Native Arbuscular Mycorrhizal Fungi Interactions with Native and Invasive Woody Plant Species

A Thesis Submitted in Partial Fulfillment of theRequirements of the Renée Crown University Honors Program at

Syracuse University

Steven G. Carlson Jr.

Candidate for Bachelor of Scienceand Renée Crown University Honors

Spring 2020

Honors Thesis in Your Major

Thesis Advisor: _______________________ Advisor’s Name and Title

Thesis Reader: _______________________ Reader’s Name and Title

Honors Director: _______________________ Dr. Danielle Smith, Director

© Steven G. Carlson Jr. 2020

ii

Abstract

Arbuscular mycorrhizal fungi (AMF) colonize the roots of a variety of land plants, providing an

alternative pathway to secure nutrients and water from the soil. In exchange, the plants provide

space for the fungi to grow while allocating a portion of the carbon they assimilate by

photosynthesis to their fungal partners. Although AMF are generally thought to be a mutualistic

partner with their plant hosts, they actually can function along a gradient from parasite to

mutualist. Invasive species may gain a competitive advantage over native species by (1) having

more beneficial relationships with their AMF, and/or (2) being less reliant on AMF giving them

an advantage when the fungal symbionts are not present. In this study, three phylogenetic pairs

of native and invasive woody shrub species were grown in pots in a greenhouse with and without

AMF in their soil. Biomass and colonization rate were measured and compared between

mycorrhizal and nonmycorrhizal treatments for all six species. The results showed an increased

dependency on AMF associations for native species compared to invasives. I found a positive

relationship between how much growing with AMF enhanced plant growth and the percent of

roots colonized by AMF for native species. These results suggest that invasive woody species

outcompete natives when AMF density is too low for native shrubs to be readily colonized. More

research is needed to determine how AMF densities may influence native and invasive shrub

establishment and growth in the field.

iii

Executive Summary

Arbuscular mycorrhizal fungi (AMF) colonize the roots of over 80% of land plant

families, forming symbiotic relationships called mycorrhizas. When colonization occurs, the

hyphal network of AMF provides greater access to nutrients and water for the plant by increasing

the volume of soil from which nutrients are taken up. In return, the plant allocates a portion of its

photosynthetically produced carbon to their fungal partners, while also providing a habitat (i.e.,

the root) in which the fungus grows. Although this relationship is generally thought to be

mutualistic, it actually functions on a gradient from parasite to mutualist. Mycorrhizal

relationships are one way an invasive plant may gain a competitive advantage over a native

species. The aim of this study was to determine if plant and AMF relations functionally differ

between native and invasive species of woody shrubs in New York to gain an understanding of

how mycorrhizal relationships may play a role in promoting plant invasion. Invasive species may

gain a competitive advantage over native species by (1) having more beneficial relationships

with their AMF, and/or (2) being less reliant on AMF, giving them an advantage when the fungal

symbionts are not present.

To test the relationships of AMF with native and invasive plants, we grew 3 native

species and 3 invasive species in a greenhouse in pots with and without AMF. The relationship

between plant and AMF was determined by 1) how the presence of AMF influenced plant

growth and 2) how AMF colonization rate of plant roots differed between native and invasive

species. To quantify growth responses, aboveground and belowground biomass measurements

were taken after a growth period of 3 months. From root material, first order fine roots were

picked to examine to what extent each plant was colonized by AMF. These fine roots were

chemically cleared of plant organelles, allowing fungal structures present to be more easily seen.

iv

The roots were then dyed with blue ink to stain existing AMF. The colonization rates were

determined by examining these cleared, stained fine roots under a microscope.

The results showed an increased dependency on AMF associations for native compared

to invasive species. Native plants that were not inoculated with AMF tended to produce less

biomass than native mycorrhizal plants, while invasive plant biomass performance did not differ

significantly between mycorrhizal and nonmycorrhizal plants. I also found that the positive

effect of growing with AMF was related to the percent that roots were colonized for native

species. These results suggest that invasive species outcompete natives when AMF density is too

low for native shrubs to be readily colonized. This could be one reason why invasive plants are

able to invade into recently disturbed habitats with low AMF abundance. More research is

needed to determine how AMF densities may influence native and invasive shrub establishment

and growth in the field. However, my results from a greenhouse experiment suggest that native

plant abundance could be limited by the availability of AMF into locales that have been recently

disturbed, where competitive invasive species are abundant and AMF density may be low.

Increasing the performance of native shrub species by providing access to their AMF symbionts

may be a method for forest managers to enhance native species abundance and diversity in New

York State forests.

v

Table of Contents

Abstract……………………………………….……………….…………… iiiExecutive Summary………………………….……………….…………... ivAcknowledgements ……………………………………………………….. vii

Introduction………………………………………………………………… 1

Methods…………………………………………………………….……...... 4

Results…………………………………………………………………….… 8

Discussion…………………………………………………………………...10

Works Cited.………………………………………………………………..14Appendix……………………………………………………………………16

vi

Acknowledgements

I would like to thank my faculty and thesis advisor and mentor Dr. Doug Frank, PhD

candidate Alex Ebert, and Dr. Louis James Lamit for helping me throughout this entire process. I

would not have been able to develop my project and write this thesis without their guidance and

expertise. I would also like to thank Dr. Kate Becklin for agreeing to be my honors reader and

providing insightful comments in the writing process. Thank you all.

vii

1

Introduction

Evidence from the fossil record indicates that associations between mycorrhizal fungi and

plants date back 460 million years (Redecker et al. 2000). These relationships, called

mycorrhizas, are generally viewed as mutualisms, meaning that both the plant and fungus benefit

from the association (Smith et al. 1997, Klironomos 2003, Chen et al 2019). When colonized

with mycorrhizal fungi, plants provide a place that the fungi can grow and reproduce (i.e., the

root), as well as a portion of their photosynthetically produced carbon, and, in exchange, the

fungi provide improved nutrient status to the plant through better absorption of minerals and

water, and defense against soil pathogens (Bonfante et al. 2010). When colonized, the extension

of fungal hyphae beyond the plant root depletion zone, the volume of soil from which a plant

root takes up nutrients, allows for greater access to nutrients, such as phosphorus, nitrogen,

sulfur, copper, and zinc (Smith et al 1997, Chen et al 2018).

Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with over 80% of land

plant families (Smith et al 1997). Intracellular AMF hyphae colonize the cortex of host roots and

penetrate the cell walls where they from arbuscules and vesicles (Picture 1). Arbuscules are

highly branches structures that increase the surface area between the plant cell membrane and the

fungus, allowing for efficient nutrient transfer between the two symbionts. Vesicles are also

incorporated within the fungal network and function mainly as storage structures for the AMF

(Chen et al 2018). Extracellular AMF hyphae, at just a few micrometers in diameter, are much

finer than the finest plant roots (Dekker 2009). With a smaller diameter, extracellular hyphae can

extend into finer spaces than the much thicker roots. The greater surface area provided by the

hyphae increases the available volume of soil from which plants can take up nutrients.

2

Although these mycorrhizal relationships are generally viewed as mutualisms, this is not

always the case. The relationships between AMF and their host plant functionally ranges along a

continuum of parasitism to mutualism (Klironomos 2003). The relationship between native and

invasive, or introduced, plant species and mycorrhizal fungi in native soils is still being

investigated, with little known about the interaction between woody plant species and AMF.

Invasive species have been shown to have a lower growth response to AMF colonization when

grown in a habitat with a native AMF community (Klironomos 2003). For a plant to successfully

invade a new area, it must have a competitive advantage over the native species in the area.

Invasive species may gain a competitive advantage over native species by (1) having more

beneficial relationships with their AMF, and/or (2) being less reliant on AMF, giving them an

advantage when the fungal symbionts are not present (Klironomos 2003, Pringle et al 2009).

Minimizing negative growth response to mycorrhizal fungal infection when the relationship is

parasitic is particularly helpful for plant invasion success. If the invasive plant forms a

mutualistic relationship with AMF, then its enhanced positive response to AMF compared to

native plants may provide it a competitive advantage. Lastly, if the invasive species is

nonmycorrhizal, meaning AMF does not readily colonize it, then the local AMF community will

not affect the plant’s growth, allowing it to take over patches of soil with particularly low AMF

densities.

The aim of this study was to understand if, and fundamentally how, native and invasive

species of shrubs in New York interact with AMF. To achieve this, we grew native and invasive

plants in a greenhouse in pots with and without native AMF. The relationship between plant and

AMF was determined by 1) how the presence of AMF influences plant growth and 2) how AMF

colonization rate of plant roots differed between native and invasive species.

3

Methods

We collected softwood cuttings of 6 shrub and liana species from a common garden at

Syracuse University, Syracuse, New York, USA in late spring 2017. The experiment included

three native species (Lonicera sempervirens, Viburnum acerifolium and Berberis canadensis)

and three nonnative species (Lonicera japonica, Viburnum opulus, and Berberis thunbergii) in

phylogenetic pairs (Table 1). Cuttings were propagated in sterilized coarse sand (Lighthouse

Pool Filter Sand, US Silica, Katy, TX, USA) and amended with 0.8% indole-3-butyric acid

(IBA) in talc as a rooting agent (Hormodin 3, OHP Inc., Mainland, PA, USA). Cuttings were

placed in a greenhouse at Syracuse University and were misted regularly during daylight hours

on a mist bench. Air temperatures were kept between 18-21 °C during daylight and 15-18 °C at

night, while the rooting media was kept at a constant 22 °C. Healthy cuttings were removed from

the propagation media, the roots were washed in DI water, and each individual was weighed

before being transplanted into a pot containing autoclaved and rinsed potting soil (Scotts

Premium Topsoil, Scotts Co., Marysville, OH, USA). Pots were 15 cm deep and contained

approximately 1 L of soil with a layer of gravel at the bottom to encourage drainage.

The arbuscular mycorrhizal (AM) treatment was created by amending half of the pots

with 20 g of live soil inoculum from an intact, maple-dominated forest near Pompey, NY, USA

(42°54’ N, 76°02’ W). Live soil was added to the pots, rooted cuttings were planted on top of the

inoculum and the roots were covered with a final layer of sterile soil. Nonmycorrhizal (NM)

treatments were created by adding 20 g of autoclaved inoculum from the same aggregate soil

sample. All pots received a microbial wash by passing a soil slurry through a 35 𝜇m nylon mesh

(Industrial Netting, Minneapolis, MN, USA) in order to exclude AMF spores (Johnson et al

4

2010). This allowed the microbial community (except for AMF) to be reestablished in the NM

treatments. All pots were fertilized with 80 mL of a half-strength Hoagland’s solution once every

2 weeks.

I harvested the plants after 3 months. Aboveground biomass was separated into stem,

leaf, and flower material, washed carefully, and placed in a drying oven for 48 hours or longer at

60 °C then weighed in grams. Aboveground biomass consisted of the sum of total stem, leaf, and

flower material for each individual. Roots were also carefully washed and a small subsample of

first order roots for each plant was reatined to determine AMF abundance. First order roots have

been shown to be the best indicator of overall mycorrhizal colonization (McCormack et al.

2015). The first order roots, as well as the remaining belowground material, were dried similarly

to aboveground material, weighed, and both were incorporated into belowground biomass.

The first order roots were stained to determine AMF colonization using the following

protocol. First order roots were placed in flip cap tubes after scanning and were then rehydrated

in DI water for minimum 24 hours. The roots were then cleared in 10% KOH (wt/vol) heated to

90 ºC to remove as many intracellular plant and non-fungal structures as possible. Cleared fine

roots were rinsed with DI water to remove excess KOH, then placed in pure white household

vinegar (5% acetic acid) for 5 minutes to acidify. Next, roots were submerged in a 5% blue ink-

vinegar solution and placed in a 95º water bath for 10-15 minutes to stain fungal structures.

Lastly, the stained roots were rinsed twice with DI water to remove excess ink, then placed in DI

water that had been slightly acidified by adding a few drops of white vinegar to de-stain the

existing plant structures, making fungal structures more visible. This was done for a minimum of

48 hours (Vierhelig et al 1998).

5

De-stained roots were mounted in Polyvinyl-Lacto-Glycerol on microscope slides to

examine fungal colonization rates. One slide was made for each sample with fine roots spread

out horizontally along the long axis of the slide and observed at a magnification of 100x. Using

the modified gridline intersect method outlined by McGonigle et al (1990), fungal colonization

rates were measured as follows: starting from the right (non-labeled) side of each slide, each

slide was moved via the stage graticule along its short axis, perpendicularly intersecting roots. At

each intersection of fine root, the presence of the AMF structures of hyphae, arbuscules, and

vesicles were tallied, using the crosshair as a reference by running the crosshair perpendicularly

along the length of the root and tallying only structures that came into contact with the crosshair

(McGonigle et al 1990). The presence of other fungal structures not belonging to AMF were also

recorded. The absence of fungal structures was also noted. Percent colonization was calculated

as the sum of transects in which AMF structures occurred divided by the total number of

transects per slide. A total of 50 root transects per slide were taken for each sample.

Statistical analysis was done in R. Linear mixed effects models were used to examine

aboveground belowground, and total biomass, root shoot ratio, and colonization rate.

Mycorrhizal growth response (MGR) and root shoot ratio (RSR) were derived for each treatment

by the following formulas:

MGR= log(average total biomass for mycorrhizal samples / average total biomass for

nonmycorrhizal samples), for each species

RSR= (belowground biomass/aboveground biomass), for each sample

The linear mixed models contained the fixed effects of nativity, mycorrhizal treatment,

and their two-way interaction. Each model also contained the random effect of species. A linear

6

mixed effects model was chosen due to the relatively small sample size of each treatment within

each treatment (Table 1). A pairwise Post Hoc test was run to determine significant differences

between groups for biomass, RSR, and colonization measurements.

7

Results

The inoculation of all six species was effective; no AMF structure was found in any of

the nonmycorrhizal samples. Colonization rates were higher for the three native species

compared to those of their phylogenetically paired invasive counterparts (p=0.0432) (Fig. 1).

Variation between each genus also occurred with each species in their phylogenetic pair having

similar colonization rates to one another.

Plant biomass differed between native and invasive plants, but mycorrhizal fungi were

more important to growth in native hosts. For aboveground biomass, native species produced

more biomass with mycorrhizas compared to without mycorrhizas (Fig. 2). The interaction

between nativity and mycorrhizal treatment was found to significantly affect aboveground

biomass (p=0.0155). Aboveground biomass of the three invasive species showed little difference

between mycorrhizal and nonmycorrhizal treatments. However, a pairwise comparison showed

that the native mycorrhizal and native nonmycorrhizal significantly differed in aboveground

biomass (5.821±1.36 vs. 1.571±0.54, average aboveground biomass per plant ± SE; p=0.0002).

For belowground biomass, AMF increased performance for native species, but not for invasive

species (Fig. 3). The interaction between nativity and mycorrhizal treatment was also found to

significantly affect belowground biomass (p=0.0183). Again, the pairwise comparison revealed

that the native mycorrhizal and native nonmycorrhizal groups significantly differed in

belowground biomass (2.705±0.52 vs. 1.075±0.33, average belowground biomass per plant ±

SE; p=0.0073). Two of the invasive species, regardless of mycorrhizal treatment, performed just

as well or better than the native mycorrhizal treatment in terms of aboveground and belowground

biomass, while Berberis thunbergii seemed to depend more on AMF inoculation for biomass

8

growth (Fig. 2 and 3). Total biomass was found to be affected significantly by an interaction

between nativity and mycorrhizal treatment (p=0.0162). There was a significant difference

between native mycorrhizal and native nonmycorrhizal groups as well (8.525±1.72 vs.

2.670±0.86, average total biomass per plant ± SE; p=0.0006) (Fig. 4).

For both native and invasive species, the introduction of AMF led to a lower RSR,

indicating more aboveground production compared to belowground. Mycorrhizal treatment was

found to significantly affect RSR for both native and invasive species (p=0.0300). The native

mycorrhizal plants had a significantly lower mean RSR compared to native nonmycorrhizal

plants (0.661±0.12 vs 1.283 ± 0.24, average RSR±SE; p = 0.0033), although the mass of

extracellular fungal hyphae was not included in belowground biomass (Fig. 5).

Lastly, mycorrhizal growth response (MGR) was greater for every native species

compared to their invasive counterpart for each native-invasive pair. MGR ranged from -0.036 to

1.52 (Fig. 6). MGR was on average 0.573 greater for native compared to invasive species.

Invasive species were also found to have a combination of lower colonization rate and MGR for

each native/invasive pair (Fig. 7).

9

Discussion

In examining the relationship between woody plants and AMF, we found that the native

species were more dependent on their mycorrhizal symbionts compared to invasive species. For

both aboveground, belowground and total biomass, AMF addition increased biomass more for

the natives than invaders. For the three invasive species, sterile treatments performed better,

although not statistically so, signifying relatively lower dependence on AMF for growth. This

result is consistent with the findings of Pringle et al. (2009), who found that invasive herbaceous

plants tended to have a lower response and sensitivity to the addition of AMF, with responses

ranging from mutualistic to parasitic (Pringle et al 2009). However, I am not aware of any study

that has investigated responses in woody plants, as this study did. Here, four species, including

all three natives, formed mutualistic relationships, while the other two species formed

commensalisms.

Differences in root shoot ratio (RSR) between mycorrhizal vs nonmycorrhizal

counterparts were present within each subset of treatments. Both native and invasive plants

belonging to the mycorrhizal treatment had a lower RSR value compared to non-mycorrhizal

samples, although only the native mycorrhizal and nonmycorrhizal groups differed statistically

speaking (Fig. 4). This is to be expected since AMF allow for plants to allocate carbon to fungi

within their roots, instead of "spending” more carbon on producing more fine roots. This

decreases the ratio of roots to shoots with the plant allocating a larger portion of its carbon

aboveground, which allows for more carbon to be procured through photosynthesis that can be

used for production of more photosynthesizing leaves. When colonized by AMF, plants will

spend less carbon by not forming new fine roots to extend beyond their own depletion zone, in

10

favor of exploring their own depletion zone and beyond through fungal hyphae gaining nutrients

in this volume of soil. The small diameter of hyphae allows the AMF to gain nutrients from the

areas in the soil where the pore size is too small for the first order roots of plants to penetrate.

The increased colonization rate found for a native species compared to their invasive

counterparts in each native-invasive pair was not expected based on the literature. AMF

colonization rates for invasive plants were found to be no lower than colonization in native

species in multiple studies (Dickie et al 2017, Bunn et al 2015), although these studies did not

differentiate based on plant functional group (i.e. only shrubs), as this study did. The three

invasive shrubs examined in this study were either less suitable hosts for the native species of

AMF in the forest that the inoculum was collected or the invasive species were able to regulate

how much colonization occurred and optimize the benefit to cost ratio (Pringle et al. 2009,

Klironomos 2003). The former is somewhat more plausible due to the coevolution of the native

plant and native fungi community, leading to increased efficiency of colonization and benefits

for both the plant and fungi when pairing with each other. The finding that the native species had

a higher MGR compared to the invasive species also was unexpected based on the literature.

When grown separately, natives and invasives have exhibited similar responses to the addition of

AMF, although the effect on woody plants was unknown (Bunn et al. 2015). For the six species

of woody shrubs examined in this study, MGR was consistently greater for the native member of

each native-invasive pair. Larger responses to AMF could be due to differences in colonization

rates because for each native species an increase in MGR was associated by an increase in

colonization rate. Whether the increased colonization rate leads to the increased MGR is subject

to further study.

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The native in each taxonomic pair of native-invasive species had a more beneficial

mycorrhizal relationship compared to the invasive species, indicating an increased dependency

upon AMF by the three native shrubs. Four species, including all three natives and the invasive

Berberis thunbergii formed mutualistic relationships with AMF: all four species had positive

MGR values. Viburnum opulus and Lonicera japonica formed commensalistic relationships with

AMF, in which the AMF benefitted in gaining a suitable habitat to grow while the plants had

slightly positive and slightly negative MGR values, respectively. For these two species, the

biomass was not significantly effected by colonization by AMF.

With densities of AMF not randomly nor homogenously distributed within habitats, the

opportunity for natives to gain a competitive advantage through forming the mutualistic

relationship with AMF is not even for any given spot within a site (Kilronomos 2003). The

uneven distribution of AMF at sites at smaller scales can lead to spatial differences in the success

of the invasion of introduced species within a habitat. In patches of soil with high AMF

abundance and density, native shrub species may be able to coexist or even outcompete invasive

species based on the competitive growth advantages in increased above and belowground

biomass and lowered RSR, which allow for increased aboveground competitiveness. Conversely,

in areas with lower native AMF density or with a differing, less advantageous AMF variety than

what native plants prefer, the competitive advantages gained from AMF colonization will be

absent for natives, potentially allowing invasive shrubs to outcompete native species.

Competitive advantages gained from plant – soil microbe interactions are particularly

important when thinking about the potential for invasive species to colonize a new area. If an

area of native plants that is also occupied by AMF varieties that are especially beneficial for

those native species is disturbed and the AMF community is altered or lost, invasive plants that

12

do not rely on AMF as much as natives will be able to colonize that disturbed area. The lack of

dependence upon AMF for competitive advantage lends the invasive species to be able to inhabit

soils with lower AMF density or witha more diverse community of AMF. Native species, on the

other hand, are confined to areas with higher AMF densities or where a specific AMF

community occurs.

More research is needed to determine how AMF densities may influence native and

invasive shrub establishment and growth in the field. My finding indicating native woody plants

in New York are more dependent upon AMF than invasive plants suggests that manipulating

mycorrhizal relationships may be a way that managers can maintain native forest integrity. Thus,

inoculating AMF in locales with increased invasive species presence and/or low AMF density,

thereby managing for AMF abundance, may help native species rebound from the invasion of

nonnative plant species in New York. Further field research needs to be conducted to determine

the AMF densities in patches of native and invasive shrubs in the forests of New York.

13

Works Cited

Bonfante, P., & Genre, A. 2010. Mechanisms underlying beneficial plant–fungus interactions

in mycorrhizal symbiosis. Nature Communications, 1(1). doi: 10.1038/ncomms1046

Bunn, R. A., Ramsey, P. W., & Lekberg, Y. (2015). Do native and invasive plants differ in their

interactions with arbuscular mycorrhizal fungi? A meta-analysis. Journal of

Ecology, 103(6), 1547–1556. doi: 10.1111/1365-2745.12456

Chen, E., Liao, H., Chen, B., & Peng, S. (2020). Arbuscular mycorrhizal fungi are a

double‐edged sword in plant invasion controlled by phosphorus concentration. New

Phytologist, 226(2), 295–300. doi: 10.1111/nph.16359

Chen M, Arato M, Borghi L, Nouri E and Reinhardt D (2018) Beneficial Services of Arbuscular

Mycorrhizal Fungi – From Ecology to Application. Front. Plant Sci. 9:1270. doi:

10.3389/fpls.2018.01270

Dickie, I. A., Bufford, J. L., Cobb, R. C., Desprez-Loustau, M.-L., Grelet, G., Hulme, P. E.,

Williams, N. M. 2017. The emerging science of linked plant-fungal invasions. New Phytologist, 215(4), 1314–1332. doi: 10.1111/nph.14657

Johnson, N. C., Wilson, G. W. T., Bowker, M. A., Wilson, J. A. & Miller, R. M. (2010). Resource limitation is a driver of local adaptation in mycorrhizal symbioses. P Natl Acad Sci USA 107, 2093-2098, doi:10.1073/pnas.0906710107.

Klironomos, J. N. 2003. Variation In Plant Response To Native And Exotic Arbuscular

Mycorrhizal Fungi. Ecology, 84(9), 2292–2301. doi: 10.1890/02-0413

McCormack, M. L. et al. 2015. Redefining fine roots improves understanding of below-ground contributions to terrestrial biosphere processes. New Phytologist 207, 505-518, doi:10.1111/nph.13363.

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McGonigle, T., Miller, M., Evans, D., Fairchild, G. & Swan, J. 1990. A new method which gives an objective measure of colonization of roots by vesicular—arbuscular mycorrhizal fungi. New phytologist 115, 495-501.

Pringle, A., Bever, J. D., Gardes, M., Parrent, J. L., Rillig, M. C., & Klironomos, J. N. 2009. Mycorrhizal Symbioses and Plant Invasions. Annual Review of Ecology, Evolution, and Systematics, 40(1), 699–715. doi: 10.1146/annurev.ecolsys.39.110707.173454

Redecker, D. 2000. Glomalean Fungi from the Ordovician. Science, 289(5486), 1920–

1921. doi: 10.1126/science.289.5486.1920

Smith S. E., Read D. J. 1997.  Mycorrhizal symbiosis. (Academic Press, London, England).

Team, R. C. 2013. R: A language and environment for statistical computing.

Vierheilig, H., Coughlan, A. P., Wyss, U. & Piché, Y. 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl. Environ. Microbiol. 64, 5004-5007.

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Appendix

Picture 1: A fine root of the shrub Viburnum acerifolium that has been colonized by arbuscular mycorrhizal fungi (AMF)

16

Table 1: Phylogenetic pairs of native and invasive species, grouped by genus, with

sample sizes.

Species Species Abbreviation

Nativity to NY # of Mycorrhizal Samples

# of Nonmycorrhizal Samples

Viburnum acerifolium

VIAC Native 3 3

Viburnum opulus

VIOPA Invasive 5 4

Lonicera sempervirens

LOSE Native 4 5

Lonicera japonica

LOJA Invasive 4 4

Berberis canadensis

BECA Native 4 2

Berberis thunbergii

BETH Invasive 5 2

Total Samples 25 20

17

VIAC VIOPA LOSE LOJA BECA BETH0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Av

erag

e %

Col

oniz

ation

Figure 1: Average percent colonization (±SE) for native and invasive shrubs. Averages

were taken for only mycorrhizal plants of each species. Sample size and species

abbreviations appear in the methods.

18

Figure 2: Aboveground biomass (±SE) for native and invasive shrubs. M+ denotes

mycorrhizal treatment, M- denotes nonmycorrhizal treatment. Sample size and species

abbreviations are provided in the methods.

19

Figure 3: Belowground biomass (±SE) for native and invasive shrubs. M+ denotes

mycorrhizal treatment, M- denotes nonmycorrhizal treatment. Sample size and species

abbreviations are provided in the methods.

20

Figure 4: Total biomass (±SE) for native and invasive shrubs. Sample size and species

abbreviations are provided in the methods.

21

Figure 5: Root: Shoot Ratio (RSR) (±SE) for native and invasive shrubs. M+ denotes

mycorrhizal Treatment, M- denotes nonmycorrhizal treatment. Sample size and species

abbreviations are provided in the methods.

Mycorrhizal Nonmycorrhizal Mycorrhizal NonmycorrhizalNative Invasive

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8Ro

ot:S

hoot

Rati

o

22

Figure 6: Mycorrhizal growth response (MGR) for native and invasive species. There

are no error bars because MGR was derived from average growth of mycorrhizal and

nonmycorrhizal plants for each species. Sample size and species abbreviations are

provided in the methods.

VIAC VIOPA LOSE LOJA BECA BETH-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8M

GR

23

Figure 7: Scatterplot of colonization (± SE) vs. mycorrhizal growth response (MGR).

Arrows denote shifts between native and invasive species within each native-invasive

pair.

Colonization Rate vs Mycorrhizal Growth Response