Invertebrates are important for restoration processes as they are key drivers of many landscape-scale ecosystem functions, including pollination, nutrient cycling and soil formation. However, invertebrates are often overlooked in restoration monitoring because they are highly diverse, poorly described, and time-consuming to survey, and require increasingly scarce taxonomic expertise to enable identification. DNA metabarcoding is a relatively new tool for rapid survey that is able to address some of these concerns, and provide information about the taxa with which invertebrates are interacting via food webs and habitat. Here we evaluate how invertebrate communities may be used to determine ecosystem trajectories during restoration. We collected ground-dwelling and airborne invertebrates across chronosequences of mine-site restoration in three ecologically disparate locations in Western Australia and identified invertebrate and plant communities using DNA metabarcoding. Ground-dwelling invertebrates showed the clearest restoration signals, with communities becoming more similar to reference communities over time. These patterns were weaker in airborne invertebrates, which have higher dispersal abilities and therefore less local fidelity to environmental conditions. Although we detected directional changes in community composition indicative of invertebrate recovery, patterns observed were inconsistent between study locations. The inclusion of plant assays allowed identification of plant species, as well as potential food sources and habitat. We demonstrate that DNA metabarcoding of invertebrate communities can be used to evaluate restoration trajectories. Testing and incorporating new monitoring techniques such as DNA metabarcoding is critical to improving restoration outcomes.

Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to detect plant and animal taxa in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. This enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and/or during ‘library preparation’, i.e. when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, the importance of ensuring that data generation is robust and fit for purpose should be at the forefront of practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms. Further, we distil the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.

Invertebrate communities provide many critical ecosystem functions (e.g. pollination, decomposition, herbivory and soil formation), and have been identified as indicators of ecological restoration. Unfortunately, invertebrates are often overlooked in restoration monitoring because they are time-consuming to survey, often require rare taxonomic expertise, and there are many undescribed species. DNA metabarcoding is a tool to rapidly survey invertebrates and can also provide information about plants with which those invertebrates are interacting. Here we evaluate how invertebrate communities may be used to determine ecosystem trajectories during restoration. We collected ground-dwelling and airborne invertebrates across chronosequences of mine-site restoration in three ecologically different locations in Western Australia, and identified invertebrate and plant communities using DNA metabarcoding. Ground-dwelling invertebrates showed the clearest restoration signals, with communities becoming more similar to reference communities over time. These patterns were weaker in airborne invertebrates, which have higher dispersal abilities and therefore less local fidelity to environmental conditions. Invertebrate community recovery was most evident in ecosystems with relatively stable climax communities, while the trajectory in the Pilbara, with its harsh climate and unpredictable monsoonal flooding, was unclear. Plant assay results indicate invertebrates are foraging locally, providing data about interactions between invertebrates and their environment. Thus, we show how DNA metabarcoding of invertebrate communities can be used to evaluate likely trajectories for restoration. Testing and incorporating new monitoring techniques such as DNA metabarcoding is critical to improving restoration outcomes, and is now particularly salient given the ambitious global restoration targets associated with the UN decade on Ecosystem Restoration.