Discussion
The development of novel high-throughput molecular methods has paved the
way for a better understanding of how environmental factors shape the
structure of species interaction networks (Nielsen et al. 2018;
Roslin et al. 2019). Using an improved and non-invasive
metabarcoding procedure based on insect feces, we reconstructed the
structure of plant–orthoptera networks across multiple sites along
elevation gradients, thus favoring technical advances in the field of
‘landscape network ecology’. We showed that networks exhibited
structural variation along the ecological gradients, as a result of both
the rewiring of species interactions and shifts in network size.
Networks of high-elevation cold environments displayed reduced levels of
specialization and increased nestedness, which are metrics theoretically
expected to be central components of network stability (Fig. 2, Fig. 4;
Table 1; Appendix S1, Fig. S4a, Table S1). We argue that when increased,
these structural properties confer higher network resilience, presumably
through a more homogeneous distribution of the herbivore interaction
over the available plant species functional space. Theoretical work on
the structure–stability relationship of ecological networks suggests a
positive association between network resilience to species extinction
and structural indices, including connectance and nestedness (Dunneet al. 2002; Memmott et al. 2004; Lafferty & Kuris 2009).
Our empirical analyses along several elevation transects support
theoretical expectations showing that networks in cold environments are
less specialized and more nested, which presumably enhance network
robustness (Burgos et al. 2007; Miller-Struttmann & Galen 2014).
Novel molecular methods enabling the monitoring of network variation in
space, as done in our study, but also in time, should provide new
perspectives for understanding the trophic architecture of species
assemblages.
The observed higher generality for alpine plant–orthoptera networks
agrees with three underlying arguments supporting biotic and abiotic
shifts along elevation gradients: (i) lower environmental
predictability, (ii) less species competition for resources, and (iii)
relaxation of plant chemical defences (Macarthur & Levins 1967;
Hodkinson 2005; Rasmann et al. 2014). First, greater
environmental stresses and variation in the alpine belt (Körner 2003;
Barry 2008) may impose constraints for insects to complete developmental
and reproduction cycles (Hodkinson 2005). In particular, environmental
fluctuation at high elevations may increase resource stochasticity,
which translates into greater spatio-temporal variation of the host
plants than at low elevations (Billings & Mooney 1968). Cooler and more
variable temperatures might also reduce search and digestive efficiency
in ectothermic animals (Hodkinson 2005). In turn, such environmental
unpredictably could be offset through the reinforcement of generalist
feeding behavior (Macarthur & Levins 1967). Overall, orthoptera are
generalist feeders (the median number of host plants in our study was
26). Hence, while food plant fluctuation should largely impact the
evolutionary specialization of more specialized clades, such as the
butterflies (median of eight host plants, Pellissier et al.2012b), orthoptera might more easily compensate for the demographic
fluctuations of food plant species by maintaining a large diet breadth
(Cates 1981) and other factors could contribute to the observed increase
in generality. Second, higher species richness of orthoptera at low
elevations (Appendix S1, Fig. S5) might pressure species to escape
competition by focusing on distinct and more specialized diets
(Macarthur & Levins 1967; Hodkinson 2005). However, we found no
relationship between the species richness of orthoptera and the overall
network specialization (Appendix S1, Fig. S6) and species niche overlap
among orthoptera did not vary along the temperature gradient, indicating
that interspecific competition for plant resources is weakened in
orthoptera (Appendix S1, Fig. S4c, Table S1). Third, it was previously
shown that alpine plant communities are less resistant to herbivores
than low-elevation plant communities (Rasmann et al. 2014;
Callis-Duehl et al. 2017). These plant defence patterns could
promote a stronger generalist feeding behavior in colder environments,
through easier digestibility of various plant materials (Moreiraet al. 2018). Our results indicating lower selectiveness of
orthoptera for alpine plants are in agreement with a generalized
reduction in defence levels in plants growing at high elevations
(Rasmann et al. 2014). We documented that orthopteran communities
from cold environments feed on a broader range of plant families and
target more intensively families such as Apiaceae, Boraginaceae,
Caryophyllaceae and Fabaceae, compared with the feeding habits of
lower-elevation orthoptera (Appendix S1, Fig. S7). In particular, we
found that some species at higher elevation were extreme generalist,
feeding on almost all plant species in the communities (Fig. 4). The
presence of those hyper-generalists might contribute to the reported
increase of nestedness with elevation (Fig. 2c, Table 1), where, more
specialist species interact more often with a proper subset of the most
generalist species (Bascompte et al. 2003; Miller-Struttmann &
Galen 2014). Russo et al. (2019) also showed higher nestedness as
a result of a prevalence of super-generalist feeders and resources.
Altogether, higher generality at the network and species level should
increase the robustness of networks to extinctions.
The increase in generality and nestedness of networks at higher
elevation (lower temperatures) were associated with an increase in
network robustness in cold environments (Fig. 2, Table 1). These results
support previously documented co-variation between network robustness
and generality (Welti et al. 2017) or nestedness (Araújo 2016) in
plant–herbivores systems, and a negative association between
temperature and network robustness (Welti et al. 2019). The
association between network specialization, nestedness and robustness
metrics with the underlying temperature gradient suggests that
ecological or evolutionary factors have led to more robust networks in
more stressful environments. Consistently, increased generality and
nestedness at high elevation may results from a greater number of plants
species involved in realized interactions thus decreasing the weight of
the keystones species (Fig. 3; Appendix S1, Table S2). In general,
orthoptera feed on multiple plant species, so the loss of one plant
species is never sufficient to cause the loss of one species of
orthoptera (averaged keystone score <1), but they still show
some degree of preference as regard to the functional traits of the
plant they are feeding on. In our study, orthoptera showed a preference
for plants with tougher leaves (Fig. 3) which typically correspond to
monocotyledons (Fig. 1; Appendix 1, Fig. S8, Table S2), some of which
were particularly dominant in the studied grasslands (e.g. Bromus,
Festuca and Nardus ), as these herbivores are equipped with
enough mandibular strength to cut through such leaves (Ibanez et
al. 2013a). We found lower and more even keystone scores for alpine
plant species, meaning that the removal of plant species at higher
elevations was associated with lower secondary extinctions (Table 3).
The decrease in the keystone score of grasses at higher sites might also
be associated with the decline in the cover of grass vegetation with
increasing elevation (Appendix S1, Fig. S9). At high-elevation sites,
keystone species also had other functional attributes, including higher
SLA but lower C/N values (Fig. 3) compared with low-elevation plants,
corresponding to more palatable and resource-rich host plants
(Pérez-Harguindeguy et al. 2013), providing herbivores with
higher nutritive content during the short growing season of the alpine
environment. These results suggest that the identity of the keystone
species in plant–orthoptera bipartite systems is determined by a
combination of factors involving plant species abundances and
co-evolutionary mechanisms between insect feeding ability and plant
defence, presumably resulting from mechanical and chemical defence
tradeoffs.
Compared with traditional methods based on visual analyses of feces or
gut content or literature-based documentation of interactions (Nielsenet al. 2018), the DNA metabarcoding procedure represents an
effective and easily adaptable method for documenting interactions
involving plants and insect species. As a compromise between the spatial
coverage of our study and the available sampling resources, potential
impacts of sampling replication and seasonality and year-to-year change
on diet composition were not assessed here (Mata et al. 2019).
Overall, our approach may open fields of investigation on the possible
spatio-temporal variation in plant–insect interactions by expanding the
means for collecting species interaction data.
Taken together, our results show an increase in plant–herbivore network
generality and nestedness with elevation, which drives variation in
network robustness along the gradient and ultimately gives lower weights
to keystone species in alpine than in lowland environments. Shifts in
abiotic components can alter the structuring of species interactions
directly or indirectly (Welti & Joern 2015; Tylianakis & Morris 2017),
by influencing the different aspects of the species interface through
both abiotic and biotic pressures. We suggest that the observed patterns
of network structural variation regarding elevation represent entangled
responses of networks to environmental predictability and plant chemical
defences, although further investigation would be required to confirm
this possibility. Generally, orthoptera are not very sensitive to
extinction, in that the loss of multiple plant species is necessary to
cause secondary extinctions. Nevertheless, intensification of land use
practices in lower-elevation mountain grasslands, for instance through
the use of fertilizers, can regularly cause the loss of multiple plant
species, which could then lead to extinctions in orthoptera (Chistéet al. 2016). Our study helps paving the way to a better
understanding of the eco-evolutionary factors underlying network
structure along large-scale ecological gradients, but also highlights
how resilient species assemblages are to the accelerated rate of species
extinction.