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

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 (R2 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: R2 = 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.