Incorporating Neglected Insect Larvae in Species Inventories: DNA Barcoding as an Effective Tool for All-Stage Invertebrate Identification in Tree Holes.

Invertebrates, especially insects, are an integral part of biodiversity. Many species live in forest ecosystems where they play a key role in decomposing wood and maintaining ecosystem functions. Nevertheless, global changes, like fires, storms, and pest outbreaks, are impacting insect diversity, reinforcing the need for long-term biomonitoring to understand and tackle these issues. Forests are heterogeneous ecosystems with tree-related microhabitats (TReMs) such as tree holes, which are important for ecosystem diversity. Conventional identification approaches for species inventories are frequently hampered by the extensive and hidden diversity of insect larval stages. Thus, there is a crucial need to develop tools that facilitate inventories of these ecological niches and allow the incorporation of such hidden diversity into long-term monitoring studies. To that end, we explored the biodiversity found in tree holes within French state forests using DNA barcoding and addressed challenges associated with traditional morphological identification methods. Results demonstrate the successful application of DNA barcoding in identifying nearly 62% of all invertebrates sampled from tree holes to the species level. Sampled invertebrates comprised 44% of larvae (566 individuals), of which nearly 50% could be assigned a species name. In total, 108 species and 173 barcode index numbers (BINs, used as species proxy) were molecularly inventoried, and 39% of these identified species were solely represented by larvae in our sampling. Our study highlights the usefulness of DNA-based identification methods and the significance of including larvae in biodiversity assessments to gain insights into species abundance and functional diversity. It also underscores the necessity of ongoing and parallel developments of DNA reference libraries to improve species molecular identification rates and accuracy, and the need to investigate potential non-destructive alternatives for biomonitoring. These efforts aim to ensure thorough and precise monitoring of invertebrate communities in tree holes and similar microhabitats.