Faculty of Science
THE EFFECT OF REDUCED SNOW COVER AND SUMMER DROUGHT ON
TEMPERATE GRASSLANDS IN THE SOUTHERN INTERIOR OF BRITISH COLUMBIA
2016 | JANELLE PAULSON

B.Sc. Honours thesis

THE EFFECT OF REDUCED SNOW COVER AND SUMMER DROUGHT ON
TEMPERATE GRASSLANDS IN THE SOUTHERN INTERIOR OF BRITISH
COLUMBIA

by

JANELLE PAULSON

A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENT OF BIOLOGICAL SCIENCES
(Ecology and Environmental Biology)

This thesis has been accepted as conforming to the required standards by:

Lauchlan Fraser (Ph.D.), Thesis Supervisor, Dept. Natural Resource Sciences

Christine Petersen (M.Sc.), Co-supervisor, Dept. Biological Sciences

Matt Reudink (Ph.D.), Examining Committee member, Dept. Biological Sciences

Dated this 9th day of May, 2016, in Kamloops, British Columbia, Canada

© Janelle Patricia Paulson, 2016

ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr. Lauchlan Fraser for support and guidance over
the course of this project, the International Drought Experiment for coordinating groups around
the globe and making this long term project possible, and my Honours committee; Christine
Petersen and Dr. Matt Reudink for reviews, suggestions and advice.
Field assistants and lab members in the Fraser Lab helped build experimental plots during
the hot summer; Jessica Hewitt, Matthew Coghill, and Sierra Rae. Lastly, a special thanks to Dr.
Heath Garris for his suggestions and encouragement throughout every step of this project.

ABSTRACT
Approximately 26% of the Earth’s total land area and 80% of agricultural land is
composed of grasslands. Lac du Bois Grasslands Protected Area near Kamloops, BC, Canada is
temperate grassland that provides recreational and economic opportunities. Due to global climate
change, Southern and central British Columbia precipitation patterns are predicted to shift with
less precipitation in the summer and more in the winter. Although winters are predicted to
become wetter, less precipitation as snowfall could lead to reduced snowpack, which is more
likely to melt earlier in the spring. Snow cover provides insulation against cold air, which helps
reduce frost stress during the winter and an earlier snowmelt may lead to water shortage in the
summer. Changing climate trends may lead to more frequent and intense droughts combined
with increased frost stress that can negatively impact plant survival and productivity. However, it
is unknown how these stress events interact with each other and if there is a positive or negative
relationship. This study looked at the effects of frost stress and drought, as well as the
combination of both, on biomass productivity. I found that snow removal increased exposure to
more variable and more negative temperatures in the winter. Plots with established rain-out
shelters showed a significant decrease in soil moisture content. Aboveground biomass did not
differ between plots, treatments types or the controls. Although no significant results were found
in biomass production, understanding how stress events interact with each other on grassland
plant communities will help predict how terrestrial ecosystems will respond in the face of global
climate change.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ............................................................................................................ ii
ABSTRACT................................................................................................................................... iii
TABLE OF CONTENTS ............................................................................................................... iv
LIST OF FIGURES ........................................................................................................................ v
Introduction ..................................................................................................................................... 1
Climate change on grassland communities ................................................................................. 2
Ecological stress memory............................................................................................................ 3
Possible mechanisms of stress imprinting................................................................................... 4
Drought tolerance and memory ................................................................................................... 5
Frost tolerance and memory ........................................................................................................ 6
Cross-stress memory ................................................................................................................... 7
Materials and Methods .................................................................................................................... 8
Study site ..................................................................................................................................... 8
Experimental design .................................................................................................................... 9
Rain-out shelter construction .................................................................................................... 10
Measurements............................................................................................................................ 13
Statistical analysis ..................................................................................................................... 13
Results ........................................................................................................................................... 14
Snow removal ............................................................................................................................ 14
Aboveground biomass ............................................................................................................... 17
Soil moisture content ................................................................................................................. 18
Discussion ..................................................................................................................................... 20
Snow removal ............................................................................................................................ 21
Aboveground biomass ............................................................................................................... 22
Soil moisture content ................................................................................................................. 23
Conclusions ............................................................................................................................... 23
LITERATURE CITED ................................................................................................................. 25

LIST OF FIGURES
Figure 1. Lac du Bois Grassland Provincial Park boundary outlined in red. The study site is
located in the upper grasslands and is shown by the marked box. ................................................. 9
Figure 2. Experimental plot design. Six 3 x 3 m plots were established, three control and three
rain-out shelters. Within each plot there are treatment and control subplots................................ 10
Figure 3. Measurements and final design of constructed rain-out shelters................................... 11
Figure 4. Rain-out shelters at the study site in the upper grasslands of Lac du Bois. Gutters move
water away from the plots and trenching helps hydrologically isolate plots from each other...... 12
Figure 5. Dates of snow removal from November 2014 to January 2015. Mean snow depth
before and after removal. .............................................................................................................. 14
Figure 6. Mean snow depth before and after removal. Asterisk indicates a significant difference
in snow depth after removal. Bars are ± Standard Error............................................................... 15
Figure 7. Temperature comparison between snow removal treatment plots and control plots.
Treatment plots have more negative and variable temperature compared to the control. ............ 16
Figure 8. Accumulated frost stress degree days of the treatment and control plots from November
2014 to August 2015. .................................................................................................................... 17
Figure 9. Aboveground biomass collected in November 2015. No difference in biomass was
found between the plots and treatment types. ............................................................................... 18
Figure 10. Pre rain-out shelter and control soil water content recorded by data loggers from
November 2014 to August 2015. This demonstrates that there is little variance in moisture
between plots. ............................................................................................................................... 19
Figure 11. Soil moisture content determined from soil cores collected in November 2015.
Different letters indicate significant differences between treatment types. .................................. 20

Introduction
Approximately 26% of the Earth’s total land area and 80% of agricultural land is
composed of grasslands (Bovel and Dixon 2012). Grasslands are defined as areas dominated by
grasses with less than 10% tree cover (Wikeem and Wikeem 2004). Lac du Bois Grasslands
Protected Area (GPA) near Kamloops, British Columbia, Canada is temperate grassland with
highly palatable forage for cattle and excellent recreational opportunities (Ministry of
Environment 2004). Its close proximity to the City of Kamloops has provided locals with easy
access for a variety of uses such as research, bird watching, all-terrain vehicle use, mountain
biking and hiking (Ministry of Environment 2000). Lac du Bois is important for conservation as
it represents the vast biodiversity of the Thompson Basin and Northern Thompson Upland
Ecosections (Ministry of Environment 2004). In the park there are lakes, ponds, wetlands, forests
and glacial landscape features which provide a diverse environment for species to inhabit
(Ministry of Environment 2000). According to the Conservation Data Centre (COSEWIC 2001)
about 45 wildlife species, 11 plant species and 13 plant communities in the Lac du Bois
ecosystem are Red or Blue-listed. Red-listed species are threatened or endangered and Bluelisted species are vulnerable (Ministry of Environment 2004). Furthermore, Lac du Bois provides
habitat for migratory birds and sustains large populations of bighorn sheep and deer (Ministry of
Environment 2004). There is an elevational gradient within the park that corresponds with
changes in grassland plant communities and increasing moisture (Lee et al. 2014). These
communities are known as the lower, middle and upper grassland; each with distinctive soil
types and species composition which contributes to high species diversity (van Ryswyk et al.
1996). It is important to protect these species to help maintain biodiversity for a healthy
ecosystem that can be enjoyed by visitors for many generations to come.

Grasslands cover about a quarter of the Earth’s ice free land surface and have high soil
carbon content, which therefore is an important factor in the global carbon balance (Jérôme et al.
2014). Agriculture, forestry and other land use (AFOLU) contribute about 12% of greenhouse
gas (GHG) emissions and fossil fuel combustion contributes about 78% of carbon emissions
(IPCC 2012). Carbon, from fossil fuels, is released into the atmosphere but forests and
grasslands can mitigate 10-40% of emissions every year (Sun et al. 2015). Not only are
grasslands important for biodiversity and livestock production, they are also crucial for reducing
the carbon imprint of greenhouse gases to help mitigate the effects of climate change (Batalla et
al. 2015).
Climate change on grassland communities
From 1906 to 2006, mean global surface air temperature has risen between 0.56 °C and
0.92 °C (IPCC 2007). Temperature changes, due to increased greenhouse gas emissions, are
predicted to cause shifts in precipitation patterns, seasonal runoff and evapotranspiration (Nalley
et al. 2013). Shifts in precipitation in Southern and Central British Columbia are predicted to
have less precipitation in the summer and more in the winter (Spittlehouse 2008). In grasslands,
water availability is a major limiting factor for above-ground biomass production (Naudts et al.
2013; Reichmann and Sala 2014). Precipitation is important in Lac du Bois Grasslands Protected
Area (GPA) as there is an elevational gradient of plant communities that correspond with
increasing elevation and moisture. With the prediction of warmer and drier growing seasons,
grassland communities may change drastically and expand northward or into forested areas (Lee
et al. 2014). Extreme weather events, such as droughts, are predicted to increase in frequency
and magnitude (Yusa et al. 2015). When exposed to drought, some plants respond by decreasing
stomatal conductance (Fraser et al. 2009). This reduces water loss by restricting the size, or

altering the density, of the stomata which limits the amount of carbon uptake during
photosynthesis (Chaves et al. 2002). With the main source of carbon limited, plants must rely on
stored reserves to meet their energy requirements (Chapin et al. 2002). This can increase plant
susceptibility to mortality after long periods of time and shift spatial distributions of plant
species. Non-native species may take advantage of gaps in plant communities and invade a
vacant niche leading to increased competition with native species (Fraser and Carlyle 2011;
Holzmueller and José 2013; Kleunen et al. 2015). Many exotic species exist in B.C’s grasslands
and may increase in abundance with changing climate to further alter community composition
and reduce diversity (Lee et al. 2014).
As a result of global climate change there is less precipitation in the form of snowfall
(Briceño et al. 2014). Although winters are predicted to become wetter, less precipitation as
snowfall leads to reduced snowpack which will melt earlier in the spring (Spittlehouse 2008).
Snow cover provides insulation against cold air which helps reduce frost stress to survive during
the winter (Briceño et al. 2014). An earlier snowmelt may lead to water shortage and drought
during the summer resulting in detrimental effects on biomass production (Choler 2015).
Independently, frost and drought may negatively impact plant survival and productivity
(Medeiros and Pockman 2011; Smith 2011). However, plants exposed to recurrent stress may be
able to adapt better to subsequent stress (Backhaus et al. 2014).
Ecological stress memory
When abiotic and biotic stresses occur, plants must be able to respond and adapt quickly
as they are immobile and unable to relocate (Kinoshita and Seki 2014). Plants show some
phenotypic plasticity as they acclimate to changing conditions (Fraser et al. 2009; Walter et al.
2011). This plasticity may prime the plant and cause a “stress imprint” that improves defence

response to future stress events (Bruce et al. 2007). Walter et al. (2013) call this phenomenon
ecological stress memory. However, it should be noted that the term “memory” does not mean
plants are cognisant but are instead stress imprinted (Bruce et al. 2007). Ecological stress
memory differs from acclimation as it remains long after the stress has been applied which
allows the plant to respond faster to recurrent stress (Walter el a. 2013). However, ecological
memory may also involve acclimation mechanisms and the accumulation of protective
substances to increase persistence (Kinoshita and Seki 2014; Walter et al. 2013).
Possible mechanisms of stress imprinting
Stress imprints are made through the process of priming by initiating a state of readiness
to allow for a faster response to an environmental cue (Bruce et al. 2007; Frost et al. 2008).
Conrath et al. (2006) propose two potential mechanisms involved with priming. The first
mechanism for priming involves the accumulation of signalling proteins or transcription factors
and the second mechanism involves epigenetic changes (Blackhaus et al. 2014; Bruce et al.
2007).

Signalling proteins that are accumulated may be activated by post-translational

modification of calcium dependent protein kinases when exposed to stress (Conrath et al. 2006;
Pastor et al. 2013). For example, a mutant with an affected cyclin-dependent kinase-like protein
has reduced salicylate defenses against microbial pathogens and abiotic stressors (Ton et al.
2005). Therefore, the accumulation of cyclin-dependent kinase may promote the salicylatedependent defence pathway (Bruce et al. 2007). However, signalling proteins are limited by
their turn-over times and are not suitable for long-lasting resistance and defence (Pastor et al.
2013). The accumulation of transcription factors may increase defence gene transcription after
being exposed to stress (Bruce et al. 2007). An example is shown when Arabidopsis is exposed
to cold stress. A stress-induced transcription factor gene, known as HOS10, encodes MYB

transcription factors that are crucial to cold acclimation but also affects drought tolerance by
controlling abscisic acid (ABA) biosynthesis (Bruce et al. 2007; Yamaguchi-Shinozaki and
Shinozaki 2006). Thus, the accumulation of transcription factors help with response time to
multiple stressors. Priming effects through epigenetic changes may lead to longer lasting stress
imprints (Bruce et al. 2007). These changes involve DNA methylation which can influence seed
development, vernalization, and organization of apical meristem all of which can be inherited
(Bruce et al. 2007; Kinoshita and Seki 2014). Modifications of chromatin may leave a record of
gene activity as epigenetic marks that can act as transgeneration stress memories for faster
responses to future attacks (Ćuk et al. 2010).
Drought tolerance and memory
When exposed to drought, plants must be able to quickly acclimate and increase their
tolerance. Some mechanisms involve the accumulation of osmoprotective proteins (dehydrins),
soluble sugars or the reduction in photosynthetic machinery (Bohnert 2000; Chaves et al. 2002).
Morphologically, the root to shoot ratio increases when undergoing drought for access to deeper
soil layers for moisture (Sanaullah et al. 2012). ABA-induced genes are important during
dehydration to partially close stomata and reduce water loss (Fleta-Soriano and Munné-Bosch
2016). Previous work found that repeated drought improved photoprotection to manage reactive
oxygen species compared to plants exposed to a single drought event (Walter et al. 2011).
Another study looking at antioxidative enzymes found that plants exposed to drought had
decreased enzymatic activity of catalase and ascorbate peroxidase which was passed on to their
progeny (Ćuk et al. 2010). This suggests that some plants are able to transmit information about
stress exposure to their progeny and that some physiological processes are modified by multistress events (Walter et al. 2013).

Frost tolerance and memory
During sub-zero temperatures, ice crystals may form within intracellular spaces or
dehydration may occur resulting in damage to the photosynthetic machinery (Roychoudhury et
al. 2015; Medeiros and Pockman 2011). Frost acclimation involves similar mechanisms to
drought acclimation such as the accumulation of osmoprotective proteins (dehydrins) and soluble
sugars (Walter et al. 2013). Transcription of late embryogenesis abundant (LEA) genes may also
occur during frost acclimation to help prevent damage from desiccation and cold (YamaguchiShinozaki and Shinozaki 2006). Frost hardening takes several weeks but dehardening can occur
within hours if the temperature increases (Walter et al. 2013). This leaves the plant vulnerable to
frost damage if the temperature is variable. Although the frequency of frost days is predicted to
decrease and mean winter temperature is predicted to increase, extreme cold events will still
occur (Kodra et al. 2011). This can be detrimental to plant communities if snow cover depth is
too low to provide enough insulation against cold air temperatures. The combination of variable
temperature, reduced snow cover and extreme cold events may intensify frost stress over the
years (Walter et al. 2013). Being able to acclimate to frost stress and stress imprint for future
winters would be advantageous especially with the predicted trends of climate change. A study
done by Tahkokorpi et al. (2007) found that after being exposed to frost stress in the winter,
newly grown stems of bilberry in the spring showed higher amounts of anthocyanin used for
protection against cold temperatures, desiccation and high light. However, a study done by Polle
et al. (1996) found that frost tolerance and resistance was lowered after exposure to frost stress.
Overall, the response to recurrent exposure of frost stress on tolerance has not been thoroughly
investigated and it remains unclear if frost stress experience helps survival (Walter et al. 2013).

Cross-stress memory
Drought and frost have similar acclimation mechanisms to help with water loss and
dehydration (Beck et al. 2007). Thus, it may be possible for the formation of stress memory to
one kind of stress that also improves the tolerance to different stress resulting in cross-stress
memory (Walter et al. 2013). A study by Medeiros and Pockman (2011) found that drought
improved freezing tolerance in leaves of Larrea tridentata. Another study by Kreyling et al.
(2012) found that prior exposure to drought improved late frost tolerance of three out of four
grass species tested. There are some studies investigating the interactions of drought on frost
tolerance but research of frost on drought tolerance has not been thoroughly investigated.
Previous studies have also been limited in time and there are few long term studies looking at
ecological stress memory (Bruce et al. 2007; Walter et al. 2013).
The International Drought Experiment has organized a vast network of scientists across
the globe to participate in a five year study that investigates the long term effects of drought
sensitivity when exposed to frost stress (International Drought Experiment 2015). This
experiment may provide more information on the mechanisms involved with ecosystem response
to recurrent stressors and if this response has a positive or negative effect on plant communities.
I asked the following questions (1) What are the effects of snow removal on biomass
productivity and soil temperature? (2) What are the effects of the rain-out shelters and drought
on biomass productivity and soil moisture? (3) Will aboveground biomass production improve
when exposed to multiple stress events compared to a single event?

Materials and Methods
Study site
This study was conducted in Lac du Bois Grasslands Protected Area, British Columbia,
Canada (Figure 1). Lac du Bois is a recreational area comprised of 15,207 ha of valley slopes
that includes an elevational gradient of lower, middle and upper grasslands (Ministry of
Environment 2004). These vegetation zones change with increasing moisture and primary
productivity over its elevational range of 300-975 m above sea level (asl) (Lee et al. 2014). Lac
du Bois represents three biogeoclimatic zones (BEC) including the Bunchgrass zone (BG),
Ponderosa Pine zone (PP) and Interior Douglas-fir zone (IDF) (Ministry of Environment 2000).
The study site is located in the upper grasslands at 900 m asl (50°47'20.4"N
120°26'53.2"W). Soils are classified as Black Chernozems and rough fescue (Festuca
campestris) is the dominate species (Ryswyk et al. 1966).

Figure 1. Lac du Bois Grassland Provincial Park boundary outlined in red. The study site is
located in the upper grasslands and is shown by the marked box.

Experimental design
Cattle exclosures (30 m x 30 m) were erected by the Agriculture and Agri-Food Canada
Kamloops office in April 2010 (McCulloch 2013). HOBO® Micro Station Data Loggers were
set up in November 2014 to record soil temperature and soil moisture every fifteen minutes.
Snow was removed from the snow removal subplots using a shovel and leaf blower in November
to January 2015 to stimulate frost stress. There were six experimental plots containing subplots
with two treatments, snow removal and the control. Three of the six plots had a rain-out shelter

and the others did not. These subplots were 1 m x 1 m to allow for a 0.5 m buffer between the
sides resulting in a 3 m wide experimental plot (Figure 2). The rain-out shelters were built within
the exclosures in May, 2015.

Figure 2. Experimental plot design. Six 3 x 3 m plots were established, three control and three
rain-out shelters. Within each plot there are treatment and control subplots.

Rain-out shelter construction
The shelters were constructed to control for the prevailing winds (North) with the slope
facing South. Post holes were dug and 4x4x8 feet pieces of commercially treated wood were
concreted in each corner of the plot. The supports of the structure consisted of 2x4x10 feet pieces
of wood with the side pieces placed at 1.3 m above grade. The upper support of the shelter was
placed at 1.6 m above grade and the lower support was placed at 1 m above grade. Two support
beams across the plot were screwed on the side supports at 80 cm and 175 cm from the top.
These cross beams provided additional support for the SUNTUF® Corrugated Polycarbonate to
gently rest on (Figure 3). All supports and posts were levelled and straightened. The

polycarbonate was cut into strips containing two troughs (roughly 14 cm) then screwed onto all
of the supports. Glue was used on the screws in the polycarbonate to prevent any leaks. The
strips were spaced 14 cm apart to reduce rainfall by 50% to stimulate drought stress. The
intensity of rainfall reduction was determined between the 1st and 5th percentile of the
precipitation historical record spanning over 50 years in Kamloops. The unmanipulated (control)
plots were not covered by rain-out shelters. These structures were built for a long-term study and
are expected to last at least five years. Construction of the shelters was completed in late August.
Finally, trenching around all of the plots occurred in September to hydrologically isolate them
from each other (Figure 4).

Figure 3. Measurements and final design of constructed rain-out shelters.

Figure 4. Rain-out shelters at the study site in the upper grasslands of Lac du Bois. Gutters move
water away from the plots and trenching helps hydrologically isolate plots from each other.

Measurements
A plant survey was conducted in June where the plant species composition was estimated
to the nearest 1% using a modified Daubenmire method with 1 x 1 m quadrats placed in the
middle of the plot. Percent cover for litter, bare soil, animal diggings and rocks was also
estimated. Slope and aspect were determined using a clinometer. Due to the delayed construction
of the plots, an aboveground biomass collection was conducted in November as an attempt to
allow for fall regrowth and some stress interactions to occur. The biomass and litter was
collected in 10 x 100 cm strips in each subplot as advised by the Nutrient Network (2008). The
collected biomass was dried at 60°C for two days then weighed. At the same time as biomass
collection, soil cores were collected from each plot and dried then weighed to determine soil
moisture content. Moisture content and temperature were recorded every fifteen minutes starting
from November 2014. The data loggers were placed in a plastic container in the center of each
plot to prevent water damage and animal interference.
Statistical analysis
The study design allowed for two-way ANOVAs to test the effect of snow removal
(present/absent) and drought (rain-out/control) on biomass and soil moisture. To meet the
ANOVA assumption of equality of variances, data were natural log or square root transformed.
A one-way ANOVA was done on mean snow depth before and after removal. Accumulated
Frost Stress Degree Days (FSDD) was calculated to determine the difference in frost stress
contribution between the control and snow removal plots. Average temperature below zero of the
snow removal and control plots were analyzed as it represents exposure to frost stress. The daily
contribution to frost stress was summed over the course of the year to show the accumulation of
frost stress. A paired t-test was done on the control and snow removal FSDD means.

Results
Snow removal
Starting in November and ending in January, snow was repeatedly removed from snow
removal subplots within experimental plots (Figure 5). Mean snow depth was square root
transformed and analyzed by one-way ANOVA.

Figure 5. Dates of snow removal from November 2014 to January 2015. Mean snow depth
before and after removal.

Removing snow showed a significant reduction in mean snow depth shown in Figure 6
(F= 19.038, df= 3, p ≤0.001). An extremely heavy snowfall occurred in January creating deep
snow cover in the control and treatment plots. It was crucial to remove snow repeatedly

throughout the winter to maintain exposure to frost stress and prevent snow build up in treatment
plots.

Figure 6. Mean snow depth before and after removal. Asterisk indicates a significant difference
in snow depth after removal. Bars are ± Standard Error.

Snow removal increased exposure to more variable and negative temperatures with a low
of -8.1 °C (Figure 7). Control plots had a low of -2.9 °C and appeared to have less variance in
temperature throughout the winter. This demonstrates increased insulation found with higher
snow cover and depth.

Figure 7. Temperature comparison between snow removal treatment plots and control plots.
Treatment plots have more negative and variable temperature compared to the control.

Accumulated FSDD was calculated between November 2014 and August 2015 (Figure
8). There was an error in one probe at a snow removal plot which inaccurately logged
temperature after the spring. Calculations were made without the defective plot temperature. The

snow removal plots accumulated FSDD for a total of 105.5 °C compared to the control plots of
34.8 °C. A high FSDD represents increased exposure to negative temperatures and potentially
more damage. Snow cover insulated control plots from sub-zero temperatures reducing the
accumulation of frost stress. A paired-t test was done on the mean FSDD of snow removal and
control plots where a significant difference was found (t4= 2.77, p= 0.022).

Figure 8. Accumulated frost stress degree days of the treatment and control plots from November
2014 to August 2015.

Aboveground biomass
Biomass data were natural log transformed to meet assumptions of equal variances. A
two-way ANOVA showed no significant effect of snow removal (F= 0.841, df= 1, p= 0.386) and
drought (F= 3.05, df= 1, p= 0.119) on aboveground biomass (Figure 9). No interactions were

found (F= 3.72, df= 1, p= 0.09). However, the drought/snow removal plots had a higher variance
(0.199) compared to the control plots (0.098). Although stress combination plots had a higher
overall biomass, no significant differences were found in the plots and treatment types.

Figure 9. Aboveground biomass collected in November 2015. No difference in biomass was
found between the plots and treatment types.

Soil moisture content
Soil moisture trends of the control and incomplete rain-out shelter plots were examined to
determine if plots differed in available moisture (Figure 10). The experimental plots show little
variance in soil water content suggesting that the plots have equal access to moisture. Soil
moisture content determined from soil cores taken in November 2015 were natural log
transformed. A two-way ANOVA was done to examine the effects of snow removal and drought

on soil moisture content. A significant difference in moisture was found between plots with a
rain-out shelter and the control (F=11.768, df= 1, p= 0.009). No differences were found between
the subplots of the snow removal treatment and control (Figure 11).

Figure 10. Pre rain-out shelter and control soil water content recorded by data loggers from
November 2014 to August 2015. This demonstrates that there is little variance in moisture
between plots.

Figure 11. Soil moisture content determined from soil cores collected in November 2015.
Different letters indicate significant differences between treatment types.

Discussion
This study investigated the effects of frost stress and drought in the temperate grassland,
Lac du Bois Grasslands Protected Area. I hypothesized that plants exposed to the combination of
frost and drought would have a higher productivity than plants exposed to either frost or drought
stress alone. This hypothesis was not confirmed as aboveground biomass was not influenced by
multiple or single stress events.
Removing snow increased temperature variance and resulted in negative temperature
spikes. Increased exposure to colder temperatures accumulated frost stress faster, and at a higher
amount. Frost stress and drought, as well as the combination of both, had no effect on

aboveground biomass. Drought was simulated by rain-out shelters which reduced soil moisture
content. No moisture differences were found between the snow removal treatment and control
subplots.
Snow removal
Snow was removed repeatedly over three months from the treatment subplots within the
experimental plots. There was a significant difference in mean snow depth after removal in
treatment plots. Over time, due to continuous snow fall, mean snow depth of treatment plots
increased to similar depths of the control plots. Repeated removal of newly fallen snow was
essential to ensure less snow cover was available for insulation against frost stress. However,
snow removal for this study may not be frequent and consistent enough to stimulate severe frost
stress to affect biomass productivity. After snow was removed on December 9th, there was a long
delay before snow was removed again on January 6th, 2015. This delay may have provided the
snow removal subplots with enough snow cover and insulation to reduce exposure to frost stress
resulting in the aboveground biomass being unaltered. With less snow cover, temperatures were
more variable and extreme in treatment plots, with a low of -8.1 °C compared to -2.9 °C in the
control plots. This clearly demonstrates how snow cover provides good insulation against subzero temperatures. Snow removal plots experienced a higher magnitude of FSDD (105.5 °C)
compared to the control (34.8 °C). This high FSDD indicates that the treatment plots were
exposed to more negative temperatures which may inhibit growth and potentially damage plants.
High stress degree day (SDD) and its relationship with increased damage is supported by a study
done on snap beans which found that increasing SDD on water stress decreased growth and
development (Helyes et al. 2006).

Aboveground biomass
Aboveground biomass was an important indicator for this study to determine if
productivity was influenced by stress events. While there was a higher overall biomass for the
snow removal/drought combination plots, there was no significant difference between the
experimental plots. This result may be due to differences in species composition between sample
sites. For example, rough fescue (Festuca campestris) is a bunchgrass which can form dense
clumps (Anderson 2006). If, by chance, a sample site included a dense clump of rough fescue
then the biomass results may be higher than a sample that did not have rough fescue. To counter
this potential variance, more replication is needed.
Contrary to previous studies, aboveground biomass was not reduced when exposed to
either frost stress or drought. Drought is a stressor that has one of the most negative effects on
plant growth and development (Fleta-Soriano and Munné-Bosch 2016). Frost stress also has
negative impacts on shoot and canopy growth as well as flowering (Briceño et al. 2014). A study
done on sub-Artic shrubs found that winter warming reduced the insulating properties of snow
cover which resulted in less summer growth and an increased frequency of dead shoots
(Bokhorst et al. 2009). However, my results are not consistent with these studies as collected
biomass productivity showed no difference when exposed to stress.
A major drawback of my study was the timing of the fully constructed rain-out shelters
which were completed at the end of August. Usually by this time, the main growing season has
already ended. Aboveground biomass was collected in November to allow for some fall regrowth
or stress interactions to occur. However, no interactions were found. Furthermore, it may be
possible that the rain-out shelters prevented fall regrowth by reducing the required amount of

moisture to initiate growth. Thus, with little to no fall regrowth, interactions and biomass
differences were not found.
Soil moisture content
Soil water content, recorded by the data loggers, was similar throughout the year among
the experimental plots. This indicates that each plot received relatively the same amount of
moisture with little variance, suggesting that plots were essentially equal and received the same
opportunity for growth regarding moisture limitations. In November, soil cores were collected
and analyzed. Soil moisture content was significantly lower in plots with rain-out shelters than
the control plots and no difference was found between treatment types. A study using a
comparable shelter design found results supporting my findings. Yahdjian and Sala (2002) found
that soil water content was higher in control plots than plots with rain-out shelters and also found
there was no significant difference between the observed and expected water interception. This is
a good indicator that the rain-out shelters for this study were performing as expected reduced
precipitation by at least 50%.
Conclusions
After conducting this study, I found no measureable effect on aboveground biomass of
frost, drought and the combination of both. Snow removal increased frost stress by exposing
plants to more variable and negative temperatures. Rain-out shelters were effective in reducing
soil moisture content for drought simulation. It appears that the frost stress was not severe
enough and drought stress was too late in the season to influence biomass production. These
results were somewhat unexpected as previous studies looking at grassland species found that
prior exposure to drought improved frost tolerance (Kreyling et al. 2012). However, we looked at
frost stress on drought tolerance so it may be possible that acclimation mechanisms differ

depending on which type of stress exposure came first. This study provides baseline data for
future and long term studies.
Long term studies are important for better understanding how ecosystems change over
time (Magnuson 1990). The International Drought Experiment (2015) is a long term study
investigating terrestrial ecosystem sensitivity to drought all around the world. This international
experiment may help us understand how and why different ecosystems respond when exposed to
drought (IDF 2015). Long term studies are especially important for understanding stress
interactions due to lagged or delayed stress effects that are not clear until after some period of
time (Walter et al. 2013). This long term study may help us better understand and predict how
terrestrial ecosystems will respond in the face of climate change. It may also provide some
insight on the consequences of ecological stress memory and if there is a positive or negative
effect on plant communities.
We plan to continue this experiment over the next five years. The rain-out shelters have
been completed and will be able to reduce precipitation throughout the entire growing season to
simulate drought. I predict that impacts on aboveground biomass will become more apparent
over the next few years as stress becomes more severe. This long term study may provide better
insights to plant responses during changing climate and be able to provide suggestions for
adapting to climate change.

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