Fig 1.
Ordination, statistical analyses and diversity measures of the river and river biofilm communities.
A. Nonmetric multidimensional scaling analysis of replicates from river and river biofilms at 4, 8 and 24 hours (T4, T8 and T24). B. Pairwise ANOSIM (left block) and PERMANOVA (right block) analyses of the river and biofilm communities at 4, 8 and 24 hours (T4, T8 and T24). Note that the R and F values are black text on white background and corresponding P values are white text on black background. C. Diversity measures of river and biofilm communities. Diversity measurements were calculated from the sum of 4 replicates for each sample. Taxa_S is the total number of taxa detected, Simpson 1_D is the inverse of the standard Simson diversity measure and Shannon H is the Shannon-Wiener index.
Fig 2.
Top 25 river and river biofilm populations.
Panel A lists the top 25 bacterial populations detected in the Red Cedar River (columns 1 and 2). The three columns to the right report the rank abundance of these populations in river biofilm at 4, 8 and 24 hrs. Panel B lists the top 25 bacterial populations detected in the 24-hour river biofilm. The column to the right reports on the rank abundance of these populations in river water. The white arrows identify the five populations that are shared in river water and 24-hour river biofilm. The four populations shaded black were previously isolated from the surface of sturgeon eggs.
Fig 3.
Resilience of single and double isolate biofilms to filtered and unfiltered river water as measured with crystal violet.
The plotted 600 nm absorbance values (abscissa) are the mean of four replicate biofilms of each of the tested strains (ordinate) and standard deviations are indicated with bars. Striped and gray bars represent biofilm challenged with filtered and unfiltered river water, respectively. Asterisks indicate responses that were statistically different (P value = 0.05 or less) from the cognate control 48-hour biofilm (black bars).
Fig 4.
Resilience of established biofilm to challenge with river bacterial populations as determined by 16S amplicon sequencing.
Panel A, percent (mean of four replicates) of the founding populations remaining in biofilms at 4, 8 and 24 hours. Panels B (F14 & F16 mixed biofilm) and C (F19 & F16 mixed biofilm), percent (mean of four replicates) of the founding two-isolate biofilms remaining after 4, 8 and 24 hours. Percent is calculated based on the entire community (11,100 sequences), including isolates. Error bars display the standard deviation based on four replicates.
Fig 5.
Cluster analysis of biofilms communities at 4, 8 and 24 hours (T4, T8, T24).
Cluster analyses (UPGMA-Bray-Curtis) were performed with (upper row) and without (lower row) the founding populations. Clusters of founding populations are color-coded systematically in all dendrograms to facilitate tracking.
Fig 6.
NMDS, ANOSIM and PERMANOVA analyses of biofilm communities from river and pre-established biofilms of Hydrogenophaga F14, Brevundimonas F16, and Acidovorax F19 at 24 hours incubation.
Sequences of the founding populations have been removed prior to comparative analyses. R values and F values are shaded and uncorrected P values are unshaded.
Fig 7.
The fate of river populations when colonizing established biofilms.
All communities from the established biofilms were compared against the river biofilm community with SIMPER (as implemented in PAST) to determine the influence the established biofilms had on community assembly. Black indicates that the population was blocked from achieving numbers comparable to the river biofilm. Green indicates that the population was selected by the established biofilm and found in substantially greater numbers than in river biofilm and Red indicates that the population size was comparable between the established and river biofilms. Only populations that accounted for 0.5% or greater dissimilarity as indicated by SIMPER with a minimum of 200 identified sequences were counted. A minimum of a 10-fold difference in population counts between the river and established biofilms was required for the blocked and selected categories. (a) Rank abundance in river biofilm, (b) OTU as determined by Mothur [53, 60], (c) number in parentheses indicates confidence of phylogenetic assignment. The OTU number is assigned by Mothur and used to track OTUs.