Phylogenetic Analysis
Maximum-likelihood (ML) phylogenetic analysis was performed using RAxML,
version 8 (Stamatakis 2014), as implemented on the CIPRES network,
version 3.3 (Miller et al . 2010). We used a GTR+G substitution
model and 1000 rapid bootstrap replicates. To facilitate comparison of
our soil-derived sequences with nodule-dwelling Frankia from the
same field locations, we sequenced the rIGS locus from 57
previously-collected nodules representing the thirteen most
commonly-observed genotypes across both A. tenuifolia andA. viridis host species (Anderson et al . 2009; 2013) that
were based on restriction fragment analysis of the nifD-K spacer locus.
In order to place our sequences within a broader phylogenetic context,
we also included reference sequences from Ghodbane-Gtari (2010), and a
set of actinobacterial outgroups downloaded from Genbank.
Operational taxonomic units (OTUs) were defined in two ways: 1) based on
well-supported clades in the phylogenetic analyses, and 2) at specific
levels of sequence similarity. For the former, clades were selected by
eye based on distance (a selected clade had a long stem branch, relative
to other clades), cohesion (a selected clade should lack long branches
within the clade), and statistical support (≥70% bootstrap). Similarity
threshold-based OTUs were generated using the average neighbor
clustering algorithm implemented in the ‘cluster’ command in mother,
version 1.0.0 (Schloss et al . 2009).
To check for sensitivity of our phylogenetic and OTU designation results
to variation in our alignment, we used the program Gblocks (Castresana
2000) to remove portions of the alignment deemed unreliable according to
the most stringent, the least stringent, and an intermediate set of
parameters. The resulting alignments were then analyzed using RAxML, and
the best tree qualitatively compared with the best tree based on the
entire alignment.