DOI: 10.5061/dryad.12jm63z7h
title: Data from: Environmental DNA metabarcoding elucidates freshwater mussel diversity and occupancy to facilitate improved management and conservation

description: 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
 various biotic and abiotic variables impact freshwater mussel eDNA
 occupancy. Results: Our eDNA metabarcoding survey largely agreed with both
 historical and contemporary surveys on Fort Johnson, while uniquely
 detecting Pleurobema riddellii, a proposed threatened species under the US
 Endangered Species Act. We also found that eDNA detections and occupancy
 had strong seasonal variation, with increased read abundance and diversity
 in the spring. Vertebrate, fish, and predator diversity, as a function of
 habitat quality, were strongly predictive of mussel occupancy, supporting
 the concept of land managers focusing on the entire ecosystem for mussel
 conservation. Lastly, we found that percent forest cover and drainage
 basin size interact to influence mussel eDNA occupancy, informing habitat
 preferences for mussel species of interest (i.e., the mussels preferred
 larger drainage sizes and perennial streams). Conclusions: Our results
 demonstrate that combining eDNA metabarcoding of target and non-target
 species with occupancy modeling can provide insights into the ecology of
 freshwater mussels and is a useful tool to improve their conservation and
 management.
description: 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 (<em>Potamilus inflatus
 </em>(Lea, 1831); 0.05 ng/μL) and European glass lizard
 (<em>Pseudopus apodus </em>(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 50
 mg/ml bovine serum albumin, 6 μL of sterile, molecular grade water, and
 1.5 μL of each 10 μM primer. The vertebrate library followed the same
 formula except each reaction used 5 μL of DNA template and 4 μL of
 sterile, molecular grade water. Thermocycling conditions of the mussel
 metabarcoding library for PCR1 included a 98°C incubation step for 5 min,
 followed by 35 cycles of 98°C for 10 s, 55°C for 1 min, and 72°C for 20 s,
 with a final extension at 72°C for 2 min. Thermocycling conditions of the
 vertebrate metabarcoding library for PCR1 included a 98°C incubation step
 for 5 min, followed by 35 cycles of 98°C for 10 s, 58°C for 30 s, and 72°C
 for 30 s, with a final extension at 72°C for 7 min. Triplicate reactions
 for each sample were pooled and amplification was verified via gel
 electrophoresis. Pooled PCR1 products were size-selected and cleaned with
 AMPure XP beads (Beckman Coulter) using a 0.7× and 0.75× bead:sample ratio
 (right side) for the mussel and vertebrate samples, respectively, followed
 by a 1.2× bead:sample ratio (left side) for both libraries, and confirmed
 via gel electrophoresis. Unique dual indexes (10 bp) and Illumina
 sequencing adapters were then annealed to the amplified products using IDT
 for Illumina UD Indexes (Table 1) with a subsequent PCR step
 (PCR2). PCR2 was performed in duplicate for each
 sample, including negative and positive controls. PCR2 reaction and
 cycling protocols were identical for both libraries with each 25 μL PCR2
 reaction consisting of 2 μL of PCR1 template, 12.5 μL of Q5® High-Fidelity
 2X Master Mix (New England Biolabs), 5.5 μL of sterile, molecular grade
 water, and 2 μL of each 10 μM IDT-Illumina UD index primer. Thermocycling
 conditions for PCR2 started with a 98°C incubation step for 3 min,
 followed by 10 cycles of 98°C for 30 s, 55°C for 30 s, and 72°C for 30 s,
 with a final extension at 72°C for 5 min. Duplicate reactions for each
 sample were pooled and amplification was verified via gel electrophoresis.
 Pooled PCR2 products were size-selected and cleaned with AMPure XP beads
 (Beckman Coulter) using a 1.0× and 1.1× bead:sample ratio (left side only)
 for mussel and vertebrate libraries, respectively, and confirmed via gel
 electrophoresis. PCR2 samples were normalized based on DNA concentrations
  quantified via Qubit dsDNA HS kit (Invitrogen) and pooled into
 sub-libraries by PCR plate. AMPure XP bead cleanups using 0.9× and 1.0×
 bead:sample ratio (left side only) for mussel and vertebrate libraries,
 respectively, were performed again on each sub-library to concentrate DNA
 and ensure primer removal prior to sequencing. Sub-libraries were then
 normalized, pooled, and submitted for sequencing at the W. M. Keck Center
 (University of Illinois, Urbana, IL) on the Illumina NovaSeq platform
 using the SP flow-cell with 2 × 250 bp paired-end reads, with an output of
 approximately 750 million reads.
description: # 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, right), and date of collection. The mussel library naming convention is more varied, but we have included a key to match the file name to the sample ID.