ARTICLE

Arbuscular mycorrhizal fungal succession in a long-lived perennial1Miranda M. Hart, Monika Gorzelak, Diane Ragone, and Susan J. Murch

Abstract: It is difficult to understand why arbuscular mycorrhizal (AM) fungal communities change over time. The role of hostidentity confounds our understanding of successional changes in AM fungal communities because hosts exert strong selectivepressure on their root-associated microbes. In this study we looked at the AM fungi associated with a long-lived perennialbreadfruit (Artocarpus altilis (Parkinson) Fosberg) to see how AM communities change over the life span of a single, long-lived host.Using 454 high-throughput sequencing, we found evidence that older trees had more AM fungal taxa than younger trees andwere associated with different AM fungal communities, but these differences were not apparent early in the life cycle. Older treeswere dominated by species of Rhizophagus P.A. Dang, whereas younger trees and genets were dominated by species of Glomus Tul.& C. Tul. Some taxa were only detected in older trees (e.g., Funneliformis C. Walker & Schuessler) or genets (e.g., Racocetra Oehl, F.A.Souza & Sieverd. and Scutellospora C. Walker & F.E. Sanders), indicating that certain AM fungal taxa may serve as “indicators” ofthe successional age of the fungal community. These results provide important information about a poorly studied system andgive insight into how AM communities change over longer time scales.

Key words: arbuscular mycorrhizal fungi, succession, breadfruit, root microbiota, community.

Résumé : Il est difficile de comprendre pourquoi les communautés fongiques de mycorhizes a arbuscules (MA) changent enfonction du temps. Le rôle de l’identité de l’hôte obscurcit notre compréhension des changements sériaux dans les communau-tés fongiques de MA car l’hôte exerce une pression sélective importante sur les microbes associés a ses racines. Dans cette étude,nous avons examiné les champignons MA associés a une espèce vivace longévive, l’arbre a pain (Artocarpus altilis (Parkinson)Fosberg), afin de voir comment les communautés de MA changent au cours de la durée de vie d’un hôte unique longévif. À l’aidedu séquençage a haut débit sur 454, nous avons trouvé des indices que les arbres plus âgés comportaient davantage de taxons deMA que les arbres plus jeunes et qu’ils étaient associés a différentes communautés de MA fongiques, mais ces différencesn’étaient pas apparentes tôt dans le cycle de croissance. Les arbres plus âgés étaient dominés par Rhizophagus P.A. Dang spp., alorsque les arbres plus jeunes et les genets étaient dominés par Glomus Tul. & C. Tul. spp. Certains taxons n’étaient détectés que chezles arbres plus âgés seulement (p.e. Funneliformis C. Walker & Schuessler) ou les genets (p.e. Racocetra Oehl, F.A. Souza & Sieverd.et Scutellospora C. Walker & F.E. Sanders), indiquant que certains taxons de MA fongiques pourraient être utilisés comme« indicateurs » de l’âge sérial d’une communauté fongique. Ces résultats fournissent une information importante sur un systèmepeu étudié et donnent un aperçu de la façon dont les communautés de MA changent en fonction d’échelles de temps pluslongues. [Traduit par la Rédaction]

Mots-clés : champignon mycorhizien a arbuscule, succession, arbre a pain, microbiote de la racine, communauté.

IntroductionThe succession of taxa in a community over time is a fundamen-

tal property of ecosystems (Clements 1916; Connell and Slatyer1977). Arbuscular mycorrhizal (AM) fungi represent one of themost important functional groups in terrestrial ecosystems. Thecommunity composition of these obligate root endophytes (phy-lum Glomeromycota) is an essential driver of plant productivity(van der Heijden et al. 1998). Thus, an understanding of communitydynamics is essential for managing landscapes over the long term.

There is a long history of successional research concerning ar-buscular mycorrhizas, including primary succession followingvolcanic eruption (Allen et al. 1992), glacier retreat (Cázares et al.2005), old fields (Boerner et al. 1996), and tropical forests (Fischeret al. 1994; Zangaro et al. 2013). However, much of this researchfocused on the role of AM fungi in plant turnover and succession(Allen et al. 1992; Fischer et al. 1994; Kikvidze et al. 2010). Studiesthat also considered dynamics in the AM fungi found mixed re-

sults. Those studies that measured total abundance of AM fungihave found no change (Jumpponen et al. 2012; Welc et al. 2012) ordecrease over time (Beauchamp et al. 2006). Others have shownincreases in root colonization or hyphal density over time(Boerner et al. 1996; Sikes et al. 2012; Zangaro et al. 2008). Simi-larly, results are mixed when considering changes in AM fungalcommunities. There is evidence of distinct communities over suc-cessional time (Johnson et al. 1991; Helgason et al. 1998) as well asdistinct “species pools” for old versus young forests (Öpik et al.2008; Davison et al. 2011). Sikes et al. (2012) showed that althoughspecies diversity was not different among AM fungal taxa along asand dune chronosequence, there was a reduction of phylogeneticdiversity in older sand dunes.

One reason for such disparate results might be due to the con-founding role of host identity in shaping AM fungal communities.The progression of plant taxa over successional time from ruderalto competitive to stress-tolerant life history strategies is well doc-umented (Clements 1916; Pianka 1970; Grime 1974). At the same

Received 22 July 2013. Accepted 7 December 2013.

M.M. Hart and M. Gorzelak. Biology Department, The University of British Columbia, Okanagan Campus, Kelowna, BC V1V 1V7, Canada.D. Ragone. Breadfruit Institute, National Tropical Botanical Garden, 3530 Papalina Road, Kalaheo, HI 96741, USA.S.J. Murch. Chemistry Department, The University of British Columbia, Okanagan Campus, Kelowna, BC V1V 1V7, Canada.Corresponding author: Miranda M. Hart (e-mail: miranda.hart@ubc.ca).1This article is part of a Special Issue entitled “The Microbiota of Plants”.

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Botany 92: 1–8 (2014) dx.doi.org/10.1139/cjb-2013-0185 Published at www.nrcresearchpress.com/cjb on 24 February 2014.

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time, there is evidence that host identity exerts a strong selectiveforce on AM fungal communities (Öpik et al. 2009; Moora et al.2011; Becklin et al. 2012). Thus, AM fungi might not undergo suc-cession per se; changes in AM communities might simply resultfrom high degrees of host specificity over successional time.

To better understand fungal community succession over time,it may be useful to consider a single plant, rather than a plantcommunity, as the “ecosystem” for an AM fungal community.Using this model, the root system of a germinating seed repre-sents primary succession. The dynamics of AM fungi within thatroot over the plant’s life span represents secondary succession.Ectomycorrhizal (ECM) researchers have used this approach tostudy fungal community turnover over time. Richard et al. (2005)found no changes among ECM fungal communities associatedwith different age classes of oaks, but Wang et al. (2012) detecteddifferences among ECM associated with young, medium-aged,and old trees. Twieg et al. (2007) found that ECM communitiesdiffered between young and older stands of birch (Betula L.) andDouglas-fir (Pseudotsuga menziesii (Mirb.) Franco), a finding that wassimilar for ECM in pines (Pinus L.) (Obase et al. 2009). Recently,Blaalid et al. (2012) found that increasing time since deglaciationwas associated with more taxa and compositional differencesamong ECM communities in a herb.

There are fewer examples using this model in the AM literature.In an early study using beachgrass (Ammophila breviligulata Fern.)along a dune chronosequence, Koske and Gemma (1997) foundthat older plants had a higher species richness. Husband et al.(2002) showed changes in tropical seedling establishment by fol-lowing the communities of AM fungi in specific trees over multi-ple growing seasons, and they found that dominant taxa shifteddramatically between seasons. This was also true of a more short-term study in Pisum sativum L., where certain AM fungal taxa dom-inated early in the growth cycle (Yu et al. 2012). In contrast withthese studies, there were no age effects on AM fungal communi-ties studied in a 35-year chronosequence of Caragana korshinskiiKom. plantations (Liu et al. 2009). Given that these few studiescover a wide range of hosts and time scales, there is clearly a needfor further research on the long-term community dynamics ofAM fungi in a single host.

In an earlier study we found breadfruit (Artocarpus spp.,Moraceae) to be highly arbuscular mycorrhizal and that rootsfrom older trees contained more AM fungal taxa (Xing et al. 2012).Breadfruit is a long-lived tree that is widespread throughout trop-ical regions (Zerega et al. 2005). It begins fruiting at 3–6 years andhas a life span of more than 100 years (Ragone 1997). The germ-plasm collection at the National Tropical Botanical Garden Bread-fruit Institute in Maui (Hawaii) presents an excellent opportunityto observe AM fungal succession within a single host. This insti-tute hosts the world’s largest curated collection of breadfruit cul-tivars consisting of 325 well-documented trees from 34 PacificIsland groups, as well as the Philippines, Indonesia, Honduras,and the Seychelles (Jones et al. 2011, 2013). The accessioned treeswere collected from 1978 to 2004 and include Artocarpus trees fromdifferent age classes (seedlings to >40 years). Further, the trees aregrown in a common garden, greatly reducing the amount of vari-ation in AM fungal communities due to site-specific differences.

In this study we used a population of breadfruit trees fromdifferent age classes as a proxy for succession within a single host(because the natural life span of a single tree can exceed 100 years).Because so little is known about changes in AM communities overthe long term, we are not yet able to make predictions about howfungal identity changes over time. Rather, this study providesdescriptive information so that future papers will be able to ad-dress how AM fungal taxa change over successional time. Here, weask whether there are changes in AM fungal communities acrossthe age classes of a single host species; more specifically, we ad-dress whether the root microbiota differ among different ageclasses of breadfruit. To answer these questions, we looked first at

broad age classes of adult trees and then at stages of seedlingestablishment because these stages represent very different phys-iological host stages. We predict that because the environmentthat the fungi experience in the host changes dramatically overthe life span of the plant, there will be selection for differentcohorts of fungi at different stages of the plant life cycle. In addi-tion, we predict that this trend would be less evident in clonallypropagated trees (ramets) because they are, in effect, an extensionof the parent root system.

MethodsThis study was made up of two separate studies. In the first

study we focused on the AM fungal communities in trees rangingfrom 5 to 40 years old. In the second study we focused on AMfungal succession during tree establishment by comparing thefungal communities of young genets, ramets, and mature parents.

Samples were collected in January 2010 (older tree study) andFebruary 2011 (seedling study) at Kahanu Garden, part of theBreadfruit Institute at the National Tropical Botanical Gardennear Hana, Maui, Hawaii (20°47=57.07==N, 156°02=18.42==W) (http://ntbg.org/breadfruit). Breadfruit trees (Artocarpus altilis (Parkinson)Fosberg) were planted from 1970 to 2004 in a grid pattern orchard(<5 ha), and these represent three age classes of trees: thoseplanted in 2003–2004 (young, 5–6 years old at the time of sam-pling), those planted in 1989–1990 (medium aged, 20–21 years oldat the time of sampling), and those planted in 1970 or 1978 (old, 32or 40 years old at the time of sampling). For the seedling study, wetargeted mature trees in the youngest age category (planted in2003–2004) to study changes in AM fungal communities associ-ated with early plant establishment. Ramets and genets could bedistinguished from each other, as attachments to the parent root(ramet) or to a seed (genet) were clearly visible. These saplingswere growing under the canopy of the parent tree, well separatedfrom other trees in the garden.

Sample collectionFor the older tree study, we randomly sampled five individual

trees from each size class, for a total of 15 trees. For each tree, wecollected roots and soil using a small shovel at a depth of approx-imately 15 cm from three random locations around the base of thetree. Young roots and any adhering rhizosphere were collectedalong with approximately 100 mL of bulk soil. For each tree, thethree samples were then pooled. Samples were refrigerated andshipped to the University of British Columbia Okanagan for fur-ther processing. For the seedling study, we randomly selected fiveparent trees with evidence of both ramet and genet reestablish-ment. In this study, a genet is a seed-propagated seedling, whereasa ramet is a seedling originating from the parent root system. Ateach parent tree we sampled roots from one genet, one ramet plusthe parent, for a total of three root samples per parent tree. Wealso sampled soil from the parent (as described above). Sampleswere kept on ice and shipped to the University of British ColumbiaOkanagan where they were then frozen until further processing.

Molecular analysis of root and soil communities

DNA extractionDNA was extracted from root and soil samples separately using

a Powersoil DNA Isolation kit (MO BIO Laboratories Inc., Carlsbad,Calif.). DNA was extracted from 150 mg of randomly selected rootsegments from each tree (first cleaned in deionized water). Soilswere sieved for root and rock fragments, and 250 mg of this ho-mogenized soil was used for DNA extraction for each sample.

Glomeromycota 18S small subunit ribosomal DNA sequenceswere amplified using NS31 (forward) (Simon et al. 1992) and AM1(reverse) (Helgason et al. 1998) primers, linked to 454 sequenc-ing adapters and linkers Primer A and Primer B, respectively,with unique 10 bp identification barcodes associated with the

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forward primer only, as per the Roche Technical Bulletin No. 9013.Identification barcodes (Roche Technical Bulletin No. 09005)were assigned to each soil and root sample. Thus, the forwardNS31 primer was 5=-CGTATCGCCTCCCTCGCGCCATCAG-{10bpMID}-TTGGAGGGCAAGTCTGGTGCC-3= and the reverse AM1 primer was5=-CTATGCGCCTTGCCAGCCCGCTCAG-GTTTCCCGTAAGGCGCCGAA-3=. PCR was carried out using 20 pmol dNTPs, 3.5 mmol·L−1 MgCl2,40 �g bovine serum albumin, 20 pmol of each primer, and 1 U GoTaqDNA polymerase with supplied buffer (Promega Corporation,Madison, Wis.). Thermocycling conditions were as follows: 95 °Cfor 1 min, 35 cycles of 95 °C for 30 s, 54 °C for 30 s, and 72 °C for1 min, followed by 72 °C for 7 min, and stored at 4 °C. PCR productswere cleaned and normalized to 1–2 ng·�L−1 using a SequelPrepNormalization Plate (96) kit (Life Technologies, N.Y.). Samplesfrom each experiment were sequenced separately on a GenomeSequencer FLX System using Titanium Series reagents (RocheApplied Science) at the Vancouver Prostate Centre. Raw sequenceswere deposited in NCBI’s BioProject database (submission IDSUB300388).

Sequence analysisSequences were analyzed using 18S reference-based pipeline in

Quantitative Insights Into Microbial Ecology (Caporaso et al. 2010).Briefly, sequences were identified by identifier tags and groupedinto operational taxonomic units (OTUs) at 97% similarity usingthe 18S pipeline (Caporaso et al. 2010).

Taxonomy was assigned using MaarjAM, a public database forGlomeromycota DNA environmental sequences (www.maarjam.botany.ut.ee) (Öpik et al. 2010). We used the database to identifyOTUs at 97% similarity, retaining sequences that found no matchin the database. OTUs with fewer than five representatives wereexcluded from the analysis. Samples were rarefied to 772 sequencesper sample (older tree study) or 545 sequences per sample (seed-ling study). Sequences that returned no match with MaarjAMwere queried against the National Institute for Health’s GenBank(http://www.ncbi.nlm.nih.gov) using BLAST (nucleotide) searches(Altschul et al. 1990). Searches that returned Glomeromycotansequences were retained, but all non-Glomeromycotan sequenceswere excluded from further analysis. The OTU tables were ex-ported to PRIMER-E (Clarke and Gorley 2006) for subsequentanalyses.

Statistical analyses

Alpha diversityWe tested for differences in species richness among our age

classes using a one-way ANOVA with “tree age” as our factor(fixed) (older tree study) or a two-way ANOVA with “tree age” as afixed factor and “parent tree” as a random factor (seedling study).Analyses were performed in SPSS 21.0.0.0 (IBM Corp 2012).

Community compositionDifferences among communities in terms of species composi-

tion were determined using the Bray–Curtis dissimilarity metric(Bray and Curtis 1957), after first performing a fourth roottransformation on abundance values. These data were used in aPrincipal Coordinates Analysis. We tested for differencesamong tree age classes using a one-way permutational multi-variate analysis of variance (PERMANOVA) (Anderson 2001) withtree age as our factor (9999 permutations; older tree study) or atwo-way PERMANOVA with tree age (fixed) and parent tree (ran-dom) as our factors (9999 permutations; seedling study).

Results

Sequence analysisOut of a total of 62 840/115 065 sequences (older tree study/

seedling study), 36 303/35 397 fulfilled the criteria for furtheranalysis. After filtering OTUs with less than five reads, we ob-tained 234/175 Glomeromycotan OTUs, 223/74 of which received aBLAST hit (similarity ≥97%) in the MaarjAM database (Glomeromy-cota small subunit rRNA gene sequences) (Öpik et al. 2010). Manyof these OTUs were associated with the same virtual taxon, so thefinal number of virtual taxa that matched the database was only66/36 (Supplementary Table S12). Several sequences did not matchMaarjAM (11/42), but were found to match Glomeromycotan se-quences in GenBank ranging from 95% to 99% similarity (http://www.ncbi.nlm.nih.gov). Non-Glomeromycotan reads were excludedfrom analyses (14%/16% of OTUs).

AM fungi abundanceIn both the older tree study and the seedling study, we did not

sample to redundancy, although we approached this more closelyin the older tree study (Fig. 1). In the older tree study, the numberof taxa identified per sample ranged from a low of five to a high of34 taxa (18–23, 95% confidence interval), whereas in the seedlingstudy, the number of taxa identified per sample ranged from a lowof nine to a high of 21 (mean = 15.98 ± 1.3, n = 11) (Fig. 2). In the oldertree study, we found that AM fungal species richness increasedwith tree age, but only for roots (F2,19 = 5.80, p = 0.015) (Fig. 2a).That is, AM fungal communities associated with trees over20 years old were different from those of trees that were 5 or6 years old (LSD, p = 0.023 and p = 0.006, respectively). We couldnot detect a difference in the number of taxa between roots andsoil (F1,19 = 0.518, p = 0.484), even though soils had consistentlyfewer taxa (Fig. 2a). These findings were reproduced when weevaluated alpha diversity using the Shannon index (H=) (data notshown). In the seedling study, we found no effect of habitat type(parent, genet, ramet, or soil) on the number of taxa in eachsample (F3,10 = 0.474, p = 0.710) (Fig. 2b). This was also true for theShannon index (data not shown).

Abundance of generaIn the older tree study, all samples were dominated by two

genera (Glomus Tul. & C. Tul. and Rhizophagus P.A. Dang) for bothroots and soil (Fig. 3a–3c, soils not shown). Glomus taxa dominatedin young trees, but were displaced by Rhizophagus in older trees.Whereas a single genotype dominated in Glomus, Rhizophagus wascomposed of multiple taxa (see below). All other taxa made up asmall fraction of sequence reads. In the seedling study, Glomus andRhizophagus also dominated in all treatments. Glomus was consis-tently the most abundant genus in all habitat types (Fig. 3d).Again, Rhizophagus was highly abundant and represented a largenumber of taxa in contrast with Glomus, which was similarly abun-dant but was represented by fewer taxa. Unlike the older treestudy, Acaulospora Trappe & Gerd. and Scutellospora C. Walker &F.E. Sanders were well represented in the roots (Fig. 3d) whileRacocetra Oehl, F.A. Souza & Sieverd. and Diversispora C. Walker& Schuessler were well represented in the soil (data not shown).

Relative abundance of taxa over age classesTo evaluate changes among the subordinate taxa among age

classes, we evaluated the occurrence of AM fungi in terms ofrelative abundance of each genus (Fig. 4a and 4b). Thus, the datarepresented are illustrative and are meant to provide insight intoAM fungal dynamics among our age classes. They do not implystatistical significance. In the older tree study, we found thatwhile Gigaspora Gerd. & Trappe was fairly evenly spread among

2Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjb-2013-0185.

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the age classes, other taxa either increased (Rhizophagus and espe-cially Funneliformis C. Walker & Schuessler that was undetectablein the youngest age class and occurred primarily in roots belong-ing to the oldest age class) or decreased (Glomus) over time orpeaked in trees belonging to the medium age class (unidentifiedGlomeromycota and Acaulospora) (Fig. 4a). In soils, additional taxawere detected (i.e., Diversispora, Racocetra, and Claroideoglomus C.Walker & Schuessler) (data not shown). In this case there weremore “age specialists”; Diversispora and Racocetra were only de-tected in soils associated with trees from the youngest age class,along with Scutellospora that occurred there 80% of the time). Aswith roots, Funneliformis was only detected in soils associated withtrees in the oldest age class. An unidentified Glomeromycota andRhizophagus both increased with tree age, whereas Acaulospora andScutellospora decreased. Claroideoglomus was found only associatedwith trees in the medium and old age classes.

Even though we could not detect compositional differencesamong genets, ramets, and parents, when we compared the rela-tive distribution of the genera across the plant age classes, generawere differently represented at each age class level (Fig. 4b). Forinstance, the abundance of Funneliformis and Gigaspora peaked insoil (not shown), whereas Acaulospora, Racocetra, and Scutellosporawere most abundant in genet roots. Diversispora dominated inparent roots. Glomus and Rhizophagus were equally distributedamong all habitat types.

Virtual taxaCertain taxa dominated in the different tree age classes (Supple-

mentary Table S22). Glomus VTX 00137 and 00140 and RhizophagusVTX 00092 and 000312 were almost exclusively detected in rootsof young trees, whereas Glomus VTX 00122, 00131, and 00160 wereonly detected in roots of the oldest trees. Glomus VTX 00166 wasonly detected in any abundance in roots of medium-aged trees. Inthe seedling study, certain taxa dominated in the early age classes(genet, ramet, and parent) (Supplementary Table S32). The bestexample of this is Acaulospora VTX 00045 that occurred at thehighest abundance overall and was only found in parent roots.Glomus VTX 00140 was also found in high abundance, but this wasa generalist and was well represented in all sample types. For theremaining sample types, Glomus VTX00166 and Rhizophagus VTX00206 dominated in ramets, Glomus VTX 00202, Scutellospora VTX

00041, and Rhizophagus VTX 00312 dominated in genets, and GlomusVTX 00222, 00315, Scutellospora VTX 00049, and Gigaspora VTX00255 dominated in soil.

Community compositionIn the older tree study, we found the AM fungal communities

differed significantly depending on the age of the trees they wereassociated with (pseudo-F[2,19] = 1.93, p = 0.0016; 9912 permuta-tions). In this case, the separation between communities was pri-marily between the oldest and youngest trees (Fig. 5). Again, wecould not detect a difference between root and soil communities(pseudo-F[1,19] = 0.8378, p = 0.59998; 9918 permutations). In theseedling study, we found that neither habitat type (pseudo-F[3,10] =1.25, p = 0.2407; 9901 permutations) nor tree identity (pseudo-F[3,10] = 1.56, p = 0.07; 9917 permutations) explained significantvariation among AM fungal communities (data not shown).

DiscussionUsing a single plant species growing in a common garden, we

found evidence for succession in AM fungal communities over the

Fig. 1. Species accumulation curve of arbuscular mycorrhizal fungifor older tree study (solid line) and seedling study (dottedline).Values are given in terms of the number of taxa. Values plottedrepresent the number of operational taxonomic units (OTUs)identified with increasing number of samples used in the analysis.For the older tree study, there were a total of 20 samples (roots andsoil), while for the seedling study, there were 12 (roots and soil).

Fig. 2. Species richness of arbuscular mycorrhizal fungalcommunities associated with root and soil samples in (a) the oldertree study and (b) the seedling study. Bars represent the number ofoperational taxonomic units in each age category. In the older treestudy, the age classes consisted of young trees (5–6 years old),medium-aged trees (20–21 years old), and old trees (32–40 years old).In the seedling study, age classes consisted of parent roots, genetroots, ramet roots, and soil collected from the parent tree. All valuesare given as the mean of n = 5 with 1 SE. Different letters above barsindicate statistical significance at p < 0.05.

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life span of a long-lived host. Older trees were associated withmore fungal taxa and had communities that were composition-ally different from younger trees. While there is evidence in theliterature about the selective force of plant development on rhi-zosphere communities (Green et al. 2006; Marschner et al. 2007;Steer and Harris 2000; Chiarini et al. 1998; Baudoin et al. 2002; Yuet al. 2012), this is the first evidence of AM fungal succession in along-lived perennial. It is unlikely that site differences played arole in our findings because a previous study found no positionaleffects (specifically soil chemistry and geographic distance fromeach other) on AM fungi and tree age (Xing et al. 2012).

These findings support previous work that showed successionalchanges in AM fungal communities, albeit with little communitydata. Husband et al. (2002) were among the first to show distinctcommunity changes in tropical seedlings, but their study fol-lowed seedlings only during initial establishment. Still, their find-ings that dominants were replaced by low abundant taxa mirrorour findings in which most taxa are shared by the age classes, butdiffer by relative abundance. This shift in relative abundance oftaxa has been reported previously (Johnson et al. 1991; Öpik et al.2009; Davison et al. 2011; Öpik and Moora 2012).

In our case, this shift in dominance is primarily due to thelarger number of Rhizophagus isolates colonizing older trees. Thisis not unexpected, because dominance by few taxa is commonin AM fungal communities (Dumbrell et al. 2010; Maherali andKlironomos 2012) and Glomus and Rhizophagus are consistently themost abundant genera in most systems.

When we focused on the earliest life stages of breadfruit, we didnot detect differences among age classes. We had predicted thatthere would be major shifts in AM fungal communities amongestablishing plants, because there is considerable evidence fromthe literature that major developmental changes in a plant’slife span are associated with changes in the fungal rhizosphere(Broeckling et al. 2008). However, we could not detect any differ-ences among our habitat types in this study. For the most part,taxa in this study overlapped considerably with the previousstudy, with few exceptions (Supplementary Table S12). As in theprevious study, all root samples were dominated by Glomus andRhizophagus taxa.

Perhaps one reason that we were unable to detect differencesamong habitat types is that the AM fungi associated with parentalroots dominated the area under the parent, given the large size of

Fig. 3. Total abundance of arbuscular mycorrhizal fungal sequences (reported at the level of genus) over three age classes in the older treestudy: (a) old (32–40 years), (b) medium-aged (20–21 years), and (c) young trees (5–6 years). All sequences for (d) the seedling study were pooledbecause there was no difference among the age classes in the relative abundance of taxa. In all cases, results are shown as a sum of allsamples in each age class.

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Fig. 4. Relative abundance of arbuscular mycorrhizal genera across all age classes in (a) the older tree study (roots only) and (b) the seedlingstudy (roots plus parent soil). These results show how each genus was represented in each age class; here the frequency of sequences for eachgenus add up to 100% across all age classes. This was done to show changes over time for all fungal taxa, including those with very lowabundance (a), and also to illustrate how changes occurred between seedlings and parent trees (b). All data shown represent sequencesoccurring in roots only.

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the parental root system compared with that of the juveniles.Thus, the parent tree may be culturing a select species pool that isavailable locally to its offspring. This may have affected our abilityto detect differences among the age classes. Similarly, it is possi-ble that the juvenile life stage requires a more “generalist” cohortof fungi. There is evidence for this in the ECM research; a distinctfungal microbiota does not become apparent until after the seed-ling has become fully established (Obase et al. 2009).

Single plant species as a proxy for secondary succession inmycorrhizal communities

It may be that age-related changes in the host plant weredriving the community differences in AM fungal communities inolder trees. There is considerable evidence that older trees behavedifferently than younger trees. For instance, old trees can haveincreased root length, thinner root diameter, more root branch-ing, and lower root and soil nutrient content than younger trees(Holdaway et al. 2011). These changes may provide more “niches”to AM fungi as the tree ages, which could manifest as increasedspecies richness.

There is also evidence for reduced resource concentration (re-duced photosynthetic rate) in long-lived trees (Yoder et al. 1994),so it may be that fungi that are successful in older trees are morestress tolerant or competitive than those in younger trees, sensuGrime (1974). There is some evidence for such strategies amongAM fungi. For example, fungi in Scutellospora and Gigaspora (andRacocetra) are known to produce more abundant absorptive myce-lia that could confer a benefit in later sere communities that aretypically nutrient stressed (Hart et al. 2001; Powell et al. 2009;Chagnon et al. 2013). Conversely, ruderal AM fungi would prolif-erate where resources (both soil and host carbon) were not limit-ing. Fungi in the Glomeraceae have been suggested as putative“ruderal” AM fungi (Hart and Reader 2002; Chagnon et al. 2013).However, our data do not show evidence of such phylogeneticspecialization; representatives of all AM fungal families are pres-ent at each age class. These results support, instead, a recent studythat found high phylogenetic diversity on roots regardless ofselection for phylogenetically conserved traits (Maherali andKlironomos 2012). So while it is very tempting to ascribe fungalmorphology with phylogenetic conservatism, high throughputdata sets reveal natural AM fungal communities are more complex.

Comparisons with other studiesBeyond general trends at broad taxonomic levels, it is difficult

to compare our results with other studies since historically AMfungal community analysis has been carried out with a variety ofmethods (spore identification, fatty acids, and fragment-basedmethods) (Gorzelak et al. 2012). Even within sequencing ap-proaches, different target genes make it difficult to compare re-sults. Nevertheless, as the sequence databases grow, our ability totrack particular isolates becomes more feasible. For example,many of the taxa detected in our study have been found in othersystems, although most of the taxa matching the database had noprevious accessions from Oceania (Supplementary Table S12). It istempting to ascribe “indicator taxa” to those that were highlyabundant in our old or young trees; however, there are notenough sequence data from systems of different ages to do this.

ConclusionOur understanding of AM fungal diversity has been greatly in-

formed by early successional or disturbed systems (agriculturalfields, old meadows, and early successional woodlands). Whilethere have been some notable exceptions, old plant hosts havebeen largely overlooked. Clearly, our results show that AM fungalcommunities change over the life span of their hosts, in terms ofspecies diversity and community composition. Still, it remains tobe seen whether these changes are due to changes in the host“environment” or to fungal interactions. To refine our under-standing of these life history strategies, we need more data fromlong-term chronosequences, using single host plants. Also, wemust renew our effort to work with biological entities of AMfungi, rather than sequence data. It would be valuable to haveisolates of these “early” and “late” stage fungi to deduce differ-ences in functioning.

ReferencesAllen, M.F., Crisafulli, C., Friese, C.F., and Jeakins, S.L. 1992. Re-formation of

mycorrhizal symbioses on Mount St Helens, 1980–1990: interactions of ro-dents and mycorrhizal fungi. Mycol. Res. 96: 447–453. doi:10.1016/S0953-7562(09)81089-7.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic localalignment search tool. J. Mol. Biol. 215: 403–410. doi:10.1016/S0022-2836(05)80360-2. PMID:2231712.

Anderson, M.J. 2001. A new method for non-parametric multivariate analysis ofvariance. Austral Ecol. 26: 32–46. doi:10.1111/j.1442-9993.2001.01070.pp.x.

Baudoin, E., Benizri, E., and Guckert, A. 2002. Impact of growth stage on thebacterial community structure along maize roots, as determined by meta-bolic and genetic fingerprinting. Appl. Soil Ecol. 19: 135–145. doi:10.1016/S0929-1393(01)00185-8.

Beauchamp, V.B., Stromberg, J.C., and Stutz, J.C. 2006. Arbuscular mycorrhizalfungi associated with Populus-Salix stands in a semiarid riparian ecosystem.New Phytol. 170: 369–380. doi:10.1111/j.1469-8137.2006.01668.x. PMID:16608461.

Becklin, K.M., Hertweck, K.L., and Jumpponen, A. 2012. Host identity impactsrhizosphere fungal communities associated with three alpine plant species.Microb. Ecol. 63: 682–693. doi:10.1007/s00248-011-9968-7. PMID:22038036.

Blaalid, R., Carlsen, T., Kumar, S., Halvorsen, R., Ugland, K.I., Fontana, G., andKauserud, H. 2012. Changes in the root-associated fungal communities alonga primary succession gradient analysed by 454 pyrosequencing. Mol. Ecol. 21:1897–1908. doi:10.1111/j.1365-294X.2011.05214.x. PMID:22590726.

Boerner, R.E.J., DeMars, B.G., and Leicht, P.N. 1996. Spatial patterns of mycorrhi-zal infectiveness of soils long a successional chronosequence. Mycorrhiza, 6:79–90. doi:10.1007/s005720050111.

Bray, J.R., and Curtis, J.T. 1957. An ordination of the upland forest communitiesof southern Wisconsin. Ecol. Monogr. 27: 325–349. doi:10.2307/1942268.

Broeckling, CD, Broz, AK, Bergelson, J, Manter, DK, and Vivanco, JM . 2008. Rootexudates regulate soil fungal community composition and diversity. Appl.Environ. Microb. 74: 738–744. doi:10.1128/AEM.02188-07.

Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D.,Costello, E.K., Fierer, N., Peña, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A.,Kelley, S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D.,Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R., Turnbaugh, P.J.,Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., and Knight, R. 2010.QIIME allows analysis of high-throughput community sequencing data. NatMethods, 7: 335–336. doi:10.1038/nmeth.f.303. PMID:20383131.

Cázares, E., Trappe, J.M., and Jumpponen, A. 2005. Mycorrhiza-plant coloniza-tion patterns on a subalpine glacier forefront as a model system of primary

Fig. 5. Principal Coordinates Analysis for all samples (root plus soil)from the older tree study. In this figure, each dot represents a singlesample from trees of different age classes (represented by symbols).Each axis is represented by the first two principal coordinates (PCO1and PCO2). Age classes are significantly different (p = 0.016).

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rich2/cjb-cjb/cjb-cjb/cjb99914/cjb0187d14z xppws S�3 3/14/14 12:45 4/Color Fig: F3-F5 Art: cjb-2013-0185 Input-1st disk, 2nd ??

succession. Mycorrhiza, 15: 405–416. doi:10.1007/s00572-004-0342-1. PMID:15772815.

Chagnon, P.-L., Bradley, R.L., Maherali, H., and Klironomos, J.N. 2013. A trait-based framework to understand life history of mycorrhizal fungi. TrendsPlant Sci. 18: 484–491. doi:10.1016/j.tplants.2013.05.001. PMID:23756036.

Chiarini, L., Bevivino, A., Dalmastri, C., Nacamulli, C., and Tabacchioni, S. 1998.Influence of plant development, cultivar and soil type on microbial coloni-zation of maize roots. Appl. Soil Ecol. 8: 11–18. doi:10.1016/S0929-1393(97)00071-1.

Clarke, K.R., and Gorley, R.N. 2006. PRIMER v6: user manual/tutorial. PRIMER-E,Plymouth.

Clements, F.E. 1916. Plant succession: an analysis of the development of vegeta-tion. Carnegie Institution of Washington, Washington.

Connell, J.H., and Slatyer, R.O. 1977. Mechanisms of succession in natural com-munities and their role in community stability and organization. Am. Nat.111: 1119–1144. doi:10.1086/283241.

Davison, J., Öpik, M., Daniell, T.J., Moora, M., and Zobel, M. 2011. Arbuscularmycorrhizal fungal communities in plant roots are not random assemblages.FEMS Microb. Ecol. 78: 103–115. doi:10.1111/j.1574-6941.2011.01103.x.

Dumbrell, A.J., Nelson, M., Helgason, T., Dytham, C., and Fitter, A.H. 2010. Rela-tive roles of niche and neutral processes in structuring a soil microbial com-munity. ISME J. 4: 337–345. doi:10.1038/ismej.2009.122. PMID:19924158.

Fischer, C.R., Janos, D.P., Perry, D.A., Linderman, R.G., and Sollins, P. 1994. My-corrhiza inoculum potentials in tropical secondary succession. Biotropica,26: 369–377. doi:10.2307/2389230.

Gorzelak, M.A., Holland, T.C., Xing, X., and Hart, M.M. 2012. Molecular ap-proaches for AM fungal community ecology: a primer. J. Microbiol. Methods,90: 108–114. doi:10.1016/j.mimet.2012.04.010.

Green, S.J., Inbar, E., Michel, F.C., Jr., Hadar, Y., and Minz, D. 2006. Succession ofbacterial communities during early plant development: transition from seedto root and effect of compost amendment. Appl. Environ. Microbiol. 72:3975–3983. doi:10.1128/AEM.02771-05. PMID:16751505.

Grime, J.P. 1974. Vegetation classification by reference to strategies. Nature, 250:26–31. doi:10.1038/250026a0.

Hart, M.M., and Reader, R.J. 2002. Taxonomic basis for variation in the coloni-zation strategy of arbuscular mycorrhizal fungi. New Phytol. 153: 335–344.doi:10.1046/j.0028-646X.2001.00312.x.

Hart, M.M., Reader, R.J., and Klironomos, J.N. 2001. Life-history strategies ofarbuscular mycorrhizal fungi in relation to their strategies successional dy-namics. Mycologia, 93: 1186–1194. doi:10.2307/3761678.

Helgason, T., Daniell, T.J., Husband, R., Fitter, A.H., and Young, J.P. 1998. Plough-ing up the wood-wide web? Nature, 394: 431. doi:10.1038/28764. PMID:9697763.

Holdaway, R.J., Richardson, S.J., Dickie, I.A., Peltzer, D.A., and Coomes, D.A. 2011.Species- and community-level patterns in fine root traits along a 120 000-yearsoil chronosequence in temperate rain forest. J. Ecol. 99: 954–963. doi:10.1111/j.1365-2745.2011.01821.x.

Husband, R., Herre, E.A., Turner, S.L., Gallery, R., and Young, J.P.W. 2002. Molec-ular diversity of arbuscular mycorrhizal fungi and patterns of host associa-tion over time and space in a tropical forest. Mol. Ecol. 11: 2669–2678. doi:10.1046/j.1365-294X.2002.01647.x. PMID:12453249.

IBM Corp. 2012. IBM SPSS Statistics for Windows, Version 21.0. [Released 2012.]IBM Corp., Armonk, New York.

Johnson, N.C., Zak, D.R., Tilman, D., and Pfleger, F.L. 1991. Dynamics of vesicular-arbuscular mycorrhizae during old field succession. Oecologia, 86: 349–358.doi:10.1007/BF00317600.

Jones, M.P., Ragone, D., Aiona, K., Lane, W.A., and Murch, S.J. 2011. Nutritionaland morphological diversity of breadfruit (Artocarpus, Moraceae): Identifica-tion of elite cultivars for food security. J. Food Comp. Anal. 24: 1091–1102.doi:10.1016/j.jfca.2011.04.002.

Jones, A.M.P., Murch, S.J., Wiseman, J., and Ragone, D. 2013. Morphologicaldiversity in breadfruit (Artocarpus, Moraceae): insights into domestication,conservation, and cultivar identification. Genet. Resour. Crop Evol. 60: 175–192. doi:10.1007/s10722-012-9824-8.

Jumpponen, A., Brown, S.P., Trappe, J.M., Cázares, E., and Strömmer, R. 2012.Twenty years of research on fungal–plant interactions on Lyman Glacierforefront – lessons learned and questions yet unanswered. Fungal Ecol. 5:430–442. doi:10.1016/j.funeco.2012.01.002.

Kikvidze, Z., Armas, C., Fukuda, K., Martínez-García, L.B., Miyata, M.,Oda-Tanaka, A., Pugnaire, F.I., and Wu, B. 2010. The role of arbuscular my-corrhizae in primary succession: differences and similarities across habitats.Web Ecol. 10: 50–57. doi:10.5194/we-10-50-2010.

Koske, R.E., and Gemma, J.N. 1997. Mycorrhizae and succession in plantings ofbeachgrass in sand dunes. Am. J. Bot. 84: 118–130. doi:10.2307/2445889.

Liu, Y., He, L., An, L., Helgason, T., and Feng, H. 2009. Arbuscular mycorrhizaldynamics in a chronosequence of Caragana korshinskii plantations. FEMSMicrob. Ecol. 67: 81–92. doi:10.1111/j.1574-6941.2008.00597.x.

Maherali, H., and Klironomos, J.N. 2012. Phylogenetic and trait-based assemblyof arbuscular mycorrhizal fungal communities. PloS ONE, 7: e36695. doi:10.1371/journal.pone.0036695. PMID:22606282.

Marschner, P., Neumann, G., Kania, A., and Weiskopf, L. 2007. Spatial and tem-poral dynamics of the microbial community structure in the rhizosphere ofcluster roots of white lupin (Lupinus albus L.). Plant Soil, 246: 167–174.

Moora, M., Berger, S., Davison, J., Öpik, M., Bommarco, R., Bruelheide, H.,Kühn, I., Kunin, W.E., Metsis, M., Rortais, A., Vanatoa, A., Vanatoa, E.,Stout, J.C., Truusa, M., Westphal, C., Zobel, M., and Walther, G.R. 2011. Alienplants associate with widespread generalist arbuscular mycorrhizal fungaltaxa: evidence from a continental-scale study using massively parallel 454sequencing. J. Biogeogr. 38: 1305–1317. doi:10.1111/j.1365-2699.2011.02478.x.

Obase, K., Cha, J.Y., Lee, J.K., Lee, S.Y., Lee, J.H., and Chun, K.W. 2009. Ectomy-corrhizal fungal communities associated with Pinus thunbergii in the easterncoastal pine forests of Korea. Mycorrhiza, 20: 39–49. doi:10.1007/s00572-009-0262-1. PMID:19557441.

Öpik, M., and Moora, M. 2012. Missing nodes and links in mycorrhizal networks.New Phytol. 194: 304–306. doi:10.1111/j.1469-8137.2012.04121.x. PMID:22428699.

Öpik, M., Moora, M., Zobel, M., Saks, U., Wheatley, R., Wright, F., and Daniell, T.2008. High diversity of arbuscular mycorrhizal fungi in a boreal herb-richconiferous forest. New Phytol. 179: 867–876. doi:10.1111/j.1469-8137.2008.02515.x. PMID:18537886.

Öpik, M., Metsis, M., Daniell, T.J., Zobel, M., and Moora, M. 2009. Large-scaleparallel 454 sequencing reveals host ecological group specificity of arbuscu-lar mycorrhizal fungi in a boreonemoral forest. New Phytol. 184: 424–437.doi:10.1111/j.1469-8137.2009.02920.x. PMID:19558424.

Öpik, M., Vanatoa, A., Vanatoa, E., Moora, M., Davison, J., Kalwij, J.M., Reier, U.,and Zobel, M. 2010. The online database MaarjAM reveals global and ecosys-temic distribution patterns in arbuscular mycorrhizal fungi (Glomeromy-cota). New Phytol. 188: 223–241. doi:10.1111/j.1469-8137.2010.03334.x. PMID:20561207.

Pianka, E.R. 1970. On r- and K-selection. Am. Nat. 104: 592–597. doi:10.1086/282697.Powell, J.R., Parrent, J.L., Hart, M.M., Klironomos, J.N., Rillig, M.C., and

Maherali, H. 2009. Phylogenetic trait conservatism and the evolution of func-tional trade-offs in arbuscular mycorrhizal fungi. Proc. R. Soc. B Biol. Sci. 276:4237–4245. doi:10.1098/rspb.2009.1015.

Ragone, D. 1997. Breadfruit: Artocarpus altilis (Parkinson) Fosberg. Promoting theconservation and use of underutilized and neglected crops. 10. InternationalPlant Genetic Resources Institute. pp. 2–72.

Richard, F., Millot, S., Gardes, M., and Selosse, M. 2005. Diversity and specificityof ectomycorrhizal fungi retrieved from an old-growth Mediterranean forestdominated by Quercus ilex. New Phytol. 166: 1011–1023. doi:10.1111/j.1469-8137.2005.01382.x. PMID:15869659.

Sikes, B.A., Maherali, H., and Klironomos, J.N. 2012. Arbuscular mycorrhizalfungal communities change among three stages of primary sand dune suc-cession but do not alter plant growth. Oikos, 121: 1791–1800. doi:10.1111/j.1600-0706.2012.20160.x.

Simon, L., Lalonde, M., and Bruns, T.D. 1992. Specific amplification of 18S fungalribosomal genes from vesicular-arbuscular endomycorrhizal fungi coloniz-ing roots. Appl. Environ. Microb. 58: 291–295. PMID:1339260.

Steer, J., and Harris, J.A. 2000. Shifts in the microbial community in rhizosphereand non-rhizosphere soils during the growth of Agrostis stolonifera. Soil Biol.Biochem. 32: 869–878. doi:10.1016/S0038-0717(99)00219-9.

Twieg, B.D., Durall, D.M., and Simard, S.W. 2007. Ectomycorrhizal fungal suc-cession in mixed temperate forests. New Phytol. 176: 437–447. doi:10.1111/j.1469-8137.2007.02173.x. PMID:17888121.

Van der Heijden, M.G., Boller, T., and Wiemken, A. 1998. Different arbuscularmycorrhizal fungal species are potential determinants of plant communitystructure. Ecology, 79: 2082–2091. doi:10.1890/0012-9658(1998)079[2082:DAMFSA]2.0.CO;2.

Wang, Q., He, X.H., and Guo, L.D. 2012. Ectomycorrhizal fungus communitiesof Quercus liaotungensis Koidz of different ages in a northern China tem-perate forest. Mycorrhiza, 22: 461–470. doi:10.1007/s00572-011-0423-x.PMID:22138969.

Welc, M., Bünemann, E.K., Fließbach, A., Frossard, E., and Jansa, J. 2012. Soilbacterial and fungal communities along a soil chronosequence assessed byfatty acid profiling. Soil Biol. Biochem. 49: 184–192. doi:10.1016/j.soilbio.2012.01.032.

Xing, X., Koch, A.M., Jones, A.M.P., Ragone, D., Murch, S., and Hart, M.M. 2012.Mutualism breakdown in breadfruit domestication. Proc. R. Soc. B Biol. Sci.279: 1122–1130. doi:10.1098/rspb.2011.1550.

Yoder, B.J., Ryan, M., Waring, R.H., Schoettle, A.W., and Kaufmann, M.R. 1994.Evidence of reduced photosynthetic rates in old trees. For. Sci. 40: 513–527.

Yu, L., Nicolaisen, M., Larsen, J., and Ravnskov, S. 2012. Succession of root-associated fungi in Pisum sativum during a plant growth cycle as examined by454 pyrosequencing. Plant Soil, 358: 225–233. doi:10.1007/s11104-012-1188-5.

Zangaro, W., de Assis, R.L., Rostirola, L.V., de Souza, P.B., Gonçalves, MC,Andrade, G., and Nogueira, M.A. 2008. Changes in arbuscular mycorrhizalassociations and fine root traits in sites under different plant successionalphases in southern Brazil. Mycorrhiza, 19: 37–45. doi:10.1007/s00572-008-0202-5. PMID:18825422.

Zangaro, W., Rostirola, L.V., de Souza, P.B., de Almeida Alves, R., Lescano, L.E.,Rondina, A.B., Nogueira, M.A., and Carrenho, R. 2013. Root colonization andspore abundance of arbuscular mycorrhizal fungi in distinct successionalstages from an Atlantic rainforest biome in southern Brazil. Mycorrhiza, 23:221–233. doi:10.1007/s00572-012-0464-9. PMID:23053578.

Zerega, N.J.C., Agone, D., and Otley, T.J. 2005. Systematics and species limits ofbreadfruit (Artocarpus, Moraceae). Syst. Bot. 30: 603–615. doi:10.1600/0363644054782134.

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