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Phylogenetic perspectives on
nodulation: evolving views of plants
and symbiotic bacteria Jeff J. Doyle
Phylogenetic studies are contributing greatly to our knowledge of relationships on both sides of
the plantÐbacteria 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. Phylogenies of 16S rRNA genes highlight the evolutionary diversity of symbiotic bacteria and appear to rule out any broad coevolution with their plant hosts, but high
levels of gene transfer might obscure the relevant pattern. 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.

N

odulation – the symbiotic partnership between flowering
plants and soil bacteria – is an ecologically and economically important plant phenomenon. From an economic
viewpoint, the ability of some plant groups to use atmospheric
nitrogen, obviating the need for massive inputs of fertilizer, is a

significant and desirable trait. A wealth of questions – ecological,
developmental and molecular – are presented by nodulation. The
process begins with signal exchange between bacteria and their
plant hosts and leads to the production of a unique organ, the nodule, which contains novel components that are not found in either
free-living bacteria or uninfected plants. From an evolutionary
perspective, the factors leading to acquisition of this novel organ
– a process that has apparently occurred several times independently – are particularly fascinating. Phylogenetic studies have been
undertaken with the aim of understanding relationships in both
plants and bacteria, and of elucidating the evolution of individual
genes and gene families within each group.

possibility that a predisposition for forming a nodulation symbiosis with rhizobial or actinorhizal bacteria has originated only
once, in all of the hundreds of thousands of flowering plant
species6. The basis for such a predisposition remains a matter of
speculation, with possible factors including unique classes of secondary metabolites or phytoalexins; specific pathogens or pathogen resistances; novel receptor molecules or hormone sensitivities;
and unique physiological traits, such as nitrogen uptake or carbon
metabolism4.
‘Predisposition’ and ‘origin’ are two quite different concepts,
however. Within this ‘nitrogen-fixing clade’, nodulating genera
are a small minority, and are scattered among non-nodulators, often

even within the same family. This observation, along with the differences seen in nodule structure between legumes and actinorhizal plants, and even among the actinorhizal families, suggests
that nodulation has originated many times independently5.
The origin(s) and evolution of nodulation in Leguminosae

Plant phylogenies: a predisposition to nodulate?

Nitrogen-fixing symbioses are best known from associations between the legume family (Leguminosae or Fabaceae) and diverse
Gram-negative soil bacteria (such as Rhizobium or Bradyrhizobium; collectively ‘rhizobia’), in which nodules are produced on
plant roots or, in some cases, on stems. However, nitrogen-fixing
symbioses in several other plant groups are also known. Parasponia, in the elm family (Ulmaceae), is the only one that is nodulated by rhizobia. Cyanobacteria (for example, Anabaena and
Nostoc) form symbioses with plants as diverse as the water fern
Azolla, cycads and the flowering plant Gunnera (Gunneraceae)1,2,
and may even contribute to the nitrogen economy of cultivated
grasses3. The Gram-positive actinomycete Frankia nodulates nearly
200 species of flowering plant belonging to eight families (Betulaceae, Casuarinaceae, Coriariaceae, Datiscaceae, Elaeagnaceae,
Myricaceae, Rhamnaceae and Rosaceae); taken together, actinorhizal symbioses fix about as much nitrogen as the legume–rhizobia
symbioses4,5.
Flowering plant families involved in these symbioses have traditionally been classified in different higher groupings, suggesting
that symbioses evolved independently in diverse plant lineages.
However, results from molecular phylogenetic studies have

necessitated a dramatic revision of this view. Studies based on the
chloroplast gene rbcL suggest that the flowering plant families
involved in rhizobial or actinorhizal symbioses belong to the same
large lineage5,6 (Fig. 1). This is interesting from a taxonomic perspective, but what is more provocative about these results is the

Although as a group, legumes are well known for their nodulation
symbiosis, not all genera nodulate. Most members of the subfamilies Papilionoideae (which includes the typical ‘beans’) and
Mimosoideae (which includes acacias and mimosas) appear to be
capable of nodulating. By contrast, very few members of the third
subfamily, Caesalpinioideae are known to nodulate7. Based on the
observation that rhizobia can infect roots of non-nodulating legumes, and that such plants show nitrogenase activity, it has been
suggested that all legumes are capable of rhizobial symbiosis,
although not all can form nodules8. This speculative hypothesis
could explain the high nitrogen content of the non-nodulating legumes9, but it leaves unanswered the question of the origin of nodules. A recent phylogenetic study, again using rbcL sequences,
supports the long-standing hypothesis that the subfamily Caesalpinioideae is a collection of diverse elements that includes the
earliest-diverging lineages of the Leguminosae, and that most of
the nodulating members of this subfamily belong to a lineage that
includes Mimosoideae10 (Fig. 2). A simple parsimonious interpretation of the distribution of nodulation on the rbcL tree suggests as
many as three independent origins of the syndrome: once in the
mimosoid lineage, once in the only remaining confirmed caesalpinoid nodulator, Chamaecrista, and a third time near the base of

the Papilionoideae.
This hypothesis may be parsimonious, but is a phenomenon as
complex as nodulation likely to have arisen independently so
many times? Loss of nodules might be expected to occur more
easily than gain, making hypotheses involving many independent

1360 - 1385/98/$ Ð see front matter © 1998 Elsevier Science. All rights reserved. PII: S1360-1385(98)01340-5

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(b)

(a)

Asterid I–V

Rosid I
Rosid II
Rosid III
Caryophyllids
Gunnera
Hamamelid II
Hamamelid I
Paleoherb I
Magnoliids
Monocots
Lauraels
Paleoherb I

Malphigiaceae
Ochnaceae
Humiriaceae
Chrysobalanaceae
Trigoniaceae
Euphorbiaceae
Erythroxylaceae

Violaceae
Linaceae
Passifloraceae
Saxifragaceae
Celastraceae
Eucryphiaceae
Saxifragaceae
Cephalotaceae
Tremandraceae
Oxalidaceae
Rosaceae (5/100)
Moraceae
Cannabaceae
Ulmaceae (1/18)
Urticaceae
Elaeagnaceae (3/3)
Rhamnaceae (7/55)
Leguminosae
Surianaceae (Stylobasium)
Polygalaceae

Rosaceae (Quillaja)
Betulaceae (1/6)
Casuarinaceae (4/4)
Fagaceae
Myricaceae (2/3)
Juglandaceae
Nothofagaceae
Coriariaceae (1/1)
Tetrameleaceae
Datiscaceae (1/3)
Cucurbitaceae
Begoniaceae

(c)
Asteridae
Rosidae
Hamamelidae
Caryophyllidae
Dilleniidae
Magnoliidae

Monocots

Other 'Rosid I' dicots

Fig. 1. Phylogeny of nodulating flowering plants. (a,b) Phylogenies based on rbcL sequence data6. Families that include nodulating genera are
shown in bold, followed in parentheses by the number of nodulating genera and the total number of genera in the family. Details of relationships in exclusively non-nodulating lineages are not shown. (c) A traditional classification scheme for flowering plants. In the rbcL sequence
phylogenies, all nodulating angiosperms and all symbiotically nitrogen-fixing angiosperms except Gunnera belong to a single extensive lineage,
the Rosid I group, whereas in the more traditional scheme families involved in nodulation are scattered throughout most of the major subclasses.
The unexpectedly close relationship revealed by rbcL suggests a predisposition to nodulation in this group of families6. However, the scattered
distribution of families and genera within the Rosid I lineage, along with structural differences in their nodules, is consistent with multiple origins
of symbioses within this group5.

losses more plausible than those invoking even a small number of
independent origins. Of course, the precedent already exists for
multiple origins, in the phylogenetically diverse actinorhizal symbioses: as already noted, actinorhizal nodules in different families are structurally diverse, as would be expected if nodules had
different origins. Legume nodules are also developmentally and
biochemically diverse, although they share many features that distinguish them from actinorhizal nodules and even from the nodules
of Parasponia. A phylogenetic assessment of the major nodule
types in the Leguminosae10 revealed that the unbranched, indeterminate ‘caesalpinioid’ nodule type is scattered throughout the family and, notably, is present in Mimosoideae, Caesalpinioideae and
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December 1998, Vol. 3, No. 12

basal Papilionoideae (Fig. 2). This distribution is consistent with a
single origin of nodulation in the family, with the caesalpinioid
nodule type evolving in the common ancestor of all nodulating
legumes.
Within Papilionoideae this basic caesalpinioid nodule type has
been modified dramatically to produce the determinate ‘desmodioid’ nodules of many Phaseoleae, such as soybean (Glycine max)
and the common bean (Phaseolus vulgaris), and the small, clustered ‘aeschynomenoid’ nodules found in the peanut (Arachis
hypogaea) and its allies. Biochemical innovations have also occurred within the subfamily, and appear to be correlated with nodule type. For example, in the determinate nodules of Phaseoleae,

trends in plant science
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nitrogen transport involves ureide compounds instead of the amides used by members of other tribes, most of which have
indeterminate nodules. Such differences
may also extend to the linkage relationships of genes involved in nodulation, although data are as yet scarce. In pea, a
number of such genes are linked11, but this
is not the case in soybean12. More precise
phylogenetic hypotheses for a wider sample of papilionoid legumes, coupled with

more detailed surveys of nodule structure
and chemistry, are needed to identify the
points at which evolutionary modifications
occurred. Phylogenetic hypotheses are already being produced for some groups of
papilionoid legumes, for example the temperate herbaceous clade that includes pea
and alfalfa13, and the Millettieae, a tribe
with many connections to Phaseoleae10,14.
Developmental conservation and
origins of legume nodules

1

Galegeae – Galega
Trifolieae – Trifolium, Melilotus, Medicago
Vicieae – Pisum, Vicia, Lens, Lathyrus
Cicereae – Cicer
Hedysareae
Carmichaelieae
Millettieae-2 – Wisteria
Loteae – Lotus, Anthyllis, Coronilla

Robinieae – Robinia, Sesbania
Phaseoleae
Phaseolus, Vigna, Lablab
Psoraleae
Glycine, Cajanus, Canavalia
Desmodieae
Millettieae-1
Indigofereae – Indigofera, Cyamopsis
Bossiaeae, Mirbelieae
Sophoreae-3
Aeschynomeneae – Arachis
Adesmieae
Dalbergieae – Dalbergia
Amorpheae
Genisteae – Genista, Lupinus
Crotalarieae, Podalyrieae
Sophoreae-2, Thermopsideae
Euchresteae
Sophoreae-2
Brongniartieae
Sophoreae-2, Swartzieae
Dipterygeae
Caesalpinieae-1
Cassieae-4 – Ceratonia
Caesalpinieae-2
MIMOSOIDEAE – Leucaena, Acacia
Cassieae-3 – Senna
Cassieae-2 – Chamaecrista
Detarieae, Macrolobieae
Cercideae – Cercis, Bauhinia
Cassieae-1 – Dialium

Regardless of whether or not nodules arose
one or more times in legumes, a key
question is, how did they evolve? Could
features of nitrogen-fixing symbiosis be
derived from other, older symbioses, such
as those involving endomycorrhizae15,16?
Both symbioses involve some induction
of the host defense response15, and recent
2
studies suggest that both phenomena share
a common signal transduction pathway17.
Mutualistic symbioses of both types could
3
have arisen from still earlier pathogenic
interactions15.
To what other structures are nodules homologous in a developmental sense? This is
an interesting question regardless of whether
or not legume nodules all have a single
Fig. 2. Phylogeny of Leguminosae, as hypothesized from rbcL sequence data10. All groups
origin (are homologous in a phylogenetic
above the broken line belong to the largest subfamily, Papilionoideae; below the line are
sense), or of the relationship between noduCaesalpinioideae and Mimosoideae. Tribal names are used, sometimes several times where
lation and other symbioses, and is irrespectribes consist of unrelated lineages (e.g. Caesalpinieae and Sophoreae). Important or characteristic genera are listed for some tribes; those shown in bold also appear in Fig. 4. Groups
tive of the ability of non-nodulating legumes
in boxes are not known to nodulate. Possible independent origins of nodulation, as sugto derive benefit from rhizobia8. However,
gested by parsimonious optimization of this trait, are shown as numbered vertical bars along
it is not a simple question, and we lack
particular branches. Drawings of the major nodule types appear next to some of the groups
sufficient information to answer it satisfacin which they occur. Unbranched, elongate, indeterminate nodules are ‘caesalpinioid’;
15
torily . By analogy, even if the flower is
branched, indeterminate nodules are ‘astragaloid’; large, round, determinate nodules are
considered to be a condensed shoot bearing
‘desmodioid’; small, clustered nodules are ‘aeschynomenoid’.
modified leaves, and is thus homologous to
the shoot, it is still a novel organ. Knowledge of this developmental homology does
not explain the evolution of flowers. The legume nodule, although
The presence of nodule-specific proteins, called nodulins, was
commonly produced on roots by rhizobial infection of root hairs, also taken as evidence that the evolutionary novelty of nodules
has a stem-like anatomy, in contrast to the anatomically more extended to the molecular level. However, it is likely that most
root-like nodules of actinorhizal plants18. The legume nodule is nodulin genes are also expressed elsewhere in the plant and have
clearly not a stem, however, regardless of whether it might have been recruited from other functions20. For example, uricase II was
been formed by recruitment of developmental programs normally thought to be expressed only in the nodules of soybean, but is now
associated with stems. Nodules contain what has been termed a known to be expressed in cotyledons too21. Thus, one challenge in
‘novel organelle’19, the symbiosome. This organelle consists of understanding the evolution of nodulation is to determine the
bacteroids (rhizobia in their modified, symbiotic phase) enclosed ancestral expression patterns of recruited nodulin genes. Data on
in a host peribacteroid membrane through which carbon and other the expression of early nodulin (ENOD) genes in mycorrhizal
nutrients are passed to the bacteria, and nitrogenous compounds symbioses17 are clearly of great interest, as are studies of genes
are transferred to the host plant.
essential for, and uniquely associated with, nodulation, but even
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tree is very complex, and it is clear that
there is still much to be learned about this
gene family. The observation of marked
phylogenetic diversity of globin genes of
the non-legume Casuarina led to the discovery of a highly diverged non-symbiotic
globin gene in soybean25. Even when this
ancient paralogue is ignored, the gene tree
for symbiotic globins of legumes is comAGL17
plicated by what appear to be varying levels of concerted evolution. This has led to
homogenization in some genera, such as
soybean and lupin, but cross-species retention of duplicate (orthologous and paralogous) relationships in genera such as alfalfa
(Medicago) and broadbean (Vicia)22. In the
latter genus, the most distantly related
member of the gene family was also found
AP3
to be induced in mycorrhizal symbiosis26.
Given the possible connections between
the two symbioses, it will be interesting to
see whether orthologues (members of the
same subfamily, divergent because of speciation rather than duplication) of this gene
can be identified in other legumes, and
whether these or other members of the
symbiotic clade of leghemoglobin genes in
Fig. 3. Phylogeny of the MADS-box transcription factor gene family. The basic topology is
other legume genera are also expressed in
from Ref. 27; nmhC5 (Ref. 28) and DEFH125 (Ref. 30) were placed in the AGL17 subfammycorrhizal associations.
ily, but have not been included together in a comprehensive phylogenetic analysis and are
A critical step in understanding the molshown as unresolved. Genes expressed in Medicago nodules belong to two subfamilies; genecular basis of development in any tissue
era from which other genes of these subfamilies were isolated are shown. One of these subor organ is the identification of specific
families (AP3) consists of genes expressed in floral organs, whereas each of the genes of the
other subfamily (AGL17) has a different expression pattern.
transcription factors. Among the most intensely studied plant transcription factors
are the members of the large MADS-box
the expression patterns of more prosaic ‘housekeeping’ genes gene family, best known for their role in floral morphogenesis, but
could contribute to our understanding of nodulation and nodule also expressed in vegetative tissues of flowering plants and in varihomology. Independent origins of nodules would mean the inde- ous tissues of conifers and even ferns27. Two MADS transcripts
pendent recruitment of ‘nodulin genes’ from within a gene family. have been identified in nodules of alfalfa, and have been found to
This could lead to the cognate nodulin genes of different legumes belong to different MADS-box gene subfamilies28,29 (Fig. 3). The
being paralogous (i.e. different duplicate copies or members of phylogenetic relationships of these genes raise more questions
different subfamilies). By contrast, the assumption is that the com- than they answer, perhaps illustrating the degree to which recruitmon ancestor of species possessing homologous nodules recruited ment to novel functions has occurred in this gene family. There
a single gene family member as its nodulin22. If there have been no appears to be no common theme among the alfalfa nodule MADSshifts in gene expression, the species that are derived from this box gene nmhC5, the root-expressed Agl17 of Arabidopsis to which
ancestor should all use the same family member (orthologue) as nmhC5 was found to be most closely related28, and the Antirtheir nodulin gene. A test for nodule homology thus involves rhinum orthologue of Agl17, which is expressed in pollen30. Simideveloping rigorous phylogenetic hypotheses for numerous gene larly, the other nodule-expressed alfalfa MADS-box gene, nmh7,
families that include nodule-expressed members.
is a member of a subfamily that includes Apetala3 of Arabidopsis
and Deficiens of Antirrhinum, both B-class floral genes known for
The phylogeny of genes: nodulin family trees
their dramatic effect on petal and stamen development. Intriguingly,
Genes involved in nodulation often belong to multigene families, it appears that no MADS-box gene with detectable sequence simiwhose evolutionary patterns can be very complex as a result of larity to nmh7 is expressed in alfalfa flowers29. Could recruitment
nested duplications, gene losses and concerted evolution23. This of the nmh7 gene product to a nodule function and its lack of
complexity is well illustrated by genes encoding leghemoglobin. expression in flowers be functionally or evolutionarily related?
Although globins in plants were originally identified in the Whether this expression pattern is generally true even of legumes
nodules of legumes (and were thus the quintessential nodulins), remains to be discovered.
globin genes have now been found in the genomes of both nonnodulating legumes and also in non-legumes24. In the nodule, the Molecular systematics of bacterial symbionts:
oxygen-binding ability of leghemoglobin contributes to maintain- bacterial taxonomy, coevolution and horizontal transfer
ing an environment that simultaneously permits bacterial growth Molecular phylogenetic studies are also revolutionizing our underand restricts oxygen availability that would inhibit bacterial nitrog- standing of the bacterial component of the symbiosis. This is not
enase. In other tissues, it is speculated that globins may serve only of great interest to bacterial taxonomists, but is also potentially
other functions such as oxygen sensing25. The plant globin gene relevant in elucidating nodule origins. At a coarse scale there is some
CRM6
CRM1
AGL12
TM8
TM3
AG
nmhC5 (Medicago) – nodule
Agl17 (Arabidopsis) – root
DEFH125 (Antirrhinum) – floral
AGL15
CRM3
GLO
nmh7 (Medicago) – nodule
Slm3 (Silene)
rad1, 2 (Rumex )
Pmads1 (Petunia)
Stdef (Solanum)
Def (Antirrhinum)
Floral
Ap3 (Arabidopsis)
Pd2 (Solanum)
Tm6 (Lycopersicon)
Cmb2 (Dianthus)
SQUA
AGL6
AGL2

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Rhizobium tropici
(Agrobacterium rhizogenes)
Rhizobium leguminosarum biovar. vicieae
Rhizobium leguminosarum biovar. trifolieae
Rhizobium leguminosarum biovar. phaseoli
Rhizobium etli
(Agrobacterium tumefaciens)
'Rhizobium' galegeae
(Agrobacterium vitis)
Sinorhizobium fredi
NGR234
Sinorhizobium meliloti
'Mesorhizobium' loti
Other 'Mesorhizobium'
(Phyllobacterium rubiacearum)

Bradyrhizobium japonicum
(Rhodopseudomonas palustris)
Bradyrhizobium elkanii

Galega orientalis
Galega spp.
Vicia
Pisum
Melilotus
Trifolium
Medicago
Lotus
Anthyllis
Sesbania
Phaseolus
Vigna
Glycine
Cajanus
Lupinus
Leucaena
Parasponia (Ulmaceae)

Azorhizobium caulinodans
(Aquabacter spiritensis)

Fig. 4. Comparison of legume and bacterial molecular phylogenies. The bacterial phylogeny31 (left) is based on 16S rRNA gene sequences, and
shows three very distinct groups of bacteria involved in nitrogen-fixing symbiosis with legumes and Parasponia (Ulmaceae). Representative
symbiotic bacteria are shown; lineages of non-symbiotic bacteria are shown by boxes, with a representative given in parentheses for lineages
closely related to symbiotic groups. On the right, phylogenetic relationships are shown for selected legume genera (reduced from the tree in Fig. 2)
nodulated by bacteria shown on the bacterial tree. Arrows connect bacterial symbionts with their plant hosts. The wide host-range of NGR234
is shown only by indicating its ability to nodulate Parasponia. Although host-ranges of individual bacteria are likely to be wider than shown
here, the lack of correlation between the 16S rRNA bacterial phylogeny and the rbcL phylogeny of Leguminosae is still apparent.

correlation between bacterial and plant relationships, with Gramnegative ‘rhizobia’ participating in symbioses with legumes, and
Gram-positive bacteria nodulating most of the non-leguminous
nodulators. At a finer scale, if there has been cospeciation of host
plants and rhizobia, then the presence of the same or closely related
rhizobial taxa in all members of a nodulating group would argue for
homology of their nodules; conversely, the presence of unrelated
rhizobia in the nodules of different species would argue for independent origins of nodules. Phylogenies inferred from 16S ribosomal RNA gene sequences emphasize the great genetic diversity
of the bacteria involved in legume symbioses31. ‘Rhizobia’ include
representatives from several major lineages of proteobacteria, with
the symbionts in each lineage being more closely related to nonsymbionts (such as Agrobacterium tumefaciens, in the case of Rhizobium itself) than to symbionts in other lineages31 (Fig. 4). The 16S
rRNA gene phylogenies indicate that there is little, if any, phylogenetic correlation between bacteria and their legume hosts. For example, Phaseolus, a member of the tribe Phaseoleae, is nodulated
by Rhizobium leguminosarum, strains of which also nodulate members of other tribes. Another member of the Phaseoleae, Glycine, is

not nodulated by Rhizobium, but instead has symbioses with members of two distinct lineages of Bradyrhizobium and with a Sinorhizobium species (Fig. 4). The wide host-ranges of some bacteria
further complicate matters: a single Rhizobium strain, NGR234,
can nodulate not only diverse legumes but also Parasponia.
It is possible that phylogenies based on 16S rRNA sequences do
not provide appropriate data for addressing nodulation origins. Bacterial genes involved in nodulation are commonly found on plasmids, and horizontal transfer might have played an important role in
the evolution of nodulation32. However, not all nodulation genes are
borne on plasmids: several key nodulation genes were found to be
absent from the fully sequenced 536 kb plasmid of the wide hostrange rhizobium strain NGR234 (Ref. 33). However, even nodulation genes that are grouped in chromosomal ‘symbiosis islands’
appear to be capable of transmission among diverse bacteria34.
Thus, phylogenetic analyses of nodulation genes can tell very different stories from the 16S rRNA genes used to construct bacterial
phylogenies32,35. The relevant information on plant–bacterial coevolution might be gained from these genes or gene complexes,
rather than from the bacteria that now bear them36.
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Conclusions and prospects

Our understanding of nodulation is indeed evolving, and phylogenetic studies are playing a significant role in this process. For
example, our view of nodulation has changed from one of multiple, completely independent origins in totally unrelated plant
groups to perhaps a single origin of a predisposition for nodulation. Similarly, nodulins are no longer considered to be uniquely
expressed in nodules, but are thought to have been recruited from
diverse ancestral functions based on pre-existing genes. Other
new insights include an improved understanding of the diversity
of relatively distantly related bacterial genera that play a part in
nodulation.
Increased phylogenetic sampling of plants will allow hypotheses on nodule evolution to be refined. Studies on the expression
and phylogenetic patterns of genes involved in nodulation, particularly in plants other than the handful already intensively studied,
will provide insights into how the nodule was assembled in the
course of evolution. In the bacterial symbiont, elucidation of the
complex relationships of both bacterial species and particular bacterial genes affords similar opportunities.
Acknowledgements

I gratefully acknowledge grant support from the US National
Science Foundation (DEB-9614984 and DEB-9420215).
References
1 Wagner, G.M. (1997) Azolla: A review of its biology and utilization, Bot. Rev.
63, 1–26
2 Ahern, C.P. and Staff, I.A. (1994) Symbiosis in cycads: The origin and
development of coralloid roots in Macrozamia communis (Cycadaceae), Am.
J. Bot. 81, 1559–1570
3 Gantar, M. et al. (1995) Colonization of wheat (Triticum vulgare L.) by
N2-fixing cyanobacteria: IV. Dark nitrogenase activity and effects of
cyanobacteria on natural 15N abundance in the plants, New Phytol. 129,
337–343
4 Swensen, S.M. and Mullin, B.C. (1997) The impact of molecular systematics
on hypotheses for the evolution of root nodule symbioses and implications for
expanding symbioses to new host plant genera, Plant Soil 194, 185–192
5 Swensen, S.M. (1996) The evolution of actinorhizal symbioses: evidence for
multiple origins of the symbiotic association, Am. J. Bot. 83, 1503–1521
6 Soltis, D.E. et al. (1995) Chloroplast gene sequence data suggest a single
origin of the predisposition for symbiotic fixation in angiosperms, Proc. Natl.
Acad. Sci. U. S. A. 92, 2647–2651
7 de Faria, S.M. et al. (1989) Occurrence of nodulation in the Leguminosae,
New Phytol. 111, 607–619
8 Bryan, J.A., Berlyn, G.P. and Gordon, J.C. (1996) Toward a new concept of
the evolution of symbiotic nitrogen fixation in the Leguminosae, Plant Soil
186, 151–159
9 McKey, D. (1994) Legumes and nitrogen: The evolutionary ecology of a
nitrogen-demanding lifestyle, in Advances in Legume Systematics, Part 5:
Nitrogen Economy (Sprent, J.I. and McKey, D., eds), pp. 211–228, Royal
Botanic Gardens, Kew
10 Doyle, J.J. et al. (1997) A phylogeny of the chloroplast gene rbcL in the
Leguminosae: Taxonomic correlations and insights into the evolution of
nodulation, Am. J. Bot. 84, 541–554
11 Kozik, A. et al. (1996) The pea early nodulin gene PsENOD7 maps in the
region of linkage group I containing sym2 and leghaemoglobin, Plant Mol.
Biol. 31, 149–156
12 Ghassemi, F. and Gresshoff, P.M. (1998) The early enod2 and the
leghemoglobin (lbc3) genes segregate independently from other known
soybean symbiotic genes, Mol. Plant–Microbe Interact. 11, 6–13
13 Sanderson, M.J. and Wojciechowski, M.F. (1996) Diversification rates in a
temperate legume clade: Are there ‘so many species’ of Astragalus
(Fabaceae)? Am. J. Bot. 83, 1488–1502

478

December 1998, Vol. 3, No. 12

14 Lavin, M. et al. (1998) Monophyletic subgroups of the tribe Millettieae
(Leguminosae) as revealed by phytochrome nucleotide sequence data, Am. J.
Bot. 85, 412–433
15 Hirsch, A.M. and LaRue, T.A. (1998) Is the legume nodule a modified root or
stem or an organ sui generis? Crit. Rev. Plant Sci. 16, 361–392
16 Gianinazzi-Pearson, V. (1996) Plant cell responses to arbuscular mycorrhizal
fungi: Getting to the roots of the symbiosis, Plant Cell 8, 1871–1883
17 Van Rhijn, P. et al. (1997) Expression of early nodulin genes in alfalfa
mycorrhizae indicates that signal transduction pathways used in forming
arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved,
Proc. Natl. Acad. Sci. U. S. A. 94, 5467–5472
18 Franche, C. et al. (1998) Actinorhizal symbioses: Recent advances in plant
molecular and genetic transformation studies, Crit. Rev. Plant Sci. 17, 1–28
19 Verma, D.P.S., Hu, C.A. and Zhang, M. (1992) Root nodule development:
origin, function and regulation of nodulin genes, Physiol. Plant. 85, 253–265
20 Nap, J-P. and Bisseling, N. (1990) The roots of nodulins, Physiol. Plant. 79,
407–414
21 Takane, K.I., Tajima, S. and Kouchi, H. (1997) Two distinct uricase II
(Nodulin 35) genes are differentially expressed in soybean plants, Mol.
Plant–Microbe Interact. 10, 735–741
22 Doyle, J.J. (1994) Phylogeny of the legume family: an approach to
understanding the origins of nodulation, Annu. Rev. Ecol. Syst. 25, 325–349
23 Clegg, M.T., Cummings, M.P. and Durbin, M.L. (1997) The evolution of plant
nuclear genes, Proc. Natl. Acad. Sci. U. S. A. 94, 7791–7798
24 Appleby, C.A., Dennis, E.S. and Peacock, W.J. (1990) A primaeval origin for
plant and animal hemoglobins, Aust. Syst. Bot. 3, 81–90
25 Andersson, C.R. et al. (1996) A new hemoglobin gene from soybean: A role
for hemoglobin in all plants, Proc. Natl. Acad. Sci. U. S. A. 93, 5682–5687
26 Frühling, M. et al. (1997) The Vicia faba leghemoglobin gene VfLb29 is
induced in root nodules and in roots colonized by the arbuscular mycorrhizal
fungus Glomus fasciculatum, Mol. Plant–Microbe Interact. 10, 124–131
27 Münster, T. et al. (1997) Floral homeotic genes were recruited from
homologous MADS-box genes preexisting in the common ancestor of ferns
and seed plants, Proc. Natl. Acad. Sci. U. S. A. 94, 2415–2420
28 Heard, J., Caspi, M. and Dunn, K. (1997) Evolutionary diversity of
symbiotically induced nodule MADS box genes: Characterization of nmhC5, a
member of a novel subfamily, Mol. Plant–Microbe Interact. 10, 665–676
29 Heard, J. and Dunn, K. (1995) Symbiotic induction of a MADS-box gene
during development of alfalfa root nodules, Proc. Natl. Acad. Sci. U. S. A. 92,
5273–5277
30 Zachgo, S., Saedler, H. and Schwarz-Sommer, Z. (1997) Pollen-specific
expression of DEFH125, a MADS-box transcription factor in Antirrhinum
with unusual features, Plant J. 11, 1043–1050
31 Young, J.P.W. (1996) Phylogeny and taxonomy of rhizobia, Plant Soil 186,
45–52
32 Mergaert, P., Van Montagu, M. and Holsters, M. (1997) Molecular
mechanisms of Nod factor diversity, Mol. Microbiol. 25, 811–817
33 Freiberg, C. et al. (1997) Molecular basis of symbiosis between Rhizobium
and legumes, Nature 387, 394–401
34 Sullivan, J.T. and Ronson, C.W. (1998) Evolution of rhizobia by acquisition
of a 500-kb symbiosis island that integrates into a phe-tRNA gene, Proc. Natl.
Acad. Sci. U. S. A. 95, 5145–5149
35 Haukka, K., Lindstrom, K. and Young, J.P.W. (1998) Three phylogenetic
groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates
from leguminous trees growing in Africa and Latin America, Appl. Environ.
Microbiol. 64, 419–426
36 Ueda, T., Suga, Y., Yahiro, N. and Matsuguchi, T. (1995) Phylogeny of sym
plasmids of rhizobia by PCR-based sequencing of a nodC segment,
J. Bacteriol. 177, 468–472

Jeff J. Doyle is at the L.H. Bailey Hortorium, 462 Mann Library
Building, Cornell University, Ithaca, NY 14853, USA (tel 11 607
255 7972; fax 11 607 255 7979; e-mail jjd5@cornell.edu).