In Press at Mycologia, preliminary version published on May 28, 2014 as doi:10.3852/13-266
Short title: Xylochrysis lucida, a new ascomycete Xylochrysis lucida gen. et sp. nov., a new lignicolous ascomycete (Sordariomycetidae) with holoblastic conidiogenesis Martina Réblová1 Department of Taxonomy, Institute of Botany of the Academy of Sciences, CZ-252 43, Průhonice, Czech Republic Václav Štěpánek Laboratory of Enzyme Technology, Institute of Microbiology of the Academy of Sciences, CZ142 20 Prague, Czech Republic René K. Schumacher Hölderlinstrasse 25, D-15517 Fürstenwalde/Spree, Germany Abstract: The monotypic genus Xylochrysis is introduced for a lignicolous perithecial ascomycete that possesses golden yellow ascomata with black glabrous necks, a three-layered ascomatal wall, persistent paraphyses, and cylindrical, long-stipitate unitunicate asci with an inamyloid apical annulus, and hyaline, ellipsoidal, unicellular ascospores. In culture it produces hyaline conidiophores with terminally arranged branches bearing metulae, conidiogenous cells and holoblastic conidia. Phylogenetic analysis of two ribosomal (nc18S and nc28S rDNA) and one protein-coding (RPB2) gene position this species within the Sordariomycetidae but without close ordinal or familial affiliation. Morphological and molecular DNA data support the recognition of this new genus and suggest that Xylochrysis is most closely related to the genera Ceratolenta, Cyanoannulus and Woswasia. Key words: holoblastic conidiogenesis, multigene analysis, Sordariomycetes, systematics, Woswasia INTRODUCTION
Copyright 2014 by The Mycological Society of America.
Wood-inhabiting Sordariomycetidae are morphologically highly variable (Zhang et al. 2006) and encompass taxa with light to dark, stromatic or nonstromatic ascomata, unitunicate asci with an amyloid or inamyloid apical ring (or that lack a ring) and paraphyses that may disintegrate early. The subclass consisted originally of three and later six orders (Eriksson and Winka 1997, Zhang et al. 2006), and it currently includes seven well characterized orders and 10 families without ordinal affiliation (Huhndorf et al. 2004, 2009; Cannon and Kirk 2007; Ferrer et al. 2012; Réblová 2013). Although the majority of Sordariomycetidae possess dark, opaque ascomata, this subclass accommodates several genera characterized by the ascomata or stroma with a brightly colored surface layer. These taxa also possess persistent paraphyses and hyaline ascospores. A recent collection made on a cultivated Rhododendron in Germany contained a perithecial ascomycete with densely aggregated golden yellow perithecia and black, glabrous necks, a three-layered ascomatal wall, cylindrical unitunicate asci with a long tapering stipe and a distinct inamyloid apical ring, and ellipsoidal, hyaline, unicellular ascospores. No conidiophores were observed on the natural substratum. The culture derived from isolated ascospores yielded whitish aerial mycelium with conidiophores that bore mono- and polyblastic conidiogenous cells and holoblastic conidia. The conidiophores grew from a palisade of cells. Species with ascomatal walls with a brightly pigmented superficial layer and hyaline to subhyaline ascospores occur in several nonstromatic members of Sordariales (e.g. Lasiosphaeria Ces. & de Not., Miller and Huhndorf 2004), Helminthosphaeriaceae (Echinosphaeria A.N. Mill. & Huhndorf; Miller and Huhndorf 2004, Puja et al. 2006, Bell and Mahoney 2008, Bell 2010), Chaetosphaeriales (Ascochalara Réblová, Chaetosphaeria Tul. & C. Tul.; Réblová 1999, Réblová and Seifert 2003, Fernández and Huhndorf 2005, Atkinson et al. 2007), Calosphaeriales (Calosphaeria Tul. & C. Tul., Jattaea Berl.; Mostert et
al. 2006, Réblová 2011), and in Lentomitella Höhn. (incertae sedis; Réblová 2006). In these genera the pigmented ascomatal layer occurs only in some species. Some species of Amplistroma Huhndorf et al. (Amplistromataceae; Huhndorf et al. 2009) and the monotypic genus Woswasia Jaklitsch et al. (incertae sedis; Jaklitsch et al. 2013) possess brightly pigmented stromata and hyaline globose ascospores. Within the Sordariomycetidae the undescribed fungus resembles Woswasia atropurpurea Jaklitsch et al. (Jaklitsch et al. 2013) in having brightly pigmented and often confluent ascomata with glabrous black necks, stipitate asci containing unicellular, hyaline ascospores, and a morphologically similar anamorph with holoblastic conidiogenesis. It also can be compared with Lentomitella tomentosa Réblová & J. Fourn. (Réblová 2006), a species with ascomata with superficial golden yellow layer and hyaline ascospores. Anamorphs with holoblastic conidiogenesis have been described for other Lentomitella. To determine the relationships of the undescribed fungus with morphologically similar fungi and other Sordariomycetes we performed phylogenetic analysis using sequences of two ribosomal RNA genes, the small and large subunit of the nuclear ribosomal RNA (nc18S and nc28S), and the second largest subunit of the RNA polymerase II gene (RPB2). MATERIAL AND METHODS Herbarium material and fungal strains.—Dry ascomata were rehydrated with water and examined with an Olympus SZX12 dissecting microscope. Centrum material was mounted in Melzer's reagent, 90% lactic acid, lactophenol with cotton blue or aqueous cotton-blue (1 mg/mL and 2 mg/mL respectively). Hand sections of the ascomatal wall were studied in 3% KOH. All measurements were taken in Melzer's reagent. Means ± standard deviations (s.d.) based on 20−25 measurements are given for dimensions of asci, ascospores, conidia and conidiogenous cells. Images were captured with differential interference (DIC) or phase contrast (PC) microscopy using an Olympus DP70 camera operated by imaging software Cell on an Olympus BX51 compound microscope. Multi-ascospore isolates were obtained from fresh material with a spore isolator (Meopta, Prague, Czech Republic). Isolates were grown on potato-dextrose agar (PDA, Oxoid) and potato-carrot agar (PCA, Gams et al.
1998). Colonies were incubated at 25 C in the dark and examined after 7, 21 and 30 d. A culture is maintained at the CBS (CBS-KNAW Fungal Biodiversity Center, Utrecht, the Netherlands). Type material is deposited in PRM (National Museum in Prague). DNA isolation, amplification and sequence alignment.—These procedures were performed as described in Réblová et al. (2011). Sequences of the undescribed ascomycete were derived from a multiple-ascospore isolate and aligned with homologous sequences retrieved from GenBank. Accession numbers of the sequences selected for phylogenetic analyses are provided in the tree after the taxon names (FIG. 1). Sequences were manually aligned in BioEdit 7.0.9.0 (Hall 1999). Alignments of the nc18S and nc28S sequences were compared to 2D structures of these genes of Saccharomyces cerevisiae Meyen ex E.C. Hansen (Gutell 1993, Gutell et al. 1993) to improve decisions regarding homologous characters and the introduction of gaps. The alignment of RPB2 sequences was performed as described in Réblová and Réblová (2013). Alignments of individual gene regions were concatenated into a single alignment. This alignment and the phylogenetic analysis are deposited in TreeBASE (study 14603). Phylogenetic analyses.—We analyzed the first two-thirds of the 5′ half of the nc28S (D1 and D2 domains corresponding to the first 1197 nucleotides of Saccharomyces cerevisiae), nearly the entire nc18S and the 5−7 segment of the RPB2. Bases 1−74 and 1−105 of the nc28S and nc18S alignments respectively and bases 1−60 of the RPB2 alignment were excluded from phylogenetic analysis because of the incompleteness of the 5′ end of the majority of the available sequences. Two outgroup taxa, S. cerevisiae and Vanderwaltozyma polyspora (van der Walt) Kurtzman (Saccharomycetes), were used to root the phylogeny. The combined dataset was partitioned into four subsets: (i) nc28S, (ii) nc18S, (iii) the first and second codon positions of RPB2 and (iv) the third codon position of RPB2. Maximum likelihood analysis (ML) was performed with RAxML-HPC 7.0.3 (Stamatakis et al. 2005, Stamatakis 2006) using a GTRCAT model of evolution, which is a combination of GTRGAMMA and GTRCAT. The GTRCAT model (a RAxML-specific alternative model, in which the alignment sites are pooled into a prespecified number of rate categories) was used for the heuristic search and the best tree was then optimized and the likelihood values were calculated under the GTRGAMMA model. Nodal support was verified by nonparametric bootstrapping (BS) with 1000 replicates. Bayesian inference was performed in a likelihood framework as implemented by MrBayes 3.0b4 software package to reconstruct phylogenetic trees (Huelsenbeck and Ronquist 2001). The program MrModeltest 2.3 (Nylander 2008) was used to infer the appropriate substitution model. Multiple Bayesian searches with Metropolis-coupled Markov chain Monte Carlo sampling were conducted. One cold and three heated Markov
chains were used in the analysis. Bayesian analysis was run for 5 000 000 generations, with trees sampled every 1000 generations. The first 20 000 trees, which represented the burn-in phase of the analysis, were discarded. To estimate posterior probabilities (PP) of recovered branches 50 % majority rule consensus trees were created from the remaining trees with PAUP* (Larget and Simon 1999, Swofford 2002).
RESULTS DNA sequencing and phylogenetic analysis.—The alignment for the combined nc18S, nc28S and RPB2 sequences was assessed for 100 taxa from 19 orders or families of Sordariomycetes. The multiple alignment consisted of 4078 sites; individual alignments consisted of 1682 (nc18S), 1270 (nc28S) and 1126 (RPB2) characters respectively. In the ML tree (FIG. 1), the Sordariomycetidae are resolved as a well supported clade (86 % ML BS and 0.99 PP) with three robust subclades (labeled clade I, II and III). The undescribed ascomycete is positioned in Clade III (92/1.0), which contains the Ophiostomatales and four other families (viz. Annulatascaceae, Magnaporthaceae, Papulosaceae and Thyridiaceae). Within Clade III the undescribed fungus is sister to Woswasia atropurpurea (94/1.0) in a weakly supported clade (−/0.58) that also contains Cyanoannulus Raja, J. Campb. & Shearer and Ceratolenta Réblová. A close relationship with the morphologically similar genus Lentomitella is not supported. TAXONOMY Xylochrysis Réblová, gen. nov. MycoBank MB805253 Etymology: Xylon (Greek = wood) refers to the lignicolous habitat; chrysós (Greek = gold) refers to the golden yellow ascomata. Ascomata superficial or erumpent through the periderm, often aggregated in small groups, rarely solitary, nonstromatic or with a stromatic base, venter globose to conical, surrounded by a pigmented layer of cells, ostiolar necks black, glabrous, papillate or beaked. Paraphyses abundant, persistent, branching, septate, longer than the asci. Asci cylindrical,
long-stipitate, with an inamyloid apical annulus. Ascospores hyaline, ellipsoidal, unicellular, thin-walled. Typus: Xylochrysis lucida Réblová Xylochrysis lucida Réblová, sp. nov.
FIGS. 2−20
MycoBank MB805254 Etymology: Lucidus (L = shiny) refers to the brightly colored ascomata. Ascomata superficial on decorticated wood under the peeled off periderm or erumpent through the periderm, often aggregated or confluent in groups of 3–10, seldom solitary, nonstromatic or with the base slightly immersed and seated on a small, basal stroma; venter 350–500 µm diam, 350–550 µm high, globose, subglobose to conical, surrounded by a golden yellow layer of cells, not changing color and without soluble pigments in 3% KOH or 98% lactic acid; ostiolar necks black, glabrous, shiny, papillate or short-beaked, 100–120 µm wide at the base, 100–250 µm long, lined with hyaline periphyses. Ascomata wall leathery to fragile, comprising three layers; outer layer 20–30 µm thick, consisting of thick-walled, irregular golden yellow cells that form a textura epidermoidea, becoming confluent among adjacent ascomata or forming a rudimentary stroma at the bases of the ascomata; middle layer 18–30 µm thick, consisting of thick-walled, brown, brick-like cells that form a textura prismatica, becoming opaque, carbonized and form thicker layer (57–77 µm) in the upper part of the ascomata and in the necks; inner layer 5–15 µm thick, composed of several layers of thin-walled, flattened, hyaline cells. Paraphyses abundant, hyaline, branching, septate, slightly constricted or not constricted, 2.5–3.0 µm wide, tapering to 2.2–2.3 µm, longer than the asci. Asci cylindrical, pars sporifera 59–70(–73) µm (m ± s.d. = 65.8 ± 6.0 µm) long, (3.7–)4–4.6 µm (m ± s.d. = 4.3 ± 0.2 µm) wide, stipe (38–)46–65(–75) µm long, tapering with bulbose base, apex obtuse with inamyloid wedge-shaped apical annulus 1.8–2.0 µm wide, ca. 1.0 µm high, eight-spored. Ascospores ellipsoidal, hyaline, unicellular, with two large drops in the
fresh material, thin-walled (6.2–)6.5–7.5(–8.4) × 2.5–3.0(–3.2) µm (m ± s.d. = 7.2 ± 0.6 × 2.8 ± 0.2 µm), l/w (2.2–)2.5(–2.8), uniseriate, rarely obliquely uniseriate in the lower part of the ascus. Characteristics in culture. Ascospores germinating after 48 h on PDA. Colony on PDA 15–20 mm diam after 30 d at 25 C, whitish, aerial hyphae abundant, with a moist (slimy) appearance in the middle, margin uneven; pale brown to beige pigment diffusing into the agar. Colony on PCA 22–30 mm diam after 30 d at 25 C, whitish, aerial hyphae scanty, center of the colony gray, wrinkled with a slightly moist appearance, margin uneven; reddish brown pigment diffusing into the agar. Conidiomata sporodochial, produced in 14 d on PDA, visible in several spots of the colony; conidiophores densely aggregated, growing from a palisade of hyaline cells; hyaline to subhyaline, 1.5–2.0 µm wide, each with a clavate apex 2.0–3.0 µm wide and bearing 2–4 metulae terminating in conidiogenous cells. Metulae 7.0–11.5 × 1.3–1.6 µm (m ± s.d. = 9.1 ± 1.6 × 1.4 ± 0.1 µm), hyaline, cylindrical. Conidiogenous cells 7.5–13(–15) × 1.3–1.8 µm (m ± s.d. = 10.1 ± 2.1 × 1.5 ± 0.2 µm), terminal, monoblastic, rarely polyblastic and sympodially proliferating, cylindrical or slightly inflated in the upper third, hyaline, with conidia developing holoblastically. Conidia 4.5–5.3(–5.6) × 1.4–1.8 µm (m ± s.d. = 4.9 ± 0.4 × 1.5 ± 0.2 µm), l/w (2.6)3.2(–4.5), ellipsoid to obovoid, unicellular, hyaline, smooth-walled, tapering toward the base to 0.6–0.7 µm. Specimen examined: GERMANY. Near Neuendorf, 50 m., old cemetery bordered by mixed forest, on decaying wood and bark of a twig of a cultivated shrubby Rhododendron sp., 14 Jan 2012, R. K. Schumacher (R.S. 044) (HOLOTYPE PRM 922621; living culture CBS 135996, ex-type).
Sequences: ITS: KF747734, nc28S: KF539911, nc18S: KF539912, RPB2: KF539913 DISCUSSION
Wood-inhabiting Sordariomycetidae comprise three strongly supported subclades; two subclades accommodate taxa in 11 well characterized orders and families (Huhndorf et al. 2004, 2009; Zhang et al. 2006; Huhndorf and Miller 2011), but the third is not clearly defined (Ferrer et al. 2012, Jaklitsch et al. 2013, Réblová 2013, present study). This third subclade includes the Ophiostomatales and other five families, namely the Amplistromataceae (not represented in our study), Annulatascaceae, Magnaporthaceae, Papulosaceae and Thyridiaceae (Clade III, FIG. 1), but the majority of its members are without familial or ordinal affiliations. Taxa belonging to Clade III are typically represented by small or monotypic genera that are not closely related and which lack conspicuous anamorph and teleomorph phenotypes. (Wong et al. 1998, Ho et al. 1999, Hyde 1992, Wong and Hyde 1999, Raja et al. 2003, Tsui et al. 2003, Campbell and Shearer 2004, Vijaykrishna et al. 2006, Arzanlou et al. 2007, Huhndorf et al. 2009, Réblová and Štěpánek 2009, Réblová 2009, Ferrer et al. 2012, Liu et al. 2012, Jaklitsch et al. 2013, Réblová 2013). Conidiogenesis within Clade III, where known, is holoblastic (Réblová 2006, Réblová 2009, Huhndorf et al. 2009, Checa et al. 2011, Jaklitsch et al. 2013), except in the Magnaporthaceae, whose members are linked with phialidic or tretic conidiogenesis (Bussaban et al. 2005, Huhndorf et al. 2008, Thongkantha et al. 2009, Zhang et al. 2011). Xylochrysis is distinctive among lignicolous Sordariomycetidae in forming conspicuous, golden yellow perithecia with black, glabrous necks. Xylochrysis lucida resembles Lentomitella tomentosa in possessing unitunicate asci and hyaline, ellipsoidal, unicellular ascospores, but species of Lentomitella differ from Xylochrysis in having cylindrical-clavate, short-stipitate asci that are rounded at the base, a pronounced wedgeshaped apical annulus and a phaeoisaria-like anamorph that produces conidia on polyblastic conidiogenous cells formed on sympodially proliferating denticles (Réblová 2006).
Lentomitella and Xylochrysis belong to Sordariomycetidae Clade III, but the molecular data do not suggest a close relationship. A close relationship of Woswasia and Xylochrysis is supported in our three-gene phylogeny. Both genera are characterized by the brightly colored layer of cells on the surface of ascomata (that are stromatic in Woswasia), long-stipitate asci, hyaline unicellular ascospores and similar conidiogenesis. Xylochrysis is easily distinguished from Woswasia in the morphology of its ascospores; those of X. lucida are ellipsoidal, smooth and thin-walled, whereas the ascospores of Woswasia atropurpurea are globose to subglobose with a thickwalled verruculose epispore. The ascomata of Xylochrysis are often densely aggregated with confluent, golden yellow-pigmented superficial layers (FIGS. 2−4), but they never form large pulvinate stromata typical of Woswasia. In W. atropurpurea two conspicuous chemical reactions were observed (Jaklitsch et al. 2013). The dark purple pigment found in surface of the stromata and the layer covering individual perithecia turns green to black after the application of 3% KOH (ionomidotic reaction) and the apical annulus of the asci of W. atropurpurea is cyanophylic, staining blue in aqueous cotton blue. Neither reaction was observed in X. lucida. The cyanophylic apical annulus was described also for Cyanoannulus petersenii Raja, J. Campb. & Shearer (Raja et al. 2003), a freshwater fungus inferred as sister of Xylochrysis and Woswasia but without significant support. Xylochrysis and Woswasia have similar holoblastic conidiogenesis, and both genera form branching conidiophores with clavate heads that bear metulae and conidiogenous cells. In axenic culture the conidiophores of W. atropurpurea develop on aerial hyphae over the entire colony, while in X. lucida the development of sporodochial conidiomata is localized (FIG. 5) and conidiophores arise from a palisade of hyaline cells (FIGS. 8−11). Sporulation in
X. lucida was observed only once on PDA, when the isolate was made directly from ascospores. Subsequent cultures yielded sterile mycelium on both PCA and PDA. ACKNOWLEDGMENTS This study was supported by the Project of the National Foundation of the Czech Republic (GAP 506/12/0038) and as a long-term research development project of the Institute of Botany, Academy of Sciences No. RVO 67985939, and by the Institute of Microbiology, Academy of Sciences No. RVO 61388971. We thank Walter Gams and Wendy A. Untereiner for their helpful editorial suggestions.
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LEGENDS FIG. 1. Phylogenetic analysis of the Sordariomycetes. Phylogram inferred from the combined nc28S-nc18S-RPB2 sequences with ML analysis with a GTRCAT model of evolution. Maximum likelihood bootstrap support (ML BS) and Bayesian posterior probability (PP) are indicated at the nodes. Branches with a black circle symbol indicate ML BS = 100%, PP values = 1.0. The GenBank accession numbers given in the tree after the names are those of nc28S/nc18S/RPB2 genes. Missing sequences are indicated by –. FIGS. 2−11. Xylochrysis lucida (PRM 922621, CBS 135996). 2−4. Ascomata on decorticated wood or erumpent through the periderm. 5. Sporulating colony on PDA. 6. Colony on PCA. 7. Conidia. 8−11. Conidiophores with metulae and conidiogenous cells on PDA. 7−11. PC. Bars: 2−4 = 500 μm, 7−11 =10 μm. FIGS. 12−20. Xylochrysis lucida (PRM 922621). 12, 13. Asci containing eight ascospores. 14. Paraphyses. 15. Ascal apex with apical annulus. 16, 17. Young asci with a bulbose base attached to ascogenous hyphae. 18−20. Ascospores. 12−15, 18−20. DIC; 16, 17. PC. Bars: 12−14 = 10 μm; 15, 18−20 = 3 μm; 16, 17 = 20 μm.
FOOTNOTES Submitted 21 Aug 2013; accepted for publication 26 Nov 2013. 1
Corresponding author. E-mail: [email protected]
Short title: Xylochrysis lucida, a new ascomycete Xylochrysis lucida gen. et sp. nov., a new lignicolous ascomycete (Sordariomycetidae) with holoblastic conidiogenesis Martina Réblová1 Department of Taxonomy, Institute of Botany of the Academy of Sciences, CZ-252 43, Průhonice, Czech Republic Václav Štěpánek Laboratory of Enzyme Technology, Institute of Microbiology of the Academy of Sciences, CZ142 20 Prague, Czech Republic René K. Schumacher Hölderlinstrasse 25, D-15517 Fürstenwalde/Spree, Germany Abstract: The monotypic genus Xylochrysis is introduced for a lignicolous perithecial ascomycete that possesses golden yellow ascomata with black glabrous necks, a three-layered ascomatal wall, persistent paraphyses, and cylindrical, long-stipitate unitunicate asci with an inamyloid apical annulus, and hyaline, ellipsoidal, unicellular ascospores. In culture it produces hyaline conidiophores with terminally arranged branches bearing metulae, conidiogenous cells and holoblastic conidia. Phylogenetic analysis of two ribosomal (nc18S and nc28S rDNA) and one protein-coding (RPB2) gene position this species within the Sordariomycetidae but without close ordinal or familial affiliation. Morphological and molecular DNA data support the recognition of this new genus and suggest that Xylochrysis is most closely related to the genera Ceratolenta, Cyanoannulus and Woswasia. Key words: holoblastic conidiogenesis, multigene analysis, Sordariomycetes, systematics, Woswasia INTRODUCTION
Copyright 2014 by The Mycological Society of America.
Wood-inhabiting Sordariomycetidae are morphologically highly variable (Zhang et al. 2006) and encompass taxa with light to dark, stromatic or nonstromatic ascomata, unitunicate asci with an amyloid or inamyloid apical ring (or that lack a ring) and paraphyses that may disintegrate early. The subclass consisted originally of three and later six orders (Eriksson and Winka 1997, Zhang et al. 2006), and it currently includes seven well characterized orders and 10 families without ordinal affiliation (Huhndorf et al. 2004, 2009; Cannon and Kirk 2007; Ferrer et al. 2012; Réblová 2013). Although the majority of Sordariomycetidae possess dark, opaque ascomata, this subclass accommodates several genera characterized by the ascomata or stroma with a brightly colored surface layer. These taxa also possess persistent paraphyses and hyaline ascospores. A recent collection made on a cultivated Rhododendron in Germany contained a perithecial ascomycete with densely aggregated golden yellow perithecia and black, glabrous necks, a three-layered ascomatal wall, cylindrical unitunicate asci with a long tapering stipe and a distinct inamyloid apical ring, and ellipsoidal, hyaline, unicellular ascospores. No conidiophores were observed on the natural substratum. The culture derived from isolated ascospores yielded whitish aerial mycelium with conidiophores that bore mono- and polyblastic conidiogenous cells and holoblastic conidia. The conidiophores grew from a palisade of cells. Species with ascomatal walls with a brightly pigmented superficial layer and hyaline to subhyaline ascospores occur in several nonstromatic members of Sordariales (e.g. Lasiosphaeria Ces. & de Not., Miller and Huhndorf 2004), Helminthosphaeriaceae (Echinosphaeria A.N. Mill. & Huhndorf; Miller and Huhndorf 2004, Puja et al. 2006, Bell and Mahoney 2008, Bell 2010), Chaetosphaeriales (Ascochalara Réblová, Chaetosphaeria Tul. & C. Tul.; Réblová 1999, Réblová and Seifert 2003, Fernández and Huhndorf 2005, Atkinson et al. 2007), Calosphaeriales (Calosphaeria Tul. & C. Tul., Jattaea Berl.; Mostert et
al. 2006, Réblová 2011), and in Lentomitella Höhn. (incertae sedis; Réblová 2006). In these genera the pigmented ascomatal layer occurs only in some species. Some species of Amplistroma Huhndorf et al. (Amplistromataceae; Huhndorf et al. 2009) and the monotypic genus Woswasia Jaklitsch et al. (incertae sedis; Jaklitsch et al. 2013) possess brightly pigmented stromata and hyaline globose ascospores. Within the Sordariomycetidae the undescribed fungus resembles Woswasia atropurpurea Jaklitsch et al. (Jaklitsch et al. 2013) in having brightly pigmented and often confluent ascomata with glabrous black necks, stipitate asci containing unicellular, hyaline ascospores, and a morphologically similar anamorph with holoblastic conidiogenesis. It also can be compared with Lentomitella tomentosa Réblová & J. Fourn. (Réblová 2006), a species with ascomata with superficial golden yellow layer and hyaline ascospores. Anamorphs with holoblastic conidiogenesis have been described for other Lentomitella. To determine the relationships of the undescribed fungus with morphologically similar fungi and other Sordariomycetes we performed phylogenetic analysis using sequences of two ribosomal RNA genes, the small and large subunit of the nuclear ribosomal RNA (nc18S and nc28S), and the second largest subunit of the RNA polymerase II gene (RPB2). MATERIAL AND METHODS Herbarium material and fungal strains.—Dry ascomata were rehydrated with water and examined with an Olympus SZX12 dissecting microscope. Centrum material was mounted in Melzer's reagent, 90% lactic acid, lactophenol with cotton blue or aqueous cotton-blue (1 mg/mL and 2 mg/mL respectively). Hand sections of the ascomatal wall were studied in 3% KOH. All measurements were taken in Melzer's reagent. Means ± standard deviations (s.d.) based on 20−25 measurements are given for dimensions of asci, ascospores, conidia and conidiogenous cells. Images were captured with differential interference (DIC) or phase contrast (PC) microscopy using an Olympus DP70 camera operated by imaging software Cell on an Olympus BX51 compound microscope. Multi-ascospore isolates were obtained from fresh material with a spore isolator (Meopta, Prague, Czech Republic). Isolates were grown on potato-dextrose agar (PDA, Oxoid) and potato-carrot agar (PCA, Gams et al.
1998). Colonies were incubated at 25 C in the dark and examined after 7, 21 and 30 d. A culture is maintained at the CBS (CBS-KNAW Fungal Biodiversity Center, Utrecht, the Netherlands). Type material is deposited in PRM (National Museum in Prague). DNA isolation, amplification and sequence alignment.—These procedures were performed as described in Réblová et al. (2011). Sequences of the undescribed ascomycete were derived from a multiple-ascospore isolate and aligned with homologous sequences retrieved from GenBank. Accession numbers of the sequences selected for phylogenetic analyses are provided in the tree after the taxon names (FIG. 1). Sequences were manually aligned in BioEdit 7.0.9.0 (Hall 1999). Alignments of the nc18S and nc28S sequences were compared to 2D structures of these genes of Saccharomyces cerevisiae Meyen ex E.C. Hansen (Gutell 1993, Gutell et al. 1993) to improve decisions regarding homologous characters and the introduction of gaps. The alignment of RPB2 sequences was performed as described in Réblová and Réblová (2013). Alignments of individual gene regions were concatenated into a single alignment. This alignment and the phylogenetic analysis are deposited in TreeBASE (study 14603). Phylogenetic analyses.—We analyzed the first two-thirds of the 5′ half of the nc28S (D1 and D2 domains corresponding to the first 1197 nucleotides of Saccharomyces cerevisiae), nearly the entire nc18S and the 5−7 segment of the RPB2. Bases 1−74 and 1−105 of the nc28S and nc18S alignments respectively and bases 1−60 of the RPB2 alignment were excluded from phylogenetic analysis because of the incompleteness of the 5′ end of the majority of the available sequences. Two outgroup taxa, S. cerevisiae and Vanderwaltozyma polyspora (van der Walt) Kurtzman (Saccharomycetes), were used to root the phylogeny. The combined dataset was partitioned into four subsets: (i) nc28S, (ii) nc18S, (iii) the first and second codon positions of RPB2 and (iv) the third codon position of RPB2. Maximum likelihood analysis (ML) was performed with RAxML-HPC 7.0.3 (Stamatakis et al. 2005, Stamatakis 2006) using a GTRCAT model of evolution, which is a combination of GTRGAMMA and GTRCAT. The GTRCAT model (a RAxML-specific alternative model, in which the alignment sites are pooled into a prespecified number of rate categories) was used for the heuristic search and the best tree was then optimized and the likelihood values were calculated under the GTRGAMMA model. Nodal support was verified by nonparametric bootstrapping (BS) with 1000 replicates. Bayesian inference was performed in a likelihood framework as implemented by MrBayes 3.0b4 software package to reconstruct phylogenetic trees (Huelsenbeck and Ronquist 2001). The program MrModeltest 2.3 (Nylander 2008) was used to infer the appropriate substitution model. Multiple Bayesian searches with Metropolis-coupled Markov chain Monte Carlo sampling were conducted. One cold and three heated Markov
chains were used in the analysis. Bayesian analysis was run for 5 000 000 generations, with trees sampled every 1000 generations. The first 20 000 trees, which represented the burn-in phase of the analysis, were discarded. To estimate posterior probabilities (PP) of recovered branches 50 % majority rule consensus trees were created from the remaining trees with PAUP* (Larget and Simon 1999, Swofford 2002).
RESULTS DNA sequencing and phylogenetic analysis.—The alignment for the combined nc18S, nc28S and RPB2 sequences was assessed for 100 taxa from 19 orders or families of Sordariomycetes. The multiple alignment consisted of 4078 sites; individual alignments consisted of 1682 (nc18S), 1270 (nc28S) and 1126 (RPB2) characters respectively. In the ML tree (FIG. 1), the Sordariomycetidae are resolved as a well supported clade (86 % ML BS and 0.99 PP) with three robust subclades (labeled clade I, II and III). The undescribed ascomycete is positioned in Clade III (92/1.0), which contains the Ophiostomatales and four other families (viz. Annulatascaceae, Magnaporthaceae, Papulosaceae and Thyridiaceae). Within Clade III the undescribed fungus is sister to Woswasia atropurpurea (94/1.0) in a weakly supported clade (−/0.58) that also contains Cyanoannulus Raja, J. Campb. & Shearer and Ceratolenta Réblová. A close relationship with the morphologically similar genus Lentomitella is not supported. TAXONOMY Xylochrysis Réblová, gen. nov. MycoBank MB805253 Etymology: Xylon (Greek = wood) refers to the lignicolous habitat; chrysós (Greek = gold) refers to the golden yellow ascomata. Ascomata superficial or erumpent through the periderm, often aggregated in small groups, rarely solitary, nonstromatic or with a stromatic base, venter globose to conical, surrounded by a pigmented layer of cells, ostiolar necks black, glabrous, papillate or beaked. Paraphyses abundant, persistent, branching, septate, longer than the asci. Asci cylindrical,
long-stipitate, with an inamyloid apical annulus. Ascospores hyaline, ellipsoidal, unicellular, thin-walled. Typus: Xylochrysis lucida Réblová Xylochrysis lucida Réblová, sp. nov.
FIGS. 2−20
MycoBank MB805254 Etymology: Lucidus (L = shiny) refers to the brightly colored ascomata. Ascomata superficial on decorticated wood under the peeled off periderm or erumpent through the periderm, often aggregated or confluent in groups of 3–10, seldom solitary, nonstromatic or with the base slightly immersed and seated on a small, basal stroma; venter 350–500 µm diam, 350–550 µm high, globose, subglobose to conical, surrounded by a golden yellow layer of cells, not changing color and without soluble pigments in 3% KOH or 98% lactic acid; ostiolar necks black, glabrous, shiny, papillate or short-beaked, 100–120 µm wide at the base, 100–250 µm long, lined with hyaline periphyses. Ascomata wall leathery to fragile, comprising three layers; outer layer 20–30 µm thick, consisting of thick-walled, irregular golden yellow cells that form a textura epidermoidea, becoming confluent among adjacent ascomata or forming a rudimentary stroma at the bases of the ascomata; middle layer 18–30 µm thick, consisting of thick-walled, brown, brick-like cells that form a textura prismatica, becoming opaque, carbonized and form thicker layer (57–77 µm) in the upper part of the ascomata and in the necks; inner layer 5–15 µm thick, composed of several layers of thin-walled, flattened, hyaline cells. Paraphyses abundant, hyaline, branching, septate, slightly constricted or not constricted, 2.5–3.0 µm wide, tapering to 2.2–2.3 µm, longer than the asci. Asci cylindrical, pars sporifera 59–70(–73) µm (m ± s.d. = 65.8 ± 6.0 µm) long, (3.7–)4–4.6 µm (m ± s.d. = 4.3 ± 0.2 µm) wide, stipe (38–)46–65(–75) µm long, tapering with bulbose base, apex obtuse with inamyloid wedge-shaped apical annulus 1.8–2.0 µm wide, ca. 1.0 µm high, eight-spored. Ascospores ellipsoidal, hyaline, unicellular, with two large drops in the
fresh material, thin-walled (6.2–)6.5–7.5(–8.4) × 2.5–3.0(–3.2) µm (m ± s.d. = 7.2 ± 0.6 × 2.8 ± 0.2 µm), l/w (2.2–)2.5(–2.8), uniseriate, rarely obliquely uniseriate in the lower part of the ascus. Characteristics in culture. Ascospores germinating after 48 h on PDA. Colony on PDA 15–20 mm diam after 30 d at 25 C, whitish, aerial hyphae abundant, with a moist (slimy) appearance in the middle, margin uneven; pale brown to beige pigment diffusing into the agar. Colony on PCA 22–30 mm diam after 30 d at 25 C, whitish, aerial hyphae scanty, center of the colony gray, wrinkled with a slightly moist appearance, margin uneven; reddish brown pigment diffusing into the agar. Conidiomata sporodochial, produced in 14 d on PDA, visible in several spots of the colony; conidiophores densely aggregated, growing from a palisade of hyaline cells; hyaline to subhyaline, 1.5–2.0 µm wide, each with a clavate apex 2.0–3.0 µm wide and bearing 2–4 metulae terminating in conidiogenous cells. Metulae 7.0–11.5 × 1.3–1.6 µm (m ± s.d. = 9.1 ± 1.6 × 1.4 ± 0.1 µm), hyaline, cylindrical. Conidiogenous cells 7.5–13(–15) × 1.3–1.8 µm (m ± s.d. = 10.1 ± 2.1 × 1.5 ± 0.2 µm), terminal, monoblastic, rarely polyblastic and sympodially proliferating, cylindrical or slightly inflated in the upper third, hyaline, with conidia developing holoblastically. Conidia 4.5–5.3(–5.6) × 1.4–1.8 µm (m ± s.d. = 4.9 ± 0.4 × 1.5 ± 0.2 µm), l/w (2.6)3.2(–4.5), ellipsoid to obovoid, unicellular, hyaline, smooth-walled, tapering toward the base to 0.6–0.7 µm. Specimen examined: GERMANY. Near Neuendorf, 50 m., old cemetery bordered by mixed forest, on decaying wood and bark of a twig of a cultivated shrubby Rhododendron sp., 14 Jan 2012, R. K. Schumacher (R.S. 044) (HOLOTYPE PRM 922621; living culture CBS 135996, ex-type).
Sequences: ITS: KF747734, nc28S: KF539911, nc18S: KF539912, RPB2: KF539913 DISCUSSION
Wood-inhabiting Sordariomycetidae comprise three strongly supported subclades; two subclades accommodate taxa in 11 well characterized orders and families (Huhndorf et al. 2004, 2009; Zhang et al. 2006; Huhndorf and Miller 2011), but the third is not clearly defined (Ferrer et al. 2012, Jaklitsch et al. 2013, Réblová 2013, present study). This third subclade includes the Ophiostomatales and other five families, namely the Amplistromataceae (not represented in our study), Annulatascaceae, Magnaporthaceae, Papulosaceae and Thyridiaceae (Clade III, FIG. 1), but the majority of its members are without familial or ordinal affiliations. Taxa belonging to Clade III are typically represented by small or monotypic genera that are not closely related and which lack conspicuous anamorph and teleomorph phenotypes. (Wong et al. 1998, Ho et al. 1999, Hyde 1992, Wong and Hyde 1999, Raja et al. 2003, Tsui et al. 2003, Campbell and Shearer 2004, Vijaykrishna et al. 2006, Arzanlou et al. 2007, Huhndorf et al. 2009, Réblová and Štěpánek 2009, Réblová 2009, Ferrer et al. 2012, Liu et al. 2012, Jaklitsch et al. 2013, Réblová 2013). Conidiogenesis within Clade III, where known, is holoblastic (Réblová 2006, Réblová 2009, Huhndorf et al. 2009, Checa et al. 2011, Jaklitsch et al. 2013), except in the Magnaporthaceae, whose members are linked with phialidic or tretic conidiogenesis (Bussaban et al. 2005, Huhndorf et al. 2008, Thongkantha et al. 2009, Zhang et al. 2011). Xylochrysis is distinctive among lignicolous Sordariomycetidae in forming conspicuous, golden yellow perithecia with black, glabrous necks. Xylochrysis lucida resembles Lentomitella tomentosa in possessing unitunicate asci and hyaline, ellipsoidal, unicellular ascospores, but species of Lentomitella differ from Xylochrysis in having cylindrical-clavate, short-stipitate asci that are rounded at the base, a pronounced wedgeshaped apical annulus and a phaeoisaria-like anamorph that produces conidia on polyblastic conidiogenous cells formed on sympodially proliferating denticles (Réblová 2006).
Lentomitella and Xylochrysis belong to Sordariomycetidae Clade III, but the molecular data do not suggest a close relationship. A close relationship of Woswasia and Xylochrysis is supported in our three-gene phylogeny. Both genera are characterized by the brightly colored layer of cells on the surface of ascomata (that are stromatic in Woswasia), long-stipitate asci, hyaline unicellular ascospores and similar conidiogenesis. Xylochrysis is easily distinguished from Woswasia in the morphology of its ascospores; those of X. lucida are ellipsoidal, smooth and thin-walled, whereas the ascospores of Woswasia atropurpurea are globose to subglobose with a thickwalled verruculose epispore. The ascomata of Xylochrysis are often densely aggregated with confluent, golden yellow-pigmented superficial layers (FIGS. 2−4), but they never form large pulvinate stromata typical of Woswasia. In W. atropurpurea two conspicuous chemical reactions were observed (Jaklitsch et al. 2013). The dark purple pigment found in surface of the stromata and the layer covering individual perithecia turns green to black after the application of 3% KOH (ionomidotic reaction) and the apical annulus of the asci of W. atropurpurea is cyanophylic, staining blue in aqueous cotton blue. Neither reaction was observed in X. lucida. The cyanophylic apical annulus was described also for Cyanoannulus petersenii Raja, J. Campb. & Shearer (Raja et al. 2003), a freshwater fungus inferred as sister of Xylochrysis and Woswasia but without significant support. Xylochrysis and Woswasia have similar holoblastic conidiogenesis, and both genera form branching conidiophores with clavate heads that bear metulae and conidiogenous cells. In axenic culture the conidiophores of W. atropurpurea develop on aerial hyphae over the entire colony, while in X. lucida the development of sporodochial conidiomata is localized (FIG. 5) and conidiophores arise from a palisade of hyaline cells (FIGS. 8−11). Sporulation in
X. lucida was observed only once on PDA, when the isolate was made directly from ascospores. Subsequent cultures yielded sterile mycelium on both PCA and PDA. ACKNOWLEDGMENTS This study was supported by the Project of the National Foundation of the Czech Republic (GAP 506/12/0038) and as a long-term research development project of the Institute of Botany, Academy of Sciences No. RVO 67985939, and by the Institute of Microbiology, Academy of Sciences No. RVO 61388971. We thank Walter Gams and Wendy A. Untereiner for their helpful editorial suggestions.
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LEGENDS FIG. 1. Phylogenetic analysis of the Sordariomycetes. Phylogram inferred from the combined nc28S-nc18S-RPB2 sequences with ML analysis with a GTRCAT model of evolution. Maximum likelihood bootstrap support (ML BS) and Bayesian posterior probability (PP) are indicated at the nodes. Branches with a black circle symbol indicate ML BS = 100%, PP values = 1.0. The GenBank accession numbers given in the tree after the names are those of nc28S/nc18S/RPB2 genes. Missing sequences are indicated by –. FIGS. 2−11. Xylochrysis lucida (PRM 922621, CBS 135996). 2−4. Ascomata on decorticated wood or erumpent through the periderm. 5. Sporulating colony on PDA. 6. Colony on PCA. 7. Conidia. 8−11. Conidiophores with metulae and conidiogenous cells on PDA. 7−11. PC. Bars: 2−4 = 500 μm, 7−11 =10 μm. FIGS. 12−20. Xylochrysis lucida (PRM 922621). 12, 13. Asci containing eight ascospores. 14. Paraphyses. 15. Ascal apex with apical annulus. 16, 17. Young asci with a bulbose base attached to ascogenous hyphae. 18−20. Ascospores. 12−15, 18−20. DIC; 16, 17. PC. Bars: 12−14 = 10 μm; 15, 18−20 = 3 μm; 16, 17 = 20 μm.
FOOTNOTES Submitted 21 Aug 2013; accepted for publication 26 Nov 2013. 1
Corresponding author. E-mail: [email protected]
