research article absence of cospeciation between the

Research ArticleAbsence of Cospeciation between the Uncultured FrankiaMicrosymbionts and the Disjunct Actinorhizal Coriaria Species

Imen Nouioui1 Faten Ghodhbane-Gtari1 Maria P Fernandez2

Abdellatif Boudabous1 Philippe Normand2 and Maher Gtari13

1 Laboratoire Microorganismes et Biomolecules Actives Universite de Tunis El Manar (FST) et Universite Carthage (INSAT)2092 Tunis Tunisia

2 Ecologie Microbienne Centre National de la Recherche Scientifique UMR 5557 Universite Lyon I 69622 Villeurbanne Cedex France3 Laboratoire Microorganismes et Biomolecules Actives Faculte des Sciences de Tunis Campus Universitaire 2092 Tunis Tunisia

Correspondence should be addressed to Maher Gtari mahergtarifstrnutn

Received 4 March 2014 Revised 25 March 2014 Accepted 27 March 2014 Published 22 April 2014

Academic Editor Ameur Cherif

Copyright copy 2014 Imen Nouioui et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Coriaria is an actinorhizal plant that forms root nodules in symbiosis with nitrogen-fixing actinobacteria of the genus FrankiaThissymbiotic association has drawn interest because of the disjunct geographical distribution of Coriaria in four separate areas of theworld and in the context of evolutionary relationships between host plants and their uncultured microsymbionts The evolutionof Frankia-Coriaria symbioses was examined from a phylogenetic viewpoint using multiple genetic markers in both bacteria andhost-plant partners Total DNA extracted from root nodules collected from five species C myrtifolia C arborea C nepalensis Cjaponica andCmicrophylla growing in theMediterranean area (Morocco and France) New Zealand Pakistan Japan andMexicorespectively was used to amplify glnA gene (glutamine synthetase) dnaA gene (chromosome replication initiator) and the nifDK IGS (intergenic spacer between nifD and nifK genes) in Frankia and the matK gene (chloroplast-encoded maturase K) andthe intergenic transcribed spacers (18S rRNA-ITS1-58S rRNA-ITS2-28S rRNA) in Coriaria species Phylogenetic reconstructionindicated that the radiations of Frankia strains and Coriaria species are not congruent The lack of cospeciation between the twosymbiotic partners may be explained by host shift at high taxonomic rank together with wind dispersal andor survival in nonhostrhizosphere

1 Introduction

The genus Frankia comprises nitrogen-fixing actinobac-teria that are able to induce perennial root nodules onwoody dicotyledonous plants called actinorhizals [1] Theactinorhizal plant families belong to three dicotyledonousorders Fagales (BetulaceaeCasuarinaceae and Myricaceae)Rosales (Elaeagnaceae Rhamnaceae and Rosaceae) andCucurbitales (Coriariaceae and Datiscaceae) [2] Analysis ofthe molecular phylogeny of members of Frankia genus con-sistently identifies four main clusters regardless of the typinglocus used [3] Three symbiotic Frankia clusters containingstrains able to establish effective nodules and fulfill Kochrsquospostulates and one atypical with strains unable to establisheffective nodulation on their host plants have been definedamong Frankia genera Cluster 1 includes Frankia strains in

association with Betulaceae Myricaceae and CasuarinaceaeCluster 2 contains Frankia nodulating species from theCoriariaceae Datiscaceae and Rosaceae families as well asCeanothus of the Rhamnaceae Frankia strains in cluster 3form effective root nodules on plants from members of theMyricaceae Rhamnaceae Elaeagnaceae andGymnostoma ofthe Casuarinaceae

Symbiotic Frankia strains have been only isolated fromFagales (Frankia cluster 1) and the families Elaeagnaceaeand Rhamnaceae (Frankia cluster 3) of the Rosales whileFrankia of cluster 2 have still not yet been isolated in culturedespite repeated attempts [2] The position in the Frankiaphylogenetic tree of cluster 2 relative to the other clusters hasvaried depending on the marker used It was proposed at thebase using glnA and 16S rRNA genes [4 5] derived with ITS16Sndash23S rRNA genes [6] and concatenated gyrB nifH and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 924235 9 pageshttpdxdoiorg1011552014924235

2 BioMed Research International

glnII genes [7] and should be clarified by the upcoming wholegenome phylogeny Nevertheless a position at the base of allsymbiotic lineages has been retained in the latest treatment ofBergeyrsquos manual [8]

Cross-inoculation studies using crushed nodules suggestthat cluster 2 strains form a separate and unique hostspecificity group [9ndash11] even though provenances from thefull geographical range have not yet been tested Despitethe high taxonomic diversity of host plants belonging tothe cross-inoculation group of cluster 2 and its disjunctrange uncultured Frankia in root nodules of several hostplants have so far shown a low level of diversity regardlessof the typing locus used [6 7 11ndash16] suggesting a recentemergence a strong and recent evolutionary bottleneck ora nonrepresentative sampling The time of emergence of allFrankia lineages is poorly documented as no convincingfossil remains An equivalence between 16S rRNA sequencesdistance and time of emergence has been proposed byOchman andWilson [17] where 1 is equivalent to 50millionyears and since 4 divergence exists between Frankia cluster2 and the other clusters one would conclude that Frankiaemerged 200 million years ago [5] which would mean thatthere is missing diversity either due to a recent evolutionarybottleneck or due to a lack of sampling [16] A possibility thusexists that the missing variability in cluster 2 strains is due tothe fact that sampling has so far been limited essentially toNorth American and Mediterranean areas

Evidence for cospeciation has been found so far only inthe case of Casuarina species growing in Australia and theirFrankia [18] that are in their immense majority resistant togrowth in pure culture Among actinorhizal plants of theCucurbitales subclade the family Coriariaceae with only onegenus Coriaria contains about 17 species [19] that occur infour disjunct areas of theworld theMediterranean SoutheastAsia Central and South America and the Pacific islands ofNew Zealand and Papua New Guinea [20ndash24] Yokoyama etal [19] considered that the Eurasian species are basal and haveemerged some 60million years agoThis date is in agreementwith the 65 million years proposed by Bell et al [25] based onmultiple genes (rbcL 18S rDNA atpB) phylogeny while thesame authors propose an emergence of the Casuarinaceae atabout 30 million years

The present study was aimed at testing the hypothesisof cospeciation between uncultured Frankiamicrosymbiontsand their Coriaria host species sampled from sites coveringthe full geographical range of the genus Coriaria myrtifolia(Morocco and France) C nepalensis (Pakistan) C arborea(New Zealand) C japonica (Japan) and C microphylla(Mexico)

2 Materials and Methods

21 DNA Extraction PCR Amplification and SequencingRoot nodules from naturally occurring Coriaria species(Table 1) were kindly provided by Dr Marıa Valdes (EscuelaNacional de Ciencias Biologicas Mexico DF Mexico) DrSajjad Mirza (National Institute for Biotechnology GeneticEngineering Faisalabad Pakistan) Dr Warwick Silvester(University of Waikato Waikato New Zealand) Dr Kawther

Benbrahim (University of Fes Fes Morocco) Dr TakashiYamanaka (Forest and Forestry Products Research InstituteIbaraki Japan) and Dr Jean-Claude Cleyet-Marel (INRA-IRD Montpellier France) Individual lobes were selectedsurface-sterilized in 30 (volvol) H

2O2 and rinsed several

times with distilled sterile water The DNA extraction fromsingle nodule lobes was performed as previously describedby Rouvier et al [26] Nodule lobes were crushed with sterileplastic mortars and pestles in 300 120583L of extraction buffer(100mMTris (pH 8) 20mM EDTA 14MNaCl 2 (wtvol)CTAB (cetyltrimethyl ammonium bromide) and 1 (wtvol)PVPP (polyvinyl polypyrrolidone)) The homogenates wereincubated at 65∘C for 60min extracted with chloroform-isoamyl alcohol (24 1 volvol) and the resulting DNA wasethanol-precipitated and resolubilized The extracted DNAwas used for PCR amplification of both bacterial and plantDNA regions using the primers listed in Table 2 The ampli-cons were then cycle-sequenced in both directions usingan ABI cycle sequencing kit (Applied Biosystem 3130) Thenucleotide sequences obtained in this studywere deposited inthe NCBI nucleotide sequence database under the accessionnumbers given in Table 1

22 Phylogenetic Analysis Frankia strainCcI3 andCasuarinaequisetifolia were used as outgroups in this study becausethey are physiologically distinct from the group studied yetphylogenetically close The data sets were completed withhomologous sequences present in the databases (Table 1)Alignments of Frankia glnA dnaA and IGS nifD-K andCori-aria matK and 18S rRNA-ITS1-58S rRNA-ITS2-28S rRNAwere generated with ClustalW [27] manually edited withMEGA50 [28] Bacterial and plant sequenceswere separatelyconcatenated and thenused to examinemaximum-likelihoodcladogram evolutionary relationships of each symbiotic part-ner using 1000 bootstraps by following the GTR + G basesubstitution model The distance between the sequences wascalculated using Kimurarsquos two-parameter model [29] Phy-logenetic trees were constructed using the Neighbor-Joiningmethod [30] with 1000 bootstraps [31] as implemented inMEGA 50 In parallel a Bayesian inference was realizedwith MrBayes [32] using the GTR + G model and 1000000generations

A statistical test for the presence of congruence betweenCoriaria and Frankia phylogenies was evaluated throughglobal distance-based fitting in ParaFit program [33] asimplemented in CopyCat [34] and tests of random associa-tion were performed with 9999 permutations globally acrossboth phylogenies for each association

An additional statistical test for correlation betweengeographical distances (obtained using httpwwwdaftlogiccomprojects-google-maps-distance-calculatorhtm) andphylogenetic distanceswasmade using Pearsonrsquos r correlationimplemented in the R software [35]

3 Results

To avoid taxonomic ambiguities DNAs from both Coriariahosts and Frankiamicrosymbionts were characterized on thesame root nodule tissuesThemethod of DNA isolation from

BioMed Research International 3

Table1Listof

Coria

riaroot

nodu

lesa

ndsequ

encesu

sedin

thisstu

dy

Species

Localitycoordinatesaltitud

e(asl)

Nod

ule

labels

Plantsequencea

ccessio

nnu

mberBa

cterialsequencea

ccessio

nnu

mberRe

ferences

ITS1-ITS

2matK

glnA

dnaA

IGSnifD

-K

Cmyrtifolia

Morocco

OuedEl

Koub

Ouezzane

35∘011015840879N

05∘201015840565E

140m

CmMs1

KC796592

KC7966

01KC

796522

KC796582

KC796555

Thisstu

dyCm

Ms2

CmMs3

CmMs4

KC796523

KC796524

KC796525

KC796583

KC796584

KC796585

KC796556

KC796557

KC796558

Thisstu

dyTh

isstu

dyTh

isstu

dy

BabBe

rredC

hefchaou

en

35∘001015840979N

04∘58101584009210158401015840E1290

m

CmM1a

KC796590

KC796599

KC796517

KC796578

KC796550

Thisstu

dyCm

M1b

KC796518

KC796579

KC796551

Thisstu

dyCm

M1c

KC796519

KC796580

KC796552

Thisstu

dyCm

M2a

KC796591

KC7966

00KC

796520

mdashKC

796553

Thisstu

dyCm

M2b

KC796521

KC796581

KC796554

Thisstu

dyFrance

Nyons44∘21101584046

5010158401015840N5∘081015840218210158401015840E259m

CmNy1

KC796598

KC7966

03KC

796531

KC796591

KC796564

Thisstu

dyCm

Ny2

CmNy3

CmNy4

CmNy5

KC796532

KC796533

KC796534

KC796535

KC796592

KC796593

KC796594

KC796595

KC796565

mdashKC

796566

KC796567

Thisstu

dyTh

isstu

dyTh

isstu

dyTh

isstu

dy

Mon

tpellier43∘361015840514810158401015840N3∘521015840239710158401015840E41

m

CmF1

KC796526

KC796586

KC796559

Thisstu

dyCm

F2KC

796593

KC7966

02KC

796527

KC796587

KC796560

Thisstu

dyCm

F3Cm

F4Cm

F5

KC796528

KC796529

KC796530

KC796588

KC796589

KC796590

KC796561

KC796562

KC796563

Thisstu

dyTh

isstu

dyTh

isstu

dyAF280102

Yang

etalun

publish

edAB0

16459

(Yokoyam

aetal2000

[19])

Cjaponica

Japan

Tosa

distr

ict+3

3∘451015840391810158401015840+

133∘27101584042891015840101584010

m

CjJA

KC7966

05KC

796536

KC796503

KC796576

Thisstu

dyCjJB

KC796594

KC796537

KC796504

KC796577

Thisstu

dyCjJC

CjJD

CjJE

KC796538

KC796539

KC796540

KC796505

KC796506

KC796507

KC796578

KC796579

KC796580

Thisstu

dyTh

isstu

dyTh

isstu

dyAF280101

Yang

etalun

publish

edAB0

16456

(Yokoyam

aetal2000

[19])

Cnepalen

sis

Pakista

n

Murree+3

3∘5410158401510158401015840N73∘2310158402510158401015840E33904

2∘N

7339

03∘E22912m

CnP1

KC796597

KC7966

07KC

796544

KC796508

KC796584

Thisstu

dyCn

P2KC

796545

KC796509

KC796585

Thisstu

dyCn

P3KC

796546

KC796510

KC796586

Thisstu

dyCn

P4AF280103

Yang

etalun

publish

ed


Table1Con

tinued

Species


e(asl)

Nod

ule

labels

Plantsequencea

ccessio

nnu

mberBa

cterialsequencea

ccessio

nnu

mberRe

ferences

ITS1-ITS

2matK

glnA

dnaA

IGSnifD

-K

Carborea

NewZealand

Hapuk

uriv

erN

orth

Canterbu

rySou

thisland

minus42∘231015840422410158401015840+

173∘41101584018071015840101584064m

CaNZ1

KC796595

KC7966

04KC

796542

KC796511

KC796581

Thisstu

dyCa

NZ2

KC796543

KC796512

KC796582

Thisstu

dyCa

NZ3

KC796544

KC796513

KC796583

Thisstu

dyAB164

54(Yokoyam

aetal2000

[19])

EF635457

Rotherham

etalun

publish

edEF

635475

Rotherham

etalun

publish

edAF277293

Yang

etalun

publish

ed

Cmicrophylla

Mexico

Morelo

s99∘30101584019∘301015840240

0m

CmicMx1

KC796596

KC7966

06KC

796547

KC796514

KC796587

Thisstu

dyCm

icMx2

KC796548

KC796515

KC796588

Thisstu

dyCm

icMx3

KC796549

KC796516

KC796589

Thisstu

dyAY

091813

Yang

etalun

publish

edAB0

16458

(Yokoyam

aetal2000

[19])

Cinterm

edia

AF280100

Yang

etalun

publish

edAB0

16455

(Yokoyam

aetal2000

[19])

Cterm

inalis

AY091817

Yang

etalun

publish

ed

Cruscifolia

AY091815

Yang

etalun

publish

edAY

091814

Yang

etalun

publish

edAF280104

Yang

etalun

publish

edAB0

16462

(Yokoyam

aetal2000

[19])

Csarm

entosa

AY091816

Yang

etalun

publish

edAB0

1646

4(Yokoyam

aetal2000

[19])

Cpapu

ana

AB0

16461

(Yokoyam

aetal2000

[19])

Datisca

glomerata

CP002801

CP002801

CP002801

(Persson

etal2011[50])

AY968449

Zhangetalun

publish

edAF4

85250

Forrestand

Hollin

gsworth

unpu

blish

ed

Casuarina

equisetifolia

CP00

0249

CP00

0249

CP00

0249

(Normandetal2007

[51])

AB0

15462

Sogo

etalun

publish

edAY

8640

57Herbertetalun

publish

ed


Table 2 Primers used for PCR amplification and DNA sequencing

Gene primers Sequence (51015840-31015840) Amplicons approximate size (bp) ReferencesglnA

DB41 TTCTTCATCCACGACCCG 500 (Clawson et al 2004 [4])DB44 GGCTTCGGCATGAAGGT

dnaAF7154 dnaAF GAGGARTTCACCAACGACTTCAT 700 Bautista et al unpublishedF7155 dnaAR CRGAAGTGCTGGCCGATCTT

IGS nifD-KF9372 nifD1 5 GTCATGCTCGCCGTCGGNG 700 This studyF9374 nifK1 5 GTTCTTCTCCCGGTAyTCCCA

F9373 nifD2 5 ACCGGCTACGAGTTCGCNCA 700 This studyF9375 nifK2 5 TGCGAGCCGTGCACCAGNG

18S-ITS1-58S-ITS2-28SITS1 TCCGTAGGTGAACCTGCGG 700 (White et al 1990 [52])ITS4 TCCTCCGCTTATTGATATGC

F9030-CJ-ITSF AGCCGGACCCGCGACGAGTTT 400 This studyF9031-CJ-ITSR CGACGTTGCGTGACGACGCCCA

matKF9249-matKF ACATTTAAATTATGTGTCAG 700 This studyF9250-matkR TGCATATACGCACAAATC

root nodules used in this study yielded PCR-amplifiable DNAfor both bacterial and plant PCR target sequences in all casesHowever in several instances it was easier to amplify Frankiathan Coriaria DNA which may have been mostly due to thespecificity of the primer sets used Thus in this study newprimers were designed (Table 2)

For the bacterial microsymbionts the average uncorrec-ted p-distances (proportion of differences between sequen-ces) were computed for each region and were found to berelatively small for dnaA (119901 = 00378) intermediate for glnA(119901 = 00625) and high for IGS nifD-K region (119901 = 00833)Blast analyses of the individual genes permitted assigningthem all to Frankia cluster 2 Nearly 3000 nucleotides wereobtained by concatenating sequences of the three DNAregions

Sequences variation for Coriaria species was small basedonmatKgene (119901 = 00205) compared to ITS1-ITS2 sequences(119901 = 00423) By concatenatingmatK and ITS1- ITS2 regiona composite sequence of 1500 nt was used for phylogeneticinference

All studied sequenceswere analyzed independently to testfor incongruence between the data sets for each symbioticpartner Similar topologies have been generally observedbetween phylogenetic trees inferred from glnA dnaA andIGS nifD-K sequences for Frankia and from matK and ITSsequences for Coriaria regardless of the used phylogeneticmethods (not shown)

The topologies of the trees obtained for the two symbioticpartners were not congruent (Figure 1) Moreover globaldistance-based ParaFit analysis recovered mostly randomassociations between Frankia and Coriaria host plant species(119901 = 033) and rejected cospeciation hypothesis On the

microbial side the New Zealand microsymbionts were atthe root (Group A) then three groups emerged groupB comprising the Pakistani Mexican and Mediterraneansymbionts from France group C comprising microsym-bionts from Morocco and then group D comprising Frenchand Japanese microsymbionts as well as the Dg1 referencesequence obtained initially from a Pakistani soil On thehost plant side group 1 at the root comprises New Zealandand South American sequences while group 2 comprises theJapanese Mediterranean and Pakistani sequences

On the other hand no significant correlations were foundfor Frankia symbionts (1199032 = 0772 Fgeneticdist = (geogdist times5830Eminus06) + 2541Eminus02) nor for the Coriaria host plants (1199032= 0883 Fgeneticdist = (geogdist times 2023Eminus06) + 6460Eminus03)(data not shown)

4 Discussion

Cospeciation has been postulated to have occurred insome Frankia actinorhizal host plants in particular in theCasuarina-Frankia cluster 1b [18] but not in Alnus-infectiveand Elaeagnus-infective Frankia strains where many isolatesable to fulfill Kochrsquos postulates have been obtained Totest if cospeciation was general or an exception it wasdecided to study uncultured Frankia microsymbionts andrepresentative Coriaria hosts a lineage where no Frankiaisolate exists and where geographic discontinuities may havelimited dispersion DNA sequences were obtained from rootnodules collected from New Zealand (C arborea) Pakistan(C nepalensis) Japan (C japonica) Mexico (C microphylla)and France and Morocco (C myrtifolia) and multiple molec-ular markers were analyzed for phylogenetic inference


Dg1

FCjJBFCjJA

FCmF1FCmF2FCmMs2FCmMs4FCmMs1FCmM1aFCmM1cFCmM1bFCmNy4FCmMs3FCmM2bFCmNy1FCmNy5FCnP1FCnP2FCmMx1FCmMx2FCaNZ2FCaNZ1100

99

99

69

100

8980

92

68

91

100

99

97

99

7581

OceaniaAsia

EuropeN AfricaAmerica

Gro

up A

G

roup

BG

roup

CG

roup

D

CmM1aCmM2aCmMs1

CmF2CmNy5

C myrtifolia C myrtifolia

CnP1

C nepalensisC terminalis C intermedia C japonica

CmicMx1C microphylla

C papuanaCaNZ1C arborea

C ruscifolia C sarmentosa

C lurida

8676

7590

7096

54

90

7083

7351

90

87

CjJA

Gro

up 2

Gro

up 1

Frankia Coriaria

Figure 1 Phylogenetic trees of the Frankiamicrosymbionts (left) and the Coriaria host plants (right)The Frankia tree was constructed usingthe glnA dnaA and the nifD-K intergenic spacer while the Coriaria tree was done using thematK and the 18S rRNA-ITS1-58S rRNA-ITS2-28S rRNA with ML method using strain CcI3 and Casuarina as outgroups respectively for Frankia and hot plant phylogenetic trees Thenumbers at branches indicate bootstrap results above 50 Lines are drawn between the microsymbionts and their hosts The color codeindicates the place of origin of the leave or of the set when homogenousThe groups numbers 1 and 2 on the right are according to Yokoyamaet al [19]

Paleontological data based on macrofossils and pollenfossils have brought several authors [36ndash40] to conclude thatthe Coriariaceae had a Laurasian origin (North Americaand Eurasia) There have been a few dissenting opinions inparticular those of Croizat [41] and Schuster [42] who con-sidered that Coriaria originated in Gondwana and migratedto the Northern Hemisphere However such paleontologicalstudies are not very convincing as it is recognizably hardto ascribe fossils to a given family and even more so to agiven genus Thus several authors have been surprised bythe results of molecular phylogeny positioning Coriariaceaeclose to the Datiscaceae Molecular approaches would thusgive support to a Gondwanan origin

Yokoyama et al [19] proposed that Coriaria species hademerged 59ndash63 million years ago which is coherent withthe date of 70 million years proposed by Bell et al [25]considerably older than that proposed (30million years) bythe same authors for the Casuarinaceae

Topology and clustering of Coriaria phylogeny obtainedin the current study are similar to those obtained byYokoyama et al [19] while the position at the base of thehost plant species from New Zealand C arborea and the

South American C ruscifolia and C microphylla species wascontrary to that of Yokoyama et al [19] who found theEurasian species at the base using rbcL (a large subunit ofribulose 15-bisphosphate carboxylaseoxygenase) and matK(maturase K) genes The present study suggests that theCoriaria ancestor may have emerged between Asia and NZand then dispersed worldwide and that the Asian lineagemay have given rise relatively recently to the Mediterraneanspecies while theNZ lineage gave rise to theNorth Americanspecies (Figure 2)

Previous studies had concluded that Frankia cluster 2 hada low genetic diversity [6 7 16] but these studies had beenfocused on only part of the full diversity of the symbioticCoriaria-Frankia essentially in North America and Mediter-ranean In this work we aimed to expand the scope of thestudy to the worldwide diversity and phylogeny ofmicrosym-bionts of Coriaria species Four microbial subgroups wereidentified that did not fit to the geographic range of the hostplants while two host plant subgroups were identified Theposition of subgroup A containing microsymbionts of NewZealand C arborea at the base of Frankia cluster 2 is inagreement with previous study [16] In view of previously


C ruscifolia

C nepalensis

Coriaria agustissima

C kingiana

C plumosaC lurida

C pteroidesC pottsiana

C sarmentosa

C papuana

C sarmentosa

CmNy1-2-3-4-5CmF1-2-3-4-5Coriaria myrtifoliaCmM1a-b-cCmM2a-bCmMs1-2-3-4-5 CnP1-2-3

Coriaria sp

C arborea

C microphyllaCmicMx1-2-3

CaNZ1-2-3

C japonicaCjJA-B-C-D-E

C terminalisC intermedia

Figure 2 Distribution of Coriaria species Root nodules have been sampled from C myrtifolia C arborea C nepalensis C japonica and Cmicrophylla growing in Mediterranean areas (Morocco and France) New Zealand Pakistan Japan and Mexico respectively Short arrowsindicate sampling sites for this study while long arrows indicate possible routes of dispersal as discussed

reported data members of cluster 2 Frankia studied herewere found to have relatively higher sequences variation (p-distance = 00625) than those reported by Vanden Heuvel etal [16] (119901 = 000454) based on the same 460 nt of the glnAgene

Molecular clock dating suggests that Frankia genus hasemerged much earlier 125Myr bp before the appearance ofangiosperm fossils in the Cretaceous period and the extantactinorhizal plants [4] Normand et al [5] using the 4divergence in the 16S rRNA between cluster 2 and otherFrankia lineages as equivalent to 50MY1 distance [17]concluded that the genus Frankia had emerged long beforethe extant dicotyledonous lineages These authors proposedFrankia cluster 2 as the proto-Frankia as nonsymbioticancestor of 62ndash130Myr bp [43] and 100ndash200Myr bp [5] Sincethe distance in the 16S rRNA gene between cluster 1a (Frankiaalni) and cluster 1b is less than 1 the date of emergence of theCasuarina-infective lineage has been proposed to be less than50 million years [5]Thus the CasuarinaFrankia 1b lineage isconsiderably younger than the CoriariaFrankia lineage andwould have had less time to migrate out of its cradle andmingle with other hosts in its new territories and lose thecospeciation signal

Symbiotic partnership often tends to become obligatoryas in the case of Casuarina host plants where Frankia is onlypresent in soils close to the host plant [44] which means thatthe bacterium loses autonomy and becomes dependent on itshost Speciation of the host could then lead to synchronousspeciation of its microsymbiont unless dispersal throughlong-distance carriers such as winds or migratory birds

occurred or if there is survival of Frankia cluster 2 in therhizosphere of nonhosts as was recently demonstrated forAlnus glutinosa in Tunisia [45] The numerous transitionsseen in the Frankia phylogenetic tree from one continent toanother would reinforce the idea

Yokoyama et al [19] concluded from their study of theCoriaria species phylogeny that the Eurasian species haddiverged earlier and are more diverse than other groups butthat nevertheless the origin of the genus could have been inNorth America whence the South America and the Pacificspecies could have originated Our study brings us to suggesta third possibility Oceania which could also be the originof this actinorhizal symbiosis which can be concluded fromphylogenetic inferences positioning both bacterial and hostplant partners as at the base to Frankia-Coriaria symbiosisAnother element that would support this hypothesis is thelarge number of extant species there according to Yokoyamaet al [19] New Zealand would be home to 8 of the 17 existingspecies A similar argument has often been made to establishSub-Saharan Africa as the cradle of humankind [46] orMexico for maize [47]

Comparison of both the plant and the microbe phyloge-netic topologies did not show any evidence for cospeciationof Frankia microsymbiontsand their Coriaria host speciesThe results obtained in this study suggest that Frankiamicrosymbionts hosted currently by Coriaria species hadprobably dispersed globally as a proto-Frankia a free livingand nonsymbiotic ancestor In parallel the proto-Coriariathen diversified into the extantCoriaria species that appear tohave been retreating given their scattered distribution a trend


possibly reinforced recently due to man uprooting becauseof the toxicity of the fruits for mammals [48 49] It canthus be hypothesized that Coriaria appeared in the PacificIslands more than 70 million years ago and presumably wassymbiotic from the start before dispersing over all continentsas they drifted apart The Coriaria species diversified intheir different biotopes as they saw the appearance of otherplants hosting the same microsymbiont of Frankia cluster 2such as Datiscaceae Rosaceae Ceanothus or even nonhostspecies such as Alnus glutinosa that was recently found tohost Frankia cluster 2 in its rhizosphere [45] Members ofthese alternative host plant species cooccur sympatricallywith Coriaria such as Ceanothus and Purshia species inMexico and Datisca cannabina in Pakistan These Frankiacluster 2 host plant species have more extended geographicdistribution and overlap in some instancesCoriariarsquos disjunctarea and as a result can compensate Frankiamicrosymbiontsremoteness which would thus obscure the cospeciationsignal Cospeciation may also occur but subsequently is lostafter bacterial mixing and fitness selection in the presence ofldquoindigenousrdquo and ldquodispersalrdquo symbionts

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work is supported by CMCU (Comite Mixte Tuniso-Francais pour la Cooperation Inter-Universitaire No10G0903) The authors are grateful to Dr Marıa Valdes(Escuela Nacional de Ciencias Biologicas Mexico Mexico)Dr Sajjad Mirza (National Institute for BiotechnologyGenetic Engineering Faisalabad Pakistan) Dr WarwickSilvester (University of Waikato Waikato New Zealand)Dr Kawther Benbrahim and Dr A Ennabili (University ofFes Fes Morocco) Mr Spick (Montpellier Botanical gardenFrance) Dr J C Cleyet-Marel (Montpellier INRA France)Mr D Moukouanga (IRD Montpellier France) and DrTakashi Yamanaka (Forest and Forestry Products ResearchInstitute Ibaraki Japan) for providing Coriaria nodules

References

[1] M P Lechevalier ldquoTaxonomy of the genus Frankia (Actinomyc-etales)rdquo International Journal of Systematic Bacteriology vol 44no 1 pp 1ndash8 1994

[2] D R Benson B D VandenHeuvel and D Potter ldquoActinorhizalsymbioses diversity and biogeographyrdquo in Plant MicrobiologyM Gillings Ed pp 97ndash127 BIOS Scientific Publishers OxfordUK 2004

[3] M Gtari L S Tisa and P Normand ldquoDiversity of Frankiastrains actinobacteria symbionts of actinorhizal plantsrdquo inSymbiotic Endophytes Soil Biology R Aroca Ed vol 37Chapter 7 pp 123ndash148 Springer Berlin Germany 2013

[4] M L Clawson A Bourret and D R Benson ldquoAssessing thephylogeny of Frankia-actinorhizal plant nitrogen-fixing root

nodule symbioses with Frankia 16S rRNA and glutamine syn-thetase gene sequencesrdquoMolecular Phylogenetics and Evolutionvol 31 no 1 pp 131ndash138 2004

[5] P Normand S Orso B Cournoyer et al ldquoMolecular phylogenyof the genus Frankia and related genera and emendation ofthe family Frankiaceaerdquo International Journal of SystematicBacteriology vol 46 no 1 pp 1ndash9 1996

[6] F Ghodhbane-Gtari I Nouioui M Chair A Boudabous andM Gtari ldquo16S-23S rRNA intergenic spacer region variability inthe genus Frankiardquo Microbial Ecology vol 60 no 3 pp 487ndash495 2010

[7] I Nouioui F Ghodhbane-Gtari N J Beauchemin L S Tisaand M Gtari ldquoPhylogeny of members of the Frankia genusbased on gyrB nifH and glnII sequencesrdquoAntonie van Leeuwen-hoek vol 100 no 4 pp 579ndash587 2011

[8] P Normand and D R Benson ldquoGenus I Frankia Brunchorst1886 174ALrdquo in Bergeyrsquos Manual of Systematic Bacteriology TheActinobacteria M Goodfellow P Kampfer H-J Busse et alEds vol 5 of Bergeyrsquos Manual Trust pp 512ndash520 Springer NewYork NY USA 2012

[9] J G Torrey ldquoCross-inoculation groups within Frankia andhost-endosymbiont associationsrdquo inThe Biology of Frankia andActinorhizal Plants C R Schwintzer and J D Tjepkema Edspp 83ndash106 Academic Press San Diego Calif USA 1990

[10] S J Kohls J Thimmapuram C A Buschena M W Paschkeand J O Dawson ldquoNodulation patterns of actinorhizal plantsin the family Rosaceaerdquo Plant and Soil vol 162 no 2 pp 229ndash239 1994

[11] M S Mirza K Pawlowski F Y Hafeez A H Chaudharyand A D L Akkermans ldquoUltrastructure of the endophyte andlocalization of nifH transcripts in root nodules of Coriarianepalensis Wall by in situ hybridizationrdquo New Phytologist vol126 no 1 pp 131ndash136 1994

[12] G Nick E Paget P Simonet A Moiroud and P NormandldquoThe nodular endophytes of Coriaria spp form a distinctlineage within the genus FrankiardquoMolecular Ecology vol 1 no3 pp 175ndash181 1992

[13] M Bosco S Jamann C Chapelon and S P Normand ldquoFrankiamicrosymbiont in Dryas drummondii nodules is closely relatedto the microsymbiont of Coriaria and genetically distinct fromother characterized Frankia strainsrdquo in Nitrogen Fixation withNon-Legumes N A Hegazi M Fayez and M Monib Eds pp173ndash183 The American University in Cairo Press 1994

[14] D R Benson D W Stephens M L Clawson and W BSilvester ldquoAmplification of 16S rRNAgenes from Frankia strainsin root nodules of Ceanothus griseus Coriaria arborea Coriariaplumosa Discaria toumatou and Purshia tridentatardquo Appliedand Environmental Microbiology vol 62 no 8 pp 2904ndash29091996

[15] D R Benson andM L Clawson ldquoEvolution of the actinorhizalplant symbiosesrdquo in Prokaryotic Nitrogen Fixation A ModelSystem for Analysis of Biological Process E W Triplett Ed pp207ndash224 Horizon Scientific Press Wymondham UK 2000

[16] B D Vanden Heuvel D R Benson E Bortiri and D PotterldquoLow genetic diversity among Frankia spp strains nodulatingsympatric populations of actinorhizal species of RosaceaeCeanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae)west of the Sierra Nevada (California)rdquo Canadian Journal ofMicrobiology vol 50 no 12 pp 989ndash1000 2004

[17] H Ochman and A C Wilson ldquoEvolution in bacteria evidencefor a universal substitution rate in cellular genomesrdquo Journal ofMolecular Evolution vol 26 no 1-2 pp 74ndash86 1987


[18] P Simonet E Navarro C Rouvier et al ldquoCo-evolutionbetween Frankia populations and host plants in the familyCasuarinaceae and consequent patterns of global dispersalrdquoEnvironmental Microbiology vol 1 no 6 pp 525ndash533 1999

[19] J Yokoyama M Suzuki K Iwatsuki and M Hasebe ldquoMolecu-lar phylogeny ofCoriaria with special emphasis on the disjunctdistributionrdquoMolecular Phylogenetics and Evolution vol 14 no1 pp 11ndash19 2000

[20] R D O Good ldquoThe geography of the genus Coriariardquo NewPhytologist vol 29 pp 170ndash198 1930

[21] H H Allan ldquoCoriariaceaerdquo in Flora of New Zealand L BMoore Ed pp 300ndash305 Government printerWellington NewZealand 1961

[22] R Melville ldquoContinental drift mesozoic continents and themigrations of the angiospermsrdquo Nature vol 211 no 5045 pp116ndash120 1966

[23] L E Skog ldquoThe genus Coriaria Coriariaceae in the WesternHemisphererdquo Rhodora vol 74 pp 242ndash253 1972

[24] R Melville ldquoVicarious plant distributions and paleogeographyof the Pacific regionrdquo inVicariance Biogeography G Nelson andE D Rosen Eds pp 413ndash435 Columbia University Press NewYork NY USA 1981

[25] C D Bell D E Soltis and P S Soltis ldquoThe age and diversi-fication of the angiosperms re-revisitedrdquo American Journal ofBotany vol 97 no 8 pp 1296ndash1303 2010

[26] C Rouvier J Schwenke Y Prin et al ldquoBiologie et diversitegenetique des souches de Frankia associees aux CasuarinaceesrdquoActa Botanica Gallica vol 143 pp 567ndash580 1996

[27] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[28] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[29] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[30] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[31] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

[32] F Ronquist and J P Huelsenbeck ldquoMrBayes 3 bayesian phylo-genetic inference under mixed modelsrdquo Bioinformatics vol 19no 12 pp 1572ndash1574 2003

[33] P Legendre Y Desdevises and E Bazin ldquoA statistical test forhost-parasite coevolutionrdquo Systematic Biology vol 51 no 2 pp217ndash234 2002

[34] J P Meier-Kolthoff A F Auch D H Huson and M GokerldquoCopyCat cophylogenetic analysis toolrdquo Bioinformatics vol 23no 7 pp 898ndash900 2007

[35] R Ihaka and R Gentleman ldquoR a language for data analysis andgraphicsrdquo Journal of Computational andGraphical Statistics vol5 no 3 pp 299ndash314 1996

[36] A N Gladkova ldquoFragments of the history of the Myricaceaefamilyrdquo Pollen and Spore vol 4 p 345 1962

[37] J A Doyle ldquoCretaceous angiosperm pollen of the Atlanticcoastal plain and its evolutionnary significancerdquo Journal of theArnold Arboretum vol 50 pp 1ndash35 1969

[38] P H Raven and D I Axelrod ldquoAngiosperm biogeography andpast continental movementsrdquo Annals of the Missouri BotanicalGarden vol 61 pp 539ndash673 1974

[39] J Muller ldquoFossil pollen records of extant angiospermsrdquo TheBotanical Review vol 47 no 1 pp 1ndash142 1981

[40] O Eriksson and B Bremer ldquoPollination systems dispersalmodes life forms and diversification rates in angiospermfamiliesrdquo Evolution vol 46 pp 258ndash266 1992

[41] L Croizat Manual of Phytogeography An Account of PlantDispersal Throughout the World Junk The Hague 1952

[42] RM Schuster ldquoPlate tectonics and its bearing on the geograph-ical origin and dispersal of angiospermsrdquo in Origin and EarlyEvolution of Angiosperms C B Beck Ed pp 48ndash138 ColumbiaUniversity Press New York 1976

[43] S-C Jeong A Liston and D D Myrold ldquoMolecular phylogenyof the genus Ceanothus (Rhamnaceae) using rbcL and ndhFsequencesrdquoTheoretical and Applied Genetics vol 94 no 6-7 pp852ndash857 1997

[44] J F Zimpfer C A Smyth and J O Dawson ldquoThe capacity ofJamaican mine spoils agricultural and forest soils to nodulateMyrica cerifera Leucaena leucocephala and Casuarina cunning-hamianardquo Physiologia Plantarum vol 99 no 4 pp 664ndash6721997

[45] I Nouioui I Sbissi F Ghodhbane-Gtari K Benbrahim PNormand and M Gtari ldquoFirst report on the occurrence of theuncultivated cluster 2 Frankia microsymbionts in soil outsidethe native actinorhizal host range areardquo Journal of Biosciencesvol 38 pp 695ndash698 2013

[46] S Ramachandran O Deshpande C C Roseman N ARosenberg M W Feldman and L L Cavalli-Sforza ldquoSupportfrom the relationship of genetic and geographic in humanpopulations for a serial founder effect originating in AfricardquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 44 pp 15942ndash15947 2005

[47] B Prasanna ldquoDiversity in global maize germplasm character-ization and utilizationrdquo Journal of Biosciences vol 37 pp 843ndash855 2012

[48] L DeHaro P Pommier L TichadouMHayek-Lanthois and JArditti ldquoPoisoning by Coriaria myrtifolia Linnaeus a new casereport and review of the literaturerdquo Toxicon vol 46 no 6 pp600ndash603 2005

[49] S F Belcher and T R Morton ldquoTutu toxicity three casereports of Coriaria arborea ingestion review of literature andrecommendations for managementrdquoThe New Zealand MedicalJournal vol 126 pp 103ndash109 2013

[50] T Persson D R Benson P Normand et al ldquoGenomesequence of ldquoCandidatus Frankia datiscaerdquo Dg1 the unculturedmicrosymbiont from nitrogen-fixing root nodules of the dicotDatisca glomeratardquo Journal of Bacteriology vol 193 no 24 pp7017ndash7018 2011

[51] P Normand P Lapierre L S Tisa et al ldquoGenome character-istics of facultatively symbiotic Frankia sp strains reflect hostrange and host plant biogeographyrdquo Genome Research vol 17no 1 pp 7ndash15 2007

[52] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


glnII genes [7] and should be clarified by the upcoming wholegenome phylogeny Nevertheless a position at the base of allsymbiotic lineages has been retained in the latest treatment ofBergeyrsquos manual [8]

Cross-inoculation studies using crushed nodules suggestthat cluster 2 strains form a separate and unique hostspecificity group [9ndash11] even though provenances from thefull geographical range have not yet been tested Despitethe high taxonomic diversity of host plants belonging tothe cross-inoculation group of cluster 2 and its disjunctrange uncultured Frankia in root nodules of several hostplants have so far shown a low level of diversity regardlessof the typing locus used [6 7 11ndash16] suggesting a recentemergence a strong and recent evolutionary bottleneck ora nonrepresentative sampling The time of emergence of allFrankia lineages is poorly documented as no convincingfossil remains An equivalence between 16S rRNA sequencesdistance and time of emergence has been proposed byOchman andWilson [17] where 1 is equivalent to 50millionyears and since 4 divergence exists between Frankia cluster2 and the other clusters one would conclude that Frankiaemerged 200 million years ago [5] which would mean thatthere is missing diversity either due to a recent evolutionarybottleneck or due to a lack of sampling [16] A possibility thusexists that the missing variability in cluster 2 strains is due tothe fact that sampling has so far been limited essentially toNorth American and Mediterranean areas

Evidence for cospeciation has been found so far only inthe case of Casuarina species growing in Australia and theirFrankia [18] that are in their immense majority resistant togrowth in pure culture Among actinorhizal plants of theCucurbitales subclade the family Coriariaceae with only onegenus Coriaria contains about 17 species [19] that occur infour disjunct areas of theworld theMediterranean SoutheastAsia Central and South America and the Pacific islands ofNew Zealand and Papua New Guinea [20ndash24] Yokoyama etal [19] considered that the Eurasian species are basal and haveemerged some 60million years agoThis date is in agreementwith the 65 million years proposed by Bell et al [25] based onmultiple genes (rbcL 18S rDNA atpB) phylogeny while thesame authors propose an emergence of the Casuarinaceae atabout 30 million years

The present study was aimed at testing the hypothesisof cospeciation between uncultured Frankiamicrosymbiontsand their Coriaria host species sampled from sites coveringthe full geographical range of the genus Coriaria myrtifolia(Morocco and France) C nepalensis (Pakistan) C arborea(New Zealand) C japonica (Japan) and C microphylla(Mexico)

2 Materials and Methods

21 DNA Extraction PCR Amplification and SequencingRoot nodules from naturally occurring Coriaria species(Table 1) were kindly provided by Dr Marıa Valdes (EscuelaNacional de Ciencias Biologicas Mexico DF Mexico) DrSajjad Mirza (National Institute for Biotechnology GeneticEngineering Faisalabad Pakistan) Dr Warwick Silvester(University of Waikato Waikato New Zealand) Dr Kawther

Benbrahim (University of Fes Fes Morocco) Dr TakashiYamanaka (Forest and Forestry Products Research InstituteIbaraki Japan) and Dr Jean-Claude Cleyet-Marel (INRA-IRD Montpellier France) Individual lobes were selectedsurface-sterilized in 30 (volvol) H

2O2 and rinsed several

times with distilled sterile water The DNA extraction fromsingle nodule lobes was performed as previously describedby Rouvier et al [26] Nodule lobes were crushed with sterileplastic mortars and pestles in 300 120583L of extraction buffer(100mMTris (pH 8) 20mM EDTA 14MNaCl 2 (wtvol)CTAB (cetyltrimethyl ammonium bromide) and 1 (wtvol)PVPP (polyvinyl polypyrrolidone)) The homogenates wereincubated at 65∘C for 60min extracted with chloroform-isoamyl alcohol (24 1 volvol) and the resulting DNA wasethanol-precipitated and resolubilized The extracted DNAwas used for PCR amplification of both bacterial and plantDNA regions using the primers listed in Table 2 The ampli-cons were then cycle-sequenced in both directions usingan ABI cycle sequencing kit (Applied Biosystem 3130) Thenucleotide sequences obtained in this studywere deposited inthe NCBI nucleotide sequence database under the accessionnumbers given in Table 1

22 Phylogenetic Analysis Frankia strainCcI3 andCasuarinaequisetifolia were used as outgroups in this study becausethey are physiologically distinct from the group studied yetphylogenetically close The data sets were completed withhomologous sequences present in the databases (Table 1)Alignments of Frankia glnA dnaA and IGS nifD-K andCori-aria matK and 18S rRNA-ITS1-58S rRNA-ITS2-28S rRNAwere generated with ClustalW [27] manually edited withMEGA50 [28] Bacterial and plant sequenceswere separatelyconcatenated and thenused to examinemaximum-likelihoodcladogram evolutionary relationships of each symbiotic part-ner using 1000 bootstraps by following the GTR + G basesubstitution model The distance between the sequences wascalculated using Kimurarsquos two-parameter model [29] Phy-logenetic trees were constructed using the Neighbor-Joiningmethod [30] with 1000 bootstraps [31] as implemented inMEGA 50 In parallel a Bayesian inference was realizedwith MrBayes [32] using the GTR + G model and 1000000generations

A statistical test for the presence of congruence betweenCoriaria and Frankia phylogenies was evaluated throughglobal distance-based fitting in ParaFit program [33] asimplemented in CopyCat [34] and tests of random associa-tion were performed with 9999 permutations globally acrossboth phylogenies for each association

An additional statistical test for correlation betweengeographical distances (obtained using httpwwwdaftlogiccomprojects-google-maps-distance-calculatorhtm) andphylogenetic distanceswasmade using Pearsonrsquos r correlationimplemented in the R software [35]

3 Results

To avoid taxonomic ambiguities DNAs from both Coriariahosts and Frankiamicrosymbionts were characterized on thesame root nodule tissuesThemethod of DNA isolation from


Table1Listof

Coria

riaroot

nodu

lesa

ndsequ

encesu

sedin

thisstu

dy

Species


e(asl)

Nod

ule

labels

Plantsequencea

ccessio

nnu

mberBa

cterialsequencea

ccessio

nnu

mberRe

ferences

ITS1-ITS

2matK

glnA

dnaA

IGSnifD

-K

Cmyrtifolia

Morocco

OuedEl

Koub

Ouezzane

35∘011015840879N

05∘201015840565E

140m

CmMs1

KC796592

KC7966

01KC

796522

KC796582

KC796555

Thisstu

dyCm

Ms2

CmMs3

CmMs4

KC796523

KC796524

KC796525

KC796583

KC796584

KC796585

KC796556

KC796557

KC796558

Thisstu

dyTh

isstu

dyTh

isstu

dy

BabBe

rredC

hefchaou

en

35∘001015840979N

04∘58101584009210158401015840E1290

m

CmM1a

KC796590

KC796599

KC796517

KC796578

KC796550

Thisstu

dyCm

M1b

KC796518

KC796579

KC796551

Thisstu

dyCm

M1c

KC796519

KC796580

KC796552

Thisstu

dyCm

M2a

KC796591

KC7966

00KC

796520

mdashKC

796553

Thisstu

dyCm

M2b

KC796521

KC796581

KC796554

Thisstu

dyFrance

Nyons44∘21101584046

5010158401015840N5∘081015840218210158401015840E259m

CmNy1

KC796598

KC7966

03KC

796531

KC796591

KC796564

Thisstu

dyCm

Ny2

CmNy3

CmNy4

CmNy5

KC796532

KC796533

KC796534

KC796535

KC796592

KC796593

KC796594

KC796595

KC796565

mdashKC

796566

KC796567

Thisstu

dyTh

isstu

dyTh

isstu

dyTh

isstu

dy

Mon

tpellier43∘361015840514810158401015840N3∘521015840239710158401015840E41

m

CmF1

KC796526

KC796586

KC796559

Thisstu

dyCm

F2KC

796593

KC7966

02KC

796527

KC796587

KC796560

Thisstu

dyCm

F3Cm

F4Cm

F5

KC796528

KC796529

KC796530

KC796588

KC796589

KC796590

KC796561

KC796562

KC796563

Thisstu

dyTh

isstu

dyTh

isstu

dyAF280102

Yang

etalun

publish

edAB0

16459

(Yokoyam

aetal2000

[19])

Cjaponica

Japan

Tosa

distr

ict+3

3∘451015840391810158401015840+

133∘27101584042891015840101584010

m

CjJA

KC7966

05KC

796536

KC796503

KC796576

Thisstu

dyCjJB

KC796594

KC796537

KC796504

KC796577

Thisstu

dyCjJC

CjJD

CjJE

KC796538

KC796539

KC796540

KC796505

KC796506

KC796507

KC796578

KC796579

KC796580

Thisstu

dyTh

isstu

dyTh

isstu

dyAF280101

Yang

etalun

publish

edAB0

16456

(Yokoyam

aetal2000

[19])

Cnepalen

sis

Pakista

n

Murree+3

3∘5410158401510158401015840N73∘2310158402510158401015840E33904

2∘N

7339

03∘E22912m

CnP1

KC796597

KC7966

07KC

796544

KC796508

KC796584

Thisstu

dyCn

P2KC

796545

KC796509

KC796585

Thisstu

dyCn

P3KC

796546

KC796510

KC796586

Thisstu

dyCn

P4AF280103

Yang

etalun

publish

ed


Table1Con

tinued

Species


e(asl)

Nod

ule

labels

Plantsequencea

ccessio

nnu

mberBa

cterialsequencea

ccessio

nnu

mberRe

ferences

ITS1-ITS

2matK

glnA

dnaA

IGSnifD

-K

Carborea

NewZealand

Hapuk

uriv

erN

orth

Canterbu

rySou

thisland

minus42∘231015840422410158401015840+

173∘41101584018071015840101584064m

CaNZ1

KC796595

KC7966

04KC

796542

KC796511

KC796581

Thisstu

dyCa

NZ2

KC796543

KC796512

KC796582

Thisstu

dyCa

NZ3

KC796544

KC796513

KC796583

Thisstu

dyAB164

54(Yokoyam

aetal2000

[19])

EF635457

Rotherham

etalun

publish

edEF

635475

Rotherham

etalun

publish

edAF277293

Yang

etalun

publish

ed

Cmicrophylla

Mexico

Morelo

s99∘30101584019∘301015840240

0m

CmicMx1

KC796596

KC7966

06KC

796547

KC796514

KC796587

Thisstu

dyCm

icMx2

KC796548

KC796515

KC796588

Thisstu

dyCm

icMx3

KC796549

KC796516

KC796589

Thisstu

dyAY

091813

Yang

etalun

publish

edAB0

16458

(Yokoyam

aetal2000

[19])

Cinterm

edia

AF280100

Yang

etalun

publish

edAB0

16455

(Yokoyam

aetal2000

[19])

Cterm

inalis

AY091817

Yang

etalun

publish

ed

Cruscifolia

AY091815

Yang

etalun

publish

edAY

091814

Yang

etalun

publish

edAF280104

Yang

etalun

publish

edAB0

16462

(Yokoyam

aetal2000

[19])

Csarm

entosa

AY091816

Yang

etalun

publish

edAB0

1646

4(Yokoyam

aetal2000

[19])

Cpapu

ana

AB0

16461

(Yokoyam

aetal2000

[19])

Datisca

glomerata

CP002801

CP002801

CP002801

(Persson

etal2011[50])

AY968449

Zhangetalun

publish

edAF4

85250

Forrestand

Hollin

gsworth

unpu

blish

ed

Casuarina

equisetifolia

CP00

0249

CP00

0249

CP00

0249

(Normandetal2007

[51])

AB0

15462

Sogo

etalun

publish

edAY

8640

57Herbertetalun

publish

ed


















4 Discussion



Dg1

FCjJBFCjJA


99

99

69

100

8980

92

68

91

100

99

97

99

7581

OceaniaAsia


Gro

up A

G

roup

BG

roup

CG

roup

D

CmM1aCmM2aCmMs1

CmF2CmNy5


CnP1





C lurida

8676

7590

7096

54

90

7083

7351

90

87

CjJA

Gro

up 2

Gro

up 1

Frankia Coriaria








C ruscifolia

C nepalensis


C kingiana

C plumosaC lurida


C sarmentosa

C papuana

C sarmentosa


Coriaria sp

C arborea


CaNZ1-2-3














Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Listof

Coria

riaroot

nodu

lesa

ndsequ

encesu

sedin

thisstu

dy

Species


e(asl)

Nod

ule

labels

Plantsequencea

ccessio

nnu

mberBa

cterialsequencea

ccessio

nnu

mberRe

ferences

ITS1-ITS

2matK

glnA

dnaA

IGSnifD

-K

Cmyrtifolia

Morocco

OuedEl

Koub

Ouezzane

35∘011015840879N

05∘201015840565E

140m

CmMs1

KC796592

KC7966

01KC

796522

KC796582

KC796555

Thisstu

dyCm

Ms2

CmMs3

CmMs4

KC796523

KC796524

KC796525

KC796583

KC796584

KC796585

KC796556

KC796557

KC796558

Thisstu

dyTh

isstu

dyTh

isstu

dy

BabBe

rredC

hefchaou

en

35∘001015840979N

04∘58101584009210158401015840E1290

m

CmM1a

KC796590

KC796599

KC796517

KC796578

KC796550

Thisstu

dyCm

M1b

KC796518

KC796579

KC796551

Thisstu

dyCm

M1c

KC796519

KC796580

KC796552

Thisstu

dyCm

M2a

KC796591

KC7966

00KC

796520

mdashKC

796553

Thisstu

dyCm

M2b

KC796521

KC796581

KC796554

Thisstu

dyFrance

Nyons44∘21101584046

5010158401015840N5∘081015840218210158401015840E259m

CmNy1

KC796598

KC7966

03KC

796531

KC796591

KC796564

Thisstu

dyCm

Ny2

CmNy3

CmNy4

CmNy5

KC796532

KC796533

KC796534

KC796535

KC796592

KC796593

KC796594

KC796595

KC796565

mdashKC

796566

KC796567

Thisstu

dyTh

isstu

dyTh

isstu

dyTh

isstu

dy

Mon

tpellier43∘361015840514810158401015840N3∘521015840239710158401015840E41

m

CmF1

KC796526

KC796586

KC796559

Thisstu

dyCm

F2KC

796593

KC7966

02KC

796527

KC796587

KC796560

Thisstu

dyCm

F3Cm

F4Cm

F5

KC796528

KC796529

KC796530

KC796588

KC796589

KC796590

KC796561

KC796562

KC796563

Thisstu

dyTh

isstu

dyTh

isstu

dyAF280102

Yang

etalun

publish

edAB0

16459

(Yokoyam

aetal2000

[19])

Cjaponica

Japan

Tosa

distr

ict+3

3∘451015840391810158401015840+

133∘27101584042891015840101584010

m

CjJA

KC7966

05KC

796536

KC796503

KC796576

Thisstu

dyCjJB

KC796594

KC796537

KC796504

KC796577

Thisstu

dyCjJC

CjJD

CjJE

KC796538

KC796539

KC796540

KC796505

KC796506

KC796507

KC796578

KC796579

KC796580

Thisstu

dyTh

isstu

dyTh

isstu

dyAF280101

Yang

etalun

publish

edAB0

16456

(Yokoyam

aetal2000

[19])

Cnepalen

sis

Pakista

n

Murree+3

3∘5410158401510158401015840N73∘2310158402510158401015840E33904

2∘N

7339

03∘E22912m

CnP1

KC796597

KC7966

07KC

796544

KC796508

KC796584

Thisstu

dyCn

P2KC

796545

KC796509

KC796585

Thisstu

dyCn

P3KC

796546

KC796510

KC796586

Thisstu

dyCn

P4AF280103

Yang

etalun

publish

ed


Table1Con

tinued

Species


e(asl)

Nod

ule

labels

Plantsequencea

ccessio

nnu

mberBa

cterialsequencea

ccessio

nnu

mberRe

ferences

ITS1-ITS

2matK

glnA

dnaA

IGSnifD

-K

Carborea

NewZealand

Hapuk

uriv

erN

orth

Canterbu

rySou

thisland

minus42∘231015840422410158401015840+

173∘41101584018071015840101584064m

CaNZ1

KC796595

KC7966

04KC

796542

KC796511

KC796581

Thisstu

dyCa

NZ2

KC796543

KC796512

KC796582

Thisstu

dyCa

NZ3

KC796544

KC796513

KC796583

Thisstu

dyAB164

54(Yokoyam

aetal2000

[19])

EF635457

Rotherham

etalun

publish

edEF

635475

Rotherham

etalun

publish

edAF277293

Yang

etalun

publish

ed

Cmicrophylla

Mexico

Morelo

s99∘30101584019∘301015840240

0m

CmicMx1

KC796596

KC7966

06KC

796547

KC796514

KC796587

Thisstu

dyCm

icMx2

KC796548

KC796515

KC796588

Thisstu

dyCm

icMx3

KC796549

KC796516

KC796589

Thisstu

dyAY

091813

Yang

etalun

publish

edAB0

16458

(Yokoyam

aetal2000

[19])

Cinterm

edia

AF280100

Yang

etalun

publish

edAB0

16455

(Yokoyam

aetal2000

[19])

Cterm

inalis

AY091817

Yang

etalun

publish

ed

Cruscifolia

AY091815

Yang

etalun

publish

edAY

091814

Yang

etalun

publish

edAF280104

Yang

etalun

publish

edAB0

16462

(Yokoyam

aetal2000

[19])

Csarm

entosa

AY091816

Yang

etalun

publish

edAB0

1646

4(Yokoyam

aetal2000

[19])

Cpapu

ana

AB0

16461

(Yokoyam

aetal2000

[19])

Datisca

glomerata

CP002801

CP002801

CP002801

(Persson

etal2011[50])

AY968449

Zhangetalun

publish

edAF4

85250

Forrestand

Hollin

gsworth

unpu

blish

ed

Casuarina

equisetifolia

CP00

0249

CP00

0249

CP00

0249

(Normandetal2007

[51])

AB0

15462

Sogo

etalun

publish

edAY

8640

57Herbertetalun

publish

ed


















4 Discussion



Dg1

FCjJBFCjJA


99

99

69

100

8980

92

68

91

100

99

97

99

7581

OceaniaAsia


Gro

up A

G

roup

BG

roup

CG

roup

D

CmM1aCmM2aCmMs1

CmF2CmNy5


CnP1





C lurida

8676

7590

7096

54

90

7083

7351

90

87

CjJA

Gro

up 2

Gro

up 1

Frankia Coriaria








C ruscifolia

C nepalensis


C kingiana

C plumosaC lurida


C sarmentosa

C papuana

C sarmentosa


Coriaria sp

C arborea


CaNZ1-2-3














Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Con

tinued

Species


e(asl)

Nod

ule

labels

Plantsequencea

ccessio

nnu

mberBa

cterialsequencea

ccessio

nnu

mberRe

ferences

ITS1-ITS

2matK

glnA

dnaA

IGSnifD

-K

Carborea

NewZealand

Hapuk

uriv

erN

orth

Canterbu

rySou

thisland

minus42∘231015840422410158401015840+

173∘41101584018071015840101584064m

CaNZ1

KC796595

KC7966

04KC

796542

KC796511

KC796581

Thisstu

dyCa

NZ2

KC796543

KC796512

KC796582

Thisstu

dyCa

NZ3

KC796544

KC796513

KC796583

Thisstu

dyAB164

54(Yokoyam

aetal2000

[19])

EF635457

Rotherham

etalun

publish

edEF

635475

Rotherham

etalun

publish

edAF277293

Yang

etalun

publish

ed

Cmicrophylla

Mexico

Morelo

s99∘30101584019∘301015840240

0m

CmicMx1

KC796596

KC7966

06KC

796547

KC796514

KC796587

Thisstu

dyCm

icMx2

KC796548

KC796515

KC796588

Thisstu

dyCm

icMx3

KC796549

KC796516

KC796589

Thisstu

dyAY

091813

Yang

etalun

publish

edAB0

16458

(Yokoyam

aetal2000

[19])

Cinterm

edia

AF280100

Yang

etalun

publish

edAB0

16455

(Yokoyam

aetal2000

[19])

Cterm

inalis

AY091817

Yang

etalun

publish

ed

Cruscifolia

AY091815

Yang

etalun

publish

edAY

091814

Yang

etalun

publish

edAF280104

Yang

etalun

publish

edAB0

16462

(Yokoyam

aetal2000

[19])

Csarm

entosa

AY091816

Yang

etalun

publish

edAB0

1646

4(Yokoyam

aetal2000

[19])

Cpapu

ana

AB0

16461

(Yokoyam

aetal2000

[19])

Datisca

glomerata

CP002801

CP002801

CP002801

(Persson

etal2011[50])

AY968449

Zhangetalun

publish

edAF4

85250

Forrestand

Hollin

gsworth

unpu

blish

ed

Casuarina

equisetifolia

CP00

0249

CP00

0249

CP00

0249

(Normandetal2007

[51])

AB0

15462

Sogo

etalun

publish

edAY

8640

57Herbertetalun

publish

ed


















4 Discussion



Dg1

FCjJBFCjJA


99

99

69

100

8980

92

68

91

100

99

97

99

7581

OceaniaAsia


Gro

up A

G

roup

BG

roup

CG

roup

D

CmM1aCmM2aCmMs1

CmF2CmNy5


CnP1





C lurida

8676

7590

7096

54

90

7083

7351

90

87

CjJA

Gro

up 2

Gro

up 1

Frankia Coriaria








C ruscifolia

C nepalensis


C kingiana

C plumosaC lurida


C sarmentosa

C papuana

C sarmentosa


Coriaria sp

C arborea


CaNZ1-2-3














Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















4 Discussion



Dg1

FCjJBFCjJA


99

99

69

100

8980

92

68

91

100

99

97

99

7581

OceaniaAsia


Gro

up A

G

roup

BG

roup

CG

roup

D

CmM1aCmM2aCmMs1

CmF2CmNy5


CnP1





C lurida

8676

7590

7096

54

90

7083

7351

90

87

CjJA

Gro

up 2

Gro

up 1

Frankia Coriaria








C ruscifolia

C nepalensis


C kingiana

C plumosaC lurida


C sarmentosa

C papuana

C sarmentosa


Coriaria sp

C arborea


CaNZ1-2-3














Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Dg1

FCjJBFCjJA


99

99

69

100

8980

92

68

91

100

99

97

99

7581

OceaniaAsia


Gro

up A

G

roup

BG

roup

CG

roup

D

CmM1aCmM2aCmMs1

CmF2CmNy5


CnP1





C lurida

8676

7590

7096

54

90

7083

7351

90

87

CjJA

Gro

up 2

Gro

up 1

Frankia Coriaria








C ruscifolia

C nepalensis


C kingiana

C plumosaC lurida


C sarmentosa

C papuana

C sarmentosa


Coriaria sp

C arborea


CaNZ1-2-3














Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


C ruscifolia

C nepalensis


C kingiana

C plumosaC lurida


C sarmentosa

C papuana

C sarmentosa


Coriaria sp

C arborea


CaNZ1-2-3














Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article absence of cospeciation between the

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