\section{Results} 
\subsection{DNA buoyant density changes in response to $^{15}$N$_{2}$} BSCs
were wetted and incubated for 4 days in transparent chambers with headspace
containing N$_{2}$ either from air or from 100 percent atom enriched
$^{15}$N$_{2}$. The chambers were illuminated with 16 h on / 8 h off cycles at
an intensity of 200 $\mu$mol photons m$^{-2}$ s$^{-1}$, which is the equivalent
of an overcast/rainy day. N$_{2}$-fixation as measured by acetylene reduction
increased from 4.8 $\mu$moles m$^{-2}$ d$^{-1}$ on day 2 to 14.5 $\mu$moles
m$^{-2}$ d$^{-1}$ on day 4. Amplicon sequences from $^{15}$N$_{2}$-labeled
samples and their corresponding unlabeled controls diverged specifically in
heavy gradient fractions (Figure~\ref*{fig:ord_heavy} and
Figure~\ref*{fig:ordination}) as assessed by Bray-Curtis dissimilarity
\citep{Bray_1957}\,, and this result was significant (Adonis test
\citep{Anderson_2001}\,; p-value: 0.001, r$^{2}$: 0.18). 

\subsection{OTUs responsive to $^{15}$N$_{2}$ are primarily
\textit{Proteobacteria} and \textit{Clostridiaceae}} 
OTUs that incorporated $^{15}$N into their DNA were detected by a differential
change in their abundance within heavy gradient fractions of
$^{15}$N$_{2}$-labeled samples relative to corresponding controls. A total
of 2,127 and 2,160 OTUs were detected in days 2 and 4, respectively, and
these OTUs were interrogated for evidence of $^{15}$N$_{2}$-labelling. Of
these OTUs, only 499 and 563, respectively, passed a sparsity threshold
applied to filter out OTUs with insufficient data for statistical analysis
(see \citet{Love_2014} for discussion of independent filtering). Of OTUs
passing the sparsity criterion, 34 were enriched significantly in heavy
fractions relative to control and this result is specifically expected for
OTUs that have $^{15}$N-labeled DNA (\textit{i.e.} $^{15}$N$_{2}$
``responders''). Of these, 19 are annotated as \textit{Firmicutes}, 12
as \textit{Proteobacteria}, 2 as \textit{Actinobacteria} and 1 as
\textit{Gemmatimonadetes} (Figure~\ref*{fig:l2fc},
Figure~\ref*{fig:trees}). If the responder OTUs are ranked by descending
enrichment in heavy gradient fractions versus control, 8 the top 10 responders
(\textit{i.e.} those most enriched in the heavy fractions of labeled gradients)
are either \textit{Firmicutes} (3 OTUs) or \textit{Proteobacteria} (5 OTUs)
(Figure~\ref*{fig:scatter_heavy}). Centroids (seed sequences) for strongly
responding \textit{Proteobacteria} OTUs all share high SSU rRNA gene sequence
identity ($>$98.48\%, Table~\ref*{tab:LTP_blast}) with isolates from genera
known to possess diazotrophs including \textit{Pseudomonas},
\textit{Klebsiella}, \textit{Shigella}, and \textit{Ideonella}. None of the
\textit{Firmicutes} OTU centroids in the top 10 responders share greater than
97\% SSU rRNA gene sequence identity with sequences in the Living Tree Project
(LTP) database of 16S rRNA gene sequences from type strains (release 115) (see
Table~\ref*{tab:LTP_blast}). OTUs that passed the sparsity threshold but were
not classified as $^{15}$N-responsive were subsequently tested with the null
hypothesis that the OTU fold enrichment in labeled gradient heavy fractions
versus control was above the selected threshold. Rejecting the second null
indicates that an OTU did \textit{not} incorporate $^{15}$N into biomass. There
were 86 and 89 ``non-responders'' at days 2 and 4, respectively. The $^{15}$N
labelling of OTUs that did not pass sparsity or could not be classified as
either a responder or non-responder cannot be determined conclusively.

Although we did not take replicate samples within a time point we can assess
the consistency of each OTUs response across the two time points. OTU fold
enrichment at day 2 and 4 was consistent (Figure~\ref*{fig:2v4}). There was
a significant correlation between OTU fold enrichment at day 2 versus day
4 (P-value 4.35e$^{-8}$). When the enrichment at day 2 is compared to day 4 via
an interaction term (day $*$ label/control, see methods), we found only two
OTUs had significantly different enrichment between time points ("OTU.227" and
"OTU.4037", Table~\ref*{tab:LTP_blast}). In addition, when day
2 and day 4 samples are treated as replicates (see methods) only five of the
OTUs we identified as responders OTUs were not significantly enriched in
labeled gradient heavy fractions versus control ("OTU.140", "OTU.4037",
"OTU.227", "OTU.137", and "OTU.263", Table~\ref*{tab:LTP_blast}). The labeling
of these OTUs should be interpreted with caution. None of the top 10 strongest
responding OTUs showed inconsistent enrichment across time points
based on the above analyses. Further, confidence in enrichment (i.e. lowest
enrichment P-values between Day 2 and Day 4) appears to be correlated with
consistency in response across both days (Figure~\ref*{fig:2v4}).

\subsection{$^{15}$N-responsive OTUs are found in low abundance in
    available environmental BSC SSU rRNA gene surveys} In total 13 of the
34 $^{15}$N-responsive OTUs have been observed previously in SSU rRNA gene
surveys of BSC communities (Figure~\ref*{fig:trees},
Figure~\ref*{fig:rspndr_dist}). Eleven of the 19 $^{15}$N-responsive
\textit{Firmicutes} OTUs are members of the \textit{Clostridiaceae}. Three
$^{15}$N-responsive \textit{Clostridiaceae} have been observed in previous
BSC SSU rRNA gene surveys.  Two $^{15}$N-responsive
\textit{Clostridiaceae} were found in "light" (\textit{i.e.} early
successional) crust during SSU rRNA gene sequence analysis of BSC
\citep{Garcia_Pichel_2013}\,, and one $^{15}$N-responsive
\textit{Clostridiaceae} OTU was found among the "below crust" BSC SSU rRNA
gene sequences described by \citet{Steven_2013} (Figure~\ref*{fig:trees}).
Five $^{15}$N-responsive proteobacterial OTUs (Table~\ref*{tab:LTP_blast})
were detected previously in BSC samples
\citep{Garcia_Pichel_2013,Steven_2013}.  The $^{15}$N-responsive
\textit{Gemmatimonadetes} OTU was observed in four
\citet{Steven_2013} samples and one $^{15}$N-responsive
\textit{Actinobacteria} OTU was found in three \citet{Steven_2013}
samples. \textit{Gemmatimonadetes} and \textit{Actinobacteria}
$^{15}$N-responsive OTUs were not observed in samples collected by
\citet{Garcia_Pichel_2013}.

\subsection{Comparison of SSU rRNA gene sequences from different BSC samples}
We compared the SSU rRNA gene sequences determined in this DNA-SIP experiment
with two previous surveys of SSU rRNA gene amplicons from BSC communities
\citep{Garcia_Pichel_2013, Steven_2013}\,. There were 3,079 OTUs
(209,354 total sequences after quality control) in the DNA-SIP data, 3,203 OTUs
(129,033 total sequences after quality control) in the
\citet{Garcia_Pichel_2013} study, and 2,481 OTUs (129,358 total sequences after
quality control) in the \citet{Steven_2013} study with a total of 4,340 OTUs
across all three datasets. Of the total 4,340 OTU centroids established
for this study, 445 have matches in the Living Tree Project (LTP) (a collection
of SSU rRNA gene sequences for all sequenced type strains \citep{Yarza_2008})
at or above a threshold of 97\% sequence identity (LTP version 115). That is,
445 of 4,340 OTUs are closely related to known isolates. The DNA-SIP data
shares 56\% OTUs with the \citet{Steven_2013} data and 46\% of OTUs with
the \citet{Garcia_Pichel_2013} data, while these latter two studies share
46\% of their OTUs. This result suggests that low frequency OTUs likely
remain undersampled in all datasets. 

Sequencing of DNA subjected to CsCl fractionation is expected to sample
a different subset of diversity than that sampled by sequencing of
unfractionated bulk DNA. For example, SIP enhances detection of OTUs that
incorporate $^{15}$N into their DNA, and these OTUs will be overrepresented in
the overall DNA-SIP sequence pool relative to their relative abundance in
unfractionated bulk community samples. In addition, the DNA-SIP sequencing
effort was directed at a relatively small number of "light" crust samples (n
= 4), while previous sequencing efforts \citep{Garcia_Pichel_2013, Steven_2013}
were spread across hundreds of samples from both "light" and "dark" crusts.
Hence, it is likely that the current study will be more likely to detect rare
OTUs present in early successional "light" crust communities, particularly
those that incorporate $^{15}$N into DNA. In all three BSC studies, most
sequences were annotated as either cyanobacteria or \textit{Proteobacteria},
though only in the DNA-SIP data did the sequences of \textit{Proteobacteria}
outnumber those of cyanobacteria. \textit{Proteobacteria} represented 29.8\% of
sequence annotations in DNA-SIP data as opposed to 17.8\% and 19.2\% for the
\citet{Garcia_Pichel_2013} and \citet{Steven_2013} data, respectively. In
addition, sequences annotated as \textit{Firmicutes} were more abundant in the
DNA-SIP data (19\%) than in the data from \citet{Steven_2013} and
\citet{Garcia_Pichel_2013} (0.21\% and 0.23\%, respectively)
(Figure~\ref*{fig:study_phy_dist}). Finally, and congruently with sampling
design sequences annotated to "Subsection IV" of cyanobacteria, which
encompasses the heterocystous cyanobacteria in the Silva taxonomic nomenclature
\citep{17947321}\,, comprised only 0.29\% of cyanobacteria sequences in the
DNA-SIP data while representing 15\%  and 23\% of cyanobacteria sequences from
the \citet{Steven_2013} and \citet{Garcia_Pichel_2013} data, respectively.
