A MORPHOLOGICAL AND VOLATILE TERPENE ANALYSIS OF PINUS

BALFOURIANA TO TEST FOR THE MOUNTAIN ISLAND EFFECT IN THE

KLAMATH MOUNTAINS

By

Ian Zacher

A Thesis

Presented to

The Faculty of Humboldt State University

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

In Biology

Committee Membership

Dr. Michael Mesler, Committee Chair; Graduate Coordinator

Dr. Andrew Eckert, Committee Member

Dr. Erik Jules, Committee Member

Dr. Alexandru Tomescu, Committee Member

December 2015

ABSTRACT

A MORPHOLOGICAL AND VOLATILE TERPENE ANALYSIS OF PINUS BALFOURIANA TO TEST FOR THE MOUNTAIN ISLAND EFFECT IN THE KLAMATH MOUNTAINS

Ian Zacher

Pinus balfouriana Grev. and Balf., foxtail pine, is endemic to California, occurring as two subspecies: P. balfouriana ssp. austrina in the southern High Sierra and

P. balfouriana ssp. balfouriana 500 km to the north in the Klamath Mountains. Previous research has shown that the northern subspecies is characterized by higher genetic diversity than the southern subspecies. This higher genetic diversity has been attributed to the mountain island effect (MIE), whereby reproductively isolated stands that are restricted to mountain peaks evolve independently via natural selection and/or genetic drift. To date, research on morphological and terpene chemistry variation in foxtail pine has been limited to analyses distinguishing the two subspecies. No work has examined the variation among stands for these traits in the northern part of the range. Here I test if there is phenotypic evidence of the mountain island effect.

Twenty anatomical, morphological, and chemical characters were sampled from twenty stands across the range of the species in the Klamath Mountains. Stands were grouped into seven regions according to which mountain they grew on. I used multivariate analysis of variance (MANOVA) to test if there were differences among ii

stands and regions. If there were differences, I used canonical discriminant analysis

(CDA) to reveal which traits contributed to the differentiation. I used cluster analysis and the Mantel test to investigate whether differences in trait expression were correlated with soil substrate and geographical distance among stands, respectively. Lastly, I used discriminant analysis to see how well trees could be classified into regions and stands within regions. Since the mountain island effect likely operates at fine scales, I expect stronger differentiation within regions than among regions. Moreover, I predicted that the set of traits that discriminates stands within each region will differ across regions.

Fifty percent or more of overall variation resided among stands for most of the individual traits, and differences among means were typically quite modest.

Nevertheless, I found significant multivariate differences in phenotypic traits among and within regions. As predicted for the MIE, differentiation was more pronounced within regions than among regions. Differences among stands were not correlated with physical distances and different characters discriminated stands within each region. Stands that grew in serpentine soil could not be distinguished from those not growing in serpentine, suggesting that serpentine was not a driving factor for differentiation among stands.

Together with previous genetic research, my findings provide support that the MIE is generating distinct mountaintop stands in the Klamath Region.

iii

Acknowledgements

I would like to thank the members of my graduate committee for all their time and patience throughout this project. I owe a debt of gratitude to Dr. Kjirsten Wayman for training me on the GC-MS in her free time as well as allowing me to use her lab to work in all summer. Her insights were invaluable to the progression of this work. I want to thank my advisor Michael Mesler for showing me some wonderful places in the Klamath

Mountains sparking a love for alpine flora that will persist in me for a long time to come.

His guidance and patience has made me a better biologist and a better writer. I would like to thank Dr. Casey Lu for training me on the SEM and allowing me to use it all summer long. I thank Dr. Mihai Tomescu for getting me interested in anatomy and morphology of plants and for his ideas and counsel for this work. I owe a huge debt to Marty Reed and

Lewis McCrigler for keeping all the instruments operational all summer long and always willing to drop what they were doing to help me. I want to thank Darrell Burlison and

Anthony Baker who provided me with a lot of equipment. I owe a lot to my field and lab assistants Brian Creeks, Nate Moy, Anne Mahr, Hannah Linville, Thomas Stanko,

Francisco Vargas, Oleksandra Gluskina, and William Penprase without whome I would still be up to my ears in needles and cones. I would like to thank Arthur Grupe for his gracious comments, and for romping in the woods with me to search for mushrooms and trees. I want to thank Angela McCartney who endured an 18 mile trek in search of trees and glory. I would like to thank Richard and Sharon Zacher for countless support and provender and who are now foxtail pine ambassadors. Charlie for his waggle tail, countless hours of free entertainment, and being the best trail companion a guy can have. iv

I want to thank Leah Sloan for walking up big mountains, searching peaks, getting me to jump into frozen lakes, and going on crazy hikes looking for trees, your love, compassion, and generosity… without you I would not be in the position I am today.

I would also like to thank the Department of Biological Sciences of Humboldt State

University and the Sawyer-Smith Botanical Field Studies Fellowship for all their support in funding this work.

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TABLE OF CONTENTS

PAGE

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

List of Tables ...... vii

List of Figures …………………………………………………………………………...vii

List of Appendices……………………………………………………………………..…ix

INTRODUCTION ...... 10

METHODS ...... 13

Field Sampling………………………………….……………………………………....13

Morphological Characters……………………………………….……………………...13

Cone characters…………………………………………………………..…………...13

Needle characters……...……………………………………………………………...14

Terpene characters..…...……………………………………………………………...15

Statistical analyses……………………………………………………………………...16

RESULTS ...... 18

DISCUSSION ...... 21

REFERENCES…………………………………………………………………………..26

vi

List of Tables

Page

Table 1:Locality and specimen collection data of all sampled foxtail pine stands……..30

Table2: Summary statistics for all 20 characters…...... …………………….…....…….38

Table 3: Summary results for MANOVA, CDA, and LDA for the differing regions………………………………………………………………………….………...39

vii

List of Figures

Page

Figure 1: Location of all 20 sampled stands ...... …..31

Figure 2: Location of the Yolla Bolly Mountains stands…..…...... …...…………………32

Figure 3: Location of the Trinity Alps stands………….....…..………………………….33

Figure 4: Location of the Scott Mountain stands …..…..………...………………….…..34

Figure 5: Location of the Mount Eddy stands… ………………...…………………...…35

Figure 6: Map of the Crater Lake stands……..………………………………………….36

Figure 7: Morphological and anatomical traits………………………………………….37

Figure 8: CDA plot of all stands within regions………………………………………....40

Figure 9: CDA plot of Trinity Alps mountain region...…………………………….……41

Figure 10: CDA plot of Crater Lake mountain region.…..……..……………….……….42

Figure 11: CDA plot of Yolla Bolly Mountain region...... 43

Figure 12: CDA plot of Mount Eddy region……………………………………………..44

Figure 13: CDA plot of Scott Mountain region……...……………………….…….……45

Figure 14: Dendrogram……………………………….………………………………….46

Figure 15: Scatterplot of Mantel test………………………………….…………………47

viii

List of Appendices

Appendix Page

A: CDA Loadings of all 20 Stands………………………………………………………48

B: CDA Loadings of Trinity Alps mountain region…………………………………...... 49

C: CDA Loadings of Crater Lake mountain region….….……………………………….50

D: CDA Loadings of Yolla Bolly Mountains region….…….………………………...…51

E: CDA Loadings of Mount Eddy region………….…………………………………….52

F: CDA Loadings of Scott Mountain region……….…….………………………………53

G: Mean values for all 20 characters for all 20 stands……….………………………...... 54

ix

1

INTRODUCTION

Pinus balfouriana Grev. and Balf., foxtail pine, is a five needle pine in the subsection Balfourianae along with the Great Basin bristlecone pine, Pinus longavea, and the Rocky Mountain bristlecone pine, Pinus aristata (Baily 1970). Foxtail pine is a long lived (up to 3000 years old), high elevation tree endemic to California (Mastrogiuseppe and Mastrogiuseppe 1980). This species occurs in two disjunct populations separated by

500 kilometers where disjunction most likely occurred during the Xerothermic period of the Holocene, approximately 8,000 years ago (Eckert and Sawyer 2002, Eckert et al.

2008). The southern population is located in the southern High Sierra Nevada, while the northern population is located in the Klamath Mountain Range (Mastrogiuseppe and

Mastrogiuseppe 1980). These two regional populations are distinguishable by differences in terpene composition, needle anatomy, and cone morphology, and are treated as two distinct subspecies, with P.balfouriana ssp. austrina in the south and P. balfouriana ssp. balfouriana in the north (Mastrogiuseppe & Mastrogiuseppe 1980, Baldwin 2012).

Trees in both subspecies typically grow on xeric slopes near tree line between

2,000 and 4,000 meters, yet stand structure and habitat are different in the two regions.

The Sierra Nevada are tall mountains where peaks average over 3000 meters high, with abundant high elevation habitat available for foxtail pines to grow in large un- fragmented, primarily monotypic stands (Eckert and Sawyer 2002), with only a single important associate, Pinus contorta (lodgepole pine). The southern subspecies grows almost exclusively on granite with the greatest stand densities on northern slopes (Eckert 2 and Sawyer 2002, Oline et al. 2000). In contrast, the Klamath Mountains are shorter with peaks never reaching 3000 meters, with few peaks reaching elevations where foxtail pines can grow (Major 1988, Eckert and Sawyer 2002). Here the species occurs as a series of relatively small, non-contiguous stands growing on mountain peaks. These northern stands typically co-occur with other conifer species including Abies magnifica var. shastensis (Shasta red fir), Tsuga mertensiana (mountain hemlock), Calocedrus decurrens (incense cedar), Pinus monticola (western white pine), Pinus contorta ssp. murrayana (lodgepole pine) and Pinus jeffreyi ( Jeffrey pine ) (Eckert and Sawyer 2002).

Northern stands grow almost exclusively on ultramafic soils, but occasionally grow on mafic soils. These stands have the greatest stand densities on south facing slopes (Eckert and Sawyer 2002). Overall, the geographic separation, small stands and topographic complexity of northern stands suggest reproductive isolation.

Recent genetic work using allozymes (Oline et al. 2000) and chloroplast, mitochondrial and nuclear DNA (Eckert et al. 2008) found more genetic heterogeneity among stands in the northern subspecies than in the southern subspecies (Oline et al.

2000, Eckert et al. 2008). This heterogeneity is consistent with stronger reproductive isolation or gene flow occurs but the effective stand sizes are small enough that there is little influence by this genetic flow. Independent evolution of geographically and reproductively isolated mountain top stands is referred to as the mountain island effect

(MIE) (Oline et al. 2000). Northern foxtail pine stands may be differentiating as a result of this effect (Oline et al. 2000).

3

To date, research on the variation in foxtail pine morphology and terpene chemistry has been limited to distinguishing the northern and southern populations. No work has examined the variation in these traits among stands in either of the two subspecies (Baily 1970, Snajberk et al. 1979, Mastrogiuseppe and Mastrogiuseppe 1980,

Eckert et al. 2008). Here I examine phenotypic differences among 20 isolated mountaintop stands of northern foxtail pine using a suite of anatomical, morphological, and chemical characters. I first grouped the stands into seven spatially isolated regions, corresponding to different mountain areas, to test for broad-scale differentiation.

Secondly, to test for the mountain island effect, I compared stands within each region. If stands are evolving independently, I expect them to differ at local scales such that degree of differentiation would be unrelated to geographical distance. In addition, I expected the specific traits that distinguish stands within each region would differ idiosyncratically depending on region.

4

METHODS

Field Sampling

I visited 20 mountaintop stands of P. balfouriana ssp. balfouriana from June to

July of 2014 across the Klamath Mountain Range of northern California (Figs. 1-6; Table

1). In each stand, I took cuttings, cones, and geo referenced 20 trees located along a meandering transect (n = 400). Eighteen of these stands were sampled previously for genetic analyses (Eckert et al. 2008). To standardize cone and foliage collection, all cuttings were made at a height of approximately 6 meters. Cuttings were taken from the tips of branches, where needles are aggregated. Because there is little crown shading of foliage in this species, differences (e.g., sun and shade needles) within a branch are minimal. Open mature cones were collected from two or more branches on each tree.

Morphological Characters

Cone Characters

I examined the same six cone traits that distinguish the two subspecies, Pinus balfouriana ssp. balfouriana and Pinus balfouriana ssp. austrina (Mastrogiuseppe and

Mastrogiuseppe 1980): cone length and width (Figure 7B); cone scale number; apophysis length, width, and thickness (Figure 7A) from four cones per tree (400 trees x 4 cones per tree = 1600 cones). Three scales from the mid-section of each cone were measured. All cones were measured to the 0.01 mm using Vernier calipers.

5

Needle Characters

I measured a set of morphological and anatomical needle characters on 10 needles from each tree (400 trees x 10 needles per tree = 4000 needles). Morphological characters measured were needle width and needle thickness, while anatomical characters measured included: resin canal diameter, distance between resin canals, and the thickness of the needle epidermal complex, which consists of the cuticle, epidermis, and the hypodermis.

In foxtail pine there are two resin ducts where one is typically larger than the other, thus the width of each resin duct was treated as a separate trait. Needle width, thickness, and resin canal traits were chosen because they were used to distinguish northern and southern populations of P. balfouriana (Mastrogiuseppe and Mastrogiuseppe 1980), and needle epidermal thickness has been used to distinguish a widely distributed pine species

Pinus mugo (Boratynska et al. 2004).

Needle characters were measured from photos taken with a scanning electron microscope (SEM). Needles were prepared for SEM by hand-cutting cross sections from the mid-section of each and immediately immersing them in a liquid nitrogen bath.

Frozen sections were then placed in 1 mL centrifuge tubes and put into a freeze dryer for

24 hours. Sets of ten were mounted on stubs, sputter coated, and photographed with the

SEM. P. balfouriana needles are triangular in cross section with two resin ducts along the base (abaxial). The width of the lumen of both resin ducts was measured. The distance between the resin ducts was measured approximately from the mid-section between the fiber cells on the outside of the canals (Figure 7C). The needle epidermal complex was

6 measured from the midpoint between the resin ducts All needle measurements were made to the 0.01 μm using Image J (version 1.48).

Volatile Terpenes

I examined the eight volatile terpenes that distinguish northern and southern populations of Pinus balfouriana (Mastrogiuseppe and Mastrogiuseppe 1980): alpha- pinene, beta-pinene, alpha-phellandrene, 3-carene, (-)-trans-caryophyllene, alpha- humulene, bornyl acetate, and methylthymol. Foliage terpenes were identified and quantified with gas chromatography–mass spectrometry (GC-MS), using a Solid Phase

Microextraction (SPME) method. Preliminary test runs indicated that 10 needles per tree were sufficient for a dependable reading on the GC-MS. Samples were analyzed within 3 days of cutting them from the tree.

Ten needles were cut into small pieces and placed into a 2 dram glass vial with a parafilm cover and allowed to sit for at least 25 minutes so the volatiles could fill the vial.

A needle with a 100 μm polydimethlsiloxene (PNMS) fiber was inserted into the vial for

1 minute to allow the volatiles to attach to the fiber. The needle was then inserted into the

GC-MS for 18 minutes. The relative amount of each compound is the proportion of the total area under all detected peaks and was calculated as the area percentage. The GC-MS was a Hewlett-Packard GCD Plus fitted with a 30 m x 0.25 mm crosslinked phenylmethyl silicone capillary column (HP-5MS). Identification of volatile compounds alpha pinene, beta pinene, and alpha phellandrene was done by comparison to published electron impact - mass spectra (EI-MS) in the National Institute of Standards and

7

Technology (NIST) 1998 computerized mass spectral library, and confirmed by comparison of spectra and retention times to commercially available standards. The remaining volatile terpenes identities were determined based on suggestion of match with a greater than 90% probability with the NIST mass spectral library.

Statistical Analysis

The 20 mountaintop stands were grouped into seven regions according to the mountain complex on which they grew. I used a combination of multivariate analyses to examine the pattern of trait variation. I used a multivariate analysis of variance

(MANOVA) to test for differences among and within regions. Canonical discriminant analysis (CDA) was then employed to reveal the degree of distinction between regions and stands within regions, as well as those traits which were responsible for these distinctions. A linear discriminant analysis (LDA) was used to determine how effectively trees could be classified into their respective stands using phenotypic traits. A Mantel test was used to determine if geographical distances among stands were correlated to degree of trait differences. Statistical significance of the Mantel test correlation was assessed using randomization (n=10,000) to get the null distribution. Trait differences were calculated by standardizing the data (mean = 0, sd = 1) and creating a Euclidian distance matrix from the trait means for each stand. Cluster analysis was used to test if stands grouped according to soil substrate. Hierarchical clustering was performed using the furthest neighbor method on Mahalanobis distances. To resolve how much total variation existed within stands, the sum of squares within was divided by sum of squares total

8

(SSw/SST). I calculated the coefficients of variation (C.V.) to compare the degree of variation among trait means across the 20 stands. All analyses were conducted in R

(version 2.13.1, 2011), using the area percentages of the volatile terpenes1, and the means of all anatomical and morphological traits from each tree.

1 Area percentage is defined as the peak’s volume divided by the total volume of all peaks of a given sample.

9

RESULTS

All 20 measured traits exhibited considerable variation, both within and among stands (Table 2). Expression of many traits ranged over an order of magnitude. Over

50% of total trait variation occurred among stands, with less than 50% of the total variation within stands. However, variation among stands for mean trait values was generally modest (C.V. less than 0.4), notable exceptions being the relative percentage of the three least common terpenes: bornyl acetate, alpha-humulene, and methylthymol

(C.V. = 6.5, 3.2, and 1.5, respectively). Production of these three compounds was sporadic within and among stands. For example, none of the trees in stand 19 produced methylthymol, but 40% of the trees in nearby stand 20 produced the compound in limited amounts.

In spite of the relatively modest among-stand variation for individual traits, there were significant multivariate differences among regions (MANOVA, Pillai= 2.6975, p<0.001) and among stands within each region (Table 3). The number of significant CDA factors differed among regions, ranging from 1 to 6 factors (Appendix B-F), but in all regions no more than two factors were required to explain over 50% of the variation.

Traits that were highly correlated with the first two axes tended to differ across regions

(Table 3, Appendix B-F). Using a LDA, trees were correctly classified into regions 62% of the time, and into stands within regions 58% - 92% of the time (Table 3).

A CDA plot including all stands reveals extensive overlap among regions, although stand 25 (Lake Mountain), and the two Mount Eddy stands (19 and 20) tend to

10 cluster apart from the other regions based on differences in the production of alpha- pinene, beta-pinene and methylthymol production (Fig. 8; Appendix A). Mount Eddy stands had higher levels of alpha-pinene (26.32% and 24.32%), lower amounts of beta- pinene (26.45% and 25.6%), and lower amounts of methylthymol (0.03% and 0%) than the Lake Mountain stand (13.46%, 46%, and 0.01% respectively) (Fig.8; Appendix A and

G). Differences among stands were more pronounced within each of the regions.

The eight Trinity Alps stands fell into three fairly distinct clusters, each corresponding to different mountain peaks. The three clusters were distinguished mainly by production of alpha-humulene and cone and apophysis length (Figure 9; Table 3;

Appendix B). Trees in stands 14 and 26 tended to produce higher amounts of alpha- humulene, and had long, wide cones with long apophyses (Fig. 11; Appendix G). In contrast, stands 4 and 5 were characterized by lower amounts of alpha-humulene, short narrow cones, and short apophyses (Fig. 9; Appendix G). Stands 6-9 were similar to stands 4 and 5, but the cones were wider (Appendix G). The two Crater Lake stands (21 and 22) fell into their own unique clusters. These clusters were distinguished by the production of alpha-phellandrene, beta-pinene, the size of the large resin duct, and needle thickness (Figure 10; Table 3; Appendix C). In contrast to stand 21, stand 22 had thicker needles, wider large resin ducts, higher levels of alpha-phellandrene, and lower levels of beta-pinene (Table 3; Appendix B and G). The two Yolla Bolly stands were separated by bornyl acetate production, needle epidermis thickness, cone length, and apophysis width

(Fig.11; Table 3; Appendix D). On average trees in stand 3 had a thicker needle epidermis, shorter cones with narrower apophyses, and higher levels of bornyl acetate

11 than trees in stand 2 (Table 3; Appendix D and G). The two Mount Eddy stands fell into two distinct clusters (Fig.12) that were dissimilar in their production of methylthymol, cone width, and needle epidermis thickness. In general, trees in stand 20 had narrower cones, a thinner needle epidermis, and produced lower amounts of methylthymol compared to stand 19 (Table 3; Appendix E and G). Neither stand produced alpha- humulene; therefore, this character was omitted when comparing differences between them. The four Scott Mountain stands grouped into four distinct clusters, which were distinguishable by the production of alpha-humulene, cone width, cone scale number, and needle epidermis thickness (Fig. 13; Table 3; Appendix F). On average stand 18 had narrower cones with fewer scales, lower levels of alpha-humulene, and a thin needle epidermis. In contrast, stand 27 had wider cones, more scales per cone, and higher levels of alpha-humulene, but also a thin needle epidermis. Stand 16 and 15 had intermediate cone widths, number of cone scales, and levels of alpha-humulene, but the needle epidermis was thin for stand 16 and thick for stand 15 (Table 3; Appendix F and G).

Across all regions stands that grew in serpentine were not distinguishable from those in non-serpentine soils (Figure 14); however, certain mountain regions serpentine may be an important factor. The two Trinity Alps stands growing in non-serpentine soil

(stands 14 and 26) could be distinguished from those stands in serpentine soil (Figure 9).

The degree of differentiation among stands was not correlated with geographic distance

(Mantel test, r = -0.088, p = 0.746, Figure 15).

12

DISCUSSION

There was evidence of morphological and terpene chemical differences of foxtail pine individuals at both regional and local scales, but differences were much more pronounced when comparing stands within regions (Fig. 9-13). The degree of phenotypic difference between stands was uncorrelated with geographic distance, and different sets of traits tended to distinguish stands in different regions. Relatively fine-scale differentiation, lack of geographic structure, and idiosyncratic differentiation are all supportive of the mountain island effect (MIE).

Genetic drift and natural selection are both potential mechanisms behind the MIE

(Oline et al. 2000, Afzal-Rafii & Dodd 1994; Weller et al. 2007; He & Jiang 2014).

These mechanisms can occur simultaneously (He & Jiang 2014), but certain characteristics of stand geography and differentiation can suggest which has the greater influence. Correlations between phenotypic traits and environmental conditions, such as moisture regime, soil composition, or level of herbivory, are indicative of natural selection. In contrast, genetic drift is consistent with differentiation of small, reproductively isolated stands that are uncorrelated with environmental conditions.

Previous research points to genetic drift as more probable in northern foxtail pine stands, because stands are relatively small and isolated (Oline et al. 2000, Mastrogiuseppe and

Mastrogiuseppe 1980). Small population sizes resulting from bottleneck events would lead to genetic drift and gene fixation, resulting in idiosyncratic trait expression across regions (Oline et al. 2000). However, natural selection is known to generate idiosyncratic

13 trait differentiation in other plants when discrete populations are shaped by complex topography, climate, and habitats (Afzal-Rafii & Dodd 1994; Weller et al. 2007; He &

Jiang 2014).

Differentiation of phenotypic traits among foxtail pine stands may have been driven by numerous selective agents, such as seed and foliage herbivory, pathogens, and moisture regime (Waillis 2007, Garcia et al. 2009, Iason et al. 2011). However, the only selective factor directly tested was the ultramafic soils (i.e., serpentine) that occur throughout the Klamath Mountain Range (Oline et al. 2000, Eckert and Sawyer 2002,

Eckert 2006). Adaptations to serpentine soils are known to cause phenotypic divergence in other plant taxa, and are one of the leading drivers of endemism of California flora

(Afzal-Rafii and Dodd 1994; Eckert and Sawyer 2002; Sawyer 2007). It has been suggested that foxtail pines may be adapting to serpentine soil as a way to escape competition with other conifers (Eckert 2006). However, in many cases stands that existed in ultramafic soils were very similar phenotypically to those that occurred on non- ultramafic soils (Figure 8, Figure 10). For example, stand 19 (Mount Eddy), which grew in ultramafic soil, was most similar to stand 22 (Crater Lake), which was on non- ultramafic soil and stand 2 (Yolla Bolly), which grew in non-ultramafic soils, was most similar to stands 16 and 27 (Scott Mountain), which grew in ultramafic soils. Only in the

Trinity Alps did I find suggestive evidence for serpentine soil as a selective agent. Stands

14 and 16 were similar phenotypically, both grew on non-ultramafic soil, and they differed substantially from the other Trinity Alps stands that grew on ultramafic soils.

However, these two stands were also the most isolated from the other stands, thus the

14 phenotypic differentiation could simply have been a result of genetic drift, with the soil substrate being merely coincidental. Stands 14 and 16 primarily differed from the other stands in alpha-humulene, yet it is unknown whether soil type can influence this secondary compound.

The patterns of differentiation and stand geography suggest that moisture regime could be an important selective agent. In the Klamath south-facing slopes tend to receive more sun than north facing slopes, and foxtail pine density is generally highest on south facing slopes (Eckert and Sawyer 2002). These slopes tend to be drier during the summer months, while north facing slopes are more shaded, thus retaining higher moisture.

Volatile terpene concentrations, along with cone and needle morphology, can vary with water availability and temperature fluctuations in other conifer species (Berland 2013;

Blanch et al. 2009; Lopez et al. 2008). South facing stands in the Crater Lake and Mount

Eddy regions either had thicker needles or a thicker needle epidermis, respectively, than the north facing stands. These traits could help trees adapt to more xeric environments, where water conservation is paramount. Concentrations of methylthymol and alpha- phellandrene were higher in north facing stands for Mount Eddy and Crater Lake, respectively. Moisture regimes could have directly selected for these secondary compounds, but more likely other factors associated with the moister environment were responsible.

Since mountaintop stands were small, it is probable that genetic drift occurred in all stands where there was sufficient isolation. The influence of genetic drift was most apparent in the Scott Mountains. The four Scott Mountain stands were all in close

15 proximity on the same mountain, but they displayed different phenotypes, especially for alpha-humulene production, cone width, cone scale number, and needle epidermis thickness (Fig. 13; Table 3). There were no apparent environmental differences that could have driven this phenotypic differentiation. Moisture regimes could affect needle epidermis thickness and potentially cone sizes, but all stands were in close proximity and located on north to northeast facing slopes, which likely had similar moisture regimes.

Herbivores or pathogens could have acted as selective agents on the production of the secondary compound alpha-humulene, but it is unlikely that these selective agents varied at such a fine scale. Since there is no evidence of selective agents causing the differentiation of Scott Mountain stands, genetic drift is likely responsible for most of these differences.

Regardless of whether genetic drift or natural selection is the primary mechanism behind the MIE within each region, reproductive isolation among stands must have been sufficient for either of these mechanisms to function. Since pine pollen can travel long distances (Bohrerova et al. 2009, Williams and von Aderkas 2011), it is difficult to understand how there was enough isolation for differentiation to occur, but fine scale physical barriers may curtail pollen dispersal (Dyer and Sork 2001). For example, in the

Scott Mountains stand 18 resided just outside of the valley bowl that the other three stands resided, while ridges separated the other stands (Fig. 4). In addition, Shasta red fir, which has been encroaching from lower elevations and, could block pollen transport, grew within stand 27 and stand 18. Moderate isolation could have caused low pollen transport among stands and enhanced inbreeding. If post-pollination success was higher

16 with resident pollen than with neighboring pollen unique genotypes could have arisen

(Varis et al. 2010, Cinget et al. 2015), furthering isolation among stands. This form of competition has not been documented for P. balfouriana, but could potentially explain why differentiation at such local scales is so prominent given the long distances pollen can travel for this genus. In addition, when pollen travels long distances through the air desiccation can occur; the viability of that pollen is unlikely (Bohrerova et al. 2009) making gene flow between stands improbable.

Future research should further explore the modes of isolation among P. balfouriana stands and the mechanisms driving the MIE. Although natural selection may have played some role in the differentiation of populations, I suspect that genetic drift has been more important. Better understanding of pre- and post-pollination processes could shed light on likely mechanisms that allow stands to remain isolated given the close proximity to each other. Also, taking core samples from stands could aid in understanding age structure of stands, seasonal weather patterns, and fire occurrences in each region.

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editors. 2012. The Jepson Manual: Vascular Plants of California, second edition.

University of California Press, Berkeley, Californaia, USA.

Berland, A. 2013. Variation in reproductive characteristics of lodgepole pine (Pinus

contorta var. latifolia) in British Columbia. Thesis, University of Victoria,

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Bohrerova, Z., M. Bohrer, K.D. Cho, M.A. Bolch, and K.G. Linden. 2009. Determining

the viability response of pine pollen to atmospheric conditions during long-

distance dispersal. Ecological Applications 19: 656–667.

Boratynska, K., Muchewicz, E., Drojma, M. 2004. Pinus mugo Turra geographic

differentiation based on needle characters. Dendrobiology 51: 9-17

Boratyńska, K. and A. Boratyński. 2007. Taxonomic differences among closely related

Pines Pinus sylvestris, P. mugo, P. uncinata, P. rotundata and P. uliginosa as

revealed in needle sclerenchyma cells. Flora 202: 555-569.

Blanch, J., J. Peñuelas, J. Sardans, and J. Llusià. 2009. Drought, warming and soil

fertilization effects on leaf volatile terpene concentrations in Pinus halepensis and

Quercus ilex. Acta Physiologiae Plantarum 31: 207-218.

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Cinget, B., S. Gérardi, J. Beaulieu, and J. Bousquet. 2015. Less pollen-mediated gene

flow for more signatures of glacial lineages: congruent evidence from balsam fir

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21

Table 1. Locality information for the stands included in the study. Substrate types: nu= non-ultramafic, u= ultramafic. With the exception of numbers 25 and 26, all stands were previously sampled by Eckert (2006).

Stand Substrate Mountain Region Longitude Latitude 2 NU Yolla Bolly 40.19563 -122.97181 3 NU Yolla Bolly 40.03787 -122.85887 4 U Trinity Alps 40.91232 -122.88764

5 U Trinity Alps 40.91408 -122.88258 6 U Trinity Alps 40.93994 -122.88221

7 U Trinity Alps 40.94016 -122.88858 8 U Trinity Alps 40.94175 -122.89847

9 U Trinity Alps 40.95049 -122.90782 14 NU Trinity Alps 41.05217 -122.91385

15 U Scott Mountains 41.22741 -122.79199 16 U Scott Mountains 41.22124 -122.78895

18 U Scott Mountains 41.23386 -122.77382 19 U Mount Eddy 41.31433 -122.48779

20 U Mount Eddy 41.31688 -122.49014 21 NU Crater Lake 41.38403 -122.58175

22 NU Crater Lake 41.37635 -122.57407 23 NU Little Duck Lake 41.30197 -122.95529

25 U Lake Mountain 41.74908 -123.13416

26 NU Trinity Alps 41.04480 -122.91080

27 U Scott Mountains 41.22820 -122.78790

22

Figure 1. Map of the 20 foxtail pine stands sampled in the Klamath Mountain Range. Colors represent mountain regions (see legend at top left).

23

Figure 2. Location of the two stands sampled in the Yolla Bolly Mountains region

24

Bee Tree Gap

Trinity Lake

Figure 3. Location of the 8 sampled stands in the Trinity Alps mountain region

25

East Bolder Lake

Figure 4. Location of the 4 sampled stands in the Scott Mountain region.

26

Figure 5. Location of the two sampled stands in the Mount Eddy region.

27

Figure 6. Location of the two sampled stands in the Crater Lake region.

28

A B

A

B

C

C D Figure 7. Morphological and anatomical traits. 7A. Apophysis thickness. 7B. Cone length. 7C. Cross section of a needle; A is the distance between the resin canals, B and C are the resin canals. 7D. Cross section of needle (SEM); A is the needle thickness, B is needle width, and C is the needle epidermis thickness. Stomatal crypts indicated by arrows in upper left.

29

Table 2. Summary statistics for 20 traits of Pinus balfouriana. Mean, range, standard deviation, coefficient of variation (C.V.), and SSw/SST (sum of squares within stands/ sum of squares total) were calculated using all 400 trees.

Character Mean SD C.V. SSw/SST Minimum Maximum α- Pinene 20.24 7.18 0.35 .24 8.11 59.39 β- Pinene 36.26 10.58 0.29 .33 4.07 56.98 3- Carene 12.88 5.06 0.39 .21 1.61 37.04 α- Phellandrene 20.15 6.01 0.30 .26 0.00 47.73 Methylthymol 0.05 0.07 1.50 .27 0.00 0.42 Bornyl acetate 0.11 0.72 6.45 .11 0.00 14.31 (-)-trans-Caryophyllene 0.32 0.18 0.55 .11 0.00 1.50 α- Humulene 0.01 0.05 3.22 .33 0.00 0.60 Apophysis Thickness (mm) 3.53 0.68 0.19 .18 2.57 10.41 Apophysis Width (mm) 14.13 1.67 0.12 .36 7.73 21.88 Apophysis Length (mm) 11.59 1.29 0.11 .37 6.61 17.83 Cone Length (mm) 91.95 17.23 0.19 .43 42.59 160.02 Cone Width (mm) 54.29 10.07 0.19 .48 29.72 81.96 Number of Cone Scales 78.68 10.77 0.14 .28 43.00 107.75 Diameter of Large Resin Duct (μm) 85.50 13.57 0.16 .26 55.51 132.57 Diameter of Small Resin Duct (μm) 68.12 11.72 0.17 .24 42.36 105.98 Distance Between Resin Ducts (μm) 292.62 58.56 0.20 .24 156.92 522.56 Needle Thickness (μm) 943.86 111.68 0.12 .34 696.52 1750.14 Needle Width (μm) 1154.13 135.72 0.12 .31 869.73 2011.67 Needle Epidermis Thickness (μm) 59.89 8.30 0.14 .41 40.38 105.67

30

Table 3. Summary of comparison of stands within each region. Factors used in CDA plots (fig. 11-15) are listed along with the total variation explained. Characters with loadings > 0.5 are shown for each factor. Correct classification is the percentage of trees correctly classified into their respective stands by the LDA.

Factors: % variation Correct Regions MANOVA Pillai Characters Loadings explained Classification

1: 39 α-Humulene -0.561 Trinity Alps p<0.0001 2.70 58% 2: 18.9 Cone Length -0.904 Cone Width -0.735 Apophysis Length -0.666

Mount Eddy p<0.0001 0.94 1: 100 Cone Width 0.764 92% Needle Epidermis Thickness 0.510 Methylthymol -0.504 Diameter of large resin duct -0.608

Crater Lake p<0.001 3.35 1: 100 Needle Height -0.572 72% β- Pinene 0.509 α- Phellandrene -0.504

Scott Mountain p<0.0001 4.41 1: 62.24 Cone Width 0.883 71% Number of Cone Scales 0.557 α- Humulene 0.532 2: 31.47 Needle Epidermis Thickness 0.858

Yolla Bolly p<0.0001 7.15 1: 100 Cone Length 0.658 90% Bornyl acetate -0.558 Apophysis Length 0.55

31

pinene decreasespinene

- Methylthymolincreases Beta Alpha-pinene decreases

Figure 8. CDA plot of all 20 sampled northern foxtail pine stands grouped by region (different colors). Each data point represents an individual tree. Factors one and two account for 33% and 23% of total variation, respectively. Arrows indicate the gradient of the most correlated traits.

32

decreases apophysis

Cone length, width, and and width, length, Cone Alpha-humulene decreases

Figure 9. CDA plot of the 8 stands sampled in the Trinity Alps mountain region. Numbers correspond to the stand; bold numbers are mean factor scores for each stand. Notice 3 clusters: stands 14 and 26, stands 4 and 5, and stands 6, 7, 8, and 9. Both factors together account for almost 60%of the variation explained. Arrows indicate the gradient of the most correlated traits. See Appendix B for loadings table.

33

Alpha-phellandrene, large resin duct, needle thickness decreases Beta-pinene increases

Figure 10. CDA plot of the 2 Crater Lake region stands. Numbers correspond to the different stands and the bold numbers are mean factor scored for each stand. Factor 1 accounted for 100% of the variation. Arrows indicate the gradient of the most correlated traits. See Appendix C for loadings table.

34

Bornyl acetate and needle epidermis thickness decreases

Cone length and apophysis width increases

Figure 11. CDA plot of the 2 stands sampled in the Yolla Bolly Mountains region using all 20 characters. Numbers correspond to the stand and the bold numbers are mean factor scores for each stand. Notice two distinct stands without overlap. Factor 1 accounted for 100% of the variation. Arrows indicate the gradient of the most correlated traits. See Appendix D for loadings

35

Methylthymol decreases

Cone width and needle epidermis thickness increases

Figure 12. CDA plot of the 2 stands sampled in the Mount Eddy region using only 19 characters. Numbers correspond to stand and the bold numbers are mean factor scores for each stand. Notice two distinct stands without overlap. Factor 1 accounted for 100% of the variation. Arrows indicate the gradient of the most correlated traits. See Appendix E for loadings.

36

Needle thickness increases epidermis Needle Cone width, number of scales, alpha-humulene increases

Figure 13. CDA plot of the 4 stands sampled in the Scott Mountain region using all 20 characters. Numbers correspond to stand number and the bolded numbers are mean factor scores for each stand. Notice 4 distinct stands with very little overlap. Both factors together accounted for over 90% of the explained variation. Arrows indicate the gradient of the most correlated traits. See Appendix F for loadings table.

37

Figure 14. Cluster analysis results. Dendrogram generated by cluster analysis using the stand means for all 20 traits. Numbers and letters represent the specific stand number and region according to Table 1. Stands that grew on non-ultramafic soils are indicated by the stars.

38

Figure 15. Scatter plot of the relationship between geographic distances and degree of trait differences, based on an Euclidian distance matrix. No relationship was found between trait differences and geographic distance (Mantel test r = -0.088, P = 0.746).

39

Appendix A. Result from the Conical Discriminant Analysis of 20 northern Pinus balfouriana stands based off of 20 characters. 12 factors were significant (P<0.0001).

Character Factor 1 Factor 2 Factor 3 Factor 4

α- Pinene -0.639 0.307 -0.147 0.162

β- Pinene 0.384 -0.577 0.157 0.197

3- Carene -0.213 -0.149 0.063 -0.025

α- Phellandrene -0.417 0.358 0.015 -0.119

Methylthymol 0.146 0.527 -0.319 0.429

Bornyl acetate -0.407 0.102 -0.284 0.198

(-)-trans-Caryophyllene -0.068 0.026 -0.141 0.284

α- Humulene 0.343 0.493 0.035 0.338

Apophysis Thickness -0.128 0.130 0.068 -0.275

Apophysis Width 0.194 0.437 0.176 -0.378

Apophysis Length -0.028 0.034 0.129 -0.200

Cone Length -0.016 -0.241 0.096 -0.027

Cone Width 0.211 0.237 -0.580 -0.309

Number of Cone Scales -0.037 0.235 -0.482 -0.268

Diameter of Large Resin Duct -0.064 -0.026 0.078 -0.383

Diameter of Small Resin Duct -0.011 0.179 0.106 -0.459

Distance Between Resin Ducts 0.180 0.021 0.379 -0.159

Needle Thickness -0.048 -0.044 -0.130 -0.135

Needle Width 0.110 0.099 0.172 0.032

Needle Epidermis Thickness 0.219 -0.054 -0.127 -0.335

% of total variance explained 33.2 23.4 18.3 13.8

40

Appendix B. Result of the Canonical Discriminant Analysis of the Trinity Alps region stands using all 20 characters. 7 factors were significant (P<0.0001).

Character Factor 1 Factor 2 Factor 3 Factor 4

α- Pinene -0.181 -0.195 0.016 -0.276

β- Pinene 0.068 0.148 0.100 0.259

3- Carene 0.223 0.403 -0.228 0.101

α- Phellandrene 0.238 -0.273 0.185 -0.408

Methylthymol 0.026 -0.105 -0.008 -0.115

Bornyl acetate -0.311 -0.262 -0.206 0.155

(-)-trans-Caryophyllene -0.043 -0.255 -0.034 -0.238

α- Humulene -0.561 -0.344 -0.054 0.119

Apophysis Thickness 0.221 -0.490 -0.081 0.315

Apophysis Width 0.244 -0.482 0.225 0.034

Apophysis Length -0.107 -0.666 -0.107 0.089

Cone Length -0.115 -0.904 -0.072 0.123

Cone Width 0.467 -0.735 -0.009 0.239

Number of Cone Scales -0.189 -0.499 0.252 0.141

Diameter of Large Resin Duct 0.224 -0.197 0.042 -0.407

Diameter of Small Resin Duct 0.265 -0.145 -0.201 -0.240

Distance Between Resin Ducts -0.081 -0.120 -0.680 -0.324

Needle Thickness 0.138 -0.134 -0.406 -0.244

Needle Width -0.012 -0.050 -0.316 -0.203

Needle Epidermis Thickness 0.194 0.137 -0.099 -0.033

% of total variance explained 39.00 18.93 16.91 9.50

41

Appendix C. Result of the Canonical Discriminate Analysis for the Crater Lake region stands using all 20 characters. Only factor 1 was significant (P<0.001).

Character Factor 1

α- Pinene -0.343

β- Pinene 0.509

3- Carene 0.227

α- Phellandrene -0.504

Methylthymol 0.022

Bornyl acetate 0.013

(-)-trans-Caryophyllene 0.154

α- Humulene 0.078

Apophysis Thickness 0.247

Apophysis Width -0.255

Apophysis Length 0.409

Cone Length 0.180

Cone Width 0.147

Number of Cone Scales -0.192

Diameter of Large Resin Duct -0.608

Diameter of Small Resin Duct -0.206

Distance Between Resin Ducts -0.190

Needle Thickness -0.572

Needle Width -0.453

Needle Epidermis Thickness 0.301

% of total variance explained 100

42

Appendix D. Result of the Canonical Discriminate Analysis for the Yolla Bolly Mountains region stands using all 20 characters. Only factor 1 was significant (P<0.0001). Character Factor 1

α- Pinene -0.111 β- Pinene -0.054 3- Carene 0.039 α- Phellandrene 0.179 Methylthymol 0.194 Bornyl acetate -0.558 (-)-trans-Caryophyllene -0.429 α- Humulene -0.368 Apophysis Thickness 0.266 Apophysis Width 0.490 Apophysis Length 0.550 Cone Length 0.658 Cone Width 0.310 Number of Cone Scales 0.488 Diameter of Large Resin Duct -0.028 Diameter of Small Resin Duct -0.035 Distance Between Resin Ducts -0.221 Needle Thickness 0.000 Needle Width 0.139 Needle Epidermis Thickness -0.674 % of total variance explained 100

43

Appendix E. Result of the Canonical Discriminate Analysis for the Mount Eddy region stands. Because neither stand had any trace amounts of alpha-Humulene, it was omitted from this analysis thus leaving 19 characters. Only factor 1 was significant (P<0.0001) Character Factor 1

α- Pinene -0.131

β- Pinene -0.050

3- Carene -0.363

α- Phellandrene 0.451

Methylthymol -0.504

Bornyl acetate 0.164

(-)-trans-Caryophyllene 0.026

Apophysis Thickness 0.063

Apophysis Width 0.198

Apophysis Length -0.172

Cone Length -0.066

Cone Width 0.764

Number of Cone Scales -0.202

Diameter of Large Resin Duct 0.116

Diameter of Small Resin Duct -0.064

Distance Between Resin Ducts 0.015

Needle Thickness 0.133

Needle Width 0.115

Needle Epidermis Thickness 0.510

% of total variance explained 100

44

Appendix F. Result of the Canonical Discriminate Analysis for the Scott Mountain region stands using all 20 characters. All three factors were significant (P<0.0001).

Character Factor 1 Factor 2 Factor 3

α- Pinene 0.263 0.169 0.335

β- Pinene -0.433 -0.136 -0.002

3- Carene -0.095 -0.179 -0.342

α- Phellandrene 0.254 0.079 -0.337

Methylthymol 0.364 0.134 0.264

Bornyl acetate 0.417 0.217 0.458

(-)-trans-Caryophyllene 0.022 -0.092 0.073

α- Humulene 0.532 -0.136 0.127

Apophysis Thickness -0.084 -0.231 0.048

Apophysis Width 0.098 -0.006 -0.726

Apophysis Length 0.092 0.155 -0.234

Cone Length 0.348 0.101 -0.278

Cone Width 0.883 0.153 -0.088

Number of Cone Scales 0.557 0.150 -0.015

Diameter of Large Resin Duct -0.002 0.156 -0.371

Diameter of Small Resin Duct -0.010 0.295 -0.429

Distance Between Resin Ducts -0.145 -0.202 -0.219

Needle Thickness 0.255 0.248 -0.453

Needle Width 0.262 0.238 -0.543

Needle Epidermis Thickness -0.134 0.858 -0.251

% of total variance explained 62.24 31.47 6.29

45

Appendix G. Means for 20 traits for all 20 stands. Terpene traits are reported as area percentages.

Pop α- Pinene β- Pinene 3- Carene α- Phellandrene Methylthymol Bornyl acetate (-)-trans-Caryophyllene

2 15.902 44.852 12.235 18.655 0.043 0.001 0.249 3 17.094 45.685 11.922 16.972 0.019 0.045 0.495 4 18.316 40.541 13.941 17.725 0.036 0.073 0.293 5 19.749 39.043 15.145 16.922 0.029 0.048 0.266 6 20.516 38.160 12.457 19.026 0.052 0.101 0.318 7 19.547 32.926 16.372 20.328 0.037 0.099 0.370 8 22.634 36.965 9.8862 23.201 0.053 0.046 0.369 9 20.411 38.993 11.241 21.344 0.047 0.063 0.359 14 24.127 33.574 10.798 19.459 0.053 0.130 0.407 15 21.528 30.375 13.350 20.439 0.106 0.093 0.287 16 19.212 34.434 14.857 18.500 0.080 0.066 0.327 18 15.458 38.544 16.468 19.161 0.027 0.015 0.291 19 24.326 25.606 10.711 29.299 0 0.820 0.284 20 26.323 26.451 15.007 21.795 0.027 0.117 0.279 21 21.222 38.279 12.778 17.824 0.110 0.107 0.411 22 26.639 27.377 11.198 24.565 0.105 0.106 0.355 23 17.221 38.431 9.948 21.363 0 0.033 0.271 25 13.463 46.062 14.670 17.678 0.012 0.066 0.267 26 21.898 37.831 9.347 17.473 0.039 0.151 0.333 27 19.033 29.039 15.910 22.627 0.092 0.067 0.304

46

Appendix G. cont.

Pop α- Humulene Apophysis Thickness (mm) Apophysis Width (mm) Apophysis Length (mm) Cone Length (mm) 2 0 3.56 15.046 11.778 86.226 3 0.088 3.337 13.430 10.238 67.370 4 0 3.319 12.797 10.510 75.732 5 0.002 3.165 12.410 10.230 67.003 6 0.002 3.640 14.037 11.785 91.997 7 0.005 3.795 14.134 11.709 93.985 8 0 3.408 14.288 11.186 83.851 9 0 4.248 13.663 11.735 90.779 14 0.020 3.822 11.857 11.769 88.299 15 0.026 3.345 14.877 12.283 105.85 16 0.030 3.829 14.588 11.816 99.035 18 0.006 3.684 15.367 12.120 98.284 19 0 3.764 14.660 11.954 96.088 20 0 3.684 14.173 12.315 98.173 21 0.024 3.517 13.697 12.176 96.265 22 0.018 3.378 14.158 16.049 97.314 23 0 3.367 14.119 11.913 91.673 25 0.001 3.387 13.852 10.684 83.753 26 0.026 3.840 14.168 12.489 105.452 27 0.039 3.565 15.778 12.339 112.294

47

Appendix G. cont.

Pop Cone Width (mm) Number of Cone Scales Diameter of Large Resin Duct (μm) Diameter of Small Resin Duct (μm) 2 48.452 74.160 83.204 67.553 3 44.728 64.250 83.902 68.325 4 51.843 74.938 73.395 56.616 5 45.317 73.396 85.157 70.198 6 59.255 76.750 87.668 72.498 7 60.301 77.446 88.354 69.476 8 54.476 77.625 90.211 67.346 9 61.768 76.913 84.538 70.380 14 52.379 78.095 80.955 63.817 15 64.216 86.586 90.218 75.432 16 57.401 81.308 83.758 65.540 18 47.637 75.400 91.649 75.287 19 57.469 74.650 91.243 71.322 20 39.879 79.600 88.701 72.803 21 53.669 74.900 78.308 62.967 22 54.312 81.350 97.527 67.164 23 49.636 71.725 75.731 60.746 25 57.300 75.413 80.861 64.740 26 57.566 88.338 81.732 64.381 27 70.415 89.075 91.696 74.607

48

Appendix G. cont.

Pop Distance Between Resin Ducts (μm) Needle Thickness (μm) Needle Width (μm) Needle Epidermis Thickness (μm) 2 312.980 1020.828 1251.831 53.656 3 331.351 1020.792 1224.411 64.625 4 267.229 836.195 1058.709 56.214 5 266.474 945.841 1142.984 64.193 6 338.093 989.889 1165.459 64.108 7 302.387 939.704 1134.774 56.136 8 253.995 898.394 1086.784 59.491 9 268.764 973.182 1143.634 62.134 14 330.188 952.675 1184.031 56.124 15 279.564 993.771 1218.28 72.816 16 306.216 909.104 1109.793 51.879 18 321.623 954.259 1178.463 65.663 19 289.335 964.127 1180.049 61.217 20 288.184 943.372 1159.568 54.899 21 256.752 845.448 1066.134 58.760 22 278.449 954.243 1164.281 55.760 23 310.246 938.976 1178.297 57.874 25 280.782 876.055 1062.748 63.635 26 262.744 888.009 1093.004 57.856 27 305.648 1021.475 1268.500 59.461