Towards a phylogenetic reappraisal of Parmulariaceae and Asterinaceae (Dothideomycetes).

Members of the Asterinaceae and Parmulariaceae are obligate biotrophic fungi with a pantropical distribution that grow in direct association with living plant tissues and produce external ascomata and bitunicate asci. These fungi are poorly known, with limited information about their taxonomic position in the Dothideomycetes. Much of what is known is conjectural and based on observation of morphological characters. An assessment of the phylogenetic position of the Asterinaceae and Parmulariaceae is provided based on a phylogenetic analysis of the nrDNA operon (ITS) and the large subunit rDNA (LSU) sequence data obtained from fresh material of selected species collected in Brazil. Three key species were included and epitypified, namely Asterina melastomatis, which is the type species for the type genus of the Asterinaceae; Prillieuxina baccharidincola (Asterinaceae); and Parmularia styracis, which is the type species for the type genus of the Parmulariaceae. An LSU rDNA phylogenetic analysis was performed indicating the correct phylogenetic placement of the Asterinales within the Dothideomycetes. From this initial analysis it is clear that the Parmulariaceae as currently circumscribed is polyphyletic, and that the Asterinaceae and Parmulariaceae are related, which justifies the maintenance of the order Asterinales. Asterotexis cucurbitacearum is recognised as distinct from other Dothideomycetes and placed in the newly proposed family and order (Asterotexiaceae, Asterotexiales), while the higher order phylogeny of Inocyclus angularis remains unresolved. Additionally, Lembosia abaxialis is introduced as a novel species and the phylogenetic placement of the genera Batistinula and Prillieuxina is clarified.

INTRODUCTION

The Parmulariaceae (Ascomycota) was informally proposed by Müller & <span class="Disease">von Arx (1962) to accommodate plant parasitic fungi with superficial, dimidiate shield-shaped or crust-like, pulvinate stromata, strongly flattened ascomata that open by irregular disintegration, or by lateral to radial, or ring-like splits. The externally visible stromata usually originate from internal hyphae or internal hypostroma (von Arx & Müller 1975). Asci in this family are ovoid to clavate, with fissitunicate or rostrate dehiscence with a hamathecium composed of pseudoparaphyses. Ascospores of members of this family are hyaline or brown, usually septate and, with or without a mucilaginous sheath. Asexual morphs of fungi in this group are poorly known. The family was formally described by Barr (1979). A more detailed account of the Parmulariaceae was provided in the monograph published by Inácio & Cannon (2008).

The Parmulariaceae together with families of foliicolous ascomycetes such as <span class="Species">Asterinaceae and Aulographaceae, has tra-ditionally been treated as a group with uncertain placement (incertae sedis) in the Dothideomycetes (Hyde et al. 2013). The Parmulariaceae differs from the supposedly closely related Asterinaceae, by having an apical stroma formed by several layers of pigmented cells, and a basal hypostroma formed by fungal hyphae, as well as by the absence of appressoria (Inácio et al. 2012a, Hongsanan et al. 2014). Superficial hyphae are absent in species of Parmulariaceae with the exception of Antoniomyces, Aulacostroma, Mintera and Symphaeophyma, although commonly found in the Asterinaceae (Inácio et al. 2012a). The taxonomic value of this feature was considered an artificial criterion for distinguishing the two families (von Arx & Müller 1975). Nevertheless as a matter of convenience, this morphological feature is still widely used to recognise whether a taxon belongs to one family or the other. The hypothesis of affinity between these two families has never been tested with modern molecular tools.

Léveillé (1845) described eight species in two genera, Asterina and Lembosia. In 1899, <span class="Species">Asterina was included in Microthyriaceae and the family was divided into two subfamilies, Asterineae and Microthyrieae, based on the presence or absence of superficial mycelium (Theissen 1913a, b). Subsequently, the family Asterinaceae was described and 18 genera were included (Hansford 1946).

Currently the Asterinaceae includes species that are either epiphytic or obligate biotrophs. Fungi in this family have dimidiate ascomata that open irregularly at maturity by means of stellar, longitudinal or irregular slits. Ascomata contain bitunicate upright asci, which are globose to oval or cylindrical. Colonies are formed on the surface of leaves or green stems of plants. When present, superficial mycelium is composed of hyphae that have opposite, alternate or irregular branches with uni- or bi-cellular appressoria that are either alternate, unilateral or a mixture of these forms and with shapes that vary between oval, ampulliform, lobate or variable. Haustoria are present in many genera (<span class="Disease">von Arx & Müller 1975, Eriksson 1981, Bezerra 2004, Hofmann et al. 2010, Hofmann & Piepenbring 2011, Hosagoudar 2012).

Recent studies have shown that morphological features alone are not a reliable basis for a natural classification that reflects true phylogenetic relationships. Some examples are found at the generic level in taxa such as Cladosporium, Microcyclosporella, Phaeomoniella, Radulidium, Ramichloridium and Septoria, among others (Arzanlou et al. 2007, Schubert et al. 2007, Frank et al. 2010, Quaedvlieg et al. 2013) and at the family level in Botryosphaeriaceae and Teratosphaeriaceae (Slippers et al. 2013, Quaedvlieg et al. 2014). Delimitation and affiliation of both the <span class="Species">Asterinaceae and Parmulariaceae and the genera they contain have relied entirely on morphological features such as ascospore septation, hamathecium reaction to iodine, presence and shape of internal stromata, plectenchyma texture and colour, ascomata and ascus dehiscence.

Morphological features are often combined with conjectured host specificity. However, the host specificity of fungi in these families has never been experimentally tested (Hofmann et al. 2010). The Asterinaceae and <span class="Species">Parmulariaceae were regarded as probably polyphyletic both by Inácio & Cannon (2008) and Hongsanan et al. (2014), respectively. Practical difficulties related to DNA extraction from old herbarium material and difficulties with recollection of type specimens have hampered a reappraisal of these two families.

Inácio & Cannon (2008) included 35 genera as members of the Parmulariaceae, while Lumbsch & Huhndorf (2010) recognised 34 genera, with the inclusion of Hemigrapha and exclusion of Apoa and Parmulariella. The latest publication mentioning this family (Hyde et al. 2013) added Antoniomyces and excluded four genera (Coccodothis, Dothidasteroma, Englerodothis and Perischizon) from the <span class="Species">Parmulariaceae based on the shape of the ascomata, reducing the total number to 31 genera. Now, with the addition of the recently described genus Rhagadolobiopsis (Guatimosim et al. 2014a), the Parmulariaceae include 32 genera and 114 synonyms (Inácio & Cannon 2008, Lumbsch & Huhndorf 2010, Inácio et al. 2012b, Hyde et al. 2013, Guatimosim et al. 2014a, b).

Lumbsch & Huhndorf (2010) included 38 genera in the Asterinaceae but, more recently, Hongsanan et al. (2014) revised the <span class="Species">Asterinaceae, and recognised only 17 genera and 42 synonyms as belonging to the family. These revisions were mostly based on morphological observations, and were not substantiated by molecular data.

Molecular phylogenetic studies of the Parmulariaceae are difficult because of their biotrophic nature as well as the difficulties involved in DNA extraction from herbarium specimens. The pioneering study of the phylogenetic placement of <span class="Species">Asterinaceae (Hofmann et al. 2010) and recent successful DNA extraction from the Meliolales (Pinho et al. 2012, 2014), shows that phylogenetic approaches can be applied to obligate biotrophs, even when only old herbarium material is available.

The aim of this study was to assess the phylogenetic placement of the Asterinaceae and <span class="Species">Parmulariaceae based on the study of newly collected epitype materials of Parmularia styracis (the type species of Parmulariacae), Asterina melastomatis (the type species of Asterinaceae) and Prillieuxina baccharidincola (Asterinaceae). Asterotexis cucurbitacearum, formerly placed in the Asterinaceae, was re-examined and found to represent a separate family, described here as new. Additionally, a new species of Lembosia is introduced and the phylogenetic placement of B. gallesiae and P. baccharidincola is elucidated.

MATERIALS AND METHODS

Sample collection and morphology

Leaf samples bearing black fungal colonies were collected in Brazil in different biomes between 2009 and 2014. These were dried in a plant press and later examined under a stereo microscope. Freehand sections of fungal colonies were prepared and fungal structures mounted in clear lactic acid, <span class="Chemical">lactophenol, lactofuchsin, and/or Melzer’s reagent. When necessary, sections were made using a Microm HM 520 freezing microtome. Observations were made with a Zeiss V20 Discovery stereo microscope and with a Zeiss Axio Imager 2 light microscope using differential interference contrast (DIC) illumination and an MRc5 camera and ZEN imaging software. Representative specimens were deposited at the herbarium of the Universidade Federal de Viçosa (VIC) and CBS Herbarium (CBS H).

Scanning electron microscopy

Samples of dried material containing fungal structures were mounted on stubs with double-sided adhesive tape and gold-coated using a Balzer’s FDU 010 sputter coater. A Carl-Zeiss Model LEO VP 1430 scanning electron microscope (SEM) was used to analyse and generate images from the samples.

DNA isolation

Leaves harbouring fertile ascomata were examined under a stereo-microscope to check for possible contamination by other fungi, including yeasts. The leaves were then soaked in sterile <span class="Chemical">water for 1 h in order to hydrate and remove the ascomata. Thirty fertile ascomata were removed from the leaves with a sterile fine pointed needle, and placed into a microcentrifuge tube (1.5 mL). Total genomic DNA was extracted by using Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, USA) following the manufacturer’s instructions and the steps described by Pinho et al. (2012).

PCR amplification

The LSU region of each fungus included in the study was sequenced with the primers LR0R + LR5 (Vilgalys & Hester 1990). For the Parmulariaceae, two additional loci, including the internal transcribed spacer regions and intervening 5.8S rDNA (ITS) and the translation elongation factor 1-alpha (EF-1 α) were amplified and sequenced with the primer pairs ITS1-F (Gardes & Bruns 1993) + ITS4 (White et al. 1990), EF2-Fd (Groenewald et al. 2013) or EF1-728F (Carbone & Kohn 1999) + EF-2 (O’Donnell et al. 1998). PCR amplifications were performed in a total volume of 12.5 μL solution containing 10–20 ng of template DNA, 1× PCR buffer, 0.63 μL <span class="Chemical">DMSO (99.9 %), 1.5 mM MgCl2, 0.5 μM of each primer, 0.25 mM of each dNTP, 1.0 U BioTaq DNA polymerase (Bioline GmbH Luckenwalde, Germany). PCR conditions for ITS and LSU were set as follows: an initial denaturation temperature of 95 °C for 5 min, followed by 35 cycles of denaturation temperature of 95 °C for 30 s, primer annealing at 52 °C for 30 s, primer extension at 72 °C for 1 min and a final extension step at 72 °C for 1 min. PCR conditions for EF-1 α were set as follows: an initial denaturation temperature of 94 °C for 5 min, followed by 45 cycles of denaturation temperature of 94 °C for 45 s, primer annealing at 52 °C for 30 s, primer extension at 72 °C for 90 s and a final extension step at 72 °C for 6 min.

DNA sequencing and phylogenetic inference

PCR amplicons of the regions targeted in this study served as templates for DNA sequencing reactions with the BigDye® Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems Life Technologies, Carlsbad, CA, USA) following the protocol of the manufacturer. DNA sequencing reactions used the same primers as those for the PCR reactions. DNA sequencing amplicons were purified through Sephadex® G-50 Superfine columns (Sigma Aldrich, St. Louis, MO) in MultiScreen HV plates (Millipore, Billerica, MA). Purified sequence reactions were run on an ABI Prism 3730xl DNA Sequencer (Life Technologies, Carlsbad, CA, USA).

DNA sequence data were analysed in MEGA (Molecular Evolutionary Genetics Analysis) v. 6.0 (Tamura et al. 2013). Consensus sequences were generated and imported into MEGA for initial alignment and the construction of sequence datasets. Sequences obtained from Schoch et al. (2009), TreeBASE study S10245, and from GenBank (www.ncbi.nlm.nih.gov) and the novel sequences generated on this study were aligned using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html; Katoh et al. 2002) and manually improved in MEGA as indicated.

Phylogenetic analysis

Appropriate gene models were selected using MrModeltest v. 2.3 (Nylander 2004) and applied to the gene partition. Based on the results of MrModeltest, a Bayesian phylogenetic analysis was performed with MrBayes v. 3.1.2 applying a general time-reversible (GTR+I+G) substitution model with inverse gamma rates and dirichlet base frequencies and a heating parameter set at 0.01. <span class="Species">Saccharomyces cerevisiae DAOM 216365 (JN938921) served as outgroup for the phylogenetic analyses. Posterior probabilities were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.1 (Ronquist et al. 2012). Six simultaneous Markov chains were run for 10 000 000 generations and trees were sampled every 100th generation and 10 000 trees were obtained. The first 2 000 trees, representing the burn-in phase were discarded, while the remaining 8 000 trees were used for calculating posterior probabilities. Bayesian posterior probabilities are presented on the left of each node (Fig. 1). Sequences derived in this study were lodged in GenBank (http://www.ncbi.nlm.nih.gov/genbank) (Table 1), the alignment and tree in TreeBASE (www.MycoBank.org) (study number 17355) and taxonomic novelties in MycoBank (www.MycoBank.org; Crous et al. 2004).

Fig. 1

A Bayesian 50 % majority rule tree based on a full length LSU alignment, containing all strains generated in this study. Bayesian posterior probabilities support values for the respective nodes are displayed in the tree. The tree was rooted to Saccharomyces cerevisiae. The scale bar indicates 0.08 expected changes per site. New sequence data are in bold.

Table 1

Strains and NCBI GenBank accessions generated in this study. Type specimens are in bold.

Species	Accession number	Host / Substrate	Locality	Collector	GenBank accessions
					LSU	ITS	EF-1 α
Asterina crysophylli	VIC 42823	Henriettea succosa	Brazil	A.L. Firmino	KP143738	–	–
A. melastomatis	VIC 42822	Miconia sp.	Brazil	A.L. Firmino	KP143739	–	–
Asterotexis cucurbitacearum	VIC 24814	Cucurbita pepo	Brazil	O.L. Pereira & A.L. Firmino	KP143734	–	–
Batistinula gallesiae	VIC 42514	Caesalpinia echinata	Brazil	A.L. Firmino, D.B. Pinho & O.L. Pereira	KP143736	–	–
Inocyclus angularis	VIC 39747	Pleopeltis astrolepis	Brazil	R.W. Barreto	KP143731	KP273233	KP289328
	VIC 39748	Pleopeltis astrolepis	Brazil	R.W. Barreto	KP143732	KP273234	KP289329
	VIC 39749	Pleopeltis astrolepis	Brazil	R.W. Barreto	KP143733	KP273235	KP289330
Lembosia abaxialis	VIC 42825	Miconia jucunda	Brazil	R.W. Barreto	KP143737	–	–
Parmularia styracis	VIC 42447	Styrax ferrugineus	Brazil	M.S. Silva & O.L. Pereira	KP143728	KP273230	KP289325
	VIC 42450	Styrax ferrugineus	Brazil	M.S. Silva & O.L. Pereira	KP143729	KP273231	KP289326
	VIC 42587	Styrax ferrugineus	Brazil	R.W. Barreto	KP143730	KP273232	KP289327
Prillieuxina baccharidincola	VIC 42817	Baccharis sp.	Brazil	O.L. Pereira	KP143735	–	–

RESULTS

Taxonomy

M.E. Barr, Mycologia 71: 944. 1979

Type species. Parmularia styracis Lév., Ann. Sci. Nat., Bot. 5: 286. 1846.

This family includes fungi forming foliicolous or lichenicolous, superficial, dark brown to black colonies. Haustoria coralloid, hyaline, numerous in each host-cell. Ascomata solitary to gregarious, superficial (or rarely immersed), shield-like, starshaped, ellipsoidal or boat-shaped, strongly flattened, membra<span class="Chemical">naceous to carbonaceous, originating from emerging hyphae or from an erumpent hypostroma, covered by a dark wall composed of often radiating rows of cells and opening by fissure or by deliquescence, containing numerous asci, dark brown to black. Asci 8-spored, thick-walled, fissitunicate, variously shaped, short stalked, with a distinct ocular chamber. Ascospores oblong, ellipsoidal or ovoid, ends rounded, 1-septate, constricted or not at the septum, hyaline to dark brown, smooth to verrucose (Inácio & Cannon 2008, Hyde et al. 2013).

Lév., Ann. Sci. Nat., Bot. 5: 286. 1846 — Fig. 2

Fig. 2

Parmularia styracis VIC 42447. a. Living leaves of Styrax ferrugineus with epiphyllous colonies; b, c. detail of the mature colony, opening by radiating fissures; d. vertical section showing entirely superficial ascoma with fertile locules; e, f. detail of the fertile locules; g, h. hyphal columns which connect the colony with the host tissue; i. horizontal section showing the detail of a tuft of internal mycelium that ruptures the cuticle and produce the initial stages of the ascostromata; j. detail of the fertile locule with fully developed asci and pseudoparaphyses; k, l. asci; m–t. ascospores. — Scale bars: d = 100 μm; e, f = 50 μm; g–m = 10 μm.

= Schneepia guaranitica Speg., Anales Soc. Ci. Argent. 19: 259. 1885.

≡ Parmularia guaranitica (S<span class="Chemical">peg.) Henn., Hedwigia 36: 230. 1897.

= Schneepia arechavaletae Speg., Bol. Acad. <span class="Chemical">Nac. Ci. 11: 581. 1889.

≡ Parmularia arechavaletae (S<span class="Chemical">peg.) G. Arnaud, Ann. Ecole Natl. Agric. Montpellier 16: 116. 1918.

= Parmularia styracis var. minor Henn., Hedwigia 34: 112. 1895.

Colonies visible as superficial, epiphyllous, black discoid structures, numerous and scattered over leaves, not associated with necrosis, 2–3 mm <span class="Chemical">diam. External mycelium absent. Internal mycelium intra- and intercellular, deeply penetrating the mesophyll, branched, 1.5–3.5 μm diam, sub-hyaline to dark brown, smooth. Haustoria coralloid, hyaline, several per host cell occupying both the subcuticular and the lacunar parenchymal cells. Internal stromata globular, 27–67 μm diam, located at the central portion of the colony, erupting through the cuticle, cells composed of a combination of textura angularis and textura prismatica, 3–8 × 2–5 μm. External stromata epiphyllous, superficial, discoid, laciniate at the edges, 1–3 mm diam, cells composed of textura prismatica, 3–7 × 1.5–3.5 μm. Ascomata, producing locules arranged in radiating lirellae-like slits, with undulated surface. In vertical section: ascomata entirely superficial, loosely connected to the leaf, delimited by a covering layer (above the fertile locules) and a lower layer. Covering layer black, 27–63 μm thick, consisting of dense dark brown radiating cells of textura angularis, 3–7 × 2–4 μm. Lower layer beneath the hymenium adjacent to the host cuticle, colourless to pale brown, intimately mingled with hyphal cells of the basal cushion, 13–46 μm thick, composed of pale brown to brown textura angularis (cells 2–7 × 1.5–4 μm). Locules with a thin basal cushion above the lower layer, asci and hamathecium immersed in a non-amyloid gelatinous stratum, 76–353 μm diam, 100–320 μm high. Pseudoparaphyses mostly colourless and pale brown at the rounded and slightly swollen, slightly verrucose tips, sometimes with brown to dark brown external material adhering, 49–115 × 1.5–3 μm, septate, thin-walled, filiform, sometimes dichotomously branched near the base. Asci bitunicate, maturing sequentially, with young and mature asci in the same locule; young asci variable in shape before spores can be distinguished, truncated at the base, subcylindrical; mature asci thick-walled (particularly in the upper portion), cylindric-clavate to clavate, 47–81 × 9–18 μm, non-amyloid, 6–8-spored, biseriate (with colourless hyaline ascospores) or unordered but becoming uniseriate at maturity (the stage containing pale brown ascospores), dehiscence through a large apical fracture in the outer wall, with the inner layer extending through it. Ascospores ellipsoidal to clavate, mostly hyaline to pale brown, thin-walled, verrucose, 1-septate, constricted at the septum, the upper cell broader and rounded, and the lower cell tappering towards a rounded end, 14–20 × 5–7 μm, smooth. Asexual morph unknown.

Type material. BRAZIL, Planaltina, on living leaves of Styrax, Clauseen, 1846 (PC!, holotype); on living leaves of Styrax ferrugineus, vicinities of the Estação Ecológica de Águas Emendadas, Cerrado biome, 16 Apr. 2013, M. Silva & O.L. Pereira (VIC 42447 = CBS H-22026, epitype designated here, MBT200333).

Additional materials examined. BRAZIL, Planaltina, on living leaves of Styrax ferrugineus, vicinities of the Estação Ecológica de Águas Emendadas, Cerrado biome, 18 Apr. 2013, M. Silva & O.L. Pereira, VIC 42450 = CBS H-22025; Minas Gerais, Capitólio, Furnas, on living leaves of <span class="Species">S. ferrugineus, S20°38’54.5" W46°13’36.8", 9 Nov. 2012, R.W. Barreto, VIC 42587 = CBS H-22027.

Notes — The ontogeny of ascomata of P. styracis resembles that recently described for the genus Rhagadolobiopsis, in that mature ascostromata are produced from several ascostromatal primordia that coalesce to form a multiloculate structure (Guatimosim et al. 2014a) (Fig. 2b, c). In contrast, Parmularia produces a column of internal mycelium in the centre of the colony that ruptures through the cuticle (Fig. 2i). When the ascomatal disk is removed, the <span class="Disease">hyphal columns are limited to the central portion of the area below the colony (Fig. 2g, h).

Hansf., Mycol. Pap. 15: 188. 1946

Type species. Asterina melastomatis Lév., Ann. Sci. Nat., Bot. 3: 59. 1845.

Foliicolous, epiphytic, obligately biotrophic. Sexual morph: Ex-ternal mycelium usually with or without appressoria, opposite, alternate or irregular branches, blackened. Appressoria uni- or bi-cellular, lateral and/or intercalary, and opposite, alternate or alternate and opposite, oval, ampulliform, lobate or variable, brown to dark brown, with penetration peg piercing through cuticle and invading the epidermic cells or on top of guard cells, forming stomatopodia. Haustoria present in various genera. Ascomata dimidiate, superficial, growing on the surface of plant leaves or stems, circular, elongate or linear, dehiscence non-ostiolate, <span class="Disease">opening by radiating star-like, longitudinal or irregular slits. Scutellum radiate, composed of isodiametric to cylindrical cells, with straight to dichotomously branched hyphae. Hypostroma (internal stroma or internal hyphae) present in some members. Pseudoparaphyses present or not, cylindrical, septate, branched or unbranched, hyaline to yellowish. Asci fissitunicate, upright and parallel, globose, ovoid or cylindrical, 4–8-spored, usually lacking a stalk, hyaline. Ascospores ellipsoidal, occasionally cylindrical, 2–6-celled, yellowish to brown (mostly brown when mature), walls smooth or with capitate ornamentation. Setae present or not on the ascomata and/or mycelium. Asexual morph hyphomycetous or coelomycetous states with pycnothyria. Conidiophores solitary, unbranched, brown. Conidiogenous cells monoblastic or proliferating percurrently, hyaline or brown. Conidia ovoid, cylindrical, conical or staurosporous, brown (von Arx & Müller 1975, Eriksson 1981, Bezerra 2004, Hofmann et al. 2010, Hofmann & Piepenbring 2011, Hosagoudar 2012, Hyde et al. 2013, Hongsanan et al. 2014).

Lév., Ann. Sci. Nat., Bot. 3: 59. 1845. — Fig. 3

Fig. 3

Asterina melastomatis VIC 42822. a. Living leaves of Miconia sp. with epiphyllous colonies; b. colony with open thyriothecia and external mycelium; c. appressoria cylindrical to long-ovate, unicellular; d. asci ovoid to slightly clavate; e. ascospores hyaline, becoming pale brown to brown at maturity. — Scale bars = 10 μm.

≡ Parasterina melastomatis (Lév.) Theiss., Syd. & P. Syd., Ann. Mycol. 15: 246. 1917.

Colonies epiphyllous, irregular to circular, single to confluent, black, 2–6 mm diam. External mycelium straight to flexuous, branching alternate to unilateral, rarely opposed, pale brown to brown, septate, hyphal cells cylindrical, 4–5 μm <span class="Chemical">diam, smooth. Appressoria numerous, entire, sessile, straight to angular, rarely crooked, rectangular to long-ovate, unicellular, alternate to unilateral, never opposed, 6–7.5 × 7–8 μm, brown, penetration peg in middle part of appressorial cell. Ascomata thyriothecia, dimidiate, superficial, developing below external mycelium, circular, single to confluent, in small clusters, fringed at margins, 165–220 μm diam, dark brown to blackish, opening by a central star-shaped fissure. Pseudoparaphyses cylindrical, septate, unbranched, hyaline to yellowish. Scutellum radiate, composed of isodiametric to cylindrical cells. Asci bitunicate, ovoid to slightly clavate, 8-spored, 47.5–57.5 × 27.5–30 μm, hyaline. Ascospores 2-celled, cylindrical, straight, constricted at the septum, hyaline initially, pale brown to brown at maturity, smooth, 19.5–21 × 9.5–11 μm. Asexual morph absent.

Type material. BRAZIL, locality unknown, on living leaves of Miconia sp., date unknown, Guillemin, (herbarium specimen not preserved); Minas Gerais, Lavras Novas, on living leaves of <span class="Species">Miconia sp., on the track of the Cachoeira das Três Quedas, S20°28’39.63" W43°29’42.27", 26 Oct. 2013, A.L. Firmino (VIC 42822, neotype designated here MBT200348). – FRENCH GUIANA, Cayene, on leaves of Melastomataceae, Nov. 1800, Leprieur (herb. Montagne 1133, Crypt. Guyan. 582); PC0084477. Referred to by Hongsanan et al. (2014) as a neotype designated by Theissen (1912 – actually 1913), but that author only referred to species being represented by that collection.

Henn., Hedwigia 48: 12. 1908. — Fig. 4

Fig. 4

Asterina chrysophylli VIC 42823. a. Living leaves of Henriettea succosa with epiphyllous colonies; b, c. SEM images: b. thyriothecium opened by a central star-shaped fissure; c. ascospore oblong, smooth, constricted at the septum; d. appressoria straight, globose to pyriform, unicellular; e. asci globose to ovoid; f. ascospores hyaline, becoming brown at maturity. — Scale bars = 10 μm.

Colonies epiphyllous, irregular to circular, solitary to confluent, black 0.5–6 mm diam. External mycelium straight to slightly flexuous, branching irregularly, pale brown to brown, septate, hyphal cells cylindrical, 4.5–5 μm <span class="Chemical">diam, smooth. Appressoria numerous, entire, sessile, straight, globose to pyriform, unicellular, alternate to unilateral, never opposed, 7.5–9.5 × 11–12.5 μm, brown, penetration peg in the middle portion of the appressorial cell. Ascomata superficial, thyriothecioid dimidiate, developing below external mycelium, circular, solitary to confluent, fringed at margins, 162–253 μm diam, opening through central star-shaped fissures, dark brown to black. Scutellum radiate, composed of somewhat isodiametric to cylindrical cells, straight. Asci bitunicate, globose to ovoid, 8-spored, 52.5–57.5 × 32.5–35 μm, hyaline, smooth. Ascospores oblong to slightly fusiform, straight to slightly curved, constricted at the septum, 27–30 × 14–15 μm, 2-celled, hyaline, becoming brown at maturity, smooth. Asexual morph absent.

Material examined. BRAZIL, Espírito Santo, Sooretama, Reserva Natural Vale, on living leaves of Henriettea succosa, 19 June 2012, A.L. Firmino, VIC 42823.

Arx, Publicações Inst. Micol. Univ. Recife 287: 6. 1960. — Fig. 5

Fig. 5

Batistinula gallesiae VIC 42514. a. Living leaves of Caesalpinia echinata with epiphyllous colonies; b–d, f, g. SEM images: b. colony with open thyriothecia and external mycelium; c. thyriothecium opened by a central star-shaped fissure; d. appressoria straight, lobate, cylindrical, unicellular; e. asci ovoid, showing immature ascospores; f. ascospores oblong, with ends broadly rounded, constricted at the septum; g. conidia of Triposporium (asexual morph) and erect conidiophore; h. ascospores with lobate appressoria. — Scale bars: b = 100 μm; c, d, f, g = 20 μm; e, h = 10 μm.

Colonies amphigenous, irregular to circular, solitary becoming confluent, black, 1–7 mm diam. External mycelium straight, branching alternate, unilateral or opposite, pale brown to brown, septate, composed of cylindrical hyphal cells, 4.5–5 μm <span class="Chemical">diam, smooth. Appressoria numerous, sessile, straight, cylindrical, 2–3 lobate, 9.5–15 × 9.5–14 μm, unicellular, alternate or unilateral, never opposed, brown, penetration peg centrally on the appressorial cell. Ascomata thyriothecioid dimidiate, isolated, superficial, developed below external mycelium, circular, fringed at margins, 152–213 μm diam, opening by a central star-shaped fissure, dark brown to black. Scutellum radiated, composed of isodiametric to cylindrical cells, straight. Asci bitunicate, globose, 50–67.5 × 32.5–47.5 μm, 4–8-spored, smooth, hyaline. Ascospores oblong, straight to slightly curved, 40–48 × 11–15 μm, base and apex broadly rounded, 4-celled, constricted at median septum, pale brown to brown, smooth. Asexual morph: Colonies superficial, developing above the external mycelium, brown to dark brown. Conidiophores arising from the hyphae, monoblastic, erect, cylindrical, unbranched, 33–60 × 9–13.5 μm, septate, brown. Conidia solitary, staurospores with three arms, 31–42.5 × 9.5–14 μm, brown, smooth, germinating at the ends of arms.

Type material. BRAZIL, Pernambuco, Recife, Poço das Maçãs, on living leaves of Gallesiae gorazemae, 7 Aug. 1960, O.S. Silva (URM 19988, holotype).

Additional material examined. BRAZIL, Espírito Santo, Sooretama, Reserva Natural Vale, on living leaves of Caesalpinia echinata, S19°19’03.28" W40°05’42.10", 15 July 2012, A.L. Firmino, D.B. Pinho & O.L. Pereira VIC 42514.

Notes — Batistinula gallesiae was originally described from living leaves of Gallesia gorazema (Phytolaccaceae) in the state of Pernambuco (Brazil). The present collection was from living leaves of Caesalpinia echinata (Fabaceae) collected in the state of Espírito Santo (Brazil). This specimen has the same morphological and biometric characteristics of the type. Caesalpinia echinata is a new host of <span class="Species">B. gallesiae and the genus remains monotypic, with distribution restricted to Brazil.

Firmino & R.W. Barreto, sp. nov. — MycoBank MB812000; Fig. 6

Fig. 6

Lembosia abaxialis VIC 42825. a. Living leaves of Miconia jucunda with hypophyllous colonies; b. colony with open hysterothecia and external mycelium; c. appressoria straight to angular, entire to irregularly lobate, unicellular; d. asci ovoid to slightly clavate; e. ascospores hyaline becoming pale brown to brown at maturity. — Scale bars: b = 20 μm; c–e = 10 μm.

Etymology. Name derived from the observation that colonies of this taxon are only formed abaxially.

Colonies hypophyllous, irregular to circular, solitary to confluent, black, 2–6 mm diam. External mycelium straight to flexuous, branching irregularly, septate, composed of cylindrical hyphal cells, 3–5 μm <span class="Chemical">diam, brown, smooth. Appressoria numerous, entire to irregularly lobate, sessile, straight to angular, 7–10 × 10–10.5 μm, unicellular, unilateral to alternate, never opposed, brown, penetration peg centrally on the appressorial cell. Ascomata hysterothecioid, superficial, developed below external mycelium, mostly linear, rarely Y-shaped, solitary to grouped, fringed at margins, 340–550 × 160–250 μm, dark brown to black, opening by longitudinal fissures. Scutellum radiated, composed of isodiametric to cylindrical cells, straight. Asci bitunicate, slightly clavate, 52.5–57.5 × 25–37.5 μm, 8-spored, hyaline. Pseudoparaphyses cylindrical, septate, unbranched, hyaline. Ascospores oblong to cylindrical, 25–29 × 12.5–15 μm, 2-celled, constricted at the septum, hyaline, becoming pale brown to brown at maturity, smooth. Asexual morph absent.

Type material. BRAZIL, Rio de Janeiro, Bosque da Barra, Barra da Tijuca, on living leaves of Miconia jucunda, 22 Mar. 2014, R.W. Barreto (VIC 42825, holotype).

Notes — Twelve species of Lembosia have been recorded on Melastomataceae (Montagne 1855, 1856, Hennings 1904, Theissen 1913c, Arnaud 1918, Petrak & Ciferri 1930, Petrak & Sydow 1931, Song & Hosagoudar 2003, Hosagoudar & Appaiah 2005, Hosagoudar 2012, Farr & Rossman 2014). Only three of these have been reported from Brazil, namely, L. catervaria, L. melastomatum and L. miconiicola. All are distinct from L. abaxialis (Table 2).

Table 2

Morphological characteristics of Lembosia spp. from Melastomataceae.

Taxon	Appressoria (μm)	Ascomata (μm)	Asci (μm)	Ascospores (μm)
Lembosia abaxialis2	7–10 × 10–10.5	340–550 × 160–250	52.5–57.5 × 25–37.5	25–29 × 12.5–15
Lembosia catervaria	6–8 diam	500–700 × 70–100	40 × 70	30–38 × 15–19
Lembosia domingensis	5–6 × 7–9	300–800 × 150–250	40–52 × 28–35	25–33 × 11–15
Lembosia gigantea	12–17 × 9	784–1064 × 302–504	84–96 × 33–41	26–29 × 14
Lembosia melastomacearum	14 × 9	784 × 336	55–72 × 41–48	26–29 × 12
Lembosia melastomatum	6–8 diam	700 × 250	70–96 × 42–52	35–40 × 16–20
Lembosia memecyli	–	200–450 × 120–150	35–55 × 26–35	20–23 × 8–10
Lembosia memecylicola	4–12 × 6–8	294–882 × 176–300	up to 45 diam	22–26 × 11–13
Lembosia miconiae-prasinae	7 wide	470–860 × 313–448	69–84 × 33–43	24–29 × 12
Lembosia miconiicola	–	500–800 high	22 × 11.5	23–28 × 11–13
Lembosia rolliniae	5–7 wide	300–350 × 100	50–60 × 30	24–26 × 10–11
Lembosia ryanii	7–17 × 5	235–425 × 145–168	36–46 × 21–31	20–21 × 9–12
Lembosia sclerolobii	–	up to 1000 × 140–180	35–50 × 30–40	17–23 × 6–9

1 Montagne (1855), Montagne (1856), Hennings (1904), Theissen (1913c), Arnaud (1918), Petrak & Ciferri (1930), Petrak & Sydow (1931), Song & Hosagoudar (2003), Hosagoudar & Appaiah (2005), Hosagoudar (2012).

2 This publication.

Based on morphological characters, L. domingensis shows similarities with <span class="Species">L. abaxialis, but differs by epiphyllous colonies, few, sparse, entire and conic appressoria, hysterothecia that are Y–X-shaped, with scarce, smaller asci, and slightly clavate ascospores (Petrak & Ciferri 1930). Additionally, L. catervaria differs from L. abaxialis by epiphyllous colonies, thicker hyphae, smaller appressoria, longer and narrower hysterothecia, wider asci and larger ascospores (Montagne 1855). Lembosia melastomatum differs from L. abaxialis by epiphyllous colonies, smaller appressoria, larger asci and ascospores (Montagne 1856). Finally, L. miconiicola differs from L. abaxialis, by epiphyllous colonies, larger hysterothecia and smaller asci (Arnaud 1918). Lembosia abaxialis is the first asterinaceous fungus reported on Miconia jucunda (Melastomataceae).

(Rehm) Petr., Sydowia 4: 536. 1950. — Fig. 7

Fig. 7

Prillieuxina baccharidincola VIC 42817. a. Living leaves of Baccharis sp. with epiphyllous colonies; b. SEM image; thyriothecium opened by a central star-shaped fissure; c. vertical section of the ascoma; d. asci ovoid to subclavate showing pseudoparaphyses; e. ascospores hyaline becoming pale brown to brown at maturity. — Scale bars = 20 μm.

Basionym. Lembosia drimydis var. baccharidincola Rehm, Ann. Mycol. 5: 532. 1907.

≡ Echidnodes baccharidincola (Rehm) Theiss. & Syd., Ann. Mycol. 15: 422. 1926.

Colonies epiphyllous, irregular to circular, solitary becoming confluent, black, 1–6.5 mm diam. External mycelium straight to flexuous, branching irregularly, septate, hyphal cells cylindrical, 3–4 μm <span class="Chemical">diam, pale brown, smooth. Appressoria absent. Ascomata thyriothecioid, single to confluent, superficial, developed below external mycelium, circular to ellipsoid, 102–160 μm diam, dark brown to blackish, opening by a central star-shaped fissure. Asci bitunicate, ovoid to subclavate, 37.5–50 × 20–30 μm, 8-spored, hyaline. Ascospores cylindrical to oblong, straight, 15–22 × 9–11.5 μm, base and apex broadly rounded, 2-celled, constricted at the septum, brown, smooth. Asexual morph absent.

Type materials. BRAZIL, São Paulo, on living leaves of Baccharis sp., unknown date, A. Usteri 8 (Z+ZT, syntype, here designated lectotype MBT200871); São Paulo, on living leaves of Baccharis sp., 5 July 1907, Usteri 41 (Z+ZT, syntype); ibid., 24 July 1907, Usteri 5 (Z+ZT, syntype); Minas Gerais, Nova Lima, on living leaves of Baccharis sp., 18 July 2012, O.L. Pereira (VIC 42817, epitype designated here MBT200345).

Additional material examined. BRAZIL, Minas Gerais, Lavras Novas, on living leaves of Baccharis sp., 10 Sept. 2012, A.L. Firmino, VIC 42818.

Firmino, O.L. Pereira & Crous, ord. nov. — MycoBank MB812001

Type family. Asterotexiaceae Firmino, O.L. Pereira & Crous, fam. nov.

Description as for the constituent family Asterotexiaceae (see below).

Notes — Representative sequences of the major orders in the Dothideomycetes support Asterotexiales as a separate entity (Fig. 1). Within Asterotexiales, two lineages can be defined, one that contains the Asterotexiaceae, and another that contains I. angularis, which is maintained as incertae sedis at the family level. The type species of <span class="Species">Inocyclus needs to be recollected and its phylogenetic position resolved.

Firmino, O.L. Pereira & Crous, fam. nov. — MycoBank MB812002

Type genus. Asterotexis <span class="Chemical">Arx, Fungus 28: 6. 1958.

Type species. Asterotexis cucurbitacearum (Rehm) <span class="Chemical">Arx (as ‘cucurbitarum’), Fungus 28: 6. 1958.

Foliar pathogens, asterinaceae-like, obligately biotrophic, colonies irregular to star-shaped, solitary to confluent, sometimes extending along the veins, dark brown to black. External mycelium growing through ascomatal cavity towards the host epidermis, connecting the neighbouring ascomata, septate, hyaline (unlike members of <span class="Species">Asterinaceae), smooth. Appressoria formed underneath the ascomata, solitary or forming in small clusters, globose, cone-shaped, ovoid to elongate, brown, with a central, hyaline penetration peg. Ascomata superficial, scutellate, dimidiate, brown to blackish. Scutellum formed by radially arranged rows of cells, opening by numerous irregular fissures, smooth. Asci bitunicate, fissitunicate, clavate to cylindrical, 8-spored, hyaline, numerous, parallel, vertically oriented within ascomata. Ascospores ellipsoidal to slipper-shaped, unequally 2-celled, slightly constricted at the septum, upper cell subglobose, lower cell smaller, subcylindrical to subcuneate, hyaline to slightly yellowish (unlike members of the Asterinaceae), smooth. Asexual morph unknown.

(Rehm) Arx, Fungus 28: 6. 1958. — Fig. 8

Fig. 8

Asterotexis cucurbitacearum VIC 42814. a, b. Symptoms on leaves of Cucurbita pepo: a. adaxial side; b. abaxial side, showing the hypophyllous colonies; c. external mycelium hyaline, connecting the ascomata in formation; d. immature ascomata in formation; e. fertile locules exposed on irregular fissures; f, g. vertical section of the ascomata, showing the appressoria with a central hyaline penetration peg, covered by the mature ascomata; h. vertical section of a fully developed ascoma, showing parallel and vertically orientated asci; i. asci; j. ascospores. — Scale bars: c–i = 10 μm; j = 5 μm.

Basionym. Dothidella cucurbitacearum Rehm, Hedwigia 36: 376. 1897.

≡ Rhagadolobium cucurbitacearum (Rehm) Theiss. & Syd., Ann. Mycol. 12: 275 .1914.

Colonies hypophyllous, irregular to star-shaped, solitary to confluent, sometimes extending along the veins, dark brown to black, 1–3 mm. External mycelium growing through ascomatal cavity towards the host epidermis, connecting the neighbouring ascomata, 3–4 μm diam, hyaline, septate, smooth. Appressoria formed underneath the ascomata, solitary or forming small groups, globose, cone-shaped, ovoid to elongate, 8–10 × 5–7 μm, brown, with a hyaline central penetration <span class="Chemical">peg. Ascomata superficial, solitary to confluent, sometimes growing to surround the basis of individual trichomes of the host, scutellate, dimidiate, circular to irregular, 1–3 mm diam, upper cells irregularly shaped and thin-walled, brown to black. Scutellum formed by radially arranged rows of cells, opening by numerous irregular fissures, pale brown, smooth. Asci bitunicate, fissitunicate, clavate to cylindrical, 40–45 × 9.5–12.5 μm, 8-spored, numerous, parallel, vertically orientated within ascomata, hyaline, smooth. Ascospores ellipsoidal to slipper-shaped, 10–14 × 4–5 μm, unequally 2-celled, slightly constricted at the septum, upper cell subglobose, lower cell smaller, subcylindrical to subcuneate, hyaline to slightly yellowish, smooth. Asexual morph unknown.

Type materials. BRAZIL, Blumenau, on living leaves of Cucurbita pepo, May 1887, E. Ule 1415 (S F47805 syntype, here designated lectotype MBT200872); Rio de Janeiro, on living leaves of <span class="Species">Cucurbita pepo, May 1887, E. Ule 676 (S F7565, syntype); Bahia, Igrapiúna, Reserva Ecológica Michelin, on living leaves of Cucurbita pepo, 15 July 2010, O.L. Pereira & A.L. Firmino, S13°49’17.90" W39°10’16.31" (VIC 42814, epitype designated here MBT200349).

Notes — Asterotexis cucurbitacearum has been recorded on living leaves of Cayaponia americana in the Dominican Republic and West Indies; on <span class="Species">Cucurbita moschata in Venezuela and West Indies; on Cucurbita pepo in Brazil, Panama, Trinidad & Tobago and West Indies; on Cucurbita sp. in Brazil and Grenada; on Gurania sp. in the Dominican Republic; on Trichosanthes sp. in the Dominican Republic and on Sechium edule in Costa Rica; Asterotexis quercina has been recorded on Quercus glauca in Nepal (Guerrero et al. 2011, Farr & Rossman 2014).

INCERTAE SEDIS

Guatimosim & R.W. Barreto, IMA Fungus 5: 52. 2014. — MB805976

Description and illustrations — Guatimosim et al. (2014b).

Materials examined. BRAZIL, Rio de Janeiro, Nova Friburgo, Mury, Sítio Colonial, on living leaves of Pleopeltis astrolepis, 30 Mar. 2013, R.W. Barreto (VIC 39747, holotype; CBS H-22028, isotype); ibid., 8 June 2013, R.W. Barreto VIC 39748, CBS H-22029; Rio de Janeiro, Nova Friburgo, Riograndina, Fazenda Barreto, on living leaves of <span class="Species">P. astrolepis, 9 June 2013, R.W. Barreto VIC 39749, CBS H-22030.

Notes — Although I. angularis is not the type species of the <span class="Disease">genus Inocyclus, it is presently the only species from which DNA is available. A fresh collection of the type species, I. psychotriae, is required to clarify the correct placement of this genus.

DISCUSSION

The order Asterinales was included within Dothideomycetes based on the SSU and LSU analyses of five species of <span class="Species">Asterina and a related asexual morph (Hofmann et al. 2010). In recent years, Asterinales was thought to comprise the families Asterinaceae, Parmulariaceae and Aulographaceae (Wu et al. 2011, Hyde et al. 2013). Recently, Hongsanan et al. (2014) provided a reassessment of the order. Based on LSU maximum likelihood and Bayesian analysis, and, despite the absence of molecular data for the Parmulariaceae, the authors concluded that only Asterinaceae should be included within Asterinales.

In the present study, we provide a robust molecular dataset that includes the type species of Asterina, as well as three other genera of <span class="Species">Asterinaceae, the type species of the Parmulariaceae and a genus formerly assigned to the Parmulariaceae. The resulting LSU rDNA tree (Fig. 1) agrees in general with recent multigene analysis of the Dothideomycetes (Schoch et al. 2009) and demonstrated that the Asterinales comprises both Asterinaceae and Parmulariaceae as proposed by Barr & Huhndorf (2001), clustering with Phaeotrichiaceae and Venturiaceae.

A second analysis (available in TreeBASE), was done aiming at verifying if the former molecular studies involving species of Asterina and Lembosia (Hofmann et al. 2010, Hongsanan et al. 2014) correctly assigned the taxa included to the <span class="Species">Asterinaceae. Based on these studies we conclude that these taxa, although considered by the authors as representative of species in the Asterinaceae, are in fact misplaced, and should be treated as incertae sedis, since they do not group with A. melastomatis – the type species of this family. The Asterinaceae, including the genera Asterina, Batistinula, Lembosia and Prillieuxina may, therefore, be polyphyletic, requiring a thorough reassessment. Nevertheless, it is important to note that all studies performed until now (Hofmann et al. 2010, Hongsanan et al. 2014), used relatively short LSU sequences (c. 490 bp) that may not provide the necessary resolution needed.

Asterotexis cucurbitacearum was initially classified in the <span class="Species">Parmulariaceae (Theissen & Sydow 1914) and then transferred to Asterinaceae (Inácio & Cannon 2008, Kirk et al. 2008, Guerrero et al. 2011). This species is clearly not a member of the Asterinaceae (contradictory to what was shown by Hongsanan et al. 2014) and is transferred here to the newly proposed family Asterotexiaceae. This new family grouped (Fig. 1) with Inocylus angularis (originally described as a member of the Parmulariaceae).

Nuclear DNA of P. styracis, the type species of the Parmulariaceae was isolated and studied for the first time here. DNA was successfully isolated from <span class="Species">I. angularis, allowing a preliminary assessment of the Parmulariaceae. Although involving only two taxa, the finding that I. angularis does not group with the type of Parmulariaceae, confirm that the Parmulariaceae is polyphyletic (Inácio & Cannon 2008, Hongsanan et al. 2014). The status of I. angularis within the genus Inocyclus requires confirmation, ideally with a molecular assessment of the type species of Inocyclus.

The molecular phylogenetic analysis presented here clearly indicates that both the Parmulariaceae and <span class="Species">Asterinaceae are polyphyletic. Only the epitypification of the taxa in these and other families of thyriothecioid ascomycetes, followed by molecular phylogenetic analysis will resolve their taxonomic placement and produce a more natural classification for these neglected tropical fungi.