 10 clutches, P = 0.0573; clutches that exhibited a significant plastic response [P < 0.05] are depicted with solid lines). Across both native and invasive populations, greater cannibal-induced plasticity in the rate of prefeeding development was associated with poorer performance during the tadpole stage (B) (clutch means, n = 22 clutches, P < 0.0001). Differences in the magnitude of the plastic response between native and invasive populations ultimately led to differences in the mean effect of cannibal exposure on tadpole viability (C); on average, prefeeding exposure to nonlethal tadpole cues did not affect subsequent performance in native populations (n = 9 clutches; development P = 0.131, growth P = 0.651), but in invasive Australian populations, exposure reduced subsequent development and growth (n = 13 clutches; development P < 0.0001, growth P < 0.0001). Means ± SE; photograph insets visually represent treatments (A) and variation in growth among 10-d-old tadpoles (C).Theoretical models predict that the ability to exhibit an adaptive plastic response should be costly, explaining why organisms do not evolve the ability to produce the optimal phenotype in all environments (40). However, despite their theoretical importance, costs of plasticity are rarely detected (41). It has therefore been suggested that the costs associated with plasticity are quickly offset by evolutionary processes, making them difficult to detect in organisms expressing well-established inducible responses (40, 42). If this is the case, plasticity costs should be more easily detected for responses that are actively evolving and have not yet been fixed in the population (SI Appendix, Fig. S4). In organisms with complex life history strategies, any costs associated with inducible defenses may be evident during the subsequent life stage as carry-over effects (43). To determine whether the ability to express this plastic response is costly, we individually raised 1,190 tadpoles from 22 of the clutches that had been monitored as hatchlings during the developmental acceleration experiment. Ten days later, we assessed their rates of survival, development (i.e., Gosner stage), and growth (measures considered appropriate proxies for “fitness” in tadpoles; see Methods). We then related each clutch’s degree of plasticity (i.e., the cannibal-induced reduction in prefeeding development time) and phenotype (i.e., the duration of prefeeding development) to its mean performance during the tadpole stage (44). We found that, in cannibal-exposed treatments, stronger adaptive plastic responses were followed by poorer tadpole performance across both native and invasive populations (Fig. 2B; effect per hour of acceleration: development: −0.53 ± 0.08 stages, df = 14.8, t = −6.93, P < 0.0001; growth: −8.94 ± 1.57 mg, df = 15.4, t = −5.70, P < 0.0001). Tadpoles from more plastic clutches also developed more slowly in control conditions (−0.171 ± 0.078 stages, df = 14.47, t = −2.19, P = 0.045). The ability to express this inducible response is therefore associated with slight performance reductions even if the threat is absent. In contrast, phenotype (i.e., the duration of prefeeding development) was not associated with tadpole fitness metrics (SI Appendix, Table S6 and Fig. S3). Therefore, poor clutch performance is not associated with rapid prefeeding development per se but with the ability to accelerate development. Ultimately, the difference in plasticity between the native and invasive range led to pronounced differences in the mean effect of exposure to conspecific cues on tadpole viability (treatment × country, development: df = 255.8, t = −6.50, P < 0.0001; growth: df = 255.6, t = −9.13, P < 0.0001; SI Appendix, Table S5). In the native range, prefeeding exposure to cues from conspecific tadpoles did not affect subsequent performance (P > 0.13 for all performance measures). However, in Australia, exposure to nonlethal cues substantially reduced tadpole development and growth rates (development: −2.34 ± 0.20 stages, df = 163.3, t = −11.7, P < 0.0001; growth: −53.8 ± 3.6 mg, df = 163.4, t = −15.1, P < 0.0001). Indeed, in Australia, the effects of prefeeding exposure to conspecific tadpoles are often so severe that they were initially attributed to intraspecific allelopathy (45) and the possibility of using conspecific tadpole cues to “poison” hatchlings has been explored as a control measure (46). These substantial costs to tadpole viability limit the adaptive value of cannibal-induced plasticity in the native range where opportunistic cannibalism poses less of a threat than the targeted cannibalism in Australia (25). Our evidence of these plasticity costs, which are theoretically predicted but infrequently detected, also supports the hypothesis that, under continued selective pressure, evolutionary processes eventually offset these costs or favor canalized defenses over costly inducible responses (SI Appendix, Fig. S4). As a result, plasticity costs that are difficult to detect for well-established inducible responses may be more overt following a shift in selective pressure.Both the regulation (i.e., plasticity) and the expression of traits may evolve in response to changes in selective pressure. In this case, targeted cannibalism of the prefeeding stages could also favor the evolution of rapid prefeeding development (47). We therefore also compared the duration of the vulnerable period in clutches from native and invasive populations. We found that Australian clutches reach the invulnerable tadpole stage more quickly than do native range clutches in both cannibal-exposed and cannibal-naïve treatments, such that the duration of prefeeding development is reduced by ∼20% in invasive populations (control: df = 5.88, t = −2.94, P = 0.027, exposed: df = 5.86, t = −3.13, P = 0.021; SI Appendix, Table S4). This difference in developmental rates is only evident during the vulnerable, prefeeding stages. In control conditions, rates of development and growth during the invulnerable tadpole stage did not differ between the native and Australian range (development: 0.747 ± 0.600 stages [SE], df = 20.01, t = 1.25, P = 0.23; growth: 9.97 ± 12.70 mg [SE], df = 20.31, t = 0.79, P = 0.44). By relating the phenotype of each clutch to its plasticity, we also found that the most extreme prefeeding developmental rates were produced by nonplastic development (Fig. 3 and SI Appendix, Table S7). However, nonplastic strategies produced remarkably different phenotypes in each country. In the native range, nonplastic clutches developed slowly, reaching the tadpole stage in ∼5 d. This fixed, slow development strategy was absent in the invasive Australian range where nonplastic clutches instead exhibited rapid development (∼3 d), an apparently derived strategy that was not observed in native range clutches. Plastic development produced intermediate phenotypes, which were present in both native and invasive populations. As a result, whereas plasticity was an effective strategy for reducing the duration of the vulnerable period within native range populations (when exposed to cannibals, the most plastic native range clutches had the shortest development times: −2.189 ± 0.862 h [SE], t = −2.54, P = 0.0347), plasticity was relatively ineffective within Australia (where the most plastic clutches had the longest development times: 2.796 ± 1.089 h [SE], t = 2.57, P = 0.0261; SI Appendix, Table S8). Interestingly, whereas the negative consequences of cannibal-induced developmental acceleration were immediately evident during the subsequent life stage, we did not detect any costs associated with canalized rapid development. However, we could not have detected any costs that do not manifest until later life stages (e.g., postmetamorphosis). In addition, maternal effects can influence offspring phenotypes, including plastic responses (48); future research should investigate the roles of genotype and maternal effects in driving phenotypic variation in this system.Open in a separate windowFig. 3.In both cannibal exposed and control conditions, the most extreme phenotypes were found in nonplastic clutches. However, the nature of the phenotype produced differed between the native and invasive range. In the native range, nonplastic clutches developed slowly, whereas in the invasive range, nonplastic clutches exhibited rapid development. Intermediate phenotypes were produced by plastic clutches and were found in both native and invasive populations. Here, quadratic regressions of plasticity (i.e., the cannibal-induced reduction in the duration of prefeeding development for each clutch) are plotted against the total duration of the vulnerable, prefeeding period. The mean observed phenotypes of the 23 clutches in control (circle; quadratic term P = 0.0116) or cannibal exposed (plus; P = 0.0044) conditions used to plot these regressions are also shown; lines connect the phenotypes observed for a certain clutch within each environment (thus indicating that clutch’s plasticity). Phenotypes only observed in the native range are shown with a gray background, whereas those only observed in the invasive range are plotted over white. The transition zone includes phenotypes observed in both native and invasive populations. Viability during the subsequent tadpole stage was significantly associated with the magnitude of the plastic response a clutch displayed during the prefeeding period; tadpole performance was poorest in the most plastic clutches, especially following cannibal exposure.Ultimately, selection for rapid development and canalization of the inducible defense may both contribute to the rapid prefeeding development of Australian clutches. The canalization of an inducible phenotype can occur where a canalized phenotype provides a fitness advantage over a plastic response [genetic assimilation (49–51)]. In this case, the inducible defenses that are common in these invasive populations, although likely initially favored in Australia as the most effective strategy from ancestral, native-range populations [i.e., Baldwin effects (34)], are both more costly and less effective at reducing the window of vulnerability than the canalized rapid development that has emerged in the invasive range [see also DeVore et al. (25)]. This inducible defense may therefore be ephemeral within these invasive populations, as a shift to the canalized rapid development already exhibited by some clutches is expected under continued selective pressure. Adaptive refinement of inducible responses may occur where the inducing environment is frequently encountered [i.e., frequency-dependent adaptation (52)]; a shift to a less costly and/or more effective defensive strategy may therefore be especially favored in parts of the invasive range where cannibals are most often present [e.g., in range-core populations (53)]. Monitoring of the stability of this inducible defense could provide insights into whether costly plastic responses that are favored following a shift in selective pressure can be either maintained via cost offsets or canalized in the induced state.Intraspecific competition can be an important source of selective pressure. In invasive populations where conspecific densities are high, this pressure may be intensified, favoring the evolution of strategies that reduce intraspecific conflict. This process may be especially important in invaders that are well defended against predators and parasites [i.e., rare enemy effects (54)]. Such adaptations can then favor further evolutionary change. Invasive cane toads display traits such as rapid prefeeding development, cannibal-induced developmental acceleration, and increased dispersal abilities during the terrestrial life stages that accelerate the colonization of new, cannibal-free habitats (10, 55). Our results reveal that the evolutionary emergence of targeted cannibalism in the invasive range may have favored these new evolutionary trajectories, demonstrating the importance of intraspecific conflict in driving adaptation in natural systems, as well as the potential for the evolutionary processes to produce mechanisms that stabilize invasive populations. These results also provide a clear example of the role of phenotypic plasticity in facilitating rapid adaptation to shifting selective pressures.