DOI: 10.5061/dryad.2jm63xsv4
title: Creating, curating, and evaluating a mitogenomic reference database to improve regional species identification using environmental DNA

description: Species detection using eDNA is revolutionizing global capacity to monitor
 biodiversity. However, the lack of regional, vouchered, genomic sequence
 information—especially sequence information that includes intraspecific
 variation—creates a bottleneck for management agencies wanting to harness
 the complete power of eDNA to monitor taxa and implement eDNA analyses.
 eDNA studies depend upon regional databases of mitogenomic sequence
 information to evaluate the effectiveness of such data to detect and
 identify taxa. We created the Oregon Biodiversity Genome Project to create
 a database of complete, nearly error-free mitogenomic sequences for all of
 Oregon's fishes. We have successfully assembled the complete
 mitogenomes of 313 specimens of freshwater, anadromous, and estuarine
 fishes representing 24 families, 55 genera, and 129 species and
 lineages. Comparative analyses of these sequences illustrate that many
 regions of the mitogenome are taxonomically informative, that the short
 (~150 bp) mitochondrial “barcode” regions typically used for eDNA assays
 do not consistently diagnose for species, and that complete single or
 multiple genes of the mitogenome are preferable for identifying Oregon’s
 fishes. This project provides a blueprint for other researchers to follow
 as they build regional databases, illustrates the taxonomic value and
 limits of complete mitogenomic sequences, and offers clues as to how
 current eDNA assays and environmental genomics methods of the future can
 best leverage this information.
description: <strong>Voucher Specimen and Tissue
 Collection</strong> The study
 area initially encompassed the state of Oregon—the region of interest for
 our eDNA monitoring program—and expanded to a few sites in northern
 California and Washington State (Fig 3). To strategize sample collection,
 we examined historical location records in fish collections such as the
 Oregon State Ichthyology Collection and conferred with local biologists to
 identify current distributions. For cases where we knew or suspected that
 deeply divergent evolutionary lineages existed in the present concept of a
 species, we aimed to include representatives of all lineages. We
 ultimately identified 146 native and nonnative freshwater fish species and
 lineages that are currently found in Oregon and strategized collections to
 span watersheds throughout the state (Appendix S1).  To
 facilitate consistent sampling, we provided sampling kits (Appendix S2,
 Box S1) to collectors that contained a 500-mL Nalgene bottle filled with
 10% formalin, a 2.0 mL cryotube filled with 95% EtOH, a sterile scalpel,
 scissors and tweezers, a bleach wipe, latex gloves, a detailed sampling
 protocol to ensure consistent tissue sampling and data collection
 (Appendix S2, Box S2), and a field notes sheet (Appendix S2, Box S3) for
 metadata collection. Collectors anesthetized and euthanized all fish
 specimens prior to tissue collection by immersion in an aqueous solution
 of Tricaine mesylate (MS-222) (400 mg MS-222, 400 mg sodium bicarbonate, 1
 L water). We instructed all partners to collect a minimum of ~0.5
 cm<sup>3</sup> of tissue from each specimen, which was then
 placed in 95% EtOH for DNA extraction and sequencing. Euthanized fish were
 placed in 10% formalin as voucher specimens, thereby ensuring preservation
 of diagnostic features. <strong>Taxonomic Verification, Accession, and Cataloging
 </strong> Fish
 biologists identified specimens provisionally in the field and then Oregon
 State Ichthyology Collection taxonomists verified or refined those
 identifications by morphological examination and reference to published
 keys (Markle &
 Tomelleri, 2016; Wydoski & Whitney, 2003). The Oregon State Ichthyology Collection is in the
 process of accessioning and cataloging all vouchers and tissues. During
 that process, the curators input the metadata associated with each
 specimen and collection event into a relational database, and the
 full-bodied voucher specimens are transferred from formalin to isopropyl
 alcohol for permanent storage in a dedicated collection facility that
 complies with modern fire and earthquake safety codes. Tissues are stored
 in 2.0 mL cryotubes in -80°C freezers. <strong>Mitogenome
 Assembly</strong> To capture
 geographic genetic variation of each resident species across its
 distribution in Oregon, we sequenced the first collected representative of
 each species and subsequently sequenced specimens collected from separate
 watersheds, when possible. We stored gzipped fastq sequencing files on 2 x
 1TB enterprise NL-SAS hard drives and performed mitogenome assemblies on 4
 x 2.30 GHz 16-core processors using 512GB ECC RAM. Mitochondrial genomes
 were assembled <em>de novo</em> from raw paired reads using
 SPAdes assembler initially (versions 3.12.0-3.15.3) (Bankevich et al., 2012) and getOrganelle 1.6.2 or 1.7.5
 (Jin et al.,
 2020) once released. We
 annotated all mitochondrial sequences using a combination of
 MITOS<sup>2</sup>WebServer (Al Arab et al., 2017; Donath et al.,
 2019) and Geneious 10.2.6
 using annotations from identical or closely-related
 species.
description: Microsoft Excel, LibreOffice, or Microsoft's free XLS Viewer
 can be used to open the Excel files and an unzip utility such as 7-Zip or
 WinZip can be used to unzip zipped fastas. For pdfs, use Adobe Acrobat
 Reader. Open Microsoft Word documents using Microsoft Word, OpenOffice
 Writer or Google Docs.