Nitrogen (N) is considered to be the major limiting factor for both microbial and plant growth in arctic and alpine biomes (Chapin et al., 1986; Wallenstein et al., 2009; Sistla et al., 2012). In particular, the amount of available soil nitrogen is very low in low organic content mineral-rich tundra soils. One example is given by recently deglaciated soils with low levels of (mineralizable) nitrogen and carbon that are colonized by patchy pioneer plant communities (Brankatschk et al., 2011). In these soils, nitrogen fixation is the primary source of bioavailable nitrogen (N) for plants (Chapin et al., 1992). Furthermore, the levels of nitrogen fixation and N cycling were shown to be correlated with vegetation cover by Brankatschk et al. (2011). These authors observed no N-fixation activity, and extremely low denitrification and nitrification rates, coupled to low nifH gene copy numbers, in the youngest (10-year old) soils in glacier forefields. In contrast, a densely plant-covered 120-year old soil in the same chronosequence showed both higher enzyme activities and higher copy numbers of the nifH, next to nirK and nirS, genes. Clearly, the older soils had gained the capacity to cycle nitrogen on the basis of a pioneer-species-based ecosystem build-up, in which plants found capacities to grow and, in turn, supported the evolved microbial communities and activity.

Arctic plants are well-adapted to low temperatures during the growing season. In fact, the photosynthesis rates in the Arctic are comparable to those at lower latitudes (Central Europe) (Chapin et al., 1987). However, to compensate for the slower enzyme kinetics at low temperatures, higher amounts of enzymes are required. For example, arctic plants have significantly higher numbers of the key photosynthesis enzyme RuBisCO (ribulose-1, 5 bisphosphate-carboxylase-oxygenase). Consequently, the tissues of arctic plants contain more N than those of plants in warmer climates (Weintraub and Schimel, 2005), underlining the demand for N availability. Biological nitrogen fixation thus constitutes a key strategy involved in initial primary production. In the emerged systems, with plants in place as primary producers, the key nitrogen input will mainly come from plant-symbiotic and/or associative nitrogen fixers. The fixed nitrogen may then be recycled/mineralized via deamination (yielding ammonia), followed by nitrification and denitrification reactions. Associative nitrogen fixing bacteria are likely vital for plant growth in the arcto-alpine pioneer communities. Yet, very little is known about these bacteria, their diversity, or forces shaping their communities.

In previous studies, we focused on two arcto-alpine pioneer plant species, Oxyria digyna and Saxifraga oppositifolia, as “models” of the primary colonizers of the nutrient-poor mineral soils that abound in arctic and alpine regions. Preliminary evidence was found for the contention that the soil bacterial communities in different arcto-alpine regions are region-specific, whereas endophytic bacterial communities (using the 16S rRNA gene sequence as a proxy) are host plant specific (Kumar et al., 2017). However, these communities also share a group of bacterial taxa (core microbiome), which are apparently associated with the host plants in all three climatic regions, including several potential nitrogen fixers (Kumar et al., 2017).

In this study, we examined the factors shaping the communities of potential nitrogen fixing bacteria (PNFB) in the two selected arcto-alpine pioneer plants and the associated soils in different climate zones. We hypothesized that the bacterial taxa with known nitrogen fixers, which we had previously identified as major members of the core microbiomes of these pioneer plants are also major part of the nifH communities of these plants. Further, we hypothesized, that, like the total bacterial communities, the PNFB communities are host plant specific, and additionally shaped by climatic regions.

Regions (350 bp long) of the nifH genes were amplified from microbial community DNA isolated from the bulk and rhizosphere soils and endospheres from O. digyna and S. oppositifolia in the three regions (Figure 1) using the optimized PCR approach (described in Materials and Methods). The quality-trimmed and translated nifH sequences were used for clustering, resulting in a total of 296 OTUs (at 90% amino acid sequence identity). These 296 OTUs were further clustered based on alignments with NifH protein reference sequences in the RDP FunGene NifH database. This resulted in 97 units, denoted “operational phylogenetic units” (OPUs).

Considering all the OPU data, a high diversity of nifH gene sequences was detected in our soil and plant microbial communities. Thus, a suite of nifH genes, affiliated with the bacterial phyla Proteobacteria (classes α-, β-, δ-, and γ-Proteobacteria and Acidithiobacillia), Firmicutes, Actinobacteria, Chlorobi, Bacteroidetes, Cyanobacteria, and Verrucomicrobia, was detected (Figure 2). The most abundant reads in the dataset clustered with nifH gene sequences from the δ-Proteobacteria (genera Geobacter and Desulfotignum), γ-Proteobacteria (Bradyrhizobium), Firmicutes (genera Clostridium, Desulfosporosinus and Acetobacterium) and β-Proteobacteria (Burkholderia [Burkholderiales] and Leptothrix [Comamonadaceae]) (Figure 2). In the further text, we use these partial sequences to characterize the populations of “PNFB.”

Using the nifH gene as a proxy, we analyzed the diversity and community composition of PNFB in two pioneer plant species growing in low-nutrient soils in three arcto-alpine climate regions. Based on our previous work, we were aware of the presence of many PNFB taxa representing Firmicutes and δ-proteobacteria, that are routinely missed by many nifH-targeting primer combinations (Gaby and Buckley, 2012), in the bacterial communities associated with O. digyna and S. oppositifolia (Nissinen et al., 2012; Kumar et al., 2017). Thus, we optimized a nested PCR approach with primers 19f/nifH3 (1st round, Yeager et al., 2004) and nifH1f/ nifH2r+nifhH2 (Zehr and McReynolds, 1989; Izquierdo and Nüsslein, 2006). The amplification system was chosen, as it reportedly yields the highest phylogenetic coverage of diazotrophic bacterial groups (Gaby and Buckley, 2012).

Overall, we detected high diversities of PNFB across all samples. Our libraries had good phylogenetic coverage, with the collective PNFB OPUs representing seven phyla (Figure 2), including representatives from nifH clusters I, II, and III. This indicates that the selected PCR approach enabled maximal coverage from all the nifH phylogenetic groupings (Gaby and Buckley, 2012).

In particular, the PNFB communities in our soils were dominated by nifH gene types from the α-, β-, γ-, and δ-Proteobacteria and the Clostridiales. The δ-proteobacterial genus Geobacter and the β-proteobacterial genera Burkholderia and Leptothrix constituted the most abundant PNFB taxa in the soil communities across all regions. The phylogenetic composition of the PNFB in our data set differs from those reported for high-organic-matter tundra soils from Arctic or alpine sites, in which aerobic proteobacterial diazotrophs dominated (Deslippe and Egger, 2006; Tai et al., 2013).

The endosphere communities in our study were characterized by high relative abundances of nifH sequences from the genera Geobacter, Clostridium, Burkholderia, and Bradyrhizobium (Figures 5A,B). Of these nifH sequences clustering with Geobacter and Clostridium have been discovered in anoxic soils, sediments and wetland rice roots (Rösch et al., 2002; Yeager et al., 2004; You et al., 2005; Wu et al., 2009; Dang et al., 2013). Further, key PNFB taxa in our study, including G. uraniireducens Rf4, G. lovleyi SZ, and Optitutaceae bacterium TAV2, were recently found to be significantly enriched in communities below the water table in thawing permafrost soils in the Canadian Arctic (Penton et al., 2016). Prevalence of anaerobic taxa, also in the plant endosphere communities could be explained by conditions enabling nitrogen fixation under anoxia in most of the soils. Supporting this contention, we observed visible water logging in soils in most of the sampling sites at the time of sampling. Further, soil humidity levels in KJ sampling sites are typically above 36% at early growing season (Kumar, personal observation). We sampled all sites early in the growing season, when the target plants were flowering, which occurred relatively shortly after snowmelt. Both O. digyna and S. oppositifolia are known to be able to tolerate waterlogging in soil, and they are common snow patch species in low-arctic and alpine soils. It is, thus, likely that the respective plant roots were in the anoxic soil zone, early in the growing season.

Interestingly, Clostridium has been reported as an active nitrogen fixer in rice roots in flooded paddy fields (Minamisawa et al., 2004), consistent with our observations. In our study, Clostridium was mainly restricted to the endosphere in NÅ and KJ. This is in agreement with our previous analysis of the total bacterial communities of the same plants (based on the16S ribosomal RNA [rRNA] gene) (Kumar et al., 2017), where we observed Clostridium spp. to be restricted to the respective endospheres, with increasing relative abundances with increasing latitude in both plant species. Interestingly, 16S rRNA gene sequences of Clostridium spp. have been observed in seeds of O. digyna (Nissinen, unpublished) and rice (Hardoim et al., 2012), indicating possible vertical transmission.

Confirming our hypothesis, we observed a clear effect of region on the PNFB community structures. Interestingly, PNFB allocated to several Geobacter species were present in higher relative abundances in the MA (2,400 m a.s.l.) derived nifH sequence data sets. This is in line with reports showing that this taxon is abundant in high mountain ranges, where it may be active under both oxic and anoxic conditions (Duc et al., 2009). Evidence for the presence of PNFB Geobacter types has also been found in nifH defined communities from switchgrass roots in oxic temperate-climate prairie ecosystems (Bahulikar et al., 2014). This indicates adjustment of this organism to temperate and environmental conditions, including plasticity to function under both aerobic and anaerobic conditions. Curiously, nifH sequences related to several Geobacter species also dominated the endosphere communities in the MA region, while they were largely replaced by those from Clostridium and other PNBF taxa in the Arctic (Figure 6). As all the main PNFB OPUs were detected in all regions, this strong regionality cannot be explained by an absence of Geobacter in the Arctic or of Clostridium in the Alps. Rather, differential selection by plants or different conditions in the endorhiza in different regions could be the driver.

One of the main differences between arctic and alpine biomes is the better drainage of alpine soils compared to Arctic ones (Körner, 2003). The NÅ sites used in this study are underlaid by permafrost, and the KJ sites are characterized by patchy permafrost, with snow often melting away as late as mid-July, leading to water-logged conditions, at least early in the growing season. The alpine MA sites, on the other hand, were not water-logged at the time of the sampling. This observation could explain the differential abundance of several PNFB taxa in the different regions, also in the endosphere.

Corroborating the 16S rRNA gene based analyses reported earlier, endophytic bacterial taxa that were previously identified as key taxa in the total endosphere bacterial communities in these same plants, e.g., Bradyrhizobium, Burkholderiales, Comamonadaceae, and Clostridia (Kumar et al., 2017), were found to also make part of the PNFB endosphere communities, indicating an important role for the nitrogen fixing bacteria for these plants common in pioneer communities.

Partially confirming our hypothesis, we detected a significant, but small effect of host plant species (O. digyna and S. oppositifolia) on the endophytic PNFB communities across regions (Table 1). The difference between PNFB communities of the two plant species was smaller than the previously reported effect on total bacterial community structures (Kumar et al., 2017). This is likely due to strong regionality in the PNFB community structures. However, the PNFB genera that showed plant species specific accumulation in the individual regions (for example Burkholderia in O. digyna and Clostridium in S. oppositifolia) were also identified as showing host plant specificity in 16s rRNA based analyses of total bacterial communities of these same plants (Kumar et al., 2017).

We provided in this paper a comprehensive overview of PNFB associated with two pioneer plant species in three arcto-alpine climate zones. Our collective data show that the PNFB communities associated with these plants in three different cold-climate regions are subjected to selective processes that operate at different scales, i.e., that of compartment and that of region and climate. On the basis of the high relative abundances of anaerobic nitrogen fixers in these communities, we hypothesize that the local conditions allowing nitrogen fixation at the plants might have been prevalently anoxic in the two arctic regions, whereas those in the alpine region may have fluctuated between prevalently oxic or anoxic conditions. These PNFB communities, representing pioneer communities that are likely to become more common in near future due to changing climate, differ clearly from plant associated PNFB communities described in different climates and in high organic matter tundra soils.

Nucleotide sequence data have been submitted to the ENA database with accession number PRJEB20618.

Study was conceptualized and designed by RN, MK, and JvE. Field work was performed by MK and RN. Sample processing was done by MK and RN. Library preparation for sequence analysis was done by MK, and data analysis was done by MK and RN. Manuscript draft was prepared by MK, RN, and JvE, and revisions were done by MK, RN, and JvE. Final version for the submission was prepared by RN and JvE.