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Phylogeny of Three East Antarctic Mosses

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Phylogeny of Three East Antarctic Mosses University of Wollongong Research Online University of Wollongong Thesis Collection 1954-2016 University of Wollongong Thesis Collections 2013 Phylogeny of Three East Antarctic Mosses Rhys A. Wyber University of Wollongong Follow this and additional works at: https://ro.uow.edu.au/theses University of Wollongong Copyright Warning You may print or download ONE copy of this document for the purpose of your own research or study. The University does not authorise you to copy, communicate or otherwise make available electronically to any other person any copyright material contained on this site. You are reminded of the following: This work is copyright. Apart from any use permitted under the Copyright Act 1968, no part of this work may be reproduced by any process, nor may any other exclusive right be exercised, without the permission of the author. Copyright owners are entitled to take legal action against persons who infringe their copyright. A reproduction of material that is protected by copyright may be a copyright infringement. A court may impose penalties and award damages in relation to offences and infringements relating to copyright material. Higher penalties may apply, and higher damages may be awarded, for offences and infringements involving the conversion of material into digital or electronic form. Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong. Recommended Citation Wyber, Rhys A., Phylogeny of Three East Antarctic Mosses, Bachelor of Medical Biotechnology Advanced - Honours thesis, , University of Wollongong, 2013. https://ro.uow.edu.au/theses/4646 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] Phylogeny of Three East Antarctic Mosses A thesis Submitted in partial fulfilment of the requirements for the award of the degree of Bachelor of Medical Biotechnology Advanced (Honours) From the University of Wollongong By Rhys A. Wyber School of Biological Sciences October 2013 ACKNOWLEDGEMENTS I would like to acknowledge all the people for whom this thesis would not have been possible without. Firstly I would like to thank Prof. Mark Dowton for his indispensable knowledge of PCR and sequencing troubleshooting, phylogenetic analysis and general advice. Also, thanks to Prof. Sharon Robinson for her help with all things related to moss and constant enthusiasm, even when bombarded with hundreds of phylogenetic trees. I wish to thank Mark Dowton’s doppelgӓnger at the University of Adelaide, Mark Stevens, for help selecting molecular markers for this project and for offering many samples for this study, which unfortunately were never received due to AQIS paperwork issues. I must acknowledge and thank all those who provided and collected samples: Jessica Bramley-Alves, Laurence Clarke, Anna Nydahl, Kengo Kato and again Sharon Robinson for Antarctic samples; Johanna Turnbull for providing some precious Heard Island moss samples; Melinda Waterman for kindly donating a number of cultured sterile Ceratodon purpureus samples; and the British Antarctic survey herbarium and New York botanic gardens herbarium, for allowing me to destructively analyse their herbarium specimens. I must thank Tracey Gibson and Meng Mao for their assistance in the laboratory, Joy Williams for teaching me the art of moss taxonomy, Alison Downing for providing assistance in the identification of troublesome samples, Margaret Phillips for sequencing all my samples and Diana King for drawing some of the maps used in this thesis. Finally, I must thank everyone who helped in the proof reading of this thesis and in particular Melinda Waterman and Julie-Ann Green for all there help and support this year. Thanks to everyone for making this one of the most rewarding years yet and of course to moss moss moss moss. iii TABLE OF CONTENTS DECLARATION ......................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................ iii TABLE OF CONTENTS ............................................................................................ iv LIST OF FIGURES .................................................................................................... vi LIST OF TABLES .................................................................................................... viii LIST OF ABBREVIATIONS ..................................................................................... ix ABSTRACT ................................................................................................................. 1 1 Introduction ............................................................................................................... 3 1.1 Antarctica ................................................................................................................... 3 1.1.1 Geobiological history ............................................................................................. 4 1.1.2 Persistence of ancient Antarctic life ...................................................................... 5 1.2 Bryophytes lifecycles ................................................................................................. 8 1.2.1 Moss identification................................................................................................. 9 1.2.2 Antarctic mosses and identification ..................................................................... 11 1.2.2.1 East Antarctic mosses .......................................................................................... 11 1.3 Molecular markers ................................................................................................... 15 1.3.1 Internal transcribed spacer ................................................................................... 16 1.3.2 Ribosomal protein subunit 4 ................................................................................ 17 1.4 Phylogenetic methods and species delimitation ...................................................... 18 2 Methods................................................................................................................... 22 2.1 Plant material and sample distributions ................................................................... 22 2.1.1 Fresh plant material.............................................................................................. 22 2.1.2 Herbarium specimens........................................................................................... 25 2.1.3 Morphological identification ............................................................................... 27 2.1.4 Geographic sampling of Bryum pseudotriquetrum .............................................. 29 2.1.5 Geographic sampling of Ceratodon purpureus ................................................... 31 2.1.6 Geographic sampling of Schistidium antarctici ................................................... 34 2.2 DNA extraction ........................................................................................................ 36 2.3 PCR amplification and gel excision ......................................................................... 36 2.3.1 PCR of DNA from fresh material ........................................................................ 37 2.3.2 PCR of DNA from herbarium material ................................................................ 37 iv 2.4 Sequencing of amplified DNA................................................................................. 38 2.5 Phylogenetic analyses of DNA sequence data ......................................................... 39 3 Results ..................................................................................................................... 43 3.1 PCR Amplification and optimisation ....................................................................... 43 3.1.1 Internal Transcribed Spacer ................................................................................. 43 3.1.2 Ribosomal protein subunit 4 ................................................................................ 47 3.2 Bryum pseudotriquetrum ......................................................................................... 48 3.2.1 Phylogeny of Bryum pseudotriquetrum ............................................................... 48 3.2.2 Ability of ITS and rps4 to identify Bryum pseudotriquetrum ............................. 51 3.3 Ceratodon purpureus ............................................................................................... 51 3.3.1 Phylogeny of Ceratodon purpureus..................................................................... 51 3.3.2 Ability of ITS and rps4 to identify Ceratodon purpureus ................................... 52 3.4 Schistidium antarctici .............................................................................................. 54 3.4.1 Phylogeny of Schistidium antarctici .................................................................... 54 3.4.2 Ability of ITS and rps4 to identify Schistidium antarctici .................................. 61 4 Discussion ............................................................................................................... 62 4.1 PCR and contaminant genes ...................................................................................
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