Diversity and Effects of the Fungal Endophytes of the Liverwort Marchantia Polymorpha

Diversity and Effects of the Fungal Endophytes of the Liverwort Marchantia Polymorpha

Diversity and Effects of the Fungal Endophytes of the Liverwort Marchantia polymorpha by Jessica Marie Nelson Department of Biology Duke University Date:_______________________ Approved: ___________________________ Arthur Jonathan Shaw, Supervisor ___________________________ Rytas Vilgalys ___________________________ François Lutzoni ___________________________ Fred S. Dietrich ___________________________ Paul S. Manos Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology in the Graduate School of Duke University 2017 i v ABSTRACT Diversity and Effects of the Fungal Endophytes of the Liverwort Marchantia polymorpha by Jessica Marie Nelson Department of Biology Duke University Date:_______________________ Approved: ___________________________ Arthur Jonathan Shaw, Supervisor ___________________________ Rytas Vilgalys ___________________________ François Lutzoni ___________________________ Fred S. Dietrich ___________________________ Paul S. Manos An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology in the Graduate School of Duke University 2017 i v Copyright by Jessica Marie Nelson 2017 Abstract Fungal endophytes are ubiquitous inhabitants of plants and can have a wide range of effects on their hosts, from pathogenic to mutualistic. These fungal associates are important drivers of plant success and therefore contribute to plant community structure. The majority of endophyte studies have focused on seed plants, but in order to understand the dynamics of endophytes at the ecosystem scale, as well as the evolution of these fungal associations, investigations are also necessary in earlier-diverging clades of plants, such as the non-vascular bryophytes (mosses, liverworts, and hornworts). This dissertation presents a survey of the diversity of fungal endophytes found in the liverwort Marchantia polymorpha L. and develops a gnotobiotic experimental system for testing the effects of these fungi on their liverwort host. The survey reveals a diverse community of fungi in M. polymorpha, with some fungi that are associated with this host across geographically distant sites. The laboratory experiments demonstrate that culturable endophytes of M. polymorpha can, in isolation, cause positive, negative, or neutral effects on host success and that these effects change in response to nutrient levels and the presence of multiple endophytes. The experimental system developed in this dissertation has great potential in the growing field of plant microbiota research to answer questions that range in scale from molecular mechanisms to ecosystem function. iv Dedication I dedicate this dissertation to my fiancé, Duncan Hauser, who has been my amazingly patient and supportive partner for the past six and a half years. Duncan encouraged my developing interest in bryophytes when we were in college together and has been encouraging me in my pursuit of science ever since. When we graduated college, he was crazy enough to pick up and move across the country with me. Over the five years of my PhD work, he has assisted me in so many ways: in the lab, in the field, as editor, scientific sounding board, and moral support. He has kept me from giving up and I could not have accomplished this dissertation without him. v Table of Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................. xi List of Figures .............................................................................................................................. xii Acknowledgements ...................................................................................................................xiv 1 Introduction ................................................................................................................................ 1 2 Chapter 1: Fungal endophyte diversity in the liverwort Marchantia polymorpha L. based on culturing and culture-independent amplicon sequencing ................................................ 8 2.1 Introduction ....................................................................................................................... 8 2.2 Materials and Methods .................................................................................................. 12 2.2.1 Collection locations ................................................................................................... 12 2.2.2 Host sampling and endophyte isolation ................................................................ 13 2.2.3 Culture identification ................................................................................................ 14 2.2.4 DNA extraction from M. polymorpha ...................................................................... 17 2.2.5 DNA extraction from substrates ............................................................................. 17 2.2.6 Amplicon sequencing ............................................................................................... 18 2.2.7 Amplicon data analysis ............................................................................................ 20 2.2.8 Fungal phylogeny ...................................................................................................... 22 2.3 Results .............................................................................................................................. 23 2.3.1 Culture collection ...................................................................................................... 23 2.3.2 Mock community recovery ...................................................................................... 26 2.3.3 Culture-independent sampling ............................................................................... 26 vi 2.3.4 Phylogeny of cultures and amplicons .................................................................... 32 2.3.5 Non-fungal associates ............................................................................................... 35 2.4 Discussion ........................................................................................................................ 36 2.4.1 Comparison of culture and culture-independent sampling of M. polymorpha endophytes .......................................................................................................................... 37 2.4.2 Factors organizing the fungal community in M. polymorpha .............................. 38 2.4.3 Dominant members of the M. polymorpha mycobiome ........................................ 41 2.4.4 Comparison of M. polymorpha fungal communities to those of other bryophytes ........................................................................................................................... 44 2.4.5 Comparison of M. polymorpha fungal communities to those of vascular plants ............................................................................................................................................... 46 2.4.6 Non-fungal associates of M. polymorpha ................................................................. 49 2.4.7 Concluding remarks .................................................................................................. 50 3 Chapter 2: A novel experimental system using the model liverwort Marchantia polymorpha L. reveals diverse effects of fungal endophytes in a bryophyte host .............. 51 3.1 Introduction ..................................................................................................................... 51 3.2 Materials and Methods .................................................................................................. 57 3.2.1 Endophyte collections ............................................................................................... 57 3.2.2 Liverwort axenic cultures ......................................................................................... 58 3.2.3 Experiment one: Fungal effect screen ..................................................................... 59 3.2.4 Experiment one data processing ............................................................................. 60 3.2.5 Experiment one data analysis .................................................................................. 63 3.2.6 Experiment two: Testing selected fungi ................................................................. 64 vii 3.2.7 Experiment two data analysis ................................................................................. 66 3.3 Results .............................................................................................................................. 67 3.3.1 Overall liverwort growth ......................................................................................... 67 3.3.2 Experiment one growth effects ................................................................................ 67 3.3.3 Experiment two growth effects ............................................................................... 70 3.3.4 Variability in isolate effects ...................................................................................... 72 3.3.5 Effects of fungi on host morphology .....................................................................

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