PHYLOGEOGRAPHY OF DOUGLAS-FIR: TESTING HYPOTHESES FROM THE FOSSIL RECORD A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY PAUL FRANCIS GUGGER IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ADVISED BY JEANNINE CAVENDER-BARES SHINYA SUGITA JUNE 2010 © Paul F. Gugger 2010 Acknowledgments I am grateful to my advisors, Jeannine Cavender-Bares and Shinya Sugita, for their continuous support and encouragement. I also thank my committee members, Ruth Shaw, Peter Tiffin, and Bob Zink, for helpful conversations and comments on manuscripts. Keith Barker provided invaluable help with analyses and software. Thank you to Nick Deacon, Kari Koehler, Jessica Savage, and Gina Quiram for feedback on manuscripts, lab assistance, and friendship. Special thanks to those who have helped with field work: Antonio González Rodríguez, Barry Jaquish, Michael Stoehr, Carlos Acosta Gómez, Ken Oyama, and Victor Rocha Ramírez. Laura Strickland assisted with the Packrat Midden Database (Chapter 1), Cathy Whitlock and Feng Sheng Hu commented on manuscripts (Chapters 1 and 2, respectively), Art Dyke and Louis Robertson provided ice sheet data used in Figures 1.2, 1.3 and 2.1 (Chapters 1 and 2), Molly Bowen and the Cornell University Computational Biology Service Unit provided computer resources for IMa analyses (Chapter 2 and 3), and Antonio González Rodríguez and Hernando Rodríguez Correa performed lab work on chloroplast microsatellites and ecological niche modeling (Chapter 3). Finally, I express gratitude to those who were influential leading up to the dissertation: John Brick (Montville Township High School), Peter Mohler, Jason McLachlan, and Paul Manos. My dissertation research was made possible by funding from the National Science Foundation Graduate Research Fellowship, Lewis and Clark Field Grant (American Philosophical Society), Sigma Xi Grants-In-Aid, and these sources from the University of Minnesota: Doctoral Dissertation Fellowship, Dayton and Wilkie Natural History Research Grants, Elmer Birney Fellowship, Carolyn Crosby Fellowship, Thesis Research Grant, Center for Community Genetics Research Grant, Florence Rothman Fellowship, and Block Grants. i Dedication To Mom and Dad for always inspiring me to follow my passion. ii Abstract Paleobotanical records and molecular data from modern forests can provide a synergistic understanding of the ecological and evolutionary history of an organism. I used the fossil record to generate hypotheses that I tested with statistical phylogeographic methods for Douglas-fir (Pseudotsuga menziesii). In Chapter 1, I describe alternative scenarios of glacial refugia and postglacial migration based on compiled fossil pollen and macrofossil evidence from the late Quaternary. In Chapter 2, I test those hypotheses using coalescent analyses of mitochondrial and chloroplast DNA sequence data. I also test the paleobotanical hypothesis that Douglas-fir’s two varieties diverged coincident with the Cascade orogeny in the late Pliocene. Finally in Chapter 3, I test whether Mexican Douglas-fir diverged from U.S. populations in the Miocene or Pleistocene, consistent with alternative interpretations of limited fossil evidence in the region. The present patterns of molecular variation in Douglas-fir are well-described by Pliocene (or early Pleistocene) divergence of its varieties, mid-Pleistocene colonization of Mexico, and restriction to multiple glacial refugia in the late Quaternary. Holocene expansion into Canada resulted in recontact among varieties and hybridization driven entirely by pollen dispersal but not seed dispersal. Douglas-fir populations have responded individualistically to past climatic and geologic change, such that some underwent expansions while others contracted to higher elevation and some diverged while others coalesced. These findings highlight the complementary insights that fossil and molecular data provide and can be used to inform the conservation and taxonomy of Douglas-fir. iii Table of Contents Acknowledgments …………………………………………………………………….… i Dedication …...……………………………………………………………………….…. ii Abstract ………………………………………………………………………………... iii Table of Contents ……………………………………………………………………… iv List of Tables ………………………………………………………………………….... v List of Figures …………………………………………………………………….……. vi Introduction …………………………………………………………………………….. 1 Chapter 1: Glacial populations and postglacial migration of Douglas-fir based on fossil pollen and macrofossil evidence ……………………………………………………….... 3 Chapter 2: Phylogeography of Douglas-fir based on mitochondrial and chloroplast DNA sequences: testing hypotheses from the fossil record ………………………………….. 37 Chapter 3: Southward Pleistocene migration of Douglas-fir into Mexico: phylogeography, ecological niche modeling, and conservation of ‘rear edge’ populations …………………………………………………………………………………………... 76 Conclusion ……………………………………………………………………...……. 111 References ……………………………………………………………………………. 113 iv List of Tables Chapter 1 Table 1.1. Conversion of 14C yr BP to cal yr BP ……………………………… 27 Table 1.2. Migration rates ……………………………………………………... 27 Table 1.S1. Fossil pollen site information …………………………………….. 36 Table 1.S2. Macrofossil site information ……………………………………… 36 Chapter 2 Table 2.1. Primers developed for this study …………………………………... 63 Table 2.2. Sequence divergence, mutation rates and molecular clock tests …... 63 Table 2.3. Diversity and neutrality measures for mtDNA and cpDNA ……….. 64 Table 2.4. SAMOVA tables …………………………………………………… 66 Table 2.5. P-values for coalescent tests of population models ………………... 66 Table 2.6. Divergence time estimates …………………………………………. 67 Table 2.S1. Sample site information and haplotype frequencies ……………… 75 Table 2.S2. Mitotype and chlorotype definitions ……………………………… 75 Chapter 3 Table 3.1. Diversity measures for chloroplast data …………………………..... 99 Table 3.2. Diversity measures for mtDNA …………………………………... 100 Table 3.3. Divergence time estimates ………………………………………... 101 Table 3.S1. Sampling site information and haplotype frequencies ………….. 108 Table 3.S2. Mitotype and chlorotype definitions …………………………….. 108 Table 3.S3. Definitions of cpSSR haplotypes ………………………………... 109 Table 3.S4. SAMOVA and AMOVA tables …………………………………. 110 v List of Figures Chapter 1 Figure 1.1. Map of study area and fossil sites …………………………………. 28 Figure 1.2. Presence/absence maps of Douglas-fir at 3,000-year intervals …… 30 Figure 1.3. Hypothesized barriers among glacial populations ………………… 32 Figure 1.4. Change in elevation through time for six regions ………………… 33 Figure 1.5. Change in elevation through time for Eastern Grand Canyon ……. 35 Figure 1.S1. Distribution of fossil pollen sites ………………………………... 36 Figure 1.S2. Distribution of macrofossil sites ………………………………… 36 Chapter 2 Figure 2.1. Hypotheses from the fossil record ………………………………… 68 Figure 2.2. Sampling sites and population groupings …………………………. 70 Figure 2.3. Population models for coalescent simulations ……………………. 71 Figure 2.4. Maps of haplotype distributions with haplotype networks ………... 72 Figure 2.5. Frequency distributions of s for tests of refugia hypotheses ……… 73 Figure 2.6. Glacial refugia and postglacial migration routes ………………….. 74 Chapter 3 Figure 3.1. Map of sample sites and range of Douglas-fir in Mexico ……….. 102 Figure 3.2. Maps of haplotype distributions with haplotype networks ………. 103 Figure 3.3. Correlation of diversity and latitude ……………………………... 104 Figure 3.4. Bayesian skyline plot of Ne through time ………………………... 105 Figure 3.5. Population structure from SAMOVA and UPGMA …………….. 106 Figure 3.6. Potential distributions based on ecological niche modeling ……... 107 vi Introduction Understanding the ecological and evolutionary history of trees is critical to understanding how forests might respond to future climate change, how to best design conservation programs, and what are the major forces driving evolutionary change. For example, some models projecting potential distributions of plants in response to future warming are parameterized with past rates of population expansion (Morin et al. 2008). More generally, the past causes of natural population divergence are central to the study of evolution. Fossil and molecular data offer complementary insights into the history of an organism. The fossil record can provide direct, dated evidence of a species presence, and sometimes abundance, at a particular site. However, fossil records are often incomplete through time and in space, most fossils are only identifiable to species or genus, and species-specific biases in pollen production, dispersal, and preservation mean that arbitrary thresholds must be used to determine presence. On the other hand, molecular data from modern forests can be used to infer historical changes in distribution or population size at the population level across an entire distribution but yield imprecise date estimates. The opposing strengths and weaknesses bring about a synergy of fossil and molecular data that can reveal population-level responses to particular past climatic and geologic events. Two major insights have come from combined fossil and molecular analysis (Petit et al. 2008; Hu et al. 2009). First, populations within a species responded individualistically to changing environmental conditions; some remained small and isolated for long durations, whereas others expanded and contracted dramatically in response to cyclical glaciations (Magri et al. 2006). Second, molecular data suggest that some populations survived the Last Glacial Maximum