A comparison of two gene regions for assessing community composition of eukaryotic marine microalgae from coastal ecosystems

Abstract

Two gene regions commonly used to characterise the diversity of eukaryotic communities using metabarcoding are the 18S ribosomal DNA V4 and V9 gene regions. We assessed the effectiveness of these two regions for characterising diverisity of coastal eukaryotic microalgae communities (EMCs) from tropical and temperate sites. We binned amplicon sequence variants (ASVs) into the high level taxonomic groups: dinoflagellates, pennate diatoms, radial centric diatoms, polar centric diatoms, chlorophytes, haptophytes and 'other microalgae'. When V4 and V9 generated ASV abundances were compared, the V9 region generated a higher number of raw reads, captured more diversity from all high level taxonomic groups and was more closely aligned with the community composition determined using light microscopy. The V4 region did resolve more ASVs to a deeper taxonomic resolution within the dinoflagellates, but did not effectively resolve other major taxonomic divisions. When characterising these communities via metabarcoding, the use of multiple gene regions is recommended, but the V9 gene region can be used in isolation to provide high-level community biodiversity to reflect relative abundances within groups. This approach reduces the cost of sequencing multiple gene regions whilst still providing important baseline ecosystem function information.

Figure 1

The high-level taxonomic groups of eukaryotic marine microalgae used in this study. The other categories include golden, yellow, and brown classes. Divisions are based on PR2 database classifications (Vaulot et al., 2022), as proposed by Adl et al., (2012) and the World Register of Marine Species (WoRMS 2023) database.

Figure 2

Diversity rarefaction curves of the 18S ribosomal DNA V4 (solid line) and V9 (dotted line) amplicon sequence variants (ASVs) from the entire eukaryotic community and subset of the eukaryotic microalgal community at the temperate and tropical sites.

Figure 3

Alpha diversity indices for the eukaryotic microalgae community (EMC) at temperate and tropical sites, comparing Amplicon sequence variants (ASV) data of the 18S ribosomal DNA (rDNA) V4 and V9 region. Diversity indices include Chao1, Shannon and Inverse Simpson. All V4 and V9 samples were rarefied to a sequence depth of 11000.

Figure 4

Eukaryotic microalgal taxonomic groups retrieved using metabarcoding of the 18S ribosomal DNA (rDNA) V4 and V9 regions, and light microscopy cell count data (LM) from a) temperate, and b) tropical sites. Eukaryotic microalgae are divided into high-level taxonomic groups as defined in methods section Bioinformatic analysis.

Figure 5

Total eukaryotic microalgae detected in environmental DNA (eDNA) extracted from 27 samples collected at the temperate site using metabarcoding from the 18S V4 and V9 regions analysed at the Order level. Each pie slice represents eukaryotic microalgal taxonomic groups, including dinoflagellates, chlorophytes, haptophytes, pennate diatoms, radial centric diatoms, polar centric diatoms, and other eukaryotic microalgae. External nodes of the dendrogram represent microalgal orders in each taxonomic group. In addition, the annotations within the external circle show the number of families detected within each of the overarching taxonomic groups.

Figure 6

Total eukaryotic microalgae detected in environmental DNA (eDNA) extracted from 27 samples collected at the tropical site using metabarcoding from the 18S rDNA V4 and V9 regions analysed at the Order level. Each pie slice represents eukaryotic microalgal high-level taxonomic groups, including dinoflagellates, chlorophytes, haptophytes, pennate diatoms, radial centric diatoms, polar centric diatoms, and other eukaryotic microalgae. External nodes represent microalgal orders in each group and the outer circle notes Families detected within each high-level taxonomic group.