Evidence of endophytic nitrogen fixation as a potential mechanism supporting colonization of non-nodulating pioneer plants on a glacial foreland

The ability of non-nodulating pioneer plants on newly exposed glacial till to obtain atmospheric N 2 was investigated using both acetylene reduction assay (ARA) and 15 N 2 gas incorporation methods. Plant tissues were also examined for the presence of endophytic diazotrophic bacteria using high-throughput nif H amplification sequencing. Both ARA and 15 N 2 gas incorporation demonstrated that non-nodulating pioneer plants can obtain atmospheric N 2 , but the amount was highly variable depending on the plant organ, with clearly higher values in leaves relative to twigs and roots. High-throughput amplification sequencing of nif H genes indicated that a great variety of diazotrophic communities developed in pioneer plant tissues under primary succession. The N 2 fixation rate (based on both 15 N 2 incorporation and ARA) was not directly linked to the abundance of any specific diazotrophs, but was correlated with the Chao1 index and the number of observed species, suggesting that different diazotrophs jointly accounted for N 2 fixation activity. Our results highlight that non-nodulating pioneer plants may acquire N through endophytic N 2 -fixing bacteria as a supplement for other potential N sources in oligotrophic environments in early stages of primary succession following glacier retreat.