Xerombrophila crystallifera, a new genus and species in the Helotiales Hans-Otto Baral, Guy Marson, Mesfin Bogale & Wendy A. Untereiner Mycological Progress ISSN 1617-416X Volume 12 Number 3 Mycol Progress (2013) 12:475-488 DOI 10.1007/s11557-012-0854-6 1 23 Your article is protected by copyright and all rights are held exclusively by German Mycological Society and Springer. This eoffprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Mycol Progress (2013) 12:475–488 DOI 10.1007/s11557-012-0854-6 ORIGINAL ARTICLE Xerombrophila crystallifera, a new genus and species in the Helotiales Hans-Otto Baral & Guy Marson & Mesfin Bogale & Wendy A. Untereiner Received: 16 May 2012 / Revised: 21 August 2012 / Accepted: 24 August 2012 / Published online: 20 September 2012 # German Mycological Society and Springer 2012 Abstract Xerombrophila, a new member of the Helotiales, is erected for a previously undescribed species that macroscopically resembles genera such as Phaeohelotium or Pezicula. The new species, for which we propose the name X. crystallifera, is characterized by a strong gelatinization of the medullary excipulum as well as the covering layer of the ectal excipulum, by the presence of abundant octahedral crystals, asci with an euamyloid apical ring that resembles the Calycina-type, and paraphyses containing refractive vacuoles. It can also be distinguished from members of both Ombrophila and Phaeohelotium in that it is desiccationtolerant by surviving several weeks in the dry state. The species appears to be confined to xeric bark of Salix and was exclusively found over waterlogged soil, irrespective of being acidic or calcareous. It is known from various planar and colline areas of temperate Europe, and can be found throughout the year. Phylogenetic analysis of partial LSU sequences positions X. crystallifera as a sister taxon of a clade within the Helotiaceae s.l. that includes Ascocoryne, Chloroscypha, Gelatinodiscus, Neobulgaria and "Sarcoleotia" turficola. Keywords Ascomycota . Ombrophila . Phaeohelotium . Phylogeny . Ribosomal DNA sequences H.-O. Baral (*) Blaihofstr. 42, Tübingen 72074, Germany e-mail: zotto@arcor.de G. Marson 45 B, rue de Bettembourg, Hesperange 5810, Luxembourg M. Bogale : W. A. Untereiner Department of Biology, Brandon University, Brandon R7A 6A9, Canada Introduction In 1988, we (G.M. and H.B.) collected a discomycete growing on the bark of Salix, characterized by its crowded, sessile, gelatinous, yellowish apothecia with a white pruina consisting of abundant octahedral crystals. Although it superficially resembles species of Pezicula Tul. & C. Tul., Phaeohelotium Kanouse, and Rodwayella Spooner, detailed study of this collection showed that it could not be identified with any of these genera. This species is also strongly reminiscent of Ombrophila in its gelatinous consistency and the presence of abundant crystals, but the comparison with members of Ombrophila revealed that it could not be easily accommodated in this genus. A thorough comparison of the descriptions of the members of the Helotiaceae led us to conclude that the species could neither be assigned to a currently recognized genus nor to a described species (Plates 1, 2, 3, 4, 5, 6). We, therefore, describe the new helotialian genus, Xerombrophila, to accommodate this species. We also explored the phylogenetic position of X. crystallifera based on the analyses of nuclear large subunit (LSU) ribosomal RNA gene sequences (Plate 8). During the past few decades, X. crystallifera has been repeatedly recorded at 15 sites so far in central and northern Europe (Plate 7). Although the species occurs in moist or wet swamps, it typically forms apothecia on dry, attached, and undecayed dead branches or sometimes still-living branches. Materials and Methods Materials examined All collections were examined in the living state in tap water (see Baral 1992), using a Zeiss Standard 14 microscope, Author's personal copy 476 Mycol Progress (2013) 12:475–488 Plate 1 Figs 1–4. Habitat of Xerombrophila crystallifera. Fig. 1. Wet willow swamp (Salix cinerea, S. pentandra, with Carex acutiformis), Radegasttal near Rehna (Mecklenburg, Germany), type locality. Fig. 2. Salicetum cinereae over Gipskeuper, Gipsweieren near Bridel (Luxembourg). Fig. 3. Ibid., corticated branch of Salix cinerea, with Parmelia sulcata, Melanelia glabratula and Exidia recisa (29.XII.2011). Fig. 4. Closeup of the latter, showing apothecia of X. crystallifera usually after rehydrating the branches bearing apothecia when collected in the dry state. The iodine reaction was tested with Lugol's solution (IKI0∼1 % I2, 2 % KI, in H2O), without potassium hydroxide pre-treatment. The presence of gel was tested using Brilliant Cresyl Blue (CRB, ∼1 % in H2O) added to a water mount. Photographic images (macroand microphotos) were obtained using a Nikon Coolpix E4500, and all drawings were done free-hand. Type material is deposited in the Botanische Staatssammlung München (M). Additional collections are held at Kew (K), the Estonian University of Life Sciences Herbarium (TAA), the Staatliches Museum für Naturkunde Stuttgart (STU), the Staatliches Museum für Naturkunde Karlsruhe (KR), and in the private herbaria of Hans-Otto Baral (H.B.) and Guy Marson (G.M.). Pure cultures of Xerombrophila crystallifera were established from ascospores shot onto the surface of plates containing 2 % malt extract agar (MEA) (CBS 128289) or one quarter strength corn meal agar (Sigma) augmented with agar (Merck) (CBS 132843). The former isolate was maintained on modified Leonian's agar (MLA) (Malloch 1981) and grown on cornmeal agar (CMA) (Gams et al. 1998) prepared using 30 g of cornmeal, filtered oatmeal agar (CBSOA) (Gams et al. 1998), MEA, MLA, and oatmeal agar (OA) (Tuite 1969) in 100 mm Petri dishes. Plates were inoculated in triplicate using 2–3 mm2 squares of agar cut from the actively growing edges of colonies on MLA and incubated at room temperature. Colony diameter was Plate 2 Figs 1–4. Xerombrophila crystallifera, apothecia in rehy-„ drated and dry state (Fig. 2a: corticated xeric branch with Physcia tenella, Parmelia sulcata, Melanelia glabratula, Orthotrichum; Fig. 3b: group of aged apothecia). Fig. 1. H.B. 7528: Luxembourg, Bridel, Gipsweieren, on Salix cinerea (scale unknown in 1b). Fig. 2. H.B. 8191: Luxembourg, Mersch, Folkent, on Salix cinerea. Fig. 3. H.B. 8192: Luxembourg, Steinsel, Kaylbaach, on Salix aurita. Fig. 4. H.B. 8046 (holotype): Germany, Mecklenburg, Rehna, Radegasttal, on Salix cinerea Author's personal copy Mycol Progress (2013) 12:475–488 477 Author's personal copy 478 Plate 3 Figs 1–3. Xerombrophila crystallifera. 1a–c. apothecia in median section (me1 0 upper layer of medullary excipulum of gelatinized textura intricata, me2 0 lower layer of gelatinized textura porrecta; ee1 0 ectal excipulum, ee2 0 gelatinized covering layer); 2a–d. octahedral crystals in medullary and ectal excipulum (2d under polarized light); 2g. hyphae of medullary excipulum, with gel sheath; 2h. dto., anchoring Mycol Progress (2013) 12:475–488 hyphae; 2i. octahedral crystal in hymenium; 2k. margin of apothecium with protruding asci; 2e–f, j. ascus apices; 3. dto., in oblique top view (apical rings blue); 2l–m. ascospores. Living state (in water, 2i, k, m in CRB, 2j in IKI) except for asci in 3 (in IKI). 1a–c. H.B. 7528 (Luxembourg, Bridel, Gipsweieren), 2a–m. H.B. 8046 (Germany, Mecklenburg, Rehna, Radegasttal), 3. H.B. 8192 (Luxembourg, Steinsel, Kaylbaach) Author's personal copy Mycol Progress (2013) 12:475–488 Plate 4 Xerombrophila crystallifera. a–c (Luxembourg, Bridel, Gipsweieren, H.B. 9587a): median section of apothecium, showing in IKI a deep red-brown cytoplasmic stain of the ectal excipulum (excluding covering layer) as well as the ascogenous hyphae in upper part of medullary excipulum; d–k (ibid., G.M. 2011-10-23#1): pure culture and 479 anamorph, d–e, g–h: multiradiate, multicellular, non-disarticulating structures; f: brown exudate in centre of older colony; i–j: conidiophores and blastoconidia; k: culture ca. 6 months after inoculation (d in CRB; d & e same scale) Author's personal copy 480 measured and descriptions of colony morphology were made at 7-day intervals for 21 days. Colour descriptions are based on Kornerup and Wanscher (1978). DNA extraction and sequence analyses DNA extraction and the amplification and sequencing of the LSU gene region of CBS 128289 were carried out as described previously (Bogale et al. 2010). Sequences were edited and assembled into larger consensus sequences using Sequencher 3.0 software (Gene Codes, Ann Arbor, MI, USA) and alignments were generated using either ClustalX, version 2.0.12 (Larkin et al. 2007), or multiple sequence alignment based on fast Fourier transform (MAFFT), version 6 (Katoh et al. 2002). Aligned sequences were corrected using Se-Al version 1.0 alpha 1 (Rambaut 1996). Multiple base indels were reduced to single characters and all ambiguously aligned sequences were excluded. The position of Xerombrophila crystallifera within the Helotiales was inferred based on the analysis of sequences of 45 taxa (Table 1). The outgroup taxa were Geoglossum glabrum, G. umbratile, and Trichoglossum hirsutum. Phylogenetic relationships were inferred employing maximum parsimony (MP) method found in PAUP* (Phylogenetic Analysis Using Parsimony *and Other Methods), version 4.0b 10 (Swofford 2003), using tree bisection-reconnection with the MulTrees option activated. Bootstrap support (BS) for branches was evaluated using heuristic searches from 1000 random addition replicates and only groups with BS greater than 50 % were retained in the bootstrap consensus. Bayesian analysis was performed using MrBayes, version 3.1.1 (Huelsenbeck and Ronquist 2001). This analysis employed TIM1 with an estimated proportion of invariable sites (I) and a gamma shape distribution of rates among site (G), which was determined as the best-fit model of sequence evolution using jModel Test (Posada 2008). Bayesian posterior probabilities (PP) were estimated using the Metropolis-coupled Markov chain Monte Carlo method by running four chains with 4,000,000 generations with rate categories and rates set to 6 and gamma, respectively, and using the program default priors for the remaining model parameters. Trees were sampled every 100th generation and those obtained before likelihoods converged were discarded. Sampled trees and parameters were summarized using the sumt and sump commands, respectively, in MrBayes. Abbreviations * 0 living state, † 0 dead state. Iodine reaction in IKI (0 Lugol’s solution): BB 0 blue at a both high and low concentrations of I2 (euamyloid). CRB 0 Brilliant Cresyl Blue. VBs 0 refractive vacuolar bodies. Lipid content: 0 0 without lipid bodies (LBs), 5 0 maximum possible lipid content relative to ascospore volume. Values in { } indicate the number of collections (or pieces of substrate bearing the fungus) that were examined. Mycol Progress (2013) 12:475–488 Plate 5 Xerombrophila crystallifera (H.B. 3495, Switzerland,„ Schaffhausen-Thayngen, Moos). a–c. fresh apothecia crowded on bast of Salix ?viminalis, erumpent below periderm (c in median section); d. part of apothecium in median section; e. dto., ectal excipulum on flanks, with octahedral crystals and druses; f. paraphyses (with VBs in terminal cells); g. mature ascospores; h. immature ascospores; i. croziers at ascus base; j. ascus apices; k. opened ascus apex. Living state except for j–k (in IKI) Results The dataset of aligned partial LSU sequences consisted of 931 characters of which 206 variable characters were parsimony-informative. A heuristic search of this data set produced two most parsimonious trees (MPT), 850 steps in length (L), with a consistency index (CI) of 0.495 and a retention index (RI) of 0.643; one of these trees is presented in Pl. 8. In this phylogeny, Xerombrophila crystallifera (GenBank accession JX454953) was inferred as the member of a strongly supported lineage (79 % BS, PP 0.95) within the Helotiaceae s.l. that also included Ascocoryne cylichnium, Neobulgaria lilacina, "Sarcoleotia" turficola, Chloroscypha enterochroma, and Gelatinodiscus flavidus. Apart from the holotype strain from the locality in Mecklenburg, another sequence was gained from an isolate from Luxembourg, which comprised only the ITS (GenBank accession JX481974, CBS 132843). In comparison to the type strain, this isolate deviated in only two basepairs. Taxonomy Xerombrophila Baral, gen. nov. MycoBank MB 800287 Xerombrophila crystallifera Baral, G. Marson & Unter., sp. nov. – Pls 1–7 MycoBank MB 800288 Descriptio generico-specifica: Apothecia 0.4–2.5 mm diam, sessilia vel (sub-)stipitata, erumpentia gregaria, subalbida vel plerumque pallide ad claro luteo-ochracea, gelatinosa, extus albide pruinosa. Asci 77–140 × 9.5–12 μm in statu vivo, octospori, apice annulo euamyloideo, e uncis nati. Ascosporae 11–21 × 3.5–4.5 μm in statu vivo, fusoideonaviculatae, homopolares, non vel leniter curvatae, guttulas paucas magnas et minutas continentes. Paraphyses cylindricae, 2.3–5 μm latae in statu vivo, multiseptatae, apice vacuolas magnas refringentas hyalinas continentes. Excipulum medullare valde gelatinosum, e textura intricata vel prismatica-porrecta. Excipulum ectale non gelatinosum, e textura globulosa(−prismatica), strato exteriore e textura intricata valde gelatinosa tectum. Crystallae octaedricae Author's personal copy Mycol Progress (2013) 12:475–488 481 Author's personal copy 482 frequens super strato exteriore, etiam in excipulo medullare. Habitat in ramis siccis vivis vel emortuis Salicis in locis udis. Typus generis Xerombrophila crystallifera Etymology Genus: named with reference to the apothecia of the type species that occur on periodically dry (xeric) branches, though always over waterlogged soil, and resemble those of species of Ombrophila in their gelatinous consistency. Species: named in reference to the abundant octahedral crystals in the excipular tissues and often in the hymenium of this species. Apothecia moist or rehydrated (0.4–)0.6–1.5(−2.5)((−4)) mm diam., (0.25–)0.5–1.5(−2.5) mm thick (receptacle 0.25– 0.6 mm), flesh soft to cartilaginous-gelatinous when hydrated, horny when dry; disc whitish to pale yellowish-cream(−cinnamon) or light yellow-ochre, turning faintly to deeply reddish- to purplish-brown with age, soon flat, sometimes eventually slightly convex, margin rather thin, not protruding, exterior whitish to pale yellowish-ochraceous, margin and flanks covered by a whitish pruina, margin not protruding; sessile or often with a more or less defined obconical or cylindrical stipe 0.5–1.3×(0.3–)0.4–0.6(−0.8) mm, erumpent through the host periderm, densely gregarious (fasciculate) in small groups or in long rows, often deformed by mutual pressure, rarely up to a hundred in a group, also scattered; in dry state margin slightly raised, thick, pruina white and more distinct, disc remaining rather flat and exposed, whitish or pale to light yellow-ochre, turning bright ochre- to dark (blackish) purplish-brown with age. Asci *(77–)85–114(−140) × (9.5–)10.5–11.5(−12) μm {4}, †(68–)75–95(−100) × (7–)7.5–10(−11) μm {4}, (*) projecting 5–15 μm beyond paraphyses (in dead state±equaling the length of the paraphyses), 8-spored, ascospores obliquely biseriate in living asci, becoming somewhat uniseriate in the lower part in dead asci, pars sporifera *32–43 μm long (immature 45–60 μm); apex (*) slightly to moderately or (†) moderately to strongly conical, immature apical ring †(2.5–)3–5(−6) × 1–2 μm, mature apical ring †1.2–2 × 2.5–3 μm {5}, of the Calycinatype, light to deep blue (BB) in IKI (without KOHpretreatment) {9}, upper part of ring at first not or only slightly widened, finally strongly expanded laterally, reactivity in IKI moderate to strong, middle part of ring cylindrical, reactivity in IKI faint when immature, later moderate; lower part of ring forming a small annular protrusion, reactivity in IKI faint or mostly very strong; base short-stalked, arising from croziers {8}; ascogenous hyphae †(3–)5–11 μm wide {1}, with strongly dextrinoid contents. Ascospores *(11–)14– 19(−21) × 3.5–4.5 μm {4}, †13.5–16×3–4 μm {2}, fusoidnaviculate, homopolar, ends obtuse or often subacute, straight to usually slightly (sometimes moderately) curved, containing 1–3 medium-sized (1–2.2 μm) and several±small LBs (lipid content 4), uninucleate; becoming (0–)1(−2)-septate and Mycol Progress (2013) 12:475–488 Plate 6 Xerombrophila crystallifera (H.B. 8046, Germany,„ Mecklenburg, Rehna, Radegasttal, holotype). a. mature ascus, paraphyses (with VBs in terminal cells), with octahedral crystals; b. mature ascospores (lower right overmature); c. apex of living immature ascus; d. apices of dead immature asci; e. apices of dead±mature asci; f. hypha of medullary excipulum, with gel sheath (c–e in IKI) eventually eguttulate when overmature, forming a germ tube but not budding conidia. Paraphyses cylindrical, terminal cells *(9–)15–36(−50) × (2.3–)3–4.5(−5) μm {3}, straight to often somewhat flexuous, lower cells *(5–)8–20(−22.5) × 2–3 (−3.5) μm {2}, rarely branched at upper septum, apices embedded in a thin, evanescent, non-refractive gel, easily separable or firmly conglutinate with the asci. Medullary excipulum hyaline, (150–)250–1000 μm thick in centre, two-layered: upper layer rather thick, of loose to medium dense, strongly gelatinized textura intricata-oblita, towards the hymenium often with larger prismatic cells forming a vertically oriented textura prismatica-porrecta, lower layer more or less delimited from upper layer, 40–70 μm thick on lower flanks, of medium to strongly gelatinized textura porrecta-oblita, extending to the margin, very sharply delimited from ectal excipulum; hyphae straight to flexuous, covered by a gelatinous sheath, inseparable even upon strong pressure, individual cells (excluding sheath) */†(10–)15–55 (−60) × 2–7(−11) μm, containing some small and mediumsized LBs, gelatinous sheath 0.5–2.5 μm thick, slightly to medium refractive, strongly so when young. Ectal excipulum hyaline to pale yellowish-greyish-cream, from base to mid flanks of thin-walled, non-gelatinized, thin-walled textura globulosa(−prismatica), 90–250 μm thick near base, oriented at a 45–90° angle to the surface, individual cells *(10–)15–30 (−43) {3} × (9–)12–22(−28) μm {2}, in young apothecia containing small to medium-sized peripheral LBs that change in dead cells to some or many small to large-sized, more centrally arranged LBs; at lower and mid flanks of textura prismatica(−angularis) oriented at a 0–45° angle to the surface, 20–30 μm thick at mid flanks, at the margin of 7–10 μm thick textura prismatica-porrecta; gelatinized covering layer 25–70 μm thick at base and lower flanks, 10 μm at mid flanks, composed of *2–4(−6) μm wide, eguttulate hyphae forming a textura intricata immersed in more or less abundant, nonrefractive gel, sometimes with abundant hair-like projections, which are thin-walled, ± straight, smooth, 40–70 × 2.5–3.3 (−4) μm. Anchoring hyphae sparse to abundant, intergrading on flanks into gelatinized covering layer, hyaline, firm-walled, externally covered by a 0.5–1 μm thick gel sheath, 2–3.5 μm wide (excluding sheath). Octahedral crystals very abundant on entire exterior of ectal excipulum outside the gel matrix, especially dense near the margin, also present throughout medullary excipulum though more scattered and mainly forming large groups or druses, abundant to nearly absent in the hymenium; crystals hyaline, rarely pale chlorinaceous, octahedric, (1–)3–22(−26) μm diam., sometimes Author's personal copy Mycol Progress (2013) 12:475–488 483 Author's personal copy 484 Mycol Progress (2013) 12:475–488 Plate 7 Known distribution of Xerombrophila crystallifera partly rhaphidic, 10–35 μm diam when forming druses, birefringent under polarized light. VBs present in living paraphyses inside the upper (8–)12–30(−55) μm of terminal cells {6}, slightly to strongly refractive, ± hyaline, globose to angular, at first multiguttulate, eventually confluent to form a single elongate-cylindrical body, rarely also inside penultimate cells, not observed in excipular cells except for some of the outermost cells of covering layer; VBs turning pale ochraceous with age (dead state); carotenoids not observed. CRB staining the non- or slightly refractive gel in medullary excipulum and covering layer light to deep lilac and the more strongly refractive gel faint lilac; also staining the very thin gel between the cells of the ectal excipulum deep lilac; the thin gelatinous layer on the surface of the ascospores stains deep lilac; the VBs turn pale turquoise by vital staining. IKI does not stain the gel or cell walls except for the ascus apical rings; glycogen in ascospores not observed, sparse in asci, but cytoplasm of ascogenous hyphae strongly and selectively stained redbrown {2}, also that of ectal excipular cells; VBs staining bright yellow to orange- or red-brown (living state). KOH causes the VBs to disappear when added in the living state, thereby not provoking a colour reaction. Cultural characteristics Ascospores germinating rapidly to form moderately rapidly growing colonies. Colonies on CBSOA 61–62 mm diam in 21 days, flat, appearing mealy, aerial mycelium absent, consisting entirely of immersed hyphae, white (1A). Colonies on MEA 60–62 mm diam in 21 days, flat, aerial mycelium absent, immersed hyphae sparse and appearing somewhat feathery toward the margins, orange white (5A2). Colony reverse orange white (5A2). Colonies on MLA 68–70 mm in 21 days, flat, appearing mealy, consisting entirely of immersed hyphae that appear feathery toward the margins, brownish grey (5C2) at the centre, becoming brownish yellow (5C7-8) toward the margin, mycelium at the margin brownish orange (5C3). Colony reverse brownish orange (5C3). Colonies on OA 65–68 mm diam in 21 days, appearing powdery, aerial mycelium cottony-floccose, forming tufts that are longer and more densely concentrated at the colony centre, hyphae at margin immersed, white to orange white (5A1–2), becoming pale orange (5A3) toward the margin. Colony reverse pale orange (5A3). Margin indistinct and irregular on all media. Hyphae on MLA and MEA hyaline, thin-walled, 2–3 μm wide and up to 4–5 μm wide, typically aggluntinated to form strands, ± eguttulate, hyaline, with scattered patches of Author's personal copy Mycol Progress (2013) 12:475–488 485 DQ227258 Catelunifera rhodogena DQ227264 Hyphodiscus hymeniophilus GU727553 Cistella spicicola 88/1.0 EU940155 Hyaloscypha vitreola AY789415 Hyaloscypha daedaleae EU940152 Hyaloscypha aureliella 73/1.0 AB481319 Lasiobelonium lonicerae AY544674 Capitotricha bicolor AB481314 Lachnum nudipes AY544646 Lachnum virgineum 85/1.0 66/0.97 DQ470957 Loramyces macrosporus 71/DQ470942 Mollisia cinerea AY789426 Vibrissea flavovirens * AY789402 Vibrissea truncorum 81/1.0 AF269219 Phialocephala fortinii 91/1.0 AY789394 Ascocoryne cylichnium 79/EU940141 Neobulgaria lilacina 80/1.0 AY789277 “Sarcoleotia” turficola AY544656 Chloroscypha enterochroma 79/0.95 * EU652381 Gelatinodiscus flavidus Xerombrophila crystallifera FJ176871 Bisporella citrina * EU940087 Bisporella citrina HQ694501 Colipila masduguana 97/0.99 AY789373 Cudoniella clavus DQ470944 Cudoniella clavus AF222491 Cyathicula coronata AY789365 Ombrophila violacea 86/0.99 AY789431 Hymenoscyphus scutula * EU940157 Hymenoscyphus fructigenus 95/0.92 HM595594 Cryptosporiopsis actinidiae 61/0.95 AY544662 Neofabraea malicorticis 91/1.0 DQ247801 Dermea acerina DQ470967 Pezicula carpinea 58/96/0.95 AY789423 Mitrula paludosa * AY789417 Mitrula elegans AY789404 Mitrula borealis EF596821 Phialea strobilina DQ470960 Bulgaria inquinans AY789359 Leotia lubrica * AY789337 Microglossum viride DQ257359 Microglossum rufum AY544653 Trichoglossum hirsutum AY789315 Geoglossum umbratile * AY789317 Geoglossum glabrum * Helotiaceae s.l. * 10 changes Plate 8 Phylogenetic relationship of Xerombrophila crystallifera and selected representatives of the Helotiales inferred from the analysis of rDNA LSU sequence data. This is one of two MPT generated from a heuristic search of 931 characters (L0850, CI00.495, RI00.643). Bootstrap values (numbers in the first position) and Bayesian posterior probabilities (numbers in the second position) are provided above or adjacent to the branches. An asterisk indicates those branches receiving significant support (BS>97 %, PP 1.0) Author's personal copy 486 bright brown exudate, occasionally becoming ossiform on MLA. In older colonies forming hyaline, multiradiate, multicellular, non-disarticulating structures composed of cells *5–7×3–4(−6) μm that are constricted at the septa and contain peripheral guttules (LBs). The surfaces of the cell walls of these structures stain lilac in CRB, especially towards their centres. Conidiomata not formed. Conidiogenous cells developing on CMA and MLA, simple, unbranched, arising from the aerial hyphae, hyaline, *10–20(−30) × 1.8–3 μm, holoblastic, sympodial, forming inconspicuous, flattened scars, surface partly pale violet in CRB. Conidia hyaline, aseptate, clavate-pyriform to almost globose, with a broadly truncate, partly cylindrical base, *(4–)5–7(−7.5) × (2.5–)3–4 (−4.5) μm, containing a few LBs 0.2–0.8(−1.2) μm diam, surface unstained in CRB. Habitat In moist to wet swamps at lakes or rivulets, in fens and peat bogs, with plant communities such as Salicetum cinereae, Salicetum albae, Alnetum glutinosae, 10–780 m alt., collected (0–)0.5–2 m above ground, on corticated, 1– 8 cm thick, attached, recently dead or sometimes still-living branches of Salix alba f. argentea {1}, S. aurita {3}, S. caprea {1}, S. cinerea {9}, S. ?fragilis {1}, S. ?purpurea {1}, S. ? viminalis {1}, and Salix sp. {3}, on hardly to medium decayed bark {15}, erumpent through very small to large holes or longitudinal splits in the host periderm, also on areas of bast exposed by the rolling apart of the loosened periderm, periderm and bast partly darkened, green algae partly abundant. Associated fungi, lichens and bryophytes include Buellia griseovirens {1}, Exidia recisa {1}, Frullania dilatata {2}, Hypogymnia physodes {1}, Melanelia glabratula {4}, Nectria ?coccinea {2}, Orbilia aurantiorubra {1}, Orthotrichum affine/sp. {5}, Parmelia sulcata {5}, Pertusaria sp. {3}, Physcia tenella {1}, Trichonectria sp. {1}, Trimmatostroma salicis {1}, Tympanis sp. {1}, Ulota crispa {1}, crustose lichens. Collected throughout the year (Dec.–Jan., May–July, Sept.–Oct.). Geology granite, Devonian slate, red sandstone, Gipskeuper, lower, middle and upper Liassic (Grès de Luxembourg), white Jurassic. Tolerance to desiccation Asci and paraphyses survive a few weeks, medullary hyphae still alive after 3 weeks, some ascospores alive after 5 weeks. Holotype: GERMANY: Mecklenburg-Vorpommern, 0.7 km SE of Rehna, E of Forstweg, NSG Radegasttal, elevation 22 m, 53°46'25"N, 11° 03'30"E, on branch of Salix cinerea (bark), 14.I.2006, T. Richter (M-0190819, ex H.B. 8046; living culture in CBS 128289). Additional specimens examined (ø0unpreserved): BELGIUM: Region wallonne, Prov. de Luxembourg, 9 km WSW of Arlon, 1 km E Vance, Villers-Tortrue, Réserve naturelle Marais de Vance, 340 m, branches of Mycol Progress (2013) 12:475–488 Salix cinerea (bark), 18.V.1989, G. Marson & H.-O. Baral (H.B. 3749). ESTONIA: Pärnumaa, 7.5 kmW of Pärnu, 2.5 kmS of Audru, Valgeranna, ∼10 m, branch of Salix (bark), 14.I.1983, K. Kalamees (H.B. 7349, TAA 120762). FRANCE: Lorraine, dépt. Vosges, 8.5 km ESE of Gérardmer, 5 km SE of Xonrupt-Longemer, Lac de Retournemer, 780 m, branch of Salix aurita (bark), 23.VI.1990, G. Marson & H.-O. Baral (ø). Alsace, dépt. Bas-Rhin, 23.5 km E of Haguenau, 3.5 km NW of Iffezheim, sailing-ship port, 116 m, branch of Salix alba f. argentea (bark), 21.I.1990, S. Philippi (H.B. 4637; KR 0024695, ex S.P. Asc003φ90, as Phaeohelotium italicum). GERMANY: Bayern, 12.5 km SSE of Deggendorf, 1 km ENE of Aicha, narrow peninsula in the Donau River, branch of Salix (bark), 17.X.1993, L.G. Krieglsteiner (STU). – 3 km NW of Gauting, 2 km NE of Pentenried, Birkenholz, 580 m, branch of Salix caprea (bark), 17.XII.2004, P. Karasch (ø). – Baden-Württemberg, 9 km WNW of Ulm, 3.5 km W of Blaustein, NSG Arnegger Ried, 495 m, branch of Salix (bark), 27.I.1990, H.-O. Baral & L.G. Krieglsteiner (STU). – 7 km SW of Balingen, Dotternhausen, Zementwerk, 660 m, branch of Salix ?fragilis (bark), 19.IX.1990, G. Marson (ø). LUXEMBOURG: Oesling, 10 km W of Ettelbruck, 1.8 km NW of Grosbous, Bruch, 380 m, branch of Salix ? cinerea (bark), 27.VII.2001, G. Marson (H.B. 7008a). – 9 km WSW of Wiltz, 1.5 km ENE of Harlange, Kuelegrouf, 475 m, branches of Salix aurita (bark), 19.VI.2004, G. Marson (H.B. 7550). Gutland, 7.5 km E of Mersch, 1.8 km E of Fischbach, Folkent, valley of ErnzBlanche, 295 m, branch of Salix cinerea (bark), 27.V.2006, G. Marson (H.B. 8191, G.M.). – 6.5 km ESE of Mersch, 1.5 km NNW of Steinsel, SW of Zapp, Kaylbaach, 255 m, branch of Salix aurita (bark), 1.VI.2006, G. Marson (H.B. 8192). – 5 km NNW of Luxembourg, 1.2 km E of Bridel, Gipsweieren, 260 m, branch of Salix cinerea (bark), 15.V.2004, G. Marson (H.B. 7528). – ibid., 29.VI.2011, G. Marson (H.B. 9587a). – ibid., 23.X.2011, G. Marson (G.M. 2011-10-23#01, H.B. 9679, CBS 132843). – ibid. 29.XII.2011, G. Marson (ø). – 5 km SE of Luxembourg, 1.5 km E of Itzig, Reimeschbaach, Salix ?viminalis, 13.I.2012, G. Marson (ø). SWITZERLAND: Kt. Schaffhausen, 2.3 km WSW of Thayngen, Moos, 430 m, branch of Salix ?purpurea (bark), 28.VII.1988, G. Marson & H.-O. Baral (H.B. 3495, duplicate in K). – ibid., branch of Salix cinerea (bark), 28.VII.1988, H.-O. Baral (ø). Author's personal copy Mycol Progress (2013) 12:475–488 Uncertain collection: AUSTRIA: Oberösterreich, Steyr-Land, Grünburg, Waldneukirchen, 450 m, Fraxinus excelsior (wood), 11.VI.1987, K. Helm (ø). Discussion Macroscopically, X. crystallifera resembles members of Phaeohelotium, a genus characterized by short-stalked, often yellow or ochraceous apothecia that is difficult to separate at the generic level from Hymenoscyphus (with usually long stalks). However, the following characters clearly exclude the present species from these two genera and suggest a relationship with the genera Neobulgaria Petrak, Ombrophila Fr., and Chloroscypha Seaver: (1) the presence of gel in the medullary excipulum and in the outer layer of the ectal excipulum, (2) the amyloid ascus apical ring of the Calycina-type which closely resembles that observed in Neobulgaria pura (Pers.) Petrak and Ombrophila rivulorum Velen., and (3) the presence of abundant octahedral crystals. Indeed, the crystals in Xerombrophila are often so abundant, especially on the apothecial surface that the structure of the ectal excipulum is difficult to observe. Characteristics of X. crystallifera that do not fit in the current concept of Neobulgaria and Ombrophila include: (1) the yellowish apothecial disc (which may turn dark purplish-brown with age due to a colour change of the VBs inside the paraphyses), and (2) the growth on periodically dry branches. Xerombrophila crystallifera is not a member of the clade that includes Ombrophila violacea (Hedw.) Fr., the type species of Ombrophila, in the phylogeny based on the analysis of partial LSU sequence (Pl. 8) but it shares morphological characteristics with some of the other members of the Helotiaceae s.l. inferred as its closest relatives. For example, Bisporella citrina (Batsch) Korf & S.E. Carp. also possesses yellowish apothecia and a very similar type of apical ring, but differs in its non-gelatinized medullary excipulum, strongly gelatinized ectal excipulum, and in lacking crystals. It must be noted that the type species of Bisporella Sacc., B. pallescens (S.F. Gray) S.E. Carp. & Korf, possesses an apical ring of the Hymenoscyphus-type, whereas B. citrina appears to be related to the genus Calycina Nees ex Gray (Baral ined.). The monotypic genus Gelatinodiscus Kanouse & A.H. Sm. as redescribed by Carpenter (1976: fig. 4) possesses a very broad apical ring of the Pezicula-type similar to Ascocoryne J.W. Groves & D.E. Wilson, "Sarcoleotia" turficola (Boud.) Dennis, and Chloroscypha, and is connected to a microconidial state (cf. Myrothecium Tode : Fr.). No crystals are mentioned in the description of the type species, G. flavidus Kanouse & A.H. Sm. The medullary excipulum is described as a textura intricata without mention of intercellular gel, 487 although the apothecia are said to be gelatinous. The microconidial state is phialidic and forms distinct sporodochia. A gelatinous covering layer is absent in B. citrina and G. flavidus, which both grow on permanently moist substrate. The general habit of G. flavidus resembles that of a Chloroscypha, i.e., yellow, stipitate apothecia on a coniferous host (Chamaecyparis), and many microscopical features are also similar, including the Pezicula-type of apical ring and the ascospores turning brown after discharge. However, the apically curled paraphyses, the thick sheath around the ascospores, and the absence of crystals deviate from the known species of Chloroscypha as does the simultaneous maturation of asci emphasized by Carpenter (1976). The author also saw a striking difference in the ectal excipulum, yet, the reported outer layer of textura porrecta in G. flavidus is possibly gelatinous and the two layered ectal excipulum could actually correspond to that observed in Chloroscypha. Our phylogenetic analysis supports the view that Gelatinodiscus might be considered as a synonym of Chloroscypha. Carpenter (1976) erected the new family Gelatinodiscaceae with the single genus Gelatinodiscus. This family could be adopted in a wider sense to comprise, besides Chloroscypha, also Ascocoryne, Neobulgaria, and "Sarcoleotia" turficola (note that S. globosa (Sommerf. : Fr.) Korf, which is considered conspecific with the type species of Sarcoleotia, clusters near Geoglossum Pers. : Fr., see Schumacher and Sivertsen 1987; Wang et al. 2006). The genus Xerombrophila might also belong in this family. Xerombrophila is also similar to a number of taxa not represented in our phylogeny. The genus Pezoloma Clem. resembles Xerombrophila in the ectal excipulum and gelatinous covering layer, but the medullary excipulum is nongelatinized, the paraphyses lack VBs, crystals are absent, the margin typically possesses long teeth, and its members occur on substrates lying on the moist ground. The genus Tatraea Svrček, which also grows on permanently moist substrates, differs from Xerombrophila, in lacking both gelatinous tissue and crystals, and in its type of apical ring which approaches that of Ascocoryne. Xerombrophila may externally also recall the genus Pezicula, which differs in its non-gelatinous excipular layers, the absence of VBs in the paraphyses, the presence of pigmented exudate over the paraphyses and excipular cells, and in possessing a broad hemiamyloid apical ring. Xerombrophila appears to share characters also with the genus Zugazaea Korf et al. with the single species Z. agyrioides Korf et al. (Iturriaga et al. 1998). Based on the reexamination of an isotype (CUP 2844), together with the study of a living specimen by M.A. Ribes (pers. comm.), this genus differs in its sessile apothecia with a convex hymenium, inamyloid asci, paraphyses that are branched in their upper part and that lack VBs, and in growing on decorticated branches on the moist ground. The gelatinized medullary excipulum of Z. agyrioides recalls Xerombrophila, but the absence of crystals and the presence of clods (or lumpy Author's personal copy 488 masses) of golden-yellow exudate in the excipulum brings into question the close relationship of these species. Helotium canum Kirschst. in Jaap (1922), a species known only from the protologue that reported it on decayed Salix branches, resembles our species at first glance. The gregarious growth of the 0.5–1 mm large, whitish, dry yellow-grey apothecia with a short and thick, whitish stalk, and also the hymenial characters would fit quite well. However, the spores are described as distinctly wider (15– 20×4–6 μm), with a granular content, and the paraphyses as narrower (1.5–2 μm). Moreover, the excipulum is of a prosenchymatic texture, and the fact that crystals are not mentioned suggests that H. canum is quite different from X. crystallifera. Judging from the protologue, H. canum might be a synonym of Hymenoscyphus laetus Boud., a species that grows on decorticated branches lying on wet ground. A single, very overmature, unpreserved collection (11.VI.1987), tentatively referred to X. crystalliferum, was found on hygric wood of Fraxinus excelsior. Its identity remains uncertain because of the stage of development of the apothecia at the time of collection. Although the ectal excipulum of this collection was covered by crystals, the cortical cells of the ectal excipulum contained VB-guttules, and the ascospores were 1–3-septate and contained a few small LBs. Regrettably, it was impossible to observe details of the ascus apex. Ecology: Apart from the single collection of uncertain identity on hygric wood of Fraxinus, Xerombrophila crystallifera appears to occur exclusively on xeric bark of Salix spp., mainly in fens and peat bogs. The species is widespread in Europe, but seems to be rather rare. We quite regularly encountered it in Luxembourg in some wet woodlands with dense Salix stands, on acidic but also calcareous soils. The species has obviously been overlooked because of its growth on still attached, corticated, living or recently dead branches that are often covered with lichens and bryophytes. The bark of the recently dead, undecayed branches attached to living trees hardly differs from bark of living branches. Various groups of desiccation-tolerant, non-lichenized Helotiales (i.e., members of the Dermateaceae and Sclerotiniaceae) are known to occur on branches at an initial stage of decay and are generally thought to live as endophytes in the living plant tissue. Nevertheless, their occurrence is often overlooked due to the prevailing focus on species growing close to the forest floor. Another example of an overlooked species growing in association with lichens on xeric bark is Proliferodiscus tricolor (Sow.: Fr.) Baral (Hofton et al. 2009). Apothecia of X. crystallifera first appear near the bases of branches and later form toward the ends of the branches, a fruiting sequence that is recognizable from the age of the ascomata. In order to detect the species in a willow hedge, sound, undecayed branches need to be sought, in which the Mycol Progress (2013) 12:475–488 bark has burst through intense exposition to direct sunlight. Branches that died long ago must be disregarded, because they are inhabited by a number of other fungi. Acknowledgments We thank Torsten Richter (Rehna) for sending the sample that served as holotype, and all the other collectors who provided us their specimens. Evi Weber (Tübingen) is thanked for preparing a pure culture of the holotype. We are also thankful to Sylvie Hermant (National Museum of Natural History, Luxembourg) for gaining a further sequence of X. crystallifera. 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