Forest canopies are dynamic interfaces between organisms and atmosphere, providing buffered microclimates and complex microhabitats. Canopies form vertically stratified ecosystems interconnected with other strata. Some forest biodiversity patterns and food webs have been documented and measurements of ecophysiology and biogeochemical cycling have allowed analyses of large-scale transfer of CO, water, and trace gases between forests and the atmosphere. However, many knowledge gaps remain. With global research networks and databases, and new technologies and infrastructure, we envisage rapid advances in our understanding of the mechanisms that drive the spatial and temporal dynamics of forests and their canopies. Such understanding is vital for the successful management and conservation of global forests and the ecosystem services they provide to the world.

Metabarcoding of arthropod communities can be used for assessing species diversity in tropical forests but the methodology requires validation for accurate and repeatable species occurrences in complex mixtures. This study investigates how the composition of ecological samples affects the accuracy of species recovery. Starting with field‐collected bulk samples from the tropical canopy, the recovery of specimens was tested for subsets of different body sizes and major taxa, by assembling these subsets into increasingly complex composite pools. After metabarcoding, we track whether richness, diversity, and most importantly composition of any size class or taxonomic subset are affected by the presence of other subsets in the mixture. Operational taxonomic units (OTUs) greatly exceeded the number of morphospecies in most taxa, even under very stringent sequencing read filtering. There was no significant effect on the recovered OTU richness of small and medium‐sized arthropods when metabarcoded alongside larger arthropods, despite substantial biomass differences in the mixture. The recovery of taxonomic subsets was not generally influenced by the presence of other taxa, although with some exceptions likely due to primer mismatches. Considerable compositional variation within size and taxon‐based subcommunities was evident resulting in high beta‐diversity among samples from within a single tree canopy, but this beta‐diversity was not affected by experimental manipulation. We conclude that OTU recovery in complex arthropod communities, with sufficient sequencing depth and within reasonable size ranges, is not skewed by variable biomass of the constituent species. This could remove the need for time‐intensive manual sorting prior to metabarcoding. However, there remains a chance of taxonomic bias, which may be primer‐dependent. There will never be a panacea primer; instead, metabarcoding studies should carefully consider whether the aim is broadscale turnover, in which case these biases may not be important, or species lists, in which case separate PCRs and sequencing might be necessary. OTU number inflation remains an issue in metabarcoding and requires bioinformatic development, particularly in read filtering and OTU clustering, and/or greater use of species‐identifying sequences generated outside of bulk sequencing.

Metabarcoding of Metazoa using mitochondrial genes may be confounded by both the accumulation of PCR and sequencing artefacts and the co-amplification of nuclear mitochondrial pseudogenes (NUMTs). The application of read abundance thresholds and denoising methods is efficient in reducing noise accompanying authentic mitochondrial amplicon sequence variants (ASVs). However, these procedures do not fully ac-

1. Metabarcoding of Metazoa using mitochondrial genes is confounded by the co-amplification of mitochondrial pseudogenes (NUMTs). Current denoising protocols have been designed to remove PCR and sequencing artefacts, but pseudogenes are not usually recognised by these procedures.Authentic mitochondrial amplicon sequence variants (ASVs), which represent the majority of reads, can be distinguished from PCR-derived errors, sequencing errors and NUMTs (non-authentic ASVs) due to their lower abundances. However, the use of simple read abundance thresholds is complicated by the highly variable DNA contribution of individuals in a metabarcoding sample.2. We show how ASVs that survive standard denoising, but are identified as non-authentic, are consistent with expectations for NUMTs with regard to patterns of phylogenetic relatedness, readabundance, and library co-occurrence. We then propose and demonstrate a new self-validating framework, named NUMT dumping, which allows NUMT filtering strategies to be evaluated by quantifying (i) the prevalence of non-authentic ASVs (NUMT and erroneous sequences) and (ii) the collateral effects on the removal of authentic ASVs (mtDNA haplotypes) in filtered data. We propose several filtering strategies within the NUMT dumping framework, based on the application of read-abundance thresholds, structured with regard to sequence library and phylogeny.3. The framework was validated using mock and natural communities, both of which showed opposing trends for the removal of authentic and non-authentic ASVs, when threshold values for minimum abundance to filter out sequences were increased. Filtering can be optimized to retain less than 5% of non-authentic ASVs while retaining more than 89% of authentic mitochondrial ASVs, or complete removal of non-authentic ASV with 77% of authentic mitochondrial ASVs retained. 4. We provide a program, NUMTdumper, that can be used to evaluate and decide upon the most adequate metabarcoding filtering strategy for specific research objectives, providing a measure of expected prevalence of non-authentic ASVs in metabarcoding datasets. In addition, this evaluation allows the user to quantify effects of taxonomic inflation when ASVs are clustered into OTUs. It improves the reliability of intraspecific genetic information derived from metabarcode data, opening the door for community-level genetic analyses requiring haplotype-level resolution.

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Disentangling the relative role of environmental filtering and spatial processes in driving metacommunity structure across mountainous regions remains challenging, as the way we quantify spatial connectivity in topographically and environmentally heterogeneous landscapes can influence our perception of which process predominates. More empirical data sets are required to account for taxon‐ and context‐dependency, but relevant research in understudied areas is often compromised by the taxonomic impediment. Here we used haplotype‐level community DNA metabarcoding, enabled by stringent filtering of amplicon sequence variants (ASVs), to characterize metacommunity structure of soil microarthropod assemblages across a mosaic of five forest habitats on the Troodos mountain range in Cyprus. We found similar β diversity patterns at ASV and species (OTU, operational taxonomic unit) levels, which pointed to a primary role of habitat filtering resulting in the existence of largely distinct metacommunities linked to different forest types. Within‐habitat turnover was correlated to topoclimatic heterogeneity, again emphasizing the role of environmental filtering. However, when integrating landscape matrix information for the highly fragmented Quercus alnifolia habitat, we also detected a major role of spatial isolation determined by patch connectivity, indicating that stochastic and niche‐based processes synergistically govern community assembly. Alpha diversity patterns varied between ASV and OTU levels, with OTU richness decreasing with elevation and ASV richness following a longitudinal gradient, potentially reflecting a decline of genetic diversity eastwards due to historical pressures. Our study demonstrates the utility of haplotype‐level community metabarcoding for characterizing metacommunity structure of complex assemblages and improving our understanding of biodiversity dynamics across mountainous landscapes worldwide.

Metabarcoding of DNA extracted from community samples of whole organisms (whole organism community DNA, wocDNA) is increasingly being applied to terrestrial, marine and freshwater metazoan communities to provide rapid, accurate and high resolution data for novel molecular ecology research. The growth of this field has been accompanied by considerable development that builds on microbial metabarcoding methods to develop appropriate and efficient sampling and laboratory protocols for whole organism metazoan communities. However, considerably less attention has focused on ensuring bioinformatic methods are adapted and applied comprehensively in wocDNA metabarcoding. In this study we examined over 600 papers and identified 111 studies that performed COI metabarcoding of wocDNA. We then systematically reviewed the bioinformatic methods employed by these papers to identify the state‐of‐the‐art. Our results show that the increasing use of wocDNA COI metabarcoding for metazoan diversity is characterised by a clear absence of bioinformatic harmonisation, and the temporal trends show little change in this situation. The reviewed literature showed (i) high heterogeneity across pipelines, tasks and tools used, (ii) limited or no adaptation of bioinformatic procedures to the nature of the COI fragment, and (iii) a worrying underreporting of tasks, software and parameters. Based upon these findings we propose a set of recommendations that we think the metabarcoding community should consider to ensure that bioinformatic methods are appropriate, comprehensive and comparable. We believe that adhering to these recommendations will improve the long‐term integrative potential of wocDNA COI metabarcoding for biodiversity science.

High‐throughput sequencing (HTS) is increasingly being used for the characterization and monitoring of biodiversity. If applied in a structured way, across broad geographical scales, it offers the potential for a much deeper understanding of global biodiversity through the integration of massive quantities of molecular inventory data generated independently at local, regional and global scales. The universality, reliability and efficiency of HTS data can potentially facilitate the seamless linking of data among species assemblages from different sites, at different hierarchical levels of diversity, for any taxonomic group and regardless of prior taxonomic knowledge. However, collective international efforts are required to optimally exploit the potential of site‐based HTS data for global integration and synthesis, efforts that at present are limited to the microbial domain. To contribute to the development of an analogous strategy for the nonmicrobial terrestrial domain, an international symposium entitled “Next Generation Biodiversity Monitoring” was held in November 2019 in Nicosia (Cyprus). The symposium brought together evolutionary geneticists, ecologists and biodiversity scientists involved in diverse regional and global initiatives using HTS as a core tool for biodiversity assessment. In this review, we summarize the consensus that emerged from the 3‐day symposium. We converged on the opinion that an effective terrestrial Genomic Observatories network for global biodiversity integration and synthesis should be spatially led and strategically united under the umbrella of the metabarcoding approach. Subsequently, we outline an HTS‐based strategy to collectively build an integrative framework for site‐based biodiversity data generation.