L. minor sampling and microbiome removal and reintroduction
Eight natural rivers, ponds, and swamps supporting populations ofL. minor were located within a 10km radius of Montreal, Canada (Table S1 & Fig. S1, Supplementary materials). From each site large samples of plants were taken, brought back to a research greenhouse at McGill University, and maintained in samples of natural pond water, also collected from each site. Microscopy revealed that the plant microbiome included a large assemblage of diverse groups of epiphytic protists, in addition to bacteria and fungi.
To test the effect of the microbiome on plant performance, we first removed the microbiome from all fronds, and then reintroduced it to a subsample from each site as a way to control for the process of microbiome removal. To sterilize the plants, individual fronds were thoroughly rinsed in deionised water, submerged for approximately 3 minutes in 10% bleach and then transferred to sterile high-nutrient Hoagland’s E-medium (recipe in Supplementary materials, Table S2). After two weeks, surviving cultures were examined with microscopy, and those that appeared to be axenic were transferred to agar plates made with Bold’s basal medium (Stein 1973) to promote algal growth, and to agar plates made with Yeast extract-peptone-dextrose growth medium (YEPD) to promote yeast and bacterial growth. After an additional two weeks of growth in Bold’s and YEPD, cultures were again examined by microscopy. This process was repeated until axenic fronds were obtained for each site.
Once sterility was confirmed, for each of the eight sites, a singleL. minor frond (here on in referred to as a genotype) was used to found a clonal population that would serve as the ancestor for all assay cultures. After one month of expansion in sterile conditions, each population (one per genotype), consisting of several hundred fronds, was split in two, one which would remain axenic, the other to which we would reintroduce the microbiome. The original samples from all sites consisting of untreated L. minor fronds with their intact microbiome, growing in their natural pond water were maintained in open 12L containers in the greenhouse (DI water added weekly to replace that lost from evaporation), and were used to reintroduce the microbiome to the experimental populations. This was done by culturing axenic fronds in their natural pond water in 1.5L culture tubs, surrounded by untreated plants from that site with intact microbiomes for an additional two weeks, or about four generations. We used a floating circular boom (10 cm diameter) to physically isolate the target fronds (of which there were roughly 50) from the others, but submerged roots were allowed to intermingle (Fig. S2, Supplementary materials). This allowed sufficient time for the reacquisition of the microbiome, which, for L. minor in similar experimental conditions, has been shown to begin within 24 hours, and to reach a stable community after 5-14 days (Acosta et al. 2020). This extra step ensured that we did not have a confounding effect of the sterilization-induced selection on more robust individuals.