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test  Biofilms, the rule rather than the exception in many aquatic environments, are a complex milieu of phototrophic and heterotrophic organisms. How environmental factors alter biofilm composition and determine which planktonic microorganisms colonize new surfaces to form biofilms is poorly understood. In aquatic plankton and biofilm communities, heterotrophic bacteria (hereafter bacteria) derive some to all of their organic carbon (C) from the photoautotrophs (hereafter algae) while simultaneously competing for inorganic nutrients such as phosphorus (P). Therefore, C inputs have the potential to alter the ecology of aquatic microbial communities by increasing the resource space available to heterotrophic bacteria (more C) while decreasing the resource space available to algae (less P). We amended a series of flow-through seawater mesocosms with C and P to achieve four target resource C:P levels. We asked if resource amendments altered the size of the biomass pool of bacteria and/or algae in both plankton and biofilm communities. We then used 454 pyrosequencing of bacterial 16S and 23S plastid genes to ask how shifts in the pool size of each community affected community composition and diversity. We saw pronounced differences between the highest carbon treatment and all other treatments. The highest carbon treatment had the lowest planktonic algal abundance yet highest biofilm biomass and highest planktonic bacterial abundance. Resource amendments did not have a significant effect on alpha diversity in either the planktonic or biofilm communities. Rather the biofilm communities consistently had higher alpha diversity than the planktonic communities for any given time point in all mesocosms. Bacterial plankton and biofilm communities were distinct in all but the highest carbon treatment where biofilm and planktonic communities increasingly resembled each other over time. Algal biofilm and plankton communities displayed distinct microbial membership and structure in all treatments including the highest carbon treatment. Our results suggest that broad ecological dynamics (e.g. shifts in dominance between algal and bacterial biomass) driven by shifts in resource availability have important underlying community membership dynamics that alter the interactions and trophic balance of both planktonic and biofilm mirobial communities.  diff --git a/Discussion.tex b/Discussion.tex  index 5fcfe24..2264e79 100644  --- a/Discussion.tex  +++ b/Discussion.tex 
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  Very few studies have previously evaluated the relationship among membership and or diversity of the plankton and the biofilm community from complex environmental microbial communities. One notable study looked at planktonic community composition and biofilm formation on glass beads placed for three weeks in two boreal freshwater streams \cite{22237539}. While that study system is markedly different than our study, the analyses and questions addressed in each study were very similar. Both studies concluded that the biofilm community membership was most likely driven by species sorting over mass effects. However, in the \citet{22237539} study the authors reported that planktonic diversity was significantly higher relative to biofilm diversity (the opposite of what we found in our study). Given the differences in the study systems, this result is not suprising. While biofilm communities were establishsed on glass beads in \citet{22237539} and glass slides (this study) over a similar time period (~21 days, \citet{22237539} and ~17 days this study) the origin of the planktonic community in each study was very different. The \citet{22237539} study was conducted in a boreal stream during snow melt when connectivity between the terrestrial and aquatic habitats was high and potentially highly variable depending on how hydrologic pathways differed among precipitation events. A separate study conducted in alpine and sub-alpine streams of the Rocky Mountains clearly showed that stream plankton communities reflected localized precipitation events and could be traced largely to sources of soil communities of drainages within the watershed \cite{22626459}. While planktonic communities in lake ecosystems can be linked to soil communities in the watershed, as residence time of the system slows the relative influence of species sorting increases. Thus, in headwater ecosystems stream plankton communities can often be composed primarily of soil organisms \cite{22378536}. In addition to the diverse source communities the \cite{22237539} study sampled the plankton community at multiple timepoints and integrated the samples before sequencing, further increasing community richness as compared to the current study where the plankton community was sampled and analyzed only at two independent timepoints. Indeed, when we pool OTU counts from all planktonic libraries and compare the rarefaction curve of the pooled planktonic libraries (algae and bacteria) against sample-wise biofilm libraries, we find more total bacterial and algal planktonic OTUs than in any given single biofilm sample. It appears, however, that some sample-wise bacterial biofilm rarefaction curves may exceed the integrated planktonic curve upon extrapolation and most exceed the integrated planktonic curve at sampling depths where data is present for the biofilm and itegrated planktonic library. This supports our conclusion that temporal heterogenetity in teh plankton was not sufficient to explain the higher diversity in the biofilm sample.  Our study provides an additional detailed analysis of biofilm community assembly mechanisms consistent with what has been previously reported. Additionally, we evaluated how resource subsidies potentially alter the seed pool (the plankton) and the biofilm community. Interestingly, biofilm community richness peaked at the intermediate treatment (C:P = 100) and appeared to decrease over time although with only two time points it was unclear how pronounced this effect was. Since biomass pools for the plankton and the biofilm increased with increasing carbon subsidies the intermediate peak in OTU richness is consistent with a classic productivity-diversity relationship that has been shown for many ecosystems and communities both microbial and otherwise (REFS). Interestingly, as diversity decreased at the high end of the carbon subsidy gradient bacterial plankton and bacteriofilm biofilm  membership became increasing increasingly  similar. This suggests that enviornments that contain high amounts of labile carbon selected for fewer dominant taxa that then also came to dominate the biofilm community. While we did not measure extracellular polymeric substances (EPS), direct microscopycounts  showed that cells in the highest carbon treatment (C:P = 500) were surrounded by what appeared to be EPS. Because biofilm EPS appeared also to increase moving from the low to high carbon treatments (Figure 3) it is possible that planktonic cells were more readily incorporated into biofilms due both to increased "stickiness" of the planktonic cells as well as the biofilm itself. While we did not observe floculating DOC which has been show shown  to dominant dominate  high DOC environment environments  in nature, we did measure a substanial increase in DOC in the C:P = 500 treatment which was more than 2-fold higher than any of the other treatments. \subsection{Biofilm and Plankton Beta Diversity}  While there are only a few studies that attempt to compare biofilm community composition and the overlyng planktonic community abundance, community,  those studies that have addressed this question illustrate community composition among the two habitats are unique with very few taxa found in both (Besemer 2007, Besemer 2012, Jackson 2001, Lyautey et al. 2005). This is consistent with our findings in this experimental system with a natural marine planktonic source commmunity. Our study also evaluates algal community composition which showed a similar result suggesting that both the algal and bacterial biofilm communities form from phylogenetically unique organisms that exist in low abundance in surrounding habitat but are readily enriched in the biofilm lifestyle. Specifically, we found that most of the biofilm enriched algal OTUs were \textit{Bacillariophyta} but there were also many \textit{Bacillariophya} OTUs enriched in the planktonic libraries. \textit{Cryptophyta} and \textit{Viridiplantae} were more uniformly enriched in the planktonic algal libraries. It appears that these broad taxonomic groups are selected against in biofilms under our experimental conditions. Alternatively, \textit{Cryptophyta} and \textit{Viridiplantae} may be selected for in the planktonic environment and exhibit growth rates sufficient for this signal to arise in our experimental residence time or they are numerically dominant taxa in the source community.  Bacterial OTUs enriched in planktonic samples displayed more dramatic differential abundance patterns than bacterial OTUs enriched in biofilm samples, but, biofilm enriched bacterial OTUs were spread across a greater phylogenetic breadth (Figure 6). This is also consistent with greater niche diversity in the biofilm environment as opposed to planktonic. The greater niche diversity would select for a more diverse set of taxa but individual taxa would not be as numerically dominant as in the more niche-uniform planktonic samples. At the order level, bacterial taxonomic groups with environment enriched OTUs tend tended  to have both plantonic and biofilm enriched members suggesting the phylogenetic coherence of this trait is not captured at the level of Order. It should be noted however that taxonomic annotations in reference databasees and therefore environmental sequence collections show little equivalency in phylogenetic breadth between groups at the same taxonomic rank \cite{Schloss_2011}. At higher taxonomic resolution (e.g. Genus-level), groups did not possess a sufficient number of OTUs to evaluate coherence between taxonomic annotation and environment type preference. Biofilm libraries were more even in shape than planktonic libraries (Figure 9) and OTUs enriched in biofilm libraries were enriched less dramatically than planktonic enriched OTUs. It's possible the planktonic environment is more uniform with respect to niche space and therefore produces more skewed rank abundance distributions. Similarly Similar  to the richness results, we found the shapecontrasts  of rank abundance distributions between biofilm and planktonic libraries in our data to be in contrast to that reported by \citet{22237539} although this may be an artifact of each study's different experimental design (see above). \subsection{Enriched Taxa in High Carbon (C:P = 500) Treatment}  Carbon amendments did not affect algal library membership and structure to the same degree as it affected bacterial library composition. As expected, bacterial OTUs enriched in the high carbon amended mesocosm (C:P = 500) include OTUS in classic copiotroph families such as \textit{Vibrionacaea} and \textit{Pseudomonadaceae}. Interestingly, the one OTU depleted in the high carbon treatments is annotated as being in the HTCC2188 order of the \textit{Gammaproteobacteria}. HTCC stands for 'high throughput culture collection' and is a prefix for strains cultured under low nutrient conditions \cite{Cho_2004, Connon_2002}.  \subsection{Conclusion}  In summary this study shows mechanistic links between large scale community level dynamics and the underlying population levels that drive them. Ulimately large scale changes in ecosystem processes are driven by composite effects of microbial communities actiing acting  as a synthesis of physiological events embedded in a complex biotic and abiotic matrix. diff --git a/Material and Methods.tex b/Material and Methods.tex  index 487f198..030c2d8 100644  --- a/Material and Methods.tex  +++ b/Material and Methods.tex 
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We placed test tube racks in one smaller (185L) and 3 larger (370L) flow-through mesocosms. Each mesocosm had an adjustable flow rate that resulted in a residence time of approximately 12h. Irregular variation in inflow rate meant that flow rate varied around that target throughout the day, however, regular monitoring ensured that the entire volume of each system was flushed approximately two times per day. To provide a surface for biofilm formation we attached coverslips to glass slides using nail polish and then attached each slide to the test tube racks using office-style binder clips. Twice daily 10 ml of 37 mM KPO$_{4}$ and 1, 5 and 50 ml of 3.7M glucose were added to each of 3 mesocosms to achieve target C:P resource amendments of 10, 100 and 500 respectively. The control mesocosm did not receive any C or P amendments.  \subsubsection{DOC and Chlorophyll Measurements}  To assess the efficacy of the carbon additions we sampled each mesocosm twice daily during the first week of the experiment to evaluate dissolved organic carbon (DOC) content. Six days after the initiation of the experiment we collected plankton on filters to evaluate planktonic Chl \textit{a} and bacterial abundance. Once it was clear (day 8) that pool size of each community had been altered we filtered plankton onto 0.2 $\mu$m filters and harvested coverslips to assess bacterial and algal community composition (16S and 23S rDNA). In addition all mesoscosms were analyzed a second time (day 17) to assess how community composition of both the plankton and biofilm communities had been altered over time. Control samples were only analyzed for community composition on day 17.  Samples for dissolved organic carbon (DOC) analysis were collected in acid washed 50 mL falcon tubes after filtration through a 0.2 polycarbonate membrane filter (Millipore GTTP GTTP02500, Sigma Aldrich P9199) attached to a 60 mL syringe. Syringes and filters were first flushed multiple times with the control sample to prevent leaching of carbon from the syringe or the filter into the sample. Samples were then frozen and analyzed for organic carbon content with a Shimadzu 500 TOC analyzer \cite{Wetzel_2000}.  diff --git a/Sequence Quality Control and Analysis.tex b/Sequence Quality Control and Analysis.tex  index f836b05..b8aec51 100644  --- a/Sequence Quality Control and Analysis.tex  +++ b/Sequence Quality Control and Analysis.tex 
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\subsubsection{Alpha and Beta diversity analyses}  Alpha diversity calculations were made using PyCogent Python bioinformatics modules \cite{17708774}. Beta diversity analyses were made using Phyloseq \cite{24699258} and its dependencies \cite{vegan}. Log$_{2}$ fold change of group mean ratios and corresponding null hypothesis based significance values were calculated using DESeq2 \cite{Love_2014}. All dispersion estimates from DESeq2 were calculated using a local fit for mean-dispersion. Native DESeq2 independent filtering was disabled in favor of explicit sparsity filtering. The sparsity thresholds that produced the maximum number of OTUs with adjusted p-values for differential abundance below a false discovery rate of 10\% were selected for biofilm versus planktonic sequence 16S/plastid 23S library comparisons. The specific sparsity threshold for plastid 23S and 16S libraries for biofilm versus plankton comparisons was 10\% (OTUs found in less than the sparsity threshold of samples were discarded from the analysis). Cook's distance filtering was also disabled when calculating p-values with DESeq2. We used the Benjamini-Hochberg method to adjust p-values for multiple testing \cite{citeulike:1042553}. Identical DESeq2 methods were used to assess enriched OTUs from relative abundances grouped into high (c:P = 500) or low (C:P < 500 and control) categories. A sparsity threshold of 25\% was used for ordination of both plastid 23S and bacterial 16S libraries. Additionally, we discarded any OTUs from the 23S data that could not be annotated as belonging in the Eukaryota. All DNA sequence based results were visualized using GGPlot2 \cite{Wickham_2009}.  Adonis tests were performed using the Bray-Curtis similarity measure for pairwise library comparisons with the default value for number of permutations (999) ("adonis" function in Vegan R package, \citet{vegan}). Principal coordinates of OTUs were found by averaging site principal coordinate values for each OTU with OTU relative abundance values (within sites) as weights. The principal coordinate OTU weighted averages were then expanded to match the site-wise variances \cite{vegan}. \subsubsection{test}  diff --git a/authors b/authors  new file mode 100644  index 0000000..e69de29  diff --git a/authors.tex b/authors.tex  new file mode 100644  index 0000000..29a80c9  --- /dev/null  +++ b/authors.tex 
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Chuck Pepe-Ranney, Edward Hall  diff --git a/figures/biplot_combined1/caption.tex b/figures/biplot_combined1/caption.tex  index f28c510..4a0e07c 100644  --- a/figures/biplot_combined1/caption.tex  +++ b/figures/biplot_combined1/caption.tex 
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\textbf{Figure 5 }Principal coordinates ordination of bray-curtis distances for 23S rRNA plastid libraries and 16S rRNA gene libraries. OTU points are weighted pricipal coordinate averages (weights are relative abundace values in each sample) and the variance along each pricipal axis is expanded to match the site variance. Point annotations denote the amended C:P ratio for the mesocosm from which each sample was derived. test  diff --git a/figures/combined_rarefaction4/caption.tex b/figures/combined_rarefaction4/caption.tex  new file mode 100644  index 0000000..a8a1b0c  --- /dev/null  +++ b/figures/combined_rarefaction4/caption.tex 
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\textbf{Figure 2} Increases in carbon resulted in decreases in A) planktonic algal biomass (estiated as Chl \textit{a} but B) not algal biomass present in the biofilm \textit{a} in each. In contrast both C) biofilm total biomass and D) number of planktonic bacterial cells increased with increasing carbon subsidies. Responses in treatments separated by different letters were statistically different from one another (p<0.05) as was the highest C:P treatment for planktonic bacterial abundance compared to the the control or the C:P = 10 treatment (p<0.05). The bacterial abudance sample for the C:P = 100 treatment was lost before analysis and is therfore not reported in panel d.  diff --git a/figures/rank_abund1/caption.tex b/figures/rank_abund1/caption.tex  index f95ec22..e716e02 100644  --- a/figures/rank_abund1/caption.tex  +++ b/figures/rank_abund1/caption.tex 
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\textbf{Figure 7} Rank abundance plots. Each panel represents a single time point and C:P. The "rank" of each OTU is based on planktonic sample relative abundance. Each position along the x-axis represents a single OTU. Both the x and y axes are scaled logarithmically. test  diff --git a/figures/rank_abund_shape1/caption.tex b/figures/rank_abund_shape1/caption.tex  new file mode 100644  index 0000000..a8a1b0c  --- /dev/null  +++ b/figures/rank_abund_shape1/caption.tex 
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test  diff --git a/title.tex b/title.tex  index 11399c1..1e451a8 100644  --- a/title.tex  +++ b/title.tex 
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The testThe  effect of carbon subsidies on planktonic niche partitioning and recruitment of bacteria to the marine biofilm lifestyle