GENETIC, GENOMIC, TAXONOMIC AND FUNCTIONAL DIVERSITY OF ENDOPHYTIC FUNGI IN CLIMATE-THREATENED BOREAL FORESTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0230936
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824

Performing Department
Plant Science

Non Technical Summary
The goal of this project is to examine the diversity of one of the most ubiquitous but least-studied groups of symbionts on earth: endophytic fungi, which occur within healthy above-ground tissues of all plants (and in lichens as endolichenic fungi) in biomes ranging from tropical forests to Arctic tundra. Defined functionally as fungi living within plants or lichens without causing detectable symptoms of disease, diverse, horizontally transmitted endophytes have been found in every plant and lichen species examined thus far, and increasingly are recognized as an accessible but under-exploited trove of ecological, genetic, and functional diversity. The species diversity of these primarily intercellular symbionts is immense: a single leaf or lichen thallus can harbor dozens of species, and individual plants may harbor hundreds to thousands of species. Overlap among biogeographic provinces is rare and endophyte communities often change markedly across hosts' geographic ranges. Primarily culture-based studies have revealed that endophytes comprise tremendous phylogenetic diversity and an enormous number of unknown fungal species. Endophytes produce metabolites of use in agriculture, medicine, and industry, and provide an array of previously understudied benefits to their hosts, including defense against pests and pathogens, and - of special relevance in the era of climate change - tolerance to heat and drought. Although abundant and diverse in all terrestrial biomes, endophytes peak in phylogenetic diversity in boreal forests -- an imperiled ecosystem of global importance, and one in which their ecological associations, genetic diversity, and functional roles are largely unknown.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	4020	1102	50%
136	0610	1070	50%

Knowledge Area
206 - Basic Plant Biology; 136 - Conservation of Biological Diversity;

Subject Of Investigation
0610 - Conifer forests of the North; 4020 - Fungi;

Field Of Science
1070 - Ecology; 1102 - Mycology;

Keywords

endophytes

fungi

symbiosis

biodiversity

boreal forest

phylogenetics

molecular systematics

genome sequencing

Goals / Objectives
The over-arching goal of this project is to study one of the planet's most prevalent but least-understood symbioses: that of hyperdiverse endophytic fungi (endophytes), which occur within healthy above-ground tissues of all plants (and in lichens as endolichenic fungi) in biomes ranging from tropical rainforests to hot deserts and Arctic tundra, and in human-influenced environments such as agricultural fields, controlled environment production systems, and managed forests and pasturelands. Hyperdiverse, ecologically important, evolutionarily dynamic, and representing an under-explored trove of taxonomic, genetic, and functional diversity, endophytes peak in phylogenetic richness in boreal forests, an imperiled ecosystem of global importance. Discovering endophyte biodiversity in boreal forests is critical because disappearance and change in these plant communities is impending, and their endophytic floras have not yet been evaluated. My preliminary research has shown that boreal endophytes comprise not only a high degree of taxonomic diversity, but genomic traits and functional diversity that is both unique relative to the endophytes in other biomes, and potentially useful for use in human sustainability. Through this project a novel, integrated, and multidisciplinary biodiversity-informatics pipeline will be developed to discover, evaluate and describe the taxonomic, functional, and genetic diversity of endophytes at multiple spatial and phylogenetic scales around the circumboreal belt. In Specific Aim 1, culture-based and culture-free approaches will unveil the biodiversity, spatial heterogeneity, environmental sensitivity, and host-associations of endophytes associated with 20 iconic boreal plant and lichen species at local, regional, and intercontinental scales. In Specific Aim 2, new analytical tools relying on reciprocally illuminating phylogenetic- and population-genetics approaches will orchestrate rapid circumscription and naming of new species, efficiently capturing and describing taxonomic diversity while framing powerful hypotheses regarding the evolution of ecological modes. The resulting framework will optimize selection and development of new model systems for fungal biology, which will be used in Specific Aim 3 to empirically assess the functional diversity of endophytes through in vitro and in vivo assays of mutualistic, pathogenic, and saprotrophic modes. In Specific Aim 4, the relative and absolute contributions of diverse genetic mechanisms to shaping that functional diversity will be examined, including mobile genetic elements, endohyphal microbial symbionts, and genome evolution. Protocols, data, results, and new bioinformatics tools will be distributed publicly through a dedicated web portal (EnDoBiodiversity.org, for Endophytic Dimensions of Biodiversity), with a focus on integrative data management, transparent data sharing, and fair release (Specific Aim 5).

Project Methods
Specific Aim 1. Unveil the scale of endophyte biodiversity. Using a broad and deep culture-based and culture-free survey of phylogenetically diverse, boreal plants and lichens at local, regional, and intercontinental scales, we will determine the scope and distribution of endophyte diversity with regard to hosts, geography, and ecologically relevant environmental factors. Specific Aim 2. Establish transformative taxonomic practices and select optimal model systems for functional and genetic studies. We will use novel and collaborative phylogenetic- and population-level approaches to efficiently, accurately, and rapidly circumscribe taxonomic diversity, cataloguing and describing new species of endophytes, inferring the evolution of their ecological modes, and guiding our selection of strains for functional and genetic analyses and development of novel model systems. Specific Aim 3. Empirically assess functional diversity of endophytes. Using phylogenetically structured sampling we will evaluate, for the first time, the diversity, breadth, and evolution of ecologically relevant functional traits of newly discovered endophytes, quantifying their substrate utilization, enzyme activity, host-symbiotic interactions, and secondary metabolite production. Specific Aim 4. Identify mechanisms underlying genetic diversity of endophytes. Using cytological, microbiological, and genomic approaches, we will comprehensively assess the roles of endohyphal viral and bacterial symbionts, nuclear genomic fluidity (variation in nuclear number, mobile genetic features), and genome evolution in shaping the genetic diversity and functional biology of endophytes. Specific Aim 5. Data management and release. With emphasis on quality control, fair release, and public access, we will share our data, protocols, results, and biodiversity informatics tools through a unified web portal (EnDoBiodiversity.org, for Endophytic Dimensions of Biodiversity), which will integrate our research with outreach, workshops, international collaborations, and mentorship of high-school, undergraduate, graduate, and postdoctoral researchers.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:This project supported research in the Arnold lab by 3 postdocs (2 female), 5 graduate students (4 female, one African American, one veteran; 2 MS and 2 Ph.D. theses defended), 20 undergraduates (11 female, 9 from under-represented groups; 3 honors theses), 6 visiting scholars (3 female), 12 high school students (9 female, 6 from under-represented groups), and 3 K-12 teachers (2 female; all from minority-serving schools). Arnold's semester-long workshops (Endophyte Discovery) reached >140 students/yr at two Hispanic- serving high schools; her citizen scientist activities engaged >400 members of the public; and her outreach presentations reached >1500 members of the public in 10 venues. She developed summer research experiences in field mycology for high school students, implemented in three summers (>40 students engaged), and coordinated a two- and three-week molecular ecology workshop for 24 teachers and high school students from minority-serving schools in the summers of 2015 and 2016. The project enhanced an undergraduate course and contributed to the development of three new courses at UA, reaching >150 students annually. Arnold authored or co-authored 57 extramural presentations (25 invited) and 35 intramural presentations. Arnold authored or co-authored 23 publications, contributed to developing biodiversity informatics pipelines for fungal community- and phylogenetic analyses, and contributed core content to the project website. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported research in the Arnold lab by 3 postdocs (2 female), 5 graduate students (4 female, one African American, one veteran; 2 MS and 2 Ph.D. theses defended), 20 undergraduates (11 female, 9 from under-represented groups; 3 honors theses), 6 visiting scholars (3 female), 12 high school students (9 female, 6 from under-represented groups), and 3 K-12 teachers (2 female; all from minority-serving schools). How have the results been disseminated to communities of interest?Arnold developed summer research experiences in field mycology for high school students, implemented in three summers (>40 students engaged), and coordinated a two- and three-week molecular ecology workshop for 24 teachers and high school students from minority-serving schools in the summers of 2015-2017. The project enhanced an undergraduate course and contributed to the development of three new courses at UA, reaching >150 students annually. Arnold authored or co-authored 57 extramural presentations (25 invited) and 35 intramural presentations. Arnold has authored or co-authored 23 publications through this project. She also contributed to developing biodiversity informatics pipelines for fungal community- and phylogenetic analyses, and contributed core content to the project website. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project aimed to discover and evaluate the taxonomic, functional, and genetic diversity of fungal endophytes at multiple spatial and phylogenetic scales in boreal forests, earth's largest and most threatened forest biome. Large-scale collections of spatially replicated sets of phylogenetically diverse plants and lichens in seven sites around the circumboreal belt generated a globally unique culture collection of >12,000 fungal strains. These provided the basis for genomics, transcriptomics, and population genetics of previously unknown endophytic lineages; functional trait analysis; multilocus phylogenetics and ancestral state reconstruction at the class, order, and family levels; assessment of endohyphal symbionts; and development and sharing of new biodiversity informatics tools for fungal systematics and species discovery. Next-gen amplicon sequencing (Illumina MiSeq) was completed for >700 host samples. Coupled with the living strain collection, this provides the largest and most comprehensive sampling to date of boreal endophytic biodiversity.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Arendt, K.R., K.L. Hockett, S.J. Araldi-Brondolo, D.A. Baltrus, A.E. Arnold. 2016. Isolation of endohyphal bacteria from foliar fungi and in vitro establishment of their symbiotic associations. Applied and Environmental Microbiology 82: 2943-2949.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Arnold, A.E., I. Carbone, F. Lutzoni, G. May. 2012. A multidimensional study of endophytic fungal diversity. IMA Fungus 2: 2-3.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Bascom-Slack, C., A.E. Arnold, S.A. Strobel. 2012. Student-directed discovery of the plant microbiome and its products. Science 338: 485-486.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Carbone, I.C., J.B. White, J. Miadlikowska, A.E. Arnold, M.A. Miller, F. Kauff, C. Schoch, J.M. URen, G. May, F. Lutzoni. 2016. Enhancing fungal species discovery and description using T-BAS: Tree-Based Alignment Selector toolkit for phylogenetic-based placement, alignment downloads, and metadata visualization. Bioinformatics 33: 1160-1168.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: URen, J.M. and A.E. Arnold. 2016. Diversity, taxonomic composition, and functional aspects of fungal communities in living, senescent, and fallen leaves at five sites across North America.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: URen, J.M., J. Miadlikowska, N. Zimmerman, F. Lutzoni, J. Stajich, A.E. Arnold. 2016. Contributions of North American endophytes to the phylogeny, ecology, and taxonomy of the Xylariaceae. Molecular Phylogenetics and Evolution 98: 210-232.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kithsiri Wijeratne, E.M., G.M.K.B. Gunaherath, V.M. Chapla, J. Tillotson, F. de la Cruz, M. Kang, J. URen, A.R. Araujo, A.E. Arnold, E. Chapman, and AA.L. Gunatilaka. 2016. Oxaspirol B with p97 inhibitory activity and other oxaspirols from Lecythophora sp. FL1375 and FL1031, endolichenic fungal strains inhabiting Parmotrema tinctorum and Cladonia evansii. Journal of Natural Products 79: 340-352.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chagnon, P.-L., J.M. URen, F. Lutzoni, J. Miadlikowska, and A.E. Arnold. 2016. Interaction type influences ecological network structure more than local abiotic conditions: evidence from fungal symbionts at a continental scale. Oecologia 180: 181-191.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wei, H., Y. Xu, P., Espinosa-Artiles, M.X. Liu, J.-G. Luo, J.M. URen, A.E. Arnold, and A.A.L. Gunatilaka. 2015. Sesquiterpenes and other constituents of Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum sp. Phytochemistry 118: 102-108.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Huang, Y.-L., M.M.N. Devan, J.M. URen, S.H. Furr, and A.E. Arnold. 2016. Pervasive effects of wildfire on foliar endophyte communities in montane forest trees. Microbial Ecology 71: 452-468.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Oono, R., F. Lutzoni, A.E. Arnold, L. Kaye, J.M. URen, G. May, I. Carbone. 2014. Genetic variation in horizontally transmitted symbionts of pine needles reveals population structure in cryptic species. American Journal of Botany 101: 1362-1374.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen, K.H., J. Miadlikowska, K. Molnar, A.E. Arnold, J.M. URen, E. Gaya, C. Gueidan, F. Lutzoni. 2015. Phylogenetic analyses of eurotiomycetous endophytes reveal their close affinities to Chaetothyriales, Eurotiales, and a new order  Phaeomoniellales. Molecular Phylogenetics and Evolution 85: 117-130.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: URen, J.M., J.M. Riddle, J.T. Monacell, I. Carbone, J. Miadlikowska, A.E. Arnold. 2014. Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community structure of endophytic fungi. Molecular Ecology Resources 14:1032-1048.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sandberg, D.C., L.J. Battista, A.E. Arnold. 2014. Fungal endophytes of aquatic macrophytes: diverse host-generalists characterized by tissue preferences and geographic structure. Microbial Ecology 67: 735-747.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nilsson, R.H., K.D. Hyde, J. Pawlowska, M. Ryberg, L. Tedersoo, A.B. Aas, S.A. Alias, A. Alves, C.L. Anderson, A. Antonelli, A.E. Arnold, B. Bahnmann, M. Bahram, J. Bengtsson-Palme, A. Berlin, S. Branco, P. Chomnunti, A. Dissanayake, R. Drenkhan, H. Friberg, T.G. Fr�slev, B. Halwachs, M. Hartmann, B. Henricot, R. Jayawardena, A. Jumpponen, H. Kauserud, S. Koskela, T. Kulik, K. Liimatainen, B. Lindahl, D. Lindner, J.K. Liu, S. Maharachchikumbura, D. Manamgoda, S. Martinsson, M.A. Neves, T. Niskanen, S. Nylinder, O.L. Pereira, D.B. Pinho, T.M. Porter, V. Queloz, T. Riit, M. Sanchez-Garc�a, F. de Sousa, E. Stefaczyk, M. Tadych, S. Takamatsu, Q. Tian, D. Udayanga, M. Unterseher, Z. Wang, S. Wikee, J. Yan, E. Larsson, K-H. Larsson, U. K�ljalg, K. Abarenkov. 2014. A distributed effort to improve the annotation of public ITS sequence data for plant pathogenic fungi. Fungal Diversity 67: 11-19.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Lau, M., N.C. Johnson, A.E. Arnold. 2013. Factors influencing communities of foliar fungal endophytes in riparian woody plants. Fungal Ecology 6: 365-378.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Hoffman, M.T., M. Gunatilaka, E.M.K. Wijeratne, A.A.L. Gunatilaka, A.E. Arnold. 2013. Endohyphal bacterium enhances production of indole-3-acetic acid by a foliar fungal endophyte. PLoS One 8: e73132.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Tedersoo, L., A.E. Arnold, K. Hansen. 2013. Novel aspects in the life cycle and biotrophic interactions in the Pezizomycetes. Molecular Ecology 22: 1488-1493.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: URen, J., F. Lutzoni, J. Miadlikowska, A. Laetsch, A.E. Arnold. 2012. Host and geographic structure of endophytic and endolichenic fungi at a continental scale. American Journal of Botany 99: 898-914.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Gazis, R., J. Miadlikowska, F. Lutzoni, A.E. Arnold, P. Chaverri. 2012. Culture-based study of endophytes associated with rubber trees in Peru reveals a new class of Pezizomycotina: Xylonomycetes. Molecular Phylogenetics and Evolution 65: 294-304.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Huang, Y.-L., E.A. Bowman, N.C. Massimo, N.P. Garber, J.M. URen, D.C. Sandberg, A.E. Arnold. 2018. Using collections data to infer biogeographic, environmental, and host structure in communities of endophytic fungi. Mycologia, in press.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Chen, K.-H., H.-L. Liao, A.E. Arnold, F. Lutzoni. 2018. Metatranscriptomic analysis of active fungal communities across a senescence gradient of the moss Dicranum scoparium. New Phytologist 218:1597-1611.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bowman, E.A., A.E. Arnold. 2018. Distributions of ectomycorrhizal and foliar endophytic fungal communities associated with Pinus ponderosa along a spatially constrained elevation gradient. American Journal of Botany, in press.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target audiences for this work include the scientific community, collaborators, and students at the UA and at partner institutions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the reporting period the project provided training and career development for two postdocs, four undergraduates, and two PhD students. Students and postdocs presented at national meetings and gave departmental seminars, led outreach activities, and published their work. The project provided mentored research for four high school students and two high school teachers. How have the results been disseminated to communities of interest?We have distributed results through the project website, our publications, and our invited and contributed presentations. What do you plan to do during the next reporting period to accomplish the goals?We will continue as above.

Impacts
What was accomplished under these goals? • We completed all processing and characterization of >12,000 cultures of endophytic fungi from the boreal realm and related ecosystems, including DNA sequencing, vouchering, and barcoding. This resulted in four publications in the current reporting period. • We published with collaborators a new biodiversity informatics package, TBAS, for phylogenetic placement of novel fungal strains and subsequent evolutionary analyses. • We leveraged this project to include new collaborators whose work has characterized the novel chemistry of selected strains, resulting in several new publications during the reporting period. • We partnered with colleagues to evaluate endohyphal bacteria in focal fungi and to describe their genomic architecture and phenotypic effects, resulting in three publications in the current reporting period. • We completed all next-generation/Illumina sequencing of field-collected material and have completed analyses, with a paper to be submitted to Science in summer 2017. • We leveraged this project to develop new genomic datasets for focal fungi in partnership with JGI. Seven genomes have been sequenced to date. • We shared strains with collaborating labs for population genomics studies and completed all relevant work, publications forthcoming. • We developed new collaborations with industry and with local research groups to expand the scope of this research in terms of functional traits, leading to two new NSF grants. • We hosted multiple high school students and two teachers for training opportunities in mentored research. • We engaged >100 high school students in citizen science projects during the reporting period. • My group gave >10 invited and contributed talks at national and international venues.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Shaffer, J.P., J.M. URen, D.A. Baltrus, R.E. Gallery, A.E. Arnold. An endohyphal bacterium (Chitinophaga, Bacteroidetes) influences carbon source use by Fusarium keratoplasticum (F. solani species complex, Nectriaceae). Frontiers in Microbiology, accepted pending revision.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Torres-Cruz!, T.J., T.L.B. Tobias, M. Almatruk, C. Hesse, C.R. Kuske, A. Desiro, G.M.N. Benucci, G. Bonito, J. Stajich, C. Dunlap, A.E. Arnold, A. Porras-Alfaro. Bifiguratus adelaidae, gen. nov. sp. nov., a new lineage of Mucoromycotina with endophytic and soil-dwelling strains. Mycologia, accepted pending revision.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Luo, J.-G.., Y.-M. Xu, D.C. Sandberg, A.E. Arnold, A.A.L. Gunatilaka. Montagnuphilones A-G, azaphilones from Montagnulaceae sp. DM0194, a fungal endophyte of submerged roots of Persicaria amphibia. Journal of Natural Products, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Baltrus, D.A., K. Dougherty, K.R. Arendt, M. Huntemann, A. Clum, M. Pillay, K. Palaniappan, N. Varghese, N. Mikhailova, D. Stamatis, T.B.K. Reddy, C.Y. Ngan, C. Daum, N. Shapiro, V. Markowitz, N. Ivanova, N. Kyrpides, T. Woyke, A.E. Arnold. 2016. Absence of genome reduction in diverse, facultative endohyphal bacteria. Microbial Genomics, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Bashyal, B.P., E.M. Kithsiri Wijeratne, J. Tillotson, A.E. Arnold, E. Chapman, A.A.L. Gunatilaka. 2016. Chlorinated dehydrocurvularins and alterperylenepoxide A from Alternaria sp. AST0039, a fungal endophyte of Astragalus lentiginosus. Journal of Natural Products, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Carbone, I.C., J.B. White, J. Miadlikowska, A.E. Arnold, M.A. Miller, F. Kauff, C. Schoch, J.M. URen, G. May, F. Lutzoni. 2016. Enhancing fungal species discovery and description using T-BAS: Tree-Based Alignment Selector toolkit for phylogenetic-based placement, alignment downloads, and metadata visualization. Bioinformatics, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: URen, J.M. and A.E. Arnold. 2016. Diversity, taxonomic composition, and functional aspects of fungal communities in living, senescent, and fallen leaves at five sites across North America. PeerJ, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Shaffer, J.S., C. Sarmiento, P.-C. Zalamea, R.E. Gallery, A.S. Davis, D.A. Baltrus, A.E. Arnold. 2016. Diversity, specificity, and phylogenetic relationships of endohyphal bacteria in fungi that inhabit tropical seeds and leaves. Frontiers in Ecology and Evolution, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Del Olmo-Ruiz, M. and A.E. Arnold. 2016. Community structure of fern-affiliated endophytes in three neotropical forests. Journal of Tropical Ecology, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Arendt, K.R., K.L. Hockett, S.J. Araldi-Brondolo, D.A. Baltrus, A.E. Arnold. 2016. Isolation of endohyphal bacteria from foliar fungi and in vitro establishment of their symbiotic associations. Applied and Environmental Microbiology, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: URen, J.M., J. Miadlikowska, N. Zimmerman, F. Lutzoni, J. Stajich, A.E. Arnold. 2016. Contributions of North American endophytes to the phylogeny, ecology, and taxonomy of the Xylariaceae. Molecular Phylogenetics and Evolution 98: 210-232.
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Chen, K.-H., H.-L. Liao, A.E. Arnold, F. Lutzoni. Metatranscriptomic analysis of active fungal communities across a senescence gradient of the moss Dicranum scoparium. The ISME Journal.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Gubiani, J., K. Wijeratne, T. Shi, A. Araujo, A.E. Arnold, E. Chapman, A.A.L. Gunatilaka. An epigenetic modifier induces production of (10S)-verruculide B, an inhibitor of protein tyrosine phosphatases by Phoma sp. nov. LG219, a fungal endophyte of Parkinsonia microphylla.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Bowman EA, Arnold AE. Fungal symbionts of Pinus ponderosa across a spatially constrained gradient of climate. The American Journal of Botany.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Van Bael, S.A., Estrada C., and Arnold, A.E. In press. Foliar endophyte communities and leaf traits in tropical trees. The Fungal Community: its organization and role in the ecosystem (J.F. White, Jr., J. Dighton, & P. Oudemans, eds). 4th Edition. Marcel-Dekker

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience for this work includes colleagues, students, members of the public, stakeholders, and collaborators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training for a postdoc in biodiversity informatics, and for another postdoc in a new project regarding transcriptomics. The project provided training in statistics and microbiology for two graduate students and in microbial sciences for four undergraduates, three high school students, and two high school teachers. All project participants have gained teaching and outreach experience. All project participants have gained experience by presenting the project in various venues. How have the results been disseminated to communities of interest?Fifteen publications were published or are in press in peer-reviewed journals. One book chapter is in review. Arnold authored or co-authored 12 scholarly, invited talks and 7 scholarly, contributed oral- and poster presentations in national and international venues. A project website has been updated with new content. Data were shared in public databases. Extensive outreach provided interactions in science with hundreds of members of the public and dozens of high school students. What do you plan to do during the next reporting period to accomplish the goals?We will continue these activities as described above.

Impacts
What was accomplished under these goals? We concluded all sequencing and vouchering of a total of 16,000 boreal endophytes and nearly 6000 endophytes and seed-associated fungi from other biomes. These are now permanently vouchered at the UA, ready for further work. We concluded all next-generation sequencing of samples representing 20 host species in each of 8 boreal sites, and an additional set of species over 1700 km transects in Quebec. We paired these with next-generation sequencing of diverse plants in temperate biomes for comparison. We concluded tool development for placing sequences bioinformatically in a phylogenetic context. We also concluded tool development for our next-gen sequencing pipeline. We formalized database/metadata input for field surveys. We initiated novel experiments across southern Arizona to evaluate climate effects on both endophytes and complementary rhizosphere communities. We initiated novel experiments across southern Arizona to explore the seed-to-seedling transition in terms of endophyte communities. We engaged teachers and high school students in summer research activities, reaching more than 30 students over a two-week, intensive workshop led and developed by Arnold, and a one-week field experience in which Arnold developed and led a two-day field workshop. We participated in AgDiscovery, a USDA program designed to engage high school students in agricultural sciences. We engaged hundreds of members of the public through outreach regarding fungal diversity (City of Knowledge, Plant Sciences Family Night). We published papers and gave professional presentations at diverse regional, national, and international venues. We submitted and garnered new grants. We shared data and cultures with cooperating partners.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: URen, J.M., J. Miadlikowska, N. Zimmerman, F. Lutzoni, J. Stajich, A.E. Arnold. 2016. Contributions of North American endophytes to the phylogeny, ecology, and taxonomy of the Xylariaceae. Accepted pending minor revision, Molecular Phylogenetics and Evolution.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Sousa, J., M. Aguilar-Perez, A.E. Arnold, N. Rios, P.D. Coley, T.A. Kursar, and L. Cubilla-Rios. 2015. Chemical constituents and their antibacterial activity from the tropical endophytic fungus Diaporthe sp. F2934. Accepted pending minor revision, Journal of Applied Microbiology.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Kithsiri Wijeratne, E.M., G.M.K.B. Gunaherath, V.M. Chapla, J. Tillotson, F. de la Cruz, M. Kang, J. URen, A.R. Araujo, A.E. Arnold, E. Chapman, and AA.L. Gunatilaka. 2015. Oxaspirol B with p97 inhibitory activity and other oxaspirols from Lecythophora sp. FL1375 and FL1031, endolichenic fungal strains inhabiting Parmotrema tinctorum and Cladonia evansii. Journal of Natural Products, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Xu, Y., B.P. Bashyal, M.X. Liu, P. Espinosa-Artiles, J.M. URen, A.E. Arnold, and A.A.L. Gunatilaka. 2015. Cytotoxic cytochalasins and other metabolites from Xylariaceae sp. FL0390, a fungal endophyte of Spanish moss. Natural Products Communications, in press.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Xu, Y., J. Mafezoli, M.C.F. Oliveira, J.M. URen, A.E. Arnold, and A.A.L. Gunatilaka. 2015. Anteaglonialides AF, Spironaphtho-1,8-dioxincyclohexa-butyrolactones and Palmarumycins CE1CE3 from Anteaglonium sp. FL0768, a fungal endophyte of sand spikemoss, Selaginella arenicola. Journal of Natural Products, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Ohkura, M., J.J. Worley, J.E. Hughes-Hallett, J.S. Fisher, B.C. Love, A.E. Arnold, and M. J. Orbach. 2015. Ophidiomyces ophiodiicola on a captive black racer (Coluber constrictor) and a garter snake (Thamnophis sirtalis) in Pennsylvania. Journal of Zoo and Wildlife Medicine, in press.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chagnon, P.-L., J.M. URen, F. Lutzoni, J. Miadlikowska, and A.E. Arnold. Interaction type influences ecological network structure more than local abiotic conditions: evidence from fungal symbionts at a continental scale. Oecologia, in press.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Huang, Y.-L., M.M.N. Devan, J.M. URen, S.H. Furr, and A.E. Arnold. 2015. Pervasive effects of wildfire on foliar endophyte communities in montane forest trees. Microbial Ecology, in press.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wei, H., Y. Xu, P., Espinosa-Artiles, M.X. Liu, J.-G. Luo, J.M. URen, A.E. Arnold, and A.A.L. Gunatilaka. Sesquiterpenes and other constituents of Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum sp. Phytochemistry, in press.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wijeratne, E.M.K., Y. Xu, A.E. Arnold, A.A.L. Gunatilaka. 2015. Pulvinulin A, graminin C, and cis-gregatin B--new natural furanones from Pulvinula sp. 11120, a fungal endophyte of Cupressus arizonica. Natural Product Communications 10: 107-111.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Martinson, E., J. Hackett, C. Machado, A.E. Arnold. 2015. Metatranscriptome analysis of fig flowers suggests mechanisms for mutualism stability and gall induction. PloS ONE 10: e0130745.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Zalamea, P.C., C. Sarmiento, A.E. Arnold, A. Davis, J. Dalling. 2015. Do microbes and abrasion by soil particles influence seed persistence and loss of physical dormancy in tropical seedbanks? Frontiers in Plant Science doi: 10.3389/fpls.2014.00799.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Corrales, A.O., A.E. Arnold, A. Ferrer, B. Turner, J.W. Dalling. 2015. Variation in ectomycorrhizal communities associated with Oreomunnea mexicana (Juglandaceae) in tropical montane forests. Mycorrhiza DOI 10.1007/s00572-015-0641-8.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Massimo, N., M.M.N. Devan, K.R. Arendt, M. Wilch, J.M. Riddle, S.H. Furr, C. Steen, J.M. URen, D.C. Sandberg, A.E. Arnold. 2015. Fungal endophytes of desert plants: infrequent in culture, but diverse and distinctive symbionts. Microbial Ecology DOI 10.1007/s00248-014-0563-6.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen, K.H., J. Miadlikowska, K. Molnar, A.E. Arnold, J.M. URen, E. Gaya, C. Gueidan, F. Lutzoni. 2015. Phylogenetic analyses of eurotiomycetous endophytes reveal their close affinities to Chaetothyriales, Eurotiales, and a new order  Phaeomoniellales. Molecular Phylogenetics and Evolution 85: 117-130.
• Type: Book Chapters Status: Under Review Year Published: 2016 Citation: Van Bael, S.A., Estrada C., and Arnold, A.E. In press. Foliar endophyte communities and leaf traits in tropical trees. The Fungal Community: its organization and role in the ecosystem (J.F. White, Jr., J. Dighton, & P. Oudemans, eds). 4th Edition. Marcel-Dekker, pp. XX-XX.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training opportunities in the Arnold lab for two postdocs (one female), six graduate students (all female, one African-American and one veteran), 16 undergraduates at the UA (five female; three Hispanic; one African, en route to US citizenship; one first-generation college student), three visiting students (one Afro-Brazilian, one Mexican; one female; one from US liberal arts college), two K-12 teachers at a Hispanic-serving high school, seven high school researchers (six female, three Hispanic), and >150 high school students at Hispanic-serving high schools. How have the results been disseminated to communities of interest? Dissemination of results in 2014 was achieved through: • One MS thesis by a female student (Arendt) • Three undergraduate honors theses (two by female undergraduates; one published with the female undergraduate as 2nd author (Battista), two ongoing: Ramos, Rosenfeld) • Projects by high school students in local and regional science fairs • Three public outreach talks, two eight-week high school workshops, Herbarium tours, multiple outreach events, together reaching hundreds of members of the public • Enhancement of curriculum in graduate and undergraduate courses at UA • Creation and teaching of a new course (Plant Microbiology) at UA • Website enhancements, Arnold laboratory website (arnoldlab.net) and EnDoBiodiversity.org, to highlight project activities • Reports to partner agencies, including Parcs Canada and the Huron Mountain Wildlife Foundation • Five intramural presentations by DOB visitors (Toledo, Ramos), K-12 teacher (Lehr) • Intramural presentation by PhD student based on rotation project (Smith; African-American) • Four presentations (2 invited, *) at international meetings (Arnold*; Arnold*; Chen; Nilsson) • Eight presentations (5 invited, *) at national meetings or in seminars (Arnold*, Arnold*, Arnold*, U'Ren*, Zimmerman*, Arendt, Arendt, Baltrus) • Peer-reviewed papers (above) • Three manuscripts to be submitted in the coming month (Arendt, Arendt, Chagnon) • At least five additional manuscripts to be completed and submitted in the coming year What do you plan to do during the next reporting period to accomplish the goals? The coming year will be especially dedicated to data analysis, presentations of results at national and international meetings, and preparation of publications, coupled with extension of our high school workshops, continued engagement with the public, and continued mentorship of diverse junior scientists. We have exceeded our proposed project goals and are on track for timely completion of major project initiatives.

Impacts
What was accomplished under these goals? 1. Completed processing of all endophytic and endolichenic fungi obtained from seven regions in the circumboreal belt. We have vouchered and sequenced 12,189 strains, far exceeding our project goal. This is the largest known collection of living boreal fungi. It is publicly available through the RLG Mycological Herbarium. This also represents the largest known collection of cultures and sequence data for cultivable endophytes in a single biome. 2. Completed DNA extractions for all host samples from seven boreal forests. We have extracted high-quality DNA from >3000 tissue collections representing ≥ 20 host species in each site. Illumina sequencing will be done in Jan. 2015 in collaboration with Argonne National Lab, yielding the largest sample of lichen- and plant DNA from boreal forests to date. 3. Completed preliminary work to establish endophyte infections in model hosts. This work leveraged an external three-year fellowship for postdoc Zimmerman. He completed preliminary surveys for focal endophytes and initiated inoculation trials for transcriptomics. 4. Completed phylogenetic and functional trait analyses for (1) focal endophyte clades and (2) model endophyte/endohyphal bacterial associations, generating a novel data set regarding substrate use by endophytic and saprotrophic fungi (in prep.) and a MS thesis. 5. Presented results at international meetings (2 invited [*], 4 total). *International Mycological Congress (IMC10). Arnold AE, J U'Ren, VL Wong, K Arendt‡, K-H Chen‡, R Oono, J Miadlikowska, I Carbone, D Baltrus, G May, F Lutzoni. What can >50,000 cultures tell us about the ecological specificity of endophytes and related fungi? *International Cooperative Biodiversity Group. Arnold AE. Using epigenetic modifiers and new substrates to enhance the pace and impact of ecologically-guided discovery of bioactive metabolites from fungi. IMC10. Chen‡ K, J Miadlikowska, K Molnar, AE Arnold, J U'Ren, E Gaya, F Lutzoni. Phylogenetic relationships of endophytic and endolichenic fungi reveal a new order within the class Eurotiomycetes. (Award, best student poster, systematics) IMC10. Nilsson RH, ten others, A.E. Arnold, and 46 others. Improving ITS sequence data for identification of plant-pathogenic fungi. (Poster) 6. Presented results at national meetings and in invited seminars (5 invited [*], 8 total). *Univ. Arizona, Plant Sciences. Arnold AE. Fungal endophytes from the tropics to the tundra: clues to the evolution of plant-fungal symbioses. *Univ. Virginia, Biology. Arnold AE. Fungal endophytes from the tropics to the tundra: clues to the evolution of plant-fungal associations. *Tulane Univ., Evolution/Ecology. Arnold AE. Fungal endophytes from the tropics to the tundra: clues to the evolution of plant-fungal associations. *Northern Arizona Univ., Biology. U'Ren JM. Geographic and temporal variation in boreal endophytic and endolichenic communities. *Univ. Arizona, Plant Sciences. Zimmerman NB. From leaves to landscapes: diversity of endophytes across environmental gradients. Univ. Arizona, Plant Sciences. Arendt K. Effects of endohyphal bacteria on fungal phenotypes. Ecological Society of America. Arendt‡ K, D Baltrus, AE Arnold. Bacterial symbionts of endophytic fungi mediate functional shifts in plant-fungal interactions. (Poster) Society for the Study of Evolution. Baltrus D, K Arendt‡, AE Arnold. Facultative endohyphal bacterial symbionts alter phenotypes of fungal endophyte hosts. (Oral) 7. Published or submitted results for publication (7 papers in revision or published). ‡, Graduate student; #, undergraduate; °, K-12 teacher; bold = Arnold lab. Chen‡ KH, J Miadlikowska, K Molnar, AE Arnold, JM U'Ren, E Gaya, C Gueidan, F Lutzoni. Phylogenetic analyses of eurotiomycetous endophytes reveal their close affinities to Chaetothyriales, Eurotiales, and a new order - Phaeomoniellales. Molecular Phylogenetics and Evolution, in revision. Lutzoni F, M Nowak, M Alfaro, J Miadlikowska, AE Arnold, D Hibbett, K Hilu, T James, D Quandt, S Magallon. Synchronized radiations in plants and fungi linked to symbiosis. Nature Communications, in revision. Massimo# N, M Devan#, K Arendt‡, M Wilch°, J Riddle#, S Furr°, C. Steen#, JM U'Ren, DC Sandberg‡, AE Arnold. 2014. Fungal endophytes of desert plants: infrequent in culture, but diverse and distinctive symbionts. Microbial Ecology, accepted pending revision. Nilsson RH, ten others, AE Arnold, and 46 others. 2014. A distributed effort to improve the annotation of public ITS sequence data for plant pathogenic fungi. Fungal Diversity 67: 11-19. Oono R, F Lutzoni, AE Arnold, L Kaye#, JM U'Ren, G May, I Carbone. 2014. Genetic variation in horizontally transmitted symbionts of pine needles reveals population structure in cryptic species. American Journal of Botany 101: 1362-1374. Sandberg‡ DC, LJ Battista#, AE Arnold. 2014. Fungal endophytes of aquatic macrophytes: diverse host-generalists characterized by tissue preferences and geographic structure. Microbial Ecology 67: 735-747. U'Ren JM, J Riddle#, JT Monacell‡, I Carbone, J Miadlikowska, AE Arnold. 2014. Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community structure of endophytic fungi. Molecular Ecology Resources14:1032-1048. 8. Developed three papers for submission in Dec. 2014: Arendt‡ K, D Baltrus, ?AE Arnold. In prep. Isolation of endohyphal bacteria from foliar fungi and in vitro re-establishment of their symbiotic associations. Applied and Environmental Microbiology. Arendt‡ K, D Baltrus, ?AE Arnold. In prep. Horizontally acquired endohyphal bacteria influence enzyme activity and plant decomposition by foliar fungi. Applied and Environmental Microbiology. Chagnon‡, P, JM U'Ren, F Lutzoni, J Miadlikowska, AE Arnold. In prep. Interaction type influences ecological network structure more than local abiotic conditions. Oikos. 9. Mentored three visiting DOB students in endophyte ecology: 2 male, 1 female; 1 Mexican, 1 Afro-Brazilian; 1 US from liberal arts college; >20 weeks total; resulted in four intramural presentations and two honors theses. 10. Hosted two eight-week-long high school workshops, serving 150 students from two Hispanic-serving high schools (Tucson High Magnet School and Sahuarita High School). Students developed research questions and assisted with data collection and analysis. 11. Mentored seven high school researchers (6 female, 3 Hispanic); all from spring 2014 won awards at local/regional science fairs. 12. Mentored K-12 teacher (male), Sahuarita High School; will develop MS thesis in General Biology program based on research project. 13. Mentored students and postdocs: 10 additional undergrads (5 female, 3 Hispanic; one honors thesis); one PhD student in a research rotation (female, African-American); two MS students (female; one veteran); 1 PhD student (female); two postdocs (one female; one externally funded). 14. Participated in additional public/academic outreach through the Tucson Biotech Pipeline; Plant Sciences Family Night (>300 members of the public); outreach talks (>100 members of the public); and tours of the UA Herbarium (>200 students and members of the public). 15. Developed new collaborations based on findings from this study, including an industrial partnership that should begin in January and two new grants: one from JGI (endophytic metatranscriptomics in a model plant/endophyte system) and one from NSF (focusing on transcriptomics of endohyphal bacteria/endophyte symbioses).

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nilsson, R.H., K.D. Hyde, J. Pawlowska, M. Ryberg, L. Tedersoo, A.B. Aas, S.A. Alias, A. Alves, C.L. Anderson, A. Antonelli, A.E. Arnold, B. Bahnmann, M. Bahram, J. Bengtsson-Palme, A. Berlin, S. Branco, P. Chomnunti, A. Dissanayake, R. Drenkhan, H. Friberg, T.G. Fr�slev, B. Halwachs, M. Hartmann, B. Henricot, R. Jayawardena, A. Jumpponen, H. Kauserud, S. Koskela, T. Kulik, K. Liimatainen, B. Lindahl, D. Lindner, J.K. Liu, S. Maharachchikumbura, D. Manamgoda, S. Martinsson, M.A. Neves, T. Niskanen, S. Nylinder, O.L. Pereira, D.B. Pinho, T.M. Porter, V. Queloz, T. Riit, M. Sanchez-Garc�a, F. de Sousa, E. Stefaczyk, M. Tadych, S. Takamatsu, Q. Tian, D. Udayanga, M. Unterseher, Z. Wang, S. Wikee, J. Yan, E. Larsson, K-H. Larsson, U. K�ljalg, K. Abarenkov. 2014. A distributed effort to improve the annotation of public ITS sequence data for plant pathogenic fungi. Fungal Diversity 67: 11-19.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Miadlikowska, J.M., F. Kauff, F. Hognabba, J.C. Oliver, K. Molnar, E. Fraker, E. Gaya, J. Hafellner, V. Hofstetter, C. Gueidan, M. Kukwa, M. L�cking, C. Bj�rk, H.J. M. Sipman, A.R. Burgaz, A. Thell, A. Passo, L. Myllys, T. Goward, S. Fernandez-Brime, G. Hestmark, J. Lendemer, H.T. Lumbsch, M. Schmull, C. Schoch, E. Serusiaux, D.R. MAddison, A.E. Arnold, F. Lutzoni, S. Stenroos. 2014. Multigene phylogenetic analysis for 1307 fungi representing 1139 infrageneric taxa, 312 genera, and 66 families of the class Lecanoromycetes (Ascomycota). Molecular Phylogenetics and Evolution 79: 132-168 (Editors Choice)
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Oono, R., F. Lutzoni, A.E. Arnold, L. Kaye, J.M. URen, G. May, I. Carbone. 2014. Genetic variation in horizontally transmitted symbionts of pine needles reveals population structure in cryptic species. American Journal of Botany 101: 1362-1374.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sandberg, D.C., L.J. Battista, A.E. Arnold. 2014. Fungal endophytes of aquatic macrophytes: diverse host-generalists characterized by tissue preferences and geographic structure. Microbial Ecology 67: 735-747.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Higginbotham, S.J., W.R. Wong, R.G. Linington, C. Spadafora, L. Iturrado, A.E. Arnold. 2014. Sloth fur as a novel source of fungi with potent anti-parasitic and anti-bacterial activity. PLoS One 9: e84549
• Type: Journal Articles Status: Published Year Published: 2014 Citation: URen, J.M., J.M. Riddle, J.T. Monacell, I. Carbone, J. Miadlikowska, A.E. Arnold. 2014. Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community structure of endophytic fungi. Molecular Ecology Resources 14:1032-1048
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Luo, J.O., X.B. Wang, Y.M. Zu, J.M. URen, A.E. Arnold, L.Y. Kong, A.A.L. 2014. Gunatilaka. Delitschiapyrone A, a pyrone-naphthalenone adduct bearing an unprecedented pentacyclic ring system from the leaf-associated fungus Delitschia sp. FL1581. Organic Letters 16: 5944-5947.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Chen, K.H., J. Miadlikowska, K. Molnar, A.E. Arnold, J.M. URen, E. Gaya, C. Gueidan, F. Lutzoni. 2014. Phylogenetic analyses of eurotiomycetous endophytes reveal their close affinities to Chaetothyriales, Eurotiales, and a new order  Phaeomoniellales. Molecular Phylogenetics and Evolution, in press.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target audiences include the public, through outreach activities; high school students, through training workshops; undergraduate students, through representation of project material in courses; graduate students, through mentorship and course presentatins; postdocs, through mentorship in projects related to this award; and colleagues/interdisciplinary scientists, through project websites, presentations, and publications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? During 2013, I mentored six graduate students (major advisor or co-advisor), four postdocs, 14 undergraduates, and six high school researchers, including members of STEM groups. I hosted several visiting graduate students, mentored Panamanian researchers in the field in Panama, and introduced ca. 210 high school students (primarily Latino) to science through two large-scale workshops. These activities are reflected in several tangible outcomes: student seminar presentations in SPLS not listed above (e.g., Shaffer, Sandberg); numerous high school research awards in local and regional science fairs; one MS thesis defense (Sandberg); and student awards (Arendt, Best Poster Presentation at 2013 Mycological Society Meetings; Shaffer, Graduate and Professional Student Council Travel Award, Mentor Travel Award from the Mycological Society of America; Sandberg, Mentor Travel Award from MSA, etc.). I oversaw student presentations at multiple national meetings, including by undergraduate- and graduate-level researchers. I am delighted to have engaged dozens of members of the public in my work through outreach presentations to potential donors (1885 Society), local businesspeople (Rotary), and members of the public and UA community interested in the Robert L. Gilbertson Mycological Herbarium. How have the results been disseminated to communities of interest? In addition to the outreach and mentorship listed above, I led or co-authored the following presentations (name of first author listed if not Arnold): A. Invited presentations (5 total): I highlighted the project in invited talks at Cornell University, the University of Georgia, Michigan State University, Northern Arizona University, and at a symposium at the British Mycological Society meetings (all Arnold, first and sole author). B. Contributed presentations (13 total): I co-authored 13 contributed presentations: American Society of Microbiologists (Shaffer), Mycological Society of America (Arendt, Chen, Corrales, Garcia, Huang, Sandberg, Shaffer, Wong, U'Ren), Fungal Genetics Conference (Oono), and the Soc. for the Study of Evolution (Chen, Oono). C. Other scholarly presentations by project personnel (intramural and regional venues; 18 total) 1. Plant Sciences seminar series, UA (two seminars: Shaffer, Sandberg) 2. Dimensions of Biodiversity annual meeting, Tucson (four: Arendt, Shaffer, U’Ren; workshop by U’Ren) 3. School of Plant Sciences Research Retreat, Tucson (three: Arendt, Shaffer, Sandberg) 4. Summer Research Institute, UA: two oral presentations (Garza, Gonzales) 5. Summer Research Institute, UA: two poster presentations (Garza, Gonzales) 6. Ecology and Evolutionary Biology Undergraduate Poster Session, UA (Battista) 7. Vision to your Future outreach event, poster session, UA (Ramos) 8. Presentations by high school researchers at local and regional events (three) What do you plan to do during the next reporting period to accomplish the goals? Continue present levels of activity

Impacts
What was accomplished under these goals? Research achievements are shown below. Completed fieldwork in eastern Russia and northern Alberta. Obtained supplemental funding to expand project; completed fieldwork at Huron Mountains, northern Michigan, USA. Obtained supplemental funding to support project-REU student, Julian Gonzales III, from UT Pan American. Led processing of 23,040 tissue segments to isolate endophytic and endolichenic fungi from those collections, and 46,080 segments for high-throughput sequencing. The original proposal focused only on collections for pyrosequencing at most of our sites. However, we recognized the potential importance of a circumboreal culturing library, and therefore processed samples for both pyrosequencing and fungal isolations in culture. Led voucher preparation and sequencing of ITSrDNA/partial LSUrDNA for 5487 new isolates of boreal endophytic and endolichenic fungi, including the last strains to emerge from cultures collected in Quebec and Alaska in 2011, and strains collected in Russia and Sweden in 2012. Developed new methods for plant inoculations and functional trait analysis with boreal endophytic fungi. Using experimental arrays of Juniperus and related plants, I developed a new method for successfully inoculating living tissue in planta with mycelial fragments. Oversaw development of new methods for evaluating substrate use in boreal endophytes via BioLog plates. Oversaw methods enhancement for DNA extraction prior to large-scale processing of tissue samples for pyrosequencing. Oversaw development of new methods for inoculation of boreal endophytic fungi with endohyphal bacteria. Oversaw new phylogenetic analyses and sampling designed to pinpoint evolutionary origins and diversity of endohyphal bacteria. Completed analyses of trial 454 pyrosequencing dataset; submitted paper for publication. Oversaw contributions of conceptual/analytical components for biodiversity informatics pipeline development. Completed data analysis for ‘citizen science’ project based on data gathered by 135 high school students in semester-long workshop. Completed transfer of bacterial cultures to collaborating lab for genome sequencing by JGI, achieved through supplemental support. Recruited new PhD student to project. Recruited new postdoctoral fellow to project. Recruited new high school teacher to project. Coordinated session of selected speakers for 2013 Fungal Genetics Conference, with Christopher Schadt. Participated in the dissertation committee activities of two project students at other institutions, Ko-Hsuan Chen (Duke) and Julien Ponchart, visiting project scholar (Universite Laval). Completed collaboration with computer science student at UA to develop in-house database for rapid downloading and organizing of sequence data. Hosted project team in Tucson, Arizona for a two-day meeting, including presentations, workshops, research planning, and research updates: February 2013. The meeting included 21 participants, including 12 from Duke, NCSU, and UMinn. Coordinated quarterly meetings for collaborating PIs by Skype and in person, including meeting at the 2013 Mycological Society of America conference.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Higginbotham, S.J., W.R. Wong, R.G. Linington, C. Spaafora, L. Iturrado, A.E. Arnold. Sloth fur as a novel source of fungi with potent anti-parasitic and anti-bacterial activity. PLoS One, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Sandberg, D.C., L.J. Battista, A.E. Arnold. Host affiliations and geographic distributions of fungal endophytes inhabiting aquatic plants in northern Arizona. Microbial Ecology, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Del Olmo* Ruiz, M. A.E. Arnold. 2013. Interannual variation and host affiliations of endophytic fungi associated with ferns at La Selva, Costa Rica. Mycologia, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Higginbotham, S.J., A.E. Arnold, A. Iba�ez, C. Spadafora, P.D. Coley, T.A. Kursar. Bioactivity of fungal endophytes as a function of their taxonomy and the taxonomy and distribution of host plants. PLoS One, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Lau, M., N. Johnson, A.E. Arnold. Fungal endophytes in riparian areas of Arizona illustrate effects of microenvironment on symbiont assemblages. Fungal Ecology, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Martinson, E.O., K.C. Jander, Y.Q. Peng, H.H. Chen, C.A. Machado, A.E. Arnold, E.A. Herre. 2013. Relative investment in egg loads and poison sacs in fig wasps: implications for physiological mechanisms underlying seed and wasp production in figs. Acta Oecologica, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Cubilla-Rios, L., P. Coley, T. Kursar, W. Gerwick, A.E. Arnold, and four others. ycoleptodiscin A and B, cytotoxic alkaloids from the endophytic fungus Mycoleptodiscus sp. F0194. Journal of Natural Products, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Wanigesekara, A., E.M. Kithsiri Wijeratne, A.E. Arnold, and A.A. Leslie Gunatilaka. 10-Deoxy-10 alpha-hydroxyascochlorin, a new cell migration inhibitor and other metabolites from Acremonium sp., a fungal endophyte in Ephedra trifurca. Natural Products Communications, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Tedersoo, L., A.E. Arnold, K. Hansen. 2013. Novel aspects in the life cycle and biotrophic interactions in the Pezizomycetes. Molecular Ecology, in press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Hoffman, M.T., M. Gunatilaka, E.M.K. Wijeratne, A.A.L. Gunatilaka, and A.E. Arnold. 2013. IAA production by endophytic Pestalotiopsis is enhanced by an endohyphal bacterium. PLoS One, in press.
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Miadlikowska, J.M., 26 others, A.E. Arnold, F. Lutzoni, S. Stenroos. Multigene phylogenetic analysis for 1307 fungi representing 1139 infrageneric taxa, 312 genera, and 66 families of the class Lecanoromycetes (Ascomycota). Molecular Phylogenetics and Evolution.
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Oono, R., Lutzoni, F., Arnold, A., Kaye, L., U'Ren, J., May, G., & Carbone, I. Genetic variation in horizontally transmitted symbionts of pine needles reveals population structure in cryptic species. The American Naturalist, in review
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Ohkura, M., Worley, J., Hughes-Hallett, J., Fisher, J., Love, B., Arnold, A., & Orbach, M. Characterization and classification of Undulatus ophiodiicola, formerly described as Chrysosporium ophiodiicola, an emerging fungal pathogen of snakes. Mycologia, in revision
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: U'Ren, J., Riddle, J., Monacell, J., Carbone, I., Miadlikowska, J., & Arnold, A.(2014). Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi. Molecular Ecology Resources, in press
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Higgins, K., Arnold, A., Coley, P., & Kursar, T. 2014. Communities of fungal endophytes in tropical forest grasses: highly diverse host- and habitat generalists characterized by strong spatial structure. Fungal Ecology(8) 1-11
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Almeida, C., Ortega, H., Higginbotham, S., Spadafora, C., Arnold, A., Coley, P., Kursar, T., Gerwick, W., & Cubilla-Rios, L. Chemical and bioactive natural products from Microthyriaceae sp., an endophytic fungus from a tropical grass. Letters in Applied Microbiology, in press
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Martinson, E., Hackett, J., Machado, C., & Arnold, A. Metatranscriptome analysis of fig flowers suggests mechanisms for mutualism stability and gall induction. Molecular Ecology, in review
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Xu, Y., Espinosa-Artiles, P., Liu, M., Arnold, A., & Gunatilaka, A.(2013). Secoemestrin D, a cytotoxic epitetrathiodioxopiperizine, and Emericellenes A-E, five sesterterpenoids from Emericella sp AST0036, a fungal endophyte of Astragalus lentiginosus. Journal of Natural Products, 76 2330-2336

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: (A) Completed the projected sampling of 20 species of lichens and plants at our third and fourth core continental sampling sites: Krasnoyarsk, Russia; Mora, Sweden. (B) Processed 11,520 tissue segments to isolate endophytic and endolichenic fungi from those collections, and 23,040 segments for high-throughput, culture-free analysis. (C) Completed fungal isolations, prepared vouchers, and sequenced ITSrDNA and partial LSUrDNA for 5427 isolates of boreal endophytic and endolichenic fungi: 1494 isolates collected in year 1 in Quebec; 2089 isolates collected in year 1 in Alaska; 1125 isolates collected in year 2 in Russia; 719 isolates collected in year 2 in Sweden. (D) Completed analyses of 454 pyrosequencing dataset for publication. (E) Enhanced techniques for screening samples for endohyphal bacteria, and evaluated their phylogenetic relationships. (F) Completed sampling and data analysis in two complementary projects: Endophyte diversity and community structure in iconic plants of the Sonoran Desert, with >550 members of the public and high school students. Fungal diversity and community structure on cactus spines and tree thorns along urban-to-wildland gradients, with three high school students. (G) Established new collaboration to sequence genomes of endohyphal bacteria, with support from the Joint Genome Institute. (H) Hosted four visiting scholars for 10-12 weeks each: three Latina/Latino undergraduates; one European PhD student. (I) Completed data entry for all field notes from Quebec, a total of 280 pages of written notes, and released these data to our online portal at EnDoBiodiversity.org. (J) Accessioned lichens from field surveys in Quebec and Alaska in the Robert L. Gilbertson Mycological Herbarium following identification by Miadlikowska, Duke. (K) Worked with Carbone lab (NCSU) to contribute to biodiversity informatics pipeline. (L) Contributed specimens of focal genus to May lab (UMinn) for genome sequencing; shared sequence data and protocols; and contributed to data analysis. (M) Contributed DNA of >200 focal fungi, and sequence data for culturing in Quebec, to Lutzoni lab (Duke). (N) Co-hosted a symposium at the 2012 International Symbiosis Congress with primary host/coordinator. (O) Co-developed a session of selected speakers for the upcoming 2013 Fungal Genetics Conference with Christopher Schadt. (P) Joined the dissertation committees of two DoB students at other institutions. (Q) Collaborated with computer science student at UA to develop in-house database for rapid downloading and organizing of sequence data. (R) Developed new collaboration with postdoc candidate, Naupaka Zimmerman, and submitted NSF Plant Genomes Fellowship proposal. (S) Submitted collaborative proposals with high school teacher Margaret Wilch to support semester-long DoB workshops at Tucson High Magnet School for 2012 and 2013. (T) Hosted four quarterly PI meetings by Skype. PARTICIPANTS: A. Elizabeth Arnold Jana M. URen Kayla Arendt Julien Ponchart Dustin C. Sandberg Justin Shaffer Nicholas Massimo Cole Steen Lorna Battista Edelio Bazan Peter Cerda MM Nandi Devan James deVore Lauren Dominick Chan Jung Thaddeus Metz Christie Moss Brittany Pena Ethan Posey Adrian Ramirez Jakob M. Riddle Thao Truong Alexia Avey Wes MacDonald Ochana Otto Amber Ross Susan Furr Margaret Wilch TARGET AUDIENCES: Students, stakeholders, the public, colleagues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This award provided the first-ever evaluation of biological richness of endophytic symbionts in boreal forest. It supported symposia, high school workshops, outreach, applications for supplemental funds, and training of one postdoc, four graduate students, five post-baccalaureate technicians, four high school students, two teachers. A total of 14 presentations were given at regional, national, or international venues (8 invited). An additional 11 presentations were given at intramural venues. High school students further presented their work in regional, national, and international competitions. The award resulted in 11 publications in 2012.

Publications

• Bascom-Slack, C., A.E. Arnold, S.A. Strobel. 2012. Student-directed discovery of the plant microbiome and its products. Science 338: 485-486.
• Tedersoo, L., A.E. Arnold, K. Hansen. 2013. Novel aspects in the life cycle and biotrophic interactions in the Pezizomycetes. Molecular Ecology, in press.
• Devan, M.M.N., S.H. Furr, A.E. Arnold. 201x. Persistent effects of wildfire on fungal endophyte communities in Quercus hypoleucoides and Juniperus deppeana. Microbial Ecology, accepted.
• Hoffman, M.T., M. Gunatilaka, E.M.K. Wijeratne, A.A.L. Gunatilaka, and A.E. Arnold. 2013. IAA production by endophytic Pestalotiopsis is enhanced by an endohyphal bacterium. PLoS ONE, accepted.
• Milani, N.A., D.P. Lawrence, A.E. Arnold, and H.D. vanEtten. 2012. Origin of pistatin demethylase (PDA) in the genus Fusarium. Fungal Genetics and Biology 49: 933-942.
• Martinson, E.O., E.A. Herre, C.A. Machado, A.E. Arnold. 2012. Culture-free survey reveals diverse fungal communities associated with figs (Ficus spp.) in Panama. Microbial Ecology 64: 1073-1084.
• Gazis, R., J. Miadlikowska, F. Lutzoni, A.E. Arnold, P. Chaverri. 2012. Culture-based study of endophytes associated with rubber trees in Peru reveals a new class of Pezizomycotina: Xylonomycetes. Molecular Phylogenetics and Evolution 65: 294-304.
• URen, J.M., F. Lutzoni, J. Miadlikowska, A. Laetsch, A.E. Arnold. 2012. Host and geographic structure of endophytic and endolichenic fungi at a continental scale. American Journal of Botany 99: 898-914.
• Higgins, K.L., A.E. Arnold, P. Coley, T. Kursar. 2012. Communities of fungal endophytes in tropical forest grasses: highly diverse host- and habitat generalists characterized by strong spatial structure. Fungal Ecology, in press.
• Varughese, T., N. Riosa, S. Higginbotham, A.E. Arnold, and 4 others. Antifungal depsidone metabolites from Cordyceps dipterigena, an endophytic fungus antagonistic to the phytopathogen Giberella fujikoroi. Tetrahedron Letters 28: 1624-1626.
• Molinar, E., N. Rios, C. Spadafora, A.E. Arnold, and 4 others. 2012. Coibanoles, a new class of meroterpenoids produced by Pycnoporus sanguineus. Tetrahedron Letters 53: 919-922.