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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.

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.

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 early population divergence. We analysed flight-height variation across speciation events inHeliconiusbutterflies representing parallel instances of divergence between low and high-altitude populations. We measured flight-height in wildH. erato venus andH. chestertonii, lowland and mountain specialists respectively, and found thatH. chestertoniiconsistently flies at a lower height. We compared these data with previously published results forH. e. cyrbiaandH. himera, the latter of which flies lower and, likeH. chestertonii, recently colonised high-altitude, dry forests. We show that these repeated trends largely result from shared patterns of selection across equivalent environments, producing parallel trait-shifts inH. himeraandH. chestertonii. Although our results imply a signature of local adaptation, we did not find an association between resource distribution and flight-height inH. e. venusandH. chestertonii. We discuss how this pattern may be explained by variation in forest structure and microclimate. Overall, our findings underscore the importance of behavioural adjustments during early divergence mediated by altitude-shifts.

The mushroom bodies, an integrative region of the insect brain involved in learning and memory, have undergone volumetric increase in several independent lineages includes bees and ants, cockroaches and some beetles. However, the selective pressures driving these expansion events are not fully understood. One promising system for investigating this question is the Neotropical butterfly genusHeliconius, which exhibits markedly enlarged mushroom bodies compared with other members of the Heliconiini tribe. Notably, this neural elaboration co-occurs with the evolution of trapline foraging behaviour and an improved capacity for learning complex visual cues and long-term memory. Here, we further investigate the behavioural consequences of this brain expansion by testing reversal learning ability, a commonly used measure of cognition and behavioural flexibility in both vertebrates and invertebrates, across threeHeliconiusand three closely-related Heliconiini species. We trained butterflies to associate a food reward with either purple or yellow flowers, before training them with the reversed associations, and then reversing the cues again. All six successfully learned the reversed cues, and, contrary to our expectations, we found no evidence thatHeliconiusperformed better than the other Heliconiini species. These results are surprising, given previous evidence linking the mushroom bodies to reversal learning in other insects, and the enhanced performance ofHeliconiusin other cognitive tests. This serves as a reminder that the functional consequences of brain expansion can be multifaceted, and do not necessarily result in an overall increase in general cognitive ability, but rather enhanced performance in specific, ecologically-relevant tasks.

The selective pressures leading to the elaboration of downstream, integrative processing centres, such as the mammalian neocortex or insect mushroom bodies, are often unclear. InHeliconiusbutterflies, the mushroom bodies are three to four times larger than their Heliconiini, and the largest known in Lepidoptera. Heliconiini lay almost exclusively onPassiflora, which exhibit a remarkable diversity of leaf shape, and it has been suggested that the mushroom body expansion ofHeliconiusmay have been driven by the cognitive demands of recognising and learning the 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, butHeliconiusare 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.

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’.

How do neural systems evolve to support new behaviors? Changes in the abundance and diversity of neural cell types, and their connectivity, shape brain composition and provide the substrate for behavioral variation. We describe a striking example of neural elaboration in an ecologically diverse tribe of Heliconiini butterflies. By building extensive new datasets of neural traits across the tribe, we identify major bursts in the size and cellular composition of the mushroom bodies, central brain structures essential for learning and memory. These expansion events are associated with increased innervation form visual centers and coincide with enhanced performance in multiple cognitive assays. This suite of neural and cognitive changes is likely tied to the emergence of derived foraging behaviors, facilitated by localized specialization of neural networks.One-Sentence SummaryMajor shifts in brain composition and behavior in butterflies with unique foraging and dietary behaviors.

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., NERC IRF; Royal Society Research Grant; Royal Commission for the Great Exhibition Research Fellowship

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.

Many species gain anti-predator 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 the 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, whilst identifying new questions about the roles of body size and toxicity in the evolution of grouping behavior.

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.

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.

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 H. 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 several transcription factor binding site candidates 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.

Heliconiusbutterflies, 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. We have generated highly contiguous genome assemblies for nine new Heliconiini, 29 additional reference-assembled genomes, and improve 10 existing assemblies. Altogether, we provide a major new 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 theHeliconiusstem. 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.

Repeated evolution can provide insight into the mechanisms that facilitate adaptation to novel or changing environments. Here we study adaptation to high altitude in two divergent tropical butterflies, H. erato and H. melpomene, which have repeatedly and independently adapted to high elevations on either side of the Andean mountains. We sequenced 518 whole genomes from elevational transects and found many regions under selection at high altitude, with repeated genetic differentiation across multiple replicates, including allopatric comparisons. In contrast, there is little ‘molecular parallelism’ between H. erato and H. melpomene. With a further 85 whole genomes of five close relatives, we find that a large proportion divergent regions have arisen from standing variation and putative adaptive introgression from high-altitude specialist species. Taken together our study supports a key role of standing genetic variation and gene flow from pre-adapted species in promoting parallel genetic local adaptation to the environment.

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 assemblyEueides isabellaand, together with the chromosome-level assembly ofDryas iulia, examine the evolutionary consequences of multiple chromosome fusions inHeliconiusbutterflies. These assemblies pinpoint fusion points on 10 of the 21 autosomal chromosomes and reveal striking differences in the characteristics of fused and unfused chromosomes. The ten smallest autosomes inD. iuliaandE. isabella, which have each fused to a longer chromosome inHeliconius, have higher repeat and GC content, and longer introns than predicted by their chromosome length. Following fusion, 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 inHeliconiuslikely had dramatic effects on population level processes shaping rates of neutral and adaptive divergence. These effects may have impacted patterns of diversification inHeliconius, a classic example of an adaptive radiation.

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.

What is the cognitive significance of variation in brain size? This question is simply put, but hard to answer, and remains one of the most enduring questions in comparative ethology. Understanding the causative links between variation in brain size and structure, and cognition requires reliable data on both neural and behavioral traits. A recent study by Horschler et al. (Anim Cogn 22(2):187-198, 2019) demonstrated the potential of citizen science and domestic dogs to provide unprecedented behavioral datasets that can be used to tackle this question. However, data on brain weight is harder to source. To test the link between performance in various cognitive tasks and variation in brain size, the authors instead relied on data for body weight, which was transformed into 'estimated brain weight' using the allometric scaling relationship between brain and body size, an approach that can be found in other papers which lack sufficient neuroanatomical data. Here, I describe some probable limitations of this approach and suggest that such transformations provide no benefit to the analyses and should be avoided.

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.

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.Heliconiusbutterflies 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,Heliconiusare 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 inHeliconius, 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 ofHeliconiusbiology 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.

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.

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 ofHeliconiusbutterflies suggest that they may have an ability to form time-dependent foraging preferences.Heliconiusare 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 allowHeliconiusto exploit temporal predictability in alternative pollen resources. Here, we provide the first experimental evidence in support of this hypothesis, demonstrating thatHeliconius hecalecan 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 toHeliconius, 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.

How an organism’s sensory system functions is central to how it navigates its environment and meets the behavioural challenges associated with survival and reproduction. Comparing sensory systems across species can reveal how facets of behaviour and ecology promote adaptive shifts in the relative importance of certain environmental cues. The insect olfactory system is 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 a macroglomerular complex (MGC), within the primary olfactory processing centre. These structures typically process pheromonal cues and provide a classic example of how variation in size can influence the functional processing of sensory cues. Though prevalent across moths, the MGC was lost during the early evolution of butterflies, consistent with evidence that courtship initiation in butterflies is primarily reliant upon visual cues, rather than long distance olfactory signals like pheromones. However, a MGC has recently been reported to be present in a species of ithomiine, Godryis zavaleta, suggesting this once lost neural adaptation has re-emerged in this clade. Here, we show that MGC’s, or MGC-like morphologies, are indeed widely distributed across the ithomiine tribe, and vary in both structure and the prevalence of sexual dimorphism. Based on patterns of variation across species with different chemical ecologies, we suggest that this structure is involved in the processing of both plant and pheromonal cues, of interlinked chemical constitution, and has evolved in conjunction with the increased importance and diversification of plant derived chemicals cues in ithomiines.

Divergence in mating behaviors plays a major role during speciation, but we know little about the genetic mechanisms underlying the evolution of such traits. Reproductive isolation between the Neotropical butterflies Heliconius melpomene and H. cydno depends on shifts in mimetic warning patterns, which also act as mating cues. Preferences for conspecific warning patterns have a strong genetic component, but unlike the warning pattern cues, the exact genes responsible remain unknown. Here, we reduce the number of candidate genes associated with a large effect QTL for changes in visual mate preference from 200 to just 6. First, we confirm that genomic regions previously associated with male courtship modulate preference behaviors, rather than other traits that may influence courtship time. We then investigate gene expression in the brains of H. melpomene, H. cydno, and their hybrids, across development. We identify two genes, regucalcin2 and an ionotropic glutamate receptor, that are differentially expressed both in species and hybrid comparisons, in a manner consistent with known patterns of dominance. We also find substitutions in the protein-coding regions of 4 genes predicted to affect protein function. These six candidates are located in regions resistant to interspecific gene flow, as expected for genes that contribute to reproductive isolation. Our candidates have been implicated in key components of neural signaling, suggesting shifts in visual mating preference do not rely on primary photoreceptor genes, but instead involve changes in visual integration or processing. Such genetic changes would allow mate preference evolution without altering perception of the wider environment. Significance statement Many species remain separate not because they fail to produce viable hybrids but because they “choose” not to mate. Although these changes in behavior can be hardwired in the genome, we know little about the specific genes involved. Heliconius butterflies display a striking diversity of warning color patterns, which they use as cues to recognize conspecifics. We analyze a genomic region associated with changes in these visual behaviors in two Heliconius species. We couple quantitative trait locus (QTL) studies with gene expression and population genomic analyses to identify candidate genes associated with these visual preferences. This is an important step towards understanding how behavioral differences, crucial to speciation, are generated both during development and across evolutionary time.

SummaryThe importance of behavioural evolution during speciation is well established, but we know little about how this is manifest in sensory and neural systems. Although a handful of studies have linked specific neural changes to divergence in host or mate preferences associated with speciation, how brains respond to broad environmental transitions, and whether this contributes to reproductive isolation, remains unknown. Here, we examine divergence in brain morphology and neural gene expression between closely related, but ecologically distinct,Heliconiusbutterflies. Despite on-going gene flow, sympatric species pairs within themelpomene-cydnocomplex 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 thatH. melpomeneandH. cydnohave 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 morphology and gene expression are heritable, exceed expected rates of neutral divergence, and result in intermediate traits in first generation hybrid offspring. This likely disrupts neural system function, leading to a mismatch between the environment and the behavioral response of hybrids. Our results suggest that disruptive selection on both neural function and external morphology result in coincident barriers to gene flow, thereby facilitating speciation.

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. Observational studies ofHeliconiusbutterflies suggest that they may have an ability to form time-dependent foraging preferences.Heliconiusare unique among butterflies in actively collecting and digesting pollen, a dietary behaviour linked to spatiotemporally faithful ‘trap-line’ foraging. Time-dependency of foraging preferences is hypothesised to allowHeliconiusto exploit temporal predictability in alternative pollen resources, as well as contributing to optimal use of learnt foraging routes. Here, we provide the first experimental evidence in support of this hypothesis, demonstrating thatHeliconius hecalecan 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 learning. However, we also show that this ability is not limited toHeliconius, as previously hypothesised, but is also present in a related genus of non-pollen feeding butterflies. This demonstrates that time learning pre-dates the origin of pollen-feeding and may be prevalent across butterflies with less specialized foraging behaviours.

This is the final version of the article. Available from The Comparative Cognition Society via the DOI in this record.

© Comparative Cognition Society. Despite prolonged interest in comparing brain size and behavioral proxies of "intelligence" across taxa, the adaptive and cognitive significance of brain size variation remains elusive. Central to this problem is the continued focus on hominid cognition as a benchmark and the assumption that behavioral complexity has a simple relationship with brain size. Although comparative studies of brain size have been criticized for not reflecting how evolution actually operates, and for producing spurious, inconsistent results, the causes of these limitations have received little discussion. We show how these issues arise from implicit assumptions about what brain size measures and how it correlates with behavioral and cognitive traits. We explore how inconsistencies can arise through heterogeneity in evolutionary trajectories and selection pressures on neuroanatomy or neurophysiology across taxa. We examine how interference from ecological and life history variables complicates interpretations of brain-behavior correlations and point out how this problem is exacerbated by the limitations of brain and cognitive measures. These considerations, and the diversity of brain morphologies and behavioral capacities, suggest that comparative brain-behavior research can make greater progress by focusing on specific neuroanatomical and behavioral traits within relevant ecological and evolutionary contexts. We suggest that a synergistic combination of the "bottom-up" approach of classical neuroethology and the "top-down" approach of comparative biology/psychology within closely related but behaviorally diverse clades can limit the effects of heterogeneity, interference, and noise. We argue that this shift away from broad-scale analyses of superficial phenotypes will provide deeper, more robust insights into brain evolution.

During speciation across ecological gradients, diverging populations are exposed to contrasting sensory and spatial information that present new behavioural and perceptive challenges. These challenges may be met by heritable or environmentally induced changes in brain function which mediate behaviour. However, few studies have investigated patterns of neural divergence at the early stages of speciation, inhibiting our understanding of the relative importance of these processes. Here, we provide a novel case study. The incipient species pair, Heliconius erato and H. himera, are parapatric across an environmental and altitudinal gradient. Despite ongoing gene flow, these species have divergent ecological, behavioural and physiological traits. We demonstrate that these taxa also differ significantly in brain composition, in particular in the relative levels of investment in structures that process sensory information. These differences are not explained solely by environmentally-induced plasticity, but include heritable, nonallometric shifts in brain structure. We suggest these differences reflect divergence to meet the demands of contrasting sensory ecologies. This conclusion would support the hypothesis that the evolution of brain structure and function play an important role in facilitating the emergence of ecologically distinct species.

Significance The new species Homo naledi was discovered in 2013 in a remote cave chamber of the Rising Star cave system, South Africa. This species survived until between 226,000 and 335,000 y ago, placing it in continental Africa at the same time as the early ancestors of modern humans were arising. Yet, H. naledi was strikingly primitive in many aspects of its anatomy, including the small size of its brain. Here, we have provided a description of endocast anatomy of this primitive species. Despite its small brain size, H. naledi shared some aspects of human brain organization, suggesting that innovations in brain structure were ancestral within the genus Homo., Hominin cranial remains from the Dinaledi Chamber, South Africa, represent multiple individuals of the species Homo naledi. This species exhibits a small endocranial volume comparable to Australopithecus, combined with several aspects of external cranial anatomy similar to larger-brained species of Homo such as Homo habilis and Homo erectus. Here, we describe the endocast anatomy of this recently discovered species. Despite the small size of the H. naledi endocasts, they share several aspects of structure in common with other species of Homo, not found in other hominins or great apes, notably in the organization of the inferior frontal and lateral orbital gyri. The presence of such structural innovations in a small-brained hominin may have relevance to behavioral evolution within the genus Homo.