Cyanobacterial Blooms Are Not a Result of Positive Selection by Freshwater Eutrophication

Long-standing cyanobacterial harmful algal blooms (CyanoHABs) are known to result from synergistic interaction between elevated nutrients and superior ecophysiology of cyanobacteria. However, it remains to be determined whether CyanoHABs are a result of positive selection by eutrophic waters. To address this, we conducted molecular evolutionary analyses on the genomes of 9 bloom-forming cyanobacteria, combined with pangenomics and metatranscriptomics. The results showed no positive selection by water eutrophication. Instead, all homologous genes in the species are under strong purifying selection based on the ratio of divergence at nonsynonymous and synonymous sites (dN/dS) and phylogeny. The dN/dS < 0.85 (median = 0.3) for all homologous genes are similar between the genes in the pathways driving CyanoHABs and housekeeping functions. Phylogenetic support for non-positive selection comes from the mixed clustering of strains: strains of the same species from diverse geographic origins form the same clusters, while strains from the same origins form different clusters. Further support lies in the codon adaptation index (CAI) and single nucleotide polymorphism (SNP). The CAI ranged from 0.42 to 0.9 (mean = 0.75), which indicates high-level codon usage bias; the pathways for CyanoHABs and housekeeping functions showed a similar CAI. Interestingly, CAI was negatively correlated with gene expression in 3 metatranscriptomes. The numbers of SNPs were concentrated around 5 to 50. As the SNP number increases, the gene expression level decreases. These negative correlations agree with the population-level dN/dS and phylogeny in supporting purifying selection in bloom-forming cyanobacteria. In summary, superior ecophysiology appears to be acquired prior to water eutrophication. IMPORTANCE CyanoHABs are global environmental hazards, and their mechanisms of action are being intensively investigated. On an ecological scale, CyanoHABs are consequences of synergistic interactions between biological functions and elevated nutrients in eutrophic waters. On an evolutionary scale, one important question is how bloom-forming cyanobacteria acquire these superior biological functions. There are several possibilities, including adaptive evolution and horizontal gene transfer. Here, we explored the possibility of positive selection. We reasoned that there are two possible periods for cyanobacteria to acquire these functions: before the onset of water eutrophication or during water eutrophication. Either way, there should be molecular signatures in protein sequences for positive selection. Interestingly, we found no positive selection by water eutrophication, but strong purifying selection instead on nearly all the genes, suggesting these superior functions aiding CyanoHABs are acquired prior to water eutrophication.