Tidewater goby and estuarine fish records from seining, qPCR and metabarcoding data for Southern California estuaries in 2023

Many studies have shown that environmental DNA (eDNA) sampling can be more sensitive than traditional sampling. For instance, past studies found a specific qPCR probe of a water sample is better than a seine for detecting the endangered tidewater goby, Eucyclogobius newberryi. Furthermore, a metabarcoding sample often detects more fish species than a seine detects. Less consideration has been given to sampling costs. To help managers choose the best sampling method for their budget, I estimated detectability and costs per sample to compare the cost-effectiveness of seining, qPCR and metabarcoding for detecting endangered tidewater gobies as well as the associated estuarine fish community in California. Five samples were enough for eDNA methods to confidently detect tidewater gobies, whereas seining required twice as many samples. Fixed program costs can be high for qPCR and seining, whereas metabarcoding had high per-sample costs, which led to changes in relative cost-effectiveness with the number of locations sampled. Under some circumstances (multiple locations visited or an already validated assay), qPCR was a bit more cost-effective than metabarcoding for detecting tidewater gobies. Under all assumptions, seining was the least cost-effective method for detecting tidewater gobies or other fishes. Metabarcoding was the most cost-effective sampling method for multiple species detection. Despite its advantages, metabarcoding still suffers from gaps in sequence databases, can yield vague results for some species, and can lead novices to serious errors. Seining is still the only way to rapidly assess densities, size distributions, and fine-scale spatial distributions. The manuscript relies on 8 separate data sets and an R file to analyze them.  Each data file has an accompanying metadata file and information file. The subset of data used is provided in the data archive (Schmelzle&Kinziger_occupancy.csv) so that analyses can be reproduced but should be cited as Schmelzle, Molly C.; Kinziger, Andrew P. (2015). Data from: Using occupancy modeling to compare environmental DNA to traditional field methods for regional-scale monitoring of an endangered aquatic species [Dataset]. Dryad. https://doi.org/10.5061/dryad.6rs23

Methods
Water samples were taken for eDNA at four sites known to contain tidewater gobies in the past: 16 samples Calleguas Creek (34 06' 42", 119 04' 54", Ventura County), 21 samples at Ormond Lagoon (34 8' 23", 119 11' 20", just west of NVBC), and 17 samples at Santa Clara River Mouth (34 4' 8", 119 15' 53", Ventura County). In addition, two samples were taken at Mugu Lagoon (34 5' 30", 119 7' 21", Ventura County) and 20 samples were taken at Devereux Slough (34 25' 4", 119 52' 27", Santa Barbara County). I used commercial aquatic eDNA kits from Jonah Ventures ® for US $90 each (this cost includes supplies, sequencing and bioinformatics). At all sites, nearshore water samples were taken for environmental DNA wearing latex gloves to reduce contamination with human DNA. Samples were then filtered through a 1-micron disk filter by pushing water through the filter with a 60cc luer-lock syringe until clogging (mean sample volume: 174 cc +/- 0.141 S.D.). Filter capsules were purged of water before filling with preservative (tris-EDTA) before refrigerating until they were express shipped back to Jonah Ventures ® for sequencing. Seines were taken at each water sampling site for one of the estuaries (Calleguas Creek) that was sampled for eDNA (tidewater gobies collection permit #PER0046428) and matched sites and effort. Seine hauls were 2.4 m wide by 6.4 m distance on average in 0.6 m water depth. Temperature was 21.2 C. conductivity was 32 (close to seawater), and DO (mg/L) was 8.3). Jonah Ventures’ ® methods for qPCR and metabarcoding are summarized as follows. After the samples were received, DNA was extracted using the DNeasy Blood & Tissue Kit. Three qPCR replicates were run for a tidewater-goby-specific primer (Schmelzle & Kinziger 2016). Metabarcoding was done for the mitochondrial 12S ribosomal RNA (rRNA) gene which was PCR amplified from each genomic DNA sample using the MiFishUF and MiFishUR primers with spacer regions. Amplicon size and PCR efficiency were visually inspected and then cleaned by incubating. A second round of PCR was performed to complete the sequencing library construct. Final indexed amplicons from each sample were cleaned and normalized using SequalPrep Normalization Plates and then pooled. Sample library pools were sent for sequencing on an Illumina NovaSeq 6000 (San Diego, CA) at the Texas A&M Agrilife Genomics and Bioinformatics Sequencing Core facility using the SP Reagent Kit v1.5 (500 cycles) (cat# 20028402). Raw sequence data were then demultiplexed using pheniqs v2.10, primers were removed with Cutadapt v3.4, and read pairs were merged, denoised and chimeras were removed with vsearch v2.15.2. Exact sequence variants observed more than 7 times were assigned with a custom best-hits algorithm and a reference database that combined Genbank and a Jonah Ventures voucher sequence record, following a consensus taxonomy with all for any taxonomic level with > 90% agreement across top hits. Raw sequences were vouchered through NCBI SRA (SRP# PRJNA924783).