Dirt cheap: An experimental test of controls on resource exchange in an ectomycorrhizal symbiosis

1. To distinguish among hypotheses on the importance of resource-exchange ratios in outcomes of mutualisms, we measured resource (carbon (C), nitrogen (N), and phosphorus (P)) transfers, and their ratios, between Pinus taeda seedlings and two ectomycorrhizal (EM) fungal species, Rhizopogon roseolus and Pisolithus arhizus in a laboratory experiment.

2. We evaluated how ambient light affected those resource fluxes and ratios over 3 time periods (10, 20, and 30 weeks), and the consequences for plant and fungal biomass accrual, in environmental chambers.

3. Our results suggest that light availability is an important factor driving absolute fluxes of N, P, and C, but not exchange ratios, although its effects vary among EM fungal species. Declines in N:C and P:C exchange ratios over time, as soil nutrient availability likely declined, were consistent with predictions of biological market models. Absolute transfer of P was an important predictor of both plant and fungal biomass, consistent with the excess resource exchange hypothesis, and N transfer to plants was positively associated with fungal biomass.

4. Altogether, light effects on resource fluxes indicated mixed support for various theoretical frameworks, while results on biomass accrual better supported the excess resource exchange hypothesis, although among-species variability is in need of further characterization.

Methods
We measured resource (carbon (C), nitrogen (N), and phosphorus (P)) transfers, and their ratios, between Pinus taeda seedlings and two ectomycorrhizal (EM) fungal species, Rhizopogon roseolus and Pisolithus arhizus in a laboratory experiment. We evaluated how ambient light affected those resource fluxes and ratios over 3 time periods (10, 20, and 30 weeks), and the consequences for plant and fungal biomass accrual, in environmental chambers. Plants and fungi were grown in chambers with a mesh barrier in the soil, which allowed only fungi (and not roots) to grow into a separate fungus-only chamber where fungal respiration rates could be measured; respiration rates were integrated over the different growth periods to estimate total amounts of C respired in each growth period. Ergosterol analyses were used to estimate fungal biomass. These methods allowed C transferred to fungi to be separated into a component respired and a component incorporated into fungal biomass. To test effects of light, fungal species, and time on absolute amounts and ratios of transferred resources (Question 1), data on C, N, and P fluxes, and N:C and P:C exchange prices were analyzed as response variables in separate univariate analyses using linear mixed-effects models using the lmerTest package in R version 3.5.2, with growth period (1, 2, and 3), light level (high and low), EM fungal species, and their interactions as fixed effects. The flux of C was also partitioned out into separate variables of C in fungal biomass and C respired by fungi, as well as carbon use efficiency (CUE), calculated as C in fungal biomass divided by total C transferred to fungi. These variables were also analyzed as above, to further understand how experimental factors may have affected carbon partitioning in the system. To test for effects of resource transfer on accumulated fungal and plant biomass (Question 2), we conducted model selection among all possible linear mixed-effect models for which the candidate variables were the main effects of the five resource flux variables representing effects of total resource transfer and exchange ratios (C, P, N, N:C, N:P).

1. 为区分关于资源交换比率在互利共生结局中重要性的各类假说，本研究通过实验室实验测定了火炬松（Pinus taeda）幼苗与两种外生菌根（ectomycorrhizal, EM）真菌物种——玫瑰须腹菌（Rhizopogon roseolus）和无足豆包菌（Pisolithus arhizus）之间的资源（碳（C）、氮（N）、磷（P））转移量及其比率。
2. 本研究在人工气候箱中设置3个培养周期（10周、20周、30周），探究了环境光照如何影响上述资源通量及其比率，以及其对植物与真菌生物量积累的效应。
3. 研究结果表明，光照可利用性是调控氮、磷、碳绝对通量的关键因子，但对资源交换比率无显著影响，且其效应因外生菌根真菌物种而异。随着培养时间延长，土壤养分可利用性大概率下降，此时氮碳（N:C）与磷碳（P:C）交换比率的降低趋势符合生物市场模型的预测。磷的绝对转移量是植物与真菌生物量的重要预测因子，这与过剩资源交换假说一致；而向植物转移的氮量与真菌生物量呈正相关关系。
4. 综合来看，光照对资源通量的效应未能完全契合任一理论框架，呈现出混合支持的结果；而生物量积累相关结果则更支持过剩资源交换假说，但物种间的差异仍需进一步表征。

实验方法
本实验测定了火炬松幼苗与两种外生菌根真菌——玫瑰须腹菌和无足豆包菌之间的资源转移量及其比率，实验在实验室环境中开展。本研究在人工气候箱中设置3个培养周期（10周、20周、30周），探究环境光照对上述资源通量及其比率的影响，以及其对植物与真菌生物量积累的效应。
实验中，植物与真菌被种植于带有土壤筛网屏障的人工气候箱内，该屏障仅允许真菌（而非植物根系）侵入单独的真菌专属培养室，以便测定真菌呼吸速率；通过对不同培养周期的呼吸速率进行积分，可估算每个周期内真菌呼吸释放的总碳量。采用麦角固醇分析法估算真菌生物量。该实验方法可将转移至真菌的碳划分为呼吸消耗组分与整合进入真菌生物量的组分。
为检验光照、真菌物种及培养时间对资源转移绝对量与比率的影响（问题1），本研究以碳、氮、磷通量以及氮碳、磷碳交换比率作为响应变量，采用R语言3.5.2版本中的lmerTest包构建线性混合效应模型，分别开展单变量分析。模型的固定效应包括培养周期（1、2、3）、光照水平（高、低）、外生菌根真菌物种及其交互作用。
本研究还将碳通量拆分为真菌生物量固持碳、真菌呼吸释放碳以及碳利用效率（carbon use efficiency, CUE）三个变量，其中碳利用效率以真菌生物量固持碳与转移至真菌的总碳量的比值计算。采用与前述一致的分析方法对上述变量开展分析，以进一步探明实验因子如何影响系统内的碳分配过程。
为探究资源转移对真菌与植物累积生物量的影响（问题2），本研究针对所有可能的线性混合效应模型开展模型选择，候选变量为代表总资源转移量与交换比率的5个资源通量变量的主效应，即碳、磷、氮、氮碳比、氮磷比。