Discussion
Larval Lepidoptera have close evolutionary relationships with their host plants, where the phenotype of either organism often influences the traits of the other. Sometimes, behavioural evolution in larvae is not just in response to predation pressure, but is instead mainly driven by an antagonistic relationship with their hosts. Our findings support those of Beltran et al. (2007) in that there is likely to have been multiple independent evolutions to gregariousness across the Heliconiini. Larval gregariousness is mostly concentrated in distinct clades across the phylogeny (Figure 1A). This pattern is expressed quantitively by the high phylogenetic signal of larval social behaviour. Additionally, our data reveal that larval gregariousness, measured by proxy of egg clutch size, increases over evolutionary time in a semi-linear pattern, whereby transitions to small clutches from single eggs tend to precede much larger clutches (Figure 3A). This suggests that there may be physiological constraints to females laying large clusters of eggs, which are incrementally overcome by increasing smaller clutch sizes over evolutionary time, and/or a selection pressure feedback loop which promotes larger clutches once gregariousness evolves.
Our analyses of host plant ecology support only one of our hypotheses, that gregarious species tend to be more specialised. Our data show that Heliconiini species with solitary larvae tend to feed on a greater variety of host species than gregarious larvae (Figure 1B). Unless they deposit their entire egg load onto one plant, females of species with solitary larvae will necessarily visit more hosts than egg-clustering species to lay a comparable number of eggs. Thus, if females need to visit a higher number of oviposition sites, potentially across multiple separate host plants, they may benefit from being comparatively less selective of these hosts. From solitary, generalist larvae, our data suggest that increased host specialisation evolves before the transition to gregariousness. A possible explanation for this evolutionary pathway is that the host plants that are utilised by species with gregarious larvae are of high nutritional quality, and are better positioned to support groups of larvae. This is supported by evidence that females of other butterfly species with gregarious larvae preferentially oviposit on higher quality hosts (Schäpers et al., 2016). We note that the findings from our focused assessment indicate that, at the local level, host plant specialisation does not significantly differ between solitary and gregarious Heliconiini, despite a similar interaction coefficient to the main model. This lack of significance is likely an effect of low power, indicated by the larger CI range in this second model, and the low numbers of hosts reportedly used by all species across this dataset.
In general, one mechanism proposed to allow host specialists to escape competition is through the evolution of toxin resistance to enable the colonisation of new resources. The Passiflora have robust chemical defences, and this toxicity can vary across species (de Castro et al., 2018, 2019). However, co-evolution alongside their hosts allows specialised larvae to minimise the fitness costs associated with metabolising their hosts’ toxins, to a greater degree than achieved by closely related generalist species (e.g. Engler-Chaouat and Gilbert, 2007; Merrill et al., 2013; de Castro et al., 2021). Increased toxicity resulting from host specialisation might explain our finding that this behaviour evolves before gregariousness for most Heliconiini (Figure 3B), given that larval toxicity most likely precedes transitions to gregariousness across the wider butterfly phylogeny (McLellan et al., 2023). Maintaining this specialisation may also be a response to high inter-specific competition for food resources (e.g. Merrill et al., 2013), as minimising additional, inter-specific competition is likely to be disproportionately important to grouped larvae. Although we were unable to account for plant chemistry in the current study, this explanation is supported by evidence that both gregarious and specialistHeliconius species are more toxic than solitary and generalist species respectively (Arias et al., 2016). Heliconius specialists are better at sequestering their host’s toxins than generalists but may be worse at synthesising their own (Engler-Chaouat and Gilbert, 2007). This could create an evolutionary feedback loop, whereby specialists become ‘locked in’ to their host, or else suffer reduced defences.
Despite evidence of an effect of host plant diversity on gregarious behaviour, none of the specific host plant traits examined in this study act as predictors of Heliconiini larval gregariousness. Whereas host plant morphology is thought to influence the evolution of other larval traits, such as anti-predator colour strategy (Prudic et al., 2007), our results do not show similar influences on social behaviour. First, we predicted that hosts with larger leaves would, on average, be preferred by gregarious larvae. Remaining as a closely aggregated group on a single plane has been shown to offer increased protection to larvae nearer the centre of the aggregation (McClure and Despland, 2011). Thus, our assumption was that leaves with larger surface areas would provide better ‘stages’ for such groupings. Additionally, evidence suggests that lepidopteran larvae develop faster on larger leaves (e.g. Potter et al., 2012), so it plausible that they might grow larger too. We found no effect of leaf size on larval social behaviour, suggesting that even small leaves may be big enough to support groups of larvae if larvae are small or the groups do not contain many individuals. Similarly, we found no evidence to suggest that larger leaves predict larger gregarious larvae, possibly because food availability may not depend on leaf size if leaves are numerous and easy to travel between. Additionally, we recorded leaf size based on mature leaf data (Ulmar and MacDougal, 2004), yet larvae may preferentially feed on young leaves (e.g. Peterson, 1987) given the potential growth benefits available (e.g. Coley et al., 2006). Our findings may nevertheless indicate that other factors, such as foliage density and ease of travel between leaves, require consideration if resource availability is to be more accurately measured.
We also predicted an evolutionary link between the presence of host leaf trichomes and larval gregariousness. This was because group-feeding can aid larvae in overcoming certain host plant defences, and in some cases may even be necessary such as for negating effects of feeding-induced toxins and leaf toughness (Denno and Benrey, 1997; Fordyce, 2003; Kawasaki et al., 2009; Despland, 2019; Rentería et al., 2022). Our hypothesis was that these feeding facilitation benefits may contribute towards a form of behavioural character displacement, where gregarious larvae specialise on well-defended hosts that solitary species struggle to feed on. Indeed, some host plant leaf trichomes act as formidable defences against larval herbivores (e.g. Gilbert, 1971; Despland, 2019) and in one system, aggregation is thought to have evolved as a response to overcome this defence (Despland, 2019). However, we found that the presence or absence of Passiflora leaf trichomes has no influence on larval social behaviour in the Heliconiini. This may be because of a lack of specificity in available vestiture data, meaning we could only record vestiture in binary format and lacked information on the length and density of trichomes on most of the pubescent plants. Other useful trichome data, particularly their structure (whether they are hooked, glandular or neither), are also absent from the literature. These features are likely to be important determinants of how difficult trichome defences are for larvae to overcome (Fürstenberg-Hägg et al., 2013; Despland, 2019). Alternatively, it may simply be the case that, in most cases, aggregating does not improve larvae’s ability to overcome trichomes to an extent that it is selected over solitary feeding.
Finally, we expected larval behaviour to vary according to their host plant’s (and by extension their own) main habitat, given the potential ecological differences between them. While we are missing habitat data for a number of host species in this study, overall, we found no evidence that habitat predicts larval behaviour. Our inclusion of habitat type was based on the assumption that it may act as a proxy for ecological factors which potentially influence larval social behaviour, such as host spatial distribution (Young, 1983; Braby and Nishida, 2010). Our negative result may indicate that this assumption is not valid. However, in both our geographically broad and focused datasets we observed that hosts growing on the edges of forest habitats are favoured by Heliconiini in general, regardless of social behaviour. It is possible that there is some aspect of this habitat that ovipositing females favour over others, although we cannot rule out a bias in how these data are recorded, such as edge habitats being easier to access than forest interiors.
In summary, larval gregariousness is widespread across the Heliconiini and has evolved repeatedly, however the specific ecological drivers of this behaviour remain unclear. Variation in host specialisation between solitary and gregarious larval Heliconiini suggests that there are certain host traits that promote aggregation, however available data has not led to their identification. We suggest a number of ecological factors which we could not include in our analyses may be relevant. First, host toxicity can vary widely (de Castro et al., 2018, 2019; Mattila et al., 2021), but the chemical defences of the larvae which sequester these toxins may be a strong selective driver of which species can afford to aggregate (Ruxton and Sherratt, 2006; McLellan et al., 2023). We did not include plant chemistry in our analyses, but this may have given us a better understanding of why larvae specialise onto certain hosts, for example to escape competition from generalists. Currently, however, data on toxicity variation across populations of larvae and their hosts is lacking. Second, host spatial density, and whether it varies between habitats, is an important factor missing from our data. We attempted to capture this by testing for an effect of host habitat on larval behaviour, on the assumption that the categories capture structural variation in the forest. However, direct data on how those habitats, or plants within them, might differ ecologically is lacking. As such, larval gregariousness may arise in response to ecological specialisation by the host plants, if the preferred host is sparsely distributed and difficult to locate, benefitting females which lay their eggs in clusters (Young 1983; Braby and Nishida 2010). Data on the relative spatial distributions of Heliconiini hosts would need to be collected if this hypothesis is to be tested, which is a challenging endeavour. Nevertheless, we have identified that increased host specialisation frequently occurs before the evolution of gregarious behaviour in Heliconiini larvae. This suggests that there are key host plant traits which may predict this behaviour, which require further study to identify.