Background In light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). Findings Using a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed Nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. Conclusions Sequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost-effective, portable, and universal approach for eukaryote DNA barcoding. Although bulk community analyses using long-amplicon approaches may introduce biases, the long rDNA amplicons approach signifies a powerful tool for enabling the accurate recovery of taxonomic and phylogenetic diversity across biological communities.

SUMMARYIslands are widely considered to be model systems for studying fundamental questions in ecology and evolutionary biology. The fundamental state factors that vary among island systems -geologic history, size, isolation and age -form the basis of mature phenomenological and predictive theory. In this review, we first highlight classic lines of inquiry that exemplify the historical and continuing importance of islands. We then show how the conceptual power of islands as 'natural laboratories' can be improved through functional classifications of both the biological properties of, and human impact on, insular systems. We highlight how global environmental change has been accentuated on islands, expressly because of their unique insular properties. We review five categories of environmental perturbation: climate change, habitat modification, direct exploitation, invasion and disease. Using an analysis of taxonomic checklists for the arthropod biotas of three wellstudied island archipelagos, we show how taxonomists are meeting the challenge of biodiversity assessment before the biodiversity disappears. Our aim is to promote discussion on the tight correlations of the environmental health of insular systems to their continued importance as singular venues for discovery in ecology and evolutionary biology, as well as to their conservation significance as hotspots of endemism.

DNA metabarcoding has developed into a routine tool for ecosystem monitoring in the past years. Recent work suggests that this powerful approach even enables community level analysis of intraspecific genetic diversity, revolutionizing our understanding of biodiversity assembly (Andújar et al., 2021). However, despite its promises, metabarcoding is subject to biases that can substantially distort estimates of diversity. Examples include external contamination of specimens, secondary predation, priming and polymerase biases, copy number variation, PCR and sequencing errors and the formation of chimeric sequences. Many of these biases can be experimentally (Kennedy et al., 2020;Krehenwinkel et al., 2017) or bioinformatically controlled (Edgar, 2016). Yet another problem that is particularly hard to control for are numts. Numts are ubiquitous components of eukaryote genomes and a long-recognized problem in phylogenetics and population genetics (Bensasson et al., 2001), but pose a particular challenge to DNA barcoding (Song et al., 2008).When a pseudogene copy is amplified it can result in incorrect inference of the evolutionary history of the lineage in question. Numts have accumulated in some lineages more than others and comparative genomic analyses are just beginning to highlight the breadth of the problem (Hazkani-Covo et al., 2010). Numt content is often evolutionarily labile (Figure 1) and can evolve quickly even within genera or species, making it particularly hard to identify them. Most evolutionary old numts can be identified by accumulated frameshift mutations and stop codons. However, the high similarity of recently evolved numts or old numts located in highly conserved DNA sequences to true mitochondrial variants makes it difficult to score intraspecific diversity. Numts usually show lower copy numbers than true mitochondrial sequences. But sequence abundance thresholds

The dynamic structure of ecological communities results from interactions among taxa that change with shifts in species composition in space and time. However, our ability to study the interplay of ecological and evolutionary processes on community assembly remains relatively unexplored due to the difficulty of measuring community structure over long temporal scales. Here, we made use of a geological chronosequence across the Hawaiian Islands, representing 50 years to 4.15 million years of ecosystem development, to sample 11 communities of arthropods and their associated plant taxa using semiquantitative DNA metabarcoding. We then examined how ecological communities changed with community age by calculating quantitative network statistics for bipartite networks of arthropod–plant associations. The average number of interactions per species (linkage density), ratio of plant to arthropod species (vulnerability) and uniformity of energy flow (interaction evenness) increased significantly in concert with community age. The index of specialization has a curvilinear relationship with community age. Our analyses suggest that younger communities are characterized by fewer but stronger interactions, while biotic associations become more even and diverse as communities mature. These shifts in structure became especially prominent on East Maui (~0.5 million years old) and older volcanos, after enough time had elapsed for adaptation and specialization to act on populations in situ. Such natural progression of specialization during community assembly is probably impeded by the rapid infiltration of non‐native species, with special risk to younger or more recently disturbed communities that are composed of fewer specialized relationships.

The bulk of models used to understand the species diversification on Madagascar have been constructed using vertebrate taxa. It is not clear how these models affect less vagile species that may interact at a variety of spatial scales. Several studies on vertebrates have divided Madagascar into east-west bioclimatic regions, suggesting there is a fundamental division between eastern wet-adapted and western dry-adapted taxa. An alternative model of ecogeographic constraints shows a north-south division. We test whether the diversification in a small arthropod with variable degrees of dispersal conform to either model of ecogeographic constraints proposed for vertebrate taxa. We employ a molecular taxonomic dataset using ~2 kilobases nuDNA (Wg, LW Rh, Abd-A, 28s) and 790 basepairs mtDNA (CO1), along with geographic and habitat data, to examine the diversification patterns of the ant genus Mystrium Roger, 1862, (Subfamily Amblyoponinae) from Madagascar. The nuclear and mitochondrial phylogenies were both congruent with morphospecies as indicated in a recent revision of the genus. Species of Mystrium practice different colony reproductive strategies (winged queens vs non-winged queens). Alternate reproductive strategies led to inequalities in female dispersal ability among species, providing an additional layer for examination of the impacts of vagility on divergence, especially when measured using a maternally inherited locus. Mystrium species distribution patterns support these models of ecogeographic constraints. Reproductive strategy effected how Mystrium mtDNA lineages were associated with large-scale habitat distinctions and various topographical features. Furthermore, in some cases we find microgeographic population structure which appears to have been impacted by localized habitat differences (tsingy limestone formations, littoral forest) on a scale much smaller than that found in vertebrates. The current system offers a finer scale look at species diversification on the island, and helps achieve a more universal understanding of the generation of biodiversity on Madagascar.