Lewis et al. (2005) divide Caesalpinioideae into four tribes (Table S2). Nodulation occurs in two, Cassieae and Caesalpinieae. Fewer genera are included in the study of Lavin et al. (2005), but comparison of the two sets of data shows that two of the groups that do not nodulate, roughly corresponding to tribes Cercideae and Detarieae, are well separated from the others (Fig. 2a, top branch) Although their nodes are relatively recent, 34 and 29 Ma, their stem clade ages show them to be early divergent lineages in the Caesalpinioideae. They are currently found principally in tropical rainforests, possibly by spreading there after evolving in dry deciduous (succulent biome) habitats (Schrire et al., 2005a, 2005b). In tribe Cassieae, the subtribe Cassiineae has three genera, Cassia, Senna and Chamaecrista– only the latter nodulates, and in the analysis of Doyle et al. (1997) it was found to be distinct from the other two. Chamaecrista is a large genus, with c. 333 species. Although mainly tropical, unusually for the subfamily, its species have extended more recently into temperate areas where herbaceous forms can be found, for instance, in grassy habitats of New England. Most species are shrubby and a few are trees. This range of habit and habitat is paralleled by a range in the structure of infected cells. In tree species, N-fixing bacteroids are retained within modified infection threads, known as fixation threads (Fig. 4d); in shrubby species, the walls of these threads are thinner; and in herbaceous species, bacteroids are released into symbiosomes (Naisbitt et al., 1992) as in all mimosoid and most papilionoid legumes (Fig. 4b). According to Lavin et al. (2005), Chamaecrista is included in a crown node of 54.5 Ma, as are some members of the large tribe Caesalpinieae, which contains all other caesalpinioid genera confirmed to nodulate. These are Campsiandra, Dimorphandra, Erythrophleum, Melanoxylon, Moldenhawera and Tachigali, which now includes the nodulating genus Sclerolobium. Depending on the particular molecular characters scored, the relatedness of the nodulating genera appears to vary considerably. In the analysis of Haston et al. (2003), based on the trnL intron of the chloroplast genome, Tachigali, Sclerolobium and Moldenhawera are close, with Dimorphandra near and Melanoxylon further away, but Haston et al. (2005) note that future molecular data may result in there being closer grouping of the nodulated genera. No nodulating genus is included in the analysis of Lavin et al. (2005). All nodulated genera in the Caesalpinieae are trees, and all have fixation threads in their active nodules. All are South American, with the exception of Erythrophleum, which is found in Africa, Asia and Australia. Thus it appears that nodulation in these genera may be an ancient character. Because of the unique nodule structure, it is most parsimonious to assume that a single nodulation event occurred, as suggested in Fig. 2a. The limited evidence available suggests that caesalpinioid legumes can nodulate with a variety of classical rhizobia (de Lajudie et al., 1998 for Chamaecrista; Moreira et al., 1998 for Dimorphandra; Parker, 2000 for Tachigali).
If it is assumed that possession of arbuscular mycorrhizas (AM) is ancestral to angiosperms (Fitter & Moyersoen, 1996 and references therein), then we may infer that all early legumes were AM. Alexander (1989) looked at the distribution of AM and ectomycorrhizas (ECT) in legumes, and found that, of the tribes of Caesalpinioideae recognized at that time, Cassieae, Caesalpinieae, Cercideae and Detarieae were AM and Amherstieae mainly ECT. The latter tribe is no longer recognized, with genera being included in Detarieae by Lewis et al. (2005). In the molecular analysis of Lavin et al. (2005), a number of known ECT genera are grouped as a branch of caesalpinioid crown node 3 dated at 29.2 Ma (Fig. 2a). However, some of these genera are also able to form AM, usually at low levels. The extent of formation of AM is location-dependent (Moyersoen & Fitter, 1999), a point that will be considered later. Some recent evidence suggests that ectotrophic mycorrhizas in legumes may be a more ancient character than generally thought (Alexander, 2006).
The mimosoid crown node is dated by Lavin et al. (2005) at approx. 42 Ma. However, there is a rather long stem (approx. 15 Ma) before the crown group starts to proliferate into most of the tribes recognizable today. Tribal phylogeny is less well resolved than in the other two subfamilies, making it difficult to map the occurrence of nodulation (Sprent, 2001). The fact that some of the putative mimosoid fossils may belong to extinct lineages adds a further complication (Lavin et al., 2005).
Three tribes are recognized, with the former Parkieae and Mimozygantheae being included in tribe Mimoseae (Luckow, 2005; Luckow et al., 2005a), which still remains one of convenience. Most of the older groups in Lavin et al. (2005) appear not to nodulate, exceptions being Entada and some species of Pentaclethra. The remaining members of tribe Mimoseae and tribes Acacieae and Ingeae (dating from approx. 30.5 Ma) are uniformly nodulated, apart from a few that have lost their nodulating ability.
Compared with the Papilionoideae, nodule characters are very uniform, all nodules studied being indeterminate with varying degrees of branching; and (with a few exceptions, detailed later) infection is through root hairs. As this involves infection threads, and as these are also the norm in caesalpinioid nodules, nodules in mimosoid legumes may have arisen from the same event as in the Caesalpinioideae, but this leaves a major problem in that the apparently most basally branching genera cannot nodulate (Fig. 2a). Species of tribe Mimoseae may be nodulated by β-rhizobia (Barrett & Parker, 2005; Elliott et al., 2007).
The taxonomy of Acacia has been in a state of flux for many years. For convenience, most people have used three subgenera, Acacia, Aculeiferum and Phyllodineae, to separate what have been known for a long time to represent at least three different genera, with Aculeiferum being polyphyletic. Molecular evidence puts Phyllodineae firmly in tribe Ingeae, with the other two retained in tribe Acacieae. At the 2005 Botanical Congress in Vienna, it was agreed by a very narrow vote to retain the name Acacia for Phyllodineae, opening up the recognition at generic level of the names Vachellia (= subgenus Acacia), Senegalia (= much of subgenus Aculeiferum), Acaciella and two as-yet unnamed genera. This overturns usual procedures in taxonomy and has upset many who believe that subgenus Acacia, rather than Phyllodineae, should have retained this name. For further details of this controversy see Lewis (2005); Luckow et al. (2005b); http://www.worldwidewattle.com). In this review, the earlier classification into subgenera will be retained. From a symbiotic rather than an purely nodulation point of view, there are clear differences between the largely Australian subgenus Phyllodineae and the other subgenera, as many of its species are able to form both AM and ECT, even when grown in Africa or Brazil, whereas local ‘acacias’ do not. This may be one reason for their being so invasive in parts of the world such as South Africa (Sprent, 2001). Although acacias can nodulate with a wide range of α-rhizobia, there are differences among the constituent subgenera (Pueppke & Broughton, 1999), each having its own spectrum of symbionts and a wide range of nodule effectiveness (e.g. Burdon et al., 1999 for Phyllodineae; Ben Romdhande et al., 2006 for Acacia tortilis ssp. radiana (Aculeiferum)). Some species of subgenus Aculeiferum, section Monacanthea, most of which are scrambling shrubs found at the edge of thickets in South America, Texas and parts of Africa, have lost their ability to nodulate (Sprent, 2001). All the nonnodulating species used in the phylogenetic analysis of Miller et al. (2003) form a very distinct clade, suggesting a single event leading to loss of nodulation: this illustrates the type of disjunction of related species found between continents (Schrire et al., 2005a, 2005b). The only nonnodulating species included by Lavin et al. (2005), Acacia greggii, is dated at approx. 15–20 Ma.
Many acacias worldwide, particularly members of subgenera Aculeiferum and Acacia, are now found in low-nutrient, dry and sometimes saline soils. Nitrogen is only one of many factors limiting plant growth, and any N fixation that does occur is restricted to periods where soil moisture is present. Where the soil is deep and sandy, such as in large parts of Africa (e.g. Sudan, Senegal), woody plants first develop extensive tap-root systems, before producing much new shoot matter, and certainly before producing root nodules. For example, Acacia senegal seedlings can have tap roots nearly 2 m long when 2 months old. In dry soils, nodules do not form even when the soil contains large numbers of compatible rhizobia, with potentially quite high rates of N fixation (Fagg & Allison, 2004 and references therein). This species, and others such as Acacia polyacantha and Acacia mellifera, belong to subgenus Aculeiferum, but are genetically distinct from the group that has lost the ability to nodulate (Harrier et al., 2002). Plants from these two groups may grow in close proximity, for example Acacia brevispica (nonnodulating) and A. mellifera (nodulating) in low-N soil of the semiarid Loruk region of Kenya (Odee et al., 2002). In these conditions, is the ability to nodulate of relatively little value? Does it matter that the plant bears nodules with a range of efficiencies? Potential rates of photosynthesis are high, so that any carbon drain on the plant by essentially parasitic nodules may not be significant. In order to nodulate with a range of rhizobia, plant roots must exude a wide range of compounds that can induce the relevant bacteria to produce their own nodule-inducing signals for transmission to the host. There is some evidence that this is the case in acacias (Shaw et al., 1997). As far as the rhizobia are concerned, it may be sufficient for them to find a niche where they get protection from adverse soil conditions, as well as a source of nutrients.
In Lewis et al. (2005), 28 tribes are retained for this subfamily. As with the other subfamilies, there are likely to be changes in the tribal affiliations in the next few years. In the analysis of Lavin et al. (2005), Papilionoideae is divided among distinct groups, and these will form the basis of the present discussion (Fig. 2b). The phylogeny and chronology are not fully congruent, so precedence will be given here to chronology, as it raises some interesting questions and also provides some answers about nodulation.
The papilionoid crown node is dated as 58.6 Ma. Before the next major crowns, the genistoid and dalbergioid, there is a rather ill-defined group of genera, most of which cannot nodulate, containing members of various tribes, including several from the Swartzieae. However, there is a distinct Swartzia node, dated at approx. 49 Ma, which contains all the genera (Ateleia, Bobgunnia, Cyathostegia and Swartzia) in tribe Swartzieae that are confirmed to nodulate. From a nodulation point of view, these genera sit uncomfortably here, as almost all other genera in the Papilionoideae (except a few that have lost the ability) can nodulate. Intuitively, it seems unlikely that there was a separate nodulation event in a small group of swartzioid legumes. It is possible that, with sampling of more species and more detailed analysis, preferably including nodulation as a character, the positions of these branches may change.
Between the genistoid and dalbergioid nodes, dated at 56.4 and 55.3 Ma, respectively (Lavin et al., 2005) are two genera, Hymenolobium and Andira, which have recently been excluded from the dalbergioid clade for many reasons, mainly molecular, but also because they do not have the aeschynomenoid type of nodule that typifies the clade (Lavin et al., 2001). Rather, they have nodules where bacteroids are retained in fixation threads (Fig. 4d), as in the Caesalpinioideae. Three other papilionoid genera share this character: Poecilanthe and Cyclolobium, both formerly in tribe Millettieae, now in Brongniarteae (Ross & Crisp, 2005), but in the genistoid crown of Lavin et al. (2005); and Dahlstedtia, which is retained in tribe Millettieae (Schrire, 2005). With the exception of Andira inermis, which is also found in West Central Africa, all five papilionoid genera with fixation threads are trees or shrubs native to South America, as are all but one of the known nodulating caesalpinioid genera. They are found in a range of habitats, from very dry to seasonally flooded. It would be interesting to sample more species of these genera to see if nodules of the shrubby species have thinner-walled fixation threads than the tree species, as found in Chaemaecrista.
The genistoid group consists of several tribes and parts of tribes. In terms of nodule characters it is rather diverse, but some patterns are beginning to emerge. The genus Ormosia (tribe Sophoreae) is separated from the rest, and it has large, often profusely branched, indeterminate nodules. In addition to Poecilanthe and Cyclolobium (see above), tribe Brongniartieae contains eight genera with unknown nodule structure. Studies of nodules on these genera could help resolve taxonomic problems. In tribe Thermopsideae, nodules are indeterminate and some have structures in which bacteroids are grouped in ‘thin-walled threads’ or other forms where they not fully released into symbiosomes (Sutherland et al., 1994). These are reminiscent of intermediates between fixation threads and symbiosomes, seen in various species of Chamaecrista. Tribe Crotalarieae has nodules that are typically indeterminate, with apical meristems that may branch or, in some species of Lotononis, grow around the root as in lupin (Corby, 1988; Fig. 3e). At least some genera appear to lack infection threads and have infected cells that can divide and give rise to patches of uniform tissue, lacking uninfected cells (Crotalaria juncea, de Rothschild, 1963; Lotononis bainsii, E. K. James, pers. comm.; Fig. 4f). These features, including indeterminate growth, are common to the next tribe, Genisteae, which includes Lupinus, a genus that has been widely studied for its nodulation characters and which is also unique in being the only legume genus known that is not mycorrhizal. In both Lupinus and Chamaecytisus (now included in Cytisus; Polhill & van Wyk, 2005), infection does not involve infection threads and, indeed, infection threads are rarely seen (Lupinus) or transient (Chamaecytisus) (James et al., 1997; Vega-Hernández et al., 2001). Infection in Lupinus albus occurs between epidermal cells adjacent to the bases of root hairs (González-Sama et al., 2004). Data on species of Cytisus, Genista, Petteria, Sarothamnus (a synonym of Cytisus) and Ulex (Lechtova-Trnka, 1931; Sajnaga et al., 2001; Kalita et al., 2006; J.I.S., unpublished data) suggest that these features may be typical of the tribe. They are nodulated by species of Bradyrhizobium with broad host range (Sajnaga et al., 2001; Kalita et al., 2006), consistent with the suggestion made below for dalbergioid legumes, that a nonhair infection process may be less well controlled.
No member of tribe Podalyrieae is included by Lavin et al. (2005), but this tribe is currently considered to be close to tribes Crotalarieae and Genisteae (van Wyk, 2005). However, nodules on Cyclopia genistoides, although indeterminate, have infection threads and a mixture of infected and uninfected cells in the central region (E. K. James, pers. comm.; Fig. 4e).
Recent studies have shown that species of three genistoid genera from Africa, Cyclopia, Crotalaria and Lotononis, can nodulate with nonclassical rhizobia. The first nodulates with Burkholderia from the β-rhizobia (Kock, 2004) and the other two with Methylobacterium from the α-group (Sy et al., 2001; Jaftha et al., 2002). A report that Aspalathus carnosa (Crotalarieae) nodulates with the β-rhizobium Burkholderia tuberum (Moulin et al., 2001) has not been confirmed.
In the dalbergioid group there are two early branches. There is little information about the smaller branch, dated at approx. 36.9 Ma, but several genera, for example Psorothamnus, are known to have indeterminate nodules, often extensively branched (Sprent, 2001). The larger group is typified by aeschynomenoid nodules, with a ‘crack’ infection pathway in which bacteria enter via breaks where lateral (adventitious in some stem nodulating species) roots emerge. Infection threads are never formed, and infected cells are not interspersed with uninfected ones. This form of infection bypasses some of the complex processes involved in infection via root hairs (Goormachtig et al., 2004), and could be regarded as primitive. The ancient origin of the genistoid and dalbergioid groups makes more sense in terms of nodulation processes than their former positions, which were thought to be derived (Polhill, 1981). As far as is known, nodulation is universal, with the possible exception of Grazielodendron and the certain exception of two closely related genera of woody climber, Chaetocalyx and Nissolia, dated at 8.5 Ma (Lavin et al., 2005), which appear to have lost their nodulating ability. Aeschynomenoid nodules are generally small (up to 5 mm in diameter), determinate in growth and short-lived, meaning that they must be replaced regularly. One advantage of this type of nodule is that it can be formed on old roots as long as they retain the ability to form laterals. This could be important for large trees, such as some species of Dalbergia and Pterocarpus, because it would allow a wider distribution of nodules across the root system. On the other hand, it could be argued that not having a root-hair infection could lead to a higher level of occupancy by less effective bacteria. There is some evidence to support this suggestion. In the economically important species Arachis hypogaea (groundnut or peanut) there is a considerable degree of promiscuity, leading to problems for introducing improved inoculant strains (Sprent, 1994b), and Rasolomampianina et al. (2005) have recently isolated seven different bacterial genera from nodules of Dalbergia spp. in Madagascar.
The smaller mirbelioid crown node originated slightly more recently (48 Ma bp). It consists of the endemic Australian tribe Bossiaeeae and the Mirbelieae, which is also found in Papua New Guinea. However, there is a comparatively long stem, the main radiation of the crown starting approx. 30 Ma, after Australia became an island (32 Ma bp), but peaking in the latter 10 Ma when the continent became increasingly arid (Crisp et al., 2004). Many of the soils that they grow in are very low in nutrients, which may explain why, outside some species of Acacia (see above), members of these tribes are the only known legumes to be dual mycorrhizal (AM + ECT), some also having cluster roots (Sprent, 1994a). All appear to have indeterminate nodules, and it is now emerging that many are nodulated by a wide range of α- and β-rhizobia, a factor thought to be advantageous in these environments (Watkin et al., 2005). Lawrie (1981) found abundant deformed root hairs on Aotus ericoides (Mirbelieae), but did not find infection threads in them. However, as threads were found in developing nodules, it was thought that a hair infection must occur. However, as discussed later, this does not necessarily follow. Not in the analysis of Lavin et al. (2005), but generally agreed to be associated with the mirbelioid clade, is the monogeneric South African tribe Hypocalypteae (Lewis et al., 2005). Although this genus is known to have indeterminate nodules (Sprent, 2001), there is no information on nodule structure or their endophytic bacteria.
Tribe Indigofereae is represented by a small crown node that appears to have separated from other papilionoid groups at least 50 Ma bp. It is mainly African, although the large type genus Indigofera has a pantropical distribution and indeterminate, often branched nodules. It has been suggested that the Indigofereae and all subsequent tribes share a polyploid event (Pfeil et al., 2005).
The millettioid crown node, dated 45 Ma bp, has two parts, one of which contains much of tribe Millettieae and the other Phaseoleae, Desmodieae and Psoraleae. This division is entirely consistent with nodule characters. With the removal of Poecilanthe and Cyclolobium to the genistoid clade (see above), the remaining genera in the older part of the millettioid node all have indeterminate nodules, often much branched and, in the one case that has been studied, Lonchocarpus muehlbergianus, infection is not via root hairs (which were absent), probably occurring between epidermal cells. Infection threads are formed later, and the central tissue has both infected and uninfected cells (Cordeiro et al., 1996). The infected tissue of Millettia laurentii is similar (T. H. A. Kiam, pers. comm.).
The generally younger part of the group, tribes Desmodieae, Phaseoleae and Psoraleae, show a range in habit, but tend towards shrubby and herbaceous forms, with some genera such as Phaseolus being relatively recent (< 10 Ma). All that have been examined typically have determinate nodules, with prominent lenticels and central tissue containing a mixture of infected and uninfected cells (Figs 3b,4a) and, where tested, the typical products exported from N fixation in nodules are ureides rather than amides (Sprent, 2001). There are occasional reports of genera with two types of nodule, for example Cajanus and Kennedia (Corby, 1988). The woody genus Erythrina is anomalous in this group on some nodule and other characters, such as nodule morphology and low levels of exported ureides (Sprent & Parsons, 2000). It is likely that the determinate nodule form has been derived from indeterminate. Its advantages and disadvantages have been discussed by Sprent (1980). Briefly, they develop rapidly and initially use the products of fixation (ureides) to fuel bacterial division and host-cell enlargement. Ureides are economical with carbon, compared with amides, but are less soluble and hence may be better for warmer regions. The lifespan of determinate nodules is usually said to be short (a few weeks), but this may apply particularly to the annual crop species that have been studied most widely. In perennial plants, nodules may last for months and also may become lobed, indicating that their meristems remain active (Sprent, 2001). When they are ephemeral, as in soybean (Glycine max), and are killed by stress, they must be replaced by new nodules. This is not true of indeterminate nodules, the apical meristems of which can survive considerable stress and may live for years, resuming growth within days of restoration of suitable growing conditions. Perhaps it is not surprising that the vast majority of all legume nodules, worldwide, are indeterminate. Rhizobia presently known to nodulate the ureide-exporting legumes are all classical rhizobia, mainly slow-growing types.
The remainder of the Papilionoideae have their origin in the Hologalegina crown node, dated at 51 Ma bp. Like the millettioid group, it is split into two main parts. The older robinioid crown node (48 Ma bp) has three sections of interest for nodule characters. First, the genus Sesbania, now included in a monogeneric tribe, Sesbanieae (Lavin & Schrire, 2005), one species of which, Sesbania rostrata, has been studied intensively as it can nodulate on both stems (associated with adventitious roots) and roots, using different infection pathways (crack entry and root-hair infection) depending on growth conditions (Goormachtig et al., 2004). Nodules may appear very similar to the aeschynomenoid type, or grow out and be indeterminate. One of its nodulating bacteria is Azorhizobium caulinodans, a classical rhizobium that can grow ex planta on nitrogen gas as its sole N source. Sister to Sesbanieae is tribe Loteae, which now includes former tribe Coronilleae. From a nodulation point of view, this change is a retrograde step, but the molecular evidence in its favour is very strong. Members of old tribe Loteae have determinate nodules, morphologically and structurally very similar to those of the Phaseoleae and related tribes described in the previous section, but exporting amides rather than ureides as a product of fixation. It appears that determinate nodules have arisen twice during legume evolution. Members of former tribe Coronilleae have indeterminate nodules. The Loteae as now understood is relatively young, herbaceous and temperate in location. The third part of this group is tribe Robineae, a complex group of woody legumes mainly in tropical and warm temperate regions, with indeterminate nodules.
The second part of this large clade is distinguished by the loss of the inverted repeat in the chloroplast genome (Doyle, 1995), and is known as the inverted repeat loss clade (IRLC). It is rooted at approx. 39 Ma. A small group of its genera, including the familiar ornamental genus Wisteria, is still included in tribe Millettieae, pending further research into its correct position. The rest of the IRLC consists of tribes Astragaleae, Galegeae, Hedysareae, Trifolieae, Cicereae and Fabeae (formerly Viceae). These tribes are largely warm temperate through temperate to arctic, and have indeterminate nodules, often branched, with root-hair infection. They generally nodulate with fast-growing α-rhizobia and usually show a high degree of specificity between symbiotic partners.