The plant genus Astragalus L. (Fabaceae) includes 2,704 species that are recognized as “accepted” in the Plant List (TPL), a dataset that serves as the taxonomic backbone of the World Flora Online.¹ It is the largest genus in the Fabaceae family, with a wide distribution worldwide (Chen et al., 2015). Many Astragalus species form nitrogen-fixing symbioses with rhizobia (Wei et al., 2003; Tian et al., 2021) that can fix atmospheric nitrogen and convert it to ammonia, which is then absorbed by their host plants, thereby reducing the need for nitrogen fertilizers (Zhou et al., 2024). More than 20 rhizobia belonging to Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and non-nodulating Agrobacterium have been isolated from the root nodules of 14 Astragalus species distributed across 12 Chinese provinces or autonomous regions, including Xinjiang, Inner Mongolia, Liaoning, Beijing, Shandong, Gansu, and Yunnan. These provinces encompass five different ecological regions, namely temperate-arid, semi-arid agricultural, semi-arid, Loess Plateau, and subtropical regions (Gao et al., 2001; Zhao et al., 2008; Chen et al., 2015; Yan et al., 2016; Zhang et al., 2020b). The genetic diversity and biogeographic distribution of rhizobia nodulating Astragalus are primarily influenced by soil contents of available phosphorus and potassium, as well as total salts, pH, and soil fertility (Wei et al., 2008; Yan et al., 2016). Mesorhizobium jarvisii is the most widespread and predominant species associated with A. sinicus in southwestern China (Zhang et al., 2020b), where the majority (53%) of isolates are non-nodulating Agrobacterium sp. Meanwhile, in the arid region of northwestern China (NWC) (Chen et al., 2015), symbiotic Mesorhizobium species comprise 41% of the rhizobial isolates. M. septentrionale and M. temperatum are the predominant species associated with Astragalus in fields with nitrogen-poor and -rich soils, respectively (Yan et al., 2016). In the Kamchatka Peninsula, genera that have been isolated from nodules of A. umbellatus and A. inopinatus are Mesorhizobium, Rhizobium, Bosea, and Bradyrhizobium (Guro et al., 2024).

A. mongholicus is a perennial plant that serves as a primary medicinal herb in the arid and semi-arid regions of NEC and NWC, where it is mainly distributed (Fu et al., 2014; Wang et al., 2023; Li et al., 2024). It can form nodules with rhizobia. The microsymbionts associated with A. mongholicus in NWC (Shanxi, Gansu, and Ningxia provinces) include M. temperatum, M. muleiense, M. septentrionale, M. ciceri, S. meliloti, and S. kummerowiae (Yan et al., 2016). The microsymbionts associated with A. mongholicus in NEC have not been reported yet. The climate of this region is temperate semi-arid with windy and dry winters and springs, warm and rainy summers, and cool autumns. The mean annual temperature is 7°C; and 70% of the annual mean precipitation of 300 mm falls between July and September. The region’s black soil is characterized by its high content of humus (Yan and Liu, 2010; Xiulian et al., 2017). These characteristics differ from those in NWC, where the climate is arid and semi-arid temperate continental monsoon; the average annual precipitation of 358.2 mm occurs mainly from July to September; and its soil is sandy with a low humus content (Meng et al., 2015).

Systematic studies on the genetic diversity and biogeographic distribution of rhizobia associated with various legumes have revealed significant differences among populations in different ecoregions (Liu G. et al., 2021; Liu L. et al., 2021). Genetic differentiation, gene flow, and allelic substitution within and between rhizobial populations may arise from mutation and recombination, resulting in specific patterns of adaptation in response to environmental variation and host selection. For instance, the biogeography of populations of M. muleiense, one of the main chickpea-nodulating rhizobial symbionts in China, reveals a pattern of chromosomal differentiation among populations from the Ningxia, Xinjiang, and Gansu provinces (or autonomic regions) (Zhang et al., 2020a). Caragana-associated mesorhizobia have evolved divergently according to the interplay between environmental conditions and host plants (Ji et al., 2015). In contrast, gene exchange and recombination among Bradyrhizobium associated with Kummerowia have occurred more frequently among the genospecies isolated from exurban and urban areas, regardless of the geographical distribution (Ji et al., 2019). However, little is known about the genetic diversity, intraregional differentiation, and gene flow among rhizobial strains associated with A. mongholicus in NEC. Moreover, the differences between the evolutionary trajectories of these strains in NEC and NWC are also unclear.

To address this knowledge gap, we collected the root nodules of A. mongholicus at a plantation. The plantation maintains a variety of Chinese herbaceous plants such as A. mongholicus, Glycyrrhiza uralensis, Sophora flavescens, Saposhnikovia divaricata, and Platycodon grandiflorus. We used multilocus sequence analysis to investigate the phylogeny and genetic diversity of rhizobial isolates associated with A. mongholicus, and we systematically estimated the intra- and inter-regional differentiation and gene flow among these isolates based on the analysis of the concatenated core genes (recA, atpD, and glnII) and the key symbiotic gene nodC (encoding N-acetylglucosaminyltransferase, NodC).

Astragalus, a primary source of traditional Chinese medicines, forms a mutualistic symbiosis with rhizobia that provides the plant with nitrogen for its growth and metabolism (Zhiyong et al., 2024). Heavy application of chemical fertilizer during cultivation affects the quality of Astragalus products. The selection of rhizobial strains that are capable of fixing nitrogen efficiently and adapting to the local soil environment is particularly important to improve the quality of Astragalus (Ji et al., 2017). However, in the absence of host plants, the majority of the free-living rhizobial strains in the soil gradually lose their ability to efficiently fix nitrogen (Ji et al., 2020). Therefore, studying the genetic differentiation and gene flow of rhizobial strains associated with Astragalus can provide a clearer understanding of the evolutionary process underlying the genetic diversity of plant-associated rhizobia. The symbiotic bacteria isolated from 14 commonly known Astragalus species have been identified as nine Mesorhizobium species (M. alhagi, M. septentrionale, M. amorphae, M. temperatum, M. tianshanense, M. ciceri, M. muleiense, M. huakuii, and M. qingshengii), two Sinorhizobium species (S. meliloti and S. fredii), eight Rhizobium species (R. mongolense, R. loessense, R. gallicum, R. huautlense, R. tropici, R. galegae, R. leguminosarum, and R. giardinii), two Bradyrhizobium species (B. elkanii and B. japonicum), and one non-nodulating Agrobacterium sp. (Wei et al., 2008; Zhao et al., 2008; Li et al., 2009; Zheng et al., 2013; Chen et al., 2015; Yan et al., 2016; Zhang et al., 2020b). However, none of the Astragalus species can establish a symbiotic relationship with all four genera of Rhizobacteria (Rhizobium, Mesorhizobium, Bradyrhizobium, and Sinorhizobium). The genotypes of plant-associated rhizobial strains depend mainly on their host Astragalus species and environmental conditions (Burghardt et al., 2022). For instance, M. septentrionale is present in most nodules, but especially in Astragalus growing on barren soils; meanwhile, M. temperatum is the predominant species in the nodules of Astragalus growing in nitrogen-rich fields (Yan et al., 2016).

A. mongholicus that is mainly distributed in NEC and NWC is one of the most important Astragalus species (Wang et al., 2023; Li et al., 2024). A systematic study of the genetic diversity and distribution of rhizobia associated with A. mongholicus in agricultural soils from NWC found that M. temperatum and M. muleiense are the predominant groups in this region (Yan et al., 2016). In NEC, however, R. yanglingense was the most predominant microsymbiont in the nodulated roots of A. mongholicus (Figure 1). Surprisingly, R. yanglingense, R. giardinii, S. fredii, and B. yuanmingense have never been reported to be present in the nodules of A. mongholicus in NWC (Yan et al., 2016). Moreover, the phylogenetic relationships among the rhizobial strains in NEC differed from those in NWC (Figure 1). A. mongholicus established symbiotic relationships with M. temperatum, M. muleiense, M. septentrionale, M. ciceri, S. kummerowiae, and S. meliloti in NWC. However, in NEC, it was associated with R. yanglingense, B. yuanmingense, S. fredii, and M. temperatum, where we observed an unexpectedly higher level of genetic diversity compared to the rhizobial isolates in NWC. An alkaline soil (pH 8.32) in NEC was detected and slightly higher than the mean pH value (pH 8.08) in NWC (Yan et al., 2016). However, organic carbon (OC), available potassium (AK), and available phosphorus (AP) of soil in NEC were lower than the mean values of corresponding OC (13.28 g/kg), AK (160.46 mg/kg), and AP (25.86 mg/kg) in NWC (Yan et al., 2016). The identities of the rhizobial strains isolated from A. mongholicus in NEC and NWC demonstrate that the distribution and community composition of rhizobia associated with this plant are dependent on the geographical location and its associated environmental conditions (Kim et al., 2022).

We were unable to detect the gene flow between rhizobial strains in NEC and NWC, as evidenced by the high Dxy values and low Nm values (Table 1; Supplementary Table S4). Our analysis indicated that the genetic materials of the rhizobial strains in NEC are derived from the ancestral lineage IV without lineage fusion (Figure 4). Moreover, no gene flow occurred throughout the strains’ evolutionary history. Our results confirmed that plant-associated rhizobial strains have evolved independently from strains that have adapted to a free-living lifestyle without intraregional recombination (Tao et al., 2021). Although these strains have been isolated from a common host, A. mongholicus, ecogeographical isolation due to the distance between NEC and NWC has restricted genetic drift and gene flow, which are major evolutionary forces underlying their distribution and community composition (Van Cauwenberghe et al., 2014; Liu et al., 2023). In addition, the high level of genetic diversity among rhizobial strains in NEC was due to the intraregional genetic differentiation rather than inter-regional genetic differentiation (Supplementary Figures S1, S2). Such diversity facilitated adaptation to the multiple factors in the local soil environment, such as nitrogen availability, rhizobial density, community complexity, and legume genotype (Burghardt et al., 2022). Finally, lower r/m and ρ/θ values (Table 2) suggested that the high genetic diversity of rhizobial strains in NEC was independent of recombination frequency, although the impact of recombination on chromosomal differentiation has been considerable among the different ecoregions of China, where it serves as the driver for mixing rhizobial populations (Tian et al., 2010; Van Cauwenberghe et al., 2014). Our results indicated that intraregional genetic differentiation by mutation, rather than recombination, is the main driver of high genetic diversity in NEC.

The nodC gene encodes N-acetylglucosaminyltransferase, which is present in all Rhizobium species and is required for the synthesis of the core structure of Nod factors (NFs). Moreover, the specific gene nodC is involved in diverse NF substitutions that confer plant specificity (Roche et al., 1996). The phylogenetic analysis classified the rhizobial strains isolated from A. mongholicus in NEC into five branches of the nodC phylogenetic tree (Figure 2). The tree branches generally corresponded to their genus and/or species definition (Figure 1). Overall, our results suggested that A. mongholicus is an extremely promiscuous legume without strict selectivity on either the symbiotic nodC gene or the species-determining core genes of rhizobia (Jiao et al., 2015). However, we also observed several cases where strains from different species or genera shared identical or highly similar nodC genes (Figure 2). This suggests that symbiotic genes were likely acquired through horizontal gene transfer among diverse rhizobial species. Frequent gene transfer within plant-associated microbial communities appears to facilitate the acquisition of symbiotic genes through horizontal gene transfer events (Laguerre et al., 2001; Msaddak et al., 2023). Furthermore, symbiotic genes are often clustered on extra-chromosomal replicons (megaplasmids and chromids) (Provorov et al., 2022) that construct the structure of the high genetic diversity of rhizobial strains in NEC. The higher nucleotide diversity (π) of nodC (Supplementary Table S3) was influenced by host-related natural selection and ecological selection pressures (Karasev et al., 2023). In the present study, the distribution of rhizobial species was influenced by the environmental conditions of the sampling sites in NEC and NWC, emphasizing the need to identify effective nitrogen-fixing strains for specific locations (Aserse et al., 2024).

We assumed that the characteristics of the conserved domains in NodC of rhizobial strains are related to A. mongholicus specificity. The conserved domain (Glyco_tranf_GTA_type superfamily) appears to be the most highly conserved and essential domain shared by all rhizobial strains that can form nodules on the roots of A. mongholicus (Figure 3). Such conservation suggests that the biosynthetic step of transferring a sugar moiety from an activated nucleotide-sugar donor to an acceptor molecule is necessary for various biological processes (e.g., biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates) of these rhizobial strains (Wang et al., 2015). In contrast, the conserved domain (Chitin_synth_2 superfamily) was found only in R. yanglingense (Figure 3), the predominant species in NEC, indicating that chitin biosynthesis (Chen et al., 2022) is not essential for the function of NodC of these rhizobial strains during their symbiosis with the host A. mongholicus.

In this study, we found that rhizobial strains associated with A. mongholicus in NEC are highly genetically diverse and consist of at least four genera. We identified Rhizobium yanglingense as the predominant microsymbiont. In addition, the microsymbiont genospecies nodulating A. mongholicus in NEC were significantly different from those in NWC. Furthermore, the evolutionary trajectory of these rhizobial strains is consistent with intraregional genetic differentiation rather than inter-regional gene flow and recombination. Ecogeographical isolation-by-distance severely restricted genetic drift and gene flow. However, we also found inter-regional symbiont genospecies, which may be the result of similar selection by hosts rather than coevolution. Finally, the Glyco_tranf_GTA_type superfamily (Glycosyltransferase family A) is the most highly conserved and essential domain in the NodC of rhizobial strains associated with A. mongholicus.