VERTICAL DISTRIRBUTION, NITROGEN CONTENT, AND NATURAL 15N AND
13C ABUNDANCE OF EPIPHYTIC MACROLICHEN FUNCTIONAL GROUPS AND
SOIL IN SUB-BOREAL SPRUCE FORESTS OF CENTRAL BRITISH COLUMBIA
by
Anna Kobylinski
B.Sc. McMaster University, 2006

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MASTER OF SCIENCE
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NATURAL RESOURCES AND ENVIRONMENTAL STUDIES - BIOLOGY

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
April 2015

© Anna Kobylinski 2015

UMI Number: 1526531

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Vertical Distribution, Nitrogen Content, and Natural 1SN and 13C Abundance of Epiphytic
Macrolichen Functional Groups and Soil in Sub-Boreal Spruce Forests of Central British
Columbia

Abstract for LAC Submission
The importance o f epiphytic cyanolichens to N inputs was evaluated in sub-boreal forest
ecosystems o f central interior BC. Four old growth sub-boreal forest sites were chosen based on
cyanolichen abundance and diversity - two High Cyano sites and two Low Cyano sites. Interior
hybrid spruce (Picea glauca x engelmannii) and subalpine fir (Abies lasiocarpa) trees were
randomly selected at these High and Low Cyano sites and access into canopies achieved through
a single-rope technique. Vertical distributions of lichen biomasses were quantified and a vertical
niche o f foliose chlorolichens was observed in the canopy where cyanolichens were deficient.
Lichen functional groups, conifer needle, and soil samples were obtained and their 15N:14N,
%N, 13C:12C, %C contents measured. Comparisons of stable isotope ratios between High and
Low Cyano sites were made. Sub-boreal forest epiphytes and foliage had isotopically lighter N
at sites with high cyanolichen abundance, indicative of greater inputs of biologically fixed-N.

Table of Contents
Abstract.......................................................................................................................................................................................i
Table o f Contents..................................................................................................................................................................... ii
List of Tables............................................................................................................................................................................iv
List o f Figures......................................................................................................................................................................... vi
Glossary................................................................................................................................................................................... ix
Acknowledgement..................................................................................................................................................................xii
Chapter 1: Literature Review and Introduction.....................................................................................................................1
1.1 Importance of forest canopies.......................................................................................................................................1
1.2 Canopy exploration........................................................................................................................................................2
1.3 Epiphytic lichens in the forest canopy..........................................................................................................................3
1.4 Factors affecting canopy lichen distribution...............................................................................................................5
1.5 Nutrient dynamics of canopy lichens...........................................................................................................................9
1.6 Cyanolichen biological N2-fixation and its contribution to the N-cycle................................................................. 13
1.7 Stable Isotopes o f Nitrogen and Carbon.....................................................................................................................16
1.8 Stable Isotope Fractionation and Discrimination......................................................................................................18
1.9 Natural Abundance Method Uses and Advantages.................................................................................................. 19
1.10 Soil Nitrogen Inputs.................................................................................................................................................. 20
1.11 Thesis overview..........................................................................................................................................................21
1.12 Literature C ited..........................................................................................................................................................22
Chapter 2: Vertical distribution and nitrogen content of epiphytic macrolichen functional groups in sub-boreal
forests of central British Colum bia...................................................................................................................................... 32
2.1 ABSTRACT................................................................................................................................................................. 32
2.2 INTRODUCTION....................................................................................................................................................... 33
2.3 M ETHODS.................................................................................................................................................................. 36
2.3.1 Study Area.............................................................................................................................................................36
2.3.2 Sample trees and lichen sampling...................................................................................................................... 37
2.3.3 Canopy Access..................................................................................................................................................... 37
2.3.4 Lichen Biomass Abundance Assessment.......................................................................................................... 38
2.3.5 Sampling of Lichens for Chemical Analyses.................................................................................................... 39
2.3.6 Statistical Analysis...............................................................................................................................................40
2.4 RESULTS.................................................................................................................................................................... 41
2.4.1 Epiphytic Lichen Abundance..............................................................................................................................41
2.4.2 Epiphytic Lichen Distribution.............................................................................................................................43
2.4.3 Functional Group Nitrogen Content................................................................................................................... 44
2.5 DISCUSSION..............................................................................................................................................................46
2.6 LITERATURE CITED................................................................................................................................................ 53
2.7 TABLES........................................................................................................................................................................58
ii

2.8

FIGURES...................................................................................................................................................................63

Chapter 3: Using stable isotope ratios 15N :14N, 13C:I2C to determine the effects o f epiphytic lichens on the nitrogen
(N) and carbon (C) cycles o f wet sub-boreal spruce and fir forests of central British Columbia.................................. 69
3.1 ABSTRACT................................................................................................................................................................. 69
3.2 INTRODUCTION........................................................................................................................................................71
3.2.1 Lichen Characteristics..........................................................................................................................................71
3.2.2 Stable Isotope Techniques................................................................................................................................... 72
3.2.3 Soil N Inputs..........................................................................................................................................................74
3.2.4 Forest Ecosystem Nutrient Cycling.................................................................................................................... 75
3.2.5 Research objectives.............................................................................................................................................. 76
3.3 M ETHODS.................................................................................................................................................................. 77
3.3.1 Study Area............................................................................................................................................................. 77
3.3.2 Sample Trees.........................................................................................................................................................78
3.3.3 Canopy Access......................................................................................................................................................78
3.3.4 Epiphytic Lichen and Needle Sampling.............................................................................................................78
3.3.5 Composite Soil Sampling.................................................................................................................................... 79
3.3.6 Stable Isotope Analyses........................................................................................................................................79
3.3.7 Natural Abundance M ethod................................................................................................................................ 81
3.3.8 Statistical analyses................................................................................................................................................ 82
3.4 RESULTS.................................................................................................................................................................... 83
3.5 DISCUSSION.............................................................................................................................................................. 87
3.6 LITERATURE CITED................................................................................................................................................ 93
3.7 TABLES........................................................................................................................................................................98
3.8 FIGURES....................................................................................................................................................................102
Chapter 4: Conclusions........................................................................................................................................................ 109
4.1

Literature C ited........................................................................................................................................................112

List of Tables
Chapter 2
Table 2.1 Latitude, longitude, and tree density with stems >25dbh for four sub-boreal conifer
forest study sites in central British Columbia. Tree stem densities (stems ha'1) data were taken
from Campbell et al. (2010b).
Table 2.2 Mean (±SD; n=4) tree diameter base height (cm), tree height (m), tree age (years) and
epiphytic lichen biomass at four study sites by functional group abundance (average g dry weight
tree'1 and average kg dry weight ha'1) on mature subalpine fir and interior hybrid spruce trees in
central British Columbia.
Table 2.3 Two-way analysis o f variance for the amount of lichen loading (total lichen grams
tree ') between spruce (S) and fir (F) and site type (High Cyano versus Low Cyano). Asterisks
represent significant difference *p < 0.05, **p < 0.01, ***p < 0.001.
Table 2.4 Two-way analysis o f variance for study tree height at peak biomass and 50% of total
biomass by lichen functional group between site type: High Cyano (H) versus Low Cyano (L)
and tree species: spruce (S) versus fir (F). Asterisks represent significant difference *p < 0.05,
**p<0.01, ***p< 0.001.
Table 2.5 One-way analysis of variance of functional group nitrogen content across both tree
types between High Cyano versus Low Cyano sites. Asterisks represent significant difference
*p < 0.05, **p < 0.01, ***p < 0.001.

iv

Chapter 3

Table 3.1 Linear Regression (OLS) coefficients with standard error for the regression model
predicting 815N ratio in lichen functional groups and needles based on host tree species (spruce
and fir), cyanolichen abundance, tree branch height, and lichen species.
Table 3.2 Linear Regression (OLS) coefficients with standard error for the regression model
predicting 513C ratio in lichen functional groups and needles based on host tree species (spruce),
cyanolichen abundance, tree branch height, the interaction between cyanolichens and site
cyanolichen abundance and lichen species.
Table 3.3 Linear Regression (OLS) coefficients with standard error for the regression model
predicting the dependent variable 8I5N in soil based on cyanolichen abundance, host tree species,
and soil horizon.
Table 3.4 Linear Regression (OLS) coefficients with standard error for the regression model
predicting the dependent variable 813C in soil based on cyanolichen abundance, host tree species,
and soil horizon.

List of Figures

Chapter 2

Figure 2.1 Map of study area including four sampling sites (High Cyano 1 and 2, Low Cyano 1
and 2) to the east of Prince George (Large inset) near Upper Fraser, British Columbia, Canada
(Small inset).
Figure 2.2 Vertical gradients in mean lichen functional group abundances (g dry weight m '1tree
height) on mature subalpine fir and interior hybrid spruce trees versus tree height (meters) in
High Cyano and Low Cyano lichen abundance sites in sub-boreal conifer forests in central
British Columbia.
Figure 2.3 Median height (±SD) where half of the total epiphytic lichen biomass is reached for
each functional group on mature fir and spruce trees at High Cyano (large symbols) and Low
Cyano (small symbols) sites in sub-boreal conifer forests of central British Columbia.
Figure 2.4 Total cyanolichen (bipartite + tripartite cyanolichen) abundance (g dry weight tree'1)
on Fir (F) and Spruce (S) study trees studied arranged from lowest to highest cyanolichen
abundance at High (H) and Low (L) Cyano sites. Mean cyanolichen biomasses tree'1±SD are
shown for fir and spruce trees at all sites.
Figure 2.5 Mean epiphytic lichen nitrogen (N: g of N tree'1±SD) across High and Low Cyano
sites for fir and spruce study trees in central British Columbia. Different letters indicate
significantly different mean values (Bonferroni multiple comparison tests: p < 0.05). Aggregate
tree-level epiphytic lichen N is shown for study trees at both High and Low Cyano sites (Figure
6 Inset).
vi

Figure 2.6 Mean epiphytic lichen nitrogen (N: kg of N ha'1±SD) at sites with (A) high (High
Cyano 1 or 2) versus low (Low Cyano 1 and 2) cyanolichen abundance on (B) either Spruce or
Fir by lichen functional group in central British Columbia. Aggregate N by functional group is
also shown (Figure 6A Inset). Dissimilar letters indicate significantly different mean values
(Bonferroni multiple comparison tests: P>0.05).

Chapter 3

Figure 3.1 Mean percentage of nitrogen (%N of lichen biomass ±SD) found in functional
groups o f lichen and foliage (needle) from 32 fir and spruce study trees at four study sites with
varying amounts of cyanolichen abundance. Different letters above error bars indicate
significant (p < 0.001) differences between lichen functional groups (Tukey multiple mean
comparison test).
Figure 3.2 Mean (+SD) N concentrations and 51SN signatures of five epiphytic lichen biomasses
and the host tree foliage (needle) biomasses across four sites and two host tree species from subboreal spruce and fir forests in central interior B.C. Half of the sites had relatively high
cyanolichen abundances (black symbols) and the other half had low cyanolichen abundances
(white symbols).
Figure 3.3 Mean (a) 815N and (b) %N of host tree foliage (needle) from lowest to highest
cyanolichen abundance (total g tree 1).

Figure 3.4 Mean 81SN o f each soil horizon, surface organic layer (F), upper most mineral
horizon (Ae) and next mineral horizon (Bf) at sites with lowest to highest cyanolichen abundance
(total g tree ‘).
Figure 3.5 Variation in the natural abundances of (a) 513C and (b) 8I5N of five epiphytic lichen
biomasses and the host tree foliage (needle) biomasses according to branch height above-groundlevel for all sites.
Figure 3.6 Dual natural abundance isotope plot of 815N and 813C of the five epiphytic lichen
biomasses and host tree foliage (needle) biomasses across four sites and two host tree species.
Figure 3.7 Mean (+SD) C concentrations and 813C signals of five epiphytic lichen biomasses
and host tree foliage (needle) biomasses across four sites and two host tree species.

Glossary
Algae: (sing, alga) The photosynthetic partner of most lichens, most are microscopic unicellular
or multicellular green algae.
Apothecia: a spore-bearing structure in many lichens and fungi consisting of a discoid or cupped
body bearing asci on the exposed flat or concave surface.
Atom Percent (Atom % ): The absolute number of atoms of a given isotope in 100 atoms of
total element. For example, the 15N content o f air N 2 is 0.3663 Atom %. For calculation, At% =
[Rs / (Rs + 1) * 100] where Rs is the ratio of the light isotope to the heavy isotope o f the sample.
Biodiversity (biological diversity): The diversity o f plants, animals, and other living organisms
in all their forms and levels of organization, including genes, species, ecosystems, and the
evolutionary and functional processes that link them.
Biogeoclimatic zone: A geographic area having similar patterns of energy flow, vegetation and
soils as a result o f a broadly homogenous macroclimate.
Biomass: The total mass of living organisms in a given area or volume. Forest autotrophic
biomass consists primarily of above-ground and below-ground tree components (stems,
branches, leaves, and roots); other woody vegetation; and mosses, lichens, and herbs.
Canopy: The uppermost layer o f vegetation formed by the crowns of trees in a forest, often
forms a distinct habitat.
Cortex: A structurally distinct outermost layer of a lichen thallus, usually with no algal cells,
present in many but not all lichens
Crustose: Crust-forming lichen with only the upper surface visible, growing on or in the
substratum.
Cyanobacteria: Prokaryotic organisms without organized chloroplasts but having
chlorophyll a and oxygen-evolving photosynthesis; capable of fixing nitrogen in heterocysts;
occurring in lichens both as primary photobionts and as internal or external cephalodia; still
commonly called blue-green algae.
Cyanobiont: The cyanobacterial photosynthetic partner in a lichen symbiosis.
Decomposition: The process by which complex organic compounds are broken down into
simpler ones which are then available for use by organisms.
Delta Units (8): Are expressed in molecules per thousand, or “per mil”. For example, 515NAir=
12 per mil means that the sample was analyzed against a reference material and found to be 12
molecules per thousand more than air - the accepted zero point for expression of l5N in per mil
notation. For calculation, 8 = [(Rs / Rr) -1 ] * 1000 where Rs is the ratio of the heavy isotope to

the light isotope o f the sample and Rr is the ratio of the heavy isotope to the light isotope of the
reference.
Ecosystem: A functional unit consisting o f all the living organisms (plants, animals, and
microbes) in a given area, and all the non-living physical and chemical factors of their
environment, linked together through nutrient cycling and energy flow. An ecosystem can be of
any size - a log, pond, field, forest, or the earth’s biosphere - but it always functions as a whole
unit. Ecosystems are commonly described according to the major type of vegetation, for
example, forest ecosystem, old-growth ecosystem, or range ecosystem.
Elemental Analyzer (EA): an automated sample preparation instrument in which samples are
automatically converted into pure gases for isotope ratio analysis. An elemental analyzer
contains the following elements: (i) furnace for combustion, reduction or pyrolysis of sample
material; (ii) chemical traps for analyte gas purification; (iii) gas chromatography for time
separation of these analyte gases.
Foliose: Leafy or flattened lichen, with upper and lower cortical layers.
Fractionation: The enrichment or depletion of a stable isotope caused by natural or artificial
processes.
Fruticose: shrubby or pendant lichen, radially symmetrical.
Isotopes: Atoms whose nuclei contain the same number of protons but a differing number of
neutrons. Two or more nuclides having the same atomic number, thus constituting the same
element, but differing in the mass number. Isotopes of a given element have the same number of
nuclear protons but differing numbers of neutrons. Naturally occurring chemical elements are
usually mixtures of isotopes so that observed (non-integer) atomic weights are average values for
the mixture.
Isotope Ratio Mass Spectrometry (IRMS): A mass spectrometer is an instrument for
separation o f molecules based upon their mass-to-charge ratio. In IRMS the mass spectrometer
used separates isotopes o f different mass within a magnetic field and precisely measures the ratio
o f two, or more, isotopes.
Isotope Ratio: The ratio o f the minor isotope over the major isotope. For example, nitrogen in
air contains 0.3663 Atom % nitrogen-15 and 99.6337 Atom % nitrogen-14, giving an isotope
ratio o f 0.3663 / 99.6337 = 0.003676466.
Macrolichen: Larger lichens of squamulose, foliose or fruticose habit.
Mycobiont: The fungal partner.
Natural Abundance: The concentration of isotopes as found in nature.
Nonsorediate: Lichens which are indistinguishable from sorediate species except for their lack
o f soredia. In these pairs o f species the sorediate lichen does not have apothecia while the
nonsorediate species produces fruiting bodies.

Old growth: A forest that contains live and dead trees of various sizes, species, composition,
and age class structure. Old-growth forests, as part of a slowly changing but dynamic ecosystem,
include climax forests but not sub-climax or mid-seral forests. The age and structure of old
growth varies significantly by forest type and from one biogeoclimatic zone to another.
Pee Dee Belemnite (PDB): A belemnite from the Cretaceous Pee Dee formation of South
Carolina, US which is used as the accepted zero point standard for expression of carbon and
oxygen isotope abundance in delta units.
Pendulous: Hanging down from a support.
Per Mil (%o): see Delta Units.
Photobiont: The photosynthetic partner, an alga or cyanobacterium.
Soredia: (sing, soredium) a flour-like or granular ball of cells of the photosynthetic partner
surrounded by fungal hyphae; these are produced from cracks or pores in the thallus or on its
surface and are asexual propagules.
Species: A singular or plural term for a population or series of populations of organisms that is
capable o f interbreeding freely with each other but not with members of other species.
Species diversity: An assessment of the number o f species present, their relative abundance in
an area, and the distribution of individuals among the species.
Stable Isotope: A non-radioactive isotope in which the number of protons and neutrons in the
atomic nucleus is constant through time.
Stand: A community of trees sufficiently uniform in species composition, age, arrangement, and
condition to be distinguishable as a group from the forest or other growth on the adjoining area.
Thallus: The vegetative part o f a lichen, containing fungal and algal cells.

Acknowledgement
I sincerely thank my supervisor, Art Fredeen, and committee members Darwyn Coxson,
Paul Sanborn, and Katherine Parker for their guidance. I am grateful to Jocelyn Campbell for
assistance in selecting field sites and for help with project design and experimental
manipulations. Kevin Jordan provided essential support in the field and assistance with canopy
access. Thanks are extended to Myles Stocki for laboratory analysis and to the many graduate
student field assistants for help in gathering samples in the canopy. Project funding from the
Natural Sciences and Engineering Research Council of Canada (NSERC 194405-2011; ALF) is
gratefully acknowledged. Finally, I am grateful for the support of my husband, family and
friends over the course of my long academic journey.

xii

Chapter 1: Literature Review and Introduction

1.1 Importance of forest canopies
Forest canopies are significant components of the environment because of their
importance to forest properties such as productivity, ecosystem health, biodiversity, and to local
as well as global climate. Canopy research was overlooked by scientists for years because o f the
inaccessibility o f most forest canopies. However, improvements in canopy access techniques
have allowed scientists in recent decades to explore forest canopies - one of the last biotic
frontiers on Earth - in order to document the importance of many processes in the forest
ecosystem that were previously unattainable (Lowman & Rinker, 2004).
Canopies perform essential functions such as photosynthetic uptake of carbon dioxide
(CO2 ), release o f water (H2 O) through transpiration and uptake and release of nutrients and other
compounds (Lowman & Rinker, 2004). Another critical role that canopies play in forest systems
is as buffers between atmosphere and soil. They provide physical protection of soils from
mechanical impacts of wind and rain and remove particles and pollutants from air and rain that
would otherwise directly impact other parts of the forest system. Canopy microclimate can also
affect epiphyte occurrence and abundance (Campbell & Coxson, 2001).
It is essential to explore the tree crowns of forests since many environmental, ecological,
and societal issues such as environmental change, acid deposition, loss of biodiversity, clean
water, timber quantity and quality, pest and disease control, food, and fiber production, are
directly influenced by the health of forest canopies (Nadkami, 2001). New discoveries resulting
from canopy research have important implications for forest management, crop production, and
medicine. Loss and fragmentation of forest canopies globally has added to the urgency for
1

increasing our understanding of canopy processes. For example, the annual volume of timber
harvested in our very own backyard, British Columbia (BC), has fluctuated around 78 million m3
in recent decades, peaking at or near 90 million m3 in 1987, 2005 and more recently in the wake
o f the mountain pine beetle epidemic (B.C. Ministry o f Forests, Lands, and Natural Resource
Operations, 2010). Since most of this harvest is still being taken from a one-time legacy of
primary forest, it stands to reason that we are losing vast regions of forest canopies yearly that
may hold untold ecological legacies.
The forest canopy is home to a wide variety o f organisms that interact with one another
and their physical environment in ways that impact the entire forest. Canopy ecosystems support
approximately 40% of the species that exist in the world today, with 10% of these specific to the
canopy (Ozanne et al,, 2003). In order to understand the significance of forest canopies to the
ecosystem, we must also understand the contributions of all canopy life forms to the forest as a
whole. A significant life form that is present in most forest canopies is tree-dwelling plants, or
epiphytes. Epiphytes can greatly contribute to the flow and input of nutrients and energy within
forest canopies and therefore represent an important and understudied group in forest
ecosystems.
1.2 Canopy exploration
Documentation and study of the world’s wealth o f epiphytes did not truly begin until
scientists began exploring the canopies of forests around the world. In the early 1970’s,
scientists such as William Denison (1973) began studying the inner workings of old growth
forests by applying direct-aid mountain-climbing techniques to access tree canopies, thus
launching a new branch of forest ecological study. Arborists further developed tree-climbing
techniques in the 1980’s and forests no longer had to be studied from the ground looking up.
2

Donald Perry also became a well-known name in canopy research but Perry cites William C.
Denison of Oregon State University as the pioneer o f canopy-climbing techniques (Perry, 1978).
Forest canopies have now been accessed with a variety of approaches including canopy
walkways, elevators, cranes, inflatable platforms, tree houses, airships, and traditional tree
climbing (Lowman & Rinker, 2004). Many of these methods offer three dimensional access into
difficult-to-reach places, however, they can also be expensive and time consuming.
Two basic styles of technical tree climbing that have emerged are tautline-hitch climbing
and Single Rope Technique (SRT). The SRT is one of the most popular ways to get into the
canopy by using a pair of ascending devices which are hand-held mechanical ascenders that slide
up the rope easily. The ascenders grip the climbing rope with cams when weighted and allow for
movement up the rope whereas rappelling descenders allow for movement down the rope (Perry
& Wiliams 1981, Risley 1981). The SRT method allows climbers to have a wide range o f
motion in the canopy by moving along branches suspended on a rope attached to an anchor
point. The SRT approach was used to gain access to conifer canopies in the present study.

1.3 Epiphytic lichens in the forest canopy
Canopies are home to a wide variety o f epiphytes, including vascular and non-vascular
plants and lichens. Vascular plant epiphytes are common in many forest types, including tropical
and temperate rainforests, but are rare in boreal conifer forests. Lichens and bryophytes are the
more common epiphytes of boreal forests, with lichens, particularly chlorolichens, being the
more common epiphytes in drier forests of interior BC.

3

Lichens form the dominant vegetation over approximately 8% of the earth’s surface. Of
the 20,000 lichen species that exist worldwide, approximately 3,600 species are found in North
America. Terrestrial lichens comprise up to 90 percent of the ground cover in northern boreal
forests (Brodo et al., 1999). In old-growth spruce and fir forests in sub-boreal BC, forests house
as many as 115 different species of epiphytic lichens (Selva, 1996) with 44 different epiphytic
macrolichen species observed previously in the wet and cool Sub-Boreal Spruce biogeoclimatic
subzone (SBS w kl) (Meidinger & Pojar, 1991) in the Aleza Lake Research Forest (Campbell &
Fredeen, 2007). British Columbia’s sub-boreal spruce (SBS) forests are home to a wide variety
of epiphytic organisms with properties and importance to conifer forest ecosystems that are
largely unknown.
Lichens are an association between a mycobiont (most commonly an ascomycete fungi
(98%), 1.6% deuteromycetes and 0.4% basidiomycetes (Rai & Bergman, 2002)) and
photobiont(s), which can be green algae (commonly Trebowcia or Trentepohlia, but also
Dichtyochloropsis reticulata such as in the tripartite cyanolichen, Lobaria pulmonaria) and/or
cyanobacteria (commonly Nostoc). Form is determined by the fungal partner, which provides a
living space for the photosynthetic partner, which in turn shares the carbohydrates it produces
through photosynthesis. The basic growth forms are crustose (flat, paint-like), fruticose
(branched and shrubby), and foliose (leafy with flat thallus). Lichens reflect the conditions of
the place they grow and enlarge approximately 1-2 mm per year (Brodo et al., 1999). They are
able to colonize rocks, trees, and soil but many factors control the distribution of these
mutualistic symbionts. Lichens are poikilohydric organisms that rely directly on the
environment for their water but they are capable o f surviving with extremely low levels of water
content.

Cyanolichens are lichens with cyanobacteria as their primary or secondary photosynthetic
partner that contribute significant amounts of nitrogen (N) to forests by converting atmospheric
N2 gas into usable forms such as ammonium (Millbank & Olsen, 1986). There are
approximately 500 lichen species where the fungus is associated with both a cyanobacterial and a
green algal biont, making them tripartite lichens (Dahlman, 2003). A significant tripartite lichen
in sub-boreal conifer forests is Lobaria pulmonaria. It has a global distribution and is the most
widely distributed and common Lobaria in North America. It is also the dominant, and
commonly the only, tripartite lichen in lower branches of forest trees in the central interior o f
BC. Its N2-fixing symbiont is the cyanobacterium Nostoc which is found in pockets within the
lichen thallus referred to as cephalodia (Brodo et al., 1999). By contrast, bipartite cyanolichens
are relatively diverse at the taxonomic level with many species occurring throughout the lower
canopies of central interior forests, but primarily in older forests, being generally absent from
younger stands.

1.4 Factors affecting canopy lichen distribution
Canopy lichen abundance and diversity can be influenced by a variety o f factors such as
climate, age of the forest stand, continuity of forest cover, gradients of temperature, moisture,
and light within the canopy, nutrient availability, and tree height.
Lichens have been widely useful as environmental indicators and are valuable for
monitoring air quality since they are sensitive to air pollution, with their disappearance often
indicative of poor air quality (Boucher & Stone, 1992). Rose (1976) introduced the idea that
lichens could be used as historical indicators when he found there was a positive correlation
between lichen diversity and forest age, but the complex relationship between lichen species
5

diversity and stand age varies across ecological gradients. Older and primary forests typically
have greater and different lichen diversity than younger forests due to a variety of factors
including a more stable canopy environment, which can be more important than the age of the
trees to some lichen species (Hilbert & Wiensczyk, 2007). The abundance o f epiphytic lichens,
specifically alectorioid lichens in coastal BC, has been shown to increase with stand age (Price &
Hochachka, 2001). There is an increase in lichen species richness with stand age (Price &
Hochachka, 2001). The abundance of N 2 -fixing cyanolichens has been shown to increase with
forest age in interior cedar-hemlock (ICH) and SBS forests, which has been proposed to
represent a significant source o f new N in older forest ecosystems of this region (Campbell &
Fredeen, 2004; Campbell & Fredeen, 2007).
Forest cover continuity and disturbance can greatly impact the distribution and
composition of canopy lichen epiphytes. A lower abundance of desiccation-sensitive lichens
was observed along edges of boreal forests than in the interior (Kivitso & Kuusinen, 2000).
Epiphytic cyanolichens are often entirely absent in the tree canopies of young PseudotsugaTsuga forests (Neitlich, 1993), an absence thought to reflect the limited dispersal abilities of
many old-forest associate lichens (Ockinger et al., 2005). Sillett and McCune (1998) showed
that old growth lichens did quite well when transplanted into clearcuts on the coast. After a fire
or logging disturbance, many lichen epiphytes don’t arrive until after colonization by
chlorolichens during the first century of stand development (Neitlich, 1993). Major disturbances
such as logging can increase irradiance and desiccation, which can cause microclimatic changes
in temperature and moisture variables important to sensitive lichen epiphytes (Stevenson &
Coxson, 2008).

6

Vertical gradients in temperature can have distinct effects on the abundance of canopy
lichens. Some species are intolerant to long periods of high or excessively low temperatures,
which can limit the distribution of these epiphytes in the canopy (Sillett & Mccune, 1998). High
temperatures lead to high respiration rates and lichens cannot maintain the high photosynthetic
rates to overcome the energy and carbon losses during these warm periods (Gaio-Oliveira et al.,
2004). Low temperatures and moisture cause ^-fixation to cease in cyanolichens, inhibiting
sufficient growth and survival o f the epiphyte (Denison, 1979; Sillett & Neitlich, 1996). Low
temperatures can also affect colonization of regenerating trees in forest gaps by chlorolichens in
northern BC (Coxson & Stevenson, 2007). Young stands in cool temperate conifer forests often
have limited populations of cyanolichens, despite abundant propagule sources available from
adjacent old-growth forests (Benson & Coxson, 2002). Small changes in temperature can also
influence the effective moisture content in poikilohydric lichen epiphytes.
Microclimate can vary greatly within a canopy by height and epiphyte abundance.
Lichen epiphytes occur along a microclimatic gradient of decreasing humidity and increasing
exposure to desiccating wind with increasing canopy height (Campbell & Coxson, 2001).
Densely packed epiphytes can reduce air circulation and trap unheated air on the undersides of
branches making the air temperature around branches with epiphytes cooler than branches after
epiphyte removal (Nadkami, 2008). Lichens must be able to tolerate frequent cycles of drying
and wetting to maintain the symbiosis (Honegger, 1998) and these cycles can in turn influence
humidity within the canopy.
As poikilohydric organisms, water content of lichen epiphytes depends on environmental
conditions. Lichens become hydrated with the receipt of precipitation and desiccated in its
absence. Lichens have no mechanisms to prevent dehydration, but can withstand extended
7

drought periods and resume physiological processes rapidly (normally minutes) after rehydration
(Lange et al., 2001). Lichen species are differentially distributed along moisture gradients. For
instance, moist and warm microclimates often support a greater amount of cyanolichen species
and dry forests support a greater abundance of chlorolichens (Sillett & Antoine, 2004). Wet and
dry periods are required by most lichens in order to maintain symbiosis (Honegger, 1998)
because prolonged hydration can reduce lichen photosynthesis by decreasing the amount of CO 2
diffusion to the photobiont (Lange et al., 1993). Chlorolichens can be physiologically activated
by water vapour alone but cyanolichens need liquid water in order to be activated (Green et al.,
2002). In addition to moisture, light and nutrient availability in forests can also affect lichen
abundance and diversity.
The amount of light that is available to lichens depends on their position within the forest
canopy and the surrounding canopy cover. In the upper canopy where there are high light
conditions, lichens have higher desiccation rates and light saturation points than lower canopy
lichens (Demmig-Adams et al., 1990; Proctor, 2000). Upper canopy zones in sub-boreal forests
are mainly comprised o f hair lichens and chlorolichens that are able to withstand these harsh
conditions. Lower canopy positions, though often less prone to desiccation, are often lightlimiting for photosynthesis. Gauslaa et al. (2008) found that genera with usnic acid such as
Alectoria decreased with height while genera with melanic pigments such as Bryoria increased
with height because dark melanic pigments have higher visible light screening efficiency.
Distribution o f epiphytic lichens is also affected by nutrients such as calcium. Spatial
variation in soil nutrient content can cause variation in cyanolichen abundance from tree to tree.
In one study, more cyanolichen appeared on trees growing in calcium-rich soils than on trees
growing in calcium-poor soils (Gauslaa, 1985) suggesting that tree canopies themselves are an
8

important ion source for epiphytic lichens (De Bruin & Hackenitz, 1986). Another study found
cyanolichens to be more abundant on branches within the drip zone of trees such as Populus that
may leach calcium, allowing cyanolichens to colonize young conifers that would otherwise be
too acidic (Sillett & Goward, 1998; Goward & Arsenault, 2000).
Lichen biodiversity in sub-boreal spruce forests changes vertically with each height zone.
In previous studies, upper canopies were found to be dominated by nonsorediate Bryoria species,
the middle canopy by Alectoria sarmentosa and Platismatia glauca and the lower canopy
primarily by cyanolichens and notably, the tripartite cyanolichen Lobaria pulmonaria (L.)
Hoffm. (Campbell & Fredeen, 2007). In some cases, lower canopy branches of young
coniferous forests have been shown to be unfavorable for chlorolichens due to the absence of
mature lower branches (Esseen et al., 1996). In some of the wetter sub-boreal spruce forests, L.
pulmonaria has been found to have greater stand level biomass than all other canopy lichen
species combined (Campbell & Coxson, 2001).

1.5 Nutrient dynamics of canopy lichens
Nutrients held in lichens positioned within the canopy are released into the forest system
by intermittent leaching (Millbank, 1982). Lichens do not provide energy, nutrients or water
directly to the vascular system of their host tree directly. Instead, rain or fog can leach out
nutrients held in lichens which can in turn enrich or deplete nutrients in lower epiphytes or
become available to other components of the ecosystem. Epiphytes also rely on atmospheric
sources for their nutrients, but lichens, particularly those in lower canopy positions, may also
gain nutrients from the leaching o f host tree tissues (Stewart et al., 1995). The rate at which
leaching of nutrients occurs from lichens depends on factors such as morphology of the lichen.
9

Finely branched hair lichens can facilitate absorption and leaching due to their large surface area
to volume ratio relative to flat foliose lichens.
One important nutrient that lichens leach, particularly N 2 -fixing cyanolichens, is N. As
water passes through the canopy it has been found to decrease in inorganic N and increase in
dissolved organic N and anions (Wania et al., 2002), indicating rapid leaching when water is
available for leaching. Even free-living N 2 -fixers may provide N to epiphytes through leaching
(Benzing, 1990), which may be of importance for chlorolichens. The most important forms of N
for plant nutrition are nitrate and ammonium but they are highly soluble in water and can be
easily lost through leaching. Ammonia volatilization or denitrification also amplifies the loss of
N to the atmosphere when ammonium is converted back into N oxides or N 2 (Peoples et al.,
1995).
Old-growth forests are highly retentive of nutrients and nutrient release rates from
decomposing litter and are dependent on mobility of nutrients in living or dead organic matter.
Portable nutrients such as magnesium (Mg), calcium (Ca), and potassium (K) are quickly
released during early decay but other elements such as N and phosphorus (P) can be fixed or
immobilized in decomposing litter with high initial C:N or C:P ratios (Manzoni et al., 2008).
Losses of limiting nutrients, such as N, are low but N can also be released from decomposing
litter with low C:N ratios during early decay (Prescott, 2005). Lichens with high N content and
low C:N ratios have been observed to have higher decomposition rates and rates were higher in
N2 -fixing lichens than in non-N 2 -fixing lichens (Crittenden & Kershaw, 1978).
Decomposition rates of lichens are controlled by a variety of factors such as
environmental effects, the quality of the litter substrate and the chemical composition of the
decomposer community (Comelissen, 1996). Increases in litter decomposition rates are often
10

associated with increasing moisture and temperature (Zhang et al., 2008). Decomposition
patterns can be strongly influenced by variations in seasonal climate and time o f litterfall can
highly impact the initial decay rate. Holub and Lajtha (2003) found that cyanolichens had a
smaller decay constant (k = 1.24 y e a r1) when placed in the field during the spring than during
the fall (k = 3.1 y e a r 1) but all lichen was thoroughly decomposed after 1 year.
Studies have found a positive relationship between snowpack and lichen decomposition
rate. One study observed slower decomposition rates of cyanolichens at drier sites in sub-boreal
forests of central BC and slower decay rates at higher elevation sites, which could be related to
deeper and more persistent snowpack (Campbell et al., 2010). The decomposition rate of hair
lichen Alectoria sarmentosa and Bryoria spp. was also influenced by snowpack. Lichen samples
lost up to two-thirds of their mass when placed on early winter snowpack as opposed to minor
amounts (approximately 10%) in mid and late-winter snowpack (Coxson & Curteanu, 2002).
Decay rates of L. pulmonaria can be inhibited by the makeup of its thick thallus and/or its
chemical composition. L. pulmonaria had higher decomposition rates than Hypogymnia spp. and
Platismatia glauca which both lack the In fix in g photobiont (McCune & Daly, 1994). Lobaria
pulmonaria and Nephroma helveticum are both N rich foliose lichen species but L. pulmonaria
decomposed slower than N. helveticum in Pacific Northwest forests (Harmon et al., 2009). The
reduced rate in decomposition could be due to the thick outer cortex or chemical resistance of the
species (Campbell et al., 2010). Lobaria oregana is a species closely related to L. pulmonaria.
Lobaria oregana decomposes slowly because it contains stictic, norstictic and constictic acids
(Culberson, 1969), which decrease decomposition rates (Hattenschwiler & Vitousek, 2000) and
N mobilization rates from chitin (Greenfield, 1993). The N content of all L. oregana samples
increased during decay to a peak o f around 2.8% N dry mass and then decreased, which
11

indicated that net N immobilization occurred in the remaining lichen during early decay,
followed by net N mineralization in later stages of decay (Holub & Lajtha, 2003).
Fixed N can become available to plants in the immediate area when lichens die and decay
or when N compounds leach from living thalli. Lobaria species contribute their fixed N to
surrounding ecosystems through leaching and decomposition. Lobaria oregana contributes 2.54.5 kg N ha ’y ' (Pike 1978; Denison 1979), which is approximately 33-67% of total new N
coming into old growth Douglas-fir (Pseudotsuga menziesii) forest ecosystems (Sollins et al.,
1980). Nitrogen made available by Lobaria spp. could increase sub-boreal forest ecosystem
productivity.
Litter from trees and epiphytes contribute to organic matter on the forest floor and add to
the decomposition energy cycle. A recent study by Campbell et al. (2010) determined that the
annual above-ground litterfall from wet sub-boreal forests in the interior of central BC was
3448-3978 kg ha ' 1 year- 1 with lichen litter making up 0.4%- 2.8% of the total litter mass
collected. They showed that 0.1 %—2.3% of the total above-ground litter biomass was from
cyanolichens contributing 0.5%—11.5% of total litterfall N. The decomposition of cyanolichens
in this study was estimated to release up to 2.1 kg N ha- 1 year- 1 of newly fixed N to mature subboreal forests (Campbell et al., 2010) making N-rich macrolichens in the interior of BC a crucial
component of the N-cycle. N itrogen-fixing epiphytes capable of biological n-fix atio n are
abundant in old-growth sub-boreal forests of central BC and may be a crucial component of the
N-cycle. This research was an attempt to quantify the importance of lichen epiphytes, and
particularly cyanolichens, to the spruce and fir forest ecosystems of central BC.

12

1.6 Cyanolichen biological N2-fixation and its contribution to the N-cycle
Nitrogen is the largest single constituent of the Earth’s atmosphere, at 78 percent by
volume, and therefore global N pools are largely dominated by the atmosphere. Nitrogen is vital
for all life since it is an essential part of proteins and nucleic acids as well as a commonly
limiting nutrient for plant growth. It can be deposited onto water and land surfaces through wet
deposition or dry deposition, the absorption of compounds by rain or the direct adsorption of
compounds to water or land surfaces, respectively. But atmospheric nitrogen (N2) is inert and
not available for use by plants and animals so it is crucial to understand the movement of N
through ecosystems.
Humans have altered the global N cycle through the use of N-rich fertilizers and the
burning of fossil fuels. Anthropogenic N can especially alter ecosystems by modifying species
diversity, plant community composition, and ecosystem function where N supply is limited
(Aber et al., 1998). Global N 2 -fixation is currently being estimated at ~240 Tg N y~'. In the next
50 years, inputs to the global N cycle are expected to continue to increase to 275 Tg N yr 1.
Currently only 40% o f all newly fixed N deposited on the Earth’s surface each year comes from
natural biological and chemical processes, whereas 60% is derived from human sources. This is
an unprecedented change on a global scale (Berman-Frank et al., 2003). It is important to
understand the N cycle, where this large amount of annually fixed N is being deposited, and if it
can be supplemented by biological N2-fixation from cyanolichens.
Certain cyanobacteria can be found in a lichenized condition, and these, under the right
ecological conditions, can fix atmospheric N into ammonia which can then be utilized by lichen
symbionts. A large amount of energy is required to break up the triple bond of N 2 into usable
forms o f N such as ammonia, so many organisms use sources of fixed N. Nitrogen fixation is an
13

energy-dependent transformation of atmospheric nitrogen gas (N 2 ) to ammonia (NH 3 ) by the
enzyme nitrogenase, which can be inhibited by O 2 .
Cyanolichens have the ability to fix atmospheric nitrogen that can then be used for
growth of higher life forms and can potentially contribute to ecosystem nutrient budgets (Becker,
1980; Forman & Dowden, 1977; (Pike, 1978). Cyanolichens have a higher N content than
lichens with green alga as their only photobiont (Rai, 1988). Green et al. (1980) reported the N
content to be 3.4% in cyanolichens and 0.5% in other lichens. Hitch and Stewart (1973) reported
similar values of 2.2% and 0.85%, respectively. About 10% of lichen species are bipartite and
contain cyanobacteria as their primary photobiont (Nash, 1996) and 3-4% are tripartite
cyanolichens, which contain green alga as their primary photobiont (Rai & Bergman, 2002).
Cyanobacteria found in bipartite lichens are either dispersed throughout the thallus or as a layer
below the upper cortex, but in tripartite lichens, the cyanobacteria occur in cephalodia. The
cyanobacteria in bipartite lichens have to provide both the fixed carbon (C) and nitrogen to the
mycobiont o f the lichen but tripartite lichens only have to provide fixed N since the fixed C
comes from the algal component of the lichen (Rai & Bergman, 2002).
The process of N 2 -fixation in cyanobacteria occurs in microaerobic (low oxygen content)
N 2 -fixing cells called heterocysts (Brodo et al., 1999) because the enzyme complex responsible
for N2-fixation is extremely sensitive to oxygen (Gallon, 1992). The rates of ^-fix atio n are
higher in tripartite lichens due to their higher frequency of heterocysts within the cephalodia of
cyanobacteria (Rai & Bergman, 2002). Heterocysts have a thick membrane that slows the
diffusion of O2 . They contain photosystem I, which supplies ATP, but they lack the watersplitting and oxygen-producing photosystem II (Ernst et al., 1983). About 5-10% of filamentous
free-living cyanobacterial cells differentiate into heterocysts under N-limited conditions
14

(Stewart, 1980). The cyanobiont in the lichen thalli continues to develop heterocysts even in the
presence of fixed-N (Bergman et al., 1992) since the cyanobiont does not incorporate the N into
its own cells. The development of more heterocysts and the exchange of metabolites are
triggered by the presence of N-starved cyanobiont cells and the rapid transfer of NH 4 into the
mycobiont, which takes place in the filaments of cyanobacteria.
Vegetative cells provide photosynthate in exchange for fixed N from the heterocysts.
Heterocysts lack Rubisco and do not fix C so they must rely on vegetative cells to supply fixed C
in order to support fixation of N 2 (Wolk et al., 1994). Once N 2 has been fixed, NH4+ ions in
isolated free-living cyanobacteria are converted to glutamine in heterocysts by the enzyme
glutamine synthetase (GS). Then the ions are exported and catalyzed to glutamate in vegetative
cells. Ammonium is then released to the mycobiont and converted to glutamate by the enzyme
glutamate dehydrogenase (GDH). The enzyme GDH converts NH 4 to glutamate because
glutamine synthase is inhibited (Wolk, 1996). Elevated concentrations of mycobiont GDH have
been shown to coincide with rates of N 2 -fixation, repression of GS synthesis, and ammonia
release by the cyanobiont along a lichen thallus (Rowell et al., 1985).
Mycobiont hyphae and cyanobiont cells in lichen thalli occur in close contact with each
other and the hyphae contain a high level of the ammonia assimilating enzyme GDH. The
mycobiont effectively absorbs ammonia released by the cyanobiont with negligible ammonia
escaping outside the thallus (Rai et al., 2002). Fixed N from the symbiotic tissues is translocated
to other parts of the host in the form of amino acids such as alanine in tripartite lichens, which
moves from cephalodia to the main thallus (Rai et al., 2000).
Nitrogen-fixing species have a competitive advantage in ecosystems where N is in low
supply. However, N2-fixation is an energy-demanding process and can be costly to an individual
15

organism so they only fix N when other sources o f N are not readily available. When the feather
moss Pleurozium schreberi (Bird.) Mitt, is exposed to high concentrations of bioavailable N, the
putative N 2 -fixing moss-cyanobacteria association will use that N instead of expending energy
on fixing new N (Zackrisson et al., 2004). Similarly, cyanolichen ^-fix atio n only occurs when
the N requirements are not met by N content in canopy throughfall. Canopy epiphytes
intercepted 8 .8 % more o f total throughfall compared with throughfall collected underneath trees
that had their epiphytes removed (Boucher & Nash 1990; Knops et al. 1991).
Biological ^-fix atio n represents a significant contributor to the global N cycle.
Cyanolichens are a crucial component of this biogeochemical cycle as they have been estimated
to contribute 3—4 kg N h a 1 year' 1 to mature conifer forests of the Pacific Northwest (Denison,
1979). Lichens represent a large portion of forest biodiversity and play an important role in the
function o f the N cycle relative to their small size. The nitrogenase enzyme complex responsible
for N 2 reduction represents the single largest contributor to the reductive portion of the global N
cycle (Christiansen et al., 2001).

1.7 Stable Isotopes of Nitrogen and Carbon
Stable isotopes can be used to increase our understanding of element cycles in
ecosystems and advance our knowledge of relationships between plants and their environment.
As elements cycle through the biosphere, the composition of isotopes changes and these changes
are being utilized by ecologists and geochemists to understand global cycles of essential
elements such as N and C (Peterson & Fry, 1987). Interest in community ecology and ecosystem
research using this technique is being facilitated by technological developments making stable

16

isotope measurements more accessible to researchers (Hobson & Wassenaar, 1999), allowing
plant ecologists to address issues that seemed problematic using other methods.
The use o f stable isotopes as a tool to investigate ecological research has increased
immensely in the last two decades. Enriched 15N tracer studies have been used in agricultural
investigations for decades (Bremner, 1965) but natural abundance studies started with Kohl,
Shearer, and Commoner (1971) and were rare until the last decade. The use of stable isotopes
will continue to increase because they can be used as nondestructive temporal integrators which
can determine interactions and responses of plants to their abiotic and biotic environments
(Dawson et al., 2002). Natural abundance (NA) is the percentage of an element occurring on
earth in a particular stable isotopic form. The abundance of an isotope remains relatively
constant in time. Stable isotope methods look at the interactions between organisms and the
resources that influence them.
Atmospheric N can only be assimilated by specialized organisms such as cyanolichens
but the nitrate and ammonia in soil are forms of inorganic N that can be assimilated by all plants
including the cyanolichen host tree. Ammonium produced by cyanolichen N 2 -fixation can have
several potential fates. It can be assimilated into soil organic microbial biomass, taken up by
plant roots, lost by denitrification into the atmosphere, leached into runoff groundwater, or
transformed into nitrate through nitrification (Nadelhoffer & Fry, 1994). The distribution of N in
forests can be identified by the contents of 1SN from N inputs and outputs.
There are two naturally abundant stable isotopes on earth of N and C. The more common
N stable isotope is l4N with 99.6337% of the total N in any system as l4N atoms. The rarer but
heavier N stable isotope is 15 N, and the remaining 0.3663% of 15N atoms (Junk and Svec, 1958).
I -y

The lighter C stable isotope is C with a natural abundance of 98.89% and the heavier C stable
17

isotope is 13C with a much lower natural abundance of 1.11% (Craig, 1953). The extra neutron
in heavier stable isotopes gives the element a greater mass without altering its chemical behavior.

1.8 Stable Isotope Fractionation and Discrimination
Isotopic fractionation is an important factor to consider in natural abundance studies
because many reactions can alter the ratio of heavy to light isotopes between a substrate and
product (Peterson & Fry, 1987). Delta (8 ) values are not absolute isotope abundances but
differences between sample and standard values in parts per thousand (i.e. per mil). Differences
in the isotope ratios can be very small so we use 8 notation which tells us how much the sample
deviates from the standard. Processes that result in differences in 8 values in products and
reactants reflect isotope fractionations and they occur because more energy is required to break
chemical bonds involving the heavier isotope than the lighter isotope. Molecules containing 14N
react faster with enzymes than molecules containing 15N (Robinson, 2001) because they are
atoms o f different sizes and weights. In other words, different atomic weights react at different
rates. Fractionations in biogeochemical reactions can provide information about elemental
processes.
The two common types of isotope fractionations are equilibrium isotope fractionation,
which occurs during isotope exchange reactions that convert one phase to another, and kinetic
isotope fractionation, which occurs when the reaction is unidirectional. Reaction rates for both
types o f fractionations are mass dependent (Dawson et al., 2002). Physical processes, such as
evaporation, discriminate against heavy isotopes. Enzymatic discrimination, or differences in
kinetic characteristics, can result in isotopically heavier or lighter products than their precursor
materials. The 15N natural abundance of ecosystem components reflects the isotopic signatures
of inputs and outputs within the system. Ultimately, the 8 15N signature of biological material is
18

the result of 8 15N signatures of the N sources and isotope fractionation during N uptake and N
assimilation, N gains and losses, and N pool mixing (Handley & Raven, 1992).
Fractionation between plant and substrate depends on physiological traits of the plant, N
form, and external N concentration (Robinson 2001). The extent of isotopic fractionation
depends on the substrate to product ratio, temperature, reaction rate, and enzymatic facilitation of
the reaction (Nadelhoffer & Fry, 1994). Patterns of I5N abundances in ecosystems can also be
influenced by a variety of factors including soil age, N 2 -fixing plants, precipitation, biochemical
processes, and mycorrhization.
In ecological studies, the degree of fractionation is typically quite small but measurable
and it is important to understand for proper data interpretation. However, not every N cycle
process always fractionates N isotopes. Fractionation depends on the process, external
conditions and the extent to which the N source has been consumed (Robinson, 2001).
Fractionation of 15N can be influenced by the N supply rate and when abundant N supplies exist,
discrimination against 15N during plant N uptake causes relative depletion in 15N of plant
biomass (Kohl & Shearer, 1980). However, under N-limited conditions, plants take up the entire
supply of N, which leaves no possibility of potential fractionation (Nadelhoffer & Fry, 1994). It
is important to consider isotopic fractionation in forest ecosystems when using the natural
abundance (NA) method as a natural integrator and tracer of ecological processes.
1.9 Natural Abundance Method Uses and Advantages
The NA method can be used to make inferences about rates and patterns of the N cycle,
the importance of N inputs and soil processes in supplying N for plant uptake, and N retention by
forests (Nadelhoffer & Fry, 1994). This method allows ecologists to use stable isotopes as
natural integrators and tracers without disturbing the natural behavior of the element in the
19

system (Handley & Raven, 1992). However, using natural abundance isotopes as tracers
requires that the different sources have repeatable and distinct 8 values that are more extensive
than the natural range of plant 5 values measured (Dawson et al. 2002). The NA technique is
more efficient if there are just two pools of N available and if the variability of l5N abundance is
small compared to the measured difference between these abundances (Boddey et al., 2000).
Natural 15N abundance values are also used for improving estimates of N fluxes and N losses
from forest ecosystems (Nadelhoffer & Fry, 1994), and are able to show how distributions of N
isotopes are linked to N cycling and other biogeochemical processes.
The NA technique has a number of advantages over 1SN enrichment procedures. Natural
abundance studies are simple, inexpensive, and non-disruptive to the study system and provide a
wide assortment o f natural experiments at a range of spatial and temporal scales. The 15N
enrichment method is costly and often disrupts the system through the addition of trace amounts
of a labeled substance such as excess N, limiting its widespread application in N cycle research
(Bedard-Haughn et al., 2003). The NA method allows for ^ -fix atio n to be monitored where
both fixing and non-N 2 -fixing reference plants are present since there are no additions or
disruptions to the system before measurements are taken (Peoples et al., 2002).
1.10 Soil Nitrogen Inputs
Approximately 50% of total N deposition from the atmosphere is comprised of the
gaseous species nitrogen monoxide (NO), nitrogen dioxide (NO2 ) and NH 3 (Stulen et al., 1998).
Ammonium is preferred by the canopy relative to atmospheric nitrate and it is more often
retained (Garten & Hanson, 1990). Most of the atmospheric N that reaches the soil surface is in
the form of nitrate. Soil nitrate is also preferentially assimilated by tree roots relative to soil NH4
(Nadelhoffer & Fry, 1988). Nitrogen dynamics in the soil are strongly controlled by biologically
20

mediated reactions such as assimilation, nitrification, and denitrification. These reactions can
result in increases in the 6 15N o f the substrate and decreases in the 6 l5N of the product.

1.11 Thesis overview
The second chapter of this thesis provides results on the patterns of variation in arboreal
lichen communities in sub-boreal spruce forests. Specifically, it focuses on how epiphytic lichen
species richness and dominance vary across tree species and height. Few studies have tried to
separate the effects of forest structure on species abundance and diversity, both spatially and
vertically within the canopy. Most research has compared the extremes of forest structure o f
managed forests to natural forests. Consequently, little information is available to guide forest
managers in assessing natural forests as a potential repository of biological diversity (Pipp et al.,
2001). This information from my findings may help guide forest management objectives that
relate to conservation of forest epiphytic lichen biodiversity in this region.
The third chapter provides an examination of the importance of N 2 -fixing epiphytic
lichens to the N budget o f SBS forests using the natural abundance of the heavy (stable) isotope
of nitrogen (I5 N) in canopy components as an in situ indicator. Cyanolichens such as Lobaria
pulmonaria are known to contribute fixed N to old-growth forests, but the amounts and
significance of these contributions are uncertain as they pertain to the N budget of mature and
old-growth spruce and fir forests.
Finally, the fourth chapter gives a brief summary o f the overall thesis findings and their
significance to sub-boreal forest management. It provides some overall conclusions from this
thesis research and recommendations for future management of sub-boreal spruce and fir forests
in central BC.
21

1.12 Literature Cited
Aber, J., McDowell, W., Nadelhoffer, K., Magill, A., Bemtson, G., Kamakea, M., McNulty, S.,
Currie, W., Rustad, L., & Fernandez, I. (1998). Nitrogen saturation in temperate forest
ecosystems— Hypothesis revisited. Bioscience, 4 8 ,921-934.
Aerts, R. (1997). Climate, leaf litter chemistry and leaf litter decomposition in terrestrial
ecosystems: a triangular relationship. Oikos, 79(3), 439—449.
B.C. Ministry of Forests, Lands, and Natural Resource Operations. (2010). The State o f British
Columbia’s Forests, 3rd ed. (pp. 1-323). Victoria, B.C. Retrieved from
http://www.for.gov.bc.ca/hfp/sof/2010/SOF_2010_Web.pdf
Becker, V. E. (1980). Nitrogen fixing lichens in forests of the Southern Appalachian Mountains
of North Carolina. Bryologist, 83, 29-39.
Bedard-Haughn, A., van Groenigen, J. W., & van Kessel, C. (2003). Tracing l5N through
landscapes: potential uses and precautions. Journal o f Hydrology, 272, 175-190.
Benson, S., & Coxson, D. S. (2002). Lichen colonization and gap structure in wet-temperate
rainforests of Northern interior British Columbia. The Bryologist, 705(4), 673-692.
Benzing, D. H. (1990). Vascular epiphytes: general biology and related biota. Cambridge:
Cambridge University Press.
Bergman, B., Rai, A. N., Johansson, C., & Soderback, E. (1992). Cyanobacterial plant
symbioses. Symbiosis, 14, 61-81.
Bergstrom, D. M., & Tweedie, C. E. (1998). A conceptual model for integrative studies of
epiphytes: nitrogen utilisation, a case study. Australian Journal o f Botany, 46, 273-280.
Berman-Frank, I., Lundgren, P., & Falkowski, P. (2003). Nitrogen fixation and photosynthetic
oxygen evolution in cyanobacteria. Research in Microbiology, 154, 157-164.
Billings, S. A., & Richter, D. D. (2006). Changes in stable isotopic signatures of soil nitrogen
and carbon during 40 years of forest development. Oecologia, 148(2), 325-33.
Boddey, R. M., Peoples, M. B., Palmer, B., & Dart, P. J. (2000). Use of the 15N natural
abundance technique to quantify biological nitrogen fixation by woody perennials. Nutrient
Cycling in Agroecosystems, 57, 235-270.

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Boucher, V. L., & Nash, T. H. I. (1990). The role of fruticose lichen Ramalina menziesii in the
annual turnover o f biomass and macronutrients in a blue oak woodland. Botanical Gazette,
1 5 1 ,114-118.
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31

Chapter 2: Vertical distribution and nitrogen content of epiphytic macrolichen functional
groups in sub-boreal forests of central British Columbia
Published in Forest Ecology and Management 329:118-128
2.1 ABSTRACT
Vertical distribution, biomass abundance and nitrogen stocks of epiphytic macrolichens
were examined in the two dominant tree species, interior hybrid spruce (Picea engelmannii Parry
ex Engelm. x glauca (Moench) and subalpine fir {Abies lasiocarpa (Hook.) Nutt.), in sub-boreal
spruce forest ecosystems of central British Columbia (BC). Lichens were contrasted between
two different site types containing either high (High Cyano) or low (Low Cyano) epiphytic
cyanolichen abundance. A single rope technique was used for canopy access and a ‘clump’
method was used to estimate the abundance of arboreal lichens at different heights within both
canopy tree species for five functional groups: Bryoria spp., Alectoria sarmentosa, foliose
chlorolichens, bipartite cyanolichens, and Lobaria pulmonaria (the only tripartite cyanolichen).
In this way, the relationship between average lichen biomasses and tree height for each tree
species were assessed. We determined that biomass was dependent on tree height and species,
with a greater abundance of lichen appearing on fir trees (mean ± SD; 1588 ± 428 g tree'1) than
the generally taller spruce trees (917 ± 422 g tree'1). Foliose chlorolichen biomass was more
abundant in trees with low abundance of L. pulmonaria (777.1 ± 365.9 g tree'1) than those with a
high abundance of L. pulmonaria (553.6 ± 400 g tree'1). Much of this increase in chlorolichen
biomass in trees with low cyanolichen abundance was a result of greater chlorolichen
abundances in lower canopy positions where cyanolichens would otherwise predominate.
Although bipartite cyanolichens had a higher %N (~3.2 %N) than L. pulmonaria (2.3 %N) of dry
mass, there was a much higher abundance of L. pulmonaria in the canopy of High Cyano sites
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containing a larger amount of N (4.04 ± 0.95 kg N h a 1) than in all bipartite cyanolichens
combined (2.33 ± 1.11 kg N ha'1) or any other lichen spp. Chlorolichens are poor competitors
for lower branch positions when cyanolichens such as Lobaria pulmonaria are abundant, the
latter of potential importance for N inputs into N-limited sub-boreal forests of central BC.

Key words: Lichen epiphytes, lichen diversity, arboreal lichen abundance, Lobaria, nitrogen,
Picea glauca x engelmannii, Abies lasiocarpa.

2.2 INTRODUCTION
Forest canopy research has revealed diverse epiphytic lichen communities in forests of
north-central BC (Botting et al., 2008; Campbell & Coxson, 2001; Campbell & Fredeen, 2007;
Coxson et al., 2003). In these forests, communities of epiphytic lichens are affixed to canopy
branches providing a critical interface between the atmosphere, tree and forest floor. Epiphytic
lichen biomass in boreal forests tends to increase with forest structural complexity (Pipp et al.,
2001), and species composition varies with temperature and moisture gradients. Sub boreal
spruce (SBS) forests of central BC can have significant biomasses of epiphytic N 2 -fixing
cyanolichens that are usually restricted to lower canopies of primarily old-growth (>140 yrs.)
forests where there is reduced stand level disturbance, adequate moisture and lower light levels.
Despite substantial epiphytic lichen diversity in sub-boreal forest canopies, a detailed
characterization o f these lichen communities by host tree species, tree and forest age and in
vertical distribution is only recently emerging.
Epiphytic lichens are significant components of boreal forest ecosystems and comprise a
large fraction of the forest’s total photosynthetic species diversity (Campbell & Fredeen, 2007).
33

Vertical gradients in epiphytic lichen diversity in forest canopies are structurally complex and
have been shown to be affected by the following factors: age of the forest stand (Rose, 1976),
continuity of forest cover (Kivitso & Kuusinen, 2000; Coxson & Stevenson, 2007), gradients in
temperature (Denison, 1979; Sillett & Neitlich, 1996), moisture (Lange et al., 2001), light
(Demmig-Adams et al., 1990; McCune, 1993; Proctor, 2000), nutrient availability (Gauslaa,
1985; De Bruin & Hackenitz, 1986), and relative tree height, host tree species, and soil type
(Campbell & Fredeen, 2007). Aspects of lichen ecology are also important. For example,
variation in fruticose arboreal lichen accumulation in old-growth balsam fir {Abies balsamea (L.)
Mill.) forests is speculated to be a reaction to microclimatic gradients, aging o f thalli, forest
fragmentation, competition, succession, and caribou grazing (Arseneau et al., 1998). Foliose
epiphytic lichens also exhibit large changes in community composition along a vertical gradient
(Campbell & Coxson, 2001) and these variations have been shown to be related to branch
position along tree height (Arseneau et al., 1998). Natural occurrence and growth of the tripartite
cyanolichen L. pulmonaria in boreal forests appear to be controlled in large part by a
compromise between adequate light and moisture availabilities where avoidance of fatal
desiccation can be achieved (Gauslaa et al., 2012).
Nitrogen that is held in epiphytic lichens, and most importantly cyanolichens, can be
released into the forest system and provide forest canopies and understory plants with N through
intermittent leaching (Millbank 1982; Benzing 1990) and decomposition of lichen litterfall
biomass (Campbell et al., 2010b). Research has established that nutrient leaching depends on
lichen structure, amount of precipitation and surface area to volume ratio, but its quantification
has not been properly assessed in sub-boreal forests of central BC. Leaching of soluble materials
occurs with mass water transport (Coxson & Curteanu, 2002) during drying and re-wetting
34

cycles. Pike (1978) showed that organic N compounds are leached from N 2 -fixing lichens
during laboratory mistings whereas N can be taken up by lichens which do not fix N. Other
epiphytes in the canopy may also rely on nutrients leached from N2 -fixing lichens.
Decomposition of lichen is also facilitated by high area to volume ratios (Esseen &
Renhom, 1998) and is highly influenced by seasonal climate variations in temperature and
moisture, time of lichen litterfall, chemical characteristics o f the lichen litter, and ground cover
(Asplund & Wardle, 2013). Nitrogen pools of epiphytic lichens in sub-boreal forests are
significant relative to atmospheric N inputs (Campbell & Fredeen, 2007) with decomposition of
cyanolichens in these forests releasing up to 2.1 kg N ha 1 year 1 (Campbell et al., 2010b).
This study had two primary objectives: to describe and quantify the vertical patterns in
arboreal macrolichen functional group abundances in sub-boreal spruce forests with varying
amounts of cyanolichen, and to quantify the impacts of this variation on the amount of N in
aggregate forest canopy lichen. To address these objectives, we assessed all epiphytic
macrolichen diversity, with a particular emphasis on cyanolichens given their N2 -fixing
properties and the importance o f N as a limiting nutrient in SBS forests. The abundance of
cyanolichens such as L. pulmonaria has been correlated with increased biodiversity (Campbell &
Fredeen, 2004), but the effects o f epiphytic cyanolichen abundance on chlorolichen functional
groups are poorly understood.
Despite the potential ecological importance o f sub-boreal epiphytic lichen communities,
there are few reliable assessments of its biomass, composition and vertical distribution in subboreal forests. Increasing fragmentation and clear-cut harvesting of northern forests may be
threatening many epiphytic lichens with limited dispersal ability and old-growth forest
preferences. This study attempts to clarify the vertical, spatial and host-tree specificities of
35

arboreal maerolichens and their biomass and N levels in mature sub-boreal spruce and fir forests
o f the BC interior.

2.3 METHODS

2.3.1 Study Area
This study was carried out on the north side of the Fraser River near the town of Upper
Fraser, BC, located approximately 70 km NE of Prince George, BC (Fig 2.1). According to the
BC Biogeoclimatic Ecosystem Classification System, the study sites were situated in Sub-Boreal
Spruce (SBS) forest ecosystems across an ecotonal region between the SBS wkl (Sub-Boreal
Spruce Willow Wet Cool) and the SBS vk (Sub-Boreal Spruce Very Wet Cool) subzones
(Meidinger & Pojar, 1991). All study sites are characterized by relatively cool, moist summers
and cold, snowy winters. Four mature sub-boreal forest sites were chosen based on having trees
with predominantly high or low epiphytic cyanolichen abundance and diversity, characterized in
greater detail in previous studies (Campbell & Fredeen, 2007; Campbell et al., 2010b). All study
sites were approximately 2-3 ha and study trees were located within 1 km of roads for safety
reasons. Site locations and spruce and fir stem densities are provided in Table 2.1.
The two dominant tree species, and therefore the primary hosts for lichen epiphytes in all
four sites, were interior hybrid spruce and subalpine fir. The two Fraser sites, spanning the SBS
wkl and the SBS vk subzones, had relatively high cyanolichen abundances and diversities
(Campbell et al., 2010b) and were termed ‘High Cyano ’ sites. These sites were located at an
elevation o f 680 m above sea level (a.s.l.) with a mean summer temperature of 11.8 ± 5.3 °C
(Campbell et al., 2010b). The two Herrick road sites had relatively low cyanolichens and
36

diversities (Campbell et al., 2010b) and are termed ‘Low Cyano ’ sites in this study. These sites
were located in the SBS vk at an elevation o f 850 m a.s.l. with mean summer temperature o f 10.8
± 5.3 °C. Soils at all sites were Orthic Humo-Ferric Podzols formed from sandy-colluvial
materials at the High Cyano sites and from sandy-skeletal glaciofluvial materials at the Low
Cyano sites. Average precipitation across the ecotonal study area is approximately 897 mm per
year (Campbell et al., 2010b). High Cyano 1 and 2 sites and Low Cyano 1 and 2 sites in this
study are equivalent to the Hi 1 and 2 and Low 1 and 2 sites in Campbell et al. (2010),
respectively.
2.3.2 Sample trees and lichen sampling
Lichen epiphytes were sampled over two summers: June to August 2008 and May to
September 2009. Four spruce and four fir study trees were selected at each of the High and Low
Cyano sites for a total of 32 trees. Healthy study trees were selected based on having a
representative form (height, diameter) and age (ranged between ~50 and 230 years old) of the
mature trees on site. All study trees were in excess of 22 m in height and 20 cm in DBH.
Needles and lichens were sampled from each canopy height zone starting at the highest
accessible point o f each o f the 32 study trees.
2.3.3 Canopy Access
Access into canopies was achieved through a single rope technique (Denison 1973; Perry
& Williams 1981). A pair of ascenders with stirrups were used to climb the tree, and a lanyard
system was used to permit movement within the tree canopy. Upon reaching the high point in a
tree, a measuring tape was dropped to the ground to measure its height. Ziploc bags were used to
collect and label lichen samples by height in the canopy. The climber descended the tree using a
37

Petzl stop descender (ZI Crolles, Cidex 105 A, Crolles, France), which allowed the hands to be
free for biomass assessment and sample collection. Selected trees were rigged, climbed and
assessed vertically for epiphytic lichen biomass.

2.3.4 Lichen Biomass Abundance Assessment
Biomass abundance of epiphytic lichens was assessed using the clump method
(Stevenson, 1978; Campbell et al., 1999). Lichen biomass was estimated separately for each
branch. The clump method relies on a correspondence between visual estimations of standard
lichen clumps (area-based for foliose lichens or length-based units for hair lichens) and accurate
clump to biomass conversion factors. The heights of all visual estimations of abundance of
species were recorded in situ so that vertical distributions of lichen biomasses could later be
calculated. Hair lichens were separated into Alectoria sarmentosa and Bryoria spp. by visual
estimation o f the percentage of Bryoria spp. in hair lichen clumps.
Biomass clumps were presumed to represent 2.5 g of hair lichens or L. pulmonaria and
1.5 g o f foliose chlorolichens and other cyanolichens (Stevenson, 1978). To corroborate the
validity of these previously measured clump to biomass conversion factors, we cut down and
assessed one percent of total branches that were visually assessed for lichen biomass: a total of
33 representative sample branches collected from 9 randomly selected trees. All lichen was
removed from branches, oven-dried and weighed to determine error for lichen biomass
estimations. Abundance estimations for hair lichens Alectoria sarmentosa and Bryoria spp. were
quite accurate but still overestimated by an average of 0.45 ±0.80 g and 0.84 ±1.37 g per branch,
respectively. Foliose chlorolichens and bipartite cyanolichens were also fairly accurate but
underestimated by an average of 1.22 ±2.54 g and 1.01 ±2.25 g per branch, respectively.
Lobaria pulmonaria biomasses were most underestimated, on average by 9.86 ±30.5 g per
38

branch. These absolute levels of over- and under-estimation of biomasses were generally small,
i.e. 1-10 % o f total biomass, for all epiphytic lichens apart from L. pulmonaria, which was
occasionally underestimated by 10-25%. Because of the large uncertainties in the correction
conversion factors, combined with their small impacts overall on biomass estimates, we chose
not to adjust the biomass estimates.

2.3.5 Sampling of Lichens for Chemical Analyses
Tree height was measured with a tape from the top of each tree and each tree canopy was
divided into three canopy height zones. Upper, middle and lower canopy were designated as the
top 1/3, middle 1/3 and lowest 1/3 by height of live tree canopy. Epiphytic lichen and needle
samples were collected throughout the tree canopy and 1 sample was collected from the upper,
middle and lower canopy height zones if they were present. Bryoria species were collected
predominantly from upper canopy and Alectoria sarmentosa from lower canopy. Samples were
placed into plastic Ziploc bags and immediately transferred into paper bags at the lab for biomass
oven-drying (55°C for 3 days). Live, green needles were collected from the highest accessible
point in the canopy within 1 m of the tree trunk. The needle cohort was exclusively from the
previous year’s foliage to keep the age of the sampled needles constant. Lichen biomass was
separated into five prominent functional groups or species: Alectoria sarmentosa, Bryoria spp.,
foliose chlorolichens, bipartite cyanolichens, and L. pulmonaria. The biomass estimates for each
site were then extrapolated to a per hectare basis using previously determined canopy tree stem
densities (Campbell et al., 2010; Table 2.1). Areal biomass estimates were likely underestimated
as snags and small trees were not included in stem density or lichen abundance estimates.
Needle and lichen % N (of dry mass) was analyzed at the University o f Saskatchewan
Stable Isotope Facilities (Saskatoon, Canada). Samples were analyzed using standardized
39

protocols with a Costech ECS4010 elemental analyzer (EA) coupled to a Delta V Advantage
mass spectrometer with Continuous Flow (Conflo) IV interface (Stocki, 2010).

2.3.6 Statistical Analysis
Statistical analyses were performed using Sigma Plot 11 (Systat Software Inc., San Jose,
CA, USA). The estimated biomasses (g dry weight) of Alectoria sarmentosa, Bryoria spp.,
foliose chlorolichens, bipartite cyanolichens, and L. pulmonaria on each branch of each tree were
compared using the Kolmogorov-Smimov and Shapiro-Wilk tests to determine if the data sets
met the assumptions of normality. Differences in total biomass of each functional group among
the tree species and two study site types were compared on each study tree using a 2 -way
analysis o f variance (ANOVA) following transformations to normalize the data where needed.
Bryoria spp. data were square root-transformed while bipartite cyanolichens and L. pulmonaria
were log-transformed to attain normality prior to analysis. Bonferroni pairwise multiple
comparisons were used to determine whether biomasses differed by tree species and site.
For each lichen functional group, the tree height was determined at which peak lichen
biomass abundance and median lichen biomass occurred. These two characteristics allowed us
to assess patterns o f vertical epiphyte distribution. A two way ANOVA was used to analyze the
data using site type (High Cyano vs. Low Cyano) and tree species (fir vs. spruce) as factors.
Mean N content was determined for each functional group and a one-way ANOVA with
Tukey post hoc test was used to evaluate differences in %N across the six lichen functional
groups. One-way ANOVAs with Bonferroni post-hoc multiple comparison tests were
subsequently conducted between lichen functional groups within sites and tree species to
determine if there was a significant difference in the amount of N (average g N tree' 1 and average
kg N ha'1) in each functional group.
40

2.4 RESULTS

2.4.1 Epiphytic Lichen Abundance
Fir study trees (-25 m) were on average 5 m shorter than spruce study trees (-30 m), but
fir trees carried greater average lichen biomass on a per tree (Table 2.2) and areal (Fig. 2.2)
basis. Fir trees also contained greater lichen biomasses for all lichen functional groups than
spruce trees at three of the four study sites (High Cyano 1 & 2, and Low Cyano 1), and for
foliose chlorolichens and bipartite cyanolichens at the fourth site, Low Cyano 2. Foliose
chlorolichens at Low Cyano study sites were in greater abundance than High Cyano study sites,
while, not surprisingly, bipartite and tripartite (L. pulmonaria) cyanolichens were both in greater
abundance at the High Cyano study sites (Table 2.2). There was a noticeable presence of foliose
chlorolichens in the upper canopies of both fir and spruce study trees (Fig. 2.2 A & B) but where
L. pulmonaria was dominant in the middle to lower canopies, we observed a dramatic decline in
foliose chlorolichen in these zones.
Hair lichens were found in upper, middle and lower canopy positions at all sites. Among
the hair lichens, Alectoria sarmentosa was generally found in the mid to lower canopy and the
greatest abundance was situated between 10 and 14 m (in the middle canopy zone: Fig. 2.2).
Bryoria species were found predominantly in the upper canopy and were highly concentrated
from 16 to 20 m. The peak abundance of Bryoria spp. was higher in the canopy on spruce trees
(Fig. 2.2D) than fir trees (Fig. 2.2C). Bryoria spp. represented a significant component of the

41

arboreal lichen in the middle and upper canopy zones with a much greater presence at Low
Cyano sites than (Fig. 2.2 C, D) High Cyano sites (Fig. 2.2 A, B).
The dominant foliose chlorolichen that was observed in the lower to upper canopy was
Platismatia glauca (L.) Culb. & C.F. Culb. and its biomass decreased with increasing tree height
within the canopy. When sites had less L. pulmonaria (i.e. at both Low Cyano sites, Fig. 2.2C,
D), foliose chlorolichens tended to dominate all three height zones and especially the middle and
lower canopy height zones on fir trees (Fig. 2.2C). Foliose chlorolichen abundance was
noticeably greater on fir (Fig. 2.2C) than on spruce trees (Fig. 2.2D).
Common bipartite cyanolichens that were found throughout the canopy (particularly at
High Cyano sites) were L. hallii (Tuck.) Zahlbr., L. scrobiculata (Scop.) D.C., Nephroma
helveticum Ach., Pseudocyphellaria anomala Brodo & Ahti., and Sticta fuliginosa (Hoffin.)
Ach. The greatest abundance of bipartite cyanolichens was found in the lower canopy from 10
m to 14 m, similar to Alectoria sarmentosa abundance (Fig. 2.2). There was also a greater
abundance o f bipartite cyanolichens on fir (Fig. 2.2A) than spruce (Fig. 2.2B). Lobaria
pulmonaria was mainly found in the lower canopy of the tree with highest abundance between 6
m and 10 m. At High Cyano sites, foliose chlorolichens were found primarily in middle and
upper tree canopy positions (Fig. 2.2A & B), except when there was little presence o f L.
pulmonaria where foliose chlorolichens became the biomass dominants in both middle and lower
canopy positions (Fig. 2.2).
The results obtained from the analysis of the lichen biomass gave information about the
variation of lichen loading between tree species and by site cyanolichen abundance. The null
hypothesis that there was no difference in the amount of lichen loading between the two tree
species was accepted for all functional groups except for foliose chlorolichens which showed a
42

significant difference between spruce and fir trees (F];3i = 12.7, p = 0.001). We analyzed this
further by looking at the difference in foliose chlorolichen abundance on spruce and fir trees at
Low Cyano and High Cyano sites. Using the Bonferroni multiple comparison test we
determined that foliose chlorolichen abundance was statistically different in both High Cyano (t
= 2.24, df =31, p = 0.033) and Low Cyano (t = 2.79, df = 31, p = 0.009) sites when controlling
for tree species (Table 2.3).
We also wanted to determine if there was a difference in lichen loading between sites.
The null hypothesis that there is no difference in the amount of lichen loading between High and
Low Cyano site locations was accepted for Alectoria sarmentosa and foliose chlorolichens when
tree species was not considered as a factor. There was a significant difference for lichen
abundance between High and Low Cyano study sites for Bryoria spp. (Fij3i = 10.4, p = 0.003),
bipartite cyanolichens (Fij3j = 27.3, p < 0.001), and L. pulmonaria (Fii3i = 51.8, p < 0.001) (Table
2.3).
2.4.2 Epiphytic Lichen Distribution
The vertical height o f peak biomass abundance (i.e. modal maximum biomass, data not
shown) and the height where half of the total biomass (i.e. mean maximum biomass) was
reached were similar in all study trees for a given functional group of lichen. Measures of
vertical peak biomass in the canopy were uniformly lower (in both spruce and fir) at Low Cyano
than at High Cyano sites (Fig. 2.3). There was a statistically significant difference between High
Cyano (HI and H2) and Low Cyano (LI and L2) sites for peak biomass height for A, sarmentosa
(Fi>3i = 10.6, p = 0.003), Bryoria spp. (Fi)3i = 9.80, p = 0.004), and L. pulmonaria (Fi,2 7 = 6.50, p
= 0.018) functional groups (Table 2.4). There was also a statistically significant difference
between High Cyano and Low Cyano abundance sites for tree height where 50 % of total
43

biomass was reached for A sarmentosa ( F i, 3i = 15.6, p < 0.001), Bryoria spp. (F p i = 7.86, p =
0.009), bipartite cyanolichens (F 1 3 0 = 4.86, p = 0.036) and L. pulmonaria within fir trees (Fi, 2 7 =
18.2, p < 0.001). In Low Cyano abundance sites, L. pulmonaria abundance was significantly
different between spruce and fir trees (Table 2.4).
Cyanolichen abundance (bipartite and the tripartite cyanolichen, L, pulmonaria) varied
greatly across study trees (Fig. 2.4). Matching our High and Low Cyano site visual selection
criteria, Low Cyano sites had relatively low cyanolichen abundances (ranging from 0 to 258 g
tree'1) while High Cyano sites, while having a much larger range of cyanolichen abundances
(from 134 to 1313 g tree'1), were also on average higher than at Low Cyano sites.
2.4.3 Functional Group Nitrogen Content
There was a higher amount of aggregate epiphytic lichen N in fir versus spruce trees, and
higher amounts of epiphytic lichen N in fir trees on High versus Low Cyano sites (Fig. 2.5,
Insert). A statistically significant difference in the amount of N coming from Bryoria spp. per
tree was found between High and Low Cyano sites (F3,4o = 71.1, p < 0.001) but not between tree
species. A greater amount of N came from Bryoria spp. in Low versus High Cyano site trees.
Alectoria sarmentosa represented the lowest amount of cumulative N per tree across the five
lichen functional groups. There was a statistically significant difference in the amount of N from
A. sarmentosa between spruce and fir trees at High Cyano sites and spruce trees at Low Cyano
sites (F3 j4 9 = 48.7, p < 0.001). A greater amount of N was contained in foliose chlorolichens on
fir than on spruce trees and there was a statistically significant difference in the amount of N in
foliose chlorolichens between site and tree species (F3 ,6 i = 190, p < 0.001). Bipartite
cyanolichens provided a greater amount of N per tree in High versus Low Cyano sites and there
was a significant difference between all sites and tree species (F 3 >9 g= 2470, p < 0.001). The
44

amount of N per tree in L. pulmonaria was significantly different between High and Low Cyano
sites and between tree species in the High Cyano sites (F3 >5 6 = 987, p < 0.001), but there was no
difference in the amount of N in L. pulmonaria between tree species in the Low Cyano sites
(Table 2.5).
On an areal basis, aggregate N from all lichen functional groups was three times higher at
High Cyano sites (8.19 ± 1.52 kg N ha'1) than at Low Cyano sites (2.68 ± 0.97 kg N ha'1) (Fig.
2.6A, Insert). Among the functional groups, the hair lichen A. sarmentosa made the smallest N
contribution at each site, and Bryoria spp. slightly greater amounts. Foliose chlorolichens
contained a similar amount of N in High and Low Cyano sites; 1.50 ± 0.09 kg N ha ' 1 versus 1.67
± 0.10 kg N ha'1, respectively. Bipartite cyanolichens contributed 6 -fold more N in High versus
Low Cyano sites; 2.33 ± 0.11 kg N ha ' 1 versus 0.39 ± 0.03 kg N ha"1, respectively.
Approximately 50 % o f the total N at High Cyano sites (4.04 ± 0.12 kg N ha'1) was contained in
one species, the tripartite cyanolichen L. pulmonaria. By contrast, only 3% of total N at Low
Cyano sites was contributed by L. pulmonaria.
In addition to site level and tree species effects on area-based N already mentioned,
Bryoria spp. ^ 7 ,4 0 = 34.2, p < 0.001), Alectoria sarmentosa (F 7 ,4 9 = 62.7, p < 0.001), foliose
chlorolichens (F 7>78 = 194, p < 0.001), bipartite cyanolichens (F 7 ,n 4 = 3095, p < 0.001), and L.
pulmonaria (F7 i62 = 710, p < 0.001) contained different aggregate N within all site and tree
species combinations (Fig. 2.6B). Combining N from all functional groups, fir trees from High
Cyano site 2 had the greatest amount of N (7.56 ± 1.58 kg N ha'1); and fir tree epiphytic lichens
from all sites generally contained more N than spruce trees.

45

2.5 DISCUSSION
Epiphytic lichen species are the primary competitors for branch or stem surfaces within
the canopies of sub-boreal forest canopies of the BC interior (Arseneau et al., 1998) where
bryophytes are typically absent or minor components of canopy epiphyte assemblages (Botting et
al., 2008). Earlier studies of epiphytic lichen species and functional groups in conifer forest
canopies have previously documented the unique vertical zonations of lichen species or
functional groups (Campbell & Coxson, 2001; McCune, 1993). Farber et al. (2014) found that
the vertical gradient of pendulous lichens is consistent with a shift in the type and function of
sun-screening pigments. Bryoria 'hair' lichens, for example, are in highest abundance in drier
upper canopy positions (Goward, 2003), while cyanolichens, with greater thallus moisture
requirements, occupy lower canopy positions (e.g. Campbell & Fredeen, 2007). Our results from
this study corroborate these general patterns of vertical distribution of epiphytic macrolichens on
both interior hybrid spruce and sub-alpine fir trees in sub-boreal conifer forests of central BC
(see Fig 2.2A, B). Since the patterns of lichen functional group biomasses are more similar to
the lower maximum heights achieved by subalpine fir than spruce trees, it may be that distance
from canopy height, not distance from ground level, is the more critical variable (i.e. Fig 2.2A
versus 2.2B). Nevertheless, the higher biomasses in fir versus spruce are consistent with
previous studies (Campbell et al., 2007), but a mechanism for this greater biomass is currently
lacking.
O f greater interest to this study was the constraining of chlorolichens to mid to highcanopy positions when cyanolichens occupied the lower canopy (i.e. in High Cyano sites). High
and Low Cyano sites were clustered (-5-10 km between sites in both cases) and no two sites
were more than 2 0 km apart, with sites differing little in climate, landscape position, host tree
46

species, composition, or density (Campbell et al., 2010a). Although the underlying reasons for
variation in cyanolichen abundance at High and Low Cyano sites were unknown, previous
studies provide some general rules. Larger (Ockinger et al., 2005) and old-growth (e.g.
Campbell & Fredeen, 2004) conifer trees and conifer under Populus spp. (Campbell et al.,
2010a; Goward & Arsenault, 2000) have a greater likelihood of harbouring diverse and abundant
arboreal cyanolichen communities. Since all High and Low Cyano sites in this study both
contained host conifer trees >140 years old and Populus spp., these wouldn't appear to explain
the variability in cyanolichen abundances in this study. Limitations in cyanolichen dispersal
capacity (Ockinger et al., 2005; Sillett & McCune, 1998) and 'site attributes' (e.g. edaphic
characteristics: Campell & Fredeen, 2007) remain possible explanations. Bark pH is another
possible factor since under similar climatic conditions, relatively high bark pH is necessary for
the development of cyanolichens on conifers (Gauslaa & Goward, 2012; Gauslaa & Holien,
1998). Ultimately, the challenge of deciphering the mechanism(s) explaining variability in
cyanolichen diversity and pattern across sub-boreal landscapes is on-going and in many ways
related to Hutchinson's 'paradox of diversity' (1957). McCune et al. (1997) have suggested that
cyanolichens are constrained to the ‘light transition zones’, but there were no significant
differences in lower canopy light levels between Low and High Cyano sites in this study. Thus,
High and Low Cyano sites afforded an excellent opportunity to examine the consequences of
naturally occurring (non-manipulative) variation in cyanolichen abundances, which varied by
over an order o f magnitude from 30 to 8 6 in Low Cyano sites to 443 to 979 (High Cyano sites)
kg tree' 1 (Table 2.2).
The greatly reduced abundances of bipartite and tripartite (L. pulmonaria) cyanolichens
at Low Cyano sites (Table 2.3) resulted in a near complete take-over of all canopy zones by
47

chlorolichens (Fig. 2.2). Foliose chlorolichens dominated much of the upper and middle canopy
zones regardless of cyanolichen abundance, but were markedly increased in lower canopy
positions in the relative absence of cyanolichens at Low Cyano sites (Fig. 2.2). Chlorolichens
extended their realized niches into lower canopy height zones in the absence o f cyanolichens,
and most notably, the absence of the cyanolichen biomass dominant, L. pulmonaria. This was
evident by the increases in Bryoria spp. biomass in the upper and mid-canopy zones at Low
Cyano sites of spruce and fir trees (Fig. 2.2C, D; Table 2.4). Overall, these results suggest that
chlorolichens can greatly extend (downward) their realized vertical niches in the absence of
cyanolichen competition in sub-boreal conifer stands. The normal preference of L. pulmonaria
for lower canopy branches o f spruce appears to be a general trait (Goward & Arsenault, 2000;
Hilmo et al., 2013). However, its presence or relative absence on the distribution of other lichens
has, to our knowledge, not previously been documented. Further manipulative experiments such
as cyanolichen removals would be helpful in further supporting the phenomenon and uncovering
possible mechanisms. We chose not to make these manipulations in this study largely over
concerns of extirpating rare bipartite lichens found at High Cyano sites.
Productivity in many conifer forest ecosystems is limited by N availability, and
particularly those in northern BC where atmospheric N inputs are relatively low (see Campbell
and Fredeen, 2007), potentially making available N from lichen leaching and decomposition
important to forest growth and/or health (e.g. Holub & Lajtha, 2003; Knowles, 2004; Caldiz et
al., 2007). In our study, the average aggregate N contained in all epiphytic lichen species at
High Cyano sites was 22.8 ± 2.0 to g N tre e 1, more than double that at Low Cyano abundance
sites: 9.4 ± 1.3 g N tree' 1 (Fig. 2.5, Insert). This range of N pool sizes brackets epiphytic lichen
N amounts estimated for another sub-boreal spruce and fir forest in central BC (Botting et al.,
48

2008). Higher cyanolichen abundances resulted in greater canopy lichen N per tree (Fig. 2.5,
Insert), largely as a result of the higher N content of bipartite and tripartite cyanolichens.
However, foliose chlorolichens represented most of the epiphytic lichen biomass (Table 2.2) and
N (Fig. 2.5) at Low Cyano abundance sites. Nevertheless, total forest lichen N was greater when
cyanolichens were abundant (i.e. at High Cyano sites) at the tree and stand (area) levels. On
average, total lichen epiphyte N totaled 8.2 ± 0.3 kg N ha ' 1 in High Cyano abundance sites
compared to only 2.6 ± 0.2 kg N ha ' 1 in Low Cyano abundance sites (Fig. 2.6, Insert).
Translation of lichen epiphyte N into N available for uptake by trees, either through
leaching of lichens from throughfall or through decomposition of fallen lichen litter, was recently
assessed at the High and Low Cyano sites (Campbell et al., 2010b). They showed that estimated
N inputs through decomposition resulted primarily from cyanolichen litter (including all bipartite
cyanolichen species plus the tripartite lichen L. pulmonaria) and were 2.64 and 0.09 kg N ha~'
y r'a t High and Low Cyano sites, respectively. These rates of decomposition represent 31% of
the cyanolichen-rich lichen epiphyte N at High Cyano sites, compared to only 3% of the
chlorolichen-rich lichen epiphyte N at Low Cyano sites, favoured by low C:N ratios (high N
content) o f cyanolichen thallus. Leaching of N from epiphytes has yet to be quantified at our
sites, but would be expected to be greater from cyanolichen-rich sites than chlorolichen-rich
sites. Supporting this notion, previous work at our sites (Campbell et al., 2010b) found that
available soil N (NO 3 ' + N H /) was 3.3-fold higher and NO 3 ' levels >60-fold higher at High than
at Low Cyano sites. Older or coastal forests ecosystems have been shown to have even greater N
inputs from epiphytic cyanolichens. For example, Denison (1979) estimated the annual N inputs
in mature conifer forests of the Pacific Northwest to be 3-4 kg N ha^yr'1. Retention efficiency
of the canopy is higher when more inorganic N is available. Lichens from the upper canopy
49

have also been shown to release minerals less readily than lichens from the lower canopy (Pike,
1978), perhaps in keeping with the low N contents of chlorolichens found in the upper canopy
zone relative to high N contents o f cyanolichen in lower canopy zones.
Tree (host) species effects on qualitative and quantitative epiphytic lichen loadings have
received relatively little examination (e.g. Botting et al., 2008). In a previous study in the same
region and general forest type in central BC, epiphyte loadings were greater in abundance on
subalpine fir than on interior hybrid spruce (Campbell & Fredeen, 2007), despite the fact that
spruce is normally taller than fir in mature forests in our region. We observed the same effects
o f host species on epiphyte abundance in the present study, with an almost doubling of epiphytic
lichens on subalpine fir (317.5 g tree'1) as opposed to the canopy dominant interior hybrid spruce
(183.5 g tree'1) (Fig. 2.2).
Lichen distribution at Low Cyano sites is lower overall than at High Cyano sites but the
heights associated with peak biomass and median biomass for individual trees were similar for
all functional groups. Peak biomass at the High Cyano sites often occurred approximately 3-5 m
higher in the tree canopy than at the Low Cyano abundance sites. This increase in height could
be a result of species competition for optimal growth conditions. There are microclimatic
gradients in moisture, temperature, light, and nutrient availability which could all have
influenced the growth o f a certain type of lichen functional groups at certain heights ( Renhom et
al., 1997; Campbell & Coxson, 2001; Gaio-Oliveira et al., 2004). Resources such as light, water,
and nutrients can be altered in the forest canopy by lichens. It is important to determine how
epiphytic cyanolichens interact with these resources to better understand the mechanisms that
contribute to the dynamics of canopy diversity. Gauslaa et al. (2006) looked at the natural
occurrence of L. pulmonaria and reported that biomass growth is controlled by a balance
50

between light availability and desiccation risk, and that the species is limited to old-growth
forests because of a physiological trade-off between growth potential and fatal desiccation
damage. Others noted that the differential interception of precipitation within the canopy can
affect lichen distribution (Coxson & Stevenson, 2007) when lichen thalli are exposed to forest
edges rather than thalli under the established forest canopy. Nutrient availability is also an
important factor since lichens are dependent on the deposition of nutrients, including N, directly
on the thallus (Nash, 1996), with the exception being N 2 -fixing cyanobacterial lichens (Rai,
1988).
Lichen growth strongly depends on the irradiance level during hydration periods
(Palmqvist, 2000; Dahlman & Palmqvist, 2003) and hydration during dark periods strongly
boosts lichen growth (L. pulmonaria and L. scrobiculata) when photosynthesis is high during
daytime (Bidussi et al., 2013). A previous study found relationships between lichen growth and
site factors reflecting the openness o f the canopy (Gauslaa et al., 2008). In that study, main
branches were cut down from two height intervals on each tree to quantify epiphytic lichen
biomass. They found that total lichen biomass was significantly higher on the lower branches
(46.2 g branch'1) compared to the branches at 5-6 m (36.6 g branch'1). The alectorioid/foliose
biomass ratio increased significantly from 0.149 at 2-3 m to 0.316 at 5-6 m, suggesting that light
can determine the balance between these two growth forms. Our observations showed a similar
Alectoria to Bryoria ratio in our sub-boreal spruce and fir canopies.
Forest stands that grow on similar sites with comparable soil parent material, topographic
position, and climate have large differences in species composition attributable to a range of
factors including historical events such as fires, droughts, or weather (Pipp et al., 2001). The
diversity and abundance of lichen biomass could be an indicator o f how long a forest has
51

developed without a major disturbance or it could give insight to a forest’s true age. In the
present study, fir trees had much higher Alectoria lichen loading than spruce trees. These trees
were o f similar size but it is unclear whether the variation in lichen abundance was a product of
age or structural characteristics that change with age.
Compounding the methodological issues that limited the validity of the mass-estimationcorrection models, the initial estimation procedure itself may have been flawed. Using the
‘clump method’, hair lichen biomasses were slightly overestimated while foliose chlorolichens
and bipartite cyanolichens biomasses were underestimated. Lobaria pulmonaria biomass was
underestimated by the largest extent; thus our estimates of canopy N contributed by L.
pulmonaria were likely considerably lower than actual amounts. One possible reason for the
greater underestimation in L. pulmonaria was that estimation accuracy appeared to decrease with
lichen abundance and thallus size. While the clump method gives a relatively accurate
estimation of the amount of lichen biomass in the canopy with minimal disturbance o f lichen
epiphytes, its accuracy and/or precision can vary with researcher, branch length and density. In
this study, lichen abundance on tree trunks was not estimated. However, it was determined
previously that more than 95% of the biomass of fruticose lichens is generally found on tree
branches with the remainder (4-5%) on trunks (Arseneau et al., 1998).
Not all factors were taken into consideration when epiphytic lichen biomass estimates
were recorded. There are microclimatic gradients in moisture, temperature, light, and nutrient
availability, all which could be influencing the growth of certain types of lichen functional
groups at a certain height (Gaio-Oliveira et al., 2004; Gauslaa et al., 2007). Branch length
measurements could have been taken, as branch size is an important predictor for epiphytic
lichen biomass (Gauslaa et al., 2008). Finally, forest structure, age, site conditions, weather
52

patterns, disturbance regimes, and ground species composition may also be determining factors
for epiphytic lichen biomass presence and abundance (Pipp et al., 2001). All considered, we are
still far from understanding the complex ecological conditions that ‘cause’ presence and
abundance of different epiphytic lichen species and communities in forest canopies. Long-term
manipulative studies where cyanolichen or chlorolichen abundance are experimentally reduced
could be one fruitful avenue to examine these relationships.

2.6 LITERATURE CITED

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Bidussi, M., Gauslaa, Y. & Solhaug, K. A. (2013). Prolonging the hydration and active
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Campbell, J., Bradfield, G. E., Prescott, C. E., & Fredeen, A. L. (2010a). The influence of
overstorey Populus on epiphytic lichens in subboreal spruce forests of British Columbia.
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Campbell, J., & Coxson, D. S. (2001). Canopy microclimate and arboreal lichen loading in
subalpine spruce-fir forest. Canadian Journal o f Botany, 79, 537-555.
Campbell, J., & Fredeen, A. L. (2004). Lobaria pulmonaria abundance as an indicator of
macrolichen diversity in Interior Cedar hemlock forests of East-Central British Columbia.
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Campbell, J., & Fredeen, A. L. (2007). Contrasting the abundance, nitrogen, and carbon of
epiphytic macrolichen species between host trees and soil types in a sub-boreal forest.
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from four epiphytic lichen litters in sub-boreal spruce forests. Canadian Journal o f Forest
Research, 40(7), 1473-1484.
Campbell, J., Stevenson, S. & Coxson, D.S. (1999). Estimating epiphyte abundance in high
elevation forest of northern British Columbia. Selbyana, 20, 261-267.
Coxson, D. S., & Curteanu, M. (2002). Decomposition of hair lichens (Alectoria sarmentosa and
Bryoria spp.) under snowpack in montane forest, Cariboo Mountains, British Columbia.
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edges on growth rates of Lobaria pulmonaria in the inland rainforest, British Columbia.
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Coxson, D. S., Stevenson, S. K., & Campbell, J. (2003). Short-term impacts of partial cutting on
lichen retention and canopy microclimate in an Engelmann spruce - subalpine fir forest in
north- central British Columbia. Canadian Journal o f Forest Research, 33, 830-841.
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arcticum and Peltigera aphthosa: empirical modelling. Functional Ecology, 821-831.
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Demmig-Adams, B., Maguas, C., Adams III, W. W., Meyer, A., Kilian, E., & Lange, O. L.
(1990). Effect of high light on the efficiency of photochemical energy conversion in a
variety of lichen species with green and blue-green phycobionts. Planta, 180, 400-409.
Denison, W. C. (1973). Life in tall trees. Scientific American, 228(6), 74—80.
Denison, W. C. (1979). Lobaria oregana, a nitrogen-fixing lichen in old-growth Douglas-fir
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in the Management o f Temperate Forests (pp. 266-275). Corvallis, Oregon: Oregon State
University Press.
Esseen, P.A. & Renhom, K.E. (1998). Mass loss of epiphytic lichen litter in a boreal forest.
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Farber, L., Solhaug, K. A., Bilger, W., &. Gauslaa, Y. (2014). Sun-screening fungal pigments
influence the vertical gradient of pendulous lichens in boreal forest canopies. Ecology, 95,
1464-1471.
Gaio-Oliveira, G., Dahlman, L., Maguas, C., & Palmqvist, K. (2004). Growth in relation to
microclimatic conditions and physiological characteristics of four Lobaria pulmonaria
populations in two contrasting habitats. Ecography, 27, 13-28.
Gauslaa, Y. (1985). The ecology of Lobarion pulmonaria and Parmelion caperatae in Quercus
dominated forests in south-west Norway. Lichenologist, 17, 117-140.
Gauslaa, Y. & Holien, H. (1998). Acidity of boreal Picea abies-canopy lichens and their
substratum, modified by local soils and airborne acidic depositions. Flora, 193,249-257.
Gauslaa, Y., Lie, M., Asbjom Solhaug, K., & Ohlson, M. (2006). Growth and ecophysiological
acclimation of the foliose lichen Lobaria pulmonaria in forests with contrasting light
climates. Oecologia, 147, 406-416.
Gauslaa, Y., Lie, M., & Ohlson, M. (2008). Epiphytic lichen biomass in a boreal Norway spruce
forest. Lichenologist, The, 40(3), 257-266.
Gauslaa, Y., Palmqvist, K., Solhaug, K. A., Holien, H., Hilmo, O., Nybakken, L., Myhre, L.C.,
& Ohlson, M. (2007). Growth of epiphytic old forest lichens across climatic and
successional gradients. Can. J. For. Res, 37, 1832-1845.
Gauslaa, Y., Coxson D. S., & Solhaug, K. A. (2012). The paradox of higher light tolerance
during desiccation in rare old forest cyanolichens than in more widespread co-occurring
chloro- and cephalolichens. New Phytologist, 195, 812-822.
Gauslaa, Y., & Goward, T. (2012). Relative growth rates of two epiphytic lichens, Lobaria
pulmonaria and Hypogymnia occidentalis, transplanted within and outside of Populus
dripzones. Botany, 90, 954-965.
Goward, T. (2003). On the dispersal of hair lichens (Bryoria) in high-elevation oldgrowth conifer
forests. Canadian Field-Naturalist, 117(1), 44—48.
Goward, T., & Arsenault, A. (2000). Cyanolichen distribution in young unmanaged forests: A
dripzone affect. The Bryologist, 103( 1), 28-37.

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Hilmo, O., Gauslaa, Y., Rocha, L., Lindmo, S., & Holien, H. (2013). Vertical gradients in
population characteristics of canopy lichens in boreal rainforests of Norway. Botany,
07(12), 814-821.
Holub, S. M., & Lajtha, K. (2003). Mass loss and nitrogen dynamics during the decomposition of
a 15 N-labeled N2 -fixing epiphytic lichen, Lobaria oregana. Canadian Journal o f Botany, 81,
698-705.
Hutchinson, G. E. (1957). Concluding Remarks (PDF). Cold Spring Harbor Symposia on
Quantitative Biology. Retrieved 24 July 2007 from
http://artifex.org/~ecoreaders/lit/Hutchinsonl957.pdf
Kivitso, L., & Kuusinen, M. (2000). Edge effects on the epiphytic lichen flora of Picea abies in
middle boreal Finland. Lichenologist, 32, 387-398.
Knowles, R. D. (2004). Peltigera, a genus of dinitrogen-fixing, terricolous lichens: Its influence
on soil processes in the northern forests of Minnesota. Doctoral dissertation. University of
Minnesota, Minneapolis, Minn., USA.
Lange, O. L., Green, T. G. A., & Heber, U. (2001). Hydration-dependent photosynthetic
production of lichens: what do laboratory studies tell us about field performance? Journal o f
Experimental Botany, 52(363), 2033-2042.
McCune, B. (1993). Gradients in Epiphyte Biomass in Three Pseudotsuga- Tsuga Forests of
Different Ages in Western Oregon and Washington. The Bryologist, 96(3), 405-411.
Meidinger, D., & Pojar, J. (1991). Ecosystems o f British Columbia. British Columbia Ministry o f
Forests Special Report Series 6. Research Branch, Ministry o f Forests, Victoria, British Co.
Millbank, J. W. (1982). The assessment of nitrogen fixation and throughput by lichens. III.
Losses o f nitrogenous compounds by Peltigera membranacea, P. polydactyla and Lobaria
pulmonaria in simulated rainfall episodes. New Phytologist, 92, 229-234.
Nash, T. (1996). Nitrogen, its metabolism and potential contribution to ecosystems. In T. H.
Nash (Ed.), Lichen biology (pp. 121-135). Cambridge, United Kingdom: Cambridge
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Lobaria pulmonaria limited by dispersal capacity or habitat quality. Biodivers. Conserv.,
14, 759-773.
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148, 11-36.

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Perry, D., & Wiliams, J. (1981). The tropical rain forest canopy: a method providing total access.
Biotropica, 75(4), 283-285.
Pike, L. H. (1978). The Importance of Epiphytic Lichens in Mineral Cycling. Bryologist, The,
81(2), 247-257.
Pipp, A. K., Henderson, C., & Callaway, R. M. (2001). Effects of forest age and forest structure
on epiphytic lichen biomass and diversity in a Douglas-fir forest. Northwest Science, 75,
12-24.
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biology (pp. 225-247). Cambridge: Cambridge University Press.
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(pp. 201-237). Boca Raton, FL, USA: CRS Press.
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D. H. Brown, D. L. Hawksworth, & R. H. Bailey (Eds.), Lichenology: Progress and
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Sillett, S. C., & McCune, B. (1998). Survival and growth of cyanolichen transplants in Douglasfir forest canopies. American Bryological and Lichenological Society, 101(1), 20-31.
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o f Saskatchewan, Saskatoon, SK.

57

2.7 TABLES

Table 2.1 Latitude, longitude, and tree density with stems >25 cm dbh for four sub-boreal
conifer forest study sites in central British Columbia. Data on tree stem densities (stems ha"1)
were taken from Campbell et al., (2010b).
Study

Fir

Spruce

Latitude

Longitude

High Cyano 1 North Fraser

54° 08’ 48.8” N

121° 49’ 25.8” W

137.5

187.5

High Cyano 2

54° 06’ 14.6” N

121° 49’ 24.4” W

250

112.5

Low Cyano 1 Herrick Road 54° 07’ 09.9” N

121° 40’ 50.7” W

150

187

Herrick Road 54° 06’ 34.9” N

121° 40’ 46.2” W

112.5

150

Site

Low Cyano 2

Location

North Fraser

58

(stems ha"1) (stems ha"1)

Table 2.2 Mean (± SD; n = 4) tree diameter base height (cm), tree height (m), tree age (years) and epiphytic lichen biomass at four
study sites by functional group abundance (average g dry weight tree ' 1 and average kg dry weight h a'1) on mature subalpine fir and
interior hybrid spruce trees in central British Columbia.
Site

Tree
Species

DBH
(cm)

Tree
Height
(m)

Tree
Age
(yrs)

Alectoria
sarmentosa

Bryoria
spp.

Foliose
chlorolichen

Bipartite
cyanolichen

Lobaria
pulmonaria

(g tre e 1)

(kg ha'1)

(g tre e ')

(kg h a ')

(g tre e 1)

(kg h a 1)

(g tree'1)

(kg ha'1)

(g tree'1)

(kg ha'1)

Fir

50.8
± 13.8

26.9
± 3 .9

103.0
± 3 .6

143.6
± 125.9

19.7
± 17.3

172.7
± 139.1

23.7
± 19.1

869.7
± 501.1

119.6
±68.9

276.0
± 135.4

38.0
± 18.6

587.6
± 115.5

80.8
± 15.9

Spruce

57.7
± 13.2

30.8
± 5.6

110.3
± 7.2

81.3
±42.1

15.2
± 7.9

123.6
± 106.4

23.2
± 20.0

582.6
± 408.8

109.2
± 76.7

70.5
± 59.4

13.2
±11.1

372.2
± 473.0

69.8
± 88.7

Fir

49.1
±6.1

26.3
± 1.0

102.0
± 14.1

47.9
± 15.3

12.0
± 3.8

50.2
±29.2

12.5
± 7 .3

605.8
± 151.2

151.5
±37.8

373.3
± 83.1

93.3
± 20.8

606.1
±251.8

151.5
± 62.9

Spruce

40.7
± 3 .3

27.5
± 2 .6

77.3
± 14.2

12.6
± 5 .3

1.4
± 0 .6

13.6
± 6.3

1.5
± 0 .7

156.2
±92.7

17.6
± 10.4

113.5
± 81.6

12.8
± 9 .2

414.5
± 169.0

46.6
± 19.0

Fir

50.3
± 5 .6

23.6
± 1.6

134.8
± 87.9

53.5
± 2 0 .7

8.0
±3.1

182.1
± 135.5

27.3
±20.3

1114.3
± 392.7

167.1
± 58.9

69.6
± 96.8

10.4
± 14.5

16.2
± 2 0 .2

2.4
± 3 .0

Spruce

44.5
±22.4

26.8
±5.1

159.0
± 1.4

48.4
± 2 3 .2

9.0
± 4 .3

179.0
± 155.5

33.5
±29.1

517.7
± 389.8

96.8
±72.9

42.6
± 38.6

8.0
± 7 .2

11.4
± 12.9

2.1
± 2 .4

Fir

54.3
± 7 .8

27.3
± 2 .7

119.7
± 32.7

38.6
± 15.3

4.3
± 1.7

212.9
±96.8

24.0
± 10.9

898.5
± 129.3

101.1
± 14.5

15.9
± 13.7

1.8
± 1.5

15.3
± 2 3 .2

1.7
± 2 .6

Spruce

59.6
± 15.3

30.1
± 6 .6

163.0
±76.9

74.1
±61.3

11.1
± 9 .2

248.0
± 151.6

37.2
± 22.7

577.9
± 180.1

86.7
± 2 7 .0

11.7
± 12.6

1.8
± 1.9

17.8
± 19.2

2.7
± 2 .9

High
Cyano 1

High
Cyano 2

Low
Cyano 1

Low
Cyano 2

59

Table 2.3 Two-way analysis of variance for the amount of lichen loading (total lichen grams
tree '*) between spruce (S) and fir (F) and site type (High Cyano versus Low Cyano). Asterisks
represent significant difference *p <_0.05, **p <_0.01, ***p <_0 .0 0 1 .
Lichen Functional Group Source
Site (H vs. L)
Alectoria sarmentosa
Tree (S vs. F)
Site*Tree
Residual
Total

Bryoria spp.

Site (H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total

Foliose chlorolichens

Site (H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total

Bipartite cyanolichens

Site (H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total

Lobaria pulmonaria

Site (H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total

Sum o f Squares

df

2511.17
2255.66
8175.71
98119.24
111061.78
252.92
10.60
13.36
681.71
958.58
900260.63
1367754.63
118830.71
2415234.25
4802080.22
7.18
0.65
0.89
7.11
15.92
1842450.08
83793.95
81949.23
995831.01
3004024.27

2511.17
1
2255.66
1
8175.71
28 3504.26
31 3582.64
1
252.92
1
10.60
1
13.36
28 24.35
31 30.92
1
900260.63
1
1367754.63
1
118830.71
28 100634.76
31 154905.81
1
7.18
1
0.65
1
0.89
27 0.26
30 0.53
1
1842450.08
1
83793.95
1
81949.23
28
31

60

1

Mean Square

F

0.717
0.644
2.333

10.388**
0.435
0.549

2.982
1 2 .6 8 8 ***
0.394

27.278***
2.479
3.391

51.805***
2.356
2.304

Table 2.4 Two-way analysis of variance for study tree height at peak biomass and 50% of total
biomass by lichen functional group between site type: High Cyano (H) versus Low Cyano (L)
and tree species: spruce (S) versus fir (F). Asterisks represent significant difference *p < 0 .0 5 ,
**p<_0 .0 1 , ***p< 0 -0 0 1 .
Lichen Functional
Group

Source

Sum of
Squares

Site(H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total
Site (H vs. L)
Tree (S vs. F)
Site’ Tree
Residual
Total
Site(H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total
Site (H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total
Site(H vs. L)
Tree (S vs. F)
Site*Tree
Residual
Total

86.46
0.02
9.25
227.75
323.48
130.82
24.33
26.83
373.66
555.63
47.05
5.95
0.85
503.14
556.98
44.99
9.98
16.83
411.46
483.86
25.25
5.18
3.18
93.15
125.75

df

Mean
Square

F

Sum of
Squares

Peak Biomass Height
Alectoria
sarmentosa

Bryoria spp.

Foliose
chlorolichens

Bipartite
cyanolichens

Lobaria pulmonaria

1
1
1
28
31
1
1
1
28
31
1
1
1
28
31

1
1
1
27
30

1
1
1
24
27

86.46
0.02
9.25
8.13
10.44
130.82
24.33
26.83
373.66
555.63
47.05
5.95
0.85
17.97
17.97
44.99
9.98
16.83
15.24
16.13
25.25
5.18
3.18
3.88
4.66

61

df

Mean
Square

F

50% Total Biomass Height
10.63**
0.002
1.137

9.803**
1.823
2.010

2.618
0.331
0.047

2.952
0.655
1.104

6.504*
1.333
0.820

80.01
1.28
13.01
143.98
238.28
88.11
15.26
7.13
313.81
424.31
30.03
12.50
3.51
268.34
314.38
62.98
1.36

1
1
1
28
31
1
I
1
28
31
1
1
1
28
31

0.11

1
27
30

350.19
415.25
32.07
8.61
9.81
42.40
90.59

1
1

1
1
1
24
27

80.01
1.28
13.01
5.14
7.69
88.11
15.26
7.13
11.21
13.69
30.03
12.50
3.51
9.58
10.14
62.98
1.36

0.11
12.97
13.84
32.07
8.61
9.81
1.77
3.36

15.56***
0.249
2.529

7.862**
1.362
0.636

3.134
1.304
0.366

4.856*
0.105
0.008

18.152***
4.876**
5.551**

Table 2.5 One-way analysis o f variance of functional group nitrogen content across both tree
types between High Cyano versus Low Cyano sites. Asterisks represent significant difference *p
<0.05, **p^0.01, ***p <0.001.
Source

Sum of

df

Mean

F

Square

Squares
Avg. g N tree
A. sarmentosa

0.712

49

0.237

48.7***

Bryoria spp.

9.206

40

3.069

71 j***

Foliose chlorolichens

209.202

61

69.734

190***

Bipartite cyanolichens

934.065

98

311.355

2470***

L. pulmonaria

1124.708

56

374.903

987***

A. sarmentosa

0.0466

49

0.00665

62.7***

Bryoria spp.

0.340

40

0.0485

3 4

Foliose chlorolichens

9.457

78

1.351

194***

Bipartite cyanolichens

60.025

114 8.575

3095***

L. pulmonaria

84.013

62

710***

Avg. kg N ha ' 1

62

1 2 .0 0 2

2

***

2.8 FIGURES
Figure 2.1 Map of study area including four sampling sites (High Cyano 1 and 2, Low Cyano 1
and 2) to the east of Prince George (Large inset) near Upper Fraser, British Columbia, Canada
(Small inset).

High 1
Study Area
V

Low1
Low 2

-Upper Fraser

High 2

GEORGE

5km

63

Figure 2.2 Vertical gradients in mean lichen functional group abundances (g dry weight m ' 1 tree
height) on mature subalpine fir and interior hybrid spruce trees versus tree height (meters) in
High Cyano and Low Cyano lichen abundance sites in sub-boreal conifer forests in central
British Columbia.
Fir

Spruce

35
AJectona sarmentosa
flryona spp
Foliose chioroiichen
a p a rtte cyano fcchens
Lobaria pulmonaria

30
25

20

High
abundance
sites

X.

15

10
5

Tree
height o
(m)
30
25

20

Low
abundance
sites

15

10
5

0
0

20

40

60

80

100

0

20

40

60

Mean lichen biomass (g per m height interval)

64

80

100

Figure 2.3 Median height (± SD) where half of the total epiphytic lichen biomass is reached for
each functional group on mature fir and spruce trees at High Cyano (large symbols) and Low
Cyano (small symbols) sites in sub-boreal conifer forests of central British Columbia.

25

Spruce
20

15

Tree
height

I

i

i

(m)
10

5

0

f

&

Lichen Functional Group

65

Figure 2.4 Total cyanolichen (bipartite + tripartite cyanolichen) abundance (g dry weight tree'1)
on Fir (F) and Spruce (S) study trees arranged from lowest to highest cyanolichen abundance at
High (H) and Low (L) Cyano sites. Mean cyanolichen biomasses tree ' 1 ± SD are shown for fir
and spruce trees at all sites.

1400

1200
1000

1200

800

Cyanolichen

1000

600

abundance
.
(Avg g tr e e ± S D )

C yanolichen
a b u n d an ce 800
(g t r e e '1)

400

600

200
Low Spruce

Low Fir

High Spruce High Fir

Site and tree species

400

200

Site & tree

66

Figure 2.5 Mean epiphytic lichen nitrogen (N: g of N tree*1 ±SD) across High and Low Cyano
sites for fir and spruce study trees in central British Columbia. Different letters indicate
significantly different mean values for each functional lichen group across the sites (Bonferroni
multiple comparison tests: p < 0.05). Aggregate tree-level epiphytic lichen N is shown for study
trees at both High and Low Cyano sites (Figure 2.5 Inset).

16 Bryoria spp.
I Alectoria sarmentosa
14 Foliose chlorolichens
Bipartite cyanolichens
■ ■ Lobaria pulmonaria
12
I

Total N
15 (g N tree-1)

-

10

Avg N

10 “

High Spruce High Fir Low Sprnce Low Fir

(g N tree ‘ ^)

8

-

6

-

4 2

-

0

-

a a

n

n

High Spruce

High Fir

Low Spruce

Site and tree species

67

Low Fir

Figure 2.6 Mean epiphytic lichen nitrogen (N: kg of N ha ' 1 ± SD) at sites with (A) high (High
Cyano 1 or 2) versus low (Low Cyano 1 and 2) cyanolichen abundance on (B) either Spruce or
Fir by lichen functional group in central British Columbia. Aggregate N by functional group is
also shown (Figure 2.6A Inset). Dissimilar letters indicate significantly different mean values
(Bonferroni multiple comparison tests: P>0.05).

I
I Bryoria spp.
I - ' . 1 Alactoriasarmentosa
■ ■ Foliose chlorolichens
m
Bipartite cyanolichens
H I Lobaria pulmonaria

H1F

H1S

H2F

H2S

L1F

L1S

L2F

Site and tree species

68

L2S

Chapter 3: Using stable isotope ratios 15N:14N, ,3C:12C to determine the effects of epiphytic
lichens on the nitrogen (N) and carbon (C) cycles of wet sub-boreal spruce and fir forests of
central British Columbia

3.1 ABSTRACT
Forest canopy research has revealed rich epiphytic lichen communities that represent a critical
interface between the atmosphere and soil. In mature sub-boreal forest ecosystems of central
British Columbia (BC), epiphytic cyanolichens make important contributions to biological
diversity, but their importance to nitrogen (N)-cycling in these forests, particularly with respect
to N 2 -fixing cyanolichens, has not yet been adequately quantified. Variations in the ratios o f the
natural abundance o f stable isotopes of N and carbon (C) (i.e. 15 N :I4 N, 1 3 C: 12 C) in forest
ecosystem components represent an important in situ method to estimate N inputs from
cyanolichens. Four mature sub-boreal spruce and fir dominated forest sites were chosen based
on relative abundance o f cyanolichens: two high (High Cyano) and two low (Low Cyano)
abundance sites. Interior hybrid spruce (Picea glauca x engelmannii) and subalpine fir (Abies
lasiocarpa) trees were randomly selected at High and Low Cyano sites and access into canopies
achieved through a single rope technique. Lichen, conifer needle, and soil samples were
obtained and their 15 N : 14N, %N, 1 3 C:12 C, %C contents determined using continuous flow gas
isotope ratio mass spectrometry. We hypothesized that High Cyano sites would elevate N levels
in host tree foliage and have isotopically heavier N (i.e. more 15 N), indicative of greater inputs of
cyanolichen fixed-N with isotopic compositions similar to atmospheric N2. Our results did show
a modest positive correlation between host tree foliage (conifer needle) N concentrations and
cyanolichen abundance. Cyanolichen abundance was a statistically significant but not an
effective predictor of lichen functional group delta (5) 15 N. By contrast, tree branch height above
69

ground level was a statistically significant predictor of needle 8 1 SN. However, site-level
cyanolichen abundance did not significantly affect 8 15N or %N of lichen or soil. Stable isotopes
of C can be used to gain further information about lichen performance across ecological
gradients in both space and time. Lichen functional groups and tree foliage fell into three
distinct groups with respect to 8 1 3 C; L. pulmonaria (lightest), needles (intermediate), and
bipartite cyanolichens, hair and chlorolichens (heaviest). Carbon isotopes were used to
determine that tree branch height was an effective predictor of needle 813C. We were able to
show how the isotopic composition of two elements changed based on various conditions such as
changes in cyanolichen abundance and canopy height. While our work did find enhancements in
host tree foliage N with increasing cyanolichen abundance, further studies are required to
determine if these result in greater tree growth.

Key Words: 815N, 8UC, stable isotope natural abundance, arboreal lichen, sub-boreal
forest, cyanolichen, Lobaria pulmonaria

70

3.2 INTRODUCTION
3.2.1 Lichen Characteristics
Tree foliage, branches, and arboreal epiphytes all intercept and regulate inputs from the
atmosphere contained in wet or dry deposition, with the amount of interception depending on
relative abundances and properties of these canopy elements (Nadkami & Sumera, 2004).
Arboreal lichens, with large specific surface areas, can play a significant role in mediating the
exchange of water and depositional constituents such as nutrients through interception,
absorption and/or assimilation processes (Pypker et al., 2006).
Nitrogen (N) is an essential growth-limiting macronutrient in plant systems and generally
limiting in forests where ambient atmospheric deposition of N is low, e.g. northwest BC,
Canada. In N-limited conifer forests, biological N 2 -fixing species have been shown to make
important contributions of N to forest by intercepting, retaining and releasing this growthlimiting nutrient (Coxson & Nadkami, 1995). Epiphytes on lower branches within a tree are
exposed to throughfall of dry and wet deposition and N sources from upper canopy epiphytes.
This interaction between upper and lower canopy zones can be further altered by vertical
gradients in the epiphyte guild to create significant variation in N concentration and 6 I5N values
of epiphytes from a communal tree (Bergstrom & Tweedie, 1998). Although the presence of
epiphytic lichens has been shown to enhance a tree’s ability to absorb and enhance atmospheric
N deposition and inputs, boosting nutrient cycling fluxes, the removal of lichens in an oak forest
system had no effects on tree growth (Knops et al., 1997). Lichens are also nutritionally
specialized fungi that derive C from algal and/or cyanobacterial photobionts (Honegger, 1993).

71

3.2.2 Stable Isotope Techniques
Stable isotopes have been used experimentally to infer many properties about elemental
cycles of important nutrients (most notably C and N) at a variety o f temporal and spatial scales
and ecological contexts. The ratio of the heavier less common stable isotope to that of the lighter
1C

1A

abundant isotope (e.g. N: N and

i 'i

i a

C: C) can be expressed as atom %. In practice, isotope

levels are typically represented in delta (5) notation which is not the absolute isotope ratio, but
the difference between the sample measurement and an internationally accepted reference
standard (Robinson, 2001) in parts per thousand or per mil (%o).
With respect to nitrogen, the natural abundance of 15N in air is constant at 0.3660% gas
and is therefore used as the reference standard for N isotope analyses. Specifically, 8 15N stable
isotope results were reported as the per mil (%o) difference (8) between the 15N :14N isotope ratio
of each sample compared to the same isotope ratio of the N 2 in air defined as 0.36637. By
contrast, the common reference material for 13C is a limestone Pee Dee Belemnite (PDB).
* -j

Specifically, 8 C stable isotope results were reported as the per mil difference (8 ) between the C
I^

19

isotope ratio of each sample and the C: C isotope ratio of PDB limestone. The absolute
isotope ratio (R) is the ratio o f 15 N :14N for N and 1 3 C:,2C for C isotope abundance. Once the R
of the sample and standard are measured, the 8 is calculated. Positive 8 values indicate the
sample contains more of the heavy isotope than the standard and negative 8 values indicate the
| c

| i

opposite. Therefore, higher levels of N or C enrichment correspond to less negative (or more
positive) 8 15 Ns and 8 1 3 Cs, respectively (Dawson et al., 2002).
In theory, natural abundances of isotopes can be used to make inferences about the
contributions o f N2-fixing species to forest trees by exploiting the naturally occurring differences
in 15 N :14N ratios between plant-available mineral N sources in the soil and that of atmospheric
72

N2 utilized by ^ -fix in g species (Boddey et al., 2000). Non-N 2 -fixing plants receive their entire
N supply from soil N pools and can be expected to be isotopically heavier than N 2 -fixing plants
and lichens, which derive a large component of their N directly from the atmosphere.
Consequently, natural abundances of stable N isotopes have frequently been used to estimate
direct or indirect contributions of ^-fix atio n to plant N content (Vitousek et al., 1989).
The distributions of stable isotopes of C and N have been widely investigated. The
I^
naturally occurring 8 C values for biological carbon compounds range from ~0%o to - 110%o
relative to the Pee Dee Belemnite standard. However, observed lichen 513C values occur over a
much narrower range (-14 to -35 %o: Lange et al., 1988), and considerably narrower still in
temperate tree species with C3 physiology (e.g. -24%o to -33%o range: O’Leary, 1988). Carbon
isotopes can inform us about interactions that occur between lichens and other organisms. A
previous review by Ehleringer & Rundel (1988) revealed that C isotopes can inform about how
species adjust their gas exchange metabolism, strategies of resource acquisition and use, and life
history patterns to ensure competitiveness as well as survival.
Most terrestrial materials have 8 I5N compositions ranging between -20%o and +30%o
(Junk & Svec, 1958), but as before, lichens have been observed to contain a more restricted
range of compositions ( -21.5%o (Fogel et al., 2008) to +18%o (Huiskes et al., 2006)), and plants
even more so (e.g. -5%o to +2.9%o: Amundson et al., 2003). The natural abundances and ratios of
stable isotope compositions are potentially useful to the researchers quantifying these processes
in that they can shift due to isotopic fractionation.
Isotopic fractionation can occur from a variety of processes. For example, ^-fix atio n in
organisms such as epiphytic cyanolichens does not discriminate between the l5N and ,4N and
therefore would normally represent N isotope ratios close to the atmospheric 8 15N standard of
73

0°/oo. By contrast, non-N2 -fixing elements and processes of forest systems such as

ectomycorrhizae (ECM), mineralization of organic nitrogen, nitrification, microbial assimilation
of inorganic N, and denitrification can fractionate N stable isotopes below ground. Nutrient
uptake in temperate and boreal trees is predominantly dependent on ECM hyphae growing into
the soil from the mycorrhizal root tips (Wallander et al., 2004). In a 1996 study, Hogberg et al.
found that ECM roots o f Norway spruce (Picea abies) and beech (Fagus sylvatica) were 2%o
enriched in l5N relative to non-mycorrhizal roots. The study also found ECM fungi were
enriched in I5N compared to their host plants, further suggesting that 15N ECM discrimination
was fungal in origin. ECM fungi were also more enriched in 13C relative to total soil C (Hogberg
et al., 1999) demonstrating 13C ECM discrimination. Biologically mediated reactions that
|f

n

control elemental dynamics in soils can result in N and C enrichment or depletion, and
therefore inferences can be difficult in complex ecosystems with a multitude of important
sources and sinks for nutrients.

3.2.3 Soil N Inputs
Soil 51SN values are higher than atmospheric N, which is isotopically lighter than
biological material, suggesting that there is discrimination against the lighter isotope during
decomposition (Peterson & Fry, 1987). Soil 5I5N values are usually positive and increase with
depth of organic horizons (‘L’, ‘F’, and ‘H’), and with mineral horizons having higher 51SN than
surficial organic horizons (Mariotti et al., 1980). This pattern occurs because there is a faster
loss o f 14N than 15N during decomposition in soils, which results in 15N increases of ~5-10%o
with increasing depth (Peterson & Fry, 1987). Thus, fractionation of isotopes during litter
decomposition in forests causes surface soils to have lower 8 15N values than deeper soils
74

(Nadelhoffer & Fry, 1988). Even slight fractionations occurring over decades of transfers of N
from mineral soil to forest biomass can be sufficient enough to increase 515N soil organic matter
by ~6-8%o (Billings & Richter, 2006). Surface soils located beneath trees have also been found
to have lower 8 15N values than those in open areas as a result of litter deposition (Shearer &
Kohl, 1986) making decomposing epiphytic lichens important to soil nutrient isotope
composition.
Since soil N is typically more abundant in l5N than the atmosphere, non-N 2 -fixing plants
typically have higher 8 15N values, and N 2 -fixing plants lower 8 15N values (i.e. closer to
atmospheric N 2 ). The difference between these values forms the basis of the 15N natural
abundance technique for estimating fixed-N contributions to plants (Shearer & Kohl, 1986).
However, attempting to trace fixed N through the ecosystems can be complicated by a myriad of
fractionations, which are caused by numerous and often serial pathways of mineralization,
nitrification, immobilization, and denitrification within the soil, plant root and mycorrhizal
fungus mediation of soil N uptake (Lajtha & Schlesinger, 1986).
3.2.4 Forest Ecosystem Nutrient Cycling
Nutrient cycling processes in the soil are known to vary with plant community
composition (Ehrenfeld et al., 2005). The processes associated with litter decomposition and
enzymatic transformations of organic substrates in particular can vary because of differences in a
variety of factors such as chemistry o f the litter material, soil biota and soil chemistry associated
with different plant species (Aerts, 1997). Both litter accumulation and stem flow can deliver N
to epiphytes from various N pools (Bergstrom & Tweedie, 1998), making the abundance and
branch position o f lichens on a tree significant. Lichens may play an important role in nutrient
cycling in forest ecosystems, but the relative impact of lichens compared to other ecosystem
75

components is not well known. The effect o f lichens on the pattern of mineral cycling could
range from major in ecosystems where lichens are abundant and contribute to the bulk of the
biomass, to minor where lichens are only present in trace amounts (Pike, 1978).

3.2.5 Research objectives
Chapter 3 builds on the findings contained in Chapter 2 by examining the importance of
the vertical distributions of epiphytic lichen species compositions and biomasses to the N
contained in wet sub-boreal spruce and fir forest ecosystems of the central interior of BC. To do
this, we measured the 15 N : 14 N, %N, 1 3 C: 12 C, and %C contents of host tree (conifer) foliage
(needles), host tree epiphytic lichen functional groups and organic and mineral soil horizons
under the tree crowns at sites containing variable amounts of cyanolichen tripartite (i.e. L.
pulmonaria) and bipartite lichens from relatively low to high levels.
We hypothesized that sub-boreal forest trees with high cyanolichen abundance should
have isotopically lighter N (i.e. more 14 N) and higher %N in foliage, indicative of greater inputs
o f biologically fixed-N into these systems. Among epiphytic lichens, cyanolichens should have
a 8 I5N closer to zero, whereas other non-N2 -fixing lichens (i.e. chlorolichens) should have a
more negative 515N (Freyer, 1991). Knowledge obtained from this canopy study provides
valuable information on the ecological function and biodiversity of epiphytic lichens on SBS
forest ecosystems in central BC.

76

3.3 METHODS
3.3.1 Study Area
The study area was previously described in Chapter 2. In brief, this study was carried out
on the north side of the Fraser River near the town of Upper Fraser, BC, located approximately
70 km NE of Prince George, BC. All study sites were characterized by having relatively cool
and moist summers and cold, snowy winters. Four mature sub-boreal forest sites were chosen
based on having trees with predominantly high (high sites) or low (low sites) epiphytic
cyanolichen abundance and diversity, characterized in greater detail by Campbell et al. (2010b).
The two dominant tree species, and therefore the primary hosts for lichen epiphytes at all
four sites, were interior hybrid spruce and subalpine fir. The two Fraser sites were selected
because o f a high cyanolichen abundance and diversity and were termed ‘High ’ sites that
spanned the SBS wkl and the SBS vk subzones (Campbell et al. 2010b). These sites were
located at an elevation of 680 m above sea level (a.s.l.) with a mean summer temperature of 1 1 . 8
± 5.3 °C and relative humidity of 78% (Campbell et al. 2010b). The two Herrick road sites were
determined to have low cyanolichen abundance and diversity and were termed ‘Low ’ sites
(Campbell et al., 2010b). These sites were located in the SBS vk at an elevation of 850 m a.s.l.
with mean summer temperature and relative humidity of 10.8 ± 5.3 °C and 78%, respectively.
Soils at all sites were Orthic Humo-Ferric Podzols formed from sandy-colluvial materials at the
High sites and from sandy-skeletal glaciofluvial materials at the Low sites. Average
precipitation in the ecotonal study area is approximately 897 mm per year (Campbell et al.,
2 0 1 0

b).

77

3.3.2 Sample Trees
Sample tree selection was previously described in Chapter 2 with the exception of the
fact that trees adjacent to Sitka alder (Alnus viridis) were excluded from the study given that
annual N 2 fixation by Sitka alder can be substantial (Sanborn et al., 2002) and would potentially
confound 15N natural abundance interpretation. In brief, all study trees were in excess of 22 m in
height and 20 cm in DBH. Needles and lichens were sampled from each canopy height zone at
the highest accessible point o f each o f the 32 study trees.
3.3.3 Canopy Access
Canopy access was previously described in Chapter 2. In brief, access into canopies was
achieved through a single rope technique (Denison, 1973; Perry & Williams, 1981). Selected
trees were rigged, climbed and assessed vertically for epiphytic lichen biomass.
3.3.4 Epiphytic Lichen and Needle Sampling
Lichen and needle sampling was previously described in Chapter 2. In brief, epiphytic lichen
and needle samples were collected over two summers: June to August 2008 and May to
September 2009 at various heights throughout the tree canopy. One random sample was sent for
isotopic analysis from the upper, middle and lower canopy height zones if they were present.
Lichen biomass was separated into five categories: Alectoria sarmentosa, Bryoria species,
foliose chlorolichen, bipartite cyanolichens, and Lobaria pulmonaria, the only tripartite
cyanolichen at the site. The needle cohort was exclusively from the previous year’s foliage to
keep the age o f the sampled needles constant.

78

3.3.5 Composite Soil Sampling
Soil samples were collected from the base of each sample tree between June and August
2009. We extracted soil cores using a soil auger (7.5 cm in diameter) by rotating the auger while
applying downward force and lifting out the full blades. Soil core samples were extracted on a
1-m distance around each sample tree base from each of the cardinal directions. Samples were
separated into three distinct layers: F - folic layer composed of organic matter rich in mycelia
(-5 cm ± 2 cm deep), Ae - grayish surface soil layer (-2-10 cm thick) and Bf - yellowish brown
to reddish brown subsoil layer (-10-20 cm). All layers were distinct and could be easily
separated from each other; the soil auger was wiped down between cores. The three layers of the
four subsamples were air dried on aluminum (Al) pie plates for two days at room temperature
(-22-25 °C). Next, roots were removed from soil and samples were sieved through a 2-mm
sieve. Then, F layer samples were separated twice more (0.85 mm and 0.3 mm sieves) to
remove small pieces of woody debris before composite samples were prepared. Composite
samples were also made from the four Ae and Bf subsamples from each site. A dry weight of 2
g from each of the three layers of the four samples was mixed into one composite sample per soil
layer ( 8 g dry weight), for a total of 96 soil samples.
3.3.6 Stable Isotope Analyses
Sample preparation of lichen, needle and soil samples took place at the University of
Northern British Columbia (UNBC) before samples were shipped to the Stable Isotope lab at the
University o f Saskatchewan (U of S) in Saskatoon, Canada for analysis. Lichen and needle
samples were oven dried for three days at 55 °C in paper bags. Samples were ball-milled
(Retsch MM301) to a particle size of less than 250 pm and inspected for fibrous matter or visible
granules that could reduce precision o f isotopic analysis. All samples were transferred, pressed
79

and encapsulated in 8 x 5-mm tin capsules (Catalog # D1008, Elemental Microanalysis Limited,
Okehampton, UK) before combustion. Each capsule was tightly crimped so that it did not leak.
The final shape of the wrapped sample was spherical or cylindrical. A list of sample weights
(accurate to 0.01 mg) was used later for mass-specific calculations. Samples were stored in
scintillation vials and weighed to an optimum weight of ~2 mg + 0.2 mg. Ball chambers were
cleaned with deionized water between samples, wiped with kimwipes, and air dried to remove all
water droplets.
Soil, needle and lichen samples were analyzed using a Costech ECS4010 elemental
analyzer (EA) coupled to a Delta V Advantage mass spectrometer with Continuous Flow
(Conflo) IV interface. The continuous flow gas isotope ratio mass spectrometer was used to
measure % N, % C and the relative abundance of isotopes (15N and I3 C) in needle, lichen, and
soil samples.
Isotope ratio analysis, 15 N :,4N and 13 C:I2 C, of solid phase samples started with
transformation to gas phase by extremely rapid and complete flash combustion of the sample
material. Ionized combustion product was then mass-analyzed by means of differing
mass:charge ratios among the various isotopic species of N 2 and carbon dioxide (CO 2 ). The 8 15N
and 513C results were optimized for maximum accuracy by the application of a 'correction curve'
to the results. However, if two different 8 I5N results were available then the 8 15N result which
had an N 2 beam area closest to 50 was used. Likewise, if two different S13C results were
available then the 8 13C result which had a CO 2 beam area closest to 70 was used. When the
stable isotopes react with the particle beam, a nuclear reaction occurs between the protons and
the target atoms creating radioactive isotopes.

80

Samples were stored in a desiccator prior to analysis and then placed into a Zero Blank
autosampler where atmospheric gases containing C and N were purged by ultra high purity
helium (He) carrier gas. The He flow rate was set to 70 ml m in '. The autosampler dropped
samples into a chromium oxide (CrO) and silvered cobaltic oxide combustion reactor set at 1040
°C in the elemental analyzer. Two seconds prior to sample entry, a pulse of high purity oxygen
(O2 ) was released to the top of the combustion column. The combustion of the O 2 pulse and high
temperature caused a flash combustion of the sample which raised the temperature to 1700 °C.
The flash ensured complete sample combustion while the CrO ensured complete oxidation of C
gases to CO2 . Nitrogen oxide gases produced were then converted to N 2 in a reduction reactor
filled with copper at 650 °C. Finally, a water trap dried out the sample gas and a 3-m gas
chromatography column separated N 2 from CO2 before gases were sent to an open split in the
Conflo III interface and then to the mass spectrometer. Isotope ratio calibrations were performed
with IAEA-N1, IAEA-N2, IAEA-NO-3, IAEA-CH6 , and USGS-24 standards (International
Atomic Energy Agency, 1995). Standards were encapsulated and periodically placed within the
run o f samples; two within-run reference samples were placed between every five samples
(Stocki, 2010).
3.3.7 Natural Abundance Method
The 6 values for 15N and l3C were calculated as follows:

8

15N [%o relative to atmospheric N2 ] = (Rsampie/Rstandard - 1) x 1000

8 I3C [%o relative to PDB] = (Rsampie/Rstandari - 1) x 1000

81

(Eq.l)
(Eq.2)

The 15N natural abundance method was used to infer the fractional contribution of
biologically fixed N to any forest system component. This temporally integrated assessment is
based on small but measurable differences in 15N abundance between atmospheric N2 and other
sources of N. The value of 0.366 atom % 15N was determined by Nier (1950) and established to
be constant by the work of Junk and Svec (1958). Measurements of 8 I5N provide time-averaged
insights into the N cycle without disturbing the system.
3.3.8 Statistical analyses
Data were analyzed using Sigma Plot 11 (Systat Software Inc., San Jose, CA, USA).
Ordinary least square (OLS) multiple linear regression analyses were performed to address the
following research questions. Was there a statistically significant difference in 8 I5N and 8 13C
between the lichen functional groups and needles controlling for tree branch height, tree species,
and lichen abundance? Was there a difference in 8 I5N and 8 13C with tree branch height
controlling for the lichen functional groups and needles? Was there a difference in 8 I5N and
8

I3C between High and Low lichen abundance sites controlling for the lichen functional groups,

needles and soil? Was there a difference in 8 ,5N and 8 13C between tree species (Spruce and Fir)
controlling for the lichen functional groups, needles and soil? Did %N in foliage (needles)
increase at sites with higher abundances of cyanolichen? Six regression models were created to
predict: i) 8 I5N in lichen functional groups; ii) 8 15N in needles; iii) 8 13C in lichen functional
groups; iv) 8 13C in needles; v) 8 15N in soil; and vi) 8 13C in soil. Correlation analyses were
conducted between all the variables in each model to check for potential collinearity. Additional
correlations were also conducted to look at the relationships between needle 8 15N and
cyanolichen abundance, % N and cyanolichen abundance, and soil 8 15N and cyanolichen
abundance.
82

Kolmogorov-Smimov and Shapiro-Wilk tests were used to determine normality of data.
Both tests showed lichen and needle 8 15N were not normally distributed (KS = 0.191, p < 0.001
and W = 0.886, p < 0.001). Mass distributions across all lichen functional groups also failed
normality tests. In addition to violating the normality assumption all variables were equally
heteroscedastic on the independent variable. Due to the bimodal distributions of the dependent
variable, data transformations were not used. Both normality tests also showed that lichen and
needle 8 13C were not normally distributed (KS = 0.122, p < 0.001 and W = 0.949, p < 0.001).
No transformations could be used to fix this multimodal distribution. The distribution of lichen
and needle 8 I5N and 8 I3C but not soil 8 15N and 8 13C showed signs of heteroscedasticity and nonlinearity across all independent variables. Since the lichen functional group and needle data
violated two of the three assumptions required by linear regression, the results of these models
may be dubious. Soil 8 I5N and 8 I3C data were normally distributed.
All ordinary least square (OLS) linear regression outcomes show an unstandardized
coefficient (B) which was the estimated change in the dependent variable associated with a oneunit change in the identified independent variable controlling for all other independent variables.

3.4 RESULTS
Nitrogen (N) contents varied by over 6 -fold among lichen epiphytes sampled. At the
extremes, Alectoria sarmentosa had the lowest % N (0.50 ±0.13) and bipartite cyanolichens had
the greatest % N (3.24 ± 0.52) on a dry weight basis (Fig. 3.1). Bryoria spp. and foliose
chlorolichens had very similar %N contents of 0.77 ±0.18 and 0.73 ±0.19, respectively.
Needles had a higher %N content (1.11 ±0.14) than hair lichens and foliose chlorolichens but
not as high as L. pulmonaria (2.26 ± 0.30). The differences in mean %N between functional
83

groups were significant (F 5 j 404 = 950.3, p < 0.001: Tukey test) except for Bryoria spp. and foliose
chlorolichens (Fig. 3.1).
All lichen functional groups or species and conifer foliage (needle) had mostly unique
8

15N signatures relative to their %N (Fig. 3.2). Bryoria spp. hair lichens at the top of the canopy

had the lowest 8 15N while the hair lichen Alectoria sarmentosa found lower in the canopy had
slightly lower N content and higher 8 I5N than Bryoria spp. Host tree needles and foliose
chlorolichens had S15N values that were intermediate between hair lichens and cyanolichens but
similar N contents. While L. pulmonaria and bipartite cyanolichens both had 8 15N values close
to atmospheric N 2 (8 15N =0), L. pulmonaria had 1.5% less N on average (Fig. 3.2). Mean 8 I5N
isotope values were slightly higher at Low abundance sites for all lichen functional groups and
host tree needles, but there was no significant difference in the 8 15N values or N contents of
lichens or needles on trees with high (black symbols) versus low (white symbols) abundance of
cyanolichens (Fig. 3.2).
When we compared 8 I5N of living conifer foliage (needle) on each of the 32 study trees
to their actual cyanolichen abundance, there was a small, but ultimately not significant (r = -0.16,
p = 0.121) negative correlation between mean needle 8 15N and increasing cyanolichen abundance
(Fig 3.3A). Host tree foliage %N showed a significant but weak positive correlation to site-level
cyanolichen abundance (r= 0.24, p = 0.019; Fig 3.3B).
There were no issues with collinearity but there were some significant correlations
between variables. Only weak to moderate relationships existed between independent variables
so we proceeded with regression analyses.
When lichen species, tree branch height, and tree species were controlled for,
cyanolichen abundance in the canopy was a statistically significant but ineffective predictor of
84

8 15N (B < 0.001, p < 0.001, Table 3.1). The regression coefficients of the dependent variable
8 15N are displayed in Table 3.1 and suggest that lichen species and cyanolichen abundance but
not tree species or tree branch height were significant predictors of 8 I5N in lichen functional
groups. The regression analysis showed that A. sarmentosa (B = -3.657, p < 0.001), Bryoria spp.
(B = -4.838, p < 0.001) and foliose chlorolichens (B = -0.482, p = 0.002) were negatively related
to S I5N levels in lichen functional groups. Conversely, bipartite cyanolichens (B = 0.561, p <
0.001) and L. pulmonaria (B = 1.608, p < 0.001) were positively related to 8 15N levels in lichen

functional groups. Both tree species (B = -0.166, p = 0.095) and tree branch height (B = 0.010, p
= 0.238) were not significantly related to 8 I5N levels in lichen functional groups (Table 3.1).
However, tree branch height was a significant predictor of 8 15N in needles (B = 0.028, p = 0.002)
but both tree species (B = -0.146, p = 0.267) and cyanolichen abundance (B < 0.001, p = 0.061)
were not significantly related to 8 I5N levels in needles.
Mean soil horizon 8 15N values were consistently higher at Low (means: F = 1.340, Ae =
5.130, Bf = 6.262) than at High (F = 0.782, Ae = 4.765, Bf = 5.383) cyanolichen abundance sites
and soil 8 I5N decreased with increasing cyanolichen abundance in soil F, Ae and B f horizons
(Fig. 3.4A-C), respectively. The abundances of cyanolichens and 8 15N were negatively correlated
in all three soil horizons, with correlations being nearly significant for the uppermost horizons
(F: r = -0.341, p = 0.056; Ae: r = -0.263, p = 0.146) and significant for the Bf horizon (r = -0.356,
p = 0.045). Mean soil horizon %N was also greater at Low than at High Cyano abundance sites
(Fig. 3.4D-F) and the uppermost soil horizon had the highest %N (Fig. 3.4D).
Cyanolichen abundance, tree species and soil horizon were all significant predictors and
all contributed to the overall relationship of both soil 8 I5N (Table 3.3) and soil 8 13C (Table 3.4).
The regression analysis showed that there was a statistically significant difference in both 8 I5N
85

(B = -0.001, p < 0.001: Table 3.3) and 8 13C (B = 0.000, p < 0.001: Table 3.4) between High and
Low cyanolichen abundance sites. There was also a statistically significant difference in soil
515N (B = -0.525, p < 0.001, Table 3.3) and soil 5,3C (B = -0.221, p = 0.009, Table 3.4) between
host tree species (spruce and fir).
When controlling for all independent variables, tree branch height was a significant
predictor of 8 I3C for lichen functional groups and needles (Fig. 3.5A, Table 3.2). There was
I^

I -j

evident clumping of 8 C into three distinct groups. Specifically, 8 C values of the lone
tripartite cyanolichen (L. pulmonaria) were lower than host tree needles which were less than
hair lichens (A. sarmentosa and Bryoria spp.), bipartite cyanolichens and foliose chlorolichens.
Tree branch height was not a significant predictor of 8 15N for lichen functional groups but was a
significant predictor of 8 I5N for needles (Fig. 3.5B, Table 3.1). Nevertheless, hair lichens A.
sarmentosa and Bryoria spp. had uniformly negative 8 I5N values that were also more negative
than all other functional groups.
A plot o f 8 i5N versus 8 I3C resulted in a visual separation of most biomass types (Fig.
3.6). Host tree foliage was intermediate to L. pulmonaria (high l5N and low l3 C) and all other
lichen functional groups. Lobaria pulmonaria had the lowest 8 I3C and the highest 8 I5N values.
Bipartite cyanolichens and foliose chlorolichens had similar ranges in both 8 15N and 8 1 3 C. Hair
lichens A. sarmentosa and Bryoria spp. had a similar range of 8 ,3C values as both bipartite
cyanolichens and foliose chlorolichens, but 8 I5N values that were more negative (from -3 to -12
%o) than all other biomass types analyzed.

Lobaria pulmonaria had a uniquely distinctive 8 I3C values (-32 and -36 %o) relative to
other lichen groups/species (-22 and -27 %o) despite relatively consistent carbon contents (Fig.
3.6). Host tree needles had 8 I3C values intermediate to L. pulmonaria and all other lichen
86

functional groups (Fig. 3.6), but at distinctly higher C contents than all fungi (Fig. 3.7). Lichen
species, cyanolichen abundance, tree branch height and the interaction between cyanolichens and
site cyanolichen abundance (but not tree species) were significant predictors of 8 13C in lichen
functional groups. The regression analyses showed that A. sarmentosa (B = 4.899, p < 0.001),
Bryoria spp. (B = 4.513, p < 0.001), foliose chlorolichens (B = 3.969, p < 0.001), and bipartite
cyanolichens (B = 4.324, p < 0.001) were positively related to 8 I3C levels in lichen functional
groups (Table 3.2). Conversely, L. pulmonaria (B = -4.358, p < 0.001) was negatively related to
8

13C levels in lichen functional groups. Both cyanolichen abundance (B = -0.001, p < 0.001) and

tree branch height (B = 0.093, p < 0.001) were significantly related to 8 13C levels in lichen
functional groups. There was also a statistically significant interaction between cyanolichens
and site cyanolichen abundance and 8 13C increased slightly as the site cyanolichen abundance
increased (B = 0.001, p = 0.005, Table 3.2). There was no significant difference in 8 13C between
sample tree species spruce and fir in lichen functional groups (B = 0.048, p = 0.594, Table 3.2).
Alternatively, both tree species (B = 0.622, p < 0.001) and tree branch height (B = 0.148, p <
0.001), but not cyanolichen abundance (B < 0.001, p = 0.942), were significant predictors of
8

I3C in needles (B = 0.028, p = 0.002).

3.5 DISCUSSION
Nitrogen-fixing lichen epiphytes can be abundant in conifer forests of central BC and are
conjectured to be an important component o f the N-cycle in these forest locations (Campbell &
Fredeen 2004, 2007; Campbell et al., 2010a). Although lichen N pool sizes and decomposition
rates in these studies were indicative of enhanced mineral N flux rates as observed in other
forests types (e.g. Knops et al., 1997)), direct links between cyanolichen abundance and
improved host tree N status have been elusive. In our study, gradients in cyanolichen abundance
87

across host trees (interior spruce and subalpine fir) in two generally high cyanolichen abundance
sites and two generally low cyanolichen abundance sites (Campbell et al., 2010b) provided us
with an opportunity to examine the potential for enhanced cyanolichen N-inputs into these
forests in central BC. We hypothesized that foliar (needle) N contents would be positively
correlated with cyanolichen abundance, and in fact this was found to be the case (Fig. 3.3b). We
further sought to more directly link increased host tree foliar N with cyanolichen N through the
unique 15 N :14N stable isotope ratios of cyanolichen N.
Our results corroborate previous findings of 8 15N values in lichen functional groups and
or species. It is already known that cyanobacterium-containing lichens (cyanolichens) that fix N 2
typically have 8 I5N values close to atmospheric N 2 , i.e. 0%o (Macko et al., 1987; Virginia &
Delwiche, 1982). This was confirmed for both bipartite and tripartite (L. pulmonaria)
cyanolichens in our study. By contrast, foliage, hair and chlorolichen epiphytes all had 8 I5N
values that were negative and less enriched in 15N than cyanolichens. Hair lichens had the lowest
515N values, while chlorolichens and host tree foliage had intermediate 8 15N values between
cyanolichen and hair lichens. Interestingly, A. sarmentosa, occurring lower in the canopy than
Bryoria, and therefore overlapping with cyanolichen canopy zones (see Chapter 2), had 8 I5N
closer to atmospheric 8 15N (0) (Fig. 3.2). This is consistent with Alectoria receiving more
leached-N from cyanolichens than Bryoria. This was supported by the fact that in High sites, a
notable increase in Alectoria 8 15N was observed, while relatively little change in 8 15N was
observed in Bryoria with increased cyanolichen abundance. In both hair lichens, relatively low N
contents and low 8 15N (~ - 6 %o in Low Cyano sites) were consistent with low atmospheric N
inputs in central BC (e.g. Hope, 2001) and the negative 8 I5N of precipitation N measured in other
studies (Freyer, 1991).
88

Although biological ^ -fix atio n may be a significant N input into ecosystems, it can be
difficult to identify since it lacks a unique 8 15N signature over and above that of background 8 15N
values due to its relative isotopic effect (Vitousek et al., 1989). Nitrogen isotope signatures of
epiphytes varied with canopy position in our study, but the reason for lichen 8 15N variability in
most studies remains unexplained. The 8 15N of hair lichens, with single sources of N (i.e.
atmospheric fixed-N), may be relatively easy to define, in contrast to other lichens (cyanolichen
and chlorolichen) lower in the canopy. At least 10 processes have been identified that can alter
8

I5N values, none of which can currently be separated out in field studies (Robinson, 2001). A

few explanations for 8 15N variability are: i) the preference for the lighter 14N isotope in uptake
may lead to 8 15N enrichment, ii) fractionation of N isotopes in gaseous phase ammonia is greater
than in the liquid phase of nitrate, and iii) transfer of organic N could result in increased l5N
depletion of the photobiont and less depletion in the mycobiont (Hobbie & Hobbie, 2008).
Variations of 8 15N values are also attributed in part to altered 15N discrimination during N
acquisition and to changes in partitioning of N isotopes (Wania et al., 2002). In general, our
results were in agreement with previous work showing that lichens with a green alga as their
photobiont (including all lichens but bipartite cyanolichens) also showed the greatest l5N
depletion (Riera, 2005) (see Fig. 3.1).
Soil 8 15N values increased with soil depth in our study, consistent with results of Gebauer
and Schulze (1991). In keeping with our expectations, we also observed decreasing trends in
8

15N with increasing cyanolichen abundance in all soil horizons (Fig. 3.4A-C) equating to less

positive soil horizon 8 15N values, more proximal to the 8 I5N of cyanolichen biomasses which
were uniformly close to zero. There are many reasons why soil 8 I5N wouldn’t necessarily be
closely correlated with tree 8 15 N. First, measured 8 I5N of soil pools may not represent the true
89

isotopic composition o f N available to plant roots, since most of the N in soils is bound in forms
that are not immediately available to plants (Jansson, 1958; Binkley & Hart, 1989). Second, only
a few % of soil total N becomes available in a year (Hogberg, 1997), and symbiotic fungi can
alter the 8 15N o f the N transferred from soil to host plant. Nevertheless, a similar downward
trend in tree foliage 8 I5N at our sites with increasing abundance o f cyanolichen (Fig. 3) was
consistent with the downward trend in soil 8 15N (Fig. 3.4A-C). Explaining the increased %N in
host tree foliage with increased cyanolichen abundance is more difficult to reconcile with total
soil N. If greater cyanolichen inputs o f N increased soil N concentrations, then a straightforward
mass action of greater plant N uptake could explain enhanced foliar %N. However, soil N
decreased in all soil horizons, though non-significantly, with higher cyanolichen abundance (Fig.
3.4D-F). Since total soil N does not in any way equate with amounts and forms of available
inorganic N, it is possible that more readily available N fractions were more available at high
abundance cyanolichen sites, even though total %N was not. Further work on soil N at these
sites would be required to test this hypothesis.
Foliar (needle) 8 I5N values in our host trees ranged between 0.6 and -2.8 %o and did not
differ significantly between High and Low lichen abundance sites or tree branch height.
However, there was a trend of decreasing foliar 8 15N with increasing cyanolichen abundance
(Fig. 3.3 A). Gebauer and Schulze (1991) reported much lower 8 1SN values of conifer needles
(between -2.5 and -4.1%o) which varied according to stand and age, with foliage from the
healthiest site having the lowest SI5N. It is unclear why S15N values observed by Gebauer &
Schulze were more negative than ours, but one possibility would be the presumed greater
atmospheric N inputs in these forests when compared to the N-limited forests of central BC.
Gebauer and Schulze (1991) also reported a similar trend of 8 I3C values of needles, which
90

ranged between -26.2 and -32.2 %o and did not differ between lichen abundance sites but did
change with canopy height. Similar to our study, needle S13C values were not significantly
different between abundance sites (Table 3.2), were more negative in the lower canopy and
increased with tree height (Fig 3.5).
Lichen 8 13C values were previously found to vary widely over a large range of habitats
and species (Batts et al., 2004). Our lichen functional groups and tree foliage (needles) fell into
three distinct groups with respect to 5I3C values: the tripartite cyanolichen L. pulmonaria with
n

m

the lowest 8 C values (-36 to -31), host tree foliage with intermediate 8 C values (-33 to -26),
and all other lichens including bipartite cyanolichens, hair lichens and chlorolichens with the
least negative 8 I3C values (-28 to -23). Our values are entirely consistent with those o f Maguas
et al. (1995), namely that tripartite lichen associations typically have the lowest and bipartite
lichens the highest 8 1 3 C. Carbon isotope discrimination in lichens (Riera, 2005) has been
attributed to the species o f photobiont present.
Interestingly, we observed positive relationships between tree branch height and 8 13C
values for both L. pulmonaria and foliage. Possible causes that may be responsible for the
observed height-specific differences in these 8 13C values are CO2 source, light level and factors
influencing diffusion resistance such as water availability. Carbon dioxide source influences the
8

I3C value of lichens (Farquhar & Ehleringer, 1989; Lakatos et al., 2007) since lichens growing

in the canopy or close to the soil will more readily assimilate CO2 from plant/atmosphere and
soil respiration, respectively (Broadmeadow et al., 1992; Maguas et al., 1993; Maguas et al.,
1995). Light levels influence photosynthesis and can alter the CO 2 gradient inside the lichen
thallus. Inorganic carbon acquisition in lichens is accomplished by the photobiont and depends
91

on moisture and light availability (Palmqvist, 2000). Lange et al. (1988) found that water
n

*-j

content influences 8 C values. Increased 8 C values have been reported for lichens in drier
habitats (Batts et al., 2004) and more positive 8 I3C values in epiphytes on thin versus thick
branches are generally used as indicators for desiccation stress (Hietz et al., 2002). Water stress
would be expected to increase with branch height. Therefore, differences in S13C likely exist due
to different microclimatic conditions in the canopy and/or the uptake of isotopically different
CO2 sources.
The major limitation of our study was that the lichen abundance data violated the basic
assumptions of the model due to methodological flaws in estimating the abundance of lichen on
tree branches (see Chapter 2). Due to this methodological estimation flaw, we could not reliably
assess that any significant relationship was present between any of the dependent and
independent variables as it pertains to lichen abundance. Although our model was inconclusive,
our results do suggest that cyanolichen abundance was a significant predictor of 8 1SN in lichen
functional groups. There were significant findings using the soil isotope data which suggest that
further exploration with lichen data is warranted. The sample size of 32 trees in this study only
provided enough power to detect large main effects. Increasing sample size of trees, though not
feasible in this study, would have provided greater power to detect moderate to small effects.

92

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Pike, L. H. (1978). The importance of epiphytic lichens in mineral cycling. The Bryologist,
81(2), 247-257.
Pypker, T. G., Unsworth, M. H., & Bond, B. J. (2006). The role of epiphytes in rainfall
interception by forests in the Pacific Northwest. II. Field measurements at the branch and
canopy scale. Canadian Journal o f Forest Research, 36(4), 819-832.
96

Riera, P. (2005). 8 13C and 8 I5N comparisons among different co-occurring lichen species from
littoral rocky substrata. Lichenologist, 57(1), 93-95.
Robinson, D. (2001). 8 I5N as an integrator of the nitrogen cycle. TRENDS in Ecology and
Evolution, 78(3), 153-162.
Sanborn, P., Preston, C., & Brockley, R. (2002). N 2 fixation by Sitka alder in a young lodgepole
pine stand in central interior British Columbia, Canada. Forest Ecology and Management,
767,223-231.
Shearer, G. B., & Kohl, D. H. (1986). ^-fix atio n in field settings: estimations based on natural
15N abundance. Australian Journal o f Plant Physiology, 13, 699-756.
Stocki, M. (2010). Costech ECS4010 Elemental Analyzer and Delta V Advantage Mass
Spectrometer with Conflo IV interface laboratory protocol. Stable Isotope Lab, University
of Saskatchewan, Saskatoon, SK.
Virginia, R. A., & Delwiche, C. C. (1982). Natural 15N abundance of presumed N 2 -fixing and
non-N2 -Fixing plants from selected ecosystems. Oecologia, 54, 317-325.
Vitousek, P. M., Shearer, G., & Kohl, D. H. (1989). Foliar 15N natural abundance in Hawaiian
rainforest: patterns and possible mechanisms. Oecologia, 78, 383-388.
Wallander, H., Goransson, H., & Rosengren, U. (2004). Production, standing biomass and
natural abundance o f N and C in ectomycorrhizal mycelia collected at different soil
depths in two forest types. Oecologia, 739(1), 89-97.
»c

n

Wania, R., Hietz, P., & Wanek, W. (2002). Natural 15N abundance of epiphytes depends on the
position within the forest canopy: source signals and isotope fractionation. Plant, Cell and
Environment, 25, 581-589.

97

3.7 TABLES
Table 3.1 Linear regression (OLS) coefficients with standard error for the regression model
predicting 8 15N in lichen functional groups (F = 220.501, p < 0.001, R2 = 0.817), and needles (F
= 4.363, p = 0.006, R2 = 0.126) based on tree species (spruce and fir), cyanolichen abundance,
tree branch height, and lichen species.

Intercept

Lichen Functional Groups
Needles
(Model i)___________________ (Model ii)
Unstandardized Std Error
Unstandardized Std Error
coefficient (B)
coefficient (B)
- 1 4 9 9 ***
0.165
-1.126***
0.171

Tree species (Spruce)

-0.166

0.099

-0.146

0.131

Cyanolichen abundance

<0 .0 0 1 ***

<0 . 0 0 1

<0 . 0 0 1

<0 . 0 0 1

Tree branch height

0 .0 1 0

0.008

0.028**

0.009

Alectoria sarmentosa

-3.657***

0.168

Bryoria spp.

-4.838***

0.183

Foliose chlorolichen

-0.482**

0.156

Bipartite cyanolichen

0.561***

0.137

Lobaria pulmonaria

1.608***

0.165

***p < 0.001, **p < 0.01, *p < 0.05

98

Table 3.2 Linear regression (OLS) coefficients with standard error for the regression model
predicting S13C in lichen functional groups (F = 711.517, p < 0.001, R2 = 0.942) and needles (F =
79.545, p < 0.001, R 2 = 0.724) based on tree species (spruce), cyanolichen abundance, tree
branch height, the interaction between cyanolichens and site cyanolichen abundance and lichen
species.

Intercept

Lichen Functional Groups
(Model iii)
Unstandardized Std Error
coefficient (B)
0.154
-30.523***

Needles
(Model iv)
Unstandardized Std Error
coefficient (B)
-31.868***
0.204

Tree species (Spruce)

0.048

0.089

0.622***

0.156

Cyanolichen abundance

-0 .0 0 1 ***

<0 . 0 0 1

<0 . 0 0 1

<0 . 0 0 1

Tree branch height

0.093***

0.008

0.148***

0 .0 1 1

.

<0 . 0 0 1

*

Cyanolichen X
Abundance interaction
Alectoria sarmentosa

4.899***

0.151

Bryoria spp.

4.513***

0.164

Foliose chlorolichen

3.969***

0.140

Bipartite cyanolichen

4 324***

0.166

Lobaria pulmonaria

-4.358***

0.149

0 0 0 1

***p < 0.001, **p < 0.01, *p < 0.05

99

Table 3.3 Linear regression (OLS) coefficients with standard error for the regression model (v)
predicting 51SN in soil based on cyanolichen abundance, tree species, and soil horizon (F =
314.044, p < 0.001, R2 = 0.932).

Intercept

Unstandardized
coefficient (B)
1.586***

Std Error
0.14

Cyanolichen abundance

-0 .0 0 1 ***

0 .0 0 0

Tree species

-0.525***

0.123

Mineral surface soil (Ae)

3.887***

0.145

Mineral subsoil (Bf)

4.777***

0.145

***p < 0.001, **p < 0.01, *p < 0.05

100

Table 3.4 Linear regression (OLS) coefficients with standard error for the regression model (vi)
11

predicting 8 C in soil based on cyanolichen abundance, tree species, and soil horizon (F =
117.897, p < 0.001, R2 = 0.838).

Intercept

Unstandardized Std Error
coefficient (B)
-26.923***
0.094

Cyanolichen abundance

0 0 0 0

***

0 .0 0 0

Tree species

-0 .2 2 1 **

0.062

Mineral surface soil (Ae)

1.523***

0.097

Mineral subsoil (Bf)

1.961***

0.097

.

***p < 0.001, **p < 0.01, *p < 0.05

101

3.8 FIGURES
Figure 3.1 Mean percentage of nitrogen (%N of lichen biomass ± SD) in functional groups of
lichen and foliage (needle) from 32 fir and spruce study trees at four study sites with varying
amounts of cyanolichen abundance. Different letters above error bars indicate significant (p <
0.001) differences between lichen functional groups (Tukey multiple mean comparison test).

4

3

•
0
▼
A
■
□

Alectoria sarm entosa
Bryoria spp.
Foliose chlordichen
Bipartite cyanolichen
Lobaria pulmonaria
Needles

A

d

I

%N 2

1

D
ca

i

I
Functional group

102

Figure 3.2 Mean (+.SD) N concentrations and 515N signatures of five epiphytic lichen
biomasses and the host tree foliage (needle) biomasses across four sites and two host tree species
from sub-boreal spruce and fir forests in central interior BC. Half of the sites had relatively high
cyanolichen abundances (black symbols) and the other half had low cyanolichen abundances
(white symbols).

4

2

0

I— 1
Mean S15N *2
(%o) ±SD
-4
• O Alectoria sarm entosa
♦ O Bryoria spp.
▼v Foliose chlorolichens
A A Bipartite cyanolichens
■ □ Lobaria pulmonaria
★ * Needles

-6

-8
-10
0

1

2

3

%N

103

4

5

Figure 3.3 Mean (A) 8 15N and (B) %N of host tree foliage (needle) from lowest to highest
cyanolichen abundance (total g tree ').

Mean
Needle ^
815N
(%o)

J

Needle
%N

200

400

600

800

1000

Cyanolichen abundance (total g tree ~1)

104

1200

1400

Figure 3.4 Mean 8 15N of each soil horizon, surface organic layer (F), upper most mineral horizon (Ae)
and next mineral horizon (Bf) at sites with lowest to highest cyanolichen abundance (total g tree ').

F Horizon

F Horizon

• •
08
06

00

Ae Horizon

Moon *

<%.)

^

*

v

„

06

00

Bf Horizon

Bf Horizon

08
06
04
02

o

00

200

400

600

800

1000

1200

1400

0

200

Cyanolichen Abundance (total g tree

105

400

)

600

800

1000

1200

1400

Figure 3.5 Variation in the natural abundances of (A) 8 13C and (B) 8 I5N of five epiphytic lichen
biomasses and the host tree foliage (needle) biomasses according to tree branch height above-groundlevel for all sites.

35

30

•
O
▼
A
a

H a r Lichen
Foliose Chlorolichene
Bipartite cyanolichens
T rp a tite cyanolichen
Needles

25

Tree 20
Height

<m> 15

tP.

10

5

0

•35

-36

-34

-32

-30

-28

-26

-24

-22

6,SC (%o)

-10

-8

-6

-4

515N (%q)

106

-2

0

2

4

Figure 3.6 Dual natural abundance isotope plot of 8 15N and S,3C of the five epiphytic lichen
biomasses and host tree foliage (needle) biomasses across four sites and two host tree species.

6
4

2
0

15

'2

8 N
(%o) -4

-6
-8

•
O
T
A

-10
□

A ledona sarm entosa
Bryoria spp.
Foliose Chlorolichens
Bipartite cyanolichens
Lobaria pulmonaria
Needles

-12

-14
-38

-36

-34

-32

-30

-28

5 13C (%o )

107

-26

-24

-22

-20

Figure 3.7 Mean (+.SD) C concentrations and 513C signals of five epiphytic lichen biomasses
and host tree foliage (needle) biomasses across four sites and two host tree species.

-22
•
Alectoria sarm eriosa
O Bryoria spp.
▼ Foliose chlorolichen
A Bipartite cyanolichen
■ Lobaria pulmonaria
□ Needles

-24

*4-26

Mean 5 13C
(%o) ±S D 'Z8
X

-30

-32

-34

-36
38

40

42

44

46
%C

108

48

50

52

54

Chapter 4: Conclusions
This research provides new insights into how epiphytic lichen species and functional
groups are distributed within canopies of sub-boreal spruce- and fir-dominated forests of central
BC where cyanolichen epiphytes are found at high or low amounts. In forest sites where
cyanolichens were in low abundance, typified by the absence of L. pulmonaria in its normal
lower-canopy positions, a flourishing canopy zone takeover was observed by another lichen
species, a foliose chlorolichen, Platismatia glauca. When cyanolichens including L. pulmonaria
were absent, the reordering of lichen communities in the bottom part of the forest canopy had an
effect on N contained in total epiphytic lichen biomass. Epiphytic lichen N was more than three
times as great in High Cyano sites as Low Cyano sites. Lichen biomass estimates of the five
lichen functional groups had distributions of growth that indicated that these epiphytes are nonuniformly distributed across the canopy. Fir trees had higher lichen loading than spruce trees
making the former tree species more favorable for colonization than the latter species. Finally,
the tree height below which half of the total lichen biomass was found was the same as the height
of peak lichen biomass across all lichen species.
Vertical distribution and quantities of lichens emphasize the importance of biodiversity
conservation in SBS forest systems. Lichens are ecologically important as food for herbivores,
shelter, nesting materials for a variety o f animals and they provide substrate and microhabitats
for other species (Ozturk et al., 2010). Epiphytic cyanolichens play an important role in
regulating the link between nutrient pathways, thus determining where N 2 -fixing cyanolichens
are present in the forest canopy contributes to understanding where biomass is being added to the
system. The vertical distribution of lichens within forest stands also enhances an understanding

109

of ecosystem function, nutrient cycling and changes in microclimatic conditions in the forest
ecosystem (Ellis, 2012).
The use of natural abundance of N stable isotopes in this sub-boreal canopy study has
shown that the abundance o f cyanolichens in the canopy can be a useful ecological indicator in
determining 815N of lichen functional groups and that tree branch height is valuable in
determining the 5I5N o f needles. 813C of lichen functional groups can be delineated by site
cyanolichen abundance, tree branch height, or the interaction between cyanolichens and site
cyanolichen abundance. Both tree species and tree branch height could be used to determine the
813C value of needles. Cyanolichen abundance, tree species and soil horizon were all significant
predictors of both soil 815N and 813C. Mineral subsoil was the only soil horizon that showed a
significant decrease in 8I5N with an increase in site-level cyanolichen abundance. Stable isotope
techniques have become important tools for ecophysiology and ecosystem research (Hogberg,
1997; Dawson et al., 2002) but have rarely been applied to lichen research. Determining explicit
values of stable isotopes is important for establishing unusual N isotopic ratios (Fogel et al.,
2008) of epiphytic lichens. Stable isotope abundances of lichens can be used for monitoring
environmental quality and change (Batts et al., 2004).
Future research should focus on improving the estimation method and investigate these
relationships with a larger sample size. Using the stable isotope approach is advantageous in
showing how components o f ecosystems are connected and which processes are most sensitive
to ecosystem-level changes such as clear cutting, fertilization or burning (Peterson & Fry, 1987).
It could be beneficial to look at isotope abundances in disturbed locations to determine if stable
isotope ratios change drastically in lichen, soil, and needles surrounding clear-cut forest sites or
sites affected by atmospheric pollutants.
110

Despite the significant limitations of this study, results suggest epiphytic lichens,
particularly those that can fix atmospheric N 2 , may provide important N-inputs into northern
interior forests of BC. Nitrogen is the most limiting nutrient for these forest ecosystems, and the
sustainability o f current forest communities may depend on the conservation of N 2 -fixing
epiphytic lichens, which are generally most abundant in mature and old growth stands.
Sustainability of social communities may also depend on the conservation of epiphytic lichens
relative to forestry. Study results could influence logging areas in the central interior of northern
BC. Composition of epiphytic lichens should be considered in forest management practices to
maintain greater species abundance and richness in the forest system in order to increase
diversity.
Cyanolichens could also be introduced to nitrogen-deficient forests. Management of
forests and lichen biodiversity influence the storage o f N in sub-boreal landscapes. Disturbances
to these sub-boreal forests can have an impact on forest diversity and function and should be
monitored closely. Shorter lived and statured subalpine fir supports larger biomasses of all
species relative to the canopy-dominant interior hybrid spruce. The mixture of spruce and fir
may provide for greater lichen epiphyte abundances as well as cyanolichen N-inputs into these
sub-boreal forests o f central BC.

I ll

4.1 Literature Cited

Batts, J. E., Calder, L. J., & Batts, B. D. (2004). Utilizing stable isotope abundances of lichens to
monitor environmental change. Chemical Geology, 204, 345 - 368.
Dawson, T. E., Mambelli, S., Plamboeck, A. H., Templer, P. H., & Tu, K. P. (2002). Stable
isotopes in plant ecology. Annual Review o f Ecology and Systematics, 35(1), 507-559.
Ellis, C. J. (2012). Lichen epiphyte diversity: a species, community and trait-based review.
Perspectives in Plant Ecology, Evolution and Systematics 14(2), 131-152.
Fogel, M. L., Wooller, M. J., Cheeseman, J., Smallwood, B. J., Roberts, Q., Romero, I., &
Meyers, M. J. (2008). Unusually negative nitrogen isotopic compositions (6I5N) of
mangroves and lichens in an oligotrophic, microbially-influenced ecosystem.
Biogeosciences, 5(6), 1693-1704.
Hogberg, P. (1997). Tansley Review No . 95 15 N natural abundance in soil - plant systems.
New Phytologist, 179-203.
Ozturk, S., Oran, S., Guvenc, S., & Dalkiran, N. (2010). Analysis of the distribution of epiphytic
lichens in the oriental beech (Fagus orientalis lipsky) forests along an altitudinal gradient in
Uludag Mountain, Bursa - Turkey. Pakistan Journal o f Botany, 42(4), 2661-2670.
Peterson, B. J., & Fry, B. (1987). Stable isotopes in ecosystem studies. Annual Review o f
Ecology and Systematics, 18( 1), 293-320.

112