DISCUSSION
The results of this study highlight the coupling of functional traits
associated with plant growth forms specific of different environmental
conditions with the differential production of glucosinolates (GSLs)
across Cardamine species. Specifically, we found thatCardamine species cluster into four main groups. Each group,
being anchored within a major climatic zone of the Alpine elevation
gradient, expressed different levels of phytochemical diversity, and
exhibited an overexpression of unique GSLs; indoles being the signature
of the alpine and low elevation groups, and aliphatics the signature of
two mid-elevation zones. Such habitat-driven phytochemical convergence
had variable consequences on herbivores belonging to different diet
breadths and feeding guilds. We thus suggest that the identity and
diversity of secondary metabolites within a given species is determined
by convergent adaptation to the local abiotic and biotic conditions,
ultimately affecting different herbivores, variously.
One major prediction for explaining variation in phytochemical diversity
across species is that phylogenetic conservatisms for phytochemical
production should result in closely-related species being more
phytochemically-similar that distantly-related species (Futuyma &
Agrawal 2009a). On the contrary, we found that the diversity of GSLs was
not explained by phylogeny. This is in contrast to the phylogenetic
conservatism reported across different families of plants (Wink 2003;
Wink & Mohamed 2003; WInkler & Mitter 2008), or within genera; such as
the production of aliphatic and branched-chain GSLs in the genusStrepthantus (Cacho et al. 2015), or the production of
cardenolides in the genus Asclepais (Agrawal et al. 2009;
Rasmann & Agrawal 2011). However, we interpret the lack of phylogenetic
signal in GSL production in our system with caution, as the reduced
number of investigated species impairs the ability to fully tease apart
potential patterns that might emerge when assessing more species-rich
clades (Swenson 2019). Nevertheless, our results are indicative of other
factors, other than shared evolutionary history, in driving the variable
production of GSLs across species having colonized different habitats.
Accordingly, previous studies also found ecological convergence in
chemical defensive profiles across species, independently of
phylogenetic relationship (Kursar & Coley 2003; Salazar et al.2016).
Here, we expanded on this previous work by integrating large-scale
ecological gradients, and we observed a significant correlation between
plant functional traits, which are associated with the specific niche of
the species within each elevation zone, and the GSLs matrix. These
results build on previous work showing, across 15 differentCardamine species, a strong correlation between climatic
variables and 10 functional traits related to abiotic tolerance, growth
and defence (Defossez et al. 2018). Taken together, these results
suggest that climatic factors force species into specific growth forms
(Wright et al. 2004; Díaz et al. 2016), and likewise
dictate the shape and structure of the phytochemicals to be produced.
However, our result are less in line with predictions of the screening
hypothesis (Berenbaum et al. 1991; Duffey & Stout 1996), but
more with the resource availability hypothesis (Coley et al.1985); alpine species, for which herbivore pressure is the lowest
(Pellissier et al. 2016), but growing in resource-poor
environments, expressed the highest number for practically all indices
of phytochemical GSL diversity. In other words, we observed a less
direct effect of herbivory pressure than that of the habitat on
phytochemistry (Richards et al. 2015). We observed that alpine
species expressed the highest phytochemical diversity, particularly when
compared to mid-elevation plant species. We argue that the higher costs
associated with replacement of biomass loss in the harsher environment,
characteristics of high elevation zones (Korner et al. 1989;
Chapin & Korner 1995), could be an explanation for the increased GSL
diversity as observed in our study. At high elevation, the cost to
recover tissue lost is strongly limited by the paucity of resources and
the cold temperatures. Therefore, for these alpine species, the fitness
costs of herbivory cannot be outweighed by the energy saved in reduced
levels of defences (Bryant et al. 1983). The production of
defence strategies is therefore more linked to the impact of herbivory
based on resources available, than solely on herbivore pressure (Coleyet al. 1985). Therefore, while alpine species (group 1) are
characterized by a combination of traits conferring high abiotic
resistance (e.g. lower SLA values, tougher leaves, and slow growth),
they also integrate higher levels of phytochemical diversity for likely
withstanding the scattered, but potentially lethal, attack of herbivores
(Rasmann et al. 2014a). Low-elevation species, on the other hand,
experience a constantly high pressure by herbivores. Thus, while
expressing traits relating to fast growth and lower abiotic resistance
(higher SLA values and softer leaves), they also express higher GSL
diversity, particularly compared to the species within the two
mid-elevation groups. Species occupying mid-elevation zones of forest
habitats are typically comprised of species with high biomass production
(especially species in group 2) and high carbon to nitrogen ratio (CN)
(Defossez et al. 2018), which suggest a preference toward
investing in tolerance instead of defenses for those species
(Núñez-Farfán et al. 2007). In sum, our results suggest that
where plant species, independently of their phylogenetic relatedness,
share a common compendium of ecological variables, such as common
herbivore pressure, similar resource levels, or similar climates, plants
are also likely to defend themselves with a similar set of chemical
molecules.
In accordance with alpine species bearing the highest chemical diversity
values, caterpillars, especially the specialist P. brassicae grew
less on those plants. Particularly, these plants produced the highest H
values. However, our results do not fully concord with the general view
that GSL are more efficient against generalist than specialists
(Schlaeppi et al. 2008; Schweiger et al. 2014; Rasmannet al. 2015). P. brassicae feeds exclusively on plants
producing GSLs (Chew 1988), also utilizing these compounds for host
recognition and as feeding stimulants (Moyes et al. 2000).
Interestingly, it has been shown that that ovipositing P. rapaefemales respond more strongly to indole GSLs, such as glucobrassicin,
(Rodman & Chew 1980; Renwick et al. 1992; Huang et al.1994), which is also a GSL characterizing the alpine species. Therefore,
the slow-growing and comparatively very small alpine Cardaminespecies needed to evolve specific GSL combinations, through high H
values, that are toxic to the specialist herbivores, but this hypothesis
needs to be tested thoroughly using mixtures of compounds.
Concerning aphids, we found that the generalist aphids M.
persicae grew more on plants with lower FDiv values (i.e. species in
group 2). Therefore, for generalist aphids, our results support the
prediction of a negative correlation between the functional chemical
diversity/divergence of GSLs and herbivore performance (Dyer et
al. 2018). That said, it has been argued that GSLs in general are less
toxic to aphids than to caterpillars, because aphids avoid the
activation of GSLs by the enzyme myrosinase
(de
Vos et al. 2007). Nevertheless, indole GSLs are thought to be
less stable, and activate spontaneously in the absence of myrosinase.
Consequently, indole GSLs alone have been shown to impair the growth of
the generalist aphid M. persicae when added to an artificial diet
or overexpressed in host plants (Kim & Jander 2007; Kim et al.2008). On the contrary, specialist aphids, such as B. brassicae ,
are able to accumulate aliphatic GSLs
(Franciset al. 2001). In line with these findings, we suggest that aphids
are impaired by the indole GSLs, which are more produced by plants in
group 1 and 4, and less produced by the plants from group 2, as well by
a GSL chemical mixture that favour functional divergence.
In summary, this study, by combining metabolomics analyses with insect
bioassays on plants growing along steep ecological gradients, provides a
novel approach for explaining the cause and consequences of variations
in phytochemical diversity across plant species. By including several
indices of phytochemical diversity, we took a step further in
mechanistically disentangling the effects of different metrics of
phytochemical diversity on insect herbivore resistance. For instance, we
observed that groups of plants bearing practically identical chemical
richness values (S) can have completely different GSLs compositions.
This indicates that focusing on arbitrarily-selected indices of
phytochemical diversity can be misleading in interpreting the
metabolomics data and their effects (Wetzel & Whitehead 2020). Taking
into account different factors determining such diversity, such as
compound class, metabolites’ molecular metrics, or biological activity,
we were able to add a functional dimension to phytochemical diversity,
as was for instance done for cardenolides in milkweeds using polarity
values (Rasmann & Agrawal 2011). We thus argue that the classical
indices of phytochemical diversity used so far (total amount, number of
compounds, Shannon diversity), should be expanded to include functional
axes of chemical diversity, in order to be able to interpret the
biological activity of secondary metabolites in a more precise and
ecologically relevant manner , and to integrate these novel axes related
to plant defenses into the functional syndrome of plant growth forms.