Phenotypic and genotypic analysis of 75 <i>Bradyrhizobium</i> strains.

1<p>Strain codes include year of isolation (05 = 2005), host species (LoA = <i>Lotus angustissimus</i>, LoM = <i>L. micranthus</i>, LoH = <i>L. heermannii</i>, LoS = <i>L. strigosus</i>), plant number, and nodule or root-surface number (the latter with R followed by root and isolate number).</p>2<p>Results of greenhouse nodulation assays in which each <i>Bradyrhizobium</i> isolate was tested on 5–8 inoculated seedlings to examine nodulation capability.</p>3<p>Results of PCR amplification assays in which we attempted to amplify each of three symbiosis loci (<i>nifD</i>, <i>nifH</i>, <i>nodDA</i>). Unsuccessful reactions were repeated thrice to confirm lack of amplification.</p

Relative host-growth effects of ancestral versus lab evolved <i>Bradyrhizobium</i>.

1<p>Mean relative growth effects of each <i>Bradyrhizobium</i> strain (Rel Grow) from ancestral (<i>ancest.</i>) and evolved strains (<i>evolv.</i>) is measured in grams (g) with standard error (se) by subtracting the dry biomass of control plants from their size-matched inoculated plant.</p>2<p>N, F and <i>p</i> values are given for a two-tailed ANOVA comparing relative growth effects of each strain in ancestral (<i>ancest.</i>) and evolved (<i>evolv.</i>) cultures.</p

Symbiotic quality measures of ancestral and evolved <i>B. japonicum</i> isolates.

<p>Relative growth effects (panels A, B) and nodulation rates (C, D) are shown for experimentally evolved strains #4 and #35 compared to their ancestors. Relative growth effects were analyzed by subtracting the size-matched control plant shoot biomass from the shoot biomass of each inoculated plant. Nodulation rates are the number of nodules per inoculated plant. Strains #4 and #35 are shown because they exhibited the greatest evolutionary increase in <i>in vitro</i> fitness as well as the largest reduction in symbiotic quality.</p

Ancestral state reconstruction of nodulation ability on <i>B. japonicum</i> phylogeny.

<p>Bayesian phylogram of 74 <i>Bradyrhizobium japonicum</i> isolates from Bodega Marine Reserve <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone.0026370-Sachs3" target="_blank">[5]</a> inferred with three loci (Its, GlnII, RecA; total 2,238 nt) and rooted with three fully sequenced <i>Bradyrhizobium</i> strains (<i>B. japonicum</i> USDA110, <i>Bradyrhizobium sp.</i> BTAi1 and <i>Bradyrhizobium sp</i> ORS278). The tree represents a single sample from the post-burnin set of trees, in which branch lengths are scaled to indicate number of nucleotide changes. Beginning from the left, taxon labels for rhizobial isolates consist of strain number (1–75), year of isolation (05 = 2005), host species (LoA = <i>Lotus angustissimus</i>, LoM = <i>L. micranthus</i>, LoH = <i>L. heermannii</i>, LoS = <i>L. strigosus</i>), plant number, and nodule or root-surface number (the latter with R followed by root and isolate number). Strain number 27 was too divergent to include on the tree as it is more closely related to <i>Methylobacterium </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone.0026370-Sachs7" target="_blank">[22]</a>. Symbiotic phenotypes on <i>L. strigosus</i> from the inoculation assays are indicated on the tips of the tree with rectangular labels (black = nodulating on <i>L. strigosus</i>, white = non-nodulating on <i>L. strigosus</i>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone.0026370-Sachs7" target="_blank">[22]</a>). Bayesian clade support values (posterior probabilities) are reported above the branches of all well-supported clades (pp≥0.80). Ancestral states are estimated for all well-supported internal nodes (pp≥0.90; labeled #s 1–30 in boxes) for the binary character of nodulating or non-nodulating on <i>L. strigosus</i> (using parsimony, maximum likelihood and Bayesian stochastic character mapping; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone-0026370-t002" target="_blank">Table 2</a>). Bayesian posterior probabilities of the ancestral states are reported using pie charts with black filling indicating the posterior probability of the ancestor being nodulating. In the parsimony analysis all 30 well-supported ancestral nodes were inferred to be non-nodulating except for #'s 12, 14 and 16.</p

Fitness evolution in <i>in vitro</i> evolved <i>Bradyrhizobium</i> isolates.

1<p>Strain codes are listed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone-0026370-t001" target="_blank">Table 1</a>.</p>2<p>Symbiotic phenotypes describe nodulating and growth effects status on <i>L. strigosus</i> in inoculation assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone.0026370-Sachs3" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone.0026370-Sachs7" target="_blank">[22]</a>).</p>3<p>Doubling (DT) time and standard error (se) are indicated for ancestral (<i>ancest.</i>) and evolved strains (<i>evolv.</i>).</p>4<p><i>F</i> and <i>p</i> values are given for a two-tailed ANOVA comparing <i>in vitro</i> doubling time in cycle 0 and 30 cultures.</p>5<p>Evolved strain #38 is not included because it was removed due to contamination.</p

Ancestral state reconstruction for supported nodes on <i>Bradyrhizobium</i> phylogeny¹.

1<p>Ancestral states are inferred on the <i>Bradyrhizobium</i> phylogeny (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026370#pone-0026370-g001" target="_blank">Figure 1</a>) for the subset of internal nodes with clade support values equal or greater than 0.9.</p>2<p>Clade support indicates the Bayesian posterior support value for each ancestral node.</p>3<p>Pr(nodulating) indicates the posterior probability of the ancestral state of nodulation estimated using both Bayesian and maximum likelihood algorithms.</p

Hollowell_S1

Table of all isolates included in analyses and associated accession numbers

Wendlandt_2018_New_Phytologist_Raw_Data

Raw data collected from individual plants (per-plant raw data) and individual nodules (per-nodule raw data) corresponding to Wendlandt et al 2018 (New Phytologist). Per-plant data include measures of leaf number at time of inoculation, shoot and root mass at time of harvest, total number and dry mass of root nodules, and nitrogen isotopic data from leaf tissue. Per-nodule data include nodule area, number of rhizobial cells per nodule (for nodules from plants inoculated with a single strain), and number of rhizobial colonies identified as particular strains (for nodules from plants co-inoculated with three strains)

Hollowell S10 Carbon Utulization Assays

Phenotypic Array data for Carbon Utilizatio