Genomic copy number variability at the genus, species and population levels impacts in situ ecological analyses of dinoflagellates and harmful algal blooms

Abstract

The application of meta-barcoding, qPCR, and metagenomics to aquatic eukaryotic microbial communities requires knowledge of genomic copy number variability (CNV). CNV may be particularly relevant to functional genes, impacting dosage and expression, yet little is known of the scale and role of CNV in microbial eukaryotes. Here, we quantify CNV of rRNA and a gene involved in Paralytic Shellfish Toxin (PST) synthesis (sxtA4), in 51 strains of 4 Alexandrium (Dinophyceae) species. Genomes varied up to threefold within species and ~7-fold amongst species, with the largest (A. pacificum, 130 ± 1.3 pg cell^-1 /~127 Gbp) in the largest size category of any eukaryote. Genomic copy numbers (GCN) of rRNA varied by 6 orders of magnitude amongst Alexandrium (10²- 10⁸ copies cell^-1) and were significantly related to genome size. Within the population CNV of rRNA was 2 orders of magnitude (10⁵ - 10⁷ cell^-1) in 15 isolates from one population, demonstrating that quantitative data based on rRNA genes needs considerable caution in interpretation, even if validated against locally isolated strains. Despite up to 30 years in laboratory culture, rRNA CNV and genome size variability were not correlated with time in culture. Cell volume was only weakly associated with rRNA GCN (20-22% variance explained across dinoflagellates, 4% in Gonyaulacales). GCN of sxtA4 varied from 0-10² copies cell^-1, was significantly related to PSTs (ng cell^-1), displaying a gene dosage effect modulating PST production. Our data indicate that in dinoflagellates, a major marine eukaryotic group, low-copy functional genes are more reliable and informative targets for quantification of ecological processes than unstable rRNA genes.

Fig. 1. Genome sizes (pg cell⁻¹, Gb cell⁻¹) of strains from four dinoflagellate species (Gonyaulacales: Alexandrium).

Images show cells using fluorescence light microscopy, stained with Calcofluor white, from [83, 84].

Fig. 2. Variation of genome size and rRNA copy number of Alexandrium species with generations in laboratory culture.

a Variation in Alexandrium spp genome size with generations in laboratory culture. Values are SD from the mean genome size. Darker background shading indicates range of deviation among Mindarie (WA) Alexandrium pacificum strains cultured for <20 generations prior to analysis. b Variation in Alexandrium species rRNA copy number with increasing generations in laboratory culture. Values are SD from the mean rRNA copy number of each species. Darker background shading indicates range of deviation among Mindarie (WA) Alexandrium pacificum strains cultured for <20 generations prior to analysis.

Fig. 3. The relationship between the CNV of marker genes and the genome size and total PSTs in Alexandrium species.

a CNV of rRNA (±SD) in strains of Alexandrium spp. b Relationship between genome size (pg cell⁻¹) and rRNA copies cell⁻¹ in Alexandrium species (F = 22.99, df=49, p < 0.0001, r² = 0.319). c CNV of sxtA4 (±SD) in Alexandrium species. d Relationship between total PSTs (ng cell⁻¹) and sxtA4 copies cell⁻¹ in Alexandrium species (F = 11.73, df=18, p = 0.003, r² = 0.395).

Fig. 4. Copy number variations comparison between A. pacificum isolated from a bloom site against strains from other sites.

a Sites where strains of A. pacificum were isolated from the Pacific region including sites across Australia. b Mindarie from where strains of A. pacificum were isolated. c CNV of sxtA4 (±SD) and rRNA (±SD) in Alexandrium pacificum from all sites and from Mindarie only.

Fig. 5. Concentration of individual PST congeners (ng PST cell⁻¹) in Alexandrium species.

The heatmap shows the inter-species and intra-species variations of each PST congeners' concentration across Alexandrium spp.

Fig. 6. Relationship between rRNA copies cell⁻¹ and the genome size and cell volume in dinoflagellates.

a Relationship between log₁₀ genome size (pg cell⁻¹) and log₁₀ rRNA copies cell⁻¹ in dinoflagellates from the current study and previous literature (F = 44.2, df=74, p < 0.0001, r² = 0.374). Regression line and 95% confidence interval shown. Purple circles= Suessiales spp, red circles= Gymnodiniales spp, green circles= Prorocentrales spp, orange circles= Peridiniales spp, blue circles= Gonyaulacales spp, yellow circles=Dinophysiales spp. Black triangles = data from the present study. b Relationship between log₁₀ cell volume (µm³) and log₁₀ rRNA copies cell⁻¹ in dinoflagellates, from the present and previous studies (F = 0.26.59, df=106, p < 0.0001, r² = 0.200). c Relationship between log₁₀ cell volume (µm³) and log₁₀ rRNA copies cell⁻¹ in dinoflagellates, from published studies only (F = 16.4, df=58, p = 0.0002, r² = 0.2204). [data from: 9, 10, 13, 19, 30, 38, 54, 55, 62, 63, 71, 74, 75, 76, 77, 78, 79, 81].