MOLECULAR PHYLOGENETICS OF MOSSES AND RELATIVES! by! Ying Chang! ! ! A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF ! DOCTOR OF PHILOSOPHY! in! The Faculty of Graduate Studies! (Botany)! ! ! THE UNIVERSITY OF BRITISH COLUMBIA! (Vancouver)! July 2011! © Ying Chang, 2011 ! ABSTRACT! Substantial ambiguities still remain concerning the broad backbone of moss phylogeny. I surveyed 17 slowly evolving plastid genes from representative taxa to reconstruct phylogenetic relationships among the major lineages of mosses in the overall context of land-plant phylogeny. I first designed 78 bryophyte-specific primers and demonstrated that they permit straightforward amplification and sequencing of 14 core genes across a broad range of bryophytes (three of the 17 genes required more effort). In combination, these genes can generate sturdy and well- resolved phylogenetic inferences of higher-order moss phylogeny, with little evidence of conflict among different data partitions or analyses. Liverworts are strongly supported as the sister group of the remaining land plants, and hornworts as sister to vascular plants. Within mosses, besides confirming some previously published findings based on other markers, my results substantially improve support for major branching patterns that were ambiguous before. The monogeneric classes Takakiopsida and Sphagnopsida likely represent the first and second split within moss phylogeny, respectively. However, this result is shown to be sensitive to the strategy used to estimate DNA substitution model parameter values and to different data partitioning methods. Regarding the placement of remaining nonperistomate lineages, the [[[Andreaeobryopsida, Andreaeopsida], Oedipodiopsida], peristomate mosses] arrangement receives moderate to strong support. Among peristomate mosses, relationships among Polytrichopsida, Tetraphidopsida and Bryopsida remain unclear, as do the earliest splits within sublcass Bryidae. A [Funariidae, [Timmiidae, [Dicranidae, Bryidae]]] arrangement is strongly supported, as are major relationships within subclasses Funariidae and Dicranidae. I also reconstructed the phylogeny of ! ""! the nonperistomate moss family Andreaeaceae, with a focus on costate taxa, using two complementary sets of plastid markers and taxa. The major subgenera (Andreaea and Chasmocalyx) and sections of Andreaea (Andreaea and Nerviae) are rejected as monophyletic. Well-supported lineages include clades comprising: (1) Andreaea nivalis and A. rigida (northern hemisphere members of subgenus Chasmocalyx) and A. blyttii (section Nerviae); (2) most of the remainder of Nerviae; (3) a mixture of costate and ecostate species from Chasmocalyx, Nerviae, all sampled members of section Andreaea, and subgenus Acroschisma. Relationships among the major lineages, including the root of the family, are all well supported. ! ! """! PREFACE! A version of Chapter 2 has been published: Chang, Y., and S. W. Graham (2011), Bryophyte-specific primers for retrieving plastid genes suitable for phylogenetic inference. American Journal of Botany 98: e109-e113. I carried out the laboratory work, designed the primers, and wrote the manuscript. Sean W. Graham provided valuable guidance with respect to primer design and writing. A version of Chapter 3 has been published: Chang, Y., and S. W. Graham (2011), Inferring the higher-order phylogeny of mosses (Bryophyta) and relatives using a large, multigene plastid data set. American Journal of Botany 98: 839-849. I carried out the laboratory work and data analyses, and wrote the manuscript. Sean W. Graham provided insights into taxonomic sampling, data analyses and contributed to the writing. A version of Chapter 4 will be submitted for publication: Chang, Y., and S. W. Graham,! Backbone phylogeny of peristomate and nonperistomate mosses based on multiple plastid markers. I carried out the laboratory work and data analyses, and wrote the manuscript. Sean W. Graham provided insights into taxonomic sampling, data analyses and contributed to the writing. A version of Chapter 5 will be submitted for publication: Chang, Y., and S. W. Graham, Molecular phylogenetics of Andreaeaceae. I carried out the laboratory work and data analyses, and wrote the manuscript. Sean W. Graham provided insights into data analyses and contributed to the writing. ! ! "#! TABLE OF CONTENTS Abstract.......................................................................................................................................... ii Preface........................................................................................................................................... iv Table of Contents .......................................................................................................................... v List of Tables ................................................................................................................................. x List of Figures.............................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Dedication .................................................................................................................................... xv 1 Introduction............................................................................................................................. 1 1.1 Overview of early land-plant phylogeny .......................................................................... 1 1.2 Overview of backbone relationships of bryophytes .......................................................... 2 1.3 Objectives of the thesis..................................................................................................... 6 2 Bryophyte-specific primers for retrieving plastid genes suitable for phylogenetic inference....................................................................................................................................... 11 2.1 Brief synopsis .................................................................................................................. 11 2.2 Introduction ..................................................................................................................... 11 2.3 Methods and results ......................................................................................................... 12 ! #! 2.3.1 “Universal” primer design and testing...................................................................... 12 2.3.2 DNA extraction, amplification and sequencing ....................................................... 13 2.3.3 Sequence editing and alignment............................................................................... 13 2.4 Conclusions ..................................................................................................................... 15 3. Inferring the higher-order phylogeny of mosses (Bryophyta) and relatives using a large, multigene plastid data set ................................................................................................ 24 3.1 Brief synopsis .................................................................................................................. 24 3.2 Introduction ..................................................................................................................... 25 3.3 Materials and methods..................................................................................................... 27 3.3.1 Taxonomic sampling strategy................................................................................... 27 3.3.2 Sequence recovery and alignment ............................................................................ 28 3.3.3 Phylogenetic analyses—Maximum likelihood and parsimony analyses.................. 29 3.3.4 Effect of rapidly evolving sites on MP inference..................................................... 31 3.4 Results ............................................................................................................................. 32 3.4.1 Phylogenetic inference—ML analysis...................................................................... 32 3.4.2 Phylogenetic inference—MP analysis...................................................................... 33 3.4.3 Exploration of conflicts between ML and MP analysis ........................................... 33 ! #"! 3.5 Discussion........................................................................................................................ 35 3.5.1 A well supported backbone of bryophyte phylogeny............................................... 35 3.5.2 Nonperistomate mosses and the earliest splits in moss phylogeny .......................... 36 3.5.3 The sister-group of land plants ................................................................................. 38 3.6 Conclusions and future work........................................................................................... 39 4. Backbone phylogeny of peristomate and nonperistomate mosses based on multiple plastid markers............................................................................................................................ 51 4.1 Brief synopsis .................................................................................................................. 51 4.2 Introduction ....................................................................................................................