INTRODUCTION
Phytochemical diversity, or the richness and abundance of the secondary
compounds produced by plants, is a key axis of the functional phenotype
that affects plant survival within its biotic and abiotic environment
(Jones & Firn 1991; Romeo et al. 1996; Hunter 2016). Ecologists
still struggle to understand, not only the origin of phytochemical
diversity, but also to quantify the consequences of
ecologically-relevant dimensions of phytochemical diversity (Richardset al. 2015), and how the functional axis of phytochemical
diversity relates to the functional axis of plant growth form (Díazet al. 2016; Durán et al. 2019). The overall assumption is
that a plant’s phytochemical make-up is the result of its evolutionary
history (Becerra 1997; Futuyma & Agrawal 2009b), as well as its
adaptations to the environment (Coley et al. 1985; Fine et
al. 2004; Defossez et al. 2018). Several ecological and
evolutionary hypotheses have been proposed for explaining variation in
phytochemical diversity, including the co-evolutionary hypothesis
(Ehrlich & Raven 1964), the screening hypothesis (Firn & Jones 2003)
and the resource availability hypothesis (Coley et al. 1985). The
aim of this study is to merge these hypotheses in order to explain
patterns of variation in the coupled plant growth form-phytochemical
phenotypes related to anti-herbivore defences across closely related
species that together have colonized large-scale climatic gradients.
From a co-evolutionary perspective, the concept of an arms race between
plants and herbivores has been proposed for explaining the ever
increasing diversity of plant secondary compounds over evolutionary
times (Ehrlich & Raven 1964). The idea being that herbivores, in
particular insects, impose strong selection pressure on plants to evolve
novel key adaptations for escaping their enemies.
Therefore, a
phylogenetic escalation for more, and more potent, phytochemical defence
traits should be observed as lineages diversify (Vermeij 1994; Farrell
& Mitter 1998). For instance, it was shown that parsnip plants evolved
more complex angular forms of furanocoumarins from more simple linear
furanocoumarins (Berenbaum & Feeny 1981), or that more complex forms of
cardenolides in milkweeds (Asclepias spp.) have emerged from more
simple forms as the consequence of the co-evolution with their
associated cerambicid beetles in the genus Tetraopes (Farrell &
Mitter 1998). Accordingly, it is predicted that first, the
presence of diverse forms of toxic phytochemicals in plants should
depend, at least partially, on the species evolutionary history, with
more recently-derived species to bear more complex levels of
phytochemical diversity compared to ancestrally-derived species, and
second, that more closely related species should be more similar in
their phytochemical make-up than distantly-related species. In other
words, we should observe a phylogenetic signal for phytochemical
diversity across species (Agrawal et al. 2009).
Along the same lines, the screening hypothesis proposes that
phytochemical diversity is maintained because it increases plants’
resistance against both generalist and specialist herbivores (Lewinsohn
& Gijzen 2009; Ali & Agrawal 2012). Accordingly, Richards et
al. (2015) showed that within the genus Piperaceae high
phytochemical diversity is associated with high diversity of herbivores,
but also with lower herbivore damage, indeed highlighting a positive
effect of phytochemical diversity in increasing resistance against
herbivores. Two mechanisms have been proposed for how phytochemical
diversity could favour plant resistance against herbivores. First, with
high phytochemical production, a plant is more likely to contain a
potent compound that is effective against a major herbivore,
cumulatively creating a selective advantage within a population (Firn &
Jones 1996). For instance, only a few of the 100-plus gibberellins have
a known biological activity, but those few that are active are potent at
nano molar amounts (Fischbach & Clardy 2007). However, Berenbaumet al. (1991) found that furanocoumarins in Pastinaca
sativa are all equally and effectively toxic to a wide variety of
herbivores. Second, high levels of phytochemical diversity might result
in effective combinations of compounds that work synergistically against
herbivores (Berenbaum & Neal 1985; Rasmann & Agrawal 2009; Richardset al. 2012), such as when the impact of nicotine on the
generalist Spodopotera exigua caterpillars is enhanced by
proteinase inhibitors in leaves of wild tobacco plants (Steppuhn &
Baldwin 2007).
Altogether, the screening hypothesis indicates that selection should
favour higher levels of phytochemical diversity, particularly in
habitats where herbivore pressure is high. Within this framework, it has
been long postulated that because warmer and more stable tropical or
lowland environments generate higher levels of plant-herbivore
interactions (Dobzhansky 1950; Schemske 2009), it should lead to
increased defence mechanisms compared to colder and less stable
environments such as temperate locations or high elevation (Coley &
Barone 1996). Nevertheless, reviews on the topic have also shown
contrasting patterns of defence investment along both latitude (Moleset al. 2011) and elevation gradients (Rasmann et al.2014b). This could be explained by other factors also influencing a
plant defensive phytochemical make-up. For instance, the resource
availability hypothesis (Coley et al. 1985) states that
environmental resources, such as soil nutrients, dictate how much a
plant can invest in growth and in defences. Specifically, it was
predicted, and later shown, that tropical plants growing in
resource-poor sandy soils, grow more slowly and are more defended
compared to their congeners that live in the nearby resource-rich clay
soils (Fine et al. 2004). Similarly, alpine Cardamine(Brassicaceae) species, living in resource poor soils, produce more
secondary metabolites (glucosinolates) than their low-elevation
congeners (Pellissier et al. 2016; Defossez et al. 2018).
Therefore, a holistic approach that encompasses environmental gradients,
and their biotic and abiotic correlates, within a phylogenetic
comparative framework is needed to tease out the intricate processes
generating chemical diversity in plants.
To this end, we performed comparative analyses of severalCardamine species growing along the elevation gradient of the
Alps. All Cardamine plants have been shown to produce a wide
array of glucosinolates (hereafter refereed to GSLs) (Pellissieret al. 2016). GSLs are sulphur- and nitrogen-containing plant
secondary metabolites that, upon tissue disruption, undergo a
myrosinase-catalysed hydrolysis generating a variety of by-products,
including nitriles, isothiocyanates, thiocyanates, oxazolidine-2-thione,
and indole, that are toxic to both specialist and generalist insect
herbivores (van Dam et al. 2009). We measured diversity of GSLs
across species, generated novel indices that functionally characterize
GSL diversity, and used a previously-collected set of growth-related
traits affiliated with each species’ growing habit (Defossez et
al. 2018). This natural system allowed asking: 1) is variation in GSL
diversity across species correlated with species’ phylogenetic distance?
We predicted to observe phylogenetic signal for glucosinolates
diversity, meaning that that closely related species are more similar in
their phytochemical make-up than distant-related species. 2) Does
variation in GSL diversity simultaneously converge with plant species
adaptation to their specific environment? Since along the elevation
gradient of the Alps, similar habitats should generate similar types and
levels of herbivory (Hodkinson 2005), we predicted that adaptation to a
specific environment, not only shapes the plant growth phenotype, but
also structures a unique chemical phenotype. 3) How are different
metrics of phytochemical diversity related to plant-herbivore
interaction? Because each metric of phytochemical diversity can only
capture a fraction of the chemical complexity, we predicted that not all
metrics of phytochemical diversity similarly predict plant resistance
against specialist and generalist herbivores (Wetzel & Whitehead 2020).
With this work, we thus expand on the ecological and evolutionary
processes that drive and maintain phytochemical diversity across space
and time, and integrate the functional axis of phytochemical diversity
with the functional axis of plant growth forms.