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Keywords:

  • British Columbia;
  • Douglas-fir;
  • ectomycorrhizas;
  • interplant carbon transfer;
  • isotopes;
  • mesh barriers;
  • mycorrhizal network pathways;
  • rhizomorphs;
  • soil disturbance;
  • source–sink

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

1. Mycorrhizal pathways are comprised of fungal hyphae that facilitate carbon transfer between plants. We determined whether net carbon transfer occurred between conspecific conifer seedlings in the field, and whether soil disturbance or access to mycorrhizal pathways affected transfer.

2. We established two soil disturbances and planted pairs of different sized Pseudotsuga menziesii var. glauca seedlings (naturally regenerated or planted) into one of four mesh treatments (0.5, 35, 250 μm or directly into soil) restricting mycorrhizal pathways. We pulse-labelled both seedlings, one with 13CO2 and the other with 14CO2, to quantify net carbon transfer. Ectomycorrhizas were identified using morphological and molecular techniques.

3. Net carbon transfers were detected and were not due to re-fixation of respired carbon. More transferred carbon accumulated in shoots than roots. In disturbed soil, there was greater net carbon transfer to natural seedlings than planted seedlings; the reverse pattern was observed in undisturbed soil. For planted seedlings only, the magnitude of net carbon gain was positively related to seedling size and height growth rate. Greater net accumulation of carbon occurred in Rhizopogon vinicolor, a long-distance ectomycorrhizal fungi exploration type (morphological character), than the two other most abundant ectomycorrhizal fungi with contact- and short-distance exploration types. Long-distance exploration types have the potential to form long-distance hyphal connections between plant roots, whereas contact- and short-distance are restricted to short-distance (c. 0–0.25 μm) connections.

4.Synthesis. These results confirm that net carbon transfer occurs through mycorrhizal pathways; however, the amount transferred was very small. Mycorrhizal pathways were facilitating net transfer of carbon to large, vigorous natural seedlings in disturbed soils, whereas smaller planted seedlings received more net carbon gain in undisturbed soils. The size variation within these planted seedlings was great enough to elicit a positive relationship between net carbon gain and seedling size and growth rate. These findings are relevant to regeneration of forests characterized by mixed severity disturbance regimes, which leave a suite of environmental conditions that may result in a greater magnitude of transfer.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Below-ground movement of carbon between plants can be viewed as a consequence of plants growing closely together and/or a phenomenon facilitated by associated soil organisms such as ectomycorrhizal (ECM) fungi (Selosse et al. 2006). Mycorrhizal pathways for interplant resource transfer have been proposed, but their relative importance has not been well studied (Newman 1988; He et al. 2005). Prominent mycorrhizal pathways for carbon transfer between two or more plants include: (i) direct transfer via mycorrhizal networks (MNs); and (ii) indirect transfer facilitated by foraging ECM mycelium (mycorrhizal–soil pathway sensuSimard & Durall 2004). Indirect transfer occurs when different fungi specific to each plant are involved or, alternatively, when a generalist fungus forming a MN is disrupted by soil fauna (Johnson et al. 2005). In either case, movement is governed by the plant’s sink strength and the ability of the fungi to scavenge resources left by a neighbouring plant. Movement through soil without mediation by ECM fungi is another important transfer pathway that is not included in mycorrhizal pathways.

Interplant resource transfer facilitated by mycorrhizal pathways has potentially far-reaching consequences for plant community dynamics (Newman 1988; Wilkinson 1998; Whitfield 2007). Carbon, nutrients or water can be transferred between plants (Selosse et al. 2006; Egerton-Warburton, Querejeta & Allen 2007; Meding & Zasoski 2008) and may benefit seedling establishment and survival under drought, nutrient-poor or light-limiting conditions (Selosse et al. 2006). Carbon has been shown to travel below-ground in both directions between two connected plants, and net transfer to one of the plants has been shown to occur where this plant is a physiological sink relative to its neighbour (Simard et al. 1997a). Typically, a so-called ‘donor’ source plant transfers more carbon than it gains from a ‘receiver’ sink plant; the magnitude of net transfer from a fully illuminated source plant has been shown to increase with receiver sink strength because of shading (Simard et al. 1997a). Plants growing under conditions that limit photosynthetic activity may benefit from mycorrhizal pathways (Simard et al. 1997a; Callaway et al. 2002). The stress-gradient hypothesis predicts that facilitation is more important to plants growing under harsh, particularly arid, conditions (Bertness & Callaway 1994; Lortie & Callaway 2006). Soil disturbance because of forest harvesting may hinder seedling establishment if heavy machinery disturbs soil structure, disrupts MNs and increases soil compaction (Senyk & Wass 1999), thus increasing water and nutrient limitations to plant growth. Soil disturbances associated with forest harvesting can accentuate the negative effects of stressful environments on seedling establishment (Fleming et al. 1998), but the role of MN facilitation under such conditions has not been studied.

There is controversy about the role of mycorrhizal pathways in interplant carbon transfer; this controversy also involves arbuscular mycorrhizal chlorophyllous plants where transferred carbon appears to remain in the fungal tissue rather than being transferred to associated plant tissue (Voets et al. 2008). Most studies examining mycorrhizal pathways in ECM plants find that carbon is transferred to the tissue of connected plants (Leake et al. 2004), with the odd exception (Wu, Nara & Hogetsu 2001). In many of these studies, however, it has been difficult to distinguish carbon transferring into root tissues from that into fungal tissues (Simard & Durall 2004). As suggested by Fitter (2001), there is a need to confirm that biologically significant amounts of carbon transferred through mycorrhizal pathways can move into the shoots of ‘receiver’ plants under field conditions (Abuzinadah & Read 1989; Robinson & Fitter 1999). The scepticism about interplant carbon transfer via mycorrhizal pathways concerns the methodologies used in previous studies (Bergelson & Crawley 1988; Robinson & Fitter 1999; Fitter 2001), even though there is unequivocal evidence of transfer to achlorophyllous plants in the field (Leake 2004; Bidartondo 2005; Selosse et al. 2006), and to shoots of chlorophyllous plants in the laboratory (Abuzinadah & Read 1989).

The morphology or exploratory type (Agerer 2001) of ECM fungi forming mycorrhizal pathways may affect the magnitude of carbon transfer (Reid & Woods 1969; Brownlee et al. 1983). Long-distance exploratory types (i.e. ECM fungi with rhizomorphs) may transfer more carbon and water between plants than short-distance or contact-exploratory types of mycorrhizal pathways, which are comprised of mycelia (Brownlee et al. 1983; Egerton-Warburton, Querejeta & Allen 2007). Close examination of rhizomorph structure and development has shown that rhizomorphs are probably responsible for most interplant resource transfer (Read, Francis & Finlay 1985; Cairney 1992), but evidence from the field demonstrating greater carbon transfer in rhizomorph-dominated mycorrhizal pathways is lacking.

The objectives of this study were to use improved methodology that addresses the concerns of Robinson & Fitter (1999) to determine whether net carbon transfer occurs between ECM seedlings, whether it occurs through mycorrhizal pathways, and whether it is affected by stress imposed by soil disturbance. We hypothesized that net carbon transfer: (i) occurs between conspecific coniferous seedlings along a source–sink gradient; (ii) predominantly occurs via the MN; (iii) is affected by soil disturbance; (iv) is experienced by both shoots and roots of ‘receiver’ seedlings under field conditions; and (v) is affected by the exploratory type of the dominant ECM.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Site description and experimental design

Characteristics of the three study sites (Dairy Creek, O’Connor Lake, Black Pines; northwest of Kamloops, British Columbia, Canada) are described in Table 1 and Hope (2006). The three sites were clear-cut between 1998 and 2000 (Hope 2006). These sites served as replicates (n = 3) for our split–split plot experiment, where the whole plot effect was soil disturbance type, the split plot effect was mesh pore size controlling for mycorrhizal pathways, and the split–split plot effect was plant type (natural or planted). To establish the soil disturbance treatments, two 5 × 25 m experimental plots were randomly selected in the middle of each 6.5-ha clearcut. On one plot, the forest floor and top 3 cm mineral soil layer were scraped off (referred to as the ‘disturbed’ soil treatment hereafter) with a plough on a small Bobcat® excavator, whereas the other plot was left undisturbed (referred to as the ‘undisturbed’ soil treatment hereafter). Twenty-four independent subplots were then created in each soil disturbance type plot using the Bobcat® excavator, where the perimeter of each square (c. 2.25 m2) subplot was trenched to a depth of 50 cm to exclude the influence of surrounding roots and to contain the carbon radioisotope.

Table 1.   Site, soil and Pseudotsuga menziesii var. glauca (interior Douglas-fir) seedling characteristics in 2006. Values for characteristics are means with 95% confidence intervals in parentheses and ranges in brackets. All seedings (labelled and non-labelled) are included in the calculations
Site characteristicsDairy Creek (DA)O’Connor Lake (OC)Black Pines (BP)
  1. *Soil type (Brunisolic Gray Luvisol), texture (silt loam) and humus form (hemimor) data mostly taken from Hope (2006).

  2. P-values for a two-sample t-test.

  3. ‡Ranges [minimum and maximum values] are presented to help in interpreting Fig. 2.

  4. [Correction added on 28 January 2010, after first online publication: Table caption expanded and soil and seedling characteristics column headings corrected].

Latitude; longitude (degrees)50.51; 120.2550.53; 120.2150.56; 120.17
Month and year of clearcutJanuary 1998January 2000January 1999
Elevation (m)109410901190
Slope (%)052
AspectNorthSouth-westSouth
Soil characteristics*Disturbed, mean (95% CI)Undisturbed, mean (95% CI)P-value†
Bulk density (g cm−3)1.36 (1.09–1.63)0.65 (0.37–0.92)0.0068
Moisture content (g H2O g soil−1)23.25 (10.60–35.90)23.33 (13.24–33.41)0.9893
pH6.19 (5.44–6.93)5.74 (5.38–6.09)0.2053
Total C (g C kg soil−1)27 (4–50)357 (243–471)0.0014
Total N (g N kg soil−1)1.2 (0.4–1.9)12.4 (9.5–15.4)0.0005
Available P (mg P kg soil−1)122 (109–134)233 (183–282)0.0038
Seedling characteristicsNatural regeneration, mean (95% CI) [range]Planted, mean (95% CI) [range]P-value
Age (years)4–63Not determined
Height (cm)30.6 (28.4–32.8) [13.0–62.5]‡26.4 (25–27.8) [14.5–36.8]0.0019
Root collar diameter (cm)0.77 (0.72–0.82) [0.30–1.35]0.60 (0.57–0.63) [0.34–0.90] <0.0001
Stem volume (cm3)17.3 (13.8–20.6) [1.2–68.9]8.2 (7.1–9.3) [1.9–23.4] <0.0001
Shoot dry biomass (g)15.6 (13.2–17.9) [1.8–47.7]8.6 (7.4–9.9) [1.1–22.5] <0.0001
Root dry biomass (g)4.8 (4.1–5.4) [1.0–13.5]3.3 (1.4–5.3) [1.0–8.0]<0.0001

To establish the mesh size and plant type treatments, naturally regenerated P. menziesii seedlings (referred to as ‘natural seedlings’ hereafter; see Table 1 for characteristics) were carefully excavated from nearby roadcuts (1–5 km away) and transplanted to the middle of each subplot in June 2004. Each subplot was then numbered and 12 of the randomly selected subplots were randomly assigned to one of four mesh treatments (mesh bags with 0.5, 35 or 250 μm pore sizes; or no mesh bag). Mesh bags were made of sturdy plain-weave nylon (Plastok Ltd., Birkenhead, UK) and were closed-bottom cylinders (seams sealed with silicone) with a volume of 6185 cm3 (15 cm in diameter and 35 cm deep). Nursery-grown P. menziesii seedlings (referred to as ‘planted seedlings’ hereafter; see Table 1 for characteristics) that were younger and smaller than the natural seedlings were then planted into the mesh bags 0.5 m away from the natural seedling in June 2004. We used two different types of seedlings (natural and planted) to establish a source–sink gradient between larger natural seedlings harbouring a well-established community of ECM fungi and smaller, non-mycorrhizal (at time of planting) planted seedlings.

Planted seedlings (seedlot no. 48520, British Columbia Ministry of Forests and Range Tree Seed Centre, Surrey, BC, Canada) were grown at the University of British Columbia (Vancouver, Canada) glasshouse for 6 months in 512B styroblocksTM (Beaver Plastics, Edmonton, AB, Canada); they were non-mycorrhizal and ranged in height from 5 to 19 cm at the time of planting.

All competing vegetation in each square subplot was clipped throughout the first growing season and then sprayed twice (2005 and 2006) with glyphosate to eliminate interspecific plant interactions. Laatikainen & Heinonen-Tanski (2002) found that glyphosate had no effect on the majority of ECM fungal species (of 64 species), with only a few species that were either stimulated or inhibited. When inserting the mesh bags, we minimized soil disturbance by carefully excavating the soil in three distinct soil layers (intact forest floor, A horizon and some of the B horizon) and replacing these layers into the bags in the same order. Similar soil excavation and replacement were carried out prior to planting the seedlings in the no mesh treatment (i.e. in the ground). Detailed characteristics of the mesh bags are outlined in Teste & Simard (2008).

Mesh treatments were used to restrict seedling access to a MN (Robinson & Fitter 1999). Seedlings planted in a 0.5-μm mesh bag could be colonized by wind- or soil-borne propagules, but not by fungi associated with nearby natural seedlings, nor could hyphae of natural seedlings anastomose with those of planted seedlings once colonized because the pores were too small for hyphal penetration based on our microscopy observations (similar to the work of He et al. (2004)) and our earlier glasshouse studies examining hyphal penetration of different mesh sizes (Teste et al. 2006). The mesh bags were carefully inserted and removed from the field soil and we did not notice any damage to the bags that could have allowed hyphal passage. Seedlings planted in 35- and 250-μm mesh bags could form a MN with individual hyphae or rhizomorphs plus hyphae, respectively, growing through the mesh from nearby natural seedlings. Field and laboratory observations demonstrated that intact rhizomorphs did not penetrate the 35-μm mesh. However, we did notice that rhizomorphs were capable of breaking down into an unstructured form (loose hyphae) at the surface of the 35-μm mesh, thus allowing penetration, but these occurrences were rare. Seedlings planted directly into soil (no mesh) could form hyphal and rhizomorph MNs, and their roots were free to intermingle with other roots. However, root intermingling between natural and planted seedlings did not occur (the closest distance separating root tips was 5 cm in the no-mesh treatment and 30 cm for the other mesh treatments), thus providing a robust opportunity to compare carbon transfer through the mycorrhiza–soil pathway (0.5-μm mesh) versus the MN pathway (no mesh, 35- and 250-μm mesh) (Simard & Durall 2004). Transfer via the simple soil-only pathway (Simard & Durall 2004) was unlikely to have occurred because seedlings were well colonized by ECM fungi by the time of labelling and the pulse–chase periods were very short (see Results and Discussion).

Carbon isotope labelling

Gas labelling bags (10 L) were custom-made with 5-ply transparent gas-tight polyethylene/nylon (FoodSaver®; Jarden Corp., Rye, NY, USA). Prior to isotope gas labelling, 1 mL gas-tight syringes (Hamilton Co., Reno, NV, USA) and a LI-6251 CO2 analyzer (LICOR Inc., Lincoln, NE, USA) were used to determine the amount of time needed for ‘donor’ seedlings to reach the compensation point inside the gas labelling bag after injecting regular CO2 gas. These preliminary data determined the ideal pulse period for complete assimilation of the 13CO2.

On each day from 26 June to 1 July 2006, 16 seedlings (on a given plot) were pulse-labelled twice (between 8:00 and 9:30 hours and between 11:00 and 12:30 hours) with 200 mL (at standard pressure and temperature) of 13CO2 (99%13C; Cambridge Isotope Laboratories, Inc., Andover, MA, USA) using a 0.5-L gas-tight super syringe (Hamilton Co.) or with 4.44 MBq gaseous 14CO2 released from 0.25 mL of Na214CO3 with lactic acid. Seedlings labelled with 14C were also injected twice with 200 mL of unlabelled CO2 gas (at standard pressure and temperature) immediately after release of 14CO2 to match the CO2 concentration inside the gas labelling bags of seedlings labelled with 13C. One of the seedlings (natural or planted) in a subplot was labelled with 13CO2 and the other seedling with 14CO2 (total of 90 seedlings labelled); these labelling treatments were fully reciprocated in replicate seedling pairs. We chose late June for labelling because previous studies in these ecosystems showed that Douglas-fir is still photosynthetically active through the day (i.e. stomata are not closed because of drought stress) and carbon is still allocated to shoots, roots and ECM fungi, thus increasing the potential for carbon transfer to various locations within the plants and fungi (Simard et al. 1997a, 2003). Late June also presented ideal labelling conditions because of the prolonged periods of cloudless days, typical during this time of year in these forests.

Six hours after labelling all neighbouring seedling shoots were carefully covered with thick plastic bags to prevent accidental aerial enrichment and gas labelling bags were removed and flushed from the labelled pairs. A few minutes later, the plastic bags were removed from all seedlings. Potted P. menziesii seedlings (referred to as ‘aerial control’ seedlings hereafter) were then placed in between the natural and planted labelled seedlings to estimate the amount of re-fixed carbon via plant or soil respiration. After a 7-day chase period, we severed shoots from roots of all seedlings (natural, planted, aerial control) and excavated root systems, removing clumps of soil only when necessary. We used a 7-day chase based on the success of previous field and laboratory studies at detecting interplant carbon (C) transfer after 6–7 days (Simard et al. 1997a; Wu, Nara & Hogetsu 2001; Lerat et al. 2002; Philip 2006). All seedlings were immediately placed into individual air-tight plastic bags and surrounded with dry ice during transport to the laboratory, where they were kept frozen at −20 °C until oven-dried. In the laboratory, remaining soil was carefully washed off root systems with running tap water in a series of tubs.

13C and 14C analysis

Shoots and roots (oven-dried at 70 °C for 48 h) were ground to 0.5 mm with a digital ED-5 mid-sized mill (Thomas Scientific ©, Swedesboro, NJ, USA), and then to 0.01-mm fine powder (c. 100 mg subsample) with a MM 200 ball mill (Retsch®, Newtown, PA, USA). For δ13C (‰) analysis, 1 mg fine powder was analysed with a Europa Hydra 20/20 isotope ratio mass spectrometer (Europa Scientific, Crewe, UK) or a Europa Integra (enriched samples) at UC Davis Stable Isotope Facility, CA, USA. For 14C analysis, the amount of 14C (dpm) of each sample was simultaneously measured by subsampling liberated CO2 (during combustion of the 1 mg fine powder prior to 13C analysis) with a gas-tight syringe and analysing it with a LS 6500 liquid scintillation counter (Beckman Coulter, Fullerton, CA, USA).

Carbon excess, bidirectional and net transfer calculations

To convert δ13C into mg of 12C-equivalent excess in seedlings, we followed a modified version of the procedure by Boutton (1991) outlined in Teste (2008). To convert ‘dpm’14C into mg 12C-equivalent excess in seedlings, we first calculated the radioactivity (RA) of the sample (sample 14C) above background:

  • image( eqn 1)

Tissue (shoots or roots) radioactivity (tissue 14C) was calculated as:

  • image(eqn 2)

This was converted to mmol to yield the tissue 14C-labeled carbon content:

  • image(eqn 3)

Finally, the tissue 14C-labelled carbon content was converted to excess tissue 12C-equivalent by the equation:

  • image(eqn 4)

where MW is molecular weight.

Excess plant 12C-equivalent was calculated as the sum of excess shoot and root 12C-equivalent.

Bidirectional transfer was the sum of excess 12C-equivalent received by both the natural and planted seedlings in a given subplot (i.e. in a seedling pair). Net transfer was based on excess plant 12C-equivalent that was received from the partner ‘donor’ seedling. Net transfer was the difference between excess 12C-equivalent received by the planted seedling and that received by the natural seedling. Positive net transfer meant that the planted seedling received more carbon than the natural seedling (i.e. a net gain by the planted seedling), and negative net transfer indicated the opposite (i.e. a net gain by the natural seedling). For calculating net carbon transfer, at least one receiver seedling (i.e. receiving 13C or 14C) in the pair required enrichment values above a 99% confidence interval (based on seedling background levels). For bidirectional transfer, however, we required that both receiver seedlings in a pair were enriched in δ13C and 14C dpm above the 99% confidence interval.

Sampling, morphotyping and molecular analyses of seedling ectomycorrhizas

From 8 to 13 July 2006, 24 non-labelled natural and planted seedlings were destructively harvested for ECM morphotyping and for estimating background carbon isotope (13C and 14C) levels of plant tissue. Natural and planted seedling root systems were severed from shoots and placed in plastic bags with loose soil and stored at 3 °C (non-labelled roots) and −20 °C (labelled roots; for determining transfer to dominant ECM, as described below in this section) until further processing. All samples were processed within 3 months after field sampling. Root samples were prepared, morphotyped using both stereo and compound microscopes (Goodman, Durall & Trofymow 1996) (200 ECM tips per seedling for a total of 9600 ECM tips), and analysed using molecular tools (PCR, sequencing (ITS region), BLAST search and microsatellite analyses of R. vinicolor ECM), for which detailed methods are described in Teste et al. (2009). For each morphotype, three samples consisting of three to five ECM tips each from different seedlings were sequenced (approximately one sample for every three seedlings) for a total of 29 sequenced samples; out of these, 18 yielded DNA for BLAST searches. Using this data, the Morisita–Horn similarity index (CMH) was calculated for the ECM community (Magurran 2004).

For every R. vinicolor ECM morphotype encountered, one sample (3–5 ECM tips) per seedling was used for microsatellite analysis. Microsatellite analysis was conducted to provide stronger evidence that MNs had formed between the seedling pairs. We used R. vinicolor because the methodologies for this species had been well established (Kretzer et al. 2003), whereas they had not for the other ECM dominant species in this study. Finally, we randomly sampled an additional 20 ECM tips (1.5–2.0 mg) per seedling from each of the three most abundant ECM taxa on all labelled and non-labelled (same seedlings as mentioned above) seedlings for isotope analysis. We then analysed these tips for isotope abundance as indicated above and calculated carbon transfer in the same manner.

Soil sampling

Soil moisture was determined in the field using a HydroSenseTM CD620 volumetric soil water meter (Campbell Scientific Inc., Edmonton, Canada) with 20 cm long probe rods. To estimate bulk density and nutrient concentrations, soil samples were excavated (0–20 cm) from five randomly selected subplots per soil disturbance treatment on 12 June 2005. Mineral soil (undisturbed and disturbed treatment) and forest floor soil (undisturbed treatment only since the forest floor was removed in the disturbed treatment) samples were air dried and sieved (2 mm). Soil analyses were carried out by the Analytical Laboratory of the British Columbia Ministry of Forests and Range, Research Branch, in Victoria, British Columbia, using the methods outlined in Tiessen & Moir (1993) for total C, McGill & Figueiredo (1993) for total N, Kalra & Maynard (1991) for available P and soil pH in H2O.

Statistical analyses

All statistical analyses were carried out using the R statistical environment for statistical computing and graphics (R Development Core Team 2007). The split-split plot design was analysed using a linear mixed-effects model (Pinheiro & Bates 2000), where sites were used as blocks (n = 3) and set as the random factor. The whole plot, split plot, split–split plot fixed factors were the soil disturbance, mesh size treatments and plant type, respectively. The effect of mesh treatment on net carbon transfer (based on C originally fixed by shoots) to the three most abundant ECM taxa was detected with one-way analysis of variance. We did not attempt to sample Amphinema, Suillus or any other taxa because they were uncommon, and it would have resulted in a very small data set with limited scope of inference. To assess the influence of seedling size and growth on net carbon transfer, we performed simple linear regression. Percentage ECM colonization was calculated as:

  • image

For all statistical comparisons, differences were considered statistically significant at  0.05 or where zero was not included in the 95% confidence intervals for differences between means (Gardner & Altman 2000). Finally, we calculated 95% confidence intervals for the mean amount of net carbon transfer to determine whether net carbon transfer was significantly greater than zero (i.e. where the interval did not include zero, then net carbon transfer was probably greater than zero) (see Di Stefano (2004) and Cumming & Finch (2005) for the interpretation and use of confidence intervals as inferential statistics).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Soil disturbance and mycorrhizal pathway effects on net carbon transfer

There was net transfer of carbon between natural and planted seedlings (Fig. 1, Table 2, see Fig. S1 in Supporting Information). Net carbon transfer occurred to 23 natural (53%) and 20 planted (47%) seedlings, indicating that neither natural nor planted seedlings acted consistently as ‘net donors’ or ‘net receivers’. For planted seedlings only, the magnitude of net carbon transferred was significantly related to seedling stem volume, shoot biomass and height growth rate, but this was not true for natural seedlings (Fig. 2). Only a few planted seedlings were mostly responsible for this relationship and an apparent size threshold existed (Fig. 2). Other plant factors appeared unimportant to patterns in net transfer. For example, the amount of carbon fixed by donors was unrelated to net carbon transfer to receivers (R2 = 0.002, = 0.38); therefore donor source strength was unrelated to transfer. The relationship between net carbon transfer and size of planted seedlings was also not influenced by the size, isotope content or isotope concentration of the neighbouring natural seedling (> 0.05). When isotope was expressed per gram of donor and per gram of receiver, patterns in transfer were not significant (> 0.05), which emphasizes the greater importance of receiver plant size (total isotope content) relative to isotope concentration on direction of transfer.

image

Figure 1.  Net carbon transfer to whole plant (shoots + roots) Pseudotsuga menziesii var. glauca seedlings (planted or natural) grown in the soil disturbance (undisturbed and disturbed) and mesh treatments in the field. Mean background δ13C (‰) and 14C (dpm) were used to calculate 99% confidence intervals (CI). Only values above the upper limit of the 99% CI for either δ13C or 14C dpm were considered enriched and used to calculate net carbon transfer (see Fig. S1 for raw data). Analysis of variance indicated that the soil disturbance (= 0.64) and mesh (= 0.74) treatments had no significant effect on net carbon transfer, but there was a significant interaction (= 0.02) between the soil treatment and plant type (natural or planted) and a significant plant type effect (= 0.01). Fig. S2 outlines the differences between the means making up this interaction. Values are means (dots) with 95% CIs and the number (n) of seedlings used to calculate these are noted at the bottom of each interval.

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Table 2.   Carbon transfer between natural and planted Pseudotsuga menziesii var. glauca (interior Douglas-fir), and carbon assimilation by aerial control seedlings in 2006 across both soil disturbance levels. Shoot and root values are means with 95% confidence intervals (CIs) in parentheses. Only shoot and root values above the upper limit of the 99% CI for either δ13C or 14C dpm (see Materials and methods section on carbon excess, bidirectional and net transfer calculations) were considered enriched and used to calculate net carbon transfer (see Fig. S1 for raw data)
 nShootnRoot95% CIs for the difference between means*
  1. nd, not determined.

  2. *These 95% confidence intervals are for the differences between the shoot and root means and if zero is included in the interval then it is concluded that the two means are not different (Gardner & Altman 2000).

  3. †Based on 14C enrichment values.

Excess12C-equivalent in plants receiving 14C (mg)400.106 (0.020–0.206)330.033 (−0.007 to 0.073)−0.020 to 0.18
Excess 12C-equivalent in plants receiving 13C (mg)360.131 (0.085–0.238)350.032 (0.019–0.045) 0.052–0.207
Aerial control excess 12C-equivalent (mg)†610.0006 (−0.0007 to 0.0020)nd –
Bidirectional transfer (mg 12C-equivalent)420.237 (0.110–0.380)410.052 (0.018–0.087) 0.054–0.331
Absolute net transfer (mg 12C-equivalent)420.134 (0.063–0.205)410.042 (0.011–0.073) 0.015–0.169
image

Figure 2.  The relationships between Pseudotsuga menziesii var. glauca planted (filled dots and thick lines) or natural (open dots and thin lines) seedlings and (a) stem volume, (b) shoot dry biomass or (c) height growth rate and net carbon transfer. Dotted lines represent 95% prediction bands.

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There was a significant interaction between soil disturbance and plant type (see Fig. S2). Net transfer was greater to the larger, natural seedlings than to the smaller, planted seedlings where soil was disturbed; however, the opposite tended to occur where soil was left undisturbed (Fig. 1, see Fig. S2). The amount of net carbon transfer was unaffected by mesh treatment. We found significantly greater gross and net carbon transfer to shoots compared with roots (Table 2). The amounts of labelled carbon found in the shoots of aerial control seedlings were so low they did not differ from zero (Table 2). Mean net carbon transfer to both types of seedlings was small relative to the amount assimilated via photosynthesis (0.0034% based on the amount of carbon isotope fixed during labelling).

Greater net accumulation of carbon occurred in R. vinicolor tips (= 0.03) than the other two abundant ECM taxa (Fig. 3). Donor hosts did not preferentially allocate carbon to R. vinicolor tips compared with other ECM tips based on excess 13C (= 0.58) or excess 14C (= 0.98).

image

Figure 3.  Net carbon gain to the three most abundant ECM taxa found on Pseudotsuga menziesii var. glauca seeedlings. Values are means with 95% confidence intervals (CI). Analysis of variance indicated that the Rhizopogon vinicolor tips had accumulated significantly more net transferred carbon than the other two ECM taxa (= 0.03). The 95% CIs indicate if net carbon transfer was significantly greater than zero, as was the case for Rhizopogon vinicolor and Wilcoxina rehmii since their CIs do not intersect the dashed zero line. These values were based on 1.5 mg of ECM tissue.

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Natural and planted seedling ECM communities

A total of five ECM taxa were found on both natural and planted seedling root systems (see Table S1). Natural and planted seedlings hosted five and four ECM taxa, respectively, with four ECM taxa in common (see Table S1). The two most abundant ECM taxa found on natural and planted seedlings were Wilcoxina rehmii and R. vinicolor (Fig. 4). Of the four shared ECM taxa, all had a relative abundance on root tips greater than 5% (Fig. 4). Natural seedlings had higher levels of ECM colonization (96%) than planted seedlings (79%) (< 0.01). The CMH similarity index indicated there was 94% similarity between the natural and planted seedling ECM communities. Unexpectedly, soil disturbance and mesh treatments did not affect colonization, richness or diversity of the seedling ECM communities (data not shown).

image

Figure 4.  Relative abundance of Pseudotsuga menziesii var. glauca natural and planted seedlings ECM taxa.

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We amplified DNA from 52 R. vinicolor samples, of which 19 produced clear fragments for four different microsatellite loci. Eight of the R. vinicolor samples allowed us to determine whether natural (four samples) and associated planted seedlings (four samples) were harbouring the same fungal genet (i.e. same individual fungus based on identical allele pattern in all target loci determined after microsatellite analysis). Of these four seedling pairs, we found one seedling pair (comprised of a natural seedling and a planted seedling in a 35-μm mesh bag) shared the same R. vinicolor genet.

Soil properties

The disturbed treatment had greater bulk density and lower total C, N and available P compared with the undisturbed treatment (Table 1), indicating that soil disturbance not only removed top soil (forest floor and upper mineral soil) but affected physical and chemical properties of the remaining mineral soil. However, there was no difference in soil moisture content and soil pH between the soil disturbance treatments (Table 1). After the seedlings were excavated, 0.1% of the total amount of 14C (and, by extension, 13C) used in the experiment remained on site in the soil (details not shown).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Seedling relationships with net carbon transfer

Net carbon transfer between P. menziesii seedlings appeared to occur along an interplant source–sink gradient governed by sink strength, agreeing with our first hypothesis (Table 2, Figs 1 and 2). However, contrary to previous studies examining effects of shading, we found that the more poorly performing seedlings did not act as stronger carbon sinks (Finlay & Read 1986; Simard et al. 1997a). Instead, net carbon transfer was greater to planted seedlings with the largest shoots and greatest height growth rates. However, there appeared to be a size threshold since only a few planted seedlings drove these results. For the natural seedlings, net carbon transfer was not governed by size or growth, perhaps because the range in seedling sizes was too narrow. The presence of a few considerably larger natural seedlings may have also produced a size threshold where a progressive increase in net carbon transfer could occur.

We think that most of the labelled carbon reaching the shoots moved via the xylem in the transpiration stream and/or as organic nitrogen compounds driven by the demands of large crowns and rapidly growing shoots (Abuzinadah & Read 1989). It is also possible that net transfer patterns were regulated by ECM fungal factors, such as degree of colonization, density of rhizomorphs, abundance of rhizomorph-forming ECM taxa, and richness and diversity of mycorrhizal pathway-forming ECM taxa. Unfortunately, we were not able to examine these possibilities for labelled seedlings because of the regulatory and logistic issues associated with molecular analyses of 14C-enriched ECM root tips.

The low net carbon transfer relative to total carbon uptake suggests that net carbon transfer between Douglas-fir seedlings was unimportant in the full sunlight conditions of our study. However, we recommend future studies be conducted under shade, including the understorey of older forests or other environmental stresses with a continuous pulse-labelling approach over longer time frames before concluding that net carbon transfer is not ecologically important for autotrophic plants. The evidence provided here, as well as by shade (Read, Francis & Finlay 1985; Simard et al. 1997a) and seasonal experiments (Lerat et al. 2002), and studies of mixotrophic (Selossse & Roy 2009) and mycoheterotrophic plants (Bidartondo 2005), suggest that the magnitude of net carbon transfer follows a source–sink gradient that can by strengthened by microclimate, soils (e.g. disturbance) or seedling nutrition factors. We also suggest that future studies focus on establishing a relationship between the magnitude of net carbon transfer and plant performance.

The variability in the magnitude of net carbon transfer reported here and elsewhere may be partly explained by various factors that would restrict the functionality of the MN. For instance, a MN may not have yet fully physically linked plants at time of pulse-labelling. Furthermore, for plants with an established MN, carbon flow may have been disrupted during the pulse-labelling and/or chase periods by soil fauna grazing on hyphae (Johnson et al. 2005). We were unable to examine these possibilities in this study.

Soil disturbance and mycorrhizal pathway effects on net carbon transfer

Soil disturbance resulted in greater net carbon transfer to natural seedlings than planted seedlings. Greater net transfer to the larger, older natural seedlings compared with the smaller planted seedlings under harsh soil conditions may reflect their richer ECM fungal community, greater ECM colonization (as shown on unlabelled seedlings), and more extensive root systems. The relative benefits of the natural seedlings’ more robust ECM community to seedling physiology were probably enhanced with increased soil environmental stress (Kranabetter, Durall & MacKenzie 2009), thus increasing the difference in relative sink strength between the natural and planted seedlings. In the undisturbed soils, there was a tendency for planted seedlings to gain more carbon than natural seedlings, but in most mesh treatments net transfer did not differ from zero for both planted and natural seedlings. These results suggest that disturbance was necessary to create a sufficient source–sink gradient between natural and planted seedlings for measurable net transfer to occur. At the same time, plant size differences appeared important for soil disturbance effects on net transfer to be expressed.

Net transfer of carbon between conspecific seedlings occurred in the 0.5-μm mesh, indicating that a direct MN pathway was not needed to transfer carbon. Almost all natural and planted seedling root tips were colonized by ECM, indicating that carbon transfer occurred via a mycorrhiza–soil pathway (Simard & Durall 2004), where carbon entered the mycorrhizosphere soil from donor ECM mycelia and was then rapidly picked up by receiver ECM mycelia. MNs have typically been thought to be comprised of intact continuous hyphal links, but in nature these links may be continuous only for short periods before hyphal fragmentation occurs (e.g. soil faunal grazing). Anastomosis allows for connections to reform, but much of interplant resource transfer may occur via the mycorrhiza–soil pathway (Simard & Durall 2004) involving the same or different fungal species. Under conditions where the magnitude of net carbon transfer is considerable (e.g. under shade), the presence of mycorrhizal fungi (i.e. mycorrhizal pathways) may be of functional importance to seedlings, regardless of whether movement of resources occurred via a continuous MN pathway or a mycorrhiza–soil pathway. In this study, the possibility for transfer through a soil-only pathway was minimized by the absence of non-mycorrhizal roots and lack of root intermingling (gaps of 5–30 cm separated interplant root tips). With a relatively large spatial gap between donor and receiver ECM root tips and short labelling chase period, carbon leaked into the soil may have been immediately immobilized by competing soil microbes in the mycorrhizosphere of roots and would have had little opportunity to move through soil pores via mass flow (Högberg & Read 2006; Högberg et al. 2008); hence, movement via the soil-only pathway (Simard & Durall 2004) probably did not occur.

Fate of carbon transferred to seedlings and ectomycorrhizas

Greater amounts of carbon were transferred to shoots than roots of seedlings, and we determined that this was not due to re-fixation of respired CO2 (from soil, hyphae or donor seedling) in the shoots, since potted aerial control seedling shoots were not enriched. These results support our fourth hypothesis that net carbon transfer occurs along mycorrhizal pathways to both shoots and roots of receiver seedlings. Our findings are not in agreement with previous arguments that carbon transferred through mycorrhizal pathways remains in plant roots because the moving carbon is a fungal resource (Robinson & Fitter 1999; Wu, Nara & Hogetsu 2001; Whitfield 2007). Since amino acids or low-weight nitrogenous compounds can move from fungal to plant tissue (Näsholm et al. 1998), it is conceivable that mycorrhizal pathway-transferred carbon moved either as nitrogenous compounds or was converted to them before moving into receiver plants (Simard & Durall 2004). Even so, it is still possible that carbon is transferred from fungal to plant tissue as fungal sugars (Bidartondo 2005).

Our findings are in contrast to Wu, Nara & Hogetsu (2001), who suggested carbon is not transferred to shoots of ECM plants, but agree with other studies demonstrating that carbon is transferred to both receiver shoots and roots (Read, Francis & Finlay 1985; Abuzinadah & Read 1989; Simard et al. 1997a,b), albeit at greater relative amounts to shoots in our study. Wu, Nara & Hogetsu (2001) covered the shoots of the receiver seedlings in aluminium foil, leading to total darkness, which would prevent photosynthetic activity and induce stomatal closure, resulting in a much-reduced transpiration flow. As carbon probably moves from roots to shoots via the xylem, Wu, Nara & Hogetsu (2001) were unlikely to find any carbon accumulating in the shoots. In this study, receiver seedlings were growing in full sunlight and at a time of year (early July) when shoots were expanding, creating high carbon and nitrogen demands. Under these conditions, shoots may have been stronger sinks than roots for mycorrhizal pathway-transferred carbon.

Rhizopogon vinicolor ECM root tips received greater amounts of transferred carbon than the other two abundant ECM taxa found on receiver seedlings in all mesh treatments. It is unlikely that this result was confounded by donor host carbon allocation differences between the ECM taxa since donors did not preferentially allocate carbon to R. vinicolor tips. This finding agrees with our fifth hypothesis that net carbon transfer is affected by the exploration type of the dominant ECM fungi; in this case, greater transfer to root tips colonized by the ECM taxon forming rhizomorphs. Using time–course autoradiography, Wu, Nara & Hogetsu (2001) observed higher radioactivity in rhizomorphs compared with the mycelium of Pisolithus tinctorius forming a MN between Pinus densiflora seedlings. Rhizomorphs potentially provide the most important pathway for carbon transfer between plants forming a MN (Brownlee et al. 1983; Read, Francis & Finlay 1985), but rhizomorph connections are more variable and may be less frequent than simple hyphal connections (Finlay & Read 1986). Because of the large soil gaps between seedling roots, our results agree with the suggestion that rhizomorphs may facilitate interplant carbon transfer over greater distances than the extending mycelia (Finlay & Read 1986).

Potential for MNs to form

The potential for MNs to form between natural and planted seedlings was high because their ECM community composition was similar and because abundance of the most common taxa was identical. This is also supported by fragment analysis (microsatellite marker analysis) showing the same R. vinicolor genet on the natural and planted seedling in one of the seedling pairs. Natural seedlings were excavated kilometres away and must have harboured different R. vinicolor genets than the genets present as resilient soil propagules (spores, sclerotia or hyphal fragments) in the subplots; this is because the largest distances found between the same genet of this species have only ranged from 2 to 12 m (Kretzer et al. 2003; Beiler et al. 2009). Given that planted seedlings were non-mycorrhizal at time of planting, we suggest that the occurrence of the same R. vinicolor genet on natural and planted seedlings arose through colonization by extraradical hyphae or rhizomorphs from a MN. Our results corroborate an associated study located in a similar, nearby forest where extensive R. vinicolor networks were observed to link multiple P. menziesii trees. This sister study also showed that the probability that two unrelated individuals could have identical multilocus genotypes by chance was extremely low (Beiler et al. 2009). In addition, other studies have shown high potential for MNs to establish among conspecific trees (Haskins & Gehring 2004; Cline, Ammirati & Edmonds 2005; Teste et al. 2009).

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

The harsh conditions created by soil disturbance increased net carbon transfer to larger, natural P. menziesii seedlings. More transferred carbon accumulated in receiver shoots than roots, possibly because sink strength in the shoots was greater than in the roots due to the phenology and full illumination of seedlings at the time of labelling. Net carbon transfer increased with planted receiver seedling shoot size and growth rate, and was significantly greater to R. vinicolor than to the other ECM taxa. These findings are relevant to forests characterized by mixed-disturbance regimes, which vary in disturbance frequency and severity and leave a suite of environmental conditions (e.g. light or soil resource availability) for seedling regeneration. Further research is needed to elucidate how soil disturbance interacts with a range of environmental conditions to affect the magnitude of intraspecific net carbon transfer and seedling performance.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

We are grateful to Graeme Hope, Amanda Schoonmaker, Wallis Johnson and Anita Norman for their aid at various stages of the field work. We are indebted to Jason Stobbe (Stobbe Excavating, Kamloops, BC, Canada) for the excavation work. We thank Leanne Philip for her advice on the isotope gas labelling. Special thanks are also due to David Harris at UC Davis SIL for conducting the dual 13C–14C isotope analyses and Ted Sedgwick and Shelley Kayfish (UBC radiation safety officers) for safety recommendations during the 14C field and laboratory work. We also thank Bill Clarke, Lenka Kudrna and MaryAnn Olson for help with the molecular work. We are grateful to Melanie Jones and the anonymous referees for their comments on earlier drafts. Funding was provided by a Forest Sciences Program of Forest Investment Innovation of British Columbia grant, a Canadian Foundation for Innovation grant, and Natural Sciences and Engineering Research Council (NSERC) Discovery Grants to S.W.S. and D.M.D., and an NSERC PGS scholarship to F.P.T. We declare that the experiments comply with the current laws of the country in which they were performed.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Table S1. List of observed ECM taxa found on Pseudotsuga menziesii var. glauca seedlings.

Figure S1.14C and δ13C of planted and natural seedlings grown under the two soil disturbance treatments and mesh treatments in the field.

Figure S2. Multiple comparisons for the soil disturbance by plant type interaction means.

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