b I ‘ (K. - 1- . . ‘43:; . .h. ‘ w“. .0539. .:§&.$.P :13... u r, ‘ . a _ , ‘ “my ll 4 153' K, ~==€a.::t: - 1.4 a . QWWW r. m t 63 . , . #JHx-r? . . 92...? fl. ....a.. r." “.5, .a Anna. ‘6 2i . a t . . . 5%.». {a S. #5. 1-“. :- Iv. 1333.» n" mafimhfl, gum? :. 35.365 ”31»: c9003 51/757577 This is to certify that the dissertation entitled PHENOTYPES AND FLORAL VARIATION: A PHYLOGENETIC APPROACH presented by ANNA KIRSTEN MONFILS has been accepted towards fulfillment of the requirements for the Doctoral degree in Plant Biol_ogy_ MajorIBlofessor’s Signature 1 0 20 a 3 Date MSU is an Affirmative Action/Equal Opportunity Institution ‘- ‘_‘ “- v '~ PHENOTYPES AND FLORAL VARIATION: A PHYLOGENETICX APPROACH By Anna Kirsten Monfils A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Department of Plant Biology 2003 ABSTRACT PHENOTYPES AND FLORAL VARIATION: A PHYLOGENETIC APPROACH By Anna Kirsten Monfils Biological diversity is created by evolution. To adequately explore biodiversity and examine processes in evolution, scientists need perspective on past evolutionary events. In this study, phenotypic diversity was investigated at several taxonomic levels by examining floral morphological characters against a backdrop of phylogenetic hypotheses. Floral traits were measured and phylogenetic hypotheses were generated across the species’ Pedicularis densiflora and Pedicularis aurantiaca, within the tropical genus Cantua, and throughout the family Brassicaceae. Floral morphological variation among populations of P. densiflora 3.1. was examined and two distinct floral morphological groups were determined. Phenotypic analysis was then combined with a phylogenetic hypothesis in order to examine the number of times different floral morphs arose, relationships among populations in different localities, and potential pollinator shifts. An investigation of the floral diversity and phylogentics among populations in the species P. densiflora s.1. warranted the splitting of the group into two distinct species, P. densiflora and P. aurantiaca. Scanning electron microscopy was used to examine pollen grain morphology within the genus Cantua. Pollen characteristics were then assessed relative to a strongly supported molecular phylogenetic hypothesis. Pollen grain morphology was found to be highly conserved within the genus and synapomorphies in pollen grain characteristics supported relationships hypothesized in the phylogenetic analysis. In addition, a potential correlation between pollen diameter and style length was examined using independent contrast and found to be insignificant. This investigation confirmed the phylogenetic utility of pollen characters and provided no support for the hypothesis that pollen grain diameter is functionally integrated with style length. Floral morphological traits in the Brassicaceae were used to examine phenotypic variance-covariance matrix (P-matrix) stability relative to a comprehensive phylogenetic hypothesis. The P-matrix stability was highly conserved among some genera in the family and more labile among others. This examination of P-matrix stability within the Brassicaceae used a phylogenetic hypothesis to confirm that matrix stability did not follow a stepwise pattern of similarity corresponding to taxonomic rank. Although each chapter of the dissertation used a different group of plants, and varied statistical techniques, they all showed the utility of combining both morphological and molecular analysis to examine patterns of floral diversity. The objectives of each study focused on different aspects of patterns of floral diversity. Ultimately, all of the investigations contained a phylogenetic hypothesis that provided a unique historical perspective to the questions being addressed. ACKNOWLEDGEMENTS This study was funded by the following sources: Botanical Society of America, California Native Plant Society, Department of Plant Biology (Michigan State University; MSU), Ecology, Evolutionary Biology and Behavior Program (MSU), The Graduate School (MSU), NSF Grant: Systematics 0f the Polemom'aceae Subfamily Cobaeoideae, Oregon Native Plant Society, Paul Taylor Funds (Department of Plant Biology, MSU), The Research Training Grant at Kellogg Biological Station (MSU) NSF DBI 9605168, and Sigma Xi. Special thanks to my major professor Alan Prather, for his guidance and assistance during this project. I would also like to thank my committee members Jeff Conner, Tao Sang, and Jim Smith. Thanks to Christy Stewart for assistance with the Brassicaceae project, Jan Szyren for her help with cultivating plants, and Shirley Owens for assistance with the scanning electron microscopy images. I would like to acknowledge and thank Jessie Keith and Chang Bao Li for their assistance in the laboratory, and Orlando Alvarez—Fuentes, Anita D’Avelos, and Eric Linton for statistical support. Thanks to Michael Monfils for editorial assistance and encouragement. Additional thanks to Tracey Bamer, Jason Kilgore, and Anne Plavonich-J ones for helping me complete this work. I would also like to acknowledge my lab mates Amanda Posto, Nate Sammons, and Rachel Williams and the assistant curators of the MSC herbarium, Alan F ryday and Deb Trock. iv TABLE OF CONTENTS LIST or TABLES ........................................................................... vii LIST OF FIGURES .......................................................................... ix INTRODUCTION ........................................................................... 1 CHAPTER 1 Floral Morphological Variation, Molecular Systematics, and Distribution In Pedicularis densiflora Benth. ex Hook. and Pedicularis aurantiaca (E. F. Sprague) Monfils & Prather (Orobanchaceae) ........................... 8 Introduction .......................................................................... 9 Materials & Methods ............................................................... 11 Results ................................................................................ 22 Conclusions ........................................................................... 56 CHAPTER 2 Phylogeny and Pollen Evolution of Cantua (Polemoniaceae subfamily Cobaeoideae): Evidence from Chloroplast and Nuclear DNA Sequence Data. CHAPTER 3 ......................................................................................... 83 Introduction .......................................................................... 84 Materials & Methods ............................................................... 88 Results ................................................................................. 97 Conclusions ........................................................................... 1 14 Evolution of Variance-Covariance Structure of Floral Morphology Among Members of the Mustard Family (Brassicaceae) ................................ 117 Introduction ........................................................................... 1 18 Materials & Methods ............................................................... 125 Results ................................................................................ 136 Conclusions ....................................... ‘ .................................... 1 52 SUMMARY ................................................................................... 157 APPENDICES ................................................................................ 161 Appendix A. Aligned sequences of Pedicularis: nuclear ribosomal internal transcribed spacer regions and 5.88 ribosomal RNA gene ..................... 162 Appendix B. Aligned sequences of Pedicularis: intergenic region between trnL3’ and trnF coding regions ................................................... 176 Table of Contents (Cont’d) APPENDICES CONT’D. Appendix C. Aligned sequences of Pedicularz’s: intergenic region between psbA and trnH coding regions ..................................................... 186 Appendix D. Aligned sequences of Cantua: intergenic region between trnTS’ and trnL coding regions .................................................... 200 Appendix E. Aligned sequences of Cantua: intergenic region between trnL3’ and trnF coding regions .................................................... 215 Appendix F. Aligned sequences of Cantua: 938 base pairs of the nth coding region ......................................................................... 226 Appendix G. Aligned sequences of Cantua: nuclear ribosomal internal transcribed spacer regions and 5.88 ribosomal RNA gene ..................... 247 Appendix H. Aligned sequences of Brassicaceae: 771 base pairs of the nth coding region .................................................................. 263 Appendix I. Pollen data for Cantua (Polemoniaceae):Light microscopy pollen diameter and style length analysis ......................................... 281 LITERATURE CITED ...................................................................... 283 vi Table 10 ll 12 LIST OF TABLES List of populations and voucher specimens for DNA sequence analysis and floral morphological analysis of Pedicularis densiflora and Pedicularis aurantz'aca. .......................................................... List of the 17 floral morphological traits of ten populations of Pedicularis densiflora and Pedicularis aurantiaca analyzed using principal component analysis .................................................. Sample size, means, standard deviations, minima, maxima, and results from ANOVA and Bonferroni (Dunn) T-tests for characters studied in the morphological analysis of ten populations of P. densiflora and P. aurantiaca sampled from California and Oregon ........................... Voucher information for material used in the molecular phylogenetic analyses of Cantua ............................................................... Voucher information for material used in palynological study of Cantua ............................................................................ Pollen diameter and style length of Cantua species included in the analysis ........................................................................... Voucher information for material used in the nth sequencing analysis of Brassicaceae ........................................................ Brassicaceae species measured for floral morphological characters, with sample locality, cultivation site, sample size, and year measured... Example of the phenotypic variance-covariance matrix for the six variables in the Nasturtium oflicinale data set ............................... The P-value results of the CPCRand analysis in the Brassicaceae ........ Results from the principal component analysis of p0pulations in the Brassicaceae. .................................................................. Results of a principal component analysis incorporating measurements from all populations measured in the Brassicaceae and Cleomaceae. . .. vii Page 12 22 3O 89 90 112 126 128 132 139 143 151 List of Tables (cont’d) Table 13 14 15 16 17 18 19 20 21 Aligned sequences of the nuclear ribosomal internal transcribed spacer regions and the 5.88 subunit of ribosomal RNA from the taxa sampled in Pedicularis .................................................................... Aligned sequences of intergenic region between trnL3’ and trnF coding regions from the taxa sampled in Pedicularis ...................... Aligned sequences of intergenic region between psbA and trnH coding regions from the taxa sampled in Pedicularis ............................... Aligned sequences of the intergenic spacer region between the trnTS’ and trnL coding regions for the taxa sampled in Cantua data set. . . . . Aligned sequences of the intergenic spacer region between the trnL3 ’ and trnF coding regions for the taxa sampled in Cantua data set. . . . . Aligned sequences of 938 base pairs of the nth coding region from the taxa sampled In the Cantua data set ...................................... Aligned sequences of the nuclear ribosomal internal transcribed spacer regions and the 5.8S subunit of ribosomal RNA from the taxa sampled in Cantua data set ............................................................... Aligned sequences of 771 base pairs of the 72th coding region from the taxa sampled in the Brassicaceae data set ............................... Pollen grain diameter and style length mean, sample size (N), standard deviation, and range for Cantua herbarium specimens used in the light microscopy pollen analysis ..................................................... viii Page 163 177 187 201 216 227 248 264 282 Figure 10 LIST OF FIGURES Map of Oregon and California representing distribution of 16 sampled populations ofP. densiflora and P. aurantiaca................................ A Pedicularis densiflora flower X 6 and schematic representation of Pedicularis densiflora corolla ................................................. Plot of factor one by factor two from the principal component analysis of 17 floral morphological characters in ten populations of Pedicularis densiflora and P. aurantiaca ................................................... Plot of factor one by factor three from the principal component analysis of 17 floral morphological characters in ten populations of Pedicularis densiflora and P. aurantiaca .................................... Plot of factor two by factor three from the principal component analysis of 17 floral morphological characters in ten populations of Pedicularis densiflora and P. aurantiaca .................................... UPGMA phenogram of taxonomic distance based on 17 continuous floral morphological character traits in ten populations of P. densiflora and P. aurantiaca ............................................................... County map of California. Map shows distribution of P. densiflora and P. aurantiaca based on morphological analysis of populations in the field and herbarium material cited ........................................ County map of Oregon. Map shows distribution of P. densiflora and P. aurantiaca based on morphological analysis of populations in the field and herbarium material cited ............................................ Strict consensus of most parsimonious trees based on anTS sequences in populations ofP. densiflora and P. aurantiaca. Strict consensus of most parsimonious trees based on psbA-trnI-I and trnL-trnF sequences in populations of P. densiflora and P. aurantiaca Page 14 17 25 27 29 40 43 45 48 52 List of Figures (cont’d) Figure 11 12 13 14 15 16 17 18 19 20 21 22 Strict consensus of most parsimonious trees based on psbA-trnH and trnL-th chloroplast sequences and nuclear ribosomal ITS sequences in populations of P. densiflora and P. aurantiaca .......................... Strict consensus of 15 most parsimonious trees based on the Cantua total ITS data set including nucleotide substitutions and indels ........ Strict consensus of two most parsimonious trees based on the Cantua combined chloroplast sequences (trnT-trnL, trnL-trnF, partial nth) including nucleotide substitutions and indels ............................. Strict consensus of four most parsimonious trees based on the Cantua combined chloroplast (trnT-trnL, trnL-trnF, partial nth) and nuclear ITS sequences including nucleotide substitutions and indels ........... Cantua tree estimate for comparative analysis ........................... SEM photographs of pollen grains in Cantua species (Polemoniaceae): (A & B) C. flexuosa; (C & D) C. cuzcoensis; (E & F) C. bicolor; (G & H) C. candelilla ......................................... SEM photographs of pollen grains in Cantua species (Polemoniaceae): (A & B) C. buxifolia; (C & D) C. pyrifolia; (E & F) C. n. sp ............................................................................ SEM photographs of pollen grains in Cantua species (Polemoniaceae): (A & B) C. volcanica (C & D) C. quercifolia. . . . . Diagram representing phylogenetic relationships and pollen synapomorphies 1n Cantua ..................................................... Schematic representation of a generic Brassicaceae flower viewed from the top. .................................................................... Schematic representation of generic Brassicaceae flower in lateral cross-section ..................................................................... A representation of the “Jump Up” approach to matrix comparisons using the F lury hierarchy. .................................................... Page 54 98 101 104 105 107 108 109 113 130 131 134 Table of Figures (Cont’d) Figure Page 23 Strict consensus of 12 most parsimonious trees from the Brassicaceae based on nth sequences ...................................................... 137 24 Strict consensus of 12 most parsimonious trees from the Brassicaceae based on nth sequences with results from common principal component analysis .............................................................. 142 xi INTRODUCTION PHENOTYPES AND FLORAL VARIATION: A PHYLOGENETIC APPROACH Biologists have long been interested in the origin, pattern, and maintenance of diversity among living organisms. Angiosperrns, the dominant plant group, are of particular interest because they have highly variable floral morphologies among species. Theories have been proposed that explain flowering plant diversity as a product of the close relationships among floral form, plant pollinator interactions, and reproductive success. A comprehensive study of flowering plant evolution is central to understanding the pattern and diversity of floral morphological variation (Darwin 1862). Even with the recent advances in statistics and molecular techniques, diversity among organisms is most often quantified using phenotypic characters. Phenotypes are the manifestation of heritable genetic variation and environmental influences. In order to study floral diversity, we need to consider morphology and variation in an evolutionary context (Felsenstein 1985, Harvey & Pagel 1991). A phylogeny is a hypothesis of the evolutionary history of a group of organisms. A robust phylogenetic reconstruction can be used to assess patterns of evolutionary changes. Phylogenies are usually displayed as a bifurcatin g tree representing the evolutionary relationships among taxa, historical relationships between common ancestors, and speciation events. Although we can never know for certain the historical events leading to the diversity currently observed among organisms, phylogenies allow us to generate testable hypotheses of relationships (Henni g 1950). Without such an historical perspective, phenotypic diversity can be interpreted to support erroneous historical relationships and the polarity of floral character traits cannot be tested. Phylogenetics has made great advances over the last twenty years with the advent of molecular methodologies. Data for cladistic analyses can be generated quickly and relatively cheaply across species, genera, families, and even kingdoms. Phenotypic characters can now be evaluated against an independent phylogenetic backdrop based on molecules. Phylogenetic approaches have increasingly been applied to questions outside the realm of taxonomy. For instance, phylogenetic hypotheses are being used in the field of community ecology to explore community assemblages and niche structure (Webb et a1. 2002). Conservation biologists use molecular systematics to define species, identify biologically important lineages, and set conservation priorities (Moritz 1995, Soltis and Gitzendanner 1999). Population biologists are starting to use comparative methods to explore quantitative genetic questions, thus initiating a new field of research, comparative quantitative genetics (Steppan et a1. 2002). Phylogenies are currently used in comparative physiology and development to choose experimental systems, determine trait polarity, study the pace of physiological and developmental change, and establish independent contrasts of traits among related groups (Monson 1996). Plant ecologists and evolutionists alike are using phylogenetic techniques to explore the evolution of plant pollination systems (Armbruster 1993, Bruneau 1997, McDade 1992), breeding systems (Rieseberg et al. 1992, Sakai et a1. 1997), hybridization (Riesberg & Ellstrad 1993, Sang et a1. 1995), polyploid evolution (Soltis and Soltis 2000), coevolution (Futuyma 1995), and evolutionary development (Albert et al. 1998). In this study, I used phylogenies to study floral morphology, functionally integrated floral characters, and variation and covariation among traits. I examined floral morphological variation in three groups: two closely related species of Pedicularis, among the species of Cantua, and across the family Brassicaceae. A molecular phylogenetic hypothesis was central to the analysis of each group. Pedicularis densiflora and P. aurantiaca were examined to detect and explain patterns of variation among populations of the two species. UPGMA, ANOVA, and PCA analyses were conducted, over 1000 herbarium specimens were studied, and a phylogenetic analysis of psbA-trnH and trnL-trnF chloroplast sequences and nuclear ribosomal ITS sequences was undertaken. Examination of all three statistical morphological analyses and review of herbarium specimens verified two distinct species, P. densiflora and P. aurantiaca. Pedicularis aurantiaca has a large calyx, floral tubes not fully exserted at anthesis, reduced lower labium, and a large opening in the galea. Pedicularis denszflora has a short calyx with a fully exerted floral tube, an enlarged lower labium, and a smaller opening in the galea. Phylogenetic analysis of the two species confirmed they were closely related taxa, sister to a clade containing Pedicularis semibarbata. Comprehensive review of the floral variation coupled with a phylogenetic hypothesis justified elevating the subspecies P. aurantiaca subsp. aurantiaca to species status. A study of Cantua (Polemoniaceae) was undertaken to estimate a phylogeny, examine pollen exine sculpturing and its utility in phylogenetic analysis, and investigate the potential functional integration between pollen size and style length within the genus. A phylogenetic analysis of the genus using chloroplast (trnT-trnL, trnL-trnF, partial nth) and nuclear ITS sequences was generated, scanning electron and light microscopy was used to view pollen morphology, and independent contrasts were used for the pollen grain diameter and style length traits. Cantua was found to be monophyletic and several relationships within the genus were resolved. Cantua volcanica was shown to be most closely related to C. quercifolia. Cantuaflexuosa, C. cuzcoensis, C. bicolor, C. . candelilla, C. buxifolz'a, C. pyrifolia and C. nov. sp. were closely related and C. pyrifolz'a was sister to C. nov. Sp. Analyses of the chloroplast and nuclear DNA sequences had conflicting results regarding the monophyly of C. bwcifolia and its relationship within the clade. Pollen characteristics within Cantua were found to be highly conserved. Spheroidal pantoporate pollen with an insulous semitectate sexine is synapomorphic for Cantua in the Cobaeoideae, giving further support to the monophyly of the genus. The relationship between C. quercifolia and C. volcanica was supported by the synapomorphy of supratectal verrucae on the sexine of the pollen grains. Neither a significant regression slope nor correlation was found using independent contrasts of pollen diameter and style length in Cantua, which rejects the null hypothesis of a morphological integration between the two traits. To summarize the Cantua results, some phylogenetic relationships within the genus were resolved, a review of pollen morphology was completed and the utility of pollen morphology in phylogenetic analyses was confirmed, and pollen diameter and style length was found to be statistically unrelated in Cantua. My research on the Brassicaceae was designed to test the hypothesis that variance- covariance matrices diverge and change following a step-wise structured pattern identical to the hierarchical family level phylogeny. Six floral morphological traits were measured in 24 Brassicaceae species to generate phenotypic variation-covariation matrices (P- matrix) for each species. A phylogenetic hypothesis of the same 24 species was generated using sequences from the nth coding region. Common principal component analysis was used to compare matrices among groups of taxa which corresponded to clades in the phylogenetic hypothesis. Equality in matrices between different species was found between Barbarea verna and B. vulgaris and proportional matrices were accepted between Raphanus raphanistrum and Raphanus sativus, as well as Nasturtium officinale and Cardamine concatenata. Proportionality among matrices in five genera was found in the A. lyrata —— Cardamine concatenata clade. Within Brassica, species had low levels of shared structure. In the Brassicae clade and its sister clade Sisymbrium altissimum /Stanleya pinnata low levels of matrix stability were found among genera. This was also true of the Hesperis matronalz's / Matthiola clade and the Lobularia / Iberis clades. The results of this study confirm that P-matrix, and potentially G-matrix, stability is highly variable and the phylogenetic level of the matrix comparison did not appear to predict the level of stability found among matrices, although generally comparisons at higher taxonomic levels resulted in lower levels of shared structure. Each chapter of my dissertation used a different group of plants to examine patterns of floral diversity using phenotypes, as well as different statistical techniques. The objectives of each study focused on different aspects of floral diversity and extended from population-level analyses ofvariation to a family-wide analysis of floral evolution. Ultimately all of the investigations were based on a phylogenetic hypothesis which provided an historical perspective. CHAPTER 1 FLORAL MORPHOLOGICAL VARIATION, MOLECULAR SYSTEMATICS, AND DISTRIBUTION IN PEDICULARIS DENSIFLORA BENTH. EX HOOK. AND PEDICULARISAURANTIACA (E. F. SPRAGUE) MONFILS & PRATHER (OROBANCHACEAE) INTRODUCTION Pedicularis L. is a large (600-800 species), monophyletic genus (Ree 2001) of hemiparasitic herbs, with a northern temperate distribution, and a tremendous amount of diversity in floral morphology. Previously, Pedicularis was placed in the family Scrophulariaceae, however recent taxonomic treatments have placed the genus in the Orobanchaceae (Olmstead et a1. 2001). Pedicularis has undergone a relatively recent diversification, likely in the Pleistocene, and Shares a center of diversity in eastern Asia with its primary pollinators, Bombus (De-Yaun 1983). Considerable research has been done to investigate the reproductive biology of Pedicularis and document the modes of plant-pollinator interaction (Sprague 1960, 1962, Grant 1966, 1967, Macior 1973, 1982, 1983, 1984, 1986a, 1986b, 1995a, 1995b, 1996, and references therein, Robart 2000, Ree 2001). This study focused on Pedicularis densiflora, a species found only in California and southern Oregon, which was first described from a communication with D. Douglas by Bentham, and published in the Flora Boreali-American_a by J. W. Hooker (1838). Sprague (1958) divided the species into two subspecies, P. densiflora subsp. densiflora Benth. ex Hook. and P.1densiflora subsp. aurantiaca E. F. Sprague. The circumscriptions of the subspecies were based on distribution, reproductive biology, phenology, host affinity, and floral morphology (Sprague 1958, 1960, 1961, and 1962). Sprague’s (1958) treatment indicated that P. densiflora subsp. densiflora is pollinated by both Bombus and hummingbirds while P. densiflora subsp. aurantiaca is pollinated exclusively by hummingbirds. Macior (1986a) documented pollinator type, frequency and mode of pollination of both subspecies. Macior’s (1986a) research supported Sprague’s (1958, 1960, and 1962) conclusions of different pollination between subspecies. Sprague (1962) and Macior (1986a) suggested that hummingbird pollination was driving a change in morphology among populations and creating a Shift within the Species from a bumblebee pollination syndrome to a hummingbird syndrome. Fundamental to their hypothesis was the concept that the species had diverged into two distinct subspecies P. densiflora subsp. densiflora and P. densiflora subsp. aurantiaca with unique floral morphologies and distinct pollinator regimes. Additionally, this hypothesis assumed that the ancestral pollination system in P. densiflora 3.1. is Bombus pollination. There have been no molecular or detailed quantitative morphological studies to either polarize character traits or definitely document either genetic or morphological divergence within the Species. An extensive examination was undertaken to detect and explain patterns of infraspecific variation within the species P. densiflora 3.1. and determine which pollination system is derived. This work revealed that P. densiflora Should be more narrowly circumscribed, and that the subspecies P. densiflora subsp. aurantiaca Should be elevated to species status as P. aurantiaca (E. F. Sprague) Monfils & Prather. The two taxa will be referred to by their species names for the remainder of this paper. MATERIALS AND METHODS Herbarium specimens — A total of 1069 herbarium Specimens ofPedicuIaris densiflora, P. aurantiaca and P. semibarbata A. Gray from CAS, DS, J EPS, MSC, ORE, OSC, POM, RSA, UC, and WILLU were studied. Specimens were screened for both morphological variation and geographic distribution. A preliminary investigation of herbarium material was used to select population study sites and floral characters for measurement. Study populations were selected from throughout the range of both P. densiflora and P. aurantiaca, sampling a broad elevational, geographic, and environmental Spectrum. Populations were representative of the full breadth of morphological diversity. Additional analyses of herbarium Specimens were conducted to evaluate utility and reliability of morphological traits in distinguishing separate species and to comprehensively review the distribution of the two taxa. Analysis of Floral Morphological Variation — During a preliminary field season in 1998 localities were screened and ten populations were chosen for the study of floral morphological divergence. Populations used in the morphological study were from the following ten localities: Bear Valley (Colusa/ Lake Co., CA), Hurles Circle (Butte Co., CA), Inskip (Butte Co., CA), McBride Springs (Siskyou Co., CA), Missouri Flats Road (Josephine Co., OR), Mount Diablo (Contra Costa Co., CA), Paradise (Marin Co, CA), Pinehurst (Jackson Co., OR), Round Valley Historical Marker (Mendocino Co., CA), and Santa Margarita Lake (San Luis Obispo Co., CA) (Table 1 and Figure 1). Voucher specimens were deposited in the Michigan State University Herbarium (MSC). ll Table 1. List of populations and voucher Specimens for DNA sequence analysis and floral morphological analysis of Pedicularis densiflora and Pedicularis aurantiaca. All voucher specimens are deposited at Michigan State University Herbarium (MSC) DNA Floral Species Location (County, State) Voucher Sequence Morphological Analysis Analysis P. aurantiaca Auberry Monfils 4 X (Fresno Co., CA) P. aurantiaca Hobo Gulch Monfils 9 X (Trinity Co., CA) P. aurantiaca Inskip MoanlS 37 X X (Butte Co., CA) P. aurantiaca McBride Springs Monfils 43 X X (Siskyou Co., CA) P. aurantiaca Pinehurst Monfils 40 X X (Jackson Co., OR) P. aurantiaca Weaver Camp Monfils 8 X (Trinity Co., CA) P. densiflora Bear Valley (Colusa/Lake Monfils 33 X X Co., CA) P. densiflora Briceland Monfils 14 X (Humbolt Co., CA) P. densiflora Hurles Circle Monfils 35 X X (Butte Co., CA) P. densiflora Missouri Flat Road Monfils 36 X X (Josephine Co., OR) P. densiflora Mount Diablo Monfils 28 X X (Contra Costa Co., CA) P. densiflora Paradise Monfils 34 X X (Marin Co., CA) P. densiflora Pinnacles National Monfrls 20 X Monument (San Benito Co., CA) P. densiflora Round Valley Historical MoanlS 32 X X Marker (Mendocino Co., CA) P. densiflora San Marcos Pass Monfils l X (Santa Barbara Co., CA) P. densiflora Santa Margarita Lake Monfils 21 X X (San Luis Obispo Co., CA) P. semibarbata A San Gorgonio Wilderness Holmgren X (San Bemardino Co., CA) 3591 P. semibarbata B East Sirretta Pass Twisselman X (Tulare Co., CA) 15760 P. cystopteridifolia A Red Lodge-Cook City Beaman 1551 X Highway (Carbon Co., WY) P. cystopteridifolia B Cody Kirkpatrick X (Park Co., WY) 3491 P. bracteosa A Absaroka Mountains Nelson 10994 X (Freemont Co., WY) P. bracteosa B Mazama Churchill X (Whatcom Co., WA) 7671512 Figure 1. Map of Oregon and California representing distribution of 16.Sampled populations of P. densi/lora and P. aurantiaca. Names refer to population localities. All populations sampled were used in the molecular analysis. Populations with astrix afier the name were used in the morphological analysis. Circles represent populations of P. densiflora and triangles represent populations of P. aurantiaca. All populations sampled were used in the molecular analysis. Populations with asterix after the name were used in the morphological analysis. 13 Missouri F lat* Hobo Gulch Weaver Cam . Bricelan 0 Round Valley" Bear Valley“ Paradise“ Mount Diablo“ Pinnacles NM Sta. Margarita Lake" San Marcos Pass 0 Pedicularis denszflora A Pedicularis aurantiaca Figure 1. KPInehurst” O '1 GON McBride Springs” Inskip“ Hurles Circle“ Auberry C . LIFORNIA When available, fifty individuals per population were studied. A total of 466 flowers were measured. Within each population, one flower each was removed from 50 plants. Plants were selected by sampling at set intervals along transects encompassing the population range. Flower sampling was based on the most apical, fully developed, intact flower with an exserted stigma. Only flowers that were Shedding pollen and had not yet begun to senesce were selected. Measurements were made using digital calipers and recorded to the nearest 0.01 mm. Floral characters used in the analysis were chosen based on the characters used by Sprague (1958) to differentiate the subspecies, floral traits involved in effective pollination as described by Macior (1986), and floral traits associated with pollination syndromes of Bombus and Hummingbird pollination as described by (Citation)Thirteen quantitative characters were measured: galea height, tube height, galea length, tube length, abaxial margin of galea, calyx length, pistil length, stamen length, labium length, angle at the axis of galea and tube, tube exsertion (extension of tube above the calyx lobes), corolla length, and labium tip to galea bend (Figure 2). Four additional characters were derived from the initial measurements, treated as ratios; and used to quantify the relationship between Shape and size: labium length/galea length, tube length/galea length, tube height/galea height, and abaxial margin of galea/galea length. Statistical analysis — Statistical Analysis System (SAS Institute, Inc. 1985) was used to perform a non- hierarchical analysis among population means for all 17 floral morphological characters Figure 2: A. Pedicularis densiflora flower X 6. Reproduced from A Manual of the F loweringPlantS of California (J epson, 1925). B. Schematic representation of Pedicularis densiflora corolla. Lines represent parameters of quantitative measurements and letters correspond to the following measurements: A. galea height, B. galea length, C. tube length, D. tube height, E. labium length, F. abaxial margin of galea, and G. labium tip to galea bend. .mm 85mm _ .<~ 2&5 tint 8:53 IV \ x x. All «030 93:30 830 IV \ a... 17 using the principal component analysis (PCA). Before analysis, all mean measurements were standardized across the populations with a mean of zero and a variance of one. Varimax rotation (Kaiser 1958) was conducted to maximize the variance of the variable loadings within a factor. The end result is a polarized loading for the variables in each factor closer to zero or one. Varimax rotation simplifies the interpretation of each factor in the PCA analysis (Johnson & Wichem 2002). Analysis of variance (ANOVA) was performed on all 17 individual characters for the ten sampled populations. The PROC UNIVARIATE and HOVTEST=LEVENE tests were conducted to assure normality and homogeneity of variance, respectively. Where necessary, data were log, square root, or arccosine transformed to fulfill the assumptions of standard variance and normality. Bonferroni (Dunn) T-tests were conducted on each variable to examine differences between all possible pairs of means and Significant groupings among means for all ten populations. A symmetric dissimilarity matrix was created fiom a rectangular data matrix of the standardized 17 morphological character traits measured in the ten selected populations using the program NTSYSpc version 2.1 (Applied Biostatistics, Inc.). The average “taxonomic” distance coefficient was utilized as a measure of dissimilarity. The unweighted pair-group method of cluster analysis (UPGMA) was performed on the dissimilarity analysis, and a UPGMA phenogram was constructed. l8 Analysis of Population Level Molecular Variation — Sampling - Leaf tissue was sampled from throughout the range of P. densiflora and P. aurantiaca for 16 populations. For the 10 populations used in the morphological analysis, floral tissue was collected and preserved in silica gel. Populations were used in the molecular study from the following sixteen localities: Auberry (Fresno Co., CA), Bear Valley (Colusa/Lake Co., CA), Briceland (Humbolt Co., CA), Hobo Gulch (Trinity Co., CA), Hurles Circle (Butte Co., CA), Inskip (Butte Co., CA), McBride Springs (Siskyou Co., CA), Missouri Flat Road (Josephine Co., OR), Mount Diablo (Contra Costa Co., CA), Paradise (Marin Co, CA), Pinehurst (Jackson Co., OR), Pinnacles National Monument (San Benito Co., CA), Round Valley Historical Marker (Mendocino Co., CA), Santa Margarita Lake (San Luis Obispo Co., CA), San Marcos Pass (Santa Barbara Co., CA), and Weaver Camp (Trinity Co., CA) (Table l and Figure 1). Additional tissue of Pedicularis taxa was collected from MSC Specimens of the following species: P. semibarbata, P. cystopteridifolia Rydb., and P. bracteosa Benth. ex Hook. (Table 1). Outgroup and Additional T axa - Pedicularis cystopteridifolia, was chosen as an outgroup and P. semibarbata and P. bracteosa were sampled to explore potential relationships to P. densiflora and P. aurantiaca. Outgroup and taxon choice was based on Sprague’s (1962) analysis of trait Similarity among California Species of Pedicularis, unpublished . nuclear ribosomal internal transcribed Spacer (ITS) phylogenies of North American Pedicularis (Robart 2000), and morphological studies of herbarium specimens. Analysis - Total DNA was extracted from either leaf or flower material using the techniques of Loockerman and Jansen (1996) and purified using the Schleicher & Schuell Elu-quick DNA Purification Kit (Keene, NH). Two sets of chloroplast primers were used: trnL-trnF primers from F ujii et al. (1997), and psbA-trnH from Sang et a1. (1997). ITSl, ITS2, and the 5.83 regions of nuclear ribosomal DNA were amplified using ITS-4 primers of White et a1 (1990) and the modified ITS-5 primer of Sang et a1. (1995). Standard amplification protocols were used, including reaction mixture components and the PCR profile (e. g. Prather et a1. 2002), and amplifications were performed on a MJ Research PTC-lOO therrnalcycler. PCR products were gel purified using the Schleicher & Schuell Elu—quick DNA Purification Kit, (Keene, NH) and sequenced in both directions using an ABI-3 73 automated sequencer. Sequencing reactions were conducted using the AmpliTaq DNA Dye Terminator Cycle Sequencing reagents (PE Applied Biosystems, Norwalk, CT). Edited sequences were aligned using the Sequencher 3.0 software (Gene Codes Corporation, Ann Arbor, MI). Termini of the trnL-trnF intergenic Spacer region and psbA-trnH intergenic spacer region were determined from Fujii (1997) and Ambrosini et al. (1992) respectively. Termini of ITSI, ITSZ, and the 5.83 regions of nuclear ribosomal DNA were determined by comparison with published sequences of Beardsley and Olmstead (2002). In the aligned sequences of the psbA-trnH intergenic Spacer region, 39 base pairs (bp237-bp276) were excluded fi'om the analysis due to ambiguity in alignment. Parsimony methods were implemented using PAUP* (version 4.0b4; Swofford 2000). Heuristic searches were performed using the TREE BISECTION RECONNECTION, and 20 MULTREES options. All insertion/deletion events, displayed as gaps in the data set, were treated as missing data. Bootstrap analyses were conducted using 10,000 replicates with 100 random addition-sequence replicates per bootstrap. A partition homogeneity test was conducted to test for homogeneity in the distribution of phylogenetic information between the nuclear and chloroplast data sets. 21 RESULTS Morphological Variation - Based on the PCA analysis, 96% of the variance in the data set was explained by the first three factors. Factor one explained 45% of the variation and was weighted for characters of galea height, tube height, abaxial margin of galea, calyx length, tube exsertion, labium tip to galea bend, abaxial margin of galea/galea length. All these features corresponded to floral Shape. Factor two explained 40% of the variation and was weighted by the following characters: tube length, pistil length, stamen length, corolla length, and tube length/galea length. These floral characteristics were predominantly attributed to flower Size and exsertion of the galea. Factor three explained I 1 % of the variation in the data set and was strongly weighted by tube height/galea height (Table 2). Table 2. List of the 17 floral morphological traits often populations of Pedicularis densiflora and P. aurantiaca analyzed using principal component analysis. The first row shows proportion of variance explained by the first three factors. Varimax rotated factor patterns for the first three factors are listed next to the floral morphological trait Factor 1 Factor 2 Factor 3 Proportion of variance explained by factor 45% 40% 11% Rotated factor pattern for Varimax rotation method GALEA HEIGHT 0.93716 0.15449 0.24154 TUBE HEIGHT 0.85129 0.31551 0.37159 GALEA LENGTH 0.46109 0.45989 0.53159 TUBE LENGTH -0.00203 0.93353 0.21847 ABAXIAL MARGIN OF GALEA 0.89167 0.23682 0.19791 CALYX LENGTH 0.98270 0.07492 0.03785 PISTIL LENGTH 0.18670 0.93626 0.02864 STAMEN LENGTH 0.24642 0.89566 0.32723 LABIUM LENGTH -0.84662 0.28518 0.15236 ANGLE AT AXIS OF GALEA AND TUBE -0.40163 0.52962 0.26179 TUBE EXSERTION -0.88867 0.42396 0.07335 COROLLA LENGTH 0.19754 0.84532 , 0.37367 LABIUM TIP TO GALEA BEND 0.93273 0.13294 0.21471 ABAXIAL MARGIN OF GALEA/GALEA 0.93363 0.15635 0.10176 LENGTH LABIUM LENGTH / GALEA LENGTH -0.70018 0.24598 0.29567 TUBE LENGTH / GALEA LENGTH -0.24703 0.93865 0.00712 TUBE HEIGHT / GALEA HEIGHT 0.19050 0.31215 0.91926 22 Factor one clearly separated two distinct clusters of populations: One group consisting of Inskip (J), McBride Springs (H), and Pinehurst (l) and the second of Bear Valley (D), Hurles Circle (F), Missouri Flats (G), Mount Diablo (C), Paradise (B), Round Valley (E), and Santa Margarita Lake (A) (Figure 3 & 4). Factor two did not separate the populations into distinct clusters; populations were broadly distributed with Bear Valley (D) and Santa Margarita Lake (A) representing the two extremes (Figure 3 & 5). Santa Margarita Lake had the smallest size measurements for overall corolla size, galea length, stamen length, tube length, pistil length, abaxial margin of galea/galea length, tube length/galea length, and abaxial margin of galea. Bear Valley had the largest values for tube length and tube exsertion. Many of these variables were heavily weighted in the second factor of the PCA analysis and provide an explanation for the separation of Santa Margarita Lake and Bear Valley in the plots with factor two. Factor three displayed a continuum of values with Missouri Flats (G) and Inskip (J) having larger values. Factor three was heavily weighted for tube height/galea height. Missouri Flats (G) and Inskip (J) exhibited the highest ratio of all populations sampled (Figure 4 & 5). Sixteen of the seventeen variables Showed Si gnificant variation among population means (p value < 0.0001) in the ANOVA (Table 3). The only character that was not Significantly different among populations Q) = 0.0363) was the angle at axis of galea and tube. This substantiated and quantified the previous reports by Sprague (1958, 1960, and 1962) and Macior (1986a) of a broad Spectrum of morphological diversity across the distribution of P. densiflora and P. aurantiaca. 23 Figure 3. Plot of factor one by factor two from the principal component analysis of 17 floral morphological characters in ten populations of Pedicularis densiflora and P. aurantiaca. Letters represent populations as follows: A. Santa Margarita Lake, B. Paradise, C. Mount Diablo, D. Bear Valley, E. Round Valley Historical Marker, F. Hurles Circle, G. Missouri Flat Road. H. McBride Springs, 1. Pinehurst, and J. Inskip. 24 0.0 —4p— mcouomm m.o- “ .m 853m m.N- : m.p Feouomu 25 Figure 4. Plot of factor one by factor three from the principal component analysis of 17 floral morphological characters in ten populations of Pedicularis densiflora and P. aurantiaca. Letters represent pOpulations as follows: A. Santa Margarita Lake, B. Paradise, C. Mount Diablo, D. Bear Valley, E. Round Valley Historical Marker, F. Hurles Circle, G. Missouri Flat Road. H. McBride Springs, 1. Pinehurst, and J. Inskip. 26 m.m MLOHomm 0.0 _ _ 0.0 m.0- 0.9- I H t m.0- 0.0 m.P wcouomu 27 Figure 5. plot of factor two by factor three from the principal component analysis of 17 floral morphological characters in ten populations of Pedicularis denszflora and P. aurantiaca. Letters represent populations as follows: A. Santa Margarita Lake, B. Paradise, C. Mount Diablo, D. Bear Valley, E. Round Valley Historical Marker, F. Hurles Circle, G. Missouri Flat Road. H. McBride Springs, 1. Pinehurst, and J. Inskip. m.N 0.N 0.F _ _ meowomu m.0 “ 0.0 m.0- mm .mntswrm 0.0 0.0 m.F «Loyomu 29 Table 3. Sample Size, means, standard deviations, minima, maxima and results from AN OVA and Bonferroni (Dunn) T-tests for characters studied in the morphological analysis of ten populations of Pedicularis densiflora and P. aurantiaca sampled from California and Oregon. In Bonferroni (Dunn) T-tests means with same letter are not significantly different. Where necessary, data were log, square root, or arcos transformed, this is denoted in the ANOVA RESULTS column as LOG, SQUARE ROOT, or ARCOS respectively. POPULATION MEAN MIN.- MAX. ANOVA BONFERRONI (Sample size) (SD) RESULTS (DUNN) T-TESTS (p-value) l. GALEA HEIGHT (mm) SANTA MARGARITA 5.27 4.43-6.56 (PR>F=0.0001) D C LAKE (N=19) (0.49) . PARADISE 5.12 4.20-6.45 LOG D (N=50) (0.44) MOUNT DIABLO 5.10 3.99-6.33 D (N=46) (0.46) BEAR VALLEY 5.31 4.20-6.53 D C (N=50) (0.53) ROUND VALLEY 5.01 4.02-6.42 D HISTORICAL MARKER (0.52) (N=51) HURLES CIRCLE 5.58 4.37-7.11 B C (N=51) (0.59) MISSOURI FLAT ROAD 5.68 4.60-6.64 B (N=50) (0.48) MCBRIDE SPRINGS 6.27 5.21-8.07 A (N=50) (0.64) PINEHURST 6.19 4.89-7.63 A (N=50) (0.61) INSKIP 6.46 5.08-8.62 A (N=50) (0.76) 2. TUBE HEIGHT (mm) SANTA MARGARITA 3.18 2.75-3.91 (PR>F=0.0001) D E LAKE (N=19) (0.31) PARADISE 3.09 2.54-3.88 LOG E (N=50) (0.32) MOUNT DIABLO 2.99 2.41-3.76 E (N=46) (0.31) BEAR VALLEY 3.39 2.67-4.3 D (N=50) (0.39) ROUND VALLEY 3.16 2.54-3.69 D E HISTORICAL MARKER (0.30) (N=51) HURLES CIRCLE 3.39 2.75-4.05 D (N=51) (0.33) MISSOURI FLAT ROAD 3.70 2.89-4.55 C (N=50) (0.35) MCBRIDE SPRINGS 4.00 3.30-4.94 A B (N=50) (0.44) PINEHURST 3.81 3 04-479 C B (N=50) (0.38) 30 Table 3 (cont’d) POPULATION MEAN MIN .— MAX. ANOVA BONFERRONI (Sample size) (SD) RESULTS (DUNN) T-TESTS (p-value) 2. TUBE HEIGHT CONTINUED (mm) INSKIP 4.26 2.97-5.12 A (N=50) (0.48) 3. GALEA LENGTH (mm) SANTA MARGARITA 18.79 15.39-21.10 (PR>F=0.0001) E LAKE (N=19) (1.41) PARADISE 18.95 15.19-22.20 LOG E (N=50) (1.70) MOUNT DIABLO 18.98 15.45-22.88 E (N=46) (1.94) BEAR VALLEY 19.72 16.39-23.19 E C D B (N=50) (1.69) ROUND VALLEY 19.25 16.70-22.40 E C D HISTORICAL MARKER (1.38) (N=51) HURLES CIRCLE 19.07 16.03-22.83 E D (N=51) (1.69) MISSOURI FLAT ROAD 21.35 18.00-24.75 A (N=50) (1.64) MCBRIDE SPRINGS 20.28 16.64-24.87 A C D B (N=50) (1.85) PINEHURST 20.41 14.29-24.03 A C B (N=50) (1.69) INSKIP 20.60 16.50-25.53 A B (N=50) (2.08) 4. TUBE LENGTH (mm) SANTA MARGARITA 10.26 7.18-13.53 (PR>F=0.0001) D LAKE (N=19) (1.69) PARADISE 12.83 8.90-18.13 LOG C B (N=50) (1.90) MOUNT DIABLO 11.90 753-1527 C (N=46) (1.52) BEAR VALLEY 15.29 10.51-20.70 A (N=50) (2.35) ROUND VALLEY 12.56 938-18.70 C HISTORICAL MARKER (1.77) (N=51) HURLES CIRCLE 13.01 9.49-17.85 C B (N=51) (1.83) MISSOURI FLAT ROAD 14.63 9.49-20.65 A (N=50) (2.37) MCBRIDE SPRINGS 12.81 860-1693 C B (N=50) (1.77) PINEHURST 11.91 905-1645 C (N=50) (1.41) INSKIP 14.18 8.28-19.41 A B §=50) (2.22) 31 Table 3 (cont’d) POPULATION MEAN MIN.— MAX. ANOVA BONFERRONI (Sample size) (SD) RESULTS (DUNN) T-TESTS (p-value) 5. ABAXIAL MARGIN OF GALEA (mm) SANTA MARGARITA l 1.36 10.05-13.01 (PR>F=0.0001) D LAKE (N=19) (0.98) PARADISE 12.30 9.86-15.91 LOG C (N=50) (1.37) MOUNT DIABLO 12.54 980-200] C (N=46) (1.63) BEAR VALLEY 12.66 9.98-15.71 C (N=50) (1.24) ROUND VALLEY 12.45 10.33-15.01 C HISTORICAL MARKER (1.13) (N=51) HURLES CIRCLE 12.87 10.36-15.99 C (N=51) (1.39) MISSOURI FLAT ROAD 14.86 12.18-17.63 B (N=50) (1.23) MCBRIDE SPRINGS 17.52 14.57-22.07 A (N=50) (1.86) PINEHURST 18.15 13.10-22.28 A (N=50) (1.59) INSKIP 17.88 14.18-23.27 A (N=50) (2.04) 6. CALYX LENGTH (mm) SANTA MARGARITA 13.99 12.36-16.78 (PR>F=0.0001) C LAKE (N=19) (1.20) PARADISE 12.67 10.13-15.11 LOG D (N=50) (1.17) MOUNT DIABLO 12.70 10.54-16.78 D (N=46) (1.41) BEAR VALLEY 14.17 10.72-17.75 C (N=50) (1.35) ROUND VALLEY 12.39 983—1609 D HISTORICAL MARKER (1.25) (N=51) HURLES CIRCLE 13.66 10.19—16.35 C (N=51) (1.45) MISSOURI FLAT ROAD 13.24 10.81-15.60 C D (N=50) (0.97) MCBRIDE SPRINGS 18.83 13.08-24.03 A (N=50) (2.31) PINEHURST 17.14 1198-21. 10 B (N=50) (1.77) INSKIP 18.64 15.57-22.92 A (N=50) (1.69) 7. PISTIL LENGTH (mm) SANTA MARGARITA 25.48 22.47-29.61 (PR>F=0.0001) E LAKE (N=19) (1.93) Table 3 (cont’d) POPULATION MEAN MIN.— MAX. ANOVA BONFERRONI (Sample size) (SD) RESULTS (DUNN) T-TESTS (p-value) 7. PISTIL LENGTH CONTINUED (mm) PARADISE 35.44 24.67-44.08 C D B (N=50) (4.79) MOUNT DIABLO 33.18 25.54-42.01 D (N=46) (3.52) BEAR VALLEY 38.73 28.50-46.00 A (N=50) (3.83) ROUND VALLEY 35.92 30.44-43.71 C A D B HISTORICAL MARKER (2.87) - (N=51) HURLES CIRCLE 34.08 24.64-43.29 C D (N=51) (4.50) MISSOURI FLAT ROAD 37.99 29.33-48.64 A B (N=50) (4.19) MCBRIDE SPRINGS 36.62 29.74-44.42 C A B (N=50) (3.30) PINEHURST 36.09 17.14-44.73 C A D B (N=50) (4.63) INSKIP 37.17 27.68-46.03 A B (N=50) (4.89) 8. STAMEN LENGTH (mm) SANTA MARGARITA 26.42 16.91-30.95 (PR>F=0.0001) E LAKE (N=19) (3.11) PARADISE 31.09 23.19-38.63 C (N=50) (3.45) MOUNT DIABLO 28.67 21.45-35.49 D (N=46) (3.45) BEAR VALLEY 33.29 24.03-40.28 C A (N=50) (3.32) ROUND VALLEY 31.16 24.39-39.42 C HISTORICAL MARKER (3.01) (N=51) HURLES CIRCLE 31.06 25.18-39.67 C (N=51) (3.54) MISSOURI FLAT ROAD 34.46 27.78-41.66 A (N=50) (3.14) MCBRIDE SPRINGS 31.92 24.62-41.15 C B (N=50) (3.02) PINEHURST 31.41 24.09-36.88 C (N=50) (2.61) INSKIP 34.13 26.51-40.94 A B (N=50) (3.19) 9. LABIUM LENGTH (mm) SANTA MARGARITA 3.82 1.12-5.49 (PR>F=0.0001) D LAKE (N=19) (1.07) PARADISE 4.09 1.84-7.01 SQUARE C D (N=50) (0.95) ROOT 33 Table 3 (cont’d) POPULATION MEAN MIN .- MAX. AN OVA BONFERRONI (Sample size) (SD) RESULTS (DUNN) T-TESTS (p-value) 9. LABIUM LENGTH CONTINUED(mm) MOUNT DIABLO 5.20 372-1223 A B (N=46) (1.32) BEAR VALLEY 5.29 3.22-8.98 A B (N=50) (1.19) ROUND VALLEY 4.65 2.88-6.77 C B HISTORICAL MARKER (0.98) (N=51) HURLES CIRCLE 4.30 2.69-7.44 C D (N=51) (0.91) MISSOURI FLAT ROAD 5.85 3.7-12.68 A (N=50) (1.34) MCBRIDE SPRINGS 2.87 1.30-4.71 E (N=50) (0.75) PINEHURST 2.86 1 .41-4.24 E (N=50) (0.70) INSKIP 3.13 164-526 E (N=50) (0.90) 10. ANGLE AT AXIS OF GALEA AND TUBE (degrees) SANTA MARGARITA 144.56 123.50-180 (PR>F=0.0363) A LAKE (N=19) (12.47) PARADISE 147.32 122.76-180 A (N=50) (10.86) MOUNT DIABLO 145.63 125.60-180 A (N=46) (9.02) BEAR VALLEY 149.90 121 .68-180 A (N=50) (10.09) ROUND VALLEY 143.73 99.37-180 A HISTORICAL MARKER (13.98) (N=51) HURLES CIRCLE 149.02 118.10-180 A (N=51) (13.63) MISSOURI FLAT ROAD 151.57 132.58-180 A (N=50) (10.14) MCBRIDE SPRINGS 144.71 117.73- A (N=50) (9.90) 163.88 PINEHURST 143.34 109.30-180 A (N=50) (15.45) INSKIP 145.90 105.92- A (N=50) (11.64) 167.81 11. TUBE EXSERTION (mm) SANTA MARGARITA -3.74 -7.14-0. l6 (PR>F=0.0001) C LAKE (N=19) (1.94) PARADISE 0.17 -4.12-5.26 A B (N=50) (1.95) MOUNT DIABLO -0.80 -4.76-2.31 B (N=46) (1.54) 34 Table 3 (cont’d) POPULATION MEAN MIN.- MAX. ANOVA BONFERRONI (Sample size) (SD) RESULTS (DUNN) T-TESTS (p-value) l l. TUBE EXSERTION CONTINUED (mm) BEAR VALLEY 1.12 -2.99-5.39 A (N=50) (2.17) ROUND VALLEY 0.16 -3.76-6.88 A B HISTORICAL MARKER (1.93) (N=51) HURLES CIRCLE -0.65 -5.74-4.31 B (N=51) (2.06) MISSOURI FLAT ROAD 1.38 -3.05-7.95 A (N=50) (2.31) MCBRIDE SPRINGS -6.02 —10.33- E (N=50) (2.00) (-l .63) PINEHURST -5.36 -9.62—12.19 E D (N=50) (1.76) INSKIP -4.46 -1 1.25-2.05 C D (N=50) (2.65) 12. COROLLA LENGTH (mm) SANTA MARGARITA 29.04 24.80-32.93 (PR>F=0.0001) E LAKE (N=19) (2.33) PARADISE 31.79 25.06-39.71 C D (N=50) (3.19) MOUNTDIABLO 30.88 23.24-37.05 E D (N=46) (2.95) BEARVALLEY 35.00 27.98-42.15 A B (N=50) (3.35) ROUNDVALLEY 31.81 27.99-41.10 C D HISTORICAL MARKER (2.70) (N=51) HURLES CIRCLE 32.08 26.62-39.99 C D (N=51) (3.23) MISSOURI FLAT ROAD 36.18 28.60-42.24 A (N=50) (3.61) MCBRIDE SPRINGS - 33.09 25.54-41.80 C B (N=50) (3.04) PINEHURST 32.32 23.97-38.18 C D (N=50) (2.50) INSKIP 34.78 293342.15 A B =50) (3.02) 13. LABIUM TIP TO GALEA BEND (mm) SANTA MARGARITA 11.26 8.90—13.83 (PR>F=0.0001) B C LAKE (N=19) (1.41) PARADISE 10.54 8.76-13.07 B C (N=50) (1.20) LOG MOUNT DIABLo 11.64 9.29-14.68 A B (N=46) (1.35) BEAR VALLEY 12.34 9.48-15.54 A (N=50) (1.28) 35 _+4 Table 3 (cont’d) POPULATION MEAN MIN.— MAX. ANOVA BONFERRONI (Sample Size) (SD) RESULTS (DUNN) T-TESTS (p-value) 13. LABIUM TIP TO GALEA BEND CONTINUED(mm) ROUND VALLEY 11.45 874-1369 A B C HISTORICAL MARKER (1.08) (N=51) . HURLES CIRCLE 10.51 828-1356 (N=51) (1.36) MISSOURI FLAT ROAD 12.34 990-1609 A (N=50) (1.28) MCBRIDE SPRINGS 5.63 3.27-7.28 D (N=50) (0.91) PINEHURST 5.11 3.62-6.88 E (N=50) (0.72) INSKIP 5.85 4.48-7.82 D (N=50) (0.84) 14. ABAXIAL MARGIN OF GALEA/GALEA LENGTH SANTA MARGARITA 0.60 0.53-0.67 (PR>F=0.0001) E LAKE (N=19) (0.05) PARADISE 0.65 0.55-0.72 ARCCOS C D (N=50) (0.04) MOUNT DIABLO 0.66 0.57-1.07 C D (N=46) (0.07) BEAR VALLEY 0.64 052-075 D (N=50) (0.05) ROUND VALLEY 0.65 0.58-0.73 C D HISTORICAL MARKER (0.04) (N=51) HURLES CIRCLE 0.68 0.53-0.79 B C (N=51) (0.05) MISSOURI FLAT ROAD 0.70 0.62-0.96 B (N=50) (0.05) MCBRIDE SPRINGS 0.86 0.79-0.95 A (N=50) (0.03) PINEHURST 0.89 0.82-0.96 A (N=50) (0.03) INSKIP 0.87 0.78-0.94 A (N=50) 0.03 15. LABIUM LENGTH / GALEA LENGTH SANTA MARGARITA 0.205 0.060-0.297 (PR>F=0.0001) B LAKE (N=19) (0.058) PARADISE 0.216 0.097-.360 B (N=50) (0.045) MOUNT DIABLO 0.276 0166-0655 A (N=46) (0.073) BEAR VALLEY 0.270 0167-0525 A (N=50) (0.063) ROUND VALLEY 0.242 0150-0378 A B HISTORICAL MARKER (0.052) fl=51) 36 Table 3 (cont’d) POPULATION MEAN MIN.-— ANOVA BONFERRONI (Sample size) (SD) MAX. RESULTS (DUNN) T-TESTS (p-value) 15. LABIUM LENGTH / GALEA LENGTH CONTINUED HURLES CIRCLE 0.226 0147-0341 B (N=51) (0.042) MISSOURI FLAT ROAD 0.275 0188-0690 A (N=50) (0.071) MCBRIDE SPRINGS 0.141 0069-0214 C (N=50) (0.034) PINEHURST 0.141 0060-0201 C (N=50) (0.035) INSKIP 0.152 0079-0239 C (N=50) (.044) 16. TUBE LENGTH / GALEA LENGTH SANTA MARGARITA 0.548 0.365-0.744 (PR>F=0.0001) E LAKE (N=19) (0.096) PARADISE 0.677 0533-0840 B (N=50) (0.081) MOUNT DIABLO 0.629 0479-0824 C (N=46) (0.079) BEAR VALLEY 0.777 0.548-1 . 106 A (N=50) (0.1 15) ROUND VALLEY 0.653 0488-0878 B HISTORICAL MARKER (0.080) (N=51) HURLES CIRCLE 0.682 0.4890877 B (N=51) (0.071) MISSOURI FLAT ROAD 0.682 0497-0977 B (N=50) (0.111) MCBRIDE SPRINGS 0.634 0491-0827 B (N=50) (0.084) PINEHURST 0.586 0.437-0.757 E (N=50) (0.072) INSKIP 0.696 0364-1042 B (N=50) (0.127) I 17. TUBE HEIGHT / GALEA HEIGHT SANTA MARGARITA 0.606 0.474-0.694 (PR>F=0.0001) C LAKE (N=19) (0.056) PARADISE 0.605 0445-0739 C (N=50) (0.060) MOUNT DIABLO 0.589 0.489-0.713 D (N=46) (0.056) BEAR VALLEY 0.642 0481-0835 C (N=50) (0.072) ROUND VALLEY 0.633 0509-0762 C HISTORICAL MARKER (0.063) (N=51) HURLES CIRCLE 0.611 0479-0771 C (N=51) (0.059) 37 Table 3 (cont’d) POPULATION MEAN MIN.- MAX. ANOVA BONFERRONI (Sample size) (SD) RESULTS (DUNN) T-TESTS (p-value) 17. TUBE HEIGHT / GALEA HEIGHT CONTINUED MISSOURI FLAT ROAD 0.654 0501-0806 A B (N=50) (0.070) MCBRIDE SPRINGS 0.639 0494-0747 C A B (N=50) (0.062) PINEHURST 0.618 0504-0733 C D B (N=50) (0.056) INSKIP 0.664 0466-0921 A (N=50) (0.078) Bonferroni (Dunn) T-tests indicated that the Inskip, Pinehurst, and McBride Springs populations Showed means not Si gnificantly different from each other, but distinct from the remaining populations for the following. five variables: galea height, abaxial margin of galea, labium length, abaxial margin of galea/galea length, and labium length/galea length. McBride Springs and Inskip had no Si gnificant difference in mean calyx length (18.83 mm and 18.64 mm, respectively) but were Significantly different from the remaining eight populations. Pinehurst had a mean calyx length of 17.14 mm, which was , also significantly different from all other populations (<14.17 mm). In the case of labium tip to galea bend, Pinehurst had the lowest mean at 5.11 mm and was significantly different from all other populations. McBride Springs and Inskip had Similar means of 5.63 mm and 5.85 mm, respectively, which were significantly different from the remaining populations and represented the lowest means besides Pinehurst. All other mean measurements of labium tip to galea bend exceeded 10.51mm. Means for tube exsertion were extreme for Inskip (-4.46 mm), Pinehurst (-5.36 mm), McBride Springs (-6.02 mm), and Santa Margarita Lake (-3.74 mm); pair-wise 38 comparisons of the population means were Si gnifieantl y different from the remaining populations. Three separate groupings of the above populations were assessed as not significantly different: Pinehurst/Inskip, Santa Margarita Lake/Inskip, and McBride Springs/Pinehurst. These taxa were Clearly larger and separated from the remaining populations, whose means for tube exsertion from -.0880 mm to 1.38 mm. No distinguishable patterns were observed for the remaining variables; however, the Bear Valley, Missouri Flat, and Inskip populations showed overall larger measurements. ANOVA results corroborated the reports by Sprague (195 8, 1960, 1962) of substantial morphological variation within the Species P. densiflora and P. aurantiaca. Sixteen of the 17 traits measured were Si gnificantly different among the ten populations in this study. Examination of population means using the Bonferroni (Dunn) T-tests indicated a cohesive morphology among the McBride Springs, Inskip, and Pinehurst populations. The UPGMA phenogram summarized potential relationships indicated by the metric of taxonomic distance. Division of the populations into two distinct clusters was substantiated by the phenogram which showed high similarity among the Inskip, McBride Springs, and Pinehurst populations. These three populations formed a Cluster while the remaining seven populations, Bear Valley, Hurles Circle, Missouri Flats, Mount Diablo, Paradise, Round Valley, and Santa Margarita Lake formed a second distinct grouping (Figure 6). 39 .0 8:me can _ 2an E wontomow 5038805 Cocos?» 98 £5083 .moEm: conflsmom .eoenzeaze N can Scranton Q 0o macaw—smog E 38b DOSE? EoEo—ofiroE Eon £53528 2 so 083 853.61% 282853 0o ESwocofi (32003 .0 2:05 ooEEQ 988980. 50.0 mm; mm .0 _ — P P h h _ b bl - I Ned 00.0 b P h K - aims T 35550 r 425.2% 8562 a: 2.5822 i l 43:52 Sum 65.85 .562 >o=m> venom - 285 Sign 80980 8:3 85$va .Sm 40 Examination of all three morphological analyses, PCA, ANOVA, and UPGMA, suggested two distinct morphological groups of populations. Pedicularis aurantiaca was represented by Inskip, McBride Springs, and Pinehurst populations which formed a well- defined group of populations with similar floral morphology. Pedicularis densiflora was represented by seven populations which formed a second distinct group, these included Bear Valley, Hurles Circle, Missouri Flats, Mount Diablo, Paradise, Round Valley, and Santa Margarita Lake populations. Observations on the ten populations combined with a review of herbarium Specimens helped Characterize floral traits, phenology and distribution in the two Species. Pedicularis aurantiaca had long calyces (12-24 mm), a reduced ratio of the labium to galea (approx. 1:7), a short labium (1-5 mm), a short region fi'om the labium tip to corolla bend (3.5-7.5 m), an enlarged abaxial margin of the galea about 7/8 as long as the galea, and a tube that is included in the calyx. Pedicularis aurantiaca flowered late April through June and grew at elevations between 100 and 7000 feet. Pedicularis aurantiaca was distributed in southeast Oregon in Klamath and Jackson Counties, northern California in the Trinity and Siskiyou Alps, and in the Sierra Nevadas as far south as Kern County (Figure 7 & 8). Pedicularis densiflora had short calyces (10-18mm), a larger labium to galea ratio (approx. 1:4), a long labium (2-12 mm), a long region from the labium tip to corolla bend (8.5 — 16 mm), an truncated abaxial margin of galea approximately 2/3 the length of the 41 Figure 7. County map of California. Map shows distribution of P. densiflora and P. aurantiaca based on morphological analysis of populations in the field and herbarium material Cited. Circle represents presence of P. densiflora herbarium material in the county. Triangle represents presence of P. aurantiaca herbarium material in the county. 42 .. 2.- ‘96.... AWE? Rana” *5. Stu. mu E52530 Figure 8. County map of Oregon. Map Shows distribution of P. densiflora and P. aurantiaca based on morphological analysis of populations in the field and herbarium material cited. Circle represents presence of P. densiflora herbarium material in the county. Triangle represents presence of P. aurantiaca herbarium material in the county. 44 3333.53 MCKSSONEK a BONUSES mteNseQmm e 4 «so .u. flu 1 . v.14.” cameo ‘. .w 65m; 45 galea, and a tube that is exserted from the calyx. The Species flowered between February and April and grew at elevations ranging from 100-4000 feet. Pedicularis densiflora was distributed along the coast from southern California into southern Oregon, with occurrences as far south as Baja, California. Populations occur in the foothills of the Sierra Nevadas in Butte, Calavaras, and Nevada counties. Field observations and a review of herbarium Specimens indicate that the distribution of Pedicularis aurantiaca is contiguous with that of P. densiflora in Jackson County in Oregon and Butte, Calavaras, Colusa, Lake, Nevada and Trinity Counties in California (Figure 7 & 8). Molecular Variation -— Six of the 16 populations examined in the molecular phylogenetic analysis were representative of P. aurantiaca: Auberry, Hobo Gulch. Inskip, McBride Springs, Pinehurst, and Weaver Camp. Pedicularis densiflora was represented by ten of the sixteen populations examined: Bear Valley, Briceland Bridge, Hurles Circle, Missouri Flat, Mount Diablo, Paradise, Pinnacles National Monument, Round Valley Historical Marker, San Marcos Pass, and Santa Margarita Lake. The ITS sequences were 603 bp long, with 74 parsimony-informative Characters. There were 16 most parsimonious trees with a length of 114, CI. excluding uninforrnative Characters (C.I.°) = 0.9032, and R1. = 0.9543. Bootstrap support, as Shown on the strict consensus tree (Figure 9), for a clade including McBride Springs, Inskip, and Pinehurst, as well as two geographically proximate sites not measured in our morphological analysis formed a monophyletic group, albeit with weak bootstrap support (bootstrap value of 46 Figure 9. Figure represents the strict consensus tree of 16 most parsimonious trees and is based on nuclear ribosomal ITS sequences 603 bp long, with 74 parsimony-informative characters. Phylogeny has a tree length of 114, C.I.c = 0.9032, and RI. = 0.9543. Numbers above the branches represent bootstrap percentages based on 10,000 replicates. Names represent locations of populations of P. densiflora/ P. aurantiaca. Populations of P. aurantiaca denoted with an asterix. Location and voucher information is available in Table l and Figure 1. 47 P. cystopteridifolia A 100 100 P. cystopteridifolia B 1—-— P. bracteosa A Figure9. 79 100 '— P. bracteosa B P. semibarbata A 98 100 64 P. semibarbata B McBride Springs“ Pinehurst* 69 Hobo GulCh* Inskip" Weaver Camp"‘ Hurles Circle Briceland Mount Diablo -— Missouri Flat Round Valley Paradise -—- Pinnacles Bear Valley r— Santa Margarita Lake San Marcos Pass Auberry Road“ 48 64%). Inclusion of Hurles Circle into the clade McBride Springs -— Hurles Circle had 100 % bootstrap support. The clade Briceland- Santa Margarita Lake had strong bootstrap support (97%). San Marcos Pass and Auberry Road populations were part of a polytomy at the base of the P. densiflora/ P. aurantiaca clade. The phylogeny Showed 98% bootstrap support for the monophyly of the P. densiflora/ P. aurantiaca clade. Independent phylogenetic analyses were initially conducted on the two Chloroplast regions. The aligned sequences ofpsbA-trnH were 605 base pairs long with 566 base pairs included in the analysis, 20 of which were phylogenetically informative. Thirty nine base pairs (bp238 through bp276) were excluded from the analysis due to ambiguity in alignment. Parsimony analysis ofpsbA-trnH intergenic Spacer resulted in 42 most parsimonious trees, with a length of 41 , a C.I.c = 1.00, and a R.I. = 1.00. The aligned sequences of trnL-trnF were 404 base pairs long with 12 substitutions, nine of which were phylogenetically informative. Parsimony analysis resulted in six most parsimonious trees, with a length of 12, a C.l.° =1, and a R.I. =1. The trnL-trnF base pair data analysis showed no resolution among the populations of the P. densiflora and P. aurantiaca species. Five P. densiflora populations (Briceland, Mount Diablo, Missouri Flat, Round Valley, and Paradise) and one P. aurantiaca population (Inskip) Share a 51 bp insertion/deletion event (Indel). This indel was treated as missing data in the parsimony analysis. 49 Data from the Chloroplast region was combined for a total 01970 base pairs with 29 parsimony informative Characters (Figure 10). Twenty eight most parsimonious trees were generated, with a length of 53, C1." = 1.00, and R. I. = 1.00. Two clades with low support were resolved. Briceland, Missouri flats, and Round Valley formed a polytomy with 63% bootstrap support. The clade that includes a more inclusive clade of those three populations and Mount Diablo had a bootstrap support of 64%. The P. densiflora/ P. aurantiaca clade has 94% bootstrap support I did not reject the null hypothesis of homogeneity in distribution of phylogenetic information between the data sets based on the partition homogeneity test (p=0.59) and the data were combined for an analysis of relationships among populations of P. densiflora and P. aurantiaca (Figure 11). The combined data set had 1573 base pairs with 103 parsimony informative characters. Eight most parsimonious trees resulted from the analysis, with a length of 168, C.1.c = 0.9194, and R. I. = 0.9600. The clade McBride Springs -— Hurles Circle had 100% bootstrap support. The Briceland-Santa Margarita Lake clade formed a monophyletic group with 99% bootstrap support. The San Marcos Pass and Auberry Road populations were part of an unresolved polytomy at the base of the P. densiflora/ P. aurantiaca clade. The phylogeny had 100% bootstrap support for the monophyly of the P. densiflora/ P. aurantiaca clade. 50 Figure 10: Figure represents the strict consensus tree of 28 most parsimonious trees and iS based on psbA-trnH and trnL-trnF sequences 970 bp long, with 29 parsimony- inforrnative Characters. Phylogeny has a tree length of 53, C.I.° = 1.00, and R. I. = 1.00. Numbers above branches are bootstrap percentages based on 10,000 replicates. . Names represent locations of populations of P. densiflora/ P. aurantiaca. Populations of P. aurantiaca denoted with an asterix. Location and voucher information is available in Table 1 and Figure 1. 51 P. cystopteridifolia A 99 75 P. cystopteridifolia B — P. bracteosa A Figure 10. 100 99 —— P. bracteosa B 1—— P. semibarbata A 95 ‘— P. semibarbata B McBride Springs* Pinehurst“ Hobo Gulch” Inskip“ Weaver Camp* 64 63 Hurles Circle Briceland Missouri Flat Round Valley Mount Diablo Paradise San Marcos Pass Pinnacles Bear Valley Auberry Road* Santa Margarita Lake Figure 11: Figure represents a strict consensus of 8 most parsimonious trees and is based on combined analysis of psbA-trnH and trnL-trnF Chloroplast sequences and nuclear ribosomal ITS sequences 1573 bp long, with 103 parsimony-informative characters. Phylogeny has a tree length of 168, C1." = 0.940, and R. l. = 0.960. Numbers above branches represent bootstrap percentages based on 10,000 replicates. Names represent locations of populations of P. densiflora/ P. aurantiaca. Populations of P. aurantiaca denoted with an asterix. Location and voucher information is available in Table 1 and Figure 1. 53 P. cystopteridifolia A 100 P. cystopteridifolia B '— P. bracteosa A 100 *— P. bracteosa B ~— P. semibarbata A 100 100 64‘ *— P. semibarbata B .6—3-E McBride Springs* Pinehurst* Hobo Gulch* & 100 99 Inskip“ Weaver Camp* ‘50 Hurles Circle Briceland 51 Missouri Flat Round Valley Mount Diablo Paradise Pinnacles Bear Valley Santa Margarita Lake San Marcos Pass Figure 11. Auberry Road“ 54 The Chloroplast and ITS primers were used to amplify regions of DNA to assess some inter-specific relationships among Closely related Pedicularis Species. Our combined analysis Showed the P. semibarbata and P. bracteosa clades were monophyletic (100% bootstrap support). Pedicularis semibarbata was Sister to the P. densiflora/ P. aurantiaca clade (100%) and P. bracteosa was Sister to the P. densiflora/P. semibarbata clade with 100% bootstrap support. 55 CONCLUSION Morphological Variation - Quantitative traits measured on ten populations, field observations, and an examination of herbarium specimens showed considerable variation in floral morphological measurements among the pOpulationS studied. These results indicated two points: the P. densiflora/ P. aurantiaca group had considerable floral morphological variation among populations, and some of this variation could be attributable to distinct groups, which herein are treated as two species, P. densiflora and P. aurantiaca. Molecular Variation -— Pedicularis densiflora and P. aurantiaca are Closely related Sister taxa. The phylogenetic analysis indicated the McBride Springs — Weaver Camp clade of P. aurantiaca populations was Closely related, genetically distinct and derived from within P. densiflora/P. aurantiaca clade. However the Hurles Circle (P. densiflora) population had a shared ancestry with this clade and the P. aurantiaca population of Auberry road and a P. densifiora population of San Marcos Pass were part of a polytomy at the base of the P. densiflora/P aurantiaca clade. In addition, in the trnL-trnF spacer region, Inskip (P. aurantiaca) shared a 51 bp indel with Briceland, Mount Diablo, Missouri Flat, Round Valley, and Paradise (P. densiflora) and this provides some evidence for a Shared Chloroplast ancestry among these six populations. Therefore no conclusions regarding the monophyly or ancestry of P. aurantiaca could be made. More informative markers may provide additional resolution for discerning the relationships among the species. 56 M Sequence data from the sampled Species indicated a common ancestor between P. densiflora, P. aurantiaca and P. semibarbata. This relationship was proposed by Li (cited as a personal communication by Sprague) and Sprague (1962). Robart’s (2000) ITS sequence phylogeny included the narrow California endemic P. howellii A. Gray. This taxon was sister to P. semibarbata, in a clade sister to the clade of P. densiflora and P. aurantiaca. This result was not surprising, P. densiflora, P. aurantiaca, P. semibarbata and P. howellii have similar floral morphologies, inhabit similar habitats, are geographically proximate, and parasitize similar woody host plants. Ancestral Pollination State- Many studies of reproductive biology of Pedicularis in the United States were conducted by Macior(1973, 1977, 1982, 1983, 1984, 1986a, 1986b, 1995a, 1995b, 1996, 1997 and references therein) who noted the Close association between Pedicularis and Bombus pollinators. With some rare exceptions noted in this study, there appeared to be a “virtually exclusive Bombus/Pedicularis reproductive association” in the genus (Macior 1997; l). Pedicularis cystopteridifolia (Macior 1983), P. howellii (Macior 1986b), and P. bracteosa (Macior 1973, Robart 2001) are Bombus pollinated. Pedicularis semibarbata is pollinated exclusively by small bees, Osmia tristella (Macior 1977). Pedicularis densiflora is pollinated by both Bombus and hummingbird pollinators and P. aurantiaca is pollinated exclusively by hummingbirds (based on overlapping populations studied by Macior 1986a, and Sprague 1960, 1962). 57 Long corolla tubes, abundant nectar, lack of floral scent, and red coloration are floral traits present in both P. densiflora and P. aurantiaca which have an association with hummingbird pollinators (Sprague 1960, Grant & Grant 1968, F aegri & van der Pijl 1966). In P. densiflora young flowers are visited by Bombus pollinators before the floral tubes are fiIlly exserted and while the nectar is accessible to the Short-tongued pollinators (Macior 1986). The lower labium of P. densiflora iS larger and capable of serving as a landing platform for Bombus pollinators. In P. aurantiaca the calyx is larger and supports the corolla tube. This trait can serve to protect the flower during the nectar probing of hummingbird visitation (Faegri & van der Pijl 1966). The labium is also significantly smaller and the abaxial margin larger in the P. aurantiaca vs. P. densiflora flowers. The labium reduction eliminates the Bombus landing platform and the enlarged abaxial margin can accommodate the larger head of the hummingbird pollinators. Detailed pollination ecology studies (Macior 1973, 1977, 1983, 1986 a and b, Robart 2001 , and Sprague 1960, 1962).were coupled with phylogenetic sequence data from the chloroplast and nuclear genome and the ITS sequence phylogeny of Robart (2000). This work supported the hypothesis of a major pollination shift in P. semibarbata with a transition from Bombus to small bee pollination. In addition, there are two potential evolutionary scenarios for pollinator shifts within P. densiflora and P. aurantiaca: A. The ancestral state of Bombus pollination in the outgroup of P. cystopteridifolia and P. bracteosa, with a partial switch to a shared pollination biology of Bombus and hummingbird pollinators in P. densiflora and a complete transition to exclusive hummingbird pollination in P. aurantiaca, or B. The ancestral pollination state of 58 Bombus pollination in the outgroup of P. cystopteridifolia and P. bracteosa, with a complete loss of Bombus pollination and a switch to hummingbird pollination in the P. aurantiaca/ P. densiflora clade, and a second Shift to shared pollination biology of regained Bombus pollination in addition to hummingbird pollination in P. densiflora. Due to the partially unresolved phylogeny among populations of P. densiflora and P. aurantiaca and the difficulty of mapping polymorphic pollination states, it is not possible to distinguish between the two evolutionary scenarios described. Taxonomic treatment — Review of herbarium Specimens, statistical analysis including ANOVA, PCA, and UPGMA, Show a clear distinction between the Species P. aurantiaca and P. densiflora. Pedicularis aurantiaca has a large calyx, floral tubes not fully exerted at anthesis, reduced lower labium and a large opening in the galea. Pedicularis densiflora has a short calyx with a fully exerted floral tube, an enlarged lower labium and a smaller opening in the galea. Flowering times and locality are different between the Species. Pedicularis aurantiaca flowers later in the season, at higher elevations and grows in southern Oregon, and northern California into the Sierra Nevadas. Pedicularis densiflora flowers early in the spring at lower elevations, and grows primarily along the coastal mountain ranges of California. A comprehensive review of over 1000 herbarium specimens confirms the morphological distinctions and diagnosability of the two Species. Previous work by Sprague ( 1958, 1960, and 1962) and Macior (1986a) on variation among pollinator regimes indicated exclusive pollination by hummingbirds in P. aurantiaca and a Shared pollination by Bombus and hummingbirds in P. densiflora. 59 A Pedicularis aurantiaca is morphologically distinct, has a later growing season, grows at higher elevations, and has a different suite of pollinators than P. densiflora. Pedicularis aurantiaca is prezygotically isolated from P. densiflora. Support for the elevation of P. aurantiaca to Species status is unambiguous. The appropriate combinations for the species are below. Pedicularis aurantiaca (E. F. Sprague) Monfils & Prather, comb. et stat. nov. Pedicularis densiflora subsp. aurantiaca E. F. Sprague, Aliso, 4:130. 1962. Type: USA: from a burn in manzanita brush, Yellow Pine Forest, between the campground and the forest, Lake Almanor, Plumas County, California, at 4300 feet, 30 May 1957, E. F. Sprague 1202 (holotype: RSA; Isotype RSA!) Description: Perennial herb with woody root. Leaves petiolate, in basal rosette, oblanceolate, bipinnate to pinnate. Inflorescence a raceme, capitate with few open flowers. Bracts of the inflorescence oblanceolate and partially laciniate. Calyx tube 12-24 mm long, hirsutulous, 5 equal, acuminate to acute, erose lobes. Corolla galeate, 23-43 mm, deep red to purple to orange-yellow, galea 14-25.5 mm long, 4.5-8.5 mm deep, abaxial margin 13-23 mm long, lower labium petals 3.5-7.5 mm long from bend in corolla to petal tips. Corolla tube not fully exserted at anthesis. Fruit a capsule. 1000-7000 feet. Flowers April through June. Pine forests. Note: Plants in diffuse patches, haustoria preferentially parasitize woody plants 60 Additional material seen: UNITED STATES. Oregon: Jackson County: 1-1.5 miles past Pinehurst Inn. N 42 ° 07.452’, W 122° 20.783’, 1167 m, 24 May 1998, Monfils 12 (MSC); 1-1.5 miles east of Pinehurst Inn. N 42 ° 07.461 ’, W 122° 20.756’, 1131 In, 11 May 2000, Monfils 40 (MSC); Pinehurst, 20 Jun 1927, Peck [5044 (DS, WILLU); Pinehurst, 27 May 1948, Peck 24973 (WILLU, UC); Near Pinehurst, 1219 m, 13 Apr 1934, Thompson 10347 (CAS, DS, POM, WILLU). Klamath County: Cascade Mnts. Near Long Prairie, along old Klamath Falls-Ashland road, 15 Jun 1895, Applegate 421a (DS); Southeast of Topsy, 1372 m, 12 May 1898, Applegate 2059 (DS); Top of Spencer Mt., Cascade MtS., 11 May 1924, Applegate 4046 (DS, WILLU); Eastern Cascades, 30 Jun 1902, Cusick 2851 (DS, ORE, POM); Klamath Falls Road “East of Mt.”, 2 Aug 1925, Henderson s. n. (ORE); Along Ashland-Klamath Falls road, 18 miles W of Keno, May 1932, Peck 9302 (DS, WILLU); MtS. Below Klamath Falls near Ore-Calif line, on Klamath river, May 1932, Sprague s.n. (OSC). County Unknown: Southern Oregon, 13 May 1907, Henderson s.n. (ORE); Location and date unknown, Bellinger s.n., (WILLU). California: Amador County: Jackson, 12 Apr 1933, Ball 18269, (RSA). Butte County, Colby, May 1890, Austin 111 (UC); Jonesville, 16 Jun 1923, Bassett s.n. (CAS); Paradise, May 1898, Bruce 2419 (DS, POM); Brush Creek, 1907, Conger 453 (CAS, POM); Chico Meadows in the Sierra Nevada, 1219 m, 11 Jun 1915, Heller s. n. (DS); Stirling, 1073 m, 18 May 1919, Heller 13170 (CAS,DS); Pentz Road, 3 miles below Paradise, 23 Mar 1939, Heller 15358 (DS); DeSabla, 853 m, 18 Apr 1978, Howell 52852 (CAS); Above the road between Paradise and Butte Meadows .1 mi. S. of Inskip. T25N, R.4E, Sec. 33, 10 May 1981, McNeal 2487 (OSC); Inskip on Skyway Road. Elevation N 61 Al 39° 59.541’, W 121° 32.389’, 1309 m, 22 May 1998, Mon/1‘15 6 (MSC); Inskip on Skyway Road. N 39 ° 59.527’, W 121° 32.461 ’, 1309 m, 9 May 2000, Monfils 37 (MSC); Durham, 17 Apr 1932, Morrison 3. n. (CAS). Fresno County: 5 miles east of Auberry, Big Sandy Bluff, 945 m, 19 Mar 1969, Ahner s. n. (CAS); Big Sandy Valley near the foot of ne. slope of Black Mnt. Big Sandy Bluff beyond Ridge View Ranch which is across the road from my place, 610 m, 20 Mar 1953,Carter 15 7 (CAS, UC); About 2 miles north of Kerckhoff Resevoir, 610 m, 29 May 1967, McClintock, Roderick &.Johnson 5. n. (CAS); Old fire trail road off Auberry Road. N 37 ° 05.144’, W 119° 26.859’, 1147 m, 20 May 1998, Monfils 4 (MSC); R. R. Grade Rd. 1+ m. w. of jet. Hiway 168 at Shaver Xing between Shaver L. & Big Crk. 1st live crk. — small intake with pipe heading down crk. — Big Creek pentstocks in View. Heavy yellow pine for. 4500’- 1000’ up s. wall Big Crk. Canyon; 6830’ Music Mt. Just sw.; 8107’ Black Pt. N. across canyon, San Joaquin R. at 2000’ 4 map miles w., 17 Jun 1955, Quibell 5102 (RSA); 5 miles east of Auberry, Big Sandy Bluff range, 945 m, 19 Mar 1969, Shannon 5. n. (RSA); Scattered along Old Railroad Grade Road. R.23E., T9S., SW1/4 section 34, 853 m, 22 Mar 1980, Shevlock 6819 (CAS, MSC); Taken near Fish Camp, 14 Apr 1938, Whilton s. n. (RSA); Along road from Shaver Lake to Big Creek Power House. W. slope of Sierra Nevada, 1829 m, 5 May 1929, Wolf 3682 (RSA); 6 mi. above Auberry on road to Pineridge. W. slope of Sierra Nevada, 18 May 1933, Wolf 4 786 (CAS, RSA, UC). Kern County: Poso Creek narrows, below Poso Flat, 762 m, 9 Mar 1963, Record 82-1 (CAS-2); Near mouth of South Fork Kern River Canyon, trail along Bartolas Creek, Domeland Wilderness, north of California highway 178 and NW of the Bloomfield Ranch. R35E, T25S, section 23. Lat/Long: 35 degrees 44’45” N, 118 degrees 11’15” W., 1006 m, 20 Apr 1991, Shevock 62 12026 (CAS); Back canyon at the cypress grove, 1265 m, 26 May 1964, T wisselmann 950 (CAS-2). Mariposa County; Mariposa, 610 m, 10 Apr 1959, Ballantyne 236 (CAS); Awahnee, 9-16 Apr 1926, Shank 1 74 71, (RSA). Placer County: Tahoe Forest, Rebel ridge Range, 701 m, 12 Apr 1926, Smith 53916 (CAS-2). Plumas County: Sw. Shore Lake Almanor, 2 mi. S. of jet. of State Hwys. 89/36, 1402 m, 23 May 1957, Balls 22519 (RSA, UC); 1 3/ 10 S. of Drakesbad,3 Jul 1938, Cantelow 2323 (CAS); Prattville, sSummer 1906, Coombs s. 11. (CAS); Woodleaf, 3000-4000’, 14 Apr 1931, Rose s. n. (CAS, DS, POM, UC); Lake Almanor, 4300’, 30 May 1957, Sprague 1214 (RSA); Growing in dry loam in Shade of Libodedrus decurrens A low cool draw 6 miles south- west of Viola, 2800’, 30 May 1957, Sprague 1231 (RSA). Shasta County: Montgomery Creek, 18 Apr 1923, Beltiel s. 11. (CAS); Near McBride Springs. On banks and under Chaparral, 24 Jun 193 8, Cooke 11098, (DS, OSC); Squaw Creek Ranger Station, Jun 1916, Drew s. 11. (DS); Mt. Shasta, 21 Jun 1893, Dudley s. n. (DS); Burney Butte, 12 Jul 1912, Eastwood 1034 (CAS); Shasta Springs, 20 May 1923, Eastwood 11854 (CAS-2); Shasta Springs, 15 May 1918, Herrin s. n. (CAS); Shasta Springs, May 1922, Herrin s. n. (CAS); Open manzanita-oak association3 miles east of Redding, 500’, 17 May 1940, Hitchcock 16466 (DS, POM); Highway 44, 5 miles west of Lassen National Park, 4600’, 9 May 1974, Keller 1301 (CAS); Mt. Shasta, McBride Springs. N 41 ° 20.687’, W 122° 16.506’, 4922’, 11 May 2000, Monfils 43 (MSC). Siskiyou County, Bare serpentine gravel hillside, Scott Mnt. 8 miles above Callahan, 5150’, 17 May 1954, Barneby 11537 (CAS); Near Mt. Shasta, 6000-7000’, 1-15 Jun 1897, Brown 356 (DS); Alpina Mnts. Goosenest foothills, 5000’, 10 May 1910, Butler 1324 (DS, POM, UC); N. Slope of Scott Mnt.,4000’, 20 May 1936, Cantelow 1435 (CAS); Scott Mountain campground, 10 miles 63 S. of Callahan, 5350’, 21 May 1949, Constance, Bonar, Holm & Wood 3288 (UC); 0.8 miles N of Scott Mnt. Summit on Hwy 3. South Slope, 5200’, 5 Jun 1975, Davidson 2684, (RSA); Bald mountain in road from Mt. Hebron to. Montague, 5500’, 15 May 1940, Gould 1242 (DS); Mi. Eddy in open gravelly places in the forest, 6800’, 15 Jul 1915, Heller 12085 (CAS, DS, UC); Sugar Creek, Salomon Mountain Range, 5500’, 26 May 1949, Parker 257 (UC); Weed, 10 Apr 1913,Smith 66 (CAS); Dunsmuir, 29 Apr 1913, Smith 151 (CAS); Shady hillside near Weed, 28 Mar 1930, T ebbe 61 (UC); Salmon Mnts., Klamath National Forest. Near South Fork of Salmon River, 5 to 9 mile southeast of Cecilville, between Lat. 41 °03’ and 41 °06’N., and Long 122°58’ and 123°03’W. Vicinity of Blind Horse Creek, 3000-3500’, 13 May 1954, Thomas 4129 (DS-2); Salmon- Trinity Mnts. about 6 miles SE of Cecilville. West Side of Rush Creek, 3800-4000’, 21 Jul 1954, Thomas & Thomas 4425 (DS). Tehama County: Along road from Viola to Mineral, 9.5 miles south of Viola, 5000’, 12 Jun 1962, Breedlove 3423 (CAS, DS); Deer Creek Rd. (Rt. 32) at milepost 12, E Side of Deer Creek ca 20 airmiles SW of Chester, 3200’, 3 May 1989, Ertter 8435 (RSA); Mill Crk. Meadows, 6 Jun 1951, Quick 51 -93 (CAS); Northern Sierra Nevada. Red Bluff — Susanville Road. 3 6/10 mi. below Mineral, 4800’, 17 May 1937, Wolf 871 2 (RSA). Trinity County: T35N, R11 W, Sec. 6. North fork of Trinity River, Hobo Gulch Camp and Vicinity. (18 miles NW of Weaverville) Along East Fork Trail, just over on the east Side of Backbone Ridge, 4000’, 25 Apr 1972, Carter 374 (GAS-2); T36N, R11W, Sec. 31. North fork of Trinity River, Hobo Gulch Camp and Vicinity. (18 miles NW of Weaverville) Along trail 0.1 miles nOrth of Hobo Gulch. (Same location as 399-27 Apr. 72), 3100’, 8 Jun 1972, Carter 399.01, (CAS-2); Scott Mnts. N. of Carrville, 25 Jun 1937, Eastwood & Howell 5013 (CAS); 3 miles from 64 Douglas City on Redding Road, 26 Apr 1954, Howell 29164 (CAS); Dry open coniferous forest in Canadian Zone, Scott Mt. Summit on road from Carrville to Callahan, 5400’, 20 May 1980, Howell, Fuller & Barbe 5354] (CAS); Weaverville, Spring 1915, Junkans s. n. (CAS); Trinity Center, 30 Apr 1928, Kildale 4605 (DS); Weaverville, 30 May 1931, Kildale 10812 (DS); Foothills, Weaverville, 4000’, 11 Apr 1880, Kleiberger s. n. (CAS); East Weaver Campground on East Weaver Creek Road. N 40 ° 46.399’, W 122° 55.371 ’, 4248’, 23 May 1998, Monfils 8 (MSC); Hobo Gulch. N 40 ° 55.723’, W 123° 09.398’, 4249’, 23 May 1998, Monfils 9 (MSC); East Weaver Public Camp, East Weaver Creek, 3000’, 16 May 1949, M1an 13256 (RSA); T32N, R9W, Secs. 28 & 33; Mt. Diablo Meridian; Southeast of confluence of Panwauket Gulch and Reading Creek; blue oak- pine woodland , 610 m, 18 May 1975, Sullivan 88 (RSA); Little East Weaver Creek, 3000’, 21 May 1914, Yates [93 70 (CAS). Tulare County: Mineral King, 2000’, 11 Apr 1958, Pawek 418 (DS); Occasional in recently disturbed road bank along Blue ridge, section 10, R.29E., T. 19S. South facing slope, 4900’, 12 May 1979, Shevock 6186 (CAS); Uncommon along Calif. Hwy 245, about one mile west from the junction with Dry Creek Road. R.27E., T.15S. section 15, 2800’, 11 Mar 1980, Shevock 6775 (CAS); Lone Pine Spring, White River, 3350’, 8 Mar 1940, Smith 51, (CAS). Tuolumne County: Priest Grade, near Big Oak Flat, Yosemite National Park, 20 Feb 1982, Botti 1489 (CAS). Groveland. Woodland edge of route 120 about 4 miles east of town, 23 Apr 1974, Churchill 744231 (MSC). Yuba County: Between Dobbins and Bullard Bar Dam, Watersth of North Fork of the Yuba River, 3.1 mi. e. of Dobbins (or 5.5 miles SW. of Bullard Bar Dam), 2570’, 19 Apr 1956, Bacigalupi, Robbins & Chisaki 5622 (RSA); 65 Comptonville, 7 Apr 1918, Eastwood 6795 (CAS). County Unknown: Sterling City, 18 May 1935, Whitaker s. n. (OSC). Pedicularis densiflora Benth. ex. Hook. Fl. Bor. Am. ii. 1 10. 1838. Type unknown. Description: Perennial herb with woody root. Leaves petiolate in basal rosette, oblanceolate, bipinnate to pinnate. Inflorescence a raceme, elongated with many open flowers. Bracts of the inflorescence oblanceolate with serrate margins. Calyx tube 10-18 mm long, hirsutulous, 5 equal, acuminate to acute, erose lobes. Corolla galeate, 23-43 mm, deep red to purple to orange-yellow, sometimes white, galea 15-25 mm long, 4-7 mm deep, abaxial margin 9.5-20 mm long, lower labium petals 8-16 mm long from bend in corolla to petal tips. Corolla tube exserted at anthesis. Fruit a capsule. 100-4000 feet. Flowers February through April. Chaparral to pine forest. Note: Plants in diffuse patches, haustoria preferentially parasitize woody plants, Additional material seen: UNITED STATES. Oregon: Jackson County: Thompson Creek, near Applegate, steep hill slope. Sec. 21 T.38S RAW, 1400’, 19 Mar 1940, Detling 3864.(ORE); Fls. Deep purple-red open pine woods on alluvium, along Applegate River, 11/2 mi. 5.6. of Provolt, 28 Apr 1948, Glowenke 11113 (U C); 5 miles S of Applegate, under madronas, 1 Jun 1951, Hitchcock 19395 (RSA); Applegate Creek, Mar 1921, Leiberg s. n. (ORE); Applegate R., Jacksonville, 27 Mar 1936, Lund s. n. 66 (OSC); Pilot Rock, 20 Apr 1932, Neiman s. n. (WILLU); Woods along Thompson Cr. 5 mi. S. of Applegate P.O., 26 Jun 1931, Peck 16423 (WILLU-2); Along the Applegate River, 12 Apr 1927, Thompson 2225 (DS,ORE). Josephine County: 4 mi. NW. of Provost, 25 Apr 1943, Bellinger s. n. (WILLU); 3 miles south of Grants Pass on New Hope Road, 9 May 1963, Curtis s. n. (OSC); Applegate Valley T37S R5W sec. 34, 21 Apr 1942, Detling 5138 (ORE); In open woods, Fruitvale, 21 Apr 1930, Henderson 12513 (ORE); Roadside in mixed Oak-Pseudotsuga woods; S. facing; on old river bench. 2 mi E of Murphy R5W; T378; SE1/4 OF NW1/4, 345 m, 20 Apr 1968, Lillico 426 (ORE); Missouri Flat Road. N 42 ° 19.288’, W 123° 13.871’, 3900’, 24 May 1998, Monfils 13 (MSC); Missouri Flat Road. N 42° 19.288’, W 123° 13.871’, 3900’, 10 Apr 1999, Monfils 36 (MSC); Applegate River, North Bank Road C. 1 mi. from Redwood Highway; S. of Grants Pass, 1300’, 23 Apr 1967, Pike 111 (ORE); Hillside near Grants Pass, 19 Mar 1918, Prescott s. n. (WILLU); Near Williams Creek highway between Provolt and Williams; also on N. Side of Applegate River on Missouri Flat. Clayey soil in woods, 13 Apr 1924, Savage s. n. (ORE); Fruit Dale, Murphy Road. In open woods, 24 Mar 1926, Savage s. n. (ORE); Mixed woods near Takilma, 21 Apr 1930, White s. n. (ORE). California: Alameda County: Laundry Harm, 29 Jan 1895, Cannon s.n. (CAS); Oakland Hills, 28 Feb 1936, Cave] 371 (CAS); Laundry Harm, 26 Apr 1891, Eastwood s.n. (ORE); Mines Road S. w. of Liverrnore, near entrance to Rancho Los Mochos Boy Scout Camp, 1500’, 3 Mar 1968, Gagné s.n. (CAS). Butte County: Near Hurleton, 1800’, 26 Mar 1980, Ahart 2060 (CAS); Cherokee mine, 29 Mar 1919, Heller 13097 (CAS, DS, POM); Hurles Circle, in center island. N 39 ° 29.764’, W 121° 22.632’, 2050’, 22 May 1998, Monfils 5 (MSC); Hurles Circle, in center island. N 39° 29.764’, W 121° 22.632’, 67 2050’, 7 Apr 1999, Monles 35 (MSC). Calavaras County: Comanche, 16 Apr 1939, Hoover 4032 (CAS). Colusa County: Wilbur Springs. TlSN, R6W, sec. 35, 3500’, 14 Apr 1979, Roth 9 (RSA); Along Brim Rd., 2.9 mi. W jct. Bear Valley Rd., 30 Mar 1996, Vincent & Rhode 7304 (RSA). Contra Costa County: Martinez, 300’, 18 Mar 1931, Benson 2662 (POM); Mount Diablo, 17 Mar 1922, Eastwood 11084 (CAS); Rocky Point, Mount Diablo. N 37° 51.814, W 121° 55.770’, 2550’, 22 Mar 1999, Monfils 28 (MSC); Rocky Point, Mount Diablo, 2000’, 5 Apr 1956, Sprague 1096 (RSA); Mount Diablo, 2000’, 11 Apr 1957, Sprague 1124 (RSA); Rocky Point, Mount Diablo, 2000’, 30 May 1957, Sprague 1134 (RSA). Glenn County: 2 miles north of Alder Springs, 3900’, 19 May 1949, Munz 13330 (RSA). Humbolt County: T7N, RSE, Sec. 29. Along highway 96, 1.9 miles north of Willow Creek, 1 May 1965, Anderson 3556 (RSA); In damp fir woods near creek, Boise Creek and Willow Creek, Trinity River, 1 Apr 1947, Brown 25 (DS); Philabiumsville, south fork of El River, 20 Mar 1927, Kildale 2909 (DS); Briceland Bridge near Garberville, 375’, 17 Apr 1925, Kildale 3096 (DS, RSA); Briceland Road. N 40° 05.678’, W 123° 51.222’, 1874’, 25 May 1998, Monfils I4 (MSC); Round Valley Historical Marker. N 39° 43.749’, W 123° 15.112’, 2160’, 25 May 1998, Monfils 16 (MSC); Kneeland Prarie, in woods in ravine, 2500’, 8 Jun 1908, Tracy 2637 (UC); Vicinity of Garberville, 400’, 17 Mar 1923, Tracy 6160 (UC); Trinity River Valley, near the south fork, 600’, 28 Feb 1926, Tracy 7369 (UC); H00pa Mnt., near summit on road west from Hoopa to Bair’s, 3500’, 15 May 1927, Tracy 8060 (UC); Willow Creek Canyon,2000’, 26 Apr 1931, Tracy 9340 (UC); Trinity River Valley, at Willow Creek, 500’, 3 Apr 1937, Tracy 1525] (UC); Trinity River Valley, at Willow Creek, 500’, 26 Mar 1941, Tracy 16818 (UC); Harris, in woods, 2500’, 31 May 1948, 68 Tracy 18006 (UC). Lake County: Lakeport, 20 Apr 1917, Bentley 5. n. (DS); Mt. Kelseyville, Middle N. Coast, Near Cold Creek Canyon River, 1700’, 30 Mar 1928, Benson 87 (POM); Bogg’s Lake, Mt. Hanna, Middle N. Coast, Clear Lake watershed, 3500’, 18 May 1935, Benson 6636 (POM); Hannah, 3000’, 8 Apr 1923, Blankinship s. n. (CAS); Mt. Konocti, 2000’, 27 Mar 1926, Blankinship s.n. (RSA); Sulphur Banks, Apr 1902, Bowman S. n. (DS); E. of Middleton, 7 Apr 1940, Cantelow 4346 (CAS); 4 mi. below Tollhouse on Middleton Road, 22 Feb 1924, Duncan 5. n. (DS); Dasheills Mt., Sanhedrin, 23 May 1925, Eastwood 12912 (CAS); Elk Mt., 17 May 193 8, Eastwood &. Howell 5703 (CAS); Clear Lake, 14 Apr 1928, Galloway s.n. (CAS), Glenbrook, near Jordan Park, 30 Mar 1931, Jussel 29 (CAS); Cobb Mt., 30 Mar 1931, Jussel s. n. (CAS); Cobb Mt., 31 Mar 1931, Jussel s. n. (CAS); Mt. St. Helena, 1 Apr 1933, La Matte s. n.(POM); Cobb Mt., 4 Jul 1893, Leitholt s. n. (DS); Bear Valley on Brim Road. N 39 ° 09.504’, W 122° 28.778’, 2665’, 26 May 1998, Monfils 17 (MSC); Bear Valley on Brim Road. N 39° 09.431’, W 1220 28.798’, 2180’, 26 May 1998, Monfils 33 (MSC); Butt’s Cyn. Rd. near Middleton, 1000’, 25 Mar 1972, Shevock 1437 (RSA); Northeast facing slope, 0.4 mile southeast of Black Oak Villa in Butts Canyon, 1.1 miles from Lake-Napa county line on Pope Valley road, 800’, 7 Mar 1953, Sweeney 971 (UC); Chaparral 11 mi. S. of Lower Lake, 13 Mar 1932, Wiggins 5769 (DS); 21/2 miles S. W. of Lakeport. Sec. 34, T.14N, R.10W, 1850’, 14 May 1937, Wilson 376 (UC). Los Angeles County: Mts. above Claremont, Johnson Pasture, 15 Feb, Bragg s. n. (POM); North slope Sta. Monica Hills, Feb 1903, Braunton 809 (DS, ORE-2); Topanga Canyon, 28 Mar 1929, Clare s.n. (RSA-2); Laurel Canyon, 24 Feb 1929, Detruers s.n. (RSA); Mulholland Drive, 6 Mar 1935, MacFadden 13246 (CAS); Laurel Canon, Mar 1943, Merritt s.n. (RSA); Franklyn 69 Canyon, Santa Monica Mts., 21 Feb, Peirson 1168 (RSA); Glendora, Little Dalton Trail, 16 Feb 1916, Perkins s.n. (RSA); 0.7 mile from Triunfo Canyon, 2 miles west of Cornell, Lobo Canyon, 800’, 31 Mar 1959, Thompson 1010 (CAS, RSA); Hwy. 23 south of Lake Elanor, Santa Monica Mtns., 1000’, no date, Wallace & Wilkin 150 (RSA). Marin County: East side of the Tiburon Peninsula just below the summit, 31 Mar 1981, Bests. n. (CAS); On trail 0.5 mi. above Phoenix Lake near jet. of Mt. Tamalpais trail, 1200’, 5 Apr 1956, Campbell 8 (RSA); Mount Tamalpais, 22 Feb 1901, Chandler 760 (POM, UC); Old hardpacked fire road, 100’, 9 Feb 1975, Edelbrock 4, (CAS); Summit Alpine Lake Trail above Deer Park, 8 Mar 1936, Ewan 9408 (RSA); Summit Alpine Lake trail above Deer Park, 8 Mar 1936, Ewan 9409 (UC); Mt. Tamalpais, 22 Apr 1930, Forests. n. (RSA); Tamalpais TlN R6W, 600’, 28 Mar 1935, French 619 (UC); South side of Mt. Tamalpais, 7 Mar 1902, Heller & Brown 5008 (DS, MSC, POM); Tiburon, 15 Feb 193 8, Hoover 2739 (UC); San Geronimo Ridge, 25 Feb 1940, Howell 15388 (CAS); Carson Ridge, 19 Apr 1942, Howell 16949A (GAS-2); San Arseloro Canyon, 25 Feb 1940, Howells. n. (CAS); Above Blythedale Canyon, east side of Mt. Tamalpais, 2 Mar 1947 Howells. n. (CAS); Mill Valley, 29 Mar 1891 , Jepson s.n. (UC); Alpine Dam Road, about 1 mile from Alpine Gulch on Fairfax side, 1040’, 10 Mar 1968, Kawahara 29 (CAS); Marin County, 1868-1869, Kellogg & Harford 713 (CAS); Ross Valley, Apr 1892, Michener & Bioletti s. n. (MSC); Tamalpais, Apr 1892, Michener & Bioletti s. n. (MSC); Fire road, south-side of Mount Tamalpais, 12 Apr 1969, Mitchell 4 (OSC); Paradise Beach Park, Tiburin Uplands Nature Preserve. N 37 ° 53.329’, W 122° 26.954’, 109’, 26 May 1998, Monfils 18 (MSC); Paradise Beach Park, Tiburin Uplands Nature Preserve. N 37° 53.309’, W 122° 27.055’, 137’, 3 Apr 1999, Monfils 34 (MSC); Mt. 7O Tamalpais, 3 Mar. 1930, Morris s. n. (RSA); Rcky and clayey bank by Paradise Dr., 0.7 mi. N. Paradise Beach Park, Tiburon Peninsula, 10 Apr 1975, Norris 2305 (RSA); Tiburon Peninsula, 200’, 15 Mar 1930, Parks 402 (POM, UC); Tiburon, Spring 1926, Parks (UC); Alpine Dam Road above Fairfax, 800’, 9 Mar 1963, Sharsmith 5194 (UC); Corte Madera, 1 Mar 1903, Sheldon 11563 (ORE); Phoenix Lake, Ross, 1000’, 4 Apr 1956, Sprague 1096 (RSA); Carson Ridge, 4 Apr 1957, Sprague 1120 (RSA); Fairfax, Mar 1928, Suttlifle s. n. (RSA); North slope of hill near Forest Knolls, 21 Mar 1936, True s. n. (RSA); Phoenix Lake, Ross, 1000’, no date, no collector (RSA); Mt. Tamalpais, Apr 1898, no collector (CAS). Mendocino County: Abt. 12 mi. e. of U. S. 101, along Calif. 20. In shade above road, 6 Apr 1954, Clarkson 300 (OSC); Red Mnt., n. Mendocino Co., 21 Jun 1937, Eastwood & Howell 4663 (CAS); Near Woodville, May 1889, Howell s. n. (UC); Ukiah, 25 Apr 1924, Jones s. n. (DS-2, POM); Kaiser District, Mar 1903, McMurphy 306 (DS); 8 miles north of Ukiah, 6 Apr 193 8, Meyer 1384 (UC); Round Valley Historical Marker. N 39° 43.749’, W 123° 15.112’, 2160’, 25 May 1998, Monfils 16 (MSC); Round Valley Historical Marker. N 39 ° 43.760’, W 123° 15.093’, 1966’, 29 Mar 1999, Monfils 32 (MSC); Mad River, 6 Jul 1890, Price s. n. (UC); Potter Valley, Apr 1894, Purpus 1009 (UC); Shady Hills, 11. Potter Vall., Mar 1894, Purpus 3089 (UC); Along rte. 128 at MP24.26, 400’, 5 Mar 1979, Smith & Wheeler 5128 (CAS); Red Flat, ‘7: mi. from wooden gate btwn, BLM & Coombs property. Red Mnt. N., 2350’, 6 Jul 1981, Smith et al. 6855 (CAS); Summit area of Red Mountain North, 7 Jul 1981, Smith et al. 6868 (CAS); Seven miles north of Laytonville near highway 101, 20 Apr 1968, Thomas 14331 (DS); Rte. 253. South of Robinson Creek Rd., Ukiah, 650’, 11 Mar 1978, Wheeler 60 (CAS); South of Leggett on the Old Redwood Hwy, 1000’, 1 Feb 1980, Wheeler 1298 (CAS); Round Valley Historical Marker, overlooking Covelo on Hwy. 162, 2000’, 12 Apr 1979, Wheeler & Smith 905 (CAS); Little River — Albion Road. Near Little River Airport, 600’, 23 May 1979, Wheeler & Smith 1026 (CAS); On Northwesterly slope, 4 miles east of Laytonville, along road to Dos Rios, 20 Mar 1948, Wiggins 11587 (DS, RSA). Monterey County: Gravel Pitt hill, 27 Jun 1905, Dudley s. n. (DS); Monterey, 1874, Abbott s. n. (CAS-2); Carmel Highlands, Peter Pan Rd., 200’, 21 Feb 1948, Balls 7834 (RSA); Monterey, 29 Mar 1933, Detling 1108 (ORE); Pine Canon, 1500’, 27 Mar 1920, Duncan 86 (DS); Near Cypress Pt., 28 May 1912, Eastwood 94a (CAS); Pacific Grove, 8 Mar 1923, Eastwood 24 71 (CAS); Monterey, 9 Mar 1913, Eastwood 2489 (CAS); Pacific Grove, Apr 1902, Elmer 3543 (DS-2, POM, UC); Two miles south of J olon on hilltop, 10 Mar 1952, Evans 5. n. (CAS); Sand Stone Cliffs near north fork of San Antonio River, 1500’, 27 Mar 1920, Ferris 1810 (DS); Landels-Hill Big Creek Reserve, Gamboa Point Section, Santa Lucia Mountains, sect.4, 2 Apr 1982, Genetti & Engles 48 (CAS); Del Monte Forest, Pacific Grove, 1 Apr 1955, Howitt 132 (CAS); Pacific Grove, 12 Apr 1933, Jussel s. n. (DS); Summit of Hesperia Mountain, north of Bryson, southern Monterey County, 1550’, 2 May 1933, Keck 2093 (DS-2); Near the school. Francis Simes Hastings Natural History Reservation, Santa Lucia Mts., 2 Apr 1944, Linsdale 71 (CAS); Santa Lucia Mountains, Apr 1898, Plaskett 75 (U C) Carmel-by-the-Sea, 3 Mar 1910, Randell 53 (DS); By trail from Carmel to Monterey, 30 Dec 1909, Randall s. n. (DS); Parkfield Road, 1.5 miles east of Vineyard Canyon Summit, 2200’, 4 Apr 1956, T wisselmann 2618 (CAS-2); Del Mouh ur. Salinias Road, 15 Apr 1912, Woodcock s. n. (POM). Napa County: Mt. St. Helena Trail, 4 May 1928, Abrams 12265 (DS); Summit of St. Helena Grade, 3 May 1928, Abrams s. n. (DS); 72 Howell Mt., 22 Mar 1936, Cantelow 1142 (CAS); East slope in the Howell Mountains, 5 miles east of Napa, North Coast Range, 1500’, 27 Mar 1938, Constance 2036 (DS, UC); Northern exposure above Putah Creek, along State Hwy. 128, about 6 miles east of Monticello, 26 Feb 1953, Crampton 989 (UC); 7.2 miles drom St. Helena on road to Pope Valley, 2 Apr 1950, F infrock 1 7 (UC); Wooded slope in the oak belt about 5 miles south of Calistoga, 12 Apr 1924, Heller 13840 (DS, POM); Near the summit of the ridges east of Napa on the Monticello road on a clay bank among shrubs, 12 Mar 1940, Heller 15514 (DS); Wooden Valley road, east side of Napa Range, 8 miles from Napa, 2 Apr 1931, Keck 1030 (DS, POM); Calistoga, 13 Apr 1929, Linsdale 257 (UC); Old Howell Mountain Road, 1500’, 31 Mar 1967, Muth 596 (RSA); 1 mile from Pacific Union College, beside road to Pope Valley, 1 May 1949, Popenoe 18 (OSC); Hills just n. of White Sulphur Ck., w. of Saint Helena, 500’, 22 Feb 1954, Raven s. n. (CAS); Base of Mt. St. Helena, 3 May 1928, Wolf 1 845 (DS); Summit of Mt. St. Helena Grade, north of Calistoga, Wolf 1 845 (RSA); Upper slopes of Mt. St. Helena, 4 May 1928, Wolf 1 85 6 (RSA). Nevada County: American Ranch Hills, 5 miles south-west of Grass Valley, on McCourtney Rd., 2200’, 8 Apr 1962, True 356 (CAS). Orange County: 14.3 miles east of San Jaun Capistrano on State 74, 1100’, 9 Mar 1962, Breedlove 1 789 (DS); Santa Ana Mountains, Cleveland Nat. Forest. Pleasants Peak (on Orange-Riverside Co. line), 4000’, 18 May 1977, Davidson 5601 (RSA); Sitton Peak Truck Trail: 0.3 mi W of ranger station near hot springs, on Ortega H’way (SR 74), 25 Apr 1990, Jaroslow B36 (RSA); Sierra Peak Trail, Santa Ana Mts, 11 Apr 1929, Johnson 1259 (RSA); 5 miles east of Trabuco Oaks, Trabuco Canyon, Cleveland National Forest, 1300’, 6 Apr 1966, Lathrop 6142 (RSA). Riverside County: Santa Ana Mountains, 3 miles above De Luz on dirt road, 73 3000’, 30 Apr 1966, Adams 5. n. (RSA); Vail Lake area, summit of “Big” Oak Mtn, N. of lake. T7S, R1W, SW ‘A sec. 34. Saddle between summits, N slope along rd heading N to Black Hills, 2600’, 30 Mar 1990, Boyd, Ross & Arnseth 3944 (RSA); Vail Lake area, saddle at N base of “Big” Oak Mtn, S of Black Hills. T7S, R1W NE %, SW '7: sec. 34, 2400’, 30 Mar 1990, Boyd, Ross & Arnseth 3953 (RSA); Santa Ana Mountains, San Mateo Cyn. Wilderness Area. Tenaja Trail from jtn. W/ Morgan Tr. S to Pigeon Spring area. T6S, RSW sec.32; T7S, R5W sec. 5, 2200-2600’, 31 Mar 1992, Boyd, Ross & Arnseth.6 763 (RSA); Aguate, So. Calif, 7 Apr 1929, Clarkk 2035 (RSA); Corona Skyline Drive, Santa Ana Mts. So. Calif, 7 Apr 1929, Crow 304 (RSA); Vicinity of Beaumont, 17 Apr 1897, Hall 476 (UC); Aguanga, 22 Dec 1925, Jaeger s. n. (POM); Corona, sec. 22, T48, R7W, 2800’, Mar 1934, Jensen 318 (UC); 18 miles S. W. of Elsinore. South slope of Tenaja Canyon, 2 Apr 1959, Lathrop 4408 (RSA); 4 miles west of Corona between Tin Mine Canyon & Santiago Peak, Skyline Drive, 4000’, 9 Apr 1969, Lathrop 6968 (RSA); Beaumont, 2000’, 25 Mar 1919, Munz, Street & Williams 2327 (DS, POM); 4 mi. SE of Oak Flat near west county line on Santiago Pk. Fire road near top of peak west of Santiago Pk., Santa Ana Mts., 4200’, 14 Apr 1959, Olmsted 3 74 (RSA); Along roadside in red clay soil, about 4 miles west of Beaumont, on road to Redlands, 19 Mar 1921, Peirson 2741 (RSA); Rancho Calif. area, ca. 8 mi. (airline) NW of Temecula, between Bruce Lane & Via View Dr. (T 78, R 1W SBBM NW /4 of SW /4, sec.27), 2400’, 22 Feb 1988, Pendelton s. n. (RSA); Santa Ana Canyon, Santa Ana Mountains, 15 Apr 1922, Pierce s. n. (POM); 1 mile south of Aguangam, Hwy 79, Agua Tibia Mts., 1940’, 12 Apr 1951, Rush 169 (POM-2); In herbosis, Lambs Canyon proper, Banning, 2300’, 25 Apr 1922, Spencer 1910 (RSA). San Benito County: Eastem 74 exposure on Peak Trail. Pinnacles National Monument, Paicines, 1500’, 22 Mar 1955, Burgess 84 (UC); Pinnacles Nat. Mon., 550 m, 6 Jun 1931, F osberg 35251 (RSA); North slope, the Pinnacles, 29 Mar 1930, Howell 4611 (CAS); Pinnacles National Monument, High Peaks Trail. N 37° 53.386’, W 1220 26.991 ’, 107’, 27 May 1998, Monfils 20 (MSC); Pinnacles National Monument, 1600-1700’, 11 May 1940, Pennell & Powell 253 70 (UC); Pinnacles National Monument. Pinnacles Loop Trail, 1200-2500’, 27 April 1975, Thomas I 7828 (DS). San Bernardino County: Devore near San Bernardino, 1900’, 12 Mar 1928, Feudge 1960 (POM); Foothills San Bemardino Mts., Apr 1882, Parish 707 (DS, ORE, UC). San Diego County: Santa Ana Mtns, San Mateo Wilderness Area, “Miller Canyon” on the south base and flank of Miller Mtn from 8802 upstream to the eastern summit area. T8S,R5W, sec. 10.15, 2100-2900’, 3 Mar 1992, Boyd & Ross 6717 (RSA); W. of Warner Hot Springs, 6 Apr 1929, Clark 2007 (RSA); South side of Gonzales Canyon, east of Del Mar, 30 Mar 1969, Copp 69-1 (CAS); Mt. Soledad, 3 Jan 1935, Gander 103 (RSA, UC); North side of San Miguel Mountain. 32°42’N 116°55 3/4’W, 900’, 24 April 1957, Moran 6000 (DS); Beaumont, 2000’, 25 Mar 1919, Munz, Street & Williams 2327 (ORE); Colorado Desert, 1500’, 9 Apr 1921, Spencer 23] (POM); Potrero Grade, 18 Mar 1917, Spencer s.n., 9 Apr. 1921 (POM); Grape Vine Canyon, 1200 m, 21 May 1930, Templeton 1625 (RSA-2); Near Santee, Feb 1915, Valentien s.n. (UC). San Louis Obispo County: Atascadero, 22 Mar 1926, Abrams 10942 (DS); 4.4 miles east of Santa Margarita; La Panza Range, 1200’, 29 Mar 1962, Breedlove 2029 (DS); 3 miles E. of P020, sec. 13, T.3OS, R.15E, 1800’, 27 Mar 1937, Giflord 801 (UC); 1 mile S. of Bee Rock, Bradley, Sec 7, T.258, R.10E, 1200’, 11 Apr 1938, Graham 305 (UC); 7X Ranch, Santa Lucia Mountains, 2200’, 2 Mar 1956, 2200’, Hardman 143 75 (CAS); 7X Ranch, Santa Lucia Mountains, 2200’, 2 Mar 1956, 2200’, Hardman 144 (CAS); Santa Rita Canyon, Santa Lucia Mountains, 13 Apr 1956, Hardman 404 (CAS); L. Delagenna Ranch, Santa Lucia Mountains, 26 Apr 1956, Hardman 533 (CAS); Calf Canyon, 5 Apr 1967, Hoover 10334 (CAS); E. side of Santa Margarita Lake, 8 Apr 1986, Keil 19136 (RSA); 3 miles N. E. of Templeton, Paso Robles, T. 278, R. 12E, 900’, 31 Mar 193 7, Lee 806 (UC); Growing in a disturbed area on a road bank beside Hwy. #229, 5.9 mi. sw. of Hwy. #41 at Creston, 29 Mar 1981, McNea12433 (OSC); Riconda Trail Head off Pozo Road. N 35 ° 17.214, W 120° 28.659’, 2200’, 18 May 1998, Monfils 2 (MSC); Riconda Trail Head off Pozo Road. N 35 ° 17.267, W 1200 28.677’, 2070’, 27 May 1998, Monfils 20, (MSC); Santa Rita Canyon, 1000’, 17 Apr 1957, Sprague 1136 (RSA); On Cayucos Rd. to Cambira on Jack Mt. Nick Marquat Ranch, 16 Apr 1957, Sprague 1137 (RSA); Hill by a spring near San Louis Obispo, May 1889, Summers 527 (CAS); 2 miles north Cuesta Pass, Santa Lucia Mountains, 2000’, 4 Apr 1963, T oschi 63:97 (CAS); Santa Margarita, Eldorado School, 20 Apr 1933, Wall s.n. (GAS-2, RSA); Roadside and hills near Adelaide, W. of Paso Robles, 24 Mar 1932, Wiggins 584 7 (CAS, DS, POM); Prefumo Canyon, no collector (DS). San Mateo County: Woodside, 16 Mar 1902, Abrams 22 78 (DS); Kings Mountain Road, Santa Cruz Mountains,500-700’, 1 Mar 1914, Abrams 5058 (POM); Woodside, 3 Mar 1895, Applegate 421 (DS); Woods on Coal Mine Ridge, 23 May 1937, Barry 155 (DS); Hill east of Lake Searsville, North Slope, 2 Mar 1929, Benson 987 (POM); Woodside, Santa Cruz Mtns, near San Fransquito Cr., 600’, 28 Feb 1931, Benson 2610 (POM); Santa Cruz Mountain Peninsula, Crystal Spring Lake, 28 Apr 1920, Borthwick 97 (DS); Near the intersection of Canada and Edgewood roads. About 3 mi. westward from Redwood City, 13 May 1974, Cahill 248 (DS); La 76 Honda Road (from Hwy. 5) to Woodside, 3 mi. from Mt. Home-Portola road, 4 Apr 1956, Campbell 1 (RSA); Big Basin of Pescadero creek, 9 May 1903, Copeland 3050 (POM),'Santa Cruz Peninsula, near Belmont, no date, Dudley s.n. (DS); Woodside, 1903 May, Elmer 4497 (CAS-2, DS, ORE, OSC, POM, UC); Near Belmont, Mar 1886, Greene 9 (ORE); San Carlos. Chaparral area north of Malabar Rd. and Melendy Dr. Lat N. 37° 29’ 38”, Long. 122° 16’ 41” W, 600-680’, 22 Apr 1973, Hemphill 737 (DS); Woods near Spring Valley Lakes, Santa Cruz Mountain Peninsula, 29 Apr 1920, Hickborn s.n. (DS); Los Trancos, 30 Apr 1908, Lewis s.n. (RSA); Kings Mt., May 1902, McMurphy s.n. (DS); Mt. above Woodside, 9 Mar 1906, McMurphy s.n. (DS); Sawyer’s Road, near Crystal Springs lake above Burlingame and Millbrae, 15 Apr 1956, 0 ’Bannon s. n. (DS); Sawyer Ridge, 17 Apr 1949, Oberlander 86 (DS); Santa Cruz Peninsula, Kings Mt., 27 Apr 1907, Patterson s.n. (RSA); Santa Cruz Peninsula, Kings Mt. Road, 27 Apr 1907, Randall 394 (DS); Santa Cruz Peninsula, Kings Mt. Road, 28 Mar 1908, Randall s. n. (DS, RSA); Belmont, 24 Feb 1935, Rose 35016 (RSA); Crystal Springs lake, 500’, 12 Apr 1939, Rose 39061 (RSA, UC); Millbrae Highlands, 300’, 31 Mar 1948, Rose 4803] (RSA); Emerald Lake, w. of Redwood City, 400’, 23 Mar 1964, Rose 64013 (DS, RSA); 2 mi. nw of Woodside, 600’, 27 Mar 1969, Rase 69009 (MSC); King’s Mountain, 27 Apr 1907, Rust 135 (RSA); King’s Mountain Road, 29 Mar 1949, Thomas 195 (DS); Jasper Ridge, about 5 miles southwest of Palo Alto. Lat N. 37° 245’, Long. 122° 14’ W, 500’, 5 Apr 1959, Thomas 7663 (MSC); l/2 way up east slope of La Honda Grade, Santa Cruz Mts., 24 Jan 1927, Wolf 1 90 (RSA); East side of La Honda Grade, 26 Feb 1927, Wolf 221 (RSA); Above Searsville Lake, 2 Mar 1927, Wolf 235 (RSA). Santa Barbara County: Purissima Hills, on road to Mission, 27 Mar 193 8, Abrams 13755, (DS); North 77 side ofthe Point Sal Ridge, 3 '/2 mi. west of Corralillos Canyon. Point Sal, 3 Mar 1958, Blakley & Muller B-2695 (CAS-2, RSA); Beside Refugio Pass Road, 5.4 miles north of U. S. 101, 24 Feb 1962, Breedlove 1778 (DS); San Marcos Pass. Old Stagecoach Road. N 34° 31.790’, W 119° 50.070’, 1402’, 17 May 1998, Monfilsl (MSC); Old Freemont Trail and Coach Rd., Santa Ynez Mts., - Area of Refugio Pass, 2200’, 18 Mar 1956, Pollard s.n. (CAS); Near summit of Refugio Pass, Santa Ynez Mts, 2200’, 19 Apr 1968, Pollard s.n. (CAS); Stagecoach Road, 1650’, 5 Apr 1970, Shevock 106 (RSA); Entrance to Foster Glen Park, along highway in San Marcos Pass, south side of Santa Ynez Mountains near summit, 11 Mar 1955, Smith 3918 (RSA, UC); Three miles north of summit of San Marcos Pass, 19 Mar 1961, Turner, Sphon & Ball C-494 (RSA). Santa Clara County: Santa Cruz Peninsula, charcoal burners, Page Mill Road, Black Mt., 6 Jul 1903, Dudley s.n. (DS); Santa Cruz Peninsula, Page Mill Road, Black Mt., 23 Apr 1904, Dudley s.n. (DS); Adelante Villa, Palo Alto, 25 Feb 1894, A. M. K. s.n. (RSA); Foothills near Stanford University, 9 Mar and 15 May 1902, Baker 283 (CAS, DS, MSC- 2, POM, UC); Black Mt., 9 Feb 1895, Burnham 9 Feb. 1895 (MSC); Eastern slope of Mount Hamilton, 3000’, 31 Mar 1933, Chambers 144 (UC); Los Gatos, 19 Mar 1897, Davy s. n. (UC); North-facing bank, south side of page Mill road, ca. 1 mile west of entrance to Palo Alto Foothills Park, Palo Alto, 1200’, 25 Mar 1969, Doty 593 (RSA); Foothills west of Los Gatos, 25 Mar 1904, Heller 7281 (DS, MSC, UC); Alpine Creek Road, northeast side of Santa Cruz Mountains, 200’, 22 Feb 1932, Keck 138 7 (DS, RSA); Foothills of the eastern side of the Santa Cruz Mountains, 5 miles south of the center of Palo Alto. Los Tracos Trail above Los Tracos Creek, 600-1800’, 3 Apr 1974, Martineau 16 (DS); 23.1 mi. w. of Interstate #5, Patterson exit, in Del Puerto canyon (2.5 mi. e. of 78 ——-——— the Mt. Hamilton-Livermore Rd.), 15 Mar 1970, McNeal 4 72 (OSC); Fire trail near Lorna base above spring,3400’, 21 Mar 1940, Nelson 71 (UC); Hills 1 ‘/2 miles south od Saratoga, Western Santa Clara County, 800’, 1 Mar 1906, Pendleton 290 (POM); 10 miles south of Black Mountain, 3. of Palo Alto, 1200-1300’, 16 May 1940, Pennell & Abrams 25429 (CAS, UC); Seeboy Ridge, Mount Hamilton Range, 2400’, 10 Feb 1934, Sharsmith 53 7 (UC); South end of Mount Day Ridge above Santa Isabella Creek, Mount Hamilton Range, 1800’, 25 Mar 1935, Sharsmith 1520 (UC); Northwest slope of Black Mountain, 1100’, 31 Jan 1948, Silva 2616 (RSA); Vicinity of Mt. Umunhum, Lat. N. 37° 095’, Long. 121° 54.2’ W, 3400’, 23 Apr 1954, Thomas 3963 (DS). Santa Cruz County: Ben Lomond Mountain area; on Eagle Rock; T9S, R3W, Sec. 16, 2200’, 23 Mar 1974, Halse 986 (OSC); Big Basin, Santa Cruz Mts., Reed s.n. (CAS); Santa Cruz Peninsula, Swanton, Spring 1912, Rich s.n. (DS); La Honda Rd. Sta. Cruz Mts., 2000’, 3 Apr 1956, Sprague 1095 (RSA); Between Eagle Rock and Locatelli Ranch. Lat. N. 37° 08.7’, Long. 122° 12’ W, 2400’, 27 Mar 1950, Thomas 1346 (DS); East side of Mill Creek Drainage about 2 miles S-SW of Eagle Rock at Lat. N. 37° 073’, Long. 122° 12.4’ W, 1800—1900’, 30 Apr 1954, Thomas 3035 (DS); Between Eagle Rock and Locatelli Ranch. Lat. N. 37° 087’, Long. 122° 12’ W, 2200’, 15 Mar 1954, Thomas 3776 (DS); Vicinity of Eagle Rock. Lat N. 37 08.8, Long. 122 11.7 W. 2200-2500’, 30 Mar 1961, Thomas 9041 (DS); Halfway down La Honda Rd. Sta. Cruz Mts., 2000’, 13 Apr 1957, no collector (RSA);. Solano County: Gates Canyon, northwest of Vacaville, 3 Mar 1951, Kehlor s. n. (OSC); 3 ‘/2 miles west of Rockville, 25 Mar 1966, Olson & Gorelick (RSA-2). Sonoma County: Pepperwood Ranch, Oak woodland between the 2 fir-Redwood canyons, 25 Mar 1981 , Denevers 565 (CAS); Calistoga,25 Mar 1922, Eastwood 11097 (CAS); Camp Meeker, 22 79 Mar 1924, Howell 287 (CAS); Near Sonoma, Mar 1962, Menzius s.n. (CAS); 2 miles from juncture of Highway 12 and Triniti Road, 750’, 27 Feb 1960, Ruckert 5 (CAS). Sonoma County, 10 Mar 1903, Rattan s.n. (DS); 0.5 mi. south of Calistoga, 400’, 29 Mar 1953, Raven 5207 (CAS); Santa Rosa Creek Canyon, 8-10 miles east of Santa Rosa, 25 Mar 1937, Robbins 10 (UC); N. side of Vine Hill School Road l/2 miles from Vine Hill Road, 250’, 19 Apr 1965, Thorne 34319 (RSA); Parker Hill Rd. ca. 2 ‘/2 mi. N of Santa Rosa and 1 mi. N of Sonoma Co. Hosp., 350’, 20 Apr 1965, Thorne 34328 (RSA). Stanislaus County: Near head of Del Puerto Canyon, 20 Apr 1941 , Hoover 4886 (UC). Trinity County: Vic. Dam at Ruth Resevoir on Mad River, 2700’, 21 May 1979, Mattoon 121 (RSA, UC); Road along Coffee Creek, above Coffee Creek Ranch, 16 Jun 1956, McClintock s. n. (CAS); T6N, R6E, Sec. 34, Found in Grays Falls Campground,21 Apr 1979, Miller 2-87 (RSA); Sec. 10, T48, R7E, West slope of Salt Cr., 2400’, 28 May 1933, Sack & Iverson 70393 (CAS); New River Trail from Grays Falls Campground on the North side of the Trinity River and west side of the New River, 800-1000’, 7 Apr 1973, Smith 6006 (RSA); Under scattered yellow pine; along Van Duzen River, about 3 miles southeast of Kuntz, 20 Apr 1950, Tracy 18633 (RSA, UC). Ventura County: Las Turas Lake, Santa Monica Mts., 14 Feb 1931, Ewan 4027 (POM); Upper Ojai, Ojai Valley and vicinity, 7 Mar 1895, Pettibone & Hubby s.n. (CAS); Hills north of Ojai, Upper Ojai, Ventura River Basin, 16 Apr 1949, Pollard s. n. (CAS); Hills north of Ojai, Upper Ojai, Ventura River Basin, 21 Apr 1949, Pollard s. n. (CAS). County Unknown: Belmont (MSC); Califomia,1853-1854, Bigelow s.n. (DS); S. Oakland Hills, Mar 1900, Carruth s.n. (CAS); Mt. Hamilton, 3000’, 3 Apr 1906, Chaualer 6014 (UC); Cuesta Summit, 2600’, 2 Apr 1908, Condit s.n. (UC); California, Coulter s.n. (UC); Mt. St. 80 Helena, 1 May 1918, Eastwood 6811 (CAS, UC); Near Woodville, May 1889, Howell 1387 (ORE); Northern California, 7 Apr 1937, Javete s.n. (OSC); Napa River Basin, Trail to beaux’s Cabin, 25 Apr 1893, Jepson s.n. (UC); Near San Jaun Hot Springs, 6 Mar 1913, Perkins s.n. (RSA); Oakland, 1903, Rattan s.n. (DS); California, 1889, Wright s.n. (UC). MEXICO: Baja California: Elev. Of peak with microwave towers at 1275m, 32°19’N-116°40’W, collections made from peak to base (ca. 600m)., Thorne et al. 62130 (CAS, RSA). Key: Key to the two species, Pedicularis densiflora and P. aurantiaca, adapted in part from the J epson Manual (Vorobik 1993): Key to Pedicularis by Linda Ann Vorobik. Steps 4’ is directly transcribed from the J epson Key. It is intended that 7,7’, 7a, and 7b fit directly into the existing key; 4’. Corolla i club-like, upper labium hooded, not beaked; lower labium not fan-like 7. Corolla deep red to red-purple (or yellow to orange, occasionally white), lower labium < 1/2 upper 7a. Lower labium >1/3rd as long as the abaxial margin of galea. Bracts of the inflorescence with serrate margins. Tube exserted beyond the calyx lobes at anthesis. .......................................... Pedicularis densiflora 81 7b. Lower labium 1/6th as long as the abaxial margin of galea. Bracts of the inflorescence partially laciniate. Tube not exserted beyond the calyx lobes at anthesis .......................................... Pedicularis aurantiaca 7’ Corolla yellowish or purplish, lower labium gen >1/2 upper Pedicularis densiflora and P. aurantiaca remain an interesting and dynamic group for the evolutionary study of character evolution, pollinator mediated selection, host specificity for parasitism, and habitat preference. Future research should focus on pollination of the two species, and the potential adaptation of floral morphology to different pollination regimes. Additional genetic work in the form of more informative sequence data (perhaps a low copy nuclear gene) or AF LPs would help resolve relationships among populations of P. densiflora and P.aurantiaca. The inferred phylogeny could be used to investigate the number of times floral morphs arose, relationships among populations in different localities, and the evolution of pollinator shifis. Pedicularis densiflora and P. aurantiaca could be used to investigate theories of speciation, pollinator mediated selection, and the rapid radiation of angiosperms. 82 CHAPTER 2 PHYLOGENY AND POLLEN EVOLUTION OF CANT UA (POLEMONIACEAE SUBFAMILY COBAEOIDEAE): EVIDENCE FROM CHLOROPLAST AND NUCLEAR DNA SEQUENCE DATA 83 INTRODUCTION Cantua J uss. is a member of the Polemoniaceae subfamily Cobaeoideae. It has approximately ten species distributed at mid to high elevations in the Andes of Bolivia, Peru, and Ecuador. Most of the species of Cantua are narrow endemics. The relatively small genus has a tremendous amount of floral diversity, with flowers varying in size, form, and color, and a variety of pollination syndromes. Bee, bird, and moth pollination are known or suspected within the genus. Cantua is one of the few genera of the Polemoniaceae that includes woody species, and the only genus that includes species with a tree habit. The pollen exine of Cantua species is pantoporate, with large tectal insulae, and is distinct from all other pollen grains in the family. The genus is the only member of the Polemoniaceae endemic to South America. One taxon, Cantua buxifolia, has the distinction of being the national flower of both Bolivia and Peru and was cultivated by the Incas and used to adorn temples. One or two species of Cantua have been included in several molecular phylogenetic analyses in an attempt to understand the relationships within the Polemoniaceae and elucidate the relationships among lineages within the angiosperrns (matK phylogeny in Steele & Vilgalys 1994, matK phylogeny in Johnson et al. 1996, anTS phylogeny in Porter 1997, NADIB phylogeny in Porter and Johnson 1998, 188 phylogeny in Johnson et a1. 1999, and nth phylogeny in Prather et al. 2000). These molecular analyses have resolved the Core Cobaeoideae (Bonplandia, Cantua and Cobaea) as a monophyletic clade distinct from the Polemonoideae with the exception of two studies: the matK phylogeny in Steele & Vilgalys (1994) and the anTS phylogeny in Porter (1997). The 84 matK phylogeny had an unresolved polytomy at the base of the Polemoniaceae that included members of the Cobaeoideae. This polytomy was resolved in a later phylogenetic analysis based on a longer region of the matK gene and more extensive taxonomic sampling (Johnson et a1. 1996). Porter’s (1997) anTS phylogeny lacked support in the basal nodes of the Core Cobaeoideae and relationships among genera changed depending upon taxon inclusion, character weighting, and alignments. Porter (1997; pg 67) commented that “it is difficult to argue that the ITS phylogeny is error free” in the relationships among the taxa that make up the Core Cobaeoideae. Overall, the molecular analyses provide sufficient evidence to accept that Cantua is a member of the Core Cobaeoideae and has a recent shared ancestry with Bonplandia and Cobaea. However, the relationships among the species of Cantua have not been explored. In the most recent classification of the Polemoniaceae, based on both morphology and molecular phylogenetics, Cantua was placed in its own tribe in the subfamily Cobaeoideae, Tribe Cantueae. The authors subsumed the small genus Huthia into Cantua (Porter & Johnson 2000). The two species previously in Huthia have been renamed C. volcanica (Brand.) J. M. Porter & Prather (=Huthia coerulea Brand), and C. mediamnis (Brand) J. M. Porter & Prather (=H. longiflora Brand). There have been no molecular phylogenetic analyses to test the monophyletic status of the genus. Previous work on pollen exine sculpturing in the Polemoniaceae indicated a congruent relationship between pollen morphology and inferred evolutionary history (Stuchlik 1967a, 1967b, and Taylor and Levin 1975). Several studies have examined pollen across 85 the family Polemoniaceae, including the Core Cobaeoideae (Stuchlik 1967a, 1967b, and Taylor and Levin 1975). Additional generic level studies have been conducted on members of the Cobaeoideae: Day and Moran (1986) described the pollen in Acanthogilia gloriosa (Brand.) Day & Moran; Prather (1999) did an extensive review of pollen in Cobaea, and a review of Bonplandia geminiflora pollen exine sculpturing is underway (Dickman and Prather, unpublished data). Until now, few species of Cantua pollen have been described (Stuchlik 1967a, 1967b, and Taylor and Levin 1975). Pollen grains in flowering plants give rise to pollen tubes, which grow through the transmitting tissue of the style and into the ovary. Once the pollen tube reaches the ovary, male gametes are released and fertilization takes place. Delpino (see Darwin 1896; 250) hypothesized pollen grain diameter is a product of the length of the style and the energy storage requirements of the pollen tube to reach the ovary. Several researchers have investigated this potential connection between style length and pollen diameter (Lee 1978, Baker & Baker 1979, Cruden and Lyon 1985, Elisens 1986, Lee 1989, Cruzan 1990, Stroo 2000, Aguilar et al 2002). Plitmann and Levin (1983) tested the correlation between pollen grain diameter and style length in the Polemoniaceae. They measured mean style length and pollen diameter for 140 species in 18 genera across the family. Using Pearson’s pair-wise correlations, they found a strong significant correlation within and among genera across the family. However, these results could be an effect of non- independence due to shared phylogenetic history instead of independent evolution (Cruden and Lyon 1985, Felsenstein 1985). 86 Plitmann and Levin (1983) sampled five Cantua specimens in their analysis of the Polemoniaceae. While they found a strong correlation between pollen diameter and style length within the family, and within other genera, they did not find a significant correlation within the genus Cantua (r = 0.39, p-value > 0.05). A broader sampling of the genus, a morphological review of the pollen, and a comprehensive phylogenetic hypothesis would add insight into the potential correlation between pollen diameter and style length among species of Cantua. This study had three primary goals: Estimate a phylogeny of the genus Cantua, examine the pollen exine sculpturing of the species, and investigate the relationship between pollen diameter and style length in Cantua in a phylogenetic context. Sequences from the nuclear ribosomal ITS gene and the chloroplast spacers trnT-trnL. trnL-trnF, and partial nth coding region were used to generate a phylogenetic hypothesis of the genus. A comprehensive review of pollen grains across Cantua completed the pollen grain sampling within the Core Cobaeids. The pollen grain study was combined with the phylogenetic analysis to address evolution of exine sculpturing within the genus and directly test the phylogenetic utility of pollen morphology. Comparative analyses were conducted to further test the potential correlation between pollen diameter and style length in Cantua. To our knowledge this was the first attempt to examine the correlation between pollen diameter and style length within a phylogenetic context. 87 MATERIALS AND METHODS Herbarium Specimens and sampling - A total of 903 herbarium specimens of Cantua were examined from the following herbaria: AAU, BM, CAS, DAV, DES, DS, F, G, GB, GH,H, HUT, JEPS, K, LL, LPB, MO, MSB, MSC, PH, QCNE, S, TEX, U, UC, UPS, and US. Permission was requested and granted to sample herbarium material for DNA extraction and/or scanning electron microscopy (S EM) and light microscopy of pollen grains from the following herbaria: F, HUH, LL, MO, MSB, MSC, NY, UC, and US. Specimens were chosen to represent the morphological and geographical variation of the species. Individuals were used in the phylogenetic and pollen analysis from the following nine species: C. bicolor, C. buxifolia, C. candelilla, C. cuzcoensis, C. flexuosa, C. pyrifolia, C. quercifolia, C. volcanica, and an as yet undescribed species (Table 4 & 5). 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E: S: ‘ . ‘ 9O distributions and/or high levels of intraspecific morphological variation, several geographically and/or morphologically distinct individuals were sampled (Table 4). Nineteen samples were included in the phylogenetic analyses. I used two sequences of the internal transcribed spacer region of nuclear ribosomal DNA (ITS) from outgroup species that were previously published. The taxa and their GenBank accession numbers are as follows: Cobaea minor (AH005678) and Cobaea pachysepala (AH005690). Additional sequences were made available for Bonplandia geminiflora, Cantua volcanica, Cobaea minor, and Cobaea pachysepala by David C. J arrell and L. Alan Prather (unpublished data). Outgroup - Bonplandia geminiflora and two species of Cobaea: C. minor and C. jachysepala were chosen as outgroups based on the nth phylogeny of Prather et a1. (2000). Phylogenetic Analysis - DNA was extracted using the techniques of Loockerman and Jansen (1996) and purified using the Schleicher & Schuell Elu-quick DNA Purification Kit (Keene, NH). Three sets of chloroplast primers were used: trnT - trnL 5’ primers fi‘om Fujii et al. (1997), trnL 3’ — trnF primers from Taberlet et a1. (1991), and primers for a partial nth coding region using the 1201 forward primer of Jansen (1992) and a reverse primer designed for this analysis. The sequence of the nth reverse primer is as f6 1 l ows: 5’-GTAGTAAATTACTAAAAAGATTRATAC-3 ’. Additional internal primers "‘ ere designed to amplify the trnT — trnL intergenic spacer. The forward primer was 5’— Q CTAAGCGGGCTCACATAA—3’, and the reverse primer sequence was 5’— 91 TGTGATGTCCI I ICCCCI 1 1—3’. ITS], ITS2, and the 5.88 regions of nuclear ribosomal DNA were amplified using ITS-4 primers of White et al (1990) and the modified ITS-5 primer of Sang et al. (1995). Standard amplification protocols were used, including reaction mixture components and the PCR profile (Prather et al. 2002), and amplifications were performed on a MJ Research PTC-IOO thermalcycler. PCR products were gel purified using the Schleicher & Schuell Elu-quick DNA Purification Kit, (Keene, NH) and then sequenced in both directions using an ABI-373 automated sequencer. Sequencing reactions were conducted using the AmpliTaq DNA Dye Terminator Cycle Sequencing reagents (PE Applied Biosystems, Norwalk, CT). Edited sequences were aligned using Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI). Termini of the trnT-trnL intergenic spacer region and trnL-trnF intergenic spacer region were determined from Fujii et al. (1995) and Morrell et a1. (1998), respectively. Termini of ITS], ITSZ, and the 5.8S regions of nuclear ribosomal DNA were determined by comparison with published sequences of Prather and Jansen (1998). file segment of nth used for the analysis began at the 1244th base pair of tobacco (Olmstead et al. 1993). P arsimony methods were implemented using PAUP* (Version 4.0b4; Swofford 2000). E1 endstic searches were performed using the TREE BISECTION RECONNECTION, and I ULTREES options. All insertion/deletion events, displayed as gaps in the data set, 92 L were treated as characters using the “Simple Indel Coding” method outlined by Simmons and Ochoterena (2000). Bootstrap analyses were conducted using 10,000 replicates with 200 random addition-sequence replicates per bootstrap replicate. A partition homogeneity test, using 10,000 replicates, was conducted to test for homogeneity in the distribution of phylogenetic information between the nuclear and chloroplast data sets. A second analysis was conducted to determine branch lengths for the independent contrast of pollen grain diameter and style length. This analysis included only one sample of each taxon. The sample was chosen by selecting the species with the most complete sequences for both the chloroplast and nuclear molecular data sets. All insertion/deletion events, displayed as gaps in the data set, were treated as missing data. Branch lengths were determined using the MinF option in PAUP* (version 4.0b4; Swofford 2000, i Swofford and Maddison 1987). Sequence termini, parsimony methods, heuristic searches, and bootstrap analysis were identical to the analysis described above. Pollen Analysis - Pollen Grain Morphology and Style Length - For the SEM study, I examined the pollen morphology of 28 individuals of nine species of Cantua. For each taxon, at least two i ndividuals were sampled from different parts of their geographical range (Table 5). P Ollen preparation for the SEM was conducted using the methods of Prather (1999). Qhopened anthers were removed from herbarium specimens, boiled in water until the D C) l len was released, and acetolyzed using the methods of Adams and Morton (1972). I acerations and critical drying point were omitted. Specimens were mounted on stubs, 93 coated with 60% palladium and 40% gold, and examined using a J EOL 64OOV scanning electron microscope at the Center for Electron Optics at Michigan State University. Light microscopy was used to measure pollen diameter and style length on a minimum of two populations for each species. Twenty three populations were sampled for a total of 555 pollen grains and 90 individual styles (Table 5, Appendix I). Whenever possible, 30 pollen grains from each population were sampled. Number of styles measured per collection varied from 1-8. Pollen grains were removed from two dehiscing anthers on herbarium specimens and stained in Cotton-blue with lactophenol for at least 24 hours prior to measurement. This assured that all pollen grains had absorbed the stain and were fully hydrated. Pollen grains were measured with a calibrated eyepiece micrometer under 4OOX magnification. Only fully stained, intact pollen grains were measured. All available styles, from fully developed, non-senescing flowers were measured on all duplicates of the same collection number. Measurements were made using digital calipers and recorded to the nearest 0.0] mm. Sale Length and Pollen Diameter Correlation—Traits in related species are not 1 hdependent. Closely related species may share common traits due to shared ancestry I: ather than independent evolution. To remove the issue of non-independence in data sets ruparative methods have been developed to examine relationships among pairs of ntinuous traits while accounting for potential effects of shared evolutionary history. To L 94 assess the relationship between style length and pollen diameter, the independent contrasts method of Felsenstein (1985) was implemented using the Comparative Analysis of Independent Contrasts (CAIC; Purvis and Rambaut 1995). CAIC is suitable for analyzing comparative data on continuous characters and can accept polytomies in a phylogeny. Mean values were calculated for pollen diameter and style length for each species, and log-transformed before analysis. In the independent contrasts analysis, two types of data were Used. The raw data set assumed every branch in the phylogeny was of equal length indicating equal amounts of evolutionary change between speciation events. The raw data analysis assumed a puntuational view of evolution with evolutionary change occurring only at the nodes. The transformed data set utilized branch lengths from the phylogenetic analysis as expected units of evolutionary change between speciation events; this data analysis assumed a gradualistic view of evolution with evolutionary change occurring along the branches between speciation events. CAIC does not accept branch lengths less than 2, so all branch lengths were multiplied by 2 to fulfill the program requirements (Purvis and Rambaut 1995). Both the raw and transformed data sets were checked to assure they conformed to the assumptions of regression models: that the residual Variation around the regression line has equal mean and variance at all points on the line. In the output, CAIC computes the standardized linear contrasts for the traits in the " halysis and forces the regression of the standardized linear contrasts through the origin. QA IC computes a slope and the significance of the linear contrasts. The value of the 95 slope describes the relationship between pollen diameter and style length based on independent contrasts (Purvis and Rambaut 1995). An additional analysis of the correlation of standardized linear contrasts was computed using the J MP statistical package (SAS Institute Inc., Cary, NC) for a direct comparison to the results reported by Plitmann and Levin (1983) who performed a Pearson pair-wise correlations correlation on non-independent data. The resultant correlation, like the $10pe in the regression analysis, is representative of the relationship between pollen grain diameter and style length minus phylogenetic effects (Pagel 1993). 96 RESULTS Phylogenetic Analysis — In the data set excluding insertion/deletion events (indels), the aligned ITS sequences were 660 bp long, with a total of 201 potentially phylo genetically informative characters. Phylogenetic analyses of the nucleotide data resulted in 15 most parsimonious trees with a length of 437, C.I.c = 0.7889, and RI. = 0.8783. Forty-five indels were scored and appended to the ITS nucleotide data, 25 of those characters were potentially phylo genetically informative. The analysis of the data set including indels resulted in 15 most parsimonious trees with a length of 489, CI.‘3 = 0.7882, and RI. = 0.8783 (Figure 12). Tree topologies were identical for the analyses including and excluding the indel characters. A clade comprising all members of the genus (Clade A, Fig. 12), including C. volcanica (formerly Huthia), had strong (100%) bootstrap support. Likewise, two major clades within Cantua were strongly supported: One clade consisting of Cantua volcanica and C. quercifolia (96%) and a second clade which encompasses the remaining Cantua species ( l 00% bootstrap support). Relationships within the second clade had weak bootstrap SUpportwith the exception of the Cantua pyrifolia / C. n. sp. clade which had strong bootstrap support (100%). 97 58 Cantuaflexuosa A 85 60 Cantuaflexuosa B 64 Cantua cuzcoensis Cantua bicolor 59 100 Cantua buxifolia A Cantua buxifolia B Cantua buxifolia C 91 l— Cantua candelrlla A I—— Cantua candelilla B 100 [—— Cantua pyrrfolia 100 96 L Cantua n. sp. 100 Cantua volcanica A Cantua volcanica B 100 Cantua quercifolia A 100 Cantua quercifolia B Cantua quercifolia C ]— Cobaea pachysepala L— Cobaea minor F G! C) $31 able 4. Bonplandia geminiflora igure l2: Strict consensus of 15 most parsimonious trees based on the Cantua total ITS ata set including nucleotide substitutions and indels. Tree length = 489, with a C.I.e = ‘ 7 882 and RI. = 0.8783. Numbers above the branches represent bootstrap support based 10,000 replicates. Location and voucher information for each sample is available in 98 Multiple taxa from the same species were in exclusive clades with strong bootstrap support (C. flexuosa 85%, C. candelilla 91%, C. volcanica 100%, and C. quercifolia 100%) with the exception of the three individuals of C. buxifolia, which had unresolved relationships at the base of a clade including C. bicolor, C. cuzcoensis and C. flexuosa. In the data set excluding indels, the aligned sequences from the trnT-trnL intergenic spacer were 642 bp long with 18 potentially phylogenetically informative characters. Parsimony analysis resulted in 207 most parsimonious trees with a length of 63, a C.I.°. = 0.9524, and a R.I. = 0.9697. The data set had five insertion-deletion events, one of which was potentially phylogenetically informative. Phylogenetic analysis of the data set including indels resulted in 441 most parsimonious trees with a length of 68, a C.I.°. = 0.9545 and a R.I. = 0.9714. I was unable to amplify Cantuaflexuosa B (Weigand 5862, see Table 4) and it was not included in trnT-trnL analyses. Analyses of the data, both including and excluding the indels, yielded a consensus trees with no resolution among the species of Cantua. In the data set excluding indels, the trnL-trnF intergenic spacer was 448 bp long with 13 potentially phylogenetically informative characters. The parsimony analysis of the nucleotide data set resulted in three most parsimonious trees, with a length of 48, a C.I.°. § 1.0, and a R.I. = 1.0. There were 14 insertion-deletion events, 9 of which were potentially phylogenetically informative. Analyses of the data set including indels found ten most parsimonious trees, with a length of 65, C.I.° = 0.8846 and a R.I = 0.9348. Ahalyses of the data set both including and excluding indels, had some resolution and 99 concordant topologies. The analysis of the data set including indels had more resolution within Cantua. The aligned sequences from the partial nth-coding region were 938 bp long, with 59 potentially phylogenetically informative characters. Two most parsimonious trees resulted from the analysis and each had a length of 119, a CI." =0.9444 and a R. I. = 0.9690. No indels were present and the strict consensus tree showed some inter- and intraspecific resolution. The chloroplast data sets including indels were combined for a total of 2047 bp and 19 indels with 100 parsimony informative characters (Figure 13). Two most parsimonious trees were generated, with a length of 256, a CI.6 = 0.9032, and a R.I. = 0.9429. The monophyly of Cantua, including C. volcanica, had strong support (100%). Cantua volcanica and C. quercifolia were strongly supported (97%) as sister taxa, and together they were sister to the remainder of the genus ( 96% bootstrap support). A clade Comprising Cantua buxifolia (A & B), C. pyrifolia and C. n. sp. had weak bootstrap SuppOrt (73%). The clade containing C. buxifolia A & B was moderately supported ( 75%) and sister to a well supported clade containing C. pyrifolia and C. n.sp. (97%). Cantuaflexuosa A & B, C. buxifolia C, both samples of C. candelilla A & B,'C. Clacoensis, and C. bicolor had strong bootstrap support (84%). The clade including C. bluifolia and C. candelilla A & B had moderate bootstrap support 100 94 I-—— Cantuaflexuosa A 58 I— Cantuaflexuosa B .—_1 Cantua buxifolia C 80 65 Cantua candelilla A 85 l Cantua candelilla B Cantua cuzcoensis , 96 Cantua bicolor 74 C antua brm'folia A 74 Cantua buxifolia B 100 Li: Cantua pyrifrflia Cantua n.sp. 100 Cantua volcanica A Cantua volcanica B 97 Cantua quercifolia A 100 Cantua quercifolia B | VT Cantua quercifolia C 100 r—— Cobaea pachysepala |—— Cobaea minor Bonplandia geminiflora F igure l3: Strict consensus of two most parsimonious trees based on the Cantua .Q‘Ornbined chloroplast sequences (trnT- trnL, trnL- trnF, partial nth) for the data sets ; hcluding indels. Tree length: 256, C. I. e = 0.9032 and a R. I. = 0.9429. Numbers above 11 e branches represent bootstrap support based on 10,000 replicates. l0l (81%). With the exception of C. bwcifolia which is paraphyletic in the phylogeny, multiple taxa from the same species were in exclusive clades with strong bootstrap support (C. flexuosa 77%, C. candelilla 66%, C. volcanica 100%, and C. quercifolia 100%). When the data sets including indels from the nuclear and chlorOplast genomes were combined, the null hypothesis of homogeneity in distribution of phylogenetic information between the data sets based on the partition homogeneity test was rejected (p = 0.0127). The data set was reviewed and the relationships of C. buxifolia in the nuclear and chloroplast phylogenies were not concordant. In the ITS phylogeny (Fig 12), Cantua buxifolia was part of an unresolved polytomy at the base of a clade including C. bicolor, C. cuzcoensis and C. flexuosa. In the chloroplast phylogeny (Fig. 13), C. buxifolia C was sister to a clade with C. candelilla, but C. buxifolia A & B were sister to the clade containing C. pyrifolia and the C. n. sp. Cantua buxifolia has a high level of intraspecific morphological variation and has been cultivated throughout much of Andean South America for centuries. The likelihood of hybridization is high, and is a likely explanation for the lack of congruence between the chloroplast and nuclear phylogenies. The S pecimen of C. buxifolia C had different relationships within Cantua in the chloroplast DNA and nuclear DNA phylogenies. This sample was removed from both data sets and a S econd partition homogeneity test was run. This test did not reject the null hypothesis of -h<)mogeneity among data sets (p = 0.2529). I 102 The total combined data set including indels had 2752 total characters: 2688 nucleotides and 64 indels. The data set had 324 potentially phylogenetically informative characters. Four most parsimonious trees resulted from the analysis, each with a tree length of 741 , a CI.6 = 0.8133, and a R.I. = 0.8903. A strict consensus tree was generated (Figure 14). The monophyly of Cantua was strongly supported (100%). The sister relationship between C. quercifolia and C. volcanica had strong bootstrap support (99%). All remaining Cantua taxa are included in a strongly supported clade (100% bootstrap support). Cantua pyrifolia and C. n. sp. are strongly supported as sister taxa (100% bootstrap support) in a polytomy at the base of the clade including the remaining Cantua species. Cantua candelilla, C. bicolor, and C. cuzcoensis, had weak bootstrap support (71%). A monophyletic group of Cantua bicolor, C. cuzcoensis, and C. flexuosa, had moderate support (81%). In this phylogenetic analysis, with the removal of C. buxifolia C, multiple taxa from the same species were in exclusive clades with strong bootstrap support (C. quercifolia 100%, C. volcanica 100%, C. buxifolia 79%, C. candelilla 100%, and C. flexuosa 97%). The tree estimated for the independent contrasts, with only one sample per species, had 2 688 total characters (Figure 15). The data set had 210 potentially phylogenetically i Ilformative characters. A single most parsimonious tree resulted from the analysis, with a l ength of650, a C.I.e = 0.7771, and a R.I. = 0.7869. The tree topology did not conflict b etween the tree from the total combined data set and the tree estimated for the Q=anparative analysis, with one exception, in the tree estimated for the comparative a*3t1zilysis a sister relationship was resolved between C. 103 97 Cantuafleruosa A 64 Cantuaflexuosa B 81 Cantua arzcaens is 71 . Cantua bicol or 100 [— Cantua cmdelilkz A '— Cantua amdelilla B 100 F— 79 r—— Cantua butrfolia A L— Cantua brocifolia B Cantua rifolia 100 I " ' W 100 t 1— Cantua n.sp. 100 Cantua volcanica A Cantua volcanica B 99 Cantua qrerci fall a A 100 Cantua quercifolia B Cantua qrerci foli a C 100 r— chaea padrys qxia L— chaea minor B aplmdia gmimflora Q i glue l4: Strict consensus of four most parsimonious trees based on the Cantua t‘ Qnibined chloroplast (trnT-trnL, trnL-trnF, and partial nth) and nuclear ITS sequences Q r the data set including indels. Tree length = 747, C.I.° = 0.8133 and a R.I. = 0.8903. N umbers above the branches represent bootstrap support based on 10,000 replicates. 104 Cantua flexuosa A 65 8 l 80 1 1 Cantua cuzcoensis 5 71 Cantua bicolor ‘ 3 12 Cantua candelilla A 100 . 7 28 4 Cantua buxifolia B 62 2 Cantua pyrifolia 100 100 49 I 1 Cantua n.sp. Cantua volcanica B 95 69 134 36 l m - 71 Cantua quercifolia B Cobaea pachysepala 100 8° 47 lnl Cobaea minor 69 Bonplandia geminiflora F i gure 15: Cantua tree estimated for comparative analysis. Single most parsimonious tree ased on the modified data set of chloroplast (trnT-trnL, trnL-trnF, partial nth) and h uclear ITS sequences excluding indels. Tree length is 650, with a CI. = 0.7771 and a - I. = 0.7869. Numbers above the branches represent bootstrap support based on 10,000 I:“elolicates, numbers below the branches indicate branch lengths based on the MINF thion in Paup“. 105 buxifolia and C. pyrifolia/ C. n.sp. This relationship, however, was weakly supported (bootstrap support of 62%). Pollen Morphology Description of Cantua Pollen — Cantua grains were large (49-93 pm), spheroidal and pantoporate with mean pore size ranging from 3.78-14.4um. The sexine was semitectate with irregularly shaped insulae, and insulae were evenly distributed over the surface of the pollen grain, with the exception of C. flexuosa where they were more sparsely and haphazardly distributed. Bacula were reticulate and the supratectal surfaces striated in all species. Cantua quercifolia and C. volcanica shared a synapomorphy of supratectal verrucae. A pollen diagnosis for each of the species of Cantua is given below (Figures 16, 17, & 18). Cantuaflexuosa (Figure 16, A & B). Pantoporate. Pollen grain spheroidal, 58 — 83 pm in diameter. Pores circular, 4.66-6.29 pm in diameter. Sexine semitectate (insulous). Insulae irregular in shape, haphazardly distributed with considerable, irregular intratectal Space. Bacula reticulate. Supratectal surface deeply striated. Cantua cuzcoensis (Figure 16, C & D). Pantoporate. Pollen grain spheroidal, 63-81 pm in diameter. Pores circular, 4.62-7.94 pm in diameter. Sexine semitectate (insulous). Insulae irregular in shape, evenly distributed. Bacula reticulate. Supratectal surface (3- eeply striated. 106 Figure 16. (A-H) SEM photographs of pollen grains of Cantua species (Polemoniaceae): (A & B) C. flexuosa; (C & D) C. cuzcoensis; (E & F) C. bicolor; (G & H) C. candelilla. Scale bars = 25 mm (A, C, E, & G) & 5 tun (B, D, F, & H). Voucher information available in Table 5. 107 Figure 17. (A-F) SEM photographs of pollen grains of Cantua species (Polemoniaceae): (A & B) C. buxifolia; (C & D) C. pyrifolia; (E & F) C. n. sp.. Scale bars = 25 um (A, C, &E)&5p.m(B,D,&F). 108 Figure 18. (A-D) SEM photographs of pollen grains of Cantua species (Polemoniaceae): (A & B) C. volcanica (C & D) C. quercifolia. Scale bars = 25 pm (A & C) & 5 [Am (B & D). Cantua bicolor (Figure 16, D & E). Pantoporate. Pollen grain spheroidal, 51.5-78.5 pm in diameter. Pores circular, 7.40-9.32 um in diameter. Sexine semitectate (insulous). Insulae irregular in shape, evenly distributed. Bacula reticulate. Supratectal surface faintly striated. Cantua candelilla (Figure 16, F & G). Pantoporate. Pollen grain spheroidal, 75.5-90.5 pm in diameter. Pores circular., 3.90-9.00 pm in diameter. Sexine semitectate (insulous). Insulae irregular in shape, evenly distributed. Bacula reticulate. Supratectal surface faintly striated. Cantua buxifolia (Figure 17, A & B). Pantoporate. Pollen grain spheroidal, 49-89 pm in diameter. Pores circular, 3.78-1 1.11 pm in diameter. Sexine semitectate (insulous). Insulae irregular in shape, evenly distributed to randomly distributed with considerable, irregular intratectal spaces. Bacula reticulate. Supratectal surface faintly striated. Cantua pyrifolr'a (Figure 17, C & D). Pantoporate. Pollen grain spheroidal, 64-86 pm in diameter. Pores circular, 4.86-14.4 um in diameter, considerable variation in pore size among individual samples. Sexine semitectate (insulous). Insulae irregular in shape, evenly distributed. Bacula reticulate, density of bacula variable among individuals sampled. Supratectal surface faintly striated. Cantua n. sp. (Figure 17, E & F). Pantoporate. Pollen grain spheroidal, 67-81 pm in diameter. Pores circular, 6.30-7.56 pm in diameter. Sexine semitectate (insulous). llO Insulae irregular in shape, evenly distributed. Bacula reticulate with occasional stray free bacula. Supratectal surface faintly striated. Cantua volcanica (Figure 18, A & B). Pantoporate. Pollen grain spheroidal, 54-69 pm in diameter. Pores circular, 4.10-8.22 pm in diameter, considerable variation in pore size among individual samples. Sexine semitectate (insulous). Insulae elongated, evenly distributed. Bacula reticulate with occasional stray free bacula. Supratectal surface faintly striated with supratectal verrucae. Cantua quercifolia (Figure 18, C & D). Pantoporate. Pollen grain spheroidal, 78.5-93 mm in diameter. Pores circular, 5.38-8.62 um in diameter, considerable variation in pore size among individual samples. Sexine semitectate (insulous). Insulae circular, evenly distributed. Stray free bacula. Supratectal surface faintly striated with supratectal verrucae. Pollen Trait Evolution-Pollen grains in Cobaea have been extensively reviewed (Prather 1999) and an analysis of pollen in Bonplandia geminiflora is currently underway (Dickman and Prather, unpublished data). Cobaea pollen grains are pantoporate and spheroidal. The grains are large (112-195 um) and have a reticulate sexine. The grains of the monotypic genus Bonplandia are also spheroidal and pantoporate. The grains are smaller in Bonplandia (60pm) and the sexine is striato reticulate. lll Cantua grains were large (49-93um), spheroidal and pantoporate, these traits are shared with the two outgroup taxa (Cobaea and Bonplandia). Cantua pollen grains share synapomorphic characters not present in the outgoup taxa: semitectate sexine with irregularly shaped insulae and reticulate bacula. Cantua quercifolia and C. volcanica shared a synapomorphy of supratectal verrucae (Figure 19). Style Length and Pollen Grain Diameter Correlation—Raw data (Table 6) did not conform to the assumption for regression analysis of equal variance of residuals (p-value < 0.05). When the data were transformed, and branch length data were utilized, data conformed to all the assumptions of the analysis. The transformed analyses did not suggest a significant relationship between pollen grain diameter and style length based on the regression of standardized linear contrasts through the origin (r2 = 0.07, slope = - 0.05, p-value = 0.48). Standardized linear contrasts were analyzed on the transformed data using Pearson pair-wise correlations and the resultant correlation values were insignificant (-0.23 Pearson’s pair-wise correlation, p-value = 0.58). Table 6. Pollen diameter and style length of Cantua taxa included in the analysis. Whenever possible, 30 pollen grains from each population were sampled. Total number of styles per collection varied from 1-10. Number of Style length (mm) Pollen diameter (um) Species populations measured Mean Range Mean Range Cantua bicolor 2 39.24 35.21-42.69 67.31 51.45-78.40 Cantua buxifolia 4 68.98 35.02-80.30 74.31 49.00-85.75 Cantua candelilla 2 65.69 56.86-73.95 81.96 73.50-90.65 Cantua cuzcoensis 2 53.54 48.50-57.96 72.80 63.70-80.85 Cantuaflexuosa 2 34.10 30.92-39.67 71.34 58.80-83.30 Cantua n. Sp. 2 42.78 38.27-45.95 75.91 68.60-80.85 Cantua pyrifolia 5 32.98 24.96-46.77 77.23 63.70-85.75 Cantua quercifolia 2 42.49 39.87-52.13 85.94 78.40-93.10 Cantua volcanica 2 21.16 16.42-24.62 62.45 53.90-68.60 Cantua bicolor, C. buxifolia, C. candelilla, C. cuzcoensis, C. flexuosa, C. n. sp., and C. pyrifolia Cantua volcanica Cantua quercifolia I: ‘y Cobaea ‘ Bonplandia geminiflora Figure 19. Diagram representing phylogenetic relationships found in the analysis of the Cantua combined chloroplast (tmT—tmL, tmL-th, partial nth) and nuclear ITS sequences and represented in Figure 14. Slash marks on the tree branches represent synapomorphic characters: A. serrritectate sexine with irregularly shaped insulae, B. reticulate bacula, C. supratectal verrucae. Representative pollen grains are shown at the ace tips: Bonplandia gemimflora (Rzedowski 18144), Cobaea (C. trianae; Panero & Clark 304-4), Cantua quercifolia (Wrigley 2), Cantua volcanica (West 7139), and seven remaining Cantua (Cantua bicolor; Cardenas 5479). Scale bar = 10 pm 113 CONCLUSIONS The phylogenetic analysis of the nuclear ribosomal ITS and chloroplast trnT-trnL, trn L- trnF and nth regions in Cantua strongly supported the inclusion of the two species formerly placed in Huthia and verified the monophyly of the genus Cantua. Cantua volcanica (=Huthia coerulea) was most closely related to C. quercifolia. These two species were the sister group to the rest of Cantua. Cantuaflexuosa, C. cuzcoensis, C. bicolor, C. candelilla, C. buxifolia, C. pyrifolia and C. n. sp were closely related and C. pyrifolia was sister to C. n. sp. Relationships between C. buxifolia and the other species in the clade were unresolved. The monophyly of C. buxifolia and its relationship to the other members of the genus Cantua could be investigated further with additional sampling within C. buxifolia and more informative molecular sequences. The work outlined here is an important link into research on the evolution of the Cobaeoideae and part of a large ongoing project to monograph the subfamily. Pollen morphological work in Cantua supported the findings from the molecular phylogenetic analysis. Pollen characteristics within Cantua were highly conserved, pollen morphology examined was consistent within populations and among species. All nine species of Cantua have spheroidal pantoporate pollen with a semitectate sexine and insulae. This is a shared derived characteristic in the Polemoniaceae, giving firrther support to the subsumption of Huthia and the monophyly of the genus. The relationship between C. quercifolia and C. volcanica was supported by the synapomorphy of supratectal verrucae on the sexine of the pollen grains. The pollen work in Cantua confirms that pollen grain morphology is highly conserved and a valuable morphological 114 character for cladistic analysis. In addition, this research completes a comprehensive review of pollen grain morphology within the subfamily Cobaeoideae. Plitmann and Levin (1983) addressed the potential relationship between pollen diameter and style length in a correlation analysis across the Polemoniaceae. They found a strong significant correlation between the traits within and among genera in the family. The results from their analysis supported the hypothesis of Delpino (Darwin 1896); pollen grain diameter is functionally integrated with style length. Lack of independence in the data set may have confounded the results of Plitmann and Levin’s research. Data were collected from several groups of related species within the Polemoniaceae, and could be affected by non-independence due to shared evolutionary history (Cruden and Lyon 1985, Felsenstein 1985, and Harvey and Pagel 1991). Plitmann & Levin (1983) found a non-significant correlation between pollen diameter and style length in five of the ten species of Cantua. My research was designed to address the correlation question using a comprehensive sample of the Cantua species and pair- wise independent comparisons of the traits. I almost doubled the representative species used in the previous study, included the recently subsumed C. volcanica, and used independent contrasts to control for the effects of shared ancestry. Independent contrasts would not result in a correlation which was not present in the non-independent data. However, the increased, more inclusive sampling could result in a correlation and the independent contrasts would be necessary to address the non-independence of data in the Cantua data set. The results from my analysis of Cantua and the analysis of Plitmann 115 and Levin (1983) are consistent. Neither significant regression nor correlation was found in the genus Cantua, which failed to find a relationship between the diameter of the pollen grain and the length of the style in Cantua. This comprehensive phylogenetic analysis of Cantua provided insight into relationships within the genus and confirmed the monophyly of the group. The investigation into pollen grain morphology substantiated the subsumption of Huthia into Cantua and provided further evidence for the monophyly of the genus. Both the pollen grain morphology and the phylogenetic analyses supported the relationship between C. volcanica and C. quercifolia. This pollen work also confirmed that pollen grain morphology is highly conserved and a valuable morphological character for cladistic analysis in Cantua. Despite comprehensive sampling and a well-supported phylogenetic hypothesis, pollen diameter and style length were found to be unrelated in Cantua. The work on pollen grain diameter and style length is the first step in a family-wide comparison of this correlation using independent contrasts to control for the effects of shared ancestry. The study outlined here is an important link into research on the evolution of the Cobaeoideae, molecular analysis using the same chloroplast and nuclear sequences and identical pollen grain preparation for SEM and light microscopy will be used in a large ongoing project to monograph this sub-family. 116 CHAPTER 3 EVOLUTION OF VARIANCE-COVARIANCE STRUCTURE OF FLORAL MORPHOLOGY AMONG MEMBERS OF THE MUSTARD FAMILY (BRASSICACEAE) ll7 INTRODUCTION Individual floral traits may not evolve independently. Flowers are complex structures made up of several functionally integrated traits which work together to aid in successful plant reproduction (Darwin 1862). Often groups of traits will evolve together due to genetically, developmentally, and functionally integrated constraints (Arnold 1992). Consequently, how diverse floral traits evolve as a unit is of tremendous interest to botanists and evolutionists alike. The matrix of genetic variance and covariance (G-matrix) represents the amount of heritable variation in a trait and its genetic covariation with other traits. It is an important concept because selective forces acting on one trait will cause evolution of a second trait even if the change infers no specific selective advantage. The G-matrix represents the type and quantity of genetic variation available to natural selection. While the G-matrix may confer a selective advantage, over time the amount of genetic variation and covariation in a population could constrain the direction of genetic drift and dictate response to selection. Genetic variation-covariation can affect both the rate and direction of evolutionary change (Lande 1976, 1979, Lande and Arnold 1983, Conner and Via I993, Steppan 1997a, 1997b, Arnold and Phillips 1999, Phillips and Arnold 1999, Badyaev and Hill 2000, Arnold et a1. 2001, and Steppan et a1. 2002). The potential constraint imposed by the G-matrix has been an issue under intense scrutiny. F utuyma (1995) studied the evolution of the host affiliation in the leaf beetle genus Ophraella and found that evolution of ecologically important traits were 118 influenced by genetic constraints. Schluter (1996) examined several vertebrate taxa to determine the long term effects of genetic variation on morphological divergence. He found that among the species he studied, morphological variation had been biased in the multivariate direction of greatest additive genetic variance for the last four million years. Both studies support the theory that quantitative genetic variance and covariance can constrain evolution over time. The degree to which the G-matrix is conserved is an important unsolved problem in evolutionary biology. Lande (1979) pointed out how consistency in the G-matrix would allow us to make inferences about patterns of selection. Lande (1979) developed an important equation for the measuring changes in traits in evolutionary time: A2 is the change in phenotypic traits across one generation. G is the matrix of genetic variances and covariances among traits. fl is the selection gradient. So the rate of A—z- is the product of additive genetic variance-covariance and the strength of selection and can be used to predict the response to selection over time, including the effects on correlated traits. Lande (1979) predicted that G-matrices would remain constant over time and could therefore be used to predict the response of natural populations to selection and drift. If the G-matrix is stable over time, then scientists can predict the response of a population to selection and reconstruct ancestral events that have lead to speciation (Arnold 1992, Arnold et al. 2001, and Steppan et al. 2002). 119 Several empirical studies have been conducted using both selection and drift experiments and comparative analyses to investigate the stability and evolution of the G-matrix (Arnold and Phillips 1999, Phillips and Arnold 1999, Roff and Mousseau 1999, Roff 2000, and Steppan et al. 2002 and references therein). Both matrix similarity and dissimilarity have been found when comparing G-matrices between populations, subspecies and species and the closer the taxonomic relationship the more stable the G- matrixes have been. Additional studies, with similar results, have been conducted using the phenotypic variance-covariance matrix (P-matrix; Steppan 1997a, 1997b, Badayeav & Hill 2000, Ackerrnann and Cheverud 2000, Dodd at al. 2000, and Baker and Wilkinson 2003). While the P-matrix is not equivalent to the G-matrix, phenotypic variance- covariance has genetic variance-covariance as a component and can give insight into the heritable genetic variation-covariation available (Lofsvold 1986, Cheverud 1988, Arnold 1992, Steppan 1997a, l997b, Wait and Levin 1988, Badyaev & Hill 2000, Arnold et al. 2001, and Steppan et al. 2002). Baker and Wilkinson (2003) and was the first to look at the evolution of the P-matrix using a phylogenetic hypothesis at the species level or higher. This study showed equality of the P-matrices among three species of Diasemopsis (stalk-eyed flies). In reviewing the findings from studies of G and P-matrices, only the Baker and Wilkinson (2003) study found proportionality or equality in the shared structure among matrices when comparing taxa at the subspecies or higher taxonomic level (see review in Steppan et al. 2002). 120 The evolution ofG-matrix structure is still not well understood, particularly in an historical context (Badyaev and Hill 2000). The question of whether the G-matrix remains stable over evolutionary time remains an unresolved issue (Arnold et al. 2001, Steppan et al. 2002). Additional investigations into the nature, stability, and historical pattern of change in the G-matrix are required. Understanding how variance-covariance structures evolve over time will provide insight into how populations evolve and species diverge (Lande 1979, 1985; Price and Grant 1985, Grant and Grant 1995, Cheverud 1996, Arnold and Phillips 1999, Camara and Pigliucci 1999, Roff et a1. 1999, Badyaev and Foresman 2000, Badyaev and Hill 2000, Arnold et al. 2001, and Steppan et al. 2002). This study examined the relationship among P-matrices across the family Brassicaceae using a strong phylogenetic hypothesis. The Brassicaceae study system was chosen for several practical reasons: investigative work on G-matrices, genetic correlations, and phenotypic correlations have already been conducted in several members of the family (Conner and Via 1993, Conner and Sterling 1995, 1996, Kercher and Conner 1996, and Conner 2003 and references therein) , preliminary research on phylogentic relationships in the Brassicaceae had been conducted by Galloway et a1. (1998) and were available for analysis, species in the family were widely distributed and easily collectable, and the floral characters were conserved and straightforward to measure across the family. The Brassicaceae includes several economically and agronomically important plants, most notably several Brassica species, which include such diverse crops as canola 121 (Brassica napus), broccoli (Brassica oleraceae var. italica), cabbage (Brassica oleraceae var. capitata), and turnips (Brassica rapa var. rapa). Several species are cultivated as omamentals including Lobularia maritima (sweet alyssum), Iberis (candy tufi), and Matthiola (stock). The Brassicaceae also contains the model species Arabidopsis thaliana and the problematic invasive plant Alliaria petiolata (garlic mustard). The family has approximately 340 genera and as many as 3350 species (Al-Shehbaz 1984). Brassicaceae are widely distributed, grow predominantly in the temperate northern hemisphere, and are most prevalent in arid regions. The largest concentrations of Brassicaceae species occur in southwestern/central Asia and the Mediterranean (A1- Shehbaz 1984). In 1998 the Angiosperm Phylogeny Group advocated lumping the Capparaceae and Brassicaceae sensu stricto into one family, the Brassicaceae. This nomenclatural change was based on the paraphyly of the Capparaceae in the morphological cladistic analysis of Judd et al. (1994) and the chloroplast rbcl molecular phylogenetic analysis of Rodman et a1 (1996). Recent nuclear 18S molecular phylogenetic analysis (Rodman et a1 1998) and chloroplast nth and trnL-trnF molecular phylogenetic analyses (Hall et al. 2002) have revisited the issue of relationship and monophyly in the Capparaceae and Brassicaceae sensu stricto. Hall et al. (2002) advocated the recognition of the three families: Capparaceae, Cleomaceae, and Brassicaceae, with the Cleomaceae most closely related to the Brassicaceae. The most recent update by the Angiosperm Phylogeny Group (2003), recognizes these studies, and advocates dissolving the Brassicaceae sensu APG 1998, and recognizing the three families as suggested by Hall et al. (2002). In addition to the molecular phylogenetic evidence for the narrowly, and traditionally, circumscribed Brassicaceae, the family has some distinct floral characteristics that distinguish the flowers from its close relatives in the Capparaceae and Cleomaceae (Hall et al. 2002). These features include four alternating clawed petals arranged in the form of a cross and tetradynamous stamens. Several attempts have been made to establish a tribal classification of the Brassicaceae (Hayek 1911, Schulz 1936, Janchen 1942, and Al-Shehbaz 1984). Most of this work has been based on morphological characters, most notably fruit morphology, pubescence, and cotyledon orientation (Al-Shehbaz 1984). In his morphological treatment of the Brassicaceae in the southeastern United States, Al-Shehbaz (1984), used a modification of the J anchen (1942) system and recognized seven major tribes, though he suspected that not all seven tribes were naturally monophyletic (Al-Shehbaz 1984, Price et al. 1994, Galloway et al. 1998). Koch et al. (2001) did the most comprehensive molecular systematic analysis of the Brassicaceae to date, using chloroplast matK and nuclear Chs sequences, and reviewed the tribal classification systems of Hayek (1911), Schulz (1936), and J anchen (1942). The study concluded that many of the tribal and subtribal classifications were artificial and “a new classification of crucifers based on molecular data would be desirable” (Koch et al. 2001; 541). No consensus has been reached on relationships among the major clades within the Brassicaceae and only a handful of studies have been conducted to investigate relationships across the family (Price et a1. 1994, Galloway et al. 1998, Koch et al. 2001, and Hall et al. 2002). 123 This study incorporates a phylogenetic hypothesis of the family Brassicaceae and a study of floral morphological traits to test Lande’s (1979) theories about stability in variance - covariance structure over time. The goal Of this research was to test the hypothesis that P- matrix stability within the Brassicaceae followed a stepwise pattern of decrease in similarity with decreased relatedness corresponding to taxonomic rank within the phylogeny. This was the first attempt to investigate P-matrix relationships across a family-level phylogeny. 124 MATERIALS AND METHODS Phylogenetic Analysis- Sampling — Fresh leaf or floral material from field collections or greenhouse material was collected, dried on silica gel, and stored at -80 degrees. A total of 25 individual specimens were used in the phylogenetic analysis. Eight DNA sequences of the partial nth coding region were previously published in Galloway et a1. (1998) and available on GENBANK (Table 7). Outgroup —Cleome spinosa was chosen as the outgroup based on the nth phylogeny of Galloway et al. (1998) and evidence from the chloroplast nth gene and trnL-trnF spacer sequences of Hall et al. (2002). Analysis - DNA was extracted using the techniques of Loockerman and Jansen (1996) and purified using the Schleicher & Schuell Elu-quick DNA Purification Kit (Keene, NH). The nth coding region was amplified using the forward primer nth 1318 and the reverse primer nth 21 10R of Olmstead and Sweere (1994). Standard amplification protocols were used, including reaction mixture components and the PCR profile (Prather et al. 2002), and amplifications were performed on a MJ Research PTC-IOO thermalcycler. PCR products were gel purified using the Schleicher & Schuell Elu—quick DNA Purification Kit, (Keene, NH) and then sequenced in both directions using an ABI-3 73 automated sequencer. Sequencing reactions were conducted 125 1 1111‘s 154:. d) N $72.; I‘ ltlu-I‘IDGFIIIIN‘P... -A V.“.~‘ndav\ ~V-~u~ i‘l wag .3 00 .33230 80¢ 00355000 0455000 M304 .3 00 330430 50¢ 0035503 0309.00 05:00:30 .. 00050;w5m ”010% 302 M304 .3 00 330—30 80¢ 05055000 0455000 ... 5&002 0% 008505. 50¢ 0000 0033030 _. 00902 0% 00350.; 50¢ 0000 0033200 .. 00302 0% 0000505. 50¢ 0000 0033200 053% 30300—054 wwozom .0034 :00 ”0050042 _.. 5&002 a. 00350.; 50¢ 000m 003>E=U DmE 40.0003 000034 .@5534 new ”5&5sz woe .3 00 330430 50¢ 00.00.5000 0455000 043m 030m 0055 033w=0m ”005342 “9023000000 0000000 005080 moi .3 00 @3230 50¢ 00355000 400980 403.033. 0030: ”5005.: ._ 000m 0034 00000.00 50¢ 00330—00 mom: .3 00 033230 50¢ 00355000 0455000 0536 30_w0_04.m wwozom .0034 :00 ”003:5: 4:00.30 305834 0000002 80¢ 0000 0033030 wag .3 00 330—30 50¢ 00655000 0455000 043m 030m 0055 xaawzmm ”503452 80% 320220 0023 .93 :5 ”case: +3005“: >232 00 0305534 5003 50¢ 0000 003256 33.83 $3092 630 a. 8580 a. 2082 630 83 e: 2 $56 a. 5550 $38“? 600 E EEO0 e 2082 40mg : 5550 a. 2082 600 s 5:80 e 2082 4030 we 3500 a. 2.082 40%,; S ego0 0. 0.082 40%,; 8% he 2 :30 a. :50 .5550 «.8383 40020 82 03w 5330 a. 0550 40020 a 5500 0. 2082 $38”? 40mg 8 ego0 a 2.082 6&0 02¢ a 2082 $3092 AUmEV QQQN L055»; thOHW q m—Gcoz £0550 A030 3% e: 533m 0. 5550 $38“? 690 we 0950 s. 2082 636 83 e: 2 .50 a ES. .3950 szv 8 0550 e 2082 0055 805m «530% .4 8250503 55.5—ng .4 00330. 0005034 .4 5350500.. magnum umd 035050 55002032 cmd 505332 5009 o4v 05535 5.33004 .4 50004 .4 5300505 5000003 .4005. 305% 0500—0 E3445 .0 320:5 «30030000 0550030 0:002 A.4V 500.000-00.50 530000 .4003 30355 530000 0:30 .4 008 0030354 .4 0300820 30835 00004 44v Ema 00400.05 .4 00000 0080054 umd area—g 00303m 503., :36 «50> 00.303m .UQ 8:5: 0:32 :5: 30 €23 03853. $520.? a. 830.0 30 see 088%? 00080 a. 0330 03—0000 53:3. .8534 A.4v 500503 05000500404 053004 <75 0.030% 5400020: 03% 53:52 3 00000500% 5 550% 00250000 0000000 ... 08500 00580— 5 0000004305 00 53300 w5000000w ”453 05 5 0000 3535 00¢ 00435.55 053004 003 000003» 4. 030.4. 126 using the AmpliTaq DNA Dye Terminator Cycle Sequencing reagents (PE Applied Biosystems, Norwalk, CT). Edited sequences were aligned using Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI). Insertion deletion events were treated as missing data. Parsimony methods were implemented using PAUP* (Version 4.0b4; Swofford 2000). Heuristic searches were performed using the TREE BISECTION RECONNECTION, and MULTREES options. Bootstrap analysis was conducted using 10,000 replicates'with 200 random addition-sequence replicates per bootstrap replicate. Floral Morphological Trait Measurements- Sampling- Floral morphological measurements were made on populations from twenty- four species within the Brassicaceae and one species from the Cleomaceae. Taxon selection was based on the phylogeny of Galloway et al. (1997) and discussions with Robert Price (personal communication). Two species, Brassica nigra and Raphanus raphanistrum, were sampled from two populations each. Study populations were selected based on availability of seeds and natural populations (Table 8). Floral measurements- Flowers were measured from wild populations whenever possible. When wild populations were unavailable, seeds from wild populations, research collections, or commercial seed companies were acquired and grown to flowering. If Tit: -2 2 3.4.2.7... .pJ Nuuu.-~.J~.unv-~ :1: 7.3.4.4317. 02.0.0... .w..r.v....-.2 .3. 07:... s 0004 mm .D 0.05 00.00002 - 0000000000 20300 0034 ”0000 00500 000050 000.00% 080 :0 - 000 85282 H000222 :0 80000220 62008020 0000 mm .3 008m 00000002 - 0000000000 ”0500.030 .4 0.5.00 0000:0004 N004 no 0005—: .00 .D I 0000.4. 05:50 0000000005 ”0400> 302 .4 00500000000 00000034 0000 9% 00054: 00 .D I 0000000000 0000000050 0400.» 302 .4 5000500000 000000004 0004 om - 000000 0.0002 0000000304 “0002002 00.04 035000.00 0050000002 0.004 :0 .0 035 0005002 - 0000000000 000002 0% 0000505. I 000m 300005500 00.2 0305002 00.0— 00 .0 008m 00wE002 - 0000000000 00002 0% 00005000. 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I 000m 30000000000 . .4 000004 4004 00 - 0000.4. 05:50 000000 “0005—: .4 0030000000 00000000 0.004 mm .0 003m 0005002 - 0000000000 00002 0% 0000500H I 000m 3000050000 .0000. 000500 05020 Noam m0 - 090000300 0300 0005002 ”00000002 000305 .0 350020 030030000 050000000 82 00 - 0220 0020220 002030 ”50000.02 20002 30 m002220.003 20800 02: 00 - 2000 250 W20=0 22000000 N00000202 .280 3200000 23500 20200 0000 cm .0 000% 0005002 - 0000000000 0300000000 00000000 00000000 .4 0000 00000000 0004 0m .0 035 0005002 - 0000000000 0300000000 00000000 00.000000 .4 0000000040 00000000 N004 wv - 25 300005 00003 00> 000003 ”0005—: 00004 2.40 0&5 00000000 52 00 - 000203 .235 28:: 2800 30 200: 020220 2004 cm 0000:: .00 .D I 0000.0 05:05 000m 00004 0000000 I 000m 3000000800 .4 00000 00000000 32 a. - 020.0 020220 002030 H0000022 00.0 200005 8000000 ooom om - 0000i 30000.05 000—4002 U00000002 000< 3:20 0000> 00002000 0004 E .3 00% 0002002 - 0000000000 0000000 30000000 0000002 .00 000005 05000. 32 t. - 0200 0020220 002.30 0000022 .0530 30 80:05 088302 32 00. - 2000 230 0250 22:00:00 H0000222 000220.200 00 20000.0 30 505 2.082002 88 00 - 020000 020220 002000 H2.000022 20:80 0 00:00 522020 00002 ooom 0m .3 035 0005002 - 0000000000 00002 00 000000084 50000 ”0000004 .5534 2.40 5003000 00000000000 00000 z 300 0203200 .0283 20000 02800 000003000 30300 00 03¢ 05 5 000000000 0003 0000000005 000030000 00 053 0000mm 05500 0003004 200000 5 00000 000 0000 000030400 00 00000005 0030mm 000000000 000% 000 050 0400000 .0000 00:02:00 503002 0400000 0003 0000000000 30E20000000 30020 00.0 000000000 0000000 000000400000 .w 030.4. 128 available, measurements were made on a minimum of 50 plants per population. A range of 22-340 plants per population were measured for a total of 1539 plants. One to three flowers per plant were measured. If more than one flower per plant was measured, the mean value was calculated and used in the analysis. Measurements were made using digital calipers and recorded to the nearest 0.01 mm. Six quantitative characters were measured: claw length, limb length, limb width, short filament length, long filament length, and pistil length (Figure 20 and 21) Populations ofRaphanus rap/zanistrum, Brassica napus, B. nigra, and Hesperis matronalis were measured according to the methods outlined in Conner & Sterling (1995). For all other populations, measurements were taken on the most apical, fully developed, intact flower. Only flowers that had not begun to senesce were measured. In wild populations, plants and seeds were sampled at set intervals along transects encompassing the population range. In the greenhouse, when the number of flowering plants exceeded 50, plants were selected for measurement using a random number table. P-matrL'x construction — The Phenotypic variance-covariance matrix was constructed from the morphological data sets of six floral measurements recorded for the plants measured in the 26 populations. Data was imported into the J MP statistical package (SAS Institute Inc., Cary, NC) to construct a variance-covariance matrix using Multivariate Analysis-Covariance Matrix. The resultant matrix has variances for the six variables on the diagonal and covariances among the six variable above and below the diagonal (Table 9). l29 at} ° TO 3 Figure 20. Schematic of generic Brassicaceae flower viewed from the top. Figure illustrates the cruciform petal arrangement. Letter A shows the floral measurement for limb width. The six tetradynamous stamens are shown: B shows one of four long antepetalous stamens located opposite each petal, C shows one of two outer short stamens borne between the petals. Letter D shows the central location of the pistil. \ I Figure 2]. Schematic representation of a generic Brassicaceae flower in lateral cross- section. Pistil is not shown and would be located in the flower center. Only three stamens are shown (two long and one short) to illustrate the tetradynamous stamens. Letters in the figure refer to floral measurements: A=short filament, B=long filament, C=c1aw length of the petal, D=Limb length. 13] Table 9. Example of the Phenotypic variance-covariance matrix for the six variables in the Nasturtium officinale data set. The variance for each variable is along the diagonal and the covariances are on the upper and lower triangle off the diagonal. Limb length Limb Width Claw length Short filament Long filament Pistil Limb length 0.1 1440 0.06869 0.07314 0.03818 0.05610 0.06642 Limb Width 0.06869 0.0636l 0.05202 0.02987 0.04101 0.05925 Claw length 0.07314 0.05202 0.13396 0.06672 0.06858 0.07521 Short filament 0.03818 0.02987 0.06672 0.12914 0.06953 0.06974 Long filament 0.05610 0.04101 0.06858 0.06953 0.08878 0.06358 Pistil 0.06642 0.05925 0.07521 0.06974 0.06358 ' 0.16584 Hierarchical Matrix Comparisons - Comparisons of phenotypic variance-covariance matrices were made using the common principal component (CPC) analysis described by Flury (1988) and implemented using CPCRand (Phillips 1998, Phillips and Arnold 1999). CPCRand allows the simultaneous comparison of multiple matrices and uses randomization procedures to circumvent problems in the data set associated with the assumption of multivariate normality and degrees of freedom. Matrices were compared among related taxa in progressively more inclusive clades based on the phylogeny of the Brassicaceae. CPCRand could not accommodate comparisons of more than ten matrices and clades including more than ten populations were not included in this analysis. A total of sixteen matrix comparisons were made. 132 The F lury hierarchy (1988) goes beyond the determination of whether matrices are equal or unequal. The “J ump-Up” approach described by Phillips and Arnold (1999) starts by testing the matrices for related structure in the partial common principal components (PCPC) and then the common principal components for all the eigenvectors (CPC). If related structure in all the eigenvectors is not rejected, then the matrices are compared for proportional eigenvalues (proportional). The final step in the hierarchy is equality of eigenvectors and eigenvalues (equality). Each level in the hierarchy is inclusive of all the lower levels and each level is tested against the model of unrelated structure. Significance testing starts at the level of PCPCl and proceeds up the hierarchy until a significant p- value is obtained. Once a significant p-value is determined, that level of the hierarchy is rejected, and the next lower level is accepted (Figure 22; Phillips & Arnold 1999). ' The “Jump Up” approach is dependent on acceptance of PCPCl in order to test any higher level in the hierarchy. In the analysis using CPCRand, eigenvectors are initially ordered by decreasing eigenvalues. A significant P-value for the first eigenvector discontinues the analysis and may preclude any investigation into matrix similarity among other eigenvectors with smaller eigenvalues. The CPCRand program has a reordering option which allows the user to change the order of the PCPCs and analyze additional eigenvectors for similarity. Houle et al. (2002) recommend reordering PCPCs to examine the possible similarity among the eigenvectors and this method was used by Baker and Wilkinson (2003) when comparing stalk-eyed flies. 133 Level in the hierarchy Testing using "Jump-Up" " Equality Proportionality- CPC * PCPC (4) + PCPC (3) + PCPC (2) * PCPC (1) ~-———— UNRELATED Figure 22. A representation of the “Jump Up” approach to matrix comparisons using the F lury hierarchy. This hierarchy represents the steps in the Brassicaceae six trait by six trait matrix comparison. Each level in the hierarchy is inclusive of all the lower levels and each level is measured against the model of unrelated structure. Testing starts with PCPC] and moves up the hierarchy. PCPC is the partial common principal component model for successively higher numbers of eigenvectors from one to four, CPC is common principal components for all six eigenvectors, proportionality indicates that the eigenvectors are identical and the eigenvalues are proportional, and equality has equal eigenvectors and eigenvalues (Phillips & Arnold 1999). 134 Reordering of the PCPC was done on all sixteen matrix comparisons in the Brassicaceae data set. In the reordered analysis, instead of proceeding through the PC in the order of highest eigenvalue to lowest, the hierarchy of matrix comparisons skipped the first PC (the PC with the largest eigenvalue) started with the 2"d PC (the PC with the second largest eigenvalue) and proceeded through the remaining smaller PCs in order of largest to smallest eigenvalue. The first PC was the final component and included in the CPC level in the hierarchy. PCA analysis was completed for each individual population and eigenvectors were compared among populations where reordering had considerable effect on the matrix comparisons in the CPC analysis. In phenotypic matrix comparisons the first PC is most often associated with size. In this analysis there was a great disparity in overall flower size across the Brassicaceae. To investigate the potential size disparity I ran a principal component analysis on the data set including all measurements in each population. The average first principal component was tabulated for each population based on the individual first PC values for measured flowers in each population. These values can be compared to examine the overall size difference among flowers assuming the first principal component is size. 135 RESULTS Phylogenetic hypothesis —The aligned nth region was 771 bp long, with 113 parsimony informative-characters. The analysis resulted in 12 most parsimonious trees with a length of 362, C.l.° = 0.5975, and a R.I. = 0.7328 (Figure 23). The Aethionema clade was strongly supported (100%) and basal to the well supported clade of the remaining Brassicaceae (99%). Three additional clades of interest were resolved in the strict consensus tree: Hesperis matronalis - Matthiola (87% bootstrap support), Arabidopsis lyrata — Cardamine concatenata (63% bootstrap support), and Brassica nigra - Stanleya pinnata (99% bootstrap support). Within the Arabidopsis lyrata — Cardamine concatenata clade, Arabidopsis lyrata was sister to A. thaliana (95%) and these taxa formed a clade sister to C. bursa-pastoris (99% bootstrap support). The clade containing Barbarea verna and B. vulgaris was strongly supported (96%) and the clade of N. oflicinale and C. concatenata had moderate support (78%). Support for the clade including B. verna, B. vulgaris, N. officinale and C. concatenata was strong (89%). The clade of Brassica nigra - Stanleya pinnata was comprised of two smaller clades: The S. altissimum and S. pinnata clade had strong support (95%), and the Brassiceae clade (Brassica nigra - Brassica rapa) had weak support (71%). The B. nigra and C. edentula clade had weak bootstrap support (52%). The clade of the two Raphanus species had moderate support (83 %) and the clade containing, B. oleraceae, B. napus, and B. rapa, was unresolved and strongly supported (94%). The clade consisting of Raphanus and the unresolved Brassica was strongly supported (93%). 136 100 C [come spinosa Aethionema arabicum 52 Aethionema grandiflorum r-— Brassica nigra 71 83 Cakile edentula Raphanus raphanistrum 93 99 94 Raphanus sativus Brassica oleracea 95 Brassica napus Brassica rapa r— Sisymbrium altissimum ‘L—— Stanleya pinnata Lobularia maritima L— Iberis Arabis hirsuta 95‘ Alliaria petiolata Arabidopsis lyrata 99 Arabidopsis thaliana 63 99 96 Capsella bursa—pastorr's r—— Barbarea vulgaris 89 Barbarea verna 78 Nasturtium oflicinale 87 Cardamine concatenata Hesperis matronalis L—— Matthiola Figure 23. Strict consensus of 12 most parsimonious trees from the Brassicaceae based on parsimony analyses of 771 base pairs of nth coding region. Phylogeny has a tree length of 362, a C.I.°. = 0.732, and a R.I. = 0.733. Numbers above the branches represent bootstrap percentages based on 10,000 replicates. 137 Matrix comparisons —Sixteen comparisons were made among matrices. Nine comparisons showed some level of matrix similarity without reordering. After reordering the first principal component, all 16 matrix comparisons had common structure at some level in the hierarchy of matrix association. Comparisons of matrix similarity were equal for two intraspecific assessments, and two of the four within genera comparisons had equal or proportional matrices. One of seven pair-wise comparisons among genera had a level of shared structure above CPC. Of the three comparisons done among more than two genera, two were proportional and one shared a common structure for only one PC (Table 10 and Figure 24). The two populations of Brassica nigra, Junkyard and Yard Waste Disposal Site, shared two principal components in common in the unordered analysis. The hypothesis of three PC in common was rejected (P=0.014). The trait loadings from the first PCs from the individual population level analysis were reviewed; Brassica nigra — Junkyard had a lower claw length weight (0.28599) on the first PC than B. nigra - Yard Waste Disposal Site (0.38854; Table 11). Principal components were reordered in the analysis and equality was found between the two P-matrices. When both populations of Raphanus raphanistrum were compared the matrices were found to be equal matrices whether the principal components were reordered or not. 138 0500.0 00500 00050.0 00500.0 00000.0 0050.0 00000.0 50000.0 50500.0 55.50 5005.0 022306230 5630.0 05* .0555: .V .335 .v. 0005 0 00000.0 00000.0 0000_.0 0500.0 00000.0 000000 50000 0 5_ _00.0 550 0 0.0 502 _.0 0:555 5300.39? 0.8 50.5 528.5030. 00000 0 000000 0800.0 0050.0 , 00000.0 00000.0 000000 50000 0 50000.0 50000.0 50000.0 020$ 05 050.55.: 5.53000 0000— 0 00000.0 05000.0 . 0502.0 00000.0 00000.0 0000—0 50000 0 502 0.0 50— 00.0 5505.0 055:0 90.6550. 05 5:52.50 5555050. 00000 0 0500.0 0055.0 00000 0 55:60» .0 05m .5805: o E .E .m .2555. 00000.0 00000.0 00000.0 50000 0 50000.0 5500.0 50000 0 x 532.5552 x .505 m .500: .m 056505 .05 0300 0 0005.0 000 5.0 00050 0 25:3 00000.0 00000.0 0050.0 50000 0 55000.0 50000.0 50000 0 x 05 5355:0005 x .05: m .500: .00 63.6350 .0 0050.0 0005.0 00000.0 00000 0 00000.0 00000.0 00000.0 50000.0 50000.0 500000 5200 0 20: 36.33 0:0 .503: 55.3% .63an5 3.55% 00000 0 05000.0 00000.0 00005.0 00000.0 00000.0 00.000 5500 0 500000 500500 55000.0 .353 055.68% 05 50053000 5355:0003 .x 00005 0 00000.0 0025.0 0300.0 06E 05w 0050 .0 0200.0 05000.0 50.005 0 50000.0 5500.0 5550.0 8325620 58055000 o»: I 53.255005 5:555 0.2 5.0 00000.0 0050 0 .0 0005.0 .. 00000.0 0005.0 00000.0 50000.0 5500.0 5500.0 5500.0 55:00» 6550.0 05“ 55055000 ENE .00 00000.0 00000.0 0503.0 00000.0 50 68 5 82;? 00000.0 05000.0 00000.0 5005.0 5030.0 50000.0 5002 .0 03> 000 0.356050 50053000 95 I EMS 053.00% 0.0 5 E 5 26:00:50 2.650500 56:00:80 50:00:80 5280500 5:5 02.00.8090 60650 606500 606500 606500 60650 080058 85.552 20.5500 coEEoU 005500 55500 55500 0550 .550 60.80 0550 .0 0500. 5 085— 50053090 8 050858 86000 05.08082 505 36> 20 .3 003260 $550058 6060.50 05.5062 59.00.» 030505 255-0 68853500 20 5 52:58 0530000 20 0c 5058 255-0 00,—. .00 0300. 139 0050.0 00050.0 00000.0 000_ _.0 00000.0 00000.0 0050.0 5050.0 50000.0 5500.0 50000.0 55.5000 05“ 5500505 550.500 00000.0 00000.0 00000.0 000—00 5582828 U 05“ 50550.0 .2 .05? 00000.0 00500.0 00000.0 5500.0 50000.0 50000.0 50000.0 .0 6.203;: .m 580000.350 U .0555: 018000 .V 00000.0 00005.0 00000.0 0050.0 . 00000.0 00505.0 00000.0 50000.0 50005.0 55000.0 50500.0 50:28:00 U 05“ 500.8%.» .2 .088; .m .5505; .m 00000.0 050— 0.0 00000.0 0250.0 00000.0 00000.0 00500.0 50000.0 50000.0 500500 500000 50:83:00 05500.50 000 502.855 55:23.02 00500.0 00500.0 00000.0 00000.0 0000— .0 00000.0 00500.0 50000.0 50000.0 50000.0 50000.0 520; 8.505% 05W 533:: 005055 E E S 5 5280500 50000500 5280500 5280500 50000500 00:00 05838080 508500 508500 308500 00085.00 3085.5 0080500 85502 008500 005500 008500 005500 005500 05:00 05080 50.80 05:00 $680 2 255 140 Figure 24. Strict consensus of tree for the Brassicaceae (see figure 23) Numbers above the branches represent bootstrap percentages based on 10,000 replicates. Notations below the branches indicate level of matrix similarity among species in the clade above the node. Matrix similarity is indicated without reordering principal components, followed by the value with reordering. Notations for similarity among matrices are as follows: U = unrelated matrix structure, PCPC # = partial common principal component followed by the number of principal components in common between the matrices, CPC = all principal components have shared structure among matrices, P = matrices are proportional and E = equality among matrices. 141 100 Cleome spinosa Aethionema arabicum 99 52 Aethionema grandiflorum Brassica nigra . PCPCZ/E 71 U/PC U/PCF PC3 83 L— Cakile edentula E/E Raphanus rapham'strum ’Cl 93 P/P 99 U/PCP C1 94 L——— Raphanus sativus Brassica oleracea 95 PCPCZW’PCPCI Brassica napus Brassica rapa Sisymbrium altissimum U/CPC Stanleya pinnata Lobularia maritima U/PCPC3 Iberis Arabis hirsuta 99 95 Alliaria petiolata Arabidopsis lyrata 63 U/PCP C2 PCPC3KBCEC2 Arabidopsis thaliana Figure 24. WP 96 Capsella bursa-pastoris Barbarea vulgaris 87 89 WP 78 Barbarea verna Nasturtium oflicinale P/P L— Cardamine concatenata Hesperis matronalis U/PCPC3 142 Matthiola Table 11. Results from the principal component analysis of populations in the Brassicaceae. Eigenvalues with percent variation explained, and eigenvectors are given. Principal components are denoted as “PC” and ordered by decreasing eigenvalues. Aethionema arabicum PC 1 PC2 PC3 PC4 PC5 PC6 Eenvalue 2.4429 1.4401 0.8811 0.6824 0.3579 0.0955 Percent 40.7150 24.0022 14.6849 11.3738 5.9654 3.2587 _l_iigenvectors Claw Iggth 0.42235 -0.51436 0.30469 -0.09881 0.19075 0.64658 Limb Width 0.29559 0.37414 0.69189 -0.45631 -0.16321 -0.24308 Limb Length 0.12228 0.77109 -0. 10780 . 0.18014 0.20898 0.55021 Anther (S) 0.48071 0.02203 -0.51354 -0.267 86 -0.65086 0.09659 Anther (L) 0.54013 0.01448 -0.32011 -0. 14104 0.65011 -0.40379 Pistil 0.44325 -0.01364 0.22503 0.81113 -0.21689 -0.21849 Alliaria petiolata PCl PC2 PC3 PC4 PC5 PC6 Eigenvalue 4.5930 0.6266 0.2830 0.2303 0.1464 0.1208 Percent 76.5497 10.4438 4.7159 3.8379 2.4402 2.0126 Eigenvectors Claw Length 0.35009 0.75867 0.46052 0.19729 0.22509 0.01405 Limb Width 0.42932 -0.15763 0.37834 -0.28156 -0.63868 -0.40064 Limb Length 0.41948 -0.36886 0.27756 -0.29492 0.23880 0.68333 Anther (S) 0.41641 0.25999 -0.60996 0.18511 -0.46159 0.37367 Anther (L) 0.42726 0.05789 -0.44232 -0.46369 0.47783 -0.41848 Pistil 0.40151 -0.43883 0.00723 0.73861 0.20742 -0.2399 Arabidopsis lyrata PC 1 PC2 PC3 PC4 PCS PC6 _E_igenvalue 3.3577 0.9194 0.8266 0.4163 0.3097 0.1704 Percent 55.9611 15.3239 13.7759 6.9375 5.1617 2.8399 Egenvectors Claw Leggth 0.32558 0.66184 0.46898 0.26566 0.11861 0.38906 Limb Width 0.36274 -0.50309 0.53501 0.21701 0.39048 -0.35988 Limb Length 0.44662 -0.47636 0.00780 -0.18331 -0.33439 0.65432 Anther (S) 0.38146 0.01362 -0.68088 0.28295 0.54191 0.11564 Anther (L) 0.47243 0.13370 -0. 17244 0.34133 -0.63800 -0.45349 Pistil , 0.44073 0.25272 -0.02054 -0.80533 0.14469 -0.26825 Arabidopsis thaliana PC 1 PC2 PC3 PC4 PC5 PC6 ___E_igenvalue 3.5445 1.2464 0.5987 0.2588 0.2013 0.1503 Percent 59.0754 20.7731 9.9778 4.3138 3.3557 2.5043 _E_igenvectors Claw Length 0.23853 0.71138 0.42324 0.39072 0.31381 0.08235 Limb Width 0.46103 0.28745 0.01912 0.21490 , -0.79829 0.14494 Limb Length 0.47159 -0.14632 0.33119 -0.42528 0.22512 -0.64418 Anther (S) 0.33116 -0.59067 0.33680 0.62752 -0. 16839 0.07663 Anther (L) 0.48235 -0.17037 -0. 14102 -0.30868 0.41846 0.66935 Pistil 0.4073 0.10949 -0.75993 0.36225 0.10052 -0.32148 Arabis hirsuta PC 1 PC2 PC3 PC4 PCS PC6 Eigenvalue 2.6684 1.2457 0.8039 0.7038 0.3268 0.2515 Percent 44.4728 20.7622 13.3975 11.7294 5.4465 4.1916 143 Table 11 (cont’d) Arabis hirsuta cont’d —————— m————— “Imam-mm MIME—W m—_—— 0.44672 mm- M [mm-”Immum-Immm ”Human-m:- Babarea vema “- — mam-mm WWW—WWW Wanna—Malawian- -———_——— WNW -0-60976 [WW—— -0-36491 —mm- W 03885 MEI—fl WWWMW man-”W mum—MIME- Barbarea vul aris W 30987 Im- 05854 02286 m1- m5! 20.8764 “-8882 “Emm- - —— _— WNW WWI—W : WEE-”fl “MI—WHEE- [mm—WWW IE!- Mammal—Malayan- Brassica na - us — “mm-E- W 16892 Iii-i. 0-7998 0-4569 mm M 443198 MIME-mm}, — -—_ ———— Wim—W—— [mm-WW” : “ESE-”Imm- KW “EH-W mall-MW m——W Brassica niy a - Junk ard ”-14- Wmmmflm m—Wfl'fi}. m ——— WWW [WW—W- : ”WI. “SEE-”W— 1 a i 1 11 111 11 Table l 1 (cont’d) Brassica nigg— Junkyard cont'd PCl PC2 PC3 PC4 PCS PC6 Anther (L) 0.43565 0.35152 0.08586 -0.68018 -0.16555 -0.43499 Pistil 0.46294 -0.03073 -0.31878 0.61320 -0.09904 -0.54526 Brassica nigra — Yard Waste Disposal Site PC 1 PC2 PC3 PC4 PCS PC6 ._Eigenvalue 3.3141 1.0112 0.5855 0.4439 0.3658 0.2796 Percent 55.2344 16.8527 9.7585 7.3983 6.0962 4.6599 _Eigenvectors Claw Length 0.38854 0.11397 0.90879 0.02315 -0.02752 0.09413 Limb Width 0.34617 0.63997 -0.28826 0.41597 -0.19557 0.41980 Limb Length 0.41096 0.43530 -0.16308 -0.52524 0.42846 -0.39442 Anther(S) 0.41653 -0.47873 -0. 16687 -0.17933 0.38034 0.62663 Anther (L) 0.43425 -0.31595 -0.10518 0.64494 0.19970 -0.49468 Pistil 0.44524 -0.24281 -0. 15972 -0.32007 -0.76999 -0. 14824 Brassica napus PC 1 PC2 PC3 PC4 PCS PC6 _Eigenvalue 2.6892 1.3756 0.7998 0.4569 0.3619 0.3165 Percent 44.8198 22.9264 13.3306 7.6157 6.0319 5.2755 jgenvectors Claw Length 0.47275 -0.31788 -0.01312 -0.50249 0.48427 -0.43390 Limb Width 0.24031 0.070726 -0.07985 -0.01004 0.54740 0.36867 Limb Length 0.44321 0.38054 -0.29018 0.36107 -0.33322 -0.57719 Anther (S) 0.44157 —0.23462 -0.56018 -0.20321 -0.33446 0.53198 Anther (L) 0.41348 -0.39778 0.28482 0.69704 0.24921 0.20422 Pistil 0.39544 0.20163 0.71716 -0.2997 -0.42525 0.13400 Barbarea oleracea PC 1 PC2 PC 3 PC4 PCS PC6 _Egenvalue 4.1835 0.9090 .4340 0.2404 .1604 0.0726 Percent 69.7250 15.1512 7.2329 4.0073 2.6732 1.2103 jigenvectors Claw Length 0.43622 -0.10026 -0.51300 -0.02993 0.68741 0.25113 Limb Width 0.34290 0.64996 0.27229 0.61234 0.10386 0.00880 Limb Length 0.39056 0.48987 0.08736 -0.74889 -0. 17634 0.08907 Anther (S) 0.44262 -0.28520 -0. 17312 0.23054 -0.62427 0.49991 Anther (L) 0.46392 -0. 16488 -0.21505 0.05252 -0.17591 -0.82319 Pistil 0.35842 -0.46797 0.76081 -0.08604 0.25481 0.03702 Brassica rapa PC 1 PC2 PC3 PC4 PCS PC6 _E_i_genva1ue 3.1750 0.9990 0.8323 0.4101 0.3296 0.2540 Percent 52.9163 16.6498 13.8721 6.8350 5.4927 4.2341 _E_i_genvectors Claw Length 0.31273 0.41859 0.73278 0.28260 0.29946 0.14308 Limb Width 0.41588 -0.50822 0.28734 -0.00759 -0.089849 —0.69147 Limb Length 0.40253 -0.60106 -0.00059 -0.08143 0.18229 0.66093 Anther (S) 0.40653 0.17204 -0.58846 0.34037 0.54994 -0.20139 Anther (L) 0.46660 0.20465 -0.17225 0.34775 -0.7S274 0.15224 Pistil 0.42922 0.36570 -0.06716 —0.82260 -0.00501 -0.02886 Cakile edentula PC 1 PC2 PC3 PC4 PCS PC6 Eigenvalue 2.7187 1.3946 0.7914 0.5300 0.3270 0.2383 145 Table 1 1 (cont’d) Cakile edentula cont’d PC 1 PC2 PC3 PC4 PCS PC6 Percent 45.3109 23.2440 13.1899 8.8339 5.4495 3.9718 iigenvectors Claw ngth 0.43903 -0.34189 -0.07112 0.69218 -0.25749 -0.37401 Limb Width 0.24453 0.62036 -0.46854 0.29079 0.50126 0.00213 Limb Legth 0.41320 0.47387 -0.10425 -0.31329 -0.70365 -0.02370 Anther (S) 0.49178 -0.22837 0.03065 -0.54749 0.39113 -0.50232 Anther (L) 0.48425 -0.36785 -0. 19795 -0.07067 0.07176 0.76214 Pistil 0.31682 0.29365 0.85113 0.18283 0.17084 0.16235 Capsella bursa-pastoris PCl PC2 PC3 PC4 PCS PC6 Eigenvalue 3.0733 1.0457 0.7345 0.5918 0.3327 0.2220 Percent 51.2210 17.4283 12.2422 9.8629 5.5452 3.7004 _E_igenvectors ' Claw Length 0.44290 -0. 13394 -0.43139 0.54102 0.05110 0.55181 Limb Width 0.47978 -0.09174 0.29296 0.34603 0.48889 -0.S6286 Limb Length 0.43523 -0.13384 0.46441 -0.53915 0.21513 0.48993 Anther (S) 0.05346 0.94966 0.18169 0.15589 0.09947 0.16758 Anther (L) 0.37709 0.22618 066730 0.52092 0.12931 -0.27069 Pistil 0.48905 0.05220 0.18465 0.03500 -0.82794 -0.19315 Cardamine concatenata PC 1 PC2 PC3 PC4 PCS PC6 Ei envalue 2.5952 1.1680 0.9131 0.6576 0.5127 0.1534 Percent 43.2542 19.4660 15.2189 10.9594 8.5445 2.5570 Eigenvectors Claw Le_ngth 0.47256 -0.01118 -0.54336 -0.18276 0.408880 -0.52992 Limb Width 0.45417 -0.55907 -0.12199 -0.15391 0.08420 0.65993 Limb Length 0.36976 0.33704 0.53540 0.58789 0.12343 —0.31966 Anther (S) 0.32369 0.29743 0.61720 -0.63702 0.13962 -0.02308 Anther (L) 0.31974 0.68100 -0.08764 0.43747 0.25911 0.40964 Pistil 0.47612 0.14661 -0. 12088 0.01093 -0.85084 -0.11471 Cleome sginosa PCl PC2 PC3 - PC4 PCS PC6 LElgenvalue 4.9859 0.5018 0.2854 0.1667 0.0457 0.0146 Percent 83.0977 8.3637 4.7564 2.7780 0.7612 0.2431 _Egenvectors Claw Leggth 0.39963 0.16972 -0.75731 0.34991 0.33711 0.04353 Limb Width 0.38354 0.51141 0.55650 0.53054 -0.01172 -0.00723 Limb Lflgth 0.39152 0.53792 -0.07493 -0.71591 -0.15404 0.03278 Anther (S) 0.42794 —0.35529 0.16720 -0.14591 0.29477 -0.74465 Anther (L) 0.41997 -0.41523 0.25266 -0.15159 0.35032 0.66458 Pistil 0.42473 ~0.34880 -0.13921 0.15773 -0.80801 0.02850 Hesperis matronalis PCl PC2 PC3 PC4 PCS PC6 figenvalue 3.6211 1.0398 0.7011 0.2753 0.2133 0.1494 Percent 60.3512 17.3300 11.6853 4.5887 3.5550 2.4898 iigenvectors Claw Length 0.28425 0.65318 0.57058 -0.01086 0.40681 0.03714 Limb Width 0.35238 -0.44962 0.62727 0.30532 -0.40882 0.14087 Limb Length 0.43914 -0.32499 0.02773 -0.82205 0.15632 —0.02392 146 Table 1 1 (cont’d) Hesperis matronalis cont’d PC 1 PC2 PC 3 PC4 PCS PC6 Anther (S) 0.45043 0.24117 -0.40684 0.07313 -0.19583 0.72782 Anther (L) 0.45463 0.32402 -0.21086 0.03446 -0.48282 -0.63997 Pistil 0.43887 -0.32003 -0.26497 0.47367 0.60949 -0.l9728 Iberis PC1 PC2 PC3 PC4 PCS PC6 Eigenvalue 3.7128 1.0840 0.4946 0.3817 0.2274 0.0994 PC 1 PC2 PC3 PC 4 PCS PC6 Eigenvectors Claw Length 0.44567 0.23196 -0.28428 -0.29278 -0.75919 0.06836 Limb Width 0.41338 -0.52935 0.13132 0.02447 -0.04319 -0.72746 Limb Length 0.38609 -0.59096 0.02085 0.25203 0.00067 0.66162 Anther (S) 0.35526 0.45835 0.67535 0.44912 -0.07666 0.00987 Anther (L) 0.40220 0.29397 -0.64070 0.41534 0.39571 -0.11055 Pistil 0.43992 0.14227 0.18684 -0.68989 0.50922 0.12675 Lobularia maritima PC 1 PC2 PC3 PC4 PCS PC6 _Eigenvalue 3.3567 0.9711 0.8468 0.3443 0.3105 0.1706 Percent 55.9444 16.1858 14.1128 5.7390 5.1745 2.8435 _E_i_genvectors Claw Leigth 0.37676 -0.00471 0.68480 0.44200 -0.43194 -0.08452 Limb Width 0.40421 0.47083 0.25824 -0.70473 -0.00871 0.22701 Limb LenALh 0.35009 0.62958 -0.29076 0.51517 0.36181 0.01479 Anther (S) 0.47148 -0. 13317 -0.37219 -0. 18774 -0.22638 -0.73141 Anther (L) 0.43532 -0.33120 -0.42386 0.08245 -0.32898 0.63727 Pistil 0.40038 -0.50448 0.24828 -0.02349 0.72316 0.00581 Matthiola PC 1 PC2 PC3 PC4 PCS PC6 Eigenvalue 2.4225 1.3719 0.8461 0.5957 0.4628 0.3010 Percent 40.3750 22.8656 14.1017 9.9282 7.7127 5.0168 _E_igenvectors Claw Length 0.46963 -0.20275 -0.48369 -0.41711 0.22627 0.52840 Limb Width 0.33143 -0.52353 0.34559 0.59416 -0.14275 0.35104 Limb Length 0.47950 -0.39637 0.17814 -0.27302 0.15142 -0.69554 Anther (S) 0.38200 0.47353 -0. 18816 0.52911 0.54189 -0. 14442 Anther (L) 0.46220 0.33204 -0.23010 0.05210 -0.77858 -0.11947 Pistil 0.28309 0.43957 0.72563 -0.34027 0.07527 0.28045 Nasturtium officinale PC 1 PC 2 PC3 PC4 PC 5 PC6 _E_i_genva1ue 3.6993 0.8981 0.5373 0.3954 0.2890 0.1810 Percent 61.6547 14.9679 8.9544 6.5906 4.8159 3.0166 Eigenvectors Claw Length 0.42110 0.05808 -0.36783 -0.81852 0.08776 -0.07950 Limb Width 0.42713 -0.47130 0.06193 0.24630 0.17950 -0.70620 Limb Lenglhk 0.41962 -0.48572 -0.21338 0.22875 0.16382 0.68067 Anther (S) 0.35307 0.67307 -0.02982 0.30304 0.57395 0.01332 Anther (L) 0.43186 0.28871 ~0.25496 0.28019 -0.76426 -0.05037 Pistil 0.39115 0.04689 0.86570 -0.21606 -0. 14049 0.17013 147 Table 1 1 (cont’d) Raphanus raphanisz Field PC 1 PC2 PC3 PC4 PCS PC6 Eigenvalue 3.4581 0.9490 0.7675 0.3710 0.3204 0.1341 Percent 57.6351 15.8161 12.7917 6.1828 5.3400 2.2343 _Eigenvectors Claw Leigth 0.45968 -0.18612 -0.23824 0.02283 -0.72975 0.40526 Limb Width 0.33444 0.68345 -0.20880 0.59998 0.05708 -0.11923 Limb Length 0.39374 0.50443 0.11157 -0.75095 0.09736 0.06826 Anther (5) 0.44456 -0.34563 -0.19896 0.09729 0.67268 0.42585 Anther (L) 0.47764 -0.34439 -0.12325 0.07345 -0.01499 -0.79526 Pistil 0.30989 -0.07577 0.91237 0.24640 -0.04473 0.05562 Raphanus raphanistrum Greenhouse PC 1 PC2 PC 3 PC4 PCS PC6 Eigenvalue 3.5331 1.0431 0.6983 0.3916 0.2424 0.0917 Percent 58.884 17.3842 11.6379 6.5263 4.0395 1.5277 _Egenvectors Claw Length 0.45013 -0.34368 -0.11313 0.10218 0.76324 0.27111 Limb Width 0.31601 0.62771 -0.35510 0.61443 -0.02284 -0.04438 Limb Length 0.36638 0.53812 -0.03202 -0.74964 0.10669 0.04269 Anther (S) 0.47064 -0.28173 -0. 14683 -0.02268 -0.61666 0.54477 Anther (L) 0.48205 -0.31726 -0.11705 -0.05442 -0.15620 -0.79115 Pistil 0.33076 0.13516 0.90820 0.21586 -0.02993 0.00403 Raphanus sativus PC 1 PC2 PC3 PC4 PCS PC6 _Eigenvalue 3.8613 0.8472 0.6630 0.3836 0.1853 0.0595 Percent 64.3557 14.1206 11.0507 6.3936 3.0885 0.9910 Eigenvectors Claw Length 0.45994 -0.00215 -0.28430 0.14226 -0.80194 -0.21043 Limb Width 0.32226 -0.67762 0.41630 0.49126 0.09617 0.11446 Limb Length 0.38139 0.07085 0.63053 -0.66139 -0.12022 -0.00799 Anther (S) 0.46508 -0.11848 -0.33148 —0. 14733 0.51800 -0.60807 Anther (L) 0.46909 0.03430 -0.39107 -0. 14125 0.18407 0.75631 Pistil 0.32064 0.72153 0.29236 0.50925 0.17603 -0.02815 Sisymbrium altissimum PC1 PC2 PC3 PC4 PCS PC6 Eigenvalue 3.2417 0.9487 1.6088 0.4861 0.4446 0.2700 Percent 54.0284 15.8117 10.1475 8.1022 7.4102 4.5001 _E_igenvectors Claw Length 0.42668 0.26772 0.20615 0.30022 -0.78087 -0.06240 Limb Width 0.41056 -0.54283 0.28431 0.05238 0.08001 0.66843 Limb Length 0.43123 -0.46600 0.14554 -O.32248 0.04507 -0.68533 Anther (8) 0.36577 0.53586 0.47000 0.15638 0.57339 —0.07028 Anther (L) 0.40556 0.35236 -0.38379 -0.70236 -0.04983 0.26130 Pistil 0.40642 -0.07212 -0.69807 0.53420 0.22483 -0.08005 Stanlela pinnata PC 1 PC2 PC3 PC4 PCS PC6 Eigenvalue 3.6190 0.8382 0.6331 0.5579 0.2883 0.0635 Percent 60.3159 13.9697 10.5515 9.2987 4.8058 1.0584 Eigenvectors Claw Length 0.41849 0.05914 -0.30768 0.59016 -0.61482 —0.02014 148 Table 1 1 (cont’d) Stanleya pinnata cont’d PC 1 PC2 PC 3 PC4 PC 5 PC6 Limb Width 0.33750 0.76137 0.05573 0.22972 0.49268 0.08831 Limb Length 0.39069 0.27976 0.46484 -0.56540 -0.48107 -0.04384 Anther (S) 0.46912 -0.l9875 -0.36687 -O.24951 0.26681 -0.68731 Anther (L) 0.46874 -0.29893 —0.28838 -0.25679 0.16462 0.71743 Pistil 0.34428 -0.45792 0.68439 0.38867 0.22260 —0.05260 Four matrix comparisons were made between among species within a genus. The analysis of Barbarea vulgaris and B. vema showed equality of matrices and comparison R raphanistrum to the R. sativus found proportionality among matrices. The comparison of Brassica oleraceae, B. napus, and B. rapa found matrices with two PC in common. Arabidopsis lyrata and A. thaliana shared a common structure in the first three PC. Reordering reduced the number of PC with shared structure by one in both the Arabidopsis clade and the Brassica polytomy (PCPC2 and PCPCl respectively). The matrices in the clade including A. lyrata, A. thaliana, and Capsella bursa-pastoris were unrelated without reordering the PCs. Reordering showed a related structure in two PC. In the first PC for the individual population analysis of PCA, C. bursa-pastorz's had an extremely low weight (0.05346) for the short anther length. This could explain why related structure in the first eigenvector was rejected. Nasturtium oflicinale and Cardamine concatenata had proportional matrices and, when included in an analysis with the Barbarea species, all four matrices were proportional. An even more inclusive clade of A. lyrata / Cardamine concatenata also was proportional. None of these values were affected by reordering PCs. 149 Several pair-wise comparisons showed significant structure only afier reordering. Sisymbrium altissimum and Stanleya pinnata had shared structure among all their principal components. Lobularia maritima and Iberis, Hesperis matronalis and Matthiola, and B. nigra and Cakile edentula had shared structure in three PCPC. The PCA analysis for the combined data from all populations had a first PC, which when averaged within populations, was closely associated with overall size of the populations’ flowers (Table 12). The first PC explained over 85% of the variation in the data set. Larger flowers had larger average PCs and the inverse was true for smaller flowers. 150 Table 12. Results of a Principal Component Analysis incorporating measurements from all 26 populations measured in the Brassicaceae and Cleomaceae including eignvalues with percent variation explained, and eigenvectors. Principal components are denoted as “PC” and ordered by decreasing eigenvalues. Species listed correspond to species listed in Table 8 followed by the average first principal component for individuals in the population. Principal Component Analysis of combined data from 26 populations in the family Brassicaceae and Cleomaceae PEA E2. E1 BEA & £96 Eigenvalue 5.0142 0.7472 0.1095 0.0928 0.0314 0.0050 Percent 85.5698 12.4531 1.8253 1.5460 0.5233 0.0825 Eigenvectors Claw Length 0.39230 0.47053 0.67419 -0.36030 -0.18095 0.08726 Limb Width 0.39454 0.46965 -0.08967 0.75023 0.22438 -0.05044 Limb Length 0.41376 0.29390 -0.71195 —0.48161 -0.00344 0.05998 Anther (S) 0.40872 -0.45219 0.10880 -0.00816 0.44415 0.64753 Anther (L) 0.42242 -0.35926 0.11803 ~0. 12671 0.31094 -0.75222 Pistil 0.41683 -0.37163 -0.06923 0.24347 -0.78927 0.03344 Species Aethionema arabicum L. Rothm. Alliaria petiolata Cavara & Grande Arabidopsis lyrata (L.) O’Kane & AL-Shehbaz Arabidopsis thaliana (L.) Heynh. Arabis hirsuta DC. Barbarea vema (Mill.) Asch. Barbarea vulgaris R.Br. Brassica napus L. Brassica nigra (L.) Koch JUNK Brassica nigra (L.) Koch YWLG Brassica oleraceae L. Brassica rapa L. Cakile edentula (Bigelow) Hook. Capsella bursa-paston's (L.) Medik. Cardamine concatenata (Michx.) O. Schwarz Cleome spinosa Jacq. Hesperis matronalis L. Iberis L. Lobularia maritima (L.) Desv. Matthiola R.Br. Nasturtium officinale R.Br. Raphanus raphanistrum L. FLD92 Raphanus raphanistrum L. SAl Raphanus sativus L. Sisymbrium altissimum L. Stanleya pinnata Britton 151 Average first principal component for individugls in efiach population -2.599 -1.581 -1 .371 -2.3208 -1.855 -1.393 -1 .443 -0.421 -0.928 -0.974 1.699 -1.074 -1.758 -2.S70 0.257 12.361 0.737 -1.871 -2.429 0.422 -1 .9 l 8 0.996 1.896 1.481 -1.425 1.062 CONCLUSION The phylogenetic analysis of the nth coding region was consistent with other molecular phylogenetic analyses within the family Brassicaceae (rbcL, Price et al. 1994; nth and Adc, Galloway et a1. 1998; ITS, Mitchell & Heenan 2000; matK and Chs, Koch et al. 2001; and nth and trnL-trnF, Hall et a1. 2002). The genus Aethionema was basal within the family Brassicaceae and a clade including Hesperis matronalis and Matthiola was basal to the remainder of the genera sampled. Good support existed for the Arabidopsis lyrata — Cardamine concatenata clade and relationships within the clade were resolved; this was the first molecular support for a clade including Nasturtium oflicz‘nale and Cardamine concatenata. S. altissimum and S. pinnata were in a clade sister to the tribe Brassiceae. The Brassiceae tribe is one of the few natural tribes described in the Brassicaceae (A1- Shehbaz 1984, Warwick & Black 1997, Gomez—Campo 1999, Koch et a1 2001). Previous phylogenetic and phenetic analyses of RF LP data have been conducted in the Brassiceae (Pradham et a1 1992, Thorman et a1. 1994, Song et al. 1988, 1990, 1992, Warwick & Black 1991 , 1993, 1997 and references therein) and a few molecular phylogenetic analyses have studied relationships in this group (Yang et a1. 1999, Wroblewski et al. 2000, and lnaba & Nishio 2002). The findings of the nth phylogenetic study were consistent with the previous investigations in finding two lineages of Brassica: one including B. oleraceae, B. napus, and B. rapa, and second including B. nigra. The comparisons of the P-matrix across the Brassicaceae found matrix proportionality at higher taxonomic levels than any study conducted to date (Steppan et a1. 2002 and references therein, Baker and Wilkinson 2003). Equality in matrices between different species was found between Barbarea vema and B. vulgaris and proportional matrices were not rejected between Raphanus raphanistrum and R. sativus, and Nasturtium ojficinale and Cardamine concatenata. Proportionality among matrices in five genera was found in the A. lyrata - Cardamine concatenata clade. The presence of matrix similarity at such high taxonomic levels was noteworthy. The study tested the hypothesis that matrix structure deteriorates at the sub-species level and higher (Steppan et a1. 2002), and found evidence for shared matrix structure among different genera in Brassicaceae. The results indicate, that in at least some cases, the P-matn'x remains proportional (eigenvectors are equal and the eigenvalues change proportionally among matrixes) over long spans of evolutionary time and if the P-matrix is a good proxy for the G-matrix, the G-matrix can be a useful tool for reconstructing past evolutionary events (Lande 1979). This supports the findings of Schluter (1996) and Futuyma (1995) regarding the potential for long term, sustained constraints of the G-matrix. Within Brassica, species had low levels of shared structure. In the Brassicae clade and its sister clade Sisymbrium altissimum /Stanleya pinnata low levels of matrix stability were found among genera. This was also true of the Hesperis matronalis / Matthiola and the Lobularia /1beris clades. This supports the premise of Steppan (1997a, 1997b, and 2002 and references therein) that variance-covariance matrix structure is not always constant. 153 The results of this study confirm that the stability of the P-matrix, and potentially the G- matrix, varies among lineages. As‘sumptions can not be made about either the stability or lack of stability in covariance structure at different taxonomic levels within a family. Within the Brassicaceae matrices were proportional among distantly related taxa, and completely unrelated among other closely related taxa. In this study the phylogenetic level of the matrix comparison did not dictate the level of stability found among matrices. More empirical studies of matrix structure are needed to investigate variance-covariance stability within a phylogenetic context. An important caveat - Measurements of the G-matrix in multiple species are not practical. Phenotypic measurements are much easier to obtain when taxa are sampled across a phylogeny. Phenotypic matrices are not identical to genetic matrices, though they may shed light on their genetic counterparts. Several studies have found that homogeneity in P-matrices may imply homogeneity in G-matrices (Cheverud 1988, Arnold 1992, Waitt & Levin 1998, Arnold et a1. 2001, and Steppan et al. 2002). The P-matrix can be a conservative estimator of G-matrix stability. P-matrices, when directly compared to G- matrices, tend to deviate more readily from shared matrix structure among more closely related taxa (Steppan et al. 2002). Both Cheverud (1988) and Waitt & Levin (1998) reviewed the literature and found phenotypic correlations were good indicators of genetic correlations. Willis et a1 (1991) questioned the findings of Cheverud (1988) and refuted the utility of phenotypic variation in determining genetic variation (for a review of the P- matrix / G-matrix debate see Steppan et al. 2002). 154 In the reordering process, the first PC was moved to the end of the PC hierarchy, and the five PCs with smaller eigenvalues were assessed for eigenvector similarity. In several cases, reordering the principal components increased the level of matrix similarity among taxa. Even though the first principal component was responsible for a large portion of the variation in the individual data sets (average 55.45%, range 40.3 8-83.10), the remaining PCs still account for approximately half the variance. The remaining PCs could account for significant shape differences between the populations and represent important principal components affecting floral morphology. It makes intuitive sense that even though populations have a high proportion of variation in their first vector the remaining vectors may share commonalities of biological significance (Houle 2002, Steppan et al. 2002, Baker and Wilkinson 2003). Analysis of the first PC for the combined data set of all 26 populations showed a strong relationship between the first factor and overall flower size. In addition, in the individual PC analysis of the populations — most populations had positive, relatively equally weighted variables. It is likely that the first vector is predominantly a size factor. If size was the only biological explanation of the first PC, and the only variance found between the populations was proportional, all the first PCs eigenvectors would be identical among populations. There were, however subtle differences in the weighting of the variables in the first eigenvector which resulted in a finding of unrelated matrix structure in the Flury Hierarchy. These subtle differences in the variable weights could be attributed to differences unrelated to proportionality, but rather shape differences in the floral morphology. This is particularly important since small differences in flower structure can 155 have a large impact on pollen deposit, pollinator attraction, and pollen removal, all key factors in reproductive success (Conner 1997). Empirical studies of patterns of P- and G-matrix similarity are important. The G-matrix plays a key role in understanding evolutionary processes (Lande 1988). Understanding how the G-matrix changes through evolutionary time helps to explain multivariate evolution (Phillips and Arnold 1999). Selection can only act on the amount of genetic variation-covariation available in a population. How this G-matrix changes dictates the potential response of the population to selection and drift. An understanding of how matrix stability changes over the course of evolutionary time may help us to understand the potential constraints caused by the G-matrix and the factors that can overcome these constraints. In understanding the leap from microevolutionary processes to macroevolutionary events, the G-matrix serves as the key component of change and evolution. 156 SUMMARY 157 This dissertation examined phenotypic diversity at several different hierarchical levels. In all studies, a phylogeny was used as the framework to interpret floral morphology. Floral traits were measured on individuals across the species Pedicularis densiflora and Pedicularis aurantiaca, within the tropical genus Cantua, and throughout the family Brassicaceae. The species Pedicularis densiflora and P. aurantiaca were previously described as sub- species of Pedicularis densiflora 5.1. A close examination of floral traits among populations revealed two distinct morphologies among the populations. This morphological variation was examined intensively using ANOVA tests, principal component analysis, UPGMA cluster analysis and a comprehensive review of over 1000 herbarium specimens. Phylogenetic analysis of the two species confirmed they were closely related taxa, sister to a clade containing Pedicularis semibarbata, but the relationships among taxa within the P. densiflora / P. aurantiaca clade were unresolved. Without resolution phylogenetic information can sometimes be of limited help. Lack of resolution within the phylogeny prevented conclusions regarding the number of times the different floral morphs arose, relationships among populations in different localities, and potential pollinator shifis. Past research on diversity in pollen morphology showed that pollen exine sculpturing was helpful in exploring phylogenetic relationships in the Polemoniaceae (Stuchlik 1967a, 1967b, Taylor and Levin 1965). Additional work examined the potential functional relationship between pollen grain size and style length (Plitmann and Levin 1983). While 158 past research had been restricted to phenotypic data, this Cantua research was able to integrate pollen and floral morphological data and a molecular phylogeny. Pollen grain morphology was highly conserved in Cantua, which supported the subsumption of Huthia into Cantua. The synapomorphy of supratectal verrucae united the C. volcanica / C. quercifolia clade and provided additional support for the hypothesized relationship. The molecular phylogeny of Cantua allowed me to confirm the phylogenetic utility of pollen morphological characters and remove the issue of non-independence when examining the relationship between pollen diameter and style length. Neither a significant regression slope nor correlation was found using independent contrasts of pollen diameter and style length in Cantua and the hypothesis for a morphological integration of these two traits in Cantua was not supported. The evolution of phenotypic variance-covariance structure is still not well understood, particularly in an historical context (Badyaev and Hill 2000). The question of whether the P-matrix remains stable over evolutionary time remains an unresolved issue (Arnold et al. 2001, Steppan et al. 2002). I studied six floral traits in the Brassicaceae to examine P- matrix stability within a phylogenetic context. The comparisons of the P-matrix across the Brassicaceae found matrix equality and proportionality at higher taxonomic levels than any study conducted to date (Steppan 2001 and references therein, Baker and Wilkinson 2003). Unrelated matrix structure also was found among species and genera in the family. The phylogenetic level of the matrix comparison did not appear to affect the level of stability found among matrices. A phylogenetic hypothesis in the Brassicaceae 159 was used to conclude that P-matrix stability does not follow a stepwise pattern of similarity corresponding to taxonomic rank within the phylogeny. Biological diversity is created by evolution. When attempting to explore biodiversity and examine processes in evolution, we need perspective on past evolutionary events. At present one of the most effective methods to investigate past evolutionary events is phylogenetics. My work on floral diversity and phenotypes in the genus Cantua and the family Brassicaceae exemplifies how phylogenetic history can clarify our understanding of biological phenomena. In contrast, phylogenetic relationships in the Pedicularis study were unresolved and consequently l was unable to add insight into past evolutionary events. Investigations of evolution benefit from the perspective of an historical context. 160 APPENDICES 161 APPENDIX A Aligned sequences of Pedicularis: Nuclear ribosomal internal transcribed spacer regions and 5.88 ribosomal RNA gene 162 wew wwe wee wew www woe eww wee ewe woe eee eww ewe eww wow eww mueumwumzlmuemwlmxmq wew wwe wee wew www woe eww wee ewe woe eee eww ewe eww www eww wmomlxuumnse wew wwe wee wew www woe eww wee ewe wwe eee eww ewe eww wow eww emHHm>lummm wew wwe wee wew www wwe eww wee ewe ooe_eee eww ewe eww www eww mmaomneem wew wwe wee wew www woe eww wee ewe wee eee eww ewe eww www eww mmmmlmoonmzlemw wew wwe wee wew www woe eww wee ewe woe eee eww ewe eww www eww wwewmumm wew wwe wee wew www wwe eww wee ewe woe eee eww ewe eww www eww eoHHm>|quom wew wwe wee wew www woe eww wee ewe wwe eee eww ewe eww woo eww umeereu20mmez wew wwe wee wew www woe eww wee ewe owe eee eww ewe eww www eww oenmewlunsoz wew wwe wee wew www woe eww wee ewe woe eee eww ewe eww woo eww enmemoeum wew wwe wew wew www woe eww wee ewe wwe eee eww ewe eww www eww odoueormmausm wew wwe wee wew www woe eww wee ewe wwe eee eww ewe eww wow eww memwlum>moz wew wwe wee wew wwo woe eww wee ewe wwe eee eww ewe eww wow eww mexmee wew wwe wee wew www woe eww wee ewe woe eee eww ewe eww www eww eoaswlonom wew wwe wee wew www woe eww wee ewe owe eee eww ewe eww www eww amusemeem wew wwe wee wew www woe eww wee ewe owe eee eww ewe eww woo eww mmceumwlmwenmoz wew www wee oew www woe eww wee ewe woe eee ewo ewe eww www ewe mrmomnumneEmml.m wew www wee wew www wwe eww wee ewe owe eee eww ewe eww wow ewe elmumnumneEmml.m wew www wee wew www woe eww wee ewe woe eee eww ewe eww woo ewe mummomoomunl.m wew www wee wew www woe eww wee ewe owe eee eww ewe eww woo ewe elmmoouomunl.m wew www wee wew www woe eww wee ewe owe eee eww ewe eww wow eee mimeHOMeweumoQOumeol.m wew www wee wew www woe eww wee ewe owe eee eww ewe eww woo eee ermeHOMeweumoQOumeol.m w v H H wee» .=-= noes wmwmoewce meme weemmee .=z= ewes Umumwevce wmeuesmenae .aus nue3 Umumoecce mmmw .H mHQMB Ge vmuwefl wemawso> Ou vcommmhuoo memz .mehmanoebmm Ge UoHQEMm mxmu on» Eoem 42m HmEowoneH mo uecsnsw mm.m may use chHmmH emommw poneeowcmeu amneouce HMEOwOQeH Hmmaosc on» no mmocmswmw omamea< .ma wHQme 163 N M. ~ § . 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coding region 263 eee eee eee eee wew eee eee ewe wwe ewe eee wew ewe eee eew eew MHOHQuumz eee eee eee eee wew eee eee ew- --- --- --- --- --- --- --- --- mmmw monmmum eee eee eee eee wew eee eee ewe wwe e-- --- --- --- --- --- --- mumemumoeoo mneemwumw eee eee eee eee wew eee eee ewe wwe eww eee wee ewe eee e ..... mumHoHummHmHnmeHHe eee eee eee eee wew eee eee eww wwe ewe eee wee ewe eee eee eew magmatmoemmmwm eee eee eee eee wew eee eee ewe wwe ewe eee wee ewe eee eee eew mnum>lmmumnemm eee eee eee eee wew eee eee eww wwe ewe eee wee ewe eee eee eew mmomemmo monmmum eee eee eee eee wew eee eee ewe wwe ewe eee wee ewe eee eee eew mHmceonuo asHuuzummz eee ewe eee eee wew eee eee ewe wwe ewe eee wee ewe eee eee eew meuoummm-MmmsnrmHHmmmmo eee ewe eee eee wew eee eee ewe wwe ewe eee wee ewe eee eee eew memeHmeu memmoanmue eee eee eee eee wew eee eee ewe wwe ewe eee wee ewe eee eee eew meummHs> mmumnmmm eee eee eee eee wew eee eee ewe wwe ewe eee wee ewe eee eee eew Edumnuemrsseuneeme 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wee eee eee ewe wwe ewe eee wee ewe eee e ..... mmoeemm meome m e H . =1: SHAH.) 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UCOUH 3N 0HQMH. 280 APPENDIX 1 Pollen data for Cantua (Polemoniaceae): Light microscopy pollen diameter and style length analysis 281 meme-cede m2 i 8.8 2.8-8.8 8e 88 8.8 $3 232 alwm> 8588; 5:56 eve—“Em aw How—om .2 .H See-8.2 e3. 5 8.8 8.8-8., 8 8e 89 8.8 63w em:- 83 case 8.585; .556 2.8-8.8 :1. 6 8.8 8.8.9..” e 2e 86 8.8 $55 a: 5 Es e 55.2232 2? 25 2880 S New 2.8-8. w Se 86 NM; 686 N .33 £885.. 5:56 2% oz 2? oz 2.8 02 8.9.8. .e 2e 8 See 920 3956 Bike 5:56 38-2.8 ooe cw 8.; 88 -88 8e 86 8.8 $55 oeee 295%; a: .2 3:56 3.3.8.8- 8e 30 :8 8.9 8.2 8.». as 8.8 one a8 862; e803 8 5;. 83.26 5:56 2e; 80 2?. 25 E 8.8 one -2 .8 8.8 a 8.9 620 88 3.826 e. a 28 mm 52553556 2 .883.” G; S 8.8 8.9 -28 8H 89 8.8 :55 088 688:: .2: £85m 5:56 2%; oz 2% oz 2?. oz 8.2 -8. e Re a: 8.8 E 23226 e 226 .23 .8 ... 3.5.6 8.8-8.8 8e 80 med. 8.8-8., 8 8.8 S 3.2 920 88 E82 e. emerge .5 ... .556 8.3-2.8 8e 6 we? nee-02 m 3:. o: 8.8 686 2.: 80m 488: 5:56 2? 2.0 28 2.0 S 8.8 08-8. e 8e cc 5.: 920 28H =§Be a. 5:8 81:50 .28 =88 .556 8.8-8.8 8..” H8 8.: 3e. e.8 8e ow 8.8 63 88m £8 86855 .556 8.8-8.8 8. 5 8.8 8.8- :8 8e 89 8.8 8.): 88 3?; 82e£ ”...-88.6 5.5.6 9.8-8.8 Re 3.0 8.8 8.8- Be 2 .m a 8.8 63 82 £ch a. 88223.. 8:858 .556 8.8-8.8 S .e 30 8.8 8.8- 2e Hem 5 3.; GB 82 .5863: 85m .5558 5:56 3:88 a: 5 8.8 8.8-8. e E... 9 8.: 955888 £8 «asexg .556 88-8.8 8.8 as See See-8. _ m we 0 8.? :55 88 225 e. 58%: 358:3 5:56 8.9-8.8 8.2 5 «N8 9; EN. 2 8 3. 5 meme Gd meow Swoatmmmm e. 5&3 page} 5:56 2.8.8.8 2.8 $0 8.8 8. ”-8.8 Be G: 2.: 8.): 2e: 82.5 e. 55m .36 .ES 55e§ 5:56 8.616 .8 one 5 8.8 9; 2:8 82 as 8.8 686 8 _ NN 9.502 a. 882x . 60m .38.; 5:56 8.8; m8 m3 3 2 .8 8.8-8.8 88 as 8.8 :55 28 355.6 .83 5:85 5:56 meE Om 20 new: ownfi Om A20 :82 A880 GE: 3:025 38on who—Em =o=om .3398 520m 2088me Em: E com: mcoHEwomm 82.836; 3250 Se 09:5 28 628.3% @5658 A20 oNHm 295m .52: Ema“: 25m 28 655% 58m 5:8 ”Hm Ban-_- 282 LITERATURE CITED 283 LITERATURE CITED Ackermann, RR, and J .M. Cheverud. 2000. Phenotypic covariance structure in Tamarins (Genus Saguinus): A comparison of variation patterns using matrix correlation and common principal component analysis. 111:489-501. Adams, R.J., and J .K. Morton. 1972. An improved technique for examining pollen under the scanning electron microscope. Pollen et Spores 14:203-212. Aguilar, R., G. Bernadello, and L. Galetto. 2002. Pollen-pistil relationships and pollen size-number trade-off in species of the tribe Lycieae (Solanaceae). J. Plant Res. 1152335- 340. Albert, V.A., M.H.G. Gustaffson, and L. Dilaurenzio. 1998. Ontogenetic systematics, molecular developmental genetics, and the angiosperrn petal. Pp. 349-374 in Molecular systematics of plants II: DNA sequencing, Soltis, D.E., P.S. Soltis, and JJ. Doyle (eds.). Norwell, Massachusetts. Al-Shehbaz, LA. 1984. The tribes of the cruciferae (Brassicaceae) in the southeastern United States. Journal of the Arnold Arboretum 65:85-111. Ambrosini, M., L.R. Ceci, S. Fiorella, and R. Gallerani. 1992. Comparison of regions coding for tRNA(His) genes of mitochondrial and chloroplast DNA in sunflower: a proposal concerning the classification of “CP-like” tRNA genes. Plant Mol. Biol. 20(1):1-4. Angiosperm Phylogeny Group. 1998. An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden 85:531-553. Angiosperm Phylogeny Group. 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141:399-436. Armbruster, WS. 1993. Evolution of plant pollination systems: hypotheses and tests with the neotropical vine Dalechampia. Evolution 47: 1480-1505. Arnold, SJ. 1992. Constraints on phenotypic evolution. Am. Nat. 140:885-8107. Arnold, S.J., M.E. Pfrender, and AG. Jones. 2001. The adaptive landscape as a conceptual bridge between micro- and macroevolution. Genetica 1 12-11329-32. Arnold, S.J., and RC. Phillips. 1999. Hierarchical comparison of genetic variance- covariance matrices. II. Coastal-inland divergence in the Garter Snake, T hamnophis elegans. Evolution 53:1516-1527. 284 Badyaev, A.V., and KR. Foresman. 2000. Extreme environmental change and evolution: stress-induced morphological variation is strongly concordant with patterns of . evolutionary divergence in shrew mandibles. Proc. R. Soc. Lond. B 267:371-379. Badyaev, A.V., and GE. Hill. 2000. The evolution of sexual dimorphism in the House finch. 1. Population divergence in morphological covariance structure. Evolution 54:1784-1794. Baker, H.G., and 1. Baker. 1979. Starch in angiosperm pollen grains and its evolutionary significance. American Journal of Botany. 66:591-600. Baker, RH, and GS. Wilkinson. 2003. Phylogenetic analysis of correlation structure in stalk-eyed flies (Diasemopsis, Diopsidae). 57:87-103. Beardsley, P.M., and R.G. Olmstead. 2002. Redefining Phrymaceae: the placement of Mimulus, tribe Mimuleae, and Phrjyma. American Journal of Botany 87(7):1093-1102. Bruneau, A. 1997. Evolution and homology of bird pollination syndromes in Erythrina (Leguminosae). American Journal of Botany 84:54-71. Camara, M.D., and M. Pigliucci. 1999. Mutational contributions to genetic variance- covariance matrices: an experimental approach using induced mutations in Arabidopsis thaliana. Evolution 53:1692-1703. Cheverud, J .M. 1988. A comparison of genetic and phenotypic correlations. Evolution 42:958-968. Cheverud, J .M. ‘1996. Quantitative genetic analysis of cranial morphology in cotton-top (Saguinus oedipus) and saddle-back (S. fuscicollis) tammarins. J. Evol. Biol. 9:5—42. Conner, J.K. 1997. Floral evolution in wild radish: the roles of pollinators, natural selection, and genetic correlations among traits. International Journal of Plant Science 158:S108-S120. Conner, J .K., R. Franks, and C. Stewart. 2003. Expression of additive genetic variances and covariances for wild radish floral traits: comparison between field and greenhouse environments. Evolution 57:487-495. Conner, J .K., and A. Sterling, 1995. Testing hypothesis of functional relationships: a comparative survey of correlation patterns among floral and vegetative traits in five insect-pollinated plants. American Journal of Botany 82: 1399-1406. Conner, J .K., and A. Sterling, 1996. Selection for independence of floral and vegetative traits: evidence from correlation patterns in five species. Canadian Journal of Botany 74: 642-644. 285 Conner, J .K., and S. Via. 1993. Patterns of phenotypic and genetic correlations among morphological and life-history traits in wild radish, Raphanus raphanistrum. Evolution 47:704-711. Cruden, R.W., and D.L. Lyon. 1985. Correlations among stigma depth, style length, and pollen grain size. Do they reflect function or phylogeny? Botanical Gazette 146:143-149. Cruzan, MB. 1990. Variation in pollen size, fertilization ability, and post fertilization siring ability in Erythronium grandiflorum. Evolution 44:843-856. Darwin, C. 1877.The various contrivances by which orchid are fertilized by insects, 2nd ed. John Murray, London (1St ed.l863). Darwin, C. 1896. The different forms of flowers on plants of the same species. Appleton, New York. Day, AG, and R. Moran. 1986 Acanthogilia, a new genus of Polemoniaceae from Baja California, Mexico. Proceedings of the California Academy of Science 44:111-126. De-Yaun, H. 1983. The distribution of Scrophulariaceae in the holoarctic with special reference to the floristic relationships between Eastern Asia and Eastern North America. Ann. Missouri Bot. Gard. 70:701-712. Dodd, R.S., Z.A. Rafii, and A. Bousquet-Mélou. 2000. Evolutionary divergence in the pan-Atlantic mangrove Avicenm'a germinans. New. Phytol. 145:1 15-125. Elisens, WJ. 1986. Pollen morphology and systematic relationships among new world species in the tribe Antirrhineae (Scrophulariaceae). American Journal of Botany 73: 1298-1311. Faegri, K. and L. van der Pijl. 1966. The principles of pollination ecology, lst ed. Pergamon Press, Oxford. F elsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791. Flury, B. 1988. Common principal components and related multivariate models. Wiley, New York. Fujii, N., K. Ueda, Y. Watano, and T. Shimizu. 1995. Intraspecific sequence variation in chloroplast DNA of Primula cunaifolia Ledeb. (Primulaceae). J. Phytogeogr. & Taxon. 43:15-24. Fujii, N., K. Ueda, Y. Watano, and T. Shimizu. 1997. Intraspecific sequence variation of chloroplast DNA in Pedicularis chamissonis Steven (Schrophulariaceae) and geographic structuring of the Japanese “Alpine” Plants. J. Plant Res. 110: 195-207. 286 Futuyma, D.J., M.C. Keese, and DJ. Funk. 1995. Genetic constraints on macroevolution: The evolution of host affiliation in the leaf beetle genus Ophraella. Evolution 49:797- 809. Galloway, G.L., R.L. Malmberg, and RA. Price. 1998. Phylogenetic utility of the nuclear gene argenine decarboxylase: An example from Brassicaceae. Mol. Biol. Evol. 1521312- 1320. Grant, K.A. 1966. A hypothesis concerning the prevalence of red coloration in California hummingbird flowers. Am. Nat. 100:85-97. Grant, K.A., and V. Grant. 1967. Effects of hummingbird migration on plant speciation in the California flora. Evolution 21 :457-465. Grant, K.A., and V. Grant. 1968. Hummingbirds and their flowers. Columbia University Press. New York. Grant, RR, and HR. Grant. 1995. Predicting microevolutionary responses to directional selection on heritable variation. Evolution 49:241-251. Gomez-Campo C. 1999. Taxonomy. Pp. 3-32 in Biology of Brassica coenospecies, Gomez-Campo, C. (ed.). Elseveir Science B.V., Amsterdam. Hall, J.C., K.J. Sytsma and EH. Iltis. 2002. Phylogeny of the Capparaceae and Brassicaceae based on chloroplast sequence data. American Journal of Botany 89: 1 826- 1842. Harvey, P.H. and MD. Pagel. 1991 The comparative method in evolutionary biology. Oxford University Press, Oxford. Hayek, A. 1911. Von. Entwurf eins Cruciferen-Systems auf phylogenetischer Grundlage. Beih. Bot. Centralbl. 27:127-335. pls. 8-12. Hennig, W. 1950. Grundziige einer theorie der phylogenetischen systematic. Deutscher Zentralverlag, Berlin. Hooker, J .W. 1838. Flora Boreali-Americana. Vol. 2. NewYork: Hafner Publishing Co. P. 110. Houle, D.H., J. Mezey, and P. Galpern. 2002. Interpretation of the results of common principal components analysis. Evolution 56:433-440. J anchen, E. 1942. Das Sytem der Cruciferen. Osterr. Bot. Zeitscher. 91:1-28. Jansen, R.K. 1992. Current research. Plant Molecular Evolution Newsletter 2:13-14. 287 J epson, W.L. 1925. Pedicularis L. Lousewort. Pp. 948-949 in A manual of the flowering plants of California. Associated Student Store, University of California, Berkeley, California. Johnson, L.A., J. L. Schultz, D.E. Soltis, and PS. Soltis. 1996. Monophyly and generic relationships of Polemoniaceae based on matK sequences. American Journal of Botany 83: 1207-1224. Johnson, L.A., D.E. Soltis, and PS. Soltis. 1999. Phylogenetic relationships of Polemoniaceae inferred from 18S ribosomal DNA sequences. Plant Systematics and Evolution 214265-68. Johnson, R.A., and D.W. Wichem. 2002. Applied Multivariate Statistical Analysis, 5th ed. Prentice Hall, Upper Saddle River, New Jersey. Judd, W.S., R.W. Sanders, and M.J. Donoghue. 1994. Angiosperm family pairs: Preliminary phylogenetic analyses. Harvard Papers on Botany 5:1-51. Kaiser, HF 1958. The varimax criterion for analytical rotation in factor analysis. Psychometrika 23: 187-200 Kercher, S., and J .K. Conner. 1996. Patterns of genetic variability within and among populations of wild radish, Raphanus Raphanistrum (Brassicaceae). American Journal of Botany 83:1416-1421. Koch, M., B. Haubold, and T. Mitchell-Olds. 2001. Molecular systematics of the Brassicaceae: evidence from coding plastic matK and nuclear Chs sequences. American Journal of Botany 88:534-544. Inaba, R., and T. Nishio. 2002. Phylogenetic analysis of Brassiceae based on the nucleotide sequences of the S-locus related gene, SLR] . Theor. Appl. Genet. 10521159- 1165. Lande, R. 1976. Natural selection and random genetic drift in phenotypic evolution. Evolution 30:314-334. Lande, R. 1979. Quantitative genetic analysis of multivariate evolution, applied to brain:body size allometry. Evolution 33:402-416. Lande, R. 1985. The dynamics of peak shifls and the patterns of morphological evolution. Paleobiology 12:34-3 54. Lande, R. and SJ. Arnold. 1983. The measurement of selection on correlated characters. Evolution 37:1210-1226. 288 Lee, S. 1978. A factor analysis study of the functional significance of angiosperm pollen. Systematic Botany 3: l-19. Lee, S. 1989. Some functional aspects of angiosperm pollen. Kor. J Plant Tax. 19: 289- 301. Lofsvold, D. 1986. Quantitative genetics of morphological differentiation in Peromyscus. 1. Test of the homogeneity of genetic covariance structure among species and subspecies. Evolution 40:559-573. Loockerman, D.J., and R.K. Jansen. 1996. The use of herbarium material for DNA studies. Pp. 205-220 in Sampling the green world, Stuessy, TE, and SH. Sohmer (eds.). Columbia University Press, New York. Macior, L.W. 1973. The pollination ecology of Pedicularis on Mount Rainier. American Journal of Botany. 60(9):863-871. Macior, L.W. 1977. The pollination ecology of Pedicularis (Scrophulariaceae) in the Sierra Nevada of California. Bulletin of the Torrey Botanical Club 104:148-154. Macior, L.W. 1982. Plant community and pollinator dynamics in the evolution of pollination mechanisms in Pedicularis (Scrophulariaceae). Pp. 29-45 in Pollination and evolution, Armstrong, J .A., J .M. Powell, and A.J. Richards (eds.). Royal Botanic Gardens, Sydney, Australia. Macior, L.W. 1983. The pollination dynamics of sympatric species of Pedicularis (Scrophulariaceae). Amer. J. Bot. 70(6):844-853 Macior, L.W. 1984. Behavioral coadaptation of Bombus pollinators and Pedicularis flowers. Vcme Symposium International sur la Pollinisation. Versailles 27-30 Septembre 1983. Ed. INRA Publ. (Les Colloques de l’INRA, n-21). Macior, L.W. 1986a. Floral resource sharing by bumblebees and hummingbirds in Pedicularis (Scrophulariaceae) pollination. Bull. Torrey Bot. Club. 113:101-109. Macior, L.W. 1986b. Pollination ecology and endemic adaptation of Pedicularis howellii Gray (Scrophulariaceae). Pl. Sp. Biol. 1:163-172. Macior, L.W. 1995a. Pollination ecology of Pedicularis in the Teton Mountain region. Plant Species Biol. 10:77-82. Macior, L.W. 1995b. Pollination ecology of Pedicularis parryi ssp. purpurea (Parry) Carr (Scrophulariaceae). Plant Species Biol. 10:163-168. Macior, L.W. 1996. Pollination ecology of Pedicularis bracteosa in the montane- subalpine ecotone. Plant Species Biol. 11:165-171. 289 Macior, L.W., and T. Ya. 1997. A preliminary study of the pollination ecology of Pedicularis in the Chinese Himilaya. Plant Species Biol. 12:1-7. McDade, LA. 1992. Pollinator relationships, biogeography, and phylogenetics. Bioscience 42:21-26. Mitchell, A.D., and PB. Heenan. .2000. Systematic relationships of New Zealand endemic Brassicaceae inferred from nrDNA ITS sequence data. Systematic Botany 25298-105. Monson, R.K. 1996. The use of phylogenetic perspective in comparative plant physiology and development biology. Ann. Missouri Botanical Garden 83:3-16. Morell, PL. and L.H. Reisberg. 1998. Molecular tests of the proposed diploid hybrid origin of Gillia achilleifolia (Polemoniaceae). American Journal of Botany 85:1439- 1453. Moritz, C. 1995. Uses of molecular phylogenies for conservation. Philosophical Transactions of the Royal Society of London 349:113-118. Olmstead, R.G., C.W. DePamphilis, A.D. Wolfe, N.D. Young, W.J. Elisons, and PA. Reeves. 2001. Disintegration of the Schrophulariaceae. American Journal of Botany 88(2):348-361. Olmstead, R.G., and J .A. Sweere. 1994. Combining data in phylogenetic systematics: an empirical approach using three molecular data sets in the Solanaceae. Syst. Biol. 43:467- 481. Olmstead, R.G., J. A. Sweere, and K.H. Wolfe. 1993. Ninety extra nucleotide in the nth gene of tobacco chloroplast DNA: A summary of revisions to the 1986 genome sequence. Plant Molecular Biology 22:1191-1193. Pagel, M. 1993. Seeking the evolutionary regression coefficient: an analysis of what comparative methods measure. Journal of Theoretical Biology 164:191-205. Phillips, PC. 1998. CPCRand: randomization test of the CPC hierarchy. Univ. of Oregon Soflware available at . Phillips, PC, and SJ. Arnold. 1999. Hierarchical comparison of genetic variance- covariance matrices. 1. Using the F lury hierarchy. Evolution 53:1506-1515. Plitmann, U., and DA. Levin. 1983. Pollen-pistil relationships in the Polemoniaceae. Evolution 37:957-967. 290 Porter, J.M. 1997. Phylogeny of the Polemoniaceae based on nuclear ribosomal internal transcribed spacer DNA sequences. Aliso 15:57-77. Porter, J .M., and LA. Johnson. 1998. Phylogenetic relationships of Polemoniaceae: inferences from nadl B intron sequences. Aliso 17:157-188. Porter, J .M., and LA. Johnson. 2000. A phylogenetic classification of Polemoniaceae. Aliso 19:55-91. Pradham A.K., S. Prakash, A. Mukhopadhyay, and D. Pental. 1992. Phylogeny of Brassica and allied genera based on variation in chloroplast and mitochondrial DNA patterns: Molecular and taxonomic classifications are incongruous. Theo. Appl. Genet. 85:331-340. Prather, LA. 1999. Systematics of Cobaea (Polemoniaceae). Systematic Botany Monographs 57:1-81. ' Prather, L.A., C.J. Ferguson, and R.K. Jansen. 2000. Polemoniaceae phylogeny and classification: Implications of sequence data from the chloroplast gene nth. Am. J. Bot. 87:1300-1308. Prather, L.A., and R.K. Jansen. 1998. The phylogeny of Cobaea (Polemoniaceae) based on sequence data from the ITS region of nuclear ribosomal DNA. Systematic Botany 23:55-70. Prather, L.A., A.K. Monfils, A.L. Posto, and RA. Williams. 2002. Monophyly and phylogeny of Monarda (Lamiaceae): Evidence from the internal transcribed spacer (ITS) region of Nuclear Ribosomal DNA. Systematic Botany 27:127-137. Price, R.A., J .D. Palmer, and LA. Al-Shehbaz. 1994. Systematic relationships of Arabidopsis: Molecular and morphological perspective. Pp. 7-19 in Arabidopsis, Meyerowitz, E.M., and CR. Somerville (eds.). Cold Spring Harbor Press, New York, New York, USA. Price, TD, and PR. Grant. 1985. The evolution of ontogeny in Darwin’s finches: A quantitative genetic approach. Am. Nat. 125:169-188. Purvis, A., and A. Rambaut. 1995. Comparative analysis by independent contrasts (CAIC): An Apple Macintosh application for analyzing comparative data. Cabios 11:247- 251. Ree, RH. 2001. Homoplasy and the Phylogeny of Pedicularis. Ph. D. dissertation. Harvard University Graduate School of Arts and Sciences, Cambridge, Massachusetts. 291 Reisberg, L.H., M.A. Hanson, and CT. Philbrick. 1992. Androdioecy is derived from dioecy in Datiscaceae: evidence from restriction site mapping of PCR-amplifies chloroplast DNA fragments. Systematic Botany 17:324-326. Reisberg, LR. and NC. Ellstrand. 1993. What can molecular and morphological markers tell us about plant hybridization. Crit. Rev. Plant Sci. 12:213-241. Robart, B.W. 2000. The systematics of Pedicularis bracteosa: Morphometrics, development, pollination ecology, and molecular phylogenetics. Illinois State University Department of Biological Science, Bloomington, Illinois. Rodman, J .E., K.G. Karol, R.A. Price, and K.J. Sytsma. 1996. Molecules, morphology, and Dahlgren’s expanded order of the Capparales. Systematic Botany 21 :289-307. Rodman, J.E., P.S. Soltis, D.E. Soltis, K.J. Sytsma, and K.G. Karol. 1998. Parallel evolution of glucosinilate biosynthesis inferred from congruent nuclear and plastid gene phylogenies. American Journal of Botany 85:997-1006. Roff, DA. 2000. The evolution of the G-matrix: selection or drift? Heredity 84:135-142. Roff, D.A., and TA. Mousseau. 1999. Does natural selection alter genetic architecture? An evaluation of quantitative genetic variation among populations of Allonemobius socius and A.fasciatus. J. Evol. Biol. 12:361-369. Roff, D.A., T.A. Mousseau, and DJ. Howard. 1999. Variation in genetic architecture of calling song among populations of Allonemobius socius, A. fasciatus, and a hybrid population: Drift or selection? Evolution 53:216-224. Sakai, A.K., S.G. Weller, W.L. Wagner, P.S. Soltis, and DE. Soltis. 1997. Phylogenetic perspective on the evolution of dioecy: Adaptive radiation in the Hawaiian genera Schicdea and Alsinidendron (Charyophylaceae: Alisinoideae). Pp. 455-473 in Molecular evolution and adaptive radiation, Givnish, T.J., and K.J. Sytsma (eds.). Cambridge University Press, New York. Sang, T., DJ. Crawford, and T.F. Stuessy. 1995. Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: Implications for biogeography and concerted evolution. Proceedings of the National Academy of Sciences 92:6813-6817. Sang, T., DJ. Crawford, and T.F. Stuessy. 1997. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). American Journal of Botany 84(8):] 120-1 136. Schluter, D. 1996. Adaptive radiation along genetic lines of least resistance. Evolution 50:1766-1776. 292 Schulz, DE. 1936. Cruciferae. In A. Engler & K. Prantl. Nat.Pflanzenfam. ed. 2. 17b: 227-658. Simmons, MP, and H. Ochoterena. 2000. Gaps as characters in sequence-based phylogenetic analysis. Systematic Biology 49:369-381. Soltis, PS, and MA. Gitzendanner. 1999. Review: Molecular systematics and the conservation of rare species. Conservation Biology 13:471-483. Soltis, PS, and DE. Soltis. 2000. The role of genetic and genomic attributes in the success of polyploids. Proceedings of the National Academy of Sciences 97:7051-7057. Song, K.M., and T.C. Osborn. 1992. Polyphyletic origins of Brassica napus: new evidence based on organelle and nuclear RF LP analyses. Genome 35:992-1001. Song, K.M., T.C. Osborn, and PH. Williams. 1988. Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs): I. Genome evolution of diploid and arnphidiploid species. Thero. Appl. Genet. 75:784-794. Song, K.M., T.C. Osborn, and PH. Williams. 1990. Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs): III Genome relationships in Brassica and related genera and the origin of B. oleracea and B. rapa (syn. campestris). Thero. Appl. Genet. 79:497-506. Sprague, E.F. 1958. A new subspecies of Pedicularis densiflora. Aliso 4:130. Sprague, E.F. 1960. Ecological life history of California species of Pedicularis. Claremont Graduate School, Claremont, California. Sprague, E.F. 1961. Parasitism in Pedicularis. Madrono 16:192-200. Sprague, E.F. 1962. Pollination and evolution in Pedicularis (Schrophulariaceae). Aliso 5:181-209. Steele, K.P., and R. Vigalys. 1994. Phylogenetic analysis of Polemoniaceae using nucleotide sequences of the plastid gene matK. Systematic Botany 19:126-142. Steppan, S.J. 1997a. Phylogenetic analysis of phenotypic covariance structure. I Contrasting results from matrix correlation and common principal component analysis. Evolution 51:571-586. Steppan, S.J. 1997b. Phylogenetic analysis of phenotypic covariance structure. 11 Reconstructing Matrix evolution. Evolution 51:587-594. Steppan, S.J., P.C. Phillips, and D. Houle. 2002. Comparative quantitative genetics: evolution of the G matrix. Trends in Ecology and Evolution 17:320-327. 293 Stroo, A. 2000. Pollen morphological evolution in bat pollinated plants. Plant Syst. Evol. 222:225-242. Stuchlik, L. 1967a. Pollen morphology in the Polemoniaceae. Grana Palynol. 7: 146-240. Stuchlik, L. 1967b. Pollen morphology and taxonomy of the family Polemoniaceae. Rev. Paleobot. Palynol. 4:325-333. Swofford, D.L. 2000. PAUP*: Phylogenetic analysis using parsimony (*and other methods). Version 4. Sunderland, Massachusetts, Sinauer Associates. Swofford, D.L., and W. P. Maddison. 1987. Reconstructing ancestral character states under Wagner parsimony. Mathematical biosciences 87: 1 99-229. Taberlet, P.L., L. Gielly, L. Pautou, and J. Bouvet. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Pl. Mol. Bio. 17:1105- 1109. Taylor, TN, and D.A. Levin. 1975. Pollen morphology of Polemoniaceae in relation to systematics and pollination: Scanning electron microscopy. Grana 15291-112. Thorman, C.E., M.E. Ferreira, L.E.A. Carnargo, J .G. Tivang, and T.C. Osborn. 1994. Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species. Theor. Appl. Genet. 88:973-980. Vorobik, LA. 1993. Pedicularis (Scrophulariaceae). Pp. 1049-1051 in The Jepson manual: Higher plants of California, Hickman, J .C. (ed.). University of California Press, Berkeley, California. Waitt, DE, and D.A. Levin. 1998. Genetic and phenotypic correlations in plants: A botanical test of Cheverud’s conjecture. Heredity 80:310-319. Warwick, 8.1., and L.D. Black. 1991. Molecular systematics of Brassica and allied genera (Subtribe Brassicinae, Brassiceae) — chloroplast genome and cytodeme congruence. Theor. Appl. Genet. 82:81-92. Warwick, SI, and L.D. Black. 1993. Molecular relationships in subtribe Brassicinae (Cruciferae, tribe Brassiceae). Canadian Journal of Botany 71 :906-918. Warwick, SI, and L.D. Black. 1997. Phylogentic implications of chloroplast DNA restriction site variation in subtribes Raphaninae and Cakilinae (Brassicaceae, tribe Brassiceae). Canadian Journal of Botany 75:960-973. 294 Webb, C.O., D.D. Ackerly, M.A. McPeek, and M.J. Donoghue. 2002. Phylogenies and community ecology. Annu. Rev. Ecol. Syst. 33:475-505. White, T.J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322 in PCR protocols: A guide to methods and applications, Innis, M., D. Gelfand, J. Sninsky, and T. White (eds.). Academic Press, San Diego. Willis, J .H., J .A. Coyne, and M. Kirkpatrick. 1991. Can one predict the evolution of quantitative characters without genetics? Evolution 45:441-444. Wroblewski, T., S. Coulibaly, J. Sadowski and CF. Quiros. 2000. Variation and the phylogenetic utility of the Arabidopsis thaliana RpsZ homolog in various aspects of the tribe Brassiceae. Mol. Phylog. Evol. 16:440-448. Yang, Y.W., K.N. Lai, P.Y. Tai, and W.H. Li. 1999. Rates of nucleotide substitution in angiosperm mitochondrial DNA sequences and dates of divergence between Brassica and other angiosperm lineages. Journal of Molecular Evolution. 74:597-604. 295 l11111111111111I