Phylogenomic Review of Root Nitrogen-Fixing Symbiont Population Nodulating Northwestern African Wild Legumes

The present review discusses the phylogenomic diversity of root nitrogen-fixing bacteria associated to wild legumes under North African soils. The genus Ensifer is a dominant rhizobium lineage nodulating the majority of the wild legumes, followed by the genus Rhizobium and Mesorhizobium. In addition, to the known rhizobial genera, two new Microvirga and Phyllobacterium genera were described as real nodulating and nitrogen-fixing microsymbiotes from Lupinus spp. The promising rhizobia related to nitrogen fixation efficiency in association with some legumes are shared. Phylogenetic studies are contributing greatly to our knowledge of relationships on both sides of the plant-bacteria nodulation symbiosis. Multiple origins of nodulation (perhaps even within the legume family) appear likely. However, all nodulating flowering plants are more closely related than previously suspected, suggesting that the predisposition to nodulate might have arisen only once. The origins of nodulation, and the extent to which developmental programs are conserved in nodules, remain unclear, but an improved understanding of the relationships between nodulin genes is providing some clues.


Introduction
Africa has a vast array of indigenous legumes, ranging from large rain forest trees to small annual herbs [1]. However, in recent years, there has been a tendency in agriculture and forestry to use exotic species for crops and wood. As has been pointed out several times over nearly 30 years, most recently [2], by the US National Academy of Sciences, this ignores the potential of the native species, which are arguably better adapted to their environment. For this review, the nodulated indigenous legume genera in Northwestern Africa with known uses have been selected to illustrate the problems and potential for their better exploitation.
The wild legume flora in Northwestern Africa is rich, with great specific and infraspecific diversity [3]. The overgrazing and expansion of agriculture has gradually

Genetics and functional genomics of legume nodulation
The interaction between rhizobia and legumes in root nodules is an essential element in sustainable agriculture, as this symbiotic association is able to enhance biological fixation of atmospheric nitrogen (N 2 ) and is also a paradigm in plant-microbe signaling [14][15][16]. The knowledge of the whole genome would allow the specific features of each rhizobium to be identified. The prominent feature of this group of bacteria is their molecular dialog with plant hosts, an interaction that is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant [17,18]. In recent years, significant progress has been made in resolving the complex exchange of signals responsible for nodulation through genome assembly, mutational and expression analysis, and proteome characterization of legumes [14,19,20] and rhizobia [15, [21][22][23].

Phylogenomic of wild legume root nitrogen-fixing symbionts
The known diversity of rhizobia increases annually and is the subject of several reviews, the most recent and comprehensive being that of [24]. It is not our intention to revisit this subject nor the genetic basis of nodulation [25,26], the horizontal transfer of symbiosis-related genes [23], or the symbiovar concept [27] but instead to attempt to link, where possible, rhizobial genotypes with their geographical locations and/or legume tribes/genera. At the time of writing, rhizobia consist of a diverse range of genera in the Alphaproteobacterial and Betaproteobacterial classes and are termed "alpha-rhizobia" and "beta-rhizobia," respectively ( Figure 1).

The genus Bradyrhizobium (Bradyrhizobiaceae)
The Bradyrhizobium genus was described by Jordan in 1982 [29]. It currently consists of nine rhizobia species.
For the Loteae tribe, previous studies found that Lotus palustris and L. purpureus species from Algeria were nodulated by Bradyrhizobium lupini, and L. pedunculatus by B. japonicum [30]. However, L. creticus ssp. maritimus is nodulated by both [30]. At Tunisia, L. roudairei microsymbiont is closely related to B. japonicum [31]. For the Acacieae tribe, two studies reported that rhizobial strains associated to the Acacia saligna, an Australian introduced species, belonged to the genus Bradyrhizobium genus under Algerian and Moroccan soils [32][33][34]. For the Genisteae tribe, it has been noticed that Bradyrhizobium is the dominant genus of Phylogenetic tree showing the relationships of currently described genera and species of alpha-and betarhizobia, based on aligned sequences of the 16S rRNA gene (1341-bp internal region) (adapted from [28]). symbiotic nitrogen-fixing bacteria associated with Retama species in North Africa: Retama monosperma, R. raetam, and R. sphaerocarpa (Algeria: [35,36]; Morocco: [37]). Recently, the novel B. retamae species, in which groups with B. elkanii and B. pachyrhizi and related B. lablabi and B. jicamae type strains are included in Bradyrhizobium group II [38], has been isolated from R. sphaerocarpa and R. monosperma in Morocco [37]. For the genus Cytisus, two studies reported that Cytisus villosus is nodulated by B. cytisi sp. nov. and B. rifense sp. nov. in Morocco [39,40] and by genetically diverse Bradyrhizobium strains in Algeria belonging to B. japonicum and B. canariense and to new lineage within the Bradyrhizobium genus [41]. Fiftytwo strains isolated from root nodules of the Moroccan shrubby legume Cytisus triflorus were genetically characterized, and results showed that it is nodulated by Bradyrhizobium strains, with 99% homology with Bradyrhizobium genosp. AD [42].  [43][44][45].

The genus Mesorhizobium (Phyllobactericeae)
The genus Mesorhizobium was described by Jarvis et al. [46]. Several Rhizobium species were transferred to this genus. It currently consists of 21 rhizobia species.
For subtribe Astragalinae (Coluteinae Clade), Guerrouj et al. [37] reported that rhizobial symbiont of Astragalus gombiformis in Eastern Morocco is closely related to M. camelthorni. A polyphasic approach analysis indicated that bacterial strains isolated from the pasture legume Biserrula pelecinus growing in Morocco belong to the genus Mesorhizobium. At Tunisia, Mahdhi et al. [47] showed that five strains isolated from Astragalus corrugatus were phylogenetically related to M. temperatum and to Mesorhizobium sp. From the tribe Galegeae (subtribe Coluteinae), Ourarhi et al. [48] reported that Colutea arborescens is nodulated by diverse rhizobia in Eastern Morocco, among them, the genus Mesorhizobium. For the Loteae tribe, M. alhagi as well as M. temperatum were isolated, at Tunisia, from Lotus creticus [49][50][51]. Zakhia et al. [31] reported that Lotus argenteus microsymbiotes are closely related to M. mediterraneum in the infra-arid zone of Tunisia. Roba et al. [52] reported that M. delmotii and M. prunaredense are two new rhizobial species nodulating Anthyllis vulneraria growing on Tunisian soils. From the Acacieae tribe, Boukhatem et al. [33] reported that rhizobial strains associated to the Acacia saligna, an Australian introduced species, to A. ehrenbergiana and F. albida belonged to M. mediterraneum under Algerian soils. From the Genisteae tribe, the genetic diversity of Genista saharae microsymbionts in the Algerian Sahara reported that they belonged to M. camelthorni [53]. For the Mimoseae tribe, root-nodulating bacteria associated to Prosopis farcta growing in the arid regions of Tunisia were assigned to the genus Mesorhizobium [54]. From the Hedysareae tribe, Zakhia et al. [31] reported that one strain isolated from Ebenus pinnata root nodules is closely related to M. ciceri in the infra-arid zone of Tunisia.

The genus Rhizobium (Rhizobiaceae)
The genus Rhizobium was the first named (from Latin meaning "root living"), and for many years this was a "catch all" genus for all rhizobia. Some species were later moved in to new genera based on phylogenetic analyses [55]. It currently consists of 49 rhizobial species.
For Galegae tribe, Zakhia et al. [31] reported that rhizobial symbionts of Astragalus gombiformis, A. armatus, and A. cruciatus are closely related to Rhizobium mongolense, R. leguminosarum, and R. galegae, in the infra-arid zone of Tunisia. From Genisteae tribe, it was shown that strains from Tunisia nodulating Argyrolobium uniflorum are closely affiliated to R. giardinii, Calicotome villosa to R. mongolense, and Genista microcephala to R. mongolense and R. leguminosarum [31]. Mahdi et al. [56][57][58] reported that strains nodulating Genista saharae and Retama retam are members of the genus Rhizobium. Nonetheless, there are reports indicating that members of the genus Rhizobium nodulate Adenocarpus decorticans and Cytisus arboreus at Morocco [59]. For the Loteae tribe, R. leguminosarum and R. mongolense were isolated, at Tunisia, from Anthyllis henoniana, R. leguminosarum from Coronilla scorpioides, and R. mongolense from Lotus creticus [31]. Rejili et al. [51] reported that Lotus creticus microsymbiotes are closely related to R. huautlense in the arid areas of Tunisia. Bacterial strains isolated from root nodules of Scorpiurus muricatus sampled from different regions of western Algeria are affiliated to R. vignae, R. radiobacter, and R. leguminosarum [60]. For the Trifolieae tribe, R. galegae species was isolated, in Tunisia and Algeria, from Medicago marima and M. truncatula [31]. In Algeria, Merabet et al. [61] reported that Medicago ciliaris and M. polymorpha are nodulated by Rhizobium sp. Similarly, genetic diversity of rhizobia from annual Medicago orbicularis showed that they are affiliated to Rhizobium tropici [62]. For the Vicieae tribe, R. leguminosarum species was isolated from Lathyrus numidicus [31]. Mahdhi et al. [63] reported that Vicia sativa isolates from Tunisia had 16S rDNA type identical to that of the reference R. leguminosarum. From Acacieae, Boukhatem et al. [33] reported that bacteria-nodulating Acacia saligna and A. seyal under Algerian soils are affiliated to the R. tropici clade and R. sullae clade, respectively. On the other hand, the same study mentioned that five bacterial isolates, all from A. saligna, formed a separate clade in the vicinity of the R. galegae-R. huautlense-R. loessense branch [33]. The same authors showed that the R. leguminosarum reference strain was represented by five A. karroo isolates and five A. seyal isolates [33]. At Tunisia, the genetic diversity of root nodule bacteria associated to Hedysarum coronarium (sulla), from Hedysareae tribe, showed that they are closely related to R. sullae [64]. Similarly, Ezzakkioui et al. [65] indicated that the strains from the Moroccan Hedysarum flexuosum legume had 99.75-100% identity with R. sullae.

The genus Ensifer (Sinorhizobium) (Rhizobiaceae)
The genera Sinorhizobium and Ensifer were recently recognized as forming a single phylogenetic clade [66,67] and are now united, and all species of the genus Sinorhizobium have been transferred to the genus Ensifer, in line with rule 38 of the Bacteriological Code [68,69] and Trigonella maritima are nodulated by E. meliloti [31,63]. Nodule rhizobia of Melilotus indicus growing in the Algerian Sahara are affiliated to E. meliloti [78]. E. meliloti and E. numidicus strains were isolated from the Genisteae tribe such as Argyrolobium uniflorum, Retama raetam, and Genista saharae [56][57][58]69]. For Galegae tribe, Mahdhi et al. [47] reported that rhizobial symbionts of Astragalus corrugatus are closely related to E. meliloti under Tunisian soils. From the Hedysareae tribe, Mahdhi et al. [35] reported that strains isolated from Hedysarum spinosissimum root nodules are closely related to E. meliloti in the infra-arid zone of Tunisia.

The genus Neorhizobium (Rhizobiaceae)
The genus Neorhizobium was proposed by Mousavi et al. [80] as an alternative to solve the issue of grouping the members of this genus with Agrobacterium and Rhizobium genera. The genetic diversity of the Algerian legume Genista saharae isolates was assessed, and results reported that they are affiliated to Neorhizobium alkalisoli, N. galegae, and N. huautlense [53]. Several studies reported that N. galegae is isolated from different legumes in Tunisia such as Astragalus sp. [31,54], Argyrolobium uniflorum [31], Anthyllis henoniana [31], Lotus creticus [31,50], Medicago marima, and M. truncatula [31]. Rejili et al. [51] reported that Lotus creticus is also nodulated by N. huautlense in the arid areas of Tunisia. For Galegae tribe, Mahdhi et al. [47] reported that rhizobial symbionts of Astragalus corrugatus are closely related to N. galegae under Tunisian soils.

The genus Phyllobacterium (Phyllobactericeae)
The Phyllobacterium genus comprises of bacteria that are well-known for their epiphytic and endophytic associations with plants [81]. Nonetheless, rootnodulating and nitrogen-fixing Phyllobacterium was described in Tunisia, in the nodules of genistoid legume Lupinus micranthus [44,45]. Prior to this finding, endophytic Phyllobacterium strains were identified on the nodules of the Tunisian legumes Genista saharae, Lotus creticus, and L. pusillus [51,57], but they are lacking the ability to form nodules.

The genus Microvirga (Methylobacteriaceae)
The genus Microvirga which comprises soil and water saprophytes was included in the alphaproteobacterial lineage of root-nodule bacteria only in 2012, although the first symbiotic strains were detected in nodules of Lupinus texensis [82][83][84]. Recently, Microvirga strains were only isolated from L. micranthus and L. luteus in Tunisia, belonging to the Genisteae tribe [44,45]. Table 1 shows the root nodule symbionts from Northwestern African wild legumes.

Promising nitrogen-fixing rhizobia
The root nodule symbiosis established between legumes and rhizobia is an exquisite biological interaction responsible for fixing a significant amount of nitrogen in terrestrial ecosystems. The success of this interaction depends on the recognition of the right partner by the plant within the richest microbial ecosystems on Earth, the soil. Recent metagenomic studies of the soil biome have revealed its complexity, which includes microorganisms that affect plant fitness and growth in a beneficial, harmful, or neutral manner. In this complex scenario, understanding the molecular mechanisms by which legumes recognize and discriminate rhizobia from pathogens, but also between distinct rhizobia species and strains that differ in their symbiotic performance, is a considerable challenge.
By symbiotic efficiency and properties, strains isolated from wild legumes varied in their symbiosis effectiveness with their host plant of origin. A great diversity among and within isolates was reported by many authors. This symbiotic diversity within and between isolates growing in diverse geographical areas was also defined by Tinick and Hadobas [85] for other legume plants. All strains were capable of nodulation. Mahdhi et al. [55,86] reported that two Retama raetam isolates RB3 and RM4 (Rhizobium) gave the highest nodule numbers per plant, 26 (±2.053) and 27 (±0.997), respectively. The effective strain LAC765 (Ensifer) was isolated from Lotus creticus with a 91.46 (±0.01%) dry biomass of the T N control [50]. The dry matter of the aerial part is considered a criterion for assessing the efficiency of a given strain; a highly significant correlation between these two parameters has been reported. Results related to symbiotic efficiency showed that among 45 tested isolates, 20 isolates are highly efficient (relative effectiveness ≥70%), 20 isolates are partially effective (60% ≤ relative effectiveness <70%), and 5 isolates are inefficient (relative effectiveness <60%). The strain GN29 isolated from Genista saharae, affiliated to Rhizobium genus, is considered inefficient (relative effectiveness = 32.29%). Among the 20 isolates considered highly efficient, 5 isolates were isolated from Retama retam, five from Lotus sp., 4 from Genista saharae, 3 from Vicia sativa, 2 from Argyrolobium uniflorum and 2 from Trigonella maritima. From the 20 highly efficient isolates, 13 isolates belong taxonomically to Ensifer sp., 6 to Rhizobium sp., and one to Mesorhizobium sp.

Conclusion
The Mediterranean basin is a hotspot place of legume diversity and the center of diversification of many of them. Our review contributes to enlarge our knowledge on the LNB-legume symbioses. We evidenced the biodiversity among bacterianodulating wild legumes in Northwestern Africa and unknown associations were found. Several groups may represent new genospecies to be further characterized to assess their taxonomical status. This work thus opens further interesting perspectives and makes new models available for evolutionary studies and for understanding mechanisms involved in nitrogen-fixing symbiosis.