\section{Discussion}
% Fakesubsubsection:BSC N-fixation has long been attributed
BSC N-fixation has long been attributed to heterocystous
cyanobacteria and the preponderance of cyanobacterial \textit{nifH} genes
observed in molecular surveys of BSCs have generally supported this hypothesis
\citep{Yeager,Yeager_2004,Yeager_2012}\,. However, in this study
$^{15}$N$_{2}$-DNA-SIP reveals that non-cyanobacterial microorganisms fix
N$_{2}$ in early successional BSC samples. \textit{Proteobacteria} and
\textit{Clostridiaceae} were most abundant among $^{15}$N$_{2}$-responsive OTUs
as revealed by a robust statistical framework for quantifying and evaluating
differential OTU abundance in microbiome studies \citep{24699258,Love_2014}\,.
Many of these OTUs (about 40\%) have been observed previously in BSC
communities. Rarefaction curves of data from \citet{Steven_2013} and
\citet{Garcia_Pichel_2013} are still sharply increasing especially for
sub-crust samples (Figure~\ref*{fig:rarefaction}) suggesting the communities
remain undersampled. Parametric richness estimates of BSC diversity indicate
that the \citet{Steven_2013} and \citet{Garcia_Pichel_2013} sequencing efforts
recovered on average 40.5\% (s.d.  9.99\%) and 45.5\% (s.d. 11.6\%) of
predicted SSU rRNA gene OTUs from crust samples (inset
Figure~\ref*{fig:rarefaction}), respectively. Therefore, it would have been
surprising if all of the $^{15}$N-responsive OTUs had been observed in prior
environmental surveys of BSCs. Nitrogenase \textit{nifH} gene sequences related
to both \textit{Proteobacteria} and \textit{Clostridiaceae} have been
previously observed in BSC samples, though typically at relative abundance that
is much lower than \textit{nifH} gene sequences from heterocystous
cyanobacteria.

% Fakesubsubsection:Notably, the DNA-SIP analysis did not
We propose three mechanisms that could bias
\textit{nifH} clone libraries against heterotrophic diazotrophs. First, extreme
polyploidy in cyanobacteria (up to 58x ploidy in stationary
phase,\citep{Griese_2011}) can be expected to inflate the representation of
cyanobacteria \textit{nifH} gene sequences in community DNA relative to the
frequency of N$_{2}$-fixing heterocysts. Although, cyanobacteria often
have relatively large cells so ploidy per cell is probably greater than ploidy
per unit volume. Second, heterocysts make up a small fraction of total cells
along a trichome, though all cells in the trichome possess the \textit{nifH}
gene. As a result of polyploidy and heterocyst frequency in a cyanobacterial
filament, the ratio of cyanobacterial \textit{nifH} gene copies to
heterotrophic \textit{nifH} gene copies may be inflated as much as 10$^{3}$
times relative to the corresponding ratio of N$_{2}$-fixing cells (i.e.
the ratio of heterocyst number to the cell number of heterotrophic
diazotrophs). Third, \textit{nifH} PCR primers, which are highly degenerate,
could be biased against heterotrophic diazotrophs. For example, the
\textit{nifH} PCR  primers used in the second round of a widely used nested PCR
protocol \citep{Yeager,Yeager_2004,Yeager_2012} have fairly low coverage for
\textit{Proteobacteria} and \textit{Clostridiales} \citep{Gaby_2012}\,. Primer
``nifH11'' is biased against ``Cluster III'' \textit{nifH} gene sequences which
includes those of the \textit{Clostridiales} (50\% \textit{in silico} coverage
of reference \textit{nifH} sequences). In addition, primer ``nifH22'' has low
coverage of reference sequences from \textit{Proteobacteria}, cyanobacteria and
``Cluster III'' \textit{nifH} gene sequences (16\%, 23\% and 21\% \textit{in
silico} coverage, respectively) \citep{Gaby_2012}\,. Hence, it is reasonable to
assume that heterotrophic diazotrophs may have been underestimated in previous
analyses of early successional BSC communities. Our DNA-SIP results, which do
not require PCR of functional genes, suggest that BSC N-fixation in early
successional BSC may include a large non-cyanobacterial component. This is
consistent with small-scale, spatially resolved functional measurements of
nitrogen fixation in BSCs \citep{15643930} that show a subsurface maximum that
does not coincide spatially with maxima in chlorophyll \textit{a}  (a proxy for
phototrophic biomass) in early-successional crusts, and a surface maximum of
N$_{2}$-fixation in mature crust that coincides with the maximum in chlorophyll
\textit{a}.  

% Fakesubsubsection:We did not observe incorporation of
We did not observe incorporation of $^{15}$N$_{2}$ into the DNA of
heterocystous cyanobacteria in the early successional BSC samples used in this
study. It is possible that $^{15}$N$_{2}$-fixation by heterocystous
cyanobacteria could go undetected in DNA-SIP. One possible explanation for this
result is that the early successional BSC samples used in this study possessed
too few heterocystous cyanobacteria to statistically evaluate their
$^{15}$N-incorporation. Indeed, heterocystous cyanobacteria represented only
0.29\% of sequences from the DNA-SIP data (see results) as opposed to 15\% and
23\% of total sequences in the \citet{Steven_2013} and
\citet{Garcia_Pichel_2013} data, respectively. OTUs that correspond to
heterocystous cyanobacteria (as defined by \citet{Yeager}), all fall below the
sparsity threshold used in our analysis (see methods). Given the sparsity of
heterocystous cyanobacteria sequences in the light crust DNA-SIP data, it is
not possible to conclusively determine whether heterocystous cyanobacteria
incorporated $^{15}$N during the incubation. Our results show that
heterotrophic diazotrophs can contribute to N$_{2}$-fixation in early
successional BSC but they do not exclude the potential for fixation by
heterocystous cyanobacteria. Indeed, heterocystous cyanobacteria if present,
active, and limited for nitrogen would be expected to form heterocysts and fix
N$_{2}$. It is likely that scarcity limits their contribution to
N$_{2}$-fixation in early successional crusts. Heterocystous
cyanobacteria form sessile colonies and they require stabilization of the crust
environment before they can successfully colonize soil; and this stabilization
is performed by other pioneering members of the crust community
\citep{Castenholz_2002}\,.  N$_{2}$-DNA-SIP would also fail to identify
N$_{2}$-fixing bacteria if N$_{2}$-fixation were uncoupled from
DNA replication over the time frame of the experiment (i.e. 4 days), that is
$^{15}$N$_{2}$-DNA-SIP will not detect bacteria that fix N$_{2}$ but do
not incorporate the $^{15}$N-label into DNA. Therefore, the contribution of
heterocystous cyanobacteria (or any other microbe) to N$_{2}$ would be
underestimated if their cell division is uncoupled from N$_{2}$-fixation
at time frames of up to 4 days. We should also note that $^{15}$N can be
incorporated into biomass from trophic interactions although in this case the
$^{15}$N labeling would likely be weaker than that for a N$_{2}$-fixer as
a result of label dilution.
 
% Fakesubsubsection:The OTUs with significant evidence of
The OTUs with significant evidence of $^{15}$N-incorporation during the
incubation were predominantly \textit{Proteobacteria} and \textit{Firmicutes}.
The \textit{Proteobacteria} OTUs with the strongest signal of
$^{15}$N-incorporation all shared high sequence identity ($>$98.5\%) with SSU
rRNA gene sequences from genera known to contain diazotrophs
(Table~\ref*{tab:LTP_blast}). In contrast the \textit{Firmicutes} that
displayed signal for $^{15}$N-incorporation (predominantly
\textit{Clostridiaceae}) were not closely related to any known cultivars
(Table~\ref*{tab:LTP_blast}). Hence, we have little knowledge of the ecology of
these organisms. Assessing the physiological characteristics of these
diazotrophic \textit{Clostridiaceae} may be useful for predicting how
environmental change will affect the development and stability of BSC. Prior
intense cultivation efforts from these crusts in separate studies did not yield
any members of the \textit{Clostridiaceae} \citep{Gundlapally_2006}\,. Although
under sampled in environmental data sets, $^{15}$N-responsive OTUs were indeed
more abundant in sub-crust or in early successional BSC samples relative to
crust surface or mature crust samples (Figure~\ref*{fig:trees} and
Figure~\ref*{fig:rspndr_dist}). While members of \textit{Clostridiaceae} have
been found in low abundance in molecular surveys of BSC, most surveys are
carried out on desicated crust samples, where thick-walled spores would
predominate relative to vegetative cells, thus increasing the likelihood for
their underrepresentation in DNA surveys. It should also be noted that crusts
were incubated in an atmosphere of He and N$_{2}$ rather than O$_{2}$ and
N$_{2}$. While cyanobacteria in the presence of light rapidly produce oxygen
super saturation in BSC relative to air \citep{JPY:JPY774}\,, and whereas
heterotrophic N$_{2}$-fixation by many microorganisms is inhibited in the
presence of atmospheric levels of O$_{2}$, it remains possible that the
conditions present in microcosm are not representative of field conditions and
may have favored N$_{2}$-fixation by crust organisms that are less active in
situ. Further experiments will need to be performed to verify that these
heterotrophic diazotrophs are contributing to the N budgets of early
successional crusts in the field.  

% Fakesubsubsection:Our results generate more refined hypotheses
Our results generate more refined hypotheses pertaining to the contribution of
diazotrophs during the development of BSC communities.  Specifically,
N$_{2}$-fixation in BSC may not be tied solely to the climax of
heterocystous cyanobacteria in mature crusts. Rather, N$_{2}$-fixation
may occur throughout crust development with the transition between early
successional and mature crusts marked by a transition between heterotrophic and
phototrophic N$_{2}$-fixation in the crust community. Therefore,
sub-biocrust soil may contribute significantly to the arid ecosystem N budget
and may be of considerable importance in the early phases of BSC establishment.
We propose that interactions between fast-growing heterotrophic diazotrophs
such as members of the \textit{Clostridiaceae} and filamentous
(non-heterocystous) cyanobacteria are important in the early establishment of
BSC communities.  During progressive desication, cyanobacteria, such as
\textit{M. vaginatus}, accumulate compatible solutes such as trehalose and
sucrose \citep{Rajeev_2013}\,. Upon wetting, microorganisms rapidly excrete
compatible solutes to prevent cell lysis due to osmotic shock
\citep{Poolman_1998}\,. Among them are dihexoses (such as sucrose and
trehalose), which are observed in natural crusts upon wetting and then are
rapidly depleted in the soil solution \citep{baran}\,.  Many
\textit{Clostridiaceae} have a saccharolytic metabolism with the potential for
rapid growth rates on substrates such as trehalose and/or sucrose
\citep{Wiegel_2006}\,. Wetting of crust may allow for rapid germination and
growth of these organisms as the time required for germination of clostridial
spores can be less than 30 minutes \citep{Stringer_2005}\,. Indeed, intense
blooms of clostridia have been detected in crusts within tens of hours of
wetting \citep{karaoz}\,. N$_{2}$-fixing clostridia are common in soils
\citep{Wiegel_2006} and it is notable that \textit{C. pasteurianum}, isolated
from soil, was the first N$_{2}$ fixing bacterium ever described
\citep{wino_1985}\,. \textit{C. pasteurianum}, though an anaerobe, grows
readily in the presence of oxygen when co-cultured with aerobic organisms that
reduce oxygen tension \citep{chester}\,. We propose that during a typical
precipitation event, water saturation and heterotrophic activity rapidly render
the interior of the crusts anoxic \citep{JPY:JPY774}, presenting optimal
conditions for growth of anaerobic, dihexose-fermenting, N$_{2}$ fixing
clostridia. Clostridial organic nitrogen would then become available to other
members of the community, including the primary producers, when carbon
limitation induces sporulation and mother cell lysis. Mother cell lysis, the
last step in sporulation, releases rich sources of P and N into the environment
in the form of nucleotides and peptides \citep{Hoch_2002}\,. 

\subsection{Conclusion} The abundance of $^{15}$N-responsive OTUs from
\textit{Clostrideaceae} and \textit{Proteobacteria} found in this study, the
\textit{nifH} gene sequences of \textit{Clostrideaceae} and
\textit{Proteobacteria} observed previously in BSC \citep{Steppe_1996}\,, and
the evidence for subsurface N$_{2}$-fixation in early successional BSC
\citep{15643930}\,, taken together, suggest that heterotrophic diazotrophs may
be important contributors to N$_{2}$-fixation in the subsurface of early
successional BSC. Heterocystous cyanobacteria are also key contributors to the
BSC N-budget, however and it is clear that heterocystous cyanobacteria increase
in abundance with BSC age \citep{Yeager_2004}\,. It is less clear if the
transition to mature crust is marked mainly by a change in the abundance and
activity of heterocystous cyanobacteria, or rather represents a succession
within the diazotroph community from early crusts where N$_{2}$-fixation
is dominated by \textit{Clostridiaceae} and \textit{Proteobacteria} to mature
crusts where it is dominated by heterocystous cyanobacteria. Predicting the
ecological response of BSC to climate change, altered precipitation regimes,
and physical disturbance requires an understanding of crust establishment,
stability, and succession. Diazotrophs are critical contributors to all of
these phenomena and their activities make critical contributions to the
N-budget of arid ecosystems worldwide.
