FOLIAR ENDOPHYTIC FUNGI OF THE NATIVE HAWAIIAN PLANT GENUS SCAEVOLA ________________ A University Thesis Presented to the FAculty of CAliforniA StAte University, EAst BAy ________________ In PArtiAl Ful(llment of the Requirements for the Degree M.S. in Biological Sciences ________________ By SeAn OmeA Imanishi Swift December 2016 Copyright © 2016 by SeAn Swift ii Abstract FoliAr endophytic fungi (FEF) hAve been found living Asymptomatically within the leAf tissues of All lAnd plAnts sampled thus fAr. HAwAii presents A unique lAndscape for eXAmining the ecology And evolution of these cryptic fungAl symbionts. The isolAtion of the HAwAiiAn ArchipelAgo provides A strong bArrier to colonization by host plAnts And their AssociAted endophytes. The nAtive plAnt genus Scaevola (GoodeniAceAe) is the result of three sepArate colonization events And hAs AdApted to A variety of hAbitAts, including coAstAl strand, rain forest, And exposed lAva :ow. This project focused on quAntitAtively Assessing FEF diversity And community structure in Scaevola through A combinAtion of culture bAsed methods And high-throughput environmentAl sequencing. LeAf samples were collected from 35 individuAls of Scaevola representing 8 species from three islAnds. Cultured endophytes were grouped into MoleculAr OperationAl TAxonomic Units (OTUs) bAsed on 97% similArity of the nucleAr ribosomal internAl transcribed spAcer region (ITS). Sequences of the nucleAr lArge ribosomal subunit (LSU) were generated from isolAtes representing eAch OTU. Phylogenies were constructed using LSU sequence dAtA And AnnotAted with ecological dAtA derived from IlluminA® sequencing. Endophyte community composition wAs compAred on the bAsis of host genetics (e.g. host species & host lineAge) And Abiotic environmentAl fActors (e.g. elevation & meAn AnnuAl temperature). iii Acknowledgements This materiAl is bAsed upon work supported by the NAtionAl Science FoundAtion under Grant No. 1360626. ThAnk you to All the people And orgAnizations thAt helped with our (eldwork in HAwAi'i, including the NAtionAl Tropical BotAnic GArden, The NAture Conservancy, PlAnt Extinction Prevention Program, And the DepArtment of LAnd And NAtural Resources. I wAnt to thAnk A greAt many people for helping me get to this point. My pArents BriAn And Yuri And my sister Erin for their constAnt support And encouragement. Dr. BriAn Perry, for freely shAring his wisdom on fungi And All other subjects. I could not hAve Asked for A more intelligent And generous mentor. ThAnk you to my committee members Dr. MariA GAllegos And Dr. Christopher BAysdorfer for their careful reAding And helpful comments. For their AssistAnce with this project, I must thAnk Dr. Anthony Amend, Gerald CobiAn, Dr. Geoffrey ZAhn, Erin DAtlof, Don Hemmes, And Alex DAnza. I Am Also indebted to the many botAnists who helped us Along the wAy, including Steve Perlman, Tim Flynn, Kristen Coelho, AdAm WilliAms, SeAnA WAlsh, And Jesse AdAms. Members of the Perry lAb Devin SchAefferkoetter And JonAthAn del Rosario provided lAbor, tolerance, And A greAt deAl of kindness, All of which I greAtly AppreciAte. Lynx GAllAgher, my erstwhile mentor in HAwAiiAn mycology And bAd behAvior is Acknowledged for his cruciAl role in my graduAte education. I would Also like to recognize Dr. DonAld Roeder for introducing me to mycology And setting me on this unorthodox pAth. FinAlly, for her endless pAtience, love, And help in the lAb, A heArtfelt thAnk you to PAtriciA Sendão, whom it is impossible to AdequAtely thAnk. v Table of Contents Abstract...................................................................................................................iii Acknowledgements................................................................................................. v List of Figures....................................................................................................... viii List of TAbles...........................................................................................................xi Introduction............................................................................................................. 1 NAture of The AssociAtion................................................................................... 1 EnvironmentAl FActors........................................................................................ 2 Host Genotype.....................................................................................................4 Evolution And Coevolution.................................................................................. 5 Implications for Scaevola.................................................................................... 6 Speci(c ReseArch Questions.............................................................................. 9 HAwAiiAn Endophytes........................................................................................12 Methodological Precedence..............................................................................13 Methods................................................................................................................ 14 SAmple Collection And Processing................................................................... 14 FungAl Culturing................................................................................................ 16 DNA Extraction, Ampli(cation, And Sequencing...............................................17 EnvironmentAl PCR And IlluminA® Sequencing...............................................19 AnAlysis............................................................................................................. 21 Results.................................................................................................................. 23 Summary DAtA from Cultured IsolAtes..............................................................23 Phylogenetics.................................................................................................... 27 Summary DAtA from IlluminA® Sequencing......................................................31 Diversity of OTUs from IlluminA® Sequencing..................................................35 SAmple DissimilArity.......................................................................................... 41 Combining Cultured IsolAte Phylogenies And IlluminA® Sequencing DAtA......48 Discussion.............................................................................................................53 Phylogenetic PlAcement And EvolutionAry Hypotheses...................................56 The DominAnt Genus Colletotrichum................................................................57 Community Ecology.......................................................................................... 60 Synthesis of Culture And EnvironmentAl Sequencing DAtA..............................64 Future Directions for Scaevola..........................................................................66 References............................................................................................................68 vi Appendix 1: SAmple MetAdAtA..............................................................................78 Appendix 2: Bioinformatics Pipeline for Processing IlluminA DAtA.......................84 Appendix 3: DAtA AnAlysis in R.............................................................................92 vii List of Figures Figure 1. A mAp showing sample sites for HAwAiiAn ScaevolA species 9 used in this project. Lines indicate regions with equAl AnnuAl rainfAll totAls ranging from 250 mm/yeAr At low elevation sites to 4000 mm/yeAr At higher elevation sites. Lines indicate increments of 500 mm/yeAr rainfAll totAls (rainfAll dAtA from Frazier, GiAmbelluca, DiAz, & NeedhAm, 2015). Figure 2. Illustration of ribosomAl cAssette showing conserved primer 19 locAtions And ApproXimAte Amplicon lengths for two relevant gene regions: nucleAr ribosomAl InternAl Transcribed SpAcer (ITS), And nucleAr ribosomAl LArge Sub-Unit (LSU). Figure 3. Graph showing distinct OTUs isolAted from eAch species. EAch 25 bAr is colored by QIIME Assigned tAXonomy of OTUs At the clAss level. Figure 4. Number of ScaevolA individuAls eAch OTU wAs isolAted from 26 (totAl individuAls successfully isolAted from = 23). Most OTUs (29) were encountered A single time. The most common And widespreAd OTU (EU552111) wAs isolAted from 11 individuAls. Figure 5. A mAximum likelihood tree of All fungAl isolAte OTUs shown in 38 the context of AscomycotA. EnlArged phylogeny on the right shows plAcement of isolAtes within SordAriomycetes. IsolAtes Are colored by genus level QIIME Assigned tAxonomy. Branch lAbels indicate bootstrap support from 100 bootstrap replicates. Figure 6. EnlArged phylogeny on the right shows plAcement of isolAtes 30 within Dothideomycetes. IsolAtes Are colored by genus level QIIME Assigned tAxonomy. Branch lAbels indicate bootstrAp support from 100 bootstrap replicates. Figure 7. A mAximum likelihood tree of isolAtes Assigned to order 31 GlomerellAles. IsolAtes Are colored by species level tAXonomy Assigned by QIIME. Branch lAbels indicate bootstrap support from 100 bootstrap replicAtes. Figure 8. Distinct OTUs recovered from eAch species through 33 environmentAl PCR And IlluminA® sequencing. DominAnt clAsses of fungAl endophytes included Dothideomycetes And SArdAriomycetes. MAny OTUs remAined unidenti(ed At the clAss level. viii Figure 9. Histogram showing the number of plAnt individuAls eAch OTU 34 wAs recovered from.