EXAMINING THE SOIL LEGACY EFFECTS OF SPOTTED
KNAPWEED (Centaurea stoebe)
by

MATTHEW GUNN COGHILL
B. Sc., Thompson Rivers University, 2015

A thesis submitted in partial fulfillment of the requirements for the degree of:
MASTER OF SCIENCE IN ENVIRONMENTAL SCIENCE

Thesis examining committee:

Lauchlan Fraser (PhD), Professor and Thesis Supervisor, Department of Natural Resource
Sciences, Thompson Rivers University

Lyn Baldwin (PhD), Associate Professor and Committee Member, Faculty of Science,
Thompson Rivers University

Wendy Gardner (PhD), Associate Professor and Committee Member, Department of Natural
Resource Sciences, Thompson Rivers University

Winter 2021
Thompson Rivers University
© Matthew Gunn Coghill, 2021

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Abstract
Spotted knapweed (Centaurea stoebe) is the most aggressive invasive forb in North
American grasslands. Since its arrival, it has spread from the west coast of North America
and reached far east. It has been able to accomplish this via the production of many small
seeds per plant, and by altering soil conditions making it difficult for native plants to grow in.
Control efforts have been extensive. Broadcast chemical controls have been applied, as well
as biological and physical controls; however, despite these interventions, spotted knapweed
continues to have negative effects on ecosystems and their functions. Spotted knapweed may
have a negative legacy effect in the soils they inhabit, which perpetuates even after removal
of this plant. To test for potential soil legacy effects, a greenhouse experiment was devised in
which C. stoebe and rough fescue (Festuca campestris) were grown in different soil types.
Activated carbon and pulp mill fly ash were used as soil amendments in each soil type in an
attempt to return soils to a pristine state, and we found that F. campestris grew best in
unamended invaded soils during a 90-day growing period. Pre-growing conditions of this soil
displayed lower levels of both carbon and nitrogen compared to other soil types, indicating
that F. campestris grew best in less hospitable conditions. As this was an unexpected result, a
field experiment was designed in which different concentrations of ash were applied to
transplanted rough fescue plugs; however, plug viability tapered off quickly after
transplanting. Conclusions drawn from this study indicate ash as a potential soil amendment
for knapweed-affected soils with directions for use in future research. Further investigation
into the use of ash as a broadscale solution to negative soil legacy effects is warranted. Ash,
an industrial waste product, could be potentially useful in areas heavily invaded by spotted
knapweed in order to deter the spread of these noxious weeds.

Keywords: invasive, spotted knapweed, fly ash, activated carbon, rough fescue, greenhouse

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Table of Contents
Abstract ..................................................................................................................................... ii
Table of Contents ..................................................................................................................... iii
Acknowledgements ................................................................................................................. vii
List of Figures ........................................................................................................................ viii
List of Tables ............................................................................................................................ x
Chapter 1: Introduction ............................................................................................................. 1
Spotted Knapweed ................................................................................................................ 2
Rough Fescue ........................................................................................................................ 6
Activated Carbon .................................................................................................................. 9
Fly Ash ................................................................................................................................ 11
Current Study ...................................................................................................................... 12
Literature Cited ................................................................................................................... 13
Chapter 2: Examining soil legacy effects of Centaurea stoebe in the context of the Lac Du
Bois Grasslands Protected Area ...................................................................................... 22
Introduction ......................................................................................................................... 22
Immediate Soil Effects and Catechin .............................................................................. 22
Soil Legacy Effects ......................................................................................................... 24
Methods .............................................................................................................................. 26
Site Selection .................................................................................................................. 26
GIS Considerations ......................................................................................................... 28
Soil, Ash, and AC Collection and Characterization ....................................................... 30

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Soil Preparation and Greenhouse Trials.......................................................................... 31
Elemental Analysis ......................................................................................................... 34
Data Analysis .................................................................................................................. 34
Field Data .................................................................................................................... 34
Greenhouse Data ......................................................................................................... 35
Biomass ................................................................................................................... 35
Plant Survival .......................................................................................................... 36
Elemental Analysis ................................................................................................. 37
Results ................................................................................................................................. 37
GIS Considerations ......................................................................................................... 37
Field Collections ............................................................................................................. 41
Greenhouse Trials ........................................................................................................... 44
Biomass ....................................................................................................................... 44
Plant Survival .............................................................................................................. 46
Germination Rate .................................................................................................... 46
Death Rate............................................................................................................... 48
Germination Survival Rate ..................................................................................... 50
Trial Survival Rate .................................................................................................. 52
Elemental Analysis ......................................................................................................... 54
Discussion ........................................................................................................................... 59
Preferential Invasion based on Biogeoclimatic Subzone ................................................ 59
Positive Soil Legacies Detected ...................................................................................... 60

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Impacts of Ash on Biomass ............................................................................................ 62
Effects of Ash and AC on Survival and Mortality .......................................................... 63
Invasive Boundary Movement ........................................................................................ 64
Limitations ...................................................................................................................... 65
Greenhouse Conditions ............................................................................................... 65
Soil Conditions............................................................................................................ 66
Potting ......................................................................................................................... 67
Elemental Analysis ..................................................................................................... 67
Literature Cited ................................................................................................................... 68
Chapter 3: Research Conclusions ........................................................................................... 75
Research Implications ......................................................................................................... 75
Management Implications................................................................................................... 77
Literature Cited ................................................................................................................... 79
Appendix A: Characterizing immediate and legacy soil effects in the field: A lesson in
transplanting .................................................................................................................... 81
Introduction ......................................................................................................................... 81
Methods .............................................................................................................................. 82
Seedling Germination ..................................................................................................... 82
Field Design .................................................................................................................... 82
Statistical Analyses ......................................................................................................... 84
Results ................................................................................................................................. 85
Trends with Survived Plugs ............................................................................................ 85
Biomass ........................................................................................................................... 86

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Leaf Height and Number of Leaves ................................................................................ 88
Discussion ........................................................................................................................... 92
Literature Cited ................................................................................................................... 94
Appendix B: Data Tables ........................................................................................................ 95

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Acknowledgements
I would first like to acknowledge my supervisory committee for being incredibly
patient with me over the years. Your support kept me going when times were tough, and I
cannot thank you all enough for being a great team. Thank you to NSERC for project
funding, Domtar Pulp for the supply of fly ash, and Thompson Rivers University for being a
second home to me for the last 9 years.
Thank you to all of the people at the Fraser Lab that have assisted me in this project,
your efforts have not gone unnoticed. There have been many research assistants that have
had their hands on this project as well as fellow Master of Science students and I hope that by
completing this project that I’ve done you all proud: Colton Stephens, Brandon Williams,
Sarah Bayliff, Kristi Gordon, Rachel Whitehouse, Piotr Dzumek, Stephen Kega, Jordann
Foster, Janelle Paulson, Chantalle Gervan, Solenn Vogel, Liam Fraser, Stephanie Jensen,
Nicholas Peterson and Sierra Rae. Thank you all for the great adventures in the field, and the
many laughs and thoughtful discussions that we all shared. You all made the Fraser Lab a
better place when you were around. Thanks to my family for their love and support to get this
thesis finished up. Finally, thank you to my wife Kirstyn for your patience and your endless
support to me.
I have to single out my supervisor, Dr. Lauchlan Fraser. It has been a true honor
working as a student of yours for the last few years. Your passion and enthusiasm for
environmental science are awe inspiring, and I’ve been lucky to watch as your lab has grown
over the years. Thank you for the endless opportunities that you’ve thrown my way,
including being a research assistant, the PlantPopNet project, the HerbDivNet project,
helping out on mine projects and other associated field work, being a lab manager, and of
course being your student. I think that I took so long to finish this because I didn’t want to
go. Thank you for everything, you are a true role model for everything that is awesome.

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List of Figures
Figure 1.1: Aboveground development of Centaurea stoebe from seed (left) to an adult
flowering plant (right). In Keil & Ochsmann (1993), courtesy of the Flora of North America
Administration, illustrated by John Myers................................................................................ 4
Figure 1.2: Flowering structures of Festuca campestris with approximate measurements. In
Darbyshire & Pavlickf (1993), courtesy of the Flora of North America Administration,
copyright Utah State University, illustrated by Cindy Roché. ................................................. 8
Figure 2.1: Provincial context of the Lac Du Bois Grasslands Protected Area and the
associated C. stoebe extent of invasion throughout the park. Further spatial context is given
for the location of the study area within the Lac Du Bois Grasslands Protected Area. The map
was created using QGIS version 3.16.0 (Hannover)............................................................... 28
Figure 2.2: Ground temperature from invaded (red) and pristine (black) sites in the Lac Du
Bois GPA from June 12 - August 30, 2017. Extended vertical bars represent actual ground
temperature readings at 2-hour intervals, and the smoothed lines represent mean daily ground
temperatures and a standard error at invaded and pristine sites.............................................. 43
Figure 2.3: Square root of biomass (mean ± SD) of Festuca campestris (left) and Centaurea
stoebe (right) when grown in different soil types and soil treatments. Comparisons of
biomass are made between control, ash, and activated carbon soil treatments for each soil
type. P-values above brackets denote significant pairwise comparisons between soil
treatments as determined by pairwise t-tests. ......................................................................... 45
Figure 2.4: The square root of germination rates (means ± SD) of Festuca campestris and
Centaurea stoebe when grown in different soil treatments and soil types. Comparisons of the
germination rates are made between control, ash, and activated carbon treated soils for each
soil type. P-values above brackets denote significant pairwise comparisons between soil
treatments as determined by pairwise t-tests. ......................................................................... 47
Figure 2.5: The aligned rank transformed death rates (means ± SD) of Festuca campestris
and Centaurea stoebe when grown in different soil treatments and soil types. Comparisons of
the death rates are made between control, ash, and activated carbon treated soils in each soil

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type. P-values above brackets denote significant pairwise comparisons between soil
treatments as determined by pairwise t-tests. ......................................................................... 49
Figure 2.6: The aligned rank transformed germination survival rates (means ± SD) of Festuca
campestris (left) and Centaurea stoebe (right) when grown in different soil types and soil
treatments. Comparisons of the germination survival rates are made between control, ash,
and activated carbon treated soils in each soil type. P-values above brackets denote
significant pairwise comparisons between soil treatments as determined by pairwise t-tests.51
Figure 2.7: The aligned rank transformed trial survival rates (means ± SD) rates of Festuca
campestris and Centaurea stoebe when grown in different soil treatments and soil types.
Comparisons of the trial survival rates are made between control, ash, and activated carbon
treated soils in each soil type. P-values above brackets denote significant pairwise
comparisons between soil treatments as determined by pairwise t-tests. ............................... 53
Figure 2.8: Total carbon, nitrogen, and hydrogen concentrations, as well as the aligned rank
transformation of the carbon to nitrogen ratio in each soil type (N = 4 for each group). Pvalues above brackets denote significant pairwise comparisons between soil treatments as
determined by pairwise t-tests. ............................................................................................... 57
Figure A.1: Design of the field grids. The crosshatches represent the 9 sample points within
the 2 m × 2 m grid. .................................................................................................................. 84
Figure A.2: Measurements of number of leaves (top) and leaf height (bottom) for each
treatment taken on the planting date and the harvest date (N = 10 for each treatment). Points
connected by lines are indicative of individuals change in either leaf height or number of
leaves....................................................................................................................................... 91

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List of Tables
Table 2.1: Environmental variables and values pertaining to optimum growth of many plants.
These conditions were replicated in the research greenhouse for the growth experiment.
Retrieved from Hendry & Grime (1993). ............................................................................... 33
Table 2.2: Expectations of the impacts to the growth of Centaurea stoebe and Festuca
campestris in each soil type based on existing literature. ....................................................... 33
Table 2.3: Description of plant survival metrics to be applied to each individual pot. In this
case, the number of seeds planted equates to 5. ...................................................................... 37
Table 2.5: Biogeoclimatic Subzones of the Lac Du Bois Grasslands Protected Area. Detailed
for each subzone are descriptions of the subzone codes, the area in km2 that the subzone
takes up in the Lac Du Bois GPA, and associated elevation descriptions gathered from
TRIM....................................................................................................................................... 38
Table 2.6: Details of plant invasion in the Lac Du Bois Grasslands Protected Area broken
down by BGC subzone. “Total” values indicate the total number of records, total physical
area, and invaded ratio of all invasive plants combined throughout the Lac Du Bois GPA in
order to highlight the same metrics for C. stoebe only. Numbers of records sum to be greater
than the total number of records throughout the Lac Du Bois GPA due to splitting of
polygons during spatial intersections. ..................................................................................... 39
Table 2.7: Province wide BGC details, only including the BGC subzones that were also
within the Lac Du Bois Grasslands Protected Area. Rank values refer to the entire list of 210
subzones with a rank of 1 being the highest rank. .................................................................. 41
Table 2.8: Details of plant invasion throughout B.C. broken down by BGC subzone. “Total”
values indicate the total number of records, total physical area, and invaded ratio of all
invasive plants combined throughout B.C. in order to highlight the same metrics for C.
stoebe only. ............................................................................................................................. 41

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Table 2.9: Summarized comparisons of field metrics between pristine and invaded sites, and
the respective T-test or Wilcox rank sum test results. Asterisks on P-values indicate
significant differences at the 0.05 level for that metric between pristine and invaded sites... 42
Table 2.4: Summarized comparisons of elevation, slope, and aspect between pristine and
invaded sites, and their associated T-test results. N = 10 for each site. .................................. 43
Table 2.10: Results of a two-way ANOVA, performed to discover differences in the square
root of plant biomass with respect to each factor and their interactions. F and p values are
reported for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK",
respectively. Significant values are bold faced. ...................................................................... 45
Table 2.11: Exploration of the main effects of soil treatment and soil type on the square root
of biomass for each species. F and p values are reported for both Festuca campestris and
Centaurea stoebe as subscripts "RF" and "SK", respectively. Significant values are bold
faced. ....................................................................................................................................... 46
Table 2.12: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the square root of the germination rate. F and p values are reported for both
Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced. ........................................................................................... 48
Table 2.13: Exploration of the main effects of soil treatment and soil type on the square root
of the germination rate of each species. F and p values are reported for both Festuca
campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively. Significant
values are bold faced. .............................................................................................................. 48
Table 2.14: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the aligned rank transformation of the death rate. F and p values are reported for
both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced. ........................................................................................... 50
Table 2.15: Exploration of the main effects of soil treatment and soil type on the aligned rank
transformation of the death rate of each species. F and p values are reported for both Festuca
campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively. ....................... 50

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Table 2.16: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the aligned rank transformation of the germination survival rate. F and p values
are reported for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK",
respectively. Significant values are bold faced. ...................................................................... 52
Table 2.17: Exploration of the main effects of soil treatment and soil type on the aligned rank
transformation of the germination survival rate of each species. F and p values are reported
for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced. ........................................................................................... 52
Table 2.18: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the aligned rank transformation of the trial survival rate. F and p values are
reported for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK",
respectively. Significant values are bold faced. ...................................................................... 54
Table 2.19: Exploration of the main effects of soil treatment and soil type on the aligned rank
transformation of the trial survival rate of each species. F and p values are reported for both
Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced. ........................................................................................... 54
Table 2.20: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on carbon, nitrogen, and hydrogen concentrations, as well as the ratio of carbon to
nitrogen. Significant values are bold faced. ............................................................................ 55
Table 2.21: Exploration of the main effects of soil treatment and soil type on carbon,
nitrogen, and hydrogen concentrations, as well as the ratio of carbon to nitrogen. Significant
values are bold faced. .............................................................................................................. 56
Table 2.22: Results from the major elements analysis. Concentration values reported are
separated by reported units for simplicity. C, H, and N values for soil blends are mean values
gathered from analysis using the FlashSmart CHNS/CHNS Elemental Analyzer whereas pure
ash and AC values were gathered from literature (see footnotes). ......................................... 58
Table A.1: Summarized field data captured from both the planting date (May 2, 2018) and
the harvest date (July 30, 2018). The values in this table are only representative from plants

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which survived the full 90-day growing period, hence the different values for N. Mean values
are appended with standard errors to indicate the spread of the data. .................................... 86
Table A.2: Summarized mean and standard deviation of biomass of F. campestris grown in
each soil treatment. ................................................................................................................. 87
Table A.3: Tukey HSD pairwise comparisons of biomass, change in number of leaves, and
leaf height change between each treatment grouping. ............................................................ 87
Table A.4: Results from a two-way repeated measures ANOVA on leaf height and number of
leaves separately. Effect of time and treatments are reported for each group. Significant p
values are bold faced. P-values on the treatment and timing effects have been adjusted using
the Bonferroni method accounting for the different number of groupings. ............................ 89
Table A.5: Results of pairwise T-tests of leaf height and number of leaves between each
treatment group, separated by planting and harvest timing. T statistics and Bonferroni
adjusted p values are reported. ................................................................................................ 90
Table A.6: Results of pairwise T-tests of leaf height and number of leaves between planting
and harvesting, separated by treatment group. T statistics and Bonferroni adjusted p values
are reported. Significant p values are bold faced. ................................................................... 91
Table B.1: X, Y coordinates for the sampling locations of each site in the Lac Du Bois GPA.
The coordinate reference system used is NAD 1983 BC Environment Albers (EPSG 3005).
................................................................................................................................................. 95
Table B.2: X, Y coordinates of the soil temperature monitors used to gather ground
temperature data. The coordinate reference system used in NAD 1983 BC Environment
Albers (EPSG 3005). .............................................................................................................. 95
Table B.3: Plant inventory for the sampled pristine and invaded areas, and respective mean
cover values in top-down percent (N = 10). A value of NULL indicates that the plant was not
found in any of the plots. Bare ground and litter covers were estimated via ground cover
measurements rather than top-down. Invasive species are denoted with an asterisk prepended
to their scientific names. All scientific and common names were retrieved from Antos et al.
(2018). ..................................................................................................................................... 96

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Chapter 1: Introduction
Invasive species are defined by three elements: 1) the species is introduced to a new
area with human assistance, 2) the species can establish and spread without human
assistance, and 3) the species is found far beyond its point of arrival (Simberloff, 2013).
Introduced species, in comparison are defined only by the first element in the invasive
species definition. Throughout the kingdom of life, there are many examples of both invasive
and introduced plants, animals, fungi, and microorganisms (Lowe et al., 2000). Introductions
of species may be intentional. For example, in re-introduction efforts to bolster wild
populations of wolves in Yellowstone National Park (Phillips & Smith, 1997), or for
aesthetic purposes to make a garden appear more vibrant or exotic. Introductions may also be
unintentional, for example during an exchange of ballast water on ships dinoflagellates may
be taken in at one international port and transported to another port where they would be
released during the next ballast exchange (Hallegraeff, 1998). No matter the intent, humans
are left to manage the consequences of these introductions, including a full-scale invasion by
an introduced species. Once introduced, invasive species increase in abundance, often at the
expense of native species (Simberloff et al., 2013). Without proper management and in more
extreme cases, invasive species may devastate the biodiversity of an area by removing the
local species to establish dominance (Simberloff et al., 2013; Vilà et al., 2011).
Plants may have traits that allow for successful establishment into new areas. This is
especially true for invasive plants. For example, small seeded invasive plants may go
unnoticed and become attached to animals, our clothing or vehicles, allowing them to be
transported long distances (Hodkinson et al., 1998). Other invasive plants can alter their
surroundings by negatively altering soil conditions, thereby impacting available forage for
grazing animals (Vilà et al., 2011; Watson & Renney, 1974). In British Columbia (BC),
Canada, there are approximately 229 plant species that exhibit invasive traits (Ministry of
Forests and Range - Range Branch, 2020). As a result, researching and developing strategies
for managing these ecological threats should be prioritized. This is especially important in
ecosystems under severe threat of collapse due to pressure from invasive species
(Klinkenberg, 2012). In BC, grasslands account for less than 1% of the total land base but
provide habitat for approximately 30% of the provinces’ species at risk, making grasslands

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one of the most critical ecosystems to protect from the threat of invasion (Wikeem &
Wikeem, 2004). Current management solutions in place for many invasive species are
reactive (i.e., invasive plants are managed after they become problematic), though proactive
approaches could allow us to prevent invasions altogether. In this way, land managers can
prevent the spread of invasives before they negatively impact ecosystems. To achieve this,
we first need a complete understanding of the invasive species in question.
Lac Du Bois Grasslands Protected Area, a provincial park located north of Kamloops,
BC, is a complex mosaic of both pristine grassland and disturbed, invaded landscape. The
undisturbed areas of the upper Lac Du Bois park are described as rough fescue (Festuca
campestris) grassland woven with layers of shrubs, forbs, grasses, and biological crusts
throughout (Delesalle et al., 2009). Many invasive plants have made their way into this
region, some forming large patches that lie adjacent to native plant communities (Invasive
Alien Plant Program, 2017). Spotted knapweed (Centaurea stoebe L.) is an invasive plant
readily found in this park despite the control efforts that have been applied since 1970 (Fraser
& Carlyle, 2011; Gayton & Miller, 2012). The impact spotted knapweed may have on
ecosystem function, its mechanisms of invasion, and its susceptibility to control methods
deserve further attention.

SPOTTED KNAPWEED
In B.C., spotted knapweed (Centaurea stoebe L.) is considered an invasive, noxious
forb (Klinkenberg, 2012). It is hypothesized to have arrived in North America in the late
1800s, likely as a contaminant in alfalfa seed shipments from Europe (Moore, 1972; Moore
et al., 1974). Spotted knapweed inhabits upper grassland regions near forest interfaces where
it roots in rich soils. Since its arrival in North America, spotted knapweed has genetically
diverged from its European ancestry, thereby becoming an invasive plant in two key ways: 1)
it is diploid at the chromosome level when found in Europe but in North America it is
tetraploid, and 2) it is a biennial plant in Europe, whereas it is a more aggressive and shortlived perennial in North America (Jacobs, 2012). As such, these changes warrant that spotted
knapweed be placed into two subspecies based on location. The native, European, diploid

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biennial is classified into subspecies Centaurea stoebe spp. stoebe, and the invasive, North
American, tetraploid perennial is subspecies Centaurea stoebe spp. micranthos (Integrated
Taxonomic Information System, 2017; Jacobs, 2012; Keil & Ochsmann, 1993). However,
there is still a lack of uniformity of the scientific name applied to North American spotted
knapweed. For example, the Encyclopedia of Life (EOL), The Plant List, and the PanEuropean Species directories Infrastructure (PESI) list North American spotted knapweed as
Centaurea stoebe ssp. australis (Pančić ex A. Kern.) Greuter, but the Integrated Taxonomic
Information System (ITIS), United States Department of Agriculture (USDA) PLANTS
Database, and E-Flora BC refer to spotted knapweed as Centaurea stoebe ssp. micranthos L.
(Gugler) Hayek (Carpinelli, 2013; Greuter, 2007; Integrated Taxonomic Information System,
2017; Klinkenberg, 2017; The Plant List, 2013; USDA NRCS, 2017). Generic and scholarly
search tools return more results for the term “Centaurea stoebe ssp. micranthos” compared to
the term “Centaurea stoebe ssp. australis". For this reason and for simplicity, C. stoebe will
refer to Centaurea stoebe spp. micranthos throughout this thesis, unless otherwise specified.
Centaurea stoebe reproduces only by seed production and may produce tens of
thousands of seeds per square meter in a single growing season (Jacobs, 2012; Schirman,
1981). This method of reproduction allows C. stoebe to continually expand within existing
stands as many of the seeds will drop in the immediate vicinity. When mature, seeds disperse
via attachment to animals or vehicles, wind, or water (Sheley et al., 1998). Seeds have a high
chance of germinating once they are established in an area, and can remain viable in soils for
eight or more years (Davis et al., 1993). Combined, these traits allow for rapid dispersal. In
North America, spotted knapweed is now found in 46 states within the United States and
eight provinces and territories of Canada (Desmet & Brouilet, 2013; EDDMapS, 2017).
Seedlings emerge during the first year of growth and share physical similarities with some
other forb species (Figure 1.1). The next growth stage is the rosette, characterized by
pinnatifid leaves with many narrow teeth. The rosette may develop directly from the seedling
within the same growing season depending on when the seedling first emerged. Finally,
flowering plants are upright with highly branched stems and pink to purple flowering heads
on the branch ends. When C. stoebe is flowering, it can be distinguished from other forbs and
knapweeds by the black-tipped involucre bracts beneath the flowering head, giving it a

4
spotted appearance and lending to its name. There may be up to 30 flowers in each composite
flowering head, all producing one seed each. With large, branching individuals, it is thus
reasonable to observe a single plant producing over 10,000 seeds.

Figure 1.1: Aboveground development of Centaurea stoebe from seed (left) to an adult
flowering plant (right). In Keil & Ochsmann (1993), courtesy of the Flora of North America
Administration, illustrated by John Myers.

Belowground, C. stoebe has a deep taproot which presents another challenge for
restoration and management of this weed (MacDonald et al., 2013). The lateral root system is
moderately sized in relation to the taproot and forms larger mats in older individuals, making
it more difficult to remove. On top of that, the root system as a whole has the ability to
negatively influence surface runoff, soil erosion, plant communities, and ecosystem
processes compared to pristine areas (Lacey et al., 1989). C. stoebe, when established, will
drain soils of available nutrients and water faster than native plants. This induces a cascade
where C. stoebe outcompetes native plants, reduces biodiversity, and increases the amount of
runoff and soil erosion that a given area receives. Furthermore, this plant is suspected to
release catechin into the soil, a phytotoxic chemical that inhibits the growth of nearby plants
(Bais et al., 2003; Reinhart & Rinella, 2011; Ridenour & Callaway, 2001). To sum up, C.

5
stoebe produces numerous seeds which remain viable for years and can outcompete other
plants for resources and space.
Currently, there are a few methods in use to eradicate C. stoebe in North America.
One method is physical removal by hand pulling (Jacobs, 2012; Sheley et al., 1998). This
method works well in certain scenarios if a few conditions are met: 1) when the soil is wet as
this allows for the entire plant with taproot to be removed, 2) individuals are immediately
placed in sealed plastic bags to prevent the spread of seeds or pollen during transportation,
and 3) the site is revisited in later years as multiple hand removal events are required. This
method is often avoided due to the reliance on suitable weather and the need for repeated
visits. Thus, biocontrol agents and herbicide treatments have been attempted for controlling
this weed. In BC, there are at least 12 biocontrol insects that have been used to target C.
stoebe (Gayton & Miller, 2012; Invasive Species Council of British Columbia, 2014;
Province of British Columbia, 2002). Some of these insects will feed on the root system of
the knapweed, which can disrupt the belowground functioning, and structural integrity of the
plant itself (e.g., Cyphocleonus achates, a wood boring weevil). Other biocontrol insects
forage the seeds and seed heads of the knapweed plant which ultimately decreases the level
of spread and invasion (ex: Larinus minutus, a seed-feeding beetle). It is also possible to
reduce C. stoebe biomass through grazing with sheep and goats, as well as cattle though it is
less palatable to them (DiTomaso, 2000; Olson et al., 1997). Targeted grazing on invasive
plants has had some positive effects in the ranching industry (Voth, 2010). Although grazing
by livestock is a form of biological control, it may not be feasible to graze all invaded areas
due to accessibility and terrain (Olson et al., 1997).
In addition to mechanical or grazing controls, chemical controls have shown to
reduce the spread of C. stoebe. Herbicide use is the most common form of control of C.
stoebe, though some precautions should be considered: 1) picloram is the most effective
active ingredient in herbicide treatments against both C. stoebe and C. diffusa, but should
avoid being used on coarse soils since it can negatively impact neighboring broadleaf herbs,
and 2) it is best to apply herbicide treatments to the rosette stage of a plants development
(Mackay, 2008). The broad use of herbicides may bring forth challenges into the future with
regards to chemical resistance. This has been first documented in 2016 in an invaded range in

6
East Kootenay, BC. This range had been historically treated with various herbicides to deal
with the spread of C. stoebe, and when individuals from this range were grown in a
greenhouse alongside plants from a historically untreated range, applications of picloram and
clopyralid did not have as pronounced of a response in plants from treated areas as opposed
to plants from untreated areas (Mangin & Hall, 2016). The implication of resistance
demonstrates that further action is required to mitigate the invasive action of C. stoebe and
perhaps long-term management methods should be developed to limit the impact of C. stoebe
on the ecosystems it invades.
Restoration efforts associated with the spread of C. stoebe are mostly tailored towards
revegetation of an affected area after treatment, usually with a native seed mix (Jacobs,
2017). Obviously, there is support for this method because it re-establishes past communities,
increases biodiversity, and improves soil conditions and ecosystem health. However, this
ignores the existing seed bank as well as the physical and chemical makeup of the affected
soils. While combinations of mechanical, biocontrol, and chemical treatments can address the
seed bank issue, these treatments do not address changes of soil structure and composition.
Specifically, the accumulation of phytotoxins in invaded soils can leave a long term or legacy
effect that persists into the future, inhibiting reseeding efforts (Del Fabbro & Prati, 2015;
Grove, 2014; Grove et al., 2012). Such legacy effects have yet to be studied in detail, and it is
unknown with C. stoebe how important these effects are or how to effectively treat them. A
better understanding of these legacy effects is thus necessary for land managers to strategize
restoration plans.

ROUGH FESCUE
Grasslands in BC can be divided based on elevation, plant communities, and climate
patterns. From valley bottoms to about 600 m above sea level, lower grassland communities
are generally dominated by drought resistant shrubs of big sagebrush (Artemisia tridentata)
and groups of bluebunch wheatgrass (Pseudoroegneria spicata) (Lee et al., 2014; van
Ryswyk et al., 1966). These regions of the grasslands are typically hotter and drier than other
grassland regions. As a result, moisture is limiting and can lead to large gaps of bare ground

7
between individual plants. A transition zone between lower and upper grassland communities
occurs between 600-900 m above sea level. These middle grasslands receive more moisture
than in the lower grasslands causing a reduction of A. tridentata, but other dryland plants
common to the lower grasslands, including P. spicata, remain present in this zone. Upper
grasslands tend to occur near Interior Douglas Fir forests between 900-1200 m above sea
level. There are smaller gaps between individual plants at this level and thus a higher total
cover of vegetation. Mild temperatures and increased precipitation ultimately allows for
greater plant diversity compared to lower elevation grasslands as well (Wikeem & Wikeem,
2004). The upper grassland communities are commonly dominated by rough fescue (Festuca
campestris Rydb.), a cool season, native, perennial bunchgrass (Fleenor, 2011). Biological
crusts and a heavy litter layer assist in moisture retention in these grasslands which assist in
the development of rich soils; however, these favorable growing conditions also allow for
invasive plant encroachment and subsequent ecosystem damage (Delesalle et al., 2009;
Fraser & Carlyle, 2011).
Festuca campestris forms large bunches from the older leaves which aid in moisture
retention. It forms a fibrous root system but does not penetrate deep soils. Leaves are
generally 10-60 cm long, 1-2 mm wide, and rough on the lower surface (Fleenor, 2011).
Stems stand 30-90 cm tall without nodes and, when mature, the inflorescence is paniculate
measuring 5-18 cm in length with 2 branches per node (Figure 1.2). Spikelets are 8-12 mm
long housing between 3-7 florets and are purplish (Douglas et al., 2001). The glumes of the
spikelet are shorter than the floret, and the lemmas, which are either sharply pointed or
awned, measure between 7-8.5 mm long. F. campestris reproduces primarily via vegetative
regeneration or seed production, though the amount of seed produced can vary by year (Hook
et al., 1994; Johnston & MacDonald, 1967). This presents further opportunity for invasive
plants to establish in years that fewer seeds are produced simply by germinant number.
Fortunately, germination rates are relatively high for this species (86-97%), thus in habitable
areas not affected by invasive plants F. campestris can successfully establish and become
forage for herbivores (Fleenor, 2011; Johnston & MacDonald, 1967).

8

Figure 1.2: Flowering structures of Festuca campestris with approximate measurements. In
Darbyshire & Pavlickf (1993), courtesy of the Flora of North America Administration,
copyright Utah State University, illustrated by Cindy Roché.

Festuca campestris is often preferred by grazing livestock (Fleenor, 2011).
Incidentally, it has high protein content which is retained even when hayed for winter
grazing, thus it has a high forage value (Alberta Prairie Conservation Forum, 2017). In turn,
livestock improve grassland conditions by providing a fertilizer (nitrogen) through their
waste which can continue to be useful even after the year it was applied (Forge et al., 2005;
Schröder et al., 2007). Despite this reciprocal relationship, research in Alberta has shown that
over-stocking a rangeland can have negative consequences including decreases in F.
campestris cover, increases in less valuable forage, and deterioration of the grasslands
(Willms et al., 1985). Overgrazing may also be a precursor to plant invasion as well,
therefore it is imperative that ranchers manage their livestock effectively to avoid these
impacts and ensure more productive grasslands in future growing seasons for their livestock
(Mack et al., 2000). With such high importance in ecosystem function and within the

9
ranching industry, rough fescue is an ideal and important plant to study alongside the
invasive, noxious spotted knapweed.

ACTIVATED CARBON
Activated carbon (AC) is a fine, porous material known for its binding properties and
adsorption abilities (Bansal & Goyal, 2005; Chiang et al., 2001; Hameed et al., 2007). More
technically, AC contains spaces enclosed by carbon atoms in between graphene layers, and
during an activation process AC will develop adsorption sites which will vary in size and
shape (Marsh & Rodríguez-Reinoso, 2006c). Activation of carbon molecules can happen
through thermal, physical, or chemical means, thus AC is always a synthetic product (Marsh
& Rodríguez-Reinoso, 2006a). The final product has an incredibly large surface area per unit
volume due to the atomic porosity of the material. AC has a wide variety of uses.
Industrially, it has uses in water purification, air pollution reduction, removal of dyes from
wastewater, and methane storage, all accomplished by adsorption processes (Marsh &
Rodríguez-Reinoso, 2006b; Pereira et al., 2003; Rodríguez-Reinoso, 2002). AC also has its
use as a consumer product where it is commonly used as an aquarium water purifier. These
adsorptive properties have spawned research on the potential removal of allelopathic
compounds from invasive plants using AC as a soil amendment (Nilsson, 1994; Prati &
Bossdorf, 2004; Ridenour & Callaway, 2001). Catechin, the suspected allelopathic
compound of C. stoebe, is also readily adsorbed by AC when added to soils (Callaway &
Aschehoug, 2000). The use of AC thus is important in studies of allelopathy; however, its use
in studies of soil legacy effects are unclear. For instance, Grove et al. (2012) studied the
effects that the invasive Cytisus scoparius (allelopathic shrub) had on Douglas-fir in the
western US. They found that Cytisus invaded soils negatively affected the growth of
Douglas-fir in comparison to soils from the nearby non-invaded forest. The invaded soils
continued to have a negative effect on Douglas-fir growth in the presence of either AC or
Cytisus litter alone, but the combination of AC and Cytisus litter had a positive effect on
Douglas-fir growth possibly caused by a fertilizing effect of the litter. These results
suggested that hand pulling an invasive plant was not enough to encourage native plant

10
growth in the area; rather, a soil amendment was required to overcome the soil legacy effects
of Cytisus scoparius. A separate study has shown that soil legacy effects can influence native
plant growth in an opposite manner as well. Del Fabbro and Prati (2015) examined the role
of soil legacy in comparison to immediate allelopathy on native plants in pairwise
monocultures and mixtures with invasive plants. They did not find evidence that invasive soil
legacies affect the growth of native plants in monocultures, but in competitive mixtures with
invasive plants the results varied: the addition of AC positively affected the growth of native
plants, while no soil amendment (i.e.; the invaded control soil condition) negatively affected
native plants. These findings are contradictory to those by Grove et al. (2012), but still
illustrate that AC can play a role in invasion ecology and that its role may vary from one
invasive species or community to the next. Results like this require further investigation
using other invasive and native plants from other areas of the world.
If AC can be an effective tool for restoring native plant communities impacted by
aggressive invasive plants, then it is important that we find solutions that are economically
feasible and environmentally beneficial. At the time of writing, laboratory grade AC from
Fisher Scientific was available at a price of $1493 for a total of 2,100 g of the product (Fisher
Scientific, 2021). For laboratory experiments, this may suffice but in terms of field studies, it
is important we find less expensive alternatives. Pulp mill fly ash, a free industrial waste
generated by the pulping process, contains some amount of carbon in the activated form and
should thus behave similarly to laboratory grade activated carbon with regards to adsorptive
properties (Ahmaruzzaman, 2010; Wang & Wu, 2006). There have not been many studies
conducted using pulp mill fly ash for adsorbing biochemical products; however, bagasse fly
ash, the waste product from sugarcane mills, has been used extensively in research due to its
ability to adsorb various chemical and biochemical compounds (Mall et al., 2005; Rafatullah
et al., 2010; Srivastava et al., 2006). The pulping processes that yield bagasse fly ash and
pulp fly ash are very similar when Kraft pulping is involved (Biermann, 1996; Rainey &
Covey, 2016), therefore I proposed the use of pulp fly ash from Domtar Pulp Mill in
Kamloops, BC, to be used as a soil amendment for treating the biochemical release of
phytotoxins by C. stoebe. The adsorption effectiveness of fly ash was compared alongside
laboratory grade AC purchased from Fisher Scientific.

11

FLY ASH
Fly ash in this experiment was sourced from the pulping process occurring at Domtar
Pulp Mill in Kamloops, British Columbia. Starting from wood chips, this mill produces
northern bleached softwood kraft (NBSK) paper products, cellulose fibres, and other
specialty pulp grades (Domtar Corporation, 2017). One of the by-products of the pulping
process is fly ash, which is generally stored on site or relocated to a landfill (Ahmaruzzaman,
2010; Pöykiö et al., 2004). Alternative uses have been found for this waste material including
being used in asphalt surfacing (Naik et al., 1994), removal of various toxic compounds and
waste materials from wastewater (Burgess et al., 2009; Rafatullah et al., 2010; Wang & Wu,
2006), and as a fertilizing soil amendment (Basu et al., 2009; Bi et al., 2009; Pöykiö et al.,
2004). For the current study, it will be used as a soil amendment with the goal of adsorbing
inhibitory biochemicals left behind by C. stoebe. We may find that it acts as a fertilizing
agent and positively affects growth, though this would be a secondary effect of the material.
It is important to note that the application rate of fly ash can have varying effects on
the soil chemistry and plant growth. Lower application rates have been associated with
greater seed germination and may also stimulate sugar production in plant tissues, whereas
higher rates may inhibit plant growth and sugar production (Singh et al., 1994, 1997). The
addition of fly ash may directly or indirectly affect soil microbial activity which could be the
reason for the varying growth effects of fly ash application rate (Gupta et al., 2002). Nitrogen
fixing bacteria can convert nitrogen gas and organic nitrogen to ammonium which is then
used by nitrifying bacteria (Fowler et al., 2013). In the absence of plants, nitrifying bacteria
have the opportunity to convert ammonium to nitrate, which is then used by denitrifying
bacteria to convert it back to nitrogen gas. Nitrate may also be assimilated in plant roots to be
used by plants during growth; however, there is a point where plants can no longer absorb
any more nitrate. At this point, denitrifying bacteria would convert the excess nitrate to
nitrogen gas. When fly ash is applied, a small amount of organic nitrogen is also added to the
soil which would stimulate the production of nitrate via the nitrification process, resulting in
the greater germination. Higher application rates of fly ash may cause denitrifying bacteria to

12
rapidly reproduce, thereby causing the negative effects towards plants since they would be
converting abundant soil nitrate to nitrogen gas. Low application rates of fly ash appear to
have greater benefits for the resulting plants (Gupta et al., 2002; Singh et al., 1994, 1997).
This should be a strong consideration for usage in research.

CURRENT STUDY
The soil legacy effects of Centaurea stoebe on Festuca campestris was characterized
in a controlled greenhouse environment. Festuca campestris is commonly found throughout
the upper grasslands in BC and has a clear negative response to invasive weed presence
(Kuang, 2015). Field collected soils were amended either with fly ash from the local Domtar
Pulp Mill in Kamloops, BC, or Fisher brand AC, or not amended at all. The growth of C.
stoebe and F. campestris was compared under these soil treatments and the following
questions were asked:
1. Are soil legacy effects from C. stoebe dominated sites impacting the
establishment and growth of rough fescue seedlings? And
2. Can activated carbon and pulp mill fly ash be used as mitigating factors on
potential allelopathic chemicals residual in soils collected from spotted
knapweed-dominated sites?
The results of the first question will allow native grassland restoration efforts
following spotted knapweed removal to become more detailed and focussed. Results from
the second question will be used to determine the feasibility of adding fly ash to the
restoration tool kit for spotted knapweed affected areas. Additionally, a field study was
conducted to determine the applicability of these amendments in a field setting. The
application of fly ash would provide further use to this waste product and would be relatively
inexpensive for restoration purposes.

13

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Chapter 2: Examining soil legacy effects of Centaurea stoebe in
the context of the Lac Du Bois Grasslands Protected Area
INTRODUCTION
Invasive plants can exploit new areas through its reproductive biology, a lack of
competitors or predators in the new area, or disturbance in the area they will inhabit (Mack et
al., 2000). Centaurea stoebe is a good example of an invasive plant, with its ability to rapidly
produce thousands of viable, small seeds that readily spread to new areas, root, develop into
new adult plants, reproduce and begin the process anew (Jacobs, 2012; Jacobs & Sheley,
1998). Furthermore, it is suspected that C. stoebe can release catechin into the soils, a
phytotoxin thought to be responsible for its invasive success (May & Baldwin, 2011; Perry et
al., 2007; Pollock et al., 2011). There is debate in the literature as to whether catechin release
occurs and whether catechin has an effect on native plant communities (Bais et al., 2003;
Blair et al., 2006; Callaway & Ridenour, 2004; Duke et al., 2009; Stermitz et al., 2009).
While it is difficult to confirm whether C. stoebe actively releases catechin into the soils, it
deserves a review of the research that has been done in the past that assesses catechin and,
more specifically, the mode in which catechin inhibits native plant growth. Finally, a
discussion on soil legacy effects is necessary to establish the grounds for this study and to
determine whether this plant does exhibit these traits.

Immediate Soil Effects and Catechin
Since arriving in North America in the late 1800s, C. stoebe has made its way across
most of the Canadian provinces and American states making it a destructive invasive weed.
Efforts to study its soil invasion properties have been met with mixed results. In 2003, Bais et
al. founded the “novel weapons hypothesis”, which states that an invaders success is due to
that invader possessing a “novel weapon”, or biochemical, against the native species it is
infringing upon (Bais et al., 2003; Callaway & Ridenour, 2004). The researchers developed
this hypothesis in an experiment using C. stoebe as their model invasive species, where they
provided evidence of C. stoebe releasing catechin through its roots into the soils. When a

23
susceptible species encounters it, it triggers root death via the generation of reactive oxygen
species at the target species’ root meristem (Bais et al., 2003). It is thought that C. stoebe
may be successful due to this release of catechin into the soils. Since this discovery, several
studies have attempted to replicate this design but have shown contradictory results (Blair et
al., 2005, 2006; Perry et al., 2007). In fact, this was addressed in 2010 when a correction for
the Bais et al. paper (2003) was published stating that only one other report (see: Perry et al.,
2007) found catechin in invaded soils at similar concentrations (“Corrections and
Clarifications,” 2010). Other labs that have attempted to replicate their design have not been
able to detect catechin in such high concentrations, or at all (Blair et al., 2005, 2006; Tharayil
et al., 2008).
The debates regarding the nature of catechin with regards to C. stoebe and its soils
continue, yet there are still vast amounts of evidence that through some means C. stoebe is
negatively impacting native plant communities (Gayton & Miller, 2012; May & Baldwin,
2011; Story, 1976; Story et al., 2008). Perhaps, the prevailing ecological conditions are all
that is driving the competitive nature of C. stoebe. It has been suggested that animal
herbivory can account for the loss of native species in areas adjacent to shrubs due to
preferential grazing (Bartholomew, 1970). Indeed, C. stoebe is naturally unpalatable to
grazing mammals and requires biological control or targeted grazing efforts for herbivory to
occur (Story, 1976; Watson & Renney, 1974). Another explanation for C. stoebe’s
competitive advantage argues that the changing soil conditions following exotic plant
invasion lead to a reduction in native plant biodiversity. A study examining C. stoebe patch
size found that the larger an invaded area is, the less available N and C are within the soils
(Fraser & Carlyle, 2011). These elements are crucial to the growth and establishment of most
plants and when they are less available, the resulting grassland will appear less productive
and healthy. The lack of vegetation on the landscape can also influence other soil properties
as well. Fraser and Carlyle (2011) found this when comparing knapweed communities to
native grassland communities: soil temperature, total phosphate, and total potassium within
the knapweed invaded soils increased while water content, litter biomass, species richness
and diversity decreased. It is plausible to assume that the removal of a large knapweed patch

24
would not result in the plant community returning to a pre-invaded state due to the altered
properties incurred from invasion.

Soil Legacy Effects
In certain cases where an invasive plant has established a large colony and existed for
an extended period of time, alterations to the soil may have occurred which make long-term
restoration plans more difficult to achieve (D’Antonio & Meyerson, 2002). One alteration
may be the addition of invaded seed to the seed bank over time compared to recently
invaded, or non-invaded areas. Holmes and Cowling (1997b, 1997a) studied this and found
that species richness, cover, and frequency of standing vegetation were inversely correlated
with timing of invasion by Acacia saligna (Labill) Wendl. They found a similar pattern when
it came to native seedling recruitment after clearing the area of the invasive Acacia (i.e.: less
seedlings established in areas that were invaded for a longer period of time compared to areas
that were invaded for shorter time periods and non-invaded areas). Ultimately, they provided
evidence of persistence of an invasive plant even after its removal from an area. Other work
has shown evidence of altered soil chemistry persisting after plant invasion. Maron and
Jefferies (2001) describe the invasive bush lupine (Lupinus arboreus) as a N-fixing shrub
that enriches the soil with N, and an increase in N has been shown to decrease diversity in
grasslands (Foster & Gross, 1998). The experimental removal of bush lupine allowed other
plants to establish, including early successional perennial forbs that were both native and
exotic. These forbs retain plant-available N in their roots and very slowly uptake N from the
soils, whereas later successional species will uptake soil N more quickly. With high amounts
of soil N, the shift back to a fully native plant community is slowed or even halted due to the
lingering presence of early successional forbs, even after a 5-year study period. In this way,
bush lupine is still affecting the resulting ecosystem in its absence. Conversely, other
invasive plants can deteriorate soils by leaving behind a nutrient deficit. Using N again as an
example, soils in the patches of C. stoebe have exhibited consistently less total N than their
native soil counterparts, which influences future restoration strategies and plant communities
(Fraser & Carlyle, 2011; Kuang, 2015). In all these examples, invasive plants are continuing

25
to negatively affect plant communities that they no longer inhabit, leaving behind soil legacy
effects which make long-term restoration more difficult to achieve.
Few studies have evaluated the hypothesis that soil legacy effects are, in fact, present
in recently invaded areas. One novel study uses a comparative approach to estimate
immediate allelopathy and soil legacy effects of eleven different invasive species on two
native species (Del Fabbro & Prati, 2015). Specifically, soil was collected from eleven sites
where an invasive species was found, and at sites adjacent to the edge of invasion (within 2-5
m). Both soil types were split in half: one half received an AC treatment to neutralize
allelochemicals, whereas the other half did not receive this treatment. Then, pairwise
treatments were set up in each soil type. Each native species was grown by themselves, the
invasive species was grown by itself, and then there were competition trials as well for a total
of 5 treatments in 4 different soil types for each invasive plant. Their results suggested that
native plant performance was determined by both immediate allelopathy and soil legacy
effects of an invasive plant, and that the physical removal of an invasive plant should trigger
the restoration of a native plant. The study did not use invasive plant members from the
genus Centaurea; however, this study provides a great model on which to base a similar
study evaluating the soil legacy effects of C. stoebe in a greenhouse setting.
Thus, in a similar manner to Del Fabbro and Prati’s work (2015), I performed a
comparative study growing Festuca campestris and Centaurea stoebe in six different soil
treatments in a controlled greenhouse environment. The role of C. stoebe’s soil legacy effects
on F. campestris was studied, and the role of ash as a tool for restoration was explored. The
following research questions were addressed throughout this study:
1. Are lingering soil legacies from Centaurea stoebe-invaded soils affecting the
germination, survival, mortality, and biomass of the native grass, Festuca
campestris? And,
2. Can pulp mill ash be used to treat Centaurea stoebe-invaded soils and foster
better growth of Festuca campestris?

26
Additionally, plant invasion in the Lac Du Bois Grasslands Protected Area is
compared to B.C. using publicly available datasets and freely open-sourced geographic
information system (GIS) tools.

METHODS
Site Selection
Prior to the greenhouse study, sites needed to be located for soil collection and, to
ensure that soils came from “invaded” or “pristine” areas. Plant surveys needed to be
conducted to objectively classify these areas. The area under investigation resides in the
upper elevation grasslands of Lac Du Bois Grasslands Protected Area (GPA), north of
Kamloops, B.C. This park has been a focal point for much research in the past due to the
interesting ecological gradient that appears with subtle changes in elevation. The Lac Du
Bois GPA spans an elevation gradient from approximately 400-1200 m a.s.l., and
encompasses an area approximately 15,000 ha (Delesalle et al., 2009). Lower elevations are
dominated by dryland shrubs and patches of bluebunch wheatgrass amidst the bare ground
and tend to occur in the bunchgrass biogeoclimatic zone where there are higher temperatures
and lower amounts of precipitation. These lower grasslands occur up to approximately 600 m
a.s.l. and due to the climatic restrictions results in lower overall vegetative cover. Middle
grasslands are generally found between 600-900 m a.s.l. and are also dominated by
bluebunch wheatgrass, though the occurrence of dryland shrubs decreases significantly.
There is a greater amount of vegetation cover in these grasslands compared to lower
grasslands, and there is also a significant presence of spotted knapweed. Upper grasslands
span an elevation gradient of approximately 900-1200 m a.s.l. and are dominated by fescues
and needlegrasses. Depending on the location, upper grasslands may be parts of the Interior
Douglas Fir, or Ponderosa Pine biogeoclimatic zones where there are cooler temperatures
and higher precipitation compared to lower grasslands in the bunchgrass zone. Additionally,
the middle and upper grassland areas contain the invasive plant under study, C. stoebe
(Figure 2.1). It occurs in varying patch sizes, with larger negative effects having been noted
in larger patch sizes (Fraser & Carlyle, 2011). A pair of sites from the upper grasslands were

27
used in this project: an area heavily invaded by C. stoebe for at least a decade, and a nearby
area within 20 m of the edge of invasion that is free of spotted knapweed. The actual invaded
area was delineated by walking along the edge of invasion using a Garmin GPS (model
GPSMAP 76CSx) and pristine sites were selected outside of the delineated area. Within the
invaded area, 10 sites were selected for plant surveys and soil collection. Invaded sites were
required to have > 80% C. stoebe cover for plant survey and soil collection purposes. Ten
pristine sites were then randomly selected within a 20 m buffer of the edge of invasion
(Appendix Table B.1). Proximity of invaded and pristine areas was important to ensure that
soil type and climatic measurements were similar between them. The point data for each
sampling site and the tracklog for the invaded area was imported into Garmin MapSource (v.
6.16.3) and exported as a shapefile for use in R for Statistical Computing (R Core Team,
2020). Permission to use the area for research purposes was granted by the BC Ministry of
Environment under Park Use Permit No. 102724.
Soil temperature monitors (Thermochrons, iButtonLink Technology) were inserted
into soils at two locations within both invaded and pristine areas on June 12, 2017 and
collected on August 30, 2017. These temperature monitors recorded the soil surface
temperature every 2 hours while they were in the field, on every even hour (i.e.: 12:00 AM,
2:00 AM, 4:00 AM, etc.). This was recorded to further distinguish invaded and pristine sites.
In July 2017, plant surveys were carried out at each of the 20 sites to quantify the total topdown percent cover and richness of all species in invaded and pristine areas (Appendix Table
B.3). To do this, 1 m × 1 m quadrat surveys were performed in each of the 20 sites and the
top-down percent cover of each species was estimated to nearest 1%. The specific location
for invaded quadrats within the invaded site were randomly chosen based on the cover of C.
stoebe when a quadrat was placed down: if there was 80% or greater top-down cover of C.
stoebe, the area within the quadrat was surveyed. Similarly, pristine areas were randomly
chosen within 20 m of the main knapweed patch in order to keep environmental variables as
constant as possible. GPS coordinates were recorded at each of the sample locations as well
as at each of the locations where soil temperature monitors were inserted using a cell phone’s
map application (Google Maps, Android version 9.57.1) (Appendix Table B.2).

28

Figure 2.1: Provincial context of the Lac Du Bois Grasslands Protected Area and the
associated C. stoebe extent of invasion throughout the park. Further spatial context is given
for the location of the study area within the Lac Du Bois Grasslands Protected Area. The map
was created using QGIS version 3.16.0 (Hannover).

GIS Considerations
Recently, a new R package was released that allowed for simple downloading of
spatial data from the BC Data Catalogue called “bcdata” (Teucher et al., 2020). There is a
separate package, “bcmaps”, that contains some commonly used spatial layers including
biogeoclimatic ecosystem classification (BEC) information (Teucher et al., 2021). These
packages were used to download the following spatial layers:
•

BC Parks, Ecological Reserves, and Protected Areas, for the boundary of the
Lac Du Bois GPA (Erlandson & MacPhail, 2020);

29
•

Invasive Alien Plant Site, for information on invasive plant sites throughout
BC (Miller & Osborne, 2020); and

•

Biogeoclimatic Ecosystem Classification (BEC) Map, for information at the
subzone level (Salkeld et al., 2020).

Additionally, the B.C. Ministry of Forests, Lands, Natural Resource Operations and
Rural Development have made a gridded digital elevation model (DEM) of the entire
province publicly available at an approximately 25 m2 resolution (2020). This data is made
available through the Terrain Resource Information Management (TRIM) base map
collection through individual tiles. The appropriate tiles for the Lac Du Bois GPA were
downloaded to gather information about the subzones within it. ClimateBC was used to
generate seasonal temperature raster images of the Lac Du Bois GPA to compare summer
mean temperatures throughout the area (Wang et al., 2016). A smaller clip of the study area
was also used to compare the elevation, slope, and aspect between invaded and pristine sites
using parametric T-tests. Slope and aspect layers were generated using the “terrain” function
in the “terra” package, utilizing bilinear interpolation of those values at each site (Hijmans,
2021).
Using R for Statistical Computing (R Core Team, 2020), the Lac Du Bois GPA was
extracted from the BC Parks layer. This extraction served as a clipping extent for the
Invasive Alien Plant Site and BEC Map layers. The Invasive Alien Plant Site layer was then
filtered to retrieve all records of C. stoebe within the Lac Du Bois GPA.
Spatial analyses included computations of the area of all invasive species within the
Lac Du Bois GPA compared to the area covered by C. stoebe only, and C. stoebe extent by
BEC subzones to determine potential trends of its location. Additional province wide
analyses were completed to compare the Lac Du Bois GPA to trends across the province. All
spatial calculations were performed using the “terra” and “sf” packages in R (Hijmans, 2021;
Pebesma, 2018).

30

Soil, Ash, and AC Collection and Characterization
Soil collection took place between October 6-9, 2017 at the same locations that plant
surveys took place earlier in the season. In other words, invaded soils came from areas that
are approximately 80-100% covered by C. stoebe, and pristine soils came from pristine areas
free of C. stoebe within 20 m of the edge of invasion. The soils in this study area are
classified as orthic black chernozems (Canadian Soil Information Service, 2013; Filatow,
2019). The collected soil was used to grow the plants from seed. In order to eliminate the
wild seed bank and mitigate the chance of other seeds sprouting the top 5 cm was scraped
away and discarded in both invaded and pristine soils (Bonis & Lepart, 1994). Sanitized
trowels were used to collect soil to a depth of approximately 20 cm, the depth at which
spotted knapweed produces most of its roots, and a total of approximately 200 L of each
invaded and pristine soils were gathered (i.e.: approximately 20 L from each site) (Story et
al., 2000). Soils were transported from the field sites directly to the Thompson Rivers
University Research Greenhouse in 4 L Ziplock freezer bags. Once the soils arrived in the
greenhouse, the bags were opened and allowed to air dry for a minimum of 30 days. After
this period, soil was sieved (1 cm mesh size) to remove stones and roots, and sieved soils
were collected in 189 L Rubbermaid Jumbo Storage tote bins (1 bin for invaded soil, and 1
bin for pristine soil). The drying and sieving steps here were based on the soil preparation
methods presented by Del Fabbro and Prati (2015). All collected soils from invaded sites
were thoroughly mixed inside this bin using a sanitized trenching shovel and motorized auger
with a sanitized 10 cm × 80 cm bit attached. These same methods were applied separately to
pristine soils. The mixing step ensures that soils are homogenous for each soil type, thus
when plants are potted, they will be using the same soils throughout. Approximately 1 L of
each soil type was collected at this stage to be sent to the BC Government’s Analytical
Laboratory in Victoria, BC for chemical analysis of major elements. Additionally,
approximately 1 L of soil was set aside for CNH analysis using a Thermo Scientific
FlashSmart CHNS/CHNS Elemental Analyzer unit at the TRU campus.
Ash was obtained from Domtar Pulp Mill in Kamloops, BC on November 27, 2017.
The bulk ash was transported from the mill in large 5-gallon (~20 L) buckets, and then
combined into a single 102 L tote bin. The bulk ash was stored until it was required for

31
preparing soils for the greenhouse trials. Again, approximately 1 L was set aside to be sent
for chemical analysis. CHN data was obtained from a concurrent students thesis (Antonelli,
2018).
Activated carbon was purchased from Fisher Scientific in September 2017.
According to its certificate of analysis, the exact substance was activated coconut charcoal.
Having arrived in a granulated form, it was ground to a finer size for homogenization later
using a cyclone mill (UDY Corporation, model no. 3010-030, mesh size 0.4 mm). Finely
ground AC was collected and stored in the airtight jars they were shipped in until they were
ready for mixing. Approximately 100 mL of AC was set aside to be sent for chemical
analysis. CHN data was gathered from multiple literature sources characterizing activated
coconut charcoal and averaged (Astuti et al., 2021; Hidayu & Muda, 2016; Iqbaldin et al.,
2013; Phan et al., 2006).

Soil Preparation and Greenhouse Trials
Both bulk pristine and bulk invaded soil types required separation and soil
amendments added. In order to create the 1% ash and 1% AC treatments, 49.5 L of the bulk
soils was removed and placed into smaller 102 L tote bins. This was done using 6 L and 500
mL Erlenmeyer flasks to accurately measure soils and amendments. Once 49.5 L of bulk
pristine or invaded soil was placed in a sanitized tote bin, 500 mL of either ash or AC was
added to create the 1% amendments. Each soil type was further mixed with a sanitized
trenching shovel and motorized auger to ensure that the amendment was spread equally
throughout the entire soil volume. This resulted in 6 soil treatment combinations:
1. Invaded soil (control)
2. Invaded soil + Ash
3. Invaded soil + AC
4. Pristine soil (control)
5. Pristine soil + Ash
6. Pristine soil + AC

32
Either C. stoebe or F. campestris was grown in each soil treatment separately for a
total of 12 treatments: thus a 2 × 3 factorial design with 2 soil types (pristine and knapweedaffected), and 3 soil treatments (control, AC and ash), repeated for 2 plant species. These
treatments were replicated 10 times and potted into 8 cm × 8 cm × 8 cm (i.e.: 512 mL) square
growing pots (Greenhouse Megastore, model SVT-400). To prevent soil from spilling
through the drainage holes, measured and cut weed barrier was inserted into the base of each
pot. Enough soil was added to each pot so that the surface of the soil was approximately 2 cm
from the top of the pot. Prior to planting any seeds, the soils were moistened with distilled
water. The pots were arranged in the research greenhouse using a randomized block design,
and 5 seeds of a species were planted in each pot (one in each corner, and one in the middle).
Individual seeds of C. stoebe were hand collected from the invaded field site on September 7,
2017 and were used in this experiment, whereas individual seeds of F. campestris needed to
be sourced from Splitrock Environmental out of Lillooet, B.C. Purchased F. campestris seeds
were found to have a much higher germination rate than field collected seeds (~80% for
purchased vs. ~15% for field collected), hence their use in this experiment. Pots were then
watered every 2-3 days using distilled water. Greenhouse conditions were kept constant for
the duration of the experiment and were controlled using Argus Controls software (Table
2.1). Individuals were grown for a period of 90 days, after which the number of successful
individuals were counted, height measurements were taken, and aboveground biomass was
clipped and placed in paper bags. Belowground biomass (i.e.: roots) was collected through
washing the soil off of the roots and was also placed in paper bags. All biomass collections
were dried in a drying oven at 70℃ for 72 hours, then weighed on a Fisher Scientific AccuSeries 4102 model scale measuring to the nearest hundredth of a gram.
Based on existing literature, the expectations of the growth response of each species
is outlined and explained (Table 2.2), and through this experiment we aim to address two
major concerns (Del Fabbro & Prati, 2015). First, the determination of whether soil legacy
effects exist in these soils is of major import. This was accomplished by comparing the
biomass of rough fescue in invaded and pristine soils. Comparing the biomass of spotted
knapweed in each soil should indicate if soil legacies from knapweed invasion improve,
inhibit, or have no effect on its growth. Second, we can determine whether ash may be used

33
as an alternative soil amendment to AC in spotted knapweed-invaded sites. The growth of F.
campestris will be compared in ash and AC treated soils to answer whether ash can be added
to the knapweed restoration toolkit. Comparing the growth of spotted knapweed in those soils
should provide valuable information as to the effects of ash on spotted knapweed, and if it is
indeed an appropriate soil amendment for knapweed infested sites.

Table 2.1: Environmental variables and values pertaining to optimum growth of many plants.
These conditions were replicated in the research greenhouse for the growth experiment.
Retrieved from Hendry & Grime (1993).
Day
Photoperiod 08:00-22:00 (Growing lights on)
Temperature 22℃
Humidity
40% R.H.

Night
22:00-08:00 (Growing lights off)
15℃
40% R.H.

Table 2.2: Expectations of the impacts to the growth of Centaurea stoebe and Festuca
campestris in each soil type based on existing literature.
Soil type
Invaded (control)
Invaded + Ash
Invaded + AC
Pristine (control)

Species
C. stoebe
C. stoebe
C. stoebe
C. stoebe

Pristine + Ash

C. stoebe

Pristine + AC
Invaded (control)

C. stoebe
F. campestris

Invaded + Ash
Invaded + AC
Pristine (control)
Pristine + Ash
Pristine + AC

F. campestris
F. campestris
F. campestris
F. campestris
F. campestris

Expected impacts to growth
None: C. stoebe is growing in soils it came from
Increase in growth due to ash acting as a fertilizer
None: AC should not inhibit invasive plant growth
Slight increase from invaded (control) soils due to
higher nutrient content
Increase in growth compared to pristine (control)
due to ash acting as a fertilizer
None: AC should not inhibit invasive plant growth
Decrease in growth compared to pristine (control)
due to C. stoebe soil legacy
None: Soil legacy effects mitigated by ash treatment
None: Soil legacy effects mitigated by AC treatment
None: F. campestris is growing in native soils
Increase in growth due to ash acting as a fertilizer
None: AC should not inhibit native plant growth

34

Elemental Analysis
Soils from the homogenized bulk collections were analyzed for total carbon, nitrogen,
hydrogen, and sulphur using a Thermo Scientific FlashSmart CHNS/CHNS elemental
analyzer. Specifically, sieved soils were dried in a Yamato drying oven (model DKN812) at
80°C for approximately 12 hours to remove any potential moisture. Next, approximately 1015 mg of soil were weighed and placed in small tin capsules and loaded sequentially into the
elemental analyzer sample wheel. This was repeated for each soil type five times for a total
of 30 soil samples. Values generated from the analysis were in percentage values of the total
sample. Additionally, homogenized soil samples were sent to the B.C. Ministry of
Environment analytical laboratory in Victoria, B.C., for further elemental analysis of the
major elements, including aluminum, boron, calcium, copper, iron, potassium, magnesium,
manganese, molybdenum, sodium, phosphorus, sulfur, and zinc. These elements were
detected using inductively coupled plasma optical emission spectrometers (ICP-OES) after
preparation using microwave acid digestion. Resulting values were returned in either mg/kg,
or total percentage depending on the element in question.

Data Analysis
FIELD DATA
Plant cover data from invaded and pristine sites was compared using Shannon and
Simpson diversity indices, along with a comparison of overall species richness using
parametric T-tests. Species richness was further explored by splitting the species into two
separate types: invasive species, and native species. Native species richness data was made
normal by a square root transformation for use in a T-test, but invasive species richness
required the use of a non-parametric Wilcoxon rank sum test. Invasive species were
identified using the most up to date invasive species list published from the Invasive Alien
Plant Program (IAPP) (Ministry of Forests and Range - Range Branch, 2020). This program
acts as a publicly accessible repository housing invasive plant information, including location

35
and control efforts. The IAPP is managed by the B.C. Ministry of Environment, but the tools
to report weeds are not restricted to government users.
Litter cover was compared between sites using a parametric T-test. Bare ground cover
was compared using a Wilcoxon rank sum test as raw and transformed data violated tests of
normality. Raw surface temperatures were compared between sites using a Wilcoxon rank
sum test. Elevation, slope, and aspect were all compared using parametric T-tests. Those
results are tabulated separately to focus on the terrain component from those sites.

GREENHOUSE DATA
Biomass
Two-factor analyses of variance (ANOVA) were performed separately for each C.
stoebe and F. campestris to compare the effect of 2 soil types (invaded soils and pristine
soils) and 3 soil amendments (control, activated carbon, and fly ash) on total biomass.
Assumptions of this test include normality of the input data, as well as homogeneity of
variances. The raw data violated normality assumptions for both species, so a square root
transformation was applied thereby fitting a normal distribution and passing a homogeneity
of variance test. By performing the same transformation to both species, the results and
effects of soil type and treatment were comparable. An analysis of the main effects of soil
types and soil treatments was then conducted for both species, and pairwise comparisons
were made between the pairings of the soil types and soil treatments. A Tukey Honest
Significant Difference (HSD) test was finally used to assess the interactions of soil type and
soil treatments.
Borrowing from Del Fabbro and Prati (2015), a continuous index of soil legacy was
measured using the raw biomass data. Essentially, it is expected that if C. stoebe exhibits
legacy effects, the biomass of F. campestris should increase more in AC soil with legacy
effects than without legacy effects. The index was calculated as:

36
Allelopathic legacy in monocultures =
(biomass in invaded soil without AC − biomass in invaded soil with AC) −
(biomass in native soil without AC − biomass in native soil with AC)
It should be noted that this is a strict measure of the strength of the soil legacy effects
exhibited by C. stoebe, and the measurement is conducted ignoring trials using ash.
Additionally, measures of immediate allelopathy are not able to be calculated since this
requires a competitive experiment which was omitted from this study. Thus, the values given
will only be a vague indication of the direction of any legacy effects. That is, any positive
result indicates a positive legacy effect, whereas negative results indicate a negative legacy
effect. All statistical analyses and figures were produced using R for Statistical Computing
(R Core Team, 2020).

Plant Survival
Various metrics were used to assess the survival of individuals through the duration
of the greenhouse trials. These included germination rate, death rate, survival rate of the
germinated seeds, and overall trial survival rate (Table 2.3). A similar application of the twofactor ANOVA was used to assess differences in these metrics between soil types and
treatments. Normality was violated in all metrics. Using a square root transformation on the
germination rate values validated the normality and homogeneity of variance assumptions of
the two-factor ANOVA. In order to properly use the other metrics in a two-factor ANOVA,
an aligned rank transformation was applied to the data in order to fit a normal distribution
(Wobbrock et al., 2011). All statistical analyses and figures were produced using R for
Statistical Computing (R Core Team, 2020).

37

Table 2.3: Description of plant survival metrics to be applied to each individual pot. In this
case, the number of seeds planted equates to 5.
Metric

Description

Equation
# Seeds Germinated
# Seeds Planted

Germination
Rate

Ratio of seeds that germinated after 30 days

Death Rate

Ratio of seedlings that died between 30 and 90
days

# Seeds Germinated

Germination
Survival Rate

Ratio of seedlings that lived between 30 and 90
days

# Survived Individuals
# Seeds Germinated

Trial Survival
Rate

Ratio of seedlings that lived to the end of the
experiment compared to the number of planted
seeds

# Survived Individuals
# Seeds Planted

Individuals –
(# Survived
)
# Seeds Germinated

Elemental Analysis
Soil carbon, nitrogen, and hydrogen results from each of the soil treatments were
compared using a parametric two-way ANOVA for each element, in which assumptions of
normality and homogeneity of variance were satisfied. Results from the major elemental
analysis were tabulated. A carbon-to-nitrogen ratio (C:N) was calculated from the C and N
data, respectively. Normality was violated on the C:N data and was unable to be transformed
to fit a normal distribution, thus an aligned rank transformation was applied to validate the
assumptions of a two-way ANOVA (Wobbrock et al., 2011).

RESULTS
GIS Considerations
The Lac Du Bois GPA encompasses an approximately 159.37 km2 area and is broken
down into six subzones and spanning an elevation gradient of approximately 334 – 1410 m
above sea level (Table 2.4). Each of the subzones are classified either as “dry” or “very dry”,

38
and most of the subzones are located on warmer aspect slopes. The range of summer
temperatures also appear to show warmer temperatures at lower elevation subzones.

Table 2.4: Biogeoclimatic Subzones of the Lac Du Bois Grasslands Protected Area. Detailed
for each subzone are descriptions of the subzone codes, the area in km2 that the subzone
takes up in the Lac Du Bois GPA, and associated elevation descriptions gathered from
TRIM.
BGC
Subzone

BGxh2

BGxw1

IDFdk1

IDFdk2

IDFxh2

PPxh2

Description
Bunchgrass:
Thompson
Very Dry
Hot
Bunchgrass:
Nicola Very
Dry Warm
Interior
Douglas Fir:
Thompson
Dry Cool
Interior
Douglas Fir:
Cascade Dry
Cool
Interior
Douglas Fir:
Thompson
Very Dry
Hot
Ponderosa
Pine:
Thompson
Very Dry
Hot

Elevation
Summer
Summer
Mean
Temperature Temperature
(m)
Range (°C)
Mean (°C)

Area
(km2)

Elevation
Range (m)

21.76

334.0 – 710.1

481.8

11.6 – 29.8

20.7

43.83

408.9 – 912.8

731.3

10.2 – 28.9

19.0

4.33

842.2 –
1312.7

1117.7

8.9 – 25.8

16.8

10.14

870.3 –
1410.5

1218.7

8.2 – 25.4

16.1

50.85

522.7 –
1301.4

924.7

8.9 – 28.3

17.8

28.46

365.8 –
1024.1

703.3

9.8 – 29.3

19.3

The IAPP found 795 total invasive plant records throughout the entire Lac Du Bois
GPA spanning 26.30 km2 (~16.5%). These records show that 32 of the 229 invasive species
listed by the IPCC have been recorded throughout the area. C. stoebe was identified in 356

39
records and covered an approximate area of 21.99 km2 (~13.8%) within the Lac Du Bois
GPA. As literature suggested, a majority of the sites invaded by C. stoebe were located in
higher elevation, cooler grasslands and forest interfaces (Table 2.5). This confirms that the
location for the study area (the IDFxh2) was appropriate. The observed invaded area at this
study site was 11,659 m2, and this area was within the bounds of an identified C. stoebe patch
by the IAPP. While the declared pristine sites appear to fall within an invaded area identified
by the IAPP, ground observations at the time of sampling showed that C. stoebe was absent
at those sites.

Table 2.5: Details of plant invasion in the Lac Du Bois Grasslands Protected Area broken
down by BGC subzone. “Total” values indicate the total number of records, total physical
area, and invaded ratio of all invasive plants combined throughout the Lac Du Bois GPA in
order to highlight the same metrics for C. stoebe only. Numbers of records sum to be greater
than the total number of records throughout the Lac Du Bois GPA due to splitting of
polygons during spatial intersections.
BGC
Subzone
BGxh2
BGxw1
IDFdk1
IDFdk2
IDFxh2
PPxh2

Total
Total
Total
C. stoebe C. stoebe
IAPP
Invaded Invaded
IAPP
Invaded
2
1
records area (km ) ratio
records area (km2)
43
1.27
5.83%
18
0.39
514
18.03
41.15%
230
15.53
6
0.010
0.24%
1
0.006
0
0
0%
0
0
236
5.96
11.71%
120
5.37
52
1.03
3.63%
27
0.69

C. stoebe
Invaded
ratio1
1.78%
35.43%
0.15%
0%
10.57%
2.44%

It is important to note that a given polygon representing an invaded area may
encompass multiple invaded species, thus while C. stoebe occupies 35.43% of the entire
BGxw1 subzone it should not be interpreted that all other invasive species within the subzone
occupy the remaining 5.72% of total invaded space. For example, C. diffusa, the next most
invasive plant in the Lac Du Bois GPA, occupies 29.47% of the same subzone due to its
close relation to C. stoebe. Indeed, C. stoebe is the most invasive plant throughout the Lac

1

Invaded ratio is calculated as the invaded area divided by total physical area of the subzone.

40
Du Bois GPA by total area and by subzone, except in the BGxh2 subzone where C. diffusa is
the most invasive plant.
Throughout B.C., there were 153,799 total invasive species recordings made through
the IAPP and 35,593 of those contained records for the presence of C. stoebe. Invasive
species make up a total of 1,369.76 km2 in B.C. (0.144%), and by itself C. stoebe covers a
total area of 521.81 km2 (0.055%). Comparatively speaking, this is greater than the city area
of Winnipeg, Manitoba (~464.33 km2).
An exploratory analysis of the same BGC subzones as the Lac Du Bois GPA
indicated that these subzones are among the most invaded of those types throughout the
province (Table 2.6). For example, the BGxh2 subzone is a comparatively smaller subzone
throughout the province being only the 171st largest; however, it is 4th most invaded subzone
when accounting for all invasive species throughout the province and contains the greatest
amount of C. stoebe cover compared to all other subzones. Comparing the invaded ratio of
the subzones within the province to within the Lac Du Bois GPA, we start to notice that the
larger subzones in the park are more heavily invaded compared to the province wide results.
For instance, the BGxw1 subzone accounts for 43.83 km2 within Lac Du Bois and was found
to be 35.43% covered by C. stoebe; however, across the province, C. stoebe invades only
5.76% of that entire subzone (Table 2.7). The same trend holds true for the IDFxh2 subzone,
but the opposite is true for the BGxh2, IDFdk2, and PPxh2 subzones where smaller
proportions are invaded in the Lac Du Bois GPA compared to provincially reported
proportions.

41
Table 2.6: Province wide BGC details, only including the BGC subzones that were also
within the Lac Du Bois Grasslands Protected Area. Rank values refer to the entire list of 210
subzones with a rank of 1 being the highest rank.
BGC
Subzone
BGxh2
BGxw1
IDFdk1
IDFdk2
IDFxh2
PPxh2

Total
Provincial Subzone
Subzone Proportion
Area
2
area (km )
of area
Rank
678.98
0.072%
171
684.02
0.072%
169
5394.87
0.569%
39
2440.03
0.257%
89
4423.58
0.467%
54
1364.74
0.144%
126

Overall
Invasive
Rank
4
5
18
13
11
6

C. stoebe
Invasive
Rank
1
2
11
7
4
3

Table 2.7: Details of plant invasion throughout B.C. broken down by BGC subzone. “Total”
values indicate the total number of records, total physical area, and invaded ratio of all
invasive plants combined throughout B.C. in order to highlight the same metrics for C.
stoebe only.
BGC
Subzone
BGxh2
BGxw1
IDFdk1
IDFdk2
IDFxh2
PPxh2

Total
Total
Total
C. stoebe C. stoebe
IAPP
Invaded Invaded
IAPP
Invaded
2
records area (km )
ratio
records area (km2)
1932
53.73
7.91%
1046
40.17
1745
45.68
6.68%
1067
39.41
4297
78.99
1.46%
2185
53.33
2127
67.01
2.75%
850
35.35
7711
167.69
3.79%
4685
140.28
2950
85.39
6.26%
1570
69.04

C. stoebe
Invaded
ratio
5.91%
5.76%
0.989%
1.45%
3.17%
5.06%

Field Collections
The results of multiple T-test and Wilcoxon rank sum tests are tabulated (Table 2.8).
An independent samples T-test did not show any statistical difference in overall species
richness between invaded and pristine sites; however, a trend was noted. When species
richness was broken down between native and invasive plant types, more clear results
emerged: a Wilcox rank sum test showed that pristine sites had significantly lower invasive
species richness than invaded sites. An independent samples T-test comparing the native

42
species richness between sites showed significantly higher native species richness in pristine
sites than in invaded sites. An independent samples T-test comparing the litter cover between
sites showed significantly higher cover in pristine sites than in invasive sites. Conversely, a
Wilcoxon rank sum test showed significantly lower bare ground cover in pristine sites
compared to invaded sites. T-tests on both Shannon-Wiener and Simpson diversity metrics
showed that pristine sites were significantly more diverse than invaded sites. Finally, a
Wilcoxon rank sum test showed significantly lower temperatures in pristine sites compared
to invaded sites (Figure 2.2).

Table 2.8: Summarized comparisons of field metrics between pristine and invaded sites, and
the respective T-test or Wilcox rank sum test results. Asterisks on P-values indicate
significant differences at the 0.05 level for that metric between pristine and invaded sites.
Metric
Overall Species Richness
Invasive Richness
Native Richness
Ground Litter Cover (%)
Bare Ground Cover (%)
Shannon-Weiner Diversity (H)
Simpson Diversity (D)
Average Daily Ground
Temperature (°C)

Mean ± SE
Pristine
Invaded
7.8 ± 0.83
5.8 ± 0.57
0.2 ± 0.13
1.5 ± 0.17
7.6 ± 0.85
4.3 ± 0.52
40.9 ± 4.69 16.3 ± 2.75
0.7 ± 0.52 11.3 ± 1.92
1.275 ± 0.14 0.569 ± 0.08
0.585 ± 0.06 0.244 ± 0.03
17.77 ± 0.20 20.86 ± 0.18

Statistic
T = -1.99
W = 98
T = -3.70
T = -4.52
W = 99
T = -4.54
T = -5.25
W = 322083

p
0.064
<0.001*
0.005*
<0.001*
<0.001*
<0.001*
0.008*
<0.001*

43

Figure 2.2: Ground temperature from invaded (red) and pristine (black) sites in the Lac Du
Bois GPA from June 12 - August 30, 2017. Extended vertical bars represent actual ground
temperature readings at 2-hour intervals, and the smoothed lines represent mean daily ground
temperatures and a standard error at invaded and pristine sites.

Comparisons of elevation, slope, and aspect between invaded and pristine sites are
tabulated (Table 2.9). Independent samples T-tests revealed no significant differences in
elevation (T = 0.005, p = 0.996), slope (T = 0.465, p = 0.647), or aspect (T = – 0.893, p =
0.384) between sites.

Table 2.9: Summarized comparisons of elevation, slope, and aspect between pristine and
invaded sites, and their associated T-test results. N = 10 for each site.
Mean ± SE
Variable
Pristine
Invaded
T
p
Elevation (m)
918.55 ± 1.01 918.56 ± 0.30
0.005 0.996
Slope (radians) 0.071 ± 0.009 0.077 ± 0.008
0.465 0.647
Aspect (radians)
4.23 ± 0.22
4.01 ± 0.11 – 0.893 0.384

44

Greenhouse Trials
Since F. campestris and C. stoebe were grown separately and not together in any
competitive scenario, the interaction between their results was not explored; however,
interactions between soil type and soil treatment were explored within each species.

BIOMASS
A two-way ANOVA was used to examine the effects of soil type and soil treatment
on biomass. A square root transformation of the biomass data was applied in order to pass the
Shapiro-Wilks normality test (p (RF) = 0.36; p (SK) = 0.14) and Levene’s test of homogeneity
of variances (p (RF) = 0.49; p (SK) = 0.61) for both species. For both C. stoebe and F.
campestris, a significant effect of the soil treatment was found (RF: F = 24.553, p = < 0.001;
SK: F = 34.195, p < 0.001; Table 2.10). There was no significant effect of soil type found
(RF: F = 1.734, p = 0.193; SK: F = 0.879, p = 0.353). A slight but nonsignificant interaction
of soil type and soil treatment was noted as well (RF: F = 2.761, p = 0.072; SK: F = 2.583, p
= 0.085).
An analysis of the main effects of soil treatment was performed using one-way
ANOVA’s grouped by soil type. It was very apparent that the significant effect of soil
treatment on biomass occurred in both invaded and pristine soil types for both C. stoebe and
F. campestris, where the addition of AC caused a reduction in biomass (Figure 2.3).
Analyses of variance were also carried out to explore the effect of soil type on biomass by
grouping data by soil treatments. A significant effect of soil type was noted in control soils;
all other soil treatments showed no effect of soil type on biomass (Table 2.11). Pairwise
comparisons between soil treatments when grouped by soil type showed biomass in soils
treated with AC was significantly lower than either a control or ash treated soils for both F.
campestris and C. stoebe.

45

Figure 2.3: Square root of biomass (mean ± SD) of Festuca campestris (left) and Centaurea
stoebe (right) when grown in different soil types and soil treatments. Comparisons of
biomass are made between control, ash, and activated carbon soil treatments for each soil
type. P-values above brackets denote significant pairwise comparisons between soil
treatments as determined by pairwise t-tests.

Table 2.10: Results of a two-way ANOVA, performed to discover differences in the square
root of plant biomass with respect to each factor and their interactions. F and p values are
reported for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK",
respectively. Significant values are bold faced.
Effect
df effect df error F RF
F SK
p RF
p SK
Soil Type
1
54 1.734 0.879 0.193 0.353
Soil Treatment
2
54 24.553 34.195 <0.001 <0.001
Soil Type × Soil Treatment
2
54 2.761 2.583 0.072 0.085

46
Table 2.11: Exploration of the main effects of soil treatment and soil type on the square root
of biomass for each species. F and p values are reported for both Festuca campestris and
Centaurea stoebe as subscripts "RF" and "SK", respectively. Significant values are bold
faced.
Soil Type
Pristine
Invaded

Main Effect
df effect df error F RF
F SK
p RF
p SK
Soil Treatment
2
54 8.411 26.697 <0.001 <0.001
Soil Treatment
2
54 18.903 10.082 <0.001 <0.001

Soil Treatment
Control
Ash
AC

Main Effect
Soil Type
Soil Type
Soil Type

df effect df error
1
54
1
54
1
54

F RF
7.157
0.009
0.09

F SK
4.359
0.432
1.256

p RF
0.01
0.925
0.766

p SK
0.042
0.514
0.267

Interestingly, the index of allelopathic legacy was positive when F. campestris was
grown in invaded soils (0.947). This result occurred due to greater biomasses in invaded soils
compared to pristine soils. In comparison, the index was negative for C. stoebe when planted
in invaded soils (‒1.712), indicating that soil legacies from C. stoebe do not allow it to grow
better in its own soils compared to when grown in pristine soils. This index further shows
that C. stoebe performed best in pristine (newly invaded) soils, though is only intended to
show relative trends of allelopathic legacy. Finally, observations of chlorosis were made in
40% of the C. stoebe pots during the growing period, but no significant trends were noted
between treatments.

PLANT SURVIVAL
Germination Rate
The germination rate for this experiment was defined as the ratio of seeds that
germinated after 30 days when 5 seeds were planted in a single pot. A two-way ANOVA was
carried out to determine any effects of soil type and soil treatment on the germination rate. A
square root transformation was applied to the germination rate data to pass the Shapiro-Wilks
normality test (p (RF) = 0.144; p (SK) = 0.149) and Levene’s test of homogeneity of variances (p
(RF) = 0.223; p (SK) = 0.082). Soil type and soil treatment were found to significantly affect the

germination of F. campestris on their own without any interacting effects (Table 2.12). The

47
germination rate of C. stoebe did not appear affected by either soil type or soil treatment by
contrast.
An analysis of the main effects of soil treatment on germination rate showed that the
germination rate of F. campestris varies significantly across soil treatments in both pristine
and invaded soils (Table 2.13). Germination rate of C. stoebe was only significantly different
across soil treatments in invaded soil types. Exploring the main effects of soil type on the
germination rate, it is clear to see that the germination rates of both F. campestris and C.
stoebe are significantly affected by soil type in ash treated soils only. In other soil treatments,
this significant effect is absent. Pairwise comparisons between soil treatments grouped by
soil type showed that the square root of F. campestris’ germination rate is significantly lower
when soils are treated with AC in both invaded and pristine soils (Figure 2.4). An ash
treatment did not appear to have any significant effects on the germination rates of either
species in either soil type when compared to a control.

Figure 2.4: The square root of germination rates (means ± SD) of Festuca campestris and
Centaurea stoebe when grown in different soil treatments and soil types. Comparisons of the
germination rates are made between control, ash, and activated carbon treated soils for each
soil type. P-values above brackets denote significant pairwise comparisons between soil
treatments as determined by pairwise t-tests.

48
Table 2.12: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the square root of the germination rate. F and p values are reported for both
Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced.
Effect
df effect df error F RF F SK
p RF
p SK
Soil Type
1
54 5.271 1.931 0.026 0.17
Soil Treatment
2
54 12.94 2.299 <0.001 0.11
Soil Type × Soil Treatment
2
54 1.048 2.464 0.358 0.095

Table 2.13: Exploration of the main effects of soil treatment and soil type on the square root
of the germination rate of each species. F and p values are reported for both Festuca
campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively. Significant
values are bold faced.
Soil Type
Pristine
Invaded

Main Effect
df effect df error F RF F SK
p RF
p SK
Soil Treatment
2
54 5.445 0.612 0.007 0.546
Soil Treatment
2
54 8.542 4.152 <0.001 0.021

Soil Treatment
Control
Ash
AC

Main Effect
Soil Type
Soil Type
Soil Type

df effect df error F RF F SK
1
54 0.054 0.708
1
54 5.116 5.225
1
54 2.196 0.926

p RF
p SK
0.817 0.404
0.028 0.026
0.144 0.34

Death Rate
The death rate for this experiment was defined as the ratio of seedlings that died
between the 30- and 90-day mark of the greenhouse trial. A two-way ANOVA was used to
determine the effects of soil type and soil treatment on the death rates of F. campestris and C.
stoebe. Prior to analysis, the death rate data was not normal and could not be transformed to
fit a normal distribution. To overcome this issue, an aligned rank transformation was used to
force the statistical tests to run a two-way ANOVA on the non-parametric data (Wobbrock et
al., 2011). Post-hoc analyses were then completed in a similar manner to the parametric twoway ANOVA post-hoc analyses. Soil type, soil treatment, and the interaction of both factors

49
all played a significant role on the death rate of F. campestris (Table 2.14). A significant
effect of soil treatment on the death rate was found in C. stoebe individuals.
An analysis of the main effects of soil treatment on the death rate revealed that the
death rate of both species is significantly affected in invaded soils but not pristine soils
(Table 2.14). When analyzing the main effects of soil type among the soil treatments, a
significant difference in the death rates of F. campestris was found in each soil treatment
(Table 2.15). For C. stoebe, soil type only affected the death rates in control treated soils.
Pairwise comparisons between soil treatments grouped by soil type showed that the death
rate of F. campestris was significantly higher in AC treated soils in invaded soils types when
compared to a control. The death rate of C. stoebe was also significantly higher in AC treated
soils in invaded soil types when compared to either a control or ash treatments (Figure 2.5).
The addition of ash did not appear to influence death rates compared to a control in any soil
treatment for either species, though the death rates were always lower in ash treated soils
when compared to AC treated soils.

Figure 2.5: The aligned rank transformed death rates (means ± SD) of Festuca campestris
and Centaurea stoebe when grown in different soil treatments and soil types. Comparisons of
the death rates are made between control, ash, and activated carbon treated soils in each soil
type. P-values above brackets denote significant pairwise comparisons between soil
treatments as determined by pairwise t-tests.

50

Table 2.14: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the aligned rank transformation of the death rate. F and p values are reported for
both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced.
Effect
df effect df error
F RF
F SK
p RF
p SK
Soil Type
1
54 29.148 2.332 <0.001 0.133
Soil Treatment
2
54 6.117 12.328 0.004 <0.001
Soil Type × Soil Treatment
2
54 6.067 0.050 0.004 0.952

Table 2.15: Exploration of the main effects of soil treatment and soil type on the aligned rank
transformation of the death rate of each species. F and p values are reported for both Festuca
campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Soil Type
Pristine
Invaded

Main Effect
df effect df error
Soil Treatment
2
54
Soil Treatment
2
54

Soil Treatment
Control
Ash
AC

Main Effect
Soil Type
Soil Type
Soil Type

F RF
F SK
1.987
2.3
5.766 14.928

df effect df error
F RF
1
54 17.069
1
54 8.552
1
54 5.268

p RF
p SK
0.147 0.110
0.005 <0.001

F SK
p RF
5.508 <0.001
0.297 0.005
0.061 0.026

p SK
0.023
0.588
0.806

Germination Survival Rate
The germination survival rate was defined as the ratio of seedlings that lived between
the 30- and 90-day mark during the greenhouse trial. Like the death rate data, the
germination survival rate was not normal and could not be transformed to fit a normal
distribution, so an aligned rank transformation was used to be able to calculate statistical
measures for this data. Soil treatment appeared to have significant effects on the germination
survival rate for both F. campestris and C. stoebe (RF: F = 9.093, p < 0.001; SK: F = 13.098,
p < 0.001; Table 2.16). The different soil types and interacting effects of soil type and

51
treatment did not have any significant impact on the germination survival rate for either
species.
An analysis of the main effects of soil treatment showed that the germination survival
rate is significantly affected in both invaded and pristine soil types across all levels of soil
treatment for both species (Table 2.17). Conversely, there was no significant main effects of
soil type on the germination survival rate across any of the soil treatments for both species.
For F. campestris, pairwise comparisons of the germination survival rate between soil
treatments when grouped by soil type revealed that invaded soils treated with ash had a
significantly greater germination survival rate when compared to either control or AC treated
soils (Figure 2.6). In pristine soils, the germination survival rate was significantly greater in
control soils when compared to either ash or AC treated soils. For C. stoebe, the germination
survival rate was significantly lower in AC treated soils in invaded soil types. An ash
treatment in pristine soil types increased the germination survival rate of C. stoebe slightly
compared to control soils, but was significantly greater than the rate when treated with AC.

Figure 2.6: The aligned rank transformed germination survival rates (means ± SD) of Festuca
campestris (left) and Centaurea stoebe (right) when grown in different soil types and soil
treatments. Comparisons of the germination survival rates are made between control, ash,
and activated carbon treated soils in each soil type. P-values above brackets denote
significant pairwise comparisons between soil treatments as determined by pairwise t-tests.

52

Table 2.16: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the aligned rank transformation of the germination survival rate. F and p values
are reported for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK",
respectively. Significant values are bold faced.
Effect
df effect df error F RF
F SK
p RF
p SK
Soil Type
1
54 0.019 0.537 0.890 0.467
Soil Treatment
2
54 9.093 13.098 <0.001 <0.001
Soil Type × Soil Treatment
2
54 0.179 0.377 0.836 0.688

Table 2.17: Exploration of the main effects of soil treatment and soil type on the aligned rank
transformation of the germination survival rate of each species. F and p values are reported
for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced.
Soil Type
Pristine
Invaded

Main Effect
df effect df error
F RF
F SK
p RF
p SK
Soil Treatment
2
54 11.672 3.331 <0.001 0.043
Soil Treatment
2
54
9.5 12.413 <0.001 <0.001

Soil Treatment
Control
Ash
AC

Main Effect
Soil Type
Soil Type
Soil Type

df effect df error
1
54
1
54
1
54

F RF
0.802
1.48
0.006

F SK
0.179
1.323
0.294

p RF
0.374
0.229
0.936

p SK
0.674
0.255
0.590

Trial Survival Rate
The trial survival rate was defined as the ratio of seedlings that lived to the end of the
experiment compared to the number of planted seeds in a pot (i.e.: 5). Again, the two-way
ANOVA required an aligned ranks transformation to bypass the tests of normality and
homogeneity of variances in order to be properly calculated. Significant differences in trial
survival rate were found to be due to soil treatments for both F. campestris and C. stoebe
(RF: F = 23.237, p < 0.001; SK: F = 8.913, p < 0.001; Table 2.18). Soil type and the
interactions between soil type and soil treatment did not have any significant effects on the
trial survival rate of either species.

53
An analysis of the main effects of the soil treatments showed significant effects of
soil treatment on trial survival rate in both invaded and pristine soil types for both F.
campestris and C. stoebe (Table 2.19). There were no main effects of soil type on the trial
survival rate of either species under any soil treatment.
For F. campestris, pairwise comparisons between soil treatments of the trial survival
rate revealed significantly lower rates in soils treated with AC compared to control and ash
treated soils in both pristine and invaded soil types (Figure 2.7). The greatest trial survival
rates were found in control soils, though this rate is not significantly greater when compared
to an ash treatment. For C. stoebe, pairwise comparisons showed significantly lower trial
survival rates in soils treated with AC when compared to the rates in ash treated soils for both
soil types. Ash treated soils had the highest trial survival rates for C. stoebe, though not
significantly greater compared to a control.

Figure 2.7: The aligned rank transformed trial survival rates (means ± SD) rates of Festuca
campestris and Centaurea stoebe when grown in different soil treatments and soil types.
Comparisons of the trial survival rates are made between control, ash, and activated carbon
treated soils in each soil type. P-values above brackets denote significant pairwise
comparisons between soil treatments as determined by pairwise t-tests.

54
Table 2.18: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on the aligned rank transformation of the trial survival rate. F and p values are
reported for both Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK",
respectively. Significant values are bold faced.
Effect
df effect df error
F RF F SK
p RF
p SK
Soil Type
1
54 1.046 1.112 0.311 0.296
Soil Treatment
2
54 23.237 8.913 <0.001 <0.001
Soil Type × Soil Treatment
2
54 1.259 1.990 0.292 0.147

Table 2.19: Exploration of the main effects of soil treatment and soil type on the aligned rank
transformation of the trial survival rate of each species. F and p values are reported for both
Festuca campestris and Centaurea stoebe as subscripts "RF" and "SK", respectively.
Significant values are bold faced.
Soil Type
Pristine
Invaded

Main Effect
df effect df error
F RF F SK
p RF
p SK
Soil Treatment
2
54 10.479 4.915 <0.001 0.011
Soil Treatment
2
54 12.873 4.02 <0.001 0.024

Soil Treatment
Control
Ash
AC

Main Effect
Soil Type
Soil Type
Soil Type

df effect df error
1
54
1
54
1
54

F RF F SK
0.09 0.031
0.807 0.214
0.329 1.413

p RF
p SK
0.766 0.862
0.373 0.645
0.528 0.240

Elemental Analysis
The FlashSmart Elemental Analyzer returns concentrations for carbon, nitrogen, and
hydrogen. Two-way ANOVA’s were performed for each of elemental contents to determine
the effects of soil type and soil treatment on the respective elemental concentrations as well
as the ratio of carbon to nitrogen (C:N). Assumptions of a two-way ANOVA were validated
using a Shapiro-Wilks test of normality (p Carbon = 0.973; p Nitrogen = 0.298; p Hydrogen = 0.786)
and a Levene’s test of homogeneity of variances (p Carbon = 0.378; p Nitrogen = 0.839; p Hydrogen =
0.365) for each of the elemental concentrations. C:N data was not normal and was unable to
be transformed to fit a normal distribution, thus an aligned rank transformation was used to
force the statistical tests to run a two-way ANOVA on the non-parametric data. Soil type

55
significantly affected carbon and nitrogen concentrations, whereas soil treatment had a more
pronounced effect on hydrogen concentrations and the C:N ratio (Table 2.20). The
interaction of soil type and soil treatment also had a significant effect on carbon
concentrations and the C:N ratio, and a nearly significant effect on nitrogen concentrations as
well.
Analyzing the main effects of soil treatments shows that the differences in carbon
concentrations can be found in both invaded and pristine soil types (Table 2.21). The effects
of soil treatment are more pronounced in pristine soil types when it comes to the differences
in nitrogen concentrations and the C:N ratio. No effects of soil treatment on hydrogen
concentrations were found in either soil type. The effects of soil type on elemental
concentrations were significant in each soil treatment for each element.
Pairwise comparisons between soil treatments among the soil types show
significantly greater carbon concentrations in soils treated with AC in invaded soil types
(Figure 2.8). In pristine soil types, soils treated with AC had the lowest amount of carbon
reported and had a significantly lower concentration compared to a control. Trends with
respect to nitrogen were less apparent, though concentrations were significantly lower in
pristine soils treated with AC compared to a control. Pairwise comparisons of hydrogen
concentrations were all insignificant. A significantly greater C:N ratio was found in pristine
soils treated with AC, and there was a trend towards a greater C:N ratio in invaded soils as
well. Concentrations of all elements were found to be greater in pristine soils compared to
invaded soil types.

Table 2.20: Results of a two-way ANOVA exploring the effects of soil type and soil
treatment on carbon, nitrogen, and hydrogen concentrations, as well as the ratio of carbon to
nitrogen. Significant values are bold faced.
Effect
Soil Type
Soil Treat.
Soil Type ×
Soil Treat.

df

df

effect

error

1
2
2

Carbon
Nitrogen
Hydrogen
C:N Ratio
F
p
F
p
F
p
F
p
18 191.6 <0.001 107.1 <0.001 1.734 0.205 1.128 0.302
18 1.469 0.256 2.356 0.123 62.99 <0.001 8.146 0.003
18 16.81 <0.001 3.53 *0.051 0.945 0.407 7.553 0.004

56

Table 2.21: Exploration of the main effects of soil treatment and soil type on carbon,
nitrogen, and hydrogen concentrations, as well as the ratio of carbon to nitrogen. Significant
values are bold faced.
Main effects of Soil Treatment
Soil Type
Carbon
Nitrogen
Hydrogen
df
df
F
p
F
p
F
p
effect error
Pristine
2 18 7.479 0.004 5.705 0.012 2.519 0.108
Invaded
2 18 10.80 <0.001 0.181 0.836 0.160 0.854

C:N Ratio
F
p
5.209 0.016
3.247 0.062

Main effects of Soil Type
Soil
Carbon
Nitrogen
Hydrogen
df
df
Treatment effect error
F
p
F
p
F
p
Control
1 18 128.2 <0.001 59.21 <0.001 21.13 <0.001
Ash
1 18 85.31 <0.001 39.20 <0.001 30.77 <0.001
AC
1 18 11.65 0.003 15.76 <0.001 12.98 0.002

C:N Ratio
F
p
0.611 0.444
0.764 0.394
0.034 0.856

57

Figure 2.8: Total carbon, nitrogen, and hydrogen concentrations, as well as the aligned rank
transformation of the carbon to nitrogen ratio in each soil type (N = 4 for each group). Pvalues above brackets denote significant pairwise comparisons between soil treatments as
determined by pairwise t-tests.

Results from the major elements analysis highlight the effects of adding either ash or
AC (Table 2.22). Interestingly, the addition of AC had differential effects on pristine and
invaded soils. For pristine soils, this addition caused decreased concentrations of aluminum,
boron, copper, potassium, manganese, sodium, phosphorus, sulfur, and zinc. Increased

58
concentrations were observed in invaded soils for those elements, as well as increases in
calcium, iron, magnesium, and sulfur. The addition of ash appeared to increase
concentrations for all elements in invaded soils except for magnesium, molybdenum, and
phosphorus, and slightly increased calcium, potassium, magnesium, and sulfur
concentrations in pristine soil types.

Table 2.22: Results from the major elements analysis. Concentration values reported are
separated by reported units for simplicity. C, H, and N values for soil blends are mean values
gathered from analysis using the FlashSmart CHNS/CHNS Elemental Analyzer whereas pure
ash and AC values were gathered from literature (see footnotes).
Element
(mg/Kg)
Al
B
Cu
Fe
Mn
Mo
Na
Zn
Element
(%)
C
Ca
H
K
Mg
N
P
S

2

Pristine Pristine Pristine Invaded Invaded Invaded Pure Pure
Control
+ AC
+ Ash Control
+ AC
+ Ash
Ash
AC
28000
27000
28000
26000
28000
27000 20000 22000
6.1
5.9
6.3
<5
6.7
6.1
52
7.1
45
43
44
47
50
48
42
100
32000
33000
32000
35000
36000
36000 17000
390
970
920
970
920
950
960 2600
11
<1
<1
<1
<1
<1
<1
2.5
<1
1200
1000
1200
820
950
920 3200
610
82
75
78
75
74
77
150
16
Pristine Pristine Pristine Invaded Invaded Invaded Pure Pure
Control
+ AC
+ Ash Control
+ AC
+ Ash
Ash
AC
2
5.8
5.0
5.4
3.6
4.4
3.6 22.5
80.43
1.3
1.3
1.4
1.3
1.4
1.4
7.8
0.06
2
1.0
0.97
1.1
0.82
0.80
0.81
NA
1.63
0.50
0.49
0.52
0.47
0.51
0.50
2
2.4
0.87
0.90
0.88
1.0
1.1
1.0
1
0.03
0.56
0.48
0.52
0.37
0.38
0.37 0.052 0.363
0.15
0.14
0.15
0.13
0.13
0.13
0.44
0.02
0.090
0.083
0.091
0.077
0.080
0.080
0.36
0.09

Data from Antonelli (2018).
Data from Astuti et al. (2021), Hidayu & Muda (2016), Iqbaldin et al. (2013), and Phan et
al. (2006).
3

59

DISCUSSION
Preferential Invasion based on Biogeoclimatic Subzone
The GIS analyses completed show that C. stoebe preferentially invades higher
elevation grasslands with cooler temperatures and forest interfaces within the Lac Du Bois
GPA. The most affected subzone in the Lac Du Bois GPA is the BGxw1, where over a third
of the area is now covered by C. stoebe. This is an important finding as it may allow directed
management of the invasive plant, as well as provide predictions for future invasion. The
publicly available IAPP database is an exciting tool for extracting invasive plant information
for both small study areas and province wide analyses. Its use is likely not harnessed as well
as it could be, and studies of plant invasions within B.C. should utilize its power in making
spatial predictions of future spread. Future studies could focus on the modelling required to
make those predictions and define what factors are important in an individual plants spread.
The finding that the BGxw1 and IDFxh2 subzones were the most negatively affected
of the subzones throughout the Lac Du Bois GPA is also important for species at risk.
Indeed, B.C. only contains only a small portion of grasslands, but they also provide important
niches for an abundance of endangered animals. The protection of grasslands should
therefore be a high priority, including invasive species management. Across B.C., C. stoebe
is the most invasive plant along with C. diffusa. The threat of habitat infringement for species
at risk by invasive species should be more closely studied and managed in order to more
directly impact the spread of invasive species.
Finally, the difference in amount of invasion by C. stoebe in the Lac Du Bois GPA
compared to similar BGC subzones throughout B.C. shows just how necessary some type of
intervention and protection is in our local grasslands. The bunchgrass zone throughout B.C.
is already at risk from climate change, woody encroachment, and anthropogenic impacts, and
the Lac Du Bois GPA is not immune to those factors either. With a high level of plant
invasion in this park compared to other areas in B.C., it is very important that this park
receives a focussed effort regarding the removal of noxious species. Such efforts may include
further biological and chemical controls that have been used in the past, as well as targeted
grazing efforts that may have a positive impact on the health of the grasslands overall.

60

Positive Soil Legacies Detected
This study aimed to answer the following questions:
•

Can we detect soil legacy effects in invaded soils?

•

Can potential legacy effects be lessened or mitigated using pulp mill fly ash as
a soil amendment?

By comparing F. campestris biomass from pristine and invaded soils with no
amendments, we may answer the first question. These greenhouse trials lead us to the
conclusion that positive soil legacy effects are found in invaded soils which aid in the growth
of a native plant. This result was unexpected: literature indicates that soil legacies in invasive
soils harbor negative effects on native plant biomass (Del Fabbro & Prati, 2015; Elgersma et
al., 2011). In certain cases, this can be visually confirmed in the very grasslands that soils
were collected from, thus this result from the greenhouse is peculiar. Moreover, results from
the AC treatments indicate that AC effectively kills most individuals. This is contradictory to
literature as well, where most studies using similar amounts of this material show that when
added to an invaded soil type, the soil condition improves for native plants, perhaps due to
the adsorption of potential allelochemicals (Del Fabbro & Prati, 2015; Nolan et al., 2015;
Perry et al., 2007). Perhaps the homogenized invaded soils did not contain potential
allelochemicals, in which case the AC addition was essentially an increase in soil carbon. As
part of the nitrogen cycle, soil microorganisms take in nitrogen to make use of available
carbon, thus depleting soils of nitrogen (Vitousek & Howarth, 1991). The apparent lack of
nitrogen therefore limits the growth of plants in that soil, which helps to explain the lack of
growth witnessed in pristine soils with AC. This can be confirmed by viewing the carbon to
nitrogen ratio analysis, where AC addition in pristine soils caused a significant increase in
the C:N ratio compared to control soils, indicating that there was more carbon left in the soils
and that nitrogen was depleted upon AC treatment. While significance didn’t transfer over to
invaded soils, there is a trend showing a greater C:N ratio in AC treated soils compared to
control and ash treated soils, which further assists in the interpretation of this result. Other
research suggests that a combination of AC and invasive plant litter is required to create soils

61
that are more hospitable for native plants with litter use being analogous to applying N
fertilizer (Grove et al., 2012). The present study made use of soil materials only and omitted
litter as part of the substrate. The results obtained from the AC treatment require further
exploration, and perhaps an investigation of the soil microbial community is necessary to
garner more conclusive results.
In order to answer the second question, we must compare the biomass of F.
campestris in invaded soils that were ash treated soils to invaded control soils. Indeed, a
legacy effect was noted in this experiment; however, the question implies that the soil legacy
effects are negative towards native plants. The addition of ash in each soil type had opposite
effects: in invaded soils, biomass decreased while biomass increased in pristine soils. While
these are not significant trends, they are worth noting since this goes against what the
expectations of this experiment were. Evidence of positive soil legacies from invasive soils
do exist (Del Fabbro & Prati, 2015; Inderjit et al., 2011), and perhaps the most logical
explanation would be that the chemicals released by the invasive plant (i.e.: the suspected
cause of legacy effects) have been degraded by soil microorganisms, and are no longer
affecting the native plants harbored within (Kaur et al., 2009; Zackrisson & Nilsson, 1992).
Additionally, Barto and Cipollini (2009) have shown that the secondary metabolites
produced by Alliaria petiolata have a very short half life even in sterile soil (~45 hours). The
metabolites produced by A. petiolata are flavonoids, which bears close resemblance to the
chemical structure of catechin, thus it may be that legacy effects are not witnessed in these
invaded soils due to the natural degradation of the suspected allelopathic compound
(Muhamad et al., 2015; vanˈt Slot & Humpf, 2009). This information may also help to
answer why conflicting results have occurred in past studies leading to the eventual
retractions of certain articles. If such temporal differences create these discrepancies, perhaps
firm methodology should be created to assist collecting data relevant to studying allelopathy
and soil legacy effects from invasive plants.

62

Impacts of Ash on Biomass
The addition of ash in this experiment did not have any significant effect on the
biomass of either species in either soil type when compared to a control; however, the
direction of biomass change is important to note for both species. Specifically, using ash
seemed to have a more neutralizing effect on the biomass of F. campestris, where it appears
to have increased biomass in pristine soils and decreased biomass in invaded soils compared
to a control. Biomass of F. campestris was equal in ash treated pristine soils and ash treated
invaded soils in the end, hence the “neutralizing” effect. A similar, yet opposite trend was
witnessed with C. stoebe where biomass was brought closer together in both soil types with
the addition of ash; however, biomass decreased in pristine soils and increased in invaded
soils with an ash amendment. In other words, it appears that adding ash in pristine soil types
incurs a benefit to native plants and not C. stoebe and adding ash in invaded soils is
beneficial to C. stoebe but not F. campestris. That addition may have altered soil conditions
such that it was no longer preferable for growing in those soil types. Regardless, this
differential effect is important to note: in the field, pristine soils are defined by the absence of
invaders, thus adding ash to a site may act to increase native biomass through a fertilizing
effect (Basu et al., 2009; Kishor et al., 2010). Indeed, ash acted to marginally increase certain
element concentrations in pristine and invaded soils, which likely accounted for the increased
biomass seen in some of the soils (Table 2.22). Interestingly, the addition of the ash
essentially had no effect on soil carbon or nitrogen values in this case (Figure 2.8). From a
management perspective, this can be positive since trends in biomass were witnessed upon
the addition of ash, but underlying soil characteristics were not largely affected. Conversely,
the results here indicate that we might wish to avoid using ash to remediate invaded soils
since it tends to aid in the growth of C. stoebe but hinders F. campestris (Figure 2.3). Further
investigation into methods of application, amounts of ash, and levels of C. stoebe invasion
should be explored to confirm these results in a field scenario.

63

Effects of Ash and AC on Survival and Mortality
This study examined four different metrics associated with seedling survival and
mortality: germination rate, mortality rate, the entire 90-day survival rate, and the survival
rate of germinated seedlings between 30- and 90-days to fully understand how longevity was
affected in each soil combination. The first observation that can be made is that activated
carbon had an overall negative impact on the survival of both F. campestris: germination
rates and both survival rates were lowest in activated carbon treated soils, and mortality rates
were highest in these treatments. This suggests that the use of activated carbon here was not
an appropriate measure against ash treatments since the results were very different. Likely,
this is an outcome of the activated carbon processing that occurred. The purchase of
granulated activated carbon was an oversight and closer results would be more likely found if
a finer form was used. Despite this, the seeds of C. stoebe appeared to have no difficulty
germinating in the presence of activated carbon. Beyond that, C. stoebe exhibited similar
outcomes to F. campestris with respect to activated carbon addition: survival rates were
lower and death rates were higher, further supporting the result that the use of granulated
activated carbon was inappropriate.
The addition of ash had interesting effects on the survival metrics of F. campestris
and C. stoebe. Mostly, there were no significant differences compared to a control; however,
the long-term survival rate of F. campestris (i.e.: the germination survival rate) was
significantly greater in invaded, ash treated soils compared to a control and AC treated soils.
This effect was not seen in pristine soil types. Moreover, C. stoebe had a slightly lower longterm survival rate in the same soils compared to a control. This implies that F. campestris
individuals that survive to the 30-day period are more likely to survive the entire duration of
the trial when grown in invaded soils treated with ash. This leads to further implications with
management: according to these results, if an invaded area is treated with ash and seeded
with F. campestris we might see limited success in the first 30 days but after that point it is
likely that those that are left are what is going to withstand. While interactions between F.
campestris and C. stoebe were not examined in this experiment, it still may be necessary to
remove C. stoebe seedlings at that 30-day period to maintain similar results in a field
application. In invaded soils that did not have any treatment, the greatest mortality of F.

64
campestris happened between the 30- and 90-day periods, suggesting that the ash may be
alleviating a negative soil effect that would otherwise cause mortality after 30-days. This
effect is not witnessed in pristine soils where the greatest survival is seen in control soils,
hence the possible alleviatory effect.

Invasive Boundary Movement
By comparing the currently invaded boundary that was walked to the old boundary
mapped by the IAPP, we are witnessing a change over time in the sizes of invaded
boundaries and their relative locations. The IAPP polygon overlapping the current study area
was created in 2006, thus it may be that the IAPP polygon is out of date. In fact, there have
been at least 1,287 recordings of either mechanical, chemical, or biological treatments
applied within the park, and a release of Cyphocleonus achates, the root boring weevil, was
applied in 2016 within 200 m of the study area. This could account for the apparently
shrunken boundary of the study area from what the IAPP had previously declared in 2006.
Another consideration may be that the IAPP identified both C. stoebe and C. diffusa in this
polygon, and that C. diffusa may be present outside of the observed polygon; however, C.
diffusa was not present in or near the study site. A final consideration may be that the IAPP
over-generalizes an invaded area, and a contiguous polygon was created where it should not
have been. Methods of the original sampling are not publicly available; thus, it is unknown
how the original polygon was created. Regardless, it is widely known that plant communities
can be drastically altered in the face of climate change, thus it is important to view future
areas of invasive expansion as well as current ones (Settele et al., 2014).
This apparent shift in the invasive site boundary also points to the need for improved
sampling of invaded areas in B.C. If the IAPP is to be the sole resource for invasive plant
information, it stands to reason that invasive areas should be monitored on a frequent basis.
Remote sensing advances in recent years has shown incredible potential for collecting a
wealth of data at a fine scale with little effort (Hung & Wu, 2018). In the future, there may be
ways to identify species from a simple image taken by unmanned aerial vehicles (UAV’s),
which would be immensely useful for more than just invasive species identification and

65
location. Indeed, efforts are underway to solve this task, though at the moment it seems this
accomplishment is limited to forest canopies (Baena et al., 2017; Santos & Ustin, 2018).
With invasive species threatening to alter landscapes, technologies should be developed for
modelling future expansions and identify areas of concern, allowing for preventative
management of invasive spread.

Limitations
GREENHOUSE CONDITIONS
As with most greenhouse studies, the climate-controlled environment within the
greenhouse is meant to promote the growth and establishment of the plants under study. As
such, these are not exactly reflective of what occurs in a field condition. In general,
greenhouse studies allow for simpler statistics and allows for better experimental
manipulation on selected variables; however, they might fail to capture the full complement
of soil and environmental processes (May & Baldwin, 2011). Consistent watering schedules
allow for plants to stay healthy throughout the duration of the study, but it may change
belowground processes and even certain aspects of plant growth which are not indicative of
field scenarios. Ideally, a field study would be conducted alongside greenhouse trials in order
to verify the applicability of the greenhouse study.
Additionally, certain aspects of the greenhouse climate are beyond human control.
The greenhouse does its best to keep within the set climatic parameters but may encounter
errors if there are external factors such as a power outage or equipment failure. In these
scenarios, climates are not being monitored or controlled and thus the greenhouse experiment
could be at risk. Even slight variations like these have been reported to have effects on the
final outcome of a greenhouse experiment on plants grown from seed (Hammer & Hopper,
1997).

66

SOIL CONDITIONS
Soils were collected in the late fall of 2017, just before snowfall. At this point, most
of the vegetation on the landscape had gone dormant, including spotted knapweed. Perhaps at
this time of year, it is not legacy effects that are impacting the growth of vegetation in the
environment, but climatic and temporal factors are playing a role. Soils in this study were
collected at one point in time, but perhaps if soils had been collected at various points
throughout the year, spotted knapweeds soil legacies would be more pronounced. In one
study, multiple samplings found varying concentrations of soil catechin throughout the year
at one study site (Perry et al., 2007). That result was fairly suspect since there was only a
single record of catechin occurrence during the entire sampling period at only a single site,
but it does indicate that some temporal sampling may need to be incorporated into future
studies of soil legacy effects on neighboring native plant species. Furthermore, litter was
discarded in this study from both invaded and pristine soils during collection, which Grove et
al. (2012) suggest is an important constituent in studies using AC.
Another consideration of the soil collection would include the depth of soil collected
as well. In this study, the top 5 cm was discarded with the intent to omit any lasting seeds in
the field seed bank (Bonis & Lepart, 1994). In doing so, however, much of the organic
material was removed as well. The top most layer of any soil horizon contains the most
mineral nutrients that a plant will draw from (Watson, 2014); however, the soils in this study
area are classified as orthic black chernozems which are reported to have an A horizon from
0 - 25 cm just below the soil surface (Canadian Soil Information Service, 2013; Filatow,
2019). Regardless, soil microorganisms can change with soil depth, and these
microorganisms can have a pronounced effect on the aboveground plant communities
(Bhattarai, 2015; Vitousek & Howarth, 1991). This removal may create a misrepresentation
of the field soils in the greenhouse study, thus the results should be taken cautiously. The
organic substances within the top 5 cm of the soils could have been more suitable for a
greenhouse trial, or oppositely, contained allelopathic catechins that would inhibit native
plant growth more effectively. The over-arching purpose of taking soils from a deeper source
was to capture potential legacy effects which would be found in older (deeper) soils;
however, in the lens of plant growth, this may not have been an optimal goal.

67

POTTING
The pots used in the greenhouse trial were 512 cm3, which are generally smaller than
what is normally used in greenhouse trials. Though crowding of individuals was not an issue
in this present study, the depth of soil (or lack thereof) may have been influential in the
results here. In nature, C. stoebe produces a large taproot, and with the limited depth of pots
there may have been a physical constraint on its growth. Having only reached a rosette life
history stage, however, the individuals did not grow large enough to produce a taproot.
Additionally, volume of soil in these pots could have influenced results as well. If 1,000 cm3
pots were used, a total of 1 L of soil could have been used in each pot. Instead, only 512 mL
could be used in the pots here. This is where restrictions may become more prevalent: the
limited volume of soil also contains a limited volume of nutrients which may be problematic
for plant growth. Initial errors in sample design were the cause of this, as well as having a
limited batch of soils to draw from.

ELEMENTAL ANALYSIS
Chemical composition of a soil can provide a wealth of information on the soils in
question, including salinity, nutrient composition, pH, and overall quality. The data that I
obtained in my study for elemental analysis represents a snapshot in time for pre-treatment
soil conditions (i.e.: amended bulk soils with no plants yet); however, it would have been
beneficial to observe changes in these nutrient compositions after the 90-day growing period.
Doing so may have provided insight into the belowground processes and aided in
explanations for why treatments using activated carbon performed differently than described
in literature.

68

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Zackrisson, O., & Nilsson, M.-C. (1992). Allelopathic effects by Empetrum hermaphroditum
on seed germination of two boreal tree species. Canadian Journal of Forest Research,
22(9), 1310–1319. https://doi.org/10.1139/x92-174

75

Chapter 3: Research Conclusions
This study showed that the removal of an invasive species promotes the growth of a
seeded native species, and the addition of pulp mill fly ash did not harbor any significant
effects to the growth of either Festuca campestris or Centaurea stoebe (though certain trends
were noted). While removal and reseeding seems like a simple solution to dealing with the
threat of invasive species spread, there are many factors that need to be considered. Soil
legacy effects from the invasive spotted knapweed were determined to be positive in this
greenhouse study; however, field experience demonstrates that left alone, recently invaded
areas do not typically regrow native vegetation naturally or when seeded (May & Baldwin,
2011; Rand et al., 2015). Taking this into account, we must explore the research and
management implications that this study has brought forward.
Another item to further discuss is the limitations that were brought on by the project
design. Standard methods for comparing the growth of various plants with respect to soil
legacy effects have been established in previous works but this project marks the first
examination of soil legacy effects of the invasive C. stoebe on a native bunchgrass (Del
Fabbro & Prati, 2015).

RESEARCH IMPLICATIONS
This study serves as a solid steppingstone for future research with invasive plants and
the use of ash as a soil amendment to alleviate the potential negative allelopathic or soil
legacy effects. Further studies should focus on a field component, where ash is broadcast
applied to an invaded site much like a chemical control. It would be interesting to see this
method alongside hand-pulling the weed when it isn’t seeding, and then observe the native
plant community in these trial areas. Other aspects to a study involving these parameters
could include the concentration of ash applied, comparisons to areas free of invasive plants,
comparisons to different BEC zones or wider regions (if applicable), and comparisons at
different times of the year. While the present study was able to control these aspects, a robust
field component could be helpful to provide a broader context for the results.

76
While the present study brought about some confounding results, we should not
immediately discount fly ash as a potential tool for invasive species management. It has been
shown to promote growth of various plants in other forms, much like that of a fertilizer
(Emilson et al., 2018; He et al., 2017). Presently, we found that adding the ash component
assisted in increasing nutrient values of B, Ca, K, Mg, and S in pristine soils and increasing
Al, B, Ca, Cu, Fe, K, Mn, Na, S, and Zn in invaded soils, albeit slightly. Perhaps with the
addition of more ash, clearer results from the ash addition could have emerged. He et al.
(2017) studied the growth of alfalfa in loessial soil and added differing concentrations of fly
ash (5%, 10%, 20%, and 40% by weight), and found that the addition of these nutrients
promoted the growth of alfalfa. Here, the researchers also noted chlorosis in their plants,
though this was at application rates of 10% and over. It is important that this study
highlighted that: with the chlorosis mentioned, we might confirm that too much AC was
added in our soils, even at a rate of 1% by volume and further tuning on the amount of AC
addition is required.
The addition of ash may also be of importance in soils with low pH: industrial fly ash
is consistently characterized as having high pH (in the range of 8 – 12), thus its application
into acidic soils would assist in increasing pH to a more suitable level (Domes et al., 2018;
Magiera et al., 2013). Studies on the remediation of Canadian forest soils using ash have
recently been of great interest. A federal working group, AshNet, is actively exploring the
potential of industrial wood ash application into forest soils and the associated publications
have shown great promise with benefits to forest health, including increased soil pH (Brais et
al., 2015; Emilson et al., 2018). As more details emerge from this group, it is likely that ash
application rates will become more refined and better characterized based on local conditions
of a given site. While invasive species management is beyond the scope of this working
group, it still points to ash being a useful remediation tool in forested soils. Future work
within this group with regards to ash’s effects on invasive species would prove useful. In
B.C., the BGC zone with the greatest amount of plant invasion by square kilometer is the
Interior Douglas Fir zone which holds a great amount of timber value within the province.
Preserving this zone from invasive species is therefore important and it would be interesting

77
to see research into the use of ash within this zone for the dual purpose of invasive species
management and forest soil remediation.
This study provided a basis for further experimentation in both the field and the
greenhouse, though the limitations addressed in Chapter 2 should be acknowledged. While
the ash additions may not have yielded the expected results, further studies on the control of
invasive plant species should consider its use as a potential control agent. In the present
study, there was no significant trend in decreasing C. stoebe biomass; however, this was a
trial using a 1% by volume ash addition. Perhaps further manipulations could be made on this
value to produce more significant results. This type of work is currently being performed in
the grasslands near Merritt, B.C. (Hampton, 2020).
There are also remote sensing and GIS considerations that can be taken into
consideration for future research as well. This study noted certain provincial BGC subzones
to have a greater proportion of C. stoebe found within them than others. While beyond the
scope of this study, it would be possible to generate spatial models that predict the presence
and potential of C. stoebe, along with other invasive species based on the existing input data
(Cutler et al., 2007). With the maps of the species’ potential, we may be able to locate areas
of rampant spread before they occur and implement management strategies that prevent
further environmental deterioration.

MANAGEMENT IMPLICATIONS
This study aimed to explore the soil legacy effects of C. stoebe and assess the use of
pulp mill fly ash as a restoration tool for invaded areas. While we found no significant trends
to indicate that using fly ash was a promising amendment, it should be noted that the data
presented are representative of a small-scale greenhouse experiment. Indeed, further efforts
to propagate the use of ash are in place on a much larger scale and show promising results in
both agricultural and forestry applications (Emilsson, 2006; Hannam et al., 2016). In many
cases, its use in agriculture may contribute to (or replace, depending on the reasoning for
application) the soil macronutrients and thus produce a healthier ecosystem. Forestry
applications of ash to bolster the soil nutrients appear to be more case specific and have many

78
factors that influence their results, including the chemical composition of the ash, soil type,
and dominant tree species (Augusto et al., 2008; Emilson et al., 2018; Pitman, 2006). The
method of application is likely a factor in this as well. In many studies of ash use, the ash is
broadcast applied whereas it was thoroughly mixed into the soils in this project. Where
successes have been noted, it is important to acknowledge the method of application in order
to have similar results in our own work. Drawing on methods from agricultural projects may
be more suitable to attempt to characterize the soil legacy effects of C. stoebe rather than a
greenhouse experiment, and further research should have a strong field component to
accomplish that goal.
The world of GIS and spatial analysis is advancing quickly, and it is important to
understand the impact that may have on the future of invasive species research. In terms of
management, we can monitor and model the advance of invasive species based on publicly
available data. It is therefore important that the dataset is updated as new data becomes
available. Additionally, investments into remote sensing hardware and software should
become more relevant since we can interpret more data from remote sensing equipment than
we could doing a standard field survey or walkabout. Innovations in remote sensing
equipment may one day allow us to differentiate invasive from native species at multiple life
stages based on imagery. If that potential could be harnessed, it would be immensely useful
for the detection of invasive species in order to mitigate their spread. For now, we must focus
on the modelling and responding to the invasive impact that is occurring on our landscapes
and especially in the grasslands.
The threat of invasive species should be taken seriously since they have an inherent
ability to cause lasting damage to an ecosystem. In BC, the grasslands account for less than
1% of the land base but provide important habitat for over 30% of the province’s species at
risk. Protecting these grasslands should be prioritized, and strategies for invasive species
mitigation should continue to be studied. The present study attempted to highlight a
phenomenon witnessed in the field, where an invasive plant may have lasting soil effects
after its removal. While this was unable to be replicated in the greenhouse trials, it is obvious
that there is something going on preventing native plant species from repopulating recently
invaded areas. There are many explanations for these results: perhaps the amount of soil in

79
the pots was not appropriate in this study, or the removal of the first 5 cm of soil prior to
collection is not standard for studies of this nature. Whatever the case, these sources of error
allow future studies to build on this and perhaps more rigorous designs will be implicated to
determine whether spotted knapweed has lasting negative effects in the grasslands of B.C.

LITERATURE CITED
Augusto, L., Bakker, M. R., & Meredieu, C. (2008). Wood ash applications to temperate
forest ecosystems—potential benefits and drawbacks. Plant and Soil, 306(1–2), 181–
198. https://doi.org/10.1007/s11104-008-9570-z
Brais, S., Bélanger, N., & Guillemette, T. (2015). Wood ash and N fertilization in the
Canadian boreal forest: Soil properties and response of jack pine and black spruce.
Forest Ecology and Management, 348, 1–14.
https://doi.org/10.1016/j.foreco.2015.03.021
Cutler, D. R., Edwards, T. C., Beard, K. H., Cutler, A., Hess, K. T., Gibson, J., & Lawler, J.
J. (2007). Random Forests for classification in ecology. Ecology, 88(11), 2783–2792.
https://doi.org/10.1890/07-0539.1
Del Fabbro, C., & Prati, D. (2015). The relative importance of immediate allelopathy and
allelopathic legacy in invasive plant species. Basic and Applied Ecology, 16(1), 28–35.
https://doi.org/10.1016/j.baae.2014.10.007
Domes, K. A., de Zeeuw, T., Massicotte, H. B., Elkin, C., McGill, W. B., Jull, M. J.,
Chisholm, C. E., & Rutherford, P. M. (2018). Short-term changes in spruce foliar
nutrients and soil properties in response to wood ash application in the sub-boreal
climate zone of British Columbia. Canadian Journal of Soil Science, 98(2), 246–263.
https://doi.org/10.1139/cjss-2017-0115
Emilson, C. E., Hannam, K., Aubin, I., Basiliko, N., Belanger, N., Brais, S., Diochon, A.,
Fleming, R., Jones, T., Kabzems, R., Laganiere, J., Markham, J., Morris, D., Rutherford,
P. M., Van Rees, K., Venier, L., Webster, K., & Hazlett, P. (2018). Synthesis of current
AshNet study designs and methods with recommendations towards a standardized
protocol. https://cfs.nrcan.gc.ca/publications?id=39388
Emilsson, S. (2006). International Handbook: From Extraction of Forest Fuels to Ash
Recycling. Swedish Forest Agency.
http://www.recash.info/uploads/documents/handbook.pdf

80
Hampton, M. (2020, July 9). Research ongoing for local grasslands. Merritt Herald, 1, 3.
https://www.merrittherald.com/research-ongoing-for-local-grasslands/
Hannam, K. D., Deschamps, C., Kwiaton, M., Venier, L., & Hazlett, P. (2016). Regulations
and guidelines for the use of wood ash as a soil amendment in Canadian forests.
https://cfs.nrcan.gc.ca/publications?id=37781
He, H., Dong, Z., Peng, Q., Wang, X., Fan, C., & Zhang, X. (2017). Impacts of coal fly ash
on plant growth and accumulation of essential nutrients and trace elements by alfalfa
(Medicago sativa) grown in a loessial soil. Journal of Environmental Management, 197,
428–439. https://doi.org/10.1016/j.jenvman.2017.04.028
Magiera, T., Gołuchowska, B., & Jabłońska, M. (2013). Technogenic magnetic particles in
alkaline dusts from power and cement plants. Water, Air, & Soil Pollution, 224(1),
1389. https://doi.org/10.1007/s11270-012-1389-9
May, L., & Baldwin, L. K. (2011). Linking field based studies with greenhouse experiments:
The impact of Centaurea stoebe (=C. maculosa) in British Columbia grasslands.
Biological Invasions, 13(4), 919–931. https://doi.org/10.1007/s10530-010-9879-4
Pitman, R. M. (2006). Wood ash use in forestry - a review of the environmental impacts.
Forestry, 79(5), 563–588. https://doi.org/10.1093/forestry/cpl041
Rand, T. A., Louda, S. M., Bradley, K. M., & Crider, K. K. (2015). Effects of invasive
knapweed (Centaurea stoebe subsp. micranthos) on a threatened native thistle (Cirsium
pitcheri) vary with environment and life stage. Botany, 93(9), 543–558.
https://doi.org/10.1139/cjb-2015-0032

81

Appendix A: Characterizing immediate and legacy soil effects in
the field: A lesson in transplanting
INTRODUCTION
Often, greenhouse trials do not correspond directly to field conditions. Field
conditions contain much variability in climate, moisture availability, soil conditions and
nutrients, herbivory, slope, and much more, thus results gained from greenhouse trials may
not always represent field conditions exactly since these types of factors are controlled for.
An obvious way to address this issue is to perform a similar study in the field, observe
growth through time, and extrapolate the information gained from the experimental process.
The native Festuca campestris and the invasive Centaurea stoebe are both found
commonly in upper elevation grasslands in the interior of British Columbia. To reiterate, C.
stoebe is an aggressive invasive forb that has caused much damage to pristine grassland areas
in North America in recent history, and its spread continues to this day despite various
control efforts. Negative effects of its invasion have been noted, including a decrease of plant
biodiversity, decrease in available forage for grazing animals, and alteration of soil
properties. Further intervention is required to diminish the presence of this invasive plant,
and with the interesting results from the greenhouse experiment, it is prudent that field
methods be developed which not only mimic the greenhouse experiment, but also have
practicality in mind on larger scales. Overall, the field trials were implemented with the
following questions in mind:
•

Are the results of the greenhouse trials representative of what would happen in
the field?

•

What concentration(s) of ash will work to mitigate the potential legacy effects
of C. stoebe?

Once again, F. campestris was chosen as the experimental plant since it is the most
common native plant found in the upper grasslands. Planting sites were located in a heavily
invaded patch of the upper grasslands of the Lac Du Bois Protected Area, North of

82
Kamloops, British Columbia where moisture is more readily available, and temperatures are
cooler relative to lower elevation grasslands.
The goal of the field trial was to support the conclusions drawn from the greenhouse
trials, and to discover the effect that ash concentration had on the growth of individual F.
campestris seedlings.

METHODS
Seedling Germination
Purchased F. campestris seeds were planted in small plug trays at the Thompson
Rivers University Research Greenhouse on March 5, 2018 with each plug measuring 4 cm in
diameter and 6 cm tall. A single seed was planted in potting soil (Sunshine Mix 4, Canadian
sphagnum peat moss) housed in each plug. A total of 1,160 seeds were planted. Growing
conditions in the greenhouse were set to follow the ISP conditions laid out by Hendry and
Grime (1993). Seedlings were allowed to grow under these constant conditions until May 2,
2018 when field planting commenced.

Field Design
This study took place in the upper grasslands of the Lac Du Bois Protected Area,
North of Kamloops, British Columbia. Specifically, the same 10 heavily invaded locations
from the greenhouse component of this project (i.e.: Chapter 2) were chosen, and grids were
formed no farther than 2 m away from the soil sampling locations. C. stoebe was physically
removed from a 2 m × 2 m square in the heavily invaded areas. Within the 4 m2 grid, 9
sample points were established in a 3 × 3 fashion, and sample points were located 0.5 m from
the edge of the 4 m2 grid, and 0.5 m from each other (Figure A.1). The 9 sample points
served as the different treatment locations for this project and were arranged in a randomized
block design. The treatments used in the field included a range of ash concentrations, a single
Activated Carbon (AC) treatment, and a control condition where no ash or AC was applied.

83
Concentrations of ash and AC were based on a 500 mL volume of soil which was the
anticipated volume that these roots would take up in a 90-day growing period, and the
individual treatments are as follows:
1. Control (no AC or Ash addition)
2. 1% (5 mL) AC addition
3. 1% (5 mL) Ash addition
4. 2% (10 mL) Ash addition
5. 5% (25 mL) Ash addition
6. 10% (50 mL) Ash addition
7. 20% (100 mL) Ash addition
Since there were only a total of 7 treatments, there were 2 “blank” locations where no
planting occurred to account for the 9 total sample points. Transplanting of the individual F.
campestris plugs occurred on May 2, 2018 and the study lasted 90 days. At each sample
point where treatments were present, a spade-type shovel was used to open the ground. The
soil treatments were then poured into the opening in the ground, and the F. campestris plugs
were planted in the openings afterwards. This method was chosen for simplicity on a larger
scale, should it be applicable. At the time of planting, the number of leaves and the height of
the tallest leaf of each F. campestris individual was recorded. Over the course of the growing
period, individuals were hand watered every 2-4 days using tap water, depending on the
weather. At the end of the growing period, number of leaves and height were once again
recorded, and aboveground biomass samples were taken from each individual. Biomass
samples were brought back to the Thompson Rivers University Research Greenhouse where
they were dried in a drying oven (Yamato DKN8132) at 70°C for 72 hours prior to weighing.
Biomass was recorded to the nearest hundredth of a gram using a Fisher Scientific top
loading scale (accu-4102).

84
2m

2m
Figure A.1: Design of the field grids. The crosshatches represent the 9 sample points within
the 2 m × 2 m grid.

Statistical Analyses
A one-way analysis of variance (ANOVA) was used to determine if soil treatments
had any effect on biomass. Assumptions of this test include normality of the input data as
well as homogeneity of variances. The raw data violated the normality assumptions; thus, a
square root transformation was applied to fit a normal distribution and pass a homogeneity of
variance test. A Tukey post-hoc analysis was completed to view the differences between each
pairing.
Two-way repeated measures ANOVA’s were used to see the differences in the
number of leaves and leaf height between the soil treatments at each time point. Normality
testing for this dataset was applied; however, the repeated measures test was carried out
regardless of the results since this test is robust against departures from normality. An
analysis of the main effects of soil type and time was conducted, and pairwise comparisons
were made between the pairings of soil treatments and time. P-values were adjusted using the
Bonferroni method to account for the unique pairwise comparisons involved in a two-way
repeated measures ANOVA. All calculations and computations were performed using R for
Statistical Computing (R Core Team, 2020).

85

RESULTS
Perhaps due to inherent issues with transplanting, many of the individual plugs did
not survive the duration of the experiment. Out of the 70 individuals planted, only 22
(31.4%) survived to the end of the 90-day growing period. The subsequent results will not
carry any real significance. Nevertheless, statistical measures were applied to examine the
plants throughout their growing period.

Trends with Survived Plugs
Both the 1% ash and control treatments had the greatest number of plugs survive to
the end of the experiment, each having 5 survivors out of 10 planted. The least successful
trial was the 1% AC treatment, which resulted in 0 survivors (Table A.1). While this
compliments the results from the greenhouse trials, we cannot rule out the possibility that
local conditions or issues with transplanting aided in this result. All survived plugs resulted
in a net loss of leaf height, with the greatest height loss occurring in 20% ash treatments.
Conversely, all trials resulted in a net gain of number of leaves with 5% ash treatments
having the greatest increase. Finally, average aboveground biomass of each living plug was
always below 0.5 g. This is not likely indicative of how F. campestris grows in the field, thus
these results should be taken lightly.

86
Table A.1: Summarized field data captured from both the planting date (May 2, 2018) and
the harvest date (July 30, 2018). The values in this table are only representative from plants
which survived the full 90-day growing period, hence the different values for N. Mean values
are appended with standard errors to indicate the spread of the data.
Mean
Planting
Soil
Height
Treatment (cm)
AC-01
NA
9.26 ±
Ash-01
0.73
9.25 ±
Ash-02
0.85
8.95 ±
Ash-05
2.35
10.05 ±
Ash-10
1.82
11.025 ±
Ash-20
1.02
7.82 ±
Control
0.63

Mean
Harvest
Height
(cm)
NA
4.24 ±
0.89
3.8 ±
1.1
4.75 ±
2.75
4.925 ±
1.01
3.875 ±
0.98
3.58 ±
0.91

Mean
Δ
Height
(cm)
NA
-5.02 ±
0.46
-5.45 ±
0.25
-4.2 ±
0.4
-5.13 ±
1.56
-7.15 ±
0.97
-4.24 ±
1.47

Mean
Planting
Leaves
(n)
NA
18 ±
2.14
15.5 ±
0.5
19 ±
5.0
15.75 ±
3.90
12.5 ±
2.53
17 ±
3.08

Mean
Harvest
Leaves
(n)
NA
20.6 ±
5.31
34 ±
5.0
51.5 ±
19.5
28.5 ±
10.90
20.75 ±
4.21
23.8 ±
4.26

Mean
Δ
Mean
Leaves Biomass
(n)
(g)
N
NA
NA 0
2.6 ± 0.174 ±
5
6.96
0.019
18.5 ± 0.175 ±
2
5.5
0.035
32.5 ± 0.185 ±
2
14.5
0.065
12.75± 0.145 ±
4
7.54
0.079
8.25 ± 0.145 ±
4
3.57
0.060
6.8 ± 0.214 ±
5
5.83
0.018

Biomass
A one-way ANOVA was performed to find any differences in biomass between the
soil treatments. A square root transformation was applied to the biomass data to pass a
Shapiro-Wilks normality test (p = 0.075) and Levene’s test of homogeneity (p = 0.234) to
allow the use of the parametric one-way ANOVA. No significant differences in biomass
were found between the treatments (F (6, 63) = 0.962, p = 0.458). The mean biomass of F.
campestris when grown in soils treated with activated carbon was slightly lower compared to
when grown in other soil treatments (Table A.2). A Tukey post-hoc analysis revealed no
significant differences between any pairing of groups (Table A.3).

87
Table A.2: Summarized mean and standard deviation of biomass of F. campestris grown in
each soil treatment.
Treatment √Biomass ± SD N
AC_01
0.180 ± 0.104 10
ASH_01
0.231 ± 0.201 10
ASH_02
0.248 ± 0.139 10
ASH_05
0.227 ± 0.151 10
ASH_10
0.281 ± 0.141 10
ASH_20
0.245 ± 0.149 10
CONTROL
0.330 ± 0.154 10

Table A.3: Tukey HSD pairwise comparisons of biomass, change in number of leaves, and
leaf height change between each treatment grouping.
Group 1
AC_01
AC_01
AC_01
AC_01
AC_01
AC_01
ASH_01
ASH_01
ASH_01
ASH_01
ASH_01
ASH_02
ASH_02
ASH_02
ASH_02
ASH_05
ASH_05
ASH_05
ASH_10
ASH_10
ASH_20

Group 2
ASH_01
ASH_02
ASH_05
ASH_10
ASH_20
CONTROL
ASH_02
ASH_05
ASH_10
ASH_20
CONTROL
ASH_05
ASH_10
ASH_20
CONTROL
ASH_10
ASH_20
CONTROL
ASH_20
CONTROL
CONTROL

p
0.987
0.949
0.992
0.743
0.96
0.3
1
1
0.99
1
0.771
1
0.999
1
0.889
0.984
1
0.729
0.998
0.991
0.869

88

Leaf Height and Number of Leaves
A two-way repeated measures ANOVA was used to determine the effects of soil
treatments over time on the maximum leaf height and number of leaves of the planted F.
campestris individuals. Normality was violated for the raw data (p < 0.001), though the test
was still used as it is robust against departures from normality. Moreover, these departures
can be explained by the fact that only 22 individuals survived to the end of the experiment,
thus biasing calculations of normality to tend towards zero. The decision was made to
proceed with a two-way repeated measures ANOVA to test for differences in leaf height and
number of leaves.
There was a significant effect of timing on the leaf heights of F. campestris between
the treatment groups (F (1, 9) = 68.49, p < 0.001, Figure A.2). There was no significant effect
of treatment on leaf height of F. campestris (F (6, 54) = 0.419, p = 0.864); however, the
interaction of treatment and timing had a slight but non-significant effect (F (6, 54) = 2.11, p =
0.067). An analysis of the main effects of soil treatment supported the conclusion that there
were no significant differences between the treatments at either time of planting or harvesting
(Table A.4). Conversely, a significant main effect of timing was found within each soil
treatment on leaf height. Pairwise comparisons of soil treatments showed no significant
difference between any two treatment groups (Table A.5). Oppositely, significant differences
were found in the pairwise comparisons of timing for each soil treatment group (Table A.6)
No significant effects on the number of leaves were found between soil treatments (F
(6, 54) = 1.136, p = 0.354) or timing (F (1, 9) = 2.879, p = 0.124, Figure A.2). Additionally, there

was no significant interaction of soil treatments or timing of sampling on the number of
leaves (F (6, 54) = 1.34, p = 0.256). There were no significant main effects of soil treatment on
the number of leaves when grouped by timing of sampling (Table A.4). A significant main
effect of timing on the number of leaves was found in soils treated with AC (F (1, 9) = 108.07,
p < 0.001). Pairwise comparisons between each soil type showed no significant differences
between any soil treatments (Table A.5), and the only significant comparison between timing
of sampling occurred in the soils treated with AC (Table A.6).

89
Table A.4: Results from a two-way repeated measures ANOVA on leaf height and number of
leaves separately. Effect of time and treatments are reported for each group. Significant p
values are bold faced. P-values on the treatment and timing effects have been adjusted using
the Bonferroni method accounting for the different number of groupings.
Two-way repeated measures ANOVA
Leaf Height
No. Leaves
Effect
df effect df error
F
p
F
p
Treatment
6
54 0.419 0.864 1.136 0.354
Timing
1
9 68.486 <0.001 2.879 0.124
Treatment × Timing
6
54 2.111 0.067
1.34 0.256

Timing
Planting
Harvest

Treatment
AC_01
ASH_01
ASH_02
ASH_05
ASH_10
ASH_20
CONTROL

Effect of soil treatment by time
Leaf Height
df effect df error
F
p
6
54 0.583
1
6
54 1.966 0.174

No. Leaves
F
p
1.23 0.612
1.21
0.63

Effect of timing by soil treatments
Leaf Height
No. Leaves
df effect df error
F
p
F
p
1
9 86.822 <0.001 108.07 <0.001
1
9 38.203 0.001 0.868
1
1
9 44.361 0.001 1.442
1
1
9 48.968 <0.001 0.584
1
1
9 41.834 0.001 0.515
1
1
9 67.953 <0.001
0.72
1
1
9 24.121 0.006
0.16
1

90
Table A.5: Results of pairwise T-tests of leaf height and number of leaves between each
treatment group, separated by planting and harvest timing. T statistics and Bonferroni
adjusted p values are reported.

Group 1
AC_01
AC_01
AC_01
AC_01
AC_01
AC_01
ASH_01
ASH_01
ASH_01
ASH_01
ASH_01
ASH_02
ASH_02
ASH_02
ASH_02
ASH_05
ASH_05
ASH_05
ASH_10
ASH_10
ASH_20

Group 2
ASH_01
ASH_02
ASH_05
ASH_10
ASH_20
CONTROL
ASH_02
ASH_05
ASH_10
ASH_20
CONTROL
ASH_05
ASH_10
ASH_20
CONTROL
ASH_10
ASH_20
CONTROL
ASH_20
CONTROL
CONTROL

Leaf Height
Planting
Harvest
T
p
T
p
0.696 1 -2.579 0.624
0.544 1 -1.427
1
1.331 1 -1.259
1
1.641 1 -2.227
1
0.176 1 -2.134
1
1.107 1 -2.435 0.792
-0.125 1 2.368 0.882
0.964 1 2.072
1
1.282 1 0.135
1
-0.508 1 0.775
1
0.328 1 0.677
1
0.607 1 -0.253
1
0.979 1 -1.198
1
-0.303 1 -0.902
1
0.315 1 -1.661
1
0.210 1 -1.204
1
-0.845 1 -1.067
1
-0.311 1 -2.060
1
-1.479 1 0.387
1
-0.674 1 0.181
1
0.669 1 -0.370
1

No. Leaves
Planting
Harvest
T
p
T
p
-0.090 1 -2.425 0.804
1.994 1 -1.480
1
-0.703 1 -1.381
1
-0.733 1 -1.861
1
1.681 1 -2.231
1
0.182 1 -2.677 0.531
1.432 1 1.496
1
-0.613 1
0
1
-0.529 1 -0.192
1
1.510 1 0.442
1
0.218 1 -0.544
1
-1.986 1 -0.631
1
-2.231 1 -0.881
1
0.285 1 -0.286
1
-0.929 1 -1.589
1
0.082 1 -0.198
1
1.775 1 0.331
1
0.942 1 -0.240
1
2.059 1 0.481
1
0.594 1 -0.073
1
-0.857 1 -0.654
1

91
Table A.6: Results of pairwise T-tests of leaf height and number of leaves between planting
and harvesting, separated by treatment group. T statistics and Bonferroni adjusted p values
are reported. Significant p values are bold faced.
Leaf Height
No. Leaves
Treatment Group 1 Group 2
T
p
T
p
AC_01
Planting Harvest 9.318 <0.001 10.396 <0.001
ASH_01
Planting Harvest 6.181 <0.001 0.931 0.376
ASH_02
Planting Harvest 6.660 <0.001 1.201 0.261
ASH_05
Planting Harvest 6.998 <0.001 0.764 0.464
ASH_10
Planting Harvest 6.468 <0.001 0.718 0.491
ASH_20
Planting Harvest 8.243 <0.001 0.849 0.418
CONTROL Planting Harvest 4.911 0.001 0.400 0.699

Figure A.2: Measurements of number of leaves (top) and leaf height (bottom) for each
treatment taken on the planting date and the harvest date (N = 10 for each treatment). Points
connected by lines are indicative of individuals change in either leaf height or number of
leaves.

92

DISCUSSION
This experiment acted as a showcase for the difficulty involved in maintaining a field
trial with transplanted grasses. Only 22 of the 70 original transplanted individuals survived to
the end of the experiment despite efforts to promote establishment in the field. When the
plugs were grown in the greenhouse, they were subjected to constant and ideal growing
conditions. Likely, the temperature fluctuation observed in the field played a critical role in
the survival of the individuals. Official climate records indicate that the lowest temperature
reached between May 1 and July 30, 2018 was on May 10, 2018 at 4:00 AM where the
temperature dropped to 5.3°C, and the highest temperature was on July 30, 2018 at 3:00 PM
where the temperature reached 37.8°C at the Kamloops Airport (LaZerte & Albers, 2018).
The greenhouse germinated F. campestris plugs may not have exhibited phenotypic plasticity
due to their controlled greenhouse climate resulting in many of the plugs initially dying. The
soils that the plugs were grown in initially was a nutrient rich potting soil and there was no
competition for resources from other plant species. This further extends the argument that the
plugs were not well suited for the environment that they would be planted in: soil nutrients
are likely to change and competition is introduced. These variables were not considered in
the initial design and lead to the poor results of this experiment.
There were no significant differences found in biomass between the different
treatment groups. In this field application, this indicates that there were no detrimental effects
of any of the soil amendments. Due to the very small individuals that managed to survive to
the end of this experiment, it was impossible to recover any belowground biomass. Perhaps
the soil amendments used in this trial impacted the root networks of the F. campestris
individuals. Future studies applying a field use of ash or activated carbon should attempt to
create a design using established natural plants as opposed to transplanting individuals. In
this manner, such an experiment could potentially highlight some effectiveness of ash as a
tool for restoration purposes. Indeed, this is being done currently in the Laurie Guichon
Memorial Grasslands by another student of the Fraser Lab (Hampton, 2020). In work being
completed by Whitehouse, attempts to limit the spread of C. stoebe are being made by
applying varying concentrations of ash and herbicides to a heavily invaded area. Other

93
studies on the remedial effects of ash are being conducted in many parts in Canada as well
through a program called AshNet (Emilson et al., 2018).
No significant differences in leaf height or number of leaves were found between
treatments at either the time of planting or harvesting. During planting, this was expected as
it shows that there was no bias towards any treatment for those variables. The harvesting data
was not significantly different between treatments either, though this is not likely what was
expected. The data shows that compared to a control, none of the soil amendments help to
create either a beneficial or harmful environment for F. campestris. This is very
contradictory to what is generally described in literature, as well as contradictory to the
results of the greenhouse study. It is very likely that the individuals that were planted became
stressed and had limited capacity to thrive in a shockingly different environment from
standard greenhouse conditions, especially at such a young life stage. Given more time and
resources, the application of ash and AC would have been better completed on a broader
scale on established plants in the field.
The significant differences in leaf height were all within treatments between the time
at planting and the time of harvest, where the leaf height decreased over time. In a more
rigorous design, we might have expected other results; however, the fact that all treatments
including the control experienced decreases in leaf height over time indicate that there were
flaws in the implementation of the field experiment.
The only significant difference in the number of leaves was found within the AC
treatments between planting and harvest. This may indicate some effect of AC on the number
of leaves; however, the data that exists for the 22 individuals that survived across the 7
treatments show sporadic results in all other treatments. The reason we see a significant
difference in the number of leaves of the AC treatment is because all individuals that
received the AC treatment died in the field. Unfortunately, since many other individuals of
other treatments also died, I cannot rule out the possibility that the reason that all individuals
of the AC treatment died was random.
Field implementations of greenhouse projects are difficult to carry out; however,
when executed properly they can have some powerful results should they share similar

94
results to the greenhouse portion. This experiment was an attempt to follow that process;
however, the transplanting procedure incurred too much stress to the planted individuals. Due
to the low number of survived individuals, there is no true significance to the data. Rather,
this portion serves as a record of my attempt at a field study and the statistical methods that
could be applied had it been successful.

LITERATURE CITED
Emilson, C. E., Hannam, K., Aubin, I., Basiliko, N., Belanger, N., Brais, S., Diochon, A.,
Fleming, R., Jones, T., Kabzems, R., Laganiere, J., Markham, J., Morris, D., Rutherford,
P. M., Van Rees, K., Venier, L., Webster, K., & Hazlett, P. (2018). Synthesis of current
AshNet study designs and methods with recommendations towards a standardized
protocol. https://cfs.nrcan.gc.ca/publications?id=39388
Hampton, M. (2020, July 9). Research ongoing for local grasslands. Merritt Herald, 1, 3.
https://www.merrittherald.com/research-ongoing-for-local-grasslands/
Hendry, G. A. F., & Grime, J. P. (Eds.). (1993). Methods in comparative plant ecology.
Springer Netherlands. https://doi.org/10.1007/978-94-011-1494-3
LaZerte, S. E., & Albers, S. (2018). weathercan: Download and format weather data from
Environment and Climate Change Canada. The Journal of Open Source Software, 3(22),
571. https://doi.org/10.21105/joss.00571
R Core Team. (2020). R: A language and environment for statistical computing. In R
Foundation for Statistical Computing (4.0.3). https://www.r-project.org/

95

Appendix B: Data Tables
Table B.1: X, Y coordinates for the sampling locations of each site in the Lac Du Bois GPA.
The coordinate reference system used is NAD 1983 BC Environment Albers (EPSG 3005).
Invaded
Site ID
Inv_01
Inv_02
Inv_03
Inv_04
Inv_05
Inv_06
Inv_07
Inv_08
Inv_09
Inv_10

X

Y

1390732.1578
1390740.7013
1390748.3212
1390775.4742
1390776.6700
1390793.4424
1390791.3640
1390786.2598
1390778.7992
1390761.0764

656644.9089
656647.7801
656654.0467
656631.4404
656620.5521
656611.4967
656600.2051
656595.1902
656591.8181
656609.7199

Pristine
Site ID
Pri_01
Pri_02
Pri_03
Pri_04
Pri_05
Pri_06
Pri_07
Pri_08
Pri_09
Pri_10

X

Y

1390786.1589
1390812.8783
1390822.1066
1390835.5155
1390819.5016
1390784.4188
1390764.6073
1390761.4072
1390743.3708
1390706.4964

656640.2280
656610.1828
656593.3971
656556.3348
656549.8116
656543.2845
656562.8504
656574.9540
656594.0062
656620.5365

Table B.2: X, Y coordinates of the soil temperature monitors used to gather ground
temperature data. The coordinate reference system used in NAD 1983 BC Environment
Albers (EPSG 3005).
Site ID
Site 17
Site 18
Site 19
Site 20

Site Association
Pristine
Pristine
Invaded
Invaded

X
1390886.9596
1390877.8708
1390792.3790
1390750.6190

Y
656607.2115
656516.0344
656569.6235
656633.3511

96
Table B.3: Plant inventory for the sampled pristine and invaded areas, and respective mean
cover values in top-down percent (N = 10). A value of NULL indicates that the plant was not
found in any of the plots. Bare ground and litter covers were estimated via ground cover
measurements rather than top-down. Invasive species are denoted with an asterisk prepended
to their scientific names. All scientific and common names were retrieved from Antos et al.
(2018).
Scientific Name

Common Name

Achillea millefolium
Agropyron spicata
Androsace septentrionalis
Antennaria neglecta
Arnica fulgens
Astragalus miser
Calochortus macrocarpus
Campanula rotundifolia
Carex praegracilis
*Centaurea stoebe
Elymus glaucus
Erigeron corymbosus
Eriogonum heracleoides
Festuca campestris
Fritillaria lanceolata
Geranium viscosissimum
Hackelia micrantha
Heuchera cylindrica
Juncus balticus
Koeleria macrantha
Lathyrus nevadensis
Lithophragma parviflorum
Lithospermum ruderale
Poa pratensis
Sisyrinchium idahoense
Stipa comata
Stipa occidentalis
Stipa richardsonii
Taraxacum officinale
*Tragopogon dubius
Zigadenus venenosus

Yarrow
Bluebunch wheatgrass
Fairy candelabra
Field pussytoes
Orange arnica
Timber milk-vetch
Sagebrush mariposa lily
Common harebell
Field sedge
Spotted knapweed
Blue wildrye
Long-leaved daisy
Parsnip-flowered buckwheat
Rough fescue
Chocolate lily
Sticky geranium
Blue stickseed
Round-leaved alumroot
Baltic rush
Junegrass
Purple peavine
Small-flowered woodland star
Lemonweed
Kentucky bluegrass
Idaho blue-eyed-grass
Needle-and-thread grass
Stiff needlegrass
Spreading needlegrass
Common dandelion
Yellow salsify
Meadow death-camas
Unknown
Bare Ground
Litter

Mean cover Mean cover
in pristine
in invaded
soils (%)
soils (%)
4
1
0.4
NULL
NULL
0.3
0.3
0.2
6.3
0.6
5
2.2
0.4
0.3
2.4
NULL
0.1
NULL
NULL
86.1
8.6
NULL
0.1
NULL
0.4
NULL
21.7
NULL
NULL
0.1
1.4
NULL
NULL
1.3
0.1
NULL
1
NULL
0.4
1.5
1.2
NULL
1.3
0.8
0.2
0.8
34.8
3.5
0.3
NULL
0.1
NULL
0.7
NULL
5.9
NULL
0.2
NULL
0.5
1.4
0.3
NULL
0.1
NULL
0.7
11.3
40.9
16.3