draft · 2017-11-09 · draft rebecca bowler: government of british columbia, ministry of forests,...

Draft

Combining leaf gas-exchange and stable carbon isotopes to

assess mycoheterotrophy in three Pyroleae species.

Journal: Botany

Manuscript ID cjb-2017-0007.R2

Manuscript Type: Article

Date Submitted by the Author: 05-Jul-2017

Complete List of Authors: Bowler, Rebecca; University of Northern British Columbia, Natural Resources and Environmental Studies Graduate Program Massicotte, Hugues; University of Northern British Columbia, Ecosystem Science and Management Fredeen, Arthur; University of Northern British Columbia, Ecosystem Science and Management

Is the invited manuscript for consideration in a Special

Issue? : N/A

Keyword: Partial mycoheterotrophy, Pyroleae, <sup>13</sup>C, photosynthesis, carbon nutrition

https://mc06.manuscriptcentral.com/botany-pubs

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Rebecca Bowler: Government of British Columbia, Ministry of Forests, Lands and Natural Resource Operations, 499 George Street, Prince George, BC, V2L 1R5

Combining leaf gas-exchange and stable carbon isotopes to assess mycoheterotrophy in 1

three Pyroleae species. 2

3

Rebecca Bowler1, Hugues B. Massicotte1,2, Arthur L. Fredeen1,2 4

5

1Ecosystem Science and Management Program, University of Northern British Columbia, 3333 6

University Way, Prince George, BC, Canada V2N 4Z9; 2Natural Resources and Environmental 7

Studies Institute, University of Northern British Columbia, 3333 University Way, Prince George, 8

BC, Canada V2N 4Z9 9

10

Rebecca Bowler: [email protected] 11

Hugues Massicotte: [email protected] 12

Corresponding author: A.L. Fredeen 13

Telephone: 250-960-5847 14

Email: [email protected] 15

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Abstract

The determination of extent of mycoheterotrophy (MH) in plants, primarily made through

the use of stable isotope methods, has gained considerable attention in the last decade. The aim

of this study was to characterize the photosynthetic rates (PS) and several gas-exchange

parameters, as well as stable carbon isotope composition (δ13C) of partially mycoheterotrophic

(PMH) Pyroleae comparative to autotrophic reference ericaceous species. An end-member

mixing model was applied to δ13C, deriving estimates of % C gained via fungi (CDF). The δ13C

was significantly enriched for Orthilia secunda and Pyrola chlorantha (relative to autotrophs)

resulting in estimates of CDF ranging from 13.8 to 20.8 %. Despite significantly lower PS rates of

O. secunda and P. chlorantha, as well as lower conductance and transpiration, there were no

significant differences in Ci:Ca ratios across all species, suggesting that the C isotope inferences

for these two species were reflective of fungal C gains. By contrast, results for all variables

indicated primarily autotrophic C nutrition for Chimaphila umbellata. Further studies such as

isotope labelling experiments or assessments of biochemical constraints to autotrophy may

resolve uncertainties in these species, allowing more accurate understanding of the complex

nutritional mode of these plants.

Key words: Partial mycoheterotrophy, Pyroleae, 13C, photosynthesis, carbon nutrition

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Introduction

In a seminal review, Leake (1994) first adequately described the nutritional strategy of

mycoheterotrophic (MH) plants. Early research into full MH species, plants that completely lack

chlorophyll and are not capable of photosynthetic C gains, led to the belief that these plants were

directly parasitic on autotrophic host plants by means of haustorial (root-like) connections on

xylem and/or phloem tissues (Leake 1994). However, it was eventually recognized that MH

plants are indirectly parasitic (epiparasites) on host autotrophs, gaining organic C through

exploitation of shared mycorrhizal networks (Leake 1994; Preiß 2009). In addition, Leake (1994)

recognized that there were probably some MH species that also contained chlorophyll and gained

some C via photosynthesis (PS). These plants are a type of mixotroph, most accurately described

as partial mycoheterotrophs (PMH). Mycoheterotrophs are thus involved in a tripartite

relationship between mycorrhizal fungi associates that are in turn linked to surrounding host

autotrophic plants which are the ultimate C sources in the system (Bidartondo 2005).

Stable isotope ratio mass spectrometry has become a powerful tool in ecological studies

allowing researchers insight into plant- and animal-environment interactions, both with abiotic

resources and other organisms (Dawson et al. 2002). The method is based on the fact that

metabolic and physical processes generally discriminate against heavy isotopes, leading to

distinct isotopic signatures between different organisms or between different organs of a single

organism (Farquhar et al. 1989; Gleixner et al. 1993; Tedersoo et al. 2007). Within the food

chain, higher trophic levels accumulate heavy isotopes, resulting in unique isotopic signatures

across trophic groups (Dawson et al. 2002). This approach has allowed researchers to document

different nutritional strategies for plants and fungi (e.g., Högberg et al. 1999; Gebauer and Meyer

2003; Trudell et al. 2004; Preiss and Gebauer 2008). In particular, analysis of natural abundance

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of stable carbon and nitrogen (13C and 15N, typically denoted as δ13C and δ15N) isotopes in

certain groups of plants, as well as their fungal symbionts, has provided strong empirical and

quantitative evidence critical in understanding both the autotrophic physiology of plants as well

as the unique nutritional mode of mycoheterotrophy.

In photosynthetic plants, overstory trees are typically more enriched in 13C than

understory plants due to exposure to greater irradiance, resulting in higher PS rates, which

reduces discrimination against 13C during the carboxylation reaction of Rubisco (Farquhar et al.

1989; Högberg et al. 1999; Courty et al. 2011). This is primarily attributed to a drawdown of

intercellular CO2 so that concentrations relative to ambient CO2 (Ci and Ca, respectively,

expressed as the ratio of Ci:Ca) are lower and a greater proportion of 13C will be assimilated

compared to plants with higher Ci:Ca (Farquhar et al. 1982; Farquhar and Sharkey 1982).

Understory plants also show lower 13C enrichment due to the incorporation of greater amounts of

13C-depleted CO2 originating from soil respiration (Farquhar et al. 1989; Courty et al. 2011;

Hynson et al. 2013). Based on these differences, Högberg et al. (1999) showed that overstory

host trees provide a greater proportion of C to ectomycorrhizal (ECM) fungi (the dominant

mycorrhizal class in boreal ecosystems) than understory autotrophs.

Simple carbohydrates transferred to ECM fungi are less enriched in 13C compared to

more complex molecules such as cellulose and lignin decomposed by saprotrophic fungi

(Gleixner et al. 1993; Badeck et al. 2005). Along with differences in N metabolism, distinct dual

isotope signatures result between ECM and saprotrophic fungi (e.g., Högberg et al. 1999; Kohzu

et al. 1999). This enrichment is mirrored by fully MH taxa relative to their fungal symbionts,

providing key evidence that ECM fungi are the principal food source for MH plants, though

numerous MH orchid species have been found to associate with saprotrophic fungi (Trudell et al.

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2003; Ogura-Tsujita et al. 2009; Hynson et al. 2013). It was the intermediate enrichment of 13C

isotopes in green plants relative to full MH species and surrounding autotrophs that provided

evidence these photosynthetic plants were gaining much of their C through fungal pathways

(e.g., Gebauer and Meyer, 2003; Julou et al. 2005; Tedersoo et al. 2007).

A common application of isotopes in ecological studies is that of end-member mixing

models, which enables quantification of the source contributions to a mixture (Dawson et al.

2002). Since there are only two potential C sources (via photosynthesis or via fungal symbionts)

contributing to the δ13C signature of PMH plants, simple two-source linear mixing models are

used to estimate the proportion of fungal-derived C (% CDF) in these plants (Gebauer and Meyer

2003; Tedersoo et al. 2007; Zimmer et al. 2007; Preiss and Gebauer 2008). The method assumes

a linear correlation between fungal-derived C and the enrichment in plant 13C, with the endpoints

of the model defined as the mean δ13C values of autotrophic reference plants (0 % CDF) and full

MH references (100 % CDF). The model makes the assumption that the reference plants fully

represent the source isotope signatures (Hynson et al. 2013).

Despite the importance of photosynthesis or other gas-exchange characteristics to δ13C

signatures, particularly PMH species, we only know of three particular studies that directly

measured gas-exchange in MH plants, all orchids, with one using a 13CO2 tracer method. In these

cases, gas-exchange showed net respiration rates due to a) a low-light environment where net

CO2 uptake could not compensate for net respiratory CO2 losses in Cephalanthera damasonium

(i.e., below light-compensation point; Julou et al. 2005), or b) inefficient photosynthetic capacity

in Limodorum abortivum (Girlanda et al. 2006), Corallorhiza trifida and Neottia nidus-avis (the

latter essentially lacking photosynthetic capacity; Cameron et al. 2009). In one other study, gas-

exchange data was used to estimate seasonal productivity in a Pyroleae species, Pyrola

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incarnata, but only via respiration measurements coupled with changes in plant biomass (Isogai

et al. 2003). This last study also did not focus on the species in the context of mycoheterotrophy.

All other studies that discuss aspects of photosynthesis or gas-exchange in full or partial MH

plants used approaches involving stable 13C data, fungal identification through molecular typing,

DNA sequencing and/or chlorophyll content and fluorescence analysis (e.g., Abadie et al. 2006;

Tedersoo et al. 2007; Preiss et al. 2010; Hynson et al. 2012; Matsuda et al. 2012), sometimes in

conjunction with environmental conditions (e.g., light levels; Preiss et al. 2010).

In this study, we investigated photosynthetic rates, as well as several other important gas-

exchange variables and 13C signatures, of three putative PMH Pyroleae species common to

central British Columbia. These were assessed in relation to fully autotrophic reference species

of the family Ericaceae in order to determine; a) if the Pyroleae exhibited gas-exchange

characteristics that would enrich 13C signatures independently of mycoheterotrophy, and b) to

apply linear mixing models to stable 13C data to derive estimates of % CDF.

Methods and Materials

Site descriptions

The study site was located adjacent to the Crooked River Provincial Park (54°28’58" N,

122°40’26" W, elevation 723 m), approximately 70 km north of the city of Prince George, British

Columbia. The site was found in the Sub-Boreal Spruce Moist Cool subzone of the

Biogeoclimatic Ecosystem Classification system, with subxeric, nutrient poor soils that were

predominantly loamy sand or fine sands.

The site was attacked by mountain pine beetle (MPB: Dendroctonus ponderosae

Hopkins) around 2003. Despite up to 95 % canopy mortality in much of the surrounding areas,

lodgepole pine (Pinus contorta Douglas ex Louden var. latifolia Engelm. ex S. Watson)

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remained the dominant canopy and understory species. There was also a considerable population

of young to mature subalpine fir (Abies lasiocarpa (Hook) Nutt.), and a minor component of

hybrid white spruce (Picea glauca (Moench) Voss x. engelmannii Parry ex. Engelm.). A wide

variety of common shrub and forb species were present, though ericaceous vegetation dominated

the understory. Four Pyroleae species were present in relatively high abundance, including

Chimaphila umbellata (L.) W.P.C. Barton, Orthilia secunda (L.) House, Pyrola chlorantha Sw

and Pyrola asarifolia Michx.; the latter was not sampled due to a smaller population and more

scattered distribution. Additionally, small populations of the fully MH Pterospora andromedea

Nutt. and Monotropa hypopitys L. occurred at the site.

Sample selection and gas exchange measurements

During the growing season of 2012, three of the four Pyroleae species were sampled for

gas-exchange measurements and natural abundance of stable 13C isotopes. Nine plots were

established with four individuals of each Pyroleae. In each plot, depending on availability of

species, three to seven samples of autotrophic Ericaceae were also measured, either Vaccinium

myrtilloides Michx., Arctostaphylos uva-ursi (L.) Spreng, or both; one plot also contained

samples of Vaccinium membranaceum Douglas ex Torr. and Vaccinium caespitosum Michx. The

leaves from these autotrophs were taken from within 20 cm or less from the ground if the plant

itself was taller.

All foliar gas-exchange measurements were performed non-destructively using a portable

gas-exchange system (model LI-6400, LiCor Inc., Lincoln, NE, USA). Sample leaves were

measured in situ under a broad range of ambient conditions approximately once per month

during the growing season (June – September), using a transparent conifer chamber (model LI-

6400-05). Net photosynthetic rates (PS: µmol m-2 s-1) and PAR (photosynthetically active

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radiation between 400-700 nm: µmol m-2 s-1), using an external quantum sensor (LI-6400 9901-

013), were measured. Full MH samples were also measured once they had emerged in late July

or August by enclosing stems within the chamber. Additional parameters investigated included

stomatal conductance (mol H2O m-2 s-1), Ci:Ca ratios (ratio of intercellular CO2 to ambient CO2),

transpiration rates (mmol H2O m-2 s-1) and water use efficiency (WUE, as a function of PS/E;

µmol CO2 mmol-1 H2O), though the latter was not calculated for full MH samples.

In August, autotroph and Pyroleae samples were also measured for light response curves

(LRCs) under controlled levels of PAR (0, 10, 25, 100, 400 and 800 µmol m-2 s-1), using a leaf

chamber with an LED light source (model LI-6400-02B). All gas-exchange measurements were

performed while controlling for CO2 concentration (400 µmol mol-1 air) and flow rate (500 µmol

s-1). Following final in situ gas-exchange measurements in September, one to three leaves were

collected for isotope analysis, including those that had been measured for gas-exchange.

Photosynthetic rates were expressed on a hemi-surface area basis (HSA: cm2). For

samples measured under ambient conditions, HSA was determined by scanning leaves or traced

leaf outlines using a flat bed scanner (Epson Expression 1640 XL), and image analyzing

software (Winfolia, v. Pro 2003d, Régent Instrument Inc., Quebec, Canada). For MH species,

surface area was calculated assuming a cylindrical stem geometry (A = 2πrh), as based on the

average of upper and lower diameters (d/2 = r) and height of the stem enclosed in the chamber

(h). All species HSA values were re-entered into the Li-6400 and final photosynthetic rates were

recalculated by the instrument prior to analysis.

Isotopic analysis and calculations to estimate % fungal-derived carbon

For isotope composition, harvested leaf samples were oven dried for 48 hours at 65 °C,

ground into a fine powder and sent for determination of natural abundances of 13C at the Stable

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Isotope Facility at the University of Saskatchewan, Saskatoon, Canada. Sample δ13C values were

determined via elemental analyzer/continuous flow isotope ratio mass spectrometry using the

following equation:

[Eq. 1] δ13C = (Rsample/Rstandard – 1) × 1000 (‰)

where Rsample and Rstandard are the ratios of heavy to light isotope (13C/12C) of the samples and

reference materials calibrated to international standards, respectively. The current international

standard for δ13C is Vienna PeeDee Belemnite (Carter and Barwick 2011).

Estimates of % fungal-derived carbon (% CDF) were calculated using linear-mixed

models as per Gebauer and Meyer (2003), and later refined by Preiss and Gebauer (2008). The

following equations were used to first determine sample enrichment (ƐSAMPLE):

[Eq. 2] ƐSAMPLE = δ13CSAMPLE – δ13CREF

where δ13CSAMPLE and δ13CREF are the δ13C for any plant and the mean of plot-specific

autotrophic reference plants, respectively. The % CDF for PMH species were then calculated

using the following equation:

[Eq. 3] % CDF = (ƐPMH/ ƐMH) x 100 %

where ƐPMH and ƐMH are individual PMH plant isotopic enrichments and ƐMH(ISO) the mean

isotopic enrichment for MH references, respectively. The MH references did not occur in sample

plots so site-specific references were used. The % CDF for each sample was also calculated using

the most recent ƐMH value of 6.177 ‰ for full MH Ericaceae published by Hynson et al. (2016)

to determine how estimates changed using MH references from the most recent and best

described value for this group.

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Statistical Analyses

Data for autotrophs and the Pyroleae were analyzed to test for species differences in

seasonal averages of in situ net PS rates, conductance, Ci:Ca ratios, transpiration, and water use

efficiency (WUE), as well as δ13C values and calculated % CDF. All the data met the assumption

of normality based on the Shapiro Wilk test. Data were initially tested using linear mixed models

to take into account any random effects of plot, but plot was not significant in any test.

Therefore, data were tested using one-way ANOVAs assuming equal variances and Tukey’s

HSD post hoc comparisons. The exceptions were PS rates, conductance and both % CDF

estimates, which did not meet homogeneity of variance assumptions; these were tested with one-

way ANOVAs assuming unequal variances, with Welch F-ratio and Dunnett’s T3 post hoc

pairwise comparisons. All ANOVAs were performed using SPSS Versions 21 or 22 (SPSS Inc.,

Chicago, IL, USA) and all p-values were considered significant at α ≤ 0.05.

In addition, using Sigmaplot v. 11.0 (Systat Software Inc., San Jose, CA), data from light

response curve measurements were fit to the following non-rectangular hyperbola function (see

Prioul and Chartier 1977):

[Eq. 4]

PS =�φPAR + PS� − � φPAR + PS�� − 4φPAR ∗ PS�θ�

2θ+ R�

where φ is the initial slope of the light response curve (µmol CO2 µmol-1 photons), PS and PSMAX

are net and light saturated photosynthetic rates (µmol m-2 s-1), respectively, PAR is

photosynthetically active radiation as described above, θ is the curvature factor for the inflection

point of the function, and Rd is the dark (metabolic) respiration rate (expressed as a negative

value as CO2 emitted; µmol m-2 s-1) as measured in the chamber when the LED lights were off.

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In all cases, the parameter θ was not significant; therefore, the value estimated for each curve

was entered in as a fixed value, and the curves were fitted again.

Results

Gas exchange variables

Comparisons of average seasonal PS rates resulted in two distinct groupings, with

autotrophs as well as C. umbellata having significantly higher PS rates than O. secunda and

P. chlorantha (p < 0.05 for all significant differences; Table 1). The only other significant

differences that were detected in any other parameter were that O. secunda had significantly

lower stomatal conductance and transpiration compared to autotrophs and C. umbellata (p ≤

0.011), while P. chlorantha had significantly lower WUE than A. uva-ursi (p = 0.011; Table 1).

While no significant differences were found in Ci:Ca ratios, O. secunda and P. chlorantha did

have slightly higher ratios compared to the other species, which was the opposite for all other

gas-exchange variables, where their values were always lower than autotrophs and C. umbellata.

Though not statistically tested, the full MH plants showed very different, though not unexpected,

physiology, with net CO2 emissions (respiration) regardless of light levels, very low g and E, and

high Ci:Ca ratios (Table 1).

Light response curves showed some similarities to the seasonal PS data, with autotrophs,

especially A. uva-ursi, having the highest PS rates at light levels above approximately 100 µmol

m-2 s-1, with C. umbellata, P. chlorantha and O. secunda having progressively lower rates

(Figure 1). These trends were the same for estimated PSMAX, with the exception that V.

myrtilloides had the same value as C. umbellata (Table 2). Respiration rates showed slightly

different relationships between species, with P. chlorantha having the lowest Rd instead of

O. secunda, while the autotrophic V. myrtilloides had lower Rd than C. umbellata, and A. uva-

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ursi had the highest respiration. Similarly, P. chlorantha had the lowest estimate for φ,

representative of quantum yield, V. myrtilloides, C. umbellata, and O. secunda were very similar,

while again A. uva-ursi had the highest value (Table 2).

Unlike seasonal PS rates, where the autotrophs were very similar to each other, in this

case V. myrtilloides showed a very similar response to light as the Pyroleae species, while

A. uva-ursi differed considerably in both the shape of the curve (Figure 1) as well as parameter

estimates (Table 2). While the fitted parameters for LRCs did not include a light saturation point,

it appears that O. secunda reached light saturation at about 400 µmol m-2 s-1, the other Pyroleae

as well as V. myrtilloides were at, or nearly at, light saturation by 800 µmol m-2 s-1, while A. uva-

ursi did not appear to become light saturated.

Stable isotope variables δ13

C and % CDF

Comparisons of δ13C values showed the same groupings as those found for seasonal PS

rates, with both autotrophic species as well as C. umbellata being significantly depleted in 13C

compared to O. secunda and P. chlorantha (p ≤ 0.005; Table 1). These relationships were

reversed compared to PS rates, however, with those species showing lower PS rates having the

more enriched δ13C values, and those with higher PS rates showing more depleted δ13C values

(Figure 2). Table 1 shows that WUE had the clearest negative relationship δ13C, where the lowest

WUE for P. chlorantha had the most enriched 13C and vice-versa for A. uva-ursi. The exceptions

were for C. umbellata and V. myrtilloides, but both variables were very similar between the two

species.

Calculated % CDF values for each of the Pyroleae species again showed the same

distinctive groups, with autotrophs and C. umbellata (0 % and 3 % CDF, respectively) differing

significantly from O. secunda and P. chlorantha (17.7% and 20.8 % CDF, respectively; Figure

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3). The use of the enrichment factor reported by Hynson et al. (2016), however, resulted in lower

% CDF estimates of 13.8 % and 16.2 %, but minimal difference for C. umbellata. Due to the

nature of the calculations, statistical values were the same for both methods (F3,70.8 = 10.056, p <

0.001).

Discussion

This study aimed to characterize the photosynthetic rates, along with important gas-

exchange variables, and stable 13C isotopes of several prevalent Pyroleae species found in the

central interior of BC. Our results support previous studies that have looked at the C nutrition of

this group (e.g., Tedersoo et al. 2007; Zimmer et al. 2007; Hynson et al. 2009; Johansson 2014).

Despite the significantly lower PS rates under ambient light levels found for O. secunda and

P. chlorantha, which would be expected to result in more depleted δ13C values, both species

were significantly enriched in 13C (Figure 2; Table 1). A summary of % CDF values reported in

the literature was presented by Hynson et al. (2013), indicating 33 % and 20 % fungal C gains in

O. secunda and P. chlorantha, respectively. In our case, δ13C data resulted in lower estimates of

% CDF of around 14 % to 18 % and 16 % to 21 % for O. secunda and P. chlorantha, respectively,

depending on whether site specific or cited ƐMH values (as per Hynson et al. 2016) were used

(Figure 3). Our findings more closely agree with those of Johansson et al. (2015), who reported

fungal C gains of 14 % for O. secunda, and 8 % to 17 % in P. chlorantha (using either Hypopitys

monotropa or Pyroleae seedling material as full MH references). In contrast, photosynthetic

rates, δ13C values and % CDF data for C. umbellata were not found to be significantly different

from autotrophs in any comparative tests (Table 1; Figure 3), agreeing with most studies on this

species which indicate primarily autotrophic nutrition (e.g., Zimmer et al. 2007; Hynson et al.

2009, Johansson et al. 2015). However, Tedersoo et al. (2007) showed significant % CDF of up to

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29 % for C. umbellata, suggesting that the species (if not all Pyroleae) is possibly facultatively

PMH.

The light response curves for each species also provide evidence the isotopic enrichment

of O. secunda and P. chlorantha were likely influenced by fungal C. The similarity in the shape

of the curves between the Pyroleae and at least V. myrtilloides, especially at lower light levels, as

well as fitted parameter estimates, indicates mostly similar photosynthetic capacity across

species that would be expected to result in more equitable 13C enrichment were there no fungal C

gains (Figure 1; Table 2).

Since there are so many factors related to gas-exchange, not to mention environmental

variability, which can influence leaf 13C enrichment, it is important to determine how those

factors may affect interpretation of that enrichment regarding mycoheterotrophic nutrition.

In the simplified model by Farquhar et al. (1982, 1989) describing photosynthetic fractionation

of 13C, the main discriminating steps are net carboxylation by Rubisco, and diffusion of CO2 into

the leaf through stomata. Stomatal diffusion is regulated by stomatal and boundary layer

conductances, and in conjunction with effects of transpiration, results in differences in Ci:Ca

ratios (Farquhar et al. 1982; Farquhar and Sharkey 1982). Hynson et al. (2013) discuss two of

these factors that could cause differential levels of 13C discrimination and assimilation between

autotrophs and MH plants, that of higher Ci:Ca ratios due to lower PS rates and/or higher

conductance, and greater use of soil-respired CO2 in the latter group.

Effects of physiology and environment on 13

C signatures

The first aspect of low PS and higher Ci in PMH plants would result in better

equilibration between CO2 concentrations of the intercellular spaces and atmosphere (via

stomatal diffusion), resulting in a better equilibrium and therefore increased discrimination

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against 13C. The significantly lower PS rates of O. secunda and P. chlorantha were not

accompanied by significantly higher Ci:Ca ratios (Table 1). Clearly higher conductance did not

lead to higher Ci:Ca ratios in the Pyroleae, since conductances were lower than autotrophs, and

significantly so for O. secunda. According to Farquhar et al. (1982), when assimilation rates are

reduced due to low stomatal conductance, Ci should actually decrease and δ13C increase.

Because either of these cases should result in depleted δ13C in the Pyroleae, these relationships

provide evidence that the 13C enrichment observed in our Pyroleae samples was reflective of

other factors, namely fungal C gains.

Lower stomatal conductance is known to occur in shade-adapted plants whereas high

conductance in sun plants facilitates rapid CO2 uptake to fulfill biochemical demand

(Kriedemann 1999). This seems to be the case, with A. uva-ursi having the highest, and

O. secunda and P. chlorantha having the lowest, stomatal conductances as well as PS rates

during both instantaneous measurements and light response curves (Figure 1; Table 1), while

maintaining nearly equal Ci:Ca ratios. We believe that, despite possible differences in

photosynthetic capacity across species (e.g., lower quantum yield of P. chlorantha), the effects

on δ13C values due to biochemical differences should be limited.

However, the Pyroleae did have slightly higher Ci:Ca ratios than autotrophs. As noted,

this should theoretically contribute to lower δ13C rather than the enriched levels in our samples.

An interesting possibility noted by Hynson et al. (2013) was that lower PS rates in PMH species

could actually mask their apparent degree of mycoheterotrophy by essentially diluting the 13C-

enriched C via mycorrhiza with more 13C-depleted photosynthetic C due the better equilibrium

described above, which we cannot discount as a possibility.

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As noted by Werner et al. (2012), the simplified model of photosynthetic isotope

discrimination is only valid if other parameters of the full model, such as internal (mesophyll)

conductance and respiratory effects, are negligible. As Ci is used as a proxy for Cc which isotope

discrimination is more accurately dependent on, it is possible that low mesophyll conductance

could result in an additional drawdown of Ci, reducing Cc and therefore discrimination, and

increasing δ13C (Ghashghaie et al. 2003; Werner et al. 2012). Generally, findings have shown

fairly consistent positive correlations between stomatal and mesophyll conductances such that Ci

and Cc are maintained at appropriate levels for biochemical CO2 optimization (Lauteri et al.

1997; Piel et al. 2002; Vrábl et al. 2009; Martins et al. 2014). However, low mesophyll

conductance has been found to increase δ13C signatures more than expected when Ci does not

adequately reflect Cc (e.g., Lauteri et al. 1997). If our PMH Pyroleae do have mesophyll

diffusional constraints, it could lead to erroneous conclusions on the degree of fungal C gains.

These parameters would need further study in the Pyroleae to determine their influence on Cc

and net assimilation rates as well as isotope signatures.

One last aspect that could influence 13C enrichment independent of mycoheterotrophy is

that of water use efficiency (Farquhar 1982, 1989; Werner et al. 2012). All things being equal,

autotrophic plants should have a negative relationship between WUE and discrimination against

13C, which would be a positive relationship between WUE and δ13C (Dawson et al. 2002; Werner

et al. 2012). It would follow that if the PMH species have lower WUE but higher 13C, this would

be an indication that something else such as fungal C is influencing 13C. The significantly lower

WUE in P. chlorantha, and O. secunda to lesser degree (not significant), along with their

significant higher δ13C, provide evidence that this is indeed the case (Table 1). The fact that the

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former is significant but not the latter may provide some explanation as to why % CDF was

higher for P. chlorantha (Table 2).

The second aspect discussed by Hynson et al. (2013) that could cause differential levels

of 13C discrimination between PMH and autotrophic species is that of incorporating more 13C-

depleted CO2 originating from soil respiration. Of the two autotrophic reference species, the low-

growing A. uva-ursi showed higher PS capacity in light response curves (Table 2; Figure 1), yet

was more depleted in 13C than the taller V. myrtilloides (Table 1). This supports the idea that A.

uva-ursi may indeed incorporate higher levels of soil-derived CO2. Since the Pyroleae have a

similar growth habit as A. uva-ursi, if 13C-depleted CO2 did contribute to lower 13C in this

species, it seems very likely the same could be occurring in the Pyroleae. Similar to the

discussion in the previous section, this could also contribute to diluting 13C-enriched fungal C,

again lowering the apparent level of PMH nutrition.

Additional considerations

Recent findings have shown that plant family is an important factor in distinguishing

isotopic patterns, which amazingly holds at the global scale (Hynson et al. 2016). We expect that

by comparing only ericaceous species in our study, we limited the influence of particular family

or guild characteristics that would potentially result in differential δ13C values between species.

Additionally, by following commonly used sampling procedures (e.g., Hynson et al. 2012), we

assume environmental variability did not substantially influence 13C discrimination. For

example, the heights of measurements and sampling occurred within approximately 20 cm of the

soil surface, and samples were measured at the plot level (though plot was not significant) to

reduce variability in light, moisture and fungal distributions. As the site was quite open due to

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substantial pine mortality following mountain pine beetle attack, we expect that turbulent mixing

of CO2 from air and soil would also reduce variability in 13C levels of the source air.

Despite all this, there were still other variables that we did not consider, such as the

remobilisation of carbohydrates from storage organs and how that might cause 13C to vary

between species as well as plant organs, particularly when estimating % CDF. Badeck et al.

(2005) found that sugars and total organic matter was around 1 ‰ to 2 ‰ more depleted than

starches in a variety of woody and herbaceous species. As noted by Hynson et al. (2013), after

dormancy periods, orchids may utilize either fungal or stored C for building new leaf tissues, but

as these C sources are more enriched in 13C than tissues formed via photosynthetic processes,

estimations of proportion of fungal C gains could be in error. Analysis of soluble sugar 13C rather

than bulk leaf tissue can help assess short term MH C gains (e.g., Hynson et al. 2012). We also

did not examine potential effects of differences in quantum yield and light saturation points in

the Pyroleae in relation to cumulative light environments, or any associated photorespiratory

processes that could influence 13C values. Future investigations into these aspects could reveal

important vernal trends in nutritional mode.

Conclusions

This is the first known study to directly evaluate δ13C values with comparative gas-

exchange measurements of PMH Pyroleae species. Our findings show that despite significantly

low PS rates in O. secunda and P. chlorantha, δ13C values were highly enriched compared to

their sister taxa C. umbellata, as well as related autotrophic references. While low PS rates are

usually expected to result in increased Ci:Ca ratios, in our case, there were no significant

differences in those ratios, suggesting that 13C enrichment due to photosynthetic processes is

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fairly similar in closely-related Ericaceous species, and thus 13C signatures are likely highly

reflective of mycoheterotrophic C gains.

Acknowledgements

The authors would like to thank summer field assistant Elaine McAloney for her

invaluable help in data collection and sample preparation in 2012. We would also like to thank

Myles Stocki at the University of Saskatchewan, who was responsible for analyzing samples for

stable isotope data, and Dr. Mike Rutherford and Dr. Marty Kranabetter, who provided valuable

feedback on the MSc thesis that led to this manuscript. This research was largely funded by

NSERC Discovery Grants to Dr. Art Fredeen and Dr. Hugues Massicotte. NSERC Canada

Graduate and UNBC scholarships provided additional support for Rebecca Bowler Finally, the

comments of two reviewers were very helpful in improving the final version of this manuscript.

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Table 1. Seasonal gas-exchange data and δ13C values of full autotrophs, partial myco-heterotrophs (PMH) and full mycoheterotrophs of the family Ericaceae. Data are means ± SD; PS = photosynthesis, and WUE = water use efficiency.

*Net PS

(µmol m-2 s-1)

*Conductance

(mol H2O m-2 s-1)

Ci:Ca

ratios

Transpiration

(mmol H2O

m-2 s-1)

WUE (umol

CO2 mmol-1

H2O) δ13C (‰)

Autotrophs

Arctostaphylos

uva-ursi 4.34 (2.06)a 0.187 (0.076)a 0.840

(0.070) 2.87 (0.74)a 1.69

(0.90)a -31.11 (1.00)a

Vaccinium

myrtilloides 4.35 (1.85)a 0.176 (0.063)a 0.833

(0.085) 3.15 (1.08)a 1.44

(0.69)ab -30.86 (0.87)a

PMH

Chimaphila

umbellata 3.96 (1.37)a 0.836

(0.048) 2.98 (0.96)a 1.46

(0.59)ab -30.71 (0.63)a

Orthilia

secunda 2.23 (0.89)b 0.114 (0.037)b 0.856

(0.057) 2.11 (0.67)b 1.23

(0.56)ab -30.00 (0.68)b

Pyrola

chlorantha 2.47 (1.05)b 0.149 (0.066)ab 0.862

(0.049) 2.57

(0.80)ab 1.12

(0.54)b -29.85 (0.77)b

†Myco-heterotrophs -0.8 (0.1) 0.03 (0.01) 1.13 (0.05) 0.76 (0.37)

-26.2 (0.1)

F-statistic 16.81 8.52 1.41 7.14 3.363 12.54

P < 0.001 < 0.001 0.234 < 0.001 0.012 < 0.001

Df 4, 57.86 4, 58.78 4, 143 4, 143 4, 142 4, 127

Note: Species sharing the same letter within a column are not significantly different at α ≤ 0.05. *Statistical values shown derive from a one-way ANOVA assuming unequal variance, with Welch F-ratios and Dunnett’s T3 post hoc comparisons. All other tests used one-way ANOVAs assuming equal variance and Tukey’s HSD post hoc comparisons. † Not included in statistical tests

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Table 2. Light response curve parameters of maximum photosynthetic rates (PSMAX), quantum yield (φ), curvature factor for the inflection point (θ), and dark respiration rates (Rd), fitted to a non-rectangular hyperbola function for two autotrophic and three Pyroleae in the family Ericaceae. Values in brackets are standard errors for parameter estimates, and all parameters were considered significant at α = 0.05.

PSMAX φ *θ Rd Chimaphila

umbellata 6.18 (0.24) 0.063

(0.010) 0.4 0.74 (0.17) p-value < 0.0001 < 0.0001 NS < 0.0001

Orthilia

secunda 3.59 (0.15) 0.066

(0.011) 0.52 0.53 (0.12) p-value < 0.0001 < 0.0001 NS < 0.0001

Pyrola

chlorantha 5.22 (0.20) 0.052

(0.008) 0.44 0.44 (0.14) p-value < 0.0001 < 0.0001 NS < 0.01

Arctostaphylos

uva-ursi 9.14 (0.79) 0.074

(0.024) -0.1045 0.96 (0.40) p-value < 0.0001 < 0.01 NS 0.022

Vaccinium

myrtilloides 6.18 (0.1) 0.067

(0.016) 0.7 0.61 (0.30) p-value < 0.0001 < 0.0001 NS 0.047

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Figure 1. Mean (± 95% CI) net photosynthesis (µmol m-2 s-1) as a non-linear regression function of photosynthetically active radiation (light response curves) for two autotrophic reference species and three Pyroleae species as measured in August 2012. Figure 2. Distribution of average seasonal net photosynthetic (PS) rates (µmol m-2 s-1) and δ13C (‰) values under ambient conditions for two autotrophic Ericaceae, putative partial mycoheterotrophic Pyroleae species and full mycoheterotrophs (MHs) in 2012. Shaded boxes represent the interquartile range (IQR) with the line at the median and the diamond representing the mean. Whiskers represent data within 1.5 x IQR and points are outliers. Figure 3. Mean (± 95% CI) estimated fungal C gains (% CDF) of three Pyroleae species in relation to two autotrophic references, Arctostaphylos uva-ursi and V. myrtilloides, calculated from mycoheterotrophic enrichment factors (ƐMH) from our site-specific reference mycoheterotrophs (Site) and those reported by Hynson et al. (2016; Literature). Different letters across panels indicate significant species differences at α = 0.05.

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draft · 2017-11-09 · draft rebecca bowler: government of british columbia, ministry of forests,...

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