Introduction
Many prey animals have evolved grouping behaviour in response to predation and resource availability. Lepidopteran larvae benefit from aggregating in a number of ways, ranging from increased protection from predators (Hunter, 2000; Reader and Houchuli, 2003; Greeney et al., 2012) to facilitated feeding (Clark and Faeth, 1997; Fordyce, 2003; Kawasaki et al., 2009; Campbell and Stastny, 2015; Rentería et al. 2022). However, larval gregariousness also imposes costs, such as greater competition for food resources between siblings (e.g. Despland and Le Huu, 2007; Pescador-Rubio, 2009), creating the context for possible evolutionary trade-offs. Identifying key biotic and ecological factors that frame these trade-offs may be critical for understanding the origin and evolution of gregarious behaviour. One of the most important of these ecological factors are larval host plants. As herbivores, lepidopteran larvae often have intimate coevolutionary relationships with their hosts. These plants can act as a major source of selection for larvae, for example due to their growth structure or by developing defences against herbivory that larvae must adapt to overcome (Clark and Faeth, 1997; Thaler et al., 2002; Wittstock and Gershenzon, 2002; Birnbaum and Abbot, 2018; de Castro et al., 2018; Karban, 2011; Despland, 2019).
Host plant traits that may influence the evolution of larval gregariousness include their relative leaf size, anti-herbivory defences and spatial distribution. For example, the average leaf size of the host may determine its suitability for group-feeding larvae. In general, and discounting foliage density, larger leaves might provide more food to support multiple larvae, and may be an indication of greater above ground biomass (e.g. Digrado et al., 2022). Larger leaves have been shown to enhance the growth rate of young lepidopteran larvae (Potter et al., 2012), and by providing greater amounts of resources, larger leaves might also allow gregarious larvae to reach larger body sizes. Large leaves also physically offer a wider surface area upon which larvae can collectively feed, which could be important if larvae benefit by remaining close to their group members, such as by reducing predation and parasitism risks (e.g. McClure and Despland, 2011).The spatial distribution, or density, of host plants might also vary, affecting how easily females locate suitable oviposition sites, and equally defining the risk of larvae moving between hosts if a food resource is exhausted. Females may therefore adjust their oviposition strategy in response to the relative difficulty of locating suitable hosts (Braby and Nishida, 2010), with clumped eggs giving rise to gregarious larvae (Clark and Faeth, 1998; Korb and Heinze, 2016).
Across both short- and long-term scales, plants are rarely passive in their coevolutionary relationships with larval herbivores, having evolved a variety of defences in response to being selected as hosts. Evolutionary adaptations such as tougher leaf surfaces can prevent larval feeding (see Fürstenberg-Hägg et al., 2013 for a review), and trichomes can physically prevent larvae from accessing the leaf tissue, significantly hinder movement, exude harmful substances, or may even cause integumental injuries (Gilbert, 1971; Fürstenberg-Hägg et al., 2013; Despland, 2019). The evolution of toxins also helps plants to escape herbivory from many generalists (Wittstock and Gershenzon, 2002; Engler-Chaouat and Gilbert, 2007; Birnbaum and Abbot, 2018). Furthermore, plants which have evolved toxins often also display more immediate responses to attack, such as the release of these concentrated toxins into sites of feeding damage (Denno and Benrey, 1997; Karban, 2011).These host plant defences, and the need to overcome them, are thought to be a main promoter of larval aggregation in some systems (Clark and Faeth, 1997; Denno and Benrey, 1997; Fordyce and Agrawal, 2001; Kawasaki et al., 2009; Despland, 2019; Rentería et al., 2022). For example, some larvae will meticulously remove leaf trichomes to reduce their harmful impact (de Castro et al., 2018), but this is likely to be a costly task for an individual. Some gregarious larvae are well-equipped to deal with trichomes, and collectively cover them in silk to avoid contact (e.g. Rathcke and Poole, 1975; Despland, 2019; Despland, 2021). Additionally, collective feeding is thought to benefit larvae against their host’s toxin release response if they can completely consume the leaf before it is flooded with toxins (Denno and Benrey, 1997).
Here, we use the Heliconiini butterfly tribe as a model system to study the influence of specific host plant traits on the evolution of larval gregarious behaviour. All Heliconiini larvae feed on vines from the Passifloraceae family (de Castro et al., 2018), which offers a shared ecological context within which specific trait differences can be interrogated. Passifloraceae are highly diverse, varying widely in their overall structure and defences against herbivory, such as egg mimicking structures to deter oviposition, extrafloral nectar rewards to attract predatory ants, and toxic chemical components in their tissues (de Castro et al., 2018). These chemicals form an important line of defence against herbivory from generalist species and, perhaps as a result, some Heliconiini have been driven into specialising on small numbers of hosts. Heliconiini larvae have evolved resistance to their host’s toxins, often in correlation with increased specialisation (Engler-Chaouat and Gilbert, 2007; Merrill et al., 2013; de Castro et al., 2021), and the ability to incorporate these toxins into their own chemical defences (Engler-Chaouat and Gilbert, 2007; Arias et al., 2016; de Castro et al., 2021). Additionally, larval social behaviour varies across the Heliconiini, even between very closely related species, with repeated shifts to grouped egg laying and gregarious larvae (Beltran et al., 2007; McLellan et al., 2023). Although little is known about the behavioural mechanisms supporting these aggregations, at least some gregarious Heliconiini are trail followers (Pescador-Rubio et al., 2011), suggesting these transitions reflect behavioural adaptations in larvae rather than simple variation in female egg laying. This variation, coupled with tribe-wide estimates of the phylogenetic structure of the Heliconiini (Kozak et al., 2015; Cicconardi et al., 2022), positions these butterflies as a highly useful system with which to study behavioural evolution in response to host plant ecology.
Here, we revisit the evolution of larval gregariousness in Heliconiini, taking a phylogenetic comparative approach to identify where transitions to larval gregariousness have taken place across the phylogeny. Then, by exploring variation in host plant use between the two behavioural phenotypes, we test hypotheses regarding the host traits that shape the evolution of gregarious behaviour. In particular, we ask i) do gregarious larvae use a narrower range of host plant species than solitary larvae? ii) Do aggregated larvae occur only on hosts with specific traits? iii) Is host plant leaf size a key constraint on the evolution of gregarious larvae and their late instar body size? And iv) do commonly used host plants occur in particular contexts or lack particular defences, rendering them more accessible to predation?