Enhanced long-term memory and increased mushroom body plasticity in Heliconius butterflies.

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.

Adult neurogenesis does not explain the extensive post-eclosion growth of Heliconius mushroom bodies.

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.

Long-term spatial memory across large spatial scales in Heliconius butterflies.

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 primates1 to Hymenopterans2. 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 longevity3. Several lines of evidence suggest that Heliconius learn the spatial location of pollen resources within an individual's home range4, 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.

Patterns of host plant use do not explain mushroom body expansion in Heliconiini butterflies.

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.

Rapid expansion and visual specialisation of learning and memory centres in the brains of Heliconiini butterflies.

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.

The evolution of adult pollen feeding did not alter postembryonic growth in Heliconius butterflies.

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.

Pollen feeding in Heliconius butterflies: the singular evolution of an adaptive suite.

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.

Heliconiini butterflies can learn time-dependent reward associations.

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.