MOLECULAR BIOLOGICAL ANALYSIS OF MORPHOLOGICAL VARIATION, POPULATIONS AND PHYLOGENY OF THE KELP COSTARIA COSTATA (PHAEOPHYTA) - Debashish Bhattacharya B.Sc. (Hons.), Dalhousie University, 1981 M.E.S., Dalhousie University, 1984 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the department of Biological Sciences Debashish Bhattacharya 1988 SIMON FRASER UNIVERSITY June 1988 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL Naue: Debashish Bhattacharya Degree : Doctor of Philosophy Title of Thesis: MOLECULAR BIOLOGICAL ANALYSIS OF MORPHOLOGICAL VARIATION, POPULATIONS AND PHYLOGENY OF THE KELP COSTARIA COSTATA (PHAEOPHYTA) Examining Committee: Chairman : Dr.%.D. Druehl, Associate ~$ofessor,Senior Supervisor Dr. D.L. Baillie, Professor Dr. A.T. BecKen~acn,HSSOCL~L~ rroressor Dr. M.J. @tn, ~rofesso?, Public Examiner ur. R.W. fiathewes, Professor, Public Examiner -- D~MTClegg, Professor, mpr. or B-a Plant Sciences, U. of California, External Examiner Date Approved June 28, 1988 PARTIAL COPYRIGHT LICENSE I hereby grant to Slmon Fraser Unlverslty the right to lend my thesis, proJect or extended essay (the Fltle of whlch is shown below) to users of the Slmon Fraser Unlverslty ~lbr&, and to m.ke partial or single copies only for such users or In response to a request from the library of any other unlverslty, or other educational Institutlon, on its own behalf or for one of Its users. I further agree that permission for multiple copying of thls work for scholarly purposes my be granted by me or the Dean of Graduate Studies. It Is understood that copylng or publlcatlon of thls work for flnanclal galn shall not be allowed without my written permlsslon. T 1 t l e of Thes l s/ProJect/Extended Essay Molecular Biological Analysis of Morphological Variation, Populations and Phylogeny of the Kelp Costaria costata (Phaeophyta). Author: (signature) Debashish Bhattacharya t name July 26, 1988 (date) ABSTRACT Molecular biological analyses of the kelp, Cost ari a cost at a (C.A. Agardh) Saunders, were undertaken to study phenotypic variation, genetic subdivision of populations and phylogeny. To study the basis of phenotypic variation in Costaria, individuals were collected from two morphologically distinct stands. Preliminary analysis of morphological variation using multivariate statistical techniques, indicated substantial differentiation between plants from the two sites. Phenotypic integration of the morphology of Costaria, on the basis of correlation analysis, was similar for wave-exposed and wave-sheltered plants. Restriction fragment length polymorphism (RFLP) analysis with 42 anonymous probes demonstrated only one polymorphism. Most of the probes (80.9%) encoded highly repeated, dispersed sequences. Primary sequence analysis of 1595 base pairs of small-subunit ribosomal DNA (~DNA,probe pCcl8) and 204 base pairs of upstream sequence failed to show any divergence between plants from the two sites. It is suggested that differentiation of developmental regulatory systems and not general sequence divergence may account for the observed high level of morphological differentiation in Costaria. Genetic subdivision of Costaria populations was inferred from usage of a polymorphic rDNA marker (pCcl8) which appeared to uncover restriction site variation in the intergenic spacer of the repeat. Twenty sites were sampled and eight distinct iii populations resolved. One variant (Variant A) identified a dominant, southern breeding group with plants from thirteen clustered sites sharing this banding pattern. The other seven variants, found in the northern portion of the study area, were unique and population-specific. It is postulated that the variant distribution was established following the last glaciation (Wisconsin). Surface water currents may have allowed the northward spread of Costaria as well as the genetic homogenization of stands sharing the predominant banding pattern. Transportation of tissues on boat hulls may also have led to the observed distribution of Variant A. The small-subunit rDNA sequence (pCcl8) of Costari~was aligned with those of other chlorophyll a+b- and chlorophyll a+c- containing vascular and nonvascular plants. Phylogenetic comparison of all sequences indicated a common ancestor for phaeophytes, chrysophytes and oomycetes. Phylogenies based on rDNA sequence data and those based on plastid characteristics were discussed and compared. ACKNOWLEDGEMENTS I would like to first thank the members of the examining committee for their helpful advice concerning this thesis. As well, I acknowledge the friendship and intellectual encouragement offered by Louis D. Bruehl and David L. Baillie. Thanks also go to the many work study students and assistants for their support in the lab. And finally, I am grateful to my parents for their ongoing love and support and Claude for her companionship and criticism. TABLE OF CONTENTS Abstract ................................................... iii Acknowledgements ............................................. v List of Tables ............................................ viii List of Figures ............................................. ix I . General ~ntroduction ...................e..O.......O.......1 Morphological and DNA Sequence Variation in Costaria .... 3 Population Analysis of Costaria ........................ 12 Evolutionary position of Costaria ...................... 16 General Summary ........................................ 18 I1 . Morphological and DNA Sequence Variation in Costaria ... 19 ater rials and Methods .........................Oe........ 19 Sampling ......................................... ... Morphometrics ....................................... Molecular ~nalysis.................................. Culture .....................~.OeOOO.....,.......... Results ......~~~e~.o~e.~~.~.O~OOeD~O~.~OO..~.~~~e~~~~. Morphometrics ...................................... Molecular ~nalysis.................................. Culture ......................,...................e.... Discussion .......................................,e..,.. 111 . Population Analysis of Costaria ........................ Materials and Methods .................................. Sampling ............................................ Detection of rDNA Variation ......................... Results ................................................ Discussion ............................................ 75 IV . Evolutionary Position of Costaria ...................... 85 Materials and Methods ................................. 85 Results .......O.*.eD....ee..OO1.............OO. 91 Discussion ............................................. 97 V . General Discussion .................................... 102 VI . References .................................OedO. ....as 107 Appendix A ............................................... 118 vii LIST OF TABLES Table Page I List of variables analysed in Costaria ................. 23 I1 Result of grouped principal components analysis run on Cape Beale and Coal Harbour plants .................. 31 I11 Results of discriminant function analysis .............. 34 IV Results of hybridization analysis . ..................... 37 V stirn nations of percentage dispersed DNA in Cost ari a .... 44 VI variation of rDNA in blade tissue of Costaria .......... 76 VII Structural distance data for study organisms based on small-subunit rRNA sequences . ...................,... 93 LIST OF FIGURES Figure Page Anatomy of Costaria . ................................... 4 ~orphologicalcomparison of wave-exposed and wave-sheltered plants of Costaria .................... 7 Location of variables measured on Costaria ............. 21 Results of cluster analysis ............................ 29 Autoradiographs showing probe sequences which are believed to exist in many forms. a few forms and in one form . ........................................... 39 Autoradiograph showing results of probing of Cape Beale and Coal Harbour plants with pCcl8 .................. 42 Geographic distibution of sites from which Costaria was sampledo ...............................~e.O~O......54 Autoradiograph showing the two size-classes of rDNA in Costaria . ......................................... 58 Restriction map of Costaria rDNA ....................... 61 Autoradiograph showing eight variants found in populations sampled in northeast pacific ............ 64 Identification of rDNAs in CsCl density gradient ....... 67 Autoradiograph showing conservation of the coding sequence of Costaria rDNA ........................... 70 Alignment of two 60 base pair repeats upstream of coding region of the small-subunit gene ............. 73 Restriction map of pCcl8 . .............................. 86 Nucleotide sequence of insert in pCcl8 ................. 89 16 Phylogeny of plants based on small-subunit rRNA sequence divergence data . ........................... 95 CHAPTER I GENERAL INTRODUCTION Species of kelp comprise the conspicuous brown seaweeds (~haeophyta)which inhabit much of the lower intertidal and subtidal regions of rocky shores in temperate climates. Though the object of many studies, there has been a dearth of data regarding molecular biological analyses of intraspecific variation, dispersal and taxonomy in kelp species. This absence of quantitative data is significant since kelps are characterized by high levels of phenotypic variation within species (Sundene 1958, Druehl 1978, Mathieson