Subterranean ecosystems host a diverse range of ancient fauna, but studying these ecosystems is challenging due to significant sampling difficulties. Environmental DNA (eDNA) metabarcoding offers a promising approach for monitoring subterranean biodiversity, yet issues such as primer bias and non-target amplification can complicate its effectiveness. Thus, thorough validation of metabarcoding primers is crucial for accurate and comprehensive assessments of subterranean faunal diversity. This study aimed to address the need for robust primer validation through in silico, in vitro and in situ analyses, shedding light on primer performance across various subterranean taxa. The primary objective was to evaluate the effectiveness of COI metabarcoding primers for assessing subterranean faunal diversity. In silico analyses involved curating COI sequences from the Barcode of Life Database (BOLD) and selecting 14 primer combinations for in vitro testing using mock communities. Results revealed varying primer performance in terms of PCR efficiency and detection limits across different taxa. One primer combination (BF1/jgHCO2198) detected 82% of taxa in the mock community, but only at high DNA concentrations of the target taxa. The highest proportion of subterranean taxa detected in a diluted mock community was 68% using the fwhF2/fwhR2n primer combination. For in situ field validation, this same primer set detected 13 out of 16 subterranean taxa identified in haul net samples, along with an additional four taxa not identified by haul net. These findings highlight the potential of COI metabarcoding and the critical importance of primer selection for eDNA studies aimed at conserving subterranean biodiversity.
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