Journal of Evolutionary Biology

Matching habitat choice provides a mechanism for individuals to maximise their expected fitness by selecting an environment that better fits their phenotype. Many animals choose their local environment by evaluating levels of perceived predation risk against possible resource gain. To test if predation risk is a major driver of habitat choice, we quantify scototaxis, or preference for dark versus light backgrounds, in juvenile guppies. As light backgrounds increase visibility to predators, this aspect of habitat choice captures variation in boldness in small fishes. By rearing and testing 586 fish descended from ten natural populations from Trinidad under common garden conditions, we first quantify (broad sense) heritable variation, i.e. evolutionary potential, within populations. Next, we test for evolutionary divergence among populations in mean preference, and if present, whether ancestral predation regime is a mediator of divergence. Finally, we ask whether families and/or populations differ in the amount of behavioural variation they contain. Habitat choice varied among families (12% of total variance), consistent with heritable variation (0.2). We also found mean preference varies among populations (11% of total variance explained). Evolutionary divergence among-populations is partly explained by ancestral predation regime, with populations from low-predation sites showing a stronger average preference for dark backgrounds than high-predation populations from the same river. Additionally, we find that within-population behavioural variation is greater in high-predation populations. We conclude that guppy populations contain heritable variation that could facilitate adaptive evolution if scototaxis is subject to natural selection. Furthermore, while genetic drift may also contribute to evolutionary divergence among-populations, observed patterns are qualitatively consistent with local adaption to predation regime. Our results suggests that high predation sites favour bolder habitat choice on average, but also that local predation regime shape the evolutionary dynamics of variation, perhaps by maintaining shy-bold variation among-individuals or by favouring individuals with less-predicable behaviour.

Conspecific sperm precedence via cryptic female choice is a post-ejaculatory selection process that reduces hybridization, and can be pronounced in sympatric species. In their native Europe, Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) exert conspecific sperm precedence under heterospecific sperm competition, which is at least partially enabled by female reproductive fluid. We examined post-ejaculatory selection of both species in Newfoundland, Canada, where Atlantic salmon evolved in absence of brown trout, but now experience hybridization threats due to anthropogenic introductions. Using split-ejaculate and split-clutch in-vitro fertilizations we evaluated whether allopatric evolution has relaxed this selection in Atlantic salmon, and found that they had no ability to bias paternity towards conspecific males, whereas naturalized brown trout retained a strong ability to do so. Female reproductive fluid influenced this, as when fluid associated with a species eggs was swapped, hybridization increased. In the artificial situation of no female reproductive fluid during sperm competition, paternity changed dramatically, but sperm swimming performance did not predict it. Our findings contribute to understanding the evolution of cryptic female choice and how the mechanisms of reproductive isolation can be reinforced through sympatry, while also highlighting a new potential conservation concern for North American Atlantic salmon.

The Red Queen Hypothesis proposes that genetic variation is maintained in populations through antagonistic coevolution of hosts and parasites. A major assumption of the Red Queen Hypothesis is tight genetic specificity for infection. However, it has been argued that this genetic interaction of host and parasite (GHxGP) is sensitive to environmental context (GHxGPxE). Environmental change could accordingly disrupt coevolutionary oscillations on relevant time scales, calling into question antagonistic coevolution as a general and robust explanation for the maintenance of genetic diversity. To evaluate this critique, we used the plant-parasitic nematode Meloidogyne arenaria and its natural bacterial parasite Pasteuria penetrans to determine if specificity is altered by temperature. We exposed six isofemale host lines to five parasite sources at three ecologically relevant temperatures. We found that, at two of three temperatures, susceptibility to infection depended on the specific combination of host line and parasite source (GHxGP). This specificity varied across temperatures, consistent with a GHxGPxE effect. This three-way interaction was driven both by quantitative changes in the strength of specificity across temperatures and shifts in the susceptibility rankings of host-parasite combinations. Our study contributes a rare experimental test of a proposed challenge to the Red Queen Hypothesis and suggests the potential for environmental context to change host-parasite specificity.

AO_SCPLOWBSTRACTC_SCPLOWEnvironmental heterogeneity across freshwater systems often promotes phenotypic variation, yet disentangling environmentally induced variation from heritable differentiation remains a central goal in evolutionary ecology. We investigated the geographic distribution and morphological differentiation, and heritability of shell traits among populations of the freshwater lymnaeid snail Pectinidens diaphanus in Patagonia. Extensive field surveys across 196 freshwater sites revealed that the species occupies a broad range of lentic and lotic habitats and constitutes the only lymnaeid inhabiting southern Patagonia. While reproductive anatomical structures were conserved across populations, shell shape differed markedly among populations from contrasting habitat types, with population identity explaining nearly 50% of total shape variation. Populations from hydrologically unstable habitats (ponds and streams) exhibited more elongated shells and relatively smaller apertures, a pattern consistent with functional responses to hydroperiod variability and desiccation risk. To assess the heritability of this differentiation, we conducted a common-garden experiment across two generations. Shell shape differences between permanent- (lagoon) and temporary- (pond) habitat-derived populations persisted into the G2 generation reared under standardized laboratory conditions, indicating that the observed variation is not solely a response to local environmental conditions but includes a heritable component. Together, our findings demonstrate that P. diaphanus constitutes the sole lymnaeid across southern Patagonia, occupying a broader range than previously documented, and that populations show heritable shell differentiation potentially associated with contrasting freshwater habitats. By integrating large-scale biogeographic surveys with morphometric and experimental approaches, this study provides new insight into how habitat variation may contribute to ecological and evolutionary differentiation in freshwater gastropods.

The evolution of alternative male reproductive strategies represents an intriguing evolutionary phenomenon. Divergent strategies are persistently at risk of local extinction or invasion, depending on the suites of traits expressed within and between morphs; hence, understanding the correlational selection that aligns reproductive strategies with behaviour, morphology and physiology is key to understanding the origin and maintenance of genetic polymorphisms. In the polychromatic painted dragon, Ctenophorus pictus, yellow, orange and red morphs are well characterised, but the blue morph has been historically absent from studied populations. Here we document the local distribution, morphology and male-contest interactions in a population where blue males are relatively common. We find that blue males express head colouration after a reaching a threshold body size, and that small blue males can reside in close proximity to other males; patterns consistent with a novel size-dependent conditional tactic within the suite of genetic strategies seen in this species. Condition-dependent, positively allometric throat bibs were non-randomly distributed among male morphs, implicating variation in correlational selection and the genetic architecture of the polymorphism. We were unable to definitively assign a morph that was superior in male competition but found that within morphs, male size was the determinant of competitive success, whilst between morphs it was not. Furthermore, contests between morphs were resolved with less aggression than contests within morphs, supporting the idea that badges resolve conflict, and that the invasion of new colour morphs may be facilitated by negative frequency dependent benefits to novel colour variants. These findings highlight the divergent phenotypic, genetic and selective environments that lead to the diversity of colour morphs.

Dormancy is a widespread adaptive strategy that allows organisms to survive in temporally varying habitats by suspending development and reproduction. Although environmental variability is expected to shape dormancy strategies, it remains unclear how differences in environmental variability and predictability influence both the production of dormant embryos and the termination of dormancy. We addressed these questions by comparing D. pulex and D. obtusa, two closely related species that inhabit environments differing in variability and predictability. We hypothesized that D. obtusa, which inhabits ephemeral environments, would exhibit a greater propensity for sexual reproduction and dormancy and would require stronger cues to break dormancy than D. pulex, which occurs in more permanent, predictable habitats. Consistent with our hypothesis, D. obtusa lineages produced significantly more males and ephippia than D. pulex when reared under identical laboratory conditions, indicating greater investment in sexual reproduction and dormancy. Contrary to our hypothesis, we found no difference in responsiveness to cues between the two species. Across species, embryos broke dormancy and hatched most readily after experiencing changes in cold and light, even if not experienced at the same time. In contrast, desiccation reduced the propensity to break dormancy. Together, these results indicate that species occupying more ephemeral environments invest more heavily in the production of dormant offspring, but that the environmental cues regulating dormancy termination appear broadly similar between species. This pattern suggests that while investment in dormancy may evolve in response to environmental variability, the mechanisms controlling dormancy termination are more conserved.

In most species, unmated individuals run the risk of dying with zero fitness. This strong selection on virgin females to mate may also explain why females subsequently remate, despite fitness costs; all that is required is a genetic correlation between virgin and non-virgin mating propensity. Despite being the null model for the evolution and maintenance of polyandry, this hypothesis has received no empirical test. We performed separate quantitative genetic and artificial selection experiments to test the presence of this cross-context genetic correlation in the cow-pea weevil, Callosobruchus maculatus. A quantitative genetic experiment did not find evidence of the hypothesised genetic correlation. However, after 13 generations of artificial selection on virgin mating latency, we found strong evidence for evolutionary divergence in remating latency. Females from lines selected for longer virgin mating latency took approximately twice as long to remate and, were less polyandrous if their virgin mating latency was longer. There was no evidence that females could mate indiscriminately and then trade-up, rather, trading up could only occur if virgin discrimination was present. Selection against virgin death will thus constrain both the evolution of non-virgin discrimination and trading up, increasing rates of polyandry. These findings reveal a genetic correlation between virgin and non-virgin latency to mate suggesting that polyandry may be maintained because of the need to breed.

Animal weapons are ecologically important traits that mediate contests over limiting resources and can strongly influence survival and reproduction. Weapon traits often exhibit substantial intraspecific morphological diversity, raising questions about the ecological drivers of this variation. Acrorhagi are weapons produced by sea anemones that are used in intraspecific territorial encounters. Although acrorhagial morphology varies widely within species, patterns of intraspecific variation remain poorly characterized, and the extent to which such variation reflects differences in local intraspecific competition is unclear. Here, we conduct morphometric analyses to characterize within-population variation and allometry in acrorhagial traits of the solitary anemone Anthopleura sola. We show that these traits covary with habitats differing in conspecific density. The number of acrorhagi scaled positively with body size, and individuals occupying a high-density habitat tended to possess more acrorhagi than did similar sized individuals from a low-density habitat. In addition, anemones from high-density habitats exhibited longer acrorhagial cnidae, a pattern that was not explained by differences in body size or acrorhagial density. Together, these results suggest that competitive context influences weapon-related traits at multiple levels of biological organization, potentially via phenotypic plasticity or selective processes. More broadly, our findings highlight how fine-scale ecological variation may contribute to the maintenance of trait diversity within and across species.

In many tropical areas, seasonal rainfall leads to distinct dry and wet seasons. Many butterflies developing under wet season conditions develop into adults with large ventral eyespots on the wing margins, whereas those developing under dry season conditions have smaller or no eyespots. In greener, wet season habitats, larger eyespots can divert predator attacks toward the wing margins, while reduced eyespot size improves camouflage in the dry leaf litter-dominated habitat during the dry season. However, the dry season is also characterised by higher desiccation stress than the wet season. We hypothesised that larvae developing under dry season conditions develop into adults with higher desiccation tolerance than those reared under wet season conditions. We tested this by rearing larvae of the butterfly Mycalesis mineus under simulated dry and wet season conditions and assaying the desiccation tolerance of the resulting adults. Butterflies reared in dry conditions survived longer under desiccation stress, lost lesser water during pupal-adult metamorphosis, and were heavier than those reared in wet conditions. We also tested the correlation between eyespot size and desiccation tolerance. A negative correlation between the traits would be expected if similar developmental pathways regulate them. Consistent with this expectation, individuals with smaller eyespots had higher desiccation tolerance. Our results demonstrate plasticity in desiccation tolerance, and suggest that predator avoidance and desiccation tolerance traits may share similar developmental pathways.

Sexual selection is a major evolutionary force, yet its demographic consequences remain unclear. While experimental studies often report positive effects of sexual selection on traits linked to population performance, comparative studies often find null or negative associations with population persistence. One explanation for this discrepancy is that the demographic consequences of sexual selection depend on ecological context, particularly variation in mortality and fecundity. Here, we used six decades of abundance data and test whether sexual selection predicts population trends across 738 bird species from Europe and North America. We quantify sexual selection using complementary proxies capturing different components of sexual selection: mating system, sexual dichromatism, sexual size dimorphism and relative testes mass. We further assess whether the effect of sexual selection in population trends is mediated by mortality and fecundity. Across all proxies, we found no evidence that sexual selection is associated with population trends. This result is consistent across continents and robust to variation in mortality and fecundity. Our findings suggest that, despite its central role in shaping phenotypic evolution, sexual selection does not translate into consistent effects on long-term population trends at macroecological scales. More broadly, these results highlight a potential disconnect between evolutionary processes and population dynamics.

The fitness of immigrants and their descendants determines the effectiveness of gene flow. Genetic incompatibilities or outbreeding depression can limit the spread of novel alleles, while highly fit immigrant lineages can hasten introgression. These fitness effects of gene flow can also differ between generations as immigrant and resident haplotypes recombine. Understanding the genetic factors that shape immigrant fitness over multiple generations is increasingly important as habitat fragmentation threatens populations by reducing genetic variation and leading to increased levels of inbreeding. Few studies have measured the multigenerational fitness of immigrant lineages, especially within populations that had histories of high gene flow. We used 33 years of life history and pedigree data on a population of Florida scrub-jays (Aphelocoma coerulescens) with historically high immigration to quantify the fitness of immigrants and their descendants. We compared the fitness of immigrants and residents as well as their resulting descendants (F1, F2, etc.) to determine the composite genetic effects responsible for fitness differences. We found evidence of additive benefits of immigrant ancestry and heterosis driven by non-additive effects that persists for multiple generations. These results are promising for conservation efforts aiming to increase connectivity and illustrate the complex dynamics that determine the rates of introgression in natural populations.

Social interactions are ubiquitous in nature and have the potential to affect trait evolution, particularly in group-living animals such as cooperative breeders. Interactions among conspecific individuals can affect the amount of additive genetic variation for a trait when the phenotype of an individual is also affected by the genotype of its social partner(s) via indirect genetic effects. Thus, quantifying both direct and indirect genetic effects of social partners is critical for understanding and predicting evolutionary trajectories. While much is known about maternal indirect genetic effects, empirical estimates of indirect genetic effects from other social partners remain limited, particularly in wild populations. Here, we use animal models to assess the contribution of indirect genetic effects from all social partners in a family group (mothers, fathers, and helpers) on juvenile morphometric traits across ontogeny in the cooperatively-breeding Florida scrub-jay (Aphelocoma coerulescens). We found indirect genetic effects of helpers and fathers on nestling weight, but no indirect genetic effect of mothers. Across ontogeny, we found increasing additive genetic variation in both weight and tarsus length. Our study provides a comprehensive assessment of within-group indirect genetic effects in a cooperative breeder and highlights the importance of considering indirect genetic effects beyond maternal effects.

Parasites can alter host populations in fundamentally different ways depending on whether exposure results in infection. Yet, most epidemiological and evolutionary inference focuses on established infections, leaving the fitness consequences of parasite exposure comparatively understudied. This gap is consequential because hosts are frequently exposed to diverse parasite genotypes, and these encounters can impose substantial fitness costs even when infection does not occur. Theory predicts that hosts may mitigate these costs when interacting with commonly encountered parasite genotypes, such that exposure to sympatric parasites incurs lower fitness consequences than exposure to novel, allopatric parasites. Here, we examine the fitness consequences of exposure and infection in the first intermediate host of the trophically transmitted tapeworm Schistocephalus solidus, a cyclopoid copepod that serves as the first host in a three-host life cycle. Using sympatric (Vancouver Island, Canada) and allopatric (Norway) host-parasite combinations, we found a striking reciprocal asymmetry. Sympatric parasites were significantly more infective, yet exposure to sympatric parasites imposed weaker fitness costs when infection did not establish. In contrast, allopatric parasites were less infective, but exposed females produced fewer eggs and had lower hatching success than both controls and females exposed to sympatric parasites, indicating substantial genotype-dependent costs of exposure. Moreover, we found that infection was highly virulent across all genotypes: a single parasite caused near-complete reproductive suppression and reduced host survival across all host-parasite pairings, confirming S. solidus as a castrating parasite in copepods. Together, these results demonstrate that exposure, not just infection, acts as a critical ecological filter with potentially large and underappreciated consequences for host population dynamics and parasite transmission.

In androdioecious species like Caenorhabditis elegans, where the primary mode of reproduction is self-fertilization, the evolutionary role of males has long puzzled biologists. One proposed benefit of males is the potential to escape inbreeding depression. We tested this by enforcing seven generations of inbreeding across nine C. elegans strains differing in baseline male frequency and measuring competitive relative fitness before and after inbreeding. We then relaxed inbreeding for four generations to assess recovery. We predicted that strains with higher male frequency, and greater opportunity for outcrossing, would exhibit faster recovery once inbreeding was relaxed. Strains varied substantially in their responses with most showing significant fitness declines and partial recovery but neither the magnitude of inbreeding depression nor the extent of recovery correlated with male frequency. These results show that male frequency is a poor predictor of inbreeding responses and does not reliably reflect realized outcrossing or its fitness consequences.

Sex ratio is a key demographic parameter shaping population dynamics and evolutionary trajectories. In biocontrol agents, demographic bottlenecks during species introduction to a new habitat and subsequent mass rearing can elevate inbreeding, potentially biasing sex ratios through sex-specific mortality associated with inbreeding depression. Moreover, reproductive endosymbionts such as Wolbachia are known to manipulate host reproduction and further skew sex ratios. However, the relative contributions of these processes to sex-ratio variation remain poorly resolved. In this study, we evaluated the effects of cross-generational full-sibling inbreeding and Wolbachia infection on sex ratio and key life-history traits in the biocontrol beetle Zygogramma bicolorata using controlled laboratory crosses across three generations. Inbreeding did not significantly alter offspring sex ratio, which remained close to parity across generations, while pupal mortality increased in later generations, consistent with delayed expression of inbreeding depression. Adult body weight remained largely unaffected by inbreeding. Wolbachia infection was detected in a subset of females and was associated with a modest but significant increase in female-biased offspring production, although the effect was variable across lineages. Strain typing identified a single supergroup A Wolbachia, consistent with previous descriptions of the wBic strain from this species. These findings indicate that sex-ratio variation in introduced populations of Z. bicolorata is not driven by inbreeding alone but instead emerges from the interaction between demographic processes and symbiont-mediated effects, providing crucial insights for optimizing biocontrol programs where sex-ratio stability is essential for population establishment and persistence. SignificanceSex ratio is a key determinant of population growth and stability - the essential parameters determining success of biocontrol programs. Yet, the mechanisms shaping sex-ratio variation remain poorly resolved. Using controlled crosses in Zygogramma bicolorata, we show that short-term inbreeding does not directly alter sex allocation, despite inducing delayed fitness costs through increased pupal mortality. In contrast, Wolbachia infection contributes to female-biased offspring production, although with variable outcome across lineages. These findings demonstrate that sex-ratio variation in Z. bicolorata arises from the interaction of demographic processes and symbiont effects, rather than a single mechanism, with important implications for predicting the establishment, persistence, and efficacy of mass-reared biocontrol populations.

Most species are geographically structured, leaving characteristic signatures in neutral regions of the genome. These signatures can be distorted when neutral regions are linked to deleterious mutations. In such regions, purifying selection can reduce genetic diversity through Background Selection (BGS) or, for recessive mutations, increase diversity through Associative Overdominance (AOD). While the effect of BGS and AOD are well characterized in panmictic populations, their effects remain largely unexplored in structured populations. Here, we investigated an Isolation with Migration model using forward simulations across a range of migration, selection, dominance, and recombination parameters. We first used a genotype-based approach to quantify the effects of deleterious mutations on standard summary statistics ({pi}, dxy, FST, DAFi). We then showed that an Ancestral Recombination Graph-based (ARG) approach, tracking tree sequences from a sample of one diploid per deme, recovers the same patterns while directly relating genetic variation to the underlying coalescent processes. When recombination is sufficiently low, we found a BGS-driven regime for weakly codominant mutations, characterized by lower diversity and increased genetic differentiation (FST). For recessive mutations, we first identified an AOD-driven regime, characterized by increased diversity and lower FST values followed by a transition to a subsequent BGS-driven regime. Genealogies were similarly impacted by deleterious mutations: BGS shrunk coalescent times and produced a shift towards lineage sorting topologies, while AOD stretched coalescent times and produced a shift toward incomplete lineage-sorting topologies. These patterns were weakened by gene flow, with FST and topologies remaining close to expected under neutrality, while diversity and coalescence times remained robust to demography. Our results provide clear evidence of BGS, AOD, and of their transition in a structured model with gene flow. Importantly, these processes leave distinct and interpretable signatures on gene trees, highlighting the potential of ARG-based approaches for inferring linked selection and dominance in structured populations. Author summaryCharacterizing how demography and selection jointly shape genomic variation is a central question in population genetics. As deleterious mutations reduce fitness, they are continuously removed from populations by purifying selection. Through linkage, this affects nearby regions of the genome, leaving signatures of selection on linked neutral genetic diversity. While these effects are well understood in random mating populations, much less is known in structured populations. Specifically, the occurrence of Background Selection (BGS), which reduces diversity, and Associative Overdominance (AOD), which increases diversity, remains underexplored. Here, we used simulations to investigate how deleterious mutations shape genomic variation in a structured two-population isolation with migration model. By combining standard population genetic analyses with a genealogical approach based on Ancestral Recombination Graphs (ARGs), we showed that BGS and AOD leave distinct and interpretable signatures on common summary statistics and the underlying genealogies. We identified clear signatures of BGS and AOD when recombination was low and revealed a transition from AOD to BGS for recessive mutations, as the strength of selection increased. Our results highlight the importance of jointly considering demography and linked selection when interpreting genomic data and demonstrate the potential of ARGs to jointly infer demography, selection, and dominance from genomic data.

Computational workshops are common in evolutionary biology and are used to share discipline-specific tools and skills with researchers. Despite the perceived importance of these workshops, there is no common set of criteria for workshop success, and there are few peer-reviewed studies investigating the efficacy of workshops or assessing the value of particular instructional techniques in this context. Here, we focused on one key element of a successful workshop: its ability to increase participants motivation to use the methods and tools presented during the workshop. We analyzed the goals, perceptions, and future plans of research practitioners engaging in a workshop on phylogenetic methods of historical biogeography using pre- and post-workshop surveys. Overall, the workshop was successful at motivating participants, and survey responses provided insights into participants perceptions of different activities, including "participatory live coding". Apart from this case study, we aim to highlight the importance of developing a common set of workshop goals in collaboration with other workshop stakeholders and the need for specialized, validated tools for assessing the efficacy of computational workshops for researchers.

Behaviour is a key yet often overlooked component of animal camouflage and how it evolves alongside colour and morphology remains poorly understood. The repeated evolution of stick-like postures in spiders offers a useful framework for investigating the importance of behaviour for concealment, since matching the environment should rely on specific body forms and postures, not just colouration. We hypothesised that when spiders behaviourally align their body with the background orientation it should influence the shape, posture and colouration that best enhances camouflage. To test this, we used a genetic algorithm and human observers to evolve digital spiders to be harder to find. We evaluated how selection under three behavioural orientation treatments (aligned, random, and evolvable orientation) influenced spider capture time, background match (lightness and colour), posture, and body (cephalothorax and abdomen) dimensions. We found that spiders that behaviourally aligned with the background took substantially longer to find through evolving a better background match, and a more elongated posture and body shape than randomly orientated spiders. Our spiders mirrored the shape and posture adopted by numerous clades, illustrating how behavioural camouflage represents a key concealment strategy in structurally complex habitats, part of an interacting suite of traits that encompass successful concealment.

Sex ratio distorters (SRDs) are heritable elements that bias offspring sex ratios to enhance their transmission. In the terrestrial isopod Armadillidium vulgare, feminization of genetic males can occur through vertical transmission of the sex ratio distorter known as the f-element, as well as through infection by Wolbachia, a maternally inherited bacterial endosymbiont that can alter host reproduction. Previous studies have focused on the distribution of SRDs and their associations with mitochondrial haplotypes in native European populations, but these patterns are poorly understood in the United States. In this study, we sampled A. vulgare in 12 states, screening individuals for Wolbachia infection, the presence of the f-element, and mitochondrial haplotypes. We found that Wolbachia shows a heterogeneous distribution across populations and haplotypes, in contrast with stronger associations in Europe. The f-element occurred in lower overall frequencies but showed a strong association with mitochondrial haplotype VI. These results indicate that patterns associated with SRD differ from those observed in Europe and suggest that multiple introductions and population mixing have shaped these distributions.

O_LITerrestrial insects are vulnerable to desiccation due to their small body size. Because insects lose most water through cuticular evaporation, cuticular traits strongly influence desiccation tolerance. Individuals with greater cuticular melanisation, i.e., darker ones, are hypothesised to tolerate desiccation better than less melanised ones. C_LIO_LIIn many butterflies, pupal colour is plastic - individuals pupating on leaves tend to be greener, while those that pupate away from leaves (off-leaf), such as on tree bark or defoliated twigs, tend to be browner. Brown pupae are hypothesised to have more cuticular melanin and are expected to experience higher desiccation stress than leaf-borne green pupae. Thus, plasticity in pupal melanisation may be an adaptation against desiccation. We tested this in the butterfly Eurema blanda. C_LIO_LIWe demonstrate that individuals pupating on on-leaf substrates are greener than those pupating on off-leaf substrates, and that desiccation stress is higher in the off-leaf substrates, a microenvironment typical of brown pupae, than in typical green pupae. Using Raman spectroscopy, we show that brown, but not green, pupal cuticles contain melanin. C_LIO_LIFollowing this, we obtained greener and browner pupae by manipulating substrate colour. When subjected to desiccation stress, browner pupae survived better than greener ones. There was no correlation between pupal colour and survival in the absence of desiccation stress. Thus, melanisation appears to confer a survival advantage to pupae by increasing desiccation tolerance. C_LIO_LISurvival under desiccating conditions was inversely related to water loss. Interestingly, melanisation did not correlate with water loss, suggesting that melanisation helps tolerate desiccation through physiological mechanisms not directly related to water loss reduction. C_LIO_LIOur findings reveal an additional, crucial, adaptive value of pupal colour plasticity, a trait that has been studied primarily from an anti-predatory perspective. C_LI