Metabarcoding analysis reveals hidden eukaryotic plankton biodiversity in the Ross Sea, Antarctica

Abstract

Background: Environmental DNA (eDNA) analysis is a highly sensitive, non-destructive method that enables the detection of various species through DNA shed into environmental samples without requiring direct organism collection. This study sought to investigate the biodiversity and community structure of eukaryotic plankton, including phytoplankton and zooplankton, in the Ross Sea marine protected area (RSR MPA) using eDNA metabarcoding analysis. By examining their spatial and vertical distributions, the study underscores the importance of continuous monitoring for the conservation of the RSR MPA.

Methods: We collected 48 seawater samples from 16 sites in the Ross Sea region for eDNA metabarcoding analysis, targeting the 18S rRNA gene region of eukaryotic plankton in Antarctica. Bioinformatic processing and taxonomic classification were conducted to assess the diversity and community composition of phytoplankton and zooplankton.

Results: Phytoplankton communities were primarily composed of six phyla with their distribution patterns and the grouping of samples with similar community structures was found to be shaped by the ocean currents of the RSR MPA and various environmental factors, such as salinity and dissolved oxygen levels. Zooplankton communities consisted of 18 major taxonomic groups, exhibiting distinct horizontal and vertical distribution patterns with differences in taxonomic community structure and species diversity across depth groups. Notably, previously undetected Antarctic species were identified in the Ross Sea region, demonstrating the effectiveness of eDNA in revealing hidden biodiversity.

Conclusions: Analyzing eukaryotic plankton communities in the vast and extreme Antarctic environment based on eDNA has proven to be highly efficient, enabling the detection of a greater number of species, including those that were difficult to identify in previous studies. It was observed that in the Ross Sea Marine Protected Area, various species form distinct community structures such as phytoplankton and zooplankton, each inhabiting the area according to different environmental variables and habitat preferences. As a designated marine protected area, the Ross Sea's unique ecosystem requires continuous monitoring and conservation efforts to address environmental changes. The genetic data obtained in this study contributes to expanding the database of Antarctic-specific species, facilitating more accurate and efficient analyses of Antarctic ecosystems in the future.

Keywords: Environmental DNA; Eukaryotic diversity; Marine protected area; Metabarcoding; Phytoplankton; Ross Sea; Zooplankton.

Figure 1. Sampling sites in the Ross Sea, an Antarctic Marine Protected Area.

A total of 48 seawater samples were collected from various depths across 16 sites and analyzed using eDNA metabarcoding targeting the 18S rRNA region to assess the biodiversity of eukaryotic organisms, including phytoplankton and zooplankton. The white line on the map represents the boundary of the MPA.

Figure 2. Taxonomic composition of phytoplankton in each surface sample at the (A) phylum and (B) genus levels.

Site numbers are indicated at the bottom of each bar in the graph. The surface samples from each site were primarily composed of six phyla and 35 genera of phytoplankton.

Figure 3. UPGMA tree of phytoplankton based on Bray-Curtis distance, combined with species composition and relative abundance of the six major species.

By comparing the grouping results of surface seawater with the flow of ocean currents (gray arrows) in the Ross Sea region, groups with similar taxonomic compositions were found to be influenced by ocean currents, with Groups 3 and 4 being significantly affected by the Ross Sea Gyre.

Figure 4. Diversity analysis results for each phytoplankton group.

(A) Alpha diversity and (B) beta diversity (based on Bray-Curtis distance) results of each group. (C) OTU Venn diagram of phytoplankton in each group. G1 to G4 represent Group 1 to Group 4, respectively. Species diversity was highest in Group 1 and lowest in Group 4. Seawater samples were distinctly clustered by group, with further classification into coastal sites (Groups 1 and 2) and open sea sites (Groups 3 and 4). The dots inside the box at the bottom right of (B) represent their size according to the number of OTUs.

Figure 5. Taxonomic composition zooplankton at the phylum level.

Each site number is indicated at the bottom of each bar in the graph. Zooplankton samples from each site were primarily composed of 21 phyla.

Figure 6. Heatmap representing the number of reads based on the composition of major zooplankton phyla in each sampling site and depth of the samples.

Color scale indicates the abundance of zooplankton (read counts). Darker red represents higher values, while lighter colors indicate lower values.

Figure 7. Diversity analysis results of each zooplankton group.

(A) Alpha diversity and (B) beta diversity (based on the Weighted UniFrac distance) results of each group. (C) OTU Venn diagram of zooplankton in each group. Group names are abbreviated as follows: S (Surface), M (Mesopelagic), E (Epipelagic), and B (Bathypelagic). Species richness was highest in the Bathypelagic group, while the Surface group exhibited the highest species evenness. Each group (Groups 1, 2, 3, and 4) showed a clustering pattern according to depth based on the Principal Coordinate Analysis (PCoA) results. The dots inside the box at the bottom right of (B) represent their size according to the number of OTUs.