host plant physiology and mycorrhizal functioningplant responses to mycorrhizal fungi,...

Host Plant Physiology and Mycorrhizal FunctioningShift across a Glacial through Future [CO2] Gradient1[OPEN]

Katie M. Becklin*, George W. R. Mullinix, and Joy K. Ward

Ecology and Evolutionary Biology Department, University of Kansas, Lawrence, Kansas 66045

ORCID ID: 0000-0003-3173-3357 (K.M.B.).

Rising atmospheric carbon dioxide concentration ([CO2]) may modulate the functioning of mycorrhizal associations by alteringthe relative degree of nutrient and carbohydrate limitations in plants. To test this, we grew Taraxacum ceratophorum andTaraxacum officinale (native and exotic dandelions) with and without mycorrhizal fungi across a broad [CO2] gradient (180–1,000 mL L21). Differential plant growth rates and vegetative plasticity were hypothesized to drive species-specific responses to[CO2] and arbuscular mycorrhizal fungi. To evaluate [CO2] effects on mycorrhizal functioning, we calculated response ratiosbased on the relative biomass of mycorrhizal (MBio) and nonmycorrhizal (NMBio) plants (RBio = [MBio 2 NMBio]/NMBio). We thenassessed linkages between RBio and host physiology, fungal growth, and biomass allocation using structural equation modeling.For T. officinale, RBio increased with rising [CO2], shifting from negative to positive values at 700 mL L21. [CO2] and mycorrhizaleffects on photosynthesis and leaf growth rates drove shifts in RBio in this species. For T. ceratophorum, RBio increased from 180 to390 mL L21 and further increases in [CO2] caused RBio to shift from positive to negative values. [CO2] and fungal effects onplant growth and carbon sink strength were correlated with shifts in RBio in this species. Overall, we show that rising [CO2]significantly altered the functioning of mycorrhizal associations. These symbioses became more beneficial with rising [CO2],but nonlinear effects may limit plant responses to mycorrhizal fungi under future [CO2]. The magnitude and mechanismsdriving mycorrhizal-CO2 responses reflected species-specific differences in growth rate and vegetative plasticity, indicatingthat these traits may provide a framework for predicting mycorrhizal responses to global change.

Atmospheric carbon dioxide concentration ([CO2])has more than doubled over the past 20,000 years,rising from a minimum value of approximately 180 mLL21 during the Last Glacial Maximum (LGM; Augustinet al., 2004) to a current value of 401 mL L21. Due toongoing fossil fuel emissions, [CO2] is expected to reach700 to 1,000 mL L21 by the end of this century (IPCC,2013). Rising [CO2] has greatly impacted plant physiol-ogy since the LGM (Sage and Coleman, 2001; Ainsworthand Rogers, 2007; Gerhart and Ward, 2010), likely al-tering interactions between plants and their microbialsymbionts over geologic and contemporary time scales.

Mycorrhizal associations are ancient plant-fungalsymbioses (Remy et al., 1994) where host plants andtheir fungal partners exchange photosynthetically

derived carbohydrates for soil nutrients (Smith andRead, 2008). These associations play a critical role inmodern ecosystems via their effects on plant physiol-ogy, species coexistence, carbon and nutrient cycling,and net primary productivity (Hodge and Fitter, 2010;Clemmensen et al., 2015; Lin et al., 2015). Temporalchanges in [CO2] since the LGM have likely influencedthe functioning of mycorrhizal associations along acontinuum from mutualism to parasitism, hereafterreferred to as the M-P continuum (Johnson et al., 1997),by altering plant carbohydrate production and nutrientdemand. Previous mycorrhizal-CO2 studies have fo-cused mainly on the effects of modern versus futureconditions (Alberton et al., 2005; Mohan et al., 2014),and little is known about mycorrhizal responses to low[CO2] of the past (Treseder et al., 2003; Procter et al.,2014). Assessing mycorrhizal responses to a broad,temporal [CO2] gradient is critical to establish a baselinefor how these symbioses functioned prior to anthro-pogenic forcing, which will provide insight into po-tential constraints on mycorrhizal responses to futureconditions (Ogle et al., 2015). In addition, rising [CO2]is known to cause nonlinear shifts in plant physiologyand growth (Gerhart and Ward, 2010); therefore, non-linear shifts in mycorrhizal functioning also are likelyto occur. Characterizing such responses is critical toaccurately predict how these symbioses will impactplant physiology and growth in the future and requiresexperimentation that manipulates mycorrhizal associ-ations at both low and elevated [CO2]. Furthermore, tofully understand the physiological mechanisms driving

1 This work was supported by the National Institutes of Health(Institutional Research and Academic Career Development Awardfellowship to K.M.B.), the National Science Foundation (grant no.IOS 1457236 to J.K.W.), and a Research Investment Council grantfrom the University of Kansas.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Katie M. Becklin ([email protected]).

K.M.B. and J.K.W. conceived the ideas and designed the study; allauthors participated in conducting the research and writing the arti-cle.

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plant responses to mycorrhizal fungi, linkages betweenhost plant physiology and mycorrhizal functioningacross the full M-P continuum need to be assessed. Abroad [CO2] gradient will likely generate both mutu-alistic and parasitic symbioses and provide insightinto physiological traits that vary with mycorrhizalfunctioning.

In this study, we examined arbuscular mycorrhizal(AM) associations in two closely related C3 plant spe-cies, Taraxacum ceratophorum and Taraxacum officinale(native and exotic common dandelions; Asteraceae),across a glacial through future [CO2] gradient. AMfungi are the predominant, ancestral type of mycor-rhizal fungi (Smith and Read, 2008), and approximately80% of modern plant species form AM associations(Brundrett, 2009). These fungi are primarily associatedwith increased phosphorus (P) uptake (Johnson, 2010),and past studies show that as much as 90% of plant P isacquired via fungal symbionts (Pearson and Jakobsen,1993; Smith et al., 2009). AM fungi also can increasenitrogen (N) uptake, especially NH4

+, although thefungal contribution to plant N uptake is challengingto measure and highly variable among studies (e.g.0%–74% of total plant N; Hodge and Storer, 2015). Inexchange for these nutrients, plants allocate an esti-mated 5% to 10% of their carbohydrates to AM fungi(Bryla and Eissenstat, 2005).

The net effect of AM fungi on plants is often mea-sured in terms of plant growth, with some associationspromoting faster growth rates and larger, more com-petitive plants (mutualism), while other associationsrestrict plant growth, resulting in smaller, less com-petitive plants (parasitism; Johnson et al., 1997). Wherean interaction falls along the M-P continuum is highlydependent on physiological tradeoffs and resourcelimitations in the host. In general, nutrient availability isthought to be the primary driver of mycorrhizal func-tioning, with AM fungi increasing plant growth whennutrients, especially P, are more limiting than carbo-hydrates (Johnson et al., 2015). Changes in [CO2] willlikely modulate nutrient effects on AM associations byaltering the relative degree of nutrient and carbohy-drate limitations in plants (Fig. 1; Johnson, 2010). Morespecifically, there is strong evidence that low [CO2]during the LGM produced major carbon (C) limitationswithin C3 plants, and modern plants grown at glacial[CO2] generally exhibit greater than 50% reductions ingrowth and photosynthetic rates relative to modern[CO2] (Polley et al., 1993; Tissue et al., 1995; Sage andColeman, 2001; Beerling, 2005; Gerhart andWard, 2010;Gerhart et al., 2012). Furthermore, the majority of C3plants show some level of increased photosyntheticrates, leaf carbohydrate levels, and biomass whengrown at future [CO2] (Ainsworth and Rogers, 2007;Prior et al., 2011). However, plants may require morenutrients in order to maintain high rates of photosyn-thesis and growth at elevated [CO2] (Campbell andSage, 2006; Lewis et al., 2010). Thus, rising [CO2] maycause mycorrhizal associations to shift along the M-Pcontinuum by reducing plant carbohydrate limitation

while simultaneously increasing plant nutrient limita-tion.

Most mycorrhizal-CO2 studies compared the effects ofcurrent (340–400 mL L21) and future (540–750 mL L21)[CO2] (Alberton et al., 2005; Mohan et al., 2014), but veryfew studies included more than two [CO2] treatments,preventing the assessment of potential nonlinear patterns.These studies indicate that AM fungi generally increaseplant growth at elevated [CO2] and that rising [CO2]promotes stronger mutualism (Mohan et al., 2014).However, the functioning of mycorrhizal associations atlow [CO2] of the past remains unclear. One possibility isthat AM fungi reduce plant growth at low [CO2] becausethese symbionts are a major sink for carbohydrates thatare expensive to produce when CO2 is limiting (Gerhartand Ward, 2010; Gerhart et al., 2012). Studies thatshow reduced AM fungal abundance in soils exposedto preindustrial [CO2] provide tentative support for thishypothesis (Treseder et al., 2003; Procter et al., 2014).Alternatively, some plants may partially compensatefor low [CO2] by increasing their investment in thephotosyntheticmachinery, including the enzymeRubisco(Sage and Coleman, 2001; Becklin et al., 2014). Thisstrategy enhances CO2 uptake but comes at the cost ofgreater demand for N (Sage and Coleman, 2001). P limi-tation also has been shown to restrict the rate of ribulosebisphosphate regeneration, with subsequent effects onphotosynthesis under preindustrial [CO2] (Campbell andSage, 2006). Thus, by alleviating nutrient limitations onplant physiology, AM fungi may promote plant growtheven under low [CO2].

Characterizing CO2 effects on AM associations iscritical for understanding the unique properties of these

Figure 1. [CO2] is predicted to mediate nutrient effects on mycorrhizalassociations by altering relative resource limitation in host plants.Specifically, the mycorrhizal response ratio (RBio) is predicted to increasewith rising [CO2] due to simultaneous increases in plant carbohydrateproduction and plant nutrient limitation. RBio is calculated from the bio-mass of mycorrhizal (MBio) and nonmycorrhizal (NMBio) plants (RBio =[MBio 2 NMBio]/NMBio). The dashed line represents a neutral association(no difference in plant size).RBio may benegative (solid line) ormarginallypositive (dotted line) at low [CO2], depending on mycorrhizal effects onplant physiological responses to a CO2-limiting environment.

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symbioses as well as for predicting how these interac-tions respond to both past and future environments.Here, we examined mycorrhizal responses across aglacial through future [CO2] gradient in a controlled-environment experiment. We predicted that mycorrhi-zal associations would become more beneficial (i.e.have a larger positive effect on plant growth) withrising [CO2] (Fig. 1). We further hypothesized twopossible outcomes for mycorrhizal functioning underlow [CO2] of the past: (1) AM fungi will exacerbatecarbohydrate constraints and restrict plant growth un-der low [CO2], resulting in parasitic mycorrhizal asso-ciations when CO2 is limiting (Fig. 1, solid line); and (2)fungal effects on nutrient uptake will alleviate nutrientconstraints on plant physiological responses to low[CO2], resulting in mutualistic mycorrhizal associationsunder glacial conditions (Fig. 1, dotted line). Addi-tionally, studies indicate that many C3 plants respondnonlinearly to rising [CO2], with stronger responses tochanges in [CO2] below the modern value comparedwith above (Gerhart and Ward, 2010). These nonlinearshifts in plant physiology and growth will likely alterplant resource limitations and mycorrhizal functioningacross a broad [CO2] gradient. Thus, we further pre-dicted that plants will be most responsive to increasesfrom glacial to modern [CO2], resulting in nonlinearshifts in plant physiology, plant growth, and mycorrhizalfunctioning across the full [CO2] gradient. To better un-derstand the mechanisms that alter plant responses tomycorrhizal fungi and overall patterns of plant produc-tivity with rising [CO2], we conducted detailed studies ofplant physiology, intraradical fungal growth, and plantbiomass allocation in mycorrhizal plants. We then testedfor causal relationships among these traits and overallplant responses to mycorrhizal fungi using structuralequation modeling (Fig. 2).

RESULTS

Our study focuses on plant physiological mecha-nisms driving shifts in mycorrhizal functioning across abroad [CO2] gradient representing glacial through fu-ture conditions. Native (T. ceratophorum) and invasive(T. officinale) host species responded differently toAM fungi, with T. ceratophorum exhibiting stronger, butmore variable, mycorrhizal responses than T. officinale.[CO2] altered the functioning of mycorrhizal associa-tions along the M-P continuum, although the patternand magnitude of these shifts depended on the hostspecies. For T. officinale, mycorrhizal associations shif-ted from parasitism at low [CO2] to mutualism at ele-vated [CO2]. In contrast, AM fungi increased the growthof T. ceratophorum at glacial to modern [CO2]; this effectwas reversed in T. ceratophorum plants grown at future[CO2]. Mechanisms contributing to shifts in mycorrhizalfunctioning with [CO2] also differed between hostspecies. [CO2] and fungal effects on plant traits relatedto C sink strength were highly correlated with shifts inmycorrhizal functioning in T. ceratophorum. In contrast,

[CO2] effects on photosynthetic rates and vegeta-tive plasticity drove shifts in mycorrhizal function-ing in T. officinale. Overall, our results indicate thatmycorrhizal associations functioned differently at thelow [CO2] of the past and that nonlinear effects couldlimit mycorrhizal responses to future [CO2] in someplant species.

Leaf-Level Physiology

[CO2] similarly affected leaf physiology in theTaraxacum spp. hosts, while the effect of AM fungi onplant-CO2 responses depended on the trait and species(Supplemental Tables S1 and S2). For T. ceratophorum,instantaneous photosynthetic rate (A) increased non-linearly with [CO2] (Fig. 3A; CO2 main effect, PC =0.0001), and this effect was similar between mycorrhi-zal and control plants (CO2 by mycorrhizal interaction,PCM = 0.07). A also increased with [CO2] in T. officinale(Fig. 3B; PC = 0.0001), although the magnitude of thisresponse was reduced in mycorrhizal plants grown atelevated [CO2] (PCM = 0.05). Stomatal conductance (gs)decreased with [CO2] in both host species, and themagnitude of this effect was greater at lower [CO2] (Fig.3, C and D; PC = 0.0001 and 0.002 for T. ceratophorumand T. officinale, respectively). Mycorrhizal treatmentdid not affect gs in T. ceratophorum (mycorrhizal maineffect, PM . 0.6). For T. officinale, gs tended to decreasemore rapidly with [CO2] in control plants comparedwith mycorrhizal plants (PCM = 0.09). Instantaneouswater use efficiency (WUE) was affected significantlyby [CO2] but not by mycorrhizal treatment. Addition-ally, the shape of the relationship between WUE and[CO2] differed between Taraxacum spp., in that WUEincreased linearly for T. ceratophorum and nonlinearlyfor T. officinale across the [CO2] gradient (Fig. 3, E and F;PC = 0.0001 for both species).

[CO2] and mycorrhizal treatment also impacted leafnutrient content and stoichiometry, although these ef-fects differed between host species (SupplementalTables S1 and S2). In T. ceratophorum, leaf N contentincreased from 180 to 390 mL L21 [CO2] and then de-creased in plants grown at elevated [CO2] (Fig. 4A; PC =0.0001). Neither [CO2] nor mycorrhizal treatment sig-nificantly affected leaf P content or leaf nitrogen-phosphorus (N-P) ratio in T. ceratophorum (Fig. 4, Cand E; P . 0.1 for both traits). However, leaf carbon-nitrogen (C-N) ratio increased across the [CO2] gradientin this host (Fig. 4G; PC = 0.0001). [CO2] had no effect onleaf N or P content in T. officinale (Fig. 4, B and D; P .0.3), but leaf N content was significantly higher inmycorrhizal plants versus control plants (PM = 0.048).Both [CO2] andmycorrhizal treatment affected leaf N-Pratio in T. officinale (Fig. 4F). Specifically, leaf N-P ratiodecreased across the [CO2] gradient, with the greatestchange occurring from 180 to 390 mL L21 [CO2] (PC =0.02). Leaf N-P ratio also was higher in mycorrhizalT. officinale plants compared with control plants (PM =0.03), although this effect was driven largely by differ-ences in the 180 to 390 mL L21 treatments. Additionally,

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Figure 2. A, Piecewise structural equationmodels describing [CO2] effects onRBio via changes in plant and fungal traits. B,Modelresults for T. ceratophorum indicate that both direct and indirect effects of [CO2] on plant traits contributed to shifts in RBio. C,Model results for T. officinale indicate that primarily direct effects of [CO2] on plant traits contributed to shifts in RBio. In B and C,boxes represent measured traits and solid arrows indicate significant pathways in the model (P , 0.05). Black and red arrowsindicate positive and negative correlations, respectively. The thickness of solid arrows was scaled to reflect the magnitude of thestandardized regression coefficients. r2 values for each component model and standardized pathway coefficients are listed inSupplemental Table S4.


Becklin et al.




leaf C-N ratio increased in T. officinale across the [CO2]gradient, and changes in this ratio were greatest atlower [CO2] (Fig. 4H; PC = 0.002).

Plant Growth and Biomass Allocation

[CO2] and mycorrhizal effects on plant growth andallocation depended on the host species (SupplementalTables S1 and S2). For T. ceratophorum, rising [CO2]significantly increased leaf, root, and total plant bio-mass (Fig. 5; PC = 0.0001 for all traits). [CO2] effects ontotal plant biomass differed between mycorrhizal andcontrol plants (Fig. 5A; nonlinear CO2 by mycorrhizalinteraction, PCCM = 0.05). In the control treatment, totalplant biomass increased linearly with [CO2] (P =0.0001). In the mycorrhizal treatment, total plant bio-mass increased from 180 to 390 mL L21 [CO2], but thenremained relatively constant with further increases in[CO2] (P = 0.01). Consequently, control plants werelarger than mycorrhizal plants at elevated [CO2] (P =0.02). Although the mycorrhizal effect was only sig-nificant at the whole-plant level, similar patterns wereobserved for leaf and total root biomass (Fig. 5, C and E;PCCM = 0.08 and 0.06, respectively). Mycorrhizal treat-ment did not significantly affect fine root biomass inT. ceratophorum (PM = 0.3 and PCCM = 0.2). Neither [CO2]nor mycorrhizal treatment affected the proportion oftotal biomass allocated to leaves and roots in this host(P . 0.6).The number of leaves produced by T. ceratophorum,

total leaf area, and total root length increased with[CO2] (Fig. 6; PC = 0.0001 for all traits). As with totalbiomass, the slope of these relationships differed be-tween mycorrhizal treatments, with control plantsproducing more leaves, leaf area, and root length thanmycorrhizal plants at elevated [CO2] (PCM , 0.05 for alltraits). Differences in leaf production by T. ceratophorumwere reflected in aboveground growth rates. Specifi-cally, the rate of change in leaf production and leaflength increased with [CO2], but this effect was reducedin mycorrhizal plants (Fig. 6, G and I; PCM , 0.05 forboth rates). Therefore, mycorrhizal T. ceratophorumplants grew at a slower rate than control plants at ele-vated [CO2]. Specific leaf area (SLA) and specific rootlength (SRL) decreased nonlinearly with [CO2] in thishost (Fig. 6, C and M; PC = 0.0002 and 0.003, respec-tively). SRL also was generally reduced in mycorrhizalplants compared with control plants at low [CO2](PCM = 0.07).For T. officinale, [CO2] had a stronger effect on plant

growth than the mycorrhizal treatment. Rising [CO2]significantly increased leaf, root, and total plant bio-mass (Fig. 5; PC , 0.05 for all traits). With the exceptionof fine roots, changes in plant biomass were greater atlow [CO2] (significant nonlinear response). [CO2] effectson total plant biomass and leaf biomass tended to differbetween mycorrhizal and control T. officinale plants(Fig. 5, B andD; PCCM = 0.06 for both traits). Specifically,total plant biomass and leaf biomass increased linearly

with [CO2] in the mycorrhizal treatment and non-linearly with [CO2] in the control treatment (P = 0.0001for all analyses). [CO2] and mycorrhizal treatment sig-nificantly affected biomass allocation to T. officinaleleaves and fine roots (PCM = 0.048). In the mycorrhizaltreatment, plants allocated relatively more biomass toleaves compared with fine roots when grown at 180 to390 mL L21 [CO2]. Relative biomass allocation to fineroots increased inmycorrhizal plants grown at elevated[CO2]. In the control treatment, [CO2] had no effect onrelative biomass allocation to aboveground and be-lowground tissues. The effects of [CO2] and mycorrhi-zal treatment on biomass allocation in T. officinale werereduced when comparing leaf biomass with total rootbiomass (PCM = 0.07).

Total leaf area and total root length increased non-linearly with [CO2] in T. officinale (Fig. 6, B and L; PC =0.01 and 0.0006, respectively), and these responses weresimilar between mycorrhizal treatments (PM . 0.3 forboth traits). Leaf growth rate (change in leaf length perday) also increased nonlinearly across the [CO2] gra-dient (Fig. 6J; PC = 0.03). Neither [CO2] nor mycorrhizaltreatment significantly affected the number of leaves orthe rate of leaf production in T. officinale (Fig. 6, F andH;P . 0.1 for both traits). SLA and SRL decreased non-linearly with [CO2] (Fig. 6, D and N; PC = 0.0002 and0.003, respectively). As with T. ceratophorum, these re-sponses were similar between mycorrhizal treatments(PCM . 0.1 and 0.7, respectively).

Fungal Growth

Fungal colonization rates in the mycorrhizal treat-ment ranged from 16% to 72% (average = 39%) ofT. ceratophorum root length and 16% to 60% (average =31%) of T. officinale root length. [CO2] had a minimaleffect on fungal growth within Taraxacum spp. roots(Supplemental Tables S1 and S2). Mycorrhizal rootlength increased linearly with [CO2] in T. ceratophorum(Fig. 7C; PC = 0.058), but this effect was not significantuntil the shift from 700 to 1,000 mL L21 [CO2]. Neithermycorrhizal colonization rate nor mycorrhizal rootlength varied with [CO2] in T. officinale (Fig. 7, B and D;PC = 0.9 and 0.3, respectively).

Mycorrhizal Response Ratio

[CO2] differentially affected the mycorrhizal responseratios based on total plant biomass of T. ceratophorumand T. officinale hosts (RBio). ForT. officinale,RBio increasedlinearly across the [CO2] gradient, and 700 mL L21 [CO2]represented a critical point abovewhichRBio shifted fromnegative to positive (Fig. 8B; PC = 0.005). Similar patternswere observed for mycorrhizal response ratios based onleaf and root biomass (Fig. 8). RBio also increased with[CO2] in T. ceratophorum plants grown at 180 to 390mL L21

[CO2]. However, further increases in [CO2] caused RBio todecrease and eventually shift from positive to negativevalues at elevated [CO2] (Fig. 8A; PC = 0.03). Mycorrhizal








response ratios based on leaf and total root biomass alsoshiftednonlinearlywith [CO2] (Fig. 8,C andE). In contrast,the response ratio based on fine root biomass was morevariable, and decreased linearly with rising [CO2](Fig. 8G).

Plant physiological mechanisms contributing to theobserved shifts in RBio depended on the host (Fig. 2). Ingeneral, models that included both [CO2] and fungaleffects on plant traits performed better thanmodels thatexcluded one of these factors (Supplemental Table S3).For T. ceratophorum, the primary traits associated withshifts in RBio were total leaf area, total root length, SRL,and SLA (Fig. 2B; Supplemental Table S4). Above-ground growth metrics also were strongly correlatedwith root-based response ratios in this host. [CO2] ef-fects on fungal growth were weakly correlated withdecreased mycorrhizal effects on aboveground growthrates and leaf area. Variation in fungal growth also wascorrelated with increased leaf N-P ratio and decreasedWUE in mycorrhizal hosts. The main traits associatedwith shifts in RBio in T. officinale plants were total leafarea, SLA, and leaf C-N ratio (Fig. 2C; SupplementalTable S4). In this case, [CO2] influenced RBio primarilythrough positive effects on root growth, which trans-lated into increased total leaf area. Response ratiosbased on photosynthetic rate also were negatively

correlated with mycorrhizal effects on total leaf area.Variation in fungal growth influenced response ratiosbased on leaf physiological traits, including gs, leaf N-Pratio, and WUE.

DISCUSSION

Long-term changes in atmospheric [CO2] are expectedto alter mycorrhizal functioning and the role of thesesymbioses in terrestrial ecosystems due to integratedshifts in plant and fungal physiology. Developments inmycorrhizal theory (Johnson, 2010; Johnson et al., 2015)have provided a framework for predicting how thesesymbioses will function as plant carbohydrate andnutrient limitations shift with rising [CO2]. Yet, ourunderstanding of mycorrhizal-CO2 responses is pri-marily limited to studies comparing modern and fu-ture [CO2] (Alberton et al., 2005; Mohan et al., 2014).The lack of information about mycorrhizal responsesto low [CO2] during the recent geologic past limits the

Figure 3. [CO2] and mycorrhizal effects on leaf gas-exchange rateswere similar between T. ceratophorum and T. officinale. Graphs showmean photosynthetic rate (A and B), gs (C and D), andWUE (E and F) formycorrhizal (black symbols) and control (white symbols) plants.

Figure 4. [CO2] and mycorrhizal effects on leaf stoichiometry differedbetween T. ceratophorum and T. officinale. Graphs show mean Ncontent (A and B), P content (C andD), N-P ratio (E and F), and C-N ratio(G and H) for mycorrhizal (black symbols) and control (white symbols)plants.


Becklin et al.







characterization of physiological mechanisms thatdrive shifts in mycorrhizal functioning prior to andafter anthropogenic influence. By examining hostplant physiology and mycorrhizal functioning acrossglacial through future [CO2], we show that plant re-sponses to mycorrhizal fungi were weaker under thelow [CO2] of the past, although in T. ceratophorum,fungal symbionts still promoted plant growth underCO2-limiting conditions. Our results also support thehypothesis that mycorrhizal associations will becomemore beneficial with rising [CO2] (Mohan et al., 2014);however, nonlinear responses may limit mycorrhizalbenefits to some plants at the elevated [CO2] of thefuture (Alberton et al., 2007). Furthermore, physio-logical mechanisms driving shifts in mycorrhizalassociations were linked to host-specific differences inplant growth rate (Koziol and Bever, 2015) and veg-etative plasticity as well as potential constraints on plantphysiology across a long-term [CO2] gradient. Overall,this work addresses key gaps in our understanding of

[CO2] effects on plant physiology and mycorrhizal as-sociations during the recent geologic past and into thefuture.

Host-Specific Differences in Mycorrhizal Functioning

The overall strength of plant responses to AM fungidiffered for T. ceratophorum and T. officinale, highlight-ing the potential for mycorrhizal associations to varyeven between closely related plant species. On average,T. ceratophorum respondedmore positively to AM fungithan T. officinale under low to modern [CO2], and thisdifference corresponds to the observed variation inmycorrhizal associations in modern Taraxacum spp.populations. In particular, previous work indicates thatT. ceratophorum is more heavily colonized by AM fungithan T. officinale in the field (Becklin and Galen, 2009)and that colonization rate more strongly predicts plantgrowth benefits in the native host (Becklin, 2010). To-gether, these data suggest overall stronger mycorrhizalassociations in the native host. Yet, T. ceratophorum alsoexhibited more variation than T. officinale in mycorrhi-zal functioning across the full [CO2] gradient. Changesin other variables, such as light and water availability,also have been shown to influence mycorrhizal func-tioning to a greater degree in T. ceratophorum comparedwithT. officinale (Becklin, 2010), indicating thatmycorrhizalassociations in the native host may be more sensitive toenvironmental conditions. Differences in mycorrhizalfunctioning between T. ceratophorum and T. officinalemay be amplified in field populations, where coloni-zation rates are generally higher andmore variable thanwhat we observed in this study.

Species-specific differences in mycorrhizal respon-siveness may reflect variable growth strategies andnative/invasive status. For example, Koziol and Bever(2015) recently showed that mycorrhizal responsive-ness was inversely related to plant growth rate in anartificial community of 30 prairie plant species. In ourstudy, T. ceratophorum had a slower growth rate thanT. officinale (Fig. 6, G and I) and tended to invest pro-portionally more biomass belowground. These growthstrategies may facilitate more beneficial mycorrhizalassociations in T. ceratophorum, especially when [CO2]limits plant growth. A recent meta-analysis furthershows that mycorrhizal responses are often dampenedin invasive plant species (Bunn et al., 2015). Further-more, the positive relationship between fungal coloni-zation and plant size that is often observed for nativehosts is generally absent in invasive hosts. The evolu-tion of reduced dependence on mycorrhizal fungi(Seifert et al., 2009) may allow invasive plants to spreadmore easily into novel environments, since they are lesslikely to be restricted by the absence of fungal partners.Mismatches between invasive plants and species ofmycorrhizal fungi in the novel range also could explaindampened mycorrhizal responses (Klironomos, 2003).Differences in fungal species composition betweencooccurring T. ceratophorum and T. officinale plants inthe field (Becklin et al., 2012) suggest that mismatches

Figure 5. The relative strength of [CO2] and mycorrhizal effects onplant biomass differed between T. ceratophorum and T. officinale.Graphs show total plant biomass (A and B), leaf biomass (C andD), totalroot biomass (E and F), and fine root biomass (G and H) for mycorrhizal(black symbols) and control (white symbols) plants. These data wereused to calculate the mycorrhizal response ratios shown in Figure 8.





could play a role during the initial invasion. Finally,many invasive plants have fast growth rates and highdegrees of plasticity that allow them to spread rapidlyinto novel habitats; these growth strategies also couldcontribute to lower mycorrhizal responsiveness inT. officinale.

Shifts in Mycorrhizal Functioning with [CO2]

Plant growth responses to AM fungi increased withrising [CO2], supporting our original hypothesis thatthese associations will become more beneficial due toreduced plant carbohydrate limitations and increasedplant nutrient limitations with rising [CO2] (Johnson,2010;Mohan et al., 2014). This patternwasmost evidentin T. officinale, which showed linear increases in RBioacross the full [CO2] gradient. In contrast, RBio increasedin T. ceratophorum only from glacial to modern [CO2].Results for T. officinale also supported the hypothesisthat AM fungi would reduce plant growth (function asparasites) under low [CO2] due to severe carbon limi-tations on plant physiology. Negative plant responsesto AM fungi, such as what we saw for T. officinale, mayhave generated negative feedbacks that reduced thefrequency or abundance of these associations duringlow [CO2] periods in the past. Indeed, studies thatexamined AM fungi under preindustrial [CO2] notedreduced fungal growth in the soil environment (Tresederet al., 2003; Procter et al., 2014). Unlike what we sawfor T. officinale, T. ceratophorum responded positivelyto AM fungi even at low [CO2]. This result supportsthe hypothesis that mycorrhizal fungi could promoteCO2 assimilation under low [CO2] by increasing theuptake of nutrients that are necessary to maximizephotosynthetic capacity in a C-limited environment.Thus, the ability to form mycorrhizal associations andthe strength of these partnerships may represent animportant adaptation that enabled some plant speciesto persist during low [CO2] periods in the past (Becklinet al., 2014).

Elevated [CO2] greatly altered mycorrhizal func-tioning in these cooccurring plant species. Specifically,AM fungi reduced the growth of T. ceratophorum plantsbut increased the growth of T. officinale plants in the1,000 mL L21 treatment. For both plant species, 700 mLL21 represented a critical point along the [CO2] gradientwhere RBio shifted from negative to positive values (orvice versa). This indicates that future anthropogenicchanges in [CO2] could drastically alter the nature ofmycorrhizal associations as well as their role in plantphysiological acclimation and adaptation to globalchange. Characterizing physiological mechanisms thatunderlie these differential shifts in mycorrhizal func-tioning at elevated [CO2] (see below) will be critical forpredicting mycorrhizal feedbacks on plant physiologyin future environments.

Both Taraxacum spp. hosts showed evidence of non-linear responses to rising [CO2] that could only beassessed across a large [CO2] gradient. As expected,

Figure 6. The relative strength of [CO2] andmycorrhizal effects on leaf androot traits differed between T. ceratophorum and T. officinale. Graphs showmean leaf area (A and B), specific leaf area (C andD), leaf number (E and F),change in leaf number per day (G and H), change in leaf length per day(I and J), total root length (K and L), and specific root length (M and N) formycorrhizal (black symbols) and control (white symbols) plants.


Becklin et al.



[CO2] caused significant nonlinear shifts in leaf gas-exchange rates, leaf stoichiometry, and plant growth,with generally greater shifts in these traits at the lowerend of the [CO2] gradient. These nonlinear shifts oftenare attributed to strong carbon limitations imposed onplant physiology at low [CO2] and the emergence ofother limiting factors (e.g. nutrient and water avail-ability) at elevated [CO2] (Gerhart and Ward, 2010;Gerhart et al., 2012). Here, we show that mycorrhizalfungi can mediate some of these nonlinear plant re-sponses across glacial through future [CO2]. Indeed,shifts in RBio and trait-specific response ratios oftenreflected differences in the shape of [CO2] responses inmycorrhizal and control plants. For example, the bio-mass of T. ceratophorum plants increased linearly with[CO2] in the control treatment but nonlinearly in themycorrhizal treatment. The reduction in mycorrhizalplant growth (relative to control plants) resulted innegative RBio under elevated [CO2]. Characterizingthese types of nonlinear responses to [CO2] is criticalto accurately predict how long-term global changewill impact mycorrhizal associations as well as thepotential constraints imposed by mycorrhizal fungion plant physiology. For example, mycorrhizal fungimay alleviate increased plant nutrient limitations atelevated [CO2]. However, if fungal growth also be-comes increasingly nutrient limited, then competitionfor nutrients between the host plant and its fungalpartners could constrain plant physiology, resultingin photosynthetic down-regulation or even growthdepressions at elevated [CO2] (Alberton et al., 2007;Johnson, 2010). Modeling these physiological feed-backs requires knowledge of potential threshold re-sponses that identify points along the [CO2] gradientthat cause critical shifts in mycorrhizal functioning

within individual hosts as well as shifts in mycorrhi-zal dynamics within the larger plant community.

In our system, shifts in the functioning ofmycorrhizalassociations with rising [CO2] could alter plant-soilfeedbacks (PSF; Mangan et al., 2010; Bever et al., 2012)that influence invasive plant dynamics (Johnson et al.,2013). PSF can facilitate the spread of invasive plants ifthe invader disrupts positive PSF in the native com-munity (Callaway et al., 2008) or if the invader experi-ences positive PSF itself (Zhang et al., 2010; Haywardet al., 2015). Our results indicate that positive PSF arelikely stronger in T. ceratophorum than in T. officinaleunder current conditions. Furthermore, previous workindicates that native AM fungi partially restrict thedistribution of T. officinale at our field site (Becklin,2010). However, if rising [CO2] enhances plant re-sponses to AM fungi in T. officinale, thereby strength-ening PSF in this host, then mycorrhizal associationscould potentially facilitate the spread of T. officinale inthe future.

Figure 7. [CO2] had minimal effects on fungal growth in T. cerato-phorum and T. officinale roots. Graphs show mean AM fungal coloni-zation (A and B) and mycorrhizal root length (C and D) for mycorrhizal(black symbols) and control (white symbols) plants.

Figure 8. [CO2] differentially affected the mycorrhizal response ratiosof T. ceratophorum and T. officinale. Graphs show response ratioscalculated based on total biomass (RBio; A and B), leaf biomass (RLeaf; CandD), total root biomass (RRoot; E and F), and fine root biomass (RFine; Gand H).





Physiological Mechanisms Driving Shifts inMycorrhizal Functioning

The strong correlation between aboveground andbelowground response ratios suggests that C sinkstrength drove shifts in RBio in T. ceratophorum. That is,T. ceratophorum plants responded more positively toAM fungi in [CO2] treatments where fungal coloni-zation stimulated root growth. Indeed, response ratiosbased on fine root biomass were highest in the [CO2]treatments that caused the greatest reductions in rootgrowth (180–390 mL L21; Figs. 5G and 8G). Thus, AMfungi had a proportionally larger influence on rootgrowth and overall plant size in these low [CO2] treat-ments.

At elevated [CO2], mycorrhizal root length increasedand RBio shifted to negative values in T. ceratophorum.This pattern may reflect resource competition betweenhost plants and their fungal symbionts, which is pre-dicted to increase under elevated [CO2] (Alberton et al.,2007; Johnson, 2010). This type of plant-fungal compe-tition could influence photosynthetic down-regulationin the host as well as constrain plant growth responsesto rising [CO2] in the future. Alternatively, if nutrientswere not limiting to either partner, then increasedfungal growth could enhance the carbohydrate sinkstrength within host plants without providing addi-tional nutrient benefits, resulting in plant growth de-pressions (Johnson, 2010). If this were the case, then wewould expect increases in fungal growth to be stronglycorrelated with decreases in RBio as well as greatergrowth depressions under low [CO2] when carbonavailability limited plant physiology and growth. Sincethat was not the case, it is unlikely that increased fungalC demand in T. ceratophorum roots explains the shiftfrom positive to negative response values at elevated[CO2]. Instead, we propose that plant-fungal competi-tion for nutrients is a more likely scenario.

Mycorrhizal response ratios in T. officinale were as-sociated primarily with fungal effects on leaf growthacross the [CO2] gradient. This species is known toadjust allocation to leaf growth based on environmen-tal conditions (Brock et al., 2005); thus, it is not sur-prising that mycorrhizal effects appear to be mediatedthrough vegetative plasticity in this host. Interestingly,mycorrhizal effects on photosynthetic rate were nega-tively correlated with shifts in leaf growth. In otherwords, AM fungi promoted leaf growth even whenphotosynthetic rate was reduced in mycorrhizal plants(relative to control plants; Figs. 3B and 8D). This sug-gests that AM fungi may have increased the C use effi-ciency ofT. officinaleplants, at least under elevated [CO2],where a relative reduction in photosynthesis betweenmycorrhizal and control plants occurred. Although[CO2] did not significantly affect fungal growth inT. officinale plants, variation in mycorrhizal root lengthwas correlated with variation in leaf-level physiology(e.g. gs and WUE). These physiological effects of AMfungi could become more important when consideringthe interactive effects of rising [CO2] and climate change

(Mohan et al., 2014). In particular, the negative correla-tion between mycorrhizal root length and WUEsuggests that AM fungi may decrease the ability ofT. officinale to tolerate drought.

CONCLUSION

A glacial through future [CO2] gradient caused dif-ferential shifts in the functioning of mycorrhizal asso-ciations within two closely related dandelion species. Inboth cases, studying mycorrhizal responses to a broad,temporal [CO2] gradient provided support for con-trasting hypotheses regardingmycorrhizal responses tolow [CO2]. Our results suggest that these critical sym-bioses were generally weaker under low [CO2] repre-senting glacial and preindustrial conditions. Exposinghost plants to the full range of [CO2] experienced overrecent geologic history also resulted in mycorrhizalassociations that spanned the M-P continuum. Sincenutrient availability was kept constant, this experimentisolated [CO2] as the driver of these shifts in mycor-rhizal functioning and identified a critical [CO2] (700mLL21) that caused mycorrhizal response ratios to shiftbetween positive and negative values. Differential re-sponses of the two plant species across the [CO2]gradient were linked to variation in plant physiologyand growth strategies, and our results suggest that keyplant traits, such as growth rate, may provide a frame-work for predicting mycorrhizal functioning. Here, wefocused primarily on host plant responses; however,significant [CO2] effects on fungal species compositionhave been noted in other studies (Treseder et al., 2003;Cotton et al., 2015). Such changes could alter the func-tional pathway by which mycorrhizal fungi impactplant physiology and growth (Sikes et al., 2010) andshould be investigated further for CO2 responses. Finally,[CO2] is an important driver of evolutionary adaptationin plants (Ward and Strain, 1999; Sage and Coleman,2001; Ward and Kelly, 2004; Beerling and Berner, 2005;Grossman andRice, 2014), and ourfindings suggest thatmycorrhizal fungi may contribute to these responses.Thus, integrating both physiological and evolutionaryperspectives intomycorrhizal-CO2 studieswill be criticalfor predicting the long-term consequences of globalchange on ecosystems.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Taraxacum ceratophorum, the native alpine dandelion, and Taraxacumofficinale, the exotic dandelion, are close congeners that cooccur at the treeline inthe Rocky Mountains (Brock, 2004; Becklin and Galen, 2009). These C3 plantsdiffer in several ecological and physiological traits that could influence theirresponses to [CO2] and AM fungi. Specifically, T. officinale has a faster growthrate and greater plasticity in biomass allocation to aboveground and belowgroundtissues (Brock and Galen, 2005; Brock et al., 2005). In contrast, T. ceratophorum isa smaller plant with a slower growth rate, higher WUE (CO2 uptake per watermolecule lost), andgreater allocation belowground.Additionally,T. ceratophorumis more heavily colonized and exhibits a larger growth response to AM fungithan T. officinale under field conditions (Becklin and Galen, 2009; Becklin, 2010).


Becklin et al.



In a field survey, AM fungi colonized 45% to 100% (average = 78%) ofT. ceratophorum roots and 0% to 97% (average = 72%) of T. officinale roots(Becklin and Galen, 2009). AM fungal community composition also varies be-tween cooccurring T. ceratophorum and T. officinale hosts (Becklin et al., 2012).Given these physiological and ecological distinctions, rising [CO2] is expected todifferentially affect plant physiology, plant growth, and mycorrhizal func-tioning in these Taraxacum spp.

To test this hypothesis, T. ceratophorum and T. officinale seeds and field soil(the source of fungal inoculum) were collected from well-studied populationson Pennsylvania Mountain (approximately 3,623 m above sea level; ParkCounty, Colorado). Both species were grown with and without AM fungi in afull factorial designwith five [CO2] treatments representing glacial (180mL L21),preindustrial (270 mL L21), modern (390 mL L21), and future (700 and 1,000mL L21) conditions. Each [CO2] treatment was replicated in two separateConviron BDR16 growth chambers. Chamber conditions were maintained at22°C/14°C and 65%/90% relative humidity (day/night). Daytime light inten-sity wasmaintained at approximately 950 mmolm22 s21 for a 14-h photoperiod.Plants were watered once daily to saturation and fertilized with 25 mL of one-half-strength Hoagland solution (modified to one-quarter-strength P concen-tration) during a 30-d growth period.

All seedswere surface sterilized andgerminated in petri dishes. Immediatelyupon germination, seedlings were transplanted into 2-Lmicrocosms containinga sterilized growth medium (2:1:1 sand:gravel:turface). Half of the microcosmswere inoculatedwith AM fungi by adding 20 g of homogenized living field soil.Control microcosms were inoculated with 20 g of sterilized field soil. Tominimize nontarget microbial differences between inoculated and controltreatments, all microcosms received 20 mL of a soil filtrate containing non-mycorrhizal soil microbes (Johnson et al., 2010). We assessed the AM fungalcolonization of mycorrhizal and control plants after harvest. A subsample ofthe fine roots from each plant in the experiment was stained with Trypan Blue(Phillips and Hayman, 1970) and analyzed for the presence of arbusculesusing the gridline intersection method (McGonigle et al., 1990). Root colo-nization rate was calculated as the percentage of root length containingarbuscules. Mycorrhizal root length was estimated by multiplying total rootlength and colonization rate. Some control plants had very low levels of AMfungi (range = 0%–15% of root length); in these cases, the intensity of colo-nization within the root also was very low compared with plants in themycorrhizal treatment and random across [CO2] treatments (P . 0.1). Theselow levels of colonization may actually represent a more realistic control,since true nonmycorrhizal Taraxacum spp. plants are unlikely to occur innature (AM fungi colonized all but one Taraxacum spp. plant examined in afield survey; Becklin and Galen, 2009). Additionally, control plants with lowcolonization levels were not statistically different in size from plants with noAM fungi (P . 0.1); thus, it is unlikely that the low colonization of controlplants affected our results.

Leaf-Level Physiology

Leaf gas-exchange measurements were made using an LI-6400XT device(Li-Cor Biosciences) between 9 AM and 12 noon on the day of harvest using afully expanded green leaf that was exposed to full light. Cuvette conditionswere set to match growth conditions. Leaves were allowed to acclimate to cu-vette conditions until the instantaneous A and gs were stable. InstantaneousWUE was calculated as the ratio of photosynthetic rate to transpiration rate.Plants were harvested immediately following gas-exchange measurements.

For most plants in the experiment, mass-based leaf N and P contents weremeasured following acid digestion of 250 mg of dry leaf biomass using aTechnicon AAII auto analyzer (Agronomy Soil Testing Laboratory, KansasState University). For samples with less than 250 mg, leaf P content was ana-lyzed followingmicrowave digestion of 50 to 100mg of dry leaf biomass using aSpectro Genesis inductively coupled plasma optical emission spectrometer(North Dakota State University). Mass-based leaf C content was analyzed on3 to 4 mg of dry leaf biomass using a Costech 4010 elemental analyzer (KeckPaleoenvironmental and Environmental Stable Isotope Laboratory, Universityof Kansas). For samples with less than 250 mg, leaf N content was analyzed inconjunction with leaf C content. Replicated samples were analyzed using allapproaches to correct for potential biases in the N and P results produced usingdifferent methods. N and P results were converted from mass-based percent-ages to nutrient content per leaf area using the ratio of total leaf biomass andtotal leaf area. Leaf N-P ratio and C-N ratio were calculated from mass-basedpercentages. In a few cases, plant biomass was too small to measure leaf N, P,andC contents on the same sample; thus, wewere not able to calculateN-P ratioand C-N ratio for all plants in the experiment.

Plant Growth and Biomass Allocation

Plant growth was monitored weekly by counting the total number of leavesand the length of the longest leaf on each plant. Leaf length is correlatedwith leafarea inTaraxacum spp. (P, 0.0001, r2. 0.75 for both species); thus, thismeasurereflects changes in photosynthetic area. Growth rates were calculated as thechange in leaf number or leaf length per day.

After 30 d, we analyzed plant size and allocation to leaf and root fractions.Root tissuewas further divided into taproot and fine root fractions. The biomassof each fraction was determined after drying the plants at 60°C for 72 h. Totalfresh leaf area was measured using an LI-3100 leaf area meter (Li-Cor Bio-sciences). SLAwas calculated as fresh leaf area per dry leaf biomass. To estimatetotal root length, we collected a random subsample of fresh fine roots from eachplant in the experiment. Root length was determined from a scanned image ofthese roots using ImageJ. We then dried the imaged roots and used regressionanalysis to determine the coefficient relating root length to root biomass for eachspecies and [CO2] treatment (mycorrhizal treatment did not affect this rela-tionship). This coefficient was used to estimate total root length from measuresof fine root biomass as well as SRL (fresh root length per dry root biomass).

Mycorrhizal Response Ratio

Shifts in mycorrhizal functioning along the M-P continuum were charac-terized using mycorrhizal response ratios (RBio). RBio represents the overall ef-fect of AM fungi on plant growth and is generally calculated by comparing thetotal biomass of mycorrhizal (MBio) and nonmycorrhizal (NMBio) plants of thesame species and age grown under similar conditions (RBio = [MBio 2 NMBio]/NMBio). Thus, RBio integrates the growth benefits of fungus-mediated nutrientuptake and the growth costs of carbohydrate allocation to fungal symbionts. Apositive RBio indicates that AM fungi increase plant growth (mutualism), whilea negative RBio indicates that AM fungi reduce plant growth (parasitism).Critical shifts in RBio include (1) shifts from positive to negative values, (2)nonlinear shifts in RBio, and (3) changes in the direction that RBio shifts across aresource gradient (e.g. increasing versus decreasing trends). For this study, wecalculated RBio using the total biomass of mycorrhizal plants and the averagetotal biomass of control plants for each host species and [CO2] treatment. Wecalculated similar response ratios using leaf gas-exchange rates, leaf carbon andnutrient contents, aboveground growth rates, total leaf area and SLA, leaf androot biomass, and total root length and SRL.

Statistical Analysis

Allplant and fungalvariableswere analyzedusinggeneralized linearmodelswith mycorrhizal treatment as a categorical variable and [CO2] as a continuousvariable. To assess the shape of plant responses to [CO2], we conducted bothlinear and quadratic models and tested for interactions between [CO2] andmycorrhizal treatment (C3M and C3C3M interaction terms in SupplementalTables S1 and S2). A significant C3M interaction indicates that the presence ofAM fungi mediated plant responses to [CO2]. Similarly, a significant C3C3Minteraction indicates that the shape (linear or nonlinear) of the response differedbetween control and mycorrhizal treatments. Models were compared using theAkaike information criterion corrected for sample size (AICc). We also con-ducted allometric analyses of biomass allocation to aboveground and be-lowground plant structures via regression with [CO2] as a covariate andmycorrhizal treatment as a categorical variable. All data were analyzedseparately for T. ceratophorum and T. officinale.

[CO2] effects on RBio were analyzed using regression with [CO2] as a con-tinuous variable. Again, we compared linear and quadratic models using AICc.To assess the mechanisms that drive shifts in RBio, we calculated response ratiosfor each plant trait measured in this study. Because these response ratios arerelative to nonmycorrhizal controls grown at the same [CO2], they minimizevariation due to inherent plant-CO2 responses and allow us to evaluate traitsthat contributed to size differences between mycorrhizal and control plantsacross the [CO2] gradient. At the leaf level, we focused on response ratios cal-culated from instantaneous gas-exchange rates that determine carbohydrateproduction and are sensitive to [CO2] in C3 plants (Ainsworth and Rogers, 2007;Gerhart and Ward, 2010). We also examined ratios based on leaf N-P ratio andC-N ratio, because these traits reflect relative resource limitations in leaves andmay be more relevant for predicting plant responses to mycorrhizal fungi thanabsolute leaf nutrient content (Johnson, 2010). Relationships among physio-logical traits, plant growth, and overall RBio were analyzed using piecewisestructural equation modeling (Lefcheck, 2016). Specifically, we compared the







following models: (1) [CO2] affected response ratios indirectly via changes infungal growth (no direct [CO2] pathways); (2) [CO2] affected response ratiosdirectly (no fungus-mediated pathways); and (3) both [CO2] and fungal growthaffected response ratios in Taraxacum spp. (Fig. 2). Conditional independencewithin each model was evaluated using Shipley’s test of direction separation(Shipley, 2009), and overall model fit was evaluated using AICc. Modelinganalyses were conducted separately for T. ceratophorum and T. officinale.

Supplemental Data

The following supplemental materials are available.

Supplemental Table S1. [CO2] and mycorrhizal effects on fungal growth,plant physiology, and plant growth in T. ceratophorum.

Supplemental Table S2. [CO2] and mycorrhizal effects on fungal growth,plant physiology, and plant growth in T. officinale.

Supplemental Table S3. Comparison of structural equation models forT. ceratophorum and T. officinale.

Supplemental Table S4. Standardized pathway coefficients and submodelr2 values for the T. ceratophorum and T. officinale models with the lowestAICc values.

ACKNOWLEDGMENTS

We thank J. Medeiros and M. Walker for help with plant care; J. Stern andW. Wang for sample preparation; and D. Jacobs for analysis of leaf nutrients.

Received May 20, 2016; accepted August 26, 2016; published August 29, 2016.

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