2 Character evolution of modern fly-speck fungi and implications … · 2020-03-13 · 3 thyriothecial fossils 4 5 Ludovic Le Renard*1, André L. Firmino2, Olinto L. Pereira3, Ruth

p. 1

1

Character evolution of modern fly-speck fungi and implications for interpreting 2

thyriothecial fossils 3

4

Ludovic Le Renard*1, André L. Firmino2, Olinto L. Pereira3, Ruth A. Stockey4, 5

Mary. L. Berbee1 6

7

1Department of Botany, University of British Columbia, Vancouver BC, V6T 1Z4, Canada 8

2Instituto de Ciências Agrárias, Universidade Federal de Uberlândia, Monte Carmelo, Minas 9

Gerais, 38500-000, Brazil 10

3Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36570-11

000, Brazil 12

4Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, 13

USA 14

15

Email: [email protected] 16

17

Manuscript received _______; revision accepted _______. 18

19

Running head: Fly-speck fungi character evolution 20

21

22

23

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 14, 2020. . https://doi.org/10.1101/2020.03.13.989582doi: bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 2

24

Abstract 25

PREMISE OF THE STUDY: Fossils show that fly-speck fungi have been reproducing with 26

small, black thyriothecia on leaf surfaces for ~250 million years. We analyze morphological 27

characters of extant thyriothecial fungi to develop a phylogenetic framework for interpreting 28

fossil taxa. 29

METHODS: We placed 59 extant fly-speck fungi in a phylogeny of 320 Ascomycota using 30

nuclear ribosomal large and small subunit sequences, including newly determined sequences 31

from nine taxa. We reconstructed ancestral character states using BayesTraits and maximum 32

likelihood after coding 11 morphological characters based on original observations and literature. 33

We analyzed the relationships of three previously published Mesozoic fossils using parsimony 34

and our morphological character matrix, constrained by the molecular phylogeny. 35

KEY RESULTS: Thyriothecia evolved convergently in multiple lineages of superficial, leaf-36

inhabiting ascomycetes. The radiate and ostiolate scutellum organization is restricted to 37

Dothideomycetes. Scutellum initiation by intercalary septation of a single hypha characterizes 38

Asterinales and Asterotexiales, and initiation by coordinated growth of two or more adjacent 39

hyphae characterizes Aulographaceae (order incertae sedis). Scutella in Microthyriales are 40

initiated apically on a lateral hyphal branch. Patterns of hyphal branching in scutella contribute 41

to distinguishing among orders. Parsimony resolves three fossil taxa as Dothideomycetes; one is 42

further resolved as a member of a Microthyriales-Zeloasperisporiales clade within 43

Dothideomycetes. 44

CONCLUSIONS: This is the most comprehensive systematic study of thyriothecial fungi and 45

their relatives to date. Parsimony analysis of the matrix of character states of modern taxa 46


https://doi.org/10.1101/2020.03.13.989582

p. 3

provides an objective basis for interpreting fossils, leading to insights into morphological 47

evolution and geological ages of Dothideomycetes clades. 48

Key words: ancestral state reconstruction; Ascomycota; Dothideomycetes; fly-speck fungi; fossil 49

fungi, leaf cuticle; morphological characters; phylogeny; scutellum; thyriothecium. 50


https://doi.org/10.1101/2020.03.13.989582

p. 4

Fly-speck fungi are ascomycetes that appear as minute (50 μm to 2 mm in diameter) black 51

reproductive structures called thyriothecia. Thyriothecia are distinguished by having scutella, 52

which are small, thin, darkly pigmented, shield-like plates that cover spore-producing cells. The 53

margins of scutella are appressed to the surfaces of living or dead leaves or stems of vascular 54

plants (Arnaud, 1917, 1918, 1925; Stevens and Ryan, 1939; Wu et al., 2011b; Wu et al., 2014). 55

Fly-speck fungi are common throughout the world (Arnaud, 1918; Doidge, 1942; Batista, 1959; 56

Batista et al., 1969; Holm and Holm, 1991), especially in tropical rainforests, where they grow 57

on leaf surfaces (Batista, 1959; Hughes, 1976; Hofmann and Piepenbring, 2011; Firmino, 2016). 58

Scutella of fly-speck fungi are also common as fossils (Elsik, 1978; Kalgutkar and Jansonius, 59

2000). Our interest in these fungi is rooted in a desire to interpret the phylogenetic significance 60

of the hundreds of fossil forms found in palynological preparations and on preserved cuticles of 61

plant fossils (Kalgutkar and Jansonius, 2000; Taylor et al., 2015). Fossil fly-speck scutella have 62

long been classified into form genera and form species that carry minimal phylogenetic 63

information. Phylogenetic classification of the fossils can potentially be improved by analysis of 64

the systematic distribution of scutellum characters among extant fly-speck fungal lineages. 65

Scutella of thyriothecia are recognizable in part because they develop above the plant 66

cuticle, in contrast to many other kinds of minute ascomycete fruiting bodies that begin within 67

host tissue and break through to the leaf surface only when spores are ready for dispersal. How 68

fly-speck fungi interact with their host plants is unclear, but some appear to be asymptomatic 69

biotrophic parasites (on living hosts) while others are found in leaf litter (Hofmann, 2009; Wu et 70

al., 2011b). Hyphae of some fly-speck fungi enter the host directly through a stoma (Hofmann, 71

2009; Firmino, 2016). Many other leaf inhabiting fungi, including fly-speck fungi, enter the host 72

using appressoria. Appressoria are specialized hyphal cells that accumulate turgor pressure and 73


https://doi.org/10.1101/2020.03.13.989582

p. 5

direct growth into the host through a penetration peg, a small melanized ring (Parbery and 74

Emmett, 1977). Appressoria then link the superficial fungal mycelium and various hyphal 75

structures inside host tissue such as haustoria, hypostromata made of densely packed or stromatic 76

hyphae, (Hofmann, 2009; Firmino, 2016), or palmettes of flattened, intercellular, dichotomous 77

hyphae (Ducomet, 1907; Arnaud, 1918). 78

Several aspects of fly-speck fungus biology make it challenging to relate their 79

morphology to phylogeny. First, thyriothecia are small, and so it can be difficult to find enough 80

individuals of the same species for morphological or molecular systematic analysis. Second, fly-81

speck fungi appear in mixtures with other epiphyllous fungi, which can complicate 82

distinguishing among species (Dilcher, 1965; Phipps and Rember, 2004). Hofmann (2009) and 83

Firmino (2016) noted mixed species of Asterina growing together, and Ellis (1976) described 84

closely related, co-occurring species in the collection of Microthyrium microscopicum that is the 85

type for its genus. For sequence analysis, species should ideally be cultured. While some fly-86

speck fungi grow in pure culture (Firmino, 2016), other species do not (Hofmann, 2009); overall, 87

they are poorly represented in culture collections. 88

A long-held view that thyriothecial fungi were monophyletic (Arnaud, 1918; Doidge, 89

1919; Clements and Shear, 1931; Gäumann, 1952; Kirk et al., 2008) has been corrected by a 90

series of molecular phylogenetic analyses. Wu et al. (2011b) found a broad sampling of 91

thyriothecial fungi to be scattered across four orders of Dothideomycetes, although with low 92

support from bootstrap values or posterior probabilities. Mapook et al. (2016) and Hernández-93

Restrepo et al. (2019) showed that species of thyriothecial and thyriothecium-like fungi in 94

Muyocopronales form a strongly supported sister group to Dyfrolomycetales, still in 95

Dothideomycetes but distant from any other thyriothecial fungi. Even more distant is a 96


https://doi.org/10.1101/2020.03.13.989582

p. 6

monophyletic group of two thyriothecial Micropeltis species nested in class Lecanoromycetes 97

rather than Dothideomycetes (Hongsanan and Hyde, 2017). 98

In spite of this recent progress, the challenges involved in collecting sufficient cells from 99

fly-speck fungi remain, and their sequences are still poorly represented in GenBank and in 100

phylogenetic studies. About 150 sequences were available, representing 18 genera, as of January 101

2019, contrasting with the more than 50 genera documented in recent publications (Hofmann, 102

2009; Wu et al., 2011a; Wu et al., 2011b; Hongsanan et al., 2014; Wu et al., 2014; Mapook et al., 103

2016), and close to ~150 generic names available in families of thyriothecial fungi 104

(Wijayawardene et al., 2014). 105

To interpret the fossil record of fly-speck fungi, our goals include increasing the sampling 106

of extant representatives and related taxa in the class Dothideomycetes. Our project provides new 107

cultures, herbarium specimens, and sequences into the public domain. Using new and previously 108

published data, our first goal is to infer a molecular phylogeny that samples as widely as possible 109

from the lineages of fly-speck fungi and their possible close relatives. Secondly, we score 110

morphological characters for selected species in each lineage, and reconstruct ancestral 111

morphological character states throughout the phylogeny. Thirdly, we use the molecular tree, 112

comparative morphology, and inferred ancestral character states to discuss the phylogenetic 113

affinities of three fly-speck fossils. 114

115

MATERIALS AND METHODS 116

Taxon sampling—We assembled a dataset of 320 ascomycete taxa including species of 117

59 fly-speck fungi. We obtained sequence data from new collections, from pure cultures where 118

possible, found in Canada, Costa Rica (Permit nºR002-2014-OT-CONAGEBIO) and France. 119


https://doi.org/10.1101/2020.03.13.989582

p. 7

Pure cultures were established by crushing individual thyriothecia under an inverted compound 120

microscope using insect pins (Austerlitz Insect pins, size 0; Slavkov u Brna, Czech Republic) and 121

then transferring individual spores or groups of spores onto water agar with 1 mM ampicillin and 122

1 mM kanamycin for germination. Germinating spores were transferred to antibiotic-free 123

nutritive media under a dissecting microscope. The resulting cultures have been deposited in the 124

Westerdijk Institute, Netherlands (Appendix S1, strains labeled with an asterisk). 125

Sequence data for nuclear ribosomal large subunit (LSU) and small subunit (SSU) DNA 126

from thyriothecial taxa and their closest relatives were recovered from GenBank using a series of 127

BLAST searches (Appendix S1). The use of these two loci was pragmatic; for most of the 128

relatives of thyriothecial fungi including other leaf-inhabiting fungi and lichenized fungi, only 129

ribosomal DNA data were available. We also included published sequences from lichenized 130

fungi or lichen associated fungi that shared the epiphyllous habitat with thyriothecial fungi. We 131

selected outgroups to represent classes related to Dothideomycetes. In addition, near full-length 132

to full-length LSU and SSU data were retrieved from 13 whole genome projects with the help of 133

BLAST searches and using the JGI MycoCosm Portal (Grigoriev et al., 2013) (Appendix S2). 134

DNA extraction, amplification, sequencing—Strains successfully isolated in pure 135

culture include Lembosina sp. CBS 144007, Lembosina aulographoides CBS 143809, 136

Microthyrium macrosporum CBS 143810, and cf. Stomiopeltis sp. CBS 143811. They were 137

harvested from culture media for genomic DNA extraction using the Qiagen DNeasy Plant 138

Minikit (QIAGEN Inc., Mississauga, Ontario). A pure culture corresponding to the voucher of 139

cf. Stomiopeltis sp. UBC-F33041 was isolated and its DNA extracted, but it could not be 140

maintained over repeated transfers to new cultures. The polymerase chain reaction (PCR) 141

reactions were performed using PureTaq Ready-To-Go PCR beads (Amersham Biosciences, 142


https://doi.org/10.1101/2020.03.13.989582

p. 8

Piscataway, New Jersey), following manufacturer instructions. Amplifications were performed 143

using fungal specific rDNA primers reported in James et al. (2006): BMB-BR, NS1, NS3, NS6, 144

NS8, NS20, NS23, NS51, ITS2, LIC1460R, LIC2197, LR0R, LR3R, LR5, LR5R, LR7R, LR8, 145

LR10, LR11, LR10R, LR12, LR13. Uncultured dried specimens Micropeltis sp. UBC-F33034, 146

Scolecopeltidium spp. UBC-F33033 and UBC-F33035, and Asterotexiaceae sp. UBC-F33036 147

were used as templates for direct DNA amplification (Lee and Taylor, 1990) of ~1 kb of the 5' 148

end of the LSU, a region known to be informative for thyriothecial fungi (Hofmann et al., 2010; 149

Wu et al., 2011b; Guatimosim et al., 2015). A cocktail of 12.5 µL of water and primers at a 150

concentration of 1.0 µM each was pipetted into a PCR reaction tube containing a PureTaq 151

Ready-To-Go PCR bead, and kept on ice. Thyriothecia were: (i) mounted in a drop of sterile 152

water, observed under the compound microscope to ascertain identity, presence of ascospores, 153

and absence of contaminant material; (ii) crushed firmly between a slide and a coverslip, and (iii) 154

~10-13 µL of the crushed ascospore material in water was pipetted from the coverslip and the 155

slide into a waiting PCR reaction tube, which was then vortexed briefly. The final volume of 156

liquid in each tube was ~25 µL, and the final concentration of each primer was 0.5 µM. The PCR 157

reactions ran for 40 cycles with an annealing temperature of 50ºC and an elongation temperature 158

of 72ºC. Direct amplifications of the 5' end of the LSU using primers LR0R-LR8 with an 159

annealing temperature of 55ºC initially yielded weak PCR product. Weak PCR products were 160

then diluted 100 to 1000 times and re-amplified with internal primers to increase product 161

concentration for sequencing (see Mitchel, 2015, for similar methods). 162

Phylogenetic inference—The LSU and SSU sequences were first aligned using the 163

iterative refinement method G-INS-I in MAFFT v7 (Katoh and Standley, 2013). Introns were 164

trimmed out manually and the resulting sequences re-aligned with MAFFT. Ambiguously 165


https://doi.org/10.1101/2020.03.13.989582

p. 9

aligned positions were excluded manually. The resulting concatenated dataset spanned 4552 bp. 166

We ran JModelTest 2 (Darriba et al., 2012) on the CIPRES portal (Miller et al., 2010) and 167

selected the GTR+G model for individual genes. We ran RAxML v.8 (Stamatakis, 2014) on two 168

partitions (one for each gene) and 1000 independent maximum likelihood searches, again 169

running on CIPRES. Branch support was assessed using 1000 bootstrap replicates (BS) 170

(Felsenstein, 1985). Bayesian analysis was performed using MrBayes 3.2 Metropolis Coupled 171

Monte Carlo algorithm (Ronquist et al., 2012). For the Bayesian analysis, we ran four 172

independent searches, each with eight MCMC chains for 160 million generations sampling every 173

5000 trees, using 128 processors of 2 Gb each on a Compute Canada cluster 174

(cedar.computecanada.ca). Following test runs, the heating parameter was adjusted to 0.03, 175

optimizing the search space so that swap frequencies were between 0.3 and 0.7, as the MrBayes 176

3.2 manual advises. We discarded 50% of the samples as burn-in. We used Tracer 1.5 to 177

determine that effective sample size exceeded 200 for all parameters, and the R package RWTY 178

(Warren et al., 2017) to verify the convergence of independent runs. We considered clades with 179

>70% likelihood bootstrap support and >0.95 Bayesian posterior probability to be moderately 180

well supported. 181

Preliminary results suggested that some taxa at various ranks were not monophyletic. To 182

explore the fit of the data to alternative hypotheses, we set monophyly constraints as single 183

individual branches and found the most likely tree given each constraint using 100 independent 184

“thorough” searches in RAxML. We then generated per-site log likelihoods of each likelihood 185

tree given the constraint, and performed an approximately unbiased (AU) test (Shimodaira, 186

2002) using CONSEL (Shimodaira and Hasegawa, 2001). We rejected hypotheses receiving a 187

probability below 0.05 in the AU test (Appendix S3). We specifically tested in turn the 188


https://doi.org/10.1101/2020.03.13.989582

p. 10

monophyly of (i) a putative clade comprising Asterinales and Asterotexiales, (ii) a putative clade 189

comprising Microthyriales and Zeloasperisporiales, (iii) Radiate thyriothecia, (iv) Asterina and 190

(v) Lembosia. 191

Observation and scoring of morphological characters—Original observations of 192

specimens were particularly important for coding thyriothecial characters that also appear in 193

fossils. Table 1 lists the 26 specimens examined directly for morphological analysis. Scoring for 194

the remaining 294 taxa was based on our interpretations of published illustrations and the 195

literature (Appendix S4). When possible, we used morphological and sequence information from 196

the same specimen (Appendix S4). 197

Table 2 shows our working definitions for scoring 11 morphological or habitat 198

characters, considering sporocarp features of both asexual, conidium-producing structures and 199

sexual ascomata. For direct observation of the hyphal structure of the dorsal surface of scutella, 200

and to look for appressoria, we peeled thyriothecia together with their surface hyphae off of leaf 201

surfaces using nail polish, as in Hosagoudar and Kapoor (1985). We examined thyriothecial 202

features using a Leica DMRB (Leitz, Wetzlar, Germany) differential interference contrast (DIC) 203

microscope, and photographed them at 400x or 1000x with a Leica DFC420 digital color camera. 204

Many scutella were more cone-shaped than strictly flat, and investigating their hyphae required 205

views from multiple focal planes. We attempted to use confocal microscopy to visualize hyphal 206

organization, staining a specimen of Microthyrium with 0.1 mg/mL calcofluor white (Sigma, St. 207

Louis, Missouri). The calcofluor revealed internal asci and some septa, but hyphae of the 208

scutellum were obscured by autofluorescence and pigmentation, and we pursued this approach 209

no further. Subsequently, we assembled stacks of bright field or DIC images at different focal 210


https://doi.org/10.1101/2020.03.13.989582

p. 11

planes to capture details of scutella and mycelia, following Harper (2015, p. 64, §4.3.1) and 211

suggestions from Bercovici et al. (2009). 212

Published written descriptions provide some character state information but are rarely 213

detailed enough to allow coding of the more subtle characters. Of the 11 scored characters, 10–214

100% could be scored per specimen, across the 320 specimens studied (Appendix S4). 215

Appressoria are an example of a character that is rarely reported or illustrated, visible in surface 216

mycelium of extant and fossil thyriothecial taxa, but rarely noted in publications on other fungi. 217

Six characters specific to sporocarps could be scored for 67–79% of the taxa included (Appendix 218

S4). Thyriothecial development could be coded in extant and fossil taxa because different stages 219

are exposed on the surfaces of leaves. Development in non-thyriothecial Dothideomycetes is 220

more difficult to observe, again leading to much missing data (Appendix S4). 221

Analysis of morphological characters—We performed maximum likelihood ancestral 222

character state reconstruction on the most likely tree in Mesquite for the scored characters. 223

Maximum likelihood reconstructions were computed using the Markov k-state 1-parameter 224

(MK1) model (Lewis, 2001). Likelihood ratio tests showed that the MK1 model, which assumes 225

equal rates of forward and reverse transitions between states for each character was more likely 226

than an asymmetrical, 2-parameter model for the five binary characters in our dataset. We 227

reconstructed character states on the single, most likely tree. 228

To take phylogenetic uncertainty into account, we also performed reconstruction using 229

BayesTraits V3 (Pagel and Meade, 2007). In BayesTraits, we used the Multistate ML search 230

algorithm, which estimates different rates for every state transition, with the option MLtries set 231

to 100, a setting which led to consistent results in preliminary runs. Each character was 232

reconstructed for 208 nodes out of the 319 nodes of our tree. The proportion of each state for 233


https://doi.org/10.1101/2020.03.13.989582

p. 12

each node was calculated from its mean value over the 5000 trees chosen randomly from the 234

among the 32,000 trees in the posterior distribution of trees in chain one of our MrBayes 235

analysis. We considered any character state reconstructions with > 0.7 of both posterior 236

probability (from BayesTraits) and proportional likelihood (maximum likelihood) to be 237

supported. 238

Analysis of fossils—We selected three fossils reported as thyriothecial from the literature 239

that were well enough illustrated to be coded for morphology. These include the oldest dispersed 240

thyriothecium-like structure (Mishra et al., 2018), a dispersed Lichenopeltella-like form (Monga 241

et al., 2015) for which cell patterns could be analyzed, and Asterina eocenica found on a leaf 242

associated with mycelium (Dilcher, 1965). As for extant taxa, we coded character states from the 243

illustrations (Appendix S4). 244

To infer the phylogenetic relationships of each of the three fossils, we used the most 245

likely, 320-taxon tree from the rDNA analysis as a topological constraint. We analyzed the 246

relationships of the fossils together and then one at a time, using the 11-character morphological 247

data set (Appendix S4). With branch-and-bound searches in PAUP 4.0a166 (Swofford 2003), we 248

found the most parsimonious positions of the fossils, given the rooted constraint tree. We did not 249

further pursue the simultaneous analysis of all three fossils because the result was a poorly 250

resolved, uninformative phylogeny (not shown). Instead, we focus on the results from adding one 251

fossil at a time. Alignments, phylograms, and cladograms are available from TreeBASE 252

(Submission ID: 25326). 253

RESULTS 254

Distribution of thyriothecium forming fungi—Thyriothecium forming fungi appear 255

polyphyletic, as expected (Fig. 1). All but Micropeltidaceae are included in class 256


https://doi.org/10.1101/2020.03.13.989582

p. 13

Dothideomycetes (Fig. 1). Within Dothideomycetes, thyriothecial fungi appear widely 257

distributed, arising from a poorly resolved backbone (Fig. 1; Appendices S5 and S6). 258

Asterotexiales form a well-supported clade rich in thyriothecial forms, including the type 259

species Asterotexis cucurbitacearum (Fig. 1; Appendices S5 and S6). Of the 35 Asterotexiales 260

taxa sampled, 20 have thyriothecial sporocarps and 19 have epiphyllous habitats (Appendix S4). 261

Clade A, one of three clades diverging near the base of the Asterotexiales, includes 19 taxa with 262

thyriothecia, and 18 of the species that are epiphyllous (Appendices S4 and S6). However, Clade 263

A also includes Buelliella poetschii, an apothecioid lichen parasite, and Mycosphaerella 264

pneumatophorae, which produces a perithecioid ascoma in (rather than superficially on) plant 265

tissue. Note that the "ioid" suffix refers to sporocarp shape, not to development of the ascomata 266

(see character state definitions in Table 2). 267

Clade B species were recently classified in Asterinales (see Ertz and Diederich, 2015) but 268

here, they group instead with Asterotexiales (Appendices S5 and S6). Clade B includes 14 269

species, most of which are apothecioid parasites of lichens, although two are corticolous, and one 270

species, Morenoina calamicola is thyriothecial and epiphyllous (Appendices S4, S5 and S6). The 271

third clade is represented by Geastrumia polystigmatis, an epiphyllous species known only by its 272

asexual state, and it has yet to be formally classified to order. Aulographaceae, represented by six 273

thyriothecial taxa, appears as the sister group of Asterotexiales, but with low branch support. 274

A clade of five orders including Microthyriales and Venturiales receives 88% bootstrap 275

support and 100% posterior probability (Fig. 1; Appendices S5 and S6). Of the orders, 276

Microthyriales and Zeloasperisporiales are thyriothecial and epiphyllous, but their close relatives 277

in Natipusillales are freshwater cleistothecioid fungi that fruit on decorticated wood (Fig. 1; 278

Appendices S4 and S6). Although Venturiales include a few thyriothecial species, most of its 279


https://doi.org/10.1101/2020.03.13.989582

p. 14

members are perithecioid leaf and stem parasites causing, for example, apple scab and black knot 280

of plum (Fig. 1; Appendices S4 and S6). 281

Most Capnodiales are perithecioid, but the order also includes thyriothecial species (Fig. 282

1; Appendix S4). The order Asterinales is nested in Capnodiales with 99% Bayesian posterior 283

probability but with negligible likelihood bootstrap support (Appendix S6). Asterinales is 284

represented here by the type species Asterina melastomatis, and by sequences from three 285

collections of Parmularia styracis, the type species of Parmulariaceae (Appendices S5 and S6). 286

Asterinales also includes anamorphic foliicolous fungi and Thyrinula eucalypti, a plant pathogen 287

that causes leaf spots around its thyriothecia. Two thyriothecial taxa in Capnodiales are not 288

closely related to one another or to Asterinales: Peltaster fructicola and Schizothyrium pomi. The 289

genus Stomiopeltis is polyphyletic and Stomiopeltis betulae is resolved in Microthyriales while 290

two species tentatively identified as Stomiopeltis form a clade with Tothia fuscella in Venturiales 291

(Fig. 1; Appendices S5 and S6). 292

Muyocopronales represents a strongly supported example of convergent origin of 293

thyriothecia (Fig. 1; Appendices S5 and S6). Muyocopronales thyriothecia have several cell 294

layers of lightly pigmented, pseudoparenchymatous cells below their outer, darkly pigmented 295

scutellum (see illustrations of Mapook et al., 2016b). The thickness of the scutellum makes 296

detailed analysis of the hyphal branching pattern challenging. 297

Micropeltidaceae provide another strongly supported example of convergent thyriothecial 298

morphology. The family is nested in Ostropomycetidae in Lecanoromycetes rather than 299

Dothideomycetes (Fig. 1; Appendices S5 and S6). The scutellum of Micropeltidaceae is 300

distinctive in that it is ostiolate and formed as a flat, compact reticulum-like network of 301


https://doi.org/10.1101/2020.03.13.989582

p. 15

overlapping hyphae. As illustrated by Hofmann and Piepenbring (2006), the hyphae do not 302

radiate from the center to the margins of the scutellum. 303

Character states of most recent common ancestors of thyriothecial clades—Bayesian vs 304

likelihood results—Different methods of character state reconstruction are generally congruent, 305

but BayesTraits more frequently gives unresolved ancestral states compared with likelihood 306

(Table 3; Appendices S6 and S7). BayesTraits reconstructs states over a posterior distribution of 307

trees, so topological conflict could have explained low support for states. Surprising, in our 308

analysis, unresolved ancestral states in BayesTraits are often associated with clades that have 309

strong topological support, indicating that conflicting branching order is not the only cause of 310

lack of resolution. Rather, the unresolved nodes are associated with missing morphological data 311

and high estimated rates of character state transitions. BayesTraits estimates the forward and 312

reverse rates separately for each character, and where data are missing, the information content is 313

insufficient to parameterize the separate rates. An example of this is evident in the reconstruction 314

of the scutellum branching pattern in the ancestor of Zeloasperisporiales and Natipusillales 315

(Table 3). For this character, BayesTraits estimated an average forward transition rate from 316

pseudoparenchymatous (0) to anisotomous branching (2) as 4.3 but it estimated the reverse rate 317

as 56. Near the reconstruction, the topology is well supported and most of the surrounding 318

branches receive ≥92% posterior probabilities but BayesTraits shows the reconstructed state as 319

completely equivocal (0.2 for each of five states) (Appendices S6 and S7). In contrast, the same 320

node is reconstructed as anisotomous with 0.85 proportional likelihood (Appendices S6 and S7) 321

because the MK1 model can take advantage of all available data to estimate the single transition 322

rate that it applies to each character. We consider results to be most reliable when supported by 323

both likelihood and Bayesian reconstructions. However, when the topology is well supported, we 324


https://doi.org/10.1101/2020.03.13.989582

p. 16

consider a reconstruction supported by likelihood to be more useful than an equivocal Bayesian 325

result. 326

Reconstruction of evolutionary origin of character states—Sporulation substrate, 327

lichenization, sporocarp type—The sporulation substrate of ancestral Dothideomycetes is 328

reconstructed with likelihood (Fig. 1; Appendix S8) as immersed in plant tissue, or with 329

BayesTraits as being saxicolous (Appendix S8). The most recent common ancestors of 330

thyriothecial lineages appear to have adapted to superficial growth on leaf or twig surfaces (Fig. 331

1; Appendix S8). In the case of Muyocropronales, adaptations to superficial growth (Appendix 332

S8) preceded origin of thyriothecia. The ancestral Dothideomycetes and the most recent common 333

ancestors of thyriothecial fungi are reconstructed as non-lichenized (Appendix S9), although 334

Micropeltidaceae is nested in the Ostropomycetidae among lichenized fungi. Reconstructions 335

suggest that Dothideomycetes initially produced apothecioid sporocarps (Appendix S10). 336

Thyriothecial Micropeltidaceae appear to have arisen from apothecioid ancestors while 337

thyriothecial Asterinales, Capnodiales and Muyocopronales appear to have originated from 338

perithecioid ancestors. In three clades, Asterotexiales Clade A, Aulographaceae, and the group of 339

Microthyriales plus Venturiales clade, ancestors are reconstructed as thyriothecial or unresolved 340

(Appendix S10). 341

Lower walls—Unlike most ascomata, thyriothecia usually lack a differentiated lower 342

wall, a layer of pigmented fungal tissue that would separate sporogenous tissue from the 343

substrate (Fig. 1; Appendix S11). Lower walls are hypothesized to be present in the ancestor of 344

Dothideomycetes based on 100% proportional likelihood and 58% support from BayesTraits 345

(Node 1 in Fig. 1; Appendix S11). Out of 59 thyriothecial species included here, only four have a 346


https://doi.org/10.1101/2020.03.13.989582

p. 17

pigmented, differentiated lower wall (Appendices S4 and S11) and ancestral species 347

reconstructed as thyriothecial are also reconstructed as lacking a differentiated lower wall. 348

Dehiscence—The mechanism of spore release from sporocarps varies among the closest 349

relatives of Dothideomycetes, and among thyriothecial taxa (Appendix S12). Likelihood 350

reconstructions suggest that ancestral Dothideomycetes opened with an ostiole (Fig. 1); 351

BayesTraits reconstructions favor opening by a regular slit and show ancestral forms with slits 352

subsequently giving rise to ostiolate clades that diversified further (Table 3; Appendices S6, S7 353

and S12). A transition to irregular slits is reconstructed somewhere before the most recent 354

common ancestor of Asterotexiales and Aulographaceae (Figs. 1 and 2; Appendix S12). A 355

convergent transition from ostioles to irregular slits appeared along a branch nested in 356

Capnodiales that leads to Asterinales. Developmentally, the irregular slits result from cracking 357

between the adjacent hyphae that make up the scutellum (Fig. 2L, 2M). The precise location of 358

the opening remains unclear until dehiscence. Slit-forming genera sometimes produce ostioles in 359

their asexual or spermatial states (Fig. 2O). In Aulographaceae, and possibly also in species of 360

Lembosia, the slit appears early in development as a line of lightly pigmented cells (Fig. 3), but 361

the crack follows longitudinal walls and propagates beyond the less pigmented area. Regular slits 362

that follow a pre-determined pattern without further propagation are uncommon among 363

Dothideomycetes. Regular slits appear to have originated twice among thyriothecial taxa, being 364

present in Parmularia in Asterinales and in Inocyclus angularis in Asterotexiales, but not in their 365

inferred common ancestor (Appendix S12). Regular slits are also found in taxa producing 366

hysterothecia and lirella (such as Farlowiella charmichaeliana, Hysterographium fraxinii, 367

Melaspileopsis cf. diplasiospora (Nyl.) Ertz & Diederich Stictographa lentiginosa (Lyell ex 368


https://doi.org/10.1101/2020.03.13.989582

p. 18

Leight.) Mudd, which at maturity may look more like apothecia than thyriothecia (Appendix 369

S12). 370

Ancestral ostioles appear to have been retained in Microthyriales and Muyocopronales 371

(Figs. 1 and 4; Appendix S12), but the mode of dehiscence and ancestral states are unknown in 372

Zeloasperisporiales. The common ancestor of Micropeltidaceae probably released spores through 373

an ostiolate thyriothecium which is hypothesized to be derived from the apothecium that is 374

shared by other members of Ostropomycetidae in Lecanoromycetes (Appendix S12). 375

Locule circumference shape—In most Ascomycota, spores are formed in a cavity called a 376

locule (Kirk et al., 2008). In Dothideomycetes, the circumference of the locule within the ascoma 377

or conidioma is commonly round but may be elongate. Elongate locules are scattered across the 378

phylogeny. Thyriothecial species with elongate locules are inferred to be closely related to 379

species with round locules (Appendix S13), contrary to predictions from the current 380

classification (Wu et al., 2011b). For example, ascoma shape, which was used to separate the 381

elongate Lembosia from circular Asterina, fails to predict relationships. Instead, species of both 382

genera appear in both Asterotexiales and in Asterinales (Fig. 1; Appendix S6). In tree topology 383

tests, constraining either Asterina or Lembosia to be monophyletic is rejected with strong support 384

(Appendix S3). 385

Radiate development—Scutella of thyriothecia are either made up of radiating hyphae 386

that can be traced most of the distance from their distal tips to their central point of origin (Figs. 387

1–5), or of hyphae that are not radiate and cannot be traced back to their origin (Figs. 1 and 5). 388

Radiating patterns of thyriothecia are restricted to Dothideomycetes (Fig. 1; Appendices S4 and 389

S14). They are reconstructed to have originated convergently from ancestors with non-radiating 390

patterns. Radiate scutella appear to have arisen independently in Asterinales and in 391


https://doi.org/10.1101/2020.03.13.989582

p. 19

Muyocopronales (Appendix S14). One or multiple additional transitions to radiate thyriothecia 392

may have occurred among Asterotexiales, Aulographaceae, and in the clade including 393

Microthyriales and Zeloasperisporiales (Table 3; Appendix S14). Non-radiating thyriothecial 394

patterns also appear to have originated convergently, being found in distantly related species in 395

Capnodiales in Dothideomycetes, and in Micropeltidaceae in Ostropomycetidae (Fig. 1; 396

Appendices S4 and S14). 397

Phylogenetic distribution of hyphal branching patterns among thyriothecial scutella—398

Likelihood consistently reconstructs pseudoparenchymatous sporocarp walls of polyhedral cells 399

as the ancestral state at the base of Dothideomycetes and Lecanoromycetes, (Figs. 1 and 5, Table 400

3; Appendix S15). However, corresponding BayesTraits reconstructions are equivocal 401

(Appendices S6, S7 and S15). Among thyriothecial fungi, branching patterns of the hyphae 402

forming the scutellum wall are usually distinctive compared with the untraceable hyphae in the 403

more widespread pseudoparenchymatous ascomatal walls of most Dothideomycetes. 404

Scutella with isotomous overlapping branching are reconstructed as possibly ancestral at 405

the base of Clade A in Asterotexiales (proportional likelihood 68%, BayesTraits 52%) and for 406

Asterinales after the divergence of Parmularia styracis (proportional likelihood 100%, 407

BayesTraits 56%), with increasing support for reconstructions with this state near the tips of the 408

tree (Table 3; Appendices S6, S7 and S15). Isotomous branching patterns in thyriothecia expand 409

the scutellum circumference mainly by duplicating files of cells through apical, nearly equal, 410

isotomous dichotomies, resulting first in a “Y” shaped cell with a basal septum, and then in three 411

cells, after additional septa delimit the two arms of the “Y” as separate cells (Figs. 2A, 2B, 2H 412

and 5B). In some cases, one of the two branches overlaps the other, growing downward and 413

disappearing below its neighbors (Figs. 2D, 2I and 5C). Septa are sometimes aligned around part 414


https://doi.org/10.1101/2020.03.13.989582

p. 20

of the circumference of the scutellum, resulting in partial concentric rings of septa (cf. Fig. 3.8 415

"d" in Hofmann 2009). Pigmentation in older parts of the scutellum contrasts with more 416

translucent tips at the margin of scutellum hyphae (Figs. 2H, 2J and 2K). 417

Scutella with pseudomonopodial branching are restricted to Aulographaceae. The 418

common ancestor of Aulographaceae is reconstructed with pseudomonopodial branching with 419

99% proportional likelihood and 28% posterior probability (Table 3; Appendices S6, S7 and 420

S15). In this growth form, the tips of dominant hyphae grow at the circumference of the 421

expanding scutellum, sequentially producing short lateral branches (Figs. 3B inset, 3D and 5E) 422

that overlap with neighboring hyphae. Hyphae curve as they grow, and septa only form in the 423

oldest hyphal segments. This results in a scutellum of irregular, multi-lobed cells (Fig. 5E). 424

Distal tips at the appressed margin are rounded, and uniform in diameter (Figs. 3B, 3D and 3H). 425

Within Aulographaceae, the construction of scutella is similar in Lembosina (Fig. 3B) and 426

Aulographum (Fig. 3D; Appendix S4). 427

Scutellum hyphae in some thyriothecial species in Venturiales and Capnodiales in 428

Dothideomycetes, and in all Micropeltidaceae in Lecanoromycetes are “untraceable” (Figs.1, 3A, 429

and 3C). Scutella may show some evidence of radiate construction, but details of hyphal 430

branching are obscured by dark pigmentation, or curved or meandering scutellum hyphae that 431

overlap and obscure one another, resulting in a “textura intricata" or “textura epidermoidea” 432

(Kirk et al., 2008; Figs. 3A, 3C and 5F). 433

In Microthyriales, scutellum hyphae usually branch dichotomously without overlap. 434

While their branching is usually isotomous, anisotomous branching also occurs, producing 435

hyphal tips of unequal width (Figs. 4B inset, 4D, 5B and 5D). A septum sometimes forms at the 436

base of only one of the two new hyphal tips, resulting in a roughly 'L' shaped proximal cell (Figs. 437


https://doi.org/10.1101/2020.03.13.989582

p. 21

4B and 5D; cf. Fig. 15E Wu et al. 2011). The anisotomous expansion is reconstructed in the 438

common ancestor of Zeloasperisporiales with 81% proportional likelihood (Fig. 1) but only 21% 439

posterior probability from BayesTraits (Appendices S6 and S7). Septa are angular (Figs. 4B, 4G, 440

4H and 5D) and constrictions at septa are infrequent, resulting in trapezoidal to nearly 441

rectangular cells with sharp angles. Partially aligned septa in adjacent hyphae sometimes result in 442

a pattern of incomplete concentric rings. Early stages of thyriothecial development may be 443

isotomous in Microthyriaceae (Figs. 4C, 4I, 4J and 5D) making the distinction between 444

anisotomy and isotomy most visible in fully grown structures (Fig. 5D). 445

Scutellum margins—Thyriothecia are unusual in that the margins of each scutellum are 446

pressed to the surface of a host--a leaf or a lichen--and the characters of the hyphae at the 447

scutellum margin differ across clades. Other kinds of sporocarps lack a direct homolog to the 448

scutellum margin, because their outer walls curve up from the substrate. In Asterotexiales, 449

Asterinales, Aulographaceae, in species of Stomiopeltis, and in Muyocopronales, the scutellum 450

margins often look crenulate or finely scalloped, because the dome-shaped hyphal tips bulge out 451

around the circumference (Figs. 2H, 2J, 3C and 3D; Appendix S16). In some Stomiopeltis-like 452

species (Fig. 3A), tips of hyphae are free; we interpret this as a variant of a crenulate margin 453

(Table 2). In other species, for example in Prillieuxina baccharidincola (Fig. 2K), the scutellum 454

hyphae anastomose with other superficial hyphae on the leaf surface, so that the margin of the 455

scutellum is poorly defined and continuous with the surface mycelium. In Microthyriales and 456

Zeloasperisporiales, scutellum margins are often entire, the scutellum hyphae ending in truncate 457

tips aligned around the circumference (Figs. 4D, 4F and 4L). In Microthyrium, the hyphal tips of 458

scutella that have finished radial expansion will narrow and continue to grow, forming a short 459


https://doi.org/10.1101/2020.03.13.989582

p. 22

fringe at the scutellum periphery (Figs. 4B and 4L). Fringe hyphae appear to be more tightly 460

appressed to the host surface than central portions of scutella. 461

Lateral appressoria—Appressoria are widely distributed across thyriothecium-forming 462

Dothideomycetes and vary in their morphology and position. Conspicuous, pigmented, lateral 463

appressoria are only produced by Asterinales and Asterotexiales (Figs. 2P–2S; Appendices S4 464

and S17). The appressoria are usually unicellular (Figs. 2P and 2R) but in some species they may 465

be two-celled (Figs. 2Q and 2S) (Hofmann, 2009, Figs. 3.8; 3.14; 3.21; 3.24; 3.31; 3.56). Lateral 466

appressoria may be swollen, lobed or minimally differentiated, and they branch from surface 467

hyphae. We coded thyriothecial taxa of Asterinales and Asterotexiales that lack appressoria or 468

produce them below the scutellum rather than on surface hyphae as absent for this character 469

(Table 1). 470

Other kinds of appressoria appear in other thyriothecial clades. All Aulographaceae 471

examined have intercalary appressoria on swollen cells with a conspicuous melanized ring (Fig. 472

3G), and strains isolated in pure culture form an appressorium regularly in each segment of 473

somatic hyphae. Species of Stomiopeltis (Venturiales) form infrequent, intercalary appressoria 474

(Figs. 3E and 3F). The hyphae bearing the appressoria may be swollen (Figs. 3E and 3G). 475

Inconspicuous, hyaline, intercalary appressoria are produced by some Microthyriales (Figs. 4E 476

and 4K). BayesTraits reconstructs lateral appressoria as present in the most recent shared 477

ancestor of Asterotexiales, and in the most recent shared ancestor of Asterinales, while 478

likelihood reconstructs them as ancestral only for lineages that have diverged more recently from 479

one another (Table 3; Appendix S17). 480

Initiation of thyriothecia—Development of thyriothecia begins with distinctive, lineage-481

specific patterns of coordinated hyphal growth and septation in the species where observation 482


https://doi.org/10.1101/2020.03.13.989582

p. 23

was possible. We observed different developmental stages of 13 taxa (Table 1) and analyzed 483

development for 14 additional taxa from published illustrations (Appendix S4). Otherwise, data 484

on development is scarce and ancestral state reconstructions are equivocal where data are 485

missing (Appendices S4, S6, S7 and S18). 486

In Asterinales and Asterotexiales a single “generator hypha” (Hofmann, 2009) initiates 487

the thyriothecia by forming multiple, closely spaced intercalary septa (Figs. 2A–2C, 2H–2J and 488

5C). Each of the delimited cells then gives rise to transverse, adjacent, dichotomizing hyphal tips 489

that together form the scutellum. The generator hypha remains above the dorsal surface of the 490

mature scutella and is visible even in mature thyriothecia (Figs. 2I–2J and 5C). Ascomata in one 491

species of Asterotexiales, Rhagadolobiopsis thelypteridis, are initiated directly over host 492

stomata, as shown by Guatimosim et al. (2014) and in Fig. 2G here; in Asterotexis 493

cucurbitacearum, they are initiated directly from ascospores (Figs. 2E and 2F) (shown 494

previously by Guerrero et al., 2011; Guatimosim et al., 2015). 495

Of all the Aulographaceae observed, three species of Lembosina and one species of 496

Aulographum initiate their ascomata with coordinated development from multiple generator 497

hyphae. Adjacent intercalary portions of the multiple generator hyphae develop abutting, 498

transverse branches that contribute to forming a single scutellum (Figs. 3H and 5E). At a later 499

stage, radially arranged hyphae form pseudomonopodial branching units that cover the upper 500

surface of the hyphal aggregate (Fig. 3D). 501

In contrast to the widespread intercalary origin among Asterinales and Asterotexiales, all 502

Microthyriales examined initiate their ascomata at the tip of a generator hypha (terminal), located 503

on a short lateral branch of a surface hypha (Figs. 4C, 4I and 5D). The generator hypha does not 504

become highly septate, but instead its tip gives rise to a succession of dichotomously branched, 505


https://doi.org/10.1101/2020.03.13.989582

p. 24

closely appressed hyphal tips that develop into a scutellum. The scutellum in Microthyriales 506

overgrows the generator hypha; the generator hypha can no longer be seen in older scutella (Figs. 507

4B, 4G and 4H). 508

Case studies using thyriothecial fossils—Morphological characters of thyriothecial 509

scutella and mycelia offer a range of variation, allowing phylogenetically informed 510

interpretations of published fossils. We looked for the oldest geological occurrence of 511

thyriothecia, and also selected two other thyriothecial fossils that have a reasonably complete set 512

of morphological characters (Fig. 6; Appendix S4). Each phylogenetic analysis that included a 513

fossil yielded more than one equally parsimonious tree, given the morphological dataset and the 514

rDNA likelihood constraint tree. All trees from the morphological datasets were the same length, 515

232 steps, whether or not one of the fossils was included. 516

Triassic dispersed scutellum–The oldest evidence of a radiate fungal scutellum is a fossil 517

from the Early Triassic of India (Induan, ~251 Ma) referred to as a "fungal thallus" but not 518

identified to species or genus. The fossil was from a palynological sample that was rich in land 519

plant cuticles and spores (Mishra et al. 2018, Fig. 8j). It shares five characters with extant 520

thyriothecial taxa (Appendix S4). Like extant radial thyriothecia, it is made up of hypha-like 521

filaments that are closely appressed along their sides. The filaments are regularly septate into 522

trapezoidal cells that form partial, concentric rings. Some of the filaments dichotomize. A central 523

circular opening appears to be an ostiole surrounded by a ring of very dark cells. Where the 524

scutellum margin is in focus, it appears to be crenulate (Fig. 6A; Appendix S4). 525

We found 28 equally parsimonious positions for this fossil relative to the constraint tree. 526

Based on its five coded characters, the fossil could cluster with thyriothecial taxa in Asterinales, 527

Asterotexiales, or Microthyriales. Missing character state data from some extant taxa increased 528


https://doi.org/10.1101/2020.03.13.989582

p. 25

the number of possible, equally parsimonious positions for the fossil to include positions among 529

the asexual taxa of Zeloasperisporiales. A strict consensus of the 28 trees resulted in a polytomy 530

of 45 lineages, all stemming from the most recent ancestor of all Dothideomycetes (Fig. 1; 531

Appendix S19). 532

Microthyriaceous fossil–Trichothyrites setifer (Cookson) Saxena & Misra, a fossil from 533

the early Eocene (Ypresian, 56–47.8 Ma) sediments of India is illustrated by Monga et al. (2015) 534

(Fig. 6B). While this specimen was from dispersed material, rather than in situ on a leaf surface, 535

it shares seven characters with extant Lichenopeltella pinophylla and six characters with other 536

thyriothecial Microthyriales (Appendix S4). It has a circular, multicellular thallus. The radiate 537

scutellum has an entire margin and shows the presence of a lower wall (Fig. 6B, arrowhead). The 538

scutellum branching pattern is mostly isotomous, but also shows anisotomous dichotomies. Its 539

central ostiole is surrounded by papillate cells. Papillate ostioles are uncommon but occur in 540

Chaetothyriothecium elegans and L. pinophylla. However, C. elegans lacks a differentiated 541

lower wall. 542

We found 16 equally parsimonious trees resolving T. setifer in our phylogeny of living 543

fungi. The strict consensus position of this fossil suggests it shares affinities with the clade 544

containing Microthyriales and Zeloasperisporiales (Fig. 1; Appendix S20). Among the 16 545

reconstructions, T. setifer is alternatively placed in Microthyriales; as sister to Microthyriales 546

(10/16); as sister to asexual taxa in Zeloasperisporiales (that are missing data for characters of 547

the sporocarp) (3/16); as the sister to the perithecioid Natipusillales (2/16); or as the sister to all 548

these lineages (Fig. 1; Appendix S20). 549

Asterina fossil–The early Eocene (56-48 Ma) Asterina eocenica was found on leaves of 550

Chrysobalanus L. (Fagaceae Dumort.) and consists of circular, multicellular thalli and mycelia 551


https://doi.org/10.1101/2020.03.13.989582

p. 26

that are preserved in different states of development (Appendix S4), allowing comparison with 552

extant Asterinales and Asterotexiales (Dilcher, 1965). The fossil thyriothecium lacks a lower 553

wall, and dehiscence was by means of irregular slits. The pattern of branching of the scutellum is 554

isotomous, with overlapping hyphae, and the margin is crenulate, as in living Asterina. The 555

sporocarp was initiated with an intercalary generator hypha (Fig. 6C). The mycelium bears 556

lateral appressoria. 557

A strict consensus of the 23 equally parsimonious trees places A. eocenica in a polytomy 558

at the base of Dothideomycetes (Appendix S21). Asterina eocenica shares all 11 morphological 559

characters with three extant taxa in Asterinales and with nine in Asterotexiales (Fig. 6C; 560

Appendix S4). Consistent with the shared characters, 18 of the individual phylogenies show A. 561

eocenica within or as sister to Asterotexiales (Appendix S22), and five show it in, or as sister to 562

Asterinales (Appendix S23). 563

564

DISCUSSION 565

Results from our molecular phylogeny of 320 species including 59 species of 566

thyriothecial taxa combined with critical analysis of 11 morphological characters of extant 567

Dothideomycetes offer grounds for cautious optimism for incorporating thyriothecial fossil data 568

into the broader picture of fungal evolution. Ancestral state reconstructions show that convergent 569

evolution of thyriothecia in Dothideomycetes likely began among fungi that produced 570

pseudoparenchymatous-walled sporocarps on surfaces of plant cuticles. Our reconstructions 571

suggest that radiate scutella arose at least three times within epiphyllous Dothideomycetes. As 572

radiate thyriothecial forms evolved, patterns of hyphal branching and septation diversified, 573

giving rise to distinctive scutella. As the following fossil case studies show, a phylogenetic 574


https://doi.org/10.1101/2020.03.13.989582

p. 27

approach takes into account morphological variation within orders and convergence among 575

orders. We apply the approach to fossils but the same methods could be used to evaluate possible 576

relationships among extant taxa that are difficult to sample using molecular techniques. 577

Case studies: interpreting characters of fossils—A dispersed scutellum as evidence of 578

Triassic Dothideomycetes—Among our sampling of extant taxa, radiate scutella are restricted to 579

Dothideomycetes. Further, ancestral state reconstructions show radiate thyriothecia as a derived 580

character state that arose convergently, but only among epiphyllous Dothideomycetes. Consistent 581

with results from the parsimony analysis, the combination of an ostiole and a scutellum with 582

dichotomous branching that is found in the fossil is ancestral for Asterinales, Microthyriales, and 583

possibly for Asterotexiales. The fossil could represent a member of any of these groups. 584

Although it cannot be classified more precisely, parsimony analysis shows that the scutellum-585

like "fungal thallus" of Mishra et al. provides evidence of Dothideomycetes. The fossil from the 586

Early Triassic (~251 Ma) (2018, Fig. 8j) likely represents the oldest evidence of thyriothecia. 587

Fossil evidence of Microthyriales—Monga (2015, Pl.2 Fig.18) illustrates the fossil 588

Trichothyrites setifer. Like the "fungal thallus" discussed above, T. setifer is from a sample rich 589

in land plant material, but its substrate is unknown. Trichothyrites specimens (synonym of 590

Notothyrites, see Kalgutkar and Jansonius, 2000), interpreted as members of Trichothyriaceae 591

have frequently been reported in the fossil record throughout the Tertiary (Kalgutkar and 592

Jansonius, 2000). Although classified in Microthyriaceae, extant Lichenopeltella shares multiple 593

characters with Trichothyriaceae Theiss., including a scutellum with isotomous and anisotomous 594

dichotomies; a scutellum margin that is entire, a papillate ostiole, and a differentiated lower wall 595

that can be seen through the translucent scutellum (Spooner and Kirk, 1990). Thyriothecia with a 596

papillate ostiole and a lower wall are characteristic of Trichothyriaceae. Anisotomous 597


https://doi.org/10.1101/2020.03.13.989582

p. 28

dichotomies were not present in Lichenopeltella pinophylla, the only extant taxon we sampled 598

with this morphology, but they do occur in the genus and are illustrated for Lichenopeltella 599

arctomiae Pérez-Ort. & T. Sprib. (Fig. 1 in Pérez-Ortega and Spribille, 2009). 600

Although T. setifer is more similar to Lichenopeltella than any other taxa in our dataset, 601

some of its shared characters including ostiolate thyriothecia are reconstructed as ancestral in the 602

larger clade encompassing Zeloasperisporiales, Natipusillales and Microthyriales. Three of six 603

taxa in Zeloasperisporiales are only known from asexual states and are missing data for the 604

sporocarp. A differentiated lower wall also occurs in non-thyriothecial ascomata of 605

Natipusillales. As a result of plesiomorphic characters and missing data, equally most 606

parsimonious placements of T. setifer were: in Microthyriales; as the sister lineage to 607

Microthyriales, Natipusillales or Zeloasperisporiales; and as sister to the clade including all of 608

these taxa. 609

At present, T. setifer (Monga et al., 2015), from the early Eocene (56–47.8 Ma), 610

represents the oldest convincing evidence of the clade including Microthyriales and Venturiales. 611

Investigating Paleocene deposits may very well yield further and older evidence of the 612

divergence of Trichothyriaceae. For more formal testing, the molecular phylogenetic positions of 613

extant taxa of Actinopeltis Höhn., Lichenopeltella, Trichothyrium Speg., and Trichothyrina 614

(Petr.) Petr. should be established using molecular data, and their morphology analyzed for 615

comparison with the fossils. 616

Asterina-like fossils could represent either Asterinales or Asterotexiales—Contrary to our 617

expectations, combining close zmorphological observations and the molecular phylogeny 618

revealed no clade-specific morphological differences between Asterinales and Asterotexiales. 619

Guatimosim et al. (2015) showed that Asterinales and Asterotexiales both include taxa with 620


https://doi.org/10.1101/2020.03.13.989582

p. 29

scutellum dehiscence by irregular cracks, and superficial hyphae bearing lateral appressoria. The 621

orders share crenulate scutellum margins. Lembosia species (characterized by elongate 622

thyriothecial locules) occur nested among both Asterinales and Asterotexiales species with round 623

locules. 624

Developmental and anatomical characters for Asterinales and Asterotexiales were 625

sometimes illustrated in great detail in early works (Arnaud, 1918; Doidge, 1919; Viégas, 1944). 626

More recently, Hofmann's (2009) thesis contains elegant drawings from diverse species, 627

documenting the common intercalary origin of thyriothecia, starting with septation in a generator 628

hypha. At the time of her thesis, the distinction between Asterinales and Asterotexiales had yet to 629

be discovered and Hofmann lacked sequence data to link her observations to clades. Our analysis 630

of species of Asterinales and Asterotexiales shows that development and scutellum branching as 631

Hofmann described are common to both orders. 632

Liu et al. (2017) interpreted Asterotexiales as "Asterinales sensu stricto" and suggested 633

that the Asterinales, represented by a clade of six species and their LSU rDNA sequences, all 634

from Brazil, should be considered "Asterinales sensu lato". However, the six Brazilian 635

Asterinales species that were sequenced by Guatimosim et al. (2015) include Asterina 636

melastomatis (from the epitype specimen of the Asterinales), which means that the ordinal name 637

applies to their clade. If a single sequence had been involved, or if the disputed Brazilian 638

Asterinales were from a clade of fungi commonly amplified from leaf surfaces in other studies, 639

contamination by non-target DNAs might seem a possible explanation for the presence of 640

morphologically similar species in phylogenetically divergent groups. However, the Brazilian 641

sequences give congruent results for species in five genera of thyriothecium forming taxa: 642

Batistinula, Prillieuxina, Parmularia, Asterina, and Lembosia. In a survey of Eucalyptus L'Hér. 643


https://doi.org/10.1101/2020.03.13.989582

p. 30

spp. Crous et al. (2019) found additional thyriothecial species of Thyrinula Petr. & Syd. to be 644

closely related to asexual species of Blastacervulus. Our analysis shows these two genera to be in 645

Asterinales, and future detailed analysis of their scutellum morphology will provide data useful 646

in reconstructing ancestral character states of Asterinales. Other thyriothecial species from leaves 647

of Eucalyptus spp. lack sequence data but are diverse in their scutellum morphology (Swart, 648

1986) and some of these may also represent Asterinales. In the absence of any contradictory 649

evidence, the sequences from Guatimosim et al. (2015) and from Crous et al. (2019) must be 650

assumed to come from their target fungi. 651

It is possible that more molecular data may resolve Asterinales and Asterotexiales as 652

sister groups, consistent with their morphological similarities. Tree topology tests did not rule 653

out a sister relationship between the two orders. Although the orders consistently appear 654

unrelated in published phylogenies, their separation never receives strong bootstrap support or 655

high posterior probabilities (Guatimosim et al., 2015; Firmino, 2016; Liu et al., 2017 and Fig. 1). 656

With cultures now available representing Asterotexiales (Asterotexiaceae sp.2 CBS 143813) and 657

Asterinales (Blastacervulus robbenensis CBS 12478) it should soon be possible to expand 658

available sequences beyond the ~1 kb of 28S ribosomal DNA per taxon that currently limits 659

phylogenetic resolution. If Asterinales and Asterotexiales are not sister groups, their thyriothecial 660

characters represent surprising convergence. If Asterinales and Asterotexiales are sister groups, a 661

phylogenetic analysis of their morphological characters, including dehiscence type, scutellum 662

branching and pattern of initiation of sporocarp, should likely allow unambiguous assignment of 663

fossils to their clade. 664

Asterina eocenica from the Middle Eocene (48.6-37.2 Ma) of western Tennessee 665

(Dilcher, 1965; Elsik and Dilcher, 1974) is the oldest fossil that combines a convincing 666


https://doi.org/10.1101/2020.03.13.989582

p. 31

Asterinales/Asterotexiales suite of characters. The exquisitely preserved scutella of A. eocenica 667

show clear isotomous dichotomies with overlap (Figs. 64 and 65 in Dilcher, 1965). This places 668

the fossil with clades of thyriothecia in which hyphae branch dichotomously at their apices to 669

produce radial scutella (Farr, 1969; Reynolds and Gilbert, 2005; Hofmann and Piepenbring, 670

2011; Guatimosim et al., 2015). Dichotomous branching is infrequent among fungi, where 671

branching is usually initiated some micrometers behind the hyphal apex. Asterina eocenica has 672

nine out of 11 characters shared by Asterinales and Asterotexiales. The development of 673

thyriothecia of A. eocenica is typical of Asterinales and Asterotexiales, with initiation by closely 674

spaced septa forming in a generator hypha. The generator hypha persists on the dorsal side of the 675

scutellum (Figs. 59, 60, 63, 64 and 65 in Dilcher, 1965). Like Asterinales and Asterotexiales, A. 676

eocenica has slit-like dehiscence (Figs. 63 and 65 in Dilcher 1965), an undifferentiated lower 677

wall (Figs. 61, 63, 65 and 66 in Dilcher, 1965), and some elongate, one-celled tapering lateral 678

hyphal projections described as appressoria (Figs. 57 and 59 in Dilcher, 1965). In their otherwise 679

detailed and useful review of fossils, Samarakoon et al. (2019) assign A. eocenica to crown 680

group Asterinales (=Asterotexiales) and with a minimum age as Paleocene, 54 Ma. The 681

Paleocene age appears to be an error because it is inconsistent with an analysis by Elsik and 682

Dilcher (1974), which assigned a more recent Middle Eocene age to the fossil's source material 683

from the Lawrence Clay Pit. None of the characters in A. eocenica justify positioning it in the 684

crown group of either order as opposed to a stem relationship of either order, and as the 685

parsimony analysis shows, it could equally well represent Asterinales and Asterotexiales. A 686

strong implication of the close similarity of Asterinales and Asterotexiales is that even a 687

beautiful, complete fossil like A. eocenica cannot be assigned to one order versus the other. 688

CONCLUSION 689


https://doi.org/10.1101/2020.03.13.989582

p. 32

Much cryptic diversity in Dothideomycetes consists of minute leaf dwelling fungi (Arx 690

and Müller, 1954; Müller and von Arx, 1962; von Arx and Müller, 1975). As our analysis shows, 691

epiphyllous fungi together with lichenized, lichenicolous and aquatic fungi are all relevant to the 692

evolution of thyriothecia, but they have been studied by different mycologists who sequenced 693

different loci for phylogenetic analysis (Dhanasekaran et al., 2006; Miadlikowska et al., 2006; 694

Etayo, 2010). The sequencing of the 5' end of the LSU was common across most studies, but 695

choices of other loci varied. We anticipate that sequencing of more Dothideomycetes genomes, 696

including some from the new isolates from this study from cultures available through the 697

Westerdijk Institute, will contribute to improved resolution of relationships among extant taxa. 698

Our molecular phylogeny of thyriothecial taxa and their closest relatives leads to testable 699

predictions about the sequence of early events in their evolutionary history. A remaining 700

limitation of this study is that we could include only a small fraction of living fly-speck species. 701

The fidelity of association of characters with extant lineages should be tested across a broader 702

range of taxa using morphological and molecular phylogenetic analysis. Undoubtedly, further 703

study will help reveal convergence and variation, and perhaps additional distinctive 704

synapomorphies. Our analyses suggest that analyzing and coding scutellum characters can link 705

fossils on leaf cuticle to phylogenetic lineages, offering rewarding insights into fungal evolution 706

through geological time. However limited fossilized characters may be, documenting equivalent 707

anatomical features in extant taxa helps communicate the significance of fossil morphologies, 708

increasing their value for paleomycology. 709

ACKNOWLEDGEMENTS 710

This research was enabled in part by support provided by WestGrid (www.westgrid.ca), 711

Compute Canada Calcul Canada (www.computecanada.ca) and CIPRES Science Gateway 712


https://doi.org/10.1101/2020.03.13.989582

p. 33

(www.phylo.org). The authors also wish to express their gratitude to UBC Bioimaging facilities 713

for use of their fluorescence microscope, UBC InterLibrary Loan service for providing crucial 714

literature, Robert Lücking for gift to UBC Herbarium of lichenized taxa in genera 715

Microtheliopsis and Asterothyrium, OTS and Conagebio, and more specifically Francisco 716

Campos Rivera and Bernal Mattarita Carranza for allowing collection of tropical thyriothecium 717

forming fungi in the research station of La Selva in Costa Rica. Funding for this research was 718

provided by a Natural Sciences and Engineering Research Council of Canada Discovery Grant 719

RGPIN-2016–03746 to MLB. 720

721

722

723

724

725

726


https://doi.org/10.1101/2020.03.13.989582

p. 34

FIGURE LEGENDS 727

Figure 1. Phylogeny and character state transitions leading to convergent evolution of fly-speck 728

fungal morphology. Numbers on boxes to the left match labeled transitions between 729

reconstructed ancestral states that received > 0.7 proportional likelihood in the maximum 730

likelihood phylogeny to the right. Thickened branches in the tree indicate bootstrap support > 731

70% and posterior probabilities > 0.95. Arrowheads indicate the most parsimonious consensus 732

positions (for A. eocenica, alternative consensus positions) of fossils. Green boxes surround 733

clades that include taxa with radiate thryiothecia. Solid black circles after taxon names indicate 734

thyriothecial species; question marks indicate possible thryiothecia still in need of anatomical 735

analysis; slashed circles indicate taxa known only from their asexual state; and tick marks 736

indicate thyriothecia with differentiated lower walls. 737

Figure 2. Morphological characters of Asterinales and Asterotexiales in face view. (A–J) 738

Development. Initiation of ascomata in Asterinales and Asterotexiales usually involves 739

intercalary septation of a superficial generator hypha but is sometimes (D) uninterpretable, (E, F) 740

from spores, or (G) from stomata. (H–J) The highly septate generator hypha usually persists 741

above the scutellum throughout development (white arrowhead). (A–D, H–J) The generator 742

hypha gives rise to the scutellum through (i) the formation of new, lateral hyphal tips that branch 743

with isotomous dichotomies, occasionally (D, I, inset) with one tip that overlaps the other (o). 744

(H, J) The appressed scutellum margin shows lightly pigmented tips and is usually crenulate at 745

maturity (black arrowhead). (K) Hyphae of the appressed marginal of scutella Prillieuxina 746

baccharidincola instead anastomose with surrounding mycelium (white arrowhead). (I, K, L–O) 747

Dehiscence. (I, K, L–M) Asterinales and Asterotexiales ascomata usually dehisce with irregular 748

slits. (K) Mature scutellum with irregular radial slits, black arrowheads. (L-M) Irregular slits 749


https://doi.org/10.1101/2020.03.13.989582

p. 35

follow the longitudinal walls of scutellum cells (white arrowheads). (N-O) Ostiolate openings are 750

less common. (N) Ostiole in young scutellum. (O) Asexual or spermatial stage, with evenly 751

pigmented cells lining ostiole. (P–S) Lateral appressoria showing characteristic melanized ring at 752

lower focal plane (insets, representing area in the dashed grey boxes). Scales (A–D, H–I) 10 µm; 753

(E–G) 20 µm; (J–K) 50 µm; (L–S) 5 µm. (A, I, J, P, Q) Asterina melastomatis VIC 52822, 1, 4, 754

6, 2 and 2 focal planes (f.p.) respectively; (B) Asterina chrysophylli VIC 42823, 4 f.p.; (C, O) 755

Asterotexiaceae sp. CBS 143813, 2 f.p. each; (D) Hemigrapha atlantica BR 14014, 1 f.p.; (E, F) 756

Asterotexis cucurbitacearum VIC 42814, 1 f.p. each; (G, L) Rhagadolobiopsis thelypteridis 757

EG156; (H, R) Batistinula gallesiae VIC 42514, 10 and 2 focal planes (f.p.) respectively; (K N), 758

Prillieuxina baccharidincola VIC 42817; (M) Asterotexiaceae sp. UBC F33036, 11 f.p.; (S) 759

Asterina sp. (VUL. 341b), 2 f.p. 760

Figure 3. Morphological characters of Aulographaceae and Stomiopeltis spp. s.l. (Venturiales) in 761

face view. (A–D) Scutella. (A) Mature Stomiopeltis-like scutellum. Hyphae are radiate but 762

untraceable due to frequent overlap of neighboring hyphae. The ostiole is wide (white 763

arrowhead) and at the distal appressed margin, hyphal tips are free (black arrowhead). (B) 764

Mature Aulographaceae scutellum with radiate, pseudomonopodial hyphal branching. Inset (m), 765

a close up of hyphal branching in the area in the dashed grey box. An irregular slit for dehiscence 766

would form in the elongate, lighter area (white arrowhead). The appressed distal margin has free 767

hyphal tips. (C) Early development of a Stomiopeltis-like scutellum. The appressed distal margin 768

is irregularly crenulate (arrowhead). (D) Young Aulographaceae scutellum showing multiple, 769

intercalary generator hyphae, pseudomonopodial hyphal branching, and an irregularly crenulate 770

appressed margin (arrowhead). (E–G) Intercalary appressoria with a melanized ring 771

(arrowheads). (H) Initiation of Aulographaceae ascomata; coordinated intercalary septation of 772


https://doi.org/10.1101/2020.03.13.989582

p. 36

multiple generator hyphae (arrowheads) gives rise to scutella. Scales, (A, B) 20 µm; (C, D) 10 773

µm; (E–H) 5 µm. (A, C, F) Stomiopeltis sp. UBC F33041, 5, 6 and 1 stacked focal planes (f.p.) 774

respectively. (E) Stomiopeltis sp. UBC F33040, 1 f.p. (B) Lembosina aulographoides CBS 775

143809, 4 f.p.; (D, G, H) Aulographum sp. CBS 143545, 1 f.p. 776

Figure 4. Morphological characters of Microthyriales in face view. (A) Calcofluor white staining 777

of Microthyrium sp. reveals radially arranged asci (arrowhead) in a hymenium below the 778

scutellum. The inset shows uneven fluorescence of scutellum hyphae due to dark pigments. (B, 779

G, H) Mature scutella are radiate and insets show details of anisotomous (a) and isotomous (i) 780

hyphal branching. Dehiscence is with an ostiole that is lined by small, darkly pigmented cells. 781

(C, D, I, J) Early development of scutella. The appressed margins of young scutella are entire. (I) 782

The scutellum is initiated at the tip of a lateral generator hypha. (C, D, J) New hyphal tips arise 783

from the generator hypha with closely spaced, isotomous, dichotomous branches. (D, J) The 784

young scutella overgrow and conceal the generator hyphae. (E, K) Intercalary appressoria in a 785

regular (E) or swollen (K) hyphal segment, showing melanized rings (arrowheads). (F, L) 786

Margins of mature scutella are considered to be entire if hyphal tips are blunt, at least in part 787

(arrowheads) even if narrow portions of the tip hyphae extend further into an irregular, 788

meandering fringe as in (B, L). Scales, (A–C) 20 µm; (D) 10 µm; (E–I) 5 µm. (A) Microthyrium 789

sp., 44 focal planes (f.p.); (B–F), Microthyrium macrosporum CBS 143810, with 4, 3, 1, 1 and 1 790

focal planes (f.p.), respectively; (G), Lichenopeltella pinophylla, CBS 143816, 14 f.p.; (H–L), M. 791

ilicinum CBS 143808, with 4, 1, 3 and 2 f.p. respectively. 792

Figure 5. Scutellum initiation and interpretation of branching. (A) Ancestral Dothideomycetes 793

had pseudoparenchymatous walls (a–b). (B) Radiate scutella in thyriothecia grow by successive 794

dichotomies followed by cross-wall formation. Isotomous dichotomies produce daughter hyphae 795


https://doi.org/10.1101/2020.03.13.989582

p. 37

of similar width. Hyphae occasionally overlap so that one file of cells seems to vanish (top); 796

anisotomous dichotomies produce daughter hyphae that differ in width and rarely overlap 797

(bottom). (C) Asterotexiales/Asterinales pattern. Scutellum hyphae show isotomous dichotomies, 798

and some hyphae overlap in some specimens (c, e) but not in all (d1-2) specimens. Overlap can be 799

missing in young specimens (e1). Initiation of the scutellum often begins with a single intercalary 800

generator hypha. The generator hypha persists above the scutellum at maturity (c, d1-2, e1-2). (D) 801

Microthyriales pattern. Young scutella are initiated at the tip of a short lateral generator hypha 802

(g1, h1). The generator hypha gives rise to scutellum hyphae that eventually overgrow it as they 803

branch with isotomous dichotomies (g1-2, h1-2). The generator hypha is not visible in the mature 804

scutellum (g2-3, h2-3). In mature scutella, hyphae show anisotomous and isotomous dichotomies 805

and hyphae do not overlap (g3, h3). (E) Aulographaceae pattern. Scutella with pseudomonopodial 806

branching (i2). The scutellum is usually initiated by coordinated growth of multiple adjacent, 807

intercalary generator hyphae (i1). (F) Stomiopeltis (UBC F33041) pattern. Scutellum hyphae are 808

generally radiate but they overlap and are irregular (j–l) and they cannot be traced to generator 809

hypha or from the center of the scutellum to the margin. Sources of drawings: (a) Fumiglobus 810

pieridicola UBC-F33205; (b) Pleospora herbarum UBC-F4461; (c) Batistinula gallesiae VIC 811

42514; (d) Asterotexiaceae sp. UBC F33036; (e) Asterina chrysophylli VIC 42823; (f) 812

Asterotexiaceae sp. CBS 143813; (g) Microthyrium ilicinum CBS 143808; (h) from M. 813

macrosporum CBS 143810, (i) Aulographum sp. CBS 143545, (j) Stomiopeltis sp. UBC F33041, 814

(k) Stomiopeltis sp. CBS 143811 and (l) Peltaster cerophilus redrawn from Fig. 2M in 815

(Medjedović et al., 2014). 816

Figure 6. Reproduction of fossil illustrations used in our case studies. (A) Triassic dispersed 817

“fungal thalli,” courtesy of Mishra et al. (2018), reproduced with the permission of Springer 818


https://doi.org/10.1101/2020.03.13.989582

p. 38

Nature; B.S.I.P. Slide No.15524, J36-4. (B) Early Eocene dispersed Trichothyrites setifer with 819

lower wall (arrowhead) seen through the translucent scutellum, reproduced with permission from 820

Monga et al. (2015); B.S.I.P. slide no. 15297, U41/3. (C) Middle Eocene Asterina eocenica on 821

cuticle of Chrysobalanus sp. described in Dilcher (1965), illustration courtesy Steven 822

Manchester, reproduced from Taylor et al. (2015), with permission from Elsevier; Florida 823

Museum of Natural history, l.f. 87. Scales, 20µm. 824

SUPPORTING INFORMATION 825

Additional Supporting Information may be found online in the supporting information section at 826

the end of the article 827

Appendix S1. Isolates included in this study; names in bold indicate newly contributed 828

collections and voucher/strain numbers beginning with 'CBS' and followed by '*' represent 829

cultures newly deposited in the Westerdijk Institute. When LSU and SSU were derived from two 830

different collections, bold indicates the voucher/strain name that is used in phylogenies. 831

Accessions labeled 'JGI' refer to sequences extracted from JGI genomes project, for which no 832

GenBank accession exists. 833

Appendix S2. JGI sequence data retrieved from MycoCosm portal. 834

Appendix S3. Results from CONSEL for six topologies tested against most likely tree. 835

Appendix S4. Matrix of characters used in ancestral character state reconstructions and in 836

parsimony analyses of fossils. 837

Appendix S5. Consensus tree from Mr. Bayes analysis with four runs of eight chains each, 838

running 160 million generations and sampling every 5000 trees, from which we discarded 50% 839

of the samples as burn-in. Posterior probabilities mapped on branches, clades are labeled and 840

ordered as in Fig. 1. 841


https://doi.org/10.1101/2020.03.13.989582

p. 39

Appendix S6. Maximum likelihood phylogeny labeled with codes for nodes reconstructed in 842

ancestral character state analyses. Taxa forming thyriothecia are indicated with pink diamonds. 843

Most likely tree obtained out of 4000 independent ML searches of a 4552 bp alignment of LSU 844

and SSU rDNA data for 320 taxa, with bootstrap support > 70% in red and a posterior 845

probability > 0.95. 846

Appendix S7. Detailed output of ancestral state reconstruction for each of 208 nodes as 847

proportional likelihoods (white rows) or posterior probabilities (grey rows). Node labels in 848

column 1 are designated in Fig. 1. Nodes labeled in Column 2 are designated in the phylogeny in 849

Appendix S6. 850

Appendix S8–S18. Ancestral character state reconstructions for 11 morphological characters, 851

given the most likely DNA sequence topology. Reconstructions from MK1 analysis are shown 852

above branches as proportional likelihoods in pie charts with red outlines. Reconstructions from 853

BayesTraits are shown below branches as posterior probabilities in pie charts with black outlines. 854

In each tree, names in grey indicate taxa that could not be coded for the character, with ancestral 855

states that could not be reconstructed. Bootstrap support >0.7 is reported in red above branches 856

and posterior probabilities >0.95 are in black below branches. 857

Appendix S8. Ancestral character state reconstructions for substrate (Sub). 858

Appendix S9. Ancestral character states reconstruction (Lic), as lichenized or non-lichenized. 859

Appendix S10. Ancestral character state reconstruction for sporocarp type (Spo). 860

Appendix S11. Ancestral character state reconstruction for differentiated or undifferentiated 861

lower wall of sporocarp (Low). 862

Appendix S12. Ancestral character state reconstruction for sporocarp dehiscence (Deh). 863

Appendix S13. Ancestral character state reconstruction for locule circumference (Loc). 864


https://doi.org/10.1101/2020.03.13.989582

p. 40

Appendix S14. Ancestral character state reconstruction for presence or absence of a radiate 865

sporocarp (Rad). 866

Appendix S15. Ancestral character state reconstruction for scutellum branching (Bra). 867

Appendix S16. Ancestral character state reconstruction for margin of sporocarp (Mar). 868

Appendix S17. Ancestral character state reconstruction for the presence or absence of lateral 869

appressoria on superficial mycelium (App). 870

Appendix S18. Ancestral character state reconstruction for Sporocarp initiation (Ini): terminal 871

on generator hypha, intercalary on generator hypha, from spore, from host stomata or from 872

multiple generator hyphae. 873

Appendix S19. Consensus of 28 equally parsimonious phylogenetic positions of the Triassic 874

“fungal thallus” from India described in Mishra et al. (2018). 875

Appendix S20. Consensus of 16 equally parsimonious phylogenetic positions of Trichothyrites 876

setifer from the Eocene of India described in Monga et al. (2015). 877

Appendix S21. Consensus of 23 equally parsimonious phylogenetic positions of Asterina 878

eocenica from the Eocene of USA, described in Dilcher (1965). 879

Appendix S22. Consensus of the 18 equally parsimonious phylogenetic trees placing Asterina 880

eocenica with taxa in Asterotexiales. 881

Appendix S23. Consensus of the 5 equally parsimonious phylogenetic trees placing Asterina 882

eocenica with taxa in Asterinales. 883

884

885


https://doi.org/10.1101/2020.03.13.989582

p. 41

LITERATURE CITED 886

887

Arnaud, G. 1917. Sur la famille des microthyriacées. Compte Rendu de l’Académie des Sciences 888

Paris 164: 574–577. 889

Arnaud, G. 1918. Les Asterinées. Annales de l'École Nationale d'Agriculture de Montpellier 16: 890

1–288. 891

Arnaud, G. 1925. Les Asterinées. IVe partie. Annales de l’École National d'Agriculture de 892

Montpellier, série 10 5: 643–722. 893

Arx, J. A. v., and E. Müller. 1954. Die Gattungen der amerosporen Pyrenomyceten. Beiträge zur 894

Kryptogamenflora der Schweiz 11: 1–434. 895

Batista, A. C. 1959. Monografia dos fungos Micropeltaceae. Publicações do Instituto de 896

Micologia da Universidade do Recife 56: 1–519. 897

Batista, A. C., J. L. Bezerra, T. T. Barros, and F. B. Leal. 1969. Sobre um novo gênero de 898

Microthyriaceae da Nova Caledônia. Instituto de Micologia, Universidade do Recife 637: 1–11. 899

Bercovici, A., A. Hadley, and U. Villanueva-Amadoz. 2009. Improving depth of field resolution 900

for palynological photomicrography. Palaeontologia Electronica 12: 1–12. 901

Clements, F. E., and C. L. Shear. 1931. The genera of fungi, second ed. New York. 902

Crous, P. W., M. J. Wingfield, R. Cheewangkoon, A. J. Carnegie, T. I. Burgess, B. A. 903

Summerell, J. Edwards, et al. 2019. Foliar pathogens of eucalypts. Studies in Mycology 94: 125–904

298. 905

Darriba, D., G. L. Taboada, R. Doallo, and D. Posada. 2012. jModelTest 2: more models, new 906

heuristics and parallel computing. Nature Methods 9: 772–772. 907


https://doi.org/10.1101/2020.03.13.989582

p. 42

Dhanasekaran, V., R. Jeewon, and K. D. Hyde. 2006. Molecular taxonomy, origins and evolution 908

of freshwater ascomycetes. Fungal Diversity 23: 351–390. 909

Dilcher, D. L. 1965. Epiphyllous fungi from Eocene deposits in western Tennessee, U.S.A. 910

Palaeontographica Abteilung B 116: 1–54. 911

Doidge, E. M. 1919. South African Microthyriaceae. Transactions of the Royal Society of South 912

Africa 8: 235–282. 913

Doidge, E. M. 1942. A revision of the South African Microthyriaceae. Bothalia 4: 273–420. 914

Ducomet, V. 1907. Recherches sur le développement de quelques champignons parasites à thalle 915

subcuticulaire. Thèse doctorale, Faculté des sciences de Paris, Rennes, France. 916

Ellis, J. P. 1976. British Microthyrium species and similar fungi. Transactions of the British 917

Mycological Society 67: 381–394. 918

Elsik, W. C. 1978. Classification and geologic history of the microthyriaceous fungi. IVth 919

International Palynological Conference (1976–77), Lucknow, India. 920

Elsik, W. C., and D. L. Dilcher. 1974. Palynology and age of clays exposed in Lawrence clay 921

pits, Henry County, Tennessee. Palaeontographica Abteilung B: 65–87. 922

Ertz, D., and P. Diederich. 2015. Dismantling Melaspileaceae: a first phylogenetic study of 923

Buelliella, Hemigrapha, Karschia, Labrocarpon and Melaspilea. Fungal Diversity 71: 141–164. 924

Etayo, J. 2010. Lichenicolous fungi from the western Pyrenees. V. Three new ascomycetes. 925

Opuscula Philolichenum 8: 131–139. 926

Farr, M. L. 1969. Some "black mildew", "sooty mold", and "fly-speck" fungi and their 927

hyperparasites from Dominica. Canadian Journal of Botany 47: 369–381. 928

Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. 929

Evolution 39: 783–791. 930


https://doi.org/10.1101/2020.03.13.989582

p. 43

Firmino, A. L. 2016. Taxonomy, phylogeny and biogeography of Asterinales on native forest 931

species from Atlantic Forest and Cerrado. Tese doutourado, Universidade Federal de Viçosa, 932

Viçosa, Minas Gerais, Brazil. 933

Gäumann, E. A. 1952. The fungi. A description of their morphological features and evolutionary 934

development. Hafner Publishing Company, New York. 935

Grigoriev, I. V., R. Nikitin, S. Haridas, A. Kuo, R. Ohm, R. Otillar, R. Riley, et al. 2013. 936

MycoCosm portal: gearing up for 1000 fungal genomes. Nucleic acids research 42: D699-D704. 937

Guatimosim, E., H. J. Pinto, R. W. Barreto, and J. Prado. 2014. Rhagadolobiopsis, a new genus 938

of Parmulariaceae from Brazil with a description of the ontogeny of its ascomata. Mycologia 939

106: 276–281. 940

Guatimosim, E., A. Firmino, J. Bezerra, O. Pereira, R. Barreto, and P. Crous. 2015. Towards a 941

phylogenetic reappraisal of Parmulariaceae and Asterinaceae (Dothideomycetes). Persoonia 35: 942

230–241. 943

Guerrero, Y., T. A. Hofmann, C. Williams, M. Thines, and M. Piepenbring. 2011. Asterotexis 944

cucurbitacearum, a poorly known pathogen of Cucurbitaceae new to Costa Rica, Grenada and 945

Panama. Mycology 2: 87–90. 946

Hernández-Restrepo, M., J. D. P. Bezerra, Y. P. Tan, N. P. Wiederhold, P. W. Crous, J. Guarro, 947

and J. Gené. 2019. Re-evaluation of Mycoleptodiscus species and morphologically similar fungi. 948

Persoonia 42: 205–227. 949

Hofmann, T., R. Kirschner, and M. Piepenbring. 2010. Phylogenetic relationships and new 950

records of Asterinaceae (Dothideomycetes) from Panama. Fungal Diversity 43: 39–53. 951

Hofmann, T. A. 2009. Plant parasitic Asterinaceae and Microthyriaceae from the Neotropics 952

(Panama). Ph.D., Johann Wolfgang Goethe-University, Frankfurt, Germany. 953


https://doi.org/10.1101/2020.03.13.989582

p. 44

Hofmann, T. A., and M. Piepenbring. 2006. New records and host plants of fly-speck fungi from 954

Panama. Fungal Diversity 22: 55–70. 955

Hofmann, T. A., and M. Piepenbring. 2011. Biodiversity of Asterina species on neotropical host 956

plants: New species and records from Panama. Mycologia 103: 1284–1301. 957

Holm, K., and L. Holm. 1991. Ascomycetes on Myrica gale in Sweden. Nordic Journal of 958

Botany 11: 675–687. 959

Hongsanan, S., and D. K. Hyde. 2017. Phylogenetic placement of Micropeltidaceae. Mycosphere 960

8: 1930–1942. 961

Hongsanan, S., Y.-M. Li, J.-K. Liu, T. Hofmann, M. Piepenbring, J. D. Bhat, S. Boonmee, et al. 962

2014. Revision of genera in Asterinales. Fungal Diversity 68: 1–68. 963

Hosagoudar, V., and J. Kapoor. 1985. New technique of mounting meliolaceous fungi. Indian 964

Phytopathology 38: 548–549. 965

Hughes, S. J. 1976. Sooty moulds. Mycologia 68: 693–820. 966

James, T. Y., F. Kauff, C. L. Schoch, P. B. Matheny, V. Hofstetter, C. J. Cox, G. Celio, et al. 967

2006. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature 443: 818–968

822. 969

Kalgutkar, R. M., and J. Jansonius. 2000. Synopsis of fossil fungal spores, mycelia and 970

fructifications. AASP Contributions Series, 351. American Association of Stratigraphic 971

Palynologists Foundation. 972

Katoh, K., and D. M. Standley. 2013. MAFFT Multiple sequence alignment software Version 7: 973

Improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. 974

Kirk, P. M., P. F. Cannon, D. W. Minter, and J. A. Stalpers. 2008. Ainsworth & Bisby's 975

Dictionary of the Fungi, 10th ed. CABI Europe, UK. 976


https://doi.org/10.1101/2020.03.13.989582

p. 45

Kumar, M. 1996. Palynostratigraphy and paleoecology of Early Eocene palynoflora of Rajpardi 977

lignite, Bharuch District, Gujarat. Palaeobotanist 43: 110–121. 978

Lee, S. B., and J. W. Taylor. 1990. Isolation of DNA from fungal mycelia and single spores. In 979

M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White [eds.], PCR Protocols: A Guide to 980

Methods and Applications, 282–287. Academic Press, San Diego. 981

Lewis, P. O. 2001. A likelihood approach to estimating phylogeny from discrete morphological 982

character data. Systematic Biology 50: 913–925. 983

Mapook, A., K. Hyde, S. Hongsanan, C. Phukhamsakda, J. Li, and S. Boonmee. 2016. 984

Palawaniaceae fam. nov., a new family (Dothideomycetes, Ascomycota) to accommodate 985

Palawania species and their evolutionary time estimates. Mycosphere 7: 1732–1745. 986

Medjedović, A., J. Frank, H.-J. Schroers, B. Oertel, and J. C. Batzer. 2014. Peltaster cerophilus 987

is a new species of the apple sooty blotch complex from Europe. Mycologia 106: 525–536. 988

Miadlikowska, J., F. Kauff, V. Hofstetter, E. Fraker, M. Grube, J. Hafellner, V. Reeb, et al. 2006. 989

New insights into classification and evolution of the Lecanoromycetes (Pezizomycotina, 990

Ascomycota) from phylogenetic analyses of three ribosomal RNA- and two protein-coding 991

genes. Mycologia 98: 1088–1103. 992

Miller, M. A., W. Pfeiffer, and T. Schwartz. 2010. Creating the CIPRES Science Gateway for 993

inference of large phylogenetic trees. Gateway Computing Environments Workshop (GCE), New 994

Orleans, LA: 1–8. 995

Mishra, S., N. Aggarwal, and N. Jha. 2018. Palaeoenvironmental change across the Permian-996

Triassic boundary inferred from palynomorph assemblages (Godavari Graben, south India). 997

Palaeobiodiversity and Palaeoenvironments 98: 177–204. 998


https://doi.org/10.1101/2020.03.13.989582

p. 46

Mitchell, A. 2015. Collecting in collections: a PCR strategy and primer set for DNA barcoding 999

of decades-old dried museum specimens. Molecular Ecology Resources 15: 1102–1111. 1000

Monga, P., M. Kumar, V. Prasad, and Y. Joshi. 2015. Palynostratigraphy, palynofacies and 1001

depositional environment of a lignite-bearing succession at Surkha Mine, Cambay Basin, north-1002

western India. Acta Palaeobotanica 55: 183–207. 1003

Müller, E., and J. A. von Arx. 1962. Die Gattungen der didymosporen Pyrenomyceten. Beiträge 1004

zur Kryptogamenflora der Schweiz 11: 1–922. 1005

Pagel, M., and A. Meade. 2007. BayesTraits v.2 Computer program and documentation 1006

available at http://www.evolution.rdg.ac.uk/BayesTraits.html. 1007

Parbery, D., and R. Emmett. 1977. Hypotheses regarding appressoria, spores, survival and 1008

phylogeny in parasitic fungi. Revue de Mycologie 41: 429–447. 1009

Phipps, C. J., and W. C. Rember. 2004. Epiphyllous fungi from the Miocene of Clarkia, Idaho: 1010

reproductive structures. Review of Palaeobotany and Palynology 129: 67–79. 1011

Reynolds, D. R., and G. S. Gilbert. 2005. Epifoliar fungi from Queensland, Australia. Australian 1012

Systematic Botany 18: 265–289. 1013

Ronquist, F., M. Teslenko, P. Van Der Mark, D. L. Ayres, A. Darling, S. Höhna, B. Larget, et al. 1014

2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large 1015

model space. Systematic Biology 61: 539–542. 1016

Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Systematic 1017

Biology 51: 492–508. 1018

Shimodaira, H., and M. Hasegawa. 2001. CONSEL: for assessing the confidence of phylogenetic 1019

tree selection. Bioinformatics 17: 1246–1247. 1020


https://doi.org/10.1101/2020.03.13.989582

p. 47

Spooner, B. M., and P. M. Kirk. 1990. Observations on some genera of Trichothyriaceae. 1021

Mycological Research 94: 223–230. 1022

Stamatakis, A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of 1023

large phylogenies. Bioinformatics 30: 1312–1313. 1024

Stevens, F. L., and M. H. Ryan. 1939. The Microthyriaceae. The University of Illinois press, 1025

Urbana. 1026

Swart, H. J. 1986. Australian leaf-inhabiting fungi XXII. Microthyrium-like fungi on Eucalyptus. 1027

Transactions of the British Mycological Society 87: 81–91. 1028

Taylor, T. N., M. Krings, and E. L. Taylor. 2015. Fossil Fungi, 1st ed. Elsevier/Academic Press 1029

Inc., San Diego. 1030

Viégas, A. P. 1944. Algun fungos do Brasil II Ascomycetos. Bragantia 4: 1–392. 1031

von Arx, J. A., and E. Müller. 1975. A re-evalutation of bitunicate ascomycetes with keys to 1032

families and genera. Studies in Mycology 9: 1–159. 1033

Warren, D. L., A. J. Geneva, and R. Lanfear. 2017. RWTY (R We There Yet): An R package for 1034

examining convergence of bayesian phylogenetic analyses. Molecular Biology and Evolution 34: 1035

1016–1020. 1036

Wijayawardene, N., P. Crous, P. Kirk, D. Hawksworth, S. Boonmee, U. Braun, D.-Q. Dai, et al. 1037

2014. Naming and outline of Dothideomycetes–2014 including proposals for the protection or 1038

suppression of generic names. Fungal Diversity 69: 1–55. 1039

Wu, H., K. D. Hyde, and H. Chen. 2011a. Studies on Microthyriaceae: placement of Actinomyxa, 1040

Asteritea, Cirsosina, Polystomellina and Stegothyrium. Cryptogamie, Mycologie 32: 3–12. 1041

Wu, H., Q. Tian, W.-J. Li, and K. Hyde. 2014. A reappraisal of Microthyriaceae. Phytotaxa 176: 1042

201–212. 1043


https://doi.org/10.1101/2020.03.13.989582

p. 48

Wu, H.-X., C. L. Schoch, S. Boonmee, A. H. Bahkali, P. Chomnunti, and K. D. Hyde. 2011b. A 1044

reappraisal of Microthyriaceae. Fungal Diversity 51: 189–248. 1045

1046

1047


https://doi.org/10.1101/2020.03.13.989582

p. 49

Table 1. Matrix of specimens and their observed character states. 1048

Specimens examined Sub Lic Spo Low Deh Loc Rad Bra Mar App Ini

Asterina melastomatis (Asterinales) VIC,

42822, BR, AL Firmino sup no thyr und irr circ yes iso cren yes int

Asterotexiaceae sp. (Asterotexiales) UBC,

F33042, CA, L Le Renard sup no thyr und irr circ yes iso cren no ?

Asterotexiaceae sp. (Asterotexiales) UBC,

F33036, CR, L Le Renard sup no thyr und ost circ yes ? cren no int

Asterotexis cucurbitacearum (Asterotexiales)

VIC, 42814, BR, OL Pereira & AL Firmino sup no thyr und irr circ yes ? cren no spo

Aulographum sp. (Aulographaceae) UBC,

F33038, CA, L Le Renard sup no thyr und irr elng yes ? cren no mult

Batistinula gallesiae (Asterinales) VIC, 42514,

BR, AL Firmino, DB Pinho & OL Pereira sup no thyr und irr circ yes iso cren yes int

Buelliella poetschii (Asterotexiales) BR,

5030020189138; 5030020188124, BE, D Ertz sup no apo dif exp circ no pseud not no ?

(which w

as not certified by peer review) is the author/funder. A

ll rights reserved. No reuse allow

ed without perm

ission. T

he copyright holder for this preprintthis version posted M

arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 50

Fumiglobus pieridicola (Capnodiales) UBC,

F23788, CA, T Bose sup no per dif ost circ no pseud not no ?

Hemigrapha atlantica (Asterotexiales) BR

5030012458426, ES, D Ertz lich no thyr und irr elng yes iso ent no ?

Inocyclus angularis (Asterotexiales) VIC,

39748, BR, RW Barreto sup no thyr dif reg elng yes ? cren no ?

Lembosina aulographoides (Aulographaceae)

UBC, F33037, CA, L Le Renard sup no thyr und irr elng

yes

mono cren no mult

Lembosina sp. (Aulographaceae) UBC,

F33044, CA, L Le Renard sup no thyr und irr elng yes mono cren no mult

Lembosina sp. (Aulographaceae) UBC,

F33045, CA, L Le Renard sup no thyr und irr elng yes mono cren no mult

Lichenopeltella pinophylla (Microthyriales)

UBC, F33032, CA, L Le Renard sup no thyr dif irr circ yes aniso ent no ter

Micropeltis sp. (Micropeltidaceae) UBC,

F33034, CR, L Le Renard sup no thyr und ost circ no untra ana no ?

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 51

Microthyrium ilicinum (Microthyriales) UBC,

F33031, FR, L Le Renard sup no thyr und ost circ yes aniso ent no ter

Microthyrium macrosporum (Microthyriales)

UBC, F33039, CA, L Le Renard sup no thyr und ost circ yes aniso ent no ter

Parmularia styracis (Asterinales) VIC, 42450,

BR, M Silva & OL Pereira sup no thyr dif irr elng yes ? cren no ?

Prillieuxina baccharidincola (Asterinales)

VIC, 42818, BR, AL Firmino sup no thyr und irr circ yes ? anast no int

Rhagadolobiopsis thelypteridis

(Asterotexiales) VIC, 31939, BR, E

Guatimosim

sup no thyr und irr elng yes iso cren no sto

Schizothyrium gaultheriae (S. pomi

(Capnodiales) was sequenced) UBC, F3143,

CA, M Barr

sup no thyr und irr circ no untra anast no ?

Scolecopeltidium sp. (Micropeltidaceae) UBC,

F33033, CR, L Le Renard sup no thyr und ost circ no untra anast no ?

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 52

Scolecopeltidium sp. (Micropeltidaceae) UBC,

F33035, CR, L Le Renard sup no thyr und ost circ no untra anast no ?

Seuratia millardetii (Lichenostigmatales)

UBC, F33043, CA, L Le Renard sup ? apo dif exp circ no pseud not no ?

cf. Stomiopeltis sp. (Venturiales) UBC,

F33040, CA, L Le Renard sup no thyr und ost circ yes untra cren no ?

cf. Stomiopeltis sp. (Venturiales) UBC,

F33041, CA, L Le Renard sup no thyr und ost circ yes untra cren no ?

1049

1050

Notes: Specimen data includes systematic affinities followed by herbarium code (BR = Meise Botanic Garden; UBC = University of 1051

British Columbia; VIC = Universidade Federal de Viçosa), specimen accession number, ISO country code and collector(s). 1052

Sub = sporulation substrate: sup = superficial on plants; lich = lichenicolous; 1053

Lic = lichenized; no = non-lichenized 1054

Spo = sporocarp: apo, apothecioid; per, perithecioid; thyr, thyriothecioid 1055

Low = sporocarp lower wall: und, undifferentiated; dif, differentiated 1056

Deh = dehiscence: ost, ostiole; reg, regular slit; irr, irregular slit; exp, exposed at maturity 1057

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 53

Loc = locule circumference: circ, circular; elng, ellipsoid to elongate 1058

Rad = radiate sporocarp: no, absent radiate; yes, present 1059

Bra = scutellum branching: pseud= pseudoparenchymatous; iso, isodichotomous; aniso, anisodichotomous; mono, pseudomonopodial 1060

dichotomies; untra, untraceable 1061

Mar = margin: not, margin not appressed; cren, crenulate, aligned; ent, entire, aligned; anast, anastomosing with surrounding 1062

mycelium 1063

App = lateral appressoria; no, absent; yes, present 1064

Ini = initiation of sporocarp, position and number of generator hyphae: int, single, intercalary; ter, single, terminal; mult, multiple, 1065

intercalary; spo, from spores; sto, from stomata1066

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 54

1067

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 55

Table 2. Definitions of characters and their states with reference to examples from illustrations 1068

in figures. 1069

Character, character states, and

examples in figures in this paper

Definition

Sub- Sporulation substrate

0–On rock or soil (= saxicolous)

Rock inhabiting (Saxomyces sp.),

ground dwelling lichen (Simonyella

variegata)

On rock or other inorganic substrates.

1–Superficial on plants (= foliicolous)

On living leaves, Asterina

melastomatis, Fig. 2J, or on dead

leaves, Microthyrium macrosporum

Fig. 4B

On leaves, stems, fruit; superficial, closely associated

with cuticle.

2–On lichens only (= lichenicolous),

Hemigrapha atlantica

On lichen thallus.

3–Immersed in plants (= lignicolous

or corticolous)

Taxa bursting through woody parts

(Dothidea sambuci), on decorticated

In bark or in woody parts of plants, in or under a plant's

epidermis.


https://doi.org/10.1101/2020.03.13.989582

p. 56

material (Natipusilla bellaspora), or

as part of corticolous lichens thalli

(Trypethelium elutheriae)

4–Other

Coenococcum geophillum,

Phaeotrichum atkisonii

Various habitats that are not the focus of this study and

that are poorly sampled here. Includes taxa that inhabit

dung, are endoparasites, or are mycorrhizal.

Lich- Lichenized

0–Non-lichenized

Asterina melastomatis Figs. 2P and

2Q

1–Lichenized

Graphis scripta

Spo- Sporocarp type

0–Apothecioid

Abrothallus usneae

Sporogenous tissue exposed at maturity.

1–Perithecioid

Acrospermum graminearum

Flask-shaped, with an ostiole

2–Thyriothecioid

Microthyrium macrosporum

Flat, appressed, sporocarp, sporogenous tissue covered

by a pigmented scutellum (comprise catathecia, which


https://doi.org/10.1101/2020.03.13.989582

p. 57

unlike most thyriothecia have a pigmented vental wall).

3–Cleistothecioid

Lepidopterella palustris

No preformed opening (include chasmothecia).

Low- Lower wall

0–Undifferentiated

Microthyrium spp.

Lower wall of sporocarp either absent or made up of

one or a few layers of unpigmented cells, contrasting

with the darker upper sporocarp wall.

1–Differentiated

Lichenopeltella pinophylla Fig. 4G

Lower wall of sporocarp present and concolorous to the

rest of the sporocarp. It does not include host tissue and

is thus not a hypostroma.

Deh- Dehiscence

0–Ostiole

Opening of most Pleosporalean fungi

Any dorsal pore by which spores are freed from an

ascigerous or pycnidial sporocarp.

1–Irregular slit

Prilleuxinia baccharidincola Fig. 2K

Elongated opening formed by cracking or splitting of

the scutellum. The splitting is irregular in that upon

opening, it usually propagates radially between lateral

walls of neighboring hyphae but it can also propagate

transversely across hyphae. The split does not appear to

follow pre-formed lines of dehiscence.


https://doi.org/10.1101/2020.03.13.989582

p. 58

2–Regular slit

Opening found in hysterothecia and

lirella, Hysteropatella elliptica,

Graphis scripta

Elongated opening formed in a regular fashion that

does not propagate irregularly.

3–No dehiscence

Piedraia hortae

Sporocarp closed when mature, no mechanism of

dehiscence as part of sporocarp growth.

4–Exposed at maturity

Abrothallus spp., Seuratia millardetii

Sporocarp mostly open, sporogenous tissue is exposed

throughout its development

Loc- Locule circumference

0–circular

Asterinaceae Figs. 2H and 2J

Microthyriaceae Figs. 4A–4D and

4G–4J

Sporocarp enlarges from a center in all direction at

about the same pace so that it appears round in dorsal

view.

1–elongate

Lembosina aulographoides Fig. 3B

Sporocarp elongates as it enlarges from a central area

into one or more 'arms.'

Rad- Radiate sporocarp wall

0–Absent

Micropeltis spp., Schizothyrium pomi

No signs of a common central origin; distal hyphae

cannot be traced back to central area of a sporocarp.


https://doi.org/10.1101/2020.03.13.989582

p. 59

1–Present

Asterina spp., Microthyrium spp.

Figs. 2, 3B, 3D and 4

Most or all of the distal hyphae of the scutellum can be

traced back to their parental hyphae at or near the

center of the sporocarp.

Bra- Branching pattern (in hyphae of sporocarp wall)

0–Pseudoparenchyma

Most pseudothecia Figs. 5Aa and

5Ab

Any wall of hyphal origin where linear hyphal

filaments can no longer be discerned. Instead, walls are

composed of polyhedral cells, and the original planes

of transverse cell divisions cannot be recognized.

1–Isotomous

Asterina spp. Fig. 5Ce

Scutellum hyphae radiate, isotomous dichotomies

predominate where in each pair of dichotomous

branches, one hyphal branch is wider than its sister.

Hyphal segments occasionally show evidence of

overlapping their neighbors.

2–Anisotomous


Fig. 5Dh

Scutellum hyphae radiate, isotomous dichotomies and

anisotomous dichotomies coexist but hyphal segments

do not show evidence of overlapping their neighbors.

3–Pseudomonopodial

Lembosia spp., Aulographum spp.

Fig. 5Ei

Scutellum hyphae radiate with few isotomous

dichotomies; abundant evidence of overlap;

pseudomonopodial dichotomies predominate and are

most evident at the periphery of the scutellum


https://doi.org/10.1101/2020.03.13.989582

p. 60

4–Untraceable

Schizothyrium sp., Micropeltis spp.,

Stomiopeltis sp.

Figs. 3A, 3C and 5Fj–5Fk

Scutellum hyphae not radiate or if radiate, they cannot

be traced from the sporocarp center to its margin.

Isotomous dichotomies are few and neighboring

hyphae frequently overlap.

Mar- Appressed margin

0–No appressed margin

Pleospora herbarum, appressed

margin not applicable; sporocarp is

flask-shaped

Sporocarp without defined, appressed margin. Instead,

the sporocarp wall usually curves upwards, forming a

flask or disc shape.

1–Crenulate to free

Batistinula gallesiae Fig. 2H

Margin of sporocarp made of well-defined hyphal tips,

each tip bulging out or elongating beyond the

longitudinal wall shared by adjacent cells.

2–Entire


Figs. 4C–4D

Hyphal tips appear truncate, blunt, at least in young

specimens. In mature specimens, the blunt tips may

give rise to a fringe of tapering hyphae that are 2-5

times narrower than their mother cells.

3–Anastomosing

Schizothyrium pomi, Prillieuxina

baccharidincola Fig. 2K

Marginal hyphae of sporocarp continue outwards,

connecting or merging with surrounding superficial

mycelium; hyphal tips not discernable.

App- Lateral appressoria

0–Absent

Aulographum sp. Fig. 3G

Appressoria absent or intercalary if present


https://doi.org/10.1101/2020.03.13.989582

p. 61

1–Present

Asterina melastomatis Figa. 2P and

2Q

Appressoria lateral, branching directly from superficial

hyphae.

Ini- Initiation of sporocarp

0–Terminal; at tip of generator hypha

Microthyrium spp.

Figs. 5Dg and 5Dh

Generator hypha is a short lateral branch of a

superficial hypha on a plant surface. The scutellum is

derived from a single cell of the generator hypha, and it

overgrows generator hyphae at maturity.

1–Intercalary; below generator hypha

Asterinales, Asterotexiales

Figs. 5Cc, 5Cd and 5Ce

Generator hypha is an intercalary (more rarely

terminal) portion of a hypha that is superficial on a

plant surface. The generator hypha remains above the

scutellum at maturity.

2–From spores

Asterotexis cucurbitacearum

Figs. 2E and 2F

Generator hypha absent; a scutellum grows directly

from each part-spore and becomes confluent with

surrounding scutella as it enlarges.

3–From host stomata

Rhagadolobiopsis thelypteridis

Fig. 2G

Generator hypha indiscernible. Scutellum arises

directly from hyphae growing up through the stoma

(likely from a hypostroma) and covers guard cells and

stomatal complex at maturity.


https://doi.org/10.1101/2020.03.13.989582

p. 62

1070

4–Multiple generator hyphae

Aulographum sp. Fig. 5Ei1

More than one intercalary generator hypha participates

in forming a single thyriothecium. A generator hypha

gives rise to the first dichotomous scutellum cells and

this differentiation propagates to adjacent portions of

the same hypha as well as to nearby hyphae, all of

which become generator hyphae and contribute to the

same scutellum.


https://doi.org/10.1101/2020.03.13.989582

p. 63

Table 3. Comparison of ancestral character states at selected nodes as reconstructed from proportional likelihoods in Mesquite 1071

(unshaded rows) and from posterior probabilities in BayesTraits (shaded rows). 1072

1073

Ancestor reconstructed Sub Lic Spo Low Deh Loc Rad Bra Mar App Ini

Asterotexiales, clade of

Asterina spp (NAstx04)

sup no thyr und irr circ yes iso cren yes int

sup no thyr und irr circ yes iso cren yes -

Asterotexiales Clade A

(NAstx03)

sup no thyr und irr circ yes iso cren no int

sup no thyr und irr - yes - cren yes -

Asterotexiales (NAstx01) sup no thyr und irr - no - not no -

- no - und irr - - - - yes -

Aulographaceae (NAulo1) sup no thyr und irr elng yes mono cren no mult

- no thyr und irr - yes - cren no -

Zeloasperisporiales (NZelo2) sup no thyr - - - yes - - no -

sup no thyr - - - yes - - no -

Zeloasperisporiales +

Natipusillales (NZelo1)

sup no thyr und - circ no aniso ent no -

- no - - - - yes - - no -

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 64

Microthyrium (NMThyr4) sup no thyr und ost circ yes aniso ent no ter

- no thyr und - - yes - - no -

Microthyriales (NMThyr2) sup no thyr und ost circ yes aniso ent no -

- no thyr - - - yes - - no -

Microthyriales + Venturiales

(NMVP)

sup no thyr - ost circ no pseud not no -

- no - - - - yes - - no -

Muyocopron +

Mycoleptodiscus (NADM08)

sup no thyr und ost circ yes - cren no -

- no thyr und ost circ yes - cren no -

Dyfrolomycetales +

Muyocopronales (NADM04)

sup no apo dif ost circ no - not no -

- no - - - - yes - - no -

Asterinales, no Parmularia

styracis (NAste2)

sup no thyr und irr circ yes iso cren no int

sup no thyr und irr - yes - - yes -

Asterinales (NAste1) sup no thyr - irr circ yes - cren no -

sup no thyr und irr - yes - - yes -

Capnodiales ss + Asterinales sup no per dif ost circ no pseud not no -

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 65

(NCapnod5) - no - - - - no - - no -

Dothideomycetes (NDothid) imm no apo dif ost circ no pseud not no -

sax no - - reg elng no - - no -

Dothideomycetes +

Arthonimycetes +

Trypetheliales (NDothTryp)

imm no apo dif exp circ no pseud not no -

sax no - - - elng no - - no -

Micropeltidaceae (NMpelt1) sup no thyr und ost circ no untra ana no -

sup no thyr und ost - no - - no -

1074

Notes: For 'Ancestor reconstructed', the node name given in parentheses is mapped onto the phylogeny in Appendix S6. 1075

Sub = sporulation substrate: sax, saxicolous; sup, superficial on plants; lich, lichenicolous; imm, immersed on plants 1076

Lic = lichenized 1077

Spo = sporocarp: apo, apothecioid; per, perithecioid; thyr, thyriothecioid 1078

Low = sporocarp lower wall: und, undifferentiated; dif, differentiated 1079

Deh = dehiscence: ost, ostiole; reg, regular slit; irr, irregular slit; exp, exposed at maturity 1080

Loc = locule circumference: circ, circular; elng, ellipsoid to elongate 1081

Rad = radiate 1082

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

p. 66

Bra = scutellum branching: pseud, pseudoparenchymatous; iso, isodichotomous; aniso, anisodichotomous; mono, pseudomonopodial 1083

dichotomies; untra, untraceable 1084

Mar = margin: not, margin not appressed; cren, crenulate, aligned; ent, entire, aligned; anast, anastomosing with surrounding 1085

mycelium 1086

App = lateral appressoria; no, absent; yes, present 1087

Ini = initiation of sporocarp, position and number of generator hyphae: int, single, intercalary; ter, single, terminal; mult, multiple, 1088

intercalary 1089

1090

1091

(which w



ed without perm

ission. T


arch 14, 2020. .

https://doi.org/10.1101/2020.03.13.989582doi:

bioRxiv preprint

https://doi.org/10.1101/2020.03.13.989582

On plants and plant surfaces

iP Ostiole

Irregular slit(s)

Dehiscence

Undifferentiated

Differentiated

Sporocarp lower wall

Pseudomonopodial

0

Pseudoparenchyma unspecified

Scutellum hyphal pattern Isotomous, overlapping

4

3

2

Untraceable 1

Isotomous, non-overlapping

Ps

Exposed

1

2

1

4Ps

9Ps

MPs

2Ps0

0

iP0

Ps

Ps0

6

73

Ps

Ps 4

5Ps ?

32

Ps

0

?

8

10

3

4

6

7

Microthyriales

Zeloasperisporiales

Dyfrolomycetales

Aulographaceae

"Schizothyriaceae"

Asterinales s.s.

Arthoniomycetes + Trypetheliales

Lecanoromycetes

Micropeltidaceae s.s.

Muyocopronales

Asterotexiales

Eurotiomycetes

Leotiomycetes

"Dothideomycetidae"

Pleosporomycetidae

Venturiales

"Capnodiales"

1

9

DOTHIDEOMYCETES

5

Abrothallales + Tubeufiales

Natipusillales

Stict is_radiata AF356663.1Acarosporina_microspora AFTOL − ID_78

Bryodiscus_arct ialpinus Baloch_SW057(S)Abscondi tel la_sphagnorum EU940095.1

Cryptodiscus_gloeocapsa TSB_30770Micropel t is_sp. UBC − F33034

Micropel t is_zingiberacicola IFRDCC_2264Scolecopel t id ium_sp.2 UBC − F33035Scolecopel t id ium_sp.1 UBC − F33033

Pel taster_f ruct icola JN573665.1

Parmularia_styracis VIC_42587Bat ist inula_gal lesiae VIC_42514

Pri l l ieuxina_baccharid incola VIC_42817Asterina_chrysophyl l i VIC_42823

Lembosia_abaxial is VIC_42825Asterina_melastomat is VIC_42822

Blastacervulus_eucalyptorum CPC_29450Blastacervulus_eucalypt i CBS_124759

Johansonia_chapadiensis CBS_H − 20484Schizothyrium_pomi CBS_228.57

Uwebraunia_commune CBS_110747Houj ia_yangl ingensis YHJN13

pStomio el t is_versicolor GA3_23C2b

Dyfrolomyces_t iomanensis NTOU3636Dyfrolomyces_rhyzophorae JK_5349A

Arxiel la_dol ichandrae CBS_138853Paramycoleptodiscus_alb izziae CPC_27552Muyocopron garethjonesi MFLU_16 − 2664

Mycoleptodiscus_indicus UAMH_8520Muyocopron_dipterocaerpi MFLUCC_14 − 110

Muyocopron_castanopsisMFLUCC_14 − 1108

Muyocopron_lithocarpi MFLUCC_10 − 41MFLUCC_14 − 1106Muyocopron_lithocarpi

cf .Stomiopel t is_sp._2 UBC − F33041Toth ia_fuscel la CBS_130266

p pcf .Stomio el t is_s ._1 CBS_143811

Lichenopel tel la_pinophyl la UBC − F33032

Stomiopel t is_betulae CBS_114420y gChaetoth riothecium_ele ans CPC_21375

pTumidis ora_shoreae MFLUCC_12 − 409Hel iocephala_graci l is MUCL_41200Hel iocephala_zimbabweensis MUCL_40019

iZ_sears ae CPC_25880Z_f icusicola MFLUCC_15 − 222Z_siamense IFRDCC_2194

Z_cl iviae CPC_25145Z_wright iae MFLUCC_15 − 215

Z_eucalyptorum CBS_124809

Geastrumia_polyst igmat is FJ147177.1Morenoina_calamicola MFLUCC_14 − 1162

Asterina_sp. MFLU13 − 619_ _Asterotexis cucurbi tacearum PMA M − 141224

Asterotexis_cucurbi tacearum VIC_42814Inocyclus_angularis VIC_39747

Lembosia_xyl iae MFLU14 − 4Lembosia_albersi i MFLU13 − 377

y p p pM cos haerel la_ neumato horae AFTOL − ID_762Asterotexiaceae_sp.1 UBC − F33036

Rhagadolobiopsis_thelyptei rid is EG_156Asterotexiaceae_sp.2 CBS_143813

Buel l iel laHemigrapha_at lant ica Ertz_14014(BR)

_poetschi i Ertz_18116(BR)

Microthyrium spp.

Asterina spp.

M

2

10

8

Ancestral character states transitions

Substrate

Immersed inplant tissue

Potential fossil calibrations

Triassic fossil 'fungal thalli'

Early Eocene Trichothyrites setifer

Middle Eocene Asterina eocenica

Ostropomycetidae

#

?

F

T

T

A

A

A

F

Aulographum spp.

Lembosina spp.

?

?


https://doi.org/10.1101/2020.03.13.989582


https://doi.org/10.1101/2020.03.13.989582


https://doi.org/10.1101/2020.03.13.989582


https://doi.org/10.1101/2020.03.13.989582

1Pse

udopare

nch

ym

a

angle

of

div

erg

ence

betw

een b

ranch

ing t

ips

Isoto

mous

dic

hoto

mie

s overl

ap

pin

g

Anis

oto

mous

dic

hoto

mie

s no o

verl

ap

Young s

poro

carp

Matu

re s

poro

carp

s

(a)

(b)

(g3)

(d1

)

(h3)

Isoto

mous

dic

hoto

mie

sExam

ple

tra

ced

onto

logic

al re

lati

onsh

ip

from

centr

e t

o m

arg

inPse

udom

onopodia

l dic

hoto

mie

sA

nis

oto

mous

dic

hoto

mie

s

Bra

nch

ing involv

ing o

verl

ap

Scu

tellu

m o

penin

g

Genera

tor

hyphae

(d2)

(c)

(e2)

(f)

(d1)

(e1)

(j)

(k)

(l)

2Tr

aci

ng r

adia

te

bra

nch

ing

4A

nis

oto

mous

Isoto

mous

3

Pse

ud

om

onop

od

ial

5U

ntr

ace

able

6

Legend

(i2)

(i1)

(g2)

(g1)

(h1)

(h2)

1

(whi

ch w

as n

ot c

ertif

ied

by p

eer

revi

ew)

is th

e au

thor

/fund

er. A

ll rig

hts

rese

rved

. No

reus

e al

low

ed w

ithou

t per

mis

sion

. T

he c

opyr

ight

hol

der

for

this

pre

prin

tth

is v

ersi

on p

oste

d M

arch

14,

202

0.

. ht

tps:

//doi

.org

/10.

1101

/202

0.03

.13.

9895

82do

i: bi

oRxi

v pr

eprin

t

https://doi.org/10.1101/2020.03.13.989582


https://doi.org/10.1101/2020.03.13.989582

Documents

2 Character evolution of modern fly-speck fungi and implications … · 2020-03-13 · 3 thyriothecial fossils 4 5 Ludovic Le Renard*1, André L. Firmino2, Olinto L. Pereira3, Ruth