! UNIVERSITY OF CALIFORNIA, MERCED Subalpine conifers host core endophytic bacteria conserved across sites and tree species A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy In Environmental Systems by Alyssa A. Carrell Committee in Charge: A. Carolin Frank, Chair Michael Beman Lara Kueppers Jason Sexton ! ! Chapter 2 © Carrell and Frank 2014 All other chapters: © Alyssa A. Carrell, 2014 All rights reserved ! ! The dissertation of Alyssa Ann Carrell is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________ Michael Beman _____________________________________ Lara Kueppers _____________________________________ Jason Sexton _____________________________________ A. Carolin Frank, Chair University of California, Merced 2014 iii! ! Table of Contents List%of%Tables............................................................................................................................ vi! List%of%Figures .........................................................................................................................vii! Acknowledgements .................................................................................................................x! Curriculum%Vitae ...................................................................................................................xii! Abstract%of%the%Dissertation..............................................................................................xiv! 1! Introduction....................................................................................................................... 1! 1.1! Organization%of%Dissertation.............................................................................................. 3! 1.2! References................................................................................................................................ 4! 2! Pinus&flexilis%and%Picea&engelmannii%share%a%simple%and%consistent%needle% endophyte%microbiota%with%a%potential%role%in%nitrogen%fixation........................... 7! 2.1! Abstract..................................................................................................................................... 7! 2.2! Introduction ............................................................................................................................ 8! 2.3! Materials%and%methods ........................................................................................................ 9! 2.3.1! Sample!collection!and!sterilization..........................................................................................9! 2.3.2! DNA!Extraction .............................................................................................................................. 10! 2.3.3! DNA!Amplification........................................................................................................................ 10! 2.3.4! Sequence!Analysis ........................................................................................................................ 11! 2.4! Results .....................................................................................................................................12! 2.4.1! Phylotypes!recovered!from!samples.................................................................................... 12! 2.4.2! Dominant!bacterial!taxa!associated!with!P.!flexilis!and!P.!engelmannii!needles ! 12! 2.4.3! Structure!of!conifer!needle!endophyte!communities ................................................... 14! 2.5! Discussion ..............................................................................................................................14! 2.6! References..............................................................................................................................18! 2.7! Tables ......................................................................................................................................27! 2.8! Figures.....................................................................................................................................28! 3! The%association%between%Pinaceae%and%an%acetic%acid%bacterial%needle% endophyte%is%persistent%across%host%species%and%geographic%locations .............34! 3.1! Abstract...................................................................................................................................34! 3.2! Introduction ..........................................................................................................................34! 3.3! Methods...................................................................................................................................36! 3.3.1! Sample!collection!and!sterilization....................................................................................... 36! 3.3.2! DNA!extraction............................................................................................................................... 36! 3.3.3! DNA!Amplification........................................................................................................................ 37! 3.3.4! Phylotype!generation!and!classification............................................................................. 37! 3.3.5! Community!Analysis.................................................................................................................... 38! 3.4! Results .....................................................................................................................................38! 3.4.1! Phylotypes!recovered ................................................................................................................. 38! 3.4.2! Dominant!bacterial!community!members......................................................................... 39! 3.4.3! Community!Diversity!and!Structure..................................................................................... 39! 3.5! Discussion ..............................................................................................................................40! iv! ! 3.5.1! All!samples!were!heavily!dominated!by!AAB1!and!most!samples!by!AAB2....... 40! 3.5.2! Remaining!community!structure!and!composition....................................................... 41! 3.6! References..............................................................................................................................42! 3.7! Tables ......................................................................................................................................46! 3.8! Figures.....................................................................................................................................47! 4! Enterobacteraceae%dominate%the%bud%and%needle%endophytic%communities% of%Pinus&contorta&in%a%subalpine%meadow .....................................................................57! 4.1! Abstract...................................................................................................................................57! 4.2! Introduction ..........................................................................................................................57! 4.3! Methods...................................................................................................................................59! 4.3.1! Sample!collection!and!sterilization....................................................................................... 59! 4.3.2! DNA!extraction!and!amplification ......................................................................................... 59! 4.3.3! Sequence!Analysis ........................................................................................................................ 60! 4.4! Results .....................................................................................................................................61! 4.5! Discussion ..............................................................................................................................61! 4.5.1! Enterobacter!and!Citrobacter!species,!not!Gluconacetobacter!species,!dominate! the!needles!of!P.!contorta!at!Tuolomne!Meadows,!CA ................................................................. 61! 4.5.2! Little!overlap!in!the!major!phylotypes!in!buds!and!needles...................................... 62! 4.6! References..............................................................................................................................63! 4.7! Figures.....................................................................................................................................67! 5! Diversity%and%Structure%of%the%Bacterial%Endophyte%Communities%in%the% Foliage%of%Giant%Trees ..........................................................................................................73! 5.1! Abstract...................................................................................................................................73! 5.2! Introduction ..........................................................................................................................74! 5.3! Methods...................................................................................................................................75! 5.3.1! Sample!collection!and!sterilization......................................................................................