The Hong Kong mayfly (Ephemeroptera) DNA barcode library: Integrative taxonomy enables regional species inventories and highlights the effect of urbanization on species loss

Freshwater biodiversity underpins ecosystem functioning but is rapidly declining due to urbanization and habitat modification, particularly in Southeast Asia where accurate species inventories remain limited. Integrative taxonomy, combining morphological and molecular approaches, provides a robust framework for generating standardized biodiversity data and biomonitoring assessments. We assessed mayfly (Ephemeroptera) biodiversity across a natural-to-urbanized gradient in Hong Kong using nymph morphology and cytochrome c oxidase subunit I (COI) barcoding to establish the first regional DNA barcode reference library. Our integrated taxonomy efforts resulted in 45 unique morphospecies (i.e. gamma-diversity). Subsequently, the Hong Kong mayfly barcode database includes 146 barcode sequences representing 53 Barcode Index Numbers (BINs), of which 67.3% were newly registered in the Barcode of Life Data System (BOLD), including seven putative undescribed lineages and four new regional records. Additionally, we found that mayfly richness declined significantly with increasing urban intensity (β = –0.308, p = 0.007) and conductivity (β = –0.427, p = 0.002), with elevation showing a marginal effect. Indicator species analysis identified Choroterpes elliptica HK01 as potentially indicative of natural sites and Tenuibaetis pseudofrequentus and Caenis lubrica likely corresponding with peri-urban habitats, while no taxa were indicative of urbanized sites. Our results demonstrate both the substantial mayfly diversity in Hong Kong and highlight the detrimental impacts of urbanization on freshwater assemblages. Critically, the barcode library presented here supports future DNA-based biomonitoring applications (e.g. eDNA metabarcoding), providing an important resource for rapid biodiversity assessment of critical habitats and helping to ensure long-term ecosystem health and stability.