Beyond conventional pollinators: advancing diversity assessments of neglected insect taxa.

Ideally, biodiversity surveys would sample insect assemblages randomly and without taxonomic bias, yet most protocols still prioritize conspicuous, charismatic, or economically important taxa (eg pollinators). Consequently, Diptera, one of the most species-rich and ecologically important insect orders, remain largely overlooked, with only a few families like Syrphidae routinely included in biodiversity assessments. To address this imbalance, here we tested a protocol designed to better capture the diversity of non-charismatic dipteran groups by comparing hand-netting and pan traps and by overcoming taxonomic impediments with cost-efficient DNA "megabarcoding" using non-destructive DNA extraction and third-generation sequencing. We applied it to brachyceran fly (Diptera: Brachycera) assemblages across four Mediterranean coastal habitats, key for coastal protection and tourism yet poorly documented. We clustered 13,877 cytochrome c oxidase subunit I barcodes into 823 molecular operational taxonomic units (MOTUs) at a 2% distance threshold, more than 10% of all Diptera species recorded for Italy. MOTUs were assigned to 48 families by morphological inspection of a single representative specimen. We then assigned ecological guilds at the family level, enabling joint assessment of taxonomic and functional diversity. Pan traps collected twice as many specimens and reached higher sample coverage than transect counts, while total MOTU richness was similar. However, assemblages differed in composition, indicating that the two methods are complementary, and still shared 27% to 40% of ecological-guild representation across habitats. Our integrative discovery framework reveals outstanding Diptera diversity and provides a scalable framework combining multiple sampling techniques with high-throughput molecular workflows, applicable to other neglected insect taxa and ecosystems.