Environmental DNA metabarcoding elucidates freshwater mussel diversity and occupancy to facilitate improved management and conservation

Aim: Freshwater mussels are considered among the most at-risk taxa in the world. As such, comprehensive monitoring assessments of what abiotic and biotic factors influence mussel occupancy will be vital for guiding effective conservation. Here, we analyzed vertebrate and mussel eDNA metabarcoding data to explore the influence of biotic (i.e., host fish diversity, predator presence, and community composition) and abiotic (i.e., drainage size, forest cover, stream order) factors on freshwater mussel populations.
Location: This study utilized water samples and tactile survey data collected from streams throughout Fort Johnson, Louisiana.
Methods: We first evaluated the effectiveness of environmental DNA (eDNA) metabarcoding for characterizing freshwater communities, based on previous conventional tactile surveys. Next, we used eDNA metabarcoding analysis for freshwater mussels and vertebrate species alongside remote sensing data to within an occupancy modeling framework to assess how vario..., Two separate eDNA metabarcoding libraries, one for freshwater mussels and one for vertebrates, were prepared using the same eDNA samples. For each library, a two-step PCR protocol was used following the Illumina 16S metagenomic sequencing library preparation guidelines, modified to target either a 180–200 bp fragment of mussel 16S ribosomal ribonucleic acid (rRNA) (Coghlan et al., 2021) or an 85–­117 bp fragment of vertebrate 12S rRNA (Riaz et al., 2011; Table 1). For the first PCR (PCR1), reactions were performed in triplicate for all samples, negative controls (sterile, molecular grade water), and positive controls consisting of Inflated heelsplitter (Potamilus inflatus (Lea, 1831); 0.05 ng/μL) and European glass lizard (Pseudopus apodus (Pallas, 1775); 0.1 ng/μL) genomic DNA for the mussel and vertebrate libraries respectfully. Each 25 μL PCR1 reaction for the mussel metabarcoding library consisted of 3 μL of DNA template, 12.5 μL of Q5® High-Fidelity 2X Master Mix (NEB), 0.5 μL of 5..., , # Environmental DNA metabarcoding elucidates freshwater mussel diversity and occupancy to facilitate improved management and conservation

[https://doi.org/10.5061/dryad.12jm63z7h](https://doi.org/10.5061/dryad.12jm63z7h)

## Description of the data and file structure

Metabarcoding results were generated from aquatic eDNA samples. For a full assessment of the methods please see \"Environmental DNA metabarcoding elucidates freshwater mussel diversity and occupancy to facilitate\". Data consists of raw demultiplexed ASV files, with each file representing a unique sample. The samples are split into results from our vertebrate (Vert_Raw_Seqs*) and mussel *(Mussel_Raw_Seqs) sequencing library.

### Files and variables

#### File: Metabarcoding\_Manuscript-selected.zip

**Description:** The raw fastq files from both the vertebrate and invertebrate metabarcoding analysis. For the vertebrate library, naming convention is as follows... site name, location of sample (control/blank, left, middle, r...