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Citation for the original published paper (version of record):
Westberg, M., Millanes, A., Knudsen, K., Wedin, M. (2015)
Phylogeny of the Acarosporaceae (Lecanoromycetes, Ascomycota, Fungi) and the evolution of
carbonized ascomata.
Fungal diversity, 73: 145-158
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1 Phylogeny of the Acarosporaceae (Lecanoromycetes,
2 Ascomycota, Fungi) and the evolution of carbonized
3 ascomata
4 5 Martin Westberg1, Ana M. Millanes2, Kerry Knudsen3 & Mats Wedin1
6 7 1
8 05 Stockholm, Sweden. Corresponding author: [email protected]
9 2
Dept. of Botany, Swedish Museum of Natural History, P.O. Box 50007, SE-104
Departamento de Biología y Geología, Universidad Rey Juan Carlos, E-28933
10 Móstoles, Spain.
11 3
12 Life Sciences, Prague, Kamýcká 129, Praha 6 - Suchdol, CZ–165 21, Czech
13 Republic.
Department of Ecology, Faculty of Environmental Sciences, Czech University of
14 15 16 1 17 Abstract. The phylogeny of the Acarosporaceae (Lecanoromycetes,
18 Acarosporomycetidae, Acarosporales) is investigated using data from three
19 molecular markers; nuclear ITS-LSU rDNA, mitochondrial SSU and β-tubulin.
20 Acarosporaceae is shown to be constituted by six main clades; Myriospora,
21 Timdalia, Pleopsidium, a clade composed by “Acarospora” rhizobola and “A.”
22 terricola, the poorly supported Sarcogyne clade (including several Polysporina and
23 Acarospora species) and the Acarospora clade (including the type of Polysporina,
24 P. simplex, and several other Polysporina species). The common ancestor of the
25 Acarosporaceae did not produce strongly black pigmented (carbonized or
26 melanized) ascomata, but this trait has arisen secondarily and independently
27 numerous times in the evolution of the group. The number of changes in character
28 states of both carbonized epihymenium and carbonized exciple are considerably
29 more than the minimum number. The genera Sarcogyne and Polysporina – largely
30 circumscribed based on the presence of black pigmented ascomata – are shown to
31 be distinctly non-monophyletic. The presence of green algae in the ascoma margin
32 (lecanorine or lecideine ascomata) may vary even within single species.
33 34 Keywords: Convergent evolution, lichens, lichenized fungi, lichenicolous
35 36 2 37 Introduction
38 39 Carbonization or melanization in fungal structures is the accumulation of various
40 degrees of a black pigmentation, which generally is presumed to consist of
41 melanins. Melanins are widely present in fungi and may have several functions,
42 including reducing the damaging effects of solar radiation (Butler & Day 1998), and
43 it is widely assumed that they have this protective function also in lichens (Rikkinen
44 1995; Gauslaa & Solhaug 2001; Hauck et al. 2007). Lichen mycobionts produce
45 various types of melanins with different metabolic pathways (Rikkinen 1995).
46 Melanin made from 1,8 dihydroxynaphtalene is a synapomorphy for the
47 Euascomycetes/Pezizomycotina (Tehler 1988), including most lichen mycobionts.
48 Little is known, however, about the molecular background and the observed
49 intraspecific variation often seen in the production of melanin (e.g. Nybakken et al.
50 2004, Rikkinen 1995). As an obvious and easy-to-recognize characteristic,
51 carbonization of different fungal fruiting body structures has been used as an
52 important character for generic circumscription in some taxonomic groups, for
53 instance the Acarosporaceae (Flotow 1851; Magnusson 1935; Vězda 1978). When
54 these genera are accepted in the current classifications, this implicitly assumes that
55 the presence of this trait characterizes natural, monophyletic groups. Understanding
56 the evolution of carbonized structures is thus crucial for our understanding of the
57 evolution of this and other fungal groups and for how we express the phylogenetic
58 relationships in our classifications.
59 Acarosporaceae is a distinct group of predominantly crustose lichenized fungi,
60 growing on exposed rocks and soil on all continents. The members are primarily
61 characterized by the multi-spored ascus (mostly >100 small, simple, colourless
3 62 spores), and they were among the earliest lichenized groups to appear (the split
63 from the rest of the Lecanoromycetes appeared in late Carboniferous-early Permian)
64 but among the most recent groups to diverge (upper Cretaceous; Prieto & Wedin
65 2013). Originally (Zahlbruckner 1907) Acarosporaceae included five genera
66 (Acarospora, Biatorella, Thelocarpon, Maronea and Glypholecia). The number of
67 genera successively increased (Magnusson 1935, Poelt 1974, Eriksson &
68 Hawksworth 1991), although it was noted that the family was heterogenous (Poelt
69 & Vězda 1981). Hafellner (1992, 1993, 1995) re-assessed the family, and suggested
70 a new family concept based on a shared ascus-type without amyloid structures and a
71 non-amyloid or weakly amyloid tholus. He excluded the re-instated Pleopsidium
72 from the family on account of its amyloid tholus. The circumscription of
73 Acarosporaceae and the relationship to Lecanorales and Lecanoromycetes has since
74 been investigated several times using molecular phylogenies. Stenroos and DePriest
75 (1998) were first to indicate that Acarosporaceae grouped outside Lecanorales s.
76 str., and several later investigations suggested that the family was a phylogenetically
77 distinct group that formed the sister-group to a large part of the Lecanoromycetes
78 (Reeb et al 2004, Wedin et al 2005, Schoch et al 2009, Bendiksby and Timdal
79 2013). Reeb et al. (2004) excluded the family from Lecanorales and described the
80 subclass Acarosporomycetidae which then included Acarospora, Glypholecia,
81 Pleopsidium, Polysporina, Sarcogyne and Thelocarpella. Wedin et al. (2005)
82 confirmed the position of Pleopsidium and in addition showed that Timdalia, a
83 recent segregate from Acarospora (Hafellner & Türk 2001) with a similar ascus-
84 type as in Pleopsidium, also belong in a well-supported monophyletic
85 Acarosporomycetidae/Acarosporaceae. Finally Crewe et al. (2006), Wedin et al.
86 (2009), and Westberg et al. (2011) suggested that the Acarospora smaragdula group
4 87 formed the sister-group to the rest of Acarosporaceae. This group was formally
88 recognized as the genus Silobia (Westberg et al. 2010), a name that was replaced by
89 the older name Myriospora (Arcadia & Knudsen 2012). The monophyletic
90 Acarosporaceae currently contains eight genera confirmed by molecular
91 phylogenies; Acarospora, Glypholecia, Myriospora, Pleopsidium, Polysporina,
92 Sarcogyne, Thelocarpella and Timdalia. Two further genera, Caeruleum and
93 Lithoglypha, are presumed to belong within Acarosporaceae, but this has so far not
94 been confirmed with molecular data. A recent analysis has also indicated that the
95 genus Eiglera is closely related to Acarosporaceae and if this is confirmed
96 Eigleraceae should be included in the Acarosporomycetidae (Miadlikowska et al.
97 2014).
98 99 In the Acarosporaceae, Acarospora, Myriospora, Pleopsidium and Timdalia have
ascomata with margins that contain green algae (the apothecia are referred to as
100 lecanorine, pseudolecanorine, or more often cryptolecanorine). Three genera,
101 Lithoglypha, Polysporina, and Sarcogyne, are characterized by algal-free lecideine
102 ascomata often with carbonized margins and/or discs (Fig. 1). The carbonization has
103 been fundamental for the current circumscription and delimitation of Sarcogyne and
104 Polysporina (Knudsen 2007b, Knudsen and Standley 2007).
105 Sarcogyne (Flotow 1851) was originally described for S. corrugata (=S. clavus)
106 a species with lecideine apothecia with very strongly carbonized margin (Fig. 1).
107 Magnusson (1935) expanded the concept of the genus and included a large number
108 of species with lecideine apothecia with or without a strongly carbonized margin.
109 Vězda (1978) later separated a number of species primarily characterized by a thick,
110 fissured, carbonized margin and a carbonized epihymenium (typically forming
111 umbonate apothecia) but also with richly branched and anastomosing paraphyses
5 112 with non-swollen apices, into Polysporina (Fig. 1). Sarcogyne in Vězda´s restricted
113 sense thus had a non-carbonized epihymenium and stouter, poorly branched
114 paraphyses with swollen apices while the margin either was strongly carbonized (as
115 in S. clavus where the interior of the margin is black pigmented and friable
116 throughout) or not (as in S. regularis which is only black pigmented at the surface)
117 and there are also examples of species with intermediary levels of carbonization as
118 in S. similis (the margin is black throughout but tar-like in texture, Knudsen and
119 Etayo 2009). Later authors, however, also included species with stout, simple
120 paraphyses in Polysporina (Ahti et al. 1987, Kantvilas 1998, Kantvilas & Seppelt
121 2006) and the only difference between the two genera at present is the carbonized
122 epihymenium (a build–up of carbonized accretions on the apothecial surface) in
123 Polysporina. The distinction between the genera is thus not clear and in need of re-
124 assessment (Hafellner 1995, Kantvilas and Seppelt 2006).
125 In this paper we investigate the phylogeny of the
126 Acarosporomycetidae/Acarosporaceae with the specific aim to clarify the evolution
127 of carbonized/melanized ascomata, with and without a carbonized epihymenium, in
128 this group. These traits are currently considered to characterize Sarcogyne and
129 Polysporina, and the monophyly of these two genera will be tested here.
130 6 131 Material & Methods
132 133 Molecular data
134 Taxon sampling
135 Species selected for sequencing were sampled broadly to cover as many different
136 genera and morphological groups as possible. We aimed at including two specimens
137 from different localities of each taxon. We obtained molecular data for 128
138 specimens (Suppl. Tab. 1 representing c. 62 taxa. The ingroup comprised 127
139 specimens, which included 57 representatives of Acarospora, 1 of Glypholecia, 11
140 of Myriospora, 3 of Pleopsidium, 36 of Polysporina, 17 of Sarcogyne, and 2 of
141 Timdalia. In spite of several attempts on fresh material we were not successful in
142 sequencing Caeruleum heppii. Fresh material of Lithoglypha and Thelocarpella was
143 not available to us in this study. Pycnora sorophora was used as outgroup.
144 145 DNA extractions, amplification, and sequencing
146 DNA was extracted from recently collected field material or from dried herbarium
147 specimens (Suppl. table 1). Total DNA was extracted using the Qiagen DNeasy
148 Plant MiniKit, according to the manufacturer’s instructions.
149 150 The selected markers for this study were the internal transcribed spacer complete
151 repeat (ITS) and the large subunit (nLSU) of the nuclear ribosomal DNA, the small
152 subunit of the mitochondrial ribosomal DNA (mtSSU), and the coding sequence of
153 the β-tubulin gene. A fragment of ca. 696 bp in the β-tubulin marker was amplified
154 using the primers BT3LM5 and BT10LM3 (Myllys et al., 2001) or with primers
155 newly designed in this study (see Suppl. table 2). The primers ITS1F (Gardes and
7 156 Bruns, 1993) and LR3 ( mycolab/primers.htm)
157 were used to amplify the internal transcribed spacer I, the 5.8 rDNA gene, the
158 internal transcribed spacer II and a fragment of approximately 600 bp in the nLSU
159 rDNA gene. The mtSSU was amplified using the primers mrSSU1 and mrSSU3R
160 (Zoller et al., 1999).
161 162 PCR amplifications were performed using Illustra™ Ready-To-Go PCR Beads,
163 according to the manufacturer’s instructions, with the following settings for ITS,
164 nLSU and mtSSU rDNA: initial denaturation 94°C for 5 min, followed by five
165 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 60 s), and finally 30 cycles (94°C
166 for 30 s, 52°C for 30 s, and 72°C for 60 s), with a final extension 72°C for 300 s;
167 and with the following settings for the gene coding for β-tubulin: initial
168 denaturation 94°C for 5 min, followed by five cycles (94°C for 30 s, 60°C for 30 s,
169 and 72°C for 60 s) and finally 30 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for
170 60 s), with a final extension 72°C for 300 s.
171 172 Before sequencing, the PCR products were purified using the PCR-M® Clean-up
173 System of Viogene or the enzymatic method Exo-sap-IT© provided by USB
174 Corporation.
175 176 The PCR-products were sequenced and purified using the DYEnamic ET terminator
177 cycle sequencing kit protocols (Amersham Biosciences, Freiburg, Germany), with
178 the following settings: 25 cycles (95°C for 20 s, 50°C for 15 s, and 60°C for 60 s).
179 The ITS rDNA sequences were produced using the PCR primers as above, plus the
180 additional primer ITS4 (White et al., 1990) when necessary. The mtSSU rDNA and
8 181 the β-tubulin sequences were produced using the PCR primers plus additional
182 internal primers (Suppl. Tab. 2) for β-tubulin when necessary. The purified samples
183 were run on an automated sequencer (ABI Prism 377).
184 185 Sequence alignment and phylogenetic analyses
186 Sequences were aligned using the multiple sequence alignment software MAFFT
187 version 7.110 (Katoh et al., 2002, Katoh and Toh 2008a). The G-INS-i algorithm
188 was used for the β-tubulin sequences and the Q-INS-i for the ITS and nLSU
189 sequences (Katoh and Toh 2008b). β-tubulin sequences were translated to amino
190 acids, and three identified introns were manually removed from the alignment.
191 Major insertions and ambiguous regions in the ITS and nLSU alignments were
192 identified and eliminated with Gblocks version 0.91b (Castresana, 2000) using the
193 relaxed parameter values suggested by Talavera and Castresana (2007).
194 We assessed congruence analysing the datasets separately by ML bootstrapping, to
195 detect possible conflicts among clades. Conflict was understood as bootstrap
196 support (≥ 70%; Hillis and Bull, 1993) for one marker, contradicted with significant
197 support by another. No incongruence was found and the data were concatenated into
198 a single dataset.
199 We used Maximum parsimony (MP), maximum likelihood (ML), and
200 Bayesian inference (BI) for the phylogenetic analyses using the combined dataset.
201 Maximum parsimony and parsimony bootstrap analyses were performed using TNT
202 1.1 1 (Tree analysis using New Technology, Goloboff et. al, 2008). We did heuristic
203 searches (‘traditional search’) collapsing ‘rule 3’ (tree collapsing = max. length 0;
204 collapsing branches with no possible support), 1000 random addition sequence
205 replications and holding up to 1000 trees during each replication, using a tree
9 206 bisection and reconnection (TBR) swapping algorithm. If the most parsimonious
207 trees (MPT) were found in only a few replications we broadened the search to
208 include more replications and holding more trees per replication. We also
209 performed a total of 1000 bootstrap replicates with the same specifications but with
210 100 random-addition sequence replicates. Parsimony-uninformative characters were
211 excluded from these analyses.
212 We performed ML analyses in RAxMLGUI 1.3, a graphical front-end for
213 RAxML (Randomized Accelerated Maximum Likelihood for High Performance
214 Computing; Stamatakis, 2006), using the GTRCAT model of nucleotide substitution
215 (a GTRGAMMA approximation with optimization of individual per-site
216 substitution rates). We partitioned the dataset by gene and by codon position in the
217 protein-coding gene (β-tubulin), which made a total of 8 partitions (ITS1, 5.8S,
218 ITS2, nLSU, mtSSU, BT 1st, BT 2nd and BT 3rd). The same model was applied to all
219 partitions because of constraints of the software RAxML. We performed a total of
220 100 runs and assessed node support via 1000 bootstrap replicates (ML + thorough
221 bootstrap; n. threads 2).
222 Bayesian analysis (Huelsenbeck et al., 2001) was achieved with the software
223 MrBayes 3.2.1 (Ronquist et al. 2011). We partitioned the dataset as in the ML
224 analyses, but in this case we selected the model of nucleotide substitution that
225 scored best for every particular partition, according to the Akaike Information
226 Criterion (AIC) in jModeltest (Posada, 2008). We used full likelihood optimization
227 and searched only among the 24 models implemented in MrBayes. Following this
228 scheme, a HKY model was selected for ITS1 and ITS2, a HKY+I+Γ model was
229 selected for the 5.8S and for the mitochondrial SSU DNA, a SYM+I+Γ model was
230 selected for the nuclear LSU rDNA, and a GTR+I+Γ, a SYM+I+Γ, and a SYM+Γ
10 231 models were selected for the 1st, 2nd and 3rd codon positions of the β-tubulin gene,
232 respectively. We linked topology across partitions but separated model parameter
233 values and proportional rates across partitions. The number of discrete gamma
234 categories was kept at default four. Bayesian prior distributions included treating all
235 tree topologies as equally likely, a uniform (0, 50) distribution for the gamma shape
236 parameter, a uniform (0, 1) distribution for the proportion of invariable sites, a flat
237 (1, 1, 1, 1, 1, 1) Dirichlet for the rate matrix, and a flat (1,1,1,1) Dirichlet for the
238 state frequencies (except when the model dictated state frequencies to be equal). We
239 performed three parallel runs, each with five chains, four of which were
240 incrementally heated with a temperature of 0.10. The analysis was diagnosed for
241 convergence every 100000 generations, measured as the average standard deviation
242 of splits across runs in the last half of the analysis. Every 100th tree was saved. The
243 first half of the run was discarded as burnin.
244 245 Hypothesis testing
246 We used Bayes factors to test the monophyly of Acarospora, Polysporina and
247 Sarcogyne, and also of the core group of Sarcogyne, i.e., those species that, like the
248 type species, S. clavus, have a strongly carbonized excipulum (e.g., Knudsen &
249 Kocourková 2008), in this analysis, S. algoviae, S. clavus, S. hypophaea, S.
250 hypophaeoides and Sarcogyne sp. 1. To calculate the Bayes factors we compared
251 the ratio of the marginal likelihoods of five hypotheses: four where each group was
252 constrained to be monophyletic, in addition to the best polygenetic hypothesis
253 recovered by MrBayes 3.2.1 (Ronquist et al., 2011) in the unconstrained analysis.
254 The Bayes factors were calculated using the modification introduced by Kass and
11 255 Raftery (1995), i.e. twice the difference between the ln harmonic mean likelihoods
256 of the two models.
257 258 Reconstruction of ancestral character states
259 We reconstructed the ancestral states of two characters, the carbonized exciple and
260 the carbonized epihymenium. The first character, carbonized exciple, has three
261 states, i.e., absence of carbonized exciple (0), presence of carbonized exciple (1),
262 and tar-like exciple (2). The second is a binary character coded as absence (0) or
263 presence (1) of carbonized epihymenium. The coding follows our own observations.
264 For the reconstruction of ancestral states we used maximum parsimony and
265 maximum likelihood as implemented in Mesquite v. 2.75 (Maddison and Maddison,
266 2011) and two Bayesian approaches, i.e., the method described by Huelsenbeck and
267 Bollback (2001) as implemented in SIMMAP v.1.5 (Bollback, 2006), following
268 Schultz and Churchil (1999), and the method described by Pagel et al. (2004) and
269 Pagel and Meade (2006), as implemented in BayesTraits (Pagel and Meade, 2007).
270 For all four methods, we used the posterior tree sample from the MrBayes analyses.
271 Branch lengths from that analysis were included in the ML and in the Bayesian
272 reconstructions. Maximum parsimony and maximum likelihood reconstructions
273 were achieved counting only unequivocal states. ML reconstructions were
274 performed under the Mk1 likelihood model for discrete morphological characters
275 (Lewis, 2001). For the SIMMAP analyses, we estimated three prior parameters: the
276 shape parameter α, and the scale parameter β, for the gamma distribution of the
277 overall substitution rate, and the shape parameter α for the beta distribution of the
278 bias parameter, this last one required only for binary morphological characters
279 (Bollback, 2006). The estimation was achieved in two steps following SIMMAP
12 280 documentation on morphology priors, and using the R package provided by
281 SIMMAP v. 1.5 (Bollback, 2006). Following this approach, the parameter values
282 for the gamma distribution of the overall substitution rate were α = 11.728 and β =
283 3.784 for character 1 (carbonization of the exciple), and α = 4.248 and β = 1.550 for
284 character 2 (carbonization of the hymenium). The single parameter value for the
285 beta distribution of the bias parameter in character 2 was α = 3.778. The number of
286 discrete gamma categories was kept at default 50. The morphological tree was
287 rescaled to a total length of 1. In the BayesTraits analyses, the prior on rates was
288 assumed to follow an exponential distribution with the mean drawn from a uniform
289 hyperprior on the interval (0, 10). The MCMC was run for 108 generations,
290 sampling parameters every 1000th generation, and discarding the first 107
291 generations as burn-in. The rate deviation of the normal distribution was set so that
292 the MCMC acceptance rate was between 20% and 40%. Each analysis was
293 conducted three times and similar harmonic mean likelihoods obtained across
294 identical runs indicated that MCMC chain had converged. To extract the marginal
295 posterior probability of each state at each node (integrated over priors, tree
296 topologies and branch lengths) from BayesTraits output, we used software written
297 by S. Ekman (Ekman et al., 2008).
298 In addition, we obtained the distribution of the number of character state
299 transformations along the Acarosporaceae using a) Maximum parsimony as
300 implemented in Mesquite (Maddison and Maddison 2011) and b) the Bayesian
301 stochastic mapping procedure described by Huelsenbeck et al. (2003) and Ronquist
302 (2004), as implemented in SIMMAP (Bollback, 2006). We used the same Bayesian
303 tree sample and the same rate priors as in the ancestral state reconstruction using
13 304 SIMMAP. The number of realizations from the prior distribution was set to 10 per
305 tree.
306 307 14 308 Results
309 We produced 112 new ITS rDNA, 126 new nLSU rDNA, 111 new mtSSU rDNA,
310 and 109 new β-tubulin sequences, and additional sequences (all produced in our
311 earlier work) were used from GenBank (Suppl. Tab. 1). We produced a combined
312 matrix of the four markers, which included a total of 512 sequences. Of the 128
313 terminals, only 5 lack β-tubulin. Therefore, sequences for all three markers were
314 available for 96% of the terminals. The combined matrix included (Suppl. Tab. 3) a
315 total of 2446 characters of aligned DNA sequences from mitochondrial and nuclear
316 genes: ITS1 rDNA (122 bp), 5.8S rDNA (117 bp), ITS2 rDNA (175 bp), nLSU
317 rDNA (584 bp), mtSSU rDNA (752 bp), and β-tubulin (696 bp).
318 The MP analysis of the combined dataset resulted in 124 most parsimonious
319 trees of 4798 steps. The best tree obtained from the ML analysis had a ln-likelihood
320 value of -24236.56. The Bayesian analyses of the combined dataset halted after
321 900,000 generations, at which time the average standard deviation of splits across
322 runs in the last half of the analysis was 0.009 (<0.01). Potential Scale Reduction
323 Factor (PSRF) values for all model parameters as well as all branch lengths were
324 close to 1 (none of the PSRF values of the model parameters was over 1.004). We
325 considered the three runs to have converged and that our sample was a valid
326 estimate of the posterior distribution. A majority rule consensus tree was
327 constructed from the 13,500 trees of the stationary tree sample. As the phylogenetic
328 reconstructions obtained by the three inference methods (MP, ML and BI) were
329 congruent, only the topology corresponding to the Bayesian analyses is shown in
330 Fig. 2.
331 332 The analyses find a number of strongly supported clades (Fig. 2) but several
branches in the backbone of the tree lack support or have support only from BI.
15 333 Myriospora form a monophyletic group as the sister-group to the rest of the family.
334 The sampled species in Pleopsidium, Timdalia and a clade formed by “Acarospora”
335 terricola and “A.” rhizobola also form distinct groups well separated from other
336 genera. The remaining species roughly form two clades, a well-supported
337 Acarospora clade including the type A. schleicheri and a clade dominated by
338 Sarcogyne species. The Acarospora clade is further composed of a well-supported
339 main clade of Acarospora and Polysporina species, the sister group of which is a
340 small clade formed by three Polysporina representatives, although this relationship
341 is only supported in the Bayesian analysis. The clade dominated by Sarcogyne
342 species has no support in any of the analyses, but the group formed by Acarospora
343 and Sarcogyne is supported by all three methods (Fig. 2). The overall support of the
344 phylogeny is high: 81% of the nodes are supported by the Bayesian analysis, 73%
345 of the nodes are supported both by Bayesian PPs and ML bootstrap, and 63% were
346 supported by all three methods (Bayesian PPs, ML, and MP bootstraps). Ten nodes
347 are supported by PPs only, including three nodes in the backbone of the phylogeny
348 (Fig. 2).
349 350 Hypothesis testing
351 The position of a few taxa such as Acarospora macrospora or Sarcogyne
352 hypophaeoides is unstable, and all hypotheses considering Acarospora,
353 Polysporina, Sarcogyne s. lat or Sarcogyne s. str. as monophyletic can be rejected.
354 The harmonic mean ln-likelihoods of each topological hypothesis are represented in
355 Tab. 1, together with the Bayes factor values resulting from the model comparisons.
356 When testing the monophyly of Sarcogyne, the Bayes factor was 173.24 in favour
357 of the best, unconstrained topology inferred by the Bayesian analysis. When testing
16 358 the monophyly of the core group of Sarcogyne, the Bayes factor was 328.12. And
359 finally, when testing the monophyly of Polysporina and Acarospora, the Bayes
360 factors were 166.09 and 167.78 respectively, in favour also of the best
361 unconstrained topology inferred by the Bayesian analysis. There is therefore very
362 strong evidence against all three hypotheses of monophyly (BF>>10; Kass &
363 Raftery, 1995, p.777).
364 365 Reconstruction of ancestral character states
366 Neither the presence of a carbonized epihymenium nor a carbonized exciple are
367 restricted to monophyletic groups (Fig. 3). We reconstructed the ancestral states of
368 these two characters in four supported nodes of the phylogeny, i.e. the node
369 including all Acarosporaceae except Myriospora (node 1, Fig. 3), the node
370 including Acarospora and Sarcogyne s. lat. (node 2, Fig. 3), the Acarospora clade,
371 which was only supported in the Bayesian analysis (node 3, Fig. 3), and finally the
372 most inclusive node within the Acarospora clade supported by all three
373 phylogenetic methods (node 4, Fig. 3). The results are summarized in Tab. 2. The
374 ancestral state of the carbonized exciple was reconstructed as ‘absence of
375 carbonized exciple’, by all methods, both for the common ancestor of the
376 Acarosporaceae excluding Myriospora at node 1, and for the common ancestor of
377 node 4 (i.e., the most inclusive clade within the Acarospora clade, supported by all
378 three reconstruction methods). Maximum parsimony and the two Bayesian methods
379 reconstruct nodes 2 and 3 as ‘absence of carbonized exciple’, although with low
380 probability, whereas maximum likelihood suggests a higher probability for the
381 common ancestor of node 2: Acarospora and Sarcogyne (s. lat.) having a non-
382 carbonized exciple (Table 2; Fig. 4). When studying the ancestral states of the
17 383 carbonized epihymenium we found that the common ancestor of the four nodes was
384 reconstructed as ‘absence of carbonized epihymenium’, by all methods.
385 BayesTraits, however, attributed very low probability to this state in the case of the
386 ancestor of the Acarospora clade, including Polysporina simplex WE30, P. simplex
387 SAR236, and Polysporina sp. 2 (Tab. 2; node 3, Fig. 3), where the probability was
388 distributed between ‘presence’ and ‘absence’ of carbonized epihymenium.
389 The total number of transformations calculated using parsimony (Mesquite)
390 and stochastic mapping (SIMMAP) are included in Tab. 3. The most probable
391 number of changes in the carbonized exciple is 12 according to parsimony, and 28
392 under the Bayesian approach, whereas the most probable number of changes in the
393 carbonized epihymenium is 7 according to parsimony or 21 according to the
394 Bayesian analysis. In all cases the numbers are several times higher than the
395 minimum possible amount of change, which is the number of states minus one (if
396 one state was plesiomorphic and the rest apomorphic) i.e. two changes in character
397 1 (carbonized exciple) and one single change in character 2 (carbonized
398 epihymenium). In the evolution of the exciple, the majority of change corresponds
399 to gains of carbonized exciple (83% under parsimony and 46% in the Bayesian
400 method). Both methods assign some probability to other transformation types, i.e.,
401 transformations from absence of carbonized exciple to tar-like exciple; from a
402 carbonized to a non-carbonized exciple, and transformations from carbonized
403 exciple to tar-like exciple. Only the Bayesian method assigns some probabilities
404 also to losses of tar-like exciple. In the second character, the gain of carbonized
405 epihymenium accounts for all the change recovered by the parsimony method. The
406 Bayesian method, however, distributes the probability between the two possible
407 transformation types (gains and losses of a carbonized epihymenium) although the
18 408 highest probability (62%) is still assigned to transformations from a non-carbonized
409 to a carbonized epihymenium (Tab. 4).
410 411 412 Discussion
413 The taxon sampling of Acarosporaceae in this study is by far the largest included in
414 a phylogenetic analysis, to date. Although the sampling possibly still is uneven and
415 clearly biased geographically with the majority of the specimens from Scandinavia
416 and to a lesser extent from North America, we think that the resulting pattern
417 reflects the phylogeny of the group well, and that the conclusions we draw will hold
418 when sampling is extended further. However, some caution is necessary when
419 considering the current phylogeny as three of the nodes in the backbone are
420 supported by PPs only. It is frequently observed that Bayesian methods of
421 phylogenetic inference produce higher probability values (PPs) for trees or clades
422 than other methods such as maximum parsimony or maximum likelihood
423 bootstrapping. This has been attributed to the so-called “Bayesian star-tree paradox
424 artefact”, i.e., most implementations of Bayesian inference do not consider
425 polytomies during the MCMC search, and can return high PPs for branches that are
426 unsupported by the data (Lewis et al., 2005; Yang 2008). It is also known that
427 “ancient rapid radiations”, detected in phylogenies as long ingroup branches
428 intercalated among short backbone internodes, cause problems in phylogenetic
429 reconstructions (Jian et al., 2008). This effect is often associated as well to a long
430 phylogenetic root (Rothfels et al., 2012).
431 As far as it is possible to compare, our results are congruent with earlier
432 analyses (Reeb et al. 2004, Wedin et al. 2005, 2009, Crewe et al. 2006). As in these
433 Myriospora is the sister-group to the rest of the family. The phylogeny furthermore
19 434 shows that species with carbonized ascomata within the Acarosporaceae are
435 distributed in two main clades, which are here referred to as the Acarospora and the
436 Sarcogyne clade respectively (Fig. 2). The Acarospora clade was retrieved in all
437 analyses with strong support although the inclusion of a small group of Polysporina
438 (P. simplex “C”) was only supported by PPs. The Sarcogyne clade lacks support for
439 the basal branches and there are several species, including the type of Sarcogyne,
440 that has an uncertain position. These results also largely agree with the recent
441 analysis by Miadlikowska et al (2014) who found one Acarospora s. str. clade and
442 one clade including both Sarcogyne and several Acarospora spp. but in addition
443 found a third main clade including a paraphyletic Pleopsidium together with the A.
444 smaragdula group (=Myriospora). That Acarospora is paraphyletic has earlier been
445 indicated by Reeb et al. (2004) and Crewe et al. (2006). However, a well-supported
446 monophyletic group corresponding to Acarospora s. str. was found in both
447 investigations. Here, we suggest that the whole Acarospora clade, which includes
448 the type of Polysporina, in the future is treated as Acarospora. There are, however,
449 to our current knowledge, no phenotypic synapomorphies unique to Acarospora.
450 This is irrespective of if Polysporina is included in Acarospora, or not.
451 Characterizing the genus morphologically is not easy but may possibly be done
452 using a suite of characters including secondary chemistry, conidia, ascus type and
453 other hymenial characters. Well-known and characteristic species and species
454 groups belonging to Acarospora s. str. include the A. schleicheri group (the type
455 species and a few other terricolous taxa with globose-subglobose spores), the A.
456 fuscata group (brown, saxicolous species containing gyrophoric acid), yellow
457 species (rhizocarpic acid), e.g., A. heufleriana and some lobate species, e.g., A.
20 458 molybdina and A. wahlenbergii although it should be noted that A. molybdina was
459 found to belong to the Myriospora clade by Miadlikowska et al (2014).
460 The Sarcogyne clade as a whole has no support and is not retrieved in all
461 individual-gene analyses (see Suppl. Figs. 1–4). The basal branches, e.g. the
462 Polysporina cyclocarpa group as well as Sarcogyne clavus and S. hypophaeoides all
463 change position (always without support) between the four different single-marker
464 ananalyses. Despite the fact that the group has no support, we believe that additional
465 data are likely to indicate that this clade is real. Reeb et al. (2004) also found a very
466 similar Sarcogyne clade, which likewise was poorly supported basally. Their
467 Sarcogyne clade included Sarcogyne regularis and S. similis together with A.
468 cervina, A. canadensis, A. laqueata, A. macrospora and Glypholecia scabra. Our
469 study thus has a similar but likewise unsupported result. In our tree (Fig. 2) several
470 other species group in this Sarcogyne clade, but without support. An additional
471 number of Acarospora spp., e.g. A. glaucocarpa, A. moenium, A. insolata and A.
472 impressula,, and a group of Polysporina specimens including P. cyclocarpa
473 characterized by comparatively stout paraphyses and broad spores may thus belong
474 here. The Sarcogyne clade contains both cryptolecanorine and lecideine taxa and
475 species with or without carbonized ascomata or carbonized epihymenium. There are
476 a couple of general phenotypic trends characterizing this clade compared to the
477 Acarospora clade. All species in the Sarcogyne clade in the tree lack pigments
478 (other than melanins) and other secondary metabolites with the exception of
479 Glypholecia scabra (gyrophoric acid). All species also have a euamyloid hymenium
480 except for the Polysporina cyclocarpa group, which have a hemiamyloid
481 hymenium. However, species without pigments and with a euamyloid hymenium
482 are also present in the Acarospora clade. Larger and broader spores, stout
21 483 paraphyses and a low hymenium are also characters that at least appear to be more
484 common in the Sarcogyne clade as compared to the Acarospora clade. With the
485 current dataset, there is in any case no support for the Sarcogyne-clade as such, and
486 little support for the relationships within it. Thus it is not meaningful at present to
487 discuss groupings in the Sarcogyne clade but we predict that in the future this group
488 will best be divided in several genera. It is, however, quite likely that the topology
489 will change considerably with more data and a revised analysis will require
490 additional markers to result in a better supported phylogeny.
491 Two species not included in our earlier phylogenies occur outside both the
492 Acarospora and the Sarcogyne clades, viz A. rhizobola and A. terricola. These two
493 terricolous taxa are both characterized by having bacilliform conidia, a character
494 that is unusual within the Acarosporomycetidae and is, in addition to these two
495 species, known from A. benedarensis (Knudsen and Fox 2010), A. convoluta
496 (Magnusson 1929), A. oligyrophorica (Aptroot 2002), A. sphaerosperma (Knudsen
497 et al. 2010), Sarcogyne crustacea (Knudsen and Kocourková 2010) and the two
498 monotypic genera Lithoglypha and Thelocarpella (Brusse 1988, Navarro-Rosinés et
499 al. 1999). A recent publication by Gueidan et al (2014), found A. rhizobola to be
500 closely related to Thelocarpella gordensis as well as the poorly understood
501 Trimmatothelopsis versipellis which was also found to have bacilliform conidia.
502 The relationship between these species will be investigated further in a forthcoming
503 paper.
504 The surprising conclusion from this investigation is that carbonized or
505 melanized ascomata is a highly plesiomorphic trait in the Acarosporaceae. Strongly
506 black pigmented ascomata have clearly appeared independently numerous times in
507 the evolution of the family. Furthermore, Polysporina with its typical umbonate,
22 508 carbonized apothecia is polyphyletic, and most species are not closely related to
509 Sarcogyne as previously believed. Instead, they are nested within Acarospora in a
510 strict sense. In addition, the species in Sarcogyne with a strongly carbonized
511 apothecium margin do not form a monophyletic group. These species were believed
512 to make up the core group of Sarcogyne and includes the type species S. clavus. The
513 results clearly show that neither the presence of a carbonized exciple nor the
514 presence of a carbonized epihymenium characterize a natural group in this lichen
515 family and thus is of very limited use to characterize genera or indeed the generic
516 placement of any formal taxa. A weak carbonization (“melanization”) of the
517 apothecial margin has also been noted to sometimes occur in several species in
518 Acarospora and Myriospora (Knudsen 2007a, Knudsen & Flakus 2009, Westberg et
519 al. 2010), which supports this.
520 The distinction between lecanorine and lecideine ascomata within
521 Acarosporaceae is usually clear-cut and has been an important character to separate
522 Acarospora (lecanorine) from Polysporina and Sarcogyne (lecideine). However,
523 some problematic cases have been observed. Magnusson placed a few taxa, today
524 recognized as Polysporina, in Acarospora based on the observed presence of green
525 algae in the margin (Magnusson 1924, 1929, 1935). These species otherwise have
526 the typical carbonized ascomata of Polysporina. Later authors have not commented
527 on this, either not observing the presence of green algae or possibly considering it
528 an occasional or aberrant occurrence and included them as Polysporina. In this
529 study we have found several specimens of P. subfuscescens s. lat., with green algae
530 within the carbonized margin (Fig. 1E–F). In the analysis they group with
531 specimens with distinctly lecideine ascomata, identified either as P. subfuscescens
532 or P. simplex depending on whether they were interpreted as lichenicolous or not.
23 533 The lecanorine specimens are all clearly lichenicolous, developing on the thallus of
534 a host. It appears to us that this character is the result of different developments or
535 different stages of the infection of the host.
536 The position of the type species of Polysporina, P. simplex has not been
537 clear in earlier studies. Polysporina cf simplex was found to be sister species to the
538 rest of the Acarosporaceae/Acarosporomycetidae by Reeb et al. (2004) but other
539 specimens sequenced and not included in that paper did not group together (Reeb,
540 pers. comm.). A different position of P. simplex was found by Crewe et al. (2006)
541 but according to our observations that specimen is a misidentified Sarcogyne cf
542 clavus. In this analysis we have identified P. simplex according to recent
543 morphological hypothesis (Knudsen & Kocourková 2008, 2009). Samples attributed
544 to Polysporina simplex are found in three different clades in our phylogeny (Fig. 2).
545 One clade (P. simplex “C”) is the sister clade to the rest of the Acarospora clade and
546 the remaining two are nested inside the Acarospora clade. The P. simplex
547 specimens differ between the clades in ecology and hymenium height (Fig. 1A–D)
548 and we conclude from our studies of the type material that it belongs to either of the
549 two closely related clades within P. simplex “A”, which are close to the generic type
550 of Acarospora, A. schleicheri. (Fig. 2)., Polysporina will be thus be treated as a
551 synonym to Acarospora. . It is also clear that also P. subfuscescens in its current
552 concept is not monophyletic and that a revision of the species taxonomy thus is
553 necessary. Our morphological studies so far indicate that it is not a case of cryptic
554 species but rather that the morphological characters have not been well understood.
555 Important characters not emphasized in the current broad concepts of some species
556 include hymenium height, spore width and paraphysis thickness. The taxonomic
557 implications will be addressed in a separate paper.
24 558 In this paper, we have again found evidence suggesting that traits seen as
559 extremely important in current generic and species classification of fungi including
560 lichens, do not necessarily characterize natural, monophyletic groups, as in so many
561 other investigations (e.g. Wedin et al. 2004; Lumbsch and Leavitt 2011; Spribille
562 and Muggia 2013; Otálora et al. 2013, 2014; Ekman et al. 2014). Strongly black
563 pigmented (carbonized or melanized) ascomata have independently arisen
564 numerous times in the evolution of the Acarosporomycetidae/Acarosporaceae, and
565 the genera Sarcogyne and Polysporina are thus distinctly non-monophyletic.
566 Ancestral state reconstructions showed that carbonization of the hymenium was
567 absent at all reconstructed nodes, and carbonisation of the exciple was absent at two
568 nodes and equivocal in the other two. The number of changes in character states of
569 both carbonized epihymenium and carbonized exciple are considerably more than
570 the minimum number, showing that these characters are highly likely to change
571 during the evolution in this group, again making them unlikely to characterize
572 natural groups on higher level. Carbonization seems to be a derived character that
573 has appeared during particular episodes in the evolution of the
574 Acarosporomycetidae/Acarosporaceae. Melanin has been connected with protection
575 against both metals and solar energy, because of its metal-binding capacity
576 (McLean et al., 1998; Purvis et al., 2004) and its ability to absorb UV radiation
577 (Gauslaa & Solhaug, 2001; Solhaug et al., 2002; Nybakken et al., 2004). If dated
578 and geographically wider phylogenies of the group become available in the future, it
579 would be interesting trying to connect the gain of strongly black-pigmented
580 ascomata with events such as colonization of more exposed or metal-rich areas,
581 where melanin-rich structures could have been of adaptive value. Finally, the
582 presence or absence of green algae in the ascoma margin (lecanorine or lecideoid
25 583 lecideine ascomata) seems to be variable even within single species. We can predict
584 future substantial re-arrangements of this very diverse and evolutionary interesting
585 group of lichenized fungi.
586 587 Acknowledgements
588 This study was funded by grants to Martin Westberg by The Swedish Taxonomy
589 Initiative (Svenska Artprojektet, administered by the Swedish Species Information
590 Centre/ArtDatabanken) and was further supported by grants to Mats Wedin from
591 the Swedish Research Council (VR 621-2009-5372, VR 621-2012-3990). The work
592 of Kerry Knudsen was financially supported by the grant “Environmental aspects of
593 sustainable development of society” 42900/1312/3166 from the Faculty of
594 Environmental Sciences, Czech University of Life Sciences Prague. We are grateful
595 to the staff at the Molecular Systematics Laboratory at the Swedish Museum of
596 Natural History for laboratory assistance, in particular Jan Ohlson and Bodil
597 Cronholm. Valerie Reeb kindly shared unpublished details from on her work on
598 Acarosporaceae. The first author would finally like to thank Ulf Arup (LD),
599 Philippe Clerc (G), Leif Tibell (UPS), Toni Berglund (Karlskoga) and members of
600 the Swedish Lichen Society for assistance during field work.
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35 Table 1. Comparison of three topological hypotheses; 1) Sarcogyne is a monophyletic
group, 2) the core group of Sarcogyne is monophyletic, and 3) Polysporina is a
monophyletic group, with 4) the best topological hypothesis inferred by the Bayesian
analysis, when using an unconstrained model (i.e., not assuming monophyly for any
of the groups). Marginal likelihoods of each model were estimated as the ln harmonic
mean likelihoods of the data. Bayes factors are calculated as twice the difference of
the ln harmonic mean likelihoods of the two models being compared.
Model ln-likelihood
Unconstrained model Constrained model
Sarcogyne monophyletic
Sarcogyne core monophyletic
Polysporina monophyletic
Acarospora monophyletic
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