Research
Evolution of symbiosis in the legume genus Aeschynomene
Clemence Chaintreuil1, Jean-Francßois Arrighi1, Eric Giraud1, Lucie Miche2, Lionel Moulin1, Bernard Dreyfus1,
Jose-Antonio Munive-Hernandez3, Marıa del Carmen Villegas-Hernandez4 and Gilles Bena1,5
1
IRD/CIRAD/UM2/Supagro, Laboratoire des Symbioses Tropicales et Mediterraneennes, F-34398, Montpellier, France; 2CNRS/IRD/Aix-Marseille Universite, Institut Mediterraneen de
Biodiversite et d’Ecologie Marine et Continentale, 13397, Marseille, France; 3Benemerita Universidad Autonoma de Puebla, Laboratorio de Ecologıa Molecular Microbiana, Mexico, Mexico;
4
Instituto Politecnico Nacional, Escuela Nacional de Ciencias Biologicas, Mexico, Mexico; 5University Mohammed V Adgal, Laboratoire de Microbiologie et Biologie Moleculaire, Rabat,
Morocco
Summary
Author for correspondence:
Gilles B
ena
Tel: +33 4 67 59 38 01
Email: gilles.bena@ird.fr
Received: 21 May 2013
Accepted: 24 June 2013
New Phytologist (2013) 200: 1247–1259
doi: 10.1111/nph.12424
Key words: Aeschynomene, Bradyrhizobium,
evolution, Nod factor-independent nodulation,
phylogeny, rhizobia, stem nodulation.
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 nodABCcontaining 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.
Introduction
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 proteinrich seeds in the absence of nitrogen fertilizer in soils. Among
the three subfamilies of the Leguminosae (Caesalpinioideae,
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
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
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1248 Research
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
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
(b)
Aeschynomene symbiotic
bacterial group
(a)
A
USDA110 *
Outgroup R. palustris
Strains with nod
genes
USDA336
USDA110@
ORS354
B. japonicum
B. elkanii
B. diazoefficiens
group A & B
ORS285,
ORS278, Btai1
groups C & D
A.
americana I
A.
schimperi
A.
crassicaulis
elaphroxylon
A.
montevidensis Φ A.
pfundii
facalta
histrix
cristata Φ
uniflora
Root nodulation
ORS336,ORS354 *
Strains with nod
genes
ORS285 *
Strains with nod
genes but still able to
nodulate
Aechynomene group
III when these genes
are disrupted
D ORS278, Btai1 *
Strains without nod
genes
STM1844 group E
Group III
nodulating bacteria
clade
A.
A.
A.
A.
A.
B
C
Bradyrhizobia
clade
Aeschynomene species
inoculation group
E
STM3844 *
Strains without nod
genes
II
A. nilotica
A. fluminensis ♣
A. afraspera
A. aspera Φ
Stem and root nodulation
III
A.
A.
A.
A.
A.
A.
rudis
pratensis
scabra
virginica Φ
filosa Φ
tambacoudensis
A.
A.
A.
A.
A.
A.
evenia
indica
sensitiva
denticulata
ciliata
deamii Φ
Stem and root nodulation
Fig. 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 Miche 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.
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New Phytologist Ó 2013 New Phytologist Trust
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(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 (Miche 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).
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
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Research 1249
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 withinspecies 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).
Plant DNA extraction, amplification, and sequencing
Total genomic DNA was isolated using the modified CTAB
extraction method (Doyle & Doyle, 1987) from new leaves of
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Table 1 Characteristics of the species and samples included in the study
Species
Aeschynomene abyssinica
Aeschynomene afraspera
Aeschynomene afraspera
Aeschynomene americana
Aeschynomene americana
Aeschynomene americana
Aeschynomene americana
Aeschynomene americana
Aeschynomene americana
Aeschynomene aspera
Aeschynomene bella
Aeschynomene brasiliana
var brasiliana
Aeschynomene brasiliana
Aeschynomene brevifolia
Aeschynomene ciliata
Aeschynomene crassicaulis
Aeschynomene cristata
Aeschynomene deamii
Aeschynomene denticulata
Aeschynomene evenia
Aeschynomene elaphroxylon
Aeschynomene falcata
Aeschynomene falcata
Aeschynomene fascicularis
Aeschynomene filosa
Aeschynomene fluitans
Aeschynomene fluminensis
Aeschynomene fluminensis
Aeschynomene histrix
Aeschynomene indica
Aeschynomene indica
Aeschynomene indica
Aeschynomene indica
Aeschynomene indica
clone a
Aeschynomene indica
clone b
Aeschynomene indica
Aeschynomene indica
Aeschynomene indica
Aeschynomene indica
Aeschynomene indica
Aeschynomene indica
Aeschynomene martii
Aeschynomene
montevidensis
Country and
locality of origin
Zimbabwe
Senegal
Senegal
Costa Rica
Guadeloupe
(West Indies)
French Guiana,
Remire
Mexico
Panama
Venezuela
Sri Lanka
Tanzanie
Brazil, S~ao Paulo
Brazil
Madagascar
Colombia
Senegal,
Niokolokoba
Democratic
Republic of
Congo
Mexico,
Tlacotalpan,
Veracruz
Brazil
USA Floride
Senegal
Brazil
South Africa
Venezuela,
Merida
Mexico
Zambia
Colombia
Brazil
French Guiana,
Kourou
USA Louisana
Voucher number
Stem nodule/root nodule a
GenBank accession
ORS278
ITS/5.8S
trnL intron
Sequence
references
ORS285
USDA110
CPI 52331B
USDA PI 544341
USDA PI 544080
USDA PI 544113
USDA PI 544122
IRRI No. 13020
ILRI 18422
V. Stranghetti 765
CIAT7589
CPI 52335
IRRI No. 13078
/
/
/
/
+/+
+/+
/
/
/+
/+
/+
/+
KC54029
FM242584
FM242585
FM242591
FM242586
KC560746
FM211217
FM211218
FM211224
FM211219
TS
TS
TS
TS
TS
/
/
/+
FM242587
FM211220
TS
/
/
/
/
/
/
/
+/+
/+
/+
/+
/+
FM242588
FM242589
FM242590
FM242623
KC540628
EF451087
FM211221
FL211222
FM211223
FM211257
KC560747
EF451126
KC540627
KC540626
FM242624
FM242594
KC560748
KC560749
FM211258
FM211227
TS
TS
TS
TS
TS
Ribeiro et al.
(2007)
TS
TS
TS
TS
/+
FM242625
KC560760
TS
/
+/+
+/+
/
+/+
/
/
/
+/+
+/+
/
KC540625
FM211238
TS
IRRI No. 13003
USDA PI 572567
+/+
+/+
+/+
+/+
/
/
USDA PI 322289
USDA PI 364378
Lavin 5730
/
/
/
/
FM242626
KC163288
KC540624
FM242596
FM242597
AF189025
FM211260
FM211228
KC560751
FM211229
FM211230
AF208929
+/+
+/+
/
/
/
/
+/+
+/+
/
/+
/+
/+
KC540623
KC540622
FM242627
FM242598
FM242599
KC560752
KC560753
FM211261
FM211231
FM211232
TS
TS
TS
TS
TS
Lavin et al.
(2001)
TS
TS
TS
TS
TS
NLU3
U59892
AF208927
Carulli 58
AF068141
IRRI No. 12146
CPI 87516
CPI 52338
IRRI No. 11009
/+
/+
USA North
Carolina
Senegal
India
Zambia
USDA PI196206
USDA PI225551
+/+
+/+
+/+
+/+
+/+
+/+
/
/
/
FM242601
KC560764
FM211233
FM211234
FM211235
Lavin et al.
(2001)
Lavin et al.
(2001)
TS
TS
TS
Zambia
USDA PI225551
+/+
+/+
/
KC560765
FM211235
TS
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
/
/
/
/
/
/
FM242603
EF451088
FM211236
FM211242
FM211254
FM211255
FM211256
FM211266
EF451127
TS
TS
TS
TS
TS
TS
Ribeiro et al.
(2007)
KC540621
KC560754
TS
Australia
Senegal, Kaolack
Senegal, Kaolack
Senegal
Senegal
Zimbabwe
Brazil, Minas
Gerais, Mato
Verde
Uruguay
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IRRI No. 13015
V.C. Souza 5455
M. Zabaleta.
Montevideo
/
/
FM242621
Ó 2013 The Authors
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Table 1 (Continued)
Species
Country and
locality of origin
Aeschynomene pfundii
Senegal
Brazil, Minas
Gerais, Belo
Horizonte
Colombia
Zimbabwe,
National Botanic
Gardens
Zimbabwe
Aeschynomene pratensis
Aeschynomene purpusii
Brazil
Mexico, Oaxaca
Aeschynomene rudis
USA, Florida
Aeschynomene rudis
Mexico, Laguna,
Veracruz
Brazil
Mexico, Juan
Campestre,
Veracruz
Mexico, Tenango,
Morelos
Mexico
Senegal
Guadeloupe,
Pointe-a-Pitre
Senegal
French Guiana
French Guiana
French Guiana
Guadeloupe,
Belle Plaine
Guadeloupe,
Pointe-a -Pitre
Senegal
Aeschynomene nilotica
Aeschynomene paniculata
Aeschynomene parviflora
Aeschynomene pfundii
Aeschynomene rudis
Aeschynomene rudis
Aeschynomene scabra
Aeschynomene scabra
Aeschynomene schimperi
Aeschynomene sensitiva
Aeschynomene sensitiva
Aeschynomene sensitiva
Aeschynomene sensitiva
Aeschynomene sensitiva
Aeschynomene sensitiva
Aeschynomene sensitiva
Aeschynomene
tambacoundensis
Aeschynomene uniflora
Aeschynomene villosa
Aeschynomene villosa
Aeschynomene villosa
Aeschynomene villosa
Aeschynomene virginica
Dem. Republic
of Congo
Mexico
Mexico
Mexico
South America
USA Virginia
Voucher number
IRRI No. 14040
P.O. Moraes. J.A.
Lombardi 2689
Stem nodule/root nodule a
GenBank accession
ORS278
/
USDA110
ITS/5.8S
trnL intron
+/+
+/+
KC560767
EF451086
KC560756
EF451125
TS
Ribeiro et al.
(2007)
KC540620
AF189026
KC560755
AF208930
TS
Lavin et al.
(2001)
/+
FM242629
FM211263
TS
IRFL 2854
Matt Lavin.
Montana
IRRI No. 13006
Lavin 5325
Matt Lavin.
Montana
USDA PI 296044
IRRI No. 12156
IRRI No. 13158
/
/
+/+
+/+
/
FM242630
FM211264
AF208928
+/+
+/+
/
FM242631
FM211265
TS
Lavin et al.
(2001)
TS
+/+
+/+
/
FM242604
FM211237
TS
+/+
+/+
+/+
+/+
/
/
FM242609
FM242615
FM211243
FM211249
TS
TS
+/+
+/+
/
FM242605
FM211239
TS
+/+
/
+/+
+/+
/
+/+
/
/+
/
FM242632
FM242633
FM242606
FM211267
KC560757
FM211240
TS
TS
TS
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
/
/
/
/
/
FM242607
FM242610
FM242611
FM242612
FM242613
FM211241
FM211244
FM211245
FM211246
FM211247
TS
TS
TS
TS
TS
+/+
+/+
/
FM242614
FM211248
TS
+/+
+/+
/
FM242634
FM211269
TS
/
/
/+
FM242635
KC560759
TS
/
/
/
/
+/+
/
/
/
/
+/+
/+
/+
/+
/+
/
FM242616
FM242617
FM242618
FM242619
KC560766
FM211250
FM211251
FM211252
FM211253
FM211272
TS
TS
TS
TS
TS
EF451089
EF451128
Ribeiro et al.
(2007)
Lavin et al.
(2001)
Lavin et al.
(2001)
Ribeiro et al.
(2007)
Lavin et al.
(2001)
Ribeiro et al.
(2007)
Adesmia lanata
Brazil, Minas
Gerais
Argentina
USDA PI 420300
Matt Lavin
Montana
J.A. Lombardi
3725
Lavin 8256
Bryaspis lupulina
Sierra Leone
Dawe 424
AF204234
AF208932
Dalbergia brasiliensis
Brazil, S~ao Paulo
In^es Cordeiro
EF451076
EF451115
Dalbergia congestiflora
El Salvador, Santa
Ana, Metapan
Brazil, Minas
Gerais, Belo
Horizonte
Hughes 1253
AF068140
AF208924
J.P. Lemos
Filho S.n.
EF451068
EF451107
Aeschynomene vogelii
Dalbergia villosa
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
Sequence
references
ORS285
AF208901
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1252 Research
Table 1 (Continued)
Species
Country and
locality of origin
Discolobium
psoraleifolium
Argentina,
Formosa,
Discolobium pulchellum
Bolivia, Santa
Cruz: Chiquitos,
Mexico, Oaxaca,
San Pedro
Totalapan
Malawi
Diphysa ormocarpoides
Geissaspis descampsii
Kotschya
aeschynomenoides
Kotschya lutea
Kotschya ochreata
Machaerium acutifolium
Machaerium nyctitans
Machaerium opacum
Ormocarpopsis
itremoensis
Ormocarpum keniense
Pictetia marginata Sauv.
Poecilanthe parviflora
Smithia abyssinica
Smithia ciliata
Soemmeringia
semperflorens
Weberbauerella
brongniartioides
Zornia sp.
Malawi
Guinea
French Guinea
Brazil, Minas
Gerais, Nova
Ponte
Brazil, Minas
Gerais, Igarape
Brazil, Minas
Gerais, S~ao
Goncßalo do Rio
Preto
Madagascar,
Fianarantsoa
Kenya, Meru
Cuba, Hoguın,
Sierra Nipe
Brazil, Rio de
Janeiro
Ethiopia
Nepal
Brazil, Roraima
Per
u, Arequipa,
Lomas de
Mollendo
Mexico,
Zacatecas,
Fresnillo
Stem nodule/root nodule a
GenBank accession
ORS278
ITS/5.8S
trnL intron
Cristobal &
Krapovickas
2167
Frey et al. 531
AF189058
AF208964
Lavin et al.
(2001)
AF189059
AF208963
Saynes V. 1286
(MEXU)
AF068168
AF208912
Lavin et al.
(2001)
Lavin et al.
(2001)
Voucher number
ORS285
USDA110
Hilliard & Burtt
4305
Salubeni
3060 (E)
AF208931
AF208934
KC560762
KC560761
AF208935
E. Tameir~ao
Neto 2190
EF451090
EF451129
C.V. Mendoncßa
455
J.A. Lombardi
4068
EF451082
EF451121
EF451097
EF451137
DuPuy 2363
AF068149
AF208918
Faden 74/958
AF068155
AF208917
Lavin 7108
AF068176
AF208910
Lima s.n.
AF187089
AF208897
ILRI 8360
Stainton
4048 (E)
Lewis 1600
KC560763
KC560758
AF208933
AF189027
AF208937
Dillon 3909
AF189028
AF208909
Lavin 5039
AF183500
AF208903
Armour 8400
Sequence
references
Lavin et al.
(2001)
Lavin et al.
(2001)
TS
Lavin et al.
(2001)
Ribeiro et al.
(2007)
Ribeiro et al.
(2007)
Ribeiro et al.
(2007)
Lavin et al.
(2001)
Lavin et al.
(2001)
Lavin et al.
(2001)
Lavin et al.
(2001)
TS
Lavin et al.
(2001)
Lavin et al.
(2001)
Lavin et al.
(2001)
Lavin et al.
(2001)
TS, this study.
Results of the inoculation tests: +/+, nodules on stems and roots; /+, nodules only on roots; / , complete lack of nodulation.
a
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,
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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
Ó 2013 The Authors
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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
Ó 2013 The Authors
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Research 1253
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.
Results
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
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1254 Research
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
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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.
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.
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
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Research 1255
Andira galeottiana
Vatairea sp.
Poecilanthe parviflora
Apoplanesia paniculata
Amorpha fruticosa
Discolobium psoraleifolium
Discolobium genus
Discolobium pulchellum
Adesmia lanata
Chaetocalyx blanchetiana
Amicia glandulosa
Zornia sp.
Weberbauerella brongniartioides
Dalbergia villosa
Dalbergia brasiliensis
Dalbergia
genus
Dalbergia congestiflora
Machaerium nyctitans
Machaerium acutifolium
Machaerium genus
Machaerium opacum
A.martii
A.fascicularis
A.purpusii
A.histrix I
Clade 1 Group I
A.paniculata
A.vogelii
Subgenus
A.brasiliana
A.brevifolia
Ochopodium
A.brasiliana var. brasiliana
A.falcata I
Pictetia marginata
Diphysa ormocarpoides
Ormocarpum coeruleum
Ormocarpopsis itremoensis
Ormocarpum keniense
A. americana I Clade 2
A. villosa I
A.fluminensis II
Group I & II
A.parviflora
A.montevidensis I
A.deamii
A.filosa
A.tambacoundensis
A.pratensis
A.sensitiva
A.ciliata
A.denticulata
A.evenia (1)
Clade 3
A.indica (2) group
A.scabra
Group III
A.rudis (3)
III
A.virginica
Soemmeringia semperflorens
Smithia abyssinica
Smithia ciliata
A.bella
1
2
: Posterior probability > 0.95
: 0.75 > Pp > 0.95
Nod-independent nodulation
Stem nodulation
Clade 4
Group I & II
+ Bryaspis genus
3
A.abyssinica
Geissaspis descampsii
Kotschya aeschynomenoides
Kotschya lutea
Kotschya ochreata
Bryaspis lupulina
A. crassicaulis I
A. pfundii I
A.elaphroxylon I
A. uniflora I (4)
A.cristata I
A. schimperi I
A. aspera II
A. afraspera II (5)
A. nilotica II (6)
A. fluitans
4
5
6
0.06
Fig. 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.
Discussion
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
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
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
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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.
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 stem nodulation character has emerged, or been
maintained, several times in the aquatic Aeschynomene
species
The phylogeny of Aeschynomene suggests that Nod
gene-independent nodulation is a derived character
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.
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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;
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Phytologist
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 geneindependent 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. NFindependent, crack entry) to sophisticated (NF, infection thread)
strategies’, although it should also be recognized that the NFindependent 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,
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
Research 1257
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.
Acknowledgements
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 Autonoma de Puebla, Mexico) 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.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 trnL maximum likelihood phylogeny.
Fig. S2 ITS maximum likelihood phylogeny.
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