Ecosystems (2012) 15: 188–199 DOI: 10.1007/s10021-011-9502-2 2011 Springer Science+Business Media, LLC
Ectomycorrhizal Networks of Pseudotsuga menziesii var. glauca Trees Facilitate Establishment of Conspecific Seedlings Under Drought Marcus A. Bingham* and Suzanne Simard Department of Forest Sciences, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
ABSTRACT d13C increased with drought due to increasing water use efficiency, but was unaffected by distance from tree or network potential. We conclude that P. menziesii seedlings may benefit from the presence of established P. menziesii trees when growing under climatic drought, but that this benefit is contingent upon the establishment of an EM network prior to the onset of summer drought. These results suggest that networks are an important mechanism for EM plants establishing in a pattern consistent with the stress-gradient hypothesis, and therefore the importance of EM networks to facilitation in regeneration of EM trees is expected to increase with drought.
Ectomycorrhizal (EM) networks are hypothesized to facilitate regeneration under abiotic stress. We tested the role of networks in interactions between P. menziesii var. glauca trees and conspecific seedlings along a climatic moisture gradient to: (1) determine the effects of climatic factors on network facilitation of Pseudotsuga menziesii (Mirb.) Franco var. glauca (Mayr) seedling establishment, (2) infer the changing importance of P. menziesii var. glauca parent trees in conspecific regeneration with climate, and (3) parse the competitive from facilitative effects of P. menziesii var. glauca trees on seedlings. When drought conditions were greatest, seedling growth increased when seedlings could form a network with trees in the absence of root competition, but was reduced when unable to form a network. Survival was maximized when seedlings were able to form a network in the absence of root competition. Seedling stem natural abundance
Key words: climate change; climatic gradients; competition; Douglas-fir; ecophysiology; facilitation; mycorrhizal networks; plant water relations; reforestation; stress-gradient hypothesis.
INTRODUCTION Ectomycorrhizas have been demonstrated to link the root systems of different plant individuals, forming ectomycorrhizal (EM) networks (Newman 1988), and recent studies have shown that they can influence establishment of Pseudotsuga menziesii (Teste and others 2010). Some basic tenets of plant community ecology were challenged in 1997 when net carbon (C) transfer between P. menziesii and paper birch (Betula papyrifera) was demonstrated in
Received 31 May 2011; accepted 21 October 2011; published online 22 November 2011 Electronic supplementary material: The online version of this article (doi:10.1007/s10021-011-9502-2) contains supplementary material, which is available to authorized users. Author Contributions: Bingham had the principal role of designing the study, writing and interpreting, analyzing data, and coordinating and conducting field and laboratory analyses. Simard coordinated funding for the project and assisted in designing the study, as well as contributing to the writing and interpretation. *Corresponding author; e-mail:
[email protected]
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Network Facilitation Under Drought the field, with more transferred through EM networks than soil pathways (Simard and others 1997b). The relative importance of these transfer pathways, nevertheless, remains debated (Robinson and Fitter 1999; Simard and Durall 2004; Philip and others 2010). If, in fact, net C transfer through EM networks is common, then traditional models of plant community dynamics (for example, Tilman 1988) need modification to allow for a multiplicity of interactions among plants, rather than simply competition determined by resource ratios (Simard and others 1997a, b; Read 2002; Brooker and Callaway 2009; van der Heijden and Horton 2009). Plant-to-plant facilitation has been shown to be most important when environmental stress is high, as predicted by the stress-gradient hypothesis (Greenlee and Callaway 1996; Callaway and others 2002; Castro and others 2004; Liancourt and others 2005; Cavieres and others 2006), and EM networks may play a role in this facilitation through either interplant transfer of nutrients or water, or through mycorrhizal fungal colonization of establishing plants (Dickie and others 2002, 2005; Querejeta and others 2003; Dickie and Reich 2005; Nara 2006; Egerton-Warburton et al 2007). Transfer of C, nitrogen (N), and water between EM seedlings is known to occur, but how it is affected by resource constraints (for example, water, nutrients, and light), biotic factors and disturbances is poorly understood. Of these factors, tree species composition, fungal species composition, mycorrhizal type, inter-tree competition for resources (especially light), tree phenology, tree size, growth rate, and soil disturbance level have all been shown to affect the magnitude or direction of net transfer (Arnebrant and others 1993; Simard and others 1997b; Teste and others 2010; Philip and others 2010). There is also evidence that both seedlings and mycorrhizal mycelia receive hydraulically redistributed water from mature trees in a number of ecosystems, that this transfer is facilitated by EM networks, and that this facilitation is likely to occur in P. menziesii var. glauca forests (Querejeta and others 2003; Brooks and others 2002, 2006; Egerton-Warburton and others 2007; Schoonmaker and others 2007; Bingham and Simard 2011a). Recent summer droughts in southern interior British Columbia (BC), and the predicted increase in average annual temperature and decrease in summer precipitation for this region with rising atmospheric CO2 concentrations, have raised concerns about forest recruitment following harvest or natural disturbance (Hamann and Wang 2006; Nitschke and Innes 2008; Spittlehouse 2008). Ensuring that forests regenerate successfully under
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shifting climate, particularly those in the most vulnerable ecosystems (for example, forest near its climatic limits (Hamann and Wang 2006)), requires that we design silvicultural systems using a sound understanding of the climatic, site and biotic factors regulating seedling recruitment. Understanding the role of EM networks in forest regeneration in stressed ecosystems is, therefore, of increased importance in designing mitigation and adaptation strategies for climate change. A better understanding of competitive and facilitative effects of residual trees on seedling recruitment across a range of stand structures and climatic regions is needed to design silvicultural systems for a range of forest types and changing climatic conditions. To elucidate the effects of climate change on interactions among plants in EM forest communities, plant–fungus–plant interactions should be examined at several field locations that differ in regional climate. In this study, we tested whether mycorrhizal networks play an increasingly important role in recruitment of P. menziesii var. glauca seedlings with increasing climatic drought stress. We chose P. menziesii var. glauca for five reasons: (1) it has a broad geographic range and environmental tolerance across the North American Cordillera; (2) its range is likely to expand upward in elevation and latitude with warming, but contract at its driest boundaries (for example, forest–grassland ecotone); (3) regeneration success has been shown to depend on mycorrhization in the field; (4) it is one of the most studied tree species in the world, as is its mycorrhizal community; and (5) it is one of the most important tree species commercially and ecologically in western North America. Current management of Pseudotsuga menziesii (Mirb.) Franco var. glauca (Mayr) (interior Douglas-fir) is based on the assumption that interactions between P. menziesii seedlings and trees are the same regardless of regional climatic differences. However, inter-tree competitive and facilitative interactions have been shown to be highly variable across precipitation gradients of P. menziesii var. glauca forests, and EM networks can mediate these interactions (Simard 2009). Furthermore, proportional colonization of P. menziesii var. glauca root tips by the predominant networking fungi, Rhizopogon spp. (Beiler and others 2010), has been found to increase with water deficiency (Kazantseva and others 2009). Although our group has shown that the same genets of Rhizopogon spp. link all mature P. menziesii var. glauca trees with understory regenerating seedlings in uneven-aged forests, and that access to both roots and mycelial networks of
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mature trees facilitates long-distance transfer of C and N to seedlings, the climatic conditions under which EM network-mediated facilitation of seedling establishment is most likely to occur have not been established (Beiler and others 2010; Twieg and others 2009; Teste and others 2010). The objective of this study was to determine whether P. menziesii var. glauca seedling establishment is affected by access to an EM network of mature residual trees, and whether this varies by regional climate and distance from the residual tree. To predict climatic effects on the interaction between EM network and distance factors, nine field sites were established along a climatic moisture gradient, where spatial climatic variability served as a proxy for climate change. The following hypotheses were tested: (1) seedling survival and growth are enhanced by EM colonization via EM networks because of greater access to soil resources, (2) EM network facilitation of seedling establishment should be greatest at intermediate distances from residual trees, where EM network facilitation outweighs competition from tree roots, and (3) facilitation of seedling recruitment by the tree via the EM network increases with drought, in keeping with the stress-gradient hypothesis, and thus should manifest in the interactive effects of regional climate, EM networks, and distance from mature trees on seedling establishment. We tested these hypotheses in very dry, dry, and moist P. menziesii var. glauca forests of BC, where a strong precipitation gradient is generated by the rain shadow of the Coast Mountain massifs in the west, and orographic lift of the Columbia Mountain highlands in the east. These forests interface with the northern extension of the sagebrush steppe of North America, and hence are predicted to experience dramatic vegetational shifts over the next century (Spittlehouse 2008).
MATERIALS
AND
METHODS
Experimental Design The field study was conducted at nine sites located across a climatic gradient that varied according to drought (that is, the ratio of mean annual potential evapotranspiration to precipitation) (Figure A1). P. menziesii var. glauca was the dominant tree species at all sites, and we attempted to establish three sites each in three subzones representing Very Dry, Dry, and Moist precipitation regimes: the Very Dry Hot P. menziesii var. glauca (IDFxh) subzone, Dry Cool P. menziesii var. glauca (IDFdk) subzone, and Moist Warm Interior Cedar Hemlock (ICHmw)
subzone (Lloyd and others 1990). Note that most but not all of the sites ultimately chosen belonged to these three biogeoclimatic units (see Table A1). The three sites within each subzone were at least 2 km apart. Each site had been ‘clearcut with reserves’ (that is, clearcut leaving mature residual trees scattered throughout the opening) within 10 years of the study initiation in 2005. Clearcut with reserves is the most common harvesting method practiced in BC, comprising 80% of cutting methods in 2009. At each site, the 14 largest, solitary, residual P. menziesii trees were selected for study, differing by as much as 2 km horizontal and 300 m vertical distance within sites. Climatic, environmental and stand history characteristics for each site are summarized in Table A1. The experiment was planned as a nested 3x3x5 factorial design, where EM network access (3 levels) and distance (5 levels) were nested within regional climate (3 levels 9 3 sites per level). Each EM network 9 distance combination was replicated seven times per site (9 sites 9 3 EM network treatments 9 5 distances 9 7 replications = 945 seedlings). Access to an EM network was controlled by planting 1-year-old nursery stock seedlings (seedlot # FDI 42192; 5001¢00¢¢N, 12049¢00¢¢W, 1000 m; Thompson Okanagan Dry seed planning zone; Figure A1) into soil or mesh bags made of sturdy plainweave nylon (Plastok, Birkenhead, UK). Seedlings were nonmycorrhizal at the time of planting. The three ‘mesh treatments’ were: (1) no mesh, where seedlings were planted directly into soil and thus could form hyphal and rhizomorph EM networks, and their roots were free to intermingle with tree roots; (2) 0.5-lm mesh, where hyphae, roots and invertebrates were restricted from accessing seedlings, and (3) 35-lm mesh, which allowed hyphae to access seedlings and form EM networks, but restricted access by roots and invertebrates larger than 35 lm in diameter (after Teste and Simard (2008) and Johnson and others (2001)). Effects on soil invertebrates were not specifically investigated; however, Teste and others (2006) found that moisture equilibrated rapidly between soil inside and outside of mesh bags of both pore sizes. Old trees and seedlings shared eight fungal morphotypes over 60% of their root tips, indicating strong networking potential (Bingham and Simard 2011b). Mesh bags were 17 cm diameter by 32 cm deep, and completely encapsulated the root system of the seedling. For the two mesh bag treatments, a cylinder of soil was removed from the ground, placed into the mesh bag, and seedlings were planted into this soil. For the no-mesh treatment, a cylinder of soil was disturbed in the same manner as that of the mesh treatments.
Network Facilitation Under Drought This method allowed us to separate soil water, EM network and root/invertebrate pathways of water and nutrient flow without creating treatment differences in the degree of soil disturbance. The five distances at which seedlings were planted from the residual trees were 0.5, 2.5, 5, 10, and 15 m from the mature tree bole. The 0.5-m treatment was immediately under the tree canopy, the 2.5-m treatment was usually at the canopy dripline, and the remaining distances were outside the mature tree canopy (that is, under open sky). One or two transects were randomly assigned to one of nine bearings from the tree (20, 60, 100, 140, 180, 220, 260, 300, or 340) (Figure A2), and mesh treatments were randomly assigned to each distance position along the transects. P. menziesii var. glauca seedlings were planted into these treatments over a period of 6 weeks in May and June of 2006, as it was not logistically possible to plant them all simultaneously. Competition from understory plants was eliminated by applying an herbicide containing glyphosate and surfactant (Credit (Nufarm Agriculture Inc., Calgary, AB, Canada) at a rate of 17 mL/ m2) in a 3-m radius around each planted seedling early every growing season. The reasons for this were threefold: (1) we wanted to isolate the interaction between the tree and seedling to determine if networks between the two were having any detectable effect, (2) some of the understory plants were ectomycorrhizal and thus confounded tree network effects, and (3) we were concerned that seedling mortality would be too high for an adequate growth analysis in the presence of herbaceous competition for water. During herbicide application, every seedling at a tree was covered with an upsidedown waste-bin. Once in the soil, glyphosate rapidly binds to soil particles and is inactivated (Andre´a and others 2003), and unbound glyphosate is rapidly degraded by microbial activity (Balthazor and Hallas 1986). It is thus unlikely the glyphosate affected mycorrhizal fungi directly. We cannot rule out that some glyphosate was taken up by networks via the EM plants it was applied to, however, material exchange between plants and their mycorrhizal symbionts is tightly physiologically regulated, and thus we think it is unlikely any glyphosate was transferred to EM fungi by their plant hosts.
Measurements Seedlings Following completion of planting in June 2006, each seedling was monitored for survival at 2-week intervals during two growing seasons, until harvest in September 2007. Following harvest, mesh bags
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were examined for tears, root penetration, and hyphal penetration. If the bag had a tear, the seedling was removed from the analysis. Roughly, half of all bags had some minor root or mycelial strand penetration at the seams, but upon inspection, it was concluded that the magnitude was low relative to the total number of root tips on the seedling and hyphae passing through 35 lm pores. Growth over two growing seasons of surviving seedlings was measured by oven-dry weight (65C for 48 h) of root and shoot biomass. A random subsample of three whole oven-dry seedling stems per treatment combination per site was ground using a Thomas Wiley mini-mill (Thomas Scientific, NJ, USA), MM 200 ball mill (Retsch Newtown, PA, USA), and mortar and pestle, then, analyzed for d13C by mass spectrometer (PDZ Europa, UK) at the University of California Davis Stable Isotope Facility. Climatic Conditions Individual tree latitude, longitude, and altitude were used to generate climate data from the webbased tool ClimateBC, which interpolates weather parameters for the period 1950–2002 (Hamann and Wang 2005; Wang and others 2006). These data are averaged and shown in Table A1 for each site. Weather and climate data for the period of 1950 to 2008 were also obtained from the Global Historical Climatology Network (GHCN) (Vose and others 1992) for the climate stations that were closest to the nine sites. We regressed the data from the GHCN against the data generated using ClimateBC to develop transfer functions to estimate monthly precipitation and mean monthly maximum temperature for each tree for the duration of the experiments. Two distinct variations on the summer heat:moisture index were then calculated, and they varied depending on the period relevant to the seedling performance variable modeled. For modeling seedling survival, the index used was: JH:M ¼ ðMean maximum 2006 JuneJuly temperatureÞ= ðð2006 MayJuly precipitationÞ=1000Þ; ð1Þ where JH:M is the June–July heat:moisture index. For modeling seedling growth and isotope abundance, the index used was: SH:M ¼ ðMean maximum 20062007 July temperatureÞ= mean 20062007 summerðMaySept:Þ precipitation =1000 ;
ð2Þ
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where SH:M is the growing season heat:moisture index. Soils Three soil pits were excavated to the bottom of the B horizon, if present, at each site to characterize the total thickness of the forest floor (L, F, and H) and mineral (A and B) horizons, as well as mineral soil texture, root abundance, root restricting layer, humus form, depth to water table, coarse fragment content, and soil order (Canadian System of Soil Classification 1998). Soil cores were also taken at the five seedling distances from three randomly selected trees per site to a depth of 32 cm and pooled for each distance by site. The pooled samples were analyzed for total C (Tiessen and Moir 1993), total N (McGill and Figueitedo 1993), and available P (Kalra and Mayhand 1991) at the BC Ministry of Forests, Research Branch Analytical Laboratory in Victoria, BC. Residual Trees Diameter at breast height, total height, crown height, slope, aspect, latitude, longitude, and elevation were recorded for each mature residual tree. All residual trees were alive and healthy when selected in May 2006. During the course of the study, a number of residual trees died at various sites due to windthrow, accidental harvest or undetermined causes. Data Analysis Many seedlings died from cattle trampling or bears pulling up mesh bags, thus reducing replication substantially. Seedlings that died of causes other than water deficiency, or that were growing near a dead residual tree, were excluded from the analysis (19% seedlings lost to these factors). Effects of the treatments on seedling survival, growth and stem natural abundance d13C were analyzed using the SAS System for Windows, V9.2 (2009). Because the sites did not fit neatly into the predetermined biogeoclimatic units, climate factors were treated as continuous variables in all statistical models. Logistic regression analysis was used to determine whether seedling survival was associated with mesh treatment, climatic variables, distance from tree, site variables or planting date (SAS PROC LOGISTIC) (Tabachnick and Fidell 2001). The general form of the model was: pðYÞ ¼ expðb0 þ b1 x1 þ b2 x2 þ . . . þ bk xk Þ=1 þ expðb0 þ b1 x1 þ b2 x2 þ . . . þ bk xk Þ;
ð3Þ
where p(Y) is the probability of survival, b0 is the intercept, b1….bk are estimated coefficients, and x1…xk are independent treatment, climatic, site, or planting date variables. The predictive parameters were allowed to enter the model if they improved the overall fit, but were ultimately removed from the model if they did not meet the criteria of P £ 0.05, with the stipulation that the treatment factors and interactions had to be retained until all remaining covariates were significant. To ensure that the best covariates were chosen for reducing experimental error, logistic regressions were also run with every covariate individually. Of all the model runs, the one with the proportionately highest reduction in error variance for error degrees of freedom lost was selected. It is common practice in multiple regressions to remove any parameters that are not significant, to increase strength of association. An odds ratio (that is, the logarithmic change in probability of survival when the predictive factor is increased by one unit) was calculated for each factor included in the final model. In logistic regression, there are no assumptions regarding normality, linearity or homogeneity of variance. The probability of correct classification of a pair of cases from every outcome category selected at random, c, is provided as a measure of strength of association. All growth and isotope analyses were performed using analysis of covariance (ANCOVA) for a factorial set of treatments using SH:M, initial seedling height (which was positively correlated to growth), final stem biomass (which was positively correlated to d13C), and site variables as covariates in a completely randomized design using SAS PROC MIXED (Milliken and Johnson 2002). By default, PROC MIXED uses the restricted maximum likelihood (REML) approach for parameter estimation, which produces unbiased parameter estimates when data are unbalanced. The general form of the model was: Yijk ¼ l þ di þ sj þ kk þ ðdsÞij þ ðdkÞik þ ðskÞjk þ ðdskÞijk þ b1 ðX1ijk X 1 ::Þ þ . . . þ bn ðXnijk X n ::Þ þ eijk ;
ð4Þ
where Yijk is the response variable (growth or d13C); l is a general mean; di, sj, and (ds)ij are the fixed effects parameters for the treatment factors, including mesh treatment and distance from tree and their interactions; b1…bn are estimated coefficients; X1…Xk are covariates, and ijk is the residual (Steel and Torrie 1980). The procedure for entry and retention of the covariates was the same as that
Network Facilitation Under Drought of the survival analysis, except that treatment factors and their interactions with each other and SH:M were retained regardless of P value. To ensure that the best covariates were chosen for reducing experimental error, ANCOVAs were also run with every covariate individually. Of all the model runs, the one with the proportionately highest reduction in error variance for error degrees of freedom lost was selected. It is standard protocol to retain all treatment factors and their interactions in ANCOVA and ANOVA because it increases the power of the test. Growth parameters were logarithmically transformed to conform to the assumptions of ANCOVA. All seedlings were assessed for proportional increase in biomass ((final biomass/initial biomass) - 1).
RESULTS Of the 766 outplanted nursery seedlings that were retained in the analysis, 412 seedlings survived to harvest. The logistic model predicting seedling survival included mesh treatment, distance from tree, JH:M, soil texture, and planting date as significant predictors of survival, along with a
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significant interaction between planting date and mesh treatment (Wald v2 = 167.5, df = 27, P < 0.0001, c = 0.883) (Table 1). All other variables and interactions tested did not enter the model at P < 0.05. Seedling survival decreased by 4% with every day after May 1st they were planted in the 35-lm mesh treatment, whereas survival increased by 4% with every day after May 1st they were planted in the 0.5-lm mesh treatment (Figure 1). Survival was not affected by planting date when seedlings were planted in the no-mesh treatment, after adjusting for other variables in the model. With the covariate adjustments, seedlings were more than seven times as likely to survive when growing in the 35-lm mesh treatment relative to the other mesh treatments combined, whereas those growing in the 0.5-lm mesh treatment were only 20% as likely to survive (Table 1; Figure 2A). Survival was substantially higher in the 35-lm treatment when seedlings were planted within the first 6 weeks of the start of the planting season, but was lower relative to the other treatments when planted in late June (Figure 1). Survival was greatest at 15 m from residual trees and lowest at 0.5 m distance, or immediately under the
Table 1. Logistic Regression Testing for the Probability of Seedling Survival in Response to Mesh Treatments, Distance from Established Tree (m), June–July Heat:Moisture Index (JH:M), Seedling Outplanting Date (number of days after May 1, 2006), and Soil Texture Logistic regression: c = 0.879
Likelihood ratio P < 0.0001
Effect
Odds ratios
df
Wald v2
P > v2
JH:M 0.5 m 2.5 m 5m 10 m 15 m 0.5 lm mesh 35 lm mesh No mesh Seedling outplanting date Planting date 9 0.5 lm mesh Planting date 9 35 lm mesh Planting date 9 no mesh Clay loam Fine Gritty silty Loam Sandy loam Silty Silty clay loam
0.953229 0.218428 0.876604 1.613329 1.539257 2.103074 0.197899 7.380933 0.684614 0.924964 1.03977 0.955997 1.006028 1.025008 0.095198 2.045414 1.46404 0.494702 21.98147 0.31468
1 4
82.8954 49.3901
<0.0001 <0.0001
2
8.7755
0.0124
1 2
33.0201 7.8328
<0.0001 0.0199
6
75.7604
<0.0001
Odds ratios are given for each continuous variable, category, and category interaction. One P value is given for each variable and variable interaction. The P value tests the null hypothesis that the odds ratio equals one.
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M. A. Bingham and S. Simard Table 2. Analysis of Covariance Results for the Natural Logarithm of Proportional Increase in Biomass of Seedlings and Seedling Stem Natural Abundance d13C Responses to Mesh Treatments, Distance from Established Tree (m), Summer Heat:Moisture Index (SH:M), and Their Interactions After Adjustment for Total Soil N (ppm extracted) and Initial Seedling Height (cm) or the Natural Logarithm of Final Seedling Stem Biomass
Figure 1. Probability of seedling survival as a function of planting date under the three mesh treatments.
Effect
Growth
d13C
Mesh Distance Mesh 9 distance SH:M SH:M 9 mesh SH:M 9 distance SH:M 9 mesh 9 distance Covariates Total soil N Initial seedling height Ln (stem biomass)
ns ns ** -*** ns ns **
ns ns ns +*** ns ns ns
+*** +*** N/A
-*** N/A +***
The direction of the relationship is provided for continuous variables. Also provided are the Akaike’s Information Criterion (AIC) value for the model and the level of significance. Ns = P > 0.1; * P £ 0.1; ** P £ 0.05; *** P £ 0.01; N/A = removed from model.
however, growth increased with SH:M close to the tree. At greater distances, growth of seedlings in 35-lm mesh decreased with SH:M. In the 0.5-lm mesh, the reverse pattern was observed. Seedling stem d13C was positively correlated with SH:M (P = 0.0034), when the natural logarithm of stem biomass and total soil N were included as covariates (Table 2). None of the other treatment factors affected d13C.
DISCUSSION Figure 2. Odds ratio values (logarithmic y-axis) for A mesh treatment and B distance in the logistic regression model predicting survival.
EM Network Enhancement of Establishment
crown of residual trees (Figure 2B). Seedling survival was highest in soils with a silty texture. The probability of survival decreased 4% for every 1 unit increase in June–July heat:moisture index. Growth of surviving seedlings was affected by an interaction of mesh treatment, distance from tree, and summer heat:moisture index, when initial seedling height and total soil N were included as covariates (Table 2). Growth of seedlings in the nomesh treatment decreased with SH:M regardless of distance from residual trees (Figures 3, A3). When seedlings were grown in the 35-lm mesh,
Seedling survival decreased when seedlings were unable to form a network and were planted early in the spring, supporting our first hypothesis that survival increases with potential access to the EM network of residual trees. Effects of EM networks on growth were more complex, however. Ectomycorrhizal networks were associated with net seedling growth only when root competition was excluded by a mesh bag, and only when seedlings were growing furthest from the tree when drought was low, and closest to the tree when drought was high. These results suggest that EM networks facilitate seedling establishment primarily when
Network Facilitation Under Drought
Figure 3. Relationship of summer heat:moisture index and the natural logarithm of proportional biomass increase after adjusting for covariates at different distances from the established tree under the no-mesh treatment (A), the 0.5-lm mesh treatment (B) and the 35-lm mesh treatment (C). All regression lines among distances under the 35-lm mesh treatment are significantly different (P £ 0.5). The regression lines in 0.5 lm and no-mesh treatments do not differ in slope or elevation.
seedlings are under drought and near residual trees, because the likelihood is greater that continuous fungal connections formed between root tips of trees and seedlings when seedlings grew close to the trees. The potential benefit of networks is further evidenced by the dramatic increase in seedling survival when they were able to form a network compared to when they were not, provided they were planted early in the growing
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season. At later planting dates, increasing drought appeared to cross a threshold where EM network facilitation was insufficient to improve survival probability. Restricted access by root or grazing hyphal invertebrates may have benefitted seedlings growing in mesh bags. However, we believe the costs of restricted soil access via roots would have outweighed any benefits due to exclusion of soil invertebrates, especially considering that mesh bags were filled with native soil that likely contained invertebrate grazers. Therefore, the lower survival of our seedlings growing in no-mesh versus 35-lm mesh is most plausibly due to root competition, because all other factors were equal (see Teste and others 2009). This contrasts with Teste and Simard (2008), who found that survival of seedlings was highest when they were grown in no-mesh. This difference may result from the fact that their experiment was conducted during wetter years, thus increasing overall resource availability to seedlings compared to our study. Greater resource availability would have increased seedling survival probability regardless of residual root competition, while potentially reducing the beneficial effects of EM fungi on the seedlings. We found no isotopic evidence that remediation of water deficiency accounted for most of the variation in growth among the mesh and distance treatments, given the lack of response of d13C to these treatments. This would be expected, however, if photosynthetic rate and stomatal conductance varied with distance and EM networks in parallel, thus leading to enhanced growth (Querejeta and others 2007). d13C was positively correlated to the drought index, and it is well established that water deficiency usually increases water use efficiency, which manifests in a decrease in photosynthetic discrimination against 13C via a reduction in stomatal conductance that precedes a reduction in photosynthetic rate (Farquhar and others 1989). Although nutrient deficiency can increase 13C discrimination via a reduction in photosynthetic capacity absent a change in stomatal conductance, total soil N was negatively correlated with d13C, which was the opposite expected if nutrient availability accounted for the substantial variation in d13C of our seedlings. In water-limited ecosystems soil N is positively correlated with soil organic matter content, and soil organic matter increases water-holding capacity and infiltration rates. Thus, in water-limited ecosystems, soil fertility is likely to be negatively correlated with plant tissue d13C. Any alleviation of water deficiency in 35-lm mesh could have occurred through EM
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uptake of soil water independent of the established tree, or through translocation of water to the seedling that was hydraulically redistributed to the EM network by the tree (Querejeta and others 2003; Egerton-Warburton and others 2007; Bingham and Simard 2011a). Other studies have shown strong evidence for hydraulically redistributed water from conifer trees to seedlings through EM networks (Warren and others 2008; Booth and Hoeksema 2010).
Network Facilitation and Distance Survival and growth generally increased with distance from residual trees, leading us to reject our second hypothesis, that seedling establishment would be greatest at intermediate distances. These results suggest that seedling performance was regulated primarily by competition from the residual trees. At the driest sites, however, seedling growth was greatest in close proximity to large trees provided they had access to the EM network in the absence of root competition (that is, in the 35-lm mesh). The benefit of networks to seedling establishment under drought and large nearby trees is consistent with the stress-gradient hypothesis of facilitation (Greenlee and Callaway 1996; Choler and others 2001; Pugnaire and Luque 2001; Bertness and Ewanchuk 2002; Callaway and others 2002; Maestre and others 2003; Castro and others 2004; Go´mez-Aparicio and others 2004; Liancourt and others 2005; Cavieres and others 2006). That we generally found increasing survival and growth with distance from mature trees in most environments contrasts with previous mycorrhizal network studies (Dickie and others 2007; McGuire 2007; Teste and Simard 2008). For example, Teste and Simard (2008) found that P. menziesii var. glauca seedling growth reached a plateau at an intermediate distance (2.5 m) from mature trees when they grew in the no-mesh treatment, but the maximum distance they tested was 5 m from residual trees, where tree root competition was still important. By contrast, our study tested distances to 15 m, where residual tree roots were no longer suppressive to seedling growth. Our results also contrast with McGuire (2007), who studied conspecific facilitation of EM Dicymbe corymbosa seedling establishment within mixed forest dominated by AM species, and she found that seedling survival decreased with distance from conspecifics. McGuire (2007) also found that survival was lowest when seedlings were unable to form a network, which is consistent with the survival difference we found between 0.5 and 35 lm mesh. Seedlings able to
form EM networks with other EM root systems may benefit from access to a larger volume of soil or nutrient transfer from connected trees (Smith and Read 1997; Read 2002; Leake and others 2004). Four reasons underlie the major differences between our experiment and McGuire’s (2007): (1) our seedlings were planted in areas with low overstory canopy coverage, whereas hers were planted in forest with high canopy coverage; (2) our seedlings were grown in greenhouses for 1 year prior to planting, while hers were naturally regenerated from seed; (3) competition from AM plants within 3 m of our seedlings was removed, whereas her seedlings were grown under native competitive conditions; and (4) only a minority of our sites were monodominant stands of EM conspecifics (at the drier end of our gradient) and had plant communities dominated by AM plants. These factors reduced the competition under which our seedlings were grown compared to McGuire’s (2007), and moreover, grass and forb density was observed to generally increase with distance from trees at our sites, whereas AM competition was likely more uniform in McGuire’s forest outside of the EM stand. This gradient may also account for the difference between our results and those of Dickie and others (2007), who found that Quercus ellipsoidalis seedling survival decreased with distance from conspecific trees in an AM plant matrix. Moreover, the maximum distance of seedlings from conspecifics in Dickie and others’ (2007) study was over twice that of ours.
Stress-Gradient When seedlings grew close to trees under high drought, those able to form networks without root competition exhibited substantially greater growth than in the other treatments. This result supports our third hypothesis that EM network facilitation of seedling establishment increases under drought due to climatic factors and increasing proximity to residual trees (from increasing root competition). The most plausible explanation for this phenomenon is that EM hyphae were facilitating uptake of hydraulically redistributed water. It has been established that Douglas-fir hydraulically redistributes water to nearby understory vegetation (Brooks and others 2002, 2006). Water translocation to Douglas-fir seedlings via EM hyphae has also been shown to occur (Plamboeck and others 2007). In contrast to their interactive effects on growth, drought, mesh treatment and distance from tree
Network Facilitation Under Drought affected survival independently of one another. When seedlings were planted early in the growing season, survival was greatest in the 35-lm mesh, all other things being equal, and lowest when growing in the 0.5-lm mesh. It is likely that survival increased when seedlings were colonized by mycorrhizal networks (that is, in the 35-lm mesh treatment) prior to the onset of the summer drought. The lack of a decrease in survival of seedlings grown in 0.5-lm mesh with later planting dates is perplexing at first glance. However, seedlings planted early may have been able to develop a more extensive root system within the 0.5-lm mesh bag prior to the onset of drought dormancy, which would cause them to deplete the water within their bag more rapidly during later periods of growth, without the compensatory effect of a mycorrhizal fungal mycelium extending out from the bag. The 0.5-lm mesh seedlings planted just prior to the summer drought, however, would deplete their soil water less rapidly during later growth periods than their early-season cohorts, while being less affected by root competition than late-season seedlings planted directly in soil. In summary, we found that EM networks of residual trees were particularly important in facilitating regeneration of P. menziesii seedlings when they were establishing under drought and root competition of nearby trees. These results suggest that the facilitative effect of solitary trees on seedlings via hydraulic redistribution may be dependent on EM connections between the root tips of trees and seedlings. Thus, maximizing regeneration in dry forests may require retention of a low density of old residual trees to ensure the maintenance of robust EM networks for colonization of new seedlings, and possibly redistribution of water or nutrients. Our results suggest that residual tree retention may be especially desirable as drought increases with climate change. Thus, managers may want to consider retaining residual trees for their facilitative effects when harvesting forests near the forest–grassland interface, where increases in recruitment difficulty are expected to be dramatic over the next decade (Hamann and Wang 2006; Spittlehouse 2008). Although caution should be taken in generalizing these results to other forest ecosystems, it is likely that the EM network effects observed in this study, and affiliated studies in the same forest type (Schoonmaker and others 2007; Teste and Simard 2008; Simard 2009; Teste and others 2009, 2010; Beiler and others 2010) apply to most dry EM coniferous forests and woodlands.
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ACKNOWLEDGMENTS We thank Robert Guy, Melanie Jones, and Sally Aitken for invaluable help in the design and implementation of the field and laboratory methods. This research was funded by an NSERC Discovery Grant and a Forest Innovation Investment-Forest Science Program grant to S.S.
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