CONTRIBUTIONS TO THE MOLECULAR BIOLOGY OF KELP Michael Keith Liptack B.Sc.. University of Washington, 199 1 THESIS SUBMlTTED Di PARTIAL FULFILLMENT OF THE REQUREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Biological Sciences O Michae! K. Liptack 1999 SMON FRASER UNIVERSITY Novembcr 1999 All rights reserved. This work rnay not be reproduced in whole or in put, by photocopy or other means. without permission of the author. 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ABSTRACT Genetic relatedness between various kelp (Order Laminariales, Class Phaeophyceae, Division Heterokontophyta) taxa was investigated using DNA sequencirig and PCR-typing. The rDNA ITS 1 region of gametophytes generated by a nanvally occurring apparent kelp hybrid of Macrocystis C. Agarâh and Pelugophycus Anschoug were sequenced to determine parentage. Al1 garnetophytes examined had only Macrocystis rDNA suggesting either a non-hybrid, or more complicated hybridization than pure equd parental contribution occwred. Laboratory-generated intergeneric hybrids of Alaria Greville and Lessoniopsis Reinke were examined for parentage based on rDNA regions arnplified using PCR. Both parental rDNA types were visible in one identified possible hybrid and non-hybnds were easily distinguished. Actin introns in both Alaria and Nereocystis Postels & Ruprecht were characterized and sequenced, representing the first actin intron sequences examined in the Heterokontophyta. The second actin intron fiom individuals of three Alaria species, spanning a geographic range of hundreds of kilometers, were sequenced to quanti@variation and to examine individual relatedness for usage in studies of gene flow and population subdivision. Relatedness seerned to correlate with oceanographic distance but not with accepted species boundaries iii Ackno wIed~mcn& 1 would like to thank the mernbers of my cornmittee for their support and reviews of this work. In particular, 1 would like to thank my senior supervisor, Louis D. Druehl. for his inspiration, support, and friendship, without which bis thesis would have been impossible. Many other lab-mates shared in field collections, technical help, and general discusions on al! mmer of phycologica! topics, and to hem 1 am etemally gntehl: lm Tan, Charlene Mayes, Handojo Kusumo, Andrew Swanson, and lan MacKenzie. I would also like to especially thank Nikita Grigoriev, Bûny Honda, and the late John D.G. Boom for al1 the intellectual encouragement, technical hel p, and fiendship. Much of this work was camed out while in residence at the Barnfieid Marine Station. 1 would like to thank the Director, Andy Spencer, and the memben of W.C.U.M.B.S. for matdal and scientific support during my studies at BMS. 1 also owe a huge debt to ail the employees of the Barnfieid Marine Station who helped me in innumerable ways: Nancy Christney, Heather Brooke, Cliff Haylock and the rest of the maintenance guys (1 promise, no more Blazer repairs! !), Shirley Pakula, Linda Mather, Leslie Rimmer, Dave Hutchison, both Karls, and anyone else 1 mighi have forgotten. Finally, I would like to thank my wife Elise for support, patience, and ever-present words of encouragement. 1 have no doubt, without her support 1 would not have made it. Thanks also to my daughters Hailey and Kyla for keeping me down to earth and my family for always believing in me. Financial support for this study came hma Parks Canada grant through the Pacific Rim National Park as well as NSERC operatine grants to Louis Druehl. Table of Contents Approval Abstract iii Acknowledgments iv Tablc of Contents v List of Tables vii ... List of Figures Vlll General Introduction 1 Chapter 1. ITS 1 NDNA Scquences of Macrocystis pyrifera, P elaguphycis porra, and a Mucrocystis x Pelugophycus Hybrid. Introduction Materials and Methods Results Discussion Chapter II. lTS1 nrDNA Fragments as Molecular Evidence for an Interfami lia1 Laminariaiean H y brid Cross Between Alaria marginata Postels & Ruprecht and Lessoniopsis littoralis (Tilden) Reinke. Introduction Materials and Methods ResuIts Discussion Chapter III. Actin Introns in Alaria and Nereocystis Introduction Materials and Methods Results Discussion Chapkr IV. Actin Introns as Markers for Phylogeopphy in Alaria Introduction Materiais and Methods Results Discussion General Conclusion Re ferences Appendix 1. Alignment of Sequences from Clones of the Actin Intron II-containing Region of Both Alaria and Nereocysfis. 169 Appendix II. Sequence alignment of Nereocystis individuals at the second actin intron. 183 Appendix III. ANS1 C++ source code created for the console program TajimaD.exe 188 Appendix IV. Alignment of Alaria actin intron II regions. 1 94 Table 1. Species, strain, general morphology. isolation locale, and culture source of gametophytes and sporophytes hmwhich sequences were either generated or obtained . 13 Table 2. Strain, species, and geneml morphology of plants fiorn which DNA was extracted. 36 Table 3. A laria marginata and Lessoniupsis 1ittoralis garnetop hyte crossing attempts and resulting sporophyte morphologies. 42 vii Figure 1. Species ranges and locations of samples. Figure 2. Nuclear ribosomal DN A (nrDNA) sequence dignrnent including regions of the 18s. ITS 1, and 5.8s regions. Figure 3. Alignment of 3' end of 18s. ITSI. and 5' end of 5.8s ruDNA fmm a number of Laminariales genera. Figure 4. Variable sites and percent idmtity matrix for the ITS 1 sequences From the alignrnent shown in Figure 2. Figure 5. 50% Majority-Rule Consensus trees generated using PHYLIP 3.5~ and the aliment from Figure 2. Figure 6. Agarose gel from PCRs of blades seen in cultures. Figure 7. Agarose gel from PCRs of Aloria marginata tissues. Figure 8. Agarose gel from PCRs of Lessoniopsis Iiftoralis tissues. Figure 9. Agarose gel from PCRs of gametophytesrosses and pseudo-crosses. Figure 10. Agarose gel of PCRs fiom bhdes in a hybrid-cross using universal primer BCI and either the Am or LI primer in adjacent pain of lanes. Figure 1 1. Designation, location. approximate position and collecter (if different fiom the author) for Alaria spp. and Nereocystis Lutkana individuals on map shown. Figure 12. Schematic of actin based on sequences hmBhattacharya et al. (1 991) and this study showing primer identity and locations. 63 viii Figure 13. Agarose gel of PCR hgrnents fiom AIaria marginufa actin introns 1 and II, 72 Figure 14. Agarosc gel from PCRs of Alaria rnarginata actin intron II. 74 Figure 15. Alignment of Alaria DNA sequences of the cloned PCR fragment including Intron 1. 77 Figure 16. Alignment of regions sunounding the second actin intron. 80 Figure 17. Alignment of regions surrounding the second actin intron. 82 Figure 18. Alignment of regions surrounding the second actin intron. 84 Figure 19. Protein alignment of actin fragments. 88 Figure 20. Alignment of Aluria actin intron II regions. 1 O5 Figure 2 1. Representation of variable sites across actin intron II of Alaria for visual determination of possible recombination between individuals 109 Figure 22. Jukes-Cantor corrected painvise distance matrices generated using PHYLIP 3,573~DNADIST.EXE. 112 Figure 23. Neighbor Joining phy logram fiom the Jukes-Cantor corrected distance matrix in Figure 22 using Neighbor.exe fiom PHYLIP 3.573~. Figure 24. Neighbor Joining phylogram fiom the Jukes-Cantor corrected distance matrix in Figure 22 minus AM03KB using Neighbot.exe fiom PHYLIP 3,573~ Figure 25. Neighbor Joining ûees using a Jukes-Cantor correctcd distance mstrix generated using PHYLIP 3.57~ Figure 26. Maximum Parsimony ûecs using PHYLIP 3.57~. Figure 27. Maximum Liklihood phylogram generated hmthe alignrnent in Figure 20 using dnarnl.exe fiom PHYLlP 3.573~with an equal probability model. 119 Figure 28. Maximum Liklihood phylogram generated hmthe alignrnent in Figure 20 minus AM03KB using dnaml.exe hmPHYLIP 3.573~ with an qua1 probability model. 120 Figure 29. Maximum Liklifood trees using an equal probability evolutionary mode1 generated using PHYLIP 3.57~ 121 Figure 30. Minimum Spanning Tree fiom alignment in Appendix IV. 125 filamentous microscopie gametophytes. The gametophytes produce eggs or spen. Upon maturity, the female proâuces a pheromone that attracts the spem (Muller 1967) to the retained egg. The resulting zygote develops into the large conspicuous sporophyte. Three families of kelp are commonly found in the northeast Pacific. Morphological features of the kelp sporophyte distinguish these families. The Alariaceae is characterized by the absence of branching and the presence of sporophylls. The Lessoniaceae is characterized by regular branching and in some cases the presence of sporophylls.