\section{Introduction} Biological soil crusts (BSC) are specialized microbial
communities that form at the soil surface in arid environments and they fill
a variety of important ecological functions. BSCs occupy plant interspaces and
cover a wide, global geographic range \citep{garcia2003estimates}\,. For
example, in some regions on the Colorado Plateau BSCs cover 80\% of the ground
\citep{karnieli2001}\,. The global biomass of BSC cyanobacteria alone is
estimated at 54 x 10$^{12}$ g C \citep{garcia2003estimates}\,. BSD nitrogen
fixation (N$_{2}$-fixation) is responsible for significant input of nitrogen
(N) to arid environments \citep{Evans_1999,belnap2003}\,. Interestingly, much
of this fixed N is exported from the crusts in dissolved form through
percolation or runoff and little is lost to volatilization
\citep{Johnson_2007}\,. The presence of BSC is positively correlated with
vascular plant survival due in part to N inputs from BSC (for review of
BSC-vascular plant interactions see \citealt{BelnapVascular}). These microbial
ecosystems are not immune to climate change and changes in precipitation and
temperature could alter BSC microbial community structure/membership and
possibly BSC diazotroph diversity and N$_{2}$-fixation
\citep{Garcia_Pichel_2013}\,.

BSC are highly susceptible to natural and anthropogenic disturbance
\citep{Garcia_Pichel_2013}. Succession in BSC communities is characterized by
transition from early successional "light" crusts to mature "dark" crusts
\citep{Yeager_2004,Belnap_2002}\,. Motile non-heterocystous
cyanobacteria(e.g.\textit{Microcoleus vaginatus} or \textit{M. steenstrupii}),
which cannot fix N$_{2}$ are pioneer colonizers of early successional crusts
and are abundant in all types of BSCs \citep{Garcia_Pichel_2013}\,.
Successional development of mature crust is accompanied by a change in color
produced by secondary colonization with non-motil N$_{2}$-fixing heterocystous
cyanobacteria which produce sunscreen compounds that reduce soil albedo
\Citep{Belnap_2002,Yeager_2004}\,. These heterocystous cyanobacteria (e.g.
\textit{Scytonema}, \textit{Spirirestis}, and \textit{Nostoc}) increase in
abundance during crust development and are more abundant in mature crusts
\citep{Yeager,Yeager_2012}. Heterocystous cyanobacteria are numerically
dominant in surveys of BSC \textit{nifH} gene diversity \citep{Yeager,
Yeager_2004, Yeager_2012}\,. For example, 89 percent of 693 \textit{nifH}
sequences derived from Colorado Plateau and New Mexico BSC were attributed to
heterocystous cyanobacteria \citep{Yeager}\,. Other BSC \textit{nifH} sequences
are attributed to \textit{Alpha-}, \textit{Beta-}, and
\textit{Gammaproteobacteria}, as well as a \textit{nifH} clade (\textit{nifH}
cluster III) that includes diverse anaerobes such as clostridia, sulfate
reducing bacteria, and anoxygenic phototrophs \citep{Steppe_1996, Yeager}\,.

Two lines of evidence suggest that nitrogen fixers other than phototrophs are
important in early-successional crusts.  First, the contributions of early
successional BSC to N$_{2}$-fixation in arid ecosystems may have been
systematically underestimated. The high abundance of heterocystous
cyanobacteria at the surface of mature crusts, where acetylene reduction assay
rates are often maximal, is generally taken as evidence that BSC
N$_{2}$-fixation occurs primarily in mature crusts and is dominated by
heterocystous cyanobacteria. However, rates of BSC N$_{2}$-fixation are
typically determined by areal measurements made at the crust surface with the
acetylene reduction assay and vary significantly across samples and studies
\citep{Evans_2001}\,. The reasons for inter-site and inter-study variability
are complex and likely include the spatial heterogeneity of BSC
\citep{Evans_2001}\,.  The acetylene reduction assay is also subject to
methodological artifacts that can complicate comparisons between samples that
differ in their physical and biological characteristics (see
\citealt{Belnap_2001} for review). In particular, N$_{2}$-fixation in early
successional BSC is maximal below the crust surface \citep{15643930} and hence
diffusional limitation (of both acetylene and ethylene) across the crust
surface can cause severe underestimates if they do not allow for sufficiently
long incubation times \citep{15643930}\,.  If BSC N$_{2}$-fixation is instead
estimated by integrating rates across a depth profile (which eliminates
constraints from diffusional limitation), then total rates of N$_{2}$-fixation
do not differ significantly between early successional and mature BSC
\citep{15643930}\,. This result suggests that diazotrophs other than
heterocystous cyanobacteria may be important contributors to N$_{2}$-fixation
in early successional BSC communities as early successional BSC possess few
heterocystous cyanobacteria and these are present near the crust surface.
Second, the bare soils that are colonized during the process of early crust
formation are unconsolidated and oligotrophic in many respects, with much lower
N content than adjacent crusts \citep{BERALDI_CAMPESI_2009}\,, and the
cyanobacteria that are typical colonization pioneers (\textit{Microcoleus}
spp., \citealt{Garcia_Pichel_2009})\,, are unable to fix nitrogen as they lack
that genetic capacity \citep{Starkenburg_2011, Rajeev_2013}\,. 

To determine the agency of nitrogen fixation in early developmental
crusts, we conducted $^{15}$N$_{2}$ DNA stable isotope probing (DNA-SIP)
experiments with early successional Colorado Plateau BSC conspicuously devoid
of significant surface populations of heterocystous cyanobacteria.  DNA-SIP
with $^{15}$N$_{2}$ has not been previously attempted with BSC. DNA-SIP
provides an accounting of \textit{active} diazotrophs on the basis of
$^{15}$N$_{2}$ assimilation into DNA whereas \textit{nifH} clone libraries
merely account for microbes with the genomic potential for N$_{2}$-fixation.
Further, we investigate the distribution of these active diazotrophs in surveys
of microbial diversity conducted on BSC over a range of spatial scales and soil
types \citep{Garcia_Pichel_2013, Steven_2013}\,. 
