Next-Generation Approaches to Understanding the Diversity and Evolution of Marine Fungi by Kathryn Therese Picard Department of Biology Duke University Date: Approved: Kathleen Pryer, Supervisor Daniele Armaleo Timothy Y. James Jason E. Stajich Jennifer J. Wernegreen 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 Abstract Next-Generation Approaches to Understanding the Diversity and Evolution of Marine Fungi by Kathryn Therese Picard Department of Biology Duke University Date: Approved: Kathleen Pryer, Supervisor Daniele Armaleo Timothy Y. James Jason E. Stajich Jennifer J. Wernegreen 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 Copyright c 2017 by Kathryn Therese Picard All rights by Kathryn Therese Picard 2017 Abstract Fungi are among the most diverse extant eukaryotic lineages, with estimates of total global diversity projecting millions of species that have yet to be cataloged. Though fungi from all phyla and habitats await discovery, marine fungi are particularly poorly understood. Historical surveys of fungi in marine habitats, which relied primarily on direct culturing or observation of fruiting bodies and other structures on incubated shore detritus, suggest that marine fungi are unexceptional in their diversity and frequency, and therefore unimportant, one might be tempted to conclude. However, with the increasing adoption of environmental sequencing as a primary tool for ex- ploring fungal diversity and ecology across disparate habitats, the discovery of novel phylotypes representing new species|and in some cases, even new phyla|demands a reappraisal of fungal diversity in marine habitats using modern molecular methods. This dissertation represents an attempt to advance our understanding of the breadth of fungal diversity, to establish a broader evolutionary context for marine fungi, and to provide some molecular tools to better study marine fungi that have, until now, eluded our detection. In Chapter1, I investigate the diversity and spatio-temporal distribution of unicellular eukaryotes in the surface waters of the English Channel using high- throughput sequencing of 18S ribosomal DNA. In addition to characterizing the taxonomic and phylogenetic diversity of planktonic protists, I also estimate the niche breadth of the taxa observed and infer ecological roles and trophic modes to examine iv if and how functional guilds change through space and time. I find that while the community observed at any given time is likely to be dominated by only a handful of species, many of them members of the Stramenopiles, Alveolates, and Rhizaria, most protistan taxa are rare specialists. I also find that the relative abundance of an individual taxon is not indicative of a specialist or generalist habit. Interestingly, I also find that fungi comprise a significant fraction of the microbial community, but the most prominent fungal taxa observed do not closely resemble phylogenetically any circumscribed species of marine fungi. Rather, they are nested within the enig- matic Cryptomycota, a recently described phylum at the base of the fungal tree. Thus, while this is the only chapter of my dissertation which does not focus primar- ily on fungal diversity or evolution, the results in many ways set the stage for my subsequent work. In Chapter2, I turn my sails back toward land to challenge long-standing my- cological lore by re-examining the diversity of coastal marine fungi. I focus on four marine habitats in coastal North Carolina (surface water, persistent wetlands, in- tertidal sand flats, and marine benthos) from which I sample water and sediments over the course of a year. Using primers designed to amplify across the entire fungal kingdom, I use the Ion Torrent platform to sequence amplicons from 28S ribosomal DNA and evaluate how successfully extant reference databases identify novel fun- gal sequences to both high and low taxonomic ranks. I find that marine fungi are far more varied than previously thought, with some early diverging fungi, and the Chytridiomycota in particular, proving to be diverse and ubiquitous. I also find that curated reference databases struggle to assign robust taxonomic identities to novel sequences across all fungal phyla, but are particularly ill-equipped to identify the marine representatives of non-Dikarya fungi. Finally, in Chapter3 I aim to address the deficiencies of curated reference databases used in taxonomic assignment by generating high-quality reference sequences from v complex environmental samples. Using a third-generation sequencing technology, PacBio circular consensus sequencing, which trades the high coverage of other se- quencing platforms for much longer read lengths, I target an approximately 2kb fragment of the ribosomal DNA operon that contains both the full fungal internal transcribed spacer (ITS) region and over 1kb of the 28S ribosomal subunit. Using a mock community approach and successive rounds of filtering, I calculate the average sequencing error for PacBio amplicons by comparing them to known sequences from axenically cultured, circumscribed species. I then revisit my samples from Chapter 2, generate amplicon sequences to be used for phylogenetic inference, and compare the accuracy of taxonomic assignments made by reference databases curated for individual loci (i.e., ITS vs. 28S) for different rDNA regions from the same oper- ational taxonomic unit. I find that stringent quality filtering of PacBio sequence data produces consensus sequences approaching the quality of MiSeq and 454, which can be used in phylogenetic analyses to provide improved taxonomic assignments. Furthermore, many of the fungal taxa observed belong to known marine lineages, while others are only distantly related to reference accessions and may represent new lineages. vi For my parents, Henry and Jossie, my husband, Christopher, and my Paw-Paw. But not for Tildepants, who's a shiftless layabout. vii Contents Abstract iv List of Tables xi List of Figures xiii Acknowledgements xv Introduction1 1 Seasonal Diversity, Distribution, and Ecological Role of Protists in the Western English Channel5 1.1 Introduction................................5 1.2 Materials and Methods..........................8 1.2.1 Sampling.............................8 1.2.2 DNA extraction and sequencing.................8 1.2.3 Sequence processing.......................9 1.2.4 Relaxed filtering datasets..................... 12 1.2.5 Statistics and community analyses............... 12 1.2.6 Estimating niche breadth and functional roles......... 13 1.3 Results................................... 14 1.3.1 Summary of sequence filtering and overall OTU composition. 14 1.3.2 Eukaryotic alpha and beta diversity............... 17 1.3.3 Inference of niche breadth and functional roles......... 20 1.4 Discussion................................. 23 viii 1.4.1 Taxonomic composition of plankton communities....... 24 1.4.2 Plankton communities through space and time......... 25 1.4.3 Methodological considerations.................. 26 1.5 Conclusions................................ 27 2 Coastal Marine Habitats Harbor Novel Early-Diverging Fungal Di- versity 28 2.1 Introduction................................ 28 2.2 Materials and Methods.......................... 31 2.2.1 Study sites and sampling regime................. 31 2.2.2 DNA extraction and sequence data generation......... 33 2.2.3 Sequence data processing..................... 35 2.2.4 Taxonomic assignment...................... 35 2.2.5 Phylogenetic placement of most abundant OTUs........ 37 2.2.6 Diversity analyses......................... 37 2.3 Results................................... 37 2.3.1 Sequence filtering and OTU clustering............. 37 2.3.2 Taxonomic assignment...................... 42 2.3.3 Per-site diversity......................... 43 2.4 Discussion................................. 50 2.4.1 Plankton sampling (Piver's Island)............... 51 2.4.2 Persistent wetland sediments (Town Marsh).......... 52 2.4.3 Intertidal sand (Bird Shoal)................... 53 2.4.4 Benthic marine sediments (Cape Lookout Bight)........ 54 2.4.5 Methodological considerations.................. 56 3 Generating Reference Sequences for Molecular Operational Taxo- nomic Units (MOTUs) with PacBio: a Case Study with the Dark ix Matter Fungi 58 3.1 Introduction................................ 58 3.2 Materials and Methods.......................... 61 3.2.1 Community DNA samples.................... 61 3.2.2 Library preparation and PacBio sequencing.......... 62 3.2.3 Sequence processing of mock community data......... 65 3.2.4 Phylogenetic analyses....................... 66 3.2.5 Comparison of fungal reference databases............ 68 3.3 Results................................... 69 3.3.1 Effect of sequence processing on observed error rate...... 69 3.3.2 Phylogenetic assignment of fungal MOTUs from marine habitats 71 3.3.3 Distribution of marine MOTUs................. 77 3.3.4 Taxonomic assignment across target loci and reference databases 77 3.4 Discussion................................. 79 3.5 Conclusions................................ 83 4 Further Insights into the Diversity and Evolution of Early-Diverging and Marine Fungi 85 Conclusions 87 A Supplementary Information for Chapter1 92 B Supplementary Information for Chapter2 98