bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.989582; this version posted March 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 Character evolution of modern fly-speck fungi and implications for interpreting 3 thyriothecial fossils 4 5 Ludovic Le Renard*1, André L. Firmino2, Olinto L. Pereira3, Ruth A. Stockey4, 6 Mary. L. Berbee1 7 8 1Department of Botany, University of British Columbia, Vancouver BC, V6T 1Z4, Canada 9 2Instituto de Ciências Agrárias, Universidade Federal de Uberlândia, Monte Carmelo, Minas 10 Gerais, 38500-000, Brazil 11 3Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36570- 12 000, Brazil 13 4Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, 14 USA 15 16 Email: [email protected] 17 18 Manuscript received _______; revision accepted _______. 19 20 Running head: Fly-speck fungi character evolution 21 22 23 p. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.989582; this version posted March 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 25 Abstract 26 PREMISE OF THE STUDY: Fossils show that fly-speck fungi have been reproducing with 27 small, black thyriothecia on leaf surfaces for ~250 million years. We analyze morphological 28 characters of extant thyriothecial fungi to develop a phylogenetic framework for interpreting 29 fossil taxa. 30 METHODS: We placed 59 extant fly-speck fungi in a phylogeny of 320 Ascomycota using 31 nuclear ribosomal large and small subunit sequences, including newly determined sequences 32 from nine taxa. We reconstructed ancestral character states using BayesTraits and maximum 33 likelihood after coding 11 morphological characters based on original observations and literature. 34 We analyzed the relationships of three previously published Mesozoic fossils using parsimony 35 and our morphological character matrix, constrained by the molecular phylogeny. 36 KEY RESULTS: Thyriothecia evolved convergently in multiple lineages of superficial, leaf- 37 inhabiting ascomycetes. The radiate and ostiolate scutellum organization is restricted to 38 Dothideomycetes. Scutellum initiation by intercalary septation of a single hypha characterizes 39 Asterinales and Asterotexiales, and initiation by coordinated growth of two or more adjacent 40 hyphae characterizes Aulographaceae (order incertae sedis). Scutella in Microthyriales are 41 initiated apically on a lateral hyphal branch. Patterns of hyphal branching in scutella contribute 42 to distinguishing among orders. Parsimony resolves three fossil taxa as Dothideomycetes; one is 43 further resolved as a member of a Microthyriales-Zeloasperisporiales clade within 44 Dothideomycetes. 45 CONCLUSIONS: This is the most comprehensive systematic study of thyriothecial fungi and 46 their relatives to date. Parsimony analysis of the matrix of character states of modern taxa p. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.989582; this version posted March 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 provides an objective basis for interpreting fossils, leading to insights into morphological 48 evolution and geological ages of Dothideomycetes clades. 49 Key words: ancestral state reconstruction; Ascomycota; Dothideomycetes; fly-speck fungi; fossil 50 fungi, leaf cuticle; morphological characters; phylogeny; scutellum; thyriothecium. p. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.989582; this version posted March 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 51 Fly-speck fungi are ascomycetes that appear as minute (50 μm to 2 mm in diameter) black 52 reproductive structures called thyriothecia. Thyriothecia are distinguished by having scutella, 53 which are small, thin, darkly pigmented, shield-like plates that cover spore-producing cells. The 54 margins of scutella are appressed to the surfaces of living or dead leaves or stems of vascular 55 plants (Arnaud, 1917, 1918, 1925; Stevens and Ryan, 1939; Wu et al., 2011b; Wu et al., 2014). 56 Fly-speck fungi are common throughout the world (Arnaud, 1918; Doidge, 1942; Batista, 1959; 57 Batista et al., 1969; Holm and Holm, 1991), especially in tropical rainforests, where they grow 58 on leaf surfaces (Batista, 1959; Hughes, 1976; Hofmann and Piepenbring, 2011; Firmino, 2016). 59 Scutella of fly-speck fungi are also common as fossils (Elsik, 1978; Kalgutkar and Jansonius, 60 2000). Our interest in these fungi is rooted in a desire to interpret the phylogenetic significance 61 of the hundreds of fossil forms found in palynological preparations and on preserved cuticles of 62 plant fossils (Kalgutkar and Jansonius, 2000; Taylor et al., 2015). Fossil fly-speck scutella have 63 long been classified into form genera and form species that carry minimal phylogenetic 64 information. Phylogenetic classification of the fossils can potentially be improved by analysis of 65 the systematic distribution of scutellum characters among extant fly-speck fungal lineages. 66 Scutella of thyriothecia are recognizable in part because they develop above the plant 67 cuticle, in contrast to many other kinds of minute ascomycete fruiting bodies that begin within 68 host tissue and break through to the leaf surface only when spores are ready for dispersal. How 69 fly-speck fungi interact with their host plants is unclear, but some appear to be asymptomatic 70 biotrophic parasites (on living hosts) while others are found in leaf litter (Hofmann, 2009; Wu et 71 al., 2011b). Hyphae of some fly-speck fungi enter the host directly through a stoma (Hofmann, 72 2009; Firmino, 2016). Many other leaf inhabiting fungi, including fly-speck fungi, enter the host 73 using appressoria. Appressoria are specialized hyphal cells that accumulate turgor pressure and p. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.989582; this version posted March 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 74 direct growth into the host through a penetration peg, a small melanized ring (Parbery and 75 Emmett, 1977). Appressoria then link the superficial fungal mycelium and various hyphal 76 structures inside host tissue such as haustoria, hypostromata made of densely packed or stromatic 77 hyphae, (Hofmann, 2009; Firmino, 2016), or palmettes of flattened, intercellular, dichotomous 78 hyphae (Ducomet, 1907; Arnaud, 1918). 79 Several aspects of fly-speck fungus biology make it challenging to relate their 80 morphology to phylogeny. First, thyriothecia are small, and so it can be difficult to find enough 81 individuals of the same species for morphological or molecular systematic analysis. Second, fly- 82 speck fungi appear in mixtures with other epiphyllous fungi, which can complicate 83 distinguishing among species (Dilcher, 1965; Phipps and Rember, 2004). Hofmann (2009) and 84 Firmino (2016) noted mixed species of Asterina growing together, and Ellis (1976) described 85 closely related, co-occurring species in the collection of Microthyrium microscopicum that is the 86 type for its genus. For sequence analysis, species should ideally be cultured. While some fly- 87 speck fungi grow in pure culture (Firmino, 2016), other species do not (Hofmann, 2009); overall, 88 they are poorly represented in culture collections. 89 A long-held view that thyriothecial fungi were monophyletic (Arnaud, 1918; Doidge, 90 1919; Clements and Shear, 1931; Gäumann, 1952; Kirk et al., 2008) has been corrected by a 91 series of molecular phylogenetic analyses. Wu et al. (2011b) found a broad sampling of 92 thyriothecial fungi to be scattered across four orders of Dothideomycetes, although with low 93 support from bootstrap values or posterior probabilities. Mapook et al. (2016) and Hernández- 94 Restrepo et al. (2019) showed that species of thyriothecial and thyriothecium-like fungi in 95 Muyocopronales form a strongly supported sister group to Dyfrolomycetales, still in 96 Dothideomycetes but distant from any other thyriothecial fungi. Even more distant is a p. 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.989582; this version posted March 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 97 monophyletic group of two thyriothecial Micropeltis species nested in class Lecanoromycetes 98 rather than Dothideomycetes (Hongsanan and Hyde, 2017). 99 In spite of this recent progress, the challenges involved in collecting sufficient cells from 100 fly-speck fungi remain, and their sequences are still poorly represented in GenBank and in 101 phylogenetic studies. About 150 sequences were available, representing 18 genera, as of January 102 2019, contrasting with the more than 50 genera documented in recent publications (Hofmann, 103 2009; Wu et al., 2011a; Wu et al., 2011b; Hongsanan et al., 2014; Wu et al., 2014; Mapook et al., 104 2016), and close to ~150 generic names available in families of thyriothecial