CONE SEROTINY AND SEED VIABILITY OF FIRE-PRONE CALIFORNIA CUPRESSUS SPECIESCUPRESSUS SPECIES
In Partial Fulfillment
Masters of Science
CUPRESSUS SPECIES
Fire-prone interior California Cupressus (cypress) species have been experiencing
low or zero seedling recruitment possibly due to decades of fire exclusion, subsequent
encroachment of shade-tolerant conifers, and unknown stand responses to different fire
severities. This study investigated the specific heating conditions required to break cone
serotiny and to promote seed dispersal by focusing on five Cupressus species of interior
California most prone to fire: Cupressus arizonica ssp. nevadensis (Piute cypress); C.
bakeri (Baker cypress); C. forbesii (Tecate cypress); C. macnabiana (McNab cypress);
and C. sargentii (Sargent cypress). A muffle furnace was used to conduct eight
temperature treatments of 250 - 700 o C, ranging in duration from 30 seconds to 5 minutes
of exposure to cones of each species. The heat-released seeds were tested for viability
using a tetrazolium red stain. Logistic regression analysis of seed viability indicated that
the duration of heating alone was highly significant (P < 0.005) for all species, regardless
of temperature. Models predicting seed viability reflected species differences in
geographic range and habitat requirements. Species comparisons revealed that C.
arizonica ssp. nevadensis and C. forbesii shared the same model for predicting seed
viability, while C. macnabiana and C. sargentii shared a different model, but C. bakeri
had a separate model. In addition, factors related to tree age and cone position on the tree
were investigated in C. sargentii. Neither factor affected seed viability. This is an
iv
important finding with regard to management in that older stands of C. sargentii may not
experience fire for many decades but still produce viable seed.
v
ACKNOWLEDGEMENTS
Funding for this project was provided by the USDA McIntire-Stennis Forestry Research
Program. Many people helped shape this project and provided valuable guidance along
the way. My advisor Dr. John Stuart remained a constant supporter and advocate while
being unafraid to ask the tough questions. He was always generous with his time and
attention, and set an example of professional decorum to which I will continually aspire.
Dr. Morgan Varner helped me to become a better scientific writer and critical thinker,
and inspired me to go further and higher with his energy and enthusiasm. Dr. Chris Edgar
generously shared his expertise and time, while helping me to become more
knowledgeable and confident with statistical analysis. Kyle Merriam greatly helped me
define my questions and focus of study in the context of land manager’s goals and
applicability to fire and vegetation management. Erin Rentz also helped me define my
methods and was always willing to answer my questions. Darrell Burlison and George
Pease were very helpful and generous with lab and field equipment needs. Gayleen Smith
was always very supportive and helpful with administrative needs.
vi
CHAPTER 1: Seed Viability and Fire-related Temperature Regimes in Interior
California Native Cupressus Species ...................................................................................1
INTRODUCTION ...............................................................................................................1
METHODS ..........................................................................................................................7
Field Data Collection ...................................................................................................... 7 Heat Treatments ............................................................................................................ 12 Seed Viability Tests ...................................................................................................... 13
Statistical Analysis ........................................................................................................ 16
RESULTS ..........................................................................................................................18
DISCUSSION ....................................................................................................................33
REFERENCES ..................................................................................................................40
CHAPTER 2: Tree Age, Cone Age and Seed Viability in Cupressus Sargentii
(Sargent Cypress) ...............................................................................................................50
CHAPTER 1
Table Page
1 Comparison of time (days) until control cones opened, number of cones open
and seed release data for all five species studied. .....................................................21
2 Percent germination of all five Cupressus species following heat treatments of
250-700° C for durations of 0.5-5 minutes. Each treatment consisted of 25
seeds. .........................................................................................................................23
3 Percent seed viability determined with tertazolium stain of all five Cupressus
species studied following heat treatments of 250-700° C for durations of 0.5-5
minutes. Each treatment consisted of 25 seeds. ........................................................25
4 Logistic models fitted for all five Cupressus species in the study along with
measures of deviance of the model terms and correct classification (%) of
predicted model outputs. The best-fitting models are designated by the
associated deviance in bold. ......................................................................................28
5 Logistic models fitted for the species interactions of the five Cupressus species
in the study along with measures of deviance of the model terms and correct
classification (%) of predicted model outputs. The best-fitting models are
designated by the associated deviance in bold. .........................................................29
CHAPTER 2
1 R 2 and P-values from a series of simple linear regression analyses for seed viability
of Cupressus sargentii (Sargent cypress) as a function of cone whorl age……….59
viii
CHAPTER 1
Figure Page
1 Range map of all five native California Cupressus species used in the study.
Circles indicate the sampling locations; C. macnabiana and C. sargentii were
collected at the same site in Lake County, California. ...............................................9
2 An example of a grove of Cupressus bakeri (Baker cypress) mixed with Pinus
jeffreyi (Jeffery pine) above Seiad Creek, Klamath National Forest, Siskiyou
County, California.....................................................................................................10
3 Cupressus arizonica ssp. nevadensis (Piute cypress) branch showing the typical
sequence of cones, 3 years and older (note the gray scale color) that was
collected from each tree. Sequoia National Forest, Tulare County, California. .......11
4 Cupressus sargentii (Sargent cypress) seeds cut longitudinally, showing the four
categories of observation: full stain of embryo (a); incomplete stain of embryo
(b); unstained embryo (c); and embryo absent (d). For all species studied, only
(a) was considered viable. .........................................................................................15
5 The cumulative proportion of seeds released immediately following heat
treatment (day 0), and at 4 and 8 days, for Cupressus bakeri (Baker cypress) and
all temperature and time combination treatments. The x-axis is temperature (°
C), the y-axis is time (min), and the z-axis is the proportion of seeds released. ......19
6 The cumulative proportion of seeds released immediately following heat
treatment (day 0), and at 4 and 8 days, for Cupressus forbesii (Tecate cypress)
and all temperature and time combination treatments. The x-axis is temperature
(° C), the y-axis is time (min), and the z-axis is the proportion of seeds released. ..20
7 Cumulative germination (%) of four Cupressus species for 250° C at 1 minute
of heat exposure for the number of days since the germination trial began.
Cupressus macnabiana was not included because no seeds germinated. .................24
8 Probability of seed viability of five serotinous California Cupressus species as a
function of heating time (model t) and temperature (model T) separately. ..............30
ix
Figure Page
9 Probability of seed viability of the best-fitting combined final models of five
serotinous California Cupressus species as a function of heating temperature,
for 0.5, 1, and 3 minutes of heating exposure time respectively. .............................31
10 Probability of seed viability of the best-fitting combined final models of five
serotinous California Cupressus species as a function of heating time at
temperatures of 300, 400, and 500° C respectively. .................................................32
CHAPTER 2
1 Location of Cupressus sargentii (Sargent cypress) study sites, located in the BLM
Knoxville Recreation Area, Lake and Napa Counties, California…………………52
2 Cone whorls on a Cupressus sargentii (Sargent cypress) branch. Cone position is
assumed to be correlated with cone age……………………………………………55
3 Sargent cypress seeds cut longitudinally, showing the four categories of
observation: full stain of embryo (a); incomplete stain of embryo (b); unstained
embryo (c); and embryo absent (d). Only seeds (a) were categorized as
viable.………………………………………………………………………………56
4 A best fit linear model of Cupressus sargentii (Sargent cypress) seed viability (%)
and tree age (years) for all trees and cone whorls sampled……………………......58
5 A best fit linear model of seed viability (%) and tree age (years) for Cupressus
sargentii (Sargent cypress) cone whorls 1 and 2…………………………………..60
6 A best fit linear model of seed viability (%) and tree age (years) for Cupressus
sargentii (Sargent cypress) cone whorls 3 and 4…………………………………..61
x
A Measured temperatures and exposure time during burning of Cupressus
macnabiana (McNab cypress) branches, for a pilot study. ..................................... 43
B The cumulative proportion of seeds released immediately following heat
treatment (day 0), at 4 and at 8 days, for Cupressus macnabiana (McNab
cypress) and all temperature and time combination treatments. The x-axis is
temperature ( o C), the y-axis is time (min), and the z-axis is the proportion of
seeds released. ...........................................................................................................44
C The cumulative proportion of seeds released immediately following heat
treatment (day 0), at 4 and at 8 days, for Cupressus sargentii (Sargent cypress)
and all temperature and time combination treatments. The x-axis is temperature
( o C), the y-axis is time (min), and the z-axis is the proportion of seeds released. ...45
D The cumulative proportion of seeds released immediately following heat
treatment (day 0), at 4 and at 8 days, for Cupressus arizonica spp. nevadensis
(Piute cypress) and all temperature and time combination treatments. The x-axis
is temperature ( o C), the y-axis is time (min), and the z-axis is the proportion of
seeds released. ...........................................................................................................46
E Cupressus macnabiana (McNab cypress) seedlings from a fire in 1999, located
in the BLM Knoxville Recreation Area, Lake and Napa Counties, California. .......47
F Cupressus macnabiana (McNab cypress) seedlings one year after the Walker
Fire in 2009, located in the BLM Knoxville Recreation Area, Lake County,
California. .................................................................................................................48
G Cupressus sargentii (Sargent cypress) seedlings in the foreground growing near
an ephemeral drainage with no evidence of recent fire, located in the BLM
Knoxville Recreation Area, Napa County, California. .............................................49
1
Cupressus Species
Fire is an important and fundamental ecological process in ecosystems
characterized by a Mediterranean climate. A long history of recurring fire has been the
primary agent of change in the vegetation composition and structure commonly
associated with this climate regime (Bond & Keeley 2005a). Many conifer species long
associated with these climates have developed reproductive adaptations to the specific
fire intensities, timing and return intervals characteristic of Mediterranean ecosystems
(Pausas et al. 2004). Cone serotiny in particular is an adaptation shared by some species
in the genera Pinus (Pine) and Cupressus (Cypress), and is indicative of a long
association with recurring fire (McMaster & Zedler 1981; Zedler 1986).
Cone serotiny entails the retention of a canopy seedbank within persistent cones
usually until conditions, such as growing space or light availability, are favorable for
regeneration (Lamont et al. 1991). In many serotinous species of North America, heat is
required for the cones to open and release seeds (Lamont et al. 1991; Vogl et al. 1977;
Zedler 1986), though the degree and mechanism of serotiny varies by species (Harvey et
al. 1980; Vogl 1973), and sometimes by population (Mallek 2009; McMaster & Zedler
1981). The degree and mechanisms of cone serotiny, and role of heat to stand
regeneration of Cupressus species of North America, and specifically California, is
largely unknown.
Native Cupressus species of California have highly restricted native ranges
(McMillan 1956; Vogl et al. 1977; Zedler 1977) and are usually dependent on fire for
regeneration. Cupressus species consist of mostly fire-dependent species that produce
serotinous cones requiring fire to open cone scales so that seed can be dispersed (Bartel
1993; Wolf 1948). Once released, the seeds germinate best with direct sunlight on
exposed mineral soil (Vogl et al. 1977). Cupressus species are often associated with
harsh, dry sites subject to extreme temperature fluctuations, and can inhabit serpentine
(some are serpentine indicators), volcanic, or granitic substrates at elevations ranging
from 300-2100 m (Stuart & Sawyer 2001). Due to decades of fire suppression and the
resulting increase in fuel loads, however, some of these scattered and remote Cupressus
populations are under threat from a lack of fire which may allow shade tolerant species to
replace them and to eventually become self-replicating canopy dominants. Other
Cupressus populations are threatened by extirpation from too-frequent stand-replacing
fires as a result of anthropogenic ignitions.
This study examined the five Cupressus species most susceptible to fire due to
their location and habitat: Cupressus arizonica ssp. nevadensis (Piute cypress), C. bakeri
(Baker cypress), C. forbesii (Tecate cypress), C. macnabiana (McNab cypress), and C.
sargentii (Sargent cypress). Both of the southern California species, C. arizonica ssp.
nevadensis and C. forbesii, are listed in the Jepson Manual (Bartel 1993) as rare and
under threat from frequent fire and from development, C. bakeri is listed as
uncommon, and C. sargentii and C. macnabiana are recognized as the most common
Cupressus species in California. Cupressus sargentii and C. macnabiana are found in
3
mostly small, widely scattered populations in Northern California, from the coast ranges
to parts of the northern Sierra Nevada for C. macnabiana (Bartel 1993).
After decades of fire exclusion, some interior California Cupressus species face
the paradox of wildfire as being deleterious to their perpetuation and, at the same time,
fire being necessary to open serotinous cone scales and to prepare receptive seed beds. If
high intensity fires occur too frequently, fire-dependent Cupressus species become
susceptible to an immaturity risk where young trees are killed before reaching
reproductive age (Keeley & Fotheringham 2000; Zedler 1977), which is estimated to be
between 10 and 15 years of age (Bartel 1993). Of particular interest to this study are C.
forbesii and C. arizonica ssp. nevadensis, listed as 1.B.1 (seriously endangered in
California) and 1.B.2 (fairly endangered in California) respectively by the California
Native Plant Society, but neither of which are currently listed for federal or state
protection (CNPS 2008). Previous studies of C. forbesii populations and seed production
have shown that they require fire return intervals longer than 40 years to develop an
adequate canopy seed bank, and are vulnerable to extirpation with fire return intervals
that are substantially less than 40 years (de Gouvenain & Ansary 2006; Zedler 1995). The
Otay Mountain population of C. forbesii in San Diego County (southern California) was
burned in 2003, and narrowly escaped burning again in the fall of 2007. If it had burned
again, the population may have been extirpated due to the lack of mature trees within the
stand.
A different situation faces the northern Cupressus species, particularly C. bakeri
and C. macnabiana. A recent study of C. macnabiana fire history found that fire return
4
intervals of 3 fires per decade did not appear to affect the persistence of C. macnabiana
populations (Mallek 2009). Cupressus bakeri is experiencing increased interspecific
competition and greater stand density in areas that have suffered a lack of fire. Evidence
of poor seedling regeneration and Abies concolor (white fir) out-competing the
established C. bakeri were observed on the Plumas and Lassen National Forests by both
Wolf (1948) and Stone (1965), and later Keeler-Wolf (2004a). Decades of fire exclusion
have left some Cupressus populations facing a risk of cone senescence before fire can
prepare the seedbed resulting in little or no regeneration when fires do occur (Keeley &
Fotheringham 2000). Therefore, C. bakeri populations may be at risk from wildfire or
inappropriate prescribed fire due to altered stand conditions.
Prescribed fire is recognized as an essential tool in restoring natural fire cycles to
historically fire-dependent ecosystems whose fire return intervals are disrupted to such an
extent that a severe fire may irrevocably alter the vegetation composition. Knowing the
type of fire behavior and prescribed fire that will result in Cupressus regeneration is
imperative. A critical step in developing burning prescriptions for Cupressus species is to
determine the temperature regimes required to break cone serotiny and to allow
subsequent seed germination. The primary goal of this study was to evaluate the heat
tolerance of cones (and their seeds) and the degree of serotiny of inland California
Cupressus species most susceptible to extirpation or ecosystem damage by altered
wildfire regimes.
The literature regarding the mechanism behind Cupressus cones opening in
response to high temperatures appears to be limited to field and lab observations.
5
Ne’eman et al. (1999) and Zedler (1986) claim that Cupressus cones open through a
combination of resin and water loss (though the mechanism has not been tested) and are
therefore prone to desiccation as the trees age or if the branch is severed from the tree.
Vogl et al. (1977) wrote that Cupressus cones open in response to high fire temperatures
melting and boiling the resin, thereby allowing the cone scales to open. A few studies
investigated the amount of heat needed to break cone serotiny in three Spanish Pinus
species. Habrouk et al. (1999) used four different time treatments with four different
temperatures to determine heat loads necessary to break serotiny. Reyes and Casal (2002)
looked at how heat treatments affect seed viability in two species of pines. Johnson and
Gutsell (1993) used heat treatments to interpret the types of fire that produce requisite
heat loads. No published reports appear to have been made on data collected for
Cupressus species in North America. Most of the knowledge regarding Cupressus species
in California has been gained through field observations made by naturalists and field
botanists such as Willis Jepson (Jepson 1923), C.B. Wolf (Wolf 1948) C.O. Stone (Stone
1965) and T. Keeler-Wolf (Keeler-Wolf 2004a, 2004b).
Currently, little information exists on the life history of Cupressus species and the
environmental conditions necessary for regeneration (Mallek 2009; Vogl et al. 1977).
The main focus of this study to determine the specific heating conditions (i.e. temperature
and duration) required to break cone serotiny and promote seed dispersal, while
minimizing seed injury. Study results will provide a better understanding of how
Cupressus regeneration is affected by fire, and wildland fire managers will be better able
to develop burn prescriptions specifically for Cupressus species.
6
This study investigates the role of fire in promoting seedling germination in fire-
prone interior California Cupressus species. In particular, specific study objectives are to
determine: 1) the minimum heat load (i.e. temperature and duration) required for
Cupressus cones to break serotiny; 2) the effect of the heat load in cones on seed
viability; 3) whether individual species respond differently to different heat loads.
7
METHODS
Field Data Collection
Five interior California Cupressus species were sampled over a 3 month period
from June to August in 2008. The study sites were located from the northern end to the
southern end of California (Figure 1). Cones were collected from 18 trees at each site, for
a total of 5 sites and 90 trees sampled (Figure 2). A branch with at least 10 cones was cut
from each tree at eye level. Live branches were collected instead of individual cones to
reduce moisture loss. The branches were transferred to plastic bags and stored in a cooler
containing ice for a maximum of 72 hours in transit to the lab. In the lab, samples were
then transferred to a refrigerator and stored at 3-5° C until the heat treatments
commenced, usually within 48 hours. All live branches were kept moist with wet paper
towels to prevent desiccation and simulate the conditions of exposure of live foliage and
cones to fire.
Cones selected for heat treatments were located near the ends of branches, while
avoiding those on the tips of branches that were brown, indicating immaturity (first to
second year cones). Cones that were gray in color with a peduncle (indicating the cones
were at least 3-5 years old) on each branch were used for the treatments (Figure 3). Cones
closer to the tree than the outer 0.5-0.8 m of the branches were avoided as these were
usually older than 5 years and sometimes invaded by burrowing insects leading to
premature desiccation.
Additionally, seeds were collected from the C. macnabiana collection site in Lake
County, California following the 2008 Walker Fire that occurred in the same population 3
8
weeks after live cone collection. The goal was to see if the seed viability from the post-
fire plots matched the seed viability measured for any specific heat treatment. The seeds
were collected from five ground plots and five canopy plots in August, 2 months after the
fire. Plots were 0.25 m 2 in size and at least 150 seeds were collected from each plot. The
seed from canopy plots was obtained by gently shaking branches with open cones over
0.25 m 2
trays. Seed density was estimated at 600-800 per m 2 from the five ground plots.
These seeds were later tested for seed viability.
9
Figure 1. Range map of all five native California Cupressus species used in the study.
Circles indicate the sampling locations; C. macnabiana and C. sargentii were collected at
the same site in Lake County, California.
10
Figure 2. An example of a grove of Cupressus bakeri (Baker cypress) mixed with Pinus
jeffreyi (Jeffery pine) above Seiad Creek, Klamath National Forest, Siskiyou County,
California.
11
Figure 3. Cupressus arizonica ssp. nevadensis (Piute cypress) branch showing the typical
sequence of cones, 3 years and older (note the gray scale color) that was collected from
each tree. Sequoia National Forest, Tulare County, California.
12
Heat Treatments
A pilot study conducted with C. macnabiana found through a combination of
burning of branches and oven treatments that cones began to open at temperatures of
250° C and above (Appendix A). The branches were burned under laboratory conditions
to measure temperature duration, using insolated iron-constantan (Type J) thermocouples
wrapped around the branch and set next to the cones, and connected to a CR1000
datalogger (Campbell Scientific Inc., Logan, UT, USA). The branches were secured to a
metal rod a few inches above the source of flaming heat (a pile of the same species’ dry
litter and foliage) on a laboratory burn platform under a 3m x 3m exhaust hood. This
technique was later repeated with C. sargentii, C. arizonica ssp. nevadensis and C.
forbesii.
Based on the results of the pilot study, the following heat treatments of 250, 300,
350, 400, 500, 600, 650, and 700° C were used. The time exposure treatments consisted
of 30 seconds, 1, 2, 3, 4, and 5 minutes for a total of 36 time/temperature combination
treatments tested on 900 total cones. Not all combinations of temperature and time were
tested due to the pattern of shorter durations of heat exposure at increased temperatures
found in the pilot study (Appendix A). At the time of treatment, cones were cut from the
branches and then randomly selected, using 5 cones per treatment combination. A control
for each species (no heat treatment) was kept at room temperature on the same starting
day as the other treatments and monitored the same length of time as the treated cones
(35 days). A muffle furnace (Thermolyne Sybron Corporation, Dubuque, Iowa, USA,
13
Model No. F-A1730) with a temperature range of 0 – 1000° C was used for all of the heat
treatments.
Seed Viability Tests
Following the heat testing of the cones, the amount of scale opening (mm) each
day was measured with a set of calipers and the number of seeds released was recorded
for a minimum of 35 days. Observed seed release over time following the treatments for
each species was then graphically analyzed and compared to the control. All collected
released seeds were tested 60 days later for germination. Lots of 25 seeds for each
treatment of each species plus the control were pre-chilled at 3-5° C for 21 days on moist
filter paper in Petri dishes, and then placed in a germination chamber (Stults Scientific
Engr. Corp., Springfield, IL, USA). The seeds were subjected to an alternating
temperature regime of 16 hours at 20° C, and 8 hours at 30° C each day for at least 30
days, following the guidelines for Cupressus species set forth by the Association of
Official Seed Analysts (2008). Germinated seeds were counted every day to quantify
total percent germination for each treatment and species.
After conducting two germination trials (50 total seeds tested per treatment), all
C. macnabiana and C. sargentii seeds failed to germinate. Tetrazolium staining was then
applied to new, untested seeds following the conclusion of germination tests in order to
get a more complete picture of seed viability in all of the Cupressus species, as not all
viable seed will always germinate. The seeds were tested with a 1% tetrazolium red
solution at 30-32° C, for 12-18 hours, following the tetrazolium testing procedures
outlined by the Association of Official Seed Analysts (2001). The stained seeds were cut
14
and visually analyzed for viability based on the staining extent and the condition of the
embryo (Figure 4a-d).
Figure 4. Cupressus sargentii (Sargent cypress) seeds cut longitudinally, showing the
four categories of observation: full stain of embryo (a); incomplete stain of embryo (b);
unstained embryo (c); and embryo absent (d). For all species studied, only (a) was
considered viable.
Statistical Analysis
Logistic regression (Hosmer & Lemeshow 2000) was used to assess the effect of
the heating duration and temperature on the probability of seed viability following heat
treatment, and to determine if these effects differed by species (after Escudero et al. 1999;
Nuñez et al. 2003). The temperature and time of exposure of the heat treatments were
selected as the predictor variables (main effects terms). The entire model which included
temperature, time, the interaction of the two, and independent terms, was tested along
with all reduced models. A species effect term, the main effects terms, and interaction
were included for all models that tested for differences between species. Logistic
relationships are expressed as the following model:
1
p = ———
1 + e -z
where p is the probability of seed viability and z is a linear function containing the
predictor variables included in the model (z = b0 + b1 × temperature + b2 × time + b3 ×
temperature × time). The coefficients of the z function were estimated using the
maximum likelihood function. The models were selected based on the significance of the
variable and the change in deviance, which is the value of the change in the – 2 log
likelihood between the model with and without predictor variables (Hosmer &
Lemeshow 2000). Testing of assumptions and residual diagnostics of the model were
conducting using procedures described by Hosmer and Lemeshow (2000). This included
assessing whether the model met the assumptions that the variables were dichotomous,
the outcomes were statistically independent, the model was correctly specified, and that
17
the categories of viable or not viable were mutually exclusive and collectively
exhaustive. Model fit was assessed by the calculation of percent correctly classified
predicted values from the models, and plotting the residuals of the deviance values. All
statistical analyses were carried out in R, an open source statistical program (R
Development Core Team 2009).
18
RESULTS
Based on qualitative graphical analysis of seed release over time following
treatment, all of the Cupressus species’ cones used in the study had a threshold at 500° C
that resulted in substantial release of seed (> 50 % of total seed release) from cones after
four days, looking at durations of 2 minutes or more (Figures 5 and 6). Cupressus forbesii
had a threshold at 500° C, but only for durations of 4 minutes and longer. Even at 600-
700° C, the proportion of C. forbesii seeds released was low relative to the other species’
cones which appeared to be nearing complete release of seeds 8 days after treatment for a
greater range of treatment combinations (Figures 6). For example, the cones of C.
sargentii and C. macnabiana exposed to 700° C released nearly 100% of their seeds after
four days (Appendices B and C). Cupressus bakeri, the most northerly species, released
more of its seeds than did C. forbesii (the most southerly species in California) at four
and at eight days following heat exposure respectively (Figure 5 and 6). Heat treatments
increased the chance and speed of cone opening and seeds released compared to control
treatments. Across species, untreated cones did not begin to open until at least 21 days
(Table 1). Even though the control for C. bakeri had the shortest time (21 days) before
the cones opened (at least 1 mm), it was approximately five more days before seeds were
released. In contrast, all C. forbesii cones and most of C. arizonica ssp. nevadensis failed
to open after 40 days (the end of the experiment period).
1 9
Day 0 Day 4 Day 8
Figure 5. The cumulative proportion of seeds released immediately following heat treatment (day 0), and at 4 and 8 days, for
Cupressus bakeri (Baker cypress) and all temperature and time combination treatments. The x-axis is temperature (° C), the y-axis is
time (min), and the z-axis is the proportion of seeds released.
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
1
2
3
4
5
Day 0 Day 4 Day 8
Figure 6. The cumulative proportion of seeds released immediately following heat treatment (day 0), and at 4 and 8 days, for
Cupressus forbesii (Tecate cypress) and all temperature and time combination treatments. The x-axis is temperature (° C), the y-axis is
time (min), and the z-axis is the proportion of seeds released.
0.5
2
4
0
0.05
0.1
0.15
0.2
0.25
0.3
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
1
2
3
4
5
21
Table 1. Comparison of time (days) until control cones opened, number of cones open
and seed release data for all five species studied.
Cypress Species
Cupressus arizonica ssp. nevadensis > 40 1 0
Cupressus forbesii > 40 0 0
22
Germination results revealed three important overall trends related to duration and
temperature of exposure to heat, and timing of germination. Seeds exposed to higher
temperatures (400° C and above) had greater germination at the shortest exposure periods
of 0.5 and 1 minute, with no germination in seeds exposed more than 2 minutes (Table
2). There was a higher percentage of seed germination for all species at the lower
temperatures than at the higher temperatures for low exposure times (Table 2). Cupressus
forbesii and C. arizonica ssp. nevadensis (the southern-most species) germination began
to occur at day 7, while C. sargentii and C. bakeri (more northern species) germination
began later, at day 14 (Figure 7). Within the first 30 days of the germination trial, the C.
forbesii control had the highest germination capacity of 36%, compared to only 8% for
the C. bakeri control, and no seeds germinated from the other species (Table 2).
Cupressus sargentii had a slow germination response, with a few seeds germinating after
30 days of treatment. Because of very low germination rates with C. macnabiana and C.
sargentii, the germination trials were complemented with a seed viability trial using
tetrazolium staining. Seed viability results obtained with tetrazolium staining yielded
more complete results than the germination trials for all five species (Table 3).
Cupressus macnabiana seeds from the Walker Fire area had an average viability
of 16.8% for the ground plots and an average viability of 12.8% for the canopy plots. The
seeds collected from the ground plots had a range of 12 - 20% seed viability, while the
canopy plots had a range of 0 - 28% seed viability.
23
Table 2. Percent germination of all five Cupressus species following heat treatments of
250-700° C for durations of 0.5-5 minutes. Each treatment consisted of 25 seeds.
Cupressus
bakeri
Cupressus
macnabiana
Cupressus
sargentii
250° C
300° C
350° C
400° C
500° C
600° C
650° C
700° C
2 min 0 0 0
24
Figure 7. Cumulative germination (%) of four Cupressus species for 250° C at 1 minute
of heat exposure for the number of days since the germination trial began. Cupressus
macnabiana was not included because no seeds germinated.
0
5
10
15
20
25
% G
25
Table 3. Percent seed viability determined with tertazolium stain of all five Cupressus
species studied following heat treatments of 250-700° C for durations of 0.5-5 minutes.
Each treatment consisted of 25 seeds.
Cupressus
bakeri
Cupressus
macnabiana
Cupressus
sargentii
250° C
300° C
350° C
400° C
500° C
600° C
650° C
700° C
2 min 0 0 0
26
For all species, the addition of the main effects terms of temperature and time
significantly reduced the amount of deviance in the models compared to the model with
just a constant term (Tables 4 and 5). In the logistic regression analysis of heat and time
treatment effects (model T, t), time was a highly significant (P < 0.005) main effect on
the probability of viability for all species tested (Table 4). For exposure times longer than
2 minutes, the probability of seed viability was very low (0.01-0.05) for C. arizonica ssp.
nevadensis, C. bakeri, and C. forbesii and approached zero at longer exposures (Figure
8). The exceptions of C. macnabiana and C. sargentii, which still had some predicted
seed viability at 5 minutes of exposure, had predicted probabilities of 0.03 and 0.02
respectively (Figure 8).
The simple model looking at the relationship between seed viability and
temperature (model T) was significant for all species except for C. arizonica ssp.
nevadensis (Table 4). Predicted C. macnabiana seed viability decreased from a maximum
of 0.12 to 0.04 between 250 and 400° C respectively (Figure 8). Cupressus forbesii, C.
arizonica ssp. nevadensis, and C. sargentii all followed a similar and gradual declining
pattern in probability of seed viability as temperature increased (Figure 8). Cupressus
bakeri was the most sensitive to higher temperatures with predicted probability of seed
viability decreasing from 0.02 to 0.01 between 250 and 400° C (Figure 8). Cupressus
bakeri also displayed a much lower probability of seed viability overall, with a maximum
predicted viability of 0.05 at 0.5 minutes and then decreasing to 0.01 at 2 minutes (Figure
8). The model with temperature and time main effects and their interaction (model T, t,
27
T*t) was significant for C. forbesii and C. arizonica ssp. nevadensis, but not for any of
the other species (Table 4).
Species comparisons were generated by combining viability data of two different
species and designating a species indicator variable to differentiate between them.
Looking at the best fitting models that were selected, three important differences were
apparent. First, the species main effects term was not significant for C. macnabiana and
C. sargentii, presumably because their seed viability responded similarly enough to
temperature and time of exposure that the same model could be used for both species
(Table 5). Second, the situation was the same for C. forbesii and C. arizonica ssp.
nevadensis. However, C. bakeri differed from C. macnabiana and C. forbesii (Table 5),
so the original model (T,t) was used (Table 4). Third, the predicted probability of
viability of C. bakeri was low compared to either the combined model for C. forbesii and
C. arizonica ssp. nevadensis or the combined model for C. macnabiana and C. sargentii
across all temperatures for specific durations (Figure 9). Cupressus bakeri also had low
predicted seed viability across all times of exposure for specific temperatures, except at
300° C (Figure 10).
All of the models predicted probabilities of seed viability less than 0.3 for all
species, as can be seen in the plots of model predicted probability against the variables of
time and temperature (Figures 8 – 10). The results of the classification tables, where the
percent correctly classified probabilities of the predicted model was compared to the
percent of the observations, were all greater than 95 % (Tables 4 and 5).
28
Table 4. Logistic models fitted for all five Cupressus species in the study along with
measures of deviance of the model terms and correct classification (%) of predicted
model outputs. The best-fitting models are designated by the associated deviance in bold.
Species Model Deviance
t 22.781 98.7
t 71.457 95.7
t 45.798 96.6
ssp. nevadensis t 24.163 97.6
T, t 11.885 97.6
t 58.209 96.5
T, t, T*t 24.089 96.5
Key: T = Temperature, t = time, model terms in italics indicate not significant using a
probability level of 0.05, deviance values in bold indicate selected models
29
Table 5. Logistic models fitted for the species interactions of the five Cupressus species
in the study along with measures of deviance of the model terms and correct
classification (%) of predicted model outputs. The best-fitting models are designated by
the associated deviance in bold.
Species Model Deviance
and Cupressus t 117.910 97.2
macnabiana T, t 63.505 97.2
T, t, T*t 59.249 97.2
T, t, S, T*t 42.979 97.2
T, t, S, T*t, T*S 40.774 97.2
T, t, S, T*t, t*S 35.832 97.2
T, t, S, T*t, t*S, T*S 35.743 97.2
Cupressus sargentii T 134.96 102.700 96.0
and Cupressus t 118.560 96.0
macnabiana T, t 56.059 96.0
T, t, T*t 53.878 96.0
T, t, S, T*t 52.868 96.0
T, t, S, T*t, T*S 50.895 96.0
T, t, S, T*t, t*S 52.459 96.0
T, t, S, T*t, t*S, T*S 50.885 96.0
Cupressus arizonica T 127.091 118.550 97.0
and Cupressus forbesii t 84.933 97.0
T, t 47.541 97.0
T, t, S, T*t 33.379 97.0
T, t, S, T*t, T*S 32.460 97.0
T, t, S, T*t, t*S 32.454 97.0
T, t, S, T*t, t*S, T*S 32.175 97.0
Cupressus bakeri T 125.389 115.230 97.5
and Cupressus forbesii t 91.495 97.5
T, t 54.864 97.5
T, t, S, T*t 34.725 97.5
T, t, S, T*t, T*S 34.621 97.5
T, t, S, T*t, t*S 33.223 97.5
T, t, S, T*t, t*S, T*S 33.054 97.5
Key: T = Temperature, t = time, S = species term, model terms in italics indicate not significant
using a probability level of 0.05, deviance values in bold indicate selected model.
3 0
Figure 8. Probability of seed viability of five serotinous California Cupressus species as a function of heating time (model t) and
temperature (model T) separately.
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
f s e
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
3 1
Figure 9. Probability of seed viability of the best-fitting combined final models of five serotinous California Cupressus species as a
function of heating temperature, for 0.5, 1, and 3 minutes of heating exposure time respectively.
200 300 400 500 600 700
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
0 0
.2 5
0 .3
f s e
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
0 0
.2 5
0 .3
f s e
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
0 0
.2 5
0 .3
f s e
3 2
Figure 10. Probability of seed viability of the best-fitting combined final models of five serotinous California Cupressus species as a
function of heating time at temperatures of 300, 400, and 500° C respectively.
0 1 2 3 4 5
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
0 0
.2 5
300 C
Time (min)
P ro
b a
f s e
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
0 0
.2 5
400 C
Time (min)
P ro
b a
f s e
0 .0
0 0
.0 5
0 .1
0 0
.1 5
0 .2
0 0
.2 5
500 C
Time (min)
P ro
b a
f s e
Results show that interior California Cupressus species have an optimal
temperature range to open their cone scales, release seeds, and maintain seed viability.
There appears to be a delicate balance between heat and exposure time for stimulating
seed release while maintaining viable seed. High proportions of seed are more frequently
released at higher temperatures and at greater exposure times, for example in the case of
700° C for 0.5, 1, and 2 minutes exposure time, very high proportions of seed were
released within 4 days (Appendix B-D), yet for nearly all species, seed viability was zero
at this temperature (Table 3). This same pattern also held for exposure times longer than
3 minutes at lower temperatures (250-400° C). Seed viability and cone serotiny of all
species were more sensitive to higher temperatures and durations of exposure, but the
degree differed by species.
There were some distinctive differences between the northern and southern
species cone responses to heat exposure and the ability of seeds to germinate. Cupressus
forbesii cones were more resistant to breaking serotiny, but were more likely to germinate
within one week of placement in the germinators. Cupressus bakeri cone serotiny was
easier to break at lower temperatures, but the seeds were less likely to germinate within
two weeks. These trends make sense given the different climates, including the onset of
precipitation and temperature regime, where these two species occur. Cupressus bakeri is
typically found at elevations of 1100-2200 m in mixed conifer forests of the mountain
ranges of northern California (Vogl et al. 1977) where it is subject to a persistent winter
snow pack. Cupressus forbesii is found at elevations of 450-1500 m growing in
34
association with chaparral in the western Peninsular Ranges of southern California
(Bartel 1993), where the onset of winter conditions is later and less severe. This pattern
highlights the findings of differences in cone serotiny between species based upon their
habitat and geographic range.
A very low to zero germination rate has been consistently found for both C.
macnabiana and C. sargentii across different studies (Ceccherini et al. 1998; McMillan
1956). However, observations in burned C. macnabiana stands (Appendix E and F) and
unburned C. sargentii stands (Appendix G) suggest that natural germination does occur
in high numbers. Cupressus macnabiana released between 600 and 800 seeds/m 2 after
the Walker fire, suggesting that the effect of a mass release of seed probably makes up
for the low germination ability. The lack of seed germination response in C. macnabiana
and C. sargentii also suggests that more germination trials need to be conducted under
different conditions. Cupressus macnabiana and C. sargentii may have different
germination requirements from the other Cupressus species studied, such as a diurnal
photoperiod, or different levels of moisture or temperatures related to climate differences,
consistent with where they occur.
The species model comparisons generated with logistic regression revealed that
C. macnabiana and C. sargentii had comparable models in predicting seed viability. The
similarities may be the result of growing in the same or similar environments where they
developed the same response to heat exposure. Second, C. arizonica ssp. nevadensis and
C. forbesii also had comparable models, which suggest that they have similar patterns in
serotiny and seed germination given that they are both southern California species that
35
grow in similar habitats. Less is known however, about the type of fire typical in the
native habitat of C. arizonica ssp. nevadensis. Finally, the model for C. bakeri was not
comparable to C. forbesii, which was expected given their very different responses to the
heat treatments and their different habitats. C. bakeri and C. macnabiana were also not
comparable, which may be caused by differences in habitat (C. macnabiana is also
associated with chaparral), even though there are known populations in close proximity
in the northern Sierra Nevada (Griffin & Critchfield 1972).
The species models predicted low seed viability across the exposure time and
temperature treatments, in all cases lower than what was observed, resulting in high
percentages (95-99%) of correctly classified outcomes for each model. The high
percentages are unsurprising given that out of 900 tested seeds (observations) for each
species, the number of viable seed ranged from 31 to 37, and since the models predict
even lower probabilities than what was observed, the margin of error is small. These
results are probably due to an overestimate of the exposure times experienced by
Cupressus cones during a wildfire. Shorter exposure times were rejected early on as the
heat loss from opening the muffle furnace could not be consistently controlled and
accounted for with exposure times less than 0.5 minute. But as was observed in the
branch burn data from the pilot study (Appendix A), brief exposure times of less than 1
minute were observed at temperatures greater than 400° C during complete cone and
foliage consumption. A series of short peaks at high temperatures were enough to trigger
cone opening and release of seed in the 1 to 4 days following heat exposure. However,
the technique of burning the branches was never standardized as the weight of the branch
36
prevented the thermocouples from remaining in a constant and repeatable position
relative to the cones. The graph, therefore, should only be taken as an approximation of
the actual temperature ranges experienced. Future explorations with Cupressus and fire
behavior may want to examine this further and develop a standardized method of burning
branches in order to make conclusions about the nature of Cupressus cone flammability.
The main conclusions from this study can be summarized that across all species,
seed viability decreased with increasing time of exposure, and to a lesser extent,
increasing temperature. From an ecological stand-point, this conclusion makes sense
given that during wildland fires, cones are heated for short periods at high temperatures,
but that protection decreases for longer heating durations (Habrouk et al. 1999).
Cupressus bakeri was less serotinous than the other four Cupressus species due to greater
seed release in a short amount of time and greater seed viability sensitivity to higher
exposure temperatures and longer durations. Cupressus forbesii was the most serotinous
species, requiring a much longer time to open and release seeds and viability being less
affected by the heat treatments. The remaining species fall in between these two species,
with C. arizonica ssp. nevadensis following C. forbesii in cone opening and seed
viability, and C. macnabiana and C. sargentii being less resilient to heat exposure, but
more so than C. bakeri. This again suggests that differences between species in cone
serotiny and seed viability may be due to different geographic locations and habitats.
The conclusions from this study, that seed viability decreases with increasing time
of exposure and increasing temperature for all five species, are reflected in the literature
of similar studies. Unlike this study, however, previous cone serotiny studies have
37
focused primarily on Mediterranean pines. Some parallels between results from
Mediterranean pines and those found in this study can still be drawn. Two studies
applying heat treatment regimes most similar to my study were conducted on the amount
of heat needed to break cone serotiny in Spanish pines using time treatments with
different temperatures to reflect the effect of ground, surface, and canopy fire behavior.
Habrouk et al. (1999) exposed cones and free seed were exposed to temperatures of 70,
120, 200, and 400° C for 2, 5, 10, and 20 minutes to simulate the conditions of a surface,
soil and canopy seed bank’s exposure to wildfire. Similar to the results of this study, a
greater percent germination at lower temperatures and exposure times, with seeds from
the treated cones having greatest success, and the free seed being the most sensitive to
higher treatment combinations. Pinus halepensis, a serotinous species, had the greatest
germination success compared to the non-serotinous species, P. sylvestris and P. nigra.
This can be compared to the results found with C. bakeri and C. forbesii, where C.
forbesii (a more serotinous species) had a greater tolerance to heat than C. bakeri (a less
serotinous species).
A second study in Spain compared Pinus pinaster and P. radiata for seed viability
following heat treatments, again focusing on the temperature regimes associated with
three different fire types (Reyes & Casal 2002). Cones were exposed to temperatures of
100, 150, 200, 250, 300, 350, 400, and 500° C for 0, 1, 5, 10, 15, 20, 25 and 30 minutes
in an experimental design that consisted of longer time treatments for the lowest
temperatures, and the shortest time treatments at higher temperatures. My treatment
design closely followed this design, but with the focus on wildfire conditions on only the
38
canopy seed bank. Reyes and Casal (2002) found that seed viability decreased slightly
with greater temperatures and increasing exposure times as was found in my study,
though they did not explore exposure times longer than 1 minute at 400° C and above.
Other studies evaluated germination response to heat exposure treatments and
analyzed their results using logistic regression to build models expressing the effect of
time, temperature and their interaction, but still used free seed to simulate wildfire’s
effect on the soil seed bank. A study on six pine species found in the Mediterranean basin
of Spain found different germination responses among species (none responded
positively to heat exposure) and developed individual species models to predict
probability of germination, but they did not compare species models (Escudero et al.
1999). A second very similar study addressing the problem of inter-specific competition
between pine and shrub species regenerating after a wildfire, found a decreased
germination response at higher temperatures and for longer exposure times, even though
they used free seed to mimic soil seed bank conditions (Nuñez et al. 2003). Another study
looked at a range of temperatures of 60 – 300° C for time periods of 1 and 5 minutes
(Torres et al. 2006). Not surprisingly and very similar to the results reported here, they
found a significant decrease in germination at 5 minutes compared to 1 minute.
Although a few studies have examined Cupressus species and fire behavior, life
history traits, or regeneration responses (de Gouvenain & Ansary 2006; Mallek 2009;
Ne'eman et al. 1999), none have studied the role of heat tolerance directly as was done in
this study. As has been shown in previous studies with pines, Cupressus species in this
study responded negatively to high heat at longer durations, but what is of more
39
ecological interest and value to management are the differences in individual species’
responses. This is the first study to compare Cupressus species responses to different heat
exposures, indicating that further research is needed examining the role of a species’
regeneration response to different fire behavior, in the context of other competing
species. That the only other comparable studies were those with Mediterranean pine
species also indicates that further research is needed for Cupressus species. As climate
change, increased human activities, and land use practices continue to cumulatively affect
fire regimes in areas where Cupressus species occur (Westerling & Bryant 2008;
Westerling et al. 2006), the need for greater knowledge of these fragmented species
increases. The results of this particular study will hopefully broaden the current
knowledge about Cupressus species in Mediterranean climate regions, particularly in
California which has such a wide diversity of Cupressus species, and their cone serotiny
responses to fire. With regard to management concerns, the findings from this study help
address the scarcity of information on Cupressus regeneration ability, and may be utilized
by forest land managers conducting active fire management, including prescribed burning
and thinning treatments on public lands where Cupressus species are found.
40
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43
Appendix A. Measured temperatures and exposure time during burning of Cupressus
macnabiana (McNab cypress) branches, for a pilot study.
4 4
Appendix B. The cumulative proportion of seeds released immediately following heat treatment (day 0), at 4 and at 8 days, for
Cupressus macnabiana (McNab cypress) and all temperature and time combination treatments. The x-axis is temperature ( o C), the y-
axis is time (min), and the z-axis is the proportion of seeds released.
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
1
2
3
4
5
Appendix C. The cumulative proportion of seeds released immediately following heat treatment (day 0), at 4 and at 8 days, for
Cupressus sargentii (Sargent cypress) and all temperature and time combination treatments. The x-axis is temperature ( o C), the y-axis
is time (min), and the z-axis is the proportion of seeds released.
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
1
2
3
4
5
Appendix D. The cumulative proportion of seeds released immediately following heat treatment (day 0), at 4 and at 8 days, for
Cupressus arizonica spp. nevadensis (Piute cypress) and all temperature and time combination treatments. The x-axis is temperature ( o
C), the y-axis is time (min), and the z-axis is the proportion of seeds released.
0.5
2
4
0
0.05
0.1
0.15
0.2
0.25
0.3
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
2
4
0
0.2
0.4
0.6
0.8
1
0.5
1
2
3
4
5
47
Appendix E. Cupressus macnabiana (McNab cypress) seedlings from a fire in 1999,
located in the BLM Knoxville Recreation Area, Lake and Napa Counties, California.
48
Appendix F. Cupressus macnabiana (McNab cypress) seedlings one year after the
Walker Fire in 2009, located in the BLM Knoxville Recreation Area, Lake County,
California.
49
Appendix G. Cupressus sargentii (Sargent cypress) seedlings in the foreground growing
near an ephemeral drainage with no evidence of recent fire, located in the BLM
Knoxville Recreation Area, Napa County, California.
50
CHAPTER 2
Tree Age, Cone Age and Seed Viability in Cupressus Sargentii (Sargent Cypress)
INTRODUCTION
Fire has long been an important ecological factor in the floristic composition,
evolution and organization of Mediterranean ecosystems. Distribution and composition of
vegetation associated with these ecosystems has been shaped by an established history of
recurring fire (Bond & Keeley 2005b). Many conifer species in these areas have
developed reproductive adaptations in response to the constant presence of fire that
enables them to remain competitive following disturbance (Pausas et al. 2004). The
conifer genera Pinus and Cupressus in the Northern Hemisphere share the particular
adaptation of cone serotiny, indicating an association with recurring fire and a
competitive resilience to disturbance (Zedler 1986). As climate change becomes more of
a factor in the shifts in frequency of fire regimes, knowledge of individual species and
plant community regeneration responses to fire becomes ever more important.
Cupressus (cypress) species native to California occur in remote, scattered and
highly restricted populations (McMillan 1956; Vogl et al. 1977; Zedler 1977). Most
Cupressus species of interior California are fire-dependent with serotinous cones (Bartel
1993; Wolf 1948), and have specific requirements for successful seedling germination,
the most notable being direct sunlight and contact with mineral soil (Vogl et al. 1977).
Harsh, dry conditions with extreme temperature fluctuations and serpentine, volcanic, or
granitic substrates at elevations of 300-2100 m characterize typical sites of Cupressus
populations (Stuart & Sawyer 2001). Cupressus sargentii (Sargent cypress) is believed to
51
be a serpentine indicator species (McMillan 1956). Some populations of C. bakeri (Baker
cypress) have been observed to have little or no seedling recruitment (Wagner & Quick
1963) possibly due to long-term fire exclusion practices, limited populations, and
unknown stand responses to different fire severities.
Years of fire suppression have resulted in some northern California Cupressus
populations facing a risk of having cones senesce before a fire can open cones and
prepare the seedbed, resulting in little or no regeneration (Keeley & Fotheringham 2000).
Ne’eman et al. (1999) hypothesized that low seedling recruitment following fire may
have been associated with low seed viability in stands of older trees. Germination
capacity has been determined to be typically low for several California native Cupressus
species (Ceccherini et al. 1998; McMillan 1956), but none of these studies explicitly
tested the relationship between tree age, cone age, and seed viability. Population age and
stand structure may be important determinants for seedling germination and
establishment, and in-so-far as these are fire-adapted species, may affect fire management
strategies. The focus of this study was to determine the effects of tree age and cone age
on seed viability of C. sargentii (Sargent cypress), an interior California Cupressus
species (Figure 1).
52
Figure 1. Location of Cupressus sargentii (Sargent cypress) study sites, located in the
BLM Knoxville Recreation Area, Lake and Napa Counties, California.
53
METHODS
Cones were obtained in the summer of 2009 from four stands of C. sargentii
(Figure 1). The cones were collected from 6 to 10 canopy dominant and co-dominant
trees in each stand, for a total of 36 trees sampled. On each tree, 3 to 5 mature cones were
collected from each whorl of cones, usually 4 to 5 whorls on a single branch, while
avoiding immature (brown) cones at the branch tips (Figure 2).
Trees were cored with an increment borer at approximately 40 cm above the litter
layer. The cores were dried, sanded and the rings were counted twice under a dissecting
microscope. Thirty C. macnabiana (McNab cypress) seedlings that were at least 40 cm
tall were collected from within 2 km of the collecting sites, in an area that had burned in
1999 (Figure 1), 10 years prior to sampling. C. macnabiana seedlings were used as
surrogates to determine seedling age as there were very few C. sargentii seedlings. Each
seedling was aged and measured for height and diameter. A slice was taken at the base, at
20 cm and at 40 cm (if greater than 40 cm in total height) of each seedling, and age and
caliper width (diameter) was recorded. The heights and age estimates were then used to
get an average of the number of years it took for a typical tree to get at least 40 cm tall,
and then added to the core ages to estimate total tree age.
The collected cones were air-dried under standard laboratory conditions (~ 25° C)
for 4 weeks to release the seeds. Seed viability was determined using a 1% solution
tetrazolium red stain following the guidelines for Cupressus species set forth by the
Association of Official Seed Analysts (2001). Stained seeds were sliced longitudinally
with a razor blade and visually analyzed for viability based on staining extent and the
54
condition of the embryo (Figure 3a-d). Linear regression and analysis of variance
(ANOVA) were used to analyze the data using the statistics program NCSS (Hintze
2007). Tree age and cone position (whorl number), a surrogate for cone age, were the
predictor variables, and seed viability was the response variable.
55
Figure 2. Cone whorls on a Cupressus sargentii (Sargent cypress) branch. Cone position
is assumed to be correlated with cone age.
56
Figure 3. Cupressus sargentii (Sargent cypress) seeds cut longitudinally, showing the
four categories of observation: full stain of embryo (a); incomplete stain of embryo (b);
unstained embryo (c); and embryo absent (d). Only seeds (a) were categorized as viable.
57
RESULTS
Seed viability ranged from 0 to 38% across all trees and cone whorls sampled.
Simple linear regression analysis was used to determine the relationship between seed
viability, tree age, and cone age (Figure 4). There was no statistically significant
difference in seed viability as a function of tree age (p= 0.0615). Nonetheless, the highest
seed viability (23-38%) was found on trees younger than 80 years old (Figure 4), and
from younger cones (2 nd
and 3 rd
year cones). The lowest seed viability (<10%) occurred
more often with trees older than 80 years.
There was no statistically significant effect of cone age, as indicated by cone
whorl number, on seed viability (p= 0.9918, F value=0.07, df= 4), as evaluated using
ANOVA. The relationship between seed viability and tree age was also evaluated for
each cone whorl number using a series of simple linear regression analyses (Figures 5
and 6). There was no statistically significant effect of tree age on seed viability for any of
the cone ages (Table 1). In sum, tree age and cone age had no statistically significant
effect on seed viability.
58
Figure 4. Cupressus sargentii (Sargent cypress) seed viability (%) and tree age (years) for
all trees and cone whorls sampled.
y = -0.0264x + 9.1803 R² = 0.0296
0
5
10
15
20
25
30
35
40
45
50
0 20 40 60 80 100 120 140 160 180
S e
e d
v ia
59
Table 1. R 2 and P-values from series of simple linear regression analyses for seed
viability of Cupressus sargentii (Sargent cypress) as a function of cone whorl age.
Cone age (yr) R 2 P-value
1 0.0145 0.4973
2 0.0708 0.1411
3 0.0735 0.1472
4 0.1176 0.1388
6 0
Figure 5. Seed viability (%) and tree age (years) for Cupressus sargentii (Sargent cypress) cone whorls 1 and 2.
0
5
10
15
20
25
30
35
40
45
50
S e
e d
v ia
b il
it y (
Whorl 2
6 1
Figure 6. Seed viability (%) and tree age (years) for Cupressus sargentii (Sargent cypress) cone whorls 3 and 4.
0
5
10
15
20
25
30
35
40
45
50
S e
e d
v ia
b il
it y (
S e
e d
v ia
b il
it y (
62
DISCUSSION
One of the main management concerns for C. sargentii is that if fire does not
occur often enough, or is completely suppressed, then seed viability in older cones and
trees declines, decreasing the potential for regeneration of the stand. However, the results
show that seed viability in C. sargentii does not appear to be affected by tree age or cone
age, as indicated by cone whorl position. Higher viability is sometimes observed in
younger trees (less than 80 years old), but at the stand level there is no statistical
difference in seed viability with stand age. The ecological implications of this are that a
lack of seedlings following fire is not related to aging C. sargentii stands, as hypothesized
by Ne’eman et al. (1999), but perhaps may be due to other environmental factors such as
competition for growing space, seed predation, direct heat injury, or post-fire seedbed
conditions. Or alternatively a lack of seedlings may be due to differences in fuel loading
and individual tree characteristics leading to different fire severities in stands of various
ages.
Goubitz and others (2003) found similar results in a study of Pinus halepensis that
investigated several combinations of presence/absence of heat exposure (80 o C for 10
minutes) and different pH levels, for young first year and old (> 4 years) cones. They
found no statistically significant difference in germination between young and old cones.
In contrast, Reyes and Casal (2001) found lower germination with older seeds (defined as
the length of time of collection until treatment, ranging from 1 to 4 years) in their study
of P. pinaster and P. radiata. Their reason for looking at these seed ages appears to be
related to the effect of length of storage time on the ability of the seeds to germinate and
63
how this may affect experiments using different seed lots, which was not the concern of
this study as fresh plant material was collected and used in one season. Length of storage
time may be an issue for Pinus species, but has not been found to have any effect on
Cupressus species (Johnson & Karrfalt 1974).
In a study of C. arizonica, C. benthamii, C. lusitanica, and C. macrocarpa De
Magistris and others (2001) found that germination capacity peaked in cones around 5
years old, but showed a significant decrease for older cones (> 7 years), and that the
results varied by species. Since our study tested cones that were most likely between 2
and 6 years of age (based on position from the branch tip), it may be that cones older than
6 years may have lower germination capacity. Further experimentation with cones older
than those used in this study may be needed to determine a more precise relationship
between seed viability and cone age. Investigating a wider range of C. sargentii
populations or other Cupressus species would also be useful. In addition, further study
should include determining the exact ages of cones tested, which would involve aging the
branches as well as the cones.
In conclusion, tree age and cone age are not important factors in C. sargentii
stands’ ability to regenerate following disturbance. This implies that a stand may not
experience wildfire for decades, and yet the level of seed viability will remain
unchanged. It may be more useful for land and fire managers of public lands to consider
other measures of stand vitality when assessing regeneration ability.
64
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