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.