Evolution of symbiosis in the legume genus Aeschynomene



  • Legumes in the genus Aeschynomene form nitrogen-fixing root nodules in association with Bradyrhizobium strains. Several aquatic and subaquatic species have the additional capacity to form stem nodules, and some of them can symbiotically interact with specific strains that do not produce the common Nod factors synthesized by all other rhizobia. The question of the emergence and evolution of these nodulation characters has been the subject of recent debate.
  • We conducted a molecular phylogenetic analysis of 38 different Aeschynomene species. The phylogeny was reconstructed with both the chloroplast DNA trnL intron and the nuclear ribosomal DNA ITS/5.8S region. We also tested 28 Aeschynomene species for their capacity to form root and stem nodules by inoculating different rhizobial strains, including nodABC-containing strains (ORS285, USDA110) and a nodABC-lacking strain (ORS278).
  • Maximum likelihood analyses resolved four distinct phylogenetic groups of Aeschynomene. We found that stem nodulation may have evolved several times in the genus, and that all Aeschynomene species using a Nod-independent symbiotic process clustered in the same clade.
  • The phylogenetic approach suggested that Nod-independent nodulation has evolved once in this genus, and should be considered as a derived character, and this result is discussed with regard to previous experimental studies.


The Leguminosae (Fabaceae) is the third largest family of flowering plants with c. 730 genera and over 19 400 species worldwide (Mabberly, 1997; Lewis et al., 2005). Legume species are particularly diverse, both in size and in ecological habitat, and include small herbs from temperate regions as well as large tropical rainforest trees. They are agriculturally and economically important, being second only to the Poaceae (e.g. cereals). In addition to the most cultivated crops, such as soybean (Glycine max), common beans (Phaseolus vulgaris), peas (Pisum sativum), and peanuts (Arachis hypogaea), that are harvested for grain or oil, legumes are also valued for timber, fuel, forage and medicines. This economic importance of the Leguminosae is mainly the result of the ability of many of its species to form a symbiotic association with soil bacteria, commonly known as rhizobia. This symbiosis usually results in the formation of root nodules, in which the rhizobia reduce atmospheric nitrogen to ammonium. This allows the plant to grow well and produce protein-rich seeds in the absence of nitrogen fertilizer in soils. Among the three subfamilies of the Leguminosae (Caesalpinioideae, Mimosoideae, and Papilionoideae), nodulation by rhizobia is rare in caesalpinioids, more common in mimosoids, and very common in papilionoids (Sprent, 2007). In all three subfamilies, nitrogen-fixing nodules are almost exclusively located on roots. However, in a very few tropical legumes that are hydrophytic and that belong to the three papilionoid genera, Aeschynomene, Sesbania, and Discolobium, (Eaglesham & Szalay, 1983; Alazard, 1985; Eaglesham et al., 1990; Ladha et al., 1992; Loureiro et al., 1994), and to the mimosoid genus Neptunia (Schaede, 1940), nodulation by rhizobia can also occur on stems. Stem nodulation was observed for the first time on Aeschynomene aspera (Heagerup, 1928), and later well documented in Sesbania rostrata (Dreyfus & Dommergues, 1981; Goormachtig et al., 2004) and Neptunia plena (James et al., 1992). Meanwhile, stem nodulation was reported on several other species of Aeschynomene (Alazard, 1985; Becker et al., 1988), and this genus contains most of the stem-nodulated species described so far.

The stem nodulation phenotype in these various legumes is, in fact, represented by a number of different ontogenies. James et al. (1992) considered that genuine stem nodules should be vascularly connected to the stem, as observed in Aeschynomene and Discolobium (Loureiro et al., 1994, 1995; James et al., 2001). If this criterion is considered to be the main one by which the term ‘stem nodules’ is used accurately, then Neptunia, which has been shown to form ‘stem nodules’ that are connected by their vascular tissue to the bases of adventitious roots (James et al., 1992; Subba Rao et al., 1995), should thus be considered as root nodules. Similarly, Goormachtig et al. (2004) also considered that stem nodules on S. rostrata were, in fact, adventitious root nodules, especially as their development is morphologically equivalent to the development of lateral root base nodules. Environmental conditions also play a major role in stem nodulation. The stem nodules of Discolobium pulchellum and Discolobium leptophyllum are compulsorily aquatic, requiring permanent submergence in water or in flooded soil (Loureiro et al., 1994; James et al., 2001). Several Aeschynomene species (Aeschynomene elaphroxylon, Aeschynomene crassicaulis, Aeschynomene americana) also form nodules on the stem (at the base or all the way up the stem) only under waterlogged conditions (Boivin et al., 1997). However, only one of these species, Aeschynomene fluminensis, has ‘flooded’ stem nodules with a vascular system connected to the stem (Loureiro et al., 1995) rather than a connection to adventitious roots.

Other species, such as Aeschynomene afraspera, Aeschynomene indica and S. rostrata, differ in their ability to readily develop stem nodules even under nonsubmerged conditions (Boivin et al., 1997). A unique feature that is found only in stem-nodulating Aeschynomene spp. is their capacity to symbiotically interact with photosynthetic bradyrhizobia (Evans et al., 1990; Giraud & Fleischman, 2004). It has been shown in Aeschynomene sensitiva that the photosynthetic activity of these bradyrhizobia facilitates ex planta survival and infectivity, and thus it could affect their biological nitrogen fixation during stem nodulation (Giraud et al., 2000).

Based on the stem and/or root nodulation ability of Bradyrhizobium isolates, three cross-inoculation groups of Aeschynomene spp. were initially defined by Alazard (1985). Group I (representative species A. americana) formed root nodules and/or adventitious root nodules on the stem, and was associated with ‘classical’ Bradyrhizobium strains in group A (Fig. 1). Species of group II (A. afraspera) formed profuse stem nodules under nonsubmerged conditions with both nonphotosynthetic (group A and B) and photosynthetic (group C) Bradyrhizobium. Group III (A. indica) formed sparse stem nodules under nonsubmerged conditions with photosynthetic strains (groups C and D). Since then, this system has become more complicated, as A. fluminensis has been shown to form stem nodules with photosynthetic strains but only in aquatic conditions (Loureiro et al., 1995). Moreover, new nonphotosynthetic isolates able to form stem nodules on group III Aeschynomene spp. have been discovered (Miché et al., 2010). Most surprisingly, sequencing of the whole genome of two group D Bradyrhizobium strains (ORS278 and BTAi1) showed that they did not contain the canonical nodABC-genes (nod) genes required for the synthesis of Nod factors (NFs), the signal molecules produced by all other rhizobia that had always been suggested as compulsory for the initiation of symbiotic nodules on legumes (Giraud et al., 2007). This result was further confirmed by a comparative genomic study of six additional strains (groups D and E) that were representative of the phylogenetic diversity of Bradyrhizobium isolated from group III plants (Fig. 1) (Mornico et al., 2012). It has finally been shown that group C strains, which form nodules on both Aeschynomene groups II and III, contain the canonical nodABC genes and produce NFs (Chaintreuil et al., 2001; Renier et al., 2011). Deletion of the nodB gene in one of these strains (ORS285) blocked nodulation of A. afraspera (group II), but did not affect nodulation of A. indica or A. sensitiva (group III), thus proving that ORS285 is able to use both Nod-dependent and Nod-independent symbiotic processes, depending on the host plant (Bonaldi et al., 2011).

Figure 1.

Evolutionary relationships among strains (a) and bacterial group of specificity and nodulation group defined among the various Aeschynomene species (b). The phylogenetic tree of strains is drawn after Miché et al. (2010). (a) @, The USDA110 strain has recently been reclassified as a new species, Bradyrhizobium diazoefficiens (Delamuta et al., 2013). Symbiotic bacteria are clustered (panel b) according to their possession (or not) of nodulation genes, photosynthetic ability, stem and root nodulation ability. The lack of nod genes in strains ORS278, Btai1 and STM3843 has been demonstrated after full genome sequencing (Giraud et al., 2007; Mornico et al., 2012). Green color, photosynthetic strains. *, representative strains of the group; Φ, species for which the inoculation group has been determined in this study; ♣, Aeschynomene fluminensis is the only species of the group that produces stem nodules only under flooded conditions.

The genus Aeschynomene contains 161 (http://www.theplantlist.org) to 180 species (Klitgaard & Lavin, 2005), half of them described from the New World, mainly South and Central America, and the other half have been found across the tropical regions of Africa, Southeast Asia, Australia and the Pacific Islands (Rudd, 1955; Verdcourt, 1971). The genus includes both herbaceous and shrubby species, annuals and perennials, some of them growing up to 8 m in height and with a basal stem width of 0.5 m (e.g. A. elaphroxylon). Half of the species are hydrophytes growing in marshes, temporary or permanent ponds, rice fields, waterlogged meadows, and along streams and riverbanks. The remaining species are more xeric and are found in savannas or dry forests.

Botanically, the genus Aeschynomene belongs to the tribe Aeschynomeneae, which has now been classified together with the Dalbergieae tribe in a monophyletic group referred to as the dalbergioid legumes, a large, mostly pantropical, group of papilionoids characterized by the presence of the Aeschynomeneae type of root nodule (Lavin et al., 2001; Sprent, 2001). Rudd (1955) published a revision of the American species of Aeschynomene, but no attempt has been made to include Old World species in this classification. The genus Aeschynomene was originally divided by Vogel (1838) into two sections: Aeschynomene L. sect. Aeschynomene, also referred to as Eu-Aeschynomene, which comprises c. 50 species with a pantropical distribution, and Aeschynomene sect. Ochopodium Vogel with > 100 pantropical species (Polhill, 1981; Rudd, 1981; Klitgaard & Lavin, 2005). This division, also retained by (Rudd, 1955), is well supported, based on morphological differences; that is, section Aeschynomene is characterized by medifixed stipules, whereas section Ochopodium has basifixed stipules. The section Ochopodium is more closely related to the genus Machaerium than to sect. Aeschynomene and to Dalbergia (Lavin et al., 2001; Ribeiro et al., 2007). Consequently, the genus Aeschynomene does not appear to be monophyletic, but this topic n eeds to be further developed using additional species from both the New and Old Worlds. The delimitation of section Aeschynomene is also problematic, as it is morphologically closely related to other genera, such as Soemmeringia, Cyclocarpa, Kotschya, Smithia, Geissaspis, Bryaspis and Humularia (Rudd, 1981). Such close relationships were more recently confirmed by DNA sequence analysis (Lavin et al., 2001; Ribeiro et al., 2007).

The aim of this study was first to conduct a molecular phylogenetic analysis including 38 species of Aeschynomene originating from both the New and Old World. By using two genomic and chloroplastic markers, ncDNA (ITS1-5.8S-ITS2) and the cpDNA (trnL) locus, respectively, we reconstructed the phylogenetic relationship among these Aeschynomene accessions, together with related species and genera. We also estimated the within-species diversity of four Aeschynomene species (A. americana, A. indica, A. sensitiva, and Aeschynomene villosa) in order to infer its possible influence on the phylogenetic reconstruction, and to link it with the geographic distribution of the accessions. We then tested most species for their ability to form root and stem nodules with various rhizobial strains, harboring nod genes or not, and consequently constructed a putative evolution of the various nodulation types found in the genus. Taxonomical and nodulation issues are discussed in the light of this phylogenetic and nodulation character-based evolution construction.

Materials and Methods

Sampling materials

This study included 38 neotropical or Old World different Aeschynomene species (Table 1), and from one to 11 accessions per species. Whenever possible, we included several individuals from the same species, sampled from the widest possible geographical area. Seventy-one different accessions were included in total, both from our experiments and via sequences retrieved from Lavin et al. (2001) and Ribeiro et al. (2007). Table 1 lists all taxa included in the study, their sources and geographic origin, their nodulation characteristics (when tested), and EMBL accession numbers. Several related genera (15) were also included in the analyses, as previous studies had suggested their close relationship and/or their phylogenetic intermingling with the genus Aeschynomene. All of these sequences, except for four from Smithia abyssinica and Kotschya lutea, were retrieved from GenBank and were originally published in Lavin et al. (2001) and Ribeiro et al. (2007).

Table 1. Characteristics of the species and samples included in the study
SpeciesCountry and locality of originVoucher numberStem nodule/root nodule aGenBank accessionSequence references
ORS278ORS285USDA110ITS/5.8StrnL intron
  1. TS, this study.

  2. a

    Results of the inoculation tests: +/+, nodules on stems and roots; −/+, nodules only on roots; −/−, complete lack of nodulation.

Aeschynomene abyssinica ZimbabweCPI 52331B    KC54029 KC560746 TS
Aeschynomene afraspera Senegal −/−+/+−/+ FM242584 FM211217 TS
Aeschynomene afraspera Senegal −/−+/+−/+ FM242585 FM211218 TS
Aeschynomene americana Costa RicaUSDA PI 544341−/−−/−−/+ FM242591 FM211224 TS
Aeschynomene americana Guadeloupe (West Indies) −/−−/−−/+ FM242586 FM211219 TS
Aeschynomene americana French Guiana, Remire −/−−/−−/+ FM242587 FM211220 TS
Aeschynomene americana MexicoUSDA PI 544080−/−−/−−/+ FM242588 FM211221 TS
Aeschynomene americana PanamaUSDA PI 544113−/−−/−−/+ FM242589 FL211222 TS
Aeschynomene americana VenezuelaUSDA PI 544122−/−−/−−/+ FM242590 FM211223 TS
Aeschynomene aspera Sri LankaIRRI No. 13020−/−+/+−/+ FM242623 FM211257 TS
Aeschynomene bella TanzanieILRI 18422    KC540628 KC560747 TS
Aeschynomene brasiliana var brasiliana Brazil, São PauloV. Stranghetti 765    EF451087 EF451126 Ribeiro et al. (2007)
Aeschynomene brasiliana BrazilCIAT7589    KC540627 KC560748 TS
Aeschynomene brevifolia MadagascarCPI 52335    KC540626 KC560749 TS
Aeschynomene ciliata ColombiaIRRI No. 13078+/++/+−/− FM242624 FM211258 TS
Aeschynomene crassicaulis Senegal, Niokolokoba −/−−/−+/+ FM242594 FM211227 TS
Aeschynomene cristata Democratic Republic of CongoIRRI No. 12146−/−−/−−/+ FM242625 KC560760 TS
Aeschynomene deamiiMexico, Tlacotalpan, Veracruz +/++/+−/− KC540625 FM211238 TS
Aeschynomene denticulata BrazilIRRI No. 13003+/++/+−/− FM242626 FM211260 TS
Aeschynomene evenia USA FlorideUSDA PI 572567+/++/+−/− KC163288 FM211228 TS
Aeschynomene elaphroxylon Senegal     KC540624 KC560751 TS
Aeschynomene falcata BrazilUSDA PI 322289−/−−/−−/+ FM242596 FM211229 TS
Aeschynomene falcata South AfricaUSDA PI 364378−/−−/−−/+ FM242597 FM211230 TS
Aeschynomene fascicularis Venezuela, MéridaLavin 5730    AF189025 AF208929 Lavin et al. (2001)
Aeschynomene filosa MexicoCPI 87516+/++/+−/− KC540623 KC560752 TS
Aeschynomene fluitans ZambiaCPI 52338    KC540622 KC560753 TS
Aeschynomene fluminensis ColombiaIRRI No. 11009−/−+/+−/+ FM242627 FM211261 TS
Aeschynomene fluminensis Brazil −/−+/+−/+ FM242598 FM211231 TS
Aeschynomene histrix French Guiana, Kourou −/−−/−−/+ FM242599 FM211232 TS
Aeschynomene indica USA LouisanaNLU3    U59892 AF208927 Lavin et al. (2001)
Aeschynomene indica USA North CarolinaCarulli 58    AF068141  Lavin et al. (2001)
Aeschynomene indica Senegal +/++/+−/−  FM211233 TS
Aeschynomene indica IndiaUSDA PI196206+/++/+−/− FM242601 FM211234 TS
Aeschynomene indica clone a ZambiaUSDA PI225551+/++/+−/− KC560764 FM211235 TS
Aeschynomene indica clone b ZambiaUSDA PI225551+/++/+−/− KC560765 FM211235 TS
Aeschynomene indica Australia +/++/+−/− FM242603 FM211236 TS
Aeschynomene indica Senegal, Kaolack +/++/+−/−  FM211242 TS
Aeschynomene indica Senegal, Kaolack +/++/+−/−  FM211254 TS
Aeschynomene indica Senegal +/++/+−/− FM242621 FM211255 TS
Aeschynomene indica Senegal +/++/+−/−  FM211256 TS
Aeschynomene indica ZimbabweIRRI No. 13015+/++/+−/−  FM211266 TS
Aeschynomene martii Brazil, Minas Gerais, Mato VerdeV.C. Souza 5455    EF451088 EF451127 Ribeiro et al. (2007)
Aeschynomene montevidensis UruguayM. Zabaleta. Montevideo−/−−/−  KC540621 KC560754 TS
Aeschynomene nilotica SenegalIRRI No. 14040−/−+/++/+ KC560767 KC560756 TS
Aeschynomene paniculata Brazil, Minas Gerais, Belo HorizonteP.O. Moraes. J.A. Lombardi 2689    EF451086 EF451125 Ribeiro et al. (2007)
Aeschynomene parviflora ColombiaIRFL 2854    KC540620 KC560755 TS
Aeschynomene pfundii Zimbabwe, National Botanic Gardens     AF189026 AF208930 Lavin et al. (2001)
Aeschynomene pfundii ZimbabweMatt Lavin. Montana−/−−/−−/+ FM242629 FM211263 TS
Aeschynomene pratensis BrazilIRRI No. 13006+/++/+−/− FM242630 FM211264 TS
Aeschynomene purpusii Mexico, OaxacaLavin 5325     AF208928 Lavin et al. (2001)
Aeschynomene rudis USA, FloridaMatt Lavin. Montana+/++/+−/− FM242631 FM211265 TS
Aeschynomene rudis Mexico, Laguna, Veracruz +/++/+−/− FM242604 FM211237 TS
Aeschynomene rudis Brazil +/++/+−/− FM242609 FM211243 TS
Aeschynomene rudis Mexico, Juan Campestre, Veracruz +/++/+−/− FM242615 FM211249 TS
Aeschynomene scabra Mexico, Tenango, Morelos +/++/+−/− FM242605 FM211239 TS
Aeschynomene scabra MexicoUSDA PI 296044+/++/+−/− FM242632 FM211267 TS
Aeschynomene schimperi SenegalIRRI No. 12156−/−−/−−/+ FM242633 KC560757 TS
Aeschynomene sensitiva Guadeloupe, Pointe-à-Pitre +/++/+−/− FM242606 FM211240 TS
Aeschynomene sensitiva Senegal +/++/+−/− FM242607 FM211241 TS
Aeschynomene sensitiva French Guiana +/++/+−/− FM242610 FM211244 TS
Aeschynomene sensitiva French Guiana +/++/+−/− FM242611 FM211245 TS
Aeschynomene sensitiva French Guiana +/++/+−/− FM242612 FM211246 TS
Aeschynomene sensitiva Guadeloupe, Belle Plaine +/++/+−/− FM242613 FM211247 TS
Aeschynomene sensitiva Guadeloupe, Pointe-à -Pitre +/++/+−/− FM242614 FM211248 TS
Aeschynomene tambacoundensis Senegal +/++/+−/− FM242634 FM211269 TS
Aeschynomene uniflora Dem. Republic of CongoIRRI No. 13158−/−−/−−/+ FM242635 KC560759 TS
Aeschynomene villosa Mexico −/−−/−−/+ FM242616 FM211250 TS
Aeschynomene villosa Mexico −/−−/−−/+ FM242617 FM211251 TS
Aeschynomene villosa Mexico −/−−/−−/+ FM242618 FM211252 TS
Aeschynomene villosa South AmericaUSDA PI 420300−/−−/−−/+ FM242619 FM211253 TS
Aeschynomene virginica USA VirginiaMatt Lavin Montana+/++/+−/− KC560766 FM211272 TS
Aeschynomene vogelii Brazil, Minas GeraisJ.A. Lombardi 3725    EF451089 EF451128 Ribeiro et al. (2007)
Adesmia lanata ArgentinaLavin 8256     AF208901 Lavin et al. (2001)
Bryaspis lupulina Sierra LeoneDawe 424    AF204234 AF208932 Lavin et al. (2001)
Dalbergia brasiliensis Brazil, São PauloInês Cordeiro    EF451076 EF451115 Ribeiro et al. (2007)
Dalbergia congestiflora El Salvador, Santa Ana, MetapanHughes 1253    AF068140 AF208924 Lavin et al. (2001)
Dalbergia villosa Brazil, Minas Gerais, Belo HorizonteJ.P. Lemos Filho S.n.    EF451068 EF451107 Ribeiro et al. (2007)
Discolobium psoraleifolium Argentina, Formosa,Cristobal & Krapovickas 2167    AF189058 AF208964 Lavin et al. (2001)
Discolobium pulchellum Bolivia, Santa Cruz: Chiquitos,Frey et al. 531    AF189059 AF208963 Lavin et al. (2001)
Diphysa ormocarpoides México, Oaxaca, San Pedro TotalapanSaynes V. 1286 (MEXU)    AF068168 AF208912 Lavin et al. (2001)
Geissaspis descampsii MalawiHilliard & Burtt 4305     AF208931 Lavin et al. (2001)
Kotschya aeschynomenoides MalawiSalubeni 3060 (E)     AF208934 Lavin et al. (2001)
Kotschya lutea Guinea     KC560762 KC560761 TS
Kotschya ochreata French GuineaArmour 8400     AF208935 Lavin et al. (2001)
Machaerium acutifolium Brazil, Minas Gerais, Nova PonteE. Tameirão Neto 2190    EF451090 EF451129 Ribeiro et al. (2007)
Machaerium nyctitans Brazil, Minas Gerais, IgarapéC.V. Mendonça 455    EF451082 EF451121 Ribeiro et al. (2007)
Machaerium opacum Brazil, Minas Gerais, São Gonçalo do Rio PretoJ.A. Lombardi 4068    EF451097 EF451137 Ribeiro et al. (2007)
Ormocarpopsis itremoensis Madagascar, FianarantsoaDuPuy 2363    AF068149 AF208918 Lavin et al. (2001)
Ormocarpum keniense Kenya, MeruFaden 74/958    AF068155 AF208917 Lavin et al. (2001)
Pictetia marginata Sauv.Cuba, Hoguín, Sierra NipeLavin 7108    AF068176 AF208910 Lavin et al. (2001)
Poecilanthe parviflora Brazil, Rio de JaneiroLima s.n.    AF187089 AF208897 Lavin et al. (2001)
Smithia abyssinica EthiopiaILRI 8360    KC560763 KC560758 TS
Smithia ciliata NepalStainton 4048 (E)     AF208933 Lavin et al. (2001)
Soemmeringia semperflorens Brazil, RoraimaLewis 1600    AF189027 AF208937 Lavin et al. (2001)
Weberbauerella brongniartioides Perú, Arequipa, Lomas de MollendoDillon 3909    AF189028 AF208909 Lavin et al. (2001)
Zornia sp.México, Zacatecas, FresnilloLavin 5039    AF183500 AF208903 Lavin et al. (2001)

Plant DNA extraction, amplification, and sequencing

Total genomic DNA was isolated using the modified CTAB extraction method (Doyle & Doyle, 1987) from new leaves of plants germinated from seeds and which were grown in our glasshouse. The chloroplast DNA trnL intron (Bakker et al., 2000) and the nuclear ribosomal DNA ITS/5.8S region (Baldwin et al., 1995) were chosen for phylogenetic analyses because they have been shown to be informative within and among closely related legume genera (Lavin et al., 2001; Ribeiro et al., 2007). The trnL (UAA) intron was amplified and sequenced using primers B49317 and A49855 (Taberlet et al., 1991). Primer pairs used for PCR to amplify and sequence the ITS region flanked the end of the 18S RNA gene ITS18 and the beginning of the 26S RNA gene ITS26 (Kass & Wink, 1997; Beyra-M & Lavin, 1999; Delgado-Salinas et al., 1999). The PCR products were purified using a Quiaquick PCR purification kit (Qiagen) according to the manufacturer's instructions. Sequencing was performed by Macrogen inc. (Seoul, Korea) using a ABI Prism 377 DNA sequencer.

Bacterial strains and culture growth conditions

For both root and stem inoculation, three rhizobial strains were used: the nod gene-lacking photosynthetic Bradyrhizobium strain ORS278, the nod gene-containing photosynthetic Bradyrhizobium strain ORS285, and the ‘classical’ nonphotosynthetic Bradyrhizobium diazoefficiens strain USDA110. All strains were grown aerobically in Yeast Mannitol (YM) medium (Vincent, 1970) on a gyratory shaker (170 rpm) at 37°C, under a light : dark cycle (16 h : 8 h) for photosynthetic Bradyrhizobium.

Root and stem inoculation

Aeschynomene seeds were surface-sterilized and dormancy was broken with 96% H2SO4 for 10–45 min, depending on the seed size for each species, and then rinsed six times in sterile distilled water to remove all traces of acid. To allow germination, seeds were placed in sterile water for 24 h at 30°C. They were then transferred either to Gibson tubes to test for root nodulation, or to glasshouse pots (plastic pots 8 cm in diameter) containing 500 g of sterilized sandy soil for stem nodulation. Inoculations of plants by the different rhizobial strains were performed both at the root level (via inoculation in hydroponic conditions, owing to the semiaquatic habit of the tested plants) and at the stem level (via inoculation of nonsubmerged stems by their careful ‘painting’ with the inocula), as already described (Giraud et al., 2000). Roots of plants grown in tubes were observed for root nodule formation 2–3 wk after inoculation. Stems of plants grown in the glasshouse were observed for stem nodulation 2–4 wk after inoculation.

Sequences alignment and phylogenetic analyses

Multiple alignments were performed for the trnL sequences with ClustalX, version 1.63b (Larkin et al., 2007), and alignments were manually corrected using GeneDoc (Nicholas et al., 1997). Phylogenetic reconstruction was performed using a maximum likelihood approach. The best model of molecular evolution for the trnL alignment was chosen using jmodeltest (Darriba et al., 2012). Most probable trees were obtained using Phyml (Guindon et al., 2009) by implementing previously estimated parameters. Statistical tests for branch supports were estimated using nonparametric bootstraps calculated on 100 replicates, implemented in Phyml.

Owing to the very high amount of nucleotide divergence among the ITS sequences, a confident alignment, including all taxa, could not be achieved easily with the former approach. We therefore conducted a Markov Chain Monte Carlo (MCMC) analysis using the software package Bali-Phy (Suchard & Redelings, 2006). Alignment uncertainties are taken into account by integrating the overall alignments in proportion to their posterior probabilities conjointly with phylogeny topology estimations. Because of the slowness of the analysis with the entire data set, we performed a first run with only 17 sequences of the 63 in the data set. These sequences were chosen according to a rough alignment obtained with ClustalX followed by NJ clustering. Each cluster was checked for the high (and alignable) similarity among sequences falling in it, and one sequence per clade was chosen. We implemented a Tamura-Nei model of molecular evolution (Tamura & Nei, 1993) with the RS07 (Redelings & Suchard, 2007) insertion/deletion model. Five independent runs were performed with this data subset, with 30 000 iterations each. Following the Bali-Phy user's guide, we estimated the SD across runs of the posterior probabilities for each run and averaged the values across splits. We also used the potential scale reduction factor to check that different runs had similar posterior distributions. The consensus alignment of the 17 sequences was then used in a second step as a guideline for the alignment of the other sequences. Based on this alignment, we estimated three different models of molecular evolution corresponding to the three ITS1, 5.8S and ITS2 regions that clearly displayed very different amounts of mutation accumulation, and reconstructed the phylogeny using these three models implemented in MrBayes software (Ronquist et al., 2012). Node supports were estimated with the posterior probabilities obtained in the MCMC analysis.

In both phylogenies, the two sequences from Andira galeaottiana and Vatairea sp. were chosen as outgroups, based on both traditional classification (Polhill, 1981) and the results of previous DNA phylogenies (Hu et al., 2000; Lavin et al., 2001; Ribeiro et al., 2007).

The topologies of the two phylogenetic trees being roughly similar, we concatenated the two data sets and reconstructed a combined tree. ITS sequences were missing for five species (Smithia ciliata, Kotschya aeschynomenoides, Kotschya ochreata, Aeschynomene purpusii, and Geissaspis descampsii), and these were indicated as missing data in the data matrix. We used MrBayes software (Ronquist et al., 2012) and applied different models of molecular evolution on each dataset partition. The four models (three for ITS and one for trnL) applied to the full dataset were used in an MCMC phylogenetic search. Searches were performed three times to check that the same equilibrium and final topology were achieved each time.

In order to study the evolution of the genus, we mapped onto the concatenated ITS-trnL phylogeny the two nodulation characters, that is, stem and Nod-independent nodulation abilities. We applied an unordered and equally weighted scheme, considering that transitions in either direction between the different states of character were equally likely, with no a priori assumptions concerning the ancestral state of the clade.

Data deposition

Sampled species, their localities, voucher specimens, and GenBank data base accession numbers for trnL intron and ITS sequences are listed in Table 1.


ITS and trnL sequences

We sequenced 65 different plant accessions (see Table 1) from Aeschynomene and other genera in this study. Together with other sequences retrieved from the databank, in total we analyzed the sequences for 71 Aeschynomene accessions and 24 from other related species. In contrast to almost the whole set of analyzed accessions for which single PCR products were obtained after amplifying the ITS and trnL markers, the electrophoregrams obtained for the ITS sequences from some A. indica accessions displayed several double peaks. As these double peaks were common and at the same position as these accessions, we cloned and sequenced the PCR product for the accession USDA PI225551, and two different sequences were subsequently obtained. A similar situation was also previously observed for an accession of Aeschynomene evenia (IRFL 6945), for which two different ITS sequences were obtained (Arrighi et al., 2013). Therefore, for these two species (A. indica and A. evenia), as the two sequences obtained were very similar each time they were analyzed (six Single Nucleotide Polymorphisms over 617 bp for both), and also fell into the same clade (data not shown), we decided to consider only one copy of ITS for each species for the subsequent phylogenetic analysis.

The Aeschynomene ITS region obtained for 70 accessions ranged in length from 597 bp (Aeschynomene filosa) to 623 bp (A. americana). The trnL regions were sequenced for 63 Aeschynomene accessions, and eight other sequences were retrieved from GenBank. Sequences ranged from 436 bp (A. villosa and Aeschynomene parviflora) to 477 bp (A. afraspera and A. nilotica). Five Aeschynomene species were represented by > two accessions: A. americana (six accessions), A. indica (11), A. sensitiva (seven), Aeschynomene rudis (four) and A. villosa (four). The within-species nucleotide divergence among sequences was low, ranging from 0 to 1% for the ITS sequences. Divergence was even lower for trnL sequences, with a maximum value of 0.4% (two mutations over the 456 bp sequence length for A. indica). Phylogenetic analyses always clustered all accessions from the same species in the same clade (data not shown). Consequently, we only kept one accession per species in all subsequent analyses.

Alignments and phylogenetic analysis

The trnL aligned matrix included 39 sequences, each from a different Aeschynomene species, and 31 other species from 22 different genera. The ITS analyses resulted in a 786 bp long matrix, with 38 Aeschynomene sequences and 27 from other genera. The two phylogenies being very similar, we combined the two data sets into a single phylogenetic analysis (the two single locus trees are given and described in Supporting Information Figs S1, S2).

For this, we reconstructed a Maximum Likelihood Phylogeny (Fig. 2). Four Aeschynomene clades were found, each supported with high posterior probabilities from the MrBayes analysis. Clade 1 grouped together 10 Aeschynomene species, all from subgenus Ochopodium, in a sister clade to the two Machaerium and Dalbergia genera. Other Aeschynomene species fell into the same monophyletic branch, but were mixed with other genera. The first emerging clade 2 grouped four Aeschynomene species, clade 3 grouped 12 species, and finally clade 4 grouped together 12 Aeschynomene species plus five other genera in a clade with an unresolved basal branching. Several species from each of the two genera Smithia and Kotschya were grouped together, suggesting a true evolutionary relationship among them within each genus. A close relationship among Aeschynomene bella, Aeschynomene abyssinica and Geissaspis descampii was observed, but this was based on a single trnL sequence of G. descampii retrieved from Genbank and thus should be verified with alternative sequences and vouchers. The grouping of Bryaspis lupulina with several Aeschynomene species in an unresolved, well supported clade is also notable. It is worth noting that the two stem-nodulating Discolobium species, which were included in the phylogeny, formed another clade that clearly belongs to the Dalbergia tribe, but it is not related to the genus Aeschynomene.

Figure 2.

ITS+trnL combined data set phylogeny. The phylogenetic reconstruction was done using Phyml (Guindon et al., 2009) (see the main text for details). Open squares indicate a posterior probability (PP) of this node > 0.95 in the Phyml analysis. Closed squares indicate a PP between 0.75 and 0.95. Groups (I, II, III) are related to Aeschynomene nodulation group (see Fig. 1). The red star indicates clade 3 within which clusters are all Nod gene-independent Aeschynomene species (i.e. group III, framed in a gray box bordered with a complete line). The group II Aeschynomene species are framed in the two gray boxes bordered with a dotted line. Species that were tested for nodulation and that were classified as belonging to groups I or II are indicated with a I or II following their name (all species from group III were tested). A crossed green circle indicates the emergence of the stem nodulation ability in a parsimonious character reconstruction evolution. The number (1–6) following a species name refers to illustrations of stem nodulation. Picture 4 (Aeschynomene uniflora) illustrates an example of group I species forming adventitious root nodules with strain USDA110 on stems under flooded conditions. A red circle frames sparse nodulation in pictures 1–3.

Evolution of symbiotic features among Aeschynomene groups

Fifty-six Aeschynomene accessions were tested for stem and root nodulation using three different strains, USDA110, ORS285 and ORS278, which permitted the distinction of the three inoculation groups defined by Alazard (1985) (cf. Fig. 1). Our tests did not reveal any within-species variation for nodulation phenotype when several accessions were used (Table 1). Among the 26 Aeschynomene species tested, 10 were nodulated only by strain USDA110, and formed only root nodules, and thus these Aeschynomene species were classified as belonging to the inoculation group I. Four species formed root nodules with USDA110, and root and stem nodules with the nod gene-containing strain ORS285, and thus belonged to the inoculation group II. Aeschynomene fluminensis could form stem nodules with ORS285, but no stem nodules in nonsubmerged conditions, which is in accordance with the previous observations of Loureiro et al. (1995), who showed that this species could form stem nodules with photosynthetic bacteria, but only under flooded conditions. Interestingly, among the Aeschynomene species, only A. crassicaulis and Aeschynomene fluitans possess the notable characteristic of having a floating stem with nodules developed on it (Fig. 2). For A. crassicaulis, we confirmed it could be nodulated only by the nonphotosynthetic strain USDA 110, and that flooding was compulsory for stem nodulation, as reported previously by Boivin et al. (1997). Finally, 12 species formed root and stem nodules with ORS285, but also with the nod gene-lacking strain ORS278, and were thus defined as belonging to inoculation group III.

We mapped the two symbiotic characters, stem nodulation and Nod-independent nodulation, onto the combined data set phylogeny. For other genera for which we did not perform any nodulation tests, and for which no published information is available with regard to their stem nodulation, we considered them as classical root-nodulating species. Similarly, none of them were considered as able to nodulate with strains lacking nod genes, even if we could not firmly exclude this option (see the 'Discussion' section). Based on an unweighted scheme, and no a priori assumptions about ancestral states, the concatenated phylogeny suggested at least three independent emergences of the stem nodulation ability, leading to A. fluminensis, clade 3, and a group of four species within clade 4 (Fig. 2).

The evolutionary pattern of the history of the Nod gene-independent character appears to be relatively simple, as all the Aeschynomene species that can form an efficient symbiosis with the nod gene-lacking strain clustered in a single clade, thus revealing a unique emergence of this ability.


All the aquatic Aeschynomene species fall into a single clade, but delineation of the genus requires revision

The genus Aeschynomene is complex, containing between 160 and 180 different species, possibly more, and is still increasing in size, as suggested by the recently described new species Aeschynomene sousae and Aeschynomene sabulicola (Queiroz & Cardoso, 2008; Delgado-Salinas & Sotuyo, 2012). As shown in previous studies (Lavin et al., 2001; Ribeiro et al., 2007), the genus Aeschynomene is polyphyletic in the two phylogenies we obtained, with species falling into two well-separated clades. The fact that the ITS and trnL sequences are retrieved from nuclear and chloroplastic genomes, respectively, reinforces the confidence we have in a true evolutionary split between the two clades. Although the aim of this study was not to redefine the borders of the genus, the phylogenies strongly suggest that the subgenus Ochopodium (clade 1 in our phylogeny) should be elevated to the genus rank, as a sister clade of Machaerium. Moreover, since A. aspera L., the type species of the genus Aeschynomene, falls into clade 4, the subgenus Ochopodium could not retain the name ‘Aeschynomene’ and would thus require renaming. All the other Aeschynomene species fall into the same main branch. The first two emerging clades (2 and 3) mostly contain American species (with the exception of Aeschynomene tambacoundensis, which is endemic to West Africa, and A. indica, which has a pantropical distribution), whereas clade 4 includes African species and one Asian species (A. aspera). America thus appears to be the center of origin of the genus, with a secondary center of diversification in Africa.

All these Aeschynomene species within this main branch (i.e. clades 2–4) share the same aquatic or semiaquatic habitat. Other related genera (Kotschya, Smithia, Geissaspis, Soemmeringia, and Bryaspis) can also be found in humid habitats, although they are not described as hydrophytes (Lewis et al., 2005). This ability to grow in or at the border of ponds (either permanent or temporary) has apparently been acquired before the diversification of all these species and genera, and might play a role in the ability of some Aeschynomene species to make stem nodules (see next paragraph).

The intermingling of the different genera makes their true delimitation unclear. Bryaspis and Soemmeringia include two and one species, respectively, whereas the others contain from three (Geissaspis) to 30 (Kotschya) different species. We cannot rule out the possibility that the molecular phylogenies, based on two sequences, might give false, or uncertain, evolutionary reconstructions. In addition, misidentification, as well as other factors that could have confused the phylogenetic pattern (i.e. lineage sorting, ancestral polymorphism) might have interfered in our results. Nevertheless, and in spite of these possibilities, whether several Aeschynomene species should be transferred to another existing genus, or conversely, whether several other genera should be included within a larger genus Aeschynomene remains an open question that should be explored further.

The stem nodulation character has emerged, or been maintained, several times in the aquatic Aeschynomene species

The scarcity of stem nodulation among the legume genera has been underlined previously, being reported in only four different genera (Aeschynomene, Discolobium, Neptunia, and Sesbania; Boivin et al., 1997), with ‘genuine’ stem nodules only recognized in the first two genera, as defined in the 'Introduction'. The distribution of stem-nodulating species, with or without genuine stem nodules, throughout the Leguminosae (Lavin et al., 2001; Wojciechowski et al., 2004; the present study) strongly suggests that the ability to form these structures on stems has evolved independently several times.

Contrary to the three other stem-nodulating genera, stem nodulation in the genus Aeschynomene is widespread, which leaves open the possibility of analyzing its evolution at a much narrower evolutionary level.

Excluding clade 1 (Ochopodium) from the analysis, the most parsimonious reconstruction of evolution suggests that the ‘stem nodulation ability’ has possibly emerged three times during the diversification of the genus. The true number of transition and/or reversions is obviously difficult to assess, especially as the probability of occurrence of each event is most certainly unequal. In addition, its distribution among all these species clearly shows that this character is not stable, with several emergences or losses occurring alternately within a short period (at least in evolutionary terms).

Interestingly, clades 2–4 include the hydrophytic Aeschynomene species, and as all stem-nodulated Aeschynomene are hydrophytes, it suggests an influence of waterlogged/flooded conditions on the emergence of the stem nodulation characteristic. All the other genera within clade 4 grow along riverbanks or ponds (Lewis et al., 2005), but none have been reported to form stem nodules. A hydrophytic ecology thus appears to be an essential requirement for the evolution of the ability to form stem nodules, but it is not on its own sufficient for it, and so it would appear to be also related to factors other than environmental ones, such as bacterial ecology, or to specific recognition mechanisms between the bacteria (rhizobia) and the host plants.

Stem nodulation in the genus Aeschynomene might have evolved in a two-step process, first with a genetic predisposition (at the base of the clade) to produce adventitious root initials all along the stem. Previous studies have shown that rhizobia colonize the stem via epidermal fissures (cracks) generated by the emergence of adventitious root primordia (Sprent, 1989; Boogerd & van Rossum, 1997) A second, still unknown, mutation that would have appeared several times will have led to the various clades in which the true stem-nodulating species fall. This is possibly linked to the ability of protruding root primordia to pierce the epidermal layer and thus to form at their base a large annular cavity in which the bacteria can easily multiply (Boivin et al., 1997; Giraud et al., 2000). Deciphering the genetic mutation(s) that drive stem nodulation in each species would help in confirming this two-step hypothesis.

The phylogeny of Aeschynomene suggests that Nod gene-independent nodulation is a derived character

All Aeschynomene species that are able to form an efficient symbiosis with bacteria lacking nodulation genes, and thus not producing NFs, fall into a single clade. Moreover, this clade does not include any other species that compulsorily require NFs to interact efficiently with symbiotic rhizobia. The most parsimonious and simplest view of evolution leads to a single emergence of the capacity to interact with bacteria without the production of NF. Consequently, Nod gene-independent nodulation should be viewed as a derived and more recent character compared with NF-mediated nodulation, as previously suggested by Okubo et al. (2012).

However, this evolutionary scenario might not be so simple. Recently, Madsen et al. (2010) showed that Lotus japonicus double mutants, affected in some determinants of the NF perception and signaling pathway, were occasionally able to form functional nodules when the plants were inoculated with a compatible rhizobial strain unable to produce NF. In such cases, nodules could be formed after a bacterial intercellular infection of the root (i.e. without the formation of infection threads). Based on these results, the authors suggested that direct intercellular infection may constitute an ancient invasion path, and that the most highly evolved state envisaged would be the root hair infection mode that requires NF receptors, which is in direct contradiction with our phylogenetic conclusions based upon the genus Aeschynomene.

The uncertainty as to whether NF independence is an ancestral or derived character cannot be dispelled easily. The symbiosis between actinorhizal plants and Frankia, which is considered to have emerged before the Rhizobium–legume symbiosis, does not involve the synthesis of NFs by the bacteria, but it does recruit some common determinants of the Nod-dependent signaling pathway described in model legumes (Normand et al., 2007; Gherbi et al., 2008). Following on from these hypotheses, Okubo et al. (2012) raised several possible scenarios, including both Nod gene-independent and Nod gene-dependent ancestries, and even an hypothesis in which the Aeschynomene ancestor was not nodulated, but later acquired the Nod gene-independent symbiotic pathways. It should also be stressed that, as underlined by Madsen et al. (2010), the two alternative invasion modes, Nod gene-independent or Nod gene-dependent, are not mutually exclusive.

The question of whether NF independence is ancestral, derived, or mixed ability remains open, and illustrates how experimental vs phylogenetic approaches might give conflicting results. This question is closely akin to that posed by Masson-Boivin et al. (2009) who, among several outstanding questions to be explored, asked if ‘symbiosis evolved from primitive (e.g. NF-independent, crack entry) to sophisticated (NF, infection thread) strategies’, although it should also be recognized that the NF-independent symbiosis should not necessarily always be viewed as being more primitive than the NF-dependent one, as, in evolution, simplest does not necessarily mean less evolved. Deciphering the details of the NF-independent strategy will be the next step for elucidating which of these alternative evolutionary possibilities is the correct one. As suggested by Arrighi et al. (2012), it might be achieved through the use of A. evenia as a model legume, as it displays all the characteristics required for genetic and molecular analyses (i.e. it is a short-perennial and autogamous diploid species with a relatively small genome).

Nod-independent nodulation occurrence and evolution

We cannot fully reject the possibility that the Nod gene-independent nodulation process was retained in genera and clades other than Aeschynomene, especially those that are infected following a crack entry process, except that it has never been demonstrated or proven before. The intercellular infection process observed in Aeschynomene species is supposedly found in > 25% of legumes (Sprent, 2007). We may then consider that within these thousands of species, among which a majority have never been studied in terms of their nodulating symbiosis, many of them might be able to use a Nod gene-independent interaction mechanism with their symbiotic rhizobial partners. At present, no specific genetic markers exist, in either the plant or the bacterial symbionts, to easily detect such ability, or to study its frequency and distribution along a wide taxonomic and phylogenetic sampling. The only alternative currently available is to search for the presence, or not, of nod genes within bacterial symbionts isolated from nodules, which is not trivial, except through full genome sequencing. Moreover, one plant species might be able to interact with Nod gene-producing bacteria, but still be able to interact in a Nod gene-independent manner, such as observed for the group III Aeschynomene species, thus making it even more complex to analyze the system.

On the other hand, it is worth noting that all rhizobial strains sampled so far, either with or without nod genes, which can make efficient nodules with group III Aeschynomene species, fall into a single clade (Fig. 1). This pattern suggests that the ability to form nodules without NF, rather than being driven solely by the plant, has also been dependent on a specific single bacterial evolution/mutation. Aeschynomene species in group III only form an efficient symbiosis with group D and E bacterial isolates, and reciprocally, nod gene-lacking strains are strictly specific to group III Aeschynomene species. This reciprocal specificity is reminiscent of the gene-for-gene interaction in phytopathology (Flor, 1942), and opens up opportunities for a coevolution process. The current associations might thus have resulted from both the bacterial acquisition of a Nod gene-independent specific recognition mechanism (e.g. one linked to an unknown receptor), and a specific mutation in the plant ancestor, that together would have mediated the high specificity observed in this interaction. The scarcity of Nod gene-independent symbiotic interactions among legumes could then be explained by the requirement for such a joint evolution of the two symbiotic partners.

Obviously several questions remain, such as the nature of the evolutionary pressures that would have driven the concordant evolution between the two partners during the emergence of Nod gene-independent nodulation, as well as the role of photosynthesis in the diversification of the symbiotic bacteria. All these questions, plus the genetic and physiological investigations underlying them, will pave the way for many fascinating new studies.


We are very grateful to M. Boursot for help with the glasshouse experiments, and P. Tisseyre for management of strain collection. We would like to thank F. Crozier and S. Gonzalez from ‘Herbier de Guyane’ (Cayenne, IRD) for their help in sampling Aeschynomene plants in French Guiana and J. L. Contreras (Universidad Autónoma de Puebla, México) for help with sampling in Mexico. We also thank M. Zabaleta (University of Montevideo, Uruguay), L. G. Santos (CIAT, Colombia), A. Jorge (ILRI, Ethiopia) and S. Norton (AusPGRIS, Australia) for providing various seeds. We are extremely grateful to E. James for a remarkable re-reading and correction of the manuscript. We finally thank three anonymous reviewers for very helpful and interesting comments on an earlier version of this manuscript.