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?