University of Florida Thesis Or Dissertation
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PHYLOGENETIC SYSTEMATICS OF SYNCEPHALIS (ZOOPAGALES: ZOOPAGOMYCOTA), A GENUS OF UBIQUITOUS MYCOPARASITES By KATHERINE LOUISE LAZARUS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016 © 2016 Katherine Louise Lazarus ACKNOWLEDGMENTS I would like to acknowledge substrate sample, culture, and sequence contributors, Gerald L. Benny, Hsiao-Man Ho, Matthew E. Smith, Kerry O’Donnel and the NRRL (Agriculture Research Service Culture Collection). I would like to thank members of the Smith Lab, Rosanne Healy, Alija Mujic, Nicole Reynolds and Arthur Grupe and I would like to thank my advisor Matthew E. Smith and committee members Jeffrey Rollins and Gerald Benny for their guidance, feedback and support. I would also like to acknowledge IFAS and the University of Florida. 3 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 3 LIST OF TABLES ............................................................................................................ 5 LIST OF FIGURES .......................................................................................................... 6 ABSTRACT ..................................................................................................................... 7 CHAPTER 1 INTRODUCTION ...................................................................................................... 9 2 MATERIALS AND METHODS ................................................................................ 14 Sampling, Isolation, Co-Culturing on Host Fungi, and Axenic Culturing ................. 14 PCR and Sequencing ............................................................................................. 16 Sequence and Phylogenetic Analysis ..................................................................... 18 3 RESULTS ............................................................................................................... 26 4 DISCUSSION ......................................................................................................... 31 Overview ................................................................................................................. 31 Clade Analysis ........................................................................................................ 31 The Hypogena Clade ....................................................................................... 31 The Vivipara Clade ........................................................................................... 33 The Obconica Clade ......................................................................................... 34 The Cornu Clade .............................................................................................. 35 The Sphaerica Clade ........................................................................................ 37 The North American and Asian Depressa Clades ............................................ 38 Species Clade A, Species Clade B, and unidentified, divergent species ......... 39 The Parvula Clade, Syncephalis nana and Syncephalis clavata ...................... 39 Sequence Analysis of the Internal Transcribed Spacer region (ITS) ...................... 40 Ecology ................................................................................................................... 42 5 CONCLUSION ........................................................................................................ 46 LIST OF REFERENCES ............................................................................................... 49 BIOGRAPHICAL SKETCH ............................................................................................ 54 4 LIST OF TABLES Table page 2-1 Collection data for the 88 Syncephalis spp. isolate sequences included in the phylogenetic analysis ......................................................................................... 23 2-2 Primers implemented in this study, with their target gene region and varying specificity ............................................................................................................ 25 3-1 Detecting Syncephalis ITS sequences in GenBank ............................................ 30 5 LIST OF FIGURES Figure page 2-1 Morphology across the genus Syncephalis ........................................................ 21 2-2 Ribosomal DNA (rDNA) primer map ................................................................... 22 3-1 Maximum Likelihood (ML) phylogeny ................................................................. 28 3-2 Length of sequences from complete internal transcribed spacer region (ITS1- 5.8S-ITS2) for 31 Syncephalis isolates from 18 species .................................... 29 6 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHYLOGENETIC SYSTEMATICS OF SYNCEPHALIS (ZOOPAGALES: ZOOPAGOMYCOTA) A GENUS OF UBIQUITOUS MYCOPARASITES By Katherine Lazarus May 2016 Chair: Matthew E. Smith Major: Plant Pathology Phylogenetic relationships among species of the mycoparasite genus Syncephalis were examined by using sequences from three nuclear encoded ribsosomal DNA genes and one protein encoded gene. The data consist of 18S, 5.8S and 28S rDNA and RPB1 for 88 Syncephalis isolates from 23 named species. I revived a culturing technique using beef liver and cellophane to grow several Syncephalis isolates without their host fungi to obtain pure DNA for some isolates. Most isolates, however, were grown in co-cultures with their host fungi and I designed Syncephalis- specific primers to obtain sequence data. Individual and combined datasets were analyzed by maximum likelihood (ML) and Bayesian analysis (BA) methods. I recovered 14 well-supported lineages and determined that most major clades contained isolates from distant localities on multiple continents. Many clades had taxonomic issues, evidently due to high phenotypic plasticity and species dimorphism. I also conducted an analysis of Syncephalis ITS sequences using complete ITS sequence data (ITS1-5.8S- ITS2) for 31 unique isolates of 18 species and determined that Syncephalis species, on average, have long ITS sequences relative to other fungi. Commonly employed 7 eukayotic and fungal primers (ITS1, ITS1-f) appear compatible across phylogentically diverse Syncephalis species, but paradoxically Syncepahlis sequences are rarely recovered in environmental molecular diversity surveys. 8 CHAPTER 1 INTRODUCTION Species of Syncephalis (Zygomycota: Piptocephalidaceae) are obligate parasites of saprobic microfungi in Mortierellomycotina and Mucoromycotina that commonly occur in soil and dung around the world. They are inconspicuous, and may superficially resemble other zygomycetes in Mortierellomycotina and Mucoromycotina as well as some anamorphic Ascomycota (Bawcutt 1983). The morphology of a typical species is a single, straight or recurved, merosporangiophore (ranging from 30 (S. hypogena R.K. Benj.) to 775 (S. obliqua H.M. Ho & Benny) µm in height) supporting an apical, fertile vesicle that produces a number of branched or unbranched, cylindrical merosporangia. Merosporangiophores arise from a base of simple rhizoids, which are used to anchor to substrates or to host hyphae. Alternatively, merosporangiophores can also be born aerially on the thin (1 µm thick), cobweb-like hyphae of the Syncephalis itself. Merospores germinate to produce more fine hyphae, which eventually develop simple haustoria to penetrate a host fungus and grow intracellularly in an obligate, biotrophic parasitism (Benjamin 1959, Embree 1963). Tieghem and Le Monnier first established the genus in 1873 and subsequently, approximately 65 species have been described. Classification has been based on morphological characters, such as merosporangiophore shape, spore ornamentation, fertile vesicle shape, number of merospores per merosporangium, the arrangement of merosporangia on the fertile vesicle, and merosporangia branching pattern (Ho 2001). Syncephalis species may be readily isolated from the environment with the use of selective media and maintained in dual culture with their host fungi. Syncephalis spp. are widespread in environments that have a rapid turnover of organic material, 9 especially woodland and pasture habitats (Richardson & Leadbeater 1972). Soil, plant debris, and dung (especially of herbivores) are prime substrates for isolation. Occasionally, a gram of soil may contain multiple Syncephalis species (K. Lazarus, personal observation). As ubiquitous mycoparasites, they likely influence the population levels of host fungi in terrestrial environments by inhibiting the vigor and sporulation of their hosts (Jeffries 1985). Typical hosts for Syncephalis species belong to the zygomycete subphyla Mucoromycotina and Mortierellomycotina (Baker et al., 1977; Richardson & Leadbeater, 1972). Hosts include several model zygomycetes, such as species of Phycomyces, Rhizopus and Mucor, as well as post-harvest decay pathogens and Choanephora cucurbitarum (Berk. & Ravenel) Thaxt., a pathogen of cucurbits and several other crops. Species of Syncephalis frequently parasitize Mucor spp. and Rhizopus spp. in nature, making these mycoparasites candidates for biocontrol research. Host range studies (Hunter & Butler 1975, Baker et al., 1977) have determined that individual