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Systematic and Applied Microbiology 37 (2014) 149–156
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Systematic and Applied Microbiology
j ourna l h omepage: www.elsev ier .de /syapm
nalysis of rhizobial strains nodulating Phaseolus vulgaris fromispaniola Island, a geographic bridge between Meso and Southmerica and the first historical link with Europe
ésar-Antonio Díaz-Alcántaraa, Martha-Helena Ramírez-Bahenab,c, Daniel Mulasd,aula García-Frailee, Alicia Gómez-Morianob, Alvaro Peixb,c, Encarna Velázqueze,∗,ernando González-Andrésd
Facultad de Ciencias Agronómicas y Veterinarias, Universidad Autónoma de Santo Domingo, Dominican RepublicInstituto de Recursos Naturales y Agrobiología, IRNASA (CSIC), Salamanca, SpainUnidad Asociada Grupo de Interacción Planta-Microorganismo, Universidad de Salamanca-IRNASA (CSIC), SpainInstituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de León, SpainDepartamento de Microbiología y Genética, Universidad de Salamanca, Spain
r t i c l e i n f o
rticle history:eceived 13 July 2013eceived in revised form5 September 2013ccepted 18 September 2013
eywords:hizobium
a b s t r a c t
Hispaniola Island was the first stopover in the travels of Columbus between America and Spain, andplayed a crucial role in the exchange of Phaseolus vulgaris seeds and their endosymbionts. The analysis ofrecA and atpD genes from strains nodulating this legume in coastal and inner regions of Hispaniola Islandshowed that they were almost identical to those of the American strains CIAT 652, Ch24-10 and CNPAF512,which were initially named as Rhizobium etli and have been recently reclassified into Rhizobium phaseoliafter the analysis of their genomes. Therefore, the species R. phaseoli is more abundant in America thanpreviously thought, and since the proposal of the American origin of R. etli was based on the analysis
haseolus vulgarisaxonomyhylogenyiogeography
of several strains that are currently known to be R. phaseoli, it can be concluded that both species havean American origin coevolving with their host in its distribution centres. The analysis of the symbiovarphaseoli nodC gene alleles carried by different species isolated in American and European countriessuggested a Mesoamerican origin of the � allele and an Andean origin of the � allele, which is supportedby the dominance of this latter allele in Europe where mostly Andean cultivars of common beans havebeen traditionally cultivated.
ntroduction
Phaseolus vulgaris is a legume indigenous to the mountainousegions of Central America and the Andes that was distributedhrough Spain and Portugal to the rest of Europe after the discov-ry of America [14]. Although this legume establishes symbiosisith different fast-growing rhizobia on mainland America, Rhizo-
ium etli is its predominant endosymbiont and an American originas been proposed for this species [1,2,6,7,36,37]. Since it has beeneported that R. etli is carried on the testa of the bean seeds [25] it
ould have been spread with common bean seeds in Europe [15,34].his fact is supported by several studies given that typical symbi-tic genes of the American R. etli biovar phaseoli were found in∗ Corresponding author at: Departamento de Microbiología y Genética, Lab. 209,dificio Departamental de Biología, Campus Miguel de Unamuno, 37007 Salamanca,pain. Tel.: +34 923 294532; fax: +34 923 224876.
E-mail address: [email protected] (E. Velázquez).
723-2020/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.syapm.2013.09.005
© 2013 Elsevier GmbH. All rights reserved.
Spain, Portugal and France linked to other chromosomal speciessuch as Rhizobium leguminosarum, Rhizobium lusitanum, Rhizobiumgallicum and Rhizobium giardinii [5,11,24,41].
Most of the studies mentioned focused on strains isolated frommainland America and Europe, but no reports are available onstrains nodulating common bean in the American islands locatedon the trade routes between these continents. This is the case ofHispaniola Island in the Caribbean Sea, which was the first islanddiscovered by Christopher Columbus on his first voyage to Americaand it became an important bridge in the trade routes betweenAmerica and Europe. In this way, P. vulgaris seeds were likelytransported from mainland America to Hispaniola after Columbus’sarrival, although it is known that common bean was cultivated onthis island before then [9]. The common bean germplasm culti-vated on Hispaniola had two different origins, the Mesoamerican
one, with small seeds, and the Andean one, with larger seeds [9].After Gepts [13] suggested that the Caribbean was a natural geo-graphic bridge between the two centres of origin, Duran et al. [9]not only found the two types, but also a sort of gradient across the
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50 C.-A. Díaz-Alcántara et al. / Systematic a
egion related to their prevalence, which was supported by RAPDnalysis and phaseolin patterns. The small-seed cultivars passedhrough the Caribbean islands of Cuba and Hispaniola on their wayrom Mexico and Central America, to the north of South Americand Brazil [13,14]. In turn, the large-seed cultivars were introducedo Hispaniola Island from the north of South America through theArawak Arc” of the Leeward Islands and the Great Antilles [9].
Since antiquity the main port in the trade routes betweenmerica and Europe within Hispaniola Island was located on thearibbean coast of the oriental part of the island, where at the endf the 15th century the city of Santo Domingo de Guzmán wasounded, which is now the capital city of the Dominican Republic.his country occupies 60% of the territory of Hispaniola Island.herefore, the port of Santo Domingo played a crucial role in theistribution of common bean seeds to Spain after the first Colum-us voyage. As the large grains were preferred to the small onesor human consumption, the large-seeded cultivars progressivelyeplaced the small-seeded ones in the region near Santo Domingo,lthough the latter remained longer in the inner and isolated moun-ainous regions of the island [9], distant from the commercial tradeoutes. As a consequence, the Andean germplasm was mostly trans-orted to Spain and Portugal and from here to the rest of Europend Africa, and for this reason the Andean germplasm is predomi-ant in these areas [14]. Therefore, Hispaniola played an essentialole in the exchange of common bean germplasm between Amer-ca and Europe, and the analysis of rhizobia nodulating this legumen the island is therefore particularly interesting for biographicaltudies.
In this study, effective rhizobial strains nodulating commonean were isolated in the Santo Domingo region near the coast,s well as from inner mountainous regions of Hispaniola Island inhe Dominican Republic zone in order to analyze their phyloge-etic relationships with rhizobia isolated from the American anduropean countries linked by trade routes since the discovery ofmerica. Several strains of Rhizobium phaseoli carrying identicalore genes to those isolated in mainland America and novel Rhi-obium chromosomal lineages not previously reported in Americar Europe to date were isolated on Hispaniola Island. This find-ng, together with the fact that several American strains initiallyamed R. etli have been reclassified into R. phaseoli, showed thathis species is as abundant in America as R. etli. Moreover, all strainsrom symbiovar phaseoli carried one of the two American nodCene alleles, indicating a common phylogenetic origin of this genen strains nodulating P. vulgaris on mainland America, Hispaniolasland and Europe.
aterials and methods
solation of rhizobial strains and nodulation tests
Rhizobial strains (Table 1) were isolated according to Vin-ent [43] on YMA medium, from effective nodules of P. vulgarisar. “José Beta” plants grown in four provinces of the Dominicanepublic in the oriental part of Hispaniola Island, in which this
ocal variety is commonly cultivated (Table 1). In each field, 15lants at least 1 m apart were randomly taken, and the most effec-ive nodules (pink-red coloured) were selected from each plantor rhizobial isolation. The sampling was carried out in two dif-erent agrosystems. The first was a lowland area near sea levelepresented by Santo Domingo Province, and the second ones
ere isolated highlands areas, represented by the rest of theampling sites (Table 1). The nodulation experiments were car-ied out on P. vulgaris var. “José Beta”, as previously described24].
plied Microbiology 37 (2014) 149–156
RAPD fingerprinting
RAPD patterns were obtained using the primer M13 (5′-GAGGGTGGCGGTTCT-3′), according to Rivas et al. [32], with thefollowing PCR conditions: preheating at 95 ◦C for 9 min; 35 cyclesof denaturing at 95 ◦C for 1 min; annealing at 45 ◦C for 1 min andextension at 75 ◦C for 2 min, and a final extension at 72 ◦C for 7 min.
Sequence analysis of rrs, recA, atpD and nodC genes
The amplification and sequencing of the rrs gene was performedaccording to Rivas et al. [30], whereas recA and atpD genes wereaccording to Gaunt et al. [12] and the nodC gene as describedby Laguerre et al. [19]. The sequences obtained were comparedwith those from GenBank using the BLASTN programme [3], andwere aligned using the Clustal W software [40]. The distances werecalculated according to Kimura’s two-parameter model [18]. Phy-logenetic trees were inferred using the neighbour-joining method[35]. Bootstrap analysis was based on 1000 resamplings. The MEGA5 package [39] was used for all analyses.
Results and discussion
Isolation of rhizobia and nodulation tests
Twenty five isolates were obtained from nodules that pre-sented the typical morphology of effective common bean nodules(pink internal colour due to leghaemoglobin). All strains were ableto induce effective nodules in P. vulgaris. The inoculated plantshad shoot weights ranging from 475 mg/plant to 932 mg/plantand plant nitrogen contents ranging from 11 to 37 mg/plant.The uninoculated controls (without added nitrogen) had aver-age shoot weights of 468 mg/plant and contained 8 mg/plant ofnitrogen.
RAPD fingerprinting
RAPD fingerprinting allows differentiation between strains ofrhizobial species nodulating common bean and provides an esti-mation of their genetic diversity [24,41,42]. In our case, the strainspresented 14 RAPD profiles, which indicated high genetic variabil-ity even in isolates originating from the same zone (Fig. 1 andTable 1). A representative strain from each RAPD profile was sub-jected to gene sequence analyses.
Analysis of chromosomal genes
The rrs gene analysis showed that strains from RAPD groupsII, VI, VII, VIII, IX, X and XI had identical sequences and thereforeonly a representative strain, VPA1100, was included in the phy-logenetic tree (Fig. S1). This strain clustered with the type strainof the species R. phaseoli ATCC 14482T, whereas the remainingstrains were located in different branches of the tree related to R.etli CFN42T or R. vallis CCBAU 65647T (Fig. S1). Nevertheless, sev-eral species of the genus Rhizobium are not easily distinguishablethrough rrs gene analysis, as occurs with some species nodulat-ing P. vulgaris (Fig. S1). To distinguish these species the analysisof the housekeeping recA and atpD genes is much more reliable[27,41]. In fast growing rhizobia nodulating P. vulgaris, the speciesdelineation limit for housekeeping gene identities has been estab-lished as 95–96% when compared with DNA-DNA hybridizationdata [27,41].
The analysis of recA and atpD genes (Figs. 2 and 3) showed thatDominican strains occupied different branches within the genusRhizobium. Strains showing the profiles II, VI, VII, VIII, IX, X andXI, represented by strains VPA1100, OLQ1051, OLQ1052, VIAD5,
C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156 151
Table 1Characteristics of strains isolated in different geographic locations from Dominican Republic at Hispaniola Island.
Strains Isolation site RAPD pattern nodC allele Species
ESC1020 Sabana Chen, Hondo Valle; Elías Pina I � Rhizobium sp.
ESC1060 Sabana Chen, Hondo Valle; Elías Pina II � Rhizobium phaseoli
ESC1110 Sabana Chen, Hondo Valle; Elías Pina III � Rhizobium sp.
ESC1120 Sabana Chen, Hondo Valle; Elías Pina III � Rhizobium sp.
ESC1129 Sabana Chen, Hondo Valle; Elías Pina III � Rhizobium sp.
OAC1020 Arroyo Cana; San José de Ocoa IV � Rhizobium sp.
OAC1031 Arroyo Cana; San José de Ocoa IV � Rhizobium sp.
OAC1140 Arroyo Cana; San José de Ocoa IV � Rhizobium sp.
OAC1150 Arroyo Cana; San José de Ocoa V � Rhizobium sp.
OLQ1051 Los Quemaos; San José de Ocoa VI � Rhizobium phaseoli
OLQ1052 Los Quemaos; San José de Ocoa VII � Rhizobium phaseoli
VIAD3 Santo Domingo VIII � Rhizobium phaseoli
VIAD5 Santo Domingo VIII � Rhizobium phaseoli
VIAD8G Santo Domingo IX � Rhizobium phaseoli
VIAD10 Santo Domingo IX � Rhizobium sp.
VIAD11 Santo Domingo X � Rhizobium phaseoli
VIAD12G Santo Domingo XI � Rhizobium phaseoli
VIAD13G Santo Domingo XII � Rhizobium sp.
VIAD14G Santo Domingo XI � Rhizobium phaseoli
VCS3041 Canada Seca, Constanza; la Vega IV � Rhizobium sp.
VLP1091 Las Palmas, Constanza; la Vega XIII � Rhizobium sp.
VPA1010 Palero, Constanza; la Vega II � Rhizobium phaseoli
VPA1100 Palero, Constanza; la Vega II � Rhizobium phaseoli
IV
X
VisAiB1
F(1(
VRI2100 El Río, Constanza, la Vega
VRI2090 El Río, Constanza, la Vega
IAD8G, VIAD11 and VIAD12G, respectively, were unambiguouslydentified as R. phaseoli because they had recA and atpD geneequence identities higher than 99.9% with respect to R. phaseoli
T
TCC 14482 . Other P. vulgaris-nodulating strains isolated in Amer-ca, such as the Mexican ch24-10, the Colombian CIAT 652 and therazilian CNPAF 512, were also located in this cluster with almost00% identity for both recA and atpD genes. These strains wereig. 1. RAPD patterns of strains isolated in this study: ESC1020 (lane 1), ESC1060 (lane 2)lane 7), OAC1140 (lane 8), OAC1150 (lane 9), OLQ1051 (lane 10), OLQ1052 (lane 11), VI6), VIAD12G (lane 17), VIAD13G (lane 18), VIAD14G (lane 19), VCS3041 (lane 20), VLP109lane 25). The different RAPD groups are indicated by Roman numerals.
� Rhizobium sp.
IV � Rhizobium sp.
initially named as R. etli but have recently been included in thespecies R. phaseoli [20].
Strain ESC1110, representing RAPD profile III, had identity val-
ues for the recA and atpD genes of lower than 95% with respectto the remaining strains isolated in this study and to the cur-rently described species of Rhizobium. The strain representing RAPDpattern XII, VIAD13G, was related to R. etli in the recA phylogenetic, ESC1110 (lane 3), ESC1120 (lane 4), ESC1129 (lane 5), OAC1020 (lane 6), OAC1031AD3 (lane 12), VIAD5 (lane 13), VIAD8G (lane 14), VIAD10 (lane 15), VIAD11 (lane1 (lane 21), VPA1010 (lane 22), VPA1100 (lane 23), VRI2100 (lane 24) and VRI2090
152 C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156
Rhiz obium etl i IE4804 (AY907457 )Rhiz obium etl i IE4876 (AY907455 )Rhiz obium etl i IE2730 (AY907456 )Rhiz obium etl i IE4810 (AY907453 )Rhiz obium etl i IE4837 (AY907463 )Rhiz obium etl i IE2704 (AY907461 )Rhiz obium etl i KIM5s (AY907360 )
Rhiz obium etl i IE4794 (AY907454 )Rhiz obium etl i GR12 (AY907361 )Rhiz obium etl i GR56 (NZ ABRB01002262 )Rhiz obium etl i CNPSO679 (JN129355 )
Rhiz obium etl i (for merly Rhiz obium etl i) CNPAF512 (AEYZ01000264 )Rhizob ium phaseo li OLQ1052 (KF63839 1)Rhizob ium phaseo li OLQ1051 (KF63839 0)
Rhizob ium pha seo li (for merly Rhiz obium etl i) Ch24 -10 (NZ_AHJU01000019 )Rhizob ium pha seo li ATCC 1448 2T (EF113136 )Rhizob ium pha seo li (for merly Rhiz obium etl i) CIAT 652 (NC_010994 )Rhizob ium phaseo li VPA1100 (KF63838 5)Rhizob ium phaseo li VIAD5 (KF63839 3)Rhizob ium phaseo li VIAD8G (KF638394 )Rhizob ium phaseo li VIAD12G (KF63839 5)Rhizob ium phaseo li VIAD11 (KF63839 6)
Rhizob ium sp. ESC1110 (KF638397 )Rhizob ium sp. BLR160 (JN649055 )
Rhizob ium sp. VLP1091 (KF638386 )Rhizob ium sp. VRI2090 (KF63838 9)
Rhizob ium sp. OAC1150 (KF63838 8)Rhizob ium sp. ESC1020 (KF63839 8)
Rhizob ium sp. OAC1020 (KF63838 7)Rhiz obium valli s CCBAU 6564 7T (GU211770 )
Rhizob ium sp. VIAD13G (KF63839 2)Rhizob ium sp. BLR62 (JN649042 )
Rhiz obium etl i CFN42T (NC 007761 )Rhiz obium etl i CNPSO666 (JN129350 )
Rhizob ium sp. RPVR24 (GQ863533 )Rhizob ium sp. LBM1123 (JF792209 )
Rhizob ium sp. RPVN06 (GQ863531 )Rhizob ium sp. RPVR32 (GQ863534 )
Rhizob ium sp. RPVR04 (GQ863532 )Rhizob ium faba e CCBAU 3320 2T (EF579941 )Rhizob ium pisi DSM 3013 2T (EF113134 )
Rhizob ium ind igo fera e CCBAU7104 2T (EF027965 )Rhizob ium legu mino sarum USDA 237 0T (AJ294376 )Rhizob ium legu mino sarum RPVF18 (GQ863530 )Rhizob ium legu mino sarum LCS0313 (JF792208 )Rhizob ium legu mino sarum BAPVPT02 (GQ863529 )
Rhizob ium oryza e Alt50 5T (FJ712274 )Rhizob ium tibe ticu m LMG 2445 3T (HQ735075 )
Rhizob ium mesoa mericanu m CC GE 50 1T (JF424620 )Rhizob ium graha mii CC GE 50 2T (JF424622 )
Rhizob ium ga llicu m R602 spT (AY907357 )Rhizob ium ga llicu m PhD12 (HQ394250 )
Rhizob ium yang lingens e SH2262 3T (AY907359 )Rhizob ium loes sen se LMG2197 5T (HQ735076 )
Rhizob ium mongo lens e USDA 184 4T (AY907358 )Rhizob ium alka liso li CCBAU0139 3T (EU672490 )
Rhizob ium lus itanu m P1-7T (DQ431674 )Rhizob ium lus itanu m p3-13 (DQ431675 )
Rhizob ium giard inii H15 2T (EU488819 )Rhizob ium leucaena e USDA 903 9T (AJ294372 )Rhizob ium multihosp iti um CCBAU 83840 1T (EF490029 )
Rhizob ium rhizogen es NCPP B 299 1T (AJ294374 )Rhiz obium freir ei PRF 81T (EU488827 )
Rhizob ium trop ici USDA903 0T (AJ294373 )Rhizob ium miluonens e CCBAU 4125 1T (HM047131 )
100
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F showic ession
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ig. 2. Neighbour-joining phylogenetic tree based on partial recA gene sequences
alculated for 1000 replications are indicated. Bar, 2 nt substitutions per 100 nt. Acc
ree and had 97% identity with respect to strain CFN42T, how-ver, the atpD genes of both strains were divergent, and showedess than 94% identity. The two mentioned strains, ESC1110 andIAD13G, were related to strains BLR160 and BRL62 nodulatingicia in Asia [28], and could represent undescribed species of Rhizo-ium, although in the latter case of VIAD13G, the identity of the recAene with R. etli CFN42T was at the limit of species differentiation.
The remaining Rhizobium strains isolated from the Dominicanepublic were not identified as described species of this genusccording to the recA and atpD gene sequence analyses. Moreover,hese strains were not related to any rhizobial strains whose recAnd atpD genes are currently available in the public databases.SC1020, representing RAPD profile I, occupied a phylogeneticallyivergent branch with identities lower than 95% for the recA andtpD genes, respectively, with respect to the remaining Dominican
trains (Figs. 2 and 3), as well as to the current species of RhizobiumFigs. 2 and 3). Strain OAC1020, displaying RAPD type IV, formedn independent branch related to the previous strain in the casef the atpD gene, but an identity of lower than 96% suggestedng the position of representative strains from each RAPD group. Bootstrap values numbers from GenBank are given in brackets.
that it did not belong to the same species, since the recA genehad an identity of lower than 92%. The strains with profiles V, XIIIand XIV, represented by strains OAC1150, VLP1091 and VRI2090,respectively, were closely related and probably belong to the samespecies, since they had more than 98% identity in both recA andatpD genes (Fig. 2). These strains were phylogenetically divergentto all described species from the genus Rhizobium (Fig. 2) andtherefore they probably belong to an undescribed species from thisgenus.
These results showed that, in addition to R. phaseoli, severalputative new species in the genus Rhizobium were endosymbiontsof P. vulgaris from Hispaniola Island and confirmed the greater use-fulness of housekeeping genes compared to the rrs gene for speciesdelineation within the genus.
Analysis of the nodC gene
The nodC gene has been widely analyzed in rhizobia, and isrelated to their host range and the degree of host promiscuity
C.-A. Díaz-Alcántara et al. / Systematic and Ap
Rhizob ium ga llicu m R602 spT (AY907371 )Rhizob ium ga llicu m PhD12 (AY929482 )
Rhizob ium yang lingens e SH2262 3T (AY907373 )Rhizob ium mongo lens e USDA 184 4T (AY907372 )
Rhizob ium loe ssen se CCB AU 7190 BT (HM142761 )Rhizob ium mesoa mericanu m CC GE 50 1T (JF424611 )
Rhizob ium tibe ticu m LMG 2445 3T (HQ735093 )Rhizob ium alka lisoli 0139 3T (EU672461 )
Rhizob ium graha mii CC GE 50 2T (JF424612 )Rhizob ium lus itanu m P1-7T (DQ431671 )Rhizob ium lus itanu m p3-13 (DQ431672 )
Rhizob ium leucaena e USDA 903 9T (AJ294396 )Rhiz obium freir ei PRF 81T (NZ AQHN01000005 )
Rhizob ium miluonens e CCBAU 4125 1T (HM047116 )Rhizobium multihospitium CCBAU 83401T (EF490019)Rhizobium tropici USDA 9030T (AJ294397)
Rhizob ium giard inii H15 2T (HQ394216 )Rhizob ium sp. ESC1110 (KF638364 )
Rhizob ium sp. BLR160 (JN648965 )Rhizob ium sp. VIAD13G (KF638365 )Rhizob ium sp. BLR62 (JN648952 )
Rhizob ium sp. OAC1020 (KF638359 )Rhizob ium sp. OAC1150 (KF638360 )Rhizob ium sp. VLP1091 (KF638358 )Rhizob ium sp. VRI2090 (KF638361 )
Rhiz obium etl i CFN42T (NC_007761 )Rhizob ium sp. ESC1020 (KF638370 )
Rhizob ium phaseo li OLQ1051 (KF638362 )Rhizob ium pha seo li (for merly Rhiz obium etl i) CNPAF512 (AEYZ01000362 )Rhizob ium phaseo li OLQ1052 (KF638363 )Rhizob ium phaseo li VIAD5 (KF638366 )Rhizob ium pha seo li ATCC 1448 2T (EF113151 )Rhizob ium pha seo li (for merly Rhiz obium etl i) CIAT 652 (NC_010994 )Rhizob ium phaseo li VIAD8G (KF638367 )Rhizob ium phaseo li VIAD12G (KF638368 )Rhizob ium phaseo li VIAD11 (KF638369 )Rhizob ium phaseo li VPA1100 (KF638357 )Rhizob ium pha seo li (for merly Rhiz obium etl i) Ch24 -10 (NZ_AHJU01000173 )
Rhizob ium faba e CCB AU 3320 2T (EF579929 )Rhizob ium pisi DSM 3013 2T (EF113149 )
Rhizob ium sp. RPVR24 (GQ863522 )Rhizob ium sp. LBM1123 (JF792200 )Rhizob ium sp. RPVN06 (GQ863520 )
Rhizob ium sp. RPVR32 (GQ863523 )Rhizob ium sp. RPVR04 (GQ863521 )
Rhiz obium valli s CCBAU 6564 7T (GU211768 )Rhiz obium etl i GR12 (AY907375 )
Rhizob ium legu minosa rum USDA 237 0T (AJ294405 )Rhizob ium legu minosa rum RPVF18 (GQ863519 )
Rhizob ium ind igo fera e CCBAU7104 2T (GU552925 )Rhizob ium legu minosa rum LCS0313 (JF792195 )Rhizob ium legu minosa rum BAPVPT02 (GQ863518 )
Rhizob ium ory zae Alt50 5T (FJ712275 )Rhiz obium etl i KIM5s (AY907374 )Rhiz obium etl i IE4794 (AY907435 )Rhiz obium etl i IE2737 (AY907439 )Rhiz obium etl i IE2704 (AY907442 )
Rhiz obium etl i IE2730 (AY907436 )Rhiz obium etl i IE4804 (AY907437 )Rhiz obium etl i IE4837 (AY907444 )
8996
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Fig. 3. Neighbour-joining phylogenetic tree based on partial atpD gene sequencessvA
[tatfds9R�tA
daTVbta
howing the position of representative strains from each RAPD group. Bootstrapalues calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt.ccession numbers from GenBank are given in brackets.
4,10,11,16,19,23,26,31,33]. P. vulgaris is a promiscuous host [22]hat is nodulated by Rhizobium strains carrying very divergent nodCnd it belongs to different symbiovars [11,19,42]. Amongst these,he biovar phaseoli is widely distributed in the world and two dif-erent nodC alleles named � and � have been found in Americanistribution centres [2]. The phylogenetic analysis of the nodC genehowed that the two types of alleles were clearly separated (about7% identity), and proved that strains from the same species, such as. phaseoli ATCC 14482T and CIAT 652, carry different nodC alleles,
and �, respectively. Also, strains from different species carriedhe same nodC allele, as occurs with the type strains of R. phaseoliTCC 14482T and R. etli CFN42T.
All Dominican strains belonged to the biovar phaseoli and wereistributed in the groups corresponding to � and � nodC alleles,lthough the � allele was slightly more abundant (Fig. 4 andable 1). Most strains identified as R. phaseoli (OLQ1051, VIAD5,
IAD8G, VIAD11 and VIAD12G) had the � allele that was carriedy the type strain of this species (ATCC 14482T), as well as byhe type strain of R. etli CFN42T, and only two strains, VPA1100nd OLQ1052, showed the � allele. Most of the remaining strainsplied Microbiology 37 (2014) 149–156 153
(OAC1150, ESC1020, ESC1110 and VIAD13G) that belonged to chro-mosomal lineages divergent to R. phaseoli had the � allele. However,two strains, VLP1091 and VRI2090, isolated at two sites from theinner La Vega region (Table 1) carried a slightly divergent nodC genephylogenetically related to the � allele (Fig. 4), suggesting a recentdiversification of this gene in some local ecosystems, since this vari-ation has not been found in the remaining strains analyzed to datefrom different continents.
Taxonomic, evolutionary and biogeography remarks
For several decades, the species R. etli was considered to be themajor endosymbiont of P. vulgaris in the distribution centres ofcommon bean in America [1,2,6,36,37]. However, the taxonomyof this species was not clearly established, mainly because rhizo-biologists considered R. phaseoli as an invalid species after Jordan’sreclassification [17]. At the beginning of rhizobiology, this specieswas defined as an endosymbiont of P. vulgaris and was included inthe validation list of Skerman et al. [38]. The discovery of the exist-ence of interchangeable symbiotic plasmids in some Rhizobiumspecies led Jordan [17] to reclassify the species R. phaseoli and R. tri-folii as R. leguminosarum. From this date, in the description of newrhizobial species, such as R. etli [36], R. phaseoli was not considered,although Jordan’s reclassification was never officially validated. Asa consequence, many strains isolated from P. vulgaris in Americawere classified as R. etli and the American origin of this species wasproposed [36]. However, in 2008, the validity of the old species R.phaseoli was recognized again, since it could be differentiated fromR. etli on the basis of housekeeping gene analysis [27] and, recently,the American P. vulgaris-nodulating strains CIAT 652, CNPAF 512and ch24-10, whose complete genomes are available, have beenreported to be R. phaseoli rather than R. etli [20]. Also, some strainsisolated in Mexico (IE6823, IE6862 and IE6760) and classified asR. etli clearly belong to the species R. phaseoli since they have beenshown to cluster with strain CIAT 652 and not with strain CFN42T
in the study of Lozano et al. [21]. Strain CIAT 151, which is used asan inoculant for common bean, and strain IPR-Pv589 [8] clusteredwith R. phaseoli instead of R. etli (Fig. S1). On the other hand, somestrains named R. etli isolated from America, such as IE2737 andIE4804 [2,21,37], IE4837 [21], IE2704, IE2730, IE4794, IE4876 andIE4810 [37], CNPSO679 and CNPSO666 [29] from Mexico and KIM5sfrom Idaho (USA) [37], as well as some Spanish strains initiallynamed as R. etli [15,34], such as G12 and GR56 [15], do not belongto this species or to R. phaseoli (Figs. 2 and 3). The same occurredwith several Spanish strains initially named as R. etli [15,34] thatdid not cluster with the type strain of R. etli CFN42T in the study ofLozano et al. [21]. This means that the taxonomic position of strainsnamed as R. etli isolated to date should be revised, particularlywhen it has only been based on partial sequences of the 16S rRNAgene.
However, based on the available data, it can be concluded thatR. phaseoli is also very abundant in American countries, and it wasfound in both coastal and mountainous regions of Hispaniola Island.All these strains belonged to the symbiovar phaseoli according tothe nodC gene analysis, and this gene seems to be exclusive forcommon bean nodulation [5,42]. Therefore, the plasmids carried bythe symbiovar phaseoli should have evolved with native commonbean plants in America [2] and R. etli sv. phaseoli is considered tobe the predominant endosymbiont of wild and cultivated commonbean in different American countries, such as Mexico, Argentina,Ecuador and USA [1,2,6,7,36,37]. In accordance with these results,coevolution of R. etli with P. vulgaris has been proposed in centres
of host diversification [2]. Nevertheless, this proposal was basedon some strains that have been reclassified recently as R. phaseoliand, therefore, we propose that this species also coevolved with P.vulgaris in its diversification centres and was disseminated in other
154 C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156
Rhizob ium giard inii sv. ph aseoli H251 (AF217264) France
Rhizobium phaseoli sv. phaseoli VPA1100 (KF638371) Dominican Republic
Rhizob ium legu minosa rum sv. phaseoli H132 (AF217263) France
Rhizob ium sp. sv. ph aseoli RPVR 32 (GQ863545 ) North Sp ain
Rhiz obium etl i sv. phaseoli CNPAF512 (AEYZ01000361) Brazil
Rhizob ium sp. sv. phaseo li ESC1110 (KF638373 ) Dominican Republi c
Rhizob ium sp. sv. phaseo li OAC1150 (KF638375 ) Dominican Republi c
Rhizob ium sp. sv. phaseo li VIAD13G (KF638377 ) Dominican Republi c
Rhizob ium sp. sv. phaseo li ESC1020 (KF638382 ) Dominican Republi c
Rhizobium phaseoli sv. phaseoli OLQ1052 (KF638384) Dominican Republic
Rhizobium etli sv. phaseoli CIAT652 (NC_010996) Colombia
Rhizobium lusitanum P3-13 (HM852099) Portugal
Rhizobium etli sv. phaseoli GR10 (GU084788) South Spain
Rhizobium etli sv. phaseoli 21PR-1 (GU084781) South Spain
Rhizob ium legu minosa rum sv. ph aseoli BAPVPT02 (GQ863540) Central Spain
Rhiz obium etl i sv. phaseoli GR56 (GU084770) South Spain
Rhizob ium legu minosa rum sv. phaseoli LBM1123 (JF792200) North Spain
Rhizob ium ga llicu m sv. phaseoli PhD12 (AF217265) France
Rhizobium sp. sv. phaseoli VLP1091 (KF638372) Dominican Republic
Rhizob ium sp. sv. phaseo li VRI2090 (KF638376 ) Dominican Republi c
Rhizobium sp. sv. phaseoli RPVR04 (GQ863543) North Spain
Rhiz obium etl i sv. ph aseoli CFN42T (NC_004041) Mexico
Rhizob ium pha seo li sv. ph aseoli ATCC 1448 2T (HM441255) USA
Rhizob ium sp. sv. phaseo li OAC1020 (KF638374 ) Dominican Republi c
Rhizobium phaseoli sv. phaseoli VIAD5 (KF638378) Dominican Republic
Rhizob ium phaseo li sv. p haseo li VIAD8G (KF638379 ) Dominican Republi c
Rhizob ium phaseo li sv. p haseo li VIAD12G (KF638380 ) Dominican Republi c
Rhizob ium phaseo li sv. phaseo li VIAD11 (KF638381 ) Dominican Republi c
Rhizob ium phaseo li sv. ph aseo li OLQ1051 (KF638383 ) Dominican Republi c
Rhizobium etli sv. phaseoli Ch24-10 (NZ_AHJU01000233) Mexico
Rhiz obium etl i sv. ph aseoli IE2730 (GU084771) Mexico
Rhiz obium etl i sv. ph aseoli GR14 (GU084756) South Spain
Rhiz obium etl i sv. ph aseoli 21 NJ-2 (GU084792) South Spain
Rhiz obium etl i sv. ph aseoli IE4810 (GU084778) Mexico
Rhiz obium etl i sv. ph aseoli IE4837 (GU084764) Mexico
Rhizob ium legu minosa rum sv. phaseoli LCS0313 (JF792202) North Spain
Ensif er fredii sv. medit erranense 16 b1 (AF481764 )
Ensif er melil oti sv. medit errane nse GVPV12 (FJ462796 )
Ensif er fredii sv. medit erranense GR-06 (AF217269 )
Rhizob ium ga llicu m sv. galli cum R602 spT (AF217266 )
Rhizob ium giard inii sv. giardinii H152T (AF217267 )
Rhizob ium yang lingens e CCB AU 7162 3T (JQ308167 )
Rhizob ium mongo lens e USDA184 4T (GQ507367 )
Rhizob ium legu minosa rum sv. trifoli i USDA 2071 (AF217271 )
Rhizob ium legu minosa rum sv. trifoli i ATC C14480 (FJ895269 )
Rhizob ium legu minosa rum sv. trifoli i WSM1325 (CP001623 )
Rhizob ium legu mino sarum sv. trifoli i WSM2304 (CP001192 )
Rhizob ium legu mino sarum sv. viciae 248 (Y00548 )
Rhizob ium legu mino sarum sv. viciae 3841 (NC_008381 )
Rhizob ium sp. sv. viciae BLR160 (JN649009 )
Rhizob ium pisi DSM 3013 2T (HQ394248 )
Rhizob ium legu mino sarum sv. viciae USDA 237 0T (FJ596038 )
Rhizob ium graha mii CC GE 50 2T (JN021932 )
Rhizob ium mesoa mericanu m CC GE 50 1T (JN021931 )
Rhizob ium leucaena e CFN29 9T (N98514 )
Rhiz obium freir ei PRF 81T (NZ AQHN01000086 )
Rhizob ium lus itanu m p1-7T (HM852098 )
Rhizob ium trop ici CIAT 89 9T (HQ824719 )
100
100100
99
6067
100
100
99
69
100
94
78
94
92
9398
99
96
87
92
100
99
0.02
Rhiz obium etl i sv. ph aseoli IE6823 (GU084774) Mexico
allele
allele
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ig. 4. Neighbour-joining phylogenetic tree based on partial nodC gene sequencesalculated for 1000 replications are indicated. Bar, 2 nt substitutions per 100 nt. Acc
eographical sites with common bean seeds. This can explain theresence of R. phaseoli on Hispaniola Island that was an impor-ant trade bridge for bean seeds and their endosymbionts betweenhe countries of North and South America. In this way, R. phaseolias found not only in Santo Domingo, nearest to South America,
ut also in inland regions of Hispaniola on the central border withaiti, which could have received bean seeds from North Americanainland countries. From the new chromosomal lineages found onispaniola Island, two of them, represented by strains ESC1110 andIAD13G, have been found recently in Asia in nodules of Vicia [28]nd the remaining ones, represented by strains OAC1020, OAC1150,LP1091 and VRI2090, have not been found to date in any otherountries and they do not cluster with the American species R. etli
nd R. phaseoli or with the strains isolated in Mexico by Silva et al.37]. These strains were isolated from remote locations of Hispan-ola and it is possible that they were present on this island prior tohe arrival of common bean seeds, and they acquired the symbioticing the position of representative strains from each RAPD group. Bootstrap values numbers from GenBank are given in brackets.
genes by horizontal transfer from the mainland America strains,since they carry the typical nodC gene alleles (� and �) of thesestrains.
The proportion of the � and � nodC alleles in strains isolatedfrom Hispaniola Island was similar to that found in mainland Amer-ican countries, although with a slight dominance of the � allele [2]that is characteristic of the type strains of R. phaseoli and R. etli, andthe Mexican strains ch24-10, IE4810 and IE2730 (Fig. 4). The cur-rently available data showed a clear prevalence of the nodC � allelein the strains from North America, including the type strains of R.phaseoli ATCC 14482T (isolated in USA) and R. etli CFN42T (isolatedin Mexico), as well as many strains isolated from P. vulgaris nodulesin different geographical locations in Mexico [2,21,29]. Therefore,
these findings suggested that this allele could have originated inNorth America and was distributed via Caribbean islands to SouthAmerica with the small-seeded Mesoamerican cultivars of commonbean. This proposal agrees with the results of Aguilar et al. [2] who
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C.-A. Díaz-Alcántara et al. / Systematic a
ound that Mesoamerican cultivars of common beans are mostlyodulated by strains carrying the nodC � allele in Mexico (Northmerica). On the contrary, Andean cultivars were mostly nodu-
ated by strains carrying the � allele in Ecuador (South America)2] and this allele is present in bean strains isolated in South Amer-ca, such as CIAT 652 isolated from Colombia and CNPAF512 fromrazil. Therefore, strains carrying the � allele could have been dis-ributed into the Caribbean islands from South America with thearge-seeded beans. This scenario is also coherent with the find-ngs in the European countries where mostly Andean cultivars areultivated, since the � nodC is the major allele found to date athe first European stops on the trade routes from America, namelypain [2,11,21,24] and Portugal [42]. In Spain, the � nodC allele washe most frequent in both north and south Spain according to theesults of several studies [2,11,21,24], and in Portugal all the strainselonging to the symbiovar phaseoli from R. lusitanum also carriedhe � nodC allele [42]. In other European countries, such as France,lthough the nodC gene is available only for a few strains, all ofhem also carry the � nodC allele of symbiovar phaseoli, regardlessf the species to which they belong, R. leguminosarum, R. gallicumr R. giardinii [5,19].
In summary, it can be concluded that the strains of R. phaseoli sv.haseoli, as well as those from R. etli sv. phaseoli, are indigenous toainland America from where they were introduced along with P.
ulgaris seeds to Hispaniola Island. The strains from other chromo-omal lineages present on Hispaniola Island could have acquiredhe symbiotic genes from these strains since they carry the twoodC alleles found in R. phaseoli in the American distribution centresf common bean. The analysis of the nodC alleles found in strainsrom Hispaniola and mainland America suggests a Mesoamericanrigin of the � allele and an Andean origin of the � allele, whichs supported by the finding of this latter allele in most of the Euro-ean strains. Consequently, the results of this study showed that thenalysis of P. vulgaris endosymbionts present in the islands locatedetween America and Spain is important for biogeographical stud-
es of these rhizobia, as well as for increasing the knowledge of theoevolution of Rhizobium-Phaseolus vulgaris symbiosis.
cknowledgements
This work was supported by the Spanish National R&D&I Pro-ramme: AECID PCI projects A/4824/06, A/7473/07, A/023132/09nd A/030020/10 to C-AD-A and FG-A, a MEC project to EVP and byhe Castilla y León Regional Government to AP.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.syapm.013.09.005.
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