Effects of urbanization and nitrogen addition on ectomycorrhizal fungal communities

Talia Michaud, Mount Holyoke College

Abstract

The relationship between ectomycorrhizal fungi (EMF) and their hosts represents an

essential facet of multiple element cycles in terrestrial systems. Anthropogenic nitrogen

deposition, however, disrupts the symbiosis between ectomycorrhizal fungi and their plant

partners, restructuring the ectomycorrhizal fungal community. I investigated the effects of

nitrogen addition and urbanization on ectomycorrhizal fungal communities along an urbanization

and nitrogen addition gradient by characterizing the ectomycorrhizal community associated with

Red oak (Quercus rubra) via DNA extraction and amplicon sequencing. With species data,

exploration type data, and percent colonization, I calculated extramatrical mycelial biomass

(EMM) along the urbanization and nitrogen addition gradients. My data indicate that EMF

communities are restructured by both urbanization and nitrogen addition, and that EMM biomass

generally declines with increasing soil nitrogen levels. This finding supports the hypothesis that

the increase of accessible nutrient supply undermines the transactional relationship between

EMF and their plant hosts.

Key Words

Ectomycorrhizal fungi, nitrogen deposition, urbanization, soil fungal communities, terrestrial

carbon cycle, exploration type, EMM biomass.

Introduction

Mycorrhizal associations are central to the evolution and survival of terrestrial vascular

plants (Brundrett 2002, Malloch et al. 1979). Mycorrhizal fungi enhance the ability of plants to

take up relatively scarce, essential nutrients, such as phosphorus and nitrogen, in return for

sugars from photosynthates. These nutrients are exchanged in the plant root via distinct,

polymorphic mechanisms. Mycorrhizal associations also protect plants against pathogens and

drought (Duchesne et al. 1987, Boyle and Hellenbrand 1991). The oldest bryophyte-like plants

discovered in the Devonian fossil record showed evidence of a mycorrhizal symbiosis much like

vesicular-arbuscular mycorrhizae, the oldest and most abundant form of mycorrhizae, even

before roots, themselves, evolved (Brundrett 2002). Currently, only 20% of plant species are

non-mycorrhizal (Tester et al. 1987).

Mycorrhizal fungi, in addition, are important mediators of terrestrial carbon cycling

(Ekblad 2013). Plants transfer up to 10-20% of their photosynthate to mycorrhizal fungi

(Treseder and Allen 2000). The fate of this flux of carbon underground and into mycorrhizal

fungi is relatively understudied.

Ectomycorrhizal fungi, comprising 5000-6000 species (Agerer 2006), associate with

about 2% of plant species (Tendersoo et al. 2010), specifically, trees and shrubs in temperate

forests (Malloch et al. 1979). Ectomycorrhizal fungi represent an important, yet understudied,

carbon sink because much of the biomass produced by these fungi, sourced from plant partners,

is not readily decomposed, contributing substantially to soil organic matter (Treseder and Allen

2000). The parameters of ectomycorrhizal fungal production and turnover, however, are largely

unknown.

Anthropogenic nitrogen deposition disrupts the symbiosis between ectomycorrhizal fungi

and their plant partners, restructuring the ectomycorrhizal fungal community (Treseder and Allen

2000, Lilleskov et al. 2002). When large amounts of labile nitrogen are available to plants,

ectomycorrhizal colonization and species richness often decreases and community composition

changes dramatically (Treseder and Allen 2000, Lilleskov et al. 2002, Morrison et al. 2016).

Species of ectomycorrhizal fungi grow at different rates, produce biomass of varying magnitudes

that varies in recalcitrance, rendering their capacity for carbon sequestration community specific

(Treseder and Allen 2000). As ectomycorrhizal fungal community composition changes in

response to nitrogen deposition, its capacity and rate of carbon sequestration changes, altering

the magnitude of this belowground carbon sink. Understanding how ectomycorrhizal fungal

communities respond to nitrogen deposition, therefore, is central to predicting how this important

carbon sink will react to accumulating nitrogen pollution (Treseder and Allen 2000).

Ectomycorrhizal fungi can be partitioned into distinct ecophysiologies according to

hyphal exploration type (Agerer 2001). Hyphal exploration type is characterized by the length of

extramatrical, radiating hyphae (contact, short, medium, long) and texture (smooth, fringe, mat).

Generally, hyphal exploration types are associated with nutrient foraging strategies. Long types,

such as Boletus, are long-distance foragers, while short and contact types, like Cenococcum and

some Russulas, respectively, gather nutrients adjacent to the root (Agerer 2001). Hyphal

exploration types are not plastic, and are generally genus specific (Agerer 2001, Hobbie and

Agerer 2010). It is important to note that only 5-10% of ectomycorrhizal fungal species have

been classified according to exploration type (Wallander et al. 2013).

Weigt et al. (2012) generated standard values for extramatrical mycelial (EMM) biomass

according to exploration type, given total ECM mantle length and percent colonization.

Extramatrical mycelial biomass increases as hyphal exploration type lengthens. Given

ectomycorrhizal species data, which yields relative abundance of exploration type, percent root

tip colonization, and average mantle length, it is possible to estimate EMM biomass in an

ecosystem.

I investigated the effects of nitrogen addition and urbanization on ectomycorrhizal fungal

communities by characterizing the ectomycorrhizal community associated with Red oak

(Quercus rubra) via DNA extraction and amplicon sequencing along a nitrogen amendment

gradient and an urbanization gradient. With species data, exploration type data, and percent

colonization, I calculated EMM biomass along the urbanization and nitrogen addition gradients.

As nitrogen deposition and urbanization intensifies, I expected dominant hyphal

exploration type to shorten, and EEM biomass to decrease, decreasing fungal inputs to soil

organic carbon.

Materials and Methods

Urbanization Gradient, Harvard Forest to Boston

The urbanization gradient was established and is maintained by Dr. Lucy Hutyra and Dr.

Pamela Templer of Boston University (Rao et al. 2014). We selected four sites along this

gradient. Two represent rural forests and two represent suburban-low urban forests.

Chronic Nitrogen Amendment Experiment, Harvard Forest

We used the three plots maintained for the Chronic Nitrogen Amendment Experiment at

the Harvard Forest since 1988 – control, low-N (50 kg/ha/yr), and high-N (150 kg/ha/yr). These

plots are treated with NH4NO3 six times each month. Morrison et al. (2016) investigated the

effects of chronic nitrogen addition on soil fungal communities in these plots. I will compare

these findings with my own.

Sample Collection

Throughout the urbanization gradient I randomly selected three individual Red oak

(Quercus rubra) trees at each site that are roughly equidistant (60 m) from the area of

disturbance to control for edge effect. I also collected soil cores from three randomly selected

Red oak (Quercus rubra) trees in the nitrogen addition plots. Within 1 m of the trunk of each

tree, I extracted three soil cores for each Red oak (Quercus rubra) tree, which are known to

partner with ectomycorrhizal fungi, that extended 10 cm under the surface of the soil. I removed

the top layer of litter to bias the sample against saprophytic fungi. I stored the soil cores at 4ºC

until further processing.

Sample Processing

I pooled and processed each tree-specific triplicate by dry sieving them through a 3 mm

sieve that was rinsed with ethanol and deionized water between triplicates. I collected the fine

roots and suspended them in deionized water. I homogenized the throughfall of each triplicate

and extracted a soil subsample, according to the advice of Dr. Jie Wei and Joseph Vineis (Wei,

Veneis pers. comm. 2018). I produced 21 soil subsamples.

DNA Extraction, Amplification, and Sequencing

I extracted DNA from the soil subsamples via the MO BIO DNeasy PowerSoil kit,

following the manufacturer’s protocol. I tested the extracted DNA for content and quality with a

NanoDrop. I then transferred my samples to Dr. Morrison at the Bay Paul Center for PCR

amplification, using the ITS1 primer to selectively amplify fungi. Generally, the ITS1 primer

alone is not sufficient to determine relative abundance of fungal taxa, yet I used it as a rough

indicator of the soil fungal community. The amplified sequences were sequenced at Dr. Hillary

Morrison’s lab at the Bay Paul Center using MiSeq Illumina Next-Gen sequencing. The

sequences were run through VAMPS (The Visualization and Analysis of Microbial Population

Structures, https://vamps.mbl.edu) yielding species data. I excluded all sequences not identified

to the species level. I also excluded all operational taxonomic units (OTUs) under 1% relative

abundance, which are generally regarded as unreliable indicators of actual relative abundance

(Lundberg et al. 2012, Smith and Peay 2014). I used the distribution of OTUs determined to the

species level as a proxy for the distribution of the greater ectomycorrhizal community.

Biomass Calculation

I calculated the average total number of root tips by extrapolating the average number of root tips

per 2 cm root segment (n=20) to the total number of root tips per unit volume given total root

length per unit volume, provided by Emma Conrad-Rooney. I calculated the number of

colonized root tips by multiplying the site-specific rate of colonization, also provided by Emma

Conrad-Rooney, by the average total number of root tips per unit volume. Given sequencing

data, I assigned exploration type to my species data using multiple literature sources, including

Agerer and Rambold (2004-2011: An information system for characterization and determination

of ectomycorrhizae). and Tedersoo and Smith (2013). When I could not assign exploration type

to the species level, I assigned it the exploration type associated with the genus (Tedersoo and

Smith 2013). For the Russula and Lactarius genera, I assigned medium exploration type to

unknown species, although exploration type varies across these genera. This assignment is

imperfect, adversely affecting the validity of my biomass estimates, yet I would rather assign an

exploration type that dominates most of the genus than exclude these prevalent species entirely.

These estimates, therefore, are subject to change given species-specific determination of

exploration type. I determined the relative abundance of each exploration type from these

exploration type assignments and the relative abundance of each species per site. I multiplied

each group of colonized tips by the average mantle length (6 mm) which I estimated from

literature values (Agerer and Rambold, 2004-2011: An information system for characterization

and determination of ectomycorrhizae). I calculated EMM biomass via the methods proposed by

Weigt et al. (2012). I multiplied the total mantle length of each exploration type by the

corresponding standard value that relates EMM biomass to mantle length. I then extrapolated

EMM biomass per unit volume to a square meter at a depth of 10 cm, which I used to compare

EMM biomass across sites.

Results

Sequencing

Out of all the sequences I received, about 60% were ectomycorrhizal (Fig. 1). Only 50%

of the ectomycorrhizal fungal sequences were identified to species level. I identified 121 species

of ectomycorrhizal fungi from 25 families. The most abundant families are Russulaceae,

Amanitaceae, and Elaphomycetaceae, although only Russulaceae (~80%) was present throughout

all the sites (Fig. 2).

Community

Species richness, determined from OTUs with relative abundance greater than 1%, like in

Morrison et al. (2016), does not change over the urbanization gradient except for a slight

decrease in SW, an urban site (Fig. 3). Species richness is lowest in the low nitrogen addition

plot and highest in the control. Species richness is slightly decreased in the high nitrogen

addition plot.

Evenness, or the relative distribution of OTUs (Hill 1973), is generally greater in the

urban sites than the rural sites (Fig. 4). Evenness in the nitrogen addition plots is highest in the

control plot, although only slightly higher than the high addition plot. Evenness in the low

addition plot, however, is greatly reduced.

Shannon Weiner diversity, used in Morrison et al. (2016) to examine changes in diversity

in the soil fungal community, is comparable between rural and urban sites, although HF1, a rural

site, demonstrated particularly low diversity (Fig, 5). The control plot has the highest diversity

over the nitrogen addition gradient, slightly higher than that of the high addition plot. The lowest

diversity across both gradients occurs in the low nitrogen addition plot.

The nitrogen addition gradient yielded high relative abundance of Lactarius and Russula

species, both of the Russulaceae family (Fig. 6). In particular, Lactarius imperceptus was

dominant in the control and low addition plot, while Russula atropurpurea was dominant in the

high addition plot.

Total Mantle Length

Total mantle length does not vary significantly over the urbanization gradient (Fig. 7).

Mantle length decreases over the nitrogen addition gradient, reflecting changes in percent root tip

colonization (Conrad-Rooney pers. comm. 2018).

Exploration Type Distribution

Medium exploration type dominates most of the sites across both gradients (Fig. 8). In

the urban sites, however, HW exhibits about 40% of short distance EMF. The other urban site,

however, is composed almost entirely of medium distance EMF. One of the rural sites also

contained low levels of short distance EMF. The control and low nitrogen addition plots are

entirely dominated by medium distance EMF. The high nitrogen addition plot, however, is

dominated by contact EMF.

Biomass

Biomass of EMM is slightly greater in the rural sites than the urban sites (Fig. 9).

Biomass decreases across the nitrogen addition gradient, with a dramatic drop between the low

and high nitrogen addition sites.

I found a significant (P<0.05) negative relationship between nitrogen mineralization and

EMM biomass (𝑟2 = 0.5051) (Fig. 10). I found a significant (P<0.05) negative relationship

between soil nitrate and EMM biomass (𝑟2 = 0.8672) (Fig. 11).

There is a strong, positive, and significant (P<0.05) relationship between EMM biomass

and soil molar C:N (provided by Maggie Anderson) along the nitrogen addition gradient (𝑟2 =

0.9961) (Fig.12). This dramatic relationship, however, is not replicated in the urbanization

gradient ( 𝑟2 = 0.0031).

Discussion

I used operational taxonomic units (OTUs) specified to species level that represented

more than 1% of the ectomycorrhizal OTUs as a proxy for the ectomycorrhizal fungal

communities throughout the gradients. Additionally, due to logistical restraints, I extracted DNA

from soil subsamples rather than pooled root samples. The species data, therefore, represents the

soil fungal community rather than the root fungal community. I did not capture the plant-

mediated filtering that occurs between the soil and the root fungal communities, although in the

nitrogen addition gradient it is likely insignificant (Morrison et al. 2016). Nevertheless, I treated

the resulting sequences as representative of the potential ectomycorrhizal fungal colonizers of

trees in mixed New England forests, much like Morrison et al. (2016).

My findings support the hypothesis that nitrogen addition restructures ectomycorrhizal

fungal communities. Although no distinct trend was found across the urbanization gradient

regarding species richness, evenness, or diversity, a pattern emerged in the nitrogen addition

plots. The low addition plot contained the lowest species richness, evenness, and diversity, due to

the dominance of one species of fungi, Lactarius imperceptus. In contrast, species richness,

evenness, and diversity were only slightly decreased in the high nitrogen addition plot. This

finding is contrary to that published in Morrison et al. (2016), wherein richness was highest in

the low nitrogen addition plot and lowest in the high addition plot, while diversity declined from

the control plot to the high addition plot. Additionally, while Morrison et al. (2016) determined

that Russula vinacea increased in abundance across the nitrogen addition gradient, I found that

Lactarius imperceptus increased from the control to the low addition plot, while Russula

atropurpurea dominated in the high addition plot. I did not find Russula vinacea in either of the

gradients. This inconsistency could be due to my use of the ITS1 primer alone, or my exclusion

of sequences that were identified at genus but not species level. If not, this finding prompts

further investigation into the dynamics and plasticity of the EMF community following chronic

nitrogen addition.

Dominant hyphal exploration type did not change over the urbanization gradient. Only in

the high addition plot did the dominant exploration type switch from medium distance to contact

type. This data support previous studies that have reported decline in medium distance types

associated with nitrogen addition (Lilleskov et al. 2011). However, this finding must be treated

with caution until all species of Russula and Lactarius captured in my data have been officially

assigned to an exploration type.

It is important to note that my estimate of EMM biomass is weakened by the framework

from which the standard values were conceived, wherein the relationship between mantle length

and biomass was derived from cultures devoid of microbial competition and herbivory (Weigt et

al. 2012). Additionally, many ectomycorrhizal fungal species reduce mycelial production in

response to nitrogen addition (Bidartondo et al. 2001). Nevertheless, my findings are a

preliminary indicator of changes in EMF mycelial biomass in response to urbanization and

nitrogen addition.

My data indicate that EMM biomass generally declines with the increasing soil nitrate

levels and nitrogen mineralization rates determined by Maggie Anderson. This finding supports

the hypothesis that the increase of accessible nutrient supply undermines the transactional

relationship between EMF and their plant hosts. To what extent these relationships are

conserved, however, is of interest to me.

A striking pattern emerged in the nitrogen addition plots regarding soil C:N (Maggie

Anderson) and EMM biomass. As C:N decreased, EMM biomass declined as well. This

relationship, however, did not hold true over the urbanization gradient, perhaps due to

confounding factors that render nitrogen addition distinct from that accompanying urbanization.

While the relationship in the nitrogen addition plots is founded on generally insufficient data,

this trend warrants further research.

The relationship between ectomycorrhizal fungi and their hosts represents an essential

facet of multiple element cycles in terrestrial systems. Investigating how these organisms react to

increasing anthropogenic stressors like nitrogen deposition, therefore, is essential to

understanding how our terrestrial systems will be molded by increasingly pervasive

anthropogenic disturbance.

Acknowledgements

Thanks to my mentors, Dr. John Hobbie and Dr. Jerry Melillo, for their encouragement

and incredible expertise. Also, I would like to thank my collaborators, Emma Conrad-Rooney

and Maggie Anderson, for their determination, organization, and relentless enthusiasm. Thanks

to Dr. Joe Vallino, Joe Vineis, and Dr. Wei Jie for their advice and support and to Dr.

Sherlynette Perez-Castro for her help on field days. Thanks to Dr. Hilary Morrison at the Bay

Paul Center and to Melissa Knorr and Dr. Serita Frey, as well as Dr. Pamela Templer and Dr.

Lucy Hutyra.

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A. 2016. Chronic nitrogen additions fundamentally restructure the soil fungal community in

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Figures and Tables

Figure 1: Proportions of sequences identified as non-EMF (Not EMF), EMF without species

identification (EMF w/o ID), and EMF identified to species level (EMF ID).

EMF ID EMF w/o ID Not EMF

Figure 2: Average relative abundance of EMF family

Russulaceae82%

Amanitaceae9%

Elaphomycetaceae6%

Other3%

Russulaceae Amanitaceae

Elaphomycetaceae Other

Figure 3: Species richness across gradients wherein NDC, NDL, and NDH are the control, low,

and high nitrogen addition plots, respectively

0

1

2

3

4

5

6

SW HW HF1 HF2 NDC NDL NDH

Nu

mb

er

of

Spec

ies

Site: Urban Rural N-Ad

Figure 4: Evenness across gradients wherein NDC, NDL, and NDH are the control, low, and

high nitrogen addition plots, respectively

0

0.2

0.4

0.6

0.8

1

SW HW HF1 HF2 NDC NDL NDH

Site: Urban Rural N-Ad

Figure 5: Shannon Weiner Diversity across gradients wherein NDC, NDL, and NDH are the

control, low, and high nitrogen addition plots, respectively

00.20.40.60.8

11.21.41.6

SW HW HF1 HF2 NDC NDL NDHSite: Urban Rural N-Ad

Figure 6: Relative abundance of two species across nitrogen addition gradient, wherein NDC,

NDL, and NDH are the control, low, and high nitrogen addition plots, respectively

0

0.2

0.4

0.6

0.8

1

NDC NDL NDH

Rel

ativ

e A

bu

nd

ance

Lactarius imperceptus Russula atropurpurea

Figure 7: Total mantle length per square meter to 10 cm depth across gradients wherein NDC,

NDL, and NDH are the control, low, and high nitrogen addition plots, respectively

0

20

40

60

80

100

HW SW HF1 HF2 NDC NDL NDH

Man

tle

Len

gth

( k

m)

Site: Urban Rural N-Ad

Commented [JEH1]: Was this a square meter 10 cm deep? If so, better add this fact here.

Figure 8: Total EMM biomass (kg/ha) across gradients wherein NDC, NDL, and NDH are the

control, low, and high nitrogen addition plots, respectively

0

100

200

300

400

500

600

HW SW HF1 HF2 NDC NDL NDH

Bio

mas

s (k

g/h

a)

Site: Urban Rural N-Ad

Figure 9: Distribution of exploration type (contact, short, medium) across gradients wherein HW

and SW are urban sites, HF1 and HF2 are rural sites, and NDC, NDL, and NDH are the control,

low, and high nitrogen addition plots, respectively.

0%

20%

40%

60%

80%

100%

HW SW HF1 HF2 NDC NDL NDH

Contact Short Medium

Figure 10: Relationship between N Mineralization (µg N/µg soil per year) and EMM biomass

(kg/ha) across gradients wherein HW and SW are urban sites, HF1 and HF2 are rural, and NDC,

NDL, and NDH are the control, low, and high nitrogen addition plots, respectively

HW

SW

HF1

HF2

NDC

NDL

NDH

R² = 0.5051

0

100

200

300

400

500

600

0 50 100 150 200

Bio

mas

s (k

g/h

a)

N Mineralization

Figure 11: Relationship between soil nitrate (µg N/g soil) and EMM biomass (kg/ha) across

gradients wherein HW and SW are urban sites, HF1 and HF2 are rural, and NDC, NDL, and

NDH are the control, low, and high nitrogen addition plots, respectively

HWSW

HF1

HF2NDC

NDL

NDHR² = 0.8672

0

100

200

300

400

500

600

0 1 2 3 4 5

Bio

mas

s (k

g/h

a)

Soil Nitrate (µg/g)

Figure 12: Relationship between soil molar C:N and EMM biomass across nitrogen addition

gradient wherein NDC, NDL, and NDH are the control, low, and high nitrogen addition plots,

respectively

NDC

NDL

NDH

R² = 0.9961

0

100

200

300

400

500

600

23 24 25 26 27 28

Bio

mas

s (k

g/h

a)

Soil C:N

Figure 13: Relationship between soil molar C:N and EMM biomass across the urbanization

gradient wherein HW and SW are urban sites, and HF1 and HF2 are rural

HWSW

HF1

HF2

R² = 0.0031

0

100

200

300

400

500

0 10 20 30 40

Bio

mas

s (k

g/h

a)

Soil C:N