The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes.

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade.

Evolution of novel strains of Ensifer nodulating the invasive legume Leucaena leucocephala (Lam.) de Wit in different climatic regions of India through lateral gene transfer.

More than 200 root-nodule bacterial strains were isolated from Leucaena leucocephala growing at 42 sampling sites across 12 states and three union territories of India. Genetic diversity was observed among 114 strains from various climatic zones; based on recA, these were identified as strains of Ensifer, Mesorhizobium, Rhizobium, and Bradyrhizobium. In multilocus sequence analysis (MLSA) strains clustered into several novel clades and lineages. Ensifer were predominant nodulating genotype isolated from majority of alkaline soils, while Mesorhizobium and Rhizobium strains were isolated from a limited sampling in North-Eastern states with acidic soils. Positive nodulation assays of selected Ensifer representing different genetic combinations of housekeeping and sym genes suggested their broad host range within the closely related mimosoid genera Vachellia, Senegalia, Mimosa, and Prosopis. Leucaena selected diverse strains of Ensifer and Mesorhizobium as symbionts depending on available soil pH, climatic, and other edaphic conditions in India. Lateral gene transfer seems to play a major role in genetic diversification of Ensifer exhibited in terms of Old World vs. Neotropical genetic make-up and mixed populations at several sites. Although Neotropical Ensifer strains were most symbiotically effective on Leucaena, the native Ensifer are promiscuous and particularly well-adapted to a wide range of sampling sites with varied climates and edaphic factors.

Selection of Bradyrhizobium or Ensifer symbionts by the native Indian caesalpinioid legume Chamaecrista pumila depends on soil pH and other edaphic and climatic factors.

Nodules of Chamaecrista pumila growing in several locations in India were sampled for anatomical studies and for characterization of their rhizobial microsymbionts. Regardless of their region of origin, the nodules were indeterminate with their bacteroids contained within symbiosomes which were surrounded by pectin. More than 150 strains were isolated from alkaline soils from the Thar Desert (Rajasthan), wet-acidic soils of Shillong (Meghalaya), and from trap experiments using soils from four other states with different agro-ecological regions. Molecular phylogenetic analysis based on five housekeeping (rrs, recA, glnII, dnaK andatpD) and two symbiotic (nodA and nifH) genes was performed for selected strains. Chamaecrista pumila was shown to be nodulated by niche-specific diverse strains of either Ensifer or Bradyrhizobium in alkaline (Thar Desert) to neutral (Tamil Nadu) soils and only Bradyrhizobium strains in acidic (Shillong) soils. Concatenated core gene phylogenies showed four novel Ensifer-MLSA types and nine Bradyrhizobium-MLSA types. Genetically diverse Ensifer strains harbored similar sym genes which were novel. In contrast, significant symbiotic diversity was observed in the Bradyrhizobium strains. The C. pumila strains cross-nodulated Vigna radiata and some wild papilionoid and mimosoid legumes. It is suggested that soil pH and moisture level played important roles in structuring the C. pumila microsymbiont community.

Legume abundance along successional and rainfall gradients in Neotropical forests.

The nutrient demands of regrowing tropical forests are partly satisfied by nitrogen-fixing legume trees, but our understanding of the abundance of those species is biased towards wet tropical regions. Here we show how the abundance of Leguminosae is affected by both recovery from disturbance and large-scale rainfall gradients through a synthesis of forest inventory plots from a network of 42 Neotropical forest chronosequences. During the first three decades of natural forest regeneration, legume basal area is twice as high in dry compared with wet secondary forests. The tremendous ecological success of legumes in recently disturbed, water-limited forests is likely to be related to both their reduced leaflet size and ability to fix N2, which together enhance legume drought tolerance and water-use efficiency. Earth system models should incorporate these large-scale successional and climatic patterns of legume dominance to provide more accurate estimates of the maximum potential for natural nitrogen fixation across tropical forests.

Biogeography of nodulated legumes and their nitrogen-fixing symbionts.

Contents 40 I. 40 II. 41 III. 44 IV. 48 V. 49 VI. 49 VII. 52 VIII. 53 53 References 53 SUMMARY: In the last decade, analyses of both molecular and morphological characters, including nodulation, have led to major changes in our understanding of legume taxonomy. In parallel there has been an explosion in the number of genera and species of rhizobia known to nodulate legumes. No attempt has been made to link these two sets of data or to consider them in a biogeographical context. This review aims to do this by relating the data to the evolution of the two partners: it highlights both longitudinal and latitudinal trends and considers these in relation to the location of major land masses over geological time. Australia is identified as being a special case and latitudes north of the equator as being pivotal in the evolution of highly specialized systems in which the differentiated rhizobia effectively become ammonia factories. However, there are still many gaps to be filled before legume nodulation is sufficiently understood to be managed for the benefit of a world in which climate change is rife.

Legumes are different: Leaf nitrogen, photosynthesis, and water use efficiency.

Using robust, pairwise comparisons and a global dataset, we show that nitrogen concentration per unit leaf mass for nitrogen-fixing plants (N2FP; mainly legumes plus some actinorhizal species) in nonagricultural ecosystems is universally greater (43-100%) than that for other plants (OP). This difference is maintained across Koppen climate zones and growth forms and strongest in the wet tropics and within deciduous angiosperms. N2FP mostly show a similar advantage over OP in nitrogen per leaf area (Narea), even in arid climates, despite diazotrophy being sensitive to drought. We also show that, for most N2FP, carbon fixation by photosynthesis (Asat) and stomatal conductance (gs) are not related to Narea-in distinct challenge to current theories that place the leaf nitrogen-Asat relationship at the center of explanations of plant fitness and competitive ability. Among N2FP, only forbs displayed an Narea-gs relationship similar to that for OP, whereas intrinsic water use efficiency (WUEi; Asat/gs) was positively related to Narea for woody N2FP. Enhanced foliar nitrogen (relative to OP) contributes strongly to other evolutionarily advantageous attributes of legumes, such as seed nitrogen and herbivore defense. These alternate explanations of clear differences in leaf N between N2FP and OP have significant implications (e.g., for global models of carbon fluxes based on relationships between leaf N and Asat). Combined, greater WUE and leaf nitrogen-in a variety of forms-enhance fitness and survival of genomes of N2FP, particularly in arid and semiarid climates.

Endemic Mimosa species from Mexico prefer alphaproteobacterial rhizobial symbionts.

The legume genus Mimosa has > 500 species, with two major centres of diversity, Brazil (c. 350 spp.) and Mexico (c. 100 spp.). In Brazil most species are nodulated by Burkholderia. Here we asked whether this is also true of native and endemic Mexican species. We have tested this apparent affinity for betaproteobacteria by examining the symbionts of native and endemic species of Mimosa in Mexico, especially from the central highlands where Mimosa spp. have diversified. Nodules were tested for betaproteobacteria using in situ immunolocalization. Rhizobia isolated from the nodules were genetically characterized and tested for their ability to nodulate Mimosa spp. Immunological analysis of 25 host taxa suggested that most (including all the highland endemics) were not nodulated by betaproteobacteria. Phylogenetic analyses of 16S rRNA, recA, nodA, nodC and nifH genes from 87 strains isolated from 20 taxa confirmed that the endemic Mexican Mimosa species favoured alphaproteobacteria in the genera Rhizobium and Ensifer: this was confirmed by nodulation tests. Host phylogeny, geographic isolation and coevolution with symbionts derived from very different soils have potentially contributed to the striking difference in the choice of symbiotic partners by Mexican and Brazilian Mimosa species.

A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms.

Symbiotic associations occur in every habitat on earth, but we know very little about their evolutionary histories. Current models of trait evolution cannot adequately reconstruct the deep history of symbiotic innovation, because they assume homogenous evolutionary processes across millions of years. Here we use a recently developed, heterogeneous and quantitative phylogenetic framework to study the origin of the symbiosis between angiosperms and nitrogen-fixing (N2) bacterial symbionts housed in nodules. We compile the largest database of global nodulating plant species and reconstruct the symbiosis' evolution. We identify a single, cryptic evolutionary innovation driving symbiotic N2-fixation evolution, followed by multiple gains and losses of the symbiosis, and the subsequent emergence of 'stable fixers' (clades extremely unlikely to lose the symbiosis). Originating over 100 MYA, this innovation suggests deep homology in symbiotic N2-fixation. Identifying cryptic innovations on the tree of life is key to understanding the evolution of complex traits, including symbiotic partnerships.

Burkholderia sp. induces functional nodules on the South African invasive legume Dipogon lignosus (Phaseoleae) in New Zealand soils.

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678(T) which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.

An invasive Mimosa in India does not adopt the symbionts of its native relatives.

BACKGROUND AND AIMS: The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species. METHODS: Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences. KEY RESULTS: Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the 'Old World' Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica. CONCLUSIONS: The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the 'local' Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.

Burkholderia diazotrophica sp. nov., isolated from root nodules of Mimosa spp.

Five strains, JPY461(T), JPY359, JPY389, DPU-3 and STM4206 were isolated from nitrogen-fixing nodules on the roots of Mimosa spp. and their taxonomic positions were investigated using a polyphasic approach. All five strains grew at 15-40 °C (optimum, 30-37 °C), at pH 4.0-8.0 (optimum, pH 6.0-7.0) and with 0-1 % (w/v) NaCl [optimum, 0 % (w/v)]. On the basis of 16S rRNA gene sequence analysis, a representative strain (JPY461(T)) showed 97.2 % sequence similarity to the closest related species Burkholderia acidipaludis SA33(T), a similarity of 97.2 % to Burkholderia terrae KMY02(T), 97.1 % to Burkholderia phymatum STM815(T) and 97.1 % to Burkholderia hospita LMG 20598(T). The predominant fatty acids of the five novel strains were summed feature 2 (comprising C(16 : 1) iso I and/or C(14 : 0) 3-OH), summed feature 3 (comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c), C(16 : 0) , C(16 : 0) 3-OH, C(17 : 0) cyclo, C(18 : 1)ω7c and C(19 : 0) cyclo ω8c. The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 63.0-65.0 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and several unidentified phospholipids. The DNA-DNA relatedness of the novel strain with respect to recognized species of the genus Burkholderia was less than 54 %. On the basis of 16S rRNA and recA gene sequence similarities, chemotaxonomic and phenotypic data, the five strains represent a novel species in the genus Burkholderia, for which the name Burkholderia diazotrophica sp. nov. is proposed with the type strain, JPY461(T) ( = LMG 26031(T) = BCRC 80259(T) = KCTC 23308(T)).

Nodulation in Dimorphandra wilsonii Rizz. (Caesalpinioideae), a threatened species native to the Brazilian Cerrado.

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N(2)-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N(2) fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N(2)-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.

Rhizobia with 16S rRNA and nifH similar to Mesorhizobium huakuii but Novel recA, glnII, nodA and nodC genes are symbionts of New Zealand Carmichaelinae.

New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus) produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.

Burkholderia symbiotica sp. nov., isolated from root nodules of Mimosa spp. native to north-east Brazil.

Four strains, designated JPY-345(T), JPY-347, JPY-366 and JPY-581, were isolated from nitrogen-fixing nodules on the roots of two species of Mimosa, Mimosa cordistipula and Mimosa misera, that are native to North East Brazil, and their taxonomic positions were investigated by using a polyphasic approach. All four strains grew at 15-43 °C (optimum 35 °C), at pH 4-7 (optimum pH 5) and with 0-2 % (w/v) NaCl (optimum 0 % NaCl). On the basis of 16S rRNA gene sequence analysis, strain JPY-345(T) showed 97.3 % sequence similarity to the closest related species Burkholderia soli GP25-8(T), 97.3 % sequence similarity to Burkholderia caryophylli ATCC25418(T) and 97.1 % sequence similarity to Burkholderia kururiensis KP23(T). The predominant fatty acids of the strains were C(18 : 1)ω7c (36.1 %), C(16 : 0) (19.8 %) and summed feature 3, comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c (11.5 %). The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 64.2-65.7 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized aminophospholipids and phospholipids. DNA-DNA hybridizations between the novel strain and recognized species of the genus Burkholderia yielded relatedness values of <51.8 %. On the basis of 16S rRNA and recA gene sequence similarities and chemotaxonomic and phenotypic data, the four strains represent a novel species in the genus Burkholderia, for which the name Burkholderia symbiotica sp. nov. is proposed. The type strain is JPY-345(T) (= LMG 26032(T) = BCRC 80258(T) = KCTC 23309(T)).

Legume-nodulating betaproteobacteria: diversity, host range, and future prospects.

Rhizobia form specialized nodules on the roots of legumes (family Fabaceae) and fix nitrogen in exchange for carbon from the host plant. Although the majority of legumes form symbioses with members of genus Rhizobium and its relatives in class Alphaproteobacteria, some legumes, such as those in the large genus Mimosa, are nodulated predominantly by betaproteobacteria in the genera Burkholderia and Cupriavidus. The principal centers of diversity of these bacteria are in central Brazil and South Africa. Molecular phylogenetic studies have shown that betaproteobacteria have existed as legume symbionts for approximately 50 million years, and that, although they have a common origin, the symbiosis genes in both subclasses have evolved separately since then. Additionally, some species of genus Burkholderia, such as B. phymatum, are highly promiscuous, effectively nodulating several important legumes, including common bean (Phaseolus vulgaris). In contrast to genus Burkholderia, only one species of genus Cupriavidus (C. taiwanensis) has so far been shown to nodulate legumes. The recent availability of the genome sequences of C. taiwanensis, B. phymatum, and B. tuberum has paved the way for a more detailed analysis of the evolutionary and mechanistic differences between nodulating strains of alpha- and betaproteobacteria. Initial analyses of genome sequences have suggested that plant-associated Burkholderia spp. have lower G+C contents than Burkholderia spp. that are opportunistic human pathogens, thus supporting previous suggestions that the plant- and human-associated groups of Burkholderia actually belong in separate genera.

Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga biomes of Brazil.

*An extensive survey of nodulation in the legume genus Mimosa was undertaken in two major biomes in Brazil, the Cerrado and the Caatinga, in both of which there are high degrees of endemicity of the genus. *Nodules were collected from 67 of the 70 Mimosa spp. found. Thirteen of the species were newly reported as nodulating. Nodules were examined by light and electron microscopy, and all except for M. gatesiae had a structure typical of effective Mimosa nodules. The endosymbiotic bacteria in nodules from all of the Mimosa spp. were identified as Burkholderia via immunolabelling with an antibody against Burkholderia phymatum STM815. *Twenty of the 23 Mimosa nodules tested were shown to contain nitrogenase by immunolabelling with an antibody to the nitrogenase Fe- (nifH) protein, and using the delta(15)N ((15)N natural abundance) technique, contributions by biological N(2) fixation of up to 60% of total plant N were calculated for Caatinga Mimosa spp. *It is concluded that nodulation in Mimosa is a generic character, and that the preferred symbionts of Brazilian species are Burkholderia. This is the first study to demonstrate N(2) fixation by beta-rhizobial symbioses in the field.

Multiple evolutionary origins of legume traits leading to extreme rhizobial differentiation.

SUMMARY: *When rhizobia differentiate inside legume host nodules to become nitrogen-fixing bacteroids, they undergo a physiological as well as a morphological transformation. These transformations are more extreme in some legume species than others, leading to fundamental differences in rhizobial life history and evolution. Here, we analysed the distribution of different bacteroid morphologies over a legume phylogeny to understand the evolutionary history of this host-influenced differentiation. *Using existing electron micrographs and new flow cytometric analyses, bacteroid morphologies were categorized as swollen or nonswollen for 40 legume species in the subfamily Papilionoideae. Maximum likelihood and Bayesian frameworks were used to reconstruct ancestral states at the bases of all major subclades within the papilionoids. *Extreme bacteroid differentiation leading to swelling was found in five out of the six major papilionoid subclades. The inferred ancestral state for the Papilionoideae was hosting nonswollen bacteroids, indicating at least five independent origins of host traits leading to swollen bacteroids. *Repeated evolution of host traits causing bacteroid swelling indicates a possible fitness benefit to the plant. Furthermore, as bacteroid swelling is often correlated with loss of reproductive viability, the evolution of bacteroid cooperation or cheating strategies could be fundamentally different between the two bacteroid morphologies.

African legumes: a vital but under-utilized resource.

Although nodulated legumes have been used by indigenous peoples in Africa for centuries, their full potential has never been realized. With modern technology there is scope for rapid improvement of both plant and microbial germplasm. This review gives examples of some recent developments in the form of case studies; these range from multipurpose human food crops, such as cowpea (Vigna unguiculata (L.) Walp.), through to beverages (teas) that are also income-generating such as rooibos (Aspalathus linearis (Burm. f.) R. Dahlgren, honeybush (Cyclopia Vent. spp.), and the widely used food additive gum arabic (Acacia senegal (L.) Willd.). These and other potential crops are well-adapted to the many different soil and climatic conditions of Africa, in particular, drought and low nutrients. All can nodulate and fix nitrogen, with varying degrees of effectiveness and using a range of bacterial symbionts. The further development of these and other species is essential, not only for African use, but also to retain the agricultural diversity that is essential for a changing world that is being increasingly dominated by a few crops such as soybean.

Burkholderia species are ancient symbionts of legumes.

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia. Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia. The symbiosis-related genes formed deep Burkholderia-specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.