Legumes represent a family of plants that are widely distributed around the world and have coevolved with nitrogen-fixing symbiotic bacteria for millions of years (Doyle, 2011; Sprent et al., 2017). The wide variety of adaptations resulting from this coevolution between plants and bacteria has allowed the occupation of diverse ecosystems, from arid regions to tropical forests (Werner et al., 2014). The geographic distribution of legumes and their close relationships with symbiotic-nodulating bacteria, such as those of the Bradyrhizobium genus, reveal patterns related to evolutionary history and biogeography, adapting to different environmental conditions and playing important roles in ecosystems and agriculture (Parker, 2012; Sprent et al., 2017).

Species belonging to the genus Bradyrhizobium are cosmopolitan bacteria that occupy diverse ecosystems and habitats around the world. The biogeography of these bacteria plays a fundamental role in adapting to a variety of global environments (Hungria et al., 2015; Sprent et al., 2017; Bouznif et al., 2019), contributing to the maintenance of ecosystems, sustainability, and agricultural productivity (Hungria et al., 2015).

Within the genus Bradyrhizobium, several species depend on nodulation (nod) genes that encode Nod factors, signaling molecules essential for host plant recognition and nodule formation in legume roots (Ormeño-Orrillo and Martínez-Romero, 2019). This genus is also an important target for studies on nif (nitrogen fixation) and fix genes that encode proteins essential for biological nitrogen fixation (BNF). The nif genes are responsible for the synthesis and functioning of nitrogenase, an enzyme complex that converts molecular nitrogen (N₂) into ammonia (NH₃ ⁺), while the fix genes act in the regulation and maintenance of nitrogenase activity (Menna and Hungria, 2011). Furthermore, the Bradyrhizobium genus is also studied in relation to nodulation genes (nod), Nod Factors, the Type III Secretion System (T3SS) and its effector proteins (T3E), which play fundamental roles in nodule formation (Giraud et al., 2007; Teulet et al., 2022).

With the establishment of symbioses, biological nitrogen fixation takes place, and it is recognized as the second most important process for plant growth after photosynthesis (Hungria and Nogueira, 2023). In general, phylogenetic and genomic analyses of rhizobial strains have provided valuable information about their evolution and geographic distribution (Stępkowski et al., 2007; Avontuur et al., 2019).

Advances in DNA sequencing technologies have enabled the generation of data from sequenced bacterial genomes, enabling pangenome analyses, which represent a valuable tool for exploring genetic diversity within bacterial species (McInerney et al., 2017; Brockhurst et al., 2019). The concept of pangenome encompasses all genes identified in a set of strains or representants of a given taxonomic group. It considers genes shared by all strains or species, known as the core genome and represented by genes predicted to encode common and vital functions, as well as genes that occur exclusively in a few organisms, representing the accessory genome and conferring the ability to adapt to different lifestyles. This approach reveals the intrinsic genomic diversity of a bacterial species or genus and the ability to adapt to different ecological environments (Tettelin et al., 2008; Brockhurst et al., 2019).

Some studies have explored the pangenomes of strains of the same species, for example, Escherichia coli (Rasko et al., 2008) and Pseudomonas aeruginosa (Mosquera-Rendón et al., 2016), of species within a genus, e.g., Burkholderia (Bach et al., 2022), Rhizobium (González et al., 2019), or even at the order level, for example, Rhizobiales (Rosselli et al., 2021), and the results obtained have advanced the understanding of these microorganisms. An analysis of the Burkholderia pangenome showed the presence of genes acquired via HGT from other microorganisms, contributing to their ability to survive in the rhizosphere (Bach et al., 2022). Similarly, a pangenome analysis of Bradyrhizobium revealed genetic traits associated with nitrogen fixation, highlighting the role of genetic diversity in shaping symbiotic efficiency (Zhong et al., 2024). Furthermore, the modular nature of symbiotic genes in Bradyrhizobium allows for evolutionary stability and flexibility, with horizontal gene transfer (HGT) playing a key role in the maintenance and reorganization of symbiotic traits (Weisberg et al., 2022).

In this study, we investigated the genetic diversity of the nif/fix, nod and T3SS genes and their effector proteins, as well as the biogeographic distribution of nod-dependent of Bradyrhizobium species isolated from legume nodules around the world. Through pangenome analysis, we sought to understand how these genes are organized and how their variations are associated with the different environments of the Northern and Southern Hemispheres. Our results provide new insights into the genetic diversity and evolution of these bacteria, highlighting the differences associated with the biogeography of the species in each hemisphere.

In this study, specific representatives of each Bradyrhizobium species capable of forming nodules from different continental regions were selected, allowing us to investigate the influence of biogeography on the evolution and genetic diversity of these microorganisms. The genetic framework of these genomic representatives includes genes involved in adaptations to different water stresses, changes in pH and temperature, and interactions with their host plants (Freitas et al., 2022; Zhang et al., 2024). These characteristics impose selective pressures on these microorganisms, capturing genomic signatures that are direct results of the environmental pressure from which the organisms were isolated. Pangenome analysis revealed that species isolated in the Northern Hemisphere showed greater sharing of accessory genes compared to the Southern Hemisphere, indicating greater genetic interaction between species in this region. This difference may be associated with ecological factors such as environmental and host diversity, which may have favored greater gene exchange. The abundance of transposases and insertion elements in species from the Northern Hemisphere suggests greater genomic plasticity, which may have facilitated the adaptation of these species to different environments. Furthermore, by selecting genomes from different biogeographic environments, it was possible to understand the conservation of genes critical for local adaptation, evidencing the evolutionary pressure on the diversification of Bradyrhizobium.

Our pangenome study revealed a reduced core genome and a large accessory genome, evidencing broad genetic variability and the ability to adapt to various environments. Our results are in line with the studies by Weisberg et al. (2022) and Zhong et al. (2024) who showed that Bradyrhizobium has a small core genome and an extensive accessory genome, which characterizes an open pangenome. This genomic profile favors the acquisition of new genes via HGT, favoring adaptation to different environmental conditions. The genomic plasticity shown in these studies demonstrates the ability of Bradyrhizobium to colonize a wide variety of hosts.

Molecular evidence from fossils indicates that legumes emerged approximately 60 million years ago (Ma) between the Cretaceous and Cenozoic periods (Herendeen, 1992). At that time, Laurasia comprised a continuous and extensive landmass in the Northern Hemisphere. On the other hand, paleogeographic evidence indicates that during the fragmentation of Gondwana, South America remained connected to Antarctica. These facts disagree with the hypothesis of an African origin of legumes, favoring the boreotropic theory that suggests a Northern Hemisphere origin of legumes, with subsequent southward dispersal (see Figure 4 of Doyle and Luckow, 2003). This interpretation is supported by terrestrial connectivity between Africa and Eurasia during the Eocene and the subsequent union of the Americas (3-4 Ma), offering a more coherent overview of migration routes.

The analysis of the Bradyrhizobium accessory genome revealed a broad interaction of orthologous clusters between Asia and North America, suggesting intense genetic exchange in the Northern Hemisphere ( Figure 4 ). Furthermore, our results revealed a putative gene flow route from B. elkanii from North America through Central America and South America that extends to southern Africa ( Figure 4 ). According to the boreotropic hypothesis, legume dispersion occurs via continental masses, and these plants act as biotic vectors for rhizobia dispersal (Doyle and Luckow, 2003). Furthermore, despite the geological separation of continents before the emergence of legumes, it is possible to observe genetic interactions between Bradyrhizobium species from southern Africa, Oceania, and South America ( Figure 4 ). According to Vinuesa and collaborators (Vinuesa et al., 2005), when studying the migration and speciation of B. canariense and B. japonicum, the most likely means for the transcontinental and hemispheric dispersal of Bradyrhizobium cells are by abiotic vectors, such as dust masses. Soil carrying Bradyrhizobium, which is transported by wind currents, has been reported in Brazil, and dust storms can reach large distances, favoring bacterial dispersal (Ferreira and Hungria, 2002). This hypothesis is reinforced by the results of our studies, which revealed genetic interactions over long distances, covering isolates from Asia, North Africa, and North America. In parallel, we observed short-range genetic interactions between Bradyrhizobium species from the B. jicamae and B. japonicum clades, which were specifically isolated from legumes of the Genistae tribe in the regions of North Africa, the Canary Islands, and Spain (See Interactive Map, Supplementary File 3 ).

We identified limited genetic interactions between species from North Africa and South Africa, possibly because they contain different biomes, raising significant questions about the dispersal routes of these species across landmasses over time and challenging the two hypotheses presented.

Schrire et al. (2005) carried out extensive work on the biogeography of legumes, revealing many differences between the hemispheres. In the Northern Hemisphere, the distribution of biomes is more homogeneous, favoring greater gene flow and facilitating dispersal and interbreeding between populations. In contrast, the Southern Hemisphere has a greater diversity of biomes, including tropical forests, savannas, and succulent and temperate biomes. This fragmentation of biomes promoted more specific selective pressures, reducing gene flow between populations. Furthermore, their work mentioned that the first legumes evolved in a semiarid region north of the Tethys Sea route, which separated the supercontinents Laurasia and Gondwana.

Our pangenome results for Bradyrhizobium indicate greater genetic sharing of the accessory genome in the Northern Hemisphere than in the Southern Hemisphere, corroborating the biogeographic patterns observed in legumes. These results indicate a possible origin of Bradyrhizobium in the Northern Hemisphere and close and adaptive coevolution with legumes over time ( Figure 3 , 4 , Supplementary Figure S1 ). Bradyrhizobium has been suggested as the ancestor of all rhizobia (Provorov and Vorob’ev, 2000; Lloret and Martínez-Romero, 2005; Hungria et al., 2015); therefore, a Northern Hemisphere origin would also indicate that bacterial symbiosis originated in this region of the world.

The geological connection between Australia and Antarctica, as part of Gondwana, allowed the migration of plant species between Australia and South America. This connection is evidenced by the presence of the Brongniartieae tribe, a monophyletic clade present on both continents (Sprent et al., 2013). This relationship may clarify the genomic interactions between members of the B. jicamae clade inhabiting Australia and South America.

Some studies have reported that several legume species found in South America, previously considered archaic, are native to North America (Taylor, 1990; Lavin and Luckow, 1993), indicating that legume species in South America are evolutionarily more recent. These studies align with the phylogenetic analysis ( Supplementary Figure S1 ), which revealed that most ancestral nodes are associated with the Northern Hemisphere, supporting the idea that Bradyrhizobium species likely originated in this region before dispersing to the Southern Hemisphere.

The genomes of Hyphomicrobiales (=Rhizobiales), including Bradyrhizobium, are marked by an abundance of insertion elements (Minamisawa et al., 1998; Barros-Carvalho et al., 2019; Bender et al., 2022). These elements are fundamental for the movement of DNA segments, contributing to genomic plasticity and diversity. Our analysis of the pangenome revealed the widespread presence of transposases and insertion elements in the Northern Hemisphere, suggesting progress in the evolutionary process and genomic adaptation compared with those in the Southern Hemisphere. Furthermore, components of the T4SS genetic framework were more abundant in the Northern Hemisphere. The identification of these genetic elements more concentrated in the Northern Hemisphere strains supports the hypothesis that they may have greater nodulation capacity through horizontal gene transfer, a key mechanism for the adaptation and evolution of these symbiotic organisms (Sprent, 1994).

The α-rhizobial T3SS genes and nod genes are coregulated, and their activation is triggered by plant and bacterial flavonoids. In the presence of flavonoids, bacterial NodD activates nod genes, and the regulator TtsL promotes the expression of T3E proteins (Dong and Song, 2020). Studies with nodule isolates belonging to the genus Bradyrhizobium have indicated that 90% of the T3SS genes are conserved (Teulet et al., 2020), confirming the identification rate of 89% of these genes in the species of this study. Species lacking the T3SS are specific to the B. japonicum clade. Furthermore, the presence of up to three noncanonical T3SS clusters in some species suggests a functional complexity that still needs to be understood (Tampakaki, 2014), and further studies are needed to elucidate these multiple T3SS clusters. Symbiovars are groups of strains that share similar symbiotic capabilities, supported by nodC and nifH phylogenies (Rogel et al., 2011). Here, species belonging to the same symbiovar generally exhibit the same T3SS and nod gene repertoire ( Figure 1 ; Supplementary Table S1 ), supporting Teulet et al. (2020)’s suggestion of a common evolutionary origin for these genes.

The NopA and NopB proteins form needle-shaped extracellular filaments (Teulet et al., 2020), whereas NopX, NopH, and NopE act as translocons anchored inside the cell, facilitating the entry of effector proteins into host cells. Although NopE is recognized as a translocon (Wenzel et al., 2010), the exact function of NopH still requires further study, although this protein has been found in the secretome of B. diazoefficiens USDA110^T (Staehelin and Krishnan, 2015). Teulet et al. (2022) proposed that the NopH and NopE proteins perform functions similar to those of NopX in T3E translocation. Our results indicate that the presence of NopX is related to the absence of NopH and NopE, suggesting that these two proteins may have compensatory functions in effector translocation, acting as alternatives when NopX is not present.

Bradyrhizobium species associated with Genisteae legumes include populations from the Canary Islands, North Africa and Spain, which exhibit different profiles regarding the type III secretion system (T3SS) and the TtsL regulator. We observed that species lacking both the T3SS and the TtsL regulator or those that simultaneously present the T3SS with the NopE and NopB proteins are associated with Genisteae legumes (see Interactive Map). In addition, these species, B. canariense, B. hipponense, B. rifense, and B. cytisi, are grouped in the symbiovar genistearum (Martinez-Romero et al., 2024). Pangenome studies with Bradyrhizobium confirmed that most isolates from Genisteae legume tribe lack the T3SS (Zhong et al., 2024). Interestingly, the species lacking the T3SS in this group possess the T4SS (see Interactive Map). A notable example is B. canariense, which lacks the T3SS and TtsL regulator but is able to nodulate several Genisteae and Loteae legumes (Vinuesa et al., 2005), suggesting an evolutionary advantage in these geographic regions. Studies involving the type IV (T4SS) and type VI (T6SS) secretion systems in Bradyrhizobium demonstrated the ability to induce nodulation in legumes, possibly compensating for the absence of the T3SS in the establishment of symbiosis (Banerjee et al., 2019; Tighilt et al., 2022; Wangthaisong et al., 2023).

The T3SS of Bradyrhizobium is encoded by a gene cluster known as Rhizobium-conserved (Rhc), whose evolution is characterized by several gains and losses, indicating specific adaptations (Teulet et al., 2020). The interaction between the NopA and NopX proteins suggests that a specific biological relationship was absent in the B. jicamae clade. These components are predominantly found in strains from the Southern Hemisphere, especially in Brazil, South Africa, and Colombia, whereas NopX is absent in strains from Canada, North Africa, and Oceania. The distribution patterns of the RhcIa and RhcIb clusters, which are associated with NopE/NopH and NopX/NopA, respectively, highlight the dynamic evolution of Bradyrhizobium (Teulet et al., 2020), suggesting that adaptations are influenced by symbiosis with host plants and are related to biogeography.

Variations in the composition of T3SS effector proteins may represent fundamental adaptations for bacteria to avoid plant immune responses or establish specific interactions and play a role in the transport and translocation of bacterial effector proteins into host plant cells (Staehelin and Krishnan, 2015; Miwa and Okazaki, 2017). The T3E proteins ErnA, NopAC, NopM, NopP, NopP2, and NopT are present in most of the genomes analyzed in our study. The NopM, NopP, and NopT proteins are found in other rhizobial genera (Kimbrel et al., 2013), and Teulet et al. (2020) proposed that these proteins were acquired horizontally by the ancestors of Bradyrhizobium lineages together with the T3SS cluster and then lost in some lineages.

In our study, NopAG and NopF proteins were predominantly found in Bradyrhizobium isolated from soybean (Glycine max), whereas the Bel2-5 and NopD proteins were exclusive to this plant. The NopAG, Bel2-5 and NopD proteins play important roles in inhibiting plant defenses, facilitating symbiosis, and influencing the processing of small plant ubiquitin proteins (Xiang et al., 2020).

The analysis of genomic relationships revealed that B. japonicum and B. daqigense, which were isolated in Asia, indicated that they have accessory genomes related to B. diazoefficiens, B. barrani, and B. ottawaense from North America. The presence of common T3E in all these genomes, with the exception of B. barranii, which lost the Bel2-5 and ErnA proteins, highlights the importance of these effectors in symbiotic interactions. These T3Es are essential to suppress plant defense responses and allow effective colonization (Teulet et al., 2022). The absence of these effectors in B. barranii suggests that this lineage may have adopted other strategies to survive and develop in the environment, possibly compensating for the loss with other symbiotic mechanisms or adjusting to different ecological niches.

Our study expands our understanding of the biogeography and evolution of symbiotic species of the genus Bradyrhizobium, providing evidence on their geographic origin and gene flow. Phylogeographic analysis of the core genome revealed that most ancestral lineages were associated with the Northern Hemisphere, indicating this region as the likely origin of these organisms.

In addition, pangenome data indicated a more intense gene flow between species from the Northern Hemisphere compared to the Southern Hemisphere. Our investigation also revealed a diversity of genes associated with symbiotic functions, including fix, nif, nod, and the T3SS and its effector proteins, demonstrating that these genes are correlated with the phylogenetic clades, hosts, and biogeographic conditions in which the species occur.