*3.1. Metabarcoding*

DNA metabarcoding [135] has emerged as a powerful technique to rapidly characterize species composition, species interactions, and—when combined with trait databases—functional aspects of biological diversity in communities. This method leverages high-throughput sequencing technology to sequence and/or extract DNA barcodes from pooled community or environmental samples. These samples represent DNA isolated from multiple species or other taxa that have been collected in bulk and targeted sequencing is performed on libraries enriched with (typically) DNA barcode amplicons [182]. Metabarcoding is an emergen<sup>t</sup> field that leverages expansive DNA barcode sequence databases and the increasingly high-throughput capacity of DNA sequencing technology.

This technique allows ecologists to explore species interactions through a new lens and is illuminating species distribution and occurrence from ecosystems and habitats that have remained all but invisible. Metabarcoding is able to provide high-resolution inventories from the hidden worlds of below-ground microbial diversity [183], freshwater [184] and marine [185] benthic communities, and the movement and dispersal of airborne fungi [186] and plants [187]; however, this method is dependent upon well-curated reference collections and databases [188].

Beyond enhanced characterization of species communities, metabarcoding has been used to explore species interactions in a variety of contexts. Some of the earliest applications of DNA metabarcoding involved the analysis of vertebrate diets [189] and this method remains a powerful tool for understanding herbivory and predation (see [125,190–192]). More recently, metabarcoding has been used to reconstruct plant–pollinator networks [146,147] and identify economically important taxa [155] or those relevant to human health [193,194].

DNA metabarcoding was developed using short-read high-throughput sequencing platforms and while these are still the norm, they pose some limitations for the technique [8], especially for longer DNA barcode loci (e.g., *matK* for plants). As the technological standard moves toward long-read sequencing approaches, new sequencing platforms and software [195] are being developed. Some recent programs (e.g., Sahlin et al. [23]) have already been used to successfully extract DNA barcode sequences from mixed samples in previously published long-read data.
