DETTWEILER-ROBINSON ET aL. Journal of Ecolog y | 895
KEYWORDS 15N translocation, Ascomycota, biological soil crusts, bunchgrass, C:N, carbon use efficiency, drylands, fungal loop hypothesis
1 | INTRODUCTION other microbially driven processes (Barger, Weber, Garcia-Pichel, Zaady, & Belnap, 2016). Similar to the microbial loop in oceans, spe- In ecosystems with low-resource availability, the retention of nutri- cies interactions that promote resource retention in drylands could ents in a living biotic pool can increase productivity. For example, slow losses, thereby increasing productivity (Bardgett & Wardle, ocean bacteria rapidly process resources from dead organisms at 2010). higher trophic levels before they sink, thereby retaining nutrients The fungal loop hypothesis proposes that networks of fungal in the photic zone (the ‘microbial loop’; Azam et al., 1983; Fenchel, hyphae link the resource dynamics of spatially and temporally dis- 2008). In tropical forests, soil fungi can translocate and retain limit- connected biocrusts and plants to promote productivity (Collins et ing nutrients in mineral-poor soils and prevent leaching during large al., 2008; Rudgers et al., 2018). Fungi are in or adjacent to both pro- rain events (Hättenschwiler, Coq, Barantal, & Handa, 2011; Lodge, ducer groups, with plant rhizospheres and biocrusts sharing 25%– 1993). In low productivity ecosystems, microbes control resources 50% of their fungal taxa (Porras-Alfaro, Herrera, Natvig, Lipinski, & in living biotic pools before releasing nutrients to primary producers; Sinsabaugh, 2011; Steven, Gallegos-Graves, Yeager, Belnap, & Kuske, in contrast, plants in mesic systems can derive nutrients from the soil 2014). Thus, the possibility exists for these shared fungi to connect solution that has been processed by microbes from the accumulated plants with biocrusts. Fungi could couple the activities of plants and soil organic matter (de Deyn, Cornelissen, & Bardgett, 2008; Knops, biocrusts temporally if they take up water or nutrients under con- Bradley, & Wedin, 2002). It is important to understand these micro- ditions when only some producers are active, such as after a light bially driven processes in low-resource ecosystems to make better rain, then transport these resources to other producers when they predictions of biogeochemical cycling and primary production. become active, such as after a large rain. Depending on the precipi- Drylands may present a unique case of microbial controls on tation regime, this fungal coupling could be more or less important to productivity because there is strong spatial and temporal heteroge- primary production. Fungi can be active at lower soil moistures than neity in resources needed for primary production. The key limiting plants (Allen, 2007; Marusenko, Huber, & Hall, 2013), supporting resource – water – is delivered in pulsed events that activate differ- their potential role as a resource reserve. Fungal hyphae may sup- ent primary producers depending on timing and amount (Collins et port more efficient translocation of resources than other potential al., 2014), potentially decoupling the activities of different producers mechanisms because movement of water and nutrients through hy- and allowing resource losses from the system. The dominant primary phae is faster than through dry soil (Frey, Six, & Elliott, 2003; Ruth, producers in drylands are vascular plants and phototrophic members Khalvati, & Schmidhalter, 2011), though diffusion and mass flow may of biological soil crusts (‘biocrusts’, diverse communities of cyano- also move resources under moist conditions. Additionally, fungi may bacteria, mosses, lichens and algae), but their activities are often act as highways that promote the movement of soil bacteria and ar- separated in both space and time. Plants occur in a patchy spatial chaea (Warmink, Nazir, Corten, & Elsas, 2011). Stable isotope tracer distribution and biocrusts occupy the soil surfaces in between plants studies have shown that N and C substrates can be translocated (interspaces). Plants generate the majority of organic matter (Aguilar between plants and biocrusts (reviewed by Rudgers et al., 2018), & Sala, 1999) and are limited secondarily by nitrogen (N; Austin et but the mechanisms of these transfers have not been documented al., 2004; Ladwig et al., 2012). Biocrusts fix atmospheric carbon (C), and could involve fungi, roots, other microbes or physical processes. and in some cases N too (Belnap, 2002). The spatial separation be- Given previous evidence that a common group of root-associated tween biocrusts on surface interspaces and plant roots deeper in soil fungi in drylands, the ‘dark septate endophytes’, generally improve means N released from biocrusts may not be accessible to plants. plant fitness (reviewed by Newsham, 2011), complex interactions Temporal separation of plant and biocrust activities may depend among plants, fungi and biocrusts may promote productivity in dry- on rainfall patterns. Only large rain events (>5 mm) sufficiently in- lands. To our knowledge, the importance of and ecological conse- crease soil moisture in the rooting zone to activate plant photosyn- quences of fungal connections for nutrient dynamics and production thesis (Huxman et al., 2004; Pockman & Small, 2010). In contrast, under different precipitation regimes have not been resolved for any biocrusts can use rain events of all sizes, although very small events dryland ecosystem. cause net losses of C because biocrusts do not remain active long We used field mesocosms in which fungal connections between enough to replace the carbon that was used to initiate photosyn- biocrusts and plant roots remained intact or were impeded for 2 years thesis (Belnap, Phillips, & Miller, 2004). N produced or immobilized to investigate three questions related to the fungal loop hypothesis. by biocrusts during times that plants are not active may be lost from (Q1) Do fungi transfer N from biocrusts to plants? N movement through the system (Belnap, 2002; Veluci, Neher, & Weicht, 2006) through or along fungal hyphae should be more rapid than through other pro- physical processes such as volatilization, photodegradation (Austin cesses, such as diffusion or independent movement by single-celled & Vivanco, 2006) and erosion (Peterjohn & Schlessinger, 1990) or microbes. (Q2) Do fungal connections improve resource reserves, and does 896 | Journal of Ecology DETTWEILER-ROBINSON ET aL. this effect vary with precipitation regime? If fungi transport N along a gra - water uptake in B. gracilis (Allen, 1982), thus fungal-mediated re- dient of availability (biocrusts with high N to plants with low N), then source transfer could also occur. C:N of plants and biocrusts should converge when fungal conn ections are present, with higher N in plants than in the absence of con nec- tions, which, in turn, will support more productivity. If C is reciprocally 2.2 | Mesocosm design transferred from plants to microbes in the biocrusts in return for N, then overall microbial carbon use efficiency should be less C limited We manipulated fungal connections (intact vs. impeded) and precipi- when intact fungal connections permit the transfer of res ources be- tation regime (small, frequent vs. large, infrequent watering events) tween biocrust and rhizosphere. If C transferred from t he rhizosphere in a full-factorial design, withn = 20 replicates per treatment combi- benefits, the photosynthetic organisms in biocrusts (pote ntially indi- nation (Figure S1). In the field, we collected biocrusts p(to 0.5–1 cm cating mixotrophy; Selosse, Charpin, & Not, 2017), the biocr usts may of soil) from around individualB. gracilis onto plastic trays then trans - be more productive and the taxa that are able to fix N (e.g. Nos tocales) planted the plants with intact rooting zone soil into plast ic pots (7.6 L) could produce more bioavailable N. Biocrust cyanobacteria may also in July–August 2013. We targeted plants that were 2–3 cm in r oot decrease their allocation to photosynthesis if plant-deri ved carbon is crown diameter (estimated shoot biomass of ~5 g) to increase t rans- available. If fungi are active during periods of low soil mois ture when plant survival compared to smaller plants. Pots were buried i nto the other organisms are not active, the benefit of fungal conn ections to ground to expose mesocosms to ambient temperature and prec ipi- resource reserves (in our study C and N) should be strongest during tation regimes. The plot was fenced to exclude large herbivo res but moist conditions when other organisms become limited by r esources was uncovered so that all pots received ambient rainfall. P ots were other than water. (Q3) Do fungal connections increase the biomass of arranged ~50 cm apart in a randomized 15 × 20 grid (an additio nal plants and/or biocrusts, and does this effect vary with precipitation regime? 200 pots in the grid were used for a related plant-biocrust i nterac- When fungal connections are intact, plant biomass and the photosyn- tion experiment; see Dettweiler-Robinson, Sinsabaugh, & Ru dgers, thetic potential of biocrusts should be higher than when connections 2018). A timeline of set-up and sampling is provided in Table S2. are impeded. If fungi ameliorate the stress of small rains to the plants and biocrusts, then fungal connections may be more important to re- source retention and productivity under precipitation regimes of many, 2.2.1 | Fungal connection treatment small rain events than regimes of few, large events. Alternatively, fungi may only benefit producers when large rains enable both plants and We used hydrophilic mesh to manipulate fungal connections t hat could biocrusts to release excess reserves of C or N. potentially form between the roots and biocrusts after mesocosm set- up. We specifically chose not to remove microbial commu nities through fungicide or sterilization because we wanted to prevent co nnectivity 2 | MATERIALS AND METHODS without reducing fungal abundance. For the intact fungal connect ions treatment, we used mesh with 50 μm pores (Small Parts, Fort Meade, 2.1 | Study system FL) to inhibit fine roots (Ares, 1976) but allow fungal hyp hae to pass through. Thus, over the 26 months of the experiment, fungal co nnec- We established a mesocosm experiment in La Puebla, NM on un- tions could re-form between the rooting zone soil and the bio crust. grazed private property (lat.: +35.978, long.: –105.995, elev.: For the impeded treatment, we used mesh with 0.45 μm pores (GE 1,800 m). This site was a one-seed juniper (Juniperus monosperma) Healthcare Life Science) to inhibit both roots and fungi (T este, Karst, savanna with scattered piñon pine Pinus( edulis). The bunchgrasses Jones, Simard, & Durall, 2006) because hyphal diameters ran ge from blue grama (Bouteloua gracilis) and Indian rice grass ( Achnatherum hy- 2 to 20 μm (Dwivedi & Arora, 1978), and thus no fungal connections menoides) dominated the groundlayer vegetation. Mature biocrusts could re-form between the rooting zone soil and the biocrust . Small contained cyanobacteria, mosses and lichens that do not f ix N bacteria/archaea could pass through both mesh sizes ( Reed & Martiny, (Microcoleus sp., Bryum argenteum, Syntrichia sp., and Pterygoneurum 2007), although the 0.45 mesh may have disrupted movement of nem- sp., Placidium sp.) as well as cyanobacteria and cyanolichens which atodes and protozoa, which range in size from ~5 to 100 μm (Swift, can fix N (Scytonema sp., and Nostoc sp., Collema sp.). Mean annual Heal, & Anderson, 1979). Because both treatments involved an initial temperature was 11°C, and mean annual precipitation was 290 mm, severing of existing fungal connections between each focal plant and ~60% of which falls in the warm months (May–October.; data from its surrounding biocrust, our 26-month fungal connections treatment 1981 to 2010, Western Regional Climate Center, 2015). may be a conservative estimate of the importance of connect ions in
We focused on B. gracilis, a widespread, dominant C 4 bunchgrass natural settings if fungal networks between plants and bioc rusts con- that hosts root-endophytic fungi including arbuscular myc orrhi- tinue to expand after 2 years of growth. zal fungi (Glomeromycota, with aseptate hyphae) and dark sep tate During set-up, mesh was placed horizontally on top of the roo t- endophytes (Ascomycota; Herrera, Poudel, Nebel, & Collins, 20 11; ing zone soil with a small hole (~5 cm diameter) in the centre to Jumpponen, Herrera, Porras-Alfaro, & Rudgers, 2017; Porra s-Alfaro allow bunchgrass shoots to pass through. Then, the biocrust was et al., 2008). Root endophytic fungi have been shown to incr ease placed on top of the mesh (~0.5 to 1 cm depth) and fungi/bacter ial DETTWEILER-ROBINSON ET aL. Journal of Ecolog y | 897 connections could then potentially re-form between the biocrust ergosterol (Olsson, Larsson, Bago, Wallander, & Aarle, 2003), and and rooting zone soil during the remainder of the experiment. ergosterol does not distinguish among mutualists, pathogens or de- Although we could not eliminate fungal connections where the composers. Samples were stored at −20°C for up to 4 months, so mesh met the pot edge or plant, we impeded most of the surface we consider these low estimates, but are comparable across treat- area between the biocrust and rooting zone. Thus, fungi were able ments. We followed methods in Wallander, Nilsson, Hagerberg, and to colonize plant roots and grow through the rooting zones soil but Bååth (2001) with the modification that no cyclohexane was added were only able to grow between the rooting zone soil and biocrust to the KOH–methanol extraction solution, and the solution was through the 50 μm mesh; fungal hyphae could not grow between heated to 80°C for 30 min. We created standards with 1 µg/ml and the rooting zone soil and biocrust through the 0.45 μm mesh. At 10 µg/ml and calculated the area under the curve at 282 nm with the end of the experiment, we visually inspected pieces of the an UltiMate 3000 high-performance liquid chromatography system mesh under the dissecting microscope and found fungal hyphae (Thermo Fisher Scientific). To determine fungal colonization of roots, growing through the 50 μm mesh but none through the 0.45 μm we stained roots with ink (Parker Quink, Parker) following Vierheilig, mesh. Coughlan, Wyss, and Piche (1998), with the modification of soaking in KOH at room temperature until roots cleared. We assessed root colonization by dark septate and aseptate hyphae via microscopy 2.2.2 | Precipitation regime following McGonigle, Miller, Evans, Fairchild, and Swan (1990). We occasionally found but did not count arbuscules because we focused To assess the effect of precipitation event size on the fungal loop, on the hyphae that are the linear transport structures. we delivered the same volume of water in different frequencies In September 2014 (1 year after mesocosm set-up), we checked a to compare small events that we expected to activate surface mi- subsample of ~15 pots for roots occurring above the mesh and found crobes to large events that we expected to activate both plants none. We measured biomass of the roots that grew above the mesh and biocrusts (Collins et al., 2014). We applied small, frequent when we harvested in October 2015 (see below). events (100 ml/pot once per week, a 2.5 mm event) or large, in- frequent events (400 ml/pot once per month, a 10 mm event) during August–October in 2013 and May–October in 2014 and 2.4.2 | Nitrogen tracer experiment (Q1) 2015 (Dettweiler-Robinson et al., 2018). These event sizes and frequencies are within historical (1981–2010) variability for this We collected ~10 leaves per plant prior to ( N = 37) and 72 hr after region (Western Regional Climate Center, 2015). The 0.45 μm (N = 26) adding the tracer to determine 15N enrichment of the fo- mesh did not have an effect on soil moisture compared to pots liar N pool. We collected roots from below the mesh ( N = 26) and with no mesh after a single watering event (Dettweiler-Robinson biocrust (N = 10) samples after ~72 hr from pots where tracer was et al., 2018). added. To obtain a natural abundance 15N value for biocrusts and roots, we additionally collected biocrusts ( N = 5) and roots (n = 3 with 0.45 μm mesh, n = 7 with no mesh [Dettweiler-Robinson et al., 2.3 | Enriched stable isotope tracer experiment 2018]) from pots that had experienced the small, frequent ateringw regime that had no tracer added (all pots with 0.50μm mesh had We only used plants from the small, frequent precipitation regime one tracer added). We dried samples for 3 days at 60°C, groundaves le day after watering (the surface was visibly dry) to ensure that microbes and roots with liquid N using a mortar and pestle and packed 4g mof had been active but to minimize the potential for direct capillary trans- leaves and 10 mg of biocrust into tin capsules (4 × 6 mm, Costech). fer of tracer through wet soil. We randomly assigned pots to three sub- Samples were run on an ECS 4010 Elemental Analyzer (Costech)and sampling periods in October 2015 to ensure that an ambient rain event a Delta V Isotope Ratio Mass Spectrometer (Thermo Scientific) at would not affect all replicates. We added a total of 800 μl of 0.86 M the University of New Mexico Center for Stable Isotopes tobtain o 15 15 N-NaNO3 (8 mg N) to two points <2.5 cm in diameter on biocrust on weight per cent C (%C), weight per cent N (%N) andδ N values opposite sides of the plant (~6 cm from root crown). The surface dried (precision < 0.3‰). within minutes and leaves did not touch the soil during application.
2.4.3 | Resource reserves (Q2) 2.4 | Responses For pots in the small, frequent precipitation regime, we us ed weight 2.4.1 | Below-ground treatment effects per cent C and N from the natural abundance samples, as above. Leaf and biocrust samples from the large, infrequent precipitation To estimate fungal abundance in the mesocosms after 26 months, regime were also collected in early October 2015, dried and packed we measured ergosterol content and hyphal colonization of roots as above, and run on an elemental analyzer (Carlo Erba NC2100, CE in October 2015. Arbuscular mycorrhizal fungi do not produce Elantech) but not analysed for stable isotopes. 898 | Journal of Ecology DETTWEILER-ROBINSON ET aL.
We also evaluated potential extracellular enzyme activity (EEA) microsite (biocrust or rooting zone soil) or morphotype (d ark septate for biocrust (separated from plant roots by mesh) and rooting or aseptate hyphae), on the ergosterol content (natural lo g trans- zone soil samples to calculate carbon use efficiency (below). Soils formed) and root colonization (logit transformed), respectively , with were collected in October 2015, sieved (2 mm) and homogenized. pot as a random effect. We compared the biomass of roots above Samples were assayed for the potential activities (nmol g−1 hr−1) of the mesh at harvest in 2015 (square root transformed) by f ungal β-1,4-glucosidase (BG) that breaks down cellulose (C-acquiring), connection and precipitation regime treatments and their int erac- β-1,4-N-acetylglucosaminidase (NAG) that hydrolyses glycosidic tion using linear models. bonds in chitin (N-acquiring), and leucine aminopeptidase (LAP) that hydrolyses amino acid residues from polypeptides (N-acquiring) using fluorogenic methylumbelliferyl-linked substrates (follow- 2.5.2 | Nitrogen tracer experiment (Q1) ing protocol in Stursova, Crenshaw, & Sinsabaugh, 2006; Table S3, Figure S4). Assays were run at pH 8. We calculated the total 15N content (μg) for leaf and root samples by calculating the atom per cent of 15N from the δ15N value, and multiplying it by the average %N for each plant and the sho ot or root 2.4.4 | Biocrust and plant production (Q3) biomass. To calculate total biocrust 15N content, we assumed that total biocrust mass in the pots averaged 140 g based on average bulk To estimate the photosynthetic capacity of biocrust phototro- density of 0.55 (from 2 cm diameter × 1 cm depth soil cores) an d total phs (Yeager et al., 2004), we sampled chlorophyll a in August 2013 area of 250 cm2. (Appendix S6) and October 2015. We aggregated two randomly placed For leaf tissue, we calculated excess 15N (μg) for each plant by 11 mm diameter × 50 mm depth samples into a 1.5 ml centrifuge tube. subtracting the natural abundance value of total 15N from the total We dried and weighed each sample then added 1 ml of dimethyl sul- 15N value 72 hr after tracer addition. Leaves collected at 8 hr a nd foxide. We used a single DMSO extraction at room temperature for 30 hr did not differ from natural abundance values (Appendi x S5). 3 days which should capture ~75% of the chlorophyll content (Castle, We did not calculate excess 15N for roots because we did not disturb Morrison, & Barger, 2011). We calculated chlorophyll a content (μg/g pots to collect root samples before tracer was added. For l eaf excess soil) by absorbance at 665 nm on a Synergy H1 Hybrid plate reader 15N and root total 15N, we tested for a fungal connection treatment (Biotek) with 750 nm as a reference wavelength (Castle et al., 2011). effect with a linear model that included the root biomass above the We non-destructively assessed shoot biomass in August 2013 mesh in each pot as a covariate. All responses were natural lo g trans- after setting up the mesocosms (Appendix S6). In October 2015, we formed to fit model assumptions. destructively harvested plants and washed roots separately from To assess the difference in water content for pots after the above and below the mesh. Shoots and roots were dried at 60°C tracer experiment, 2.5 cm diameter soil cores were taken under for 3 days and weighed. We estimated total seed biomass in 2015 the mesh to a depth of 5 cm when plants were harvested 72 hr by multiplying average seed mass per inflorescence (from seven after tracer was added. Soils were weighed immediately then dried randomly chosen inflorescences per plant) by the total number of for 3 days at 60°C and reweighed to calculate gravimetric wat er inflorescences. content.
2.5 | Analyses 2.5.3 | Resource reserves (Q2)
All analyses were run in R version 3.3.1 (2016-06-21; R Core Team, We assessed biocrust, soil and plant responses from destructive 2016) with October 2015 data. We used Q–Q plots to assess nor- harvest October 2015. We analysed molar C:N with mixed-ef- mality of residuals and plotted residuals versus fitted va lues to as- fects models that included the fixed effects of fungal connection, sess homogeneity of variances. All general linear models w ere run in precipitation regime and sample type (leaf or biocrust), all inter- base R. General linear mixed-effects models were run with pac kage actions, and pot identity as a random effect. We decompose d
C:N of 7.2 and for molar C:N of rooting zone soil of 10.5 (Xu, (mean % of total root biomass above the mesh = 27.11 ± 1.8 SE; Thornton, & Post, 2013). We used mixed-effects models with fun- median = 25.0%). gal connection, precipitation regime and sample type (biocrust or rooting zone soil), as above. 3.2 | Q1: 15N content of roots trended higher when fungal connections were intact 2.5.4 | Biocrust and plant production (Q3) Only plants in the small, frequent precipitation regime were used We assessed biocrust and plant responses from October 2015 for Q1, so there are no stable isotope results for the effect of pre - after 26 months of fungal connection treatment and 15 months of cipitation regime treatment. The fungal connection treatment rainfall regime treatment. All models included the fixed effects of did not affect natural abundance leaf or root 15N content (leaf: 2 fungal connection, precipitation regime, their interaction and initial mean = 1.09 ± 0.10 SE mg, F1,35 = 0.63, p = .434, R = .0; root: 2 plant shoot mass or biocrust chlorophyll a content as a covariate mean = 0.259 ± 0.027SE mg, F1,8 = 1.91, p = .204, R = .09). to account for initial differences (Appendix S6). Chlorophyll a con- There was significant excess 15N in leaf samples 72 hr after the tent and biomasses were square root-transformed. Multivariate tracer was added (intercept F1,23 = 226.94, p < .001, Figure 1) and mixed-effects models for plants accounted for correlated biomass a positive relationship with biomass of root above the mesh (coef- responses among roots, shoots and seeds (Appendix S7, TableS8). ficient = 0.105, F1,23 = 5.28, p = .031), but there was no significant 2 Because treatments interacted with plant tissue type in multivari- effect of connection treatment (F1,23 = 0.22, p = .636; R = .12). ate models, we decomposed individual biomass variables in eparates Because leaf 15N did not respond to impeded fungal connections, linear models. we do not know whether fungi inside roots were sequestering N,r o if plants were primarily storing N in below-ground tissue,a possible strategy because we harvested at a time close to plant escencesen 3 | RESULTS in October. Root total 15N (collected below the mesh) averaged 20% lower 3.1 | Below-ground treatment effects when connections were impeded than when they were intact 2 (F1,23 = 2.23, p = .148, R = .37). The biomass of roots above the Fungal abundance metrics responded to both precipitation e- r mesh was a strong, positive predictor of the total15N of roots below gime and connection treatment after 26 months (Table 1). Inbi- the mesh (coefficient = 0.061,F 1,23 = 15.02,p = .001). We cannot ocrust, there was 54% lower ergosterol in the large, infrequent determine if 15N was in root tissue or inside fungal structures within (mean = 0.16 μg per g, 95% CI = 0.11–0.21) than the small, frequent the root tissue. 15 regime (0.34 μg per g, 95% CI = 0.26–0.44;t 145 = 3.81, p < .001), but Natural abundance total N in biocrusts averaged 1.85 ± 1.06 no difference in ergosterol by precipitation treatment in the rooting SE mg, and after 3 days, biocrusts retained substantial amounts 15 zone soil (0.04 μg per g, 95% CI = 0.03–0.06,t 145 = −0.42, p = .679). of the tracer, with N content = 1.24 ± 0.38SE mg. On average, The fungal connection treatment had no effect on ergosterol con- less than 1% of the tracer added was found in the leaf or root tent in either microsite. In the small, frequent regime, septatea hy- tissue. 64% of the15 N added remained in the biocrust after 72 hr. phal colonization was 50% lower when connections were impeded Gravimetric water content did not differ by connectioneatment tr
(8%, 95% CI = 4–13) than intact (16%, 95% CI = 16–23;t134 = 2.15, (F1,52 = 1.47,p = .230) and averaged 1.9% ± 1.2SE at harvest after p = .034). In the large, infrequent regime, there was no difference 72 hr. in aseptate hyphal colonization between connection treatment (7%,
95% CI = 4–10,t 134 = −0.87, p = .387). Neither connection or precipi- tation regime had an effect on dark septate colonization (20%, 95% 3.3 | Q2: Fungal connections altered the C:N of CI = 16–24). plants and microbial carbon use efficiency but only The roots that grew above the mesh after October 2014 had under a large events rainfall regime potentially unimpeded connections with the biocrust andthere - fore should have decreased the effectiveness of this treatment. Under the large, infrequent precipitation regime, leaf C:N was However, this decreased effectiveness did not override treatment 18% higher when fungal connections were impeded than intact effects on plant biomass (see below). Roots were in a shallow soil (t108 = −3.45, p = .008, Figure 2, Table 1), indicating stoichiometric layer below the biocrust filaments. There were no treatment fe- convergence of plants on the higher N content of biocrusts when fects on the biomass of roots above the mesh (mean = 4.02 ± 0.41SE connections were intact. This shift was driven more by N content in g, median = 2.88g; Connection: F1,71 = 0.51, p = .475; Precipitation: plants (43% lower N in impeded than intact) than by C content (32%
F1,71 = 1.05, p = .301; Connection × Precipitation: F1,71 = 1.03, lower C in impeded than intact). Fungal connections had no effect p = .314) and most of the biomass of roots was below the mesh under small, frequent rains (Figure 2, Table 1). 900 | Journal of Ecology DETTWEILER-ROBINSON ET aL.
.205 .032 .001 .779 p
1,69 n.i. n.i. 1.64 4.79 11.31 0.08 Total n.i. n.i. .20 n.i. n.i.
.089 .177 .629 .247 .449 p F
1,68 n.i. 2.98 1.85 0.24 1.36 0.58 Root n.i. n.i. .07 n.i. n.i. ≤ .05 are shown in bold and rows with ‘n.i.’ egime on fungal abundance, nitrogen p
.115 .005 .017 .239 p F
eaf. 1,58 n.i. n.i. 2.56 8.49 6.06 Seed 1.42 n.i. n.i. .10 n.i. n.i. .351 .004 .763 <.001
p F
1,72 n.i. n.i. 1.72 8.99 23.95 0.09 Plant Shoot n.i. n.i. .28 n.i. n.i.
.304 .113 .728 .290 p F
a 1,70 n.i. n.i. 1.07 2.58 0.12 1.14 Biocrust Chl. n.i. n.i. <.01 n.i. n.i. .004 .007 .359
p F <.001 .432 .505 .334 sting for effects of fungal connection treatment and precipitation r 2 n.i. n.i. n.i. 8.24 7.33 0.84 CUE Biocrust versus Root Zone Soil 45.09 0.62 0.44 0.93 , for colonization were aseptate and septate, and for C:N were biocrust or l .024
.742 .978 .915
p X <.001 .213 .039 .001 2 n.i. 5.03 n.i. 0.11 0.00 0.01 N dynamicsN C:N Biocrust versus Leaf 356.06 1.55 4.27 10.57 .035 .005 .030
p X .439 .050 .085 .090 2 n.i. n.i. n.i. 4.44 7.97 4.61 Colonization Aseptate versus Septate 0.60 3.82 2.96 2.88
.946 .005 .920 p X <.001 .885 .034 .939 2 3.00 .083 n.i. n.i. 0.00 7.83 0.01 Fungal abundance Fungal Ergosterol X Biocrust versus Root zone Soil 61.23 0.02 4.50 .54 (marginal) .20 (marginal) .90 (marginal) .54 (marginal) 0.00 Results from general linear mixed-effects and general linear models te Sample types for ergosterol and CUE were biocrust and rooting zone soils 2 Precip. Row in grid Col. in grid Value in 2013 Conn. × Precip. Precipitation Connection Factor
Sample type Type × Conn.Type Type × Precip.Type Type × Conn.Type × R Note: mean the factor was not included. TABLE 1 TABLE dynamics, biocrust production and plant biomass DETTWEILER-ROBINSON ET aL. Journal of Ecolog y | 901
connection or precipitation regime (p > .10; Table 1). Although we intended to provide similar biocrusts and plants to each treatment combination, there were some random differences in production within the first month of transplanting in 2013 (Appendix S6), which is why we included the productivity in 2013 as a covariate in the models of productivity at harvest in 2015.
4 | DISCUSSION
Our results were consistent with the fungal loop hypothesis because fungal connections were important for long-term plant and microbial resource dynamics and plant productivity. We found little evidence of shifts in fungal abundance or composition, and because of the differences in plant performance, our results support the hypoth- esis that intact fungal connections improve resource acquisition, transfer or retention. Additionally, over the short term, fungal con- nections may move available N to deeper plant roots more rapidly than through other processes, and thus prevent photodegradation or leaching loss. Given the potential growth of roots above the mesh for up to 1 year before harvest, we consider our results conservative in estimating the role of fungi in plant productivity. However, we did not find strong benefits of fungal connections to biocrusts. Thus, FIGURE 1 Estimated marginal means ± 95% CI of (a) leaf excess 15N and (b) root total 15N 72 hr after 8 mg 15N-nitrate tracer was the ‘loop’ was incomplete due to the lack of apparent reciprocal ex- added to the biocrust by fungal connection (intact = light grey; change of plant C for biocrust N. impeded = dark grey)
Although biocrust C:N was not affected by the fungal or pre- 4.1 | Q1: Fungi transferred N from biocrusts cipitation treatments, microbial carbon use efficiency in biocrusts to roots and rooting zone soil did respond to treatments. Under the large, infrequent precipitation regime, biocrust CUE was 7% greater When fungal connections were intact, more N moved from bi-
(t128 = −1.94, p = .055) and rooting zone CUE was 10% greater ocrusts to plant roots. The biological effect size was large (20% 15 (t128 = −3.11, p = .002) when fungal connections were impeded than less total N in roots when connections were impeded than in- intact (Figure 2, Table 1). Fungal connections had no effect on CUE tact), but due to high variability among samples, we did not find a under the small, frequent precipitation regime. statistically significant effect (p = .148). The distance between the tracer and the plant base was small (<10 cm), and the root-free bi- ocrust layer was small (~1 cm thick), and thus diffusion through the 3.4 | Q3: Plant biomass, but not biocrust soil cannot be ruled out. Diffusion should have occurred equally photosynthetic capacity, increased with fungal between the two treatments because the mesh treatment did not connections affect water content, and thus there may have been some role of fungal connections between biocrusts and roots that enhanced N Under the large, infrequent precipitation regime, impeding fungal transfer in some cases. Results support prior observations that N connections reduced shoot biomass by 24% (t72 = 3.56, p = .001), moves faster than can be explained by physical processes alone seed biomass by 46% (t58 = 2.64, p = .011) and total biomass by (Rudgers et al., 2018). Although there was no evidence of transfer
25% (t69 = 3.05, p = .003) compared to intact connections (Figure 3, to leaves by 30 hr, at 72 hr, there were trends that showed re- Table 1). In contrast, under the small, frequent precipitation regime, duced transfer to roots when fungal connections were impeded. the presence of fungal connections did not affect plant biomass re- The tracer would have had to move through physical processes (or sponses. The plants in the small, frequent watering regime had 10% bacteria/archaea) from the biocrust to the rooting zone soil in the smaller shoots and 14.7% lower total biomass than in the large, in- impeded connections treatment, and transport in this treatment frequent regime, indicating the small, frequent was a less beneficial was slower than in the intact connections treatment. We did not regime. Root biomass did not respond to any treatment (p > .10; conduct this experiment in the large, infrequent precipitation re- Table 1). Biocrust chlorophyll content also did not respond to fungal gime treatment pots because of concerns with diffusion through 902 | Journal of Ecology DETTWEILER-ROBINSON ET aL.
FIGURE 2 Estimated marginal means ± 95% CI of fungal connection (intact = light grey; impeded = dark grey) and precipitation regimes (small, frequent vs. large, infrequent) treatments for (a) biocrust and (b) leaf tissue C:N ratio and (c) biocrust and (d) carbon use efficiency (CUE) calculated from extracellular enzyme activity. Different letters within each level of precipitation regime indicate significant differences between fungal connection treatments at p ≤ .05, while a prime symbol indicates differences at .05 < p ≤ .10 (false discovery rate corrected)
FIGURE 3 Estimated marginal means ± 95% CI of fungal connection (intact = light grey; impeded = dark grey) and precipitation regime (small, frequent vs. large, infrequent) treatments of plant (a) shoot, (b) seed, (c) root and (d) total biomass. Different letters within each level of precipitation regime indicate significant differences between fungal connection treatments at p ≤ .05 (false discovery rate corrected)
wet soil. Given that the plant response to fungal connection was We cannot yet determine which fungal taxa provided translo- stronger under large, infrequent than small, frequent rains, we cation services or whether C was rewarded to the fungi (Hortal et consider these tracer results to be a conservative estimate of how al., 2017), but our design ruled out any organisms >50 μm in both much fungal connections mediate resource transfer. connection treatments. Thus, we conclude that soil microbiota DETTWEILER-ROBINSON ET aL. Journal of Ecolog y | 903
>0.45 μm in size may be key regulators of N transfer in drylands. the plants being smaller (Figure 3) and therefore providing less C Microarthropods can affect soil resource cycling (Darby & Neher, via above- and below-ground litter or root exudates. Additionally, 2016), and their movement between biocrusts and rooting zone given that C from plants can move to biocrusts (Green, Porras- soil may have been impeded by our mesh. Prokaryotes that trav- Alfaro, & Sinsabaugh, 2008), impeding the fungal connections may elled along fungal hyphae (Warmink et al., 2011) could still pass reduce that direct input of C to biocrust microbes. Although we do through both mesh types, but these microbes would lack fungal not have evidence to support that mechanism, if we had observed highways in the 0.45 μm mesh treatment and any movement using that impeded connections reduced biocrust CUE but did not affect the fungal network would be impeded. Therefore, we cannot rule rhizosphere CUE, we could conclude that transfer of below-ground out the potentially important role of fungal networks for transport plant inputs to biocrust were impeded by the mesh. Instead, we of bacteria or archaea involved in resource transfer or process- found altered CUE in both the rooting zone soil, which is in di rect ing. Fungi remain the best candidate taxa for translocating water contact with roots, and the biocrust, which is separated by the mesh and nutrients because of their small diameter, linear networks of treatment. Thus, we did not find support for a reciprocal, fu ngal-me- hyphae, shared presence in both the plants and biocrusts (Porras- diated transfer of C from plants to biocrusts in exchange for t he N Alfaro et al., 2011), and rapid cytoplasmic movement within hy- transfer from biocrusts to plants. phae (Lew, 2005). Fungi are generally considered more active at lower soil moistures than bacteria, but soil communities with different fungal-to-bacterial ratios did not differ dramatically in 4.3 | Q3: Fungi increased plant biomass only under respiration activity (Manzoni, Schimel, & Porporato, 2012), sug- a large events precipitation regime gesting similarly active fungi and bacteria. Thus again, we cannot rule out the role of bacteria in resource transfers in low moisture Previous research has shown that fungi can redistribute water periods. (Allen, 2007; Prieto, Armas, & Pugnaire, 2012) and translocate C The plant response to our treatment indicates that only fungal and N between plants (He, Critchley, & Bledsoe, 2003). Although connectivity between biocrust and plant was disrupted, rather than we have not parsed the effects of different mechanisms, intact fun- major shifts in the root endophyte community (as determined by gal connections benefitted plants, with effects that depended on morphotypes) or total non-mycorrhizal fungal abundance (as deter- the climate context. Similar context dependency on resource avail- mined by ergosterol). However, although there was a 50% reduction ability has been found in other plant–microbe systems (reviewed in aseptate hyphae when connections were impeded compared to by Hoeksema et al., 2010). For example, in mesic grasslands, ferti- intact in the small, frequent rainfall regime, there were no differ- lization with P changed the plant–AMF interaction from beneficial ences in the plant productivity in these treatment combinations. to negative or neutral (Johnson, Wilson, Wilson, Miller, & Bowker, 2015). Similarly, greater availability of simple sugars and organic N sources increased the growth of plants colonized by dark septate 4.2 | Q2: Fungal connections improved endophytes compared to those without endophytes (Mayerhofer, resource reserves Kernaghan, & Harper, 2013). In drylands, where water is the pri- mary resource that drives productivity, regimes dominated by Further strengthening the idea that N is a key resource affected by large rain events may be critical for fuelling plant–fungal symbi- fungal connections, after 26 months C:N in plants was more similar oses. For example, fungi have been shown to take up to 30% of to that of biocrusts when fungal connections were intact than im- plant C (reviewed in Brüggemann et al., 2011), and thus plants peded, consistent with the hypothesis that fungi transport N along may need sufficient rainfall inputs to produce enough biomass to a gradient (Boberg, Finlay, Stenlid, & Lindahl, 2010) from the high N maintain beneficial root-associated fungi. In turn, under large rain content biocrusts to the low N content plants (Collins et al., 2014). events, fungi may accelerate uptake of limiting resources, allow- However, this effect was only evident in the large, infrequen t precip- ing plants to build more biomass relative to regimes of many, small itation regime. Thus, if future climate shifts to small, frequent events events (Mandyam & Jumpponen, 2005; Smith & Read, 2008) as ob- (Petrie, Collins, Gutzler, & Moore, 2014), there may be less reten- served in our study. Biocrusts were less responsive to either fungal tion in these already resource-limited systems. The lack of change connection or precipitation treatments than were plants, suggest- in biocrust C:N or chlorophyll, despite altered CUE, may reflect a ing their greater resilience to future changes in precipitation event shift in microbial composition (e.g. Ramirez, Craine, & Fierer, 2012) sizes. However, a recent study in a similar ecosystem showed or in allocation by the same community that compensates for overall declines in biocrusts, particularly N-fixing cyanobacteria, dur- changes in resource availability. ing a prolonged, extreme experimental drought (Fernandes et al., Microbial carbon use efficiency increased under impeded fun- 2018), indicating high sensitivity of biocrusts to the total amount gal connections under the large, infrequent precipitation regime, of precipitation. These context dependencies are important to un- suggesting that microbes in both biocrust and rooting zone soils ex- derstand because drought duration (Maloney et al., 2014) and the perience relatively greater C limitation than N limitation when con- frequency of extreme precipitation event sizes (Polley et al., 2013) nections are impeded than intact. This may be an indirect effect of are predicted to increase in drylands of the southwestern United 904 | Journal of Ecology DETTWEILER-ROBINSON ET aL.
States and globally (IPCC & Core Writing Team, 2014). However, who assisted with laboratory and field work. Lee Taylor, Matthew recent regional trends show an increased frequency of small Bowker, Scott Collins, Anny Chung, Lukas Bell-Dereske, the events in our region (Petrie et al., 2014). Given the uncertainty in Florida State University ecology reading group and six review- future scenarios, understanding how climate variables affect spe- ers provided valuable manuscript feedback. We thank Eva's dad, cies interactions and species’ nutrient cycling roles is essential to Creighton Robinson, for letting us sample on his property. Support improving predictions for the future. provided by NSF awards 1557135, 1557162 and 1456955, UNM In our previous publication to determine whether fungal con- Department of Biology and Biology Graduate Student Association nections mediate net competition or mutualism between plants grants, and NSF Awards to the Sevilleta Long-Term Ecological and biocrusts (Dettweiler-Robinson et al., 2018), we compared Research Program. mesocosms with no mesh (all fungal, root, microarthropod, etc. connections intact; not included in this current publication) to AUTHORS' CONTRIBUTIONS 0.45 μm mesh (fungal connections impeded; the same pots as in E.D.-R collected and analyzed data and wrote the manuscript. J.A.R this current publication) and crossed these with plant and bio- and R.L.S. assisted with the design and implementation of the field crust removal treatments (not included in this current publication, work, training for lab work, and data analysis. All authors contrib- N = 200) to simulate loss of producers from the ecosystem. In that uted substantially to writing the manuscript. study, total plant biomass was similarly lower (16%) when fungal connections were impeded than when all connections were intact. DATA AVAILABILITY STATEMENT However, in both the previous and the current analysis, the initial Context dependency of effect of fungal connections between effect of impeding fungal connections (year 1, 2013–2104) was to plants and biocrusts. Environmental Data Initiative. https ://doi. increase rather than reduce plant growth (data not shown), even o r g / 1 0 . 6 0 7 3 / p a s t a / b 0 c 9 4 0 7 7 e 8 4 4 c a 6 2 9 e b e d c 8 b c c e 5 f 5 5 0 though impeded connections reduced biomass by the time of har- (Stricker, 2019). vest. Pots with all connections intact (Dettweiler-Robinson et al., 2018) or with fungal connections intact (this manuscript) may have ORCID allowed passage of pathogenic or herbivorous organisms from the Eva Dettweiler-Robinson https://orcid.org/0000-0002-9742-3434 surface to the roots, and those organisms may have dispropor- Jennifer A. Rudgers https://orcid.org/0000-0001-7094-4857 tionately consumed plants that were initially larger. Alternatively, microbes may have switched from detrimental to beneficial (e.g. REFERENCES Johnson & Graham, 2013) during the initial disruption of com- Aguilar, M. R., & Sala, O. E. (1999). Patch structure, dynamics and Trends in munities required to create the mesocosms, but once conditions implications for the functioning of arid ecosystems. Ecology & Evolution, 14(7), 273–277. https ://doi.org/10.1016/ stabilized, the fungal connections between plants and biocrusts S0169-5347(99)01612-2 became net beneficial. Allen, M. F. (1982). 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