Nanopore sequencing of nested nrDNA barcodes reliably identifies orchid bees (Euglossini, Apidae)

Orchid bees (Euglossini, Apidae) are Neotropical insects whose species-level identification can depend on minute morphological characters, some difficult to see or analyze. In such cases, DNA barcoding may facilitate identification by comparing standardized DNA sequences with reference libraries. The mitochondrial cytochrome c oxidase I (COI) marker widely used in animals does not, however, provide uniform species-level resolution across bee lineages. We developed an adaptive-length nuclear ribosomal DNA (nrDNA) barcoding framework based on overlapping Nanopore-sequenced markers spanning approximately 500 to 5500 bp for 114 Euglossini species. By matching barcode length to specimen quality, material with varied preservation histories was processed within a single analysis. Leave-k-out validation with IDTAXA achieved more than 96% identification success for two longer barcodes, while performance was lower for the shortest. Combining barcode lengths within one reference library maintained high identification success, and confidence filtering reduced overclassification when species were absent from the reference library. For orchid bees, this framework permits affordable high-throughput identification and supports targeted taxonomic verification and revision. Combining adaptive barcode lengths in one analytical framework offers a general design principle for long-read reference-library construction. Its performance must now be tested in other groups.