Building a Comprehensive and Accessible DNA Barcode Database for Fish and Invertebrates in the San Francisco Estuary

Ravi Nagarajan1, Ann Holmes1, Danielle Myers1, Julia Mouat2, Alisha Goodbla1, Mallory Bedwell3, Melinda Baerwald3, Brian Schreier3, Andrew Rypel4, Larry Brown5, & Andrea Schreier1

Background Target Species

Barcode Genes • Environmental DNA (eDNA) methods rely on DNA Mitogenome Common Name Accession COI 12S 16S CytB 18S Scientific Name barcode reference sequences for each species. Yellowfin Goby Acanthogobius flavimanus Green Sturgeon NC_028405 Acipenser medirostris White Sturgeon NC_004743 Acipenser transmontanus American Shad NC_014690 Alosa sapidissima • Some SFE species already have DNA barcode reference sequences White Catfish NC_037012 Ameiurus catus Sacramento Perch Archoplites interruptus available in public databases, but some do not have have barcode data and Sacramento Sucker Catostomus occidentalis Prickly Sculpin NC_036145 Cottus asper many have only partial data. Riffle Sculpin Cottus gulosus Common (European) Carp NC_001606 Cyprinus carpio Threadfin shad NC_009580 Dorosoma petenense • We are building a custom DNA barcode reference sequence database for SFE Western Mosquitofish NC_004388 Gambusia affinis Three-spined Stickleback NC_041244 Gasterosteus aculeatus fish and invertebrates. California roach Hesperoleucus symmetricus Wakasagi Smelt NC_015242 Hypomesus nipponensis Delta Smelt Hypomesus transpacificus • This database is the first phase of a multiyear Prop 1 funded project to develop Tule Perch Hysterocarpus traskii River Lamprey Lampetra ayresi Pacific Lamprey Lampetra tridentata/Entosphenus tridentatus eDNA metabarcoding methods to complement existing SFE monitoring. Hitch NC_033930 Lavinia exilicauda Bluegill NC_015984 Lepomis macrochirus

Fish Staghorn Sculpin Leptocottus armatus • The custom database will be made public as a resource to all scientists and Bluefin Killifish Lucania goodei Inland (Mississippi) Silverside Menidia beryllina will enable accurate taxonomic assignments from eDNA metabarcoding Smallmouth Bass NC_011361 Micropterus dolomieu Spotted Bass Micropterus punctulatus data. The data will also be useful for other genetic applications. Largemouth Bass NC_008106 Micropterus salmoides Striped Bass NC_014353 Morone saxatilis Hardhead Mylopharodon conocephalus Steelhead/Rainbow Trout NC_001717 Oncorhynchus mykiss Kokanee Salmon NC_008615 Oncorhynchus nerka Chinook Salmon NC_002980 Oncorhynchus tshawytscha Sacramento Blackfish Orthodon microlepidotus Largescale Loach NC_023803 Paramisgurnus dabryanus Bigscale Logperch NC_008111 Percina macrolepida Starry Flounder NC_010966 Platichthys stellatus Sacramento Splittail NC_033942 Pogonichthys macrolepidotus Publicly available Sacramento Pikeminnow Ptychocheilus grandis reference sequence Longfin Smelt Spirinchus thaleichthys Shimofuri Goby NC_015992 Tridentiger bifasciatus (not necessarily from Approach TOTALS: 23 39 31 27 37 8 SFE)

No reference Extract PCR amplify Mitogenome sequence Collect SFE ~5 barcode DNA Assemble Common Name Accession COI 12S 16S CytB 18S Scientific Name specimens sequencing database Bay barnacle Amphibalanus improvisus DNA regions Amphibalanus subalbidus Amphibalanus subalbidus Yukon floater Anodonta beringiana California floater mussel Anodonta californiensis Partial reference Woebegone floater Anodonta dejecta sequence Western floater Anodonta kennerlyi Oregon floater Anodonta oregonensis Willamette floater Anodonta wahlamatensis Chinese mystery snail NC_035734.1 Cipangopaludina chinensis Collection of invasive Japanese mystery snail LC514194.1* Cipangopaludina japonica zebra mussels Asian clam NC_046410.1 Corbicula fluminea Corophium Corophium spp. (Dreissena polymorpha) California bay shrimp Crangon franciscorum from San Justo Quagga mussel Dreissena bugensis Reservoir, CA. Zebra mussel KY091877.1* Dreissena polymorpha Eogammarus confervicolus Eogammarus confervicolus Photos credit: Chinese mitten crab NC_006992.1 Eriocheir sinensis Heather Perry, CDFW Siberian prawn MF687349.1 Exopalaemon modestus Gammarus daiberi Gammarus daiberi Western ridged mussel Gonidea angulata Hyalella azteca NC_039403.1 Hyalella azteca Hyperacanthomysis shrimp Hyperacanthomysis longirostris • Specimen collection and DNA extractions ongoing Atlantic Macoma Macoma petalum Western pearlshell NC_015476.1 Margaritifera falcata

Invertebrates False dark mussel Mytilopsis leucophaeata • PCR primer testing complete (see below) Neomysis shrimp Neomysis mercedis Oriental shrimp Palaemon macrodactylus Pea cockle or pea clam Pisidium casertanum • Goal for database is to sequence ~5 PCR products for each specimen Ridged-beak peaclam Pisidium compressum Golden apple (mystery) snail NC_037691.1 Pomacea bridgesii Channeled apple snail NC_024586.1 Pomacea canaliculata • Some barcoding genes contain multiple barcodes Overbite clam Potamocorbula amurensis New Zealand mud snail NC_020790.1 Potamopyrgus antipodarum • eDNA metabarcoding accuracy is improved by sequencing >1 barcode for each sample Synidotea laticauda Synidotea laticauda Banded mystery snail Viviparus georgianus • For reference database sequencing, we are prioritizing PCR primers that will TOTALS: 12 34 12 12 12 15 give us sequence information for multiple barcodes from one PCR/sequencing reaction • DNA sequencing started July 2020 (delayed due to COVID-19 lab shutdown) • Most SFE fish and invertebrate species have existing COI reference sequences • Some extracted will also be used to create known DNA mixtures to • References for other commonly used barcode genes (12S, 16S, Cytb, and 18S) are still eDNA metabarcoding protocol prior to using with eDNA samples needed for many SFE species • Our objective is to fill in as many of these gaps as possible PCR primers tested for Sanger DNA sequencing:

Gene Type Primer names (Forward/Reverse) Reference Cytochrome C Oxidase 1 (COI) mitochondrial jgLCO1490/jgHCO2198 Geller et al 2013

12S rRNA mitochondrial tRNA-Phe/teleo_R Doble et al 2019 12S rRNA mitochondrial MiFish-U-F/MiFish-U-R Miya et al. 2015 Summary 16s rRNA mitochondrial Ac16s-F/Ac16s-R Evans et al. 2015 • Database expected completion by December 2020. 16s rRNA mitochondrial 16s_Metazoa_fw/16s_Metazoa_rev Shelton et al 2016 16s rRNA mitochondrial 16S-V5-F/16S-V5-F Riaz et al. 2011 • The database will improve the accuracy and utility of eDNA Cytochrome B (CytB) mitochondrial L14912/H15149c Burgener and Hubner 1998 metabarcoding. 18S rRNA nuclear SSU-F1/SSU-R568 Tanabe et al 2015 • Will enable monitoring using eDNA metabarcoding, 18S rRNA nuclear SSU-F1289/SSU-R1772 Tanabe et al 2015 complementing and augmenting existing IEP monitoring.

Author Affiliations References Acknowledgements Funding: CDFW/CA Prop 1 Cory Saltzman (CDFW) Ted Grosholz (UC Davis) • Burgener and Hubner (1998) Mitochondrial DNA enrichment for species identification and evolutionary analysis. European Food Research and Technology 207(4) 261-3. Sakura Evans (CDFW) Betsy Wells (DWR) 1. Genomic Variation Laboratory, UC Davis • Doble et al (2019) Testing the performance of environmental DNA metabarcoding for surveying highly diverse tropical fish communities: A case study from Lake Tanganyika. Environmental DNA 2(1) 24-41 DOI: 10.1002/edn3.43. Martha Volkoff (CDFW) Jesse Adams (DWR) 2. Integrative Genetics and Genomics Graduate Group, UC Davis • Evans et al (2015) Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding. Mol Ecol Res 16, 29-41. • Geller et al (2013) Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys. Mol Ecol Res 13, 851–861 Heather Perry (CDFW) Suisun Marsh Fish Survey • Miya et al (2015) MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R. Soc. open sci. 2: 150088. Ian Ralston (CDFW) Teejay O’Rear (UC Davis) 3. CA Dept of Water Resources • Riaz et al (2011) ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis. Nuc Acids Res 39(21). doi:10.1093/nar/gkr732. • Shelton et al (2016) A framework for inferring biological communities from environmental DNA. Ecological Applications, 26(6), 1645–1659. Chelsea Callahan (CDFW) Dylan Stompe (UC Davis) 4. Department of Wildlife, Fish, and Conservation Biology, UC Davis • Tanabe et al (2015) Comparative study of the validity of three regions of the 18S-rRNA gene for massively parallel sequencing-based monitoring of the planktonic eukaryote community. Mol Ecol Res 16(2) 402-414 doi: 10.1111/1755-0998.12459. Jessica Weidenfeld (UC Davis) John Durand (UC Davis) 5. US Geological Survey Shayan Kaveh (UC Davis) Lisa Thompson (Sacramento Area Sewer Khyana Yearwood (UC Davis) District & Sacramento Regional County Serra Perry (UC Davis) Sanitation District) Victor Martinez (UC Davis) Cara Wademan (UC Davis) Samantha Barnum (UC Davis) Grace Auringer (UC Davis)