2.3.2. Herbivory and Food Webs

The accurate and repeatable identifications of species is imperative if we are to fully understand the ecology and evolution of interactions among partners in natural and humanaltered environments. This requirement is especially true for specialized interactions, including mutualisms and antagonisms. The application of DNA barcodes as species-level markers has revolutionized our ability to track species interactions and the community networks they form, in boreal, temperate, and tropical habitats.

Food web interactions have been greatly clarified with the application of DNA barcodes. Smith et al. [116] using the CO1 DNA barcode marker were able to verify the food web structure of the spruce budworm and its numerous parasitoids to understand the population dynamics of this major pest of trees in boreal forests. The utility of DNA barcodes to identify the diversity of host plants for herbivorous beetles have been demonstrated in both neotropical [62,117] and Asian tropical forests [118]. These early studies used a limited number of molecular markers and were only able to identity the hosts at the generic or familial level.

The most comprehensive analyses between herbivorous beetles and their host plants have been conducted by García-Robledo and colleagues [72,73,119]. The host-specific relationships between rolled-leaf beetles in the genera *Cephaloleia* and *Chelobasis* (Chrysomelidae) and plants in the order Zingiberales have been well-studied by ecologists [120], but the application of DNA barcodes to both the beetles and the hosts have provided a much more detailed and quantitative measure of these interactions [74]. One of the advantages of using an easily extracted DNA barcode is that the beetles can be identified at any of their life stages and not only as adults as in most previous investigations using morphological features [119]. Once the basic network of food web interactions is established using DNA barcodes, comparisons can be made across habitats, elevations, and temperature gradients. Most recently Palmer et al. [121] have extended this methodology to the interactions among katydids and their host plants in a wet forest habitat in Panama. They found that, in general, these insects consumed a broad range of flowering plants and were rarely specialists on only a few species. It has been shown in numerous cases (e.g., Hebert et al. [70]) that

DNA barcodes can detect the presence of cryptic species, especially in insects. This power of DNA barcoding has greatly improved the understanding of species boundaries in the rolled-leaf beetles, allowing for more precise mapping of the insect–host networks. The detection of these cryptic species clearly demonstrated that the elevational distributions and thermal tolerances of the beetles was much narrower than previously thought, which will have an impact on the food web networks as climate change differentially impacts both host and herbivore migrations [73].

DNA barcodes have also altered our view of why tropical biomes are so diverse. It has long been held that specialized ecological interactions, which are common in tropical forests, will lead to reproductive isolation and speciation, and hence greater biotic diversity in the tropics. One such specialization is that between tropical flowers and the nectar-robbing floral mites that are caried from plant to plant on the bills of hummingbird pollinators [122]. This specialization allows floral mites to easily find mates and reproduce, because many conspecifics accumulate in the flowers of only a few species of plants. This "mating rendezvous hypothesis" [123] accounted for the host specialization in these mites. However, using DNA barcode markers to identify the mites, rather than morphological identification, has now shown that most floral mites are generalists and not specialists [124]. The mating rendezvous hypothesis is no longer supported, at least for mite diversification.

This detailed understanding of herbivore–host interactions using DNA barcodes has also been applied to large mammalian herbivores. In a semiarid African savannah, Kartzinel et al. [125] determined the extent that sympatric mammalian browsers and grazers could partition their diets. After building a library of plant DNA barcodes for the local flora, they quantified the diet breadth, composition, and overlap for seven co-occurring mammal species, ranging in size from dik-diks to elephants using DNA metabarcoding. Earlier conclusions on competition and coexistence in these habitats based on low-resolution analyses were shown to be misleading, according to the more high-resolution taxonomic data provided by the metabarcoding results. This work in Africa has now been extended to demonstrate that the abundance and diversity of food plants is negatively associated with their mammalian herbivores, apparently to avoid consumption [126]. The same type of DNA barcoding protocol has also been adapted to tracking and identifying the vectors of bird-dispersed fruits and seeds in the field [127] in order to build a quantifiable network of frugivores and seed dispersal interactions.

### 2.3.3. Symbiotic Relationships and Plant-Pollinator Interactions

Symbioses, perhaps the most characteristic of "species interactions," entail very close relationships between two or more species living together, and DNA barcodes have facilitated researchers studying such close interactions [128]. In some groups (e.g., fungi, [129]), DNA barcoding has revolutionized the field, especially where symbiotic partners are very closely associated and interactions often exist at a cellular level (e.g., in lichens [130]). The use of DNA barcodes to understand symbioses is common in all major clades, including arthropods [131], vertebrates [132], green plants [133], and fungi [134]. An especially powerful tool for symbiosis-based research is metabarcoding [135], which allows for pooled sequencing from closely associated, symbiotic organisms that are otherwise difficult to isolate. The application of DNA barcodes to more closely track and untangle symbiotic relationships is still in its infancy (see below Section 3.1).

The interactions between plants and pollinators is a symbiotic mutualism that is critical for the survival of both partners. An understanding of the dynamics of these interactions is a priority for plant and insect ecologists to conserve biodiversity and to protect the agricultural crop supply chain. DNA barcodes have been explored for more than two decades as a means to identify plants from the insects that have visited them as pollinators [136]. Given the nature of pollination dynamics, samples removed either from plants or their pollinators can include a mixed community of pollen and, therefore, metabarcoding approaches provide a unique tool to identify the diversity contained in these mixed samples [137,138].

Clare et al. [139] were among the first to apply metabarcoding to study plant–pollinator interactions, extending the concepts earlier proposed by Valentini et al. [68] and Soininen et al. [140]. A key threshold for advancing these methods is a comprehensive DNA barcode sequence reference database. For example, the first national DNA barcode sequence reference database of Wales [141] has provided a benchmark for DNA metabarcoding studies of plant– pollinator interactions and this has recently grown into a comprehensive database for all of the United Kingdom [59]. Together, these databases have proven powerful for reconstructing bee foraging behavior [142–144]. These and other studies [145–147] have built a strong foundation for using DNA metabarcoding to study plant–pollinator interactions.
