The nodA sequences of the Lotononis s. l. rhizobia are polyphyletic, forming
four separate clades in which the nodA lineages are associated with the rhizobial
tree is not well supported. The nodA sequences of the Lotononis s. l. rhizobia are
intermingled with nodA of rhizobia isolated from host plants that are not closely
related to Lotononis s. l. (Figure 2.11). This indicates firstly, that the symbiotic genes
of Lotononis s. l. rhizobia were derived from different sources or have evolved
divergently and secondly, that those Leobordea and Lotononis s. str. hosts which are
promiscuous nodulators are not stringently selecting the rhizobial symbiotic genotype.
The nodA sequences of the methylobacterial strains WSM2598 and WSM2667
are related to nodA of Methylobacterium sp. 4-46 (isolated from L. bainesii) and M.
nodulans ORS 2060 (which specifically nodulates Crotalaria podocarpa, Crotalaria
glaucoides and Crotalaria perrottetti (Renier et al., 2008; Sy et al., 2001)). Both
WSM2667 and ORS 2060 are unable to nodulate L. bainesii and L. heterophylla,
although they do form occasional ineffective nodules on L. angolensis (this study).
The nodA and nod box sequences of the Methylobacterium rhizobia (4-46,
WSM2598, ORS 2060 and WSM2667) suggest that while there is a close phylogenetic relationship between the nodulation genes of M. nodulans and the
Listia methylobacteria, the branch depth indicates their divergence occurred some
time ago. It has also been suggested that the nodulation genes of the
Methylobacterium rhizobia have been acquired by horizontal gene transfer (HGT)
from Bradyrhizobium strains (Moulin et al., 2004). Their considerable divergence
again suggests an ancient origin for this putative HGT event. In this study, the nodA
sequences of the Methylobacterium strains appear to be more closely related to the
The ORS 2060 nod genes are organised in a single operon that encodes NodA,
NodB, NodC, NodI, NodJ and NodH proteins and is almost identical to the nod
operon found in the sequenced genome of Methylobacterium sp. 4-46 (Renier et al.,
2008). It can be hypothesised from this that Listia and Crotalaria hosts that are
nodulated by methylobacteria have similar Nod factor requirements. Crotalaria
species are postulated to belong to two inoculation groups: most are nodulated by bradyrhizobia that may produce fucosylated Nod factors, while C. podocarpa, C.
glaucoides and C. perrottetti are specifically nodulated by M. nodulans, which
produces sulfated Nod factors (Renier et al., 2008) The Methylobacterium nodA
sequences are in a sister clade to those of several Burkholderia tuberum strains that
nodulate South African fynbos legumes (Elliott et al., 2007; Garau et al., 2009).
Interestingly, Burkholderia tuberum STM678 (previously named as Bradyrhizobium
aspalati; see Elliott et al. (2007)) does not produce sulfated Nod factors (Boone et
al., 1999).
The nodA sequences of the Bradyrhizobium, Ensifer and Microvirga strains do
not group with rhizobia that nodulate other crotalarioid legumes. This may, however, be more a reflection of the current lack of data on rhizobia associated with this group of host plants. The nodA sequences of the E. meliloti strains WSM2653 and
WSM3040 are clearly grouped with diverse Ensifer and Mesorhizobium strains that
form a nodulation group for Acacia, Prosopis and Leucaena (Ba et al., 2002). The
nodA lineages of the bradyrhizobial isolates WSM2632 and WSM2783 are grouped
apart from the Ensifer strains. Interestingly, they are also well separated from the
clade containing all other Bradyrhizobium sequences, including ORS 1816, isolated
found that the nodA phylogeny of African bradyrhizobia placed them in the large,
pan-tropical Clade III (Steenkamp et al., 2008). Moulin et al. (2004) considered
bradyrhizobia to form a monophyletic branch within the nodA tree and their deduced
NodA proteins contain 209–211 amino acids (with the exception of the
photosynthetic stem-nodulating Bradyrhizobium strains, which encode 197 amino-
acid proteins). The published Methylobacterium strains 4-46 and ORS 2060, and
WSM2598 and WSM2667 from this study, along with Burkholderia tuberum strain
STM678 also contain deduced NodA proteins of over 200 residues. In contrast, NodA in other rhizobial genera is usually composed of 195–198 amino acids. The
greater length of the bradyrhizobial NodA is the result of a 12–13 amino acid
segment at the N-terminal end (Moulin et al., 2004). The primers used in this study
did not amplify this section of nodA, but it would be interesting to obtain the
complete nodA sequences for WSM2632 and WSM2783, to determine whether, as
the phylogenetic tree suggests, they are more closely related to Ensifer and
Mesorhizobium nodA, rather than other bradyrhizobial strains.
The position of the Microvirga strain WSM3557 in the nodA phylogenetic tree
is intriguing. It forms a sister clade to the bradyrhizobial isolates WSM2632 and WSM2783and is separate from the only other nodulating Microvirga nodA sequence
described to date, that of Lut6, which specifically nodulates Lupinus texensis, a
species endemic to Texas, USA (Andam & Parker, 2007). Notably, WSM3557 is also separate from the nodA lineages of the Methylobacterium strains that
specifically nodulate the remaining Listia species. Microvirga has not previously
symbiotic genes that confer the ability to nodulate Listia angolensis have been
acquired from another rhizobial lineage via HGT, but due to the low bootstrap values for the higher branches of the nodA tree, the source of the donor and the phylogeny
of these nod genes are unclear.
Although the nodA gene is a host range determinant (Debellé et al., 1996;
Lortet et al., 1996), the congruence between nodA phylogeny and the taxonomy of
the legume host is stronger in some associations than others. Host plants in the
Galegeae, Trifolieae and Vicieae tribes (the galegoid clade) are nodulated by diverse
rhizobia with similar nodA sequences; which is related to the requirement of these
legumes for Nod factors that are N-acylated with unsaturated fatty acids (Debellé et
al., 2001; Suominen et al., 2001). Several studies of other legume species that are
nodulated by phylogenetically diverse rhizobia have also found the nod genes to be
highly similar, regardless of rhizobial chromosomal background (Ba et al., 2002;
Haukka et al., 1998; Laguerre et al., 2001; Lu et al., 2009). Conversely, Han et al.
(2010) and Zhao et al. (2010), in studies of rhizobia isolated from wild legumes in
China, concluded that the relations between the microsymbiont chromosomal background, nod genes and host plant were promiscuous and the nodA or nodC
lineages were clearly associated with the rhizobial genomic background. Canary Island Lotus species also have Ensifer meliloti and Mesorhizobium microsymbionts
in which distinct nodC lineages are related to the different chromosomal genotypes,
although in this case the E. meliloti strains have a restricted host range (Lorite et al.,
Symbiotic relationships between the Lotononis s. l. legumes and their
associated rhizobia appear to follow the latter model, where nodulation gene lineages are related to the microsymbiont chromosomal background. It is not possible, from the data on nodA presented in this study, to trace the lines of descent of the symbiotic
genes that allow nodulation of Lotononis s. l. hosts, or say whether they came from a
single, or multiple, ancestors. There appears to be no evidence for a “Lotononis s. l.”
or a “Listia” nodulation genotype as such, although the very high nodA sequence
identity seen within, rather than between, the Lotononis s. l.-associated
Bradyrhizobium, Ensifer, M. nodulans and pigmented Methylobacterium strains
argues for selection pressure on nod genes and possible HGT within those
chromosomal backgrounds. The collection and study of more rhizobial isolates from
Leobordia and Lotononis s. l. species and the acquisition of complete nodA
sequences for all Lotononis s. l. rhizobia could provide more robust phylogenies of
symbiotic loci and a greater understanding of the evolution of symbiotic relationships within Lotononis s. l. species.
If there is no evidence for selection pressure on Lotononis s. l.-associated
rhizobial for a particular nodulation genotype, what other factors could account for the range of microsymbiont diversity and specificity seen in this group of host plants?
2.4.5 Environmental factors that may be effectors of diversification in