Evolution of lifestyles in Capnodiales

The Capnodiales, which includes fungi known as the sooty moulds, represents the second largest order in Dothideomycetes, encompassing morphologically and ecologically diverse fungi with different lifestyles and modes of nutrition. They include saprobes, plant and human pathogens, mycoparasites, rock-inhabiting fungi (RIF), lichenised, epi-, ecto- and endophytes. The aim of this study was to elucidate the lifestyles and evolutionary patterns of the Capnodiales as well as to reconsider their phylogeny by including numerous new collections of sooty moulds, and using four nuclear loci, LSU, ITS, TEF-1α and RPB2. Based on the phylogenetic results, combined with morphology and ecology, Capnodiales s. lat. is shown to be polyphyletic, representing seven different orders. The sooty moulds are restricted to Capnodiales s. str., while Mycosphaerellales is resurrected, and five new orders including Cladosporiales, Comminutisporales, Neophaeothecales, Phaeothecales and Racodiales are introduced. Four families, three genera, 21 species and five combinations are introduced as new. Furthermore, ancestral reconstruction analysis revealed that the saprobic lifestyle is a primitive state in Capnodiales s. lat., and that several transitions have occurred to evolve lichenised, plant and human parasitic, ectophytic (sooty blotch and flyspeck) and more recently epiphytic (sooty mould) lifestyles.

The Capnodiales represent the second largest order in Dothideomycetes after the Pleosporales. The Capnodiales is included in the subclass Dothideomycetidae along with the Dothideales and Myrangiales ). The taxonomic concept of this order was expanded from the original description by Luttrell (1955), based on a multigene phylogeny and the presence of ostiolar periphyses as a synapomorphic feature (Schoch et al. 2006). Taxa in this order lack pseudoparaphyses, but include several species with periphysoids and periphyses (Lumbsch & Lindemuth 2001).
As discussed by Schoch & Grube (2015), the Capnodiales was established based on the sooty moulds in three families, Antennulariaceae, Capnodiaceae, and Coccodiniaceae. However, phylogenetic analyses revealed that the sooty moulds are polyphyletic and include species residing in two different classes, Dothideomycetes and Eurotiomycetes (Crous et al. 2007a).
During the course of the past decade, considerable attention has been paid to the phylogeny and systematics of genera and families in the Capnodiales. Presently the order accommodates fungi having highly diverse ecological niches, lifestyles and modes of nutrition (Crous et al. 2007a, 2009, Ruibal et al. 2009, Hyde et al. 2013, Chomnunti et al. 2014, Quaedvlieg et al. 2014, Ismail et al. 2016, Hongsanan et al. 2017, Videira et al. 2017, Crous et al. 2018. Although the Capnodiales s. str. are epiphytic sooty moulds, the presently applied circumscription also includes ectophytes and plant pathogens. Previous studies have, however, not addressed this ecological divergence adequately. This is due to a limited sampling of sooty moulds, and a poorly resolved phylogenetic backbone mainly based on nuclear ribosomal RNA genes. The aim of this study was therefore to reconsider the phylogenetic backbone of the Capnodiales by including numerous new collections of sooty moulds, thus also providing a more robust phylogeny using four nuclear loci, LSU, ITS, TEF-1α and RPB2.

Isolates
The sooty mould isolates studied here were obtained from the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute (WI), Utrecht, the Netherlands, and the working collection of Pedro Crous (CPC) housed at the WI (Table 1). Sequences of other strains were retrieved from GenBank (Tables 1,  S1). Representative cultures of the new species described in this study were deposited in the CBS culture collection.
PCR conditions for LSU, ITS and TEF-1α were: an initial denaturation step of 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 45 s at 52°C (ITS, LSU) or 55°C (TEF-1α) and 1 min at 72°C, and a final elongation step of 7 min at 72°C. Touchdown PCR was performed for amplification of RPB2 as follows: an initial denaturation at 95°C for 5 min followed by 35 cycles of 30 s at 95°C, 30 s at 60°C (5-10 cycles)/56°C (5-10 cycles)/52°C (15-25 cycles) and 1 min at 72°C, and a final elongation step of 7 min at 72°C.
The PCR products were sequenced with both forward and reverse primers using an Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific). The DNASTAR Lasergene SeqMan Pro v. 8.1.3. software was used to obtain consensus sequences. All new sequences were submitted to GenBank (Tables 1, S1).

Phylogenetic analyses
Generated sequences were aligned with sequences retrieved from GenBank (http://www.ncbi.nlm.nih.gov) using the online interface of MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/ index.html), and manually edited in MEGA v. 7.0.21. Maximum Likelihood (ML) and Bayesian analysis (BA) were implemented for phylogenetic inferences of both single locus and concatenated alignments on the CIPRES Science Gateway portal (https://www.phylo.org/; Miller et al. 2012) using RAxML-HPC BlackBox v. 8.2.10 (Stamatakis 2014) and MrBayes v. 3.2.6 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003, respectively. The ML analyses were performed using a GTR+GAMMA substitution model and four rate classes with 1 000 bootstrap iterations. For the Bayesian analyses the optimal nucleotide substitution models were determined for each locus using MrModelTest v. 2.3 (Nylander 2004). Bayesian analyses were computed under the optimal nucleotide substitution models with four simultaneous Markov Chain Monte Carlo chains, 10 M generations and a sampling frequency of 1 000 generations, ending the run automatically when standard deviation of split frequencies dropped below 0.01. Burn-in was set to remove 25 % of the first sampled trees, after which the 50 % majority rule consensus trees and posterior probability (PP) values were calculated. The resulting trees were plotted using FigTree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree). Alignments and trees were deposited in TreeBASE (www.treebase.org; S25414) and taxonomic novelties in MycoBank (www.MycoBank.org; Crous et al. 2004).
Ancestral states were reconstructed using Mesquite v.3.6 (Maddison & Maddison 2018). Character history was inferred using the Bayesian tree (see above) as phylogenetic framework. Ancestral states were determined based on a maximum likelihood approach with a MK1 model of evolution. Characters states were defined as saprobe, epiphyte, parasite or lichen. The character state for taxa with an uncertain lifestyle was coded as "?".

Morphology
Isolates stored in liquid nitrogen or lyophilised were reactivated on 2 % malt extract agar (MEA) or oatmeal agar (OA). Colonies were sub-cultured onto MEA, OA, cornmeal agar (CMA), potato dextrose agar (PDA), and synthetic nutrient-poor agar (SNA) supplemented with pine needles at room temperature. Culture media were prepared as described by Crous et al. (2019a, b). Cultures were examined periodically for the development of reproductive structures. Slide preparations were made with clear lactic acid or Shear's mounting fluid. Morphological observations of fungal structures were made using a Nikon SMZ1000 dissecting microscope and a Zeiss Axioscope 2 compound microscope with differential interference contrast (DIC) illumination. Measurements and images were taken using a Nikon DS-Ri2 high definition colour digital camera. Measurements and descriptions of microscopic structures were made from cultures grown on SNA. A few strains that were sterile on SNA were described from other media (indicated in text). The mean, standard deviation, maximum and minimum values of at least 30 fungal structures were calculated where possible. Dimensions are presented as a range with extremes in parentheses. Growth rates were measured on MEA after 2 wk and colony characters were noted. Colony colours were rated according to the colour chart of Rayner (1970).

Phylogeny
Amplification of the partial sequences of LSU, ITS and TEF-1α was successful but RPB2 proved difficult to amplify using normal    PCR, and therefore a touchdown PCR program was used. For most of the isolates the combination of fRPB2-f5F and fRPB2-7cR primers (Liu et al. 1999) was more successful than the primer combination fRPB2-f5F2 and fRPB2-7cR (Sung et al. 2007). For a few isolates, we used the forward primer fRPB2-f5F2 instead of fRPB2-f5F. Two datasets were analysed in this study. The first dataset consisted of combined LSU, TEF-1α and RPB2, including 193 taxa representing three orders Capnodiales s. lat., Myrangiales, Dothideales, with Venturia inaequalis (CBS 594.70) as outgroup. After alignment the dataset contained a total of 3 168 characters (LSU: 837, TEF-1α: 1 176, RPB2: 1 147), including alignment gaps. MrModelTest revealed that the general time-reversible model of evolution (Rodríguez et al. 1990), including estimation of invariable sites and assuming a discrete gamma distribution (GTR+I+G) with six rate categories (lsetnst = 6, rates = invgamma) and dirichlet (1,1,1,1) base frequencies is the best nucleotide substitution model for all loci (LSU, TEF-1α and RPB2). The Bayesian analyses of the concatenated alignments of three loci generated 7 292 trees from which 1 822 trees were discarded as burn-in. The consensus tree and posterior probability values (PP) were calculated from the remaining 5 470 trees. The average standard deviation of split frequencies was 0.009987 at the end of the run. The RAxML search of the dataset with 1 767 distinct alignment patterns produced a best-scoring ML tree (lnL = -66287.001595). The bootstrap values equal to or higher than 50 % were mapped on the Bayesian tree (Figs 1, S1). The same phylogenetic tree was obtained from both RAxML and Bayesian analyses. Capnodiales s. lat. was split into seven distinct clades representing seven orders. Sooty mould fungi constituted Capnodiales s. str., a single highly supported clade (ML-BS = 90 %, PP = 1). Mycosphaerellales with high support in both analyses (ML-BS = 100 %, PP = 1) proved clearly distinct from Capnodiales s. str., and was thus resurrected here as a separate order containing eight families: Mycosphaerellaceae, Dissoconiaceae, Phaeothecoidiellaceae, Schizothyriaceae, Extremaceae, Cystocoleaceae, Neodevriesiaceae and Teratosphaeriaceae (Figs 1, S1). In the Bayesian analysis Mycosphaerellales grouped with Capnodiales s. str. in a well-supported clade (PP = 0.98), while in the RAxML analysis it was a sister group of Cladosporiaceae with low support (ML-BS < 50 %). Therefore, Cladosporiaceae was elevated to ordinal level, and Cladosporiales introduced. In both RAxML and Bayesian analyses four new orders were recognised: Phaeothecales, Neophaeothecales, Racodiales (for Racodium rupestre), and Comminutisporales (for Comminutispora agavaciensis).
The second dataset consisted of aligned sequences of four loci (LSU, ITS, TEF-1α and RPB2), and included 102 taxa belonging to Capnodiales s. str., and two species, Myriangium hispanicum (CBS 247.33) and Elsinoe phaseoli (AFTOL-ID-1855), as the outgroup taxa. The aligned dataset contained 3 603 characters (LSU: 849, ITS: 553, TEF-1α: 1 035, RPB2: 1 154), including alignment gaps. Results from MrModelTest indicated a GTR+I+G as the best fit model for the ITS sequence data, as was the case for three other loci (LSU, TEF-1α and RPB2) in dataset 1. The RAxML search of the second dataset detected 1 463 distinct alignment patterns and yielded a tree with lnL = -30052.650187 (Fig. 2). The Bayesian analyses generated 4 622 trees from which 1 154 trees were discarded as burn-in. The consensus tree and posterior probability values (PP) were calculated from the remaining 3 468 trees. The average standard deviation of split frequencies was 0.009980 at the end of the run. Posterior probability values were mapped on the ML tree ( Fig. 2). Three families, namely Capnodiaceae, Neoantennariellaceae and Readerielliopsidaceae were recognised in Capnodiales s. str. In Capnodiaceae seven morphologically and phylogenetically well-supported genera including Capnodium, Chaetocapnodium, Conidiocarpus, Heteroconium, Leptoxyphium, Phragmocapnias and Polychaeton were identified. Eleven new species were recognised in this family. Readerielliopsidaceae contained four genera (Phaeoxyphiella, Readerielliopsis, Scolecoxyphium and Scorias) and Neoantennariellaceae three genera (Fumiglobus, Neoantennariella and Neoasbolisia).

Readerielliopsis
Ancestral state reconstruction revealed a saprobic lifestyle as the ancestral state of the Capnodiales s. lat. included in this study (see Neophaeothecales; Fig. 3), while whole genome sequences of a more diverse set of taxa also supported the ancestral state of Dothideomycetes to be saprobic (Haridas et al. 2020). In the dataset included in the present study the saprobic lifestyle emerged during the evolution of diverse taxa, with several reversals back to parasitism. All Capnodiales emerged from an ancestor that had an ectophytic lifestyle. The analyses also revealed that all Mycosphaerellales shared an ancestor that was saprobic. From this ancestor ectophytes and lichen associated fungi emerged, while the ancestors of several species in this group reverted to a parasitic lifestyle. The ancestor of all Cladosporiales and Comminutisporales were saprobes but some species in the Cladosporiales reverted back to parasitism. Results showed that the ancestor of the Racodiales was a lichen-associated fungus, and that of the Dothideales was a saprobe.

Taxonomy
Based on the phylogenetic analyses of the two datasets generated in this study, combined with the differences in morphology and ecology, the Capnodiales requires redefinition, and the Mycosphaerellales must be resurrected. Furthermore, five new orders, four new families, three new genera, 21 new species and five new combinations are introduced below. Etymology: Name refers to the genus Cladosporium.
Etymology: Name refers to the fact that it is related to Ca. coffeicola.     Notes: Chaetocapnodium is a hitherto monotypic genus introduced based on the morphology of its sexual morph and supported by LSU sequence data (Liu et al. 2015). The phylogenies generated in the present study (Figs S1, 2), however, revealed that Antennariella placitae and Phragmocapnias philippinensis are congeneric with Chaetocapnodium. Nine other isolates clustered in six distinct clades representing six species, five of which are recognised as taxonomic novelties. Two new combinations are proposed and five new species described in Chaetocapnodium.
Etymology: Name refers to Indonesia where this fungus was collected.
Etymology: Name reflects the fact that it was collected from an island.
Culture characteristics: Colonies, leathery, appressed, with fluffy aerial mycelium, with creamy conidial exudates in centre of colony; surface folded, edge sinuate, pale mouse grey to olivaceous black after 2 wk in the dark at 25°C. Colonies reaching 29 mm diam on MEA after 2 wk in the dark at 25°C.  Notes: Chaetocapnodium summerellii resembles Ch. thailandense and Ch. tanzanicum in morphology, but is phylogenetically distinct, forming a separate clade (Figs S1, 2). Furthermore, its radial growth rate on MEA at 25°C (29 mm diam/2 wk) is slower than that of the latter two species (38 mm diam/2 wk).
Etymology: Name refers to Tanzania where this fungus was collected.
Culture characteristics: Colonies, leathery, metallic, appressed, with fluffy aerial mycelium at the centre, with creamy exudates of pycnidia containing conidia, folded, edge sinuate, smoke grey to greenish black, edge sienna to cinnamon after 2 wk in the dark at 25°C. Colonies reaching 38 mm diam on MEA after 2 wk in the dark at 25°C.  Fig. 13.
Etymology: Name refers to Thailand where this fungus was collected.
Culture characteristics: Colonies, leathery, appressed, with fluffy aerial mycelium, glaucous grey to pale olivaceous grey after 2 wk in the dark at 25°C. Colonies reaching 38 mm diam on MEA after 2 wk in the dark at 25°C. Notes: Phylogenetically, Ch. thailandense constitutes a distinct lineage (Figs S1, 2). Morphologically Ch. thailandense resembles Ch. tanzanicum and Ch. summerellii. It is distinguished Saprobic on sugary exudates from insects, with dark mycelium forming a soot-like coating on the upper surface of leaves. Thallus composed of black, pelliculose, reticulately branched, dense, cylindrical, radiating, septate hyphae. Ascomata not observed. Conidiomata pycnidial, supported on black, long, narrow, cylindrical stalks composed of tightly compacted, anastomosed, synnematous cylindrical hyphae, lageniform with a brown oval or ellipsoid part, which produces a long neck and conidia. The pycnidium wall is composed of two or more layers, the outer one being more or less pseudoparenchymatous although the short cells tend to be arranged linearly, indicating their origin from longitudinally fused hypha. Ostiole surrounded by hyaline, subulate, hyphal extensions. Conidia small, ellipsoid, continuous, aseptate, hyaline, smooth-walled, arranged in a droplet at the apex of pycnidial neck (adapted from Hughes 1976, Chomnunti et al. 2011.

Type species: Conidiocarpus caucasicus Woron.
Notes: Conidiocarpus with the type species Co. caucasicus was introduced by Woronichin in Jaczewski (1917). However, Hughes (1976) stated that Batista & Ciferri (1963a) considered Co. penzigii, the second oldest species introduced in 1926, as the type species. Conidiocarpus has been reported as the asexual morph of Phragmocapnias (Hughes 1976). Phragmocapnias betle is the type species of Phragmocapnias. Following the ICN code based on the priority rule and one fungus = one name principles, Bose et al. (2014) chose Conidiocarpus and transferred species of Phragmocapnias to Conidiocarpus.
In this study based on phylogenetic analyses, the type species of Conidiocarpus and Phragmocapnias clustered in two distinct clades representing two different genera (Figs S1, 2). As discussed by Hughes (1976), pycnidia of Co. caucasicus are elongated, 540-650 μm long including a stalk, swollen part and a neck. In morphological studies, we found that all of the species that grouped with C. caucasicus produced a Conidiocarpus pycnidial type, typified by having a long neck (Fig. 14). Hughes (1976) mentioned that in cases where the sexualasexual connections have been confirmed, the pycnidial morphs of Phragmocapnias were the tall Conidiocarpus conidiomata that lacked necks (Figs 14, 16). Chomnunti et al. (2011) designated an epitype (MFLU09-0650, living culture MFLUCC10-0053) for the type species Phragmocapnias betle, and re-described this species based on the sexual morph. They did not observe the asexual morph. Based on Hughes (1976), asexual conidiomata are Conidiocarpus pycnidia (150-700 μm long) that lack a neck. To observe the pycnidial morph of Phragmocapnias betle, we used different culture media and were able to introduce the asexual morph on PDA, PCA and CMA. Conidiomata were pycnidia with short stalks, ellipsoidal swellings, and lacking necks. Therefore, following the views of Hughes (1976), we chose to resurrect Phragmocapnias for species with conidiocarpus-like pycnidia lacking necks.
In our phylogenetic analyses based on four loci (LSU, ITS, TEF-1α and RPB2), Conidiocarpus isolates clustered in two subclades (Figs S1, 2). The ex-type isolates of Co. asiaticus and Co. siamensis together with Co. caucasicus and the nine isolates sequenced in this study clustered in the first subclade strongly supported in the RAxML analysis, but with no support from the Bayesian analyses. These species are morphologically different but phylogenetically unresolved, which may be due to missing data. Only LSU sequence data are available for the ex-type strains of Co. caucasicus (GUMH937) and Co. siamensis (MFLUCC10-0064), and LSU/ITS sequences for Co. asiaticus (MFLUCC10-0062). Two isolates CPC 20464 and CPC 20468 for which four genes sequenced in this study and an isolate belonging to Co. siamensis (MFLUCC10-0061) with LSU and ITS sequences clustered in the second sub-clade, representing a putatively new Conidiocarpus species supported by both RAxML and Bayesian analyses (ML-BS = 100 %, PP = 0.82). However, the identity of the other Conidiocarpus isolates included in this study can only be resolved once additional gene regions have been sequenced. ). Leptoxyphium cacuminum and L. glochidion are identical based on LSU sequences, but they are morphologically different. These species clustered in the same clade together with 14 isolates considered in this study (Figs S1, 2). Some variation in nucleotide sequences (especially RPB2) was observed within this clade, which may indicate intra-or interspecific variation. Isolates CBS 451.66 and CPC 26196 clustered in a distinct clade (Figs S1, 2) representing a new species described below.
Etymology: Name refers to Citrus, the host genus from which it was collected.
Culture characteristics: Colonies, appressed, with fluffy aerial mycelium, with creamy conidial exudates, smoke grey to pale olivaceous grey after 2 wk in the dark at 25°C. Colonies reaching 39 mm diam on MEA after 2 wk in the dark at 25°C. Notes: Phylogenetically, L. citri is closely related to L. kurandae (Figs S1, 2). These two species differ in 1, 4 and 10 bp in LSU,
Culture characteristics: Colonies leathery, appressed, with fluffy aerial mycelium, somewhat folded at the middle, pale olivaceous grey to pale greenish grey after 2 wk in the dark at 25°C. Colonies reaching 32-34 mm diam on MEA after 2 wk in the dark at 25°C. Notes: This species was recently described by Hongsanan et al. (2015b) based on its sexual morph. Phylogenetically, it is closely related to P. betle (Figs S1, 2).
Mycelium superficial or immersed, hyaline to brown, branched, consisting of smooth, thin-walled, septate hyphae, constricted at septa, with a mucilaginous outer wall layer. Conidiomata pycnidial, supported on a black mycelial network, narrow and cylindrical stalks composed of tightly compacted, anastomosed, synnematous cylindrical hyphae, lageniform with a brown oval or ellipsoid part which produces conidia and a long neck. The pycnidium wall is composed of two or more layers, the outer one being more or less pseudoparenchymatous although the short cells tend to be arranged linearly, indicating their origin from longitudinally fused hypha. Ostiole surrounded by hyaline, subulate, hyphal extensions. Conidia small, oblong to ellipsoid, continuous, aseptate, hyaline, smooth-walled, arranged in a droplet at the apex of pycnidium.

Type species: Polychaeton quercinum (Pers.) Kuntze
Notes: Polychaeton was introduced by Persoon as a sub-genus in Fumago, and raised to generic rank by L eveill e (1847). The taxonomy of Polychaeton was discussed by Hughes (1976) and Chomnunti et al. (2011). Hughes (1976) considered Po. citri and Po. quercinum suitable as generic types, and designated Po. quercinum as lectotype species of Polychaeton. Chomnunti et al. In this study, we examined isolate CBS 116435 deposited in CBS as Po. citri (Pers.) L ev. from Iran on Citrus aurantium, isolated by Walter Gams. In the phylogenetic analyses (Figs S1, 2) this isolate clustered in a distinct clade close to Conidiocarpus and Phragmocapnias. Hughes (1976) mentioned that in Polychaeton pycnidia are supported on a stalk, have an ellipsoidal pycnidial cavity, with no conspicuous swelling, and terminate in a neck with hyaline hyphal extensions. On SNA isolate CBS 116435 produced pycnidia with a swollen body, a long neck, and a short stalk. On OA pycnidia tended to have much longer necks with no conspicuous swelling, while in Conidiocarpus pycnidia have a conspicuous swelling, a long neck, and are supported on a long stalk on both OA and SNA (Fig. 14). Therefore, we designate this clade as Polychaeton, although further studies are required to resolve the taxonomy of the various species described in the genus.
Etymology: Name refers to the genus Neoantennariella.
Mycelium superficial or immersed, pale brown to brown, branched, consisting of smooth, thin-walled, septate hyphae, constricted at septa, with a mucilaginous outer wall layer.
Pycnidia superficial or immersed, mostly globose or cylindrical, pale to dark brown, intercalary, lateral or terminal on erect hyphal branches. Ostiole absent, or not well-developed, mostly releasing conidia by means of irregular rupture. Conidia hyaline, aseptate, ellipsoid to ovoid, with minute guttules, smooth, thin-walled.   Etymology: Name reflects its morphological similarity to the genus Antennariella.
Mycelium superficial or immersed, pale brown to brown, branched, consisting of smooth, thin-walled, septate hyphae, constricted at septa, with a mucilaginous outer wall layer. Pycnidia superficial or immersed, mostly globose or cylindrical, pale to dark brown, mostly intercalary, lateral or terminal on erect hyphal branches. Ostiole absent, or not well-developed, mostly releasing conidia by means of irregular rupture. Conidia hyaline, aseptate, ellipsoid to ovoid, with minute guttules, smooth, thinwalled.
Etymology: Name reflects the host genus Phylica from which it was isolated.
Etymology: Name reflects its morphological similarity to the genus Asbolisia Bat. & Cif.
Etymology: Name reflects the host genus Phylica from which it was isolated.
Culture characteristics: Colonies leathery, appressed, with fluffy aerial mycelium, edge sinuate, glaucous grey to olivaceous grey after 2 wk in the dark at 25°C. Colonies reaching 14 mm diam on MEA after 2 wk in the dark at 25°C. Etymology: Name refers to the genus Readerielliopsis.
Mycelium superficial or immersed, hyaline to pale brown, branched, hyphae smooth, thin-walled, septate, constricted at septa, with a mucilaginous outer wall layer. Pycnidia superficial, globose to pyriform, cylindrical to flask-shaped, short to long, straight to irregular, occurring singly or in groups, medium to dark brown, if synnematous, then with a hyaline to pale brown stalk forming a long neck. Ostiole absent, or present, with or without hyphal hairs. Conidia small, hyaline, smooth, aseptate, oblong to ellipsoid or obdeltoid, or pale to medium brown, transversely euseptate, filiform, fusoid-ellipsoidal. Type species: Fumagospora capnodioides G. Arnaud As discussed by Hughes (1976) Fumagospora with hyaline and continuous conidia that become brown and transversely septate with one or more longitudinally septate cells, is the asexual morph of some Capnodium species (e.g. Capnodium salicinum). In this study Ca. salicinum (CBS 131.34) constitutes a distinct clade in Readerielliopsidaceae separate from other Capnodium species that grouped in Capnodium sensu Chomnunti et al. (2011). In a study on capnodiaceous sooty molds in Iran, phylogenetic analysis based on ITS sequence data placed isolate GUM 1315 with Fumagospora morphology (fig. 2

LIFESTYLES IN CAPNODIALES
www.studiesinmycology.org ellipsoidal, 3-septate phragmoconidia, which are at first hyaline, slowly becoming brown. The seventh species, Ph. callitris, has 5septate conidia, but is a nomen nudum because it lacks a Latin diagnosis. There are no cultures or sequences available for any of these species. In this study, we examined five isolates which based on phylogenetic inference clustered in two distinct clades (Figs S1, 2). Etymology: Name reflects Phylica, the host genus from which it was collected.
Culture characteristics: Colonies, leathery, appressed, with fluffy aerial mycelium, folded, edge sinuate, glaucous grey to pale olivaceous grey after 2 wk in the dark at 25°C. Colonies reaching 18 mm diam on MEA after 2 wk in the dark at 25°C.  Notes: Phaeoxyphiella phylicae differs from all seven species described by Batista & Ciferri (1963a) by producing 11-19-celled phragmoconidia. Conidial dimensions in Ph. phylicae are close to those of Ph. walteri, but conidia are 11-19-celled in Ph. phylicae, and 3-15-celled in Ph. walteri. Mycelium superficial or immersed, hyaline to brown, branched, hyphae smooth, thin-walled, septate, constricted at septa, with a mucilaginous outer wall layer. Pycnidia irregularly cylindrical, straight or flexuous, short or long, sessile, with no swollen part to indicate the location of the pycnidial cavity; wall is composed of linearly arranged, fused hyphae. Ostiole present, without hyaline hyphal extensions; around the ostiole the hyphae are brown and obtusely rounded. Conidia ellipsoidal, hyaline and continuous (adapted from Hughes 1976).

Readerielliopsis
Notes: A search in Index Fungorum and MycoBank (March 2019) revealed four names in Scolecoxyphium. This genus was established by Ciferri and Batista based on the type species, S. fraserae (Ciferri et al. 1956). Three additional species were introduced by Batista & Ciferri (1963a). No cultures or sequence data exist for any of these species. In the phylogenies generated here, a highly supported clade was resolved resembling Scolecoxyphium (Figs S1, 2). The taxa studied here differed from all previously described species of Scolecoxyphium, and are therefore described as new. Etymology: Name reflects the host genus Blechnum from which it was isolated.
Culture characteristics: Colonies, leathery, appressed, with fluffy aerial mycelium, folded, edge sinuate, smoke grey to olivaceous grey after 2 wk in the dark at 25°C. Colonies reaching 12 mm diam on MEA after 2 wk in the dark at 25°C. Notes: Scolecoxyphium leucadendri formed a distinct clade in the phylogenetic trees (Figs S1, 2), but is morphologically similar to S. blechni. The two species can be differentiated based on the pycnidial size on OA, geography and substrate.
Etymology: Name reflects the host genus Phylica from which it was isolated.
Culture characteristics: Colonies, leathery, appressed, with fluffy aerial mycelium, folded, edge sinuate, smoke grey to olivaceous grey with a pink pigment after 2 wk in the dark at 25°C. Colonies reaching 15 mm diam on MEA after 2 wk in the dark at 25°C. Notes: Genetically S. phylicae constitutes a separate clade within Scolecoxyphium (Figs S1, 2). It is morphologically distinguishable based on its short cylindrical pycnidia and longer conidia.
Notes: A search in Index Fungorum and MycoBank (March 2019) revealed 13 names in Scorias. Chomnunti et al. (2011) reexamined the type species, Sc. spongiosa, and designated an epitype MFLU10-0013 (ex-epitype MFLUCC10-0084) from Thailand. Cultures and molecular data are presently available for three species: Sc. leucadendri (LSU, ITS, TEF-1α and RPB2), Sc. mangiferae (LSU and ITS) and Sc. spongiosa (LSU). In this study we examined and sequenced isolates CBS 201.30 and CBS 325.33, which are representatives of a new species described here.
Etymology: Name reflects the fact that it was isolated from an aphid.
Etymology: Name reflects the host genus Camillia from which it was collected.
Previous phylogenetic studies on Capnodiales all suffered from a limited sampling of true sooty moulds, an aspect that we have addressed in the present study. In addition, we have included two protein-coding genes (TEF-1α and RPB2) together with rDNA sequence data (LSU and ITS) to achieve a more stable and robustly supported phylogeny for this extremely diverse group of fungi.
Sooty moulds are presently classified in seven families, with some miscellaneous genera in either Dothideomycetes or Eurotiomycetes . Dothideomycetous sooty moulds belong to four families, namely Antennulariellaceae, Capnodiaceae, Euantennariaceae and Metacapnodiaceae. Although many studies have focused on the taxonomy of sooty moulds (Hughes 1951(Hughes , 1966(Hughes , 1972(Hughes , 1976, isolated as endophytes, being ecologically distinct from the sooty moulds, which are epiphytes. Xu et al. (2017) and Ismail et al. (2016) showed that the ectophytic sooty blotch and flyspeck fungi (Dissoconiaceae, Phaeothecoidiellaceae, Schizothyriaceae) have evolved from ancestral phytopathogenic relatives. Lichenisation has occurred once in Cystocoleaceae, while plant pathogens are found in Mycosphaerellaceae, Neodevriesiaceae and Teratosphaeriaceae and human pathogens have evolved in Teratosphaeriaceae. To better elucidate the general evolutionary pattern, a greater number of samples and genome-wide comparative analyses will be conducted in future studies.
Members of Cladosporiaceae are chiefly saprobic and endophytic, with a few species that are fungicolous, lichenicolous, or plant pathogenic. In a series of phylogenetic studies, members of Cladosporiaceae were resolved as a distinct clade apart from Capnodiaceae, Mycosphaerellaceae and allied families: Dissoconiaceae, Extremaceae, Neodevriesiaceae, Phaeothecoidiellaceae, Schizothyriaceae and Teratosphaeriaceae , Schoch et al. 2006, 2009, Suetrong et al. 2009, Bensch et al. 2012, Hyde et al. 2013, Van Nieuwenhuijzen et al. 2016, Videira et al. 2017). In the present study, Cladosporiaceae clustered apart from Mycosphaerellales and Capnodiales s. str. and formed a distinct clade sister to Comminutisopora agavaciensis. Therefore, Cladosporiaceae has been elevated to ordinal level as Cladosporiales. Morphologically, members of Cladosporiales are quite distinct from those of Mycosphaerellales, having long, solitary, flexuous conidiophores with chains of dry, pigmented conidia. Ecologically, conidia of Cladosporiales can rehydrate, germinate and grow within hours, while members of Mycosphaerellales are generally slow to reactivate, and far less hardy to extremes in temperature and moisture conditions. Endoconidial taxa within Capnodiales s. lat. that belong to Comminutispora and Phaeotheca have received considerable attention (Sigler et al. 1981, Ramaley 1996, de Hoog et al. 1997, Crous et al. 2009. These saprobic fungi , Crous et al. 2009) were found to occupy a basal position in the phylogenetic tree (Figs S1, 1, 3), representing three new orders, namely Comminutisporales, Neophaeothecales and Phaeothecales. Furthermore, our results have resolved Phaeotheca as polyphyletic, representing two distinct clades. Phaeotheca fissurella (Phaeothecales ord. nov.) clustered apart from P. salicornia and P. triangularis in a clade together with the lichen Racodium rupestre. Racodium rupestre clusters in a separate clade apart from another lichen species, Cystocoleus ebeneus (Cystocoleaceae, Mycosphaerellales), and represents a new order, Racodiales ord. nov. Phaeotheca salicornia and P. triangularis occupied the basal position in the phylogenetic tree (Figs S1, 1, 3) as one of the earliest lineages in Capnodiales s. lat., for which we introduced a new genus Neophaeotheca (Neophaeothecaceae fam. nov. and Neophaeothecales ord. nov.). Members of Phaeothecales, Racodiales and Neophaeothecales are commonly isolated under more dry, extreme conditions. The present study has provided a more stable backbone for the phylogeny of sooty moulds and allied taxa formerly classified in what was circumscribed as "Capnodiales s. lat." Many families are not yet represented in our phylogenetic analysis, pending further collections. Although our results revealed Capnodiales s. lat. as polyphyletic, including seven different orders, this remains a work in progress. Furthermore, phylogenetic ancestral reconstruction analysis has revealed the saprobic lifestyle to be a primitive state in Capnodiales s. lat. (see Neophaeothecales; Fig. 3), while Haridas et al. (2020) also showed the ancestral state of Dothideomycetes to be saprobic. Several transitions have occurred to evolve lichenised, epiphytic and plant and human pathogenic lifestyles (Hongsanan et al. 2016, Ametrano et al. 2019, with the sooty mould ecology apparently having evolved more recently. A more robust sampling of the unexplored or little-known clades of Dothideomycetes, and genome-wide comparative analyses will provide greater clarity on the evolutionary patterns of lifestyles and modes of nutrition, that has made it possible for communities of Dothideomycetes to adapt to changing environmental conditions.