1
Restoration Strategies for a Native Perennial: Germination and Seedling
Physiology of Sphaeralcea Munroana
A Thesis
Presented in Partial Fulfillment of the Requirements for the Degree of
Master of Science
with a
Major in Forest Resources
in the
College of Graduate Studies
University of Idaho
by
Olga A. Kildisheva
May 4, 2011
Major Professor: Anthony S. Davis
2
AUTHORIZATION TO SUBMIT THESIS
This thesis of Olga A. Kildisheva, submitted for the degree of Master of Science
with a major in Forest Resources and titled “Restoration Strategies for a Native
Perennial: Germination and Seedling Physiology of Sphaeralcea Munroana” has been
reviewed in final form. Permission, as indicated by the signatures and dates given below,
is now granted to submit final copies to the College of Graduate Studies for approval.
Major Professor ______________________________Date______________
Anthony S. Davis
Committee
Members
_______________________________Date______________
R. Kasten Dumroese
________________________________Date______________
James L. Kingery
Department
Administrator________________________________Date______________
Jo Ellen Force
Discipline's
College Dean________________________________Date_______________
Kurt S. Pregitzer
Final Approval and Acceptance by the College of Graduate Studies
_______________________________Date_______________
Nilsa A. Bosque-Pérez
3
Abstract
The Great Basin region of the western United States has undergone significant
disturbance and fragmentation because of overgrazing for livestock production,
disruption of the natural fire regimes, and the introduction of non-native species. At
present, habitat loss greatly surpasses the rate of system recovery, making restoration
integral to ecosystem function and resilience. To date, restoration research and practice
have focused largely on keystone plant species, however, to be effective, restoration must
include myriad plant forms and guilds. Munro’s globemallow (Sphaeralcea munroana
(Douglas) Spach) is a perennial, cool-season forb, endemic to the Great Basin that serves
as an important host for native pollinators, provides soil stabilization, and is a source of
food for numerous mammals. For these reasons, as well as its ability to tolerate
disturbance, temperature extremes, and drought, it is an important candidate for broad
scale ecosystem restoration across its native range. Little information regarding seed
germination and seedling establishment is available for Munro’s globemallow, and what
does exist is synthesized in the first chapter of this thesis. The second chapter is a detailed
examination of the seed dormancy mechanisms. The results of 4 experiments identified
physical dormancy, which inhibits water imbibition, to be responsible for poor
germination. Seed coat scarification is essential for germination and can be achieved
through mechanical or boiling water scarification. The third chapter of this thesis
describes an experiment conducted to evaluate a suite of morphological and physiological
responses of Munro’s globemallow to a range of temperature and moisture conditions
during seedling establishment. Results indicate that seedlings are able to tolerate drought
and relative high temperature conditions early in their establishment, however, low
temperatures substantially limited seedling development; thus, a later sowing date may
optimize plant establishment. Overall, nursery production, seed increase projects, and
outplanting success of Munro’s globemallow should be improved through the
incorporation of the results of this thesis.
4
Acknowledgements
This project would not have been possible without the financial support from the Idaho
Transportation Department, the Great Basin Native Plant Selection and Increase Project,
the University of Idaho Seed Grant Program, and the University of Idaho Center for
Nursery and Seedling Research. In particular, I’m grateful to Cathy Ford and Nancy
Shaw, who saw merit in this research direction and worked to facilitate funding.
I thank my major professor, Dr. Anthony S. Davis for encouraging me to work
independently, pushing me to achieve my goals, and always being only an email away. I
extend my thanks to Dr. Kas Dumroese for his editorial support, direction, and practical
wisdom. I also thank Dr. Jim Kingery for introducing me to intermountain flora and
sharing with me a bounty of stories.
I’m grateful to Timothy Johnson for his statistical support, Nalin Suranjith GamaArachchige for his assistance in electron microscopy, as well as Nancy Shaw, Matt Fisk,
and Erin Denney for aiding with seed collection. I’m obliged to everyone at the Pitkin
Forest Nursery for teaching me the ways of nursery management and for their help during
the course of my education at the University of Idaho.
I extend my gratitude to Rob Keefe for sparking my interest in seed ecology. I would also
like to thank Emily Overton, Bridget McNassar, Sasha Podolak, Jake Kleinknecht, Eric
Creeden, Sara Ashkannejhad, Amy Whitcomb, and Jen Lennon for their research
involvement, humor, and camaraderie.
I offer my sincere gratitude to Matthew Aghai for always being the voice of reason and
for his endless support through this process. Finally, I’m grateful to my parents for their
whole-hearted encouragement.
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Table of Contents
Title Page
i
Authorization to Submit Thesis
ii
Abstract
iii
Acknowledgements
iv
Table of Contents
v
List of Tables
vii
List of Figures
viii
Chapter One
Sphaeralcea munroana: Restoration Potentials and Challenges
General Introduction
1
Species Overview
2
Seed Dormancy
3
Temperature and Moisture as Limiting Factors to Seedling Development
9
Conclusions
12
Literature Cited
13
Chapter Two
Seed Coat Morphology, Physical Dormancy, and the Use of
Scarification for Dormancy Break in Sphaeralcea munroana
Abstract
19
Introduction
20
Materials and Methods
25
Statistical Analysis
27
Results
28
Discussion
31
Conclusions
34
6
Literature Cited
Chapter Three
35
Individual and Synergistic Influence of Temperature and
Moisture on Sphaeralcea munroana Seedling Growth
Abstract
44
Introduction
45
Materials and Methods
Plant Establishment
47
Temperature and Moisture Treatments
47
Statistical Analysis
49
Results
49
Discussion
50
Conclusions
53
Literature Cited
54
Appendix A
67
Appendix B
68
7
List of Tables
Chapter Two
Table 2.1.
The effects of mechanical scarification, soaking duration in GA3, or pure
water solution on the germination behavior of Sphaeralcea munroana at
the end of the 21-d observation period. Parameter values were generated
using a non-linear regression model (Equation 2.2), where G (t) is the
cumulative germination percentage at time (t) expressed in days (d), Gc is
cumulative germination, or the germination asymptote at the end of the
testing period (%), GC50 is the time (d) required to reach 50%
germination, and Gl is the time (d) until first germination. Multiple
comparisons were obtained using Tukey’s HSD (α = 0.05).
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Table 2.2.
The effects of “non-traditional” scarification techniques on the
germination behavior of Sphaeralcea munroana at the end of the 21-d
observation period. Parameter values were generated using a non-linear
regression model (Equation 2.1), where G (t) is the cumulative
germination percentage at time (t) expressed in days (d), Gc is cumulative
germination, or the germination asymptote at the end of the testing period
(%), GC50 is the time (d) required to reach 50% germination, and Gd is the
germination rate (% /d). Multiple comparisons were obtained using
Tukey’s HSD (α = 0.05).
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Chapter Three
Table 3.1.
Sphaeralcea munroana seed collection sites.
56
Table 3.2.
Results of the mixed model analysis for the effects of temperature,
moisture, and their interaction on the morphological and physiological
responses of Sphaeralcea munroana seedlings 35 d after sowing.
56
Appendix B
Table 3.3.
Sphaeralcea munroana gas exchange responses to 2 temperature and 4
moisture regimes 35 d after sowing.
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8
List of Figures
Chapter One
Figure 1.1.
General outline of the Great Basin region of the western United States. 18
Chapter Two
Figure 2.1.
Scanning electron micrographs of a Sphaeralcea munroana seed; (A) top
view of the chalazal region of a dormant seed showing the chalazal slit;
(B) formation of water gap following dormancy break; (C) the underside
of the dislodged cap-like blister covering the water gap; (D) palisade cells
of the cap-like structure. Abbreviations: Chsl, chalazal slit; Chsl*,
remaining part of the chalazal slit after dormancy break; Ll, light line; Mi,
micropyle; Mt, maternal tissue; Pa, palisade; Spl, subpalisade cells; Wg,
water gap.
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Figure 2.2.
Mass increase percentage (on a fresh mass basis) of control, mechanically,
and boiling water scarified seeds of Sphaeralcea munroana during a 24-hr
incubation. Each point scatter represents the mean (±s.e.) of 10 replicates.
Different letters indicate significant differences (p < 0.05) at the end of the
24-hr period.
40
Figure 2.3.
Cumulative germination percentage of Sphaeralcea munroana seed
subject to 4 seed treatments during a 21-d observation period. Each line
represents the mean (±s.e.) response of 5 replicates. Different letters
indicate significant differences between treatments.
41
Figure 2.4.
Germination capacity of Sphaeralcea munroana seeds 21 d following
exposure to 8 treatments. Each bar represents the mean (±s.e.) of 5
replicates. Different letters indicate significant differences (α = 0.05).
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Cumulative germination percentage of Sphaeralcea munroana seeds
subject to 6 treatments during a 21-d observation period. Each line
represents the mean (±s.e.) response of 5 replicates. Different letters
indicate significant differences between treatments.
43
Figure 2.5.
Chapter Three
Figure 3.1.
Number of true leaves (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature regimes.
Each bar represents the mean response of 5 replicates. Different letters
indicate significant (p < 0.005) differences between treatments.
57
9
Figure 3.2.
Total leaf area (mean±s.e.) 35 d after sowing, produced by Sphaeralcea
munroana seedlings grown under 2 temperature regimes. Each bar
represents the mean response of 5 replicates. Different letters indicate
significant (p < 0.005) differences between treatments.
58
Figure 3.3.
Aboveground biomass (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature regimes.
Each bar represents the mean response of 5 replicates. Different letters
indicate significant (p < 0.005) differences between treatments.
59
Figure 3.4.
Belowground biomass (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature regimes.
Each bar represents the mean response of 5 replicates. Different letters
indicate significant (p < 0.005) differences between treatments.
60
Figure 3.5.
Belowground biomass (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 4 irrigation regimes. Each
bar represents the mean response of 5 replicates. Different letters indicate
significant (p < 0.005) differences among treatments.
61
Figure 3.6.
Root-to-shoot ratios (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 4 irrigation regimes. Each
bar represents the mean response of 5 replicates. Different letters indicate
significant (p < 0.005) differences among treatments.
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Figure 3.7.
Total leaf area (mean±s.e.) 35 d after sowing, produced by Sphaeralcea
munroana seedlings grown under 2 temperature and 4 irrigation regimes.
Each bar represents the mean response of 5 replicates. Different letters
indicate significant (p < 0.005) differences among treatments.
63
Figure 3.8.
Aboveground biomass (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature and 4
irrigation regimes. Each bar represents the mean response of 5 replicates.
Different letters indicate significant (p < 0.005) differences among
treatments.
64
Figure 3.9.
Belowground biomass (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature and 4
irrigation regimes. Each bar represents the mean response of 5 replicates.
Different letters indicate significant (p < 0.005) differences among
treatments.
65
10
Figure 3.10.
Root to shoot ratios (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature and 4
irrigation regimes. Each bar represents the mean response of 5 replicates.
Different letters indicate significant (p < 0.005) differences among
treatments.
66
Appendix A
Figure 3.11.
Light response curves of photosynthetic carbon fixation measured on
individual leaves of 5 mature Sphaeralcea munroana plants at the
University of Idaho Shattuck Arboretum. Measurements were made
between 1000 and 1300 on 02 October 2011 using an LI-6400 Portable
Photosynthesis System (Li-Cor, Inc., Lincoln, NE). Constant chamber
CO2 (400 μmol mol-1), temperature (20±0.16° C), and relative humidity
(79±0.58%) were maintained to avoid confounding the assimilation
response. The initial leaf equilibration was set to high photon flux density
(2000 μmol quanta m-2s-1), followed by subsequent light decreases in steps
of 200 μmol quanta m-2s-1, until a negative photosynthetic rate was
reached. Each point represents a mean±s.e. of 5 replicates.
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11
Chapter 1:
Sphaeralcea munroana: Restoration Potentials and Challenges
General Introduction
The arid-steppe of North America’s Great Basin is delineated by the Colorado and
Columbia Plateaus, the Sierra Nevada Mountains, and the Mojave Desert (Figure 1.1.).
The region’s unique geomorphology has a considerable effect on the climate, which is
dominated by temperature extremes and low, primarily winter, precipitation (Knapp,
1996). The vegetative communities of this area are characterized by the presence of
shrubs, perennial bunchgrasses, and forbs (Holmgren, 1972).
As an ecological unit, the Great Basin has suffered from substantial disturbance and
fragmentation due to overgrazing, shrub removal, and non-native species introduction
(Mack, 1981). Decades of natural fire suppression and the aggressive spread of
cheatgrass (Bromus tectorum) have been linked to a 4 to 10 fold increase in fire incidence
in the course of the last century (D'Antonio and Vitousek, 1992). Frequent fires have
promoted an extensive system conversion from sagebrush- to annual grass-dominated
communities, which has reduced the available soil moisture, promoted nutrient depletion,
and intensified resource competition (Billings, 1990;Whisenant, 1990; D'Antonio and
Vitousek, 1992; Obrist, et al., 2003). By recent estimates, the rate of habitat loss greatly
surpasses the rate of system recovery, further jeopardizing the populations of sagebrushsteppe obligates, such as pygmy rabbit (Brachylagus idahoensis), greater sage-grouse
(Centrocercus urophasianus), and Brewer’s sparrow (Spizella breweri) (Wisdom et al.,
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2005). The predicted rise in CO2 has been projected to enhance biomass production of C3
annual grasses, which could further exacerbate ecosystem conversion (Smith et al., 1987,
Smith et al., 2000; Grunzweig and Korner, 2001). Thus, the use of endemic plant species
is critical in promoting ecological recovery.
Herbaceous perennials comprise a substantial portion of the Great Basin floristic
communities and are an integral component of these systems. The use of native species in
restoration is optimal because they are evolutionarily adapted to withstand severe climate
conditions, provide long-term soil stabilization, and foster habitat biodiversity. Despite
the importance of their role, the use of forbs in habitat restoration is relatively novel and
largely unexplored (Parkinson, 2008). One such species, Sphaeralcea munroana
(Douglas) Spach) (Malvaceae), is a desirable candidate for revegetation; however, its use
is constrained by the lack of information regarding the species requirements for
successful germination and establishment.
Species Overview
Munro’s globemallow is a cool-season herbaceous perennial endemic to the Great Basin.
Plants initiate growth from a caudex in the form of multiple, unbranched stems, typically
reaching 20 to 80 cm in height. Showy, orange inflorescences are produced from May to
August. Subsequent seeds are arranged in a schizocarp, composed of 10 to 12 mericarps
that form a ring, each containing 1 to 2 pubescent seeds 1.5 mm in length (Rydberg,
1917; Lyons, 1995). S. munroana is able to establish on disturbed sites and survive
drought and temperature extremes. In addition to its resilience, this plant is an essential
13
forage source for numerous rodents, lagamorphs, and ungulates (Beale and Smith, 1970;
Pendery and Rumbaugh, 1986; Rumbaugh et al,. 1993; Pavek, et al. 2011). Furthermore,
it provides nutrition for 20 generalist and 3 specialized (Diadasia diminuta, D. lutzi, and
Colletes sphaeralcea) bee species (Cane, 2007). These attributes have made S. munroana
an important candidate for broad scale restoration across its native range.
Seed dormancy, however, severely limits in and ex situ germination of S. munroana. Few
studies explore the dormancy mechanisms and methods for dormancy break in the
Sphaeralcea genus (Page, et al. 1966; Roth et al., 1987). Moreover, little is known about
seedling tolerance to temperature and moisture stress. Ehleringer and Cooper (1998)
suggest that S. ambiuga, native to the Mojave Desert, is “a short-lived, opportunistic
species that establishes during wet years but demonstrates higher mortality during dry
years due to relatively low water use efficiency”. It is unclear if this life strategy is shared
by Sphaeralcea munroana. Plant establishment is the most critical phase in determining
future survival and persistence (Harper, 1977); thus, seedling post-germination responses
to temperature and moisture stress are important and must be specifically addressed.
Seed Dormancy
Dormancy is characterized by Vleeshouwers et al. (1995) as “a seed characteristic, the
degree of which defines what conditions should be met to make the seed germinate”. This
is an evolutionary response to temporal variation in environmental conditions, which
allows for different species to be favored over time (Rees, 1996). Several types of
dormancy exist and are defined based on the mechanisms that prevent germination.
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Physical dormancy is common among desert species (including several in the
Sphaeralcea genus) and is thought to have developed to prolong seed longevity (Page et
al., 1966; Sabo et al., 1979; Roth et al., 1987; Baskin and Baskin, 1998; Smith and
Kratsch, 2009). Physically dormant seeds often possess a palisade layer of lignified cells
that prevent water entry into the seed (Corner, 1951; Vazquez-Yanes and Perez-Garcia,
1976). Water imbibition is critical because it drives seed expansion (necessary for
germination) and is dependent on the interaction between the growth potential of the
embryo and the constraints imposed by the seed coat (Kucera et al., 2005). Imbibition in
many seeds with physical dormancy is regulated by a specialized anatomical structure,
defined as the “water gap”, which is located within the seed coat. The water gap becomes
permeable when exposed to temperature flux, drying, or scarification, thus allowing
imbibition into an otherwise impermeable seed (Baskin and Baskin, 1998; Baskin et al.,
2000; Baskin, 2003). The location, anatomy, and morphology of the water gap
demonstrate intra- and extra-family variability (Baskin et al., 2000). The chalazal region
is a critical site for water entry in a number of Malvacea species, including Abelmoschus
esculentus, Gossypium hirsutum, and Sida spinosa (Christiansen and Moore, 1959; Egley
and Paul, 1981; 1982; Serrato-Valenti et al., 1991). In these species the water gap is
obstructed by 2 tissue types. Maternal tissue forms a cap that project downward into the
chalazal slit and mesophyll tissue projects upward. At the initiation of dormancy break, a
section of these tissues that radially surround the chalazal slit) become partially
permeable and allow imbibition, which leads to a separation of the palisade and subpalisade layer of cells causing the formation of a blister (Serrato-Valenti et al., 1991).
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Once water surrounds the entire palisade layer of the seed coat, the mesophyll cracks,
allowing the upper portion of the palisade cells to continue swelling. This process
eventually causes a detachment of the blister roof, allowing for full imbibition (SerratoValenti et al., 1991).
Natural phenomena, such as abrasion by sand and stones (in rapidly moving washes
during periods of flooding), temperature fluctuations, fires, and animal digestion are
thought to alleviate physical dormancy (Went, 1955; Gutterman, 1993; Baskin and
Baskin, 1998). A number of ex situ techniques, primarily chemical and mechanical
scarification, have been used to promote germination of physically dormant seeds (Page
et al., 1966; Roth et al., 1987; Hoffman et al., 1989; Baskin and Baskin, 1998). For
example, Page et al. (1966) observed 30 to 40% germination of S. grossulariifolia
following submergence in sulfuric acid, a substantial improvement compared to the
control (0%). Similarly, submergence of Sphaeralcea seeds in 18 M sulfuric acid for 10
min improved germination of S. coccinea and 2 accessions of S. grossulariifolia
compared with the control (77, 69, 62% versus 5, 14, and 32%, respectively), but failed
to do so for S. munroana (8%) relative to the control (2%)(Roth et al., 1987).
Additionally, organic solvents have been used to promote germination of physically
dormant seeds. A 4-hr submergence in diethyl dioxide improved germination of S.
grossulariifolia to 67% compared with 0% reported for untreated seeds (Page et al.,
1966). Roth et al. (1987) also found a 3-hr submergence of S. coccinea, S. munroana, and
2 accessions of S. grossulariifolia in diethyl dioxide significantly enhanced germination
compared with the control (36, 53, 89, and 68% versus 5, 2, 14, and 32%, respectively).
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Despite the effectiveness of chemical scarification, chemicals can be hazardous, difficult
to obtain, and present serious health risks (Mallinckrodt Baker, 2008 a, b). As a result,
alternative methods of seed treatment may be superior.
Another technique for improving imbibition of impermeable seeds is immersion in hot
water. Boiling water improved seed permeability of several Malvaceae species,
presumably by unblocking the water gap (Christiansen and Moore, 1959; Baskin and
Baskin, 1997). For example, seeds of Iliamna corei (Malvaceae) germinated to 93% (as
compared to 0% germination of the control), following a 5-sec submergence in boiling
water (Baskin and Baskin, 1997).
Higher germination has also been achieved by mechanical scarification. Seeds of I. corei
germinated to 100% following manual scarification with a single edge razor blade
(Baskin and Baskin, 1997). The International Seed Testing Association (ISTA, 1976)
rules suggest scarification with a file to be a successful measure for relieving dormancy
in Althaea officinalis and A. rosea (Malvaceae). Although effective, these techniques are
time consuming and unrealistic for use as operational seed treatments.
A number of mechanical scarification methods designed for treating large seed quantities
exist; however, few have proven to be successful in alleviating dormancy in Sphaeralcea
species. Primarily a result of scarification severity, embryo damage often overshadows
the dormancy-breaking effects of the treatment. Page et al. (1966) report decreases in
germination of S. grossulariaefolia with the duration of scarification time in a sandpaperlined rotating drum, while Roth et al. (1987) suggest that seeds of S. grossulariaefolia, S.
17
coccinea, and S. munroana perished following mechanical scarification, irrelevant of
treatment duration. More recently, rotating rock tumblers filled with abrasive media have
been used to promote germination of some physically dormant species and provide a
potential alternative to traditionally used scarification techniques (Dreesen, 2004).
Unfortunately, I found no information regarding the effects of tumbling on the
germination behavior of Sphaeralcea munroana. Traditionally used chemical and
mechanical scarification techniques are effective, but these methods are hazardous and
can result in severe embryonic damage. Thus, other alternatives are needed in order to
promote the effective use of S. munroana in restoration.
To meet that need, other less traditional techniques, such as fire and heating treatments,
may be employed to effectively induce permeability in physically dormant seeds. For
example, I. corei demonstrated increased germination following simulated annual
summer burning (1 to 2 min), with the highest germination achieved after 6 years (39 ±
6%) compared with the control (0%)(Baskin and Baskin, 1997). Moreover, physically
dormant seeds of 8 Fabaceae species germinated to a greater extent after being ignited
with a torch (Sugii, 2003). In some cases, dry heat may be a substitute for fire, often
achieving better results. For example, Baskin and Baskin (1997) found that several dry
heat temperatures and exposure durations optimized the germination of I. corei.
Although physical dormancy has been documented in a number of Malvaceae species,
reported germination of Sphaeralcea munroana has failed to exceed 53% following
scarification, even when dormancy is presumably broken (Roth et al., 1987; Smith and
18
Kratsch, 2009). Thus, it is unclear whether the seeds of this species have additional
dormancy types. Physiological dormancy, characterized by the presence of chemical
inhibitors that prevent embryonic growth, is commonly found in cold desert herbaceous
perennials, and can be relieved by stratification (Baskin and Baskin, 1998). Gibberellic
acid (GA3) has successfully served as a proxy for stratification for a number of
physiologically dormant species (Timson, 1966; Pinfield et al., 1972). The exogenous
application of GA3 is thought to enhance germination by increasing the growth potential
of the embryo and by overcoming the mechanical constraints that prevent radical
emergence (Karssen et al., 1989; Hilhorst and Karssen, 1992; Hilhorst, 1995; Bewley,
1997; Koornneef et al., 2002; Leubner-Metzger, 2003). In addition, exogenous
application of GA3 alone, or in combination with scarification, significantly amplified the
germination capacity of several species in the Cactaceae, including Cereus spp.,
Echinocactus grusonii, Leuchtenbergia principis, Sclerocactus mariposensis, and
Harrisia fragrans (Krulik, 1981; Moreno et al., 1991; De La Rosa-Ibarra and Garcia,
1994; Dehgan and Perez, 2005).
Although most physically dormant species do not exhibit additional dormancy, some do,
including several members of Malvaceae (Baskin and Baskin, 1998). The coupling of
physical and physiological dormancy is termed combined dormancy and requires both
types to be broken for germination to occur (Nikolaeva, 1969; Baskin and Baskin, 1998).
Dunn (2011) reports increased germination of Sphaeralcea ambigua and S. coccinea (to
45.3 and 85.3% relative to the control 18.0 and 4.7%, respectively) following a 30-d
stratification of scarified seeds. Similarly, Smith and Kratsch (2009) report that following
19
the mixture of scarification and a 6-wk stratification, seeds of S. grossulariifolia, S.
parvifolia, and S. munroana germinated to a greater extent than from either treatment
alone, suggesting that seeds of S. munroana may also exhibit combined dormancy.
Temperature and Moisture as Limiting Factors to Seedling Development
In regions where the growing conditions are restrictive, plant establishment is the most
vulnerable stage in vegetative community development (Call and Roundy, 1991). In the
arid-steppe ecosystem, wide diurnal temperature fluctuations, episodic precipitation
pulses, and extensive droughts impose key restraints to post-germination survival. The
maximum temperature and soil moisture changes occur in close proximity to the soil
surface, making seedlings more susceptible to environmental fluxes than mature plants
(Bazzaz and Mezga, 1973; Raynal and Bazzaz, 1973; Regehr and Bazzaz, 1976). Thus,
opportunities for plant establishment in the Great Basin are strongly limited by the
environmental conditions.
Diurnal temperature can vary by 20 °C, while seasonal differences can be on the order of
40 °C (Smith and Nowak, 1990). These oscillations can be further enhanced throughout
the year by the topography-induced nocturnal cold aid drainage (Osmond et al., 1990).
The Great Basin-Mojave region is described as the most arid habitat in North America,
with annual precipitation averaging 50 to 300 mm. Due to low humidity and abundant
irradiance the potential evapotraspiration in this region is high, ranging between 1100
mm in the northern and 2000 mm in the southern portion of the basin (Flaschka et al.,
1987). On an inter-annual basis, summer precipitation is highly variable, typically
20
representing only 20 to 30% of total annual precipitation (Bell, 1979). A strong disparity
exists between maximum water availability and the ability of plants to use it, because a
substantial portion of available moisture is considerably reduced by the time air and soil
temperatures become suitable for plants to be fully physiologically active (Caldwell,
1985). The interaction between precipitation and temperature patterns bear considerable
implications on the physiological ecology of the native floristic communities. The
beginning of the growing season in this region is directly correlated with the amount of
winter-spring precipitation and the rise in air and soil temperatures (Turner and Randall,
1987). The seasonal reduction in rainfall amplifies the importance of the spring growing
season; as a result, most species initiate growth in March and April, when maximum
daily temperatures range from 5 to 15 °C and night temperatures remain near freezing
(Thornthwaite, 1948; Comstock and Ehleringer, 1992). Furthermore, maximum
temperature and soil moisture changes occur in close proximity to the soil surface, which
limit seedling establishment following disturbance (Bazzaz and Mezga, 1973; Raynal and
Bazzaz, 1973; Regehr and Bazzaz, 1976).
Plant photosynthetic rates demonstrate substantial temperature dependence. At low
temperatures photosynthetic rates are reduced due to the decline in the enzyme-catalyzed
reaction rates. As temperatures rise, carbon assimilation rises, until a maximum (defined
as a “temperature optimum”) is reached, and declines once the optimum has been
exceeded (Comstock and Ehleringer, 1992). The maximum photosynthetic rates exhibit
considerable variability, and are primarily governed by environmental conditions. At the
beginning of the growing season, when day-time temperatures do not exceed 20 °C, the
21
temperature optimum form many Great Basin shrubs is 15 °C, increasing by 5 to 10 °C
later in the season (Caldwell, 1985). The maximum CO2 assimilation rates of shrubs
native to this region, under natural or irrigated conditions, range from 14 to 19 µmol CO2
m-2 s-1 (Depuit and Caldwell, 1975; Caldwell et al., 1977). At this time, most research has
focused on examining the photosynthetic behavior of perennial shrubs, with little
attention given to herbaceous species. However, the phenological cycles of herbaceous
and woody perennials are different, and may be expresses through variation in
photosynthetic behavior.
The increasing prominence of native forbs in restoration requires a clear understanding of
the physiological ecology of these species. Furthermore, outside agricultural research,
knowledge regarding the photosynthetic behavior of members of the Malvaceae family is
lacking. In a cultivation setting, Sida spinosa, Gossypium herbaceum, and Gossypium
arboreum (Malvaceae), demonstrate relatively high CO2 compensation points that are
associated with large net CO2 fixation and faster growth rates (Chen et al., 1970). In the
Great Basin, however, rapid growth rates can create severe internal moisture deficits,
making the ability of plants to effectively regulate moisture loss imperative.
In general, low spring temperatures allow for higher water use efficiency as cold soils
reduce leaf conductance, but the freezing night temperatures prevent the full reduction in
stomatal opening during the day, when moisture loss is high (Smith and Novak, 1990).
As soil moisture deficits increase with seasonal warming, plant water content declines
resulting in a lower turgor pressure. Under these conditions, turgor-dependent processes
22
such as leaf expansion and root elongation are suppressed. Decreases in turgor have been
linked to reductions in aboveground plant area and overall growth rates (Taiz and Zeiger,
2006). Moisture restrictions cause a greater portion of photosynthates to be distributed to
the root system, promoting belowground biomass production. Thus, root-to-shoot ratios
(R:S) are a direct result of a dynamic equilibrium between water uptake and
photosynthesis (Taiz and Zeiger, 2006). R:S of desert perennials exhibit considerable
variability among species, ranging from 0.15 to 6.77, with high R:S values characteristic
of plants in the northern Great Basin as compared to a much lower ratios for plants found
in the Mojave and Sonoran Deserts (Barbour, 1973; Caldwell et al., 1977). Most research
has characterized mature perennial shrub and grass species, while little is known about
growth dynamics of forbs, especially shortly following establishment.
Conclusions
Considering the rate of habitat degradation in the Great Basin, revegetation with native
plants is integral to ecosystem function and resilience. Munro’s globemallow is a species
with a high restoration potential; but its use is limited by the failure to achieve successful
in and ex situ germination as a result of seed dormancy. Furthermore, wide diurnal
temperature fluctuations, episodic precipitation pulses, and extensive droughts pose key
constraints to post-germination survival. Effective use of S. munroana depends on a clear
understanding of dormancy mechanisms and seedling response to environmental stress
following germination.
23
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27
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28
Figure 1.1. General outline of the Great Basin region of the western United States.
29
Chapter Two:
Seed Coat Morphology, Physical Dormancy, and the Use of Scarification for
Dormancy Break in Sphaeralcea munroana Seeds
Abstract
The results of a series of experiments involving a variety of dormancy-breaking
treatments indicate that Sphaeralcea munroana seeds are physically dormant, possess a
water gap responsible for water uptake, and that dormancy is best relieved by mechanical
or boiling water scarification. Mechanical scarification resulted in the highest water
uptake (86% ) compared to boiling water scarification (22%), and the control (12%),
suggesting that boiling resulted in only partial water gap opening. Exogenous application
of GA3 and stratification failed to enhance germination compared with scarification
alone, indicating an absence of additional dormancy types. These results should improve
the usefulness of this cool-season perennial for restoration in the Great Basin, where its
effectiveness in soil stabilization, tolerance of disturbed sites, drought, extreme
temperatures, and importance as a food source for myriad animals makes it an important
species.
30
Introduction
Munro’s globemallow (Sphaeralcea munroana (Douglas) Spach) (Malvaceae), a
perennial, cool-season forb endemic to the Great Basin, is an important candidate for
broad-scale ecosystem restoration across its native range. It serves as an important host
for native pollinators, provides soil stabilization, and is a source of food for myriad
mammals (Beale and Smith, 1970; Pendery and Rumbaugh, 1986; Rumbaugh et al.,
1993; Pavek et al., 2011). In addition, the species is commonly found on disturbed sites
and is able to tolerate drought and extreme temperature conditions. Currently, the lack of
successful in and ex situ germination due to seed dormancy limits its use in restoration.
Few sources explore the dormancy mechanisms and methods that induce germination in
the Sphaeralcea genus (Page et al., 1966; Roth et al., 1987).
Dormancy is described as “a seed characteristic, the degree of which defines what
conditions should be met to make the seed germinate” (Vleeshouwers et al., 1995).
Several types of dormancy exist and are characterized based on the mechanisms that
prevent germination. Physically dormant seeds have a palisade layer of lignified cells that
prevent water imbibition (Corner, 1951; Vazquez-Yanes and Perez-Garcia, 1976).
Although a number of species in the Sphaeralcea genus have been observed to benefit
from scarification, the cause of dormancy has not been examined directly (Page et al.,
1966; Sabo et al., 1979; Roth et al. 1987; Smith and Kratsch, 2009). In these species,
imbibition (critical for germination) is regulated by a water gap structure located within
the seed coat. The water gap can become permeable following exposure to temperature
31
flux, drying, or scarification, thus allowing imbibition into an otherwise impermeable
seed (Baskin and Baskin, 1998; Baskin, 2003; Baskin et al., 2000).
Ex situ, chemical and mechanical scarification have been used to improve germination of
physically dormant seeds (Page et al., 1966; Roth et al., 1987; Hoffman et al., 1989;
Baskin and Baskin, 1998). For example, Page et al. (1966) reported increased
germination (30 to 40%) of S. grossulariifolia after submergence in sulfuric acid, a
substantial improvement compared to the control (0%). Similarly, submergence of
Sphaeralcea seeds in 18 M sulfuric acid for 10 min improved germination of S. coccinea
and 2 accessions of S. grossulariifolia relative to the control (77, 69, 62% versus 5, 14,
and 32%, respectively), but failed to do so for S. munroana (8%) compared to the control
(2%)(Roth et al., 1987). Organic solvents have also been used to promote germination of
physically dormant seeds. Page et al. (1966) reported 67% germination of treated S.
grossulariifolia seeds following a 4-hr submergence in diethyl dioxide versus 0%
germination of untreated seeds. Roth et al. (1987) found a 3-hr submergence of S.
coccinea, S. munroana, and 2 accessions of S. grossulariifolia in diethyl dioxide to
significantly enhance germination compared with the control (36, 53, 89, and 68% versus
5, 2, 14, and 32%, respectively). Despite the effectiveness of chemical scarification,
chemicals can be hazardous, difficult to obtain, and present serious health risks
(Mallinckrodt Baker, 2008 a, b). As a result, alternative methods of seed treatment may
be superior.
32
Boiling water promoted seed permeability and subsequent germination of several
Malvaceae species, presumably through opening of the water gap (Christiansen and
Moore, 1959; Baskin and Baskin, 1997). For example, seeds of Iliamna corei
(Malvaceae) germinated to 93% (as compared to 0% germination of the control),
following a 5-sec submergence in boiling water (Baskin and Baskin, 1997).
Mechanical scarification has also been reported to boost germination rates. The
International Seed Testing Association recommends scarification with a metal file to
break dormancy of Althaea officinalis and A. rosea (Malvaceae) (ISTA, 1976). However,
manual seed treatment techniques are time consuming and unrealistic for operational use
(Baskin and Baskin, 1998). Mechanized techniques can result in embryo damage due to
scarification severity, often overriding the benefits of the treatment. Page et al. (1966)
report decreases in germination S. grossulariaefolia with the duration of scarification
time in a sandpaper-lined rotating drum, while Roth et al. (1987) suggest that seeds of S.
grossulariaefolia, S. coccinea, and S. munroana perished after mechanical scarification
irrelevant of treatment duration.
Less traditional techniques, such as rock tumbling, fire, and heating have been effectively
used to increase seed coat permeability in some physically dormant species. For example,
Dreesen (2009) recommends the use of seed abrasion in rotating rock tumbler to improve
germination of physically dormant species. I found, however, no information regarding
the effects of tumbling on the germination behavior of Sphaeralcea munroana. Abrasion
through fire has been observed to benefit germination. For example, I. corei
33
demonstrated increased germination following a 1 to 2 min simulated annual summer
burning, with the highest germination achieved after 6 years (39 ± 6% ) compared with
the control (0%) (Baskin and Baskin, 1997). Moreover, germination of physically
dormant seeds of 8 Fabacea species was substantially amplified after ignition with a
torch (Sugii, 2003). Dry heat may be a substitute for fire, often achieving superior results.
Baskin and Baskin (1997) found that several dry heat temperatures and exposure
durations optimized I. corei germination.
Despite evidence for the presence of physical dormancy, reported germination of S.
munroana has failed to exceed 53%, even when dormancy was presumably broken (Roth
et al., 1987; Smith and Kratsch, 2009). Thus, it is unclear whether these seeds possess
additional dormancy types. Physiological dormancy, characterized by the presence of
chemical inhibitors that prevent embryonic growth, is commonly found in cold desert
herbaceous perennials and can be relieved by stratification (Timson, 1966; Pinfield, et al.,
1972; Baskin and Baskin, 1998). Gibberellic acid can serve as a proxy for stratification
by alleviating the chemical constraints that prevent radical emergence and increasing
embryonic growth (Hilhorst, 1995; Bewley, 1997; Koornneef et al., 2002; LeubnerMetzger, 2003).
Although less common, the coupling of physical and physiological dormancy (combined
dormancy) requires both types to be broken before germination can occur (Nikolaeva,
1969; Baskin and Baskin, 1998; Emery, 1987). Dunn (2011) reports increased
34
germination of Sphaeralcea ambigua and S. coccinea (to 45.3 and 85.3%, respectively)
compared to the control (18.0 and 4.7%, respectively), following a 30-d stratification of
scarified seeds. Similarly, Smith and Kratsch (2009) report that following the mixture of
scarification and a 6-wk stratification, seeds of S. grossulariifolia, S. parvifolia, and S.
munroana germinated to a greater extent than from either treatment alone, suggesting that
seeds of S. munroana may also exhibit combined dormancy. Smith and Kratsch (2009)
report that pairing scarification with a 6-wk stratification resulted in higher germination
of S. grossulariifolia, S. parvifolia, and S. munroana than either treatment alone,
suggesting that seeds of S. munroana may exhibit combined dormancy.
Successful use of Munro’s globemallow in restoration depends on a clear understanding
of the underlying mechanisms governing seed germination. The process of seed
imbibition and the site of water entry are critical to our comprehension of the germination
dynamics and treatment effects. To address these questions, 4 experiments were initiated.
The first experiment compared (a) water uptake of non-treated, scarified, and boiling
water scarified seeds and (b) identified the primary site of water uptake. The second
experiment, investigated the germination response of fresh S. munroana seeds to
mechanical scarification, 6-wk stratification, and their combination. The third experiment
evaluated the germination behavior of stored seeds following mechanical scarification,
submergence duration in gibberellic acid or plain water, and several combinations of
these treatments. The use of native species for restoration is limited by the high seed
procurement cost and low establishment rates (when compared to the use of non-native
cultivars). Thus, the economically feasible use S. munroana is dependent on the
35
development of large scale seed pre-treatment techniques. In order to address this issue,
the final experiment assessed the effectiveness of boiling water, tumbling, burning,
heating, and burning + heating scarification on germination.
Materials and Methods
In general, the experiments were conducted at the University of Idaho, Center for Forest
Nursery and Seedling Research, Moscow. Prior to each experiment, seeds were sterilized
with a 0.5% NaOCl solution for 15 min and double-rinsed with deionized (DI) water. The
general procedure included placing seeds onto germination paper (soaked with DI water)
inside randomly arranged Petri dishes, remoistening germination paper with 5 ml of DI
water every 3 d to avoid a potential reduction in dormancy break due to substrate
desiccation (Flemion, 1931; Stokes, 1965), and monitoring germination (defined by ≥ 5
mm radical length) daily for 21 d.
Specifically, for the first 2 experiments, seeds were collected in August 2010 near
Payette, Idaho (N 43° 52’ 49.6” W 116° 47’ 01.8”) and kept at 21±1 °C to avoid the
possibility of stratification during refrigerated storage. Experiments 3 and 4 used seeds
obtained from native stands throughout the Wasatch Mountains of northern Utah (Great
Basin Seeds LLC, Ephraim, UT) and stored at 1.5±0.5 °C. All treatments had 5, 50-seed
replicates except those in the first experiment, which contained 10, 15-seed replicates.
The first experiment, compared water uptake of seeds after exposure to 3 treatments: (1)
control, (2) mechanical scarification with a sharp blade in the region opposite the chalaza,
and (3) boiling water scarification achieved by a10-sec submergence in 100 °C water.
36
Seeds in each replicate were weighed to the nearest 0.1 mg and re-weighed (after being
blotted to reduce the presence of free water) at 1-hr intervals for 10 hr and once at the end
of the 24-hr observation period (Gama-Arachchige et al., 2010). Mass increase,
expressed as a mean percentage gain on a dry weight basis, was calculated. Subsamples
of control and boiling water scarified seeds were sent to the Electron Microscopy Center
at the University of Kentucky, Lexington and scanned with a S-3200 Hitachi (Hitachi
High Technologies America, Inc. Pleasanton, CA) scanning electron microscope
(acceleration voltage of 5.0 kV) (Gama-Arachchige et al. 2010). The chalazal region and
the dislodged chalazal cap were observed and photomicrographed.
The second experiment assessed the presence of physical, physiological, and combined
dormancy by comparing the germination following exposure to a (1) control, (2)
mechanical scarification (described above), (3) stratification, and (4) combined
scarification + stratification treatments. Seeds were stratified at 4.6±0.02 °C for 6 wk on
moistened germination paper inside sealed Petri dishes. Seeds in the combination
treatment were scarified first (Smith and Kratsch, 2009).
The third experiment evaluated the effects of GA3 on germination. Seeds were subject to
8 treatments: (1) control, a (2) 24- and (3) 48-hr soak in DI water, and a (4) 24- and (5)
48-hr soak in 100 ppm GA3 solution (90% gibberellin A3 basis and DI water). The final 3
treatments were applied to mechanically scarified seeds soaked for (6) 0, (7) 24 or (8) 48
hr in 100 ppm GA3. The final experiment looked at the effects of “non-traditional”
scarification, including (1) control, (2) boiling water (described above), (3) tumbling, (4)
37
burning, (5) dry-heating, and (6) burning + heating. Seeds were tumble-scarified in a
rotary rock tumbler (Model AR-1) with dry aluminum oxide grit (12 Mesh, Kramer
Industries, Inc., Piscataway, New Jersey) for 72 hr. Following tumbling, seeds were
separated from grit using a series of sieves (Dreesen, 2004). For burning scarification,
seeds were placed in single layer onto a metal mesh screen, submerged uniformly in 95%
ethyl alcohol for 1 min. Seeds were removed from the alcohol and placed on a fireresistant surface, ignited with a hand-held butane torch, and allowed to burn for 10 sec
before being extinguished with DI water (Sugii, 2003). For dry-heat scarification seeds
were placed into a laboratory oven (80 °C) for 60 min (Baskin and Baskin 1997). Seeds
subject to burning + heating scarification were burned first.
Statistical Analysis
Although germination capacity is conventionally used to assess seed performance, other
factors (i.e. germination rate and uniformity) are important (Ching, 1959; Thomson and
El-Kassaby, 1993; El-Kassaby, 2007). Combining multiple parameters into a single
variable (e.g. Czabator, 1962) in an attempt to better describe germination performance
has failed to provide a holistic representation of germination. To avoid this, I fit daily
germination data to 2 mathematical models, the parameters of which give a detailed
description of seed behavior. In experiments 2 and 4, a 3-parameter model was used
(Equation 2.1); where G (t) is the cumulative germination percentage at time (t)
expressed in days (d), Gc is cumulative germination, or the germination asymptote at the
end of the testing period (%), GC50 is the time in days required to reach 50% germination,
38
G (t ) 
Gc
1 e

GC50 t
(Equation 2.1)
Gd
G(t )  Gc 1  e
e
GC50( t  Gl )

(Equation 2.2)
and Gd is the germination rate (% /d). Due to differences in germination behavior in
experiment 3, an alternative model was used (Equation 2.2); where Gd was replaced with
Gl, defined as the time in days to first germination. All remaining parameters were
identical to those described above. Parameters (Gc, GC50, Gd, Gl) for each replication
were generated by curve-fitting. Expected mean squares, components of variance, and R2
values were estimated for all parameters. In all experiments, specific treatment
differences were explored with one-way ANOVA and Tukey’s HSD (α = 0.05) (SAS
Institute Inc., Cary, NC).
Results
In the first experiment (evaluating imbibition), mechanical and boiling water scarification
resulted in significant increases (p < 0.0001 and p = 0.0347, respectively) in water uptake
compared with the control (Figure 2.2.). The measurements for 3 of the 10 replicates
were systematic outliers and were excluded from this analysis. Mechanically scarified
39
seeds exhibited greater (p < 0.0001) mass gain compared to the boiling water
scarification at the end of the 24-hr observation period (Figure 2.2.). The
photomicrographic evaluation revealed that the chalazal region of the seed possesses a
water gap that is covered by a cap-like structure made up of maternal tissues and palisade
cells (Figure 2.1. A, B). Boiling water scarification caused a contiguous crack to form
around the chalazal region, which lead to the water gap dislodgement and subsequent
imbibition (Figure 2.1. B, C, D) (Serrato-Valenti et al., 1992).
In the second experiment (investigating presence of physical, physiological, or combined
dormancy in fresh seeds), Gc was significantly different among all treatments, while
GC50 and Gd were not (p < 0.0001, p = 0.3530, and p = 0.4526, respectively). Most of the
variation in Gc (R2 = 0.77) was explained by the differences in treatment. Germination
was enhanced (p = 0.0030, p = 0.0002) with scarification (35%) and the combined
scarification and stratification (44%) (Figure 2.3.). Combining both treatments failed (p =
0.5191) to improve Gc when compared with scarification alone.
The results of experiment 3 (evaluating germination following mechanical scarification,
as well as soaking duration in GA3, and water) indicate that Gc, GC50, and Gl were
different among all treatments (p < 0.0001, p = 0.0005, and p = 0.0386, respectively).
Most of the variation in Gc (R2 = 0.93) was explained by differences in treatment. The
correlation was not as strong for GC50 (R2 = 0.53) and Gl (R2 = 0.35) parameters. Results
confirm that S. munroana seeds are physically dormant, with scarified seeds exhibiting a
higher (p < 0.0001) germination compared to non-scarified seeds (Table 2.1., 2.4.).
40
Exogenous addition of GA3 did not promote germination of the non-scarified seeds.
Scarification, scarification + water (24 hr), and scarification + GA3 (24 hr) yielded the
greatest Gc (87, 93, and 88%, respectively) at the end of 21 d. Gc was higher (p = 0.0328)
for scarification + water (24 hr) than for scarification + water (48 hr) treated seeds.
However, a similar relationship did not exist (p = 0.9669) between scarified + GA3
treatments. GC50 was significantly lower for GA3 (48 hr), scarified, and scarified seeds +
GA3 (24 and 48 hr) compared with the control (Table 2.1.), suggesting that seeds
germinated more succinctly. Seeds in the scarification + GA3 (24 hr) treatment began
germination later (p = 0.0497) than their cohorts in the scarification + water (48 hr)
treatment.
In experiment 4, Gc, GC50, and Gd varied significantly (p < 0.0001, p < 0.0001, and p =
0.0115, respectively) among treatments (Table 2.2.). Most variation in GC50 and Gc (R2 =
0.75, R2 = 0.64) could be explained by differences in treatment, with a weaker correlation
for Gd (R2 = 0.44). Gc was highest (49%) after boiling water scarification compared to all
other treatments (p < 0.0001) (Table 2.2., Figure. 2.5.). The remaining treatments did not
enhance germination (Gc < 20%). Boiling water scarification produced a different
germination behavior than the other treatments. For example, until day 7, seed
germination was low, demonstrated by the larger (p < 0.0001) GC50 parameter estimate
(8.2 d). However, following day 7 (when the Gd of the remaining treatments began to
slow), the Gd of boiling water scarified seeds increased (p = 0.0165) without reaching an
asymptote within the test duration.
41
Discussion
Results suggest that seed coat impermeability in Munro’s globemallow is indicative of
physical dormancy. Occurrence of the water gap structure in the chalazal region of the
seed, responsible for water uptake following natural dormancy break, is analogous to
other Malvaceae species (Christiansen and Moore, 1959; Egley and Paul, 1981; 1982;
Egley et al., 1986). Permeability can be achieved artificially (e.g. mechanical
scarification) or by stimulating natural dormancy release (e.g. boiling water
scarification). Presumably a result of the separation of the palisade and subpalisade layers
of cells, boiling water scarification caused the detachment of the cap-like structure in the
occlusion of the water gap, allowing imbibition to occur (Serrato-Valenti et al., 1992).
Because seed mass increased to 86% following mechanical and to 22% after boiling
water scarification, it appears that boiling water scarification only partially opens the
water gap. Even so, treatments such as boiling water that induce a natural dormancy
break are less likely to damage the embryo and are thus more desirable (Page et al., 1966;
Roth et al., 1987).
My findings that mechanical scarification amplified Gc coincide with Baskin and Baskin
(1997) who observed 100% germination of mechanically scarified seeds of I. corei.
However, when large quantities of seeds must be treated, boiling water scarification can
be a safe alternative to the use of other chemical scarification treatments, including
sulfuric acid and diethyl dioxide (Roth et al., 1987). With boiling water scarification, all
3 germination parameters had large standard errors, which may again indicate an
42
incomplete opening of the water gap, resulting in lower total water uptake (Table 2.2.).
Because the germination curve never reached its asymptote within the duration of
thestudy, a more accurate assessment of the effects of boiling water scarification would
require a longer observation period. Neither heating nor burning yielded significant
germination improvements, which is inconsistent with Baskin and Baskin (1997), who
found that I. corei germination was optimized following a 60-min dry heat (80 °C)
application. Thus, although general patterns of dormancy may be similar across members
of the same genus, explicit temperature and moisture requirements seem to be species
specific.
I found a lack of evidence for the presence of additional dormancy types, with both
stratification and GA3 failing to boost Gc compared with scarification alone. The addition
of GA3 (24 hr) to scarified seeds amplified Gc over the control, but was not different from
water (24 hr), and was likely a result of soaking itself. It is possible that a higher
concentration of GA3 was needed to influence germination behavior. For example, in a
study comparing the effects of 500 and 1000 ppm of GA3, only the 1000 ppm solution
enhanced germination of Harrisia fragrans (Dehgan and Pérez, 2005). Considering,
however, that a Gc of 93% was achieved by the scarification + water, gibberellins are
unlikely to play an important role in alleviating dormancy of S. munroana.
The germination of S. munroana reported for experiment 3 (93 %) was much higher than
previously observed (51% for S. munroana and 88% for other Sphaeralcea spp.) (Roth et
al., 1987; Smith and Kratsch, 2009). Despite the 95.5 % purity reported for the seed lot
43
used in experiment 3, seeds were additionally cleaned in an air column seed separator
(Model B, E.L. Erickson, Brookings, SD) to reduce the presence of empty seeds. The
satisfactory level of cleaning was determined by X-ray images (MX-20 Radiography
System, Faxitron X-ray Corp., Lincolnshire, IL) which allowed me to monitoring seed fill
until a desired level was reached. Through this process a substantial amount of seeds,
mostly light brown in color, was removed. It is possible that simply increasing seed lot
purity and viability through additional cleaning could improve germination of
Sphaeralcea spp., and should be considered in future research and production.
While some authors (Smith and Kratsch, 2009; Dunn, 2011) report germination
improvements of Sphaeralcea seeds following the combination of scarification and
stratification, it is possible that their results a are not a feature of the stratification
temperature but of the duration of time seeds spend in moist conditions, which allows for
optimal imbibition to occur even in partially permeable seeds.
Additionally, after-ripening or a period of dry storage (24±1 °C) of freshly collected
seeds, used to promote dormancy release, could play a role in the reported differences in
treatment effects. For example, loss of physiological dormancy in Geraniaceae seeds
occurs within 2 mo of dry storage (Baskin and Baskin, 1974; Van Assche and
Vanderlook, 2006). Furthermore, several cold desert perennials have been reported to
benefit from after-ripening (Baskin and Baskin, 1998). This process has been suggested
to increase the acceptable germination temperature range, promote the loss of ABA,
diminish the GA3 requirement, improve germination rate, and impact seed sensitivity to
44
light (Bewley, 1997; Finch-Savage and Leubner-Metzger, 2006; Kucera et al., 2005).
However, seed storage conditions are rarely reported, making comparisons difficult.
Additionally, collection timing is strongly coupled with seed maturity and moisture
content, which have a strong effect on dormancy strength and germination behavior
(Dornobos et al., 1989; Michael et al., 2007). For example, Michael et al. (2007) found
that seeds of Malva parviflora (Malvaceae) decreased in moisture content and
germination capacity with seed development until complete physiological maturity.
Germination declines are associated with the development of physiological dormancy and
the enhancement of physical dormancy as a result of declining seed moisture content
(Michael et al., 2007). Thus, high temperature and low moisture content during the
growing season may contribute to the degree of dormancy development in seeds of S.
munroana.
Conclusions
My findings suggest that seeds of Sphaeralcea munroana exhibit strong physical
dormancy due to the impermeability of the seed coat and the presence of a cap-like
structure in the occlusion of the water gap, which inhibits water imbibition and prevents
germination. Seed coat scarification is essential for germination, following which seeds
may benefit from submergence in water. Although mechanical scarification produced the
greatest germination, boiling water scarification can be an effective and operationally
feasible method for breaking dormancy in large quantities of seeds.
45
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Gama-Arachchige, N. S., J. M. Baskin, R. L. Geneve, and C. C. Baskin. 2010.
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48
Table 2.1. The effects of mechanical scarification, soaking duration in GA3, or pure water
solution on the germination behavior of Sphaeralcea munroana at the end of the 21-d
observation period. Parameter values were generated using a non-linear regression model
(Equation 2.2), where G (t) is the cumulative germination percentage at time (t) expressed in
days (d), Gc is cumulative germination, or the germination asymptote at the end of the testing
period (%), GC50 is the time (d) required to reach 50% germination, and Gl is the time (d) until
first germination. Multiple comparisons were obtained using Tukey’s HSD (α = 0.05).
Treatment
Gc (%)
Control
15.9±4.5 c
Mechanical Scarification
87.1±3.8 ab
Gibberellic Acid (24 hr)
15.9±2.3 c
Gibberellic Acid (48 hr)
15.1±2.4 c
Mechanical Scarification + GA3 (24 hr)
88.1±3.9 ab
Mechanical Scarification + GA3 (48 hr)
81.7±1.8 ab
Mechanical Scarification + Water (24 hr)
93.4±2.9 a
Mechanical Scarification + Water (48 hr)
71.9±9.3 b
0.0001
P values
Different letters indicate significant differences among treatments
GC50 (d)
2.7±0.5 a
0.7±0.2 b
1.7±0.6 ab
0.9±0.3 b
0.6±0.1 b
0.6±0.1 b
1.5±0.3 ab
1.8±0.4 ab
0.0005
Gl (d)
0.1±0.1 ab
0.3±0.1 ab
0.4±0.2 ab
0.2±0.2 ab
0.6±0.1 b
0.4±0.2 ab
0.5±0.1 ab
0.0±0.0 a
0.0386
Table 2.2. The effects of “non-traditional” scarification techniques on the germination behavior
of Sphaeralcea munroana at the end of the 21-d observation period. Parameter values were
generated using a non-linear regression model (Equation 2.1), where G (t) is the cumulative
germination percentage at time (t) expressed in days (d), Gc is cumulative germination, or the
germination asymptote at the end of the testing period (%), GC50 is the time (d) required to reach
50% germination, and Gd is the germination rate (% /d). Multiple comparisons were obtained
using Tukey’s HSD (α = 0.05).
Treatment
Gc (%)
GC50 (d)
Gd (% /d)
Control
10.7±1.1 b
1.6±0.2 b
1.4±0.5 b
Boiling water
49.0±12.9 a
8.2±1.2 a
4.2±0.8 a
Tumbling
20.3±1.3 b
2.1±0.1 b
2.3±0.3 b
Burning
17.0±1.6 b
2.0±0.3 b
2.0±0.6 b
Heating
10.7±1.3 b
4.4±0.2 b
2.1±0.5 b
Burning + Heating
4.2±1.8 b
4.2±0.1 b
1.2±0.5 b
0.0001
0.0001
0.0115
P values
Different letters indicate significant differences among treatments.
49
Figure 2.1. Scanning electron micrographs of a Sphaeralcea munroana seed; (A) top view of the
chalazal region of a dormant seed showing the chalazal slit; (B) formation of water gap following
dormancy break; (C) the underside of the dislodged cap-like blister covering the water gap; (D) palisade
cells of the cap-like structure. Abbreviations: Chsl, chalazal slit; Chsl*, remaining part of the chalazal
slit after dormancy break; Ll, light line; Mi, micropyle; Mt, maternal tissue; Pa, palisade; Spl,
subpalisade cells; Wg, water gap.
50
100
Control
Mechanical
Boiling water
a
Mass Increase (%)
80
60
40
b
20
c
0
0
1
2
3
4
5
6
7
8
9
10 24
Time (h)
Figure 2.2. Mass increase percentage (on a fresh mass basis) of control,
mechanically, and boiling water scarified seeds of Sphaeralcea munroana
during a 24-hr incubation. Each point scatter represents the mean (±s.e.) of
10 replicates. Different letters indicate significant differences (p < 0.05) at
the end of the 24-hr period.
51
100
Control
Scarified
Stratified
Scarified + Stratified
90
Cumulative Germination (%)
80
70
60
50
b
40
b
30
20
a
a
10
0
0
5
10
15
20
Time (d)
Figure 2.3. Cumulative germination percentage of Sphaeralcea munroana
seed subject to 4 seed treatments during a 21-d observation period. Each
line represents the mean (±s.e.) response of 5 replicates. Different letters
indicate significant differences (p < 0.05) between treatments.
52
a
100
90
Germination Capacity (%)
80
Control
GA3 (24 h)
GA3 (48 h)
Scarified
Scarified + water (24 h)
Scarified + water (48 h)
Scarified + GA3 (24 h)
Scarified + GA3 (48 h)
ab
ab
ab
b
70
60
50
40
30
c
20
c
c
10
0
Treatment
Figure 2.4. Germination capacity of Sphaeralcea munroana seeds 21 d
following exposure to 8 treatments. Each bar represents the mean
(± s.e.) response of 5 replicates. Different letters indicate significant
differences (α = 0.05).
53
50
Control
Boiling
Tumbling
Burning
Heating
Burning + Heating
Cumulative Germination (%)
40
a
30
b
b
20
b
b
10
b
0
0
5
10
15
20
Time (d)
Figure 2.5. Cumulative germination percentage of Sphaeralcea munroana
seeds subject to 6 treatments during a 21-d observation period. Each line
represents the mean (±s.e.) of 5 replicates. Different letters indicate
significant differences (p < 0.05) between treatments.
54
Chapter Three:
Individual and Synergistic Influence of Temperature and Moisture on Sphaeralcea
munroana Seedling Growth
Abstract
For Munro’s globemallow (Sphaeralcea munroana) seedlings, temperature affects
growth immediately after germination more than available moisture. In a growth chamber
study, germinants grown at colder temperatures 17/3 °C had 47% fewer leaves, 21% less
leaf area, and less shoot (31%) and root (31%) biomass than their cohorts grown at 23/9
°C. Decreasing irrigation frequency favored biomass allocation to roots and a subsequent
increase in root-to-shoot ratio. Neither temperature nor moisture had an effect on gas
exchange. These results suggest that although this perennial, cool-season forb shows
considerable potential for restoration use on arid sites, it may not be the best candidate for
early competition with cool season grasses during its establishment phase. Because
growth is hindered by cool temperatures, a later sowing date may improve establishment
in nurseries, seed production areas, and restoration sites.
55
Introduction
Plant establishment is the most vulnerable stage in plant community development,
especially in regions with restrictive growing conditions (Call and Roundy, 1991). Wide
diurnal temperature fluctuations, episodic precipitation pulses, and extensive droughts
present major limitations to post-germination survival. Seedlings are more susceptible to
environmental fluxes than mature plants because the maximum temperature and soil
moisture changes occur in close proximity to the soil surface (Bazzaz and Mezga, 1973;
Raynal and Bazzaz, 1973; Regehr and Bazzaz, 1976).
In the Great Basin, diurnal temperatures can fluctuate by 20 °C (Smith and Nowak,
1990), in part due to topographically-induced formation of large nocturnal cold aid
drainage throughout the year (Osmond et al., 1990). The Great Basin-Mojave region has
been characterized as the most arid habitat in North America, with precipitation
averaging 50 to 300 mm annually. As a result of low humidity and abundant irradiance
the potential evapotranspiration in this region is high, ranging between 1100 mm in the
northern and 2000 mm in the southern portion of the basin (Flaschka et al., 1987). On an
inter-annual basis, summer precipitation is highly variable, typically representing only 20
to 30% of total annual precipitation (Bell, 1979). Thus, strong disparity exists between
maximum water availability and the ability of plants to use it because a substantial
portion of available moisture is lost by the time air and soil temperatures become suitable
for plants to be fully physiologically active (Caldwell, 1985). For example, despite being
abundant, winter precipitation may not be used during a considerable portion of the year
56
as a result of cold temperatures, which create a reduction in the physiological plant
activity. Moreover, in early spring, moisture loss can still occur via sublimation and
evaporation. Therefore, the beginning of the growing season is directly correlated with
the amount of winter-spring precipitation and the increase in air and soil temperatures
(Turner and Randall, 1987). The seasonal reduction in rainfall enhances the importance
of the cool spring growing season. As a result, most species initiate growth in March and
April, when maximum daily temperatures range from 5 to 15 °C and night temperatures
remain near freezing (Thornthwaite, 1948; Comstock and Ehleringer, 1992). At the soil
surface, especially on sites where recent disturbance has considerably reduced the
presence of plant cover, diurnal temperature differences are more pronounced. Clearly,
the interaction between precipitation and temperature patterns bear considerable
implications on the physiological ecology of the native floristic communities.
One important component of Great Basin plant communities is Munro’s globemallow
(Sphaeralcea munroana (Douglas) Spach). This endemic, perennial forb provides soil
stabilization and is a source of nutrition for myriad animals (Beale and Smith, 1970;
Pendery and Rumbaugh, 1986; Rumbaugh et al., 1993; Pavek et al., 2011). For these
reasons, as well as its ability to tolerate disturbed sites, drought, and extreme
temperatures, it is an important candidate for broad scale restoration across its range.
While native plants are fundamental in maintaining ecosystem function, the use of forbs
in restoration is relatively novel and vastly unexplored (Parkinson, 2008). As a result,
little is known about the range of tolerance to environmental conditions that allows for
successful establishment and growth of S. munroana. Therefore, the study objective was
57
to evaluate a suite of morphological and physiological characteristics of S. munroana to a
range of temperature and moisture conditions during establishment.
Materials and Methods
Plant Establishment
Sphaeralcea munroana seeds were collected from 5 locations throughout Oregon and
Idaho (Table 3.1.). All seeds were hand scarified with a scalpel in order to break physical
seed dormancy (Kildisheva, 2011) and were sown directly into 66 ml containers (Model
RLC4, Ray Leach®, Stuewe and Sons, Inc., Tangent, OR). Containers, augmented with
mesh liners to prevent media loss, were filled with autoclaved sand and placed into 4
environmental growth chambers (Model E-30B, Percival Scientific, Inc., IA). A 10-d
period was allowed for germination, during which chambers were set to a 24 °C (8 hr)/17
°C (16 hr) diurnal cycle (Sabo et al., 1979). Seedlings were thinned to 1 plant per
container 10 d after sowing (DAS) and randomly assigned to a temperature and moisture
treatment. At this point, a one-time application of 18-24-16 (N-P-K) solution of MiracleGro® Water Soluble Rose Plant Food (Scotts Co., Marysville, OH) was administered to
all containers at a rate of 3.8 mg N per plant.
Temperature and Moisture Treatments
Because S. munroana typically germinates in April (Parkinson, 2008), climate records for
April from 1950 through 2010 were obtained using PRISM Data Explorer
(http://prismmap.nacse.org/nn/) for each coordinate and averaged across sites. The
58
resultant temperature regime was 17/3 °C, which I contrasted with a 23/9 °C regime that
represented a 6 °C increase in temperature. The diurnal transition followed a 13-h day
and an 11-h night. Irradiance was set to 950 µmol m-²s-¹ based on values provided by
light response curves developed for field-planted specimens of S. munroana (Appendix A
Figure 3.11.). Relative humidity was ambient and ranged between 50 and 60%. Within
each chamber, plants were subject to 1 of the 4 moisture availability treatments (3, 6, 9
and 12-d intervals between recharging each container to field capacity). Each temperature
× moisture combination was randomly assigned 20 seedlings. Containers were
subirrigated by placing them into deionized water for 1 hr. To prevent the effects of
environmental heterogeneity within chambers (Lee and Rawlings, 1982), plants were
randomized every 3 d.
Mortality, physiological, and morphological assessments were made at the end of the 25
d period (35 DAS). For the purpose of uniformity, 5 seedlings from each temperature ×
moisture regime were randomly selected for physiological and morphological
measurements. Gas exchange rates (i.e. photosynthesis, stomatal conductance, and
transpiration) were evaluated within 4 hr of light period initiation using an LI-6400
Portable Photosynthesis System (Li-Cor, Inc., Lincoln, NE). Plants were gently removed
from containers and separated by tissue type (i.e. root, shoot) from which leaf area and
number of true leaves were determined. After drying tissues at 80 °C for 24 hr, the aboveand belowground biomass and the root-to-shoot ratios (R:S) were determined.
Cumulative plant mortality for each treatment was recorded upon the termination of the
experiment.
59
Statistical Analysis
A split-plot design with 2 temperature regimes (whole plot) and 4 moisture levels (subplot) was used. Individual growth chambers were treated as blocks and moisture
treatments were completely randomized within chambers. Statistical analysis was
conducted with SAS version 9.2 software (SAS Institute Inc., Cary, NC). A mixed effects
procedure (PROC MIXED) was used to test the main effects of temperature and moisture
by nesting the block (chamber) within temperature. Between-chamber replication was
attained by repeating the study during 2 distinct time periods. The testing period was not
a significant variable, and was thus eliminated from the analysis. Pair-wise comparisons
(a = 0.05) of the least square mean estimates were made for all temperature, moisture,
and temperature × moisture interactions.
Results
Plant mortality, photosynthesis, conductance, and transpiration were unaffected by
temperature, moisture, or the temperature × moisture interaction (Table 3.2., Appendix
B). Seedlings grown at 17/3 °C produced less true leaves, leaf area, above- and
belowground biomass (47, 21, 31, and 31%, respectively) than their cohorts grown at
23/9 °C (Table 3.2., Figure 3.1.-3.4.). Belowground biomass and R:S were the only
variables affected by temperature, exhibiting higher values (p = 0.0127 and p = 0.0317)
under the most moisture limiting conditions (12-d irrigation interval) (Table 3.2., Figure
3.5., 3.6.). Leaf area, above- and belowground biomass, and R:S were significantly
influenced by the temperature × moisture interaction (Table 3.2.). At low temperatures
60
(17/3 °C), these parameters remained constant, irrelevant of moisture availability.
However, under the warmest, driest conditions (23/9 °C, 12 d-irrigation interval) leaf area
and aboveground biomass decreased relative to more frequent (≤ 9 d) irrigation (Figure
3.7., 3.8.). This trend was not as strong for the aboveground biomass, as plants grown
under a 6-d irrigation interval exhibited a similar response as plants subject to the driest
regime. Finally, seedlings grown under the warmest, driest conditions had greater (p =
0.0268 and p = 0.0034) belowground biomass and R:S (Figure 3.9., 3.10.).
Discussion
In general, the imposed average April temperatures and moisture availability regimes
applied in this study were sufficient for survival. This suggests that following
germination, S. munroana plants are relatively resilient to temperature increases and
moisture fluctuations. However, short-term resilience is not necessarily indicative of
long-term persistence. It is possible that survivorship may differ during the course of an
entire growing season, because development was influenced by temperature differences.
Due to the brevity of the period suitable for growth in the Great Basin, plants must
establish adequate root systems early in the season as available water shifts downwards in
the soil profile in order to ensure survival prior to entering mid-summer dormancy
(Fernadez and Caldwell, 1975; Sturges, 1977). Thus, further research, focused on the
relation of seedling growth rates early in the season and their relation to subsequent
phenology and survival, is necessary.
61
Plants grown under the 17/3 °C regime developed slower, producing roughly half the
number of true leaves as those grown at 23/9 °C. This combination of reduced leaf
number and leaf area resulted in 29% less biomass production. Moisture availability
alone did not elicit substantial effects on plant growth, but the combined influence of
warmer temperature and limited moisture caused a predicted decline in leaf area.
Decreases in turgor have been linked to reduced aboveground biomass production and
overall growth rates in a number of species (Taiz and Zeigler, 2006). For example, Smith
and Nobel (1978) report a direct link between leaf size and leaf water potential in a Great
Basin perennial, Encelia farionsa. In my study, seedlings exposed to the warmest, driest
regime produced less aboveground biomass than plants brought to field capacity every 3
and 9 d. Thus, plants were able to curtail moisture demands by reducing their
transpirational surface area, as opposed to decreasing stomatal conductance. However,
this response may be a result of low night air temperatures that prevent a full reduction in
stomatal opening during the day (Anderson and McNaughton, 1973).
I saw a 28% reduction in biomass for seedlings grown at 17/3 °C compared with their
warmer cohorts which is analogous to the results reported for the native bunch grass,
Agropyron spicatum at temperatures below 8 °C (Harris, 1967; Harris and Wilson, 1970).
The capacity to initiate root extension at low temperatures can, however, offer an
advantage in the Great Basin. For example, the competitive abilities of the invasive
Bromus tectroum have been largely attributed to the species capability to maintain high
levels of belowground production at temperatures below 3 °C (Harris, 1967; Harris and
62
Wilson, 1970). Thus, if restoration goals require direct competition with Bromus
tectorum early in the growing season, the use of S. munroana alone may not be optimal.
The decline in moisture availability encouraged root production in the 2 most waterlimited treatments (9 and 12-d irrigation interval). Higher temperatures amplified the
effects of moisture deficit, inducing the highest belowground production under the
warmest, driest conditions. When considering water availability alone, the most moisturelimited seedlings produced the highest R:S, reflecting greater resource partitioning to
belowground production. R:S ranged from 0.68 to 1.17, which correspond to those
reported for a number of mature Great Basin perennials (Barbour, 1973; Caldwell et al.,
1977). The imposed environmental conditions did influence the rates of gas exchange. In
general, photosynthetic rates ranged from 7.98 to 13.18 mol CO2 m-2s-1 from the warmest,
driest to the coolest, wettest conditions. These rates are similar to those observed in
several Great Basin woody perennials, suggesting the existence of physiological
similarities between the 2 groups (Depuit and Caldwell, 1975; Caldwell, et al., 1977).
Conductance conveyed a similar relationship, ranging from 0.096 mol m-2s-1 under the
warmest, driest treatment to 0.208 mol m-2s-1 in the coolest most frequently saturated
environment. An identical correlation was observed with regard to transpiration, which
varied from 0.0019 to 0.0040 (mmol m-2 s-1).
Conclusions
Under the tested conditions, temperature was the single largest driver of plant behavior.
Although moisture limitations are likely to become more pronounced later in the growing
63
season, moisture alone did not strongly influence the physiological and morphological
response of S. munroana during initial establishment. Low temperatures impeded plant
growth, presumably through the reduction in belowground biomass. Root growth was
driven by moisture availability and temperature, but under cool edaphic conditions the
temperature influence tended to supersede the role of moisture. The warmest, driest
conditions elicited a reduction in aboveground biomass and an increase in belowground
production, without affecting gas exchange rates. This implies that seedlings of S.
munroana are reasonably drought tolerant even during early development. Further
research is needed in order to evaluate in situ plant responses to environmental conditions
during early establishment. Because my results indicate that cool night temperatures pose
a stricter growth limitation than moisture, a later sowing date (which corresponds more
closely with the 23/9 °C regime) may optimize S. munroana establishment.
64
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66
Table 3.1. Sphaeralcea munroana seed collection sites.
Location
Elevation
Collection
(m)
Date
N 43° 45.799’ W 117° 07.825’
779
04 June 2010
N 43° 46.156’ W 117° 19.090’
749
06 June 2010
N 43° 13.010’ W 119° 00.267’
1302
20 June 2010
N 43° 47.356’ W 117° 37.859’
899
22 June 2010
N 43° 52.827’ W 116° 47.030’
811
08 July 2010
State
OR
OR
OR
OR
ID
Table 3.2. Results of the mixed model analysis for the effects of temperature, moisture, and their
interaction on the morphological and physiological responses of Sphaeralcea munroana seedlings 35 d
after sowing.
Temperature × Moisture
Source of Variation
Temperature
Moisture
p
p
p
Mortality
True Leaves
Leaf Area
Aboveground Biomass
Belowground Biomass
Root:Shoot
Photosynthesis
Transpiration
Conductance
α = 0.05
0.2202
0.0052
0.0005
0.0013
0.0062
0.9416
0.0649
0.1450
0.2872
0.2948
0.0817
0.1370
0.2878
0.0127
0.0317
0.5699
0.1041
0.0706
0.6515
0.0915
0.0026
0.0140
0.0268
0.0034
0.8260
0.9977
0.8899
67
5
True Leaves (number)
4
B
3
2
A
1
0
17/9 °C
23/9 °C
Diurnal Temperature Regime
Figure 3.1. Number of true leaves (mean±s.e.) 35 d after sowing, produced
by Sphaeralcea munroana seedlings grown under 2 temperature regimes.
Each bar represents the mean response of 5 replicates. Different letters
indicate significant (p < 0.005) differences between treatments.
68
3.0
B
2.5
2
Leaf Area (cm )
2.0
1.5
1.0
A
0.5
0.0
17/3 °C
23/9 °C
Diurnal Temperature Regime
Figure 3.2. Total leaf area (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature regimes. Each
bar represents the mean response of 5 replicates. Different letters indicate
significant (p < 0.005) differences between treatments.
69
0.020
Aboveground Biomass (g)
B
0.015
0.010
A
0.005
0.000
17/3 °C
23/9 °C
Diurnal Temperature Regime
Figure 3.3. Aboveground biomass (mean±s.e.) 35 d after sowing, produced
by Sphaeralcea munroana seedlings grown under 2 temperature regimes.
Each bar represents the mean response of 5 replicates. Different letters
indicate significant (p < 0.005) differences between treatments.
70
0.020
Belowground Biomass (g)
B
0.015
0.010
0.005
A
0.000
17/3 °C
23/9 °C
Diurnal Temperature Regime
Figure 3.4. Belowground biomass (mean±s.e.) 35 d after sowing, produced
by Sphaeralcea munroana seedlings grown under 2 temperature regimes.
Each bar represents the mean response of 5 replicates. Different letters
indicate significant (p < 0.005) differences between treatments.
71
Figure 3.5. Belowground biomass (mean±s.e.) 35 d after sowing, produced
by Sphaeralcea munroana seedlings grown under 4 irrigation regimes. Each
bar represents the mean response of 5 replicates. Different letters indicate
significant (p < 0.005) differences among treatments.
72
Figure 3.6. Root-to-shoot ratios (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 4 irrigation regimes. Each
bar represents the mean response of 5 replicates. Different letters indicate
significant (p < 0.005) differences among treatments.
73
Figure 3.7. Total leaf area (mean±s.e.) 35 d after sowing, produced by
Sphaeralcea munroana seedlings grown under 2 temperature and 4
irrigation regimes. Each bar represents the mean response of 5 replicates.
Different letters indicate significant (p < 0.005) differences among
treatments.
74
Figure 3.8. Aboveground biomass (mean±s.e.) 35 d after sowing, produced
by Sphaeralcea munroana seedlings grown under 2 temperature and 4
irrigation regimes. Each bar represents the mean response of 5 replicates.
Different letters indicate significant (p < 0.005) differences among
treatments.
75
Figure 3.9. Belowground biomass (mean±s.e.) 35 d after sowing, produced
by Sphaeralcea munroana seedlings grown under 2 temperature and 4
irrigation regimes. Each bar represents the mean response of 5 replicates.
Different letters indicate significant (p < 0.005) differences among
treatments.
76
Figure 3.10. Root-to-shoot ratios (mean±s.e.) 35 d after sowing, produced
by Sphaeralcea munroana seedlings grown under 2 temperature and 4
irrigation regimes. Each bar represents the mean response of 5 replicates.
Different letters indicate significant (p < 0.005) differences among
treatments.
77
Appendix A
-1 -1
Photosynthetic Rate (mol CO2m s )
25
20
15
10
5
0
-5
0
500
1000
1500
2000
2500
-2 -1
Photon Flux Density (mol quanta m s )
Figure 3.11. Light response curves of photosynthetic carbon fixation
measured on individual leaves of 5 mature Sphaeralcea munroana plants at
the University of Idaho Shattuck Arboretum. Measurements were made
between 1000 and 1300 on 02 October 2011 using an LI-6400 Portable
Photosynthesis System (Li-Cor, Inc., Lincoln, NE). Constant chamber CO 2
(400 μmol mol-1), temperature (20±0.16 °C), and relative humidity
(79±0.58%) were maintained to avoid confounding the assimilation response.
The initial leaf equilibration was set to high photon flux density (2000 μmol
quanta m-2s-1), followed by subsequent light decreases in steps of 200 μmol
quanta m-2s-1, until a negative photosynthetic rate was reached. Each point
represents a mean±s.e. of 5 replicates.
78
Appendix B
Table 3.1. Sphaeralcea munroana gas exchange responses to 2 temperature and 4 moisture regimes 35 d
after sowing.
Photosynthesis
Conductance
Transpiration
(mol CO2 m-2s-1)
(mol m-2s-1)
(mmol m-2s-1)
17/3 °C
23/9 °C
17/3 °C
23/9 °C
17/3 °C
23/9 °C
Moisture
13.12±1.27
8.90±0.65
0.21±0.02
0.18±0.03
0.004±0.0006
0.003±0.0003
3 (d)
13.18±1.75
8.23±0.68
0.19±0.03
0.15±0.02
0.004±0.0007
0.003±0.0003
6 (d)
12.27±1.44
9.07±0.86
0.19±0.03
0.16±0.02
0.004±0.0006
0.003±0.0003
9 (d)
10.95±1.86
7.97±0.85
0.16±0.03
0.10±0.015
0.003±0.0006
0.002±0.0003
12 (d)
None of the values are significantly different between or within columns.