Cone Serotiny and Seed Viability of Fire-Prone California Cupressus Species

Home , Cupressus arizonica, Cupressus bakeri, Cupressus forbesii, Cupressus macnabiana, Cupressus sargentii

CONE SEROTINY AND SEED VIABILITY OF FIRE-PRONE CALIFORNIA CUPRESSUS SPECIES

Kate L. Milich

A Thesis

Presented to

The Faculty of Humboldt State University

In Partial Fulfillment

Of the Requirements for the Degree

Masters of Science

In Natural Resources: Forestry

May, 2010

ABSTRACT

CONE SEROTINY AND SEED VIABILITY OF FIRE-PRONE CALIFORNIA CUPRESSUS SPECIES

Kate L. Milich

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 - 700o 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

iii 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.

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.

TABLE OF CONTENTS

Page

ABSTRACT ...... iii

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

LIST OF APPENDICES ...... x

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

INTRODUCTION ...... 50

METHODS ...... 53

RESULTS ...... 57

DISCUSSION ...... 62

REFERENCES ...... 64

LIST OF TABLES

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 R2 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

vii

LIST OF FIGURES

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

viii

LIST OF FIGURES (Continued)

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

LIST OF APPENDICES

CHAPTER 1

APPENDIX Page

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

CHAPTER 1

Seed Viability and Fire-related Temperature Regimes in Interior California Native Cupressus Species

INTRODUCTION

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.

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.

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.

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 m2 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 m2 trays. Seed density was estimated at 600-800 per m2 from the five ground plots.

These seeds were later tested for seed viability.

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.

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.

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.

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,

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).

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).

Day 0 Day 4 Day 8

1 1 1 0.8 0.5 0.8 0.8 0.6 1 0.6 0.6 2 0.4 0.4 0.4 3 0.2 0.2 0.2 4 4 4 4 0 0 5 0 2 2 2 250 250 250 300 350 0.5 300 350 0.5 300 350 0.5 400 500 400 500 400 500 600 600 600 650 650 700 650 700 700

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.

Day 0 Day 4 Day 8

1 0.3 1 0.8 0.25 0.8 0.5 0.2 1 0.6 0.6 0.15 2 0.4 0.4 0.1 3 0.2 0.2 0.05 4 4 4 4 0 5 0 2 2 0 2 250 250 250 300 300 0.5 300 0.5 350 400 0.5 350 400 350 400 500 600 500 600 500 600 650 700 650 700 650 700

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.

Table 1. Comparison of time (days) until control cones opened, number of cones open and seed release data for all five species studied. Time for cones # of cones # seeds released Cypress Species to open (days) open out of once cones 5 opened Cupressus bakeri 21 3 0 Cupressus macnabiana 35 1 3 Cupressus sargentii 35 3 15 Cupressus arizonica ssp. nevadensis > 40 1 0 Cupressus forbesii > 40 0 0

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 Cupressus Cupressus Cupressus arizonica Cupressus bakeri macnabiana sargentii ssp. nevadensis forbesii Control 8 0 0 0 36 1 min 4 0 8 6 24 2 min 4 0 0 8 24 250° C 3 min 0 0 4 4 8 4 min 0 0 0 0 0 5 min 0 0 0 0 0 1 min 0 0 0 8 8 2 min 0 0 0 8 4 300° C 3 min 0 0 0 4 0 4 min 0 0 0 0 0 5 min 0 0 0 0 0 1 min 0 0 0 20 8 2 min 0 0 0 0 0 350° C 3 min 0 0 0 4 0 4 min 0 0 0 0 0 5 min 0 0 0 0 0 0.5 min 4 0 0 6 16 1 min 0 0 4 6 8 2 min 0 0 0 0 0 400° C 3 min 0 0 0 0 0 4 min 0 0 0 0 0 5 min 0 0 0 0 0 0.5 min 0 0 0 6 20 1 min 0 0 0 8 4 2 min 0 0 0 0 0 500° C 3 min 0 0 0 0 0 4 min 0 0 0 0 0 5 min 0 0 0 0 0 0.5 min 4 0 0 4 4 600° C 1 min 0 0 0 2 12 2 min 0 0 0 0 0 0.5 min 0 0 0 0 0 650° C 1 min 0 0 0 0 8 2 min 0 0 0 0 0 0.5 min 0 0 0 4 12 700° C 1 min 0 0 0 0 0 2 min 0 0 0

C. forbesii 15 C. arizonica ssp.nevadensis

% Germination% 10 C. sargentii

C. bakeri 5

Days since trial began

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.

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 Cupressus Cupressus Cupressus arizonica Cupressus bakeri macnabiana sargentii ssp. nevadensis forbesii Control 20 16 8 12 24 1 min 4 20 8 12 12 2 min 8 24 12 8 32 250° C 3 min 0 16 8 4 8 4 min 0 8 4 0 0 5 min 0 4 0 0 0 1 min 4 12 20 8 12 2 min 0 16 8 4 4 300° C 3 min 0 4 8 0 0 4 min 0 0 4 0 0 5 min 0 0 0 0 0 1 min 8 16 12 12 12 2 min 0 0 0 0 0 350° C 3 min 0 0 0 0 0 4 min 0 0 0 0 0 5 min 0 0 0 0 0 0.5 min 8 8 4 8 8 1 min 4 4 8 4 4 2 min 0 4 0 0 0 400° C 3 min 0 0 0 0 0 4 min 0 0 0 0 0 5 min 0 0 0 0 0 0.5 min 8 4 4 4 4 1 min 0 4 8 4 4 2 min 0 0 0 0 0 500° C 3 min 0 0 0 0 0 4 min 0 0 0 0 0 5 min 0 0 0 0 0 0.5 min 4 0 4 4 12 600° C 1 min 0 0 0 4 4 2 min 0 4 4 0 0 0.5 min 0 4 4 8 4 650° C 1 min 0 0 0 0 4 2 min 0 4 4 0 0 0.5 min 0 0 0 4 0 700° C 1 min 0 0 0 0 0 2 min 0 0 0

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,

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).

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 Deviance (model) Correct (constant) classification (%) Cupressus bakeri T 38.116 35.996 98.7 t 22.781 98.7 T, t 11.175 98.7 T, t, T*t 8.862 98.7 Cupressus macnabiana T 78.544 52.244 95.7 t 71.457 95.7 T, t 27.402 95.7 T, t, T*t 25.654 95.7 Cupressus sargentii T 55.462 47.303 96.6 t 45.798 96.6 T, t 25.418 96.6 T, t, T*t 24.998 96.6 Cupressus arizonica T 47.133 45.754 97.6 ssp. nevadensis t 24.163 97.6 T, t 11.885 97.6 T, t, T*t 8.027 97.6 Cupressus forbesii T 78.375 70.197 96.5 t 58.209 96.5 T, t 32.213 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 Deviance Correct (constant) (model) classification (%) Cupressus bakeri T 134.37 106.500 97.2 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, T*t 35.059 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, T*t 44.123 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.

0.20 0.20 Tecate Tecate Tecate Piute Piute McNab McNab McNab Baker Baker

Sargent Sargent Sargent

0.15 0.15

0.10 0.10

Probability of seed viabilityseed Probability of viabilityseed Probability of

0.05 0.05

0.00 0.00

0 1 2 3 4 5 200 300 400 500 600 700

Time (min) Temperature (C)

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.

0.5 min 1 min 3 min

0.30 0.30 0.30 McNab - Sargent McNab - Sargent McNab - Sargent Tecate - Piute Tecate - Piute Tecate - Piute

Baker Baker Baker

0.25 0.25 0.25

0.20 0.20 0.20

0.15 0.15 0.15

0.10 0.10 0.10

Probability of seed viabilityseed Probability of viabilityseed Probability of viabilityseed Probability of

0.05 0.05 0.05

0.00 0.00 0.00

200 300 400 500 600 700 200 300 400 500 600 700 200 300 400 500 600 700 Temperature (C) Temperature (C) Temperature (C)

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.

300 C 400 C 500 C

0.25 0.25 0.25 McNab - Sargent McNab - Sargent McNab - Sargent Tecate - Piute Tecate - Piute Tecate - Piute

Baker Baker Baker

0.20 0.20 0.20

0.15 0.15 0.15

0.10 0.10 0.10

Probability of seed viabilityseed Probability of viabilityseed Probability of viabilityseed Probability of

0.05 0.05 0.05

0.00 0.00 0.00

0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 Time (min) Time (min) Time (min)

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.

DISCUSSION

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/m2 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.

REFERENCES

Association of Official Seed Analysts (2001) Tetrazolium Testing Handbook #29, Cupressaceae 2001 update. Association of Official Seed Analysts, Inc., Stillwater, Oklahoma

Association of Official Seed Analysts (2008) AOSA Rules for Testing Seeds. Association of Official Seed Analysts, Inc., Stillwater, Oklahoma

Bartel JA (1993) Cupressus. In: Hickman JC (ed), The Jepson manual: higher plants of California. University of California Press, Berkeley, California, pp 111-114

Bond WJ and Keeley JE (2005) Fire as a global 'herbivore': the ecology and evolution of flammable ecosystems. Trends Ecol Evol 20: 387-394

Ceccherini L, Raddi S and Andreoli C (1998) The effect of seed stratification on germination of 14 Cupressus species. Seed Sci Technology 26: 159-168

CNPS (2008) Inventory of rare and endagered plants (online edition v7-08a). California Native Plant Society, Sacramento. Available online http://www.cnps.org/inventory/. Accessed 13 Feb 2008. de Gouvenain RC and Ansary AM (2006) Association between fire return interval and population dynamics in four California populations of Tecate cypress (Cupressus forbesii). The Southwest Nat 51: 447-454

Escudero A, Sanz MV, Pita JM and Pérez-García F (1999) Probability of germination after heat treatment of native Spanish pines. Ann For Sci 56: 511-520

Griffin JR and Critchfield WB (1972) The distribution of forest trees in California. USDA Forest Service Research Paper PSW-82, Berkeley, California.

Habrouk A, Retana J and Espelta JM (1999) Role of heat tolerance and cone protection of seeds in the response of three pine species to wildfires. Plant Ecol 145: 91-99

Harvey HT, Shellhammer HS and Stecker RE (1980) Giant sequoia ecology. Scientific Monograph Series 12. U.S. Department of the Interior, National Park Service Washington, D.C.

Hosmer DW and Lemeshow S (2000) Applied Logistic Regression. John Wiley and Sons, New York

Jepson WL (1923) The trees of California. University of California, Berkeley, California

Johnson EA and Gutsell SL (1993) Heat budget and fire behavior associated with the opening of serotinous cones in two Pinus species. J Veg Sci 4: 745-750

Keeler-Wolf T (2004a) Mud Lake. In: Cheng S (ed), Forest Service Research Natural Areas in California. USDA Forest Service General Technical Report PSW-GTR- 188, Albany, California, pp 208-211

Keeler-Wolf T (2004b) Timbered Crater. In: Cheng S (ed), Forest Service Research Natural Areas of California. USDA Forest Service General Technical Report PSW-GTR-188, Albany, California, pp 285-289

Keeley JE and Fotheringham CJ (2000) Role of fire in regeneration from seed. In: Fenner M (ed), Seeds: the ecology of regeneration in plant communities. CABI International, New York, pp 311-330

Lamont BB, Le Maitre DC, Cowling RM and Enright NJ (1991) Seed storage in woody plants. Botanical Rev 57: 277-317

Mallek CR (2009) Fire history, stand origins, and the persistence of McNab cypress, northern California, USA. Fire Ecol 5: 100-119

McMaster GS and Zedler PH (1981) Delayed seed dispersal in Pinus torreyana (Torrey pine). Oecologia 51: 62-66

McMillan C (1956) The edaphic restriction of Cupressus and Pinus in the Coast Ranges of central California. Ecol Monogr 26: 177-212

Ne'eman G, Fotheringham CJ and Keeley JE (1999) Patch to landscape patterns in post fire recruitment of a serotinous conifer. Plant Ecol 145: 235-242 Nuñez MR, Bravo F and Calvo L (2003) Predicting the probability of seed germination in Pinus sylvestris L. and four competitor shrub species after fire. Ann For Sci 60: 75-81

Pausas JG, Bradstock RA, Keith DA, Keeley JE and Network GF (2004) Plant functional traits in relation to fire in crown-fire ecosystems. Ecology 85: 1085-1100

R Development Core Team (2009), R: a language and environment for statistical computing, version 2.7.2. R Foundation for Statistical Computing, Vienna, Austria. Available online http://www.R-project.org. Accessed 04 Feb 2010.

Reyes O and Casal M (2002) Effect of high temperatures on cone opening and on the release and viability of Pinus pinaster and P. radiata seeds in NW Spain. Ann For Sci 59: 327-334

Stone CO (1965) Modoc cypress, Cupressus bakeri Jepson does occur in Modoc County. Aliso 6: 77-87

Stuart JD and Sawyer JO (2001) Trees and shrubs of California. Univeristy of California Press, Berkeley, California

Torres O, Calvo L and Valbuena L (2006) Influence of high temperatures on seed germination of a special Pinus pinaster stand adapted to frequent fires. Plant Ecol 186: 129-136

Vogl R, Armstrong K, White K and Cole K (1977) The closed-cone pines and cypresses. In: Barbour MG and Major J (eds), Terrestrial vegetation of California. Wiley- Interscience, New York

Vogl RJ (1973) Ecology of knobcone pine in the Santa Ana Mountains, California. Ecol Monogr 43: 125-143

Westerling AL and Bryant BP (2008) Climate change and wildfire in California. Climate Change 87 (Suppl 1): S231-S249

Westerling AL, Hidalgo HG, Cayan DR and Swetnam TW (2006) Warming and earlier spring increase Western U.S. forest wildfire activity. Science 313: 940-943

Wolf CB (1948) Taxonomic and distributional status of the New World cypresses. Aliso 1: 70-91

Zedler PH (1977) Life history attributes of plants and the fire cycle: a case study in chaparral dominated by Cupressus forbesii [in California]. Gen. Tech. Rep. WO- 3. In: Mooney HA and Conrad CE (eds), Proceedings of the symposium on the environmental consequences of fire and fuel management in mediterranean ecosystems. USDA Forest Service, pp 451-458.

Zedler PH (1986) Closed-cone pines of the chaparral. Fremontia 14: 14-17

Zedler PH (1995) Plant life history and dynamic specialization in the chaparral/coastal sage shrub flora in southern California. In: Arroyo MTK, Zedler PH and Fox MD (eds), Ecology and biogeography of Mediterranean ecosystems in Chile, California, and Australia. Springer-Verlag, New York

Appendix A. Measured temperatures and exposure time during burning of Cupressus macnabiana (McNab cypress) branches, for a pilot study.

Day 0 Day 4 Day 8

1 1 1

0.8 0.8 0.8 0.5

0.6 0.6 0.6 1 2 0.4 0.4 0.4 3 0.2 0.2 0.2 4 4 4 4 0 5 0 2 2 0 2 250 250 300 250 300 300 350 0.5 350 400 0.5 350 0.5 400 500 500 400 500 600 600 650 600 650 700 700 650 700

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.

Day 0 Day 4 Day 8

1 1 1 0.8 0.5 0.8 0.8 0.6 1 0.6 0.6 2 0.4 0.4 0.4 3 0.2 0.2 0.2 4 4 4 4 0 5 0 2 2 0 2 250 250 250 300 300 0.5 300 0.5 350 400 0.5 350 400 350 400 500 600 500 600 500 600 650 700 650 700 650 700

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.

Day 0 Day 4 Day 8

1 1 0.3 0.8 0.25 0.8 0.5

0.2 0.6 0.6 1 0.15 2 0.4 0.4 0.1 3 0.2 0.2 0.05 4 4 4 4 0 0 5 0 2 2 2 250 250 300 250 300 300 350 0.5 350 400 0.5 350 0.5 400 500 500 400 500 600 600 650 600 650 700 700 650 700

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.

Appendix E. Cupressus macnabiana (McNab cypress) seedlings from a fire in 1999, located in the BLM Knoxville Recreation Area, Lake and Napa Counties, California.

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.

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.

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).

Figure 1. Location of Cupressus sargentii (Sargent cypress) study sites, located in the BLM Knoxville Recreation Area, Lake and Napa Counties, California.

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.

Figure 2. Cone whorls on a Cupressus sargentii (Sargent cypress) branch. Cone position is assumed to be correlated with cone age.

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.

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 (2nd and 3rd 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.

45 y = -0.0264x + 9.1803 40 R² = 0.0296

Seed viabilitySeed (%) 15

0 0 20 40 60 80 100 120 140 160 180

Tree age (yrs)

Figure 4. Cupressus sargentii (Sargent cypress) seed viability (%) and tree age (years) for all trees and cone whorls sampled.

Table 1. R2 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) R2 P-value 1 0.0145 0.4973 2 0.0708 0.1411 3 0.0735 0.1472 4 0.1176 0.1388

50 50 Whorl 1 Whorl 2 45 45

40 40

35 35

30 30

25 25

20 20

15 viability Seed(%) 15 Seed viability Seedviability (%) 10 10

5 5

0 0 0 50 100 150 0 50 100 150 200 Tree age (yrs) Tree age (yrs)

Figure 5. Seed viability (%) and tree age (years) for Cupressus sargentii (Sargent cypress) cone whorls 1 and 2.

50 Whorl 3 50 45 Whorl 4 45 40 40 35 35 30 30 25 25

20 20

15 Seed viability (%) Seed viability (%) 15

10 10

5 5

0 0 0 50 100 150 200 0 50 100 150 200 Tree age (yrs) Tree age (yrs)

Figure 6. Seed viability (%) and tree age (years) for Cupressus sargentii (Sargent cypress) cone whorls 3 and 4.

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 (80o 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.

REFERENCES

Association of Official Seed Analysts (2001) Tetrazolium Testing Handbook #29, Cupressaceae 2001 update. Association of Official Seed Analysts, Inc., Stillwater, Oklahoma

Bartel JA (1993) Cupressus. In: Hickman JC (ed), The Jepson manual: higher plants of California. University of California Press, Berkeley, California, pp 111-114

Bond WJ and Keeley JE (2005) Fire as a global 'herbivore': the ecology and evolution of flammable ecosystems. Trends in Ecology and Evolution 20: 387-394

Ceccherini L, Raddi S and Andreoli C (1998) The effect of seed stratification on germination of 14 Cupressus species. Seed Sci Technology 26: 159-168

De Magistris AA, Hashimoto PN, Masoni SL and Chiesa A (2001) Germination of serotinous cone seeds in Cupressus ssp. Israel J Plant Sci 49: 253-258

Goubitz S, Werger MJA and Ne'eman G (2003) Germination response to fire-related factors of seeds from non-serotinous and serotinous cones. Plant Ecol 169: 195-204

Hintze J (2007) Number Crunching Statistical Systems (NCSS), version 2007. NCSS, LLC, Kaysville, Utah.

Johnson LC and Karrfalt RP (1974) The Woody Plant Seed Manual, Cupressus L. cypress. In: Bonner FT and Karrfalt RP (eds), Agricultural Handbook No. 727. U.S. Department of Agriculture, Forest Service, Washington D.C., pp 1- 13

Keeley JE and Fotheringham CJ (2000) Role of fire in regeneration from seed. In: Fenner M (ed), Seeds: the ecology of regeneration in plant communities. CABI International, New York, pp 311-330

McMillan C (1956) The edaphic restriction of Cupressus and Pinus in the Coast Ranges of central California. Ecol Monogr 26: 177-212

Ne'eman G, Fotheringham CJ and Keeley JE (1999) Patch to landscape patterns in post fire recruitment of a serotinous conifer. Plant Ecol 145: 235-242

Pausas JG, Bradstock RA, Keith DA, Keeley JE and Network GF (2004) Plant functional traits in relation to fire in crown-fire ecosystems. Ecology 85: 1085-1100

Reyes O and Casal M (2001) The influence of seed age on germinative response to the effects of fire in Pinus pinaster, Pinus radiate and Eucalyptus globules. Ann For Sci 58: 439-447

Stuart JD and Sawyer JO (2001) Trees and shrubs of California. Univeristy of California Press, Berkeley, California

Vogl R, Armstrong K, White K and Cole K (1977) The closed-cone pines and cypresses. In: Barbour MG and Major J (eds), Terrestrial vegetation of California. Wiley-Interscience, New York

Wagner WW and Quick CR (1963) Cupressus bakeri -- an extension the known botanical range. Aliso 5: 351-352

Wolf CB (1948) Taxonomic and distributional status of the New World cypresses. Aliso 1: 70-91

Zedler PH (1977) Life history attributes of plants and the fire cycle: a case study in chaparral dominated by Cupressus forbesii [in California]. Gen. Tech. Rep. WO-3. In: Mooney HA and Conrad CE (eds), Proceedings of the symposium on the environmental consequences of fire and fuel management in mediterranean ecosystems. USDA Forest Service, pp 451-458.

Zedler PH (1986) Closed-cone pines of the chaparral. Fremontia 14: 14-17