Metabarcoding and Metagenomics 5: 35–42 DOI 10.3897/mbmg.5.62823 Research Article Metabarcoding of environmental samples suggest wide distribution of eelgrass (Zostera marina) pathogens in the north Pacific Damian M. Menning1, Hunter A. Gravley2, Melissa N. Cady3, Daniel Pepin4, Sandy Wyllie-Echeverria5,6, David H. Ward1, Sandra L. Talbot1 1 US Geological Survey, Alaska Science Center, 4210 University Dr., Anchorage, AK 99508, USA 2 US Fish and Wildlife Service, 1011 E. Tudor Rd. #200, Anchorage, AK 99503, USA 3 US Fish and Wildlife Service, Pacific Islands Fish and Wildlife Office, 154 Waianuenue Avenue Suite 103, Hilo, Hawaiʻi 96720-2452, USA 4 US Fish and Wildlife Service, Alaska Peninsula/Becharof National Wildlife Refuge, King Salmon, AK 99613, USA 5 Friday Harbor Laboratories, College of the Environment, University of Washington, 620 University Road, Friday Harbor, WA 98250, USA 6 College of Science and Mathematics, University of the Virgin Islands, 2 John Brewers Bay, St. Thomas, VI 00802, USA Corresponding author: Damian M. Menning ([email protected]) Academic editor: Jan Pawlowski | Received 5 January 2021 | Accepted 24 March 2021 | Published 9 April 2021 Abstract Seagrass meadows provide important ecological services to the marine environment but are declining worldwide. Although eel- grass meadows in the north Pacific are thought to be relatively healthy, few studies have assessed the presence of known disease pathogens in these meadows. In a pilot study to test the efficacy of the methods and to provide foundational disease biodiversity data in the north Pacific, we leveraged metabarcoding of environmental DNA extracted from water, sediment, and eelgrass tissue samples collected from five widely distributed eelgrass meadows in Alaska and one in Japan and uncovered wide prevalence of two classes of pathogenic organisms – Labyrinthula zosterae and other associated strains of Labyrinthula, and the Phytophthora/ Halophytophthora blight species complex – known to have caused decline in eelgrass (Zostera marina) elsewhere in the species’ global distribution. Although the distribution of these disease organisms is not well understood in the north Pacific, we uncovered the presence of at least one eelgrass pathogen at every locality sampled. Key Words Alaska, eDNA, disease, Japan, seagrass 1. Introduction angiosperm adapted to the cold waters of northern high latitudes. Eelgrass meadows provide valuable habitat for Seagrass meadows comprise the most widespread coastal diverse animal assemblages – including salmon (Salmo- ecosystems in the world (Green and Short 2003), provid- nidae), Dungeness crab (Metacarcinus magister), herring ing important ecological services to the marine environ- (Clupeidae), and gunnels (Pholidae) to name a few – and ment and promoting ecosystem health (Costanza et al. function as an important primary producer and erosion sta- 1997; Nordlund et al. 2016). They act as nurseries that bilizer in coastal ecosystems (Asmala et al. 2019; Olson shelter young animals, provide habitat for fish, crabs, and et al. 2019; Marin-Diaz et al. 2020). In Alaska, eelgrass other invertebrates, sequester carbon (Hemminga and Du- meadows also provide critical foraging habitat for mi- arte 2000; Duarte et al. 2005; Macreadie et al. 2014), and grating avian species, including most of the world’s pop- mitigate the impact of terrestrial pollution on the marine ulation of Pacific black brant Branta( bernicla nigricans) environment (Fernandes et al. 2009; Lamb et al. 2017). (Ward et al. 2005). Eelgrass, like other seagrass species, In the north Pacific, the predominant seagrass species has been declining globally (Waycott et al. 2009). Major is eelgrass, Zostera marina, a widely distributed marine declines of eelgrass meadows in North America have been Copyright Damian M. Menning et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 36 Damian M. Menning et al.: eDNA metabarcoding shows wide distribution of eelgrass pathogens in the Pacific attributed to both localized and general human-induced recently documented in Alaska waters (Menning et al. events, such as the release of oil, farming induced eutro- 2020), supporting the hypothesis of Govers et al. (2016) phication, residential expansion, climate change (Short that the distribution of Phytophthora is likely wider than and Burdick 1996; Short and Wyllie-Echeverria 1996; currently known. Orth et al. 2006; Waycott et al. 2009), and natural events, The spatial extent of the eelgrass beds in Izembek La- such as anoxia and disease (Short et al. 1987; Muehlstein goon, the most extensively studied eelgrass bed in Alaska, 1992; Short and Wyllie-Echeverria 1996; Plus et al. 2003). is considered stable (Ward et al. 1997; Hogrefe et al. 2014). Baseline abundance and distribution data collected from a Nevertheless, its aboveground biomass has fluctuated number sites suggest eelgrass beds are stable in Alaska widely in since 2012 (Ward and Amundson 2019), possibly (Ward 2021; Ward et al. 2021), but trend data are limited in response to the recent increased water temperatures in to a single location (Izembek Lagoon; Ward et al. 1997; the north Pacific (Bond et al. 2015) and Eastern Bering Sea Hogrefe et al. 2014; Ward and Amundson 2019) and little (Gamito et al. 2015). Although prior work by Menning et al. is known regarding broader patterns of eelgrass across the (2020) found both pathogen classes in Izembek Lagoon in state and elsewhere in the north Pacific. Alaska, the prevalence of eelgrass pathogens – which may Diseases specific to eelgrass have played roles in eel- increase in virulence with increasing water temperatures grass meadow losses in Europe and the Atlantic and have (Harvell et al. 2002) – has not been characterized across the potential to emerge elsewhere (Govers et al. 2016; Alaska coastal regions. To better understand the presence Lindholm et al. 2016). The most severe declines of eelgrass and potential extent of these pathogens in the eastern north in North America occurred in the 1930s, along the Atlan- Pacific, we leveraged the prior eDNA approach (Menning tic coast, where the species suffered an almost complete et al. 2020) to assay environmental samples collected from die-off attributed to a virulent pathogenic strain of protist, five broadly distributed eelgrass beds in Alaska. For com- Labyrinthula zosterae (Short et al. 1987). The loss of eel- parative purposes that would allow us to better understand grass meadows promoted declines in fish and waterfowl the distribution of these pathogens in the broader north species along the Atlantic coasts of North America and Eu- Pacific, we also sampled one site in Japan. In addition to rope (Cottam et al. 1944). Martin et al. (2016) provided Izembek Lagoon, in Alaska, we extended our sampling evidence that both pathogenic and non-pathogenic strains to the east to include two meadows in the Gulf of Alaska of Labyrinthula are distributed globally, suggesting similar Large Marine Ecosystem (Kupreanof Island, in Frederick outbreaks could occur elsewhere. Lesions on eelgrass, at- Sound, and Chignik Lagoon, on the southern coast of the tributable to the presence of Labyrinthula sp., were recently central Alaska Peninsula) and three meadows in the East- documented in eelgrass beds in Puget Sound, Washington ern Bering Sea Large Marine Ecosystem (Port Moller Spit (Groner et al. 2014; Groner et al. 2016), but disease lesions and Port Moller Hot Spring, on the northern coast of the have yet to be reported from Alaska, the northwesternmost middle Alaska Peninsula, and to the north to include Safety distribution of eelgrass in the eastern north Pacific. Lagoon near Nome on the southside of the Seward Penin- Nevertheless, Martin et al. (2016) provided evidence sula; Fig. 1). Our sampling in Japan was conducted in an that both pathogenic and non-pathogenic strains of eelgrass bed in Notsuke Bay, in the western north Pacif- Labyrinthula are distributed globally and developed a ic. Further, we augmented prior research (Menning et al. cladogenic context for both subtypes; they reported the 2020) from Izembek Lagoon to test our initial hypothesis presence of a species of Labyrinthula in eelgrass mead- that there are seasonal differences in the detection of patho- ows in Kasitsna Bay on the Kenai Peninsula, Alaska, genic and non-pathogenic strains of Labyrinthula sp. and that it belonged to a non-pathogenic clade. More re- cently, Menning et al. (2020) leveraged next-generation sequencing of environmental DNA (eDNA; Peters et al. 2. Methods 2017) in a metabarcoding approach designed to detect the presence of Labyrinthula sp. DNA – including strains hy- 2.1 Sample collection pothesized to be virulent (Martin et al. 2016). Relatedly, Govers et al. (2016) reported the presence Environmental samples were collected from the water col- of two closely related fungi-like oomycetes species in umn, sediment, and eelgrass leaves periodically between the Atlantic: Phytophthora gemini and a previously un- January 2016 and August 2018. Water samples were col- described species, Halophytophthora sp. Zostera. Both lected in 500 mL volumes and filtered through 0.22 micron species are potent pathogens closely related to the potato filters (GTTP 04700, Millipore), which were then stored in blight (P. infestans), and both may hinder sexual repro- 5 mL