Insects are the most diverse terrestrial organisms, yet they are underrepresented in large-scale conservation assessments. To address this gap, the California Insect Barcoding Initiative is developing a publicly accessible, statewide DNA-barcode reference library for California insects to support scalable surveys, establish baseline biodiversity measurements, and generate potential distribution maps that inform conservation planning. Specimens are collected with a hybrid strategy that combines standardized Malaise trapping, to enable replicable sampling, and opportunistic collecting, to maximize taxonomic coverage. To date, we have barcoded over one million specimens; preliminary completeness analyses suggest that current sampling captures roughly 65–69% of the fauna, implying a conservative minimum of circa 61,000 insect species in California. Using all sequenced specimen records, we generated rule-based spatial range interpolations constrained by ecoregion and vegetation type, and used these to infer spatial patterns in insect species richness across the state. We identify areas of both high and low potential species richness, with current peaks in the Southern California Mountains ecoregion and the Mojave Basin and Range ecoregion. Our species richness estimates and spatial patterns are explicitly provisional and are expected to evolve as sampling gaps are addressed. Finally, we make all sequence data, specimen images, and occurrence records publicly available via the Barcode of Life Datasystem. This ongoing effort constitutes the first large-scale DNA barcode-based survey for California insects, providing an expandable foundation for tracking temporal change, testing drivers of insect diversity, and prioritizing regions for conservation and targeted inventory.

Arthropod communities globally are declining while undergoing taxonomic and functional homogenization, with agricultural activity being a strong contributory factor. Here we use DNA metabarcoding to quantify how variation in climate, agricultural intensity, and plant community composition shape spatiotemporal variation in a metacommunity of > 10,000 arthropod species sampled from 29 Malaise traps across 15 sites in southern Ontario, Canada. Local variation in plant community composition and canopy cover best explained arthropod community dissimilarity. Climatic variables followed closely as explanatory factors, driven primarily by seasonal variation in temperature. The proportion of agricultural land at the landscape scale had no detectable effect. Our results suggest that plant community composition, microclimate, and seasonality structured the arthropod metacommunity to considerable degree, factors that are rarely incorporated into assessments of biodiversity loss due to agriculture. We conclude that habitat restoration on marginal lands is likely an effective strategy for promoting arthropod biodiversity in agroecosystems.

The nuclear genomes of most animal species include segments of the mitogenome, but the count of these NUMTs varies greatly. This study examines the incidence of NUMTs derived from a 658 bp region of the cytochrome c oxidase I (COI) gene as a proxy for other coding regions of the mitochondrial genome. Analysis focuses on the most diverse group of terrestrial organisms, insects, because COI-based identification systems play a key role in clarifying their diversity, an essential antecedent to genome sequencing. Nearly 10,000 COI NUMTs ≥ 100 bp were detected in the genomes of 1,002 insect species with a range from 0–443. NUMT counts were similar among congeners, but differences among genera in a family were often large with genome size explaining 56% of the mitogenome-wide variation in counts. While many of these NUMTs possessed an indel or premature stop codon allowing their exclusion, the others could complicate species diagnosis as they averaged 10.1% divergence from their mitochondrial homologue. The count of NUMTs varies widely among insect lineages, peaking in groups that employ direct development or incomplete metamorphosis. They can raise the apparent species count by up to 22% when the 658 bp barcode region is examined while shorter targets (300 bp, 150 bp) elevate exposure (58–111%) to “ghost” species. As a result, NUMTs represent a particular complication for protocols (e.g., eDNA, metabarcoding) which employ short amplicons for biodiversity assessments.

Because DNA metabarcoding typically employs sequence diversity among mitochondrial amplicons to estimate species composition, nuclear mitochondrial pseudogenes (NUMTs) can inflate diversity. This study quantifies the incidence and attributes of NUMTs derived from the 658 bp barcode region of cytochrome c oxidase I (COI) in 156 marine animal genomes. The number of NUMTs meeting four length criteria (>150 bp, >300 bp, >450 bp, >600 bp) was determined, and they were examined to ascertain if they could be recognized by their possession of indels or stop codons. In total, 389 NUMTs <100 bp were detected, with an average of 2.49 per species (range = 0–50) and a mean length of 336 bp +/- 208 bp. Among NUMTs lacking diagnostic features, 52.5% were ≤300 bp, 63.9% were ≤450 bp, and 76.2% were ≤600 bp. Studies examing 150 bp amplicons inflate the OTU count by 1.57x compared to the true species count and increase perceived intraspecific variation at COI by 1.19x (when sequence variants with >2% sequence divergence are recognized as different OTUs). There was a weak positive correlation between genome size and NUMT count but no variation among phyla, trophic groups or life history traits. While bioinformatic advances will improve NUMT detection, the best defense involves targeting long amplicons and developing reference databases that include both mitochondrial sequences and their NUMT derivatives.

To associate specimens identified by molecular characters to other biological knowledge, we need reference sequences annotated by Linnaean taxonomy. In this paper, we 1) report the creation of a comprehensive reference library of DNA barcodes for the arthropods of an entire country (Finland), 2) publish this library, and 3) deliver a new identification tool based on this resource. The reference library contains mtDNA COI barcodes for 11,275 (43%) of 26,437 arthropod species known from Finland, including 10,811 (45%) of 23,956 insect species. To quantify the improvement in identification accuracy enabled by the current reference library, we ran 1,000 Finnish insect and spider species through the Barcode of Life Data system (BOLD) identification engine. Of these, 91% were correctly assigned to a unique species when compared to the new reference library alone, 85% were correctly identified when compared to BOLD with the new material included, and 75% with the new material excluded. To capitalize on this resource, we used the new reference material to train a probabilistic taxonomic assignment tool, FinPROTAX, scoring high success. For the full-length barcode region, the accuracy of taxonomic assignments at the level of classes, orders, families, subfamilies, tribes, genera, and species reached 99.9%, 99.9%, 99.8%, 99.7%, 99.4%, 96.8%, and 88.5%, respectively. The FinBOL arthropod reference library and FinPROTAX are available through the Finnish Biodiversity Information Facility (www.laji.fi). Overall, the FinBOL investment represents a massive capacity-transfer from the taxonomic community of Finland to all sectors of society.