Combining multi-marker metabarcoding and digital holography to describe eukaryotic plankton across the Newfoundland Shelf

Abstract

The planktonic diversity throughout the oceans is vital to ecosystem functioning and linked to environmental change. Plankton monitoring tools have advanced considerably with high-throughput in-situ digital cameras and genomic sequencing, opening new challenges for high-frequency observations of community composition, structure, and species discovery. Here, we combine multi-marker metabarcoding based on nuclear 18S (V4) and plastidial 16S (V4-V5) rRNA gene amplicons with a digital in-line holographic microscope to provide a synoptic diversity survey of eukaryotic plankton along the Newfoundland Shelf (Canada) during the winter transition phase of the North Atlantic bloom phenomenon. Metabarcoding revealed a rich eukaryotic diversity unidentifiable in the imaging samples, confirming the presence of ecologically important saprophytic protists which were unclassifiable in matching images, and detecting important groups unobserved or taxonomically unresolved during similar sequencing campaigns in the Northwest Atlantic Ocean. In turn, imaging analysis provided quantitative observations of widely prevalent plankton from every trophic level. Despite contrasting plankton compositions portrayed by each sampling method, both capture broad spatial differences between the northern and southern sectors of the Newfoundland Shelf and suggest complementary estimations of important features in eukaryotic assemblages. Future tasks will involve standardizing digital imaging and metabarcoding for wider use and consistent, comparable ocean observations.

Figure 1

The Bonavista Banks (BB) and SE Grand Banks (SEGB) sampling transects. Stations (white and black) contain full CTD profiles, white dots indicate Niskin bottle samples for DNA and imaging at 5 m, and the asterisks indicate additional Niskin samples from 20 and 50 m. Note that the ship did not sample every original station, but original station names are kept here, thus SEGB-19 was the 15^th station sampled at the SE Grand Banks. Maps were generated using the marmap R package (v. 1.0.6).

Figure 2

The full set of observations for both transects including chlorophyll-a fluorescence (Fluor.), imaging concentrations, ASV richness, physicochemical data (temperature, salinity, oxygen) and the multi-marker taxonomic composition. Richness error bars represent standard errors under the breakaway model. The x-axis is oriented by station order from shore to shelf and depth (5–50 m). Comparing transects reveals a contrast between higher plankton richness and lower abundance on Bonavista Banks and vice versa on the SE Grand Banks. Physical gradients spanning on-shelf to off-shelf water masses are also evident in salinity on the Bonavista Banks and temperature on the SE Grand Banks; however the 18S and 16S taxonomic compositions across these gradients are more complex during the early winter sampling period.

Figure 3

Principal Component Analysis (PCA) of 18S and chloroplast (cps) 16S rRNA markers showing the first (PC1) and second (PC2) most important components (axes) with shapes indicating sample depth (m). The ordination is calculated with an Aitchison’s distance a compositionally valid Euclidean distance of the clr-transformed ASVs.

Figure 4

A collection of eukaryotic taxa observed on the Newfoundland Shelf. Groups are broadly divided into trophic level with respective scale bars, labelled at image 1, 14, and 21. Heterotrophs included adult copepods (1–3), larval nauplii (4–5), the saprophytic phylum Labyrinthulomycetes (6–8), the Amoebozoa Platyamoeba (9), tintinnids (10–12), and appendicularians (13). Mixotrophic dinoflagellates (14–19) included several genera of Tripos (14–16), with Tripos fusus (15) and Tripos lineatum (16), Gyrodinium (17), Prorocentrum (18), and Protoperidinium (19). The heterotrophic radiolarian Acantharia (20) commonly bears photosynthetic symbionts, creating mixotrophic nutrition. The photosynthetic autotrophs included diatoms (21–35), and the silicoflagellate genera Dictyocha (35–36). The diatom genera included Proboscia (21), Chaetoceros (22–24), plus taxonomically unresolved chain-forming (25–26) and centric groups (27–30), Thalassionema (31), Pseudo-nitzschia (32), rod-shaped groups (33), and Nitzschia (34). To highlight additional data-driven benefits of holography, partially focused images (e.g., 21 and 23) produced from our methods can be refocused from raw holograms using, for example, an oblique reconstruction to recover obscured features outside the plane perpendicular to the optical axis.