Geographic isolation substantially contributes to species endemism on oceanic islands when speciation involves the colonisation of a new island. However, less is understood about the drivers of speciation within islands. What is lacking is a general understanding of the geographic scale of gene flow limitation within islands, and thus the spatial scale and drivers of geographical speciation within insular contexts. Using a community of beetle species, we show that when dispersal ability and climate tolerance are restricted, microclimatic variation over distances of only a few kilometres can maintain strong geographic isolation extending back several millions of years. Further to this, we demonstrate congruent diversification with gene flow across species, mediated by Quaternary climate oscillations that have facilitated a dynamic of isolation and secondary contact. The unprecedented scale of parallel species responses to a common environmental driver for evolutionary change has profound consequences for understanding past and future species responses to climate variation.

Using a series of standardised sampling plots within forest ecosystems in remote oceanic islands, we reveal fundamental differences between the structuring of aboveground and belowground arthropod biodiversity that are likely due to large-scale species introductions by humans. Species of beetle and spider were sampled almost exclusively from single islands, while soil dwelling Collembola exhibited more than tenfold higher species sharing among islands. Comparison of Collembola mitochondrial metagenomic data to a database of more than 80,000 Collembola barcode sequences revealed almost 30% of sampled island species are genetically identical, or near identical, to individuals sampled from often very distant geographic regions of the world. Patterns of mtDNA relatedness among Collembola implicate human-mediated species introductions, with minimum estimates for the proportion of introduced species on the sampled islands ranging from 45-88%. Our results call for more attention to soil mesofauna to understand the global extent and ecological consequences of species introductions.

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.