The Pennsylvania State University The Graduate School Eberly College of Science SYSTEMATICS AND PLASTID GENOME EVOLUTION IN THE PARASITIC PLANT GENUS CUSCUTA (DODDER) A Thesis in Biology by Joel R. McNeal © 2005 Joel R. McNeal Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2005 The thesis of Joel R. McNeal was reviewed and approved* by the following: Claude W. dePamphilis Associate Professor of Biology Thesis Adviser Chair of Committee Stephen W. Schaeffer Associate Professor of Biology Andrew G. Stephenson Professor of Biology David M. Geiser Associate Professor of Plant Pathology Douglas R. Cavener Professor of Biology Head of the Department of Biology *Signatures are on file in the Graduate School. iii ABSTRACT Parasitism has evolved independently many times within the course of angiosperm evolution. One of the most economically damaging of these parasitic lineages is the genus Cuscuta, which is derived from within the Morning Glory Family (Convolvulaceae). All species of Cuscuta are epiphytic stem parasites that lack roots and expanded leaves at maturity. Their hosts include a wide array of non-grass land plants, and a single Cuscuta individual may parasitize dozens of host species simultaneously. Although almost 200 described species exist, identification and taxonomy are difficult within the genus, as few morphological characteristics exist outside of the inflorescence in these reduced parasites. Although only about 10% of the genes necessary for photosynthesis are transcribed from the plastid chromosome, analyses of plastid genome structure, gene content, and sequence evolution are an excellent and efficient way to study the changes in photosynthetic ability and organellar function that accompany the transition from autotrophy to heterotrophy in angiosperms. This work presents a useful method for acquiring complete plastid genome sequences from parasitic plants, analyses of full plastid genome sequences from two Cuscuta species and a nonparasitic relative, and a well-resolved and highly-supported phylogeny of Cuscuta using 3 plastid genes (rbcL, rps2, and matK) and the nuclear ribosomal internal transcribed spacer locus. A molecular phylogenetic approach is used to address hypotheses involving taxonomy, biogeography, morphological evolution, photosynthetic ability, and plastid genome evolution within the genus. iv TABLE OF CONTENTS LIST OF FIGURES................................................................................................... vi LIST OF TABLES.....................................................................................................vii ACKNOWLEDGMENTS......................................................................................... viii Chapter 1: Overview..................................................................................................1 References......................................................................................................5 Chapter 2: Utilization of Partial Genomic Fosmid Libraries for Sequencing Complete Organellar Genomes.............................................................................6 Abstract..........................................................................................................7 Introduction....................................................................................................8 Materials and Methods...................................................................................10 DNA Isolation and Purification......................................................... 10 Partial Genomic Library Construction...............................................11 Identifying Plastid Clones..................................................................11 Selecting Clones for Sequencing....................................................... 13 Results and Discussion.................................................................................. 14 Acknowledgments..........................................................................................19 References......................................................................................................20 Chapter 3: Disappearance of Introns Promotes Adaptive Change and Loss of a Highly Conserved Maturase...............................................................................24 Abstract..........................................................................................................25 Methods......................................................................................................... 32 PCR and Sequencing..........................................................................32 Data Analyses.................................................................................... 33 References..........................................................................................34 Acknowledgments..............................................................................36 Chapter 4: Complete Plastid Genome Sequences Suggest Strong Selection for Retention of Photosynthetic Genes in the Parasitic Plant Genus Cuscuta............41 Abstract..........................................................................................................42 Introduction....................................................................................................44 Materials and Methods...................................................................................48 Plastid Genome Sequencing, Assembly, and Annotation..................48 Molecular Evolutionary Analyses......................................................49 Results and Discussion.................................................................................. 51 v Acknowledgments..........................................................................................65 Literature Cited.............................................................................................. 66 Chapter 5: Systematics and Plastid Genome Evolution of the Cryptically Photosynthetic Parasitic Plant Genus Cuscuta (Convolvulaceae).............................85 Acknowledgments..........................................................................................86 Abstract..........................................................................................................87 Materials and Methods...................................................................................92 Plant Material.....................................................................................92 PCR and Sequencing..........................................................................93 Phylogenetic Analyses.......................................................................95 Genome Size Estimates......................................................................96 Rates Analyses...................................................................................96 Results............................................................................................................97 Phylogeny.......................................................................................... 97 Genome Size Results......................................................................... 99 Plastid Genome Variation Assays......................................................100 Tests of Selective Constraint............................................................. 101 Discussion......................................................................................................102 Morphological and Biogeographical Interpretation of Phylogeny.... 102 Genome Sizes and Speciation............................................................104 Plastid Genome Evoluton in Cuscuta................................................ 106 Loss of Photosynthesis in Cuscuta.................................................... 107 References......................................................................................................109 Appendix........................................................................................................129 Chapter 6: Future Direction and Conclusion............................................................. 130 References......................................................................................................133 vi LIST OF FIGURES Chapter 2: Figure Legends............................................................................................................22 Figure 1: Macroarray screen of fosmid clones using pooled plastid probes.............. 23 Figure 2: Map of end-sequenced clone coverage on plastid genomes........................23 Chapter 3: Figure Legends...........................................................................................................38 Figure 1: Results of PCR assays for presence or absence of two group IIB introns contained in ycf3 (a) and a group IIA intron in 3' rps12 (b)........ 39 Figure 2: Phylogenies of (a) Convolvulaceae and (b) Orobanchaceae inferred from nucleotide sequence of full and partial matK sequences..................39 Chapter 4: Figure Legends...........................................................................................................72 Figure 1: Circular map of the complete plastid genome of Ipomoea purpurea Inset: Genomes scaled to relative size......................................................73 Figure 2: Circular map of the complete plastid genome of Cuscuta exaltata............74 Figure 3: Circular