ECOLOGICAL GENETICS OF THE MOSS PHYSCOAHTRELLA PATENS Erica Jane HooPer Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences September2008 The candidateconfirms that the work submittedis his/her own and that appropriate credit hasbeen given where referencehas been made to the work of others. This copy hasbeen suppliedon the understandingthat it is copyright material and that no quotation from the thesismay be publishedwithout proper acknowledgement. Acknowledgements Firstly I would like to thankmy supervisorsAndy Cumingand Bill Kunin, for all their support,guidance and encouragement. This hasbeen immensely valuable to mewhilst conductingand completing this project,and without which I wouldhave beenunable to finish.I amalso extremely grateful to YasukoKamisugi, for all her helpand advice with laboratorywork, particularly the AFLP analysis.Thanks also go to ClaireSmith, for herhelp with planttissue culture. I am very grateful to all the bryologists who have assistedin finding bryophyte populations,without which I would have beencompletely unable to undertakethis research.Particular thanks go to SamBosanquet, David Chamberlain,Gorden Rothero,John Blackburn, HansBlom and RachelO'Hara for accompanyingme on my hunt for populations.I am also very grateftil to Chris Preston,David Long, David Holyoak, Mark Pool, Paul King, Tom Blockeel, Ron Porley, Angela Newton and Mark Lawley, for supplying details of populations.Thanks go to all the landownersand wardenswho gavetheir permissionfor plant collectionsto take place. Many thanksgo to Jeff Duckett at QueenMary, University of London, for his help with identifying Physcomitrella readeri, without whose expertise the moss would remain a mystery. Thanks also go to both Jeff Duckett and Sylvia Pressel, for supplying beautiftil SEM imagesof the P. readeri plants. I would like to thank my colleaguesat the University of Leeds,for providing both their friendship and support,along with a stimulating environmentin which to work. Lastly I will thank my family for their enthusiasmand endlessencouragement, and for accompanyingme on cold, wet, field excursions.I particularly thank my partner for all his crucial supportand understanding,and for spotting the odd sporophyte when I was aboutto give up. iii Abstract Molecular geneticstudies using the model bryophytePhyscomitrella patens have advancedthe body of knowledgesurrounding plant functional geneticsand molecularbiology, yet very little is known aboutthe ecology and population geneticsof this species.Although the bryophytesare the secondlargest group of plants, there is little information regardingthe population geneticsof bryophytesin general.To addressthese issues I have conductedthe first study into the population geneticsof P. patens, whereplants from eight populationsin Britain havebeen collected. Samplingwithin thesepopulations was conductedaccording to a hierarchicalscale, so as to assessnot only the level of geneticvariation within populations,but how this is structuredspatially. Analysis of the plants collectedwas conductedusing amplified fragmentlength polymorphism (AFLP) analysis.No spatial geneticstructure was found within populationsof P. patens, and it is hypothesisedthat the natureof the ephemeralaquatic habitats that P. patens occupiesmay accountfor this finding. In this thesisa novel method for studying the mating systemsoperating within bryophytepopulations has been proposed, which exploits the dominanthaploid stageof the bryophyte life cycle. This methodology hasbeen applied to natural populationsof P. patens, and evidenceof mixed mating has beenobserved. Bryophytes are often overlookedor under-recordedin their natural environment,and distribution datawithin Great Britain is likely to be inaccuratefor a large numberof species.This issueis highlighted in this thesis,as a bryophyte speciesnew to Europehas been discovered. iv Table of Contents Acknowledgments i ...................................................................................................... Abstract iii ...................................................................................................................... List Tables of .............................................................................................................vii List Figures of ...........................................................................................................vii List Appendices of ...................................................................................................... x List Abbreviations Used of ........................................................................................ xi Chapter I GeneralIntroduction I ........................................................................ 1.1 Introduction 1 to project ............................................................................... 1.2 Genetic analysis of population structure and evolutionary processes 3 1.2.1 Fine-scale 3 spatial structure ............................................................... 1.2.2 Mating 5 systems within populations .................................................. 1.2.2.1 The AFLPs in 6 use of population genetic analysis ............. 1.3 Bryophyte biology life history 8 and ........................................................... 1.3.1 Geneticdiversity in bryophyte 10 populations................................... 1.3.2Bryophyte 11 mating systems............................................................. 1.4 Population 12 geneticsof model species..................................................... 1.4.1 P. 14 patens -a useftil ecological model ............................................ 1.4.1.1 Biology P. 15 of patens ........................................................ 1.4.1.2 Phylogenetics 16 and taxonomy........................................... 1.4.1.3 Historical 19 perspective...................................................... 1.4.1.4 Genomics 20 ......................................................................... 1.5 Aims the 22 of project .................................................................................. Chapter 2 General Materials Methods 24 and ..................................................... 2.1 Study 24 sites ............................................................................................... V 2.2 Sample DNA 30 culture and extraction ....................................................... 2.3 AFLP 30 analysis......................................................................................... Chapter3 Geneticstructure within and betweenpopulations of P. 32 patens ......................................................................................................... 3.1 Introduction 32 ............................................................................................. 3.2 Materials 33 and methods............................................................................ 3.2.1 AFLP 34 profile scoring and error-ratetesting ................................... 3.2.2 Data 35 analysis.................................................................................. 3.2.2.1 Geneticdiversity linkage disequilibrium 35 and .................. 3.2.2.2 Genetic between 36 structurewithin and populations.......... 3.3 Results 38 ..................................................................................................... 3.3.1 Geneticdiversity linkage disequilibrium 39 and ................................ 3.3.2 Genetic between 42 structurewithin and populations........................ 3.4 Discussion 45 ............................................................................................... Chapter4 P. in Europe 51 readeri ....................................................................... 4.1 Introduction 51 ............................................................................................. 4.2 Materials 53 and methods............................................................................ 4.2.1 Populationdetails 53 and samplecollection ....................................... 4.2.2 Sample 53 culture ................................................................................ 4.2.3 Nuclear ITS 57 region analysis ........................................................... 4.2.3.1 Amplification 57 and sequencing ......................................... 4.2.3.2 Sequence 58 alignment and analysis .................................... 4.2.4 Morphologcial 59 analysis .................................................................. 4.3 Results .....................................................................................................59 4.3.1 ITS 59 sequenceanalysis .................................................................... 4.3.2 Morphological 63 analysis.................................................................. vi 4.4 Discussion 65 ............................................................................................... Chapter 5 A method for estimation of the mating systems operating P. 68 within populations of patens ........................................................................ 5.1 Introduction 68 ............................................................................................. 5.2 Materials 71 and methods ............................................................................ 5.2.1 Trial known 71 using progeny ............................................................ 5.2.1.1 DNA AFLP 72 extraction