Introduction

Members of the slow-growing genus Bradyrhizobium constitute an important group of rhizobia [1, 2]. They symbiotically fix N2 with diverse legume tribes and are the predominant symbionts of a wide range of nodulating legumes [3, 4]. Recent studies, however, have provided accumulating evidence for the existence of close relatives of rhizobia including Bradyrhizobium that do not carry nod genes which are essential for the establishment of nodulation, and thus are not able to nodulate legumes [5]. Increasingly, Bradyrhizobium strains without nod have been found to be particularly abundant in soils [6,7,8]. VanInsberghe et al. [8] showed that non-symbiotic Bradyrhizobium was the dominant bacterial lineage in North America coniferous soils. A global atlas of the dominant soil-dwelling bacteria based upon 16S rRNA gene amplicon sequencing indicated that the genus Bradyrhizobium was the most abundant bacterial lineage in soils across the world [6]. A recent study suggested that ancestral lineages of Bradyrhizobium might adapt to a free-living lifestyle, highlighting the importance of the free-living lifestyle in the evolution of Bradyrhizobium [9].

Although the majority of N2 fixation in terrestrial ecosystems is generally thought to be performed by symbiotic rhizobia [10,11,12], free-living N2-fixing bacteria may be important contributors to the nitrogen budgets in a number of environments, for example soil ecosystems that lack leguminous plants [13]. Free-living N2 fixation also occurs in understudied ecosystems such as deep soil and canopy soil [14, 15], which may lead to the underestimation of its global rates. However, rhizobia are generally thought to be capable of N2 fixation only in nodules [16, 17]. The exceptions are found in some members of Bradyrhizobium and Azorhizobium which were shown to be able to fix N2 in both the symbiotic and free-living states [18, 19]. Such cases in Bradyrhizobium were mostly explored in photosynthetic members [18, 19], but have recently been found in other strains. For instance, Bradyrhizobium sp. AT1, a non-symbiotic strain isolated from the root of sweet potato was reported to fix N2 with a rate comparable to the photosynthetic strain Bradyrhizobium sp. ORS278 under the molecular oxygen (O2) concentrations at 1–5% (for both) in the free-living state [20]. Another example is Bradyrhizobium sp. DOA9, which harbors a nif cluster on its chromosome and another on a symbiotic plasmid: while both could participate in N2 fixation during symbiosis, only the chromosomal one participated in N2 fixation in the free-living state [21].

Compared with the nodules, the condition of soils is not favorable for N2 fixation for several reasons. First, in contrast to being at nanomolar levels in nodules, the O2 concentration in soils could be up to atmospheric levels [22], which is highly detrimental to N2 fixation, a process that requires an anaerobic microenvironment [23]. Second, unlike symbiotic rhizobia, free-living rhizobia lack readily available organic matter provided by the host plants in exchange for fixed nitrogen. Last, free-living soil bacteria are frequently exposed to stresses like droughts, high osmolarity, and high temperature. It is therefore imperative to investigate the strategies that free-living N2-fixing Bradyrhizobium may use to deal with the harsh condition in soils.

Despite the potential ecological significance, free-living Bradyrhizobium, particularly those able to fix N2, have been poorly characterized. To this end, we isolated 88 Bradyrhizobium and five related strains from soils of soybean cropland, artificial park, forest, and grassland at several geographic locations in China, and had their genomes sequenced. By building a phylogenomic tree and reconstructing the ancestral lifestyles with additional 252 genomes where 42 are free-living Bradyrhizobium strains deposited in public databases, we revealed complex lifestyle shifts along the evolutionary history of Bradyrhizobium. Notably, we showed that horizontal gene transfer (HGT) of a unique nif island may have facilitated their transitions from symbiotic to free-living lifestyle and adaptation to soil habitats.

Methods and materials

Soil and plant tissue sampling and processing

Samples were collected from five different sites in China (Supplementary Fig. S1A) that cover several ecosystem types: soybean cropland (Heihe and Lvliang), artificial park (Shenzhen), undeveloped forest (Lanzhou), and grassland (Hefei). In Heihe, Lvliang and Hefei sites, intact Glycine max (soybean) and Erigeron annuus (annual fleabane) plants were excavated from soil, and shaken mildly to remove soil loosely adhering to the roots. The remaining adherent soil was separated from the roots as rhizosphere soil. For Shenzhen and Lanzhou sites, 0–5 cm and 5–10 cm depth bulk soil was collected from the vicinity of plant root. At the Shenzhen sampling site, the soil was collected near Acacia confusa (legume), Calliandra haematocephala (legume) and Bambusoideae (non-legume). At the Lanzhou site, the soil was collected near a non-leguminous plant Picea asperata. Detailed information about sampling sites was provided in Supplementary Dataset S1. Soil samples were placed in sterile bags, kept on ice, and immediately transported to the laboratory for further processing.

Bradyrhizobium isolation and identification

To prepare soil inoculum, 5.0 g of fresh soil was put in a 50 mL conical tube with 45 mL of sterile deionized water. After mixing with a vortex mixer, 1 mL of soil suspension was serially diluted, and the dilutions were used for inoculation. Roots of Erigeron annuus were processed according to Coombs and Franco [24]. One gram of surface-sterilized roots was grinded in a mortar and diluted with PBS buffer, and the dilutions of the root slurry were used for inoculation. Five different media were applied to retrieve target bacteria from the samples, including modified arabinose-gluconate (MAG) media adapted from the study Sachs et al. [25], 10- and 100-fold diluted MAG, ρMAG (MAG supplemented with ρ-coumaric acid) and vanillic acid media (detailed in Supplementary Text S1.2). After the sterilized medium was cooled to 60 °C, 55 mg/L cycloheximide was added to inhibit fungal growth. All agar plates were incubated at 28 °C. Colonies with small, white and raised morphology formed after 7 days were picked for species identification.

Colonies were identified by a 1465 bp fragment PCR product using universal bacteria 16S rRNA primers 27F and 1492R. Chelex 100 resin [Bio-rad, USA] was used to extract DNA from bacterial colonies for PCR reaction, and the recipe of PCR was prepared using Premix Taq [Takara Bio, USA]. The PCR conditions were as follows: denaturation at 95 °C for 5 min, followed by 32 cycles (95 °C for 45 s, 55 °C for 45 s and 72 °C for 90 s), final extension at 72 °C for 10 min. The taxonomic information of the isolates was obtained by comparing the 16S rRNA gene sequences using EzBioCloud [26], which is a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.

Phylogenomics analysis and reconstruction of ancestral lifestyles

We obtained 93 isolates that likely resembled Bradyrhizobium, and had their genomes sequenced with the HiSeq X platform (Illumina), followed by genome assembly and gene identification using SPAdes v3.10.1 [27] and Prokka v1.12 [28], respectively. Completeness of these genomes was calculated using CheckM v1.0.7 [29] with default parameters.

The phylogenomic tree of Bradyrhizobium was constructed using IQ-Tree v1.6.2 [30] with the 93 strains isolated in the current study and 252 genomes deposited in the NCBI Genbank database based on amino acid sequences of 123 shared single-copy genes identified by OrthoFinder v2.2.7 [31]. Bradyrhizobium members were divided into three lifestyle types based on the presence or absence of nod and nif genes (Supplementary Datasets S1 and S2). Strains that possess nifBDEHKN and nodABCIJ genes (at most one missing gene allowed; the same thereafter) were classified as symbiotic (Sym), strains possessing only nifBDEHKN genes were classified as free-living nif-carrying (FLnif), and those lacking both nod and nif genes were classified as free-living non-nif-carrying (FLnonnif). The lifestyle of ancestral nodes in the phylogenomic tree was inferred using Mesquite v3.61 [32] by the maximum parsimony reconstruction method, which infers the ancestral lifestyles by minimizing the number of steps of lifestyle change along the phylogenomic tree. Similar to a recent study [9], we did not apply the methodologically complex maximum likelihood method because the frequent lifestyle transitions across short branches in the Bradyrhizobium phylogeny likely leads to overestimates of the transition rate and hence inaccurate reconstruction of ancestral lifestyles.

Amplicon sequence analysis

Metadata of 4958 sequence files (runs) were retrieved from the NCBI Sequence Read Archive (SRA) using the term “nifH metagenome” (last accessed in December 2020), which actually means nifH amplicon datasets. Raw data were downloaded using the SRA Toolkit (https://github.com/ncbi/sra-tools) and were further processed with QIIME2 [33]. We applied evolutionary placement methods to assign the short sequence reads to the four phylogenetically distinguishable types of nif clusters (i.e., FL, PB, Sym and SymBasal) using PaPaRa v2.5 [34] and EPA-ng v0.3.8 [35]. The normalized abundance of each type of Bradyrhizobium nifH genes was calculated as the number of reads assigned to the corresponding nifH type divided by the total number of reads in the amplicon sequencing dataset assigned to Bradyrhizobium (Supplementary Dataset S3).

Identification of potential transposons, prophages and direct sequence repeats of the nif island

We searched for potential transposable elements and prophages on the nif island using MGEfinder v2.3.0 [36] and phigaro v.1.0.6 [37] with default parameters, respectively. Potential direct sequence repeats surrounding the island were searched using BLASTN with 10 kb sequences at the left and right boundaries of the island as query and subject, respectively (“blastn –query left_10kb.fasta –subject right_10kb.fasta –word_size 7 –evalue 10”).

Results and discussion

Newly isolated free-living strains expand ecological diversity of Bradyrhizobium

We isolated and sequenced the genomes of 93 Bradyrhizobium strains initially identified by 16S rRNA gene (five later shown to belong to Afipia based on phylogenomic construction) isolated from diverse types of soils, i.e., lateritic red earths, black soils, brown coniferous forest soils, cinnamon soils, and yellow-brown earths in different sites of China (Supplementary Fig. S1A). Among these isolates, 81 are non-symbiotic strains, as evidenced by the lack of the nodulation-determining nod genes, four of which carry nif genes. The remaining twelve are potential symbiotic strains: 11 of them carry nod genes and the other one (B. denitrificans SZCCT0094) is phylogenetically clustered with photosynthetic members which use a nod-independent strategy for nodulation [38] (Fig. 1, Supplementary Fig. S2); all of the twelve were isolated from the rhizosphere or bulk soil of leguminous plants (Supplementary Dataset S1). Together with the 215 publicly available Bradyrhizobium genomes, our dataset included 168 symbiotic (Sym), 21 free-living nif-carrying (FLnif), and 119 free-living non-nif-carrying (FLnonnif) strains (Supplementary Datasets S1 and S2).

Fig. 1: The maximum-likelihood phylogenomic tree of Bradyrhizobium and inferred lifestyle evolutionary history.
figure 1

Strains from Xanthobacteraceae were used as an outgroup. Ancestral lifestyles were inferred using the parsimony method in Mesquite. Purple circles on the nodes indicate ultrafast bootstrap values higher than or equal to 95% calculated by IQ-Tree. The red and green triangles on the outer layer indicate symbiotic members of the basal lineages of the B. japonicum supergroup and free-living nif-carrying strains, respectively.

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Despite a significantly expanded dataset, all the Bradyrhizobium isolates fall into seven phylogenetic supergroups (Fig. 1) named in a recent study [2]. These are the Soil 1, Soil 2, B. jicamae, B. elkanii, Kakadu, Photosynthetic, and B. japonicum supergroups (Fig. 1). Among the 93 newly isolated strains, 88 contribute to all these supergroups except for the Soil 2 and Kakadu supergroups, and the remaining five strains are related to the genus Afipia in the outgroup. The newly isolated strains are phylogenetically nested within those that have publicly available genomes, except for the 13 strains in the B. jicamae supergroup which form a sister clade to the previously sequenced strains of that supergroup (Fig. 1).

Our ancestral lifestyle reconstruction analysis inferred that the last common ancestor (LCA) of Bradyrhizobium adapted to a free-living lifestyle, consistent with our prior study based on a very limited taxon sampling [9] (Fig. 1). This is evident by the pattern that a vast majority of the outgroup lineages as well as all members of the basal Bradyrhizobium clade (Soil 1 and Soil 2 supergroups) are non-symbiotic lineages. The symbiotic lifestyle independently originated eight times based on the sampled lineages, including three major origins: one in the B. jicamae supergroup (FLnonnif-Sym1), one in the B. elkanii supergroup (FLnonnif-Sym3), and another one at the LCA of the Kakadu, Photosynthetic, and B. japonicum supergroups (FLnonnif-Sym4). These transitions occurred in relatively deep phylogenetic positions, suggesting that they represent evolutionarily ancient events. Because the ancestors of both the B. jicamae and B. elkanii supergroups were inferred to be free-living non-nif-carrying bacteria (Fig. 1), it is no surprise to observe that nearly half of the analyzed strains from these two supergroups are free-living non-nif-carrying strains. Free-living lifestyles originated from symbiotic strains 23 times: 13 of these events gave rise to non-nif-carrying strains and 10 gave rise to nif-carrying members (Fig. 1). Transitions from symbiotic to free-living lifestyle (both Sym-FLnif and Sym-FLnonnif) likely occurred recently as indicated by their shallow phylogenetic positions (Fig. 1). Hence, the loss of the ability to nodulate legume plants occurred more frequently than the reverse process in Bradyrhizobium, consistent with a prior study based on the analysis of phenotypic markers [7]. This pattern remains when we combined FLnif and FLnonnif as a single free-living lifestyle (Supplementary Fig. S3). Furthermore, the free-living nif-carrying lifestyle originated from free-living non-nif-carrying ancestors four times (Fig. 1). The infrequency of this type of lifestyle transition does not necessarily mean that this type of transition is rare, as it may result from a potential bacterial cultivation bias against those carrying the nif cluster under aerobic conditions with readily available N sources (see also Caveats and concluding remarks). The above patterns held true based on a condensed phylogeny with 90% ultrafast bootstrap as the cutoff (Supplementary Fig. S4, branches that split in <90% of the sampled trees were condensed).

HGT of the nif island drives the expansion of the free-living nif-carrying lifestyle

Free-living nif-carrying Bradyrhizobium is of much interest, because these Bradyrhizobium members may play a previously unrecognized role in the N cycle in soils, especially given the reportedly high abundance of Bradyrhizobium in soils [6]. We asked where the N2-fixing (nif) genes in free-living Bradyrhizobium came from and whether the nif genes differ between free-living and symbiotic members. It is apparent from Fig. 1 that most free-living strains evolved from their symbiotic ancestors, hence one possibility is that free-living nif-carrying strains inherited the same version of nif genes from their nodulating ancestors. Alternatively, free-living nif-carrying Bradyrhizobium might have lost the entire symbiosis island which includes both nod and nif clusters among other genes, and instead recruited a new set of nif genes from an external donor by HGT. To test these competing hypotheses, we constructed the phylogenetic tree of the nif cluster based on the concatenated alignment of the genes nifABDEHKNX for those from the Bradyrhizobium and several other rhizobia including Azorhizobium, Rhizobium, Sinorhizobium and Mesorhizobium from Alphaproteobacteria, and Burkholderia and Cupriavidus from Betaproteobacteria (see Materials and methods).

The nif genes of all Bradyrhizobium and the two strains from Azorhizobium form a monophyletic group (Fig. 2, Supplementary Fig. S5). The topology of nif tree suggests a single origin of nif genes in Bradyrhizobium, and the nif genes likely transmitted to Azorhizobium by HGT (Fig. 2). Within the Bradyrhizobium clade, the nif tree (Fig. 2) displays substantial topological incongruence with the species tree (Fig. 1), suggesting different evolutionary histories between nif genes and the core genome. Following its origin, the nif genes diverged into two major clades: the Sym1 clade consisting of mainly symbiotic strains and the other clade composed of photosynthetic and free-living strains as well as some symbiotic strains. For the latter clade, the Photosynthetic Bradyrhizobium supergroup together with a strain from the B. jicamae supergroup (SZCCT0283) and the two Azorhizobium sequences branched off first, followed by a group of strains from the B. japonicum supergroup (Fig. 2) with mixed evolutionary relatedness in the species tree (Fig. 1). These mixed B. japonicum supergroup members further diverged into four clades in the nif tree, three of which are constituted by symbiotic strains: SymBasal is largely comprised by several early-split lineages of the B. japonicum supergroup and spans over the species tree, whereas Sym2 and Sym3 are each composed of strains at shallow phylogenetic positions clustered in the species tree (Fig. 1). Apparently, HGT occurred within SymBasal and between these three clusters. The other clade is mostly composed of free-living nif-carrying members (Cluster FL) (Fig. 2) traversing the phylogenomic tree but restricted to the B. japonicum supergroup (Fig. 1), which is strong evidence for HGT of the nif genes among these free-living Bradyrhizobium members.

Fig. 2: Nif gene phylogeny of Bradyrhizobium.
figure 2

The phylogeny was constructed using the concatenated alignments of nifABDEHKNX. Nif genes from Rhizobium, Sinorhizobium, Mesorhizobium, Azorhizobium and Beta-rhizobia (rhizobia from Betaproteobacteria) are used as the outgroup. The purple circles on the nodes indicate ultrafast bootstrap values higher than or equal to 95% calculated by IQ-Tree. The ultrafast bootstrap of some key nodes related to Cluster FL is also indicated. The tips with a black box around within Cluster FL denote those with experimental evidence for their N2 fixation in the free-living state. Note that three symbiotic strains, namely DOA9, p9-20 and CCBAU 53363, carried a nif cluster similar to the one on the free-living nif island (NI) in addition to another one on the symbiosis island (SI) or plasmid. Members of Cluster FL, SymBasal, Sym2 and Sym3 are also marked on the phylogenomic tree (Fig. 1). The geographic locations of nif-carrying strains in Cluster FL are displayed in Supplementary Fig. S1B.

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Strikingly, despite a scattered distribution in the species phylogeny (Fig. 1), 17 free-living nif-carrying strains group on the nif phylogeny (Cluster FL in Fig. 2). Also grouping in Cluster FL are the nif genes from three symbiotic strains (DOA9, CCBAU 53363 and p9-20). These three strains encode another nif cluster located in the symbiotic plasmid (DOA9) or symbiosis island on the chromosome (CCBAU 53363 and p9-20), which group with other symbiotic strains in the nif phylogeny (Fig. 2). Remarkably, as introduced above, two strains (Bradyrhizobium sp. AT1 and Bradyrhizobium sp. DOA9) whose nif genes from Cluster FL in the nif phylogeny were previously shown to perform free-living N2 fixation under micro-aerobic conditions (marked by a box around the strain name in Fig. 2) [20, 21]. The above evidence together indicates that nif genes from Cluster FL, which is mainly comprised by free-living Bradyrhizobium, are likely to be a special group of nif genes that specifically take part in free-living N2 fixation.

A unique nif island likely contributes to the oxygen tolerance of free-living nif-carrying Bradyrhizobium

We sought more evidence for HGT of the nif cluster by analyzing genes surrounding it. This led to the identification of a ~50 kb genomic region containing nif genes (nif island) conserved among all members of Cluster FL (Fig. 3A). The regions flanking the nif island are not conserved across most free-living strains (Supplementary Fig. S6), providing further evidence for HGT of the nif island. Note that four strains in Cluster FL (BM-T, Y-H1, R2.2-H and W) share highly conserved genomic contexts (Supplementary Fig. S6). These strains are closely related and comprise a monophyletic group in the phylogenomic tree (marked by a box around the strain names in Supplementary Fig. S2), indicating that the shared genomic context of their nif islands was inherited from their LCA. Likewise, the conserved genomic context of the nif island among strains from the Photosynthetic Bradyrhizobium supergroup (Supplementary Fig. S6) is consistent with their vertical descent shown in the species phylogeny (Fig. 1).

Fig. 3: The comparison of the gene arrangement of the nif gene cluster located in the nif island and in the symbiosis island.
figure 3

Gene functions are distinguished by different colors. The visualization of gene arrangement is performed with dna-features-viewer v3.0.3 [74]. The structures of the gene arrangement classified the nif clusters into three types: those belonging to the Cluster FL or Cluster PB (A), those found in the symbiosis island in most symbiotic strains or in FLnif (free-living nif-carrying) strains that cluster with symbiotic strains (B). In panel B, the free-living strain SEMIA 6399 whose phylogenetic position is nested within symbiotic strains, labeled as “FLnifwithinSym”. “SymBasal” indicate basal lineages of the B. japonicum supergroup (referred to Fig. 2). “Sym1, 2, 3” stands for different cluster of symbiotic Bradyrhizobium denoted in Fig. 2. Note that on the symbiosis island of symbiotic Bradyrhizobium, nod genes are often separated far away from nif genes [43] so they are usually not shown in the figure.

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The upstream boundary of the nif island in Cluster FL Bradyrhizobium (Fig. 3A) is marked by nifA, which codes for a transcriptional activator required for the expression of nif operons [39]. Adjacent to nifA is a suf cluster, which is responsible for synthesizing and inserting Fe-S clusters into nitrogenase [40]. The core genes encoding the nitrogenase are located downstream of the suf cluster, and are mainly separated into two operons, nifDKENX and nifHQ. FixABCX which encode a membrane complex involved in electron transfer to nitrogenase are located downstream of the nif genes [41]. Extensively studied in rhizobia, the role of fixABCX in N2 fixation by providing electrons for nitrogenase has been suggested for free-living diazotrophs [42]. At the downstream boundary of the nif island is a mod cluster encoding molybdate transporter. In general, the nif island conserved in free-living Bradyrhizobium is similar to that in photosynthetic strains (Fig. 3A), as also noted in previous studies based on genomic analysis of only a few photosynthetic and free-living strains [43, 44], and that in a newly sequenced soil-dwelling strain (SZCCT0283) from the B. jicamae supergroup (Supplementary Fig. S7A). A few differences are that those from Cluster PB carry an additional copy of nifH but fewer genes in the mod operon compared with those from Cluster FL (Fig. 3).

The nif clusters of the nif island conserved in Cluster PB and FL (Fig. 3A) share many genes with those on the symbiosis island (Fig. 3B), such as nifDKENBH and fixBCX, agreeing with their essential roles in N2 fixation. However, they also display notable differences. For most symbiotic Bradyrhizobium, nifH gene is located together with nifQ on their genomes (Fig. 3B), but in SymBasal nifH is located in the upstream of the nifDKENX cluster (Fig. 3B, Supplementary Fig. S7B). A notable feature of Sym1 is that a gene cluster encoding hya and hyp, which is responsible for hydrogenase synthesis (hyaABCDF) and maturation (hypVABFCDE), is inserted between fixJL and nifDKENX gene clusters (Fig. 3B, Supplementary Fig. S7B). Although nod genes are usually separated far away from nif genes on the symbiosis island, these genes are located relatively close to nif genes in Sym3 (Fig. 3B, Supplementary Figs. S6, S7B).

Apparently, the gene arrangement of the nif island is more compact and more conserved across different free-living and photosynthetic strains than across symbiotic strains (Fig. 3). The nif island additionally carries a nifV gene, which encodes homocitrate synthase to synthesize homocitrate, a component of the Fe-Mo cofactor of nitrogenase [45]. A previous study reported that the vast majority of symbiotic rhizobia (not limited to Bradyrhizobium) do not harbor nifV [45], which is known to be compensated for by the homocitrate provided by their legume host during the symbiotic stage [46]. Here, we found that nifV is present in around half of the Bradyrhizobium genomes, not restricted to the free-living and photosynthetic strains (Fig. 2). Phylogenetic analysis suggests that nifV was presumably present at the LCA of Bradyrhizobium, but lost within the B. japonicum supergroup (Supplementary Fig. S8). This is consistent with the hypothesis that nifV was likely required in early Bradyrhizobium ancestors which took a free-living lifestyle (Fig. 1), and became dispensable later when the descendant Bradyrhizobium switched to a symbiotic environment in which homocitrate is readily available.

We further identified that several genes involved in O2 tolerance and stress response are also specific to the nif island of free-living and photosynthetic members (Fig. 3), including glbO and hspQ. Specifically, glbO encodes a 17-kDa group-II truncated hemoglobin that binds O2 with high affinity [47, 48]. The expression of truncated hemoglobin glbO is induced upon exposure to oxidative stress [49]. The product of glbO might therefore play an important role in protecting nitrogenase from O2 inactivation, endowing the free-living Bradyrhizobium with higher tolerance to O2. HspQ encodes a chaperone protein, which combats the detrimental effects on proteins caused by stressors such as increased temperature, oxidative stress, and heavy metals [50]. We also observed copy number differences between the free-living and symbiotic members, such as the nifZ which has a function in the maturation of the Fe-Mo protein nitrogenase [51], but whether the additional copies contribute to adaptation to the free-living lifestyle is unknown. In summary, we predict that in addition to nifV, the genes involved in oxygen tolerance and stress response in the nif island (e.g., glbO, hspQ and mod) may play a role in the adaptation to free-living lifestyle. Intriguingly, the nif island from Azorhizobium, which is embedded in the Bradyrhizobium nif tree (Fig. 2) and is able to perform free-living N2 fixation [16], shares some of the above genes like nifV, glbO and mod, and a generally similar gene arrangement with the free-living nif island in Bradyrhizobium (Supplementary Fig. S7C). It would be interesting to conduct experiments to assess the detailed functions of the genes on the nif island under O2 concentrations resembling those of soil environments.

An interesting finding is that despite the high similarity in gene arrangement of the nif island between Cluster PB and Cluster FL, these two groups cluster in different clades in the nif phylogeny (Fig. 2). Given the high similarity of the nif island between Cluster PB and Cluster FL strains (Fig. 3), it seems unlikely that the nif island is the result of convergent evolution between photosynthetic and free-living Bradyrhizobium lineages. One possibility is that early in evolution Cluster FL acquired the nif island from Cluster PB or an even unsampled lineage related to Cluster PB that carries the nif island, and that the nif genes on the nif island might be later replaced by those derived from symbiotic lineages related to Sym3, Sym2 and SymBasal through recombination. To gain insights into this evolutionary process, it would be useful to sequence more genomes from related lineages in Bradyrhizobium.

Note that, for simplicity, we defined free-living strains based on the absence of nod. However, four strains claimed as free-living nif-carrying Bradyrhizobium in the current study, namely B. mercantei SEMIA 6399, B. yuanmingense P10 130, B. liaoningense CCBAU 83689 and B. amphicarpaeae 39S1MB, were originally isolated from nodules (Supplementary Dataset S2). Interestingly, except the last isolate whose nif genes were found in Cluster FL, the nif genes of the other three are embedded in symbiotic lineages in the nif phylogeny (Fig. 2), indicating that the nif genes were inherited from nodulating ancestors. Consistent with this idea, genes encoding hydrogenase are adjacent to nif, which is similar to Sym1 (Fig. 3, Supplementary Fig. S7B). These three strains also carry genes encoding components of T3SS and the effectors it injects (Supplementary Dataset S4), suggesting that they are likely nodule-inhabiting strains that may not nodulate legumes but with the potential to interact with plants. This is in contrast to other free-living nif-carrying strains where symbiosis genes were commonly lost during lifestyle transitions Sym-FLnonnif 1, 4, 7 and 10 (Supplementary Fig. S9C; Supplementary Text S2; see also Supplementary Datasets S5–S8 for gene gains/losses during different lifestyle transitions). In general, the arrangement of nif genes and their surrounding regions of these strains are similar to that of symbiotic strains, particularly when compared with their closest relatives: P10 130 shows a very high similarity to its closest nod-carrying relative CCGE-LA001 in the genomic context of the nif genes, SEMIA 6399 is also highly similar to its closest nod-carrying relative BR 10303, in addition to the absence of nifV in BR 10303 (Supplementary Fig. S7B). We did not analyze CCBAU 83689 as the contig carrying nif genes is too short. The results indicate the different origins of the free-living nif-carrying Bradyrhizobium: while some were derived from their symbiotic ancestors, most analyzed strains of this lifestyle originated via HGT of the free-living nif island (Fig. 3).

Putative mechanisms of the horizontal transfer of the nif island

Genomic islands are defined as large genomic segments that have probably been horizontally acquired by prokaryotes, which are usually 10–200 kb in length [52, 53]. Clearly, the 50-kb region containing the genes necessary for N2 fixation conserved among free-living nif-carrying Bradyrhizobium (nif island) well meets this criterion, as the genes it contains, not only nif genes (Fig. 2) but fix genes at the other end of the island (Supplementary Fig. S10), show a similar phylogeny that is very different from the species tree. Other features of mobile islands include different G + C content from the surrounding regions, the flanking of direct repeats or tRNA genes which serve as the sites for site-specific recombination [54, 55], and the presence of genes coding for transposable elements, integrases, or prophages (reviewed in [53]). However, we found that the nif island is rarely associated with any of the above features (Supplementary Fig. S11; see also Methods). This hints that the free-living nif-carrying Bradyrhizobium-specific nif island likely uses a mechanism distinct from other genomic islands for its movement across bacteria.

Intriguingly, we noticed that the flanking regions of the nif island (Fig. 3) are conserved in the symbiotic relatives of some free-living nif-carrying members (denoted by purple triangles in Fig. 1). As shown in Supplementary Fig. S12, this conserved flanking region consists of a gene encoding xanthine dehydrogenase (XDH), fabG which participates in fatty acid biosynthesis, a short gene of unknown function, and modD presumably involved in molybdate transport. In the strains carrying the nif island, these genes are separated by the nif island, with modD located on one side, and fabG and XDH located on the other side (Fig. 3). This opens up the possibility that homologous recombination is responsible for the spread of nif island among free-living Bradyrhizobium: recombination at both ends of the XDH-fabG-modD cluster can well lead to the insertion of the nif island. This mechanism, if true, suggests that the nif island can be horizontally transferred only between strains with the XDH-fabG-modD gene cluster. Such strains in the genomes analyzed in the current study were only found in the B. japonicum supergroup (Fig. 1), consistent with the limited distribution of free-living nif-carrying strains in this supergroup. This mechanism has been shown to be responsible for the gains and losses of genomic islands associated with pathogenic and commensal lifestyles among Pseudomonas lineages [56]. Further, other scenarios, such as illegitimate recombination, which could be mediated by non-homologous end joining as shown in a recent study [57], or hijacking the machinery produced by other genomic islands (e.g., by using the integrase and conjugation system encoded by other genomic island) [58], might also be possible.

Widespread distribution of the unique free-living nif island in diazotrophic communities

We further asked how prevalent the nif island specific to free-living Bradyrhizobium is in the diazotrophic communities of different environments and how it compares to the nif genes of symbiotic members. We therefore assessed the normalized abundances of nifH, the most commonly used marker gene to identify N2-fixing microbes [59], that potentially correspond to free-living (Cluster FL in Fig. 2), photosynthetic (Cluster PB in Fig. 2), and symbiotic Bradyrhizobium (Sym and SymBasal in Fig. 2) in 4958 amplicon sequencing datasets (see Materials and methods). We divided these datasets into four categories according to the environments where the samples were collected: soils (e.g., bulk soil, rhizosphere and freshwater lake sediment), plants (root and phyllosphere samples), marine (e.g., seawater, marine sediment, coral and mangrove samples), and others (e.g., bioreactor and unidentified samples) (Supplementary Dataset S3). Amplicon reads were assigned to different types of Bradyrhizobium nifH according to their phylogenetic placements (see Materials and methods).

Amplicon analysis showed that Bradyrhizobium constitute an important group of diazotrophic communities in different habitats, and the relative abundance of Bradyrhizobium was 7.52 ± 18.57%, 6.67 ± 12.75%, 17.39 ± 18.32%, 14.54 ± 18.85% (mean ± standard deviation) in marine, soil, plant, and other environmental types, respectively (Supplementary Dataset S3). The nifH that resembles those belonging to Cluster FL displayed the highest normalized abundance in samples from marine, soil, and habitats classified as “others” (Fig. 4; p < 0.001 for all comparisons, paired Wilcoxon–Mann–Whitney test). Specifically, Cluster FL accounts for 56 ± 47%, 46 ± 38%, and 51 ± 39% of reads that were assigned to Bradyrhizobium in these three types of habitats (marine, soil, and others), respectively. In particular, for marine samples, nearly all Bradyrhizobium nifH were assigned to Cluster FL (Fig. 4; Supplementary Dataset S3). For the datasets of plant samples, while symbiotic nifH displays the highest abundance (Fig. 4), nifH assigned to Cluster FL also shows a considerably high normalized abundance in plants (29 ± 39%), and is even the dominant ecotype in diazotrophic communities associated with some Asteraceae species and the roots of several perennial grass species (Supplementary Dataset S3). Hence, though the presence of free-living Bradyrhizobium-specific nifH does not necessarily indicate the occurrence of a complete nif island, it is tempting to suggest that Bradyrhizobium members carrying the free-living nif island i) are distributed in a variety of ecosystems and geographic locations (Supplementary Fig. S13), and ii) are in general more abundant than that of symbiotic strains in the genus in most habitats except plants, hinting at a previously unrecognized role of their nif island in the free-living lifestyle.

Fig. 4: Normalized abundance of free-living and symbiotic nifH of Bradyrhizobium in amplicon sequencing samples collected from different types of environments.
figure 4

The values shown as percentage in the y-axis in the violin plots denote the normalized abundance (see Materials and methods) of different types of nifH. The width of each curve represents the kernel estimation showing the frequency of samples at different normalized abundances. The P value is derived from a paired Wilcoxon–Mann–Whitney test (two-sided).

Full size image

It is interesting that the relative abundance of nifH often exhibits a bimodal distribution (Fig. 4). To examine whether this is a potential result of combining different sample types in individual categories, e.g., soils contain bulk soils, rhizosphere and lake sediment, we analyzed the relative abundance of nifH in different subtypes of each environment category. As shown in Supplementary Fig. S14 stacked histogram, there is not a strong association between the relative abundance of a particular type of nifH and the specific environment subtype, although in some cases, for example, FL nifH appears specifically enriched in “coral metagenome” samples of marine amplicon sequencing datasets but are almost depleted from “mangrove metagenome” samples. Similarly, for the plant-associated datasets, nifH from symbiotic members (Sym) is enriched in “root metagenome” samples, while FL nifH exhibits a high proportion in phyllosphere samples.

Caveats and concluding remarks

Adding the 88 soil-dwelling strains greatly expands our knowledge of the ecology and evolution of Bradyrhizobium. However, because of the bias towards symbiotic strains in previous studies and the difficulties in cultivating those dwelling in soil environments, the free-living strains analyzed here may underestimate the real diversity of wild diazotrophic Bradyrhizobium that adapt to a free-living lifestyle. For example, prior studies have shown that Bradyrhizobium is the dominant genus in the diazotrophic communities in many soil ecosystems, most of which have not been sampled for Bradyrhizobium cultivation however [60, 61]. Of the limited types of soil ecosystems sampled here, the Bradyrhizobium strains we collected are not necessarily representative of the wild populations in these ecosystems owing to the potential cultivation bias. For example, our cultivation process was under aerobic condition and the cultivation medium was rich in fixed nitrogen, conditions disfavoring the isolation of diazotrophic members. Therefore, it is not clear whether important free-living diazotrophic lineages occupying distinct phylogenetic positions are missing. Another caveat to the current conclusion is that some of our analyses built on an over-simplified classification of Bradyrhizobium lifestyles, which was based on the presence/absence of certain genes like nif and nod. In fact, isolates with a symbiosis island could be ineffective (i.e., those form nodules but fix little N2 within nodules) [62]. Likewise, whether the free-living strains possessing the nif island could perform N2 fixation, and if they could, the N2 fixation efficiency under different O2 concentrations, remains to be studied. Another overlooked issue related to lifestyle analysis is that lifestyle transitions may cover intermediate state, which cannot be accounted for by state-of-the-art ancestral reconstruction methods. Specifically, one possible route may include a non-nif-carrying free-living intermediate during the transition from a symbiotic ancestor to a nif-carrying free-living descendant, which is represented as Sym → FLnonnif → FLnif. An alternative trajectory can be Sym → (Sym + FLnif) → FLnif, in which the intermediate state was a free-living strain that carries a symbiosis island, as represented by the three strains carrying both a nif island and a symbiosis island or plasmid (AT1, DOA9, and p9-20). Besides, two of the nif-carrying strains that lack nod (SEMIA 6399 and P10 130) analyzed here were reported to nodulate legumes despite a high host specificity [63, 64]. However, this contradicts with the current understanding that Bradyrhizobium members that lack nod but can nodulate legumes are only found in the Photosynthetic supergroup [65]. Hence, whether they can use a nod-independent strategy for nodulation, and if so, how this mechanism works, remain further investigation.

It is also important to note that it is the relative abundance that was used to measure the prevalence of free-living nifH (Fig. 4). Hence, a high relative abundance of a certain type of Bradyrhizobium nifH only indicates its high abundance relative to other N2-fixing members in the sampling sites. Actually, in some environments, the overall rates of N2 fixation were very low [66]. Further, amplicon analysis suffers from biases ranging from sample preparation, primer selection, and chimeric sequences generated by sequencing and bioinformatics analysis. This could make the results based on different samples collected from various habitats difficult to compare. Besides, it would be interesting to compare the abundances of nif-carrying and non-nif-carrying Bradyrhizobium members from the same sampling sites, which could be done by measuring the abundances of both nifH and housekeeping genes that can provide a high taxonomic resolution.

In spite of the above caveats, our study reveals an interesting pattern of origins of free-living nif-carrying Bradyrhizobium from their symbiotic ancestors. Though the nested phylogenetic positions within symbiotic lineages (Fig. 1) might leave an impression that free-living nif-carrying members could have inherited the nif genes from their symbiotic ancestors, we provided compelling evidence that this is unlikely the case. Rather, it is the HGT of a conserved nif island that drives the independent transitions from symbiotic to free-living nif-carrying lineages. This might serve as a classic example of independent transitions in lifestyle driven by HGT of certain genes in bacteria, which, although mostly explored in pathogens or symbionts in prior studies [67,68,69], may play a prominent role in free-living bacteria. Given the global dominance of Bradyrhizobium in the soil microbiota, even a small proportion of them being diazotrophic members could potentially bring a considerable amount of fixed nitrogen to the bulk soils and other nitrogen-limited terrestrial ecosystems. Our results therefore have implications for understanding nitrogen fixation. Because one of the major aims of modern agricultural research is to transfer the N2 fixation ability to non-legume crops [70, 71], the capacity of certain free-living Bradyrhizobium to fix N2 and their association with non-leguminous plants may imply potential application in agriculture.