Digital Commons @ Michigan Tech Digital Commons @ Michigan Tech Biogeographic Patterns in Members of Globally Distributed and Biogeographic Patterns in Members of Globally Distributed and Dominant Taxa Found in Port Microbial Communities. Dominant Taxa Found in Port Microbial Communities.

We conducted a global characterization of the microbial communities of shipping ports to serve as a novel system to investigate microbial biogeography. The community structures of port microbes from marine and freshwater habitats house relatively similar phyla, despite spanning large spatial scales. As part of this project, we collected 1,218 surface water samples from 604 locations across eight countries and three continents to catalogue a total of 20 shipping ports distributed across the East and West Coast of the United States, Europe, and Asia to represent the largest study of port-associated microbial communities to date. Here, we demonstrated the utility of machine learning to leverage this robust system to character-ize microbial biogeography by identifying trends in biodiversity across broad spatial scales. We found that for geographic locations sharing similar environmental conditions, subpopulations from the dominant phyla of these habitats ( Actinobacteria , Bacteroidetes , Cyanobacteria, and Proteobacteria ) can be used to differentiate 20 geographic locations distributed globally. These results suggest that despite the over-whelming diversity within microbial communities, members of the most abundant and ubiquitous microbial groups in the system can be used to differentiate a geospatial location across global spatial scales. Our study provides insight into how microbes are dispersed spatially and robust methods whereby we can interrogate microbial biogeography. ﬁndings indicate that the members of globally dominant phyla are important for differentiating locations, which reduces the reliance on rare taxa to probe geography. Further, this study displays how global biogeographic patterning of aquatic microbial communities (and other systems) can be assessed through populations of the highly abundant and ubiquitous taxa that dominant the system. 1.5 mM MgCl , and 1 U high-ﬁdelity polymerase per reaction in 96-well VWR polypropylene plates. The primer pair 515f and 926r was used at a concentration of 0.4 (cid:4) M to amplify a construct that spans the variable regions 4 and 5 (V4–V5) of the 16S rRNA gene (49). The PCR thermal cycler settings were as 95°C for 3 min; 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; 72°C cleanup was performed after ﬁrst-stage ampliﬁcation PCR to remove residual primers and excess reagents from PCR mixtures. For this cleanup, we followed the MiSeq library preparation guide (Illumina, San Diego, CA) and deviated from the standard protocol by using AxyPrep Mag PCR cleanup beads (Corning, Big Flags, NY, USA), using 10 mM Tris at a pH of 8 (down from 8.5) and by using 28 (cid:4) l AxyPrep beads in the second-stage cleanup since the PCR volume was 25 (cid:4) l (down from 50 (cid:4) l). Second-stage indexing PCRs took place under the same mixture conditions as ﬁrst-stage ampliﬁcation PCR and with primers that contained a unique index sequence for each sample and the Illumina sequencing adaptors. An additional PCR cleanup was done after second-stage PCR, eluting to a ﬁnal volume of 50 (cid:4) l. Library preparation and sample pooling were performed according to the MiSeq 16S sequencing library preparation guide (Illumina). The products from the second-stage indexing PCR and subsequent cleanup stages were pooled into a library for sequencing at an equimolar concentration of 10 nM after ensuring that primer contamination was absent or at a minimum using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). Denaturation and dilution pooled 16S rRNA gene library the to agglomerate all 3,214 ASVs into their respective levels of taxonomy (phylum to genus). Annotation of environmental conditions. All 3,214 ASVs were used to identify which environmental conditions were considered signiﬁcant in explaining beta diversity in our microbial community across spatial scales. PERMANOVA (42) was conducted using distance matrices (Bray-Curtis) with 999 permu-tations in vegan (56), and signiﬁcance ( P (cid:3) 0.001) was assessed through F testing based on the sequential sums of squares between the physiochemical parameters chosen and the ﬁve geographic regions to which the local ports were assigned. To account for the trends in environmental conditions and their correlation to each region, these same physiochemical parameters were used to annotate an ANOVA of each condition across all regions ( P (cid:3) 0.001). In order to detect the biotic relationships of the taxa and their association to the six physiochemical parameters, we used our ASVs to identify correlations using Pearson coefﬁcient | r| (63), and associated P value s were adjusted for multiple comparisons for environmental variables (Benjamini-Hochberg). Finally, to deﬁne how well these six physiochemical parameters could explain the total sample variance in the microbial community, a constrained analysis of principal coordinates (CAP; Bray-Curtis) was applied to all 3,214 ASVs using vegan (56). a Bray-Curtis the visualized through the plot_ordination ellipses t level 0.95.