Swabbing as a Non‐Lethal <scp>DNA</scp> Collection Method for Earthworm Barcoding: Performance and Citizen Science Perspectives

Lethal specimen collection followed by destructive tissue sampling is employed routinely in earthworm DNA barcoding studies. However, this approach may be ecologically disadvantageous and risks public disengagement with biodiversity and barcoding research due to negative emotional associations, particularly in Citizen Science (CS) projects. The study describes the performance of swab sampling as a non-lethal and emotionally-adaptive alternative to tissue sampling in earthworm barcoding. Three DNA collection methods, namely swab sampling with silica-based DNA extraction (Swab-QIAGEN), tissue sampling with silica-based DNA extraction (Tissue-QIAGEN), and tissue sampling with resin-based DNA extraction (Tissue-Chelex) were compared for generating cytochrome c oxidase I (COI) barcodes from 40 adult earthworms. All methods showed comparably high PCR amplification success rates (Tissue-Chelex = 100%; Tissue-QIAGEN = 100%; Swab-QIAGEN = 97.5%), except for one recalcitrant Swab-QIAGEN sample. Considering the pool of successfully amplified samples, the sequencing success rate for the Tissue-Chelex, Tissue-QIAGEN, and Swab-QIAGEN methods was 82.5% (33/40), 87.50% (35/40), and 66.7% (26/39), respectively-with 25 of 119 barcodes flagged as sequencing/methodological failures due to unusable contigs, stop codons/indels, or contamination. Although swabs showed lower success rates overall, they produced the greatest proportion of reference-quality barcodes among functional sequences (80.8%; 21/26), indicating their potential for DNA collection compared to Tissue-Chelex (75.8%; 25/33) and Tissue-QIAGEN (68.6%; 24/35). All intra-specimen barcodes, excluding contaminations, received consistent homology-based taxonomic assignments with average similarity percentage above 99%, corresponding to a 90.4% morphospecies-genetic data concordance rate. The average intra-specimen pairwise sequence percentage identity was above 99% (i.e., < 0.1% average sequencing error) across all methods, and no method-specific clustering was observed in the COI barcode tree. Overall, the results indicated a high degree of interoperability and agreement between swab and tissue sampling methods for recovering the same or nearly-identical haplotypes with consistent taxonomic identities from each specimen. However, the swabbing methodology requires standardization before it can be confidently utilized in CS-based earthworm DNA barcoding projects.