A Morphological and Volatile Terpene Analysis of Pinus
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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. v 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