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Systematic and Applied Microbiology 37 (2014) 140–148

Contents lists available at ScienceDirect

Systematic and Applied Microbiology

j ourna l ho mepage: www.elsev ier .de /syapm

ore and symbiotic genes reveal nine Mesorhizobium genospeciesnd three symbiotic lineages among the rhizobia nodulatingicer canariense in its natural habitat (La Palma, Canary Islands)�

atalia Armas-Capotea,1, Juan Pérez-Yépeza,1, Pilar Martínez-Hidalgob,íctor Garzón-Machadoc, Marcelino del Arco-Aguilarc, Encarna Velázquezb,ilagros León-Barriosa,∗

Departamento de Microbiología y Biología Celular, Universidad de La Laguna, 38071 La Laguna, Tenerife, Canary Islands, SpainDepartamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, SpainDepartamento de Biología Vegetal (Botánica), Universidad de La Laguna, 38071 La Laguna, Tenerife, Canary Islands, Spain

r t i c l e i n f o

rticle history:eceived 1 July 2013eceived in revised form 30 July 2013ccepted 3 August 2013

eywords:icer canarienseild chickpeaesorhizobium

ore-gene phylogenyymbiotic-gene phylogenyovel symbiotypes

a b s t r a c t

Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhi-zobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised.The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequenc-ing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genusand distinguished nine different ribotypes. Four branches included minority ribotypes (3–5 isolates),whereas another five contained the predominant ribotypes that clustered with reference strains of M.tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense.The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M.caraganae group and outlined several potential novel species. The RAPD profiles showed a high diver-sity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbioticlineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were onlydetected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was

associated with two particular chromosomal backgrounds. However, nodC sequences closely related tosymbiovar loti predominated in most isolates, and they were detected in several chromosomal back-grounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is apromiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which meansit will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis.

ntroduction

Genus Cicer (Leguminosae) [5] comprises 44 species, 9 annualsnd 35 perennials that have a centre of diversity in south-westernsia, with remote, endemic species found in Morocco and theanary Islands [46]. The genus is the only member of the tribe

� Nucleotide sequence data reported are available in the EMBL databasender the accession numbers: HF931040–HF931081 for 16S rRNA genes,F933967–HF934005, HF954915 and HG003676–HG003677 for nodC genes,G323883–HG323909 for recA genes and HG323910–HG323936 for glnII genes.∗ Corresponding author at: Departamento de Microbiología y Biología Celular,niversidad de La Laguna, Facultad de Farmacia, Avd. Astrofísico Fco. Sánchez s/n,pain. Tel.: +34 922318481; fax: +34 922318666.

E-mail address: mileonba@ull.es (M. León-Barrios).1 These authors have equally contributed to this work.

723-2020/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.syapm.2013.08.004

© 2013 Elsevier GmbH. All rights reserved.

Cicereae, subfamily Papilionoideae. The most widely known speciesis the cultivated chickpea, Cicer arietinum L., owing to the factthat it is one of the most important grain legume crops in theworld, especially in several Mediterranean countries. It has tradi-tionally been considered a restrictive host for nodulation because,until recently, only two species had been recognised as specificsymbionts: Mesorhizobium ciceri [30] and M. mediterraneum [29].However, this situation changed with the detection of chickpea rhi-zobia belonging to several other Mesorhizobium species [6,20,36].Recently, the focus of interest has broadened to the wild Cicerspecies, since increasing C. arietinum productivity is constrictedby various abiotic and biotic stresses. For instance, C. arietinumis very susceptible to a number of pathogens that seriously affect

crop yield [4,38]. In contrast, the wild Cicer are resistant to severalof these stresses, and are therefore potential sources of a diversegene pool that could provide greater tolerance for C. arietinum viainterspecific hybridisation [4,24,42]

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N. Armas-Capote et al. / Systematic an

Cicer canariense Santos and Lewis [39] is a wild chickpeandemic to the Canary Islands and has been found mainly on Laalma, as well as in one location in the southwest of Tenerife. It isound growing in the dry mesocanarian bioclimatic belt character-stic of the local pine forests (Pinus canariensis), although it tendso seek out the more humid areas of these habitats [39]. In theine forest understorey, it is found sharing the habitat with otherlants that include several other legumes, such as Lotus campylo-ladus ssp. hillebrandii which is one of the most common abundantccompanying species. This perennial legume is the only nativeicer species in the Canaries and natural populations of C. canariensere fragmented and contain a low number of individuals, whichre currently catalogued as “endangered” (EN) in the Red List ofhe Spanish Vascular Flora [27]. Herbivores have probably been the

ost important threat for C. canariense (mouflons and rabbits onenerife, and Barbary sheep, goats and rabbits on La Palma) [8].oreover, it is used by local people as cattle forage. Apart from

ts biological importance and fodder potential, C. canariense couldlso be a good source of resistance genes, since it has shown resis-ance to infections by fungi such as Fusarium oxysporum, a majorause of wilt in chickpea [13], and viruses (pea streak carlavirus,14]) or to caterpillar attack, Helicoverpa armigera [40]. Therefore, C.anariense has also attracted attention for the genetic improvementf the commercial chickpea [25].

Despite the growing interest in wild Cicer species, there isot much information concerning the diversity of microsymbiontsodulating these legumes. To our knowledge, there is only oneeport concerning the characterisation of a rhizobia collectionsolated from perennial wild chickpea C. anatolicum in eastern Ana-olia, Turkey [31]. However, only rep-PCR fingerprints (ERIC-, REP-nd BOX-PCR), were used to assign them to the doubtful taxonomicffiliation of Rhizobium leguminosarum ssp. ciceri.

In this current study, the genetic diversity of the rhizobia nodu-ating Cicer canariense was investigated for the first time in naturalnd some reinforced populations on La Palma (Canary Islands).ppropriate inoculation with good nitrogen-fixing rhizobia couldelp in the recovery of C. canariense populations in the Caldera deaburiente National Park, and might also be a possible source ofhizobial inoculants that could be used potentially for improvedybrid chickpea cultivars of C. arietinum-C. canariense.

aterials and methods

ocalities and isolation of the rhizobia

Localities for rhizobia isolations were selected in areas with nat-rally growing or planted populations of C. canariense (Table 1).en sample sites were selected that included six locations withinhe Caldera de Taburiente National Park (CdTNP): (Miradore las Chozas (MdC) (UTM coordinate 28R220323/3177809,280 m.a.s.l.), Roque de los Cuervos (RdC) (28R220530/3177444;480 m.a.s.l.), Fuente de las Mulas (FdM) (28R217896/3180655,50 m.a.s.l.), Risco Liso (RL) (28R218196/3181403, 1020 m.a.s.l.),iachuelo (Ria) (28R221606/3177050, 1110 m.a.s.l.) and southejenado (Bej) (28R0220744/3176348, 1288 m.a.s.l.). Another fourreas were selected outside the Park: Barranco (Bco.) de los Hom-res (BdH) (28R219475/3165577, 911 m.a.s.l.), Bco. de BriestasBdB) (28R214671/3186655; 1375 m.a.s.l.), Bco. de Dornajito (BdD)28R213842/3187105; 1186 m.a.s.l.) and Bco. de Izcagua (BdI)28R211180/3186279, 798 m.a.s.l.). The localities outside the Parkere included because they were optimal areas for C. canariense

ccording to a modelling map built as described. A chorologicalap of C. canariense was built with ArcGIS [3] from the coordinates

f the known wild populations. Taking into account geographicalnd ecological features and parameters of these localities, the

lied Microbiology 37 (2014) 140–148 141

Maximum Entropy (Maxent) algorithm [33] was run in order tobuild a modelling map which predicted the optimal areas for C.canariense populations. The Maxent algorithm is used to induc-tively interpolate or extrapolate fundamental niches outside thelocations where a species is present (i.e. realised niches) by relatingspecies presence to environmental predictors. The environmentalvariables were selected between the most common features influ-encing the Canary distribution of the plants (altitude, slope, aspect,soil type, average annual rainfall and temperature). A digital eleva-tion model (DEM) for the area was used directly to obtain altitude,and indirectly (using spatial analyst techniques) to obtain slopeand aspect. In addition, interpolation techniques applied to mete-orological stations operated by the State Meteorological Agency ofSpain (AEMET) were used to generate both average annual rainfalland temperature. A map of soil type was also taken into account inorder to build the modelling map. The model was calibrated usinga random 70% of the data as a training sample and evaluated withthe remaining 30%. Ten replicates were performed to improve themodel. To evaluate the accuracy of the model, measurements of thereceiver operating characteristic (ROC) curve and the area underthe curve (AUC) were used. The ROC curve represents the relation-ship between 1 and the correctly predicted percentage presence(sensitivity) minus the correctly predicted percentage absences(specificity). The area under the curve (AUC) measures the abil-ity of the model to classify a species correctly as present or absent[32].

To isolate the rhizobia, sterilised and germinated seeds weresown as trap plants in the soil samples. Seed germination requiredprevious removal of seed hardness by 120 min exposure to concen-trated sulphuric acid [11]. The bacteria were recovered from theroot nodules eight weeks later. Alternatively, when possible, thebacteria were isolated directly from root nodules of wild seedlings.Nodules were crushed separately and purified by repeated streak-ing on yeast mannitol agar (YMA) [43]. The isolates were grownand purified at 28 ◦C on YMA and stored at −80 ◦C in 20% glycerolYM (v/v).

Infectivity tests

To test infectivity on their original legume, a large sample ofisolates were selected that represented all genospecies and sym-biotypes at different locations. However, seed availability did notpermit infectivity to be tested for all isolates. The isolates weregrown to high cell density in YEM broth and this suspension wasused to inoculate C. canariense seedlings. Three or four replicatesfor each strain were infected and checked for nodulation eightweeks later. Non-inoculated plants were used as negative con-trols. Nitrogen-free medium [35] was used with trace elements,as previously described [43].

DNA isolation

Total genomic DNA was obtained from bacterial batch culturesgrown until late exponential phase using the AquaPure GenomicDNA isolation kit (Bio-Rad). DNA concentrations were visually esti-mated from a 1% agarose gel electrophoresis by comparing theDNA samples with a lambda-DNA HindIII digest or alternatively bymeasurement in a NanoDrop 1000 spectrophotometer.

Random amplified polymorphic DNA (RAPD) fingerprint

The M13-RAPD-PCR technique was used to produce a molecu-lar fingerprint of each isolate following the procedure previouslydescribed [34]. TIFF files were analysed using GelComparII 4.0

142 N. Armas-Capote et al. / Systematic and Applied Microbiology 37 (2014) 140–148

Table 1Isolates, locations and some genetic characteristics of Cicer canariense.

Isolate Locationa 16S RFLPgroupb

16S phylogeny(closest species)

Accession numbers nodC gene symbiotype Accession numbers Phenotype onC. canariense

CCANP1 MdC (d) Maus M. australicum HF931040 loti HF933967 Nod+/Fix+CCANP2 MdC (d) MoppCCANP3 MdC (d) Mopp M. opportunistum HF931041 new HF933968 Nod+/Fix+CCANP5 MdC (d) Mtian loti HF933969 Nod+/Fix+CCANP7 MdC (d) MtianCCANP8 MdC (d) MtianCCANP9 MdC (d) Mtian loti HF933970 Nod+/Fix+CCANP10 MdC (d) MtianCCANP11 MdC (d) Mtian M. tianshan-SGr HF931042 loti HF933971 Nod+/Fix+CCANP12 MdC (d) Mtian lotiCCANP13 MdC (d) Mtian loti HF933972CCANP14 MdC (d) Mci M. ciceri HF931043 loti HF933973 Nod+/Fix+CCANP15 MdC (d) MciCCANP16 MdC (d) Mtian Nod+/Fix+CCANP17 MdC (d) Mcarag M. caraganae-IIa

CCANP18 MdC (d) MtianCCANP19 MdC (d) MtianCCANP20 MdC (d) Mloti M. ciceri HF931044 loti HF933974 Nod+/Fix+CCANP21 MdC (d) MtianCCANP22 MdC (d) MlotiCCANP23 MdC (d) Mloti loti HF933975 Nod+/Fix+CCANP24 MdC (t) Mci M. ciceri HF931045 loti HF933976 Nod+/Fix+CCANP25 MdC (t) Mci loti HF933977 Nod+/Fix+CCANP26 MdC (t) MausCCANP27 MdC (t) Mloti M. ciceri HF931046 loti HF933978 Nod+/Fix+CCANP28 RL (t) MoppCCANP29 MdC (t) Mtian M. tianshan-SGr HF931047 loti HF933979 Nod+/Fix+CCANP30 MdC (t) MtianCCANP31 MdC (t) MtianCCANP32 MdC (t) MtianCCANP33 MdC (t) Mtian M. tianshan-SGr HF931048 loti HF933980 Nod+/Fix+CCANP34 FdM (t) Mcarag M. caraganae-I HF931049 new HF933981 Nod+/Fix+CCANP35 FdM (t) Mcarag M. caraganae-I HF931050 new HF933982 Nod+/Fix+CCANP36 FdM (t) Mcarag M. caraganae-Ia

CCANP37 FdM (t) Mcarag M. caraganae-Ia

CCANP38 FdM (t) Mcarag M. caraganae-I HF931051 new HF933983 Nod+/Fix+CCANP40 MdC (t) MtianCCANP41 MdC (t) Mtian Nod+/Fix+CCANP42 MdC (t) MtianCCANP43 MdC (t) MtianCCANP44 MdC (t) Mtian Nod+/Fix+CCANP45 MdC (t) McaragCCANP47 Ria (t) MciCCANP48 Ria (t) Mci M. ciceri HF931052 ciceri HF933984 Nod+/Fix+CCANP53 BdH (d) Mcarag M. caraganae-IIa

CCANP54 BdH (d) Mcarag M. caraganae-IIa

CCANP55 BdH (d) Mlo-SBr M. loti-SBr HF931053 loti HF933985 Nod+/Fix+CCANP56 BdH (d) McaragCCANP58 BdH (d) Mcarag M. caraganae-II HF931054 loti HF933986 Nod+/Fix+CCANP59 BdH (d) Mcarag M. caraganae-IIa

CCANP61 BdH (d) Mlo-SBr M. loti-SBr HF931055 loti HF933987 Nod+/Fix+CCANP62 BdH (d) Mcarag M. caraganae-IIa

CCANP63 BdH (d) Mcarag M. caraganae-II HF931056 loti HG003676 Nod+/Fix+CCANP64 Bej (d) Maus M. australicum HF931057 loti HF954915 Nod+/Fix+CCANP65 Bej (d) Mcarag M. caraganae-II HF931058 –b Nod+/Fix+CCANP66 Bej (d) Mlo-SBr Nod+/Fix+CCANP67 Bej (d) MoppCCANP68 Ria (d) Mtian loti HF933988 Nod+/Fix+CCANP70 Ria (t) Mam/Mhua M. amorphae-like HF931059 new HF933989 Nod+/Fix+CCANP73 Ria (d) Mopp M. opportunistum HF931060 new HF933990 Nod+/Fix+CCANP75 Bej (t) Maus Nod+/Fix+CCANP76 Bej (t) Mtian M. tianshan-SGr HF931061CCANP77 Bej (t) Mcarag M. caraganae-II HF931062 –b

CCANP78 RdC (t) Mtian M. tianshan-SGr HF931063 Nod+/Fix+CCANP79 RdC (t) Mci M. ciceri HF931064 ciceri HF933991 Nod+/Fix+CCANP80 RdC (t) Mci ciceri HF933992 Nod+/Fix+CCANP82 RdC (t) Mci M. ciceri HF931065 ciceri HF933993 Nod+/Fix+CCANP83 RdC (t) MtianCCANP84 RdC (t) Mam/Mhua loti HF933994 Nod+/Fix+CCANP85 RdC (t) Mci M. ciceriCCANP86 RdC (t) MciCCANP87 RdC (t) Mam/Mhua M. amorphae HF931067 loti HF933995 Nod+/Fix+CCANP88 RdC (t) Mcarag M. caraganae-Ia

CCANP89 RdC (t) Mtian

N. Armas-Capote et al. / Systematic and Applied Microbiology 37 (2014) 140–148 143

Table 1 (Continued)

Isolate Locationa 16S RFLPgroupb

16S phylogeny(closest species)

Accession numbers nodC gene symbiotype Accession numbers Phenotype onC. canariense

CCANP90 FdM (t) Mtian M. tianshan-SGr HF931068 HG003677 Nod+/Fix+CCANP91 FdM (t) Mcarag M. caraganae-Ia

CCANP92 FdM (t) McaragCCANP93 FdM (t) Mci M. ciceri HF931069 ciceri HF933996 Nod+/Fix+CCANP94 FdM (t) Mcarag M. caraganae-I HF931070CCANP95 FdM (t) Mcarag M. caraganae-I HF931071 new HF933997 Nod+/Fix+CCANP96 MdC (t) Mtian M. tianshan-SGr HF931072 loti HF933998 Nod+/Fix+CCANP98 MdC (t) Mcarag M. caraganae-Ia

CCANP99 Bej (t) Mopp M. opportunistum HF931073 –b Nod+/Fix+CCANP100 Bej (t) Mopp M. opportunistum HF931074 –b Nod+/Fix+CCANP101 Bej (t) Mopp –b

CCANP102 Bej (t) Mopp Nod+/Fix+CCANP103 Bej (t) MausCCANP104 Bej (t) MoppCCANP105 Bej (t) Mcarag M. caraganae-II HF931075 Nod+/Fix+CCANP106 Bej (t) Mcarag M. caraganae-II HF931076CCANP107 Bej (t) Mcarag M. caraganae-IIa Nod+/Fix+CCANP108 BdH (t) Mcarag M. caraganae-IIa

CCANP109 BdH (t) Mopp M. opportunistum-like HF931077 loti HF933999 Nod+/Fix+CCANP110 BdH (t) Mopp Nod+/Fix+CCANP113 BdD (d) Mopp loti HF934000CCANP114 BdD (d) MoppCCANP115 BdD (t) McaragCCANP117 BdD (t) Mcarag M. caraganae-I HF931078 loti HF934001 Nod+/Fix+CCANP119 BdB (t) MtamaCCANP121 BdB (t) MtamaCCANP122 BdB (t) Mtama M. tamadayense HF931079 loti HF934002 Nod+/Fix+CCANP123 BdB (t) MtamaCCANP124 BdB (t) MtamaCCANP125 BdB (t) MtamaCCANP126 BdB (t) MtamaCCANP127 BdB (t) Mtama loti HF934003 Nod+/Fix+CCANP128 BdB (t) MtamaCCANP130 BdB (t) Mtama M. tamadayense HF931080 loti HF934004 Nod+/Fix+CCANP131 BdB (t) MtamaCCANP133 BdI (t) Mcarag M. caraganae-I HF931081 loti HF934005 Nod+/Fix+CCANP134 BdI (t) Mcarag M. caraganae-Ia

CCANP135 BdI (t) Mcarag M. caraganae-Ia

CCANP138 BdI (t) Mcarag

(a) Locations: MdC: Mirador de las Chozas, FdM: Fuente de la Mula, RdC: Roque de los Cuervos, Ria: El Riachuelo, Bej: Bejenado sur, RL: Risco Liso alto, BdH: Barranco de losHombres, BdD: Barranco de Dornajito, BdB: Barranco de Briestas, BdI: Barranco de Izcagua. In brackets: (d) direct isolation; (t) trap plant.(b) Mcarag: M. caraganae; Mloti: M. loti; Mam/Mhua: M. amorphae/M. huakuii; Maus: M. australicum; Mopp: M. opportunistum; Mtian: M. tianshanense; M. tianshan-SGr: M.t tamad

succe

(i

R(

dv(aa(gwaMMaLa(f

ianshanense-subgroup; Mlo-Sbranch: M. loti-subbranch; Mci: M. ciceri; Mtama: M.a Deduced from sequencing of a 16S rDNA partial fragment (400 nt).b Several attempts to amplify the nodC gene with the two pair of primers were un

Applied Maths, Belgium) with the Pearson correlation coefficientn order to generate a UPGMA dendrogram.

estriction fragment length polymorphism of 16S rRNA genes16S-RFLP)

Near full-length 16S rRNA genes were amplified as previouslyescribed [12] and 8 �L aliquots of the PCR products were indi-idually restricted with three endonucleases, MspI, RsaI and HinfIPromega), following the manufacturer’s recommendations withn excess of enzyme (5U). Gel electrophoreses were photographednd the digitalised TIFF files were analysed using GelComparII 4.0Applied Maths, Belgium) in order to construct a UPGMA dendro-ram. The combined restriction patterns from three endonucleasesere used to classify the isolates at the genus level and for

n approximation to species level. Reference strains used were:. ciceri USDA 3383T, Mesorhizobium mediterraneum USDA3392T,esorhizobium loti NZP 2213T, M. opportunistum WSM 2075T, M.

morphae LMG 18977T, M. huakuii LMG 14107T, M. metallidurans

MG 24485T, M. caraganae LMG 24397T, M. chacoense PR5T, M.ustralicum WSM2073T and Mesorhizobium tianshanense A-1BST

patterns for these latter two species were deduced respectivelyrom their sequences: AY601516 and AF041447); Ensifer meliloti

ayense.

ssful.

LMG 6133T, E. medicae USDA 1037T, Rhizobium leguminosarumUSDA2370T, R. tropici II B CIAT889T, Bradyrhizobium japonicumUSDA 6T and B. canariense BTA-1T.

Sequencing of 16S rRNA, recA and nodC genes

The 16S rRNA genes (rrs) were amplified as described above. TherecA and glnII gene sequences were amplified by using the primerpairs recA6/recA555 [9], and glnII 12F and glnII 689R [48], respec-tively, and the nodC genes by using the primer pairs nodCF/nodCI[18] or nodCMesoF and nodCMesoR [36]. The PCR-amplified prod-ucts were purified (Qiaquick Extraction Kit, Qiagen) and sequencedin an ABI3730XL (Macrogen, Inc.) or, alternatively, in a Genetic Ana-lyzer 3500 (Applied Biosystems; Servicio de Genómica, Universidadde La Laguna).

Phylogenetic analyses

Sequence alignments (ClustalW) and phylogenetic analyses

were conducted using the MEGA5 software package (version 5.10)[45] using the neighbour-joining (NJ) and maximum likelihood(ML) methods. Neighbour-joining (NJ) [41] trees were built usingKimura’s 2-parameter model [17]. For ML reconstructions, the

144 N. Armas-Capote et al. / Systematic and Applied Microbiology 37 (2014) 140–148

samp

btiwaw(

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L

nc(z((FaRtfunoswmbbc

Fig. 1. Map (La Palma, Canary Is.) showing the distribution of

est-fitting evolutionary model of nucleotide substitutions for eachype of sequence was determined using the MEGA5 package ands detailed in the figure legends. Confidence of the tree branches

as estimated with 1000 and 500 bootstrap replications for NJnd ML, respectively. The sequences of rrs genes were comparedith those of bacterial type strains using the EzTaxon-e server

http://eztaxon-e.ezbiocloud.net; [16]).

esults

ocations and isolations

A collection of 113 isolates were recovered from the rootodules of Cicer canariense plants growing at ten locations withurrently known populations of this legume species on La PalmaTable 1; Fig. 1). Since the main aim was to characterise the rhi-obia populations inside the Caldera de Taburiente National ParkCdTNP), most isolates (83) were recovered from six localities thereMdC, RdC, FdM, RL, Ria and Bej; Table 1). Three of these, Bej,dM, and RdC, contained natural populations of C. canariense with

very low number of individuals. The other three locations, MdC,L and Ria, were experimental populations (planted plots) insidehe Park (Table 1). At MdC, the relatively large number of seedlingsound around this plot permitted direct isolations from root nod-les (Table 1). Although a similar situation was found in the RL plot,o nodules were found in situ on the roots of these seedlings, andnly one nodule was obtained from the trap plants grown in theseoils. This locality has a high nitrogen content [7] that, togetherith the thick layer of pine needle litter (about 25 cm deep) accu-

ulated over this soil and a well developed fungus layer, could

e responsible for inhibition of nodulation. A modelling map wasuilt taking into account environmental variables prevalent at theurrently known locations of this species. The variables slope and

le sites where root nodules of Cicer canariense were collected.

aspect were not found to be informative and were eliminated fromthe modelling reconstruction, and the final model thus retainedaltitude, soil type, the mean annual rainfall and temperature as themost significant parameters. Table S1 shows the habitat values suit-able for the presence of C. canariense species, which were used toconstruct the suitability map for the distribution of this species (Fig.S1). The high AUC value of 0.926 guarantees modelling reliability[2]. The jackknife test (incorporated in Maxent software) showedthat the average annual rainfall was the most informative param-eter for species distribution. The modelling showed that only thepopulations in the south of the Park (Ria, MdC, RdC and Bej, redand orange areas on the map; Fig. S1) were included inside optimalareas for C. canariense. However, several other optimal areas werepredicted outside the Park and a group of 30 isolates was recoveredfrom four natural populations there (BdH, BdI, BdB and BdD) (Fig.S1), since we were interested in knowing whether specific rhizo-bia were associated with each locality. This could be useful whenintroducing C. canariense plants during reinforcement campaignsin order to inoculate them with the appropriate rhizobia strain fora specific area.

Genetic diversity of the isolates: 16S-RFLP and RAPD fingerprints

16S-RFLP analysis (Fig. S2) grouped the isolates within theMesorhizobium genus and distinguished nine different ribotypes(ribosomal genotypes) that were either closely grouped or occurredwithin clades containing several Mesorhizobium reference species,indicating that they were taxonomically diverse. Four branchesrepresented minor ribotypes (3–5 isolates) and they showed pat-terns identical or close to type strains of M. australicum (5 isolates),

M. loti (4), M. loti-subbranch (3) and M. amorphae/M. huakuii (3).The other branches contained the five predominant ribotypes thatgrouped with species M. caraganae (32), M. tianshanense/M. gob-iense/M. metallidurans (the “M. tianshanense-subgroup”) (29), M.

d Applied Microbiology 37 (2014) 140–148 145

obttnTpllRoMdsrgip

g(blrrrfsMrwactts

1

nsgricMcRtotfCsR9ls361abd1

Fig. 2. Maximum likelihood (Jukes-Cantor model with gamma distribution andinvariant sites) phylogenetic tree based on 16S rRNA gene sequences (1308 nt) ofCicer canariense isolates and type strains of Mesorhizobium. Accession numbers are

N. Armas-Capote et al. / Systematic an

pportunistum (14), M. ciceri (12) and M. tamadayense (11). A fewranches contained just one isolate and were not considered fur-her in this work. The presence of the major ribotypes varied athe different geographical sites: some were restricted or predomi-ated at a single location, whereas others were widely distributed.he M. tamadayense ribotype was exclusively detected in a naturalopulation of C. canariense located outside the Park (BdB). The iso-

ates with an M. ciceri ribotype predominated in two neighbouringocations (RdC and MdC). The members of the M. tianshanense-FLP group were recovered almost exclusively (23 out of 29) inne experimental plot in the MdC locality. The isolates from the. opportunistum and M. caraganae-RFLP groups were more widely

istributed, and were recovered in several locations inside and out-ide the Park. However, these two ribotypes were predominantlyecovered in localities where natural populations of C. canarienserew. This was especially remarkable for the M. caraganae ribotypesolates, since, out of 32 in total, 29 were recovered from naturalopulations compared to 3 isolates from experimental plots.

At the infraspecific level, the RAPD fingerprints showed a wideenetic diversity with almost all isolates being different strainsFig. S3). Ten were redundant strains (a strain was considered toe a clone when it showed ≥98% similarity; Fig. S3). Highly simi-

ar isolates belonged to the same RFLP group and, in general, wereecovered from the same location. The isolates with the M. ciceriibotype showed little diversity (85.3% similarity and 4 out of 12edundant strains) and only distantly clustered with a group ofour isolates with an M. loti-RFLP pattern that, according to theequences, belonged to M. ciceri. Likewise, most isolates from the. tianshanense group clustered together (80.1% similarity and 4

edundant strains). The M. caraganae and M. opportunistum isolatesere genetically more diverse, which correlated with their wider

rea of distribution inside and outside the Park, and only smalllusters were delineated, usually from the same location. The M.amadayense isolates, despite being exclusive to one single loca-ion outside the Park (BdB), were also diverse (only one redundanttrain being detected).

6S rRNA, recA and glnII gene phylogenies

To infer the phylogenetic relationships with all currently recog-ised species within the genus Mesorhizobium, representativetrains from the 16S-RFLP groups were chosen for 16S rRNAene sequencing. The ML (Fig. 2) and NJ (Fig. S4) phylogeneticeconstructions were congruent and confirmed the RFLP group-ngs, but they resolved more internal divergence within the M.araganae-RFLP group (Fig. 2). The representative isolates from the. ciceri-RFLP group had 16S sequences 99.9–100% similar to M.

iceri UPM-CaT. Moreover, CCANP20 and CCANP27 from the M. loti-FLP group, were closer to M. ciceri UPM-CaT (99.9% similarity)han to M. loti NZP2213T (99.8%) (Fig. 2). The isolates from the M.pportunistum-RFLP group were 99.8–99.9% similar to M. oppor-unistum WSM2075T and clustered congruently (Fig. 2), exceptor CCANP109 (99.6% similarity) that clustered separately. IsolatesCANP1 and CCANP64 (from a small RFLP group of five) were 100%imilar to M. australicum WSM2073T. From the M. tamadayense-FLP group, CCANP122 and CCANP130 were respectively 100% and9.1% similar to the type strain and clustered together (Fig. 2). The

argest RFLP group of M. caraganae isolates was resolved into twoubgroups (Fig. 2) that we referred to as M. caraganae-I (CCANP:4, 35, 38, 94, 95, 117 and 133) and M. caraganae-II (CCANP: 58,3, 65, 77, 105 and 106). Isolates from group I had sequences00% similar to M. caraganae CCBAU 11299T, whereas group II

lso shared the highest similarity (99.7%) with the type strain,ut clustered apart in a well-supported branch without previouslyescribed Mesorhizobium species (Fig. 2). A partial fragment of the6S rRNA gene obtained to distinguish between another 14 isolates

given in parentheses. Numbers at the nodes are bootstrap support values (≥50%) for500 pseudoreplicates. The scale bar indicates the number of substitutions per site.

within the M. caraganae-RFLP group showed that approximately50% of isolates belonged to each subgroup (Fig. S5). Representativeisolates from the M. tianshanense-RFLP group shared the highestsimilarities (99.7–99.9%) with three related species: M. tiansha-nense, M. metallidurans and M. gobiense. Although the referencespecies were not resolved in the ribosomal phylogeny, our isolates(except CCANP90) formed a homogeneous clade (here referred toas the M. tianshanense-subgroup) (Fig. 2). Isolates CCANP55 andCCANP61 from the minor RFLP group called the M. loti-subbranch,were placed in a subbranch that had M. shangrilense, M. qingshengiiand M. australicum as closest relatives (Fig. 2). Finally, CCANP70and CCANP87 from the M. amorphae/M. huakuii-RFLP group sharedthe highest similarities, 99.8% and 100%, respectively, to M. amor-phae ACCC19665T, although, while CCANP87 grouped closely to M.amorphae, CCANP70 formed a subbranch on its own (Fig. 2).

Partial sequences from two protein codifying genes, recA and

glnII, were obtained in order to contrast the ribosomal phylogeny.They were mostly congruent with each other and showed good sup-port for the genomic groups delineated in the ribosomal phylogeny

146 N. Armas-Capote et al. / Systematic and App

Fig. 3. Maximum likelihood (Tamura 3-parameter with gamma distribution andinvariant sites) phylogenetic tree based on nodC sequences (672 nt) of Cicercct

(staafRMdfNctt

P

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anariense isolates and reference strains. The significance of each branch is indi-ated by a bootstrap value (≥60%) calculated for 500 subsets. The scale bar indicateshe number of substitutions per site.

Figs. S6A and B show the ML trees; NJ reconstructions producedimilar topologies and are not shown). Minor discrepancies withhe ribosomal phylogeny were found for two isolates: CCANP73nd CCANP117, which occupied branches on their own in the glnInd recA phylogenies, respectively. The greatest discrepancy wasound in the housekeeping gene phylogenies for the M. caraganae-FLP group. Although both phylogenies delineated the subgroups. caraganae-I and M. caraganae-II, the glnII phylogeny was coinci-

ent with the ribosomal phylogeny and showed a close relationshipor both subgroups with M. caraganae CCBAU 11299T (Fig. S6B).evertheless, in the recA phylogeny the two subgroups clusteredlose to each other but were distantly placed from the M. caraganaeype strain (Fig. S6A). Thus, further work will be needed to resolvehe definitive taxonomic affiliation of these isolates.

hylogeny from the nodC genes

Representative strains from the different ribosomal lineagesere chosen for partial sequencing of the nodC gene. ML (Fig. 3)

nd NJ (Fig. S7) phylogenic trees were coincident and showed

lied Microbiology 37 (2014) 140–148

that the mesorhizobia nodulating C. canariense belonged to threedistant symbiotic lineages (A–C; Fig. 3). The larger lineage (A;Fig. 3) included isolates with highly similar nodC gene sequences(94.7–95.3%) to those of reference strains such as M. loti NZP2213T,M. loti R7A, and MAFF303099, which are the typical symbionts ofLotus species. This symbiotype was the most widely distributedbetween the C. canariense root nodule bacteria, since it wasdetected in isolates from the nine genomic groups. Within thislarge branch, several subbranches could be distinguished, onecontained only M. loti reference strains (A4; Fig. 3) and anotherthree had several groups of isolates. The largest one was mis-cellaneous (A1; Fig. 3) and included isolates from seven genomicgroups (M. caraganae-I, M. caraganae-II, M. ciceri, M. opportunistum,M. australicum, M. loti-subbranch and M. amorphae); however, theother two subbranches contained isolates from only one genomiclineage: the M. tianshanense-subgroup (A3; Fig. 3) or the M. tama-dayense group (A2; Fig. 3). Isolate CCANP117 formed a separatesubbranch. The second symbiotic lineage (B; Fig. 3) was formedby isolates with nodC sequences 100% similar to those of symbio-var ciceri. This symbiotype was only found in M. ciceri isolates. Athird symbiotype (C; Fig. 3) was detected in M. caraganae-I and M.opportunistum groups and isolate CCANP70. These nodC sequenceswere divergent from all those previously known, and formed a sep-arate well-supported branch whose closest relatives belonged to acongruent group of several Mesorhizobium species (Fig. 3).

Representative strains from the three nodC symbiotypes andthose belonging to different genomic groups were checked forinfectivity on the original legume host C. canariense. All isolatesshowed a nodulating phenotype (Table 1), although differenceswere observed in the growth and greenness of plants. However,since the number of replicates was low, it will be necessary toextend the number of infectivity assays to draw conclusions aboutthe nitrogen fixation efficiency of the three symbiotic lineages.

Discussion

Genetic diversity and taxonomy of the Cicer canariense rhizobia

In this study, the genetic diversity of the rhizobia that nodu-late the wild chickpea Cicer canariense has been identified andcharacterised for the first time, and the genospecies distributionin their natural habitats of La Palma was demonstrated. It wasfound that the C. canariense rhizobia were highly diverse, withmesorhizobial strains scattered within up to nine lineages of theMesorhizobium genus. Some of these genotypes could be assignedto already recognised Mesorhizobium species, whereas others mightrepresent novel species. The operational taxonomic units (OTUs)were defined according to the consensus of the genomic group-ings defined by the 16S-RFLP analysis and phylogenies from therrs, recA and glnII genes. Thus, nine genomic groups or genospecieswere considered: M. caraganae-I, M. caraganae-II, M. tianshanense-subgroup, M. opportunistum, M. ciceri, M. australicum, M. amorphae,M. loti-subbranch and M. tamadayense. In addition, a few iso-lates formed unique branches that could represent novel lineages.Although the definitive affiliation to a named species or descrip-tion of a new taxon will require further studies, several taxonomicgeneralisations can be made regarding the rhizobial diversity ofthis wild Cicer. The diversity detected in the C. canariense rhi-zobia is in some ways comparable to that of the cultivated C.arietinum rhizobia in Portuguese soils, where nodulation by six dif-ferent Mesorhizobium species, M. ciceri, M. mediterraneum, M. loti,

M. amorphae, M. tianshanense and M. opportunistum, has alreadybeen described [1,19,20]. This is therefore far from the former tax-onomic outline that contained only two specific species, M. ciceriand M. mediterraneum. The endemic Cicer shared some rhizobial

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N. Armas-Capote et al. / Systematic an

ymbionts with the cultivated chickpeas but lacked others, while itlso nodulated with rhizobia not previously linked with the genusicer. Thus, M. ciceri constituted a minority genotype (10% of iso-

ates) among the C. canariense symbionts, and M. mediterraneumas not detected. However, M. opportunistum, recently described as

common C. arietinum root nodule bacteria [20], has also now beenound as the third predominant genotype among the C. canariensehizobia. The second most predominant genotypes among the C.anariense rhizobia had M. tianshanense and other closely relatedpecies as the closest relatives. However, our group was clearlyifferentiated in all the phylogenies from the reference species,uggesting that they represent a novel Mesorhizobium species. Thisovel lineage was almost restricted to one locality inside the ParkMdC), and was isolated from a C. canariense population in a plantedlot. However, some C. arietium rhizobia isolated in Portugal, whichere assigned to M. tianshanense based on ribosomal sequences

36], could also belong to this novel lineage (Figs. 2, S4 and S6A). Theargest group of isolates belonged to, or were closely related to, M.araganae [10], a species not previously related to the Cicer genus.he M. caraganae-I group was recovered mainly in a natural pop-lation inside the Park (FdM) and, according to the data presentedere, might belong to M. caraganae. The M. caraganae-II isolatesredominated in another two natural populations inside (Bej) andutside (BdH) the Park, and could be a novel related species. Finally,mong the minority genospecies detected, M. amorphae [49] haslready been linked with C. arietinum [36], whereas it is the firstime that M. australicum [28] and M. tamadayense [34] have beenited as Cicer symbionts.

ymbiotic characteristics of the C. canariense rhizobia

It is commonly found that rhizobia from distinct species andven genera that nodulate the same legume host harbour highlyimilar symbiotic genes [6,18,23,48,51]. This is also the situationor the C. arietinum rhizobia, which all share the same commonenes: symbiovar ciceri [19,20,36,50]. However, in the C. canariensehizobia a more complex picture was found with three differentymbiotypes. Two symbiotypes showed correlations to particularesorhizobium chromosomal backgrounds. The symbiotype cicerias strictly associated with isolates with an M. ciceri chromosome,

s in all the C. arietinum rhizobia. The novel symbiotype was onlyetected in M. caraganae-I and M. opportunistum isolates (onexception was CCANP70), and the divergence of these sequencesuggested a specific symbiovar for C. canariense. However, most ofhe isolates harboured symbiotype loti genes (about 95% similaro symbiovar loti), widely spread across the nine chromosomalackgrounds or genospecies of Mesorhizobium detected here,

ncluding some M. ciceri isolates. The high similarity between theodC sequences of our isolates and the Lotus rhizobia, together withhe fact that in M. loti these genes are situated on mobile symbioticslands that can be transferred in nature [15,44], suggested thatotus rhizobia were probably the source symbiotic genes for most ofhe C. canariense symbionts. It is interesting that while C. canarienseopulations are scarce and fragmented, L. campylocladus ssp. hille-randii grows abundantly in the same habitats, offering a possibleource of symbiotic genes for the C. canariense rhizobia. However,. loti, the typical Lotus rhizobium and supposedly the origin of the

ymbiovar loti-type genes, was not found among the C. canarienseymbionts. It may be that certain core-genome genes absent in. loti are needed in order to support nodulation on C. canariense

r, contrary to previous hypotheses, M. loti is in fact an infrequentymbiont of Lotus, as has already been suggested [21,22].

The symbiovars in rhizobia reflect bacterial adaptation toegumes [37]. Therefore, the divergence in the nodC sequences

ithin the large cluster of “symbiovar loti” (approximately 4–5%mong subbranches; Fig. 3) could reflect specific adaptations to a

lied Microbiology 37 (2014) 140–148 147

new niche (from the Lotus root nodule to C. canariense). Neverthe-less, co-evolution between the symbiotic genes and the chromoso-mal core genes also seems to be observed, since two subbranchesonly contained isolates with the same chromosomal background.Several cases of co-evolution between the symbiotic and the coregenes have also been described in other symbioses [26,47,52].

Variations in nodule colour and plant development were noticedwhen inoculated with different isolates, suggesting differences inthe symbiotic capabilities among the three lineages. To describethe symbiotic characteristics of the symbiovars associated withthe C. canariense rhizobia will require other symbiotic genes tobe sequenced and more extensive assays to estimate and comparetheir N2-fixation effectiveness, as well as infectivity tests in otherlegumes in order to determine their host range and host specificity.In summary, these results show that C. canariense is a promiscuouslegume permitting nodulation by many different Mesorhizobiumspecies and by three different symbiotypes, which makes it impor-tant to determine the combination of core and symbiotic genes thatproduce the most effective symbiosis.

Genospecies distribution in optimal areas for C. canariense

The modelling map (Fig. S1) produced in this study showedseveral areas outside the Park that were optimal for C. canariensenatural populations (BdH, BdI, BdB and BdD). It was found thatthe isolates were less diverse at these localities and only fourgenospecies were detected, but with M. caraganae-I and M. tama-dayense being predominant. Inside the Park only the southernpopulations (Ria, MdC, RdC and Bej) were included within the opti-mal areas. Within these areas, the rhizobia diversity was high with8 out of 9 genospecies detected, although their preponderance var-ied at the different locations. Optimal areas for C. canariense shouldbe taken into account in future reinforcement or reintroductionprogrammes in the Park. The availability of a rhizobia collectionfrom most of the currently known C. canariense populations andthe knowledge of the territorial genospecies distribution on LaPalma will be very useful in such C. canariense reinforcement cam-paigns, since inoculation with the appropriate rhizobial strainscould greatly benefit the establishment and development of thislegume species.

Acknowledgments

This work has been supported by the Ministerio de Medio Ambi-ente y Medio Rural y Marino, Organismo Autónomo de ParquesNacionales (Ref. 111/2010). We would like to thank Angel Palo-mares (Director of the Parque Nacional de la Caldera de Taburiente)for facilitating the work in the Park and providing seeds, and toAntonio Rodriguez Lerín (Biologist Technician, TRAGSA group) forhelping in the field work, localising C. canariense populations, andsharing his knowledge and experience of the Park.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.syapm.2013.08.004.

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