Lichen-symbiotic cyanobacteria associated with <i>Peltigera</i> have an alternative vanadium-dependent nitrogen fixation system

<div><p>In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the well-studied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genus <i>Nostoc</i> associated with four samples from different geographical regions, representing different lichen-forming fungal species in the genus <i>Peltigera</i>. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N₂ fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylene-reduction assay data.</p></div

Pollen dispersal distributions.

<p>In each stand, observed pollen dispersal is represented by dotted line, confidence intervals are delimited by solid lines. ________ confidence limits ------------- observed distribution.</p

Location of the eight studied white oak stands across Europe.

<p>Location of the eight studied white oak stands across Europe.</p

Description of oak stands.

<p>5 <i>Q. robur</i> islands (11.24 ha, 1 ha, 4 ha, 4.4 ha, trees along a 500m road),</p><p>1 <i>Q. faginea</i> island (26.8 ha).</p><p>Q. pubescens.</p><p>Q. faginea.</p

Variation of the percentage of hybrid acorns per mother tree.

<p>In each stand, each bar is a mother tree, bar height represents the percentage of acorns collected from the mother tree with a father assigned from a species different from the species of the mother tree (hence a hybrid). □ <i>Quercus petraea</i> ▪ <i>Quercus robur </i> Undetermined species.</p

Results of seedlings parentage analyses.

<p><i>Ns</i>: number of seedlings. <i>s</i>: percentage of selfing. <i>h</i>: percentage of hybrids, SD: standard deviation.</p><p>The three seedlings with two parents assigned were the result of selfing</p

Seed dispersal distributions.

<p>In each stand, observed seed dispersal is represented by dotted line, confidence intervals are delimited by solid lines. ________ confidence limits ------------- observed distribution.</p

Variation of the percentage of fathers assigned per mother tree.

<p>In each stand, each bar is a mother tree, bar height represents the percentage of acorns collected from the mother tree with a father assigned. □ <i>Quercus petraea</i> ▪ <i>Quercus robur </i> Undetermined species.</p

Variation of the relative reproductive success of male parents.

<p>The number of different fathers assigned after paternity analysis is given for each stand. In each stand, each bar is a father, fathers are ordered by decreasing reproductive success, bar height represents the percentage of the total number of acorns sired by the father. □ <i>Quercus petraea</i> ▪ <i>Quercus robur </i> Undetermined species.</p

Results of paternity analyses.

<p><i>Nof</i>: number of offspring with a father assigned (%). <i>pi</i>: pollen immigration. <i>Nf</i>: number of different fathers assigned. <i>Ngp</i>: Number of genotyped parents. <i>Nf</i>/<i>Ngp</i> (%). <i>Nof</i>/<i>Nf</i>: mean number of offspring per father (standard deviation (SD)). (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085130#pone-0085130-g002" target="_blank">Fig. 2:</a> male reproductive success). <i>Ne</i>: effective number of fathers (1/Σ(<i>f_i</i>²), where <i>f_i</i> is the relative reproductive success of each father).</p