1. Systematics of Clavicipitaceous Fungi Associated with Morning Glories Based on rpb1 Sequence Data Saroj Simkhada, Alyssa M. Brown, and Richard E. Miller Department of Biological Sciences Southeastern Louisiana University, Hammond, LA

2. Grasses contain ergot-alkaloid producing Clavicipitaceous fungi Classification Kingdom: Fungi Division: Ascomycota Class: Sordariomycetes Order: Hypocreales Family: “Clavicipitaceae” Ergot alkaloids Mycotoxin Amine-based compound Secondary metabolite Biologically active alkaloids Epichloe on grass photo: David Kennard

3. Grasses contain ergot-alkaloid producing Clavicipitaceous fungi Advantage to grasses: Decreased herbivory Drought tolerance Enhanced growth Competitive ability

4. Periglandula New fungal genus of Clavicipitaceae Symbionts with morning glory hosts Periglandula turbinae Host: Turbina corymbosa Periglandula ipomoeae Host: Ipomoea asarifolia Clavicipitaceae is not monophyletic Animal pathogen and fungal parasites Clavicipitaceae clades A, B, C Plant-associated Clavicipitaceae clade A Epiphytic fungi

5. Clavicipitaceae A, B, C Nutritional mode shift Clavicipitaceae A Grass endophytes

6. Objective 1 Determine the placement of Periglandula associated with morning glories within the order Hypocreales using rpb1 DNA sequence data. Examine the pattern of nutritional mode shift with the addition of new Periglandula sequences. Objective 2 Construct phylogeny estimating evolutionary relationships among Periglandula derived from different morning glory hosts Determine whether Periglandula phylogeny: (a) follows plant host phylogeny, or (b) exhibits biogeographic pattern. Research Objectives

7. Methods to obtain new Periglandula sequence data: Ergot-positive host morning glories were sampled. - chemical analysis (D. Pannicione) Ergot-positive accessions grown in green house DNA extraction using Qiagen DNeasy plant minikit PCR using Periglandula specific primers rpb1 gene region (developed by A. Brown) PCR-based sequencing -- Beckman-Coulter Genomics

8. Methods: Objectives 1 – Placement of Periglandula within Hypocreales: Integrate new Periglandula sequences with examplar sample of published sequences for Hypocreales Redo the phylogenetic analyses with these new data Alignment Automated alignment using Sate II Bayesian phylogenetic analyses 45 taxa (528 sites) 3 million generations used to reach convergence burnin of 50,000 generations removed Standard deviation of split frequencies of 0.0061 indicating convergence has been reached

9. Methods: Objective 2 – Molecular phylogenetics of Periglandula Phylogeny reconstruction of Periglandula sequence data using new results and one published sequence Alignment and Bayesian analysis similar to above

10. Objective 1: Determine the placement of Periglandula associated with morning glories within the order Hypocreales using rpb1 DNA sequence data. Examine the pattern of nutritional mode shift with the addition of new Periglandula sequences.

11. Genus Periglandula Fall in Clavicipitaceae clade A Sister to grass associated fungi Form a monophyletic group Phylogeny of Hypocreales (adapted from Spatofora 2007) rpb1 sequence data nutritional shifts noted

12. Objective 2: Construct phylogeny estimating evolutionary relationships among Periglandula derived from different morning glory hosts Determine whether Periglandula phylogeny: (a) follows plant host phylogeny, or (b) exhibits biogeographic pattern

13. Periglandula Phylogeny Periglandula turbinae distantly related Periglandula from other related hosts are closely related Clade resolved that included Periglandula from I. argillicola, I. pes-caprae, I. muelleri

14. Morning Glory Phylogeny Sample of Ipomoeeae Host species from study ITS sequence data Eserman & Miller, in prep. Adapted from larger Bayesian analysis Posterior probs. indicated colors correspond to host distributions on previous slide

15. Periglandula phylogeny essentially congruent with morning glory phylogeny Morning Glory Phylogeny

16. Periglandula phylogeny essentially congruent with biogeography (note phylogeny and biogeography are confounded) Morning Glory Phylogeny

17. Objective 1: Determine the placement of Periglandula associated with morning glories within the order Hypocreales using rpb1 DNA sequence data. Periglandula form a monophyletic group within the Hypocreales and within Clavicipitacea A, the plant-associated fungal clade Examine the pattern of nutritional mode shift with the addition of new Periglandula sequences. Addition of Periglandula retains clade of plant-associated fungi RESEARCH OBJECTIVES

18. Objective 2: Reconstructed phylogeny estimating evolutionary relationships among Periglandula derived from different morning glory hosts Determine whether Periglandula phylogeny: (a) follows plant host phylogeny, or (b) exhibits biogeographic pattern. Results supported both hypotheses. Further sampling needed from more host species from within Ipomoeeae to resolve competing hypotheses

19. References Spatafora J, Sung G, Sung J, Hywel-Jones N, White J. Phylogenetic evidence for an animal pathogen origin of ergot and the grass endophytes. Molecular Ecology.April 15, 2007;16(8):1701-1711. Clay K. Fungal endophytes of grasses. Annual Review of Ecology and Systematics.1990, 21, 275–297.