Stable isotope probing implicates a species of <i><scp>C</scp>ortinarius</i> in carbon transfer through ectomycorrhizal fungal mycelial networks in <scp>A</scp>rctic tundra

Mycorrhizal networks (MNs) occur when the mycelium of one or more mycorrhizal fungus colonizes two or more nearby plants (Molina et al., 1992). MNs can serve as pathways for the transfer of carbon (C), nitrogen (N), phosphorus (P), water, defense signals and allelochemicals among plants (see Simard et al., 2012, for a review) but, to the best of our knowledge, the fungi involved in these resource transfers among green plants in the field have never been directly identified. This information is important because it would reveal the unique roles of mycorrhizal fungal species, shedding light on some of the functions of these complex belowground networks, and potentially providing additional ecological context for the increasingly large body of molecular community data that is accumulating for mycorrhizal fungi (Blaalid et al., 2014; Horn et al., 2014; Morgado et al., 2015). MNs mediate plant–plant interactions with potential implications for plant diversity at local and regional scales (Perry et al., 1989; McGuire, 2007; Deslippe & Simard, 2011) and are likely to have foundational roles in the structure and regeneration of terrestrial ecosystems (Simard, 2009; Simard et al., 2012). Thus, an improved understanding of MNs may lead to more appropriate and effective land conservation and ecological restoration practices. In recent decades, regional warming associated with anthropogenic climate change has led to increased plant biomass across the Arctic tundra biome (Jia et al., 2003; Chapin et al., 2005; Macias-Fauria et al., 2012). Differences in the relative productivity of plant species have led to altered plant community compositions, with ectomycorrhizal (EM) shrub species increasingly dominating in many regions (Myers-Smith et al., 2011; Bonfils et al., 2012). In the moist-acidic tundra of Arctic Alaska, the EM shrub Betula nana has increased most strongly (Sturm et al., 2001) and this effect is further enhanced through experimental warming (Chapin et al., 1995; Sistla et al., 2013). When warmed, the EM fungal (EMF) community associating with B. nana shifts from being dominated by members of the Russulaceae to one dominated by Cortinarius spp. (Deslippe et al., 2011). Cortinarius spp. are C-demanding, rhizomorph-forming basiodimycetes that grow extensive mycelia in soil (Agerer, 2001, 2006). Some Cortinarius species produce highly efficient oxidative enzymes (Bodeker et al., 2009, 2014), which they utilize to mobilize growth-limiting N for their host from complex soil organic matter (Lilleskov et al., 2002; Hobbie & Agerer, 2010; Hobbie et al., 2013). Recent work has highlighted the importance of N mobilization by Cortinarius spp. in maintaining rates of C-cycling in other high-latitude ecosystems (Clemmensen et al., 2013, 2015; Lindahl & Tunlid, 2015) and similar processes could be involved where shrubs are spreading in Arctic tundra as climate warms. Indeed, the transition from ericaceous tundra heath to EM shrub tundra is accompanied by significantly higher rates of fungal hyphal growth and C turnover and lower soil organic C stocks in Swedish Lapland (Parker et al., 2015). Studies of C-transfer through EMF mycelial networks have often utilized stable or radiocarbon isotopes and employed ‘pulse-chase’ methodology to trace photosynthetic C from a labeled ‘donor’ plant to nearby ‘receiver’ plants. These studies have revealed that the magnitude of C transfer through EMF mycelial networks is highly variable in nature, ranging from < 1% to 10% of the donor plant's net photosynthesis (Simard et al., 1997a; Teste et al., 2010). This variability reflects plant physiological factors that affect the magnitude of source–sink gradients for C among networked plants, and fungal factors, such as the extent of fungal colonization of roots, the composition of EMF community and the continuity of the hyphal pathway (see Simard et al., 2012, for a review). Indeed, small but statistically significant C-transfer to receiver plants through soils and a discontinuous hyphal pathway have been observed (Philip et al., 2010; Deslippe & Simard, 2011), suggesting a possible additional role for bacteria, saprotrophic fungi, or other members of the rhizosphere community in C-transfer among plants. Bacterial-mediated C transfer among plants could occur, for example, if a donor plant's rhizodeposit C was acquired by a rhizosphere bacterium which subsequently became an endophyte of the receiver plant (Rosenblueth & Martinez-Romero, 2006). Previously, through 13CO2 pulse-chase labeling of B. nana plants in Arctic tundra we showed significant transfer of C through EMF mycelial networks to aboveground tissues and rhizomes of B. nana receivers in conspecific pairs only. We also found low but nonzero C transfer among B. nana pairs through soil pathways. Here we report the use of stable isotope probing (SIP) of phospholipid fatty acids (PLFAs) paired with a DNA-SIP-pyrosequencing approach of root samples collected in that study to test the hypotheses that: (1) EMF were the primary conduits for C among B. nana individuals, and (2) Cortinarius were more important conduits for C than other members of the EMF community associating with B. nana. A significant and unique role for one or more species of Cortinarius in C-transfer among B. nana individuals would constitute evidence that EMF species in mixed communities perform particular functions for their host, one step towards elucidating the functions of these diverse relationships. This study took place in Low Arctic tussock tundra at Toolik Lake, Alaska, USA (68°38ʹN, 149°34ʹW). The plant community at this site is co-dominated by the EM dwarf shrub B. nana L. and the nonmycorrhizal sedge Eriophorum vaginatum, with an understory mainly of ericaceous plant species and mosses (Supporting Information Table S1). B. nana ‘donor’ plants were sealed in gas-tight chambers and pulsed with 3.2 mmol 13CO2 (Fig. 1a). B. nana donors were removed from labeling chambers when CO2 concentrations inside the chamber fell below ambient concentrations (c. 4 h; Fig. 1b). After a 7-d chase period, stems, leaves and rhizomes of the B. nana donor and all potential ‘receiver’ plant species present in the 55 cm-diameter study plots were harvested and 13C-tissue content analyzed (Fig. 1c). We found statistically significant 13C-transfer only among pairs of B. nana plants (Deslippe & Simard, 2011). We sampled the roots of each donor B. nana plant as well as one independent receiver B. nana plant per plot. Independent receiver B. nana plants were not connected to the donor plant via belowground plant tissues (e.g. rhizomes or root grafts). The independence of the receiver plant was determined at the time of destructive sampling of the plot. Where more than one B. nana receiver occurred, the largest plant was selected for root sampling. Likewise, we sampled the roots of an unlabeled B. nana plant growing a minimum distance of 2 m upwind from each 13C-CO2 labeling plot (12C control plant; Fig. 1c). Fine roots occurred predominantly in the organic soil horizon. We collected B. nana roots by tracing the main stem to belowground rhizomes and these to clusters of root tips, which could then be sampled in an intact state. Considerable effort was made to sample the entire fine root system of each plant, but it is possible that some fine root clusters were missed. Each sample consisted of a minimum of three terminal root clusters and adhering organic soil particles, these were 5–10 cm3 in volume, and all were visibly EM. Samples were placed immediately on ice and frozen at −80°C within 2 h. They remained frozen during transport to the laboratory. For PLFA-SIP, we selected root samples from pairs of B. nana (one donor, one receiver) in six experimental plots from the Deslippe & Simard (2011) study, selecting the plots where the greatest total 13C-enrichment of the receiver plant tissues (sum of leaf, stem and rhizome 13C-contents) had been observed. We also sampled the roots of 12C control plants adjacent to each plot (Fig. 1c). The purpose of the 12C-control plant root sample was to provide estimates of the natural abundance of 13C in microbial PLFAs. Total lipids were extracted from c. 1 g freeze-dried rhizosphere soil, fractionated, and the phospholipid fraction was trans methylated as described by Bengtson et al. (2009). Fatty acid methyl esters (FAMEs) were analyzed by capillary GC-combustion-isotope ratio mass spectrometry (GC-C-IRMS) at the Stable Isotope Research Unit in the Department of Crop and Soil Science, Oregon State University, Corvallis, OR, USA. FAMEs were identified as described by Williams et al. (2006) and Butler et al. (2003) and quantified using 19:0 methyl ester as the internal standard. We identified 21 FAMEs containing 14–20 C atoms. The δ13C values of individual PLFAs were determined according to Williams et al. (2006), where the atomic 13C excess of PLFAs in samples was calculated relative to the mean δ13C value for the corresponding PLFA for the root samples of the six unlabeled control B. nana plants. Values were expressed as ng PLFA 13C incorporation g−1 dry weight soil. We used the 13C enrichment of 18:2ω6,9 as an indication of fungal 13C incorporation in each sample. As a measure of bacterial 13C incorporation, we used the sum of the enrichment of the following PLFAs: i15:0, a15:0, 15:0, i16:0, 10Me16:0, i17:0, a17:0, cy17:0, 17:0, 18:1n7, 10Me18:0 and cy19:0 (Frostegard & Baath, 1996). We assessed correlations among the 13C-enrichments of fungal and bacterial PLFAs and dry weight of plant tissue 13C content using Pearson's product-moment correlation in SPSS v.22 (IBM SPSS Statistics, Armonk, NY, USA). For each PLFA, the proportion of 13C-enrichment per sample was then calculated. Means are reported ± 1 standard error (SE). For DNA-SIP, we selected root samples from the receiver B. nana in three experimental plots together with root samples from each adjacent unlabeled 12C-control plant (Fig. 1c). DNA was extracted twice from 0.5 g of fresh-frozen root sample using a FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA). The replicate DNA extractions were then pooled and quantified using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). DNA-SIP followed the ‘fractionation’ method without EtBr described by Neufeld et al. (2007). Gradients were formed using a Beckman Coulter Ultracentrifuge fitted with a Vti 65.2 rotor (Beckman Coulter Canada LP, Mississauga, ON, Canada). Following Gallagher et al. (2005), we included 10 μg ‘13C-carrier DNA’ within each ultracentrifuge tube. 13C-DNA was generated by extracting DNA from Escherichia coli grown in Minimal Media liquid culture, with 13C-glucose as a sole C source. Gradients were displaced and separated into 12 fractions with water colored with toludine blue using a Razel Clinical Syringe Pump (Razel Scientific Instruments Inc., Georgia, VT, USA). The total DNA content of each fraction was quantified by agarose gel electrophoresis through comparison to a known quantity of 1 kb Trackit™ quantification ladder (Life Technologies, Waltham, MN, USA) using AlphaView imaging software (Protein Simple, Toronto, ON, Canada). 13C-enriched ‘heavy’ DNA was contained in fractions 7 and 8, while fraction 10 contained ‘light’ 12C-DNA. We pooled fractions 7 and 8 from each receiver or control plant for a single ‘heavy’ 13C DNA fraction per plant. Eukaryotic ribosomal internal transcribed spacers (ITS2) were amplified from the heavy and light fractions using the primers ITS3/ITS4 (White et al., 1990) and sequenced using 454-pyrosequencing GS-FLX Titanium technology (Roche 454 Life Sciences, Branford, CT, USA) at the Genome Quebec Innovation Centre, Montreal, Canada. We pyrosequenced heavy and light fractions of the receiver plants from the three experimental plots individually, but pooled the heavy fractions (7 and 8) of all three unlabeled control plants for a total of seven pyrosequencing samples (i.e. ‘Biosamples’). The fungal community of the biosample derived from the pooled heavy fractions of the unlabeled control plant roots was compared to that derived from the heavy fractions of receiver plants from experimental plots. This served to confirm that the fungi identified as being enriched in the heavy fractions of receivers from experimental plots did not also occur in significantly higher abundances in samples with only natural abundance of 13C. This situation could arise, for example, for taxa with higher than average G + C contents as the lower buoyant densities of G + C content could to to the of the and are described by et al. DNA-SIP pyrosequencing approach which were to the the study for and to are described by et al. were into at an of using the et al., 2011). were the et al., and using the et al., and a minimum of in et al., These were used for all the through of the among We then used of to additional to the of that in abundance among the heavy and light we all unique with that were derived from Cortinarius and a using et al., (Fig. S1). We used species to the of to the heavy and light values were calculated with the method of & using of with as in & In we used species to the of the sum of all to heavy or light we the of the sum of all that did not to the Cortinarius to heavy and light We on members of the Russulaceae and on Cortinarius spp. because they are members of the EMF community associating with B. nana at this site (Deslippe et al., 2011) and because the of and Cortinarius could have our to significant 13C-enrichment of these are reported ± 1 of the root samples of six B. nana pairs greatest enrichment of the fatty acid which is to and (Fig. and reflects by plant roots as well as by soil The fungal 18:2ω6,9 showed the greatest with the roots of receiver plants containing on of the 13C-enrichment ± ng g−1 dry weight as 18:2ω6,9 in the roots of donor plants ± ng g−1 For all 18:2ω6,9 showed 13C-enrichment than did the sum of 13C-enrichment of all bacterial PLFAs. 13C-enrichment of the fungal was that of the bacterial and these did not significantly root samples of donor and receiver plants (Fig. These a role for fungi relative to in 13C-transfer among pairs of B. nana in the providing for our tissue 13C enrichment of donors and receivers was significantly to the 13C enrichment of 18:2ω6,9 but not significantly to the sum of 13C-enrichment of all bacterial or to the sum of total PLFA These that than bacterial 13C enrichment is more to plant 13C tissue In the of donor plants, this is with the that plant C is acquired by mycorrhizal fungi being through the saprotrophic fungal and bacterial community et al., et al., 2010; et al., et al., 2015). In the of receiver plants, this for a hyphal as a pathway for C-transfer among B. nana The 13C enrichment of 18:2ω6,9 was also significantly to total and bacterial PLFA 13C-enrichment < that fungi dominated of 13C from plants and suggesting that they may have of 13C by the bacterial a that with other recent work et al., and additional for our DNA-SIP pyrosequencing approach fungal that into at a A in of all fungal generated in this study is to this S1). the rhizosphere fungal community was with the most only of all fungal while the seven most and each The abundances of most were similar in the heavy and light fractions of The most an of the with to the fungus from was a of this (Fig. Table the most was on average two of magnitude more in the heavy than light fractions ± ± Fig. Table while it was from the heavy fractions derived from the unlabeled 12C-control plant (Fig. was as the sole significant of the heavy fraction at Fig. Table This for our that Cortinarius was an important for C among B. nana plants relative to other members of the fungal The that into which had to derived from of Cortinarius and with as the for of that is among these species (Fig. and we are to provide more and are to be associated with Betula et al., We observed Cortinarius spp. at the study of which had a a with Cortinarius et al., was more than the (Fig. This Cortinarius occurred in abundance from each Here we that one or more species of Cortinarius was likely the pathway for C-transfer among B. nana plants in Arctic C-transfer through EM networks to receiver plant tissues has been well for two (Simard et al., et al., & Simard, 2011; Deslippe & Simard, 2011), ecological has been the of et al., Simard et al., 2002; & The of this has on the that a soil fungus would growth-limiting C to host plant (Simard et al., 2012). this situation is the for fungi and plants in green plants are relative to their fungi and the of C this is to be (Simard et al., 2012). EMF are often N relative to their host plants, and significant C-transfer among green plants through MNs would be if organic N was a in which C We that C-transfer among B. nana through EM networks may the of acid N from Cortinarius to B. nana. In the tussock tundra Toolik soil N in and acid is < 4 μg g−1 soil the growing & and soil organic matter et al., In these in with of root when soil and acid concentrations are at their values & EMF oxidative (Bodeker et al., 2009, are likely to significantly to these a proportion of the of N that EMF to plants & 2006). by plant and EMF acids a of within roots et al., and These may via the In plant roots, most is to acids transport et al., 2009, and similar occur within the EMF it that an EMF with could mycelial concentrations of formed that acids containing N and recent plant These acids could then gradients to be acquired by plants connected in the studies to the of N from EMF to their host plants are which C for two N was found to be the primary through which N is from members of the Russulaceae to et al., acid are in plant et al., and strongly during with EMF et al., 2010). Thus, while the 13C enrichment of Cortinarius spp. DNA we observed in the roots of receiver B. nana plants evidence that these plants are to donors through Cortinarius the transfer of C through these MNs into plant tissues (Deslippe & Simard, 2011) likely reflects mycelial N and the transfer of formed to B. nana This situation with that of mycorrhizal fungi which N and it to but transfer it to the plant without C et al., acids are the of N from EMF to their host plants, then in at the among EMF and may the C-transfer through as studies that have found large and significant C-transfer through MNs to plant tissues have been in EM (Simard et al., 2012). Previously, we found that on average of a donor plant's net photosynthetic C was to receiver B. nana tissues through MNs (Deslippe & Simard, 2011). C-transfer occurred only within pairs of B. nana and not B. nana and other plant species, the other EM plants we observed large variability in C-transfer through the pathway relative to other belowground pathways root and the soil that Cortinarius was among fungi in being highly enriched in and that the abundance of EMF on B. nana can from to of the in to warming at this site (Deslippe et al., 2011), it that in the relative abundance of Cortinarius on B. nana roots may for at some proportion of the variability in C-transfer that we observed. the of hyphal among EM plants is significantly to through MNs et al., further for this we used species to the 13C-enrichment of all and of all that did not into when the abundances of these were they did not higher for the heavy This significant 13C-enrichment among members of the Russulaceae or among members of other species of Cortinarius Thus, in to the more and EMF on B. nana receivers that were not enriched in 13C in this study (e.g. it that members of the Russulaceae and other Cortinarius species were not conduits of C-transfer among B. nana. The unique enrichment of which is likely a single Cortinarius the that MNs dominated by rhizomorph-forming fungal species are of resource transfer than dominated by such as we identified in the Here we that a Cortinarius was the likely pathway for C-transfer among B. nana plants in Arctic data that a Cortinarius a particular for host that other EMF not to significant the for C-transfer through MNs has to be one possible is that C-transfer among B. nana individuals through MNs reflects the of acid N from Cortinarius to B. nana. This is with the evidence that some Cortinarius species (Bodeker et al., 2009, are important in growth-limiting N for their host from complex soil organic matter (Lilleskov et al., 2002; Hobbie & Agerer, 2010; Hobbie et al., which in is important in maintaining rates of C-cycling (Clemmensen et al., 2013, 2015; Lindahl & Tunlid, 2015). C-transfer among B. nana individuals through mycelial networks of Cortinarius may to the evidence EMF to the of in ecosystems (Clemmensen et al., 2015). the of Arctic EM shrubs et al., 2011; et al., 2011; et al., and EMF communities (Deslippe et al., Morgado et al., 2015) to climate it is possible that these unique of Cortinarius are The are to the and to for on of this The of the of for of Cortinarius The are to and of the Stable Isotope Research Oregon State University, for of microbial PLFAs. is to Neufeld at the of for in DNA-SIP The and Hobbie and the Arctic for This work was made possible by a and Research of Canada to and and a of to and the the and and analyzed the the with from and are not for the content or of information by the than be to the Fig. containing and other Fig. abundances of the fungal in 12C-control plant root Fig. Cortinarius at the study site at the time of sampling. Table of all plant species in study plots Table species for fungal Table abundances and of fungal derived from samples Table abundances and of Russulaceae and fungal A of the fungal generated in this study in The is not for the content or of information by the than be to the corresponding for the

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