The ISME Journal (2016) 10, 2118–2128 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 www.nature.com/ismej ORIGINAL ARTICLE Multi-year assessment of coastal planktonic fungi reveals environmental drivers of diversity and abundance Joe D Taylor1,3 and Michael Cunliffe1,2 1Marine Biological Association of the United Kingdom, The Laboratory, Plymouth, UK and 2Marine Biology and Ecology Research Centre, Marine Institute, Plymouth University, Plymouth, UK Mycoplankton have so far been a neglected component of pelagic marine ecosystems, having been poorly studied relative to other plankton groups. Currently, there is a lack of understanding of how mycoplankton diversity changes through time, and the identity of controlling environmental drivers. Using Fungi-specific high-throughput sequencing and quantitative PCR analysis of plankton DNA samples collected over 6 years from the coastal biodiversity time series site Station L4 situated off Plymouth (UK), we have assessed changes in the temporal variability of mycoplankton diversity and abundance in relation to co-occurring environmental variables. Mycoplankton diversity at Station L4 was dominated by Ascomycota, Basidiomycota and Chytridiomycota, with several orders within these phyla frequently abundant and dominant in multiple years. Repeating interannual mycoplank- ton blooms were linked to potential controlling environmental drivers, including nitrogen availability and temperature. Specific relationships between mycoplankton and other plankton groups were also identified, with seasonal chytrid blooms matching diatom blooms in consecutive years. Mycoplank- ton α-diversity was greatest during periods of reduced salinity at Station L4, indicative of riverine input to the ecosystem. Mycoplankton abundance also increased during periods of reduced salinity, and when potential substrate availability was increased, including particulate organic matter. This study has identified possible controlling environmental drivers of mycoplankton diversity and abundance in a coastal sea ecosystem, and therefore sheds new light on the biology and ecology of an enigmatic marine plankton group. Mycoplankton have several potential functional roles, including saprotrophs and parasites, that should now be considered within the consensus view of pelagic ecosystem functioning and services. The ISME Journal (2016) 10, 2118–2128; doi:10.1038/ismej.2016.24; published online 4 March 2016 Introduction (Caporaso et al., 2012; Logares et al., 2014; de Vargas et al., 2015). Plankton are the foundation of marine ecosystems Even though planktonic fungi (mycoplankton) because they sustain food webs and drive global have been known to be present in marine ecosystems biogeochemical cycles (Worden et al., 2015). Some for several decades (Vishniac, 1956), they have plankton groups, such as phytoplankton and zoo- received considerably less attention compared with plankton, have been studied extensively for decades, other plankton groups (Wang et al., 2012). Marine with earlier research founded on microscope-based mycoplankton exist as free-living filamentous assessments of diversity and abundance (Russell et al., and yeast forms, or as parasites of other plankton 1971; Southward, 1974). More recently, through the (Richards et al., 2012; Wang et al., 2012). The application of DNA-based tools that incorporate ecosystem functioning roles that marine myco- previously poorly described plankton groups, such plankton may undertake have been largely assumed as bacterioplankton and protists, marine plankton from comparison with their terrestrial and fresh- diversity has been shown to be vast, complex and influenced by a wide range of environmental drivers water analogues (Richards et al., 2012). For example, Richards et al. (2012) proposed that, similar to terrestrial fungi, saprotrophic marine fungi probably Correspondence: M Cunliffe, Marine Biological Association of the attach to particulate organic material, produce extra- United Kingdom, The Laboratory, Citadel Hill, Plymouth PL1 2PB, cellular enzymes and utilise resulting lower mole- UK. cular weight compounds. Supporting this theory, E-mail: [email protected] evidence from the coastal waters off Chile suggest 3Current address: Department of Biology, University of York, York, UK. that mycoplankton have a major role in processing Received 5 August 2015; revised 7 January 2016; accepted phytoplankton-derived biopolymers using extra- 21 January 2016; published online 4 March 2016 cellular enzymes (Gutiérrez et al., 2011). Marine mycoplankton diversity and abundance JD Taylor and M Cunliffe 2119 Relative to other plankton groups, such as the The environmental metadata used in this study bacterioplankton, there have been a limited number were collected at the same time as the seawater of assessments of marine mycoplankton diversity. So samples for DNA extraction (above) also as part far, studies of marine mycoplankton diversity have of the regular activities at the WCO (Table 1 and been largely based on samples collected from one-off Supplementary Figure 1). Temperature and salinity sampling campaigns that have provided important and were determined via a rosette-mounted SeaBird SBE insightful ‘snap shots’ of diversity, but with limited 19+ CTD (Sea-Bird Electronics, St Bellevue, WA, temporal resolution (Bass et al., 2007; Gao et al., 2010; USA). Nutrients were measured in seawater samples Richards et al., 2012; Manohar and Raghukumar, 2013; collected via a sampling bottle attached to the rosette Taylor and Cunliffe, 2014). Time series assessments of and using established protocols for nitrate (Brewer and other plankton groups have proven invaluable in Riley, 1965), nitrite (Grasshoff et al., 1999), ammonia developing our understanding of their biology and (Mantoura and Woodward, 1983), phosphate (Zhang ecology (Karl and Church, 2014; Fuhrman et al., 2015). and Chi, 2002) and silicate (Kirkwood, 1989). Spatially defined and reoccurring assessments of The concentrations of particulate organic carbon, total plankton diversity and abundance that are aligned particulate nitrogen and particulate organic nitrogen with co-occurring environmental variables (tempera- were determined by filtering 250 ml seawater samples ture, nutrients and so on) have been used to identify (prefiltered through 200 μm mesh) onto ashed glass the key controlling environmental drivers that influ- fibre filters (GF/F) and analysed using an FlashEA ence planktonic organisms, and to form the basis of 1112 elemental analyser (Thermo Scientific, Lough- establishing the underpinning principles of their borough, UK). For chlorophyll a determination, ecologies and ecosystem functional roles. For example, 100 ml of sea water was filtered onto GF/F, followed multi-year assessments of bacterioplankton diversity at by extraction in 90% acetone at 4 °C, and fluorescence the time series site Station L4 in the Western English measured using a fluorometer (Welschmeyer, 1994). Channel has shown that bacterioplankton have To determine diatom abundance and diversity, 200 ml annually repeating seasonal-scale patterns in diversity seawater samples were fixed with Lugol’siodine that relate to changes in a variety of environmental solution (2% final concentration) and examined drivers, including seawater temperature, seasonal by light microscopy via the Utermöhl counting diatom blooms and organic carbon availability technique (Widdicombe et al., 2010). Metadata are (Gilbert et al., 2009, 2012; Taylor et al., 2014). available from the WCO (www.westernchannelobser The coastal time series site Station L4 is located vatory.org.uk). 13kmoffPlymouthintheWesternEnglishChannel and has a depth of 50 m (Figure 1). The site is dynamic and influenced by periodic estuarine incursions from DNA extraction, PCR and sequencing the neighbouring Tamar and Plym rivers. During the Filters were transferred to 2 ml microcentrifuge tubes summer, the water column becomes thermally strati- and 1 ml phenol/chloroform/isoamyl alcohol (25:24:1) fied at Station L4 and nutrient depletion occurs was added. The tubes were vortexed for 2 min, heated (Smyth et al., 2010). Here we present a multi-year for 10 min at 60 °C, before being vortexing again and time series assessment of mycoplankton diversity and centrifuged for 1 min at 13 000 g. The aqueous phase abundance, correlating observed changes with co- (~0.5 ml) was transferred to a new 1.5 ml tube contain- occurring environmental parameters. We have ing 0.5 ml chloroform/isoamyl alcohol, briefly vortexed focussed on the mycoplankton communities at Station and centrifuged again for 1 min at 13 000 g.The L4 because previous studies have indicated that aqueous phase was transferred again to a new 1.5 ml mycoplankton diversity is greater in coastal waters microcentrifuge tube followed by 10% (v/v) 3 M sodium than in the open ocean (Gao et al., 2010; Wang et al., acetate and 2 volumes of molecular-grade ethanol. 2014), and the station has been proven to be an DNA was precipitated for 1 h at − 20 °C, followed by exemplar site for elucidating the ecology and biology centrifugation at 13 000 g for 25 min. The supernatant of other plankton groups (Eloire et al., 2010; was removed and the DNA pellet washed with 0.5 ml Widdicombe et al., 2010; Gilbert et al., 2012). 75% ethanol, before being dissolved in 50 μl nuclease free molecular-grade water and stored at − 80 °C. The fungi-specific internal transcribed spacer Materials and methods (ITS) region (ITS1,5.8S, ITS2) was amplified