Chapter 89

Symbiotic Nitrogen Fixation in Legumes: Perspectives on the Diversity and Evolution of Nodulation by Rhizobium and Burkholderia Species

Robert Walker School of Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, Australia Christina M. Agapakis Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA Elizabeth Watkin School of Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, Australia Ann M. Hirsch Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA

89.1 INTRODUCTION Burkholderia,andtheirrelevancetothiswell-studied, benefcial interaction between bacterium and plant. More than a decade ago, we wrote an update on the In particular, we examine the concept of host range, Rhizobium–legume symbiosis asking the question “what biogeography, as well as evolutionary history with respect makes this symbiosis so special?” (Hirsch et al., 2001). to nodulation by Burkholderia and Rhizobium (see also Since that time, we have learned a great deal more about this Chapter 17). Phylogenetic analyses strongly indicate that important plant–microbe association. However, the story of both genera acquired the nodulation genes at approximately symbiotic nitrogen fxation in legumes has become more the same time as the legumes were evolving (Bontemps complicated due to the discovery of several Betaproteobac- et al., 2010). We elaborate on this evolutionary theme as we teria, namely species of Burkholderia and Cupriavidus, delve into the even earlier history of these two nodulating which also establish nitrogen-fxing nodules on legumes. and nitrogen-fxing bacterial families, with the goal of estab- In this chapter, we expand our analysis of the Rhizobium– lishing an evolutionary description of this still incompletely legume symbiosis to include the betarhizobia, specifcally understood symbiosis.

Biological Nitrogen Fixation, Volume 2, First Edition. Edited by Frans J. de Bruijn. © 2015 John Wiley & Sons, Inc. Published 2015 by John Wiley & Sons, Inc. 913 914 Chapter 89 Symbiotic Nitrogen Fixation in Legumes: Perspectives on the Diversity and Evolution

89.2 THE LEGUMES into most of their major lineages within a relatively short period of time (ca. 55–50 Mya) (Herendeen et al., 1992; The legumes (Fabaceae or Leguminosae) are the third Doyle and Luckow, 2003; Lewis et al., 2005; Sprent, 2007, largest family of fowering plants (>19,000 species); only 2008). This is approximately the same time that the rest Asteraceae and Orchidaceae are larger (Lewis et al., 2005). of the angiosperm families were evolving (Lewis et al., The family is monophyletic, but comprises three subfamilies 2005). The boreotropical forest migration hypothesis has (36 tribes), namely Caesalpinioideae, Mimosoideae, and replaced the older Gondwanan theory in which legumes Papillionoideae, which represent 22%, 10%, and 67%, moved northward from their presumed sites of origin in respectively, of the family (Sprent, 2001). Of the three Africa to South America and then, before the land masses subfamilies, caesalpinioid legumes are less likely to be drifted apart 165–145 Mya, to North America. Nonetheless, nodulated, but this is not because they evolved frst (Sprent, the boreotropical hypothesis leaves much unexplained, 2007). Several caesalpinioid legumes considered to be especially with regard to the evolution of legume nodulation “basal” are not found in the fossil fora in the early history (Sprent, 2008; see Chapter 3). of the family (Herendeen et al., 1992). Although nodules have not been found in the fossil The structure of the fowers of the subfamily members record, it is extremely likely that many of the legumes were differs signifcantly (Fig. 89.1), but legumes are incredibly already developing root nodules inhabited by nitrogen-fxing diverse in many other features, including phytochemical rhizobia by the time that the major lineages were established. profles, nodulation status, and habitats. Legumes occupy The partnership between rhizobia and legumes is based on deserts and grass-poor, succulent-rich, dry environments apre-existingandancientrelationship,datingfromthe (S-biomes); tropical rain forests (R-biomes); grass-rich early Devonian (416–350 Mya) between arbuscular myc- prairies and savannahs (G-biomes); temperate ecosystems orrhizal fungi (AMF) and plants. The evolutionary origin (T-biomes); and agricultural systems derived from anthro- of the genes important for root nodulation that was derived pogenic input. Numerous books and papers have described from the arbuscular-mycorrhizal (AM) symbiosis has been the biology, nodulation, and evolution of the legumes, so we extensively studied and reviewed (Hirsch et al., 2001; Szcz- describe only a few salient features in this chapter (Sprent, glowsky and Amyot, 2003; Streng et al., 2011; and others; 2001; Doyle and Luckow, 2003; Lewis et al., 2005). see also Chapters 42, 54, 55, 108, 110). The legumes are believed to have evolved ca. 65–50 million years ago (Mya) in the late Cretaceous/early Tertiary after the K/T extinction (Lavin et al., 2005). This 89.3 THE RHIZOBIA hypothesis is in part based on the discovery of legume fossils dating from the Paleocene (ca. 66–56 Mya; Herendeen et al., “Rhizobia” is the collective term given to bacteria capa- 1992) and also on molecular analysis developed from matK ble of inducing the development of and then populating and rbcL phylogenies (Lavin et al., 2005). The evolutionary nitrogen-fxing nodules (Sprent, 2001). A long-standing migration of legumes southward is postulated to have started debate exists over whether legumes and rhizobia coevolved north of the Tethys Sea, under warmer climate conditions, or whether the plants selected for symbiotic bacteria, but after the continents drifted apart (see Doyle and Luckow, the presence of horizontal gene transfer and the lack of par- 2003; Sprent, 2007). This boreotropical origin, deduced allel speciation between legumes and rhizobia might argue in part from the current habitats of legumes, is consistent against a coevolutionary trajectory (Martínez-Romero, with the migration of legumes from an S-biome to the R-, 2009; Young and Johnston, 1989). G-, and T-biomes (Lewis et al., 2005). Both molecular Up to 2001, only the Alphaproteobacteria in the family and fossil data suggest that legumes rapidly diversifed Rhizobiaceae had been classifed as rhizobia. These bacteria

(a) (b) (c)

Figure 89.1 Examples of fowers from the three legume subfamilies. (a) Caesalpinoideae: Chamaecrista fasciculata (N.A. Fujishige). (b) Mimosoideae: Caliandra hematocephala (A.M. Hirsch). (c) Papillionoideae: Swainsona formosa (W. Deng). 89.5 The Root-Nodulating Burkholderia Species 915 were frst isolated from root nodules by Beijerinck (1890), 89.4 THE BETARHIZOBIA and the name Rhizobium was suggested shortly afterwards by Frank (1889) (see additional references in Gyaneshwar In 2001, two genera of the family Burkholderiaceae, et al., 2011; see Chapter 88). Until the 1980s, all the legume Burkholderia and Cupriavidus,werereportedtonodu- root-nodulating bacteria (RNB) were classifed in the genus late legumes, but at that time were not shown to induce Rhizobium,whichhadbeendividedintofastandslow effective, that is, nitrogen-fxing nodules on their hosts growers. After it became apparent that signifcantly greater (Moulin et al., 2001; see Chapter 17). Much of the original differences than just growth rate existed, Bradyrhizobium work on the Burkholderia symbiotic species focused on (Bradyrhizobiaceae)wasproposedbyJordan(1982)to B. tuberum and B. phymatum,butitwasquicklyshownthat encompass the slow-growing species whereas the name Rhi- neither species re-infected the hosts from which they were zobium was retained for the fast growers. Using a molecular originally isolated (Aspalathus carnosa and Machaerium clock approach, the split between Rhizobium and Bradyrhi- lunatum,respectively).Laterstudiesshowedthatbothgen- zobium is estimated to have taken place 800–300 Mya era established nitrogen-fxing nodules with a number of depending on the gene analyzed (Turner and Young, 2000). legume hosts. The majority of Burkholderia species have Rhizobium has also been subdivided into a number been isolated from nodules of Mimosoideae, especially the of distinct genera. Chen et al. (1988) isolated fast- and genus Mimosa. Mimosa spp. are native to the Neotropics slow-growing rhizobia from nodules of soybean (Glycine although a few endemics live in the Paleotropics, mainly in soja)andfromsoil,andafteranalyzingcellularcomposition Madagascar, and also extend into southeast tropical Africa and by using methods such as DNA–DNA hybridization, and India (Lewis et al., 2005; see Chapter 17). serology, and phage typing, proposed a new genus, Sinorhi- Some Burkholderia species are free-living and function zobium. Thus, the fast-growing isolate Rhizobium fredii as soil-dwelling microbes in bioremediation or act as plant was renamed Sinorhizobium fredii.In2008,however,tax- growth-promoting bacteria (PGPB) living as epiphytes on plants (de Bruijn, 2013). Still others appear to be endophytes onomists questioned whether Sinorhizobium was distinct based on their isolation from both legume and nonlegume from the genus Ensifer,andtheJudicialCommissionof hosts following surface sterilization of plant tissues the International Committee on Systematics of Prokaryotes (Compant et al., 2008). However, many of these species (2008) decided that it was not, and thus the name Ensifer, have not been rigorously confrmed as either endophytes or which had priority, was retained (see also Chapter 3). PGPB based on experimental studies, with the exception of Later, other genera were described, including Azorhizobium B. phytofrmans (Sessitsch et al., 2005). (Dreyfus et al., 1988), which was placed in the family Yabuuchi et al. (1992) split Burkholderia from Pseu- Xanthobacteraceae,andMesorhizobium (Jarvis et al., 1997) domonas homology group II and named the genus for W.H. in the Phyllobacteriaceae.ThegenusPhyllobacterium Burkholder, who isolated the etiological agent of onion rot contains three rhizobial species that are closely related to (Burkholder, 1942). Burkholderia species, other than the Mesorhizobium (Valverde et al., 2005; Mantelin et al., 2006). RNB (shown in Fig. 89.3), are ubiquitous in the environ- Concurrently, additional RNB from Rhizobiales were ment and are found in soil, rhizosphere, clinical samples, added to the list: Devosia neptunia (family Hyphomicrobi- plants, fungus, insects, and animals (Compant et al., 2008). aceae (Rivas et al.,2003));Ochrobactrum (Trujillo et al., Burkholderia species have long been associated with plant 2005; Zurdo-Piñeiro et al., 2007) (family Brucellaceae); and animal pathogenesis, and all of the initial species moved Microvirga (Ardley et al., 2012; see Chapter 23); and Methy- from Pseudomonas to Burkholderia demonstrated virulence lobacterium (family Methylbacteriaceae)(Jourandet al., toward numerous plants and animals (Compant et al., 2008). 2004). Each family that contains RNB also houses nonrhi- However, efforts have been initiated to differentiate the zobial species, some of which are pathogenic on animals symbiotic and environmental Burkholderia species from the or plants. For example, most of the Xanthobacteraceae are opportunistic, mammalian, and plant pathogens, and have nonnodulating species and many Brucellaceae are human led to the proposal that the former be assigned to a new pathogens. As another example, Bradyrhizobium is closely group/genus because it is phylogenetically distinct from related to the genus Apifa,whichincludeshumanpathogens the Burkholderia group (Angus et al., 2014, Estrada-de Los (La Scola et al.,2002). Santos et al., 2013, Suárez-Moreno et al., 2012; see also Other novel species of rhizobia recently isolated include Chapter 17). Shinella kummerowiae from the herbal legume Kummerowia stipulacea in China (Lin et al., 2008). This genus is closely 89.5 THE ROOT-NODULATING related to Rhizobium and the bacteria are often found as nod- Burkholderia SPECIES ule occupants and in soil samples, but had not been shown previously to induce nodules. The diversity of organisms that To study the evolution of symbiotic genes, Bontemps function as RNB is summarized in Figure 89.2. et al. (2010) prepared a phylogenetic analysis using partial 916 Chapter 89 Symbiotic Nitrogen Fixation in Legumes: Perspectives on the Diversity and Evolution

Figure 89.2 Unrooted phylogenetic reconstruction of 16S ribosomal RNA of rhizobial and related nonrhizobial species by Neighbor Joining. Bootstrap percentage after 1000 replications is shown on nodes. Scale bar represents the number of substitutions per site. Colored nodes represent each of the main families with genera containing rhizobia (in bold). Purple, Rhizobiaceae;green,Brucellaceae;pink,Phyllobacteriaceae;olive, Xanthobacteraceae;turquoise,Hyphomicrobiaceae;blue,Methylobacteriaceae;orange,Bradyrhizobiaceae;red,Burkholderiaceae (see also Chapter 17). 89.5 The Root-Nodulating Burkholderia Species 917

Figure 89.2 (Continued)

sequences of the 16S, recA, nodC,andnifH regions of iso- partial nodC sequences place South American Burkholde- lates from nodules of 47 species of Mimosa spp. growing in ria symbiotic genes in a monophyletic group that is highly various regions of Brazil. All sequences were highly homol- divergent from all other nodC sequences and further sug- ogous to Burkholderia species from seven distinct clusters gest that any nod gene transfer event that occurred must have (clades). Four of the seven clusters observed by Bontemps taken place at least 50–60 Mya (Bontemps et al., 2010). This et al. (2010) contained named species of Burkholderia transfer of symbiotic genes coincides with the period when RNB (cluster 2, B. phymatum;cluster4,B. mimosarum; the legumes themselves were diversifying into their major cluster 5, B. nodosa; and cluster 6, B. tuberum). However, lineages (Fig. 89.4). three clusters represented novel species (clusters 1, 3, and The legume genus Mimosa is almost exclusively nodu- 7). Based on recent evidence, cluster 3 is closely related to lated by species of Burkholderia (see also Chapter 17). the recently described B. diazotrophica (Sheu et al., 2013); Mimosa most likely followed a boreotropical migration one isolate within this cluster, Burkholderia sp. mpa3.10 from north to south and this theory is supported by fossil shares over 99% 16S and recA sequence homology to B. evidence in North America and Europe, but palynological diazotrophica (Walker & Watkin, unpublished). Isolates data have also been collected in Africa (Herendeen et al., from cluster 7 may be related to the recently sequenced B. 1992). However, disjunct populations of Mimosa in South phenoliruptrix (de Oliveira et al., 2012), and are also closely Eastern Africa, Madagascar, and India may have originated related to B. fungorum.Cluster1maycontaintherecently from long-distance oceanic dispersal (Simon et al., 2011). described B. symbiotica, which is the frst Burkholderia Some of these introduced populations might be coincident sp. RNB characterized that is not closely related to other with the southern hemisphere locations of the isolated RNB Brazilian species (Sheu et al., 2012). Burkholderia species (Fig. 89.3). The study by Bontemps et al. (2010) gave us important In contrast to the South American Burkholderia species, insight into the evolution of nodulation in Burkholderia by the South African B. tuberum strains nodulate papillionoid showing that it had a long and stable genetic history (Angus legumes. This nodulation preference is correlated with and Hirsch, 2010), which is most likely as old as that of nodulation (nod)andnitrogen-fxation genes (nif)gene the nodulating Rhizobiales. Phylograms reconstructed from sequence organization, which differ between the South 918 Chapter 89 Symbiotic Nitrogen Fixation in Legumes: Perspectives on the Diversity and Evolution

B. phymatum

B. mimosarum B. mimosarum B. phymatum

Endemic South American species B. phymatum B. mimosarum B. tuberum B. mimosarum B. nodosa B. diazotrophica B. sabiae B. phenoliruptrix B. diazotrophica B. symbiotica Endemic South African species B. phenoliruptrix B. tuberum B. rhynchosiae B. sprentiae

Described species Undescribed isolates Isolated from invasive Mimosa spp. Novel undescribed isolate

Figure 89.3 Distribution of characterized type strains and undescribed isolates of Burkholderia. Note the mostly southern hemisphere distribution of the strain isolations. Strain CCGE1002 (Mexico) is described as B. tuberum in Mishra et al., (2012).

African and South American RNB. One hypothesis posits described (Beukes et al., 2013; De Meyer et al., 2014). Such that the establishment of Mimosa from long-distance oceanic studies will increase our understanding of this group of dispersal may have led to the introduction of B. tuberum into Burkholderia spp. South Africa, where it could have lost the ability to nodulate Mimosa in favor of the endemic Papillionoideae population following the lateral transfer of local nod genes (from 89.6 REGARDING THE alpha rhizobia?). Mimosa introduction has been observed in EVOLUTION OF NODULATING Australia with the accidental importation of South Amer- Burkholderiaceae AND Rhizobiaceae ican Burkholderia species, including B. diazotrophica and B. mimosarum (Walker and Watkin, unpublished), on Alimitedfossilrecordexistsforbacteria,exceptfor introduced M. pigra seeds (Parker et al., 2007). cyanobacteria where fossils resembling these organisms The idea of introduced Mimosa species, however, does have been reported from the Archean period of Earth history not adequately explain the distinct lineages of Burkholderia (Schopf, 2006; Javaux et al., 2010). Nonetheless, ques- species found in South Africa that nodulate papillionoid tions remain as to their actual identity and whether or not cyanobacterial photosynthesis evolved that early. In any legumes. The frst lineage described is typifed by the type case, most agree that marine bacteria probably evolved strain B. tuberum STM678T, originally isolated from A. about 3.5 billion years ago (Gya). Undisputed microfossils carnosa,whereasasecondlineagecontainstherecently from 1.2 Gya have been found (Horodyski and Knauth, described B. rhynchosiae (De Meyer et al., 2013a), which 1994) (Fig. 89.4). Due to the debate about the exact nature nodulates species of Rhynchosia (Garau et al., 2009). So of the fossils from 3.5 Gya, conclusions regarding bacterial far, no information is available about nod and nif gene orga- evolution are based mainly on geochemical evidence, the nization in B. rhynchosiae,norabouthowthesesymbiotic detection of biomarkers, and isotope ratios (Fischer, 2008). T genes compare to those of B. tuberum STM678 except for The accumulation of such information has strongly sug- the fact that the nodA genes are 96% identical (Garau et al., gested that bacteria achieved a terrestrial lifestyle (became 2009). Another lineage, B. sprentiae, is more closely related terrabacteria) some 2.7–2.6 Gya (Watanabe et al., 2000) to B. tuberum STM678T,butitnodulatesLebeckia spp. (De (Fig. 89.4). Moreover, it appears that many prokaryotic Meyer et al., 2013b). Numerous South African species have lineages were already evolved as early as 2.7–2.5 Gya, been recently isolated and many are in the process of being supporting the even older history for the earliest bacteria. 89.6 Regarding the Evolution of Nodulating Burkholderiaceae and Rhizobiaceae 919

Present 50 Evolution of legumes; hypothesized horizontal transfer of nod genes Breakup of Gondwana Phanerozoic 0.5 200

Rise of terrestrial Azospirillum spp.

Oldest eukaryotic fossils Rise of vascular plants; 500 arbuscular mycorrhizal fungal fossils 1.5 Mya Divergence of Rhizobium and Bradyrhizobium

Increasing oxygen 2.5 Evidence for “terrabacteria” Archaean Proterozoic Evidence for marine bacterial fossils 3.5 Hadean 4.5 Gya Origin of Earth Figure 89.4 Timeline showing some key points in the evolution of the legumes and their associated nitrogen-fxing bacteria. (Source: Modifed from Fischer (2008).)

More recently, genome analysis has been used to study ancient mycorrhizal signaling pathway to ensure root cell bacterial evolution. Wisniewsky-Dyé et al. (2011) proposed entry and subsequent nodulation for the proliferation and the divergence of terrestrial azospirilla from their aquatic protection of the bacteria is well accepted. Rhodosporillaceae relatives had occurred ca. 200–400 Mya Thus, this brings up the question as to whether ancestral based on their distinct genomes. Almost half of the ter- Rhizobiales and/or Burkholderiales could have been copar- restrial azospirilla genomes appear to have been derived ticipants in the origin of both plant–mycorrhizal associations by horizontal gene transfer from Rhizobiales and other and nitrogen-fxing symbioses. For the Betaproteobacte- Alphaproteobacteria (greatest percentage) and also from ria, the answer is probably yes based on Burkholderia Burkholderiales, suggesting that these groups evolved long symbionts being present within AMF hyphae. Candida- before Azospirillum diverged. Therefore, Rhizobiales and tus Glomeribacteria gigasporarum (CGG) is an obligate Burkholderiales were likely to have been among the bacte- endobacterium that lives surrounded by a membrane within rial lineages that colonized the land prior to the evolution of the cytoplasm of AMF cells, which themselves are obligate the vascular plants, the vast majority of which established a biotrophs and dependent on their plant hosts (Jargeat et al., symbiotic association with AMF (Fig. 89.4). Both fossil and 2004). However, no evidence for nif genes was uncovered molecular evidence date the AMF association with plants to (Jargeat et al., 2004), suggesting that the CGG ancestor have been present ca. 430–350 Mya in the early Devonian was unlikely to be a diazotroph although it is sister to (Simon et al., 1993; Taylor et al., 1995). Because many of the free-living Burkholderia species that fxnitrogenaswell plant genes involved in establishing the AM symbiosis and as to B. rhizoxinica HKI 0454T (Ghignone et al., 2004), the nitrogen-fxing nodule are conserved and also because an endophyte of the phytopathogenic fungus Rhizopus,the the initial microbial signal molecules are chemically related causal agent of rice-seedling blight (Partida-Martinez et al., (see references in Hirsch and Fujishige, 2012; see Chapters 2007). In spite of its small genomes and lack of nif genes, 55, 110), the idea that nitrogen-fxing rhizobia coopted the some 250,000 CGG cells are estimated to live within one 920 Chapter 89 Symbiotic Nitrogen Fixation in Legumes: Perspectives on the Diversity and Evolution fungal cell (Bianciotto et al., 1996) and at least 20,000 per and based on timing of the divergence of Bradyrhizobium spore (Lumini et al., 2007), strongly suggesting that these and Rhizobium and the presence of Burkholderia in AMF microbes provide benefts to their host. Indeed, the bacteria (Fig. 89.4), it is a formal possibility that the two families were found to improve the ftness of Gigasporaceae by were members of the Gondwanan microfora. Thus, it can be promoting presymbiotic hyphal expansion and branching deduced that certain Burkholderia species such as B. tuberum (Lumini et al., 2007). originated when South Africa and South America were still Based on studies of various betaproteobacterial connected, which might explain the disparate localization genomes, the difference in mutation accumulation in of B. tuberum with the mimosoid (South American) and CGG differs from free-living Burkholderia species and papillionoid (South African) legumes after the continents from the Burkholderia endosymbionts of Rhizopus,strongly split. The example of a South American B. tuberum is the suggesting an ancient interaction with the fungus (Castillo strain Burkholderia CCGE1002, which was isolated from and Pawlowska, 2010). Similar to insect symbionts, CGG nodules of Mimosa occidentalis growing in Mexico and has and B. rhizoxinica have reduced genomes, but the fungal characteristics typifying both the South African and South pathogen has a large number of genes associated with vir- American strains (Mishra et al., 2012; Ormeño-Orrillo et al., ulence (Lackner et al., 2011). Moreover, B. rhizoxincia can 2012). Strain CCGE1002 is B. tuberum based on a concate- be grown in culture and re-infect its fungal host (Moebius nated sequence of 16S RNA and four housekeeping genes et al., 2014). In contrast, CGG is unable to synthesize several (Agapakis et al., unpublished), but not if based on nodA or essential amino acids and vitamins, and is totally dependent nifH (Mishra et al., 2012), However, in contrast to South on its host for carbon, nitrogen, and phosphorous (Ghignone African B. tuberum STM678T and B. tuberum related strains et al., 2012). The complex network seen in this tripartite such as B. rhynchosiae, B. dilworthii, and B. sprentiae (De symbiosis strongly suggests a different pattern of molecular Meyer et al., 2013a,b, 2014), CCGE1002 has a nod-nif gene evolution than that observed in the free-living Burkholde- arrangement upon a symbiotic plasmid that is similar to that ria species or B. rhizoxincia (Castillo and Pawlowska, of other Mimosa-nodulating Burkholderia strains except 2010). Whether or not an ancient Burkholderia–AMF–plant it has an integrase gene terminating the operon (Agapakis symbiosis is the ancestor of the nodulation pathway (see et al., unpublished). In addition, the nod and nif genes of Chapters 51, 59, 110) may be diffcult to prove, but it is not the Mimosoideae-nodulating Burkholderia strains are not clear whether any better candidate presents itself. To our closely related to those of the Papillionoideae-nodulating knowledge, the evidence for a Rhizobiales endosymbiont South African B. tuberum strains (Mishra et al., 2012). living in association with AMF cells has not been reported. Bontemps et al. (2010) placed the acquisition of the sym- biotic genes by RNB at about the same time as when the legumes were diversifying. We propose that the presence of 89.7 FINAL COMMENTS AND different legume subfamilies, Mimosoid versus Papillionoid, PERSPECTIVES as well as the infuence of different biogeographies on the two once connected continents facilitated the divergence Strong evidence for distinct Burkholderia populations in of Burkholderia RNB into separate lineages, account- South America and Southern Africa exists, but so far not ing for the Mimosa-nodulating strain CCGE1002 versus much is known about the Indian Burkholderia populations the Papillionoideae-nodulating B. tuberum STM678T and (Fig. 89.3). The Indian Burkholderia RNB may be closely related species (see also Chapter 19). related to RNB isolated from Dalbergia legume nodules in Finally, which of the RNB evolved frst, the alpharhi- Madagascar although this strain was identifed at the time as zobia or betarhizobia? If Burkholderia species inhabited B. cepacia and lacks nod genes (Rasolomampianina et al., fungal cells at the time of the emergence of the vascular 2005). Although Burkholderia strains have been isolated plants, this strongly suggests their ancient origin. The time- from nodules of legumes growing in Australia, so far no nod line implies that Burkholderia may have associated with genes have been detected in these (R. Walker, unpublished). plants some 500 Mya via the AM symbiosis, and in the For China and Europe, no reports of Burkholderia RNB process, recruited the capacity for an intimate interaction from endemic legumes have been described. Clearly more with the plant from AMF via an unknown mechanism. This isolations need to be made. Thus, it appears that South- hypothesis suggests that Burkholderia could have been the ern Africa and South America are the major centers for source of the nodulation genes via lateral transfer of genes Burkholderia evolution (see also Chapter 17). if a nonreduced genome ancestor associated with the fungi. To explain these apparently disparate centers of origin However, an alternative hypothesis, as previously mentioned of the RNB Burkholderiacae,itisimportanttoremem- (Hirsch et al., 2001), is that the original source of these ber that ca. 200 Mya, South America, Africa, India, and genes was outside either Burkholderiaceae or Rhizobiaceae Australia were all part of the single continent Gondwana. because of their lower G + Ccontentcomparedtotherest If Burkholdericeae and Rhizobiaceae date from this time, of the genome. For both groups of RNB, the genome G + C References 921 content is higher overall than that of the nodulation genes. Angus AA, Hirsch, AM. 2010. Perspective: insights into the history of the Potential recruitment of nodB and nodC from other bacteria legume-betaproteobacterial symbiosis. Mol. Ecol. 19: 28–30. or fungi is a distinct possibility, but more elusive is the Ardley JK, Parker MA, De Meyer SE, O’Hara GW, Reeve WG, Yates RJ, et al. 2012. Microvirga lupini sp. nov., Microvirga lotononidis sp. nov., source of the gene-encoding NodA, the protein responsible and Microvirga zambiensis sp. nov. are alphaproteobacterial root nodule for adding a fatty acyl chain onto the resulting free amino bacteria that specifcally nodulate and fxnitrogenwithgeographically group of the nascent Nod factor. and taxonomically separate legume hosts. Int. J. Syst. Evol. Microbiol. Based on the isolations of Burkholderia RNB so far 62: 2579–2588. (Fig. 89.3), it appears that the RNB group is confned to the Beijerinck MW. 1890. Künstiliche infection von Vicia faba mit Bacil- southern hemisphere except for CCGE1002, which was iso- lus radicicola: Ernärungsbedingungen deiser Bacterie. Botan. Ztg. 48: 837–843. lated from nodules in Mexico, as well as some undescribed Beukes CW, Venter SN, Law IJ, Phalane FL, Steenkemp ET. 2013. isolates from China. However, based on fossil data, the South African papilionoid legumes are nodulated by diverse Burkholderia distribution of many legume genera is very different from with unique nodulation and nitrogen-fxation loci. PLoS One 9: e68406. what it was predicted to be 65 Mya. The greatest diversity doi: 10.1371/journal.pone.0068406. at the generic level then and now is in tropical America and Bianciotto V, Bandi C, Minerdi D, Sironi M, Tichy HV, Bonfante Africa/Madagascar (Herendeen et al., 1992). Coincidentally, P. 1996. An obligately endosymbiotic mycorrhizal fungus itself har- bors obligately intracellular bacteria. Appl. Environ. Microbiol. 62: this distribution overlaps with that of the Burkholderia RNB. 3005–3010. Of the North American fossil fora, many extant relatives Bontemps C, Elliott GN, Simon MF, dos Reis FB Jr.,, Gross E, Lawton are now restricted to South America possibly as a result RC, et al. 2010. Burkholderia species are ancient symbionts of legumes. of climatic constraints (Herendeen et al., 1992). A more Mol. Ecol. 19: 44–52. extensive focus on Burkholderia RNB and their hosts is de Bruijn FJ. (2013) Molecular Microbial Ecology of the Rhizosphere. v.I needed to obtain a more complete knowledge of legume and and II, Hoboken, NJ: Wiley-Blackwell Publishers, pp. 1–1328. symbiont biogeography. Burkholder WH. 1942. Three bacterial plant pathogens. Phytomonas caryophylli sp.n., Phytomonas alliicola sp.n. and Phytomonas manihotis Exploring the origins of nodulation provides insight into (Artaud, Berthet and Bondar) Viégas. 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De Meyer SED, Cnockaert M, Ardley JK, Trengove RD, Garau G, Howieson JG, Vandamme P. 2013a. Burkholderia rhynchosiae sp. nov. ACKNOWLEDGMENTS isolated from Rhynchosia ferulifolia root nodules from South Africa. Int. J. Syst. Evol. Microbiol.. 63: 3944–3949. The National Science Foundation, the Sol Leshin Program De Meyer SED, Cnockaert M, Ardley JK, Maker G, Yates R, for Collaboration between the Ben Gurion University of Howieson JG, Vandamme P. 2013b. Burkholderia sprentiae sp. nov. iso- lated from Lebeckia ambigua root nodules from South Africa. Int. J. Syst. the Negev, Israel, and the Shanbrom Family Fund, fund Evol. Microbiol.. 63: 3950–3957. research on plant–microbe interactions in the Hirsch lab. De Meyer SED, Cnockaert M, Ardley JK, Christina M. Agapakis was supported by a L’Oreal USA Van Wyk B-E, Vandamme P. Howieson JG. 2014. Burkholderia Fellowship for Women in Science. An Australian Grains dilworthii sp. nov., isolated from Lebeckia ambigua root nodules. Int. J. Research and Development Council Travel award supported Syst. Evol. Microbiol.. 64: 1090–1095. Robert Walker. Doyle JJ, Luckow MA. 2003. The rest of the iceberg. Legume diversity We thank Dr. Stefan Kirchanski for his helpful com- and evolution in a phylogenetic context. Plant Physiol. 131: 900–910. Dreyfus B, Garcia JL, Gillis M. 1988. Characterization of Azorhizobium ments on the manuscript. We appreciate the insights of Profs. caulinodans gen. nov., sp. nov., a stem-nodulating nitrogen-fxing bac- Janet I. Sprent (Dundee) and J. William Schopf (UCLA). terium isolated from Sesbania rostrata.Int.J.Syst.Bacteriol.38:89–98. Janet motivated us to think about the “big picture” of the Estrada-de los Santos P, Vinuesa P, Martínez-Aguilar L, Hirsch evolution of legumes while Bill inspired us to think about AM, Caballero-Mellado J. 2013. Phylogenetic analysis of Burkholde- the even “bigger picture,” that is, microbial evolution. ria species by Multilocus Sequence Analysis. Curr. 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