Symbiotic Frankia bacteria in Alnus forests inMexico and the United States of America: isgeographic location a good predictor ofassemblage structure?

Logan Massie Higgins and Peter Gault Kennedy

Abstract: While the biogeography of Alnus species is well characterized, that of their microbial symbionts remains lesswell understood. Little is known, for example, about how the genotypic richness of Alnus-associated Frankia bacteria variesat the continental scale, and the richness of Alnus-associated Frankia at tropical latitudes has yet to be explored. In thisstudy, we conducted sequence-based analyses of the nifH gene comparing Frankia found in root nodules of two Alnus spe-cies in central Mexico with those associated with two Alnus species in the northwestern United States of America (USA).Similar to Frankia assemblages in northwestern USA and other geographic locations, genotypic richness within the Mexicansamples was low, with five genotypes total using a ≥97% nifH sequence similarity cutoff. The vast majority of Mexican se-quences belonged to genotypes also very common in northwestern USA Alnus forests, although two novel Mexican geno-types were identified. Phylogenetic analyses confirmed that all of the genotypes present in Mexico belong to larger cladesof Alnus-associated Frankia. Genotype- and distance-based community analyses indicated that neither geographic locationnor the phylogenetic relationships among hosts are strong predictors of Frankia assemblage structure. Our results suggestthat factors other than classic biogeography are more influential in determining the continental-scale distribution and diver-sity of Alnus-associated Frankia.

Key words: Frankia, Alnus, microbial biogeography, symbiosis, host association.

Résumé : Bien que la biogéographie des espèces d’Alnus soit bien documentée, celle de leurs symbiotes microbiens de-meure moins bien comprise. On sait peu de choses par exemple sur la façon avec laquelle la richesse génotypique des bacté-ries Frankia associées aux Alnus varie à l’échelle continentale; la richesse des Frankia associés aux Alnus aux latitudestropicales reste à explorer. Dans cette étude, l’auteur a effectué des analyses basées sur le séquençage du gène nifH, en com-parant les Frankia trouvés dans les nodules racinaires de deux espèces d’Alnus du centre du Mexique avec ceux associés àdeux espèces d’Alnus du nord-ouest américain. Tout comme pour les assemblages des Frankia du nord-ouest américain etautres localités géographiques, la richesse génotypique dans les échantillons du Mexique est faible avec un total de cinq gé-notypes, en utilisant une coupure de similarité à ≥97 % de la séquence du gène nifH. La grande majorité des séquencesmexicaines appartient aux génotypes également communs dans les forêts d’Alnus du Nord-ouest américain, bien qu’on aitidentifié deux nouveaux génotypes pour le Mexique. Les analyses phylogénétiques confirment que tous les génotypes pré-sents au Mexique appartiennent à des clades plus larges de Frankia associés aux Alnus. Les analyses de communautés ba-sées sur les génotypes et les distances indiquent que ni la localisation géographique ni les relations phylogénétiques parmiles hôtes ne constituent un moyen robuste de prédiction pour la structure d’assemblage des Frankia. Les résultats suggèrentque des facteurs autres que la biogéographie classique exercent plus d’influence sur la détermination de la distribution et dela diversité des Frankia associés aux Alnus, à l’échelle continentale.

Mots‐clés : Frankia, Alnus, biogéographie microbienne, symbiose, hôte et associé.

[Traduit par la Rédaction]

Introduction

A central tenet of biogeography is that taxonomic similarityamong ecological communities declines with increasing dis-tance owing to both historical factors and contemporary envi-ronmental constraints (Wiens and Donoghue 2004). While thistenet has been well demonstrated for many plants and animals

(Morrone and Crisci 1995), there is a lack of agreement as towhether it also holds true for microorganisms (Fenchel andFinlay 2004; Foissner 2006; Martiny et al. 2006). The BaasBecking hypothesis regarding microbial community composi-tion (i.e., “everything is everywhere, but the environment se-lects”) (Baas Becking 1934) has been increasingly called intoquestion as molecular-based studies reveal evidence of biogeo-

Received 29 September 2011. Accepted 10 January 2012. Published at www.nrcresearchpress.com/cjb on 14 May 2012.

L.M. Higgins and P.G. Kennedy. Department of Biology, Lewis & Clark College, 0615 SW Palatine Hill Road, Portland, OR 97219,USA.

Corresponding author: Peter Gault Kennedy (e-mail: pkennedy@lclark.edu).

423

Botany 90: 423–431 (2012) doi:10.1139/B2012-006 Published by NRC Research Press

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graphical structure in a diverse array of microbial communitiesat global and continental scales, including thermophilic arch-aea (Whitaker et al. 2003), soil-dwelling pseudomonads (Choand Tiedje 2000), and ammonia-oxidizing bacteria in saltmarshes (Martiny et al. 2011). Thus, it now appears that bothdispersal limitation and environmental conditions can be im-portant factors that determine how microbial communities arestructured.While the distributions of many groups of free-living mi-

croorganisms do have a clear biogeography (Martiny et al.2006), the extent to which the symbiotic microorganisms fol-low the same pattern is less well understood. Some recentstudies suggest that the distribution of symbiotic microbes re-flects the biogeography of their hosts, but others indicate thathost biogeography is of little importance. For example, a re-cent study of conifer-root associated microfungi revealed noevidence of biogeographical sorting based on host species(Queloz et al. 2011), but other researchers have been able tolink the phylogeography of the human pathogen Heliobacterpylori to prehistoric human colonization of the islands of thePacific Ocean (Moodley et al. 2009). Taken together, theseresults suggest the geographic distribution of symbiotic mi-croorganisms may be driven by either (i) host biogeography,(ii) dispersal limitation, or (iii) environmental factors.In this study, we investigated geographic assemblage pat-

terns of bacteria in the genus Frankia. These diazotrophic ac-tinobacteria live independently in soils and form symbioticassociations with over 200 host plant species worldwide(Huss-Danell 1997). Plant capture bioassays suggest thatFrankia are frequent members of soil microbial communities,even where no actinorhizal (i.e., Frankia-associated) host ispresent (Welsh et al. 2009a; Benson and Dawson 2007). Be-cause these bacteria can have both free-living and symbioticlifestyles, their biogeography may reflect a complex mix ofhost and nonhost associated factors. At local and regionalspatial scales, factors other than geographic proximity aretypically the best predictors of Frankia genotype composi-tion. For example, abiotic factors such as soil pH (Huguet etal. 2004), climate (Igual et al. 2006), and elevation (Khan etal. 2007) along with biotic factors such as host identity (Li-pus and Kennedy 2011) have all been identified as importantdeterminants of Frankia assemblage structure. Frankia geno-types can be grouped into three clusters, each with strong af-finities for specific host lineages (Benson and Dawson 2007);for example, cluster 1 Frankia associate with members of thehigher Hammelid families Betulaceae, Myricaceae, and Casu-arinaceae. Since certain host lineages (e.g., casuarinas) haverestricted distributions themselves, the global biogeographyof Frankia bacteria is primarily driven by host association(Benson and Dawson 2007).Members of the genus Alnus are the dominant Frankia

hosts in the Northern Hemisphere (Benson and Dawson2007). While many studies of Alnus-associated communitieshave been conducted at the local and regional scales, therehave been few studies conducted at the continental scale, andto date none have investigated Alnus-associated Frankia attropical latitudes. To address this gap, we used molecularanalysis of the nifH gene region to study communities ofFrankia associated with four different Alnus species in mon-

tane central Mexico and the northwestern United States ofAmerica (USA). Using a range of methods, we sought to dif-ferentiate among host-, dispersal-, and environment-relatedfactors as potential determinants of Alnus-associated Frankiaassemblage structure at the continental scale. In this study,we estimated Frankia genotypic richness at the different sitesusing a range of nifH sequence similarity cutoff levels, andthen used both genotype- and phylogenetic-distance basedmethods to compare the Frankia assemblages recoveredfrom each site. In addition, we used maximum likelihood(ML) and Bayesian inference phylogenetic analyses to deter-mine how the sequences we recovered fit into the greaterAlnus-associated Frankia phylogeny.

Materials and methods

Nodule collection

MexicoIn June and July 2010, Frankia in Alnus root nodules were

collected from four Mexican field sites. Two sites were lo-cated at La Malinche National Park in the state of Tlaxcalaand two other sites at the Acatlán volcano in the state of Ve-racruz (for site maps, see supplemental Fig. S11). La Ma-linche and Acatlán are separated by 132 km. The two LaMalinche sites were located 10 km apart on opposite sidesof the 4460 m volcano. Both sites were co-dominated by Al-nus jorullensis Kunth and Pinus montezumae Lamb., a nativeconifer species. The two Acatlán sites, hereinafter referred toas Naolinco sites 1 and 2, were located 1 km apart on oppo-site sides of the 1900 m volcano. Both Naolinco sites weredominated exclusively by Alnus acuminata Kunth. No otherknown Frankia hosts were present at any of the four sites.Additional geographic and climatic data for each site is pre-sented in Table 1.At La Malinche sites 1 and 2, and Naolinco site 1, mature

Alnus trees separated by ≥2 m along two parallel transectswere numbered and 20 trees were randomly selected for sam-pling. At each tree, three Frankia nodules were collected inthe immediate vicinity of the trunk (≤1 m) from the top20 cm of soil. At Naolinco site 2, steep terrain made estab-lishing transects untenable. As such, 20 trees separated by≥2 m were haphazardly selected for the same sampling asthe other sites. Nodules from all sites were placed in individ-ual 25 mL screw-cap tubes and stored on ice for a maximumof 72 h during transport to the laboratory. In the laboratory,nodules were surface-sterilized by manual agitation in a 10%bleach solution for 2 min, rinsed multiple times with deion-ized water, and stored at –20 °C.

USAIn 2008 and 2009, Frankia in nodules from four sites in

Oregon and Washington, USA (two each of Alnus rubraBong. and Alnus viridis subsp. sinuata Regel), were collectedusing a similar sampling design to that used in Mexico (forsite maps, see supplemental Fig. S11). DNA extraction, PCRamplification, and sequencing procedures for these sampleswere identical to those used for the Mexican samples. Forsite and sampling details, see Table 1 and Lipus and Ken-nedy (2011).

1Supplementary data are available with the article through the journal Web site (http://nrcresearchpress.com/doi/suppl/10.1139/b2012-006).

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Molecular identificationTotal DNA was extracted from a single lobe of each nod-

ule with a DNEasy blood and tissue kit (Qiagen, Carlsbad,USA). A 606 bp portion of the nifH gene was PCR amplifiedusing the Frankia-specific primers nifHf1 and nifHr (Welshet al. 2009a). Twenty-micolitre PCR reactions contained0.5 µL of DNA, 0.4 µL of 10 µmol/L nifHf1, and nifHr,10 µL MasterAmp F PCR buffer (Epicentre Technologies,Madison, USA), and 0.75 U Taq polymerase (New EnglandBioLabs, Ipswich, USA). PCR conditions were 96 °C for5 min; 35 cycles of 96 °C for 30 s, 64 °C for 30 s, and72 °C for 45 s; and 72 °C for 7 min. PCR products were vi-sualized by electrophoresis on 1.5% agarose gels. For sam-ples that did not yield any product, a second PCR wasperformed using 1 µL of DNA and 0.2 µL bovine serum al-bumin added. All positive PCR products were cleaned withExoSap-IT (USB, Cleveland, USA) (0.75 µL ExoSap,0.25 µL water, and 7.5 µL PCR product, incubated at 37 °Cfor 45 min and then 80 °C for 15 min) and sequenced in asingle direction on a 3730xl DNA analyzer at the GeneticsCore facility at the University of Arizona, USA.All of the Mexican and USA nifH chromatograms were

visually assessed and corrected in Sequencher 4.8 (Gene Co-des, Ann Arbor, USA). Clean sequences were trimmed to574 bp and aligned using MUSCLE (Edgar 2004). Sampleswere sorted into operational taxonomic units (OTUs) at≥95%, ≥97%, ≥99%, and 100% sequence similarity usingthe mean neighbor clustering method in MOTHUR version1.15 (Schloss et al. 2009). Hereinafter, the term genotype isused to refer to an OTU group at a given sequence similaritythreshold. The term KL is used to distinguish among theOTU groups recovered at ≥97% similarity, a threshold thathas been commonly used in previous studies of Frankia ecol-ogy and results in readily reproducible clusters using variousmethods (Mirza et al. 2009; Welsh et al. 2009a; Kennedy etal. 2010b; Lipus and Kennedy 2011). Sequence distance ma-trices were constructed in MEGA 5 (Tamura et al. 2011).Representatives of each of the resulting unique nifH sequen-ces were deposited in GenBank under the accession numbersJN099752–JN099781. A representative of each unique se-quence was also sequenced in the reverse direction and con-firmed to be identical.

Community and phylogenetic analysesSimilarities in the structure of the Mexican and USA

Frankia assemblages were assessed using a nonmetric multi-dimensional scaling (NMDS) plot created in PRIMER ver-sion 5 (Clarke and Gorley 2001). Preliminary analysesindicated that results were similar across different OTU simi-larity thresholds, so only the ≥97% threshold is presented.The NMDS plot was generated from Bray–Curtis similarityvalues that were calculated from a square-root transformedsite-by-genotype abundance matrix. Similarities among thesame assemblages were also compared using the Fast Uni-Frac platform for assessing microbial communities (Hamadyet al. 2010; http://bmf2.colorado.edu/fastunifrac/). Fast Uni-Frac uses phylogenetics-based analyses that differ from theOTU-based NMDS approach, which disregards the differen-ces among sequences within any given OTU. An ML tree ofthe 46 unique Mexican and USA sequences was used to cal-culate non-normalized, abundance-weighted Fast UniFrac

Tab

le1.

Site

details

forAlnus

forestsat

which

Frankianodulesweresampled.

Hostspecies

Country

Site

name

Latitu

de,longitu

deElevatio

n(m

)Meantemperature

(°C)a

Annualprecipitatio

n(m

m)a

No.

ofsequences

No.

ofFrankia

genotypesb

Alnus

acum

inata

Mexico

Naolin

cosite

119.6748°N,9

6.8520°W

1816

16.0

2414

544

Mexico

Naolin

cosite

219.6830°N,9

6.8580°W

1880

15.6

2413

593

Alnus

jorullensis

Mexico

LaMalinchesite

119.2678°N,9

8.0355°W

3283

9.6

928

542

Mexico

LaMalinchesite

219.1882°N,9

7.9828°W

2929

11.9

620

561

Alnus

rubra

USA

FoxCreek

45.5668°N,1

23.5687°W

154

10.1

2138

463

USA

Mt.Hood

45.1515°N,1

12.1417°W

556

9.0

1973

542

Alnus

viridis

USA

Barlow

45.2838°N,1

21.6700°W

1222

5.8

1140

482

USA

St.H

elens

46.2323°N,1

22.1522°W

1232

4.9

2588

562

a Estim

ated

usingtheUnitedStates

Deptartmentof

Agriculture

/Fo

restServicecurrentclim

atemodel

(http

://forest.m

oscowfsl.w

su.edu/clim

ate/).

b Genotypedefinedas

allindividualssharing≥97%

nifH

sequence

similarity.T

hetotalnumberof

Frankiagenotypesacross

allsiteswas

seven.

Higgins and Kennedy 425

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significance values for all pairs of host species. Differencesamong sites were also assessed in Fast UniFrac using anabundance-weighted jackknife cluster analysis with 1000 re-starts.ML and Bayesian methods were used to construct a nifH

phylogenetic tree using sequence data from five sources: (i)28 sequences representing the five published Alnus-infectiveFrankia clusters (Welsh et al. 2009a); (ii) 16 sequences col-lected from root nodules of Alnus oblongifolia Torr. in cen-tral Arizona (Welsh et al. 2009b); (iii) 19 sequencesrepresenting Alnus-infective Frankia genotypes from thenorthwestern USA (Kennedy et al. 2010b; Lipus and Ken-nedy 2011); (iv) 6 sequences representing the two novelFrankia genotypes recovered in Mexico (see below); and (v)4 sequences representing the four Elaeagnus-infectiveFrankia groups (since Alnus species are known to occasion-ally host Frankia strains that typically infect members of thegenus Elaeagnus (Bosco et al. 1992)). An uncultured FrankianifH sequence from a Datisca cannabina L. nodule was usedas an outgroup. A General Time Reversible (GTR) modelwith five rate categories, gamma shape parameter (+G) 0.82,and proportion of invariable sites (+I) 0.69 was determinedto be the most appropriate substitution model according toModeltest (Posada and Crandall 1998). The ML analysis wasperformed using PhyML 3.0 (Guindon et al. 2010; http://www.atgc-montpellier.fr/phyml/). The Bayesian analysis wasperformed using MrBayes version 3.2 (Ronquist and Huel-senbeck 2003) and run until the mean standard deviation ofsplit frequencies was below 0.01. A consensus tree was con-structed following a visually determined burn-in of 10%.

ResultsOf the 240 Frankia in nodules collected in Mexico, 223

(93%) yielded clear nifH sequences. Of the 230 nodules col-lected in the USA, 203 (89%) yielded clear sequences (Ta-ble 1). Among the Mexican sequences, 31 were unique.When grouped at ≥99% sequence similarity there were 10genotypes; at ≥97% five; and at ≥95% three. Mean sequencesimilarity between any two Mexican sequences was 97.7%(SD = 1.7%), which corresponded to ca. 13 single nucleotidedifferences. Among the USA sequences, 15 were unique. At≥99% similarity there were seven genotypes; at ≥97% four;and at ≥95% three. Mean similarity between USA sequenceswas 98.0% (SD = 1.7%; ca. 12 single nucleotide differences).Mean similarity between Mexico and USA sequences was97.6% (SD = 1.2%; ca. 14 single nucleotide differences).The overlap in Frankia genotype composition between

Mexico and the USA varied depending on sequence similar-ity threshold (Fig. 1). When analyzed at 100% similarity,only 2 of the 44 clusters contained nodule samples fromboth Mexico and USA (Table 2). Those two clusters, how-ever, contained 22% of all samples, the majority of which be-longed to clone xvii of genotype KL01. At 97% sequencesimilarity, the vast majority of Frankia samples belonged toKL genotypes present in both Mexico and the USA. Four-hundred-five samples belonged to either KL01 or KL02,while the remaining two belonged to KL04. Two genotypes,KL07 and KL08, represented by 17 samples, were novel toMexico. Among the 259 KL01 samples, there was a total of28 nonidentical sequences, whereas there were only eight

nonidentical sequences among the 147 KL02 samples. Someof the less common genotypes contained considerable micro-sequence diversity, with genotypes KL07 and KL08 contain-ing two and four nonidentical sequences despite being repre-sented by a much smaller number of samples (Table 2).Genotype KL04 was represented by two identical sequences,one each from Mexico and the USA, while KL06 was repre-sented by a single USA sequence. Genotypes KL03 andKL05, which have been recovered in low numbers in thenorthwestern USA (Kennedy et al. 2010b), were not presentat any of the sites included in these analyses.Similarity in the structure of Frankia assemblages in Mex-

ico and USA was not strongly related to geographic proxim-ity. In the NMDS plot, the A. rubra (USA) and A. acuminata(Mexico) sites clustered close together, while the A. viridis(USA) and A. jorullensis (Mexico) sites formed a separatecluster (Fig. 2A). Clustering corresponded with hosts domi-nated by Frankia genotypes KL01 (A. viridis and A. jorullen-

Fig. 1. Distribution of Frankia genotypes recovered from root no-dules of four Alnus host species in Mexico and the northwesternUnited States of America. For each of the four host species, noduleswere collected from two different study sites (there were only minordifferences between paired sites, so pooled results shown). The innercircles represent the 15 genotypes recovered by using a ≥99% nifHsequence similarity cutoff. The middle circles represent the six gen-otypes recovered from clustering the same sequences at ≥97% simi-larity, and the outer circles represent the four genotypes recoveredfrom clustering at ≥95% similarity. The number of genotypes recov-ered at the 95%, 97%, and 99% levels were Alnus acuminata (Mex-ico): 3, 5, and 8, respectively; Alnus jorullensis (Mexico): 1, 2, and5, respectively; Alnus rubra (USA): 2, 3, and 6, respectively; andAlnus viridis (USA): 2, 3, and 6, respectively. At the ≥97% level,the two most common genotypes are named KL1 and KL2 and weredistributed as indicated.

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sis) and KL02 (A. rubra and A. acuminata). Fast UniFracsignificance tests also indicated that differences in assem-blage structure were not strongly influenced by geographicproximity. Frankia assemblages associated with the two Mex-ican host species were significantly different from each other

(P < 0.05), while the assemblages on the two USA host spe-cies were not (P > 0.05). Alnus jorullensis associatedFrankia assemblages were significantly different from theA. rubra assemblages (P < 0.05) but not from A. viridis(P > 0.05). Alnus acuminata associated Frankia assemblages

Table 2. GenBank accession numbers and occurrence data for Frankia nifH genotypes in nodules of four Alnusspp. in central Mexico and northwestern United States of America.

No. of nodules

Mexico USA

KL genotypea Subtypeb Accession no.Alnusjorullensis

Alnusacuminata

Alnusviridis

Alnusrubra Total

1 xvii GU810478 4 87 911 ii JN099753 38 9 471 vii JN099758 19 191 xviii JN099768 11 111 v JN099756 10 101 ix JN099760 9 91 xxii JN860055 9 91 i JN099752 8 81 xxi GU810473 8 81 vi JN099757 6 61 xxviii JN860056 6 61 xxiv GU810477 4 1 51 iv JN099755 3 31 xxiii JN860057 1 2 31 xxv JN860058 1 2 31 iii JN099754 2 21 viii JN099759 2 21 x JN099761 2 21 xi JN099762 2 21 xiv JN099765 2 21 xv JN099766 2 21 xvi JN099767 2 21 xx JN099770 2 21 xii JN099763 1 11 xiii JN099764 1 11 xix JN099769 1 11 xxvi JN860059 1 11 xxvii JN860060 1 12 v GU810474 70 702 iv JN099774 44 442 ii JN099772 13 132 iii JN099773 11 112 vi JN860061 3 32 vii JN860062 3 32 viii JN860063 2 22 i JN099771 1 14 i GU810476 1 1 26 i GU810482 1 17 i JN099776 4 47 ii JN099777 1 18 i JN099778 6 68 iv JN099781 4 48 ii JN099779 1 18 iii JN099780 1 1Total 110 113 103 100 426

aGenotypes are based on a 574-bp portion of the nifH gene. Sequences are listed first by genotype, then prevalence, i.e., ≥97%nifH sequence similarity using the mean neighbor clustering algorithm in mothur (Schloss et al. 2009).

bDefined at 100% similarity along the 574-bp sequence analyzed.

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were not significantly different from the assemblages foundon either USA host species (P > 0.05). Similar to theNMDS analysis, Fast UniFrac jackknife clustering showedthat A. acuminata assemblages associated more closely withA. rubra, and A. jorullensis with A. viridis (Fig. 2B).ML and Bayesian phylogenetic trees were both fairly well

resolved and showed no major inconsistencies, although theML tree reported several weakly resolved relationships re-ported as polytomies in the Bayesian tree. The largest clade,designated clade 1, contained members of genotypes KL01and KL02, as well as AI, the largest Alnus-infective Frankiacluster recognized by previous researchers (Welsh et al.2009a). The two novel Mexican genotypes, KL07 and

KL08, both grouped within known Alnus-infective clades.The four KL08 genotype sequences fell into clade 1, whilethose of genotype KL07 fell into clade 2. Of the six KL gen-otypes present at the northwestern USA sites, five belongedto clade 1, while KL04 belonged to clade 2 (Fig. 3).

DiscussionIn this first study of Alnus-associated Frankia in Mexico,

we found assemblages that were characterized by low geno-type richness and had high compositional overlap with those3500 km away in the northwestern USA. These data suggestthat the common Alnus-associated Frankia genotypes appearto have a widespread distribution throughout western NorthAmerica. While the criteria used for defining Frankia nifHgenotypes had some effect on the level of compositional over-lap, multiple analytical approaches indicated that geographicproximity was not the major factor in determining assemblagestructure. Phylogenetic analysis of nifH sequences from uncul-tured Alnus-associated Frankia recovered from diverse sitesworldwide further confirmed that if Alnus-associated Frankiahave a clear biogeography driven by dispersal limitation, thenit is most likely to become apparent at spatial scales largerthan the one investigated here.The low genotype richness of the Mexican sites is similar

to assemblages associated with diverse Alnus species world-wide. We have previously documented that Frankia genotyperichness in the northwestern USA forests is typically low (i.e.,2–5 genoytpes) (Kennedy et al. 2010a, Kennedy et al. 2010b,Lipus and Kennedy 2011), and a recent study of Frankia rich-ness in root nodules of A. oblongifolia at three locationsthroughout the state of Arizona also yielded only threeFrankia nifH genotypes at the ≥97% sequence similarity level(Welsh et al. 2009b). Outside the contiguous 48 states, usinga wide array of different molecular methods and gene regions,Alnus glutinosa L. in Spain (Igual et al. 2006), Alnus incanasubsp. tenuifolia Moench and A. viridis in Alaska (Andersonet al. 2009), and Alnus nepalensis D. Don in China (Dai et al.2004) were also found to harbor at most a handful of Frankiagenotypes. Although there is currently little information avail-able concerning Frankia richness in Mexico, Cabrera andValdés (2010) found only six genotypes associated with Cas-uarina equisetifolia L. across a broad geographical and eleva-tional gradient in the Gulf Coast region.In molecular diversity analyses of Frankia using nifH, the

cutoff level of ≥97% sequence similarity results in taxonomicgroups that reliably correlate to distinct, reproducible cladesin phylogenetic analyses (Mirza et al. 2009; Welsh et al.2009a; Kennedy et al. 2010b; Lipus and Kennedy 2011). Inaddition, Lipus and Kennedy (2011) found evidence of hostspecies-level associations among genotypes defined at thissequence similarity level. Thus, we have chosen to focus onAlnus-infective Frankia genotypes defined at ≥97% nifHidentity. Nevertheless, because the ≥97% cutoff is somewhatarbitrary, we also compared the Frankia assemblages at dif-ferent sites using both more and less stringent genotype defi-nitions. Overall, our genotype-based results were very similarto those obtained using phylogenetic distance-based UniFracanalyses, which also consistently showed that geographicproximity was a nonsignificant factor in determining similar-ity among Frankia assemblages. Since UniFrac analyses take

Fig. 2. (A) Operational taxonomic unit (OTU)-based nonmetricmultidimensional scaling (NMDS) plot and (B) sequence distance-based Fast UniFrac jackknife cluster diagram of Frankia assem-blages at eight Alnus sites in Mexico and the northwestern UnitedStates of America. In (A), symbols closer together represent moresimilar Frankia assemblages, while in (B), assemblage similarity isindicated by branch length. Numbers at nodes indicate the fractionof iterations in which the given node was recovered. Symbols indi-cate the following: triangles, Mexico sites; circles, USA sites; closedsymbols, assemblages dominated by Frankia genotype KL01; opensymbols, assemblages dominated by KL02. Site names are abbre-viated as follows: M1 and M2, La Malinche sites 1 and 2 (Alnus jor-ullensis); N1 and N2, Naolinco sites 1 and 2 (Alnus acuminata);BA, Barlow (Alnus viridis); SH, St. Helens (A. viridis); FC, FoxCreek (Alnus rubra); MH, Mt. Hood (A. rubra).

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into consideration all of the sequence variation among sam-ples, they circumvent the problem of potentially arbitrarygenotype definitions. Given that the genotype-based NMDSplot grouped the sampling sites in a manner similar to theUniFrac jackknife clustering analysis, we are confident thatthe ≥97% criterion is sufficient to accurately represent thedifferences among Alnus-associated Frankia assemblages.Genotypes KL01 and KL02 comprised 92% and 99% of all

nodules sampled in Mexico and the USA, respectively, buteach genotype was dominant on only one of the two hostspecies sampled in each region. Lipus and Kennedy (2011)demonstrated that the observed host-association pattern in

USA Alnus forests was reproducible in reciprocal soil trans-plant experiments, and therefore appears to reflect a prefer-ence on the part of either the host or the microsymbiont forcertain partners over others. We believe that this preference islikely also the explanation for the dominance patterns inMexican Alnus–Frankia symbioses as well. We can onlyspeculate as to the mechanism of preference within theAlnus–Frankia symbiosis. If it were an instance of long-termco-evolution between particular Alnus species and Frankiagenotypes, we would expect more closely related Alnus spe-cies to host more similar Frankia assemblages. Our resultsrefute this hypothesis, since A. acuminata and A. jorullensis

Fig. 3. Bayesian inference tree of nifH sequences from diverse Alnus-associated Frankia isolates. Sequences are labeled with country or re-gion of origin (i.e., Hungary, Alaska, Peru, Japan, or Rwanda), host species information (i.e., AO, Alnus oblongifolia), or genotype name (i.e.,KL1–KL8) and GenBank accession number. Four Eleagnus-associated sequences were included, as Frankia genotypes that typically infectEleagnus species have been reported to occasionally colonize Alnus (Bosco et al. 1992). An uncultured Datisca-associated isolate was in-cluded as the outgroup. Where appropriate, clades are labeled according to the clusters defined by Welsh et al. (2009a). Lineages that arenovel to this study (KL7 and KL8) are identified by bold underlined text. Where available, branches are labeled with Bayesian posteriorprobabilities and, in parentheses, maximum likelihood aLRT scores.

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are sister taxa, yet their Frankia assemblages are less similarto each other than they are to the more distantly related A. ru-bra and A. viridis, respectively (Chen and Li 2004) (see sup-plemental Fig. S21).In our phylogenetic analysis using sequences from diverse

nifH-based studies, we found that our Mexican and USAsamples fell into one of two major clades within the largerAlnus-associated clade. Members of the larger clade (clade1), which includes KL01 and KL02, included samples col-lected on five different continents. The second clade (clade2) contains mostly North American isolates and one recov-ered from a site in Japan. Since clade 2 has relatively fewmembers that may represent minor components in localFrankia assemblages, this observation could simply reflectinadequate sampling outside of North America. Alternatively,it could also signify a biogeographical influence, in that thelarge, diverse clade 1 appears to be globally distributed,whereas members of clade 2 may have a more limited range.Interestingly, ancestors of the Alnus species native to westernNorth America and Central America are thought to have ori-ginated in Asia, having migrated to North America via theBering land bridge (Chen and Li 2004), providing a possibleshared biogeographical history for members of clade 2 andtheir hosts.Our results largely corroborate the Baas Becking hypothesis

for microbes capable of both free-living and host-associatedlife histories. In western North America, most Alnus-associatedFrankia genotypes appear to be widely distributed, with localhost species or environments selecting certain genotypes ofbacteria, for reasons that we do not yet fully understand. Thereis some evidence that soil chemistry may be a contributingfactor (Anderson et al. 2009; Lipus and Kennedy 2011).Although not formally analyzed in this study, soil chemicaldata for the Mexican field sites was obtained for a concurrentstudy of ectomycorrhizal fungal diversity in the same forests(Kennedy et al. 2011). In general, soil chemistry was similaracross all sites, with the striking exception of phosphorus,which was ca. 50–90 times higher at the La Malinche sitesthan at the Naolinco sites. Since phosphorus level in the soilis correlated with nodulation levels (Gentili and Huss-Danell2003) and nodule phenotype (Markham and Chanway 1998),it would be interesting to investigate the degree to which vary-ing soil chemistry affects Frankia assemblage composition inthe future. In addition, future studies of Frankia biogeographywould benefit from examining Frankia diversity at spatialscales beyond continents, since if Alnus-associated Frankia as-semblages are to have clear biogeographic patterning causedby dispersal limitation, it is most likely to become apparentonly at the global scale.

AcknowledgementsWe thank R. Garibay Orijel and R. Angeles Argáiz for as-

sistance with field collections in Mexico. A. Lipus, E. Oppelt,R. Rogers, and J. Schouboe participated in field collections inthe USA, and A. Lipus, E. Oppelt, and J. Schouboe assistedwith laboratory work in the USA. G. Binford provided supportwith phylogenetic analyses. We also gratefully acknowledgetwo anonymous reviewers for their constructive comments onearlier versions of this manuscript. The research was fundedby a grant from the Lewis & Clark College Student Academic

Affairs Board and the U.S. National Science Foundation(Grant No. DEB-1020735).

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