Plants within communities can be interconnected and exchange resources through a common hyphal network1,2,3,4,5,6,7,8,9,10,11,12,13,14, and form guilds based on their shared mycorrhizal associates15,16,17. Consequently, the theory that plant community dynamics operate mainly within the constraints of resource supply18 should be reformulated to consider mutualism between plants and their mycorrhizal fungi, as well as microbially mediated resource sharing15,17,19.

We have studied shared compatibility of ectomycorrhizal fungi and interspecific carbon transfer in Betula papyrifera Marsh. and Pseudotsuga menziesii (Mirb.) Franco. Seven ectomycorrhizal morphotypes were common between B. papyrifera and P. menziesii, covering over 90% of their root tips, indicating a high potential for interspecific hyphal connections20. We used reciprocal labelling with 13C and 14C in the field to examine below-ground transfer between the two tree species. To our knowledge, this is the first study in which reciprocal labelling has been used to investigate bidirectional and net carbon transfer.

We examined carbon transfer during two growing seasons, when seedlings planted in forest soil were two or three years old. Ectomycorrhizal B. papyrifera and P. menziesii and vesicular–arbuscular (VA) mycorrhizal Thuja plicata D. Don. were planted 0.5 m apart in three-seedling groups. These three tree species occur naturally together in the experimental area. Thuja plicata served as a marker for indirect isotope movement through soil pathways (that is, in respired gaseous CO2, exudates, and sloughed root and fungal cells). Four or six weeks before labelling, P. menziesii seedlings in the replicate groups were exposed to three light treatments: deep shade, partial shade, and full ambient sunlight (Table 1). After the shading period, B. papyrifera and P. menziesii in each group were pulse-labelled with gaseous 13CO2 or 14CO2. This approach enabled detection of the carbon isotope that was received by one seedling from the other. Reciprocal labelling schemes were applied to replicate seedling groups in the first year to account for differences in absolute isotope fixation between 13C and 14C. Pulse-labelling for 2 h in full ambient light with a minimum photosynthetically active radiation level of 800 µmol m−2 s−1 was followed by a 9-day chase period under the light treatments. Seedlings were then harvested and separated into foliage, stems, coarse roots and fine roots. Ground tissue was combusted to CO2 and analysed for 13C abundance by mass spectrometry and 14C by liquid scintillation.

Table 1 Light treatment, photosynthetic rate and carbon transfer

Carbon transfer between B. papyrifera and P. menziesii occurred in both directions in the first and second year, and, although there was no net transfer in the first year, there was a net gain in carbon by P. menziesii in all three light intensities in the second year (Table 1and Fig. 1). In the first year (two-year-old seedlings), isotope transfer to P. menziesii was balanced by reciprocal transfer to B.papyrifera (so there was no net transfer) in all light intensities (P = 0.10 for light treatment effects). Bidirectional transfer between B. papyrifera and P. menziesii represented 4% of total isotope fixed by both species together, and transfer through soil pathways to T.plicata was <1% of the total transfer between the ectomycorrhizal species20.

Figure 1: Mean (± s.e.) net transfer from B. papyrifera to P. menziesii in deep shade, partial shade and full ambient sunlight in the first (two-year-old seedlings, open circles) and second (three-year-old seedlings, filled circles) experiment years.
figure 1

Within years, means denoted by the same letter (a or b) do not differ significantly (P < 0.05). Asterisks indicate treatments in which net transfer was greater than zero (P < 0.05).

In the second year (three-year-old seedlings), net transfer to P. menziesii and bidirectional transfer between B. papyrifera and P. menziesii represented on average 6% and 7%, respectively, of the total isotope fixed by the two tree species together, and both were over twice as great in deep shade as in partial shade or full light (P < 0.05; Table 1and Fig. 1). Transfer to VA mycorrhizal T. plicata averaged 18% of the total transfer between B. papyrifera and P.menziesii, suggesting that most transfer between the ectomycorrhizal tree species occurred through hyphal connections20. The greater effect of shading on magnitude of bidirectional transfer and the occurrence of net transfer in the second rather than the first experiment year coincided with greater root extension and potential for interplant hyphal linkages, as well as increased vigour of seedlings in the second year, and indicates that transfer varies according to seedling status and environmental conditions.

The greater transfer in deep shade in the second year was due to increased transfer from B. papyrifera to P. menziesii, indicating that net transfer was affected by changes in the sink strength of P.menziesii. Net transfer from B. papyrifera to P. menziesii was associated with whole-seedling net photosynthetic rates 1.5 and 4.3 times greater for B. papyrifera than for P. menziesii in full light and deep shade, respectively, and foliar nitrogen concentrations twice as great for B. papyrifera as for P. menziesii20. These results suggest that carbon was transferred between species down carbon and nitrogen gradients. Fully developed leaves are strong sinks for nitrogen and are sources rather than sinks for carbon21, and amino acids rather than sugars have been shown to pass from ectomycorrhizal fungi into host plants22,23. Consequently, it has been suggested that transfer may be regulated more strongly by a nitrogen gradient than a carbon gradient6,22.

The 3–10% net transfer from B. papyrifera to P. menziesii measured in these experiments represents a substantial carbon gain by P. menziesii. It is similar to the 10% 14C transfer one way from clonal parent plants to connected ramets that has been suggested to be of sufficient magnitude to increase survival and growth of the ramets24. It is also similar to the 5–15% transfer of fixed gaseous 15N2 from Alnus glutinosa to Pinus contorta through ectomycorrhizal connections6, and to the 0–10% of carbon in Cynodon dactylon roots that was derived from Plantago lanceolata through VA mycorrhizal connections25. Carbon transfer of this magnitude, especially if it is associated with nitrogen, would be expected to affect performance under critically harsh conditions or over the lifetime of a P. menziesii seedling10,11.

In our experiments, the average amount of received isotope that was retranslocated from roots to foliage was 13% for P. menziesii and 45% for B. papyrifera. The amounts retranslocated from roots to foliage of receivers exceed those observed in previous ectomycorrhizal carbon-transfer experiments (usually <10%)5,10, and could either supplement photosynthate or, if carbon is transferred as amino acids, the foliar nitrogen pools23. The pattern of carbon transfer in these ectomycorrhizal plants therefore contrasts with that reported for some VA mycorrhizal systems, where relatively little of the transferred carbon was onwardly mobilized into shoots of receiver plants, and may represent carbon restricted to fungal tissue in roots of receiver plants3,25,26.

The amount of carbon exchanged between B. papyrifera and P. menziesii is indicative of a tightly linked plant–fungus–soil system. Our study extends earlier laboratory results3,4,5,6,10,11,24,25 to the field, providing direct evidence for both bidirectional and net carbon transfer between plant species, for the occurrence of hyphal as well as soil pathways, and for source–sink regulation of net transfer in field conditions. When P. menziesii seedlings are establishing and growing in the shade of illuminated B. papyrifera in natural, mixed communities, they may benefit directly from their association with B. papyrifera through supplemental gains in transferred carbon. There is a further possibility that carbon is distributed below-ground among plants across resource gradients, other than light, that affect relative photosynthetic potential within a mycorrhizally linked plant community. Such a mechanism would offer one explanation for the ability of species-rich communities to maintain productivity during drought or where nutrients are limiting27. If our results reflect the magnitude of carbon transfer in natural systems, then the net competitive effect of one species on another cannot be predicted without a better understanding of interplant carbon transfer through shared mycorrhizal fungi and soil pathways. A more even distribution of carbon among plants as a result of below-ground transfer may have implications for local interspecific interactions28, maintenance of biodiversity11,15,17, and therefore for ecosystem productivity, stability and sustainability16,19,27.


Seedling groups. In both experiment years, each experimental unit consisted of a seedling group. In the first year (two-year-old seedlings), a 3 × 2 factorial set of treatments was replicated four times in a completely randomized design (24 experimental units). The treatments consisted of three P. menziesii light treatments (full ambient light, partial shade and deep shade) and two labelling schemes (14C-B. papyrifera with 13C-P. menziesii; 13C-B. papyrifera with 14C-P. menziesii). In the second year (three-year-old seedlings), each of the three light treatments was applied to five replicate groups of seedlings in a completely randomized design (15 experimental units). Owing to a shortage of replicate seedling groups, only one labelling scheme was applied in the second year: 14C-B. papyrifera with 13C-P. menziesii.

Light treatments. In the partial and deep shade treatments, P. menziesii seedlings were shaded with lightly and heavily clothed cone-shaped tents four weeks before labelling on 14–16 July 1993 (first year), and six weeks before labelling on 4–5 August 1994 (second year). The shade tents were removed from P. menziesii during the 2-h pulse for maximum labelling efficiency. Each B. papyrifera and P. menziesii seedling was sealed inside flexible, airtight, 5-mm thick × 60-cm wide × 90-cm tall fluoropolymer gas sampling bags (Norton Performance Plastics). The shoot of one partner seedling was then pulsed with 200 ml gaseous 13CO2 (99%, equivalent to 107.42 mg 13C), and the shoot of the other partner was pulsed with 7.4 MBq gaseous 14CO2 (equivalent to 52.86 µg 14C, released from Na214CO3 with lactic acid).

Bidirectional and net transfer calculations. Sample δ13C (‰) and 14C (Bq) values were converted to excess carbon isotope (mg) for bidirectional and net transfer calculations. The conversions were based on previously described procedures29,30. Conversion of δ13C (‰) to the absolute isotope ratio (13C/12C) of the sample was based on the PeeDee Belemnite (PDB) standard. Fractional abundance (13C/(13C + 12C)) and total carbon content (mg) of the sample were used to calculate 13C content (mg) of the sample. Conversion of 14C (Bq) to 14C content (mg) was based on the batch-specific activity (λ) of Na2 14CO2 (λ = 1.96 GBq mmol−1; Amersham Canada). Excess isotope (13C or 14C) content (mg) of the tissue was calculated as the product of excess isotope content of the sample and tissue biomass, and excess isotope (mg) of the whole plant was determined by summing excess isotope content of the four tissue types. Bidirectional and net transfer calculations were based on whole-plant levels of excess isotope that were received from partner donor seedlings. Bidirectional transfer was the sum of isotope received by both P. menziesii and B. papyrifera in a seedling group. Conversely, net transfer was the difference between isotope received by P. menziesii and that received by B. papyrifera. Positive net transfer indicated that a greater amount of isotope was received by P. menziesii than by B. papyrifera, and negative net transfer indicated the opposite. Bidirectional and net transfer were expressed as proportions of total isotope assimilated by P. menziesii and B. papyrifera together.

Statistical analysis. Significant labelling scheme effects in the first year were detected using two factor analysis of variance (ANOVA) (α set at 0.05). For calculations of bidirectional and net carbon transfer, labelling scheme effects were first removed by applying correction factors to excess 14C content (mg) on a treatment–species–tissue basis. The correction factors were the species–tissue-specific ratios of excess 13C (mg) to excess 14C (mg) measured in the reciprocal labelling schemes of the same light treatment. The treatment–species–tissue-specific correction factors were averaged over the four replicates per labelling scheme in the first year. The correction factors derived in the first year were applied to data collected in the second year, because only one labelling scheme was applied in the second year. Using the corrected excess 14C values (analogous to excess 13C equivalent values), data from the first and second years were subject to one-factor ANOVA for comparisons of net and bidirectional transfer among P. menziesii light treatments (7 degrees of freedom for the first year, 4 for the second). Net transfer estimates were compared with zero using t-tests.