mutualism persistence and abandonment during the …izt.ciens.ucv.ve/ecologia/archivos/eco_pob...

vol . 1 8 8 , no . 5 the amer ican natural i st november 20 16

E-Article

Mutualism Persistence and Abandonment during

the Evolution of the Mycorrhizal Symbiosis

Hafiz Maherali,1,* Brad Oberle,2 Peter F. Stevens,3 William K. Cornwell,4 and Daniel J. McGlinn5

1. Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada; 2. Division of Natural Sciences, NewCollege of Florida, Sarasota, Florida 34243; 3. Missouri Botanical Garden, St. Louis, Missouri 63110; 4. Evolution and Ecology ResearchCentre, School of Biological Earth and Environmental Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia;5. Department of Biology, College of Charleston, Charleston, South Carolina 29424

Submitted April 21, 2016; Accepted July 13, 2016; Electronically published September 27, 2016

Online enhancements: supplemental figures. Dryad data: http://dx.doi.org/10.5061/dryad.n8bm9.

abstract: Mutualistic symbioses with mycorrhizal fungi are wide-spread in plants. The majority of plant species associate with arbus-cular mycorrhizal (AM) fungi. By contrast, the minority associate withectomycorrhizal (EM) fungi, have abandoned the symbiosis and are non-mycorrhizal (NM), or engage in an intermediate, weakly AM symbiosis(AMNM). To understand the processes that maintain the mycorrhi-zal symbiosis or cause its loss, we reconstructed its evolution using a∼3,000-species seed plant phylogeny integrated with mycorrhizal stateinformation. Reconstruction indicated that the common ancestor ofseed plants most likely associated with AM fungi and that the EM, NM,and AMNM states descended from the AM state. Direct transitionsfrom the AM state to the EM and NM states were infrequent and gen-erally irreversible, implying that natural selection or genetic constraintcould promote stasis once a particular state evolved. However, the evo-lution of the NM state was more frequent via an indirect pathwaythrough the AMNM state, suggesting that weakening of the AM sym-biosis is a necessary precursor to mutualism abandonment. Neverthe-less, reversions from the AMNM state back to the AM state were anorder of magnitude more likely than transitions to the NM state, sug-gesting that natural selection favors the AM symbiosis over mutualismabandonment.

Keywords: adaptation, arbuscular mycorrhizal fungi, ectomycorrhizalfungi, mutualism, symbiosis.

Introduction

Mutualisms, in which different species interact in close prox-imity and provide resources, services, and other benefits toeach other, are widespread in nature, occurring in everykingdom of life and in every biome (Bronstein 2015). Mu-tualisms can define organism structure and function, in-

* Corresponding author; e-mail: [email protected]: Oberle, http://orcid.org/0000-0002-4227-3352; Cornwell, http://

orcid.org/0000-0003-4080-4073.

Am. Nat. 2016. Vol. 188, pp. E000–E000. q 2016 by The University of Chicago.0003-0147/2016/18805-56949$15.00. All rights reserved.DOI: 10.1086/688675

This content downloaded from 136.1All use subject to University of Chicago Press Term

fluence the distribution and abundance of the partners,and control the cycling of nutrients and primary produc-tivity of ecosystems (Janzen 1985; Vitousek and Walker1989; Herre et al. 1999; Culley et al. 2002; Bronstein 2015).Though mutualism is common in nature, theory conflicts onwhether it should be stable once it evolves or be abandoned.For instance, natural selection is expected to favor exploita-tion over cooperation because individuals that derive bene-fits from a mutualistic partner can maximize their own fit-ness by avoiding the costs of cooperation (Bronstein 1994;Herre et al. 1999). Similarly, the benefit-to-cost ratio for eachmutualist might decline or flip to parasitism in certain eco-logical contexts, leading to natural selection for abandon-ment (Sachs and Simms 2006). Despite these possibilities,a mutualism can persist if there is selection to promote co-operation (Pellmyr and Muth 1994; Kiers et al. 2011; Fred-erickson 2013) or if mutations that reduce cooperation ariseinfrequently (Sachs and Simms 2006). The likelihood of mu-tualism persistence could also depend on the proximity ofthe interacting partners. For example, in symbiotic mutual-isms, where one organism inhabits the other, the declinein host performance caused by a less cooperative symbiontwould have immediate negative fitness consequences for thesymbiont and therefore limit the evolution of abandonmentin comparison to less intimate mutualisms (Herre et al. 1999;Sachs and Simms 2006; Douglas 2015).One way to examine whether mutualisms are stable over

evolutionary time or tend toward abandonment is to re-construct ancestral states using phylogenies and informa-tion about the character states of extant species (Sachs andSimms 2006; Sachs et al. 2011; Werner et al. 2014). Suchreconstructions have been carried out for only a handfulof mutualisms (Sachs et al. 2011), and there is no consen-sus on whether mutualisms are more likely to persist or beabandoned (Weiblin and Treiber 2015). For example, pol-lination mutualisms in angiosperms appear to be lost fre-

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E000 The American Naturalist

quently (Culley et al. 2002; Friedman 2011), whereas themutualistic symbiosis between plants and nitrogen-fixingbacteria appears to persist over evolutionary time (Werneret al. 2014).

One of the most taxonomically widespread and ecolog-ically important mutualisms in nature is the symbiosis be-tween plants and root-inhabiting mycorrhizal fungi (Brun-drett 1991; Smith and Read 2008), which occurs in up to∼90% of vascular plant species (Brundrett 2009). The fungiare obligate symbionts because they require sugars fromphotosynthesis for metabolism, growth, and reproduction(Smith and Read 2008). Though the symbiosis can be fac-ultative for plants, mycorrhizal fungi provide plants withimproved access to soil nutrients (Smith and Read 2008)and other services that enhance fitness, such as increased re-sistance to environmental stresses and pathogens (Newshamet al. 1995; Veresoglou andRillig 2012). Themycorrhizal sym-biosis is therefore generally mutualistic, but the appearanceof species that are not colonized by fungi in several angio-sperm lineages suggests that it has been abandoned severaltimes in the evolutionary history of plants (Peat and Fitter1993; Fitter and Moyersoen 1996; Wang and Qiu 2006). Aformal ancestral reconstruction of the mycorrhizal symbio-sis has not been done, however, and so little is known aboutthe tendency for the symbiosis to persist as a mutualism orto be abandoned.

The mycorrhizal symbiosis is dominated by plant inter-actions with arbuscular mycorrhizal (AM) fungi in the Glo-meromycota, which is present in nearly every seed planttaxonomic division (∼67% of species; Wang and Qiu 2006;Brundrett 2009). The AM symbiosis is also considered tobe the ancestral condition in terrestrial plants based on fos-sil evidence (Simon et al. 1993; Remy et al. 1994; Taylor et al.1995). An alternate form of the mycorrhizal symbiosis takesplace between ∼2% of seed plant species and ectomycorrhi-zal (EM) fungi in the Dikarya (Ascomycota and Basidiomy-cota; Wang and Qiu 2006; Brundrett 2009). A substantialnumber of plant species have lost the capacity to form thesymbiosis, where fungi are not observed in roots, and aretherefore categorized as nonmycorrhizal (NM; ∼10% of spe-cies; Brundrett 2009). There are also many plant species thatare considered to be weak AMmutualists, able to grow eitherwith or without the presence of AM fungi in nature, depend-ing on ecological context (AMNM; ∼12% of species; Fitterand Moyersoen 1996; Brundrett 2009; Hempel et al. 2013).Last, ∼10% of seed plants engage in plant family-specific in-teractions with mycorrhizal fungi, including those found inthe Orchidaceae and Ericaceae (orchid and ericoid mycor-rhizas; Brundrett 2009).

The evolutionary processes that maintain a certain typeof mutualistic mycorrhizal symbiosis or cause its loss arenot well understood (Peat and Fitter 1993; Fitter and Moy-ersoen 1996; Wang and Qiu 2006; Brundrett 2009). For


example, the evolution of a mutualistic symbiosis likely ben-efited ancient terrestrial plants, whose simple root structureswould not have been able to take up recalcitrant ions such asphosphate from mineral substrates (Pirozynski and Malloch1975; Brundrett 2002; Kenrick and Strullu-Derrien 2014).However, modern terrestrial plants have evolved extensiveand complex root systems, suggesting that the mechanismsresponsible for the origin of the symbiosis differ from themechanisms that maintain it (Fitter and Moyersoen 1996;Maherali 2014). Identifying potential evolutionary processesresponsible for variation in the mycorrhizal symbiosis canprovide direction for future experimental studies of the causesof plant adaptation to the mycorrhizal symbiosis at the pop-ulation level, where evolution occurs (Wade and Kalisz 1990;Ng and Smith 2014).Ancestral reconstruction of the mycorrhizal symbiosis can

assist in distinguishing among several non–mutually exclu-sive evolutionary hypotheses for the extant distribution ofmycorrhizal states in seed plants. First, NM and EM statesmay be favored by natural selection, but the ancestral AMsymbiosis may dominate because there has not been enoughtime for the alternate states to increase in frequency. If nat-ural selection does indeed favor the evolution of the NM andEM states, as plants evolved either specialized root struc-tures for nutrient uptake or Dikarya-mediated resource ac-quisition (Trappe 1987; Fitter and Moyersoen 1996), thentransition rates from the AM state to the alternate NM orEM state should be higher than reversions back to the AMstate. This prediction is supported in part by phylogenomicanalyses indicating that the evolution of the NM or EMstate involves losses of AM symbiosis genes (Delaux et al.2014; Garcia et al. 2015). Though this implies that the lossof the AM symbiosis is irreversible, it is not known whethertransitions from the NM or EM state back to the AM stateare generally absent. Second, the dominance of the AM sym-biosis could be explained by consistent natural selection toevolve the AM symbiosis from alternate states despite lossesto those states. If this was the case, then reversion rates fromthe NM and EM states back to the AM state should be higherthan transition rates out of the AM state. Third, the AMsymbiosis could be maintained by stabilizing or purifyingselection, a lack of suitable mutations to facilitate the evo-lution of alternate states, or other evolutionary constraintsthat promote stasis. In this case, transition rates among theAM, NM, and EM states should be low.If direct transition rates between the AM state and the

alternate NM and EM states are low, one possible pathwayto mutualism abandonment is through an intermediate statewhere the AM symbiosis has neutral or negative effects onplants, which could result in stronger selection against it(Sachs and Simms 2006). A neutral or negative net effect ofthe AM symbiosis on plants is commonly observed, and anumber of putatively mycorrhizal plants can grow and re-


Evolution of the Mycorrhizal Symbiosis E000

produce in the absence of the symbiosis (Johnson et al.1997; Klironomos 2003; Hoeksema et al. 2010; Johnsonand Graham 2013). If the AMNM state adequately describesthe phenotype of plants that gain little mutualistic benefitfrom the symbiosis (e.g., Hempel et al. 2013), then it couldbe an intermediate stage between a mutualism with AM fungiand the abandonment of the symbiosis (the NM state).

To examine the evolutionary dynamics of mutualism per-sistence and loss in the mycorrhizal symbiosis, we recon-structed its evolutionary history in seed plants. To do this,we used a ∼3,000-taxon fossil-calibrated molecular phylog-eny and mycorrhizal state database. We examined whethertransition rates (in events per million years [myr]) were bi-ased toward or against particular states or whether stasis oc-curred once a particular state evolved. In addition, we testedwhether the AMNM state is a necessary intermediate be-tween a mutualistic interaction with AM fungi and an alter-nate mutualism with EM fungi or the abandonment of themutualism (the NM state). Because evolutionary rates ofchange are known to differ within the seed plants (Beaulieuet al. 2013; Zanne et al. 2014), the analysis was done acrossall seed plants and within major the divisions (superrosids,superasterids, monocots, and gymnosperms). Observing sim-ilar patterns in the different seed plant divisions, despite dif-ferences in evolutionary rates and constituent species, strength-ens conclusions about the processes responsible for the lossor gain of the mycorrhizal symbiosis (Rabosky and Gold-berg 2015).

Material and Methods

Assembly of the Database

To construct a database for phylogenetic analysis of the my-corrhizal symbiosis, we used species-level reports of mycor-rhizal status from previously published lists and surveys(Siquiera 1998; Klironomos 2003; Tawaraya 2003; Wangand Qiu 2006; McGuire et al. 2008; Hoeksema et al. 2010;Akhmetzhanova et al. 2012; Hempel et al. 2013; Maherali2014; Peay et al. 2015). Overlapping records (i.e., thosefrom the same original source) between published databaseswere removed. Somepublished records includedmultiple ob-servations for a single species, and these observations wereretained. Species were binned into one of four categories:AM, EM, NM, and AMNM. Species were designated as AMor NM only when all records were unanimous in this as-sessment. In situations where a species was listed as bothAM and NM (i.e., plants in nature were observed to be col-onized by AM in some contexts but uncolonized in others),it was placed in the AMNM state. The number of speciesthat had both AM and EM records was low (∼0.1% of data),and this sample size was too small to be used in the analy-sis (Rabosky and Goldberg 2015). Therefore, when a species


was recorded as both AM and EM, it was placed in the EMcategory to distinguish species that were potentially capa-ble of forming EM symbiosis from those that could not. Asmall number of species listed as forming symbioses witharbutoid mycorrhiza were designated EM species based onfunctional similarity and prior convention (Smith and Read2008). Species forming symbiosis with ericoid or orchid my-corrhizas, listed as mycoheterotrophs, or associating withdark septate endophytic fungi were excluded. The databasecontained 12,549 published records comprising 5,284 uniqueseed plant species. Collated data are deposited in the DryadDigital Repository: http://dx.doi.org/10.5061/dryad.n8bm9(Maherali et al. 2016).A majority of species were assigned a state based on a

single observation, whereas a minority had multiple obser-vations (table 1; Maherali et al. 2016). The relative propor-tion of species assignments based on single observations wasconsistent among the different seed plant divisions in theanalysis. For example, ∼65%–70% of species state assign-ments in the superrosids, superasterids, and monocots werebased on single observations. For AM and NM states, ∼68%of species state assignments were based on single observa-tions, whereas for the EM state, 42% of assignments werebased on single observations. The AMNM state, by defini-tion, had at least two observations for each species, and 31%of species fell into this category. Despite the preponderanceof single-observation state assignments, designations for eachstate in the database at the genus and family in our databasewere generally consistent with the genus- and family-levellist for these states in Brundrett (2009), which was devel-oped independently from ours. Thus, state assignments re-flected the consensus view of the current literature.

Ancestral Reconstruction of the Mycorrhizal Symbiosis

To reconstruct the evolution of the symbiosis, the databasewas combined with a recently published datedmolecular phy-logeny (32,223 species; Zanne et al. 2013). This tree was con-structed from seven gene regions (18S rDNA, 26S rDNA,ITS, matK, rbcL, atpB, and trnL-F) obtained from searchesof vascular plant species accessions contained in GenBank(Zanne et al. 2014). To increase the number of matches be-tween the database and the tree, taxon names in our data-base were cleaned using the Taxonomic Name ResolutionService (Boyle et al. 2013). This process included updatingtaxon names as well as eliminating synonyms. The treematched 2,979 seed plant species in the database to yield atime-calibrated tree with mycorrhizal state information atthe tips.To examine character evolution along the tree, we used

a continuous-time Markov chain model (Pagel’s multistate;Pagel 1994; O’Meara 2012) as implemented in R (ver. 3.20;R Core Team 2015) using the package corHMM (Beaulieu



et al. 2013). To assign the probability that a particular nodein the phylogeny was in one of the four mycorrhizal statesand calculate transition rates (in events per myr), we usedthe marginal reconstruction method (Yang et al. 1995;Koshi and Goldstein 1996) given the phylogeny and tipstates. The first model fitted to the data was fully variable;it did not fix the ancestral state on the tree and allowed tran-sition rates between states to be asymmetric. To test varioushypotheses of character evolution, we restricted transitionrates between states and calculated the likelihoods of theresulting models. For example, to test whether the fully var-iable model was a better fit than a model with equal transi-tion rates between states, we calculated log likelihoods foreach model and compared them using a likelihood ratio x2

test (Pagel 1994). To test whether bidirectional transitionrates (q) between pairs of states (e.g., q1 and q2) differed fromequality, we ran iterations of the model where q1 p q2 foreach pairwise state combination and compared the log like-lihood to the fully variable model. If the constrained modelwas a significantly poorer fit, then we concluded that tran-sition rates between the two states were asymmetric. If therewas no change in model fit, then we concluded that the tran-sition rates between the two states were equal. Similarly, totest whether transition rates in both directions between stateswere required (i.e., different from zero), we ran iterations of


the model where q1 p q2 p 0 for each pairwise state com-bination and tested it against the fully variable model. If theconstrained model was a poorer fit, then we concluded thatthe transition rates between the two states were significantlydifferent from zero and thus necessary. If there was no changein model fit, then we concluded that the transition rates be-tween the two states were equal to zero. In this way, we couldevaluate whether there were direct transitions between theprimary AM and NM states or the EM state or whether theAMNM state was required to account for transitions betweenthe primary states. In addition, in some situations the valueof a transition rate was estimated as zero, even if the bidi-rectional rates were significantly different from zero and/or asymmetric. When this occurred, we assumed that a rateestimated to be zero did not differ significantly from zero.To determine whether transition rates among states dif-

fered among the major divisions of seed plants, the anal-ysis was run for the entire seed plant tree, as well as forthe superrosids, superasterids, monocots, and gymnospermsseparately. This was done to account for the possibility thatrates of evolution differ between these groups (e.g., Zanneet al. 2014). In addition, these taxonomic divisions differin their global distributions and functional characteristics(Cornwell et al. 2014) as well as contain different subsetsof various mycorrhizal states (Wang and Qiu 2006). Ob-

Table 1: Summary of the number of species with either multiple or single observations of mycorrhizal state in the data set, organizedby mycorrhizal state and taxonomic division

Taxonomicdivisionand state

No. AM, NM, and EM species with1 observation and no. AMNMspecies with 2 observations

No. AM, NM, and EM species with11 observation and no. AMNMspecies with 12 observations

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Total

04:21:33 urnals.uch

Proportion single(AM, NM, EM) or double(AMNM) observations

Seed plants:
AM 1,193 564 1,757 .68 EM 114 156 270 .42 NM 291 139 430 .68 AMNM 160 362 522 .31
Gymnosperms:
AM 44 29 73 .60 EM 42 68 110 .38
Monocots:
AM 250 127 377 .66 NM 96 52 148 .65 AMNM 35 108 143 .25
Superasterids:
AM 423 182 605 .70 NM 112 47 159 .70 AMNM 65 142 207 .31
Superrosids:
AM 412 195 607 .68 EM 71 82 153 .46 NM 77 35 112 .69 AMNM 53 101 154 .34
Note: See text for description of mycorrhizal states.

AMicago.edu/t-and-c).


serving similar patterns of evolutionary change in the dif-ferent seed plant divisions would strengthen inferences aboutthe mechanisms responsible for the loss or gain of each state(Rabosky and Goldberg 2015). Because reconstructions oftransition rates can be inaccurate when the frequency ofa particular state is extremely rare, we eliminated the EMstate from the analysis of superasterids, where it was foundin !10 species.

Estimating transition rates between states relies on theassumption that they reflect actual evolutionary transitionsfrom one state to another, rather than other processes. Forexample, the continuous-time Markov chain model may re-cover a high net directional transition from one state to an-other, but if the latter state also promotes diversification,then the transition rate may be overestimated (Maddison2006; Maddison et al. 2007; Ng and Smith 2014). Thoughwe initially used binary and multistate speciation and ex-tinction models (Maddison et al. 2007; FitzJohn 2012) to es-timate transition rates and diversification simultaneously,our findings indicated that such models were not appropri-ate for the phylogenetic scale of our sample (fig. S1; figs. S1–S3 available online). This is because when rate heterogene-ity is high, as is the case for an analysis that includes all ofthe seed plants (Rabosky 2016), state-dependent speciationand extinction (SSE)models can produce spuriously high ex-tinction rates that generally exceed speciation rates (Rabosky2010, 2016; Pyron and Burbrink 2013). When high extinc-tion rates cause net diversification rates to be negative for aparticular character state, the resulting model overestimatestransitions to that state (Rabosky and Goldberg 2015). Onesolution to the challenge posed by a high rate of heteroge-neity is to carry out analyses at smaller phylogenetic scales,where evolutionary rates could be less variable. Neverthe-less, rate heterogeneity was still high enough within super-rosids, superasterids, and monocots to produce spuriouslyhigh extinction rates, which meant that SSE models couldnot be used for these groups. Rate heterogeneity is lowerin the gymnosperms (e.g., Buschiazzo et al. 2012; Wang andRan 2014), and the SSE model did not produce spuriouslyhigh extinction rates in this group. However, there was nosignificant difference in net diversification between the AMand EM states in the gymnosperms, and transition rates wereindistinguishable from those produced by the continuous-time Markov chain model (figs. S2, S3), and so only transi-tion rates from the Markov chain model are presented inthe results.

Results

Of the 2,979 species sampled, 1,026 were superrosids, 971were superasterids, 668 were monocots, and 183 were gym-nosperms. The remaining 131 species were in the basal eu-dicots, the basal angiosperms, and Magnoliids. The AM state


occurred in 59% of species, whereas EM, NM, and AMNMspecies constituted 9.1%, 14.4%, and 17.5%, respectively. Mar-ginal reconstruction of the mycorrhizal symbiosis (fig. 1)showed that the AM state is found in every major divisionof seed plants and is the most likely ancestral condition (i.e.,was in the plurality or majority for node probabilities) inthe crown of each taxonomic division and the seed plantsas a whole. The NM and AMNM states were found in everydivision of the angiosperms, whereas the EM state occurredprimarily in the superrosids and the gymnosperms. Of thenon-AM states, the EM state appeared first, more than 200myrago in the gymnosperms and more recently in the super-rosids (∼100 myr; Fagales). The NM state appears to haveevolved more recently, !100 myr ago for some groups (Al-ismatales, Caryophyllales, Crassulaceae) and ∼50 myr forothers (Brassicaceae, Cyperaceae, Juncaceae, Lamiales). TheAMNM state was widely dispersed in the angiospermsand appeared to have evolved within the same time frameas the NM state.For seed plants, transition rates between states were gen-

erally asymmetric. A model that allowed all rates to varywas a significantly better fit to the data, by 859 log-likelihoodunits, than a model that constrained all transition rates toequality (likelihood ratio x2 test statistics; table 2). The onlypairwise transition rates that differed significantly from equal-ity were those between the primary AM or NM state andthe intermediate AMNM state. Transition rates from AMto AMNM were 73% lower than reversions, whereas transi-tion rates from NM to AMNM were 2.8 times higher thanreversions. In addition, transition rates between the AM andAMNMstates were at least an order of magnitude higher thanany other transition rates (fig. 2a). Bidirectional transitionrates between the AM-AMNM and NM-AMNM states werealso significantly different from zero; models that set thesetransitions to zero were significantly worse fits to the datathan the full model (table 2). Though transition rates be-tween the AM andNM states were also significantly differentfrom zero, only the transition rate from AM to NM was pos-itive, whereas the reversion rate was zero. Transitions be-tween the EM and NM states were significantly differentfrom zero but were lower than transition rates between otherpairs of states.In the superrosids, a model with fully asymmetric tran-

sition rates also had a much higher (i.e., less negative) log-likelihood ratio than a model with equal transition rates(table 2). As with seed plants as a whole, the transition ratebetween the AM and AMNM states was higher than be-tween any other pairs of states (fig. 2b), and transitionrates between the AM or NM state and the AMNM inter-mediate state were significantly asymmetric. The transi-tion rate from the AM state to the AMNM state was 78%lower than the reverse, whereas the transition rate from theNM state to the AMNM state was three times higher than



the reverse. Transition rates between AM and NM stateswere equal, however, and among the lowest observed. Tran-sition rates between AM and EM states were asymmetric,with the AM to EM rate 79% lower than the reverse. Allpairwise transition rates between states were significantlydifferent from zero (table 2), though we note that the tran-sition rate from AMNM to EM and from NM to EM wasestimated as zero.

For both the superasterids and the monocots, modelsthat constrained transition rates to equality were also sig-nificantly poorer fits to the data than models that allowedtransition rates to be asymmetric (likelihood ratio x2 teststatistics; table 3). In addition, direct transition rates be-tween the primary AM and NM states were not different


from zero. However, in a majority of cases, transition ratesbetween the primary AM or NM state and the intermedi-ate AMNM state were both asymmetric and significantlydifferent from zero. For the superasterids, the transitionrate from AM to AMNM was 68% lower than the reverse.The transition rate fromNM to AMNMwas higher than thereverse, which was estimated as zero (fig. 2c). For monocots,the transition rate from AM to AMNM was 64% lower thanthe reversion rate, whereas transitions between NM andAMNM were not significantly different from equality (table 3;fig. 2d).In the gymnosperms, where only AM and EM states were

observed, the transition rate from AM to EM was positive,whereas the reversion rate was zero (fig. 2e). However, a

Figure 1: A time-calibrated phylogeny for 2,979 seed plant species with tips painted in one of four color-coded mycorrhizal states:arbuscular mycorrhizal (AM; blue), ectomycorrhizal (EM; green), nonmycorrhizal (NM; red), and weakly AM (AMNM; purple). The my-corrhizal state of interior nodes was assigned to the state that was most likely (i.e., when a state was in plurality or majority) based on mar-ginal reconstruction. The names of major divisions are printed in black, and the names of majority EM and NM clades are printed in theirrespective colors. To illustrate the timescale of the phylogeny, a 50-million-year-long reference bar is included.


4


model that held both rates equal was not significantly dif-ferent from the full model (table 3). A model that held thetransition rates between states to zero did not converge ona solution, and we therefore tested the hypothesis that tran-sition rates differed from zero by constraining each rate insequence. Forcing the transition rate from AM to EM to bezero worsened model fit, indicating that this transition wasdifferent from zero (table 3). By contrast, a model that forcedthe transition rate from EM to AM to be zero had no effecton model fit, which was consistent with the estimated value(0) for that transition rate in the full model (fig. 2e).

Discussion

We present the first formal ancestral reconstruction of themycorrhizal symbiosis in seed plants and its major taxo-nomic divisions and show that the symbiosis is more likelyto persist than be abandoned. This pattern was found atthe level of the seed plants and also replicated in each tax-onomic division where both mycorrhizal and nonmycor-rhizal states were present (fig. 2a–2d), raising confidencein the inferences drawn from ancestral reconstruction (Ra-bosky and Goldberg 2015). Our findings support the pre-diction that the AM symbiosis persists because direct tran-sitions between the primary AM, EM, and NM states are


rare, as would be expected if there was stabilizing selectionto maintain a particular state once it evolved, if mutationsthat allow transitions between states occur at a low rate, orif other evolutionary constraints promote stasis. The re-sults also support the hypothesis that loss of symbiosis genescould prevent NM lineages from directly regaining the AMsymbiosis (Delaux et al. 2014; Garcia et al. 2015). Loss ofsymbiosis genes has been documented in the superrosids(Brassicales), superasterids (Lamiales, Caryophyllales), andgymnosperms (Pinaceae), but the observation that rever-sion rates from the NM state to the AM state were also esti-mated to be zero in the monocots implies that the evolutionof the NM state in this division could involve irreversiblesymbiosis gene loss. In addition, we show that the mostlikely pathway for losing the ancestral AM symbiosis isthrough the AMNM state (fig. 2), suggesting that a weaken-ing of the mutualism is a necessary step toward abandon-ment. However, the AM symbiosis was much more likelyto be regained from the AMNM state rather than proceedingto mutualism abandonment, implying that natural selectionis more likely to favor the evolution of mutualism rather thanmutualism abandonment. Though the mycorrhizal state datain the analysis reflect the consensus in the literature, our workhighlights the fact that only a fraction of seed plant specieshave been sampled for mycorrhizal state with replication.

Table 2: Summary of log-likelihood scores, likelihood ratio x 2 test statistics, and the associated probability values for different continuous-time Markov chain models of character evolution for the seed plants and the superrosids

Seed plants (N p 2,979)

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Superrosids (N p 1,026)

Model type
Loglik x 2 P Loglik
2016 04:21:33 AMw.journals.uchic

x2

ago.edu/t-and-c

P

All transitions asymmetric
22,410.4 2760.9 All transitions equal 23,269.4 1,720.25 !2.2#10299 2970.9 20 2.46#10293
AM-NM transitions equal
22,410.2 .422 .52 2761.4 1.07 .3 AM-EM transitions equal 22,409.7 1.50 .22 2763.6 5.38 .02 AM-AMNM transitions equal 22,543.5 266.2 7.64#10260 2795.8 69.9 6.11#10217
EM-NM transitions equal
22,409.6 1.65 .20 2761.5 1.34 .25 EM-AMNM transitions equal 22,409.9 1.13 .28 2761.6 1.36 .24 NM-AMNM transitions equal 22,419.2 17.5 2.8#1025 2767.2 12.7 3.67#1024
AM-NM transitions p 0
22,414.2 7.63 .0058 2763.4 4.97 .026 AM-EM transitions p 0 22,411.3 1.69 .9 2768.8 15.9 6.7#1025
AM-AMNM transitions p 0
22,597.3 354.1 5.46#10279 2806.6 91.5 1.11#10221
EM-NM transitions p 0
22,413.6 6.34 .011 2764.6 7.37 .0066 EM-AMNM transitions p 0 22,411.7 2.58 .11 2764.9 8.04 .0046 NM-AMNM transitions p 0 22,432.2 43.5 4.2#10211 2786.5 51.2 8.41#10213
Note: To test hypotheses about character evolution, comparisons were made between a model that allowed transition rates between all states to vary (fullyasymmetric rates) and several constrained models that forced all transitions to be equal, iteratively held pairwise transitions between states as equal, or droppedthem from the model (i.e., made them equal to zero). In cases where there was no change between the log-likelihood fit of the fully asymmetric model and thatof a particular constrained model, it can be concluded that the transition rates between the specified states do not differ from equality (if the constraint wasequality) or do not differ from zero (if the constraint was zero). In cases where a constrained model was a significantly worse fit to the data, it can be concludedthat the transition rates between the specified states differ from equality (if the constraint was equality) or zero (if the constraint was zero). Statistically sig-nificant differences between the full model and each constrained model are indicated in bold. See text for description of model types.

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Consequently, our results and conclusions could be altered iffuture sampling efforts change the consensus onmycorrhizalstate assignment.

An important inference from the ancestral reconstructionis that the AMNM state appears to be a necessary interme-diate along the pathway to the evolution of the NM state.This inference is based on the observation that (i) transitionrates between the primary AM and NM states and the in-termediate AMNM state were often an order of magnitudemore frequent than between any other pair of states (fig. 2)and (ii) setting these transitions to zero resulted in poorermodel fits to the data (tables 2, 3). If the AMNM state rep-resents plants that can be negatively affected by AM coloni-zation in certain ecological contexts (Johnson et al. 1997;Klironomos 2003; Hempel et al. 2013; Johnson and Graham2013), then our result suggests that a reduction in thebenefit-to-cost ratio of the mutualism is a requirement formutualism abandonment. This is because reduced benefitsand higher costs of the symbiosis should result in strongernatural selection to limit root colonization by fungi (Koideand Schreiner 1992). Though no studies have examinednatural selection on the mycorrhizal symbiosis (Baskett andSchemske2015), reciprocal transplant and commongarden ex-periments show that plant adaptation to novel soil habitatscan involve the ability to reduce both fungal colonizationof roots (Sherrard and Maherali 2012) and mycorrhizal de-


pendence (Seifert et al. 2009), implying that selection forabandonment can occur in contemporary populations. Nev-ertheless, transition rates from the AMNM state to the NMstate were low overall and absent in the superasterids, andreversions to the AM state from the AMNM state were morelikely than transitions to abandonment at all taxonomic lev-els. Therefore, the evolution of mutualism abandonment, evenvia the AMNM state, was likely infrequent.Given that recovery of the mutualism from the NM state

should be rare because necessary genes may have been lost(Delaux et al. 2014), an interesting result was that the tran-sition rate from NM to the AMNM intermediate state washigher than the reverse. This finding implies that loss of themycorrhizal symbiosis can be recovered through the AMNMintermediate. However, it is also possible that this result re-flects challenges in categorizing the AMNM phenotype. Be-cause they are obligate symbionts, AM fungi are able to col-onize characteristically NM species, even though a functionalsymbiosis may not be formed (Veiga et al. 2013; Lekberget al. 2015). If the AMNM state included such species, it ispossible that the higher NM to AMNM transition rate wascaused by the presence of NM species that were categorizedas AMNM because of aggressive colonization by Glomero-mycota, rather than functional reversions from the NMstate to the AMNM state. More detailed studies that verifythe functionality of the AM symbiosis in lineages where the

Table 3: Summary of log-likelihood scores, likelihood ratio x 2 test statistics, and the associated probability values for different continuous-time Markov chain models of character evolution for the superasterids, monocots, and gymnosperms

Superasterids (N p 971)

59.235.223 on October 0s and Conditions (http://

Monocots (N p 668)

Model type
Loglik x 2 P Loglik
1, 2016 04:21:33www.journals.uc

x2

AMhicago.edu/t-and

P

2814.4 2593.2 All transitions equal 2949.8 270.8 7.45#10261 2660.7 135 !3.37#10231
AM-NM transitions equal
2815.5 2.3 .13 2594 1.63 .20 AM-AMNM transitions equal 2866 103.2 3.06#10224 2619.4 52.3 4.66#10213
NM-AMNM transitions equal
2816.4 4.08 .04 2594.1 1.77 .18 AM-NM transitions p 0 2815.7 2.68 .10 2594.6 2.75 .10 AM-AMNM transitions p 0 2900.8 172.9 1.72#10239 2659.1 131.7 1.75#10230
NM-AMNM transitions p 0
2823.1 17.5 2.8#1025 2595.9 5.25 .022
Gymnosperms (N p 183)

Loglik
x 2 P
210.83 AM-EM transitions equal 212.41 3.16 .076 AM to EM transition p 0 220.46 19.25 1.14#1025
EM to AM transition p 0
210.83 0 1
Note: To test hypotheses about character evolution, comparisons were made between a model that allowed transition rates between all states to vary (fullyasymmetric rates) and several constrained models that forced all transitions to be equal, iteratively held pairwise transitions between states as equal, or droppedthem from the model (i.e., made them equal to zero). In cases where the there was no change between the log-likelihood fit of the fully asymmetric model andthat of a particular constrained model, it can be concluded that the transition rates between the specified states do not differ from equality (if the constraint wasequality) or do not differ from zero (if the constraint was zero). In cases where a constrained model was a significantly worse fit to the data, it can be concludedthat the transition rates between the specified states differ from equality (if the constraint was equality) or zero (if the constraint was zero). Statistically sig-nificant differences between the full model and each constrained model are indicated in bold. See text for description of model types.

-c).


NM state is ancestral but that also contain species that arecategorized as AM or AMNM (e.g., Asteraceae, Brassicaceae,Caryophyllaceae, Cyperaceae, Lamiaceae, Poaceae; fig. 1) arerequired to determine whether and how frequently the AMsymbiosis can evolve from a NM ancestor via the AMNMstate.

Our results indicate that the only pathway to the EMstate is directly from the AM symbiosis. This result is notsurprising for gymnosperms, where only the AM and EMstates occur (table 3) and fossil evidence indicates that theAM state is ancestral (Remy et al. 1994). In the superrosids,where other states are present, we also found that the onlynonzero transition rate to the EM state was from the AMstate. Our findings also suggest that the EM state in the gym-nosperms is more stable (i.e., there were no transitions backto the AM state) than in the superrosids, where the rever-sion rate from EM to AM was higher than the transitionrate from AM to EM. One explanation for reversions to theAM symbiosis in superrosids is that unlike in gymnosperms,a small fraction of superrosid species are capable of form-ing functional symbiosis with both AM and EM fungi (e.g.,Gehring et al. 2006). The presence of species that can simul-taneously engage in both the EM and AM symbiosis in thesuperrosids suggests that contrary to gymnosperms (Garciaet al. 2015), loss of AM symbiosis genes does not necessarilyoccur when the EM symbiosis evolves.

The low transition rate to the NM state, either directlyfrom the AM state or indirectly through the AMNM state,does not explain the relatively high frequency of nonmycor-rhizal seed plants (∼10% overall and 14.4% in the analyzeddata set; Brundrett 2009). When it evolves, abandonmentof the mutualism appears to give rise to entire lineages thatalso lack the ability to form the symbiosis (fig. 1). Becauselosses of symbiosis genes could preclude a reversion to theAM state (Delaux et al. 2014), the most likely explanationfor the prevalence of the NM state is that it is associatedwith elevated speciation rate. Though we were unable to in-corporate state-dependent diversification into the presentanalysis, previous studies of speciation rates in seed plantsare consistent with this explanation. For example, character-istically NM lineages such as the Caryophyllales, Lamiales,Cyperaceae, and Brassicales have among the highest speci-ation rates in the angiosperms (Magallón and Sanderson2001; Magallón and Castillo 2009). Nevertheless, this pat-tern does not imply that the NM state causes increased spe-ciation. It may instead be caused by other characteristics ofthe lineages in which mutualism abandonment has occurred(Maddison and FitzJohn 2015). Specifically, NM species areprimarily herbaceous, with some notable exceptions (e.g.,Proteaceae), whereas the other mycorrhizal states containhigher proportions of woody species (Fitter and Moyersoen1996; Hempel et al. 2013). The herbaceous habit, because offaster generation times, can confer faster rate of molecular


evolution and increased speciation (Eriksson and Bremer1992; Verdu 2002; Smith and Donoghue 2008). Thus, NMlineages may have a higher speciation rate not because theyhave abandoned the mutualism with mycorrhizal fungi butbecause they are largely short lived and herbaceous. Moregenerally, these findings suggest that the ancestral recon-structions described here may not be strongly sensitive toexcluding the effects of diversification. If the potentiallyhigher rates of diversification in NM lineages had been in-cluded in the model, this would have further reduced thetransition rate to the NM state (Maddison 2006; Maddisonet al. 2007; Ng and Smith 2014), which was already esti-mated to be low (fig. 2).One critical source of uncertainty in probabilistic recon-

structions of ancestral character states is error in the stateassignment of extant species (Cunningham et al. 1998).Though such errors are not unique to this study, assigningmycorrhizal state to any given species from literature datacan be challenging because misidentification of fungi canlead to a false positive or a false negative for the symbiosis(Dickie et al. 2007; Brundrett 2009). In the data set, sucherrors were most likely for species in the AM and NM statesbecause 68% of assignments in these groups were based ona single literature observation. By contrast, species listed asAMNM were less likely to be assigned in error because atleast two observations were required to identify them andbecause 69% of AMNM species assignments were basedon more than two literature observations (table 1). Thoughstate assignments were consistent with the literature con-sensus (Brundrett 2009), it cannot be determined how manysingle observations are incorrect. Therefore, some transitionrates between the AM and AMNM states and between theNM and AMNM states could be biased by errors in state as-signment. For example, incorrectly categorizing an AMNMspecies as AM could inflate the reversion rate from theAMNM state to the AM state. The transition rates reportedhere should therefore be viewed as contingent upon furthersampling.Given that a majority of state assignments in the literature

are based on single observations, our analysis highlights theneed for more thorough evaluations of mycorrhizal state as-signments if progress is to be made in testing hypothesesabout the evolutionary dynamics of the symbiosis. Our find-ings can guide some of these future efforts at finer phyloge-netic scales. For example, though transition rates betweenthe AM and AMNM states favored the former in all threeangiosperm divisions, they were often an order of magnitudehigher in the monocots and superasterids than in the super-rosids. Because the relative proportions of single-species ob-servations for the AM and NM states were high among allthree divisions (0.65–0.70; table 1), differences in transitionrates between these states and the AMNM state among an-giosperm divisions could reflect errors in state assignment



rather than differences in the evolutionary lability of thesymbiosis. To distinguish between sampling error and evo-lutionary lability as causes of this pattern, future work couldfocus on increasing the accuracy of mycorrhizal state assign-ment in lineages that appear to contain substantial pro-portions of both AM and NM as well as AMNM speciesin the monocots (Alismatales), superasterids (Caryophyllales,Lamiales), and superrosids (Crassulaceae, Saxifragaceae). Be-cause rate heterogeneity is expected to be more modest atfiner phylogenetic scales, focusing sampling efforts on suchgroups would also enable hypothesis tests about differencesin state-dependent diversification (Rabosky 2016).

An additional source of uncertainty is that the proportionof species in each state may not be representative of theactual distribution of mycorrhizal states in the seed plants.Using Brundrett’s (2009) estimates as a reference point, welikely underestimated the proportion of seed plant speciesin the AM state (59%; fig. 2a), whereas the proportions ofspecies in the EM, NM, and AMNM states (9.1%, 14.4%,17.5%, respectively) were likely overestimated (see fig. 3in Brundrett 2009; AM p 67.4%; EM p 1.9%; NM p9.7%; AMNM p 11.7%). This bias could have caused anoverestimate of the transitions from the AM state to the EM,NM, and AMNM states and underestimated reversions fromthese states to the AM state. Given that direct transitionsamong the three primary AM, EM, and NM states were es-timated to be zero or very close to zero and that transitionsbetween the AM and AMNM states were strongly biased tothe former state, the effect of overestimating EM, NM, andAMNM abundance on our conclusions may be modest.

In conclusion, this study presents the first formal ances-tral reconstruction of the mycorrhizal symbiosis in seedplants. Though only a small number of broad-scale ancestralreconstructions of mutualism have been carried out (Sachsand Simms 2006; Sachs et al. 2011; Werner et al. 2014), theobservation that mutualism is more likely to persist than beabandoned in the mycorrhizal symbiosis is consistent withpatterns detected for other types of symbioses (Sachs et al.2011; Werner et al. 2014). Our findings suggest that persis-tence of themycorrhizal symbiosis could occur via two path-ways. First, direct transition rates out of the AM symbiosiswere low, implying that stabilizing selection, a low mutationrate, or another constraint promotes stasis. Second, the highrate of reversion from the intermediate AMNM state to theAM state suggests that natural selection favors the evolutionof a stronger mutualism rather than abandonment. Strongselection for mutualism could occur because plants obtainmultiple services from the symbiosis (Newsham et al. 1995),and so a decline in benefit-to-cost ratio for any one servicewould not necessarily eliminate the fitness benefits that plantsobtain from cooperation (Sachs and Simms 2006; Veresoglouand Rillig 2012). Similarly, because reduced plant perfor-mance has negative consequences for root-inhabiting fungi,


exploitation of plants by mycorrhizal fungi might be infre-quent, which would limit the evolution of mutualism aban-donment (Douglas 2015). To test these predictions, studiesthat quantify the magnitude and direction of natural selec-tion on the mycorrhizal symbiosis at the population level arenecessary. Though such studies are rare (Hoeksema 2010;Baskett and Schemske 2015), genetic variation for plant re-sponse to mycorrhizal colonization exists in natural popula-tions (Garrido et al. 2010; Ramos-Zapata et al. 2010; Ronsheim2016), suggesting that evolutionary responses to natural se-lection on the mycorrhizal symbiosis are likely.

Acknowledgments

This work was supported by a Discovery grant from theNatural Sciences and Engineering Research Council, theUniversity of Guelph, and the National Evolutionary Syn-thesis Center (NESCent; National Science Foundation EF-0423641) through support for the Tempo and Mode ofPlant Trait Evolution Working Group and the sabbaticaland short-term visitor program. We thank J. M. Beaulieu,P. S. Manos, B. C. O’Meara, K. G. Peay, G. D. Werner, andA. E. Zanne for helpful advice and discussions and C. M.Caruso and two anonymous reviewers for comments thatimproved the manuscript. J. M. Beaulieu also assisted withthe construction of figure 1. We also thank the scholarlycommunity and administrative staff at NESCent for pro-viding a superb environment for synthesis research.

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