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How do neural circuits accommodate changes that produce cognitive variation? We explore this question by analyzing the evolutionary dynamics of an insect learning and memory circuit centered within the mushroom body. Mushroom bodies are composed of a conserved wiring logic, mainly consisting of Kenyon cells, dopaminergic neurons, and mushroom body output neurons. Despite this conserved makeup, there is huge diversity in mushroom body size and shape across insects. However, empirical data on how evolution modifies the function and architecture of this circuit are largely lacking. To address this, we leverage the recent radiation of a Neotropical tribe of butterflies, the Heliconiini (Nymphalidae), which show extensive variation in mushroom body size over comparatively short phylogenetic timescales, linked to specific changes in foraging ecology, life history, and cognition. To understand how such an extensive increase in size is accommodated through changes in lobe circuit architecture, we combined immunostainings of structural markers, neurotransmitters, and neural injections to generate new, quantitative anatomies of the Nymphalid mushroom body lobe. Our comparative analyses across Heliconiini demonstrate that some Kenyon cell sub-populations expanded at higher rates than others in Heliconius and identify an additional increase in GABA-ergic feedback neurons, which are essential for non-elemental learning and sparse coding. Taken together, our results demonstrate mosaic evolution of functionally related neural systems and cell types and identify that evolutionary malleability in an architecturally conserved parallel circuit guides adaptation in cognitive ability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Spatial learning is vital in foraging ecology. Many hymenopteran insects are adept spatial foragers that rely on visual cues contained within broader wide-field scenes for central place foraging from a central nest. By contrast, for butterflies, which lack central nest sites, visual cue use during spatial foraging is less understood. Heliconius butterflies, however, exhibit stable nocturnal roosts, strong site fidelity and a sophisticated capacity for spatial navigation. This study furthers our understanding of Heliconius spatial learning by testing whether H. erato can associate a spatially informative visual cue with artificial feeders. We explored the relative importance of a visual local landmark compared with broader, wide-field visual cues, through experiments with (i) a fixed rewarded feeder with a local landmark; (ii) a mobile rewarded feeder with the landmark as the sole reliable cue; (iii) the same setup while blocking visual access to external landscape features. Our data suggest that Heliconius butterflies learn static feeder locations without relying on a local individual landmark. Instead, we suggest they integrate broader landscape and celestial cues. This suggests that Heliconius butterflies and central place foraging hymenopterans likely share similar visual navigation strategies, using wide-field, low-resolution views rather than focusing on specific individual landmarks., Competing Interests: We declare we have no competing interests., (© 2024 The Author(s).)

The olfactory sense is crucial for organisms, facilitating environmental recognition and interindividual communication. Ithomiini butterflies exemplify this importance not only because they rely strongly on olfactory cues for both inter- and intra-sexual behaviors, but also because they show convergent evolution of specialized structures within the antennal lobe, called macroglomerular complexes (MGCs). These structures, widely absent in butterflies, are present in moths where they enable heightened sensitivity to, and integration of, information from various types of pheromones. In this study, we investigate chemosensory evolution across six Ithomiini species and identify possible links between expression profiles and neuroanatomical. To enable this, we sequenced four new high-quality genome assemblies and six sex-specific antennal transcriptomes for three of these species with different MGC morphologies. With extensive genomic analyses, we found that the expression of antennal transcriptomes across species exhibit profound divergence, and identified highly expressed ORs, which we hypothesize may be associated to MGCs, as highly expressed ORs are absent in Methona, an Ithomiini lineage which also lacks MGCs. More broadly, we show how antennal sexual dimorphism is prevalent in both chemosensory genes and non-chemosensory genes, with possible relevance for behavior. As an example, we show how lipid-related genes exhibit consistent sexual dimorphism, potentially linked to lipid transport or host selection. In this study, we investigate the antennal chemosensory adaptations, suggesting a link between genetic diversity, ecological specialization, and sensory perception with the convergent evolution of MCGs. Insights into chemosensory gene evolution, expression patterns, and potential functional implications enhance our knowledge of sensory adaptations and sexual dimorphisms in butterflies, laying the foundation for future investigations into the genetic drivers of insect behavior, adaptation, and speciation., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Integrating information across sensory modalities enables animals to orchestrate a wide range of complex behaviours. The relative importance placed on one sensory modality over another reflects the reliability of cues in a particular environment and corresponding differences in neural investment. As populations diverge across environmental gradients, the reliability of sensory cues may shift, favouring divergence in neural investment and the weight given to different sensory modalities. During their divergence across closed-forest and forest-edge habitats, closely related butterflies Heliconius cydno and Heliconius melpomene evolved distinct brain morphologies, with the former investing more in vision. Quantitative genetic analyses suggest that selection drove these changes, but their behavioural consequences remain uncertain. We hypothesized that divergent neural investment may alter sensory weighting. We trained individuals in an associative learning experiment using multimodal colour and odour cues. When positively rewarded stimuli were presented in conflict, i.e. pairing positively trained colour with negatively trained odour and vice versa , H. cydno favoured visual cues more strongly than H. melpomene . Hence, differences in sensory weighting may evolve early during divergence and are predicted by patterns of neural investment. These findings, alongside other examples, imply that differences in sensory weighting stem from divergent investment as adaptations to local sensory environments.

Introduction: Advance care planning (ACP), a critical component of quality dementia care, is underutilized due to lack of clinician comfort and the challenging nature of ACP in this context. The Serious Illness Conversation Guide (SICG) is a well-validated clinician-facing tool, developed with patient and clinician input, to facilitate ACP. The aim of this project was to adapt the SICG for dementia for the first time to promote high-quality ACP. Methods: This study uses a mixed-methods approach to adapt the SICG tool for use in dementia care. Experts with relevant clinical, ethical, and topical knowledge were interviewed to develop alterations to the SICG for dementia care. Patients and caregivers were shown a mock interview of the adapted SICG for dementia (SICG-D) to elicit feedback. Results: 8 relevant experts were interviewed. Adaptations included topical alterations to make the conversation more applicable to dementia as well as alterations to the structure of the conversation to accommodate the patient-caregiver dyad. Twenty interviews were conducted with 14 patients and 18 caregivers (either together or separately). A thematic content analysis of interview transcripts demonstrated positive impressions of the tool. In anonymous survey results, 94% reported a positive impression of the conversation and 89% endorsed incorporation of the adapted guide into dementia healthcare. Conclusion: This paper presents the SICG-D, an adapted version of the SICG for use in dementia care. This guide leverages the strengths of the SICG to promote values-based ACP conversations and has been adapted to better facilitate patient-caregiver-clinician triadic communication., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

When populations experience different sensory conditions, natural selection may favor sensory system divergence, affecting peripheral structures and/or downstream neural pathways. We characterized the outer eye morphology of sympatric Heliconius butterflies from different forest types and their first-generation reciprocal hybrids to test for adaptive visual system divergence and hybrid disruption. In Panama, Heliconius cydno occurs in closed forests, whereas Heliconius melpomene resides at the forest edge. Among wild individuals, H. cydno has larger eyes than H. melpomene, and there are heritable, habitat-associated differences in the visual brain structures that exceed neutral divergence expectations. Notably, hybrids have intermediate neural phenotypes, suggesting disruption. To test for similar effects in the visual periphery, we reared both species and their hybrids in common garden conditions. We confirm that H. cydno has larger eyes and provide new evidence that this is driven by selection. Hybrid eye morphology is more H. melpomene-like despite body size being intermediate, contrasting with neural trait intermediacy. Overall, our results suggest that eye morphology differences between H. cydno and H. melpomene are adaptive and that hybrids may suffer fitness costs due to a mismatch between the peripheral visual structures and previously described neural traits that could affect visual performance., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Neural circuits govern the interface between the external environment, internal cues and outwardly directed behaviours. To process multiple environmental stimuli and integrate these with internal state requires considerable neural computation. Expansion in neural network size, most readily represented by whole brain size, has historically been linked to behavioural complexity, or the predominance of cognitive behaviours. Yet, it is largely unclear which aspects of circuit variation impact variation in performance. A key question in the field of evolutionary neurobiology is therefore how neural circuits evolve to allow improved behavioural performance or innovation. We discuss this question by first exploring how volumetric changes in brain areas reflect actual neural circuit change. We explore three major axes of neural circuit evolution-replication, restructuring and reconditioning of cells and circuits-and discuss how these could relate to broader phenotypes and behavioural variation. This discussion touches on the relevant uses and limitations of volumetrics, while advocating a more circuit-based view of cognition. We then use this framework to showcase an example from the insect brain, the multi-sensory integration and internal processing that is shared between the mushroom bodies and central complex. We end by identifying future trends in this research area, which promise to advance the field of evolutionary neurobiology.

Larval Lepidoptera gain survival advantages by aggregating, especially when combined with aposematic warning signals, yet reductions in predation risk may not be experienced equally across all group members. Hamilton's selfish herd theory predicts that larvae that surround themselves with their group mates should be at lower risk of predation, and those on the periphery of aggregations experience the greatest risk, yet this has rarely been tested. Here, we expose aggregations of artificial 'caterpillar' targets to predation from free-flying, wild birds to test for marginal predation when all prey are equally accessible and for an interaction between warning coloration and marginal predation. We find that targets nearer the centre of the aggregation survived better than peripheral targets and nearby targets isolated from the group. However, there was no difference in survival between peripheral and isolated targets. We also find that grouped targets survived better than isolated targets when both are aposematic, but not when they are non-signalling. To our knowledge, our data provide the first evidence to suggest that avian predators preferentially target peripheral larvae from aggregations and that prey warning signals enhance predator avoidance of groups.

Insect herbivores, such as lepidopteran larvae, often have close evolutionary relationships with their host plants, with which they may be locked in an evolutionary arms race. Larval grouping behaviour may be one behavioural adaptation that improves host plant feeding, but aggregation also comes with costs, such as higher competition and limited resource access. Here, we use the Heliconiini butterfly tribe to explore the impact of host plant traits on the evolution of larval gregariousness. Heliconiini almost exclusively utilise species from the Passifloraceae as larval host plants. Passifloraceae display incredible diversity in leaf shape and a range of anti-herbivore defences, suggesting they are responding to, and influencing, the evolution of Heliconiini larvae. By analysing larval social behaviour as both a binary (solitary or gregarious) and categorical (increasing larval group size) trait, we revisit the multiple origins of larval gregariousness across Heliconiini. We investigate whether host habitat, leaf defences and leaf size are important drivers of, or constraints on, larval gregariousness. Whereas our data do not reveal links between larval gregariousness and the host plant traits included in this study, we do find an interaction between host plant specialisation and larval behaviour, revealing gregarious larvae to be more likely to feed on a narrower range of host plant species than solitary larvae. We also find evidence that this increased specialisation typically precedes the evolutionary transition to gregarious behaviour. The comparatively greater host specialisation of gregarious larvae suggests that there are specific morphological and/or ecological features of their host plants that favour this behaviour., Competing Interests: The authors declare no competing interests., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Vertical gradients in microclimate, resource availability, and interspecific interactions are thought to underly stratification patterns in tropical insect communities. However, only a few studies have explored the adaptive significance of vertical space use during the early stages of reproductive isolation. We analysed flight-height variation across speciation events in Heliconius butterflies, representing parallel colonizations of high-altitude forest. We measured flight-height in wild H. erato venus and H. chestertonii, parapatric lowland and mountain specialists, respectively, and found that H. chestertonii consistently flies at a lower height. By comparing our data to previously published results for the ecologically equivalent H. e. cyrbia (lowland) and H. himera (high altitude), we found that the species flying closest to the ground are those that recently colonized high-altitude forests. We show that these repeated trends largely result from shared patterns of ecological selection producing parallel trait-shifts in H. himera and H. chestertonii. Although our results imply a signature of local adaptation, we did not find an association between resource distribution and flight-height in H. e. venus and H. chestertonii. We discuss how this pattern may be explained by variations in forest structure and microclimate. Overall, our findings underscore the importance of behavioural adjustments during early divergence mediated by altitude-shifts., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology.)

Heliconius butterflies exhibit expanded mushroom bodies, a key brain region for learning and memory in insects, and a novel foraging strategy unique among Lepidoptera - traplining for pollen. We tested visual long-term memory across six Heliconius and outgroup Heliconiini species. Heliconius species exhibited greater fidelity to learned colors after eight days without reinforcement, with further evidence of recall at 13 days. We also measured the plastic response of the mushroom body calyces over this time period, finding substantial post-eclosion expansion and synaptic pruning in the calyx of Heliconius erato , but not in the outgroup Heliconiini Dryas iulia . In Heliconius erato , visual associative learning experience specifically was associated with a greater retention of synapses and recall accuracy was positively correlated with synapse number. These results suggest that increases in the size of specific brain regions and changes in their plastic response to experience may coevolve to support novel behaviors., Competing Interests: The authors declare no competing interest., (© 2024 The Authors.)

In this work, we explore the potential influence of sensory ecology on speciation, including but not limited to the concept of sensory drive, which concerns the coevolution of signals and sensory systems with the local environment. The sensory environment can influence individual fitness in a variety of ways, thereby affecting the evolution of both pre- and postmating reproductive isolation. Previous work focused on sensory drive has undoubtedly advanced the field, but we argue that it may have also narrowed our understanding of the broader influence of the sensory ecology on speciation. Moreover, the clearest examples of sensory drive are largely limited to aquatic organisms, which may skew the influence of contributing factors. We review the evidence for sensory drive across environmental conditions, and in this context discuss the importance of more generalized effects of sensory ecology on adaptive behavioral divergence. Finally, we consider the potential of rapid environmental change to influence reproductive barriers related to sensory ecologies. Our synthesis shows the importance of sensory conditions for local adaptation and divergence in a range of behavioral contexts and extends our understanding of the interplay between sensory ecology and speciation., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

The striking structural variation seen in arthropod visual systems can be explained by the overall quantity and spatio-temporal structure of light within habitats coupled with developmental and physiological constraints. However, little is currently known about how fine-scale variation in visual structures arises across shorter evolutionary and ecological scales. In this study, we characterise patterns of interspecific (between species), intraspecific (between sexes) and intraindividual (between eye regions) variation in the visual system of four ithomiine butterfly species. These species are part of a diverse 26-million-year-old Neotropical radiation where changes in mimetic colouration are associated with fine-scale shifts in ecology, such as microhabitat preference. Using a combination of selection analyses on visual opsin sequences, in vivo ophthalmoscopy, micro-computed tomography (micro-CT), immunohistochemistry, confocal microscopy and neural tracing, we quantify and describe physiological, anatomical and molecular traits involved in visual processing. Using these data, we provide evidence of substantial variation within the visual systems of Ithomiini, including: (i) relaxed selection on visual opsins, perhaps mediated by habitat preference, (ii) interspecific shifts in visual system physiology and anatomy, and (iii) extensive sexual dimorphism, including the complete absence of a butterfly-specific optic neuropil in the males of some species. We conclude that considerable visual system variation can exist within diverse insect radiations, hinting at the evolutionary lability of these systems to rapidly develop specialisations to distinct visual ecologies, with selection acting at the perceptual, processing and molecular level., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

In evolutionary terms, life is about reproduction. Yet, in some species, individuals forgo their own reproduction to support the reproductive efforts of others. Social insect colonies for example, can contain up to a million workers that actively cooperate in tasks such as foraging, brood care and nest defence, but do not produce offspring. In such societies the division of labour is pronounced, and reproduction is restricted to just one or a few individuals, most notably the queen(s). This extreme eusocial organisation exists in only a few mammals, crustaceans and insects, but strikingly, it evolved independently up to nine times in the order Hymenoptera (including ants, bees and wasps). Transitions from a solitary lifestyle to an organised society can occur through natural selection when helpers obtain a fitness benefit from cooperating with kin, owing to the indirect transmission of genes through siblings. However, this process, called kin selection, is vulnerable to parasitism and opportunistic behaviours from unrelated individuals. An ability to distinguish kin from non-kin, and to respond accordingly, could therefore critically facilitate the evolution of eusociality and the maintenance of non-reproductive workers. The question of how the hymenopteran brain has adapted to support this function is therefore a fundamental issue in evolutionary neuroethology. Early neuroanatomical investigations proposed that social Hymenoptera have expanded integrative brain areas due to selection for increased cognitive capabilities in the context of processing social information. Later studies challenged this assumption and instead pointed to an intimate link between higher social organisation and the existence of developed sensory structures involved in recognition and communication. In particular, chemical signalling of social identity, known to be mediated through cuticular hydrocarbons (CHCs), may have evolved hand in hand with a specialised chemosensory system in Hymenoptera. Here, we compile the current knowledge on this recognition system, from emitted identity signals, to the molecular and neuronal basis of chemical detection, with particular emphasis on its evolutionary history. Finally, we ask whether the evolution of social behaviour in Hymenoptera could have driven the expansion of their complex olfactory system, or whether the early origin and conservation of an olfactory subsystem dedicated to social recognition could explain the abundance of eusocial species in this insect order. Answering this question will require further comparative studies to provide a comprehensive view on lineage-specific adaptations in the olfactory pathway of Hymenoptera., (© 2023 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Among butterflies, Heliconius have a unique behavioural profile, being the sole genus to actively feed on pollen. Heliconius learn the location of pollen resources, and have enhanced visual memories and expanded mushroom bodies, an insect learning and memory centre, relative to related genera. These structures also show extensive post-eclosion growth and developmental sensitivity to environmental conditions. However, whether this reflects plasticity in neurite growth, or an extension of neurogenesis into the adult stage, is unknown. Adult neurogenesis has been described in some Lepidoptera, and could provide one route to the increased neuron number observed in Heliconius. Here, we compare volumetric changes in the mushroom bodies of freshly eclosed and aged Heliconius erato and Dryas iulia , and estimate the number of intrinsic mushroom body neurons using a new and validated automated method to count nuclei. Despite extensive volumetric variation associated with age, our data show that neuron number is remarkably constant in both species, suggesting a lack of adult neurogenesis in the mushroom bodies. We support this conclusion with assays of mitotic cells, which reveal very low levels of post-eclosion cell division. Our analyses provide an insight into the evolution of neural plasticity, and can serve as a basis for continued exploration of the potential mechanisms behind brain development and maturation., Competing Interests: We have no competing interests., (© 2023 The Authors.)

The study of neurogenesis is critical to understanding of the evolution of nervous systems. Within invertebrates, this process has been extensively studied in Drosophila melanogaster, which is the predominant model thanks to the availability of advanced genetic tools. However, insect nervous systems are extremely diverse, and by studying a range of taxa we can gain additional information about how nervous systems and their development evolve. One example of the high diversity of insect nervous system diversity is provided by the mushroom bodies. Mushroom bodies have critical roles in learning and memory and vary dramatically across species in relative size and the type(s) of sensory information they process. Heliconiini butterflies provide a useful snapshot of this diversity within a closely related clade. Within Heliconiini, the genus Heliconius contains species where mushroom bodies are 3-4 times larger than other closely related genera, relative to the rest of the brain. This variation in size is largely explained by increases in the number of Kenyon cells, the intrinsic neurons which form the mushroom body. Hence, variation in mushroom body size is the product of changes in cell proliferation during Kenyon cell neurogenesis. Studying this variation requires adapting labelling techniques for use in less commonly studied organisms, as methods developed for common laboratory insects often do not work. Here, we present a modified protocol for EdU staining to examine neurogenesis in large-brained insects, using Heliconiini butterflies as our primary case, but also demonstrating applicability to cockroaches, another large-brained insect., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Anton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Heliconius butterflies, a speciose genus of Müllerian mimics, represent a classic example of an adaptive radiation that includes a range of derived dietary, life history, physiological and neural traits. However, key lineages within the genus, and across the broader Heliconiini tribe, lack genomic resources, limiting our understanding of how adaptive and neutral processes shaped genome evolution during their radiation. Here, we generate highly contiguous genome assemblies for nine Heliconiini, 29 additional reference-assembled genomes, and improve 10 existing assemblies. Altogether, we provide a dataset of annotated genomes for a total of 63 species, including 58 species within the Heliconiini tribe. We use this extensive dataset to generate a robust and dated heliconiine phylogeny, describe major patterns of introgression, explore the evolution of genome architecture, and the genomic basis of key innovations in this enigmatic group, including an assessment of the evolution of putative regulatory regions at the Heliconius stem. Our work illustrates how the increased resolution provided by such dense genomic sampling improves our power to generate and test gene-phenotype hypotheses, and precisely characterize how genomes evolve., (© 2023. Springer Nature Limited.)

Endangered species have small, unsustainable population sizes that are geographically or genetically restricted. Ex-situ conservation programmes are therefore faced with the challenge of breeding sufficiently sized, genetically diverse populations earmarked for reintroduction that have the behavioural skills to survive and breed in the wild. Yet, maintaining historically beneficial behaviours may be insufficient, as research continues to suggest that certain cognitive-behavioural skills and flexibility are necessary to cope with human-induced rapid environmental change (HIREC). This paper begins by reviewing interdisciplinary studies on the 'captivity effect' in laboratory, farmed, domesticated and feral vertebrates and finds that captivity imposes rapid yet often reversible changes to the brain, cognition and behaviour. However, research on this effect in ex-situ conservation sites is lacking. This paper reveals an apparent mismatch between ex-situ enrichment aims and the cognitive-behavioural skills possessed by animals currently coping with HIREC. After synthesizing literature across neuroscience, behavioural biology, comparative cognition and field conservation, it seems that ex-situ endangered species deemed for reintroduction may have better chances of coping with HIREC if their natural cognition and behavioural repertoires are actively preserved. Evaluating the effects of environmental challenges rather than captivity per se is recommended, in addition to using targeted cognitive enrichment., Competing Interests: We have no competing interests., (© 2023 The Authors.)

Locating food in heterogeneous environments is a core survival challenge. The distribution of resources shapes foraging strategies, imposing demands on perception, learning and memory, and associated brain structures. Indeed, selection for foraging efficiency is linked to brain expansion in diverse taxa, from primates 1 to Hymenopterans 2 . Among butterflies, Heliconius have a unique dietary adaptation, actively collecting and feeding on pollen, providing a source of essential amino acids as adults, negating reproductive senescence and facilitating an extended longevity 3 . Several lines of evidence suggest that Heliconius learn the spatial location of pollen resources within an individual's home range 4 , and spatial learning may be more pronounced at these large spatial scales. However, experimental evidence of spatial learning in Heliconius, or any other butterfly, is so far absent. We therefore tested the ability of Heliconius to learn the spatial location of food rewards at three ecologically-relevant spatial scales, representing multiple flowers on a single plant, multiple plants within a locality, and multiple localities. Heliconius were able to learn spatial information at all three scales, consistent with this ability being an important component of their natural foraging behaviour., Competing Interests: Author contributions All authors contributed to the experimental design. P.A.M. and F.J.Y. conducted the experiments and analyzed the data, under the supervision of M.Z.C. and S.H.M., and contributed equally. P.A.M., F.J.Y. and S.H.M. wrote the draft manuscript and all authors edited and approved the final version. Declaration of interests The authors declare no competing interests, (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Changes in ecological preference, often driven by spatial and temporal variation in resource distribution, can expose populations to environments with divergent information content. This can lead to adaptive changes in the degree to which individuals invest in sensory systems and downstream processes, to optimize behavioural performance in different contexts. At the same time, environmental conditions can produce plastic responses in nervous system development and maturation, providing an alternative route to integrating neural and ecological variation. Here, we explore how these two processes play out across a community of Heliconius butterflies. Heliconius communities exhibit multiple Mullerian mimicry rings, associated with habitat partitioning across environmental gradients. These environmental differences have previously been linked to heritable divergence in brain morphology in parapatric species pairs. They also exhibit a unique dietary adaptation, known as pollen feeding, that relies heavily on learning foraging routes, or trap-lines, between resources, which implies an important environmental influence on behavioural development. By comparing brain morphology across 133 wild-caught and insectary-reared individuals from seven Heliconius species, we find strong evidence for interspecific variation in patterns of neural investment. These largely fall into two distinct patterns of variation; first, we find consistent patterns of divergence in the size of visual brain components across both wild and insectary-reared individuals, suggesting genetically encoded divergence in the visual pathway. Second, we find interspecific differences in mushroom body size, a central component of learning and memory systems, but only among wild caught individuals. The lack of this effect in common-garden individuals suggests an extensive role for developmental plasticity in interspecific variation in the wild. Finally, we illustrate the impact of relatively small-scale spatial effects on mushroom body plasticity by performing experiments altering the cage size and structure experienced by individual H. hecale. Our data provide a comprehensive survey of community level variation in brain structure, and demonstrate that genetic effects and developmental plasticity contribute to different axes of interspecific neural variation., (© 2023 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

The selective pressures leading to the elaboration of downstream, integrative processing centres, such as the mammalian neocortex or insect mushroom bodies, are often unclear. In Heliconius butterflies, the mushroom bodies are two to four times larger than those of their Heliconiini relatives, and the largest known in Lepidoptera. Heliconiini lay almost exclusively on Passiflora , which exhibit a remarkable diversity of leaf shape, and it has been suggested that the mushroom body expansion of Heliconius may have been driven by the cognitive demands of recognizing and learning leaf shapes of local host plants. We test this hypothesis using two complementary methods: (i) phylogenetic comparative analyses to test whether variation in mushroom body size is associated with the morphological diversity of host plants exploited across the Heliconiini; and (ii) shape-learning experiments using six Heliconiini species. We found that variation in the range of leaf morphologies used by Heliconiini was not associated with mushroom body volume. Similarly, we find interspecific differences in shape-learning ability, but Heliconius are not overall better shape learners than other Heliconiini. Together these results suggest that the visual recognition and learning of host plants was not a main factor driving the diversity of mushroom body size in this tribe.

Changes in the abundance and diversity of neural cell types, and their connectivity, shape brain composition and provide the substrate for behavioral evolution. Although investment in sensory brain regions is understood to be largely driven by the relative ecological importance of particular sensory modalities, how selective pressures impact the elaboration of integrative brain centers has been more difficult to pinpoint. Here, we provide evidence of extensive, mosaic expansion of an integration brain center among closely related species, which is not explained by changes in sites of primary sensory input. By building new datasets of neural traits among a tribe of diverse Neotropical butterflies, the Heliconiini, we detected several major evolutionary expansions of the mushroom bodies, central brain structures pivotal for insect learning and memory. The genus Heliconius, which exhibits a unique dietary innovation, pollen-feeding, and derived foraging behaviors reliant on spatial memory, shows the most extreme enlargement. This expansion is primarily associated with increased visual processing areas and coincides with increased precision of visual processing, and enhanced long term memory. These results demonstrate that selection for behavioral innovation and enhanced cognitive ability occurred through expansion and localized specialization in integrative brain centers., (© 2023. The Author(s).)

AbstractMany species gain antipredator benefits by combining gregarious behavior with warning coloration, yet there is debate over which trait evolves first and which is the secondary adaptive enhancement. Body size can also influence how predators receive aposematic signals and potentially constrain the evolution of gregarious behavior. To our knowledge, the causative links between the evolution of gregariousness, aposematism, and larger body sizes have not been fully resolved. Here, using the most recently resolved butterfly phylogeny and an extensive new dataset of larval traits, we reveal the evolutionary interactions between important traits linked to larval gregariousness. We show that larval gregariousness has arisen many times across butterflies, and aposematism is a likely prerequisite for gregariousness to evolve. We also find that body size may be an important factor for determining the coloration of solitary, but not gregarious, larvae. Additionally, by exposing artificial larvae to wild avian predation, we show that undefended, cryptic larvae are heavily predated when aggregated but benefit from solitariness, whereas the reverse is true for aposematic prey. Our data reinforce the importance of aposematism for gregarious larval survival while identifying new questions about the roles of body size and toxicity in the evolution of grouping behavior.

Prey animals typically try to avoid being detected and/or advertise to would-be predators that they should be avoided. Both anti-predator strategies primarily rely on colour to succeed, but the specific patterning used is also important. While the role of patterning in camouflage is relatively clear, the design features of aposematic patterns are less well understood. Here, we use a comparative approach to investigate how pattern use varies across a phylogeny of 268 species of cryptic and aposematic butterfly larvae, which also vary in social behaviour. We find that longitudinal stripes are used more frequently by cryptic larvae, and that patterns putatively linked to crypsis are more likely to be used by solitary larvae. By contrast, aposematic larvae are more likely to use horizontal bands and spots, but we find no differences in the use of individual pattern elements between solitary and gregarious aposematic species. However, solitary aposematic larvae are more likely to display multiple pattern elements, whereas those with no pattern are more likely to be gregarious. Our study advances our understanding of how pattern variation, coloration and social behaviour covary across lepidopteran larvae, and highlights new questions about how patterning affects larval detectability and predator responses to aposematic prey.

Parallel evolution of morphological traits is widely reported, providing evidence for the role of local conditions in driving adaptive divergence. Comparatively, fewer studies have tested for parallelism in behavior, and it is less clear to what extent heritable behavioral shifts contribute to adaptive divergence. We exploit repeated incipient speciation across altitudinal gradients to explore behavior and physiology in Heliconius butterflies adapted to high-elevation. We performed common garden experiments with H. chestertonii, a high-altitude specialist from the Colombian Cordillera Occidental, and H. erato venus, a low-elevation proxy for the ancestral population, and compared our results to existing data for an equivalent Ecuadorian taxa-pair. Using broad-scale climatic data, we show that both pairs diverge across similar ecological gradients, confirmed using localized data loggers in the ranges of H. chestertonii and H. e. venus. We further show that H. chestertonii and H. e. venus have divergent activity patterns, attributable to different responses to microclimate, and life histories. Finally, we provide evidence for parallelism in these traits with H. himera and H. e. cyrbia. We propose that this is a result of selection associated with independent colonizations of high-altitude forests, emphasizing the importance of heritable behavioral and physiological adaptations during population divergence and speciation., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)

Insects may acquire social information by active communication and through inadvertent social cues. In a foraging setting, the latter may indicate the presence and quality of resources. Although social learning in foraging contexts is prevalent in eusocial species, this behaviour has been hypothesized to also exist between conspecifics in non-social species with sophisticated behaviours, including Heliconius butterflies. Heliconius are the only butterfly genus with active pollen feeding, a dietary innovation associated with a specialized, spatially faithful foraging behaviour known as trap-lining. Long-standing hypotheses suggest that Heliconius may acquire trap-line information by following experienced individuals. Indeed, Heliconius often aggregate in social roosts, which could act as 'information centres', and present conspecific following behaviour, enhancing opportunities for social learning. Here, we provide a direct test of social learning ability in Heliconius using an associative learning task in which naive individuals completed a colour preference test in the presence of demonstrators trained to feed randomly or with a strong colour preference. We found no evidence that Heliconius erato , which roost socially, used social information in this task. Combined with existing field studies, our results add to data which contradict the hypothesized role of social learning in Heliconius foraging behaviour.

To evolve, and remain adaptive, collective behaviours must have a positive impact on overall individual fitness. However, these adaptive benefits may not be immediately apparent owing to an array of interactions with other ecological traits, which can depend on a lineage's evolutionary past and the mechanisms controlling group behaviour. A coherent understanding of how these behaviours evolve, are exhibited, and are coordinated across individuals, therefore requires an integrative approach spanning traditional disciplines in behavioural biology. Here, we argue that lepidopteran larvae are well placed to serve as study systems for investigating the integrative biology of collective behaviour. Lepidopteran larvae display a striking diversity in social behaviour, which illustrates critical interactions between ecological, morphological and behavioural traits. While previous, often classic, work has provided an understanding of how and why collective behaviours evolve in Lepidoptera, much less is known about the developmental and mechanistic basis of these traits. Recent advances in the quantification of behaviour, and the availability of genomic resources and manipulative tools, allied with the exploitation of the behavioural diversity of tractable lepidopteran clades, will change this. In doing so, we will be able to address previously intractable questions that can reveal the interplay between levels of biological variation. This article is part of a discussion meeting issue 'Collective behaviour through time'.

Bottlenose dolphins are highly social, renowned for their vocal flexibility, and possess highly enlarged brains relative to their body size. Here, we discuss some of the defining features of bottlenose dolphin social and vocal complexity and place this in the context of their cognitive evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite insertions and deletions being the most common structural variants (SVs) found across genomes, not much is known about how much these SVs vary within populations and between closely related species, nor their significance in evolution. To address these questions, we characterized the evolution of indel SVs using genome assemblies of three closely related Heliconius butterfly species. Over the relatively short evolutionary timescales investigated, up to 18.0% of the genome was composed of indels between two haplotypes of an individual Heliconius charithonia butterfly and up to 62.7% included lineage-specific SVs between the genomes of the most distant species (11 Mya). Lineage-specific sequences were mostly characterized as transposable elements (TEs) inserted at random throughout the genome and their overall distribution was similarly affected by linked selection as single nucleotide substitutions. Using chromatin accessibility profiles (i.e., ATAC-seq) of head tissue in caterpillars to identify sequences with potential cis -regulatory function, we found that out of the 31,066 identified differences in chromatin accessibility between species, 30.4% were within lineage-specific SVs and 9.4% were characterized as TE insertions. These TE insertions were localized closer to gene transcription start sites than expected at random and were enriched for sites with significant resemblance to several transcription factor binding sites with known function in neuron development in Drosophila We also identified 24 TE insertions with head-specific chromatin accessibility. Our results show high rates of structural genome evolution that were previously overlooked in comparative genomic studies and suggest a high potential for structural variation to serve as raw material for adaptive evolution., (© 2022 Ruggieri et al.; Published by Cold Spring Harbor Laboratory Press.)

Repeated evolution can provide insight into the mechanisms that facilitate adaptation to novel or changing environments. Here we study adaptation to altitude in two tropical butterflies, Heliconius erato and H. melpomene, which have repeatedly and independently adapted to montane habitats on either side of the Andes. We sequenced 518 whole genomes from altitudinal transects and found many regions differentiated between highland (~ 1200 m) and lowland (~ 200 m) populations. We show repeated genetic differentiation across replicate populations within species, including allopatric comparisons. In contrast, there is little molecular parallelism between the two species. By sampling five close relatives, we find that a large proportion of divergent regions identified within species have arisen from standing variation and putative adaptive introgression from high-altitude specialist species. Taken together our study supports a role for both standing genetic variation and gene flow from independently adapted species in promoting parallel local adaptation to the environment., (© 2022. The Author(s).)

Microhabitat partitioning in heterogenous environments can support more diverse communities but may expose partitioned species to distinct perceptual challenges. Divergence across microhabitats could therefore lead to local adaptation to contrasting sensory conditions across small spatial scales, but this aspect of community structuring is rarely explored. Diverse communities of ithomiine butterflies provide an example where closely related species partition tropical forests, where shifts in mimetic coloration are tightly associated with shifts in habitat preference. We test the hypothesis that these mimetic and ecological shifts are associated with distinct patterns of sensory neural investment by comparing brain structure across 164 individuals of 16 species from three ithomiine clades. We find distinct brain morphologies between Oleriina and Hypothyris, which are mimetically homogenous and occupy a single microhabitat. Oleriina, which occurs in low-light microhabitats, invests less in visual brain regions than Hypothyris, with one notable exception, Hyposcada anchiala, the only Oleriina sampled to have converged on mimicry rings found in Hypothyris. We also find that Napeogenes, which has diversified into a range of mimicry rings, shows intermediate patterns of sensory investment. We identify flight height as a critical factor shaping neuroanatomical diversity, with species that fly higher in the canopy investing more in visual structures. Our work suggests that the sensory ecology of species may be impacted by, and interact with, the ways in which communities of closely related organisms are adaptively assembled., (© 2022 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.)

For many animals, the availability and provision of dietary resources can vary markedly between juvenile and adult stages, often leading to a temporal separation of nutrient acquisition and use. Juvenile developmental programs are likely limited by the energetic demands of many adult tissues and processes with early developmental origins. Enhanced dietary quality in the adult stage may, therefore, alter selection on life history and growth patterns in juvenile stages. Heliconius are unique among butterflies in actively collecting and digesting pollen grains, which provide an adult source of essential amino acids. The origin of pollen feeding has therefore previously been hypothesized to lift constraints on larval growth rates, allowing Heliconius to spend less time as larvae when they are most vulnerable to predation. By measuring larval and pupal life-history traits across three pollen-feeding and three nonpollen-feeding Heliconiini, we provide the first test of this hypothesis. Although we detect significant interspecific variation in larval and pupal development, we do not find any consistent shift associated with pollen feeding. We discuss how this result may fit with patterns of nitrogen allocation, the benefits of nitrogenous stores, and developmental limitations on growth. Our results provide a framework for studies aiming to link innovations in adult Heliconius to altered selection regimes and developmental programs in early life stages., Competing Interests: We declare no conflicts of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Microhabitat choice plays a major role in shaping local patterns of biodiversity. In butterflies, stratification in flight height has an important role in maintaining community diversity. Despite its presumed importance, the role of behavioural shifts in early stages of speciation in response to differences in habitat structure is yet to be established. Here, we investigated variation in flight height behaviour in two closely related Heliconius species, H. erato cyrbia and H. himera, which produce viable hybrids but are isolated across an environmental gradient, spanning lowland wet forest to high-altitude scrub forest. Speciation in this pair is associated with strong assortative mating, but ecological isolation and local adaptation are also considered essential for complete reproductive isolation. We quantified differences in flight height and forest structure across the environmental gradient and tested the importance of resource distribution in explaining flight behaviour. We then used common garden experiments to test whether differences in flight height reflect individual responses to resource distribution or genetically determined shifts in foraging behaviour. We found that the two species fly at different heights in the wild, and demonstrated that this can be explained by differences in the vertical distribution of plant resources. In both the wild and captivity, H. himera choose to fly lower and feed at lower positions, closely mirroring differences in resource availability in the wild. Given expectations that foraging efficiency contributes to survival and reproductive success, we suggest that foraging behaviour may reflect local adaptation to divergent forest structures. Our results highlight the potential role of habitat-dependent divergence in behaviour during the early stages of speciation., (© 2022 British Ecological Society.)

How an organism's sensory system functions is central to how it navigates its environment. The insect olfactory system is a prominent model for investigating how ecological factors impact sensory reception and processing. Notably, work in Lepidoptera led to the discovery of vastly expanded structures, termed macroglomerular complexes (MGCs), within the primary olfactory processing centre. MGCs typically process pheromonal cues, are usually larger in males, and provide classic examples of how variation in the size of neural structures reflects the importance of sensory cues. Though prevalent across moths, MGCs were lost during the origin of butterflies, consistent with evidence that courtship initiation in butterflies is primarily reliant on visual cues, rather than long distance chemical signals. However, an MGC was recently described in a species of ithomiine butterfly, suggesting that this once lost neural adaptation has re-emerged in this tribe. Here, we show that MGC-like morphologies are widely distributed across ithomiines, but vary in both their structure and prevalence of sexual dimorphism. Based on this interspecific variation we suggest that the ithomiine MGC is involved in processing both plant and pheromonal cues, which have similarities in their chemical constitution, and co-evolved with an increased importance of plant derived chemical compounds., (© 2021 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.)

Major changes in chromosome number and structure are linked to a series of evolutionary phenomena, including intrinsic barriers to gene flow or suppression of recombination due to chromosomal rearrangements. However, chromosome rearrangements can also affect the fundamental dynamics of molecular evolution within populations by changing relationships between linked loci and altering rates of recombination. Here, we build chromosome-level assembly Eueides isabella and, together with a recent chromosome-level assembly of Dryas iulia, examine the evolutionary consequences of multiple chromosome fusions in Heliconius butterflies. These assemblies pinpoint fusion points on 10 of the 20 autosomal chromosomes and reveal striking differences in the characteristics of fused and unfused chromosomes. The ten smallest autosomes in D. iulia and E. isabella, which have each fused to a longer chromosome in Heliconius, have higher repeat and GC content, and longer introns than predicted by their chromosome length. When fused, these characteristics change to become more in line with chromosome length. The fusions also led to reduced diversity, which likely reflects increased background selection and selection against introgression between diverging populations, following a reduction in per-base recombination rate. We further show that chromosome size and fusion impact turnover rates of functional loci at a macroevolutionary scale. Together these results provide further evidence that chromosome fusion in Heliconius likely had dramatic effects on population level processes shaping rates of neutral and adaptive divergence. These effects may have impacted patterns of diversification in Heliconius, a classic example of an adaptive radiation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In butterflies and moths, which exhibit highly variable sex determination mechanisms, the homogametic Z chromosome is deeply conserved and is featured in many genome assemblies. The evolution and origin of the female W sex chromosome, however, remains mostly unknown. Previous studies have proposed that a ZZ/Z0 sex determination system is ancestral to Lepidoptera, and that W chromosomes may originate from sex-linked B chromosomes. Here, we sequence and assemble the female Dryas iulia genome into 32 highly contiguous ordered and oriented chromosomes, including the Z and W sex chromosomes. We then use sex-specific Hi-C, ATAC-seq, PRO-seq, and whole-genome DNA sequence data sets to test if features of the D. iulia W chromosome are consistent with a hypothesized B chromosome origin. We show that the putative W chromosome displays female-associated DNA sequence, gene expression, and chromatin accessibility to confirm the sex-linked function of the W sequence. In contrast with expectations from studies of homologous sex chromosomes, highly repetitive DNA content on the W chromosome, the sole presence of domesticated repetitive elements in functional DNA, and lack of sequence homology with the Z chromosome or autosomes is most consistent with a B chromosome origin for the W, although it remains challenging to rule out extensive sequence divergence. Synteny analysis of the D. iulia W chromosome with other female lepidopteran genome assemblies shows no homology between W chromosomes and suggests multiple, independent origins of the W chromosome from a B chromosome likely occurred in butterflies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Vertebrate brain structure is characterised not only by relative consistency in scaling between components, but also by many examples of divergence from these general trends.. Alternative hypotheses explain these patterns by emphasising either 'external' processes, such as coordinated or divergent selection, or 'internal' processes, like developmental coupling among brain regions. Although these hypotheses are not mutually exclusive, there is little agreement over their relative importance across time or how that importance may vary across evolutionary contexts., Results: We introduce an agent-based model to simulate brain evolution in a 'bare-bones' system and examine dependencies between variables shaping brain evolution. We show that 'concerted' patterns of brain evolution do not, in themselves, provide evidence for developmental coupling, despite these terms often being treated as synonymous in the literature. Instead, concerted evolution can reflect either functional or developmental integration. Our model further allows us to clarify conditions under which such developmental coupling, or uncoupling, is potentially adaptive, revealing support for the maintenance of both mechanisms in neural evolution. Critically, we illustrate how the probability of deviation from concerted evolution depends on the cost/benefit ratio of neural tissue, which increases when overall brain size is itself under constraint., Conclusions: We conclude that both developmentally coupled and uncoupled brain architectures can provide adaptive mechanisms, depending on the distribution of selection across brain structures, life history and costs of neural tissue. However, when constraints also act on overall brain size, heterogeneity in selection across brain structures will favour region specific, or mosaic, evolution. Regardless, the respective advantages of developmentally coupled and uncoupled brain architectures mean that both may persist in fluctuating environments. This implies that developmental coupling is unlikely to be a persistent constraint, but could evolve as an adaptive outcome to selection to maintain functional integration.

The importance of behavioral evolution during speciation is well established, but we know little about how this is manifest in sensory and neural systems. A handful of studies have linked specific neural changes to divergence in host or mate preferences associated with speciation. However, the degree to which brains are adapted to local environmental conditions, and whether this contributes to reproductive isolation between close relatives that have diverged in ecology, remains unknown. Here, we examine divergence in brain morphology and neural gene expression between closely related, but ecologically distinct, Heliconius butterflies. Despite ongoing gene flow, sympatric species pairs within the melpomene-cydno complex are consistently separated across a gradient of open to closed forest and decreasing light intensity. By generating quantitative neuroanatomical data for 107 butterflies, we show that Heliconius melpomene and Heliconius cydno clades have substantial shifts in brain morphology across their geographic range, with divergent structures clustered in the visual system. These neuroanatomical differences are mirrored by extensive divergence in neural gene expression. Differences in both neural morphology and gene expression are heritable, exceed expected rates of neutral divergence, and result in intermediate traits in first-generation hybrid offspring. Strong evidence of divergent selection implies local adaptation to distinct selective optima in each parental microhabitat, suggesting the intermediate traits of hybrids are poorly matched to either condition. Neural traits may therefore contribute to coincident barriers to gene flow, thereby helping to facilitate speciation., Competing Interests: The authors declare no competing interest.

Butterflies display incredible ecological and behavioural diversity. As such, they have been subject to intense study since the birth of evolutionary biology. However, with some possible exceptions, they are underused models in comparative and functional neurobiology. We highlight a series of areas, spanning sensory ecology to cognition, in which butterflies are particularly promising systems for investigating the neurobiological basis for behavioural or ecological variation. These fields benefit from a history of molecular and quantitative genetics, and basic comparative neuroanatomy, but these strands of research are yet to be widely integrated. We discuss areas for potential growth and argue that new experimental techniques, growing genomic resources, and tools for functional genetics will accelerate the use of butterflies in neurobiology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Major evolutionary transitions can be triggered by behavioural novelty, and are often associated with 'adaptive suites', which involve shifts in multiple co-adapted traits subject to complex interactions. Heliconius butterflies represent one such example, actively feeding on pollen, a behaviour unique among butterflies. Pollen feeding permits a prolonged reproductive lifespan, and co-occurs with a constellation of behavioural, neuroanatomical, life history, morphological and physiological traits that are absent in closely related, non-pollen-feeding genera. As a highly tractable system, supported by considerable ecological and genomic data, Heliconius are an excellent model for investigating how behavioural innovation can trigger a cascade of adaptive shifts in multiple diverse, but interrelated, traits. Here, we synthesize current knowledge of pollen feeding in Heliconius , and explore potential interactions between associated, putatively adaptive, traits. Currently, no physiological, morphological or molecular innovation has been explicitly linked to the origin of pollen feeding, and several hypothesized links between different aspects of Heliconius biology remain poorly tested. However, resolving these uncertainties will contribute to our understanding of how behavioural innovations evolve and subsequently alter the evolutionary trajectories of diverse traits impacting resource acquisition, life history, senescence and cognition.

Many animal species remain separate not because their individuals fail to produce viable hybrids but because they "choose" not to mate. However, we still know very little of the genetic mechanisms underlying changes in these mate preference behaviours. Heliconius butterflies display bright warning patterns, which they also use to recognize conspecifics. Here, we couple QTL for divergence in visual preference behaviours with population genomic and gene expression analyses of neural tissue (central brain, optic lobes and ommatidia) across development in two sympatric Heliconius species. Within a region containing 200 genes, we identify five genes that are strongly associated with divergent visual preferences. Three of these have previously been implicated in key components of neural signalling (specifically an ionotropic glutamate receptor and two regucalcins), and overall our candidates suggest shifts in behaviour involve changes in visual integration or processing. This would allow preference evolution without altering perception of the wider environment.

For many pollinators, flowers provide predictable temporal schedules of resource availability, meaning an ability to learn time-dependent information could be widely beneficial. However, this ability has only been demonstrated in a handful of species. Observations of Heliconius butterflies suggest that they may have an ability to form time-dependent foraging preferences. Heliconius are unique among butterflies in actively collecting pollen, a dietary behaviour linked to spatio-temporally faithful 'trap-line' foraging. Time dependency of foraging preferences is hypothesized to allow Heliconius to exploit temporal predictability in alternative pollen resources. Here, we provide the first experimental evidence in support of this hypothesis, demonstrating that Heliconius hecale can learn opposing colour preferences in two time periods. This shift in preference is robust to the order of presentation, suggesting that preference is tied to the time of day and not due to ordinal or interval learning. However, this ability is not limited to Heliconius , as previously hypothesized, but also present in a related genus of non-pollen feeding butterflies. This demonstrates time learning likely pre-dates the origin of pollen feeding and may be prevalent across butterflies with less specialized foraging behaviours.