Unravelling Bulk Ichthyoplankton Diversity in Vietnam: Metabarcoding Validation With Controlled Mock Samples

ABSTRACT

The sustainability of Southeast Asian fisheries hinges on high‐throughput tools for monitoring early life‐stage fish biodiversity. However, applying DNA metabarcoding to hyper‐diverse tropical ichthyoplankton requires rigorous calibration to ensure quantitative reliability. We systematically evaluated the metabarcoding workflow using controlled mock communities, revealing that taxonomic recovery is governed by a stochastic limit of detection at a normalised proxy biomass threshold of ≤ 0.05. Quantitative analysis confirmed a significant linear relationship between specimen size and read abundance ( R ² up to 0.817), demonstrating that biomass‐driven template competition induces frequent false negatives for low‐biomass taxa, a phenomenon exacerbated by increasing community complexity (ANOVA: p  < 0.001). To mitigate these systemic biases, we applied a size‐stratified specimen‐balancing strategy intended to increase the representation of small‐bodied components in natural bulk samples. Applying this optimised workflow to field samples from Khanh Hoa, Vietnam, we identified 139 species and unmasked a North–South biogeographic dichotomy (PERMANOVA: R ²  = 53%, p  = 0.001) driven by transect‐scale environmental gradients and local hydrography. Notably, we identified diversity hotspots requiring > 200,000 reads for saturation, suggesting these sites act as critical larval retention zones. The contrast between functional management zones was highly significant ( p  = 0.002), with the conservation area (Zone B) exhibiting higher alpha richness and a nine‐fold increase in unique indicator species compared to high‐activity areas (18 vs. 2). Our work demonstrates that comprehensive validation is vital for accurate metabarcoding, offering a robust framework to understand how ecological gradients and localised human pressures shape Vietnam's critical marine spawning sites and nursery grounds.