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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: ludovic.lerenard@botany.ubc.ca 16
17
Manuscript received _______; revision accepted _______. 18
19
Running head: Fly-speck fungi character evolution 20
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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
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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
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Fly-speck fungi are ascomycetes that appear as minute (50 m 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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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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
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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
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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
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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
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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
(Medjedovi 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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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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
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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
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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
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p. 41
LITERATURE CITED 886
887
Arnaud, G. 1917. Sur la famille des microthyriacées. Compte Rendu de l’Académie des Sciences 888
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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 ?
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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 ?
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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 ?
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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
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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
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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.
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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
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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.
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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.
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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
Microthyrium macrosporum
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
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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
Microthyrium macrosporum
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
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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.
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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.
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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 -
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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 -
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(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
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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
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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
4
Ps
9
Ps
M
Ps
2
Ps
0
0
iP
0
Ps
Ps
0
6
7
3
Ps
Ps 4
5
Ps
?
3
2
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
D
O
T
H
I
D
E
O
M
Y
C
E
T
E
S
5
Abrothallales + Tubeufiales
Natipusillales
St ict is _rad iat a AF356663.1
Acarosporina_microsporaAFTOL −ID_78
Br yod isc us_a rct ial pi nus Baloch_SW057(S)
Ab sco ndi te lla _sph agn oru m EU940095.1
Cr ypt odi scu s_gl oeo cap sa TSB_30770
Mi cro pel ti s_sp . UBC−F33034
Mi cro pel ti s_zi ngi ber aci col a IFRDCC_2264
Sc ol ec op el t id i um _sp .2 UBC −F33035
Sc ol ec op el t id i um _sp .1 UBC −F33033
Peltaster_fructicolaJN573665.1
Pa rmu lar ia_s tyr aci s VIC_42587
Bat ist inu la _gal les iae VIC_42514
Pr ill ie uxi na_b acc har id inc ola VIC_42817
As ter ina _chr yso phy ll i VIC_42823
Le mbo sia _aba xi ali s VIC_42825
As ter ina _mel ast oma ti s VIC_42822
Bl ast ace rvu lus _euc aly pto rum CPC_29450
Bla sta cer vul us_e uca lyp ti CBS_124759
Jo han son ia_c hap ad ien sis CBS_H −20484
Sc hiz ot hyr ium _pom i CBS_228.57
Uw ebr aun ia_c omm une CBS_110747
Houj ia _yan gli nge nsi s YHJN13
pS tom io e lt is_v ers ico lor GA3_23C2b
Dy fro lom yce s_ti oma nen si s NTOU3636
Dy fro lom yce s_rh yzo pho rae JK_5349A
Ar xie ll a_do lic ha ndr ae CBS_138853
Pa ram yco lep tod is cus_a lb izz iae CPC_27552
Muyocopron garethjonesi MFLU_16 −2664
My col ept odi sc us_i ndi cus UAMH_8520
Muyocopron_dipterocaerpi MFLUCC_14 −110
Muyocopron_castanopsis MFLUCC_14 −1108
Muyocopron_lithocarpi MFLUCC_10 −41
MFLUCC_14 −1106
Muyocopron_lithocarpi
cf .St o mi op el t is _sp ._2 UBC −F33041
Tot hia _fus cel la CBS_130266
p p
cf .St o mi o el t is _s ._1 CBS_143811
Li che nop el tel la_p ino ph yll a UBC−F33032
St omi ope lt is_b etu la e CBS_114420
y g
Ch aet ot h ri oth eci um_e le an s CPC_21375
p
Tu mid is or a_sh ore ae MFLUCC_12 −409
He lio cep ha la_g rac ili s MUCL_41200
Hel ioc eph al a_zi mba bwe ens is MUCL_40019
i
Z_s ear s ae CPC_25880
Z_f icu si col a MFLUCC_15−222
Z_s iam ens e IFRDCC_2194
Z_c liv iae CPC_25145
Z_w rig hti ae MFLUCC_15 −215
Z_e uca lyp tor um CBS_124809
Gea str umi a_po lys tig mat is FJ147177.1
Mo ren oin a_ca lam ico la MFLUCC_14 −1162
As ter ina _sp. MFLU13 −619
__
As ter ote xis cu cur bit ace aru m PMA M −141224
As ter ote xis _cuc urb it ace aru m VIC_42814
Ino cycl us _angu la ris VIC_39747
Le mbo sia _xyl ia e MFLU14−4
Le mbo sia _alb er sii MFLU13 −377
y p p pM co s ha ere ll a_ ne uma to ho rae AFTOL −ID_762
As te ro te xi a ce ae _sp. 1 UBC −F33036
Rha gad olo bi ops is_t hel yp tei rid is EG_156
As te ro te xi a ce ae _sp. 2 CBS_143813
Bu el li e ll a
He mig rap ha_a tl ant ica Ertz_14014(BR )
_poe ts chi i Ertz_18116(BR)
Microthyrium spp.
Asterina spp.
M
2
10
8
Ancestral character
states transitions
Substrate
Immersed in
plant 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.
?
?
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1Pseudoparenchyma
angle of divergence
between branching tips
Isotomous dichotomies overlapping
Anisotomous dichotomies no overlap
Young sporocarp Mature sporocarps
(a) (b) (g3)
(d1)
(h3)
Isotomous dichotomies
Example traced
ontological relationship
from centre to margin Pseudomonopodial dichotomies
Anisotomous dichotomies
Branching involving overlap
Scutellum opening
Generator hyphae
(d2)
(c)
(e2)(f)
(d1)
(e1)
(j) (k)
(l)
2Tracing radiate
branching
4Anisotomous
Isotomous
3
Pseudomonopodial
5Untraceable
6
Legend
(i2)
(i1)
(g2)(g1)
(h1)(h2)
1
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