Bacterial communities of F. distichus across the entire dataset
Our results from four datasets showed bacterial communities on F. distichus significantly differed from environmental samples (rock surface and seawater; Adonis2: Pseudo-F(2:740) = 66.009, R2 = 0.15, p < 0.001) [Fig. 1A]. This indicates F. distichus host unique microbiomes, but within F. distichus samples there were significant differences by site and time point [Fig. 1B]. The dispersion within the largest dataset, “2017 Quadra” is a result of seasonal variation in bacterial communities across this one-year timeseries.
Core bacterial taxa of F. distichus
To identify which taxa are tightly associated with F. distichus, we first used a two-step indicator species analysis (IndVal) approach to identify the core bacteria. In step 1 we identified bacterial ASVs that were enriched on F. distichus and prevalent across individuals at each site and time point (indicator taxa) at a threshold of > 0.7 index value. Then in step 2 we asked which of these indicator taxa were consistently identified across sites and time (core taxa), using the criteria that an ASV had to be an indicator taxon in all datasets and in at least 7 of 11 sampling events. In step 1 we identified a total of 263 ASVs within 76 bacterial genera and found that indicator taxa are highly variable across sites and time points [Supplementary Table 1]. In step 2, we identified 15 core bacterial ASVs. These 15 core ASVs belong to the genera Granulosicoccus, Blastopirellula, Litorimonas, Rubidimonas, Hellea, Roseibacillus, Rubritalea, and Nonlabens [Supplementary Table 2]. Interestingly, no ASVs were present in every sample of F. distichus across sites and over time, although these taxa meet our definition of core as they are significantly prevalent and predominant in F. distichus samples across all datasets.
We compared the IndVal method to the commonly used frequency threshold method, which does not consider the occurrence of host-associated bacteria in the surrounding environment. At a frequency threshold of 50% we identified 28 ASVs as F. distichus-core taxa across our datasets. The choice of a threshold is somewhat arbitrary across core microbiome studies, and we present the frequency (prevalence) of each ASV that was an indicator in at least one sampling event in Supplementary Table 2 for ease of comparison. For example, 80% frequency threshold captured 3 taxa (ASV1, ASV2 and ASV5) that are the most dominant taxa in our datasets (Supplementary Table 2). We found that the core identified using a simple frequency threshold of 50% resulted in overlap of 11 ASVs identified by our IndVal analysis. As expected, a simple frequency method also captured taxa (e.g., ASV10, ASV41, ASV43 and ASV52) that are also prevalent in environmental samples [Fig. 2]. On the other hand, our two-step IndVal approach detected 4 core ASVs that are highly specific to F. distichus, but less prevalent in the environment, overall [Fig. 2–4].
Core bacteria of F. distichus over time
We further investigated the “2017 Quadra” dataset longitudinally sampled between 2017 March and 2018 January to ask whether the core bacterial taxa are persistently present and stable in relative abundance over this seasonal timeseries. The full Quadra 2017 dataset was used to identify the core, so it was expected that our core bacteria are present in this dataset. Our results show that the relative abundances of most core ASVs fluctuate over time, but a few are stable [Fig. 3]. For example, Blastopirellula ASV2 and Granulosicoccus ASV4 were strikingly overrepresented on F. distichus between March and June but decreased in relative abundance during the summer season (July-October), while Granulosicoccus ASV1 was stable in relative abundance across all months. Dokdonia ASV6 identified by a simple frequency threshold of 50% was also relatively stable across all seasons, and commonly found on rocks. We also observed that a few of core taxa (ASV72, ASV84, ASV126, ASV130) by IndVal were nearly absent (< 0.1%) in F. distichus samples collected between July and January [Fig. 3]. Because the 16S amplicon data used in this study is compositional, it is not clear if changes in the prevalence of core taxa are driven by decreases in absolute abundance or influenced by blooms of other taxa on F. distichus.
Macroalgal host specificity of F. distichus-core bacteria
We then asked whether the F. distichus-core ASVs associate predominately with Fucus (Fucus specialists) or with diverse seaweed species (seaweed generalists). We assessed the specificity of the Fucus core bacterial ASVs by determining their distribution on F. distichus and 35 sympatric seaweed species sampled at West Beach, Calvert Island, BC [Fig. 4]. Bacterial communities were found to vary among the diverse sympatric seaweed species , presumably because these macroalgae differ in their morphology, production of secondary metabolites, and physiological functions which can act as selective filters for different bacterial communities [76, 77]. Our data showed that many of F.distichus-core bacterial taxa are still present on other seaweed species in sympatry, but a few were exclusively enriched on F. distichus [Fig. 4]. For instance, Granulosicoccus sp. (ASV1 and ASV4) and Litorimonas sp. (ASV5, ASV23 and ASV72) were found on most sympatric brown, green, and red macroalgae at West Beach [Fig. 4]. On the other hand, core bacterial taxa Rubritalea sp. (ASV3) and Roseibacilus sp. (ASV67 and ASV169), were specifically enriched on F. distichus and nearly absent on other seaweeds. Blastopirellula sp. (ASV2) was associated with seven macroalgal species and five of them were brown algae, suggesting a possible niche within brown macroalgae. Because the sample size for each seaweed species is uneven, we must be cautious in interpreting differences in their relative abundance and prevalence of the core ASVs. However, it is clear that many F. distichus-core taxa identified across our datasets associate with diverse macroalgae and are likely macroalgal generalists, while only Rubritalea sp. (ASV3) is a candidate for being a Fucus specialist.
Phylogenetic placement of core bacteria
We used broadly sampled phylogenetic trees to identify the closest relatives of the F. distichus-core ASVs. We annotated the tree with information on the isolation source from GenBank reporting the environment or host or from which sequences were isolated. This allowed us to determine whether the F. distichus core fall within macroalgal-associated clades or clades that have been broadly characterized from other hosts and abiotic environments. In cases where we identified multiple core ASVs belonging to the same genus, this allowed us to determine whether the core ASVs are clustered together or fall within distinct clades.
We found the three core ASVs within Granulosicoccus genus fall within distinct clades [Supplementary Fig. 1]. The closest relatives of ASV4 and ASV130 were previously detected on brown macroalgae. On the other hand, close relatives of ASV11 and ASV1 were isolated from the environment (i.e, marine sediment and ice) [Supplementary Fig. 1].
Closely related bacteria of core ASVs within Litorimonas were mostly detected with brown and green macroalgae, although a few of them were still associated with environment (i.e., seawater and marine surface) [Supplementary Fig. 2]. Core ASVs within Granulosicoccus and Litorimonas genus are strong indicator bacteria of F. distichus and widespread on diverse seaweed species in our dataset. This is likely to reflect that they are generally well-adapted on seaweeds but facultative bacteria that can be acquired from surrounding environments.
The closest relatives of Blastopirellula (Planctomycetes), core ASV2 were particularly detected with Fucus species [Supplementary Fig. 3], supporting our results that ASV2 maybe specialist on Fucus or brown algal clade. Another specialist, Rubritalea, core ASV3 is one that enriched in F. distichus samples but nearly absent in other 34 seaweed samples. We found that a sequence that closely match core ASV3 and ASV38 was also found on Fucus vesiculosus [Supplementary Fig. 4]. Overall, the core taxa, ASV2 and ASV3 maybe specialists on Fucus or brown algae, respectively, but we could not find evidence of co-diversification with seaweed hosts from the phylogenetic trees of the F. distichus-core bacteria because host and symbiont phylogenies are not concurrent.