Supplementary Materials for:A preliminary assessment of the biodiversity of inner-coastal zooplankton of southwestern Puerto Rico using a DNA metabarcoding approach

Abstract— Zooplankton communities are fundamental to marine food webs and ecosystem functioning, yet their biodiversity in tropical regions like Puerto Rico remains understudied. It is crucial to understand the zooplankton biodiversity given the current risks posed by climate change and anthropogenic pressures. This study provides the first metabarcoding assessment of zooplankton diversity in La Parguera, southwestern Puerto Rico, combining CO1 metabarcoding with traditional microscopy to offer a comprehensive overview. Zooplankton samples were collected during autumn and winter (2019–2020), across diel cycles and using three mesh sizes (63 µm, 202 µm, 500 µm). Microscopy results confirmed the dominance of calanoid copepods, particularly Paracalanus and Acartia spp., consistent with previous studies. In contrast, metabarcoding revealed a more diverse community than the microscopy methods, with high relative abundance of meroplanktonic mollusks (e.g., Caenogastropoda) and arthropod larvae (e.g., Mithrax hispidus), likely due to primer biases and enhanced detection of larval stages. The CO1 dataset enabled the detection of cryptic taxa (e.g., Aglaophenia latecarinata) using a customized bioinformatics pipeline. Permutational Multivariate Analysis of Variance (PERMANOVA) detected no significant differences in community composition across diel, seasonal, or mesh-size gradients, suggesting short-term stability. This work establishes a critical baseline for zooplankton research in Puerto Rico after two decades of limited study, highlighting the value of integrative approaches to monitor biodiversity shifts in vulnerable tropical ecosystems.Here are the supplementary materials for this research, mostly focusing on the DNA metabarcoding pipeline for the bioinformatics portion of the research (Appendix A) and data obtained from it (Appendix B and Appendix C).