# Absence of Cospeciation between the UnculturedFrankia Microsymbionts and the Disjunct Actinorhizal Coriaria Species

Authors: Imen Nouioui; Faten Ghodhbane-Gtari; Maria P. Fernandez; Abdellatif Boudabous; Philippe Normand; Maher Gtari
Journal: BioMed Research International
 (2014)

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## Abstract

Coriaria is an actinorhizal plant that forms root nodules in symbiosis with nitrogen-fixing actinobacteria of the genus Frankia. This symbiotic association has drawn interest because of the disjunct geographical distribution of Coriaria in four separate areas of the world and in the context of evolutionary relationships between host plants and their uncultured microsymbionts. The evolution of Frankia-Coriaria symbioses was examined from a phylogenetic viewpoint using multiple genetic markers in both bacteria and host-plant partners. Total DNA extracted from root nodules collected from five species: C. myrtifolia, C. arborea, C. nepalensis, C. japonica, and C. microphylla, growing in the Mediterranean area (Morocco and France), New Zealand, Pakistan, Japan, and Mexico, respectively, was used to amplify glnA gene (glutamine synthetase), dnaA gene (chromosome replication initiator), and the nif DK IGS (intergenic spacer between nifD and nifK genes) in Frankia and the matK gene (chloroplast-encoded maturase K) and the intergenic transcribed spacers (18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA) in Coriaria species. Phylogenetic reconstruction indicated that the radiations of Frankia strains and Coriaria species are not congruent. The lack of cospeciation between the two symbiotic partners may be explained by host shift at high taxonomic rank together with wind dispersal and/or survival in nonhost rhizosphere.


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## Body

## 1. Introduction

The genusFrankia comprises nitrogen-fixing actinobacteria that are able to induce perennial root nodules on woody dicotyledonous plants called actinorhizals [1]. The actinorhizal plant families belong to three dicotyledonous orders: Fagales (Betulaceae,Casuarinaceae, and Myricaceae), Rosales (Elaeagnaceae, Rhamnaceae, and Rosaceae), and Cucurbitales (Coriariaceae and Datiscaceae) [2]. Analysis of the molecular phylogeny of members ofFrankia genus consistently identifies four main clusters regardless of the typing locus used [3]. Three symbioticFrankia clusters containing strains able to establish effective nodules and fulfill Koch’s postulates and one atypical with strains unable to establish effective nodulation on their host plants have been defined amongFrankia genera. Cluster 1 includesFrankia strains in association with Betulaceae, Myricaceae, and Casuarinaceae. Cluster 2 containsFrankia nodulating species from the Coriariaceae, Datiscaceae, and Rosaceae families as well asCeanothus of the Rhamnaceae.Frankia strains in cluster 3 form effective root nodules on plants from members of the Myricaceae, Rhamnaceae, Elaeagnaceae, andGymnostoma of the Casuarinaceae.SymbioticFrankia strains have been only isolated from Fagales (Frankia cluster 1) and the families Elaeagnaceae and Rhamnaceae (Frankia cluster 3) of the Rosales, whileFrankia of cluster 2 have still not yet been isolated in culture despite repeated attempts [2]. The position in theFrankia phylogenetic tree of cluster 2 relative to the other clusters has varied depending on the marker used. It was proposed at the base usingglnA and 16S rRNA genes [4, 5], derived with ITS 16S–23S rRNA genes [6] and concatenatedgyrB,nifH andglnII genes [7] and should be clarified by the upcoming whole genome phylogeny. Nevertheless, a position at the base of all symbiotic lineages has been retained in the latest treatment of Bergey’s manual [8].Cross-inoculation studies using crushed nodules suggest that cluster 2 strains form a separate and unique host specificity group [9–11], even though provenances from the full geographical range have not yet been tested. Despite the high taxonomic diversity of host plants belonging to the cross-inoculation group of cluster 2 and its disjunct range, unculturedFrankia in root nodules of several host plants have so far shown a low level of diversity regardless of the typing locus used [6, 7, 11–16], suggesting a recent emergence, a strong and recent evolutionary bottleneck, or a nonrepresentative sampling. The time of emergence of allFrankia lineages is poorly documented as no convincing fossil remains. An equivalence between 16S rRNA sequences distance and time of emergence has been proposed by Ochman and Wilson [17] where 1% is equivalent to 50 million years, and since 4% divergence exists betweenFrankiacluster 2 and the other clusters, one would conclude thatFrankia emerged 200  million years ago [5], which would mean that there is missing diversity either due to a recent evolutionary bottleneck or due to a lack of sampling [16]. A possibility thus exists that the missing variability in cluster 2 strains is due to the fact that sampling has so far been limited essentially to North American and Mediterranean areas.Evidence for cospeciation has been found so far only in the case ofCasuarina species growing in Australia and theirFrankia[18] that are in their immense majority resistant to growth in pure culture. Among actinorhizal plants of the Cucurbitales subclade, the family Coriariaceae, with only one genus,Coriaria, contains about 17 species [19] that occur in four disjunct areas of the world: the Mediterranean, Southeast Asia, Central and South America, and the Pacific islands of New Zealand and Papua New Guinea [20–24]. Yokoyama et al. [19] considered that the Eurasian species are basal and have emerged some 60 million years ago. This date is in agreement with the 65 million years proposed by Bell et al. [25] based on multiple genes (rbcL, 18S rDNA,atpB) phylogeny, while the same authors propose an emergence of the Casuarinaceae at about 30 million years.The present study was aimed at testing the hypothesis of cospeciation between unculturedFrankia microsymbionts and theirCoriaria host species sampled from sites covering the full geographical range of the genus:Coriaria myrtifolia (Morocco and France),C. nepalensis (Pakistan),C. arborea (New Zealand),C. japonica (Japan), andC. microphylla (Mexico).


## 2. Materials and Methods

### 2.1. DNA Extraction, PCR Amplification, and Sequencing

Root nodules from naturally occurringCoriaria species (Table 1) were kindly provided by Dr. María Valdés (Escuela Nacional de Ciencias Biológicas, México, DF, México), Dr. Sajjad Mirza (National Institute for Biotechnology Genetic Engineering, Faisalabad, Pakistan), Dr. Warwick Silvester (University of Waikato, Waikato, New Zealand), Dr. Kawther Benbrahim (University of Fes, Fes, Morocco), Dr. Takashi Yamanaka (Forest and Forestry Products Research Institute, Ibaraki, Japan), and Dr. Jean-Claude Cleyet-Marel (INRA-IRD, Montpellier, France). Individual lobes were selected, surface-sterilized in 30% (vol/vol) H2O2, and rinsed several times with distilled sterile water. The DNA extraction from single nodule lobes was performed as previously described by Rouvier et al. [26]. Nodule lobes were crushed with sterile plastic mortars and pestles in 300 μL of extraction buffer (100 mM Tris (pH 8), 20 mM EDTA, 1.4 M NaCl, 2% (wt/vol) CTAB (cetyltrimethyl ammonium bromide), and 1% (wt/vol) PVPP (polyvinyl polypyrrolidone)). The homogenates were incubated at 65°C for 60min, extracted with chloroform-isoamyl alcohol (24 : 1, vol/vol) and the resulting DNA was ethanol-precipitated and resolubilized. The extracted DNA was used for PCR amplification of both bacterial and plant DNA regions using the primers listed in Table 2. The amplicons were then cycle-sequenced in both directions using an ABI cycle sequencing kit (Applied Biosystem 3130). The nucleotide sequences obtained in this study were deposited in the NCBI nucleotide sequence database under the accession numbers given in Table 1.Table 1
List ofCoriaria root nodules and sequences used in this study.
Species
Locality coordinates/altitude (asl)
Nodule labels
Plant sequence accession number
Bacterial sequence accession number
References
ITS1-ITS2
matK
glnA
dnaA
IGSnifD-K
C. myrtifolia
Morocco
Oued El Koub, Ouezzane: 35°01′879N/05°20′565E/140 m
CmMs1
KC796592
KC796601
KC796522
KC796582
KC796555
This study
CmMs2CmMs3CmMs4
KC796523KC796524KC796525
KC796583KC796584KC796585
KC796556KC796557KC796558
This studyThis studyThis study
Bab Berred, Chefchaouen: 35°00′979N/04°58′092′′E/1290 m
CmM1a
KC796590
KC796599
KC796517
KC796578
KC796550
This study
CmM1b
KC796518
KC796579
KC796551
This study
CmM1c
KC796519
KC796580
KC796552
This study
CmM2a
KC796591
KC796600
KC796520
—
KC796553
This study
CmM2b
KC796521
KC796581
KC796554
This study
France
Nyons,  44°21′46.50′′N/5°08′21.82′′E/259 m
CmNy1
KC796598
KC796603
KC796531
KC796591
KC796564
This study
CmNy2CmNy3CmNy4CmNy5
KC796532KC796533KC796534KC796535
KC796592KC796593KC796594KC796595
KC796565—KC796566KC796567
This studyThis studyThis studyThis study
Montpellier, 43°36′51.48′′N/3°52′23.97′′E/41 m
CmF1
KC796526
KC796586
KC796559
This study
CmF2
KC796593
KC796602
KC796527
KC796587
KC796560
This study
CmF3CmF4CmF5
KC796528KC796529KC796530
KC796588KC796589KC796590
KC796561KC796562KC796563
This studyThis studyThis study
AF280102
Yang et al., unpublished
AB016459
(Yokoyama et al., 2000 [19])
C. japonica
Japan
Tosa district, +33°45′39.18′′, +133°27′42, 89′′/10 m
CjJA
KC796605
KC796536
KC796503
KC796576
This study
CjJB
KC796594
KC796537
KC796504
KC796577
This study
CjJCCjJDCjJE
KC796538KC796539KC796540
KC796505KC796506KC796507
KC796578KC796579KC796580
This studyThis studyThis study
AF280101
Yang et al., unpublished
AB016456
(Yokoyama et al., 2000 [19])
C. nepalensis
Pakistan
Murree, +33°54′15′′N 73°23′25′′E/33.9042°N 73.3903°E/2291.2 m
CnP1
KC796597
KC796607
KC796544
KC796508
KC796584
This study
CnP2
KC796545
KC796509
KC796585
This study
CnP3
KC796546
KC796510
KC796586
This study
CnP4
AF280103
Yang et al., unpublished
C. arborea
New Zealand
Hapuku river, North Canterbury, South island: −42°23′42.24′′, +173°41′18.07′′/64 m
CaNZ1
KC796595
KC796604
KC796542
KC796511
KC796581
This study
CaNZ2
KC796543
KC796512
KC796582
This study
CaNZ3
KC796544
KC796513
KC796583
This study
AB16454
(Yokoyama et al., 2000 [19])
EF635457
Rotherham et al., unpublished
EF635475
Rotherham et al., unpublished
AF277293
Yang et al., unpublished
C. microphylla
Mexico
Morelos, 99°30′, 19°30′/2400 m
CmicMx1
KC796596
KC796606
KC796547
KC796514
KC796587
This study
CmicMx2
KC796548
KC796515
KC796588
This study
CmicMx3
KC796549
KC796516
KC796589
This study
AY091813
Yang et al., unpublished
AB016458
(Yokoyama et al., 2000 [19])
C. intermedia
AF280100
Yang et al., unpublished
AB016455
(Yokoyama et al., 2000 [19])
C. terminalis
AY091817
Yang et al., unpublished
C. ruscifolia
AY091815
Yang et al., unpublished
AY091814
Yang et al., unpublished
AF280104
Yang et al., unpublished
AB016462
(Yokoyama et al., 2000 [19])
C. sarmentosa
AY091816
Yang et al., unpublished
AB016464
(Yokoyama et al., 2000 [19])
C. papuana
AB016461
(Yokoyama et al., 2000 [19])
Datisca glomerata
CP002801
CP002801
CP002801
(Persson et al., 2011 [50])
AY968449
Zhang et al., unpublished
AF485250
Forrest and Hollingsworth unpublished
Casuarina equisetifolia
CP000249
CP000249
CP000249
(Normand et al., 2007 [51])
AB015462
Sogo et al.,  unpublished
AY864057
Herbert et al.,  unpublishedTable 2
Primers used for PCR amplification and DNA sequencing.
Gene primers
Sequence (5′-3′)
Amplicons approximate size (bp)
References
glnA
DB41
TTCTTCATCCACGACCCG
500
(Clawson et al., 2004 [4])
DB44
GGCTTCGGCATGAAGGT
dnaA
F7154_dnaAF
GAGGARTTCACCAACGACTTCAT
700
Bautista et al. unpublished
F7155_dnaAR
CRGAAGTGCTGGCCGATCTT
IGSnifD-K
F9372_nifD1 5
GTCATGCTCGCCGTCGGNG
700
This study
F9374_nifK1 5
GTTCTTCTCCCGGTAyTCCCA
F9373_nifD2 5
ACCGGCTACGAGTTCGCNCA
700
This study
F9375_nifK2 5
TGCGAGCCGTGCACCAGNG
18S-ITS1-5.8S-ITS2-28S
ITS1
TCCGTAGGTGAACCTGCGG
700
(White et al., 1990 [52])
ITS4
TCCTCCGCTTATTGATATGC
F9030-CJ-ITSF
AGCCGGACCCGCGACGAGTTT
400
This study
F9031-CJ-ITSR
CGACGTTGCGTGACGACGCCCA
matK
F9249-matKF
ACATTTAAATTATGTGTCAG
700
This study
F9250-matkR
TGCATATACGCACAAATC


### 2.2. Phylogenetic Analysis

Frankia strain CcI3 andCasuarina equisetifolia were used as outgroups in this study because they are physiologically distinct from the group studied yet phylogenetically close. The data sets were completed with homologous sequences present in the databases (Table 1). Alignments ofFrankia glnA,dnaA, and IGSnifD-K andCoriaria matK and 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA were generated with ClustalW [27], manually edited with MEGA 5.0 [28]. Bacterial and plant sequences were separately concatenated and then used to examine maximum-likelihood cladogram evolutionary relationships of each symbiotic partner using 1000 bootstraps by following the GTR + G base substitution model. The distance between the sequences was calculated using Kimura’s two-parameter model [29]. Phylogenetic trees were constructed using the Neighbor-Joining method [30] with 1000 bootstraps [31] as implemented in MEGA 5.0. In parallel, a Bayesian inference was realized with MrBayes [32] using the GTR + G model and 1,000,000 generations.A statistical test for the presence of congruence betweenCoriaria andFrankia phylogenies was evaluated through global distance-based fitting in ParaFit program [33] as implemented in CopyCat [34] and tests of random association were performed with 9999 permutations globally across both phylogenies for each association.An additional statistical test for correlation between geographical distances (obtained usinghttp://www.daftlogic.com/projects-google-maps-distance-calculator.htm) and phylogenetic distances was made using Pearson’sr correlation implemented in the R software [35].


## 2.1. DNA Extraction, PCR Amplification, and Sequencing

Root nodules from naturally occurringCoriaria species (Table 1) were kindly provided by Dr. María Valdés (Escuela Nacional de Ciencias Biológicas, México, DF, México), Dr. Sajjad Mirza (National Institute for Biotechnology Genetic Engineering, Faisalabad, Pakistan), Dr. Warwick Silvester (University of Waikato, Waikato, New Zealand), Dr. Kawther Benbrahim (University of Fes, Fes, Morocco), Dr. Takashi Yamanaka (Forest and Forestry Products Research Institute, Ibaraki, Japan), and Dr. Jean-Claude Cleyet-Marel (INRA-IRD, Montpellier, France). Individual lobes were selected, surface-sterilized in 30% (vol/vol) H2O2, and rinsed several times with distilled sterile water. The DNA extraction from single nodule lobes was performed as previously described by Rouvier et al. [26]. Nodule lobes were crushed with sterile plastic mortars and pestles in 300 μL of extraction buffer (100 mM Tris (pH 8), 20 mM EDTA, 1.4 M NaCl, 2% (wt/vol) CTAB (cetyltrimethyl ammonium bromide), and 1% (wt/vol) PVPP (polyvinyl polypyrrolidone)). The homogenates were incubated at 65°C for 60min, extracted with chloroform-isoamyl alcohol (24 : 1, vol/vol) and the resulting DNA was ethanol-precipitated and resolubilized. The extracted DNA was used for PCR amplification of both bacterial and plant DNA regions using the primers listed in Table 2. The amplicons were then cycle-sequenced in both directions using an ABI cycle sequencing kit (Applied Biosystem 3130). The nucleotide sequences obtained in this study were deposited in the NCBI nucleotide sequence database under the accession numbers given in Table 1.Table 1
List ofCoriaria root nodules and sequences used in this study.
Species
Locality coordinates/altitude (asl)
Nodule labels
Plant sequence accession number
Bacterial sequence accession number
References
ITS1-ITS2
matK
glnA
dnaA
IGSnifD-K
C. myrtifolia
Morocco
Oued El Koub, Ouezzane: 35°01′879N/05°20′565E/140 m
CmMs1
KC796592
KC796601
KC796522
KC796582
KC796555
This study
CmMs2CmMs3CmMs4
KC796523KC796524KC796525
KC796583KC796584KC796585
KC796556KC796557KC796558
This studyThis studyThis study
Bab Berred, Chefchaouen: 35°00′979N/04°58′092′′E/1290 m
CmM1a
KC796590
KC796599
KC796517
KC796578
KC796550
This study
CmM1b
KC796518
KC796579
KC796551
This study
CmM1c
KC796519
KC796580
KC796552
This study
CmM2a
KC796591
KC796600
KC796520
—
KC796553
This study
CmM2b
KC796521
KC796581
KC796554
This study
France
Nyons,  44°21′46.50′′N/5°08′21.82′′E/259 m
CmNy1
KC796598
KC796603
KC796531
KC796591
KC796564
This study
CmNy2CmNy3CmNy4CmNy5
KC796532KC796533KC796534KC796535
KC796592KC796593KC796594KC796595
KC796565—KC796566KC796567
This studyThis studyThis studyThis study
Montpellier, 43°36′51.48′′N/3°52′23.97′′E/41 m
CmF1
KC796526
KC796586
KC796559
This study
CmF2
KC796593
KC796602
KC796527
KC796587
KC796560
This study
CmF3CmF4CmF5
KC796528KC796529KC796530
KC796588KC796589KC796590
KC796561KC796562KC796563
This studyThis studyThis study
AF280102
Yang et al., unpublished
AB016459
(Yokoyama et al., 2000 [19])
C. japonica
Japan
Tosa district, +33°45′39.18′′, +133°27′42, 89′′/10 m
CjJA
KC796605
KC796536
KC796503
KC796576
This study
CjJB
KC796594
KC796537
KC796504
KC796577
This study
CjJCCjJDCjJE
KC796538KC796539KC796540
KC796505KC796506KC796507
KC796578KC796579KC796580
This studyThis studyThis study
AF280101
Yang et al., unpublished
AB016456
(Yokoyama et al., 2000 [19])
C. nepalensis
Pakistan
Murree, +33°54′15′′N 73°23′25′′E/33.9042°N 73.3903°E/2291.2 m
CnP1
KC796597
KC796607
KC796544
KC796508
KC796584
This study
CnP2
KC796545
KC796509
KC796585
This study
CnP3
KC796546
KC796510
KC796586
This study
CnP4
AF280103
Yang et al., unpublished
C. arborea
New Zealand
Hapuku river, North Canterbury, South island: −42°23′42.24′′, +173°41′18.07′′/64 m
CaNZ1
KC796595
KC796604
KC796542
KC796511
KC796581
This study
CaNZ2
KC796543
KC796512
KC796582
This study
CaNZ3
KC796544
KC796513
KC796583
This study
AB16454
(Yokoyama et al., 2000 [19])
EF635457
Rotherham et al., unpublished
EF635475
Rotherham et al., unpublished
AF277293
Yang et al., unpublished
C. microphylla
Mexico
Morelos, 99°30′, 19°30′/2400 m
CmicMx1
KC796596
KC796606
KC796547
KC796514
KC796587
This study
CmicMx2
KC796548
KC796515
KC796588
This study
CmicMx3
KC796549
KC796516
KC796589
This study
AY091813
Yang et al., unpublished
AB016458
(Yokoyama et al., 2000 [19])
C. intermedia
AF280100
Yang et al., unpublished
AB016455
(Yokoyama et al., 2000 [19])
C. terminalis
AY091817
Yang et al., unpublished
C. ruscifolia
AY091815
Yang et al., unpublished
AY091814
Yang et al., unpublished
AF280104
Yang et al., unpublished
AB016462
(Yokoyama et al., 2000 [19])
C. sarmentosa
AY091816
Yang et al., unpublished
AB016464
(Yokoyama et al., 2000 [19])
C. papuana
AB016461
(Yokoyama et al., 2000 [19])
Datisca glomerata
CP002801
CP002801
CP002801
(Persson et al., 2011 [50])
AY968449
Zhang et al., unpublished
AF485250
Forrest and Hollingsworth unpublished
Casuarina equisetifolia
CP000249
CP000249
CP000249
(Normand et al., 2007 [51])
AB015462
Sogo et al.,  unpublished
AY864057
Herbert et al.,  unpublishedTable 2
Primers used for PCR amplification and DNA sequencing.
Gene primers
Sequence (5′-3′)
Amplicons approximate size (bp)
References
glnA
DB41
TTCTTCATCCACGACCCG
500
(Clawson et al., 2004 [4])
DB44
GGCTTCGGCATGAAGGT
dnaA
F7154_dnaAF
GAGGARTTCACCAACGACTTCAT
700
Bautista et al. unpublished
F7155_dnaAR
CRGAAGTGCTGGCCGATCTT
IGSnifD-K
F9372_nifD1 5
GTCATGCTCGCCGTCGGNG
700
This study
F9374_nifK1 5
GTTCTTCTCCCGGTAyTCCCA
F9373_nifD2 5
ACCGGCTACGAGTTCGCNCA
700
This study
F9375_nifK2 5
TGCGAGCCGTGCACCAGNG
18S-ITS1-5.8S-ITS2-28S
ITS1
TCCGTAGGTGAACCTGCGG
700
(White et al., 1990 [52])
ITS4
TCCTCCGCTTATTGATATGC
F9030-CJ-ITSF
AGCCGGACCCGCGACGAGTTT
400
This study
F9031-CJ-ITSR
CGACGTTGCGTGACGACGCCCA
matK
F9249-matKF
ACATTTAAATTATGTGTCAG
700
This study
F9250-matkR
TGCATATACGCACAAATC


## 2.2. Phylogenetic Analysis

Frankia strain CcI3 andCasuarina equisetifolia were used as outgroups in this study because they are physiologically distinct from the group studied yet phylogenetically close. The data sets were completed with homologous sequences present in the databases (Table 1). Alignments ofFrankia glnA,dnaA, and IGSnifD-K andCoriaria matK and 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA were generated with ClustalW [27], manually edited with MEGA 5.0 [28]. Bacterial and plant sequences were separately concatenated and then used to examine maximum-likelihood cladogram evolutionary relationships of each symbiotic partner using 1000 bootstraps by following the GTR + G base substitution model. The distance between the sequences was calculated using Kimura’s two-parameter model [29]. Phylogenetic trees were constructed using the Neighbor-Joining method [30] with 1000 bootstraps [31] as implemented in MEGA 5.0. In parallel, a Bayesian inference was realized with MrBayes [32] using the GTR + G model and 1,000,000 generations.A statistical test for the presence of congruence betweenCoriaria andFrankia phylogenies was evaluated through global distance-based fitting in ParaFit program [33] as implemented in CopyCat [34] and tests of random association were performed with 9999 permutations globally across both phylogenies for each association.An additional statistical test for correlation between geographical distances (obtained usinghttp://www.daftlogic.com/projects-google-maps-distance-calculator.htm) and phylogenetic distances was made using Pearson’sr correlation implemented in the R software [35].


## 3. Results

To avoid taxonomic ambiguities, DNAs from bothCoriaria hosts andFrankia microsymbionts were characterized on the same root nodule tissues. The method of DNA isolation from root nodules used in this study yielded PCR-amplifiable DNA for both bacterial and plant PCR target sequences in all cases. However, in several instances it was easier to amplifyFrankia thanCoriaria DNA, which may have been mostly due to the specificity of the primer sets used. Thus, in this study, new primers were designed (Table 2).For the bacterial microsymbionts, the average uncorrectedp-distances (proportion of differences between sequences) were computed for each region and were found to be relatively small fordnaA (p=0.0378), intermediate forglnA (p=0.0625), and high for IGSnifD-K region (p=0.0833). Blast analyses of the individual genes permitted assigning them all toFrankia cluster 2. Nearly 3000 nucleotides were obtained by concatenating sequences of the three DNA regions.Sequences variation forCoriaria species was small based onmatK gene (p=0.0205) compared to ITS1-ITS2 sequences (p=0.0423). By concatenatingmatK and ITS1- ITS2 region, a composite sequence of 1500 nt was used for phylogenetic inference.All studied sequences were analyzed independently to test for incongruence between the data sets for each symbiotic partner. Similar topologies have been generally observed between phylogenetic trees inferred fromglnA,dnaA, and IGSnifD-K sequences forFrankia and frommatK and ITS sequences forCoriaria regardless of the used phylogenetic methods (not shown).The topologies of the trees obtained for the two symbiotic partners were not congruent (Figure1). Moreover, global distance-based ParaFit analysis recovered mostly random associations betweenFrankia andCoriaria host plant species (p=0.33) and rejected cospeciation hypothesis. On the microbial side, the New Zealand microsymbionts were at the root (Group A); then three groups emerged, group B comprising the Pakistani, Mexican, and Mediterranean symbionts from France, group C comprising microsymbionts from Morocco, and then group D comprising French and Japanese microsymbionts as well as the Dg1 reference sequence obtained initially from a Pakistani soil. On the host plant side, group 1 at the root comprises New Zealand and South American sequences, while group 2 comprises the Japanese, Mediterranean, and Pakistani sequences.Figure 1
Phylogenetic trees of theFrankia microsymbionts (left) and theCoriaria host plants (right). TheFrankia tree was constructed using theglnA,dnaA, and thenifD-K intergenic spacer, while theCoriaria tree was done using thematK and the 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA with ML method using strain CcI3 and Casuarina as outgroups respectively for Frankia and hot plant phylogenetic trees. The numbers at branches indicate bootstrap results above 50%. Lines are drawn between the microsymbionts and their hosts. The color code indicates the place of origin of the leave or of the set when homogenous. The groups numbers 1 and 2 on the right are according to Yokoyama et al. [19].On the other hand, no significant correlations were found forFrankia symbionts (r2 = 0.772; Fgeneticdist = (geogdist × 5.830E-06) + 2.541E-02) nor for theCoriaria host plants (r2 = 0.883; Fgeneticdist = (geogdist × 2.023E-06) + 6.460E-03) (data not shown).


## 4. Discussion

Cospeciation has been postulated to have occurred in someFrankia actinorhizal host plants, in particular in theCasuarina-Frankia cluster 1b [18] but not inAlnus-infective andElaeagnus-infectiveFrankia strains where many isolates able to fulfill Koch’s postulates have been obtained. To test if cospeciation was general or an exception, it was decided to study unculturedFrankia microsymbionts and representativeCoriaria hosts, a lineage where noFrankia isolate exists and where geographic discontinuities may have limited dispersion. DNA sequences were obtained from root nodules collected from New Zealand (C. arborea), Pakistan (C. nepalensis), Japan (C. japonica), Mexico (C. microphylla), and France and Morocco (C. myrtifolia) and multiple molecular markers were analyzed for phylogenetic inference.Paleontological data based on macrofossils and pollen fossils have brought several authors [36–40] to conclude that the Coriariaceae had a Laurasian origin (North America and Eurasia). There have been a few dissenting opinions, in particular those of Croizat [41] and Schuster [42] who considered thatCoriariaoriginated in Gondwana and migrated to the Northern Hemisphere. However, such paleontological studies are not very convincing, as it is recognizably hard to ascribe fossils to a given family and even more so to a given genus. Thus, several authors have been surprised by the results of molecular phylogeny positioning Coriariaceae close to the Datiscaceae. Molecular approaches would thus give support to a Gondwanan origin.Yokoyama et al. [19] proposed thatCoriaria species had emerged 59–63 million years ago, which is coherent with the date of 70 million years proposed by Bell et al. [25], considerably older than that proposed (30 million years) by the same authors for the Casuarinaceae.Topology and clustering ofCoriaria phylogeny obtained in the current study are similar to those obtained by Yokoyama et al. [19], while the position at the base of the host plant species from New Zealand,C. arborea, and the South AmericanC. ruscifolia andC.microphyllaspecies was contrary to that of Yokoyama et al. [19] who found the Eurasian species at the base usingrbcL (a large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase) andmatK (maturase K) genes. The present study suggests that theCoriariaancestor may have emerged between Asia and NZ and then dispersed worldwide and that the Asian lineage may have given rise relatively recently to the Mediterranean species, while the NZ lineage gave rise to the North American species (Figure 2).Figure 2
Distribution ofCoriaria species. Root nodules have been sampled fromC. myrtifolia,C. arborea,C. nepalensis,C. japonica, andC. microphyllagrowing in Mediterranean areas (Morocco and France), New Zealand, Pakistan, Japan, and Mexico, respectively. Short arrows indicate sampling sites for this study while long arrows indicate possible routes of dispersal as discussed.Previous studies had concluded thatFrankia cluster 2 had a low genetic diversity [6, 7, 16] but these studies had been focused on only part of the full diversity of the symbioticCoriaria-Frankia, essentially in North America and Mediterranean. In this work we aimed to expand the scope of the study to the worldwide diversity and phylogeny of microsymbionts ofCoriaria species. Four microbial subgroups were identified that did not fit to the geographic range of the host plants, while two host plant subgroups were identified. The position of subgroup A containing microsymbionts of New ZealandC. arborea at the base ofFrankia cluster 2 is in agreement with previous study [16]. In view of previously reported data, members of cluster 2Frankia studied here were found to have relatively higher sequences variation (p-distance = 0.0625) than those reported by Vanden Heuvel et al. [16] (p=0.00454) based on the same 460 nt of theglnA gene.Molecular clock dating suggests thatFrankia genus has emerged much earlier, 125 Myr bp before the appearance of angiosperm fossils in the Cretaceous period and the extant actinorhizal plants [4]. Normand et al. [5] using the 4% divergence in the 16S rRNA between cluster 2 and otherFrankialineages as equivalent to 50 MY/1% distance [17] concluded that the genusFrankia had emerged long before the extant dicotyledonous lineages. These authors proposedFrankia cluster 2 as the proto-Frankia as nonsymbiotic ancestor of 62–130 Myr bp [43] and 100–200 Myr bp [5]. Since the distance in the 16S rRNA gene between cluster 1a (Frankia alni) and cluster 1b is less than 1%, the date of emergence of theCasuarina-infective lineage has been proposed to be less than 50 million years [5]. Thus theCasuarina/Frankia 1b lineage is considerably younger than theCoriaria/Frankia lineage and would have had less time to migrate out of its cradle and mingle with other hosts in its new territories and lose the cospeciation signal.Symbiotic partnership often tends to become obligatory, as in the case ofCasuarina host plants, whereFrankia is only present in soils close to the host plant [44], which means that the bacterium loses autonomy and becomes dependent on its host. Speciation of the host could then lead to synchronous speciation of its microsymbiont unless dispersal through long-distance carriers such as winds or migratory birds occurred or if there is survival ofFrankia cluster 2 in the rhizosphere of nonhosts as was recently demonstrated forAlnus glutinosa in Tunisia [45]. The numerous transitions seen in theFrankia phylogenetic tree from one continent to another would reinforce the idea.Yokoyama et al. [19] concluded from their study of theCoriariaspecies phylogeny that the Eurasian species had diverged earlier and are more diverse than other groups, but that nevertheless the origin of the genus could have been in North America, whence the South America and the Pacific species could have originated. Our study brings us to suggest a third possibility, Oceania, which could also be the origin of this actinorhizal symbiosis, which can be concluded from phylogenetic inferences positioning both bacterial and host plant partners as at the base toFrankia-Coriaria symbiosis. Another element that would support this hypothesis is the large number of extant species there; according to Yokoyama et al. [19] New Zealand would be home to 8 of the 17 existing species. A similar argument has often been made to establish Sub-Saharan Africa as the cradle of humankind [46] or Mexico for maize [47].Comparison of both the plant and the microbe phylogenetic topologies did not show any evidence for cospeciation ofFrankia microsymbiontsand theirCoriariahost species. The results obtained in this study suggest thatFrankia microsymbionts hosted currently byCoriaria species had probably dispersed globally as a proto-Frankia, a free living and nonsymbiotic ancestor. In parallel, the proto-Coriariathen diversified into the extantCoriaria species that appear to have been retreating given their scattered distribution, a trend possibly reinforced recently due to man uprooting because of the toxicity of the fruits for mammals [48, 49]. It can thus be hypothesized thatCoriaria appeared in the Pacific Islands more than 70 million years ago and presumably was symbiotic from the start, before dispersing over all continents as they drifted apart. TheCoriariaspecies diversified in their different biotopes, as they saw the appearance of other plants hosting the same microsymbiont ofFrankiacluster 2 such as Datiscaceae, Rosaceae, Ceanothus, or even nonhost species such asAlnus glutinosa that was recently found to hostFrankia cluster 2 in its rhizosphere [45]. Members of these alternative host plant species cooccur sympatrically withCoriaria such asCeanothus andPurshia species in Mexico andDatisca cannabina in Pakistan. TheseFrankia cluster 2 host plant species have more extended geographic distribution and overlap in some instancesCoriaria’s disjunct area and as a result can compensateFrankia microsymbionts remoteness, which would thus obscure the cospeciation signal. Cospeciation may also occur but subsequently is lost after bacterial mixing and fitness selection in the presence of “indigenous” and “dispersal” symbionts.



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*Source: 924235-2014-04-22.xml*