Are Oligotypes Meaningful Ecological and Phylogenetic Units? A Case Study of Microcystis in Freshwater Lakes

Abstract

Oligotyping is a computational method used to increase the resolution of marker gene microbiome studies. Although oligotyping can distinguish highly similar sequence variants, the resulting units are not necessarily phylogenetically and ecologically informative due to limitations of the selected marker gene. In this perspective, we examine how oligotyping data is interpreted in recent literature, and we illustrate some of the method's constraints with a case study of the harmful bloom-forming cyanobacterium Microcystis. We identified three Microcystis oligotypes from a western Lake Erie bacterial community 16S rRNA gene (V4 region) survey that had previously clustered into one OTU. We found the same three oligotypes and two additional sequence variants in 46 Microcystis cultures isolated from Michigan inland lakes spanning a trophic gradient. In Lake Erie, shifts in Microcystis oligotypes corresponded to spatial nutrient gradients and temporal transitions in bloom toxicity. In the cultures, Microcystis oligotypes showed preferential distributions for different trophic states, but genomic data revealed that the oligotypes identified in Lake Erie did not correspond to toxin gene presence. Thus, oligotypes could not be used for inferring toxic ecotypes. Most strikingly, Microcystis oligotypes were not monophyletic. Our study supports the utility of oligotyping for distinguishing sequence types along certain ecological features, while it stresses that 16S rRNA gene sequence types may not reflect ecologically or phylogenetically cohesive populations. Therefore, we recommend that studies employing oligotyping or related tools consider these caveats during data interpretation.

Keywords: 16S rRNA gene sequencing; Microcystis; dada2; ecotypes; microbial species; oligotypes.

FIGURE 1

Spatiotemporal distribution of Microcystis oligotypes in western Lake Erie. (A) The relative abundance of Microcystis oligotypes, as fraction of total bacterial reads, from three sites in Western Lake Erie over time. The offshore site had lower median total phosphorus and chlorophyll a levels than the two nearshore sites. Samples were taken from the retentate of 2 L lake water filtered through a 100 μm filter. M denotes missing samples. (B) Particulate Microcystin-LR concentrations over sites and time.

FIGURE 2

RAxML tree for cultured Microcystis strains based on five concatenated housekeeping genes (pgi, gltX, ftsZ, glnA, gyrB). Presence of microcystin biosynthesis gene was determined from assembly and retrieval of Microcystis genes from the Microcystis-heterotroph co-culture metagenome. Trophic status of the lake was determined from total phosphorus levels (Supplementary Tables S1, S2).