Introduction
It is expected that global warming and the ensuing sea ice melt will strongly alter the Arctic pelagic environment. This could eventually result in a modification in the species composition and biomass of unicellular plankton, changing matter fluxes affecting the entire pelagic and even benthic systems (Wassmann, 2015). Thus, it is necessary to generate information on the temporal occurrences of planktonic species to also better understand their variability and responses towards different environmental conditions. Acknowledging this, in 1999, the Alfred-Wegener-Institute Helmholtz Centre for Polar and Marine Research established the ‘Long-Term Ecological Research Site (LTER) HAUSGARTEN to carry out regular observations of the ecosystem in the eastern Fram Strait. This involves an extended sediment trap program based on annual deployments of moored sediment traps at key stations of the observation area (Soltwedel et al. 2005; 2016). Despite uncertainties related to the use of these tools, such as trapping efficiency, validity of results etc. (Butman 1986; Gust et al. 1994; Buesseler et al. 2007), they remain useful in gaining insights of vertical particle flux patterns. Moreover, the deployment of sediment traps facilitates an understanding of plankton dynamics in the upper water column all year-round, even in remote areas such as the Arctic Ocean.
Measurements of bulk parameters like sinking matter and its components combined with light microscopy can provide an estimate of the pelagic eukaryotic microbial community in the catchment area above the traps (Bauerfeind et al., 2009). Unfortunately, these assessments are mainly focused on larger organisms from the micro-plankton fraction, as surveys of small eukaryotic microbial species from the nano- and picoplankton fractions in sediment traps are almost impossible owing to their size and a simple morphology. For example, besides micro-eukaryotic stramenopiles (mainly diatoms), pico- and nano-eukaryotic Mamiellophyceae (mainly Micromonas ) and haptophyceae (mainlyPhaeocystis sp. ) are key Arctic phytoplankton taxa. They are well known to be major contributors to phytoplankton communities and biomass (Lovejoy and Potvin, 2011; Metfies, von Appen, Kilias, Nicolaus, & Nothig, 2016), although their contributions to vertical flux and particle export are not well understood. Thus, it is particularly important to elucidate this black box, because changes in the abundance of these key phytoplankton taxa have been observed in the area of LTER HAUSGARTEN. These changes have been associated with a warm water anomaly from 2005 to 2007 (Beszczynska-Moller, Fahrbach, Schauer, & Hansen, 2012; Nöthig et al., 2015), while some of them even remained after the warm-water event. During this warm period, higher phytoplankton biomass was observed in the water column, eukaryotic microbial plankton >3 µm changed in composition, and diatoms that dominated the summer period before the warm period significantly decreased (Nöthig et al., 2015). In 2006, Phaeocystis pouchetii started to dominate the eukaryotic microbial community and remained prominent in the region since then, while diatom concentrations remained low and small flagellates increased in abundance (Nöthig et al., 2015).
Over the recent years, 18S rRNA gene meta-barcoding using high throughput sequencing (HTS) platforms has become an indispensable tool to generate cultivation-independent and in-depth information on the biodiversity and community composition of eukaryotic microbes, including all size fractions, dominant and rare taxa (de Vargas et al., 2015; Sunagawa et al., 2015). A considerable number of marine surveys have also taken advantage of ribosomal sequence information to broaden our understanding of protist diversity and community structure (e.g., Gescher et al., 2008; Metfies et al., 2010), and revolutionized the field of microbial ecology into a semi quantitative method that allowed testing and modeling of assemblages across time and space (e.g., Vernet et al., 2017), and even determining links between and among communities and/or functions (e.g., Lima-Mendez et al., 2015). The 18S rRNA V4 region is the most frequently used marker for this purpose. It is best suited for HTS-based surveillance of microbial eukaryotes (Dunthorn et al., 2014), as the combination of V4 with the V5 region provides the most detailed phylogenetic information on the 18S rRNA gene (Hugerth et al., 2014). However, different primer-evaluation studies also showed that most of the known primer sets amplifying the V4 region are limited in their potential to provide comprehensive biodiversity information as they discriminate against certain taxa (Bradley, Pinto, & Guest, 2016). Among these, the primers reported by Stoeck et al. (2010) have been widely used in assessing microbial eukaryotic biodiversity in different marine habitats including the Arctic region (e.g., Comeau et al., 2013; Hardge et al., 2017). Although a truthfully ‘universal’ primer that can amplify all known representative sequences has not yet been described, many efforts have been done to at least minimize or lessen potential PCR biases. Hugerth et al. (2014) for example has systematically designed primers that greatly performed in silico but were not tested using mock communities. Their results showed that the majority of primer sets were able to amplify most but not all taxonomic groups. This is important since our capacity to describe and predict patterns in the communities depend on our ability to amplify and detect most members of the community. Such task has become more challenging since many of the existing primers were primarily designed based on available sequences of known species, and accumulating evidence show that many previously unknown taxa are increasingly detected with more environmental surveys (Massana et al., 2011). Thus, there is a need to carefully select for primers to be used depending on the question being asked or the targeted taxonomic groups, or even use complementary primer sets to retrieve realistic information on microbial biodiversity in a sample.
This study aimed at elucidating changes in the composition of exported pelagic phytoplankton communities in response to a warm anomaly in Fram Strait from 2005-2007. The study is based on using a set of published and newly developed primer-sets considering their complementarity, limitations and advantages deduced. We explored major taxonomic phytoplankton groups including the Chlorophyta, Haptophyta and Bacillariophyta, and their contribution to the exported eukaryotic microbial communities during the phases of maximum particulate organic carbon (POC) flux in spring and autumn from 2000-2011 in the area of LTER HAUSGARTEN. This study provides new insights on how primer biases could limit or improve our understanding of ecological dynamics of microbial communities and their contribution to carbon transport in the changing oceans, such as the Arctic.