Alternative approaches to identify core bacteria in Fucus distichus microbiome and assess their distribution and host-specificity

BACKGROUND: Identifying meaningful ecological associations between host and components of the microbiome is challenging. This is especially true for hosts such as marine macroalgae where the taxonomic composition of the microbiome is highly diverse and variable in space and time. Identifying core taxa is one way forward but there are many methods and thresholds in use. This study leverages a large dataset of microbial communities associated with the widespread brown macroalga, Fucus distichus, across sites and years on one island in British Columbia, Canada. We compare three different methodological approaches to identify core taxa at the amplicon sequence variant (ASV) level from this dataset: (1) frequency analysis of taxa on F. distichus performed over the whole dataset, (2) indicator species analysis (IndVal) over the whole dataset that identifies frequent taxa that are enriched on F. distichus in comparison to the local environment, and (3) a two-step IndVal method that identifies taxa that are consistently enriched on F. distichus across sites and time points. We then investigated a F. distichus time-series dataset to see if those core taxa are seasonally consistent on another remote island in British Columbia, Canada. We then evaluate host-specificity of the identified F. distichus core ASVs using comparative data from 32 other macroalgal species sampled at one of the sites. RESULTS: We show that a handful of core ASVs are consistently identified by both frequency analysis and IndVal approaches with alternative definitions, although no ASVs were always present on F. distichus and IndVal identified a diverse array of F. distichus indicator taxa across sites on Calvert Island in multiple years. Frequency analysis captured a broader suit of taxa, while IndVal was better at identifying host-specific microbes. Finally, two-step IndVal identified hundreds of indicator ASVs for particular sites/timepoints but only 12 that were indicators in a majority (> 6 out of 11) of sites/timepoints. Ten of these ASVs were also indicators on Quadra Island, 250 km away. Many F. distichus-core ASVs are generally found on multiple macroalgal species, while a few ASVs are highly specific to F. distichus. CONCLUSIONS: Different methodological approaches with variable set thresholds influence core identification, but a handful of core taxa are apparently identifiable as they are widespread and temporally associated with F. distichus and enriched in comparison to the environment. Moreover, we show that many of these core ASVs of F. distichus are found on multiple macroalgal hosts, indicating that most occupy a macroalgal generalist niche rather than forming highly specialized associations with F. distichus. Further studies should test whether macroalgal generalists or specialists are more likely to engage in biologically important exchanges with host.

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