Discussion
In this study, we investigated how long-term protection from herbivory
by overabundant white-tailed deer affected the metabolomic profiles of
long-lived woody or semi-woody plant species in a suburban forest
understory. Furthermore, by studying two indigenous and two
nonindigenous, invasive plant species that are subject to different deer
preferences, we could consider how metabolomic responses to deer browse
may affect the ecology of invasion in suburban forests. Below, in light
of the results, we discuss our hypotheses that 1) the metabolomic
profiles of fenced and unfenced plants diverge, and 2) this divergence
is more pronounced for species most affected by deer in our site. First,
we discuss the overall metabolomic variation among the species.
We studied four diverse species, including the indigenous tree speciesN. sylvatica , the indigenous shrub L. benzoin , the
nonindigenous shrub E. alatus , and the nonindigenous semi-woody
shrub R. multiflora . All four species are potentially subject to
severe negative effects from deer herbivory; they are all long-lived and
are therefore exposed to chronic deer browse in from overabundant deer
while growing in the deer browse zone. Their shared understory forest
environment has very limited light , so losing photosynthetic tissue to
deer herbivory can be a serious problem for any of these species.
Although the four species share these morphological and ecological
similarities, they are rather distant phylogenetically. All belong to
different plant Orders (Figure 9 ), which could impose
metabolomic variation among them based solely on genetic distance .
Additionally, they possess some distinct chemical or morphological
features that could affect their metabolomes. Lindera benzoin is
notably chemically defended , with very aromatic foliage, R.
multiflora is defended with large prickles, and E. alatusproduces tough, corky protrusions from its branches. In contrast,N. sylvatica possesses none of these features. Given this
diversity, it was not surprising that the four species’ metabolomes were
quite distinct, with only 1.1% of the 2,333 detected metabolite
features shared among them, and just 23% of the putative metabolic
pathways shared among all four species (Figure 2A, D ).
The dendrogram based on all metabolites produced four distinct clusters
for the four species, but the two nonindigenous species, E.
alatus and R. multiflora , clustered together (Figure
2C ). In a recent study in eastern US forests, demonstrated metabolomic
divergence between a large group of indigenous and nonindigenous species
in the same plant community, along with evidence for greater regional
invasion frequency in nonindigenous species with greater chemical
novelty. In our case, with just two indigenous and two nonindigenous
species, it is not possible to confidently attribute the clustering to
their indigenous status, although our result is consistent with Sedio et
al’s observation. Also, E. alatus and R. multiflora happen
to be more closely related phylogenetically than are the two indigenous
species we studied (Figure 9 ); so their greater metabolomic
similarity could be due simply to genetic relatedness rather than some
similar physiology related to being successful invasive species. We also
note that the two indigenous species, N. sylvatica and L.
benzoin , shared the largest number of putative metabolic pathways that
the detected metabolites may be part of, possibly suggesting greater
similarity in their physiological ecologies. While variation among the
four species based on the metabolite features is quite clear, any
prediction that divergence in these metabolites indicates differences in
the pathways is rather speculative, since the connection from
metabolites to pathways is based on the only knowledge available at this
point, which is from the model plant Arabidopsis thaliana . We
must be very cautious when applying such evidence to quite different
species and contexts , such as woody species in a forest understory.
Even given the differences among the species enumerated above, we had
predicted that their shared suburban forest understory environment, with
its overabundance of deer, would lead to an across-the-board divergence
of the plants’ metabolomes in fenced versus unfenced plots. In support
of this hypothesis, we did indeed observe a global divergence
(Figure 3A, 3B ). We suspect that the difference was caused
mainly by protection from deer herbivory in the fencing treatment, which
is widely considered the common effect of deer exclosure . However,
preventing deer access also may alter other ecological variables that
could affect plant stress and influence a plants’ metabolome, i.e.
trampling of plants; soil compaction and therefore more difficult root
penetration that negatively affects the soil microbial community and/or
access to soil water and nutrients; elimination of deer fecal
deposition, thereby altering soil nutrients; and release from herbivory
for the entire plant community, creating increased competition with
other plants . An important aim of ongoing research in deer-related
plant ecometabolomics will be to disentangle all of these possible
effects of deer on plant metabolomes in natural communities.
The overall divergence in the metabolomes of fenced and unfenced plants
is by no means the entire story; rather, there were notable distinctions
among the species/fencing treatment combinations in metabolites that
were the main drivers of the fenced/unfenced divergence (Figure
3C ). For the top five of these metabolites, a significant difference
between fenced and unfenced samples within a species appeared in just
some: N. sylvatica for metabolites 1, 2, 4, and 5; L.
benzoin for metabolites 2 and 3; R. multiflora and E.
alatus for metabolite 5. It should be noted that these were
conservative pairwise tests, and the means in Figure 3C suggest
additional marked differences, i.e. metabolite 3 in R. multifloraand N. sylvatica and metabolites 2 and 5 in E. alatus . In
any case, the species’ responses to protection from deer were variable
for these five metabolites, and there was variation in whether the five
increased or decreased when exposed to deer. For metabolites 1, 2, and
4, the significant differences were due to greater production in plants
exposed to deer, but for metabolites 3 and 5, plants protected from deer
by fencing had greater amounts. These findings on the top five
metabolites responsible for the global fenced/unfenced divergence
therefore partially support the idea that metabolites involved in plant
defense responses would be downregulated in fenced plots. More detailed
knowledge of these chemicals’ roles in the defense physiology of woody,
understory plants is needed to fully understand why some were
upregulated and some downregulated.
The separate analyses on each species provides further insight on the
responses of defense-related metabolites to deer fencing and also
enables discussion of our second hypothesis, that the effect of fencing
on a plant’s metabolomic profile is stronger for plant species that are
more preferred by deer. Support for this hypothesis would add credence
to the idea that it is herbivory by deer, rather than other
fencing-related impacts, that affects the metabolome. Out of the four
species, N. sylvatica stood out as the one with a metabolomic
profile most affected by protection from deer; only for this species did
the fenced and unfenced samples clearly cluster into separate groups on
both the PCA based on all of its detected metabolites and in the HCA
based on its top 30 significantly different metabolites (Figure
4 vs. Figures 5, 6, and 7 ). Some of these top metabolites are
putatively involved in pathways that produce intermediate compounds that
can be used to produce defense secondary metabolites, e.g. pentose
phosphate pathway and cyanoamino acid metabolism , or are involved in
indirect defenses, e.g. monoterpenoid biosynthesis . Although fencing
did not separate the other three species into distinct treatment-based
clusters on the PCA plot, a significant numbers of metabolite features
exhibited significant changes in their accumulation based on fencing
treatment (Figures 5D, 6D, and 7D ).
Deer did, indeed, browse N. sylvatica frequently, but the other
species also were commonly browsed (Figure 1 ). In the summer of
2018, the same season when the samples were taken, N. sylvaticaand E. alatus were browsed the most and at very similar rates,
but the differences among the four species was not statistically
significant. The browse rates from all observations pooled across all
observed individuals from 2012-2019 provides more comprehensive
knowledge of deer preference for these four species in the site, and
revealed that the two indigenous species, N. sylvatica andL. benzoin , were preferred over the two non-indigenous species,E. alatus and R. multiflora . Full support for our second
hypothesis would therefore require that both N. sylvatica andL. benzoin exhibit more divergent metabolomic profiles between
the fenced and unfenced plants than did the less-preferred nonindigenous
species, yet such divergence was only clear in N. sylvatica .
There are several aspects of herbivory that our data did not address and
which may be key to understanding the variation among species in their
metabolomic responses to protection from deer; these are all worth
further study. First, as in other studies, e.g. , we used the proportion
of observed plants with presence of browse signs as our metric for deer
browse. A more fine-grained metric that captures the intensity, rather
than presence, of browsing on a species could be a better predictor of
how metabolomes respond to overabundant deer.
Second, plants are affected by herbivory not just by whether they are
attacked, but also by their tolerance of any herbivory that does occur ,
and the four species we studied may differ in tolerance. For example,N. sylvatica is the only tree of the four species we studied, and
this plant architecture, with one central stem and terminal bud, could
make its seedlings less tolerant of a deer browse event that removes
that bud, compared to more branched shrub species with more meristems .
We may then expect a lack of tolerance to be correlated with stronger
chemical defenses against herbivory , although some recent evidence for
this idea is equivocal or negative ). In any case, variation in
tolerance to deer herbivory may correlate to variation in a species’
metabolomic profile.
Finally, our framework for investigating the effect of deer on the
plants’ metabolomes relied on comparing plants that had been protected
from chronic deer herbivory for years (in fenced plots) versus those
exposed to deer (in unfenced plots). Given the overall high browse rates
on these species, it was reasonable to assume that at least some of the
exposed plants would have experienced herbivory in the recent past,
leading to metabolomic profiles different from protected plants.
However, little is known about the timing of herbivory-induced
metabolite production in long-lived woody plants. While there is
evidence that woody plants maintain increased levels of some induced
defense chemicals for months following herbivory , other studies in
herbaceous plants showed that the metabolomic response to herbivory can
be very rapid and then quickly wane . Similarly, the priming of the
metabolome caused by an herbivory event, which readies the plant or
neighboring plants to rapidly defend against subsequent herbivory, may
persist for months in woody plants, and even throughout the plant life
cycle of herbaceous plants, or may last only days .
The four species included in this study exhibited variation in their
metabolomes, but also in their ecological success in response to
protection from deer. The connection between these responses is worth
consideration. Nyssa sylvatica was the only species of the four
that, in both measures of ecological success, percent cover and height,
showed statistically significantly greater values in fenced vs. unfenced
plots after 6.5 years (Figure 8 ). This suggests that N.
sylvatica is particularly vulnerable to deer pressure in this forest.
It was indeed browsed at one of the highest rates, suffering tissue
loss, but it also was the one species that showed a clear metabolomic
difference in fenced vs. unfenced plots, with increased production in
deer-exposed plants of potentially costly secondary metabolites that are
involved in defense-related pathways. The increased metabolite
production did not appear to protect it from browse relative to the
other three species, since it had among the higher browse rates in
unfenced plots. Thus, we may expect this indigenous tree species to
decline under severe, chronic deer herbivory, as in this suburban
forest.
In contrast, one of the invasive species, R. multiflora , actually
did better in the unfenced plots exposed to deer pressure, with a
significantly greater mean height and a trend of increased cover.
Although R. multiflora was browsed, it had among the lower browse
rates and much less difference between metabolites in fenced vs.
unfenced plants. This all suggests that R. multiflora is
resistant and/or tolerant of deer browsing and, under the severe deer
pressure in suburban forests, even may gain a competitive advantage over
other, more susceptible species. Thus, we may expect this nonindigenous,
invasive species to increase in the plant community.
It is important to note that species’ responses to deer in a community
are specific and likely cannot be generalized into, for example,
indigenous vs. nonindigenous invasive species. Of the other two species,
the indigenous L. benzoin did not show a strong difference among
the metabolites in fenced vs. unfenced plants and exhibited only a weak
positive effect on growth when protected from deer in the fences, but it
had among the highest browse rates. The nonindigenous, invasive E.
alatus was as highly browsed as the indigenous N. sylvatica in
the summer of 2018, yet like the indigenous L. benzoin , had only
a weak positive growth response to protection from deer, and did not
have clear difference in its metabolites in fenced vs unfenced plants.
This study has set the stage for further research on how severe, chronic
deer pressure affects the metabolomes of long-lived species in forests,
with possible consequences for the ecology of communities that are now
composed of a mix of indigenous and nonindigenous species, as in many
suburban forests. While our research clearly indicates that protection
from overabundant deer in suburban forests can influence a plants’
metabolome, including affecting metabolites putatively involved in plant
defense and stress pathways, it is only a first step. Needed next are
ecometabolomic studies that include quantification of deer preference
and herbivory intensity in various ways, measurements of tolerance to
deer herbivory, documentation of the timing of metabolomic responses to
deer herbivory in long-lived plants, and determination of the chemical
identities of significant metabolite features.