Mycol Progress (2015) 14: 3 DOI 10.1007/s11557-015-1025-3

ORIGINAL ARTICLE

Curviclavula, a new genus of anamorphic Helotiales (Leotiomycetes) isolated from air

Gregorio Delgado & Andrew N. Miller & Fernando A. Fernández

Received: 24 July 2014 /Revised: 27 September 2014 /Accepted: 8 October 2014 /Published online: 3 February 2015 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2015

Abstract A new genus Curviclavula,withC. anemophila as Introduction the type species, is established to accommodate a hyphomy- cetous anamorph isolated from a culturable air sample collect- During a routine analysis of culturable air samples received in ed outdoors in New Mexico, USA. The fungus is morpholog- our laboratory for indoor air quality examination, a colony of ically distinct in having 1–5 times dichotomously branched an unusual fungus was recovered and isolated. The source conidiophores and integrated, terminal or intercalary, indeter- sample was collected outdoors at the townsite of Los minate, irregularly sympodial conidiogenous cells with 1–2 Alamos, New Mexico, where the surrounding vegetation is a flattened, inconspicuous, non-protuberant, and neither thick- lower montane coniferous forest dominated by the widespread ened nor darkened conidiogenous loci. The conidia are variable Ponderosa pine (Pinus ponderosa P. Lawson & C. Lawson)/ in shape, occasionally didymosporous or phragmosporous, but Gambel oak (Quercus gambelii Nutt.) community type (Dick- usually minutely cheiroid at first and composed of a Peddie 1993;Morrow1995). The fungus was first tentatively subcylindrical to obconical, often inflated, straight or curved identified as a species of Cheiromycella Höhn. based on the basal cell that bears a subglobose lateral cell and a short row presence of a variable conidial morphology that includes of 2–4 cells. The apical cell of the developing conidium curves didymosporous, phragmosporous, and minute cheirosporous and fuses with the lateral and adjacent cells once in contact to conidia (Ellis 1971). After careful observations, however, the eventually forming a tightly appressed, subglobose to broadly isolate showed an unusual combination of morphological fea- clavate conidium. Conidial secession is schizolytic, but the co- tures, particularly certain aspects of conidial development, nidial basal cells often show minute marginal frills and dark- which are considerably different from any previously known ened hila. Phylogenetic analyses using DNA sequences from taxa of hyphomycetous anamorphs. In order to further confirm the nuclear ribosomal large subunit and internal transcribed the fungus identity and to explore its possible phylogenetic spacer region also supported Curviclavula as distinct and and taxonomic relationships, DNA sequences of the nuclear placed it within the Helotiales (Leotiomycetes, Ascomycota) ribosomal large subunit (LSU) and internal transcribed spacer with strong affinity to the family Hyaloscyphaceae. (ITS) region were obtained and analyzed. Molecular data also supported the recognition of a novel taxon, and, therefore, a new genus and species is introduced here to accommodate it Keywords Airborne fungi . Microfungi . Phylogeny . based on morphological and molecular characters. Taxonomy

Material and methods

G. Delgado (*) : F. A. Fernández EMLab P&K North Phoenix, 1501 West Knudsen Drive, Isolation and morphological studies Phoenix, AZ 85027, USA e-mail: [email protected] The original colony was recovered from an air sample collect- ed with an Andersen sampler on an agar plate (Andersen A. N. Miller Illinois Natural History Survey, University of Illinois, 1816 South 1958) after seven days of incubation at 25 °C. It was trans- Oak Street, Champaign, IL 61820, USA ferred aseptically to 2 % Malt Extract Agar (MEA: Healthlink, 3 Page 2 of 7 Mycol Progress (2015) 14: 3

Jacksonville, FL) and incubated as previously described. length consisting of 17 taxa in the Hyaloscyphaceae and Single-spore cultures were obtained following Choi et al. incertae sedis Helotiales. GenBank accession numbers are (1999) and colony features were observed and measured after shown on the phylogenetic tree after species names. 21 days. Color codes in parentheses are from Kornerup and Alignments of each region were created manually by eye in Wanscher (1978). Microscopic slides were mounted in lacto- Sequencher 5.1 and ambiguous regions were removed using cotton blue and microphotographs were taken using an the online version of Gblocks v. 0.91b (Castresana 2000). The Olympus BX-45 compound microscope. A total of 100 mea- settings were set for less stringent selection by allowing small- surements were made at 1,000× magnification for each fungal er final blocks, gap positions within the final blocks, and less structure, and minimum, maximum, fifth and 95th percentile strict flanking positions. The Akaike Information Criterion values were calculated using Microsoft Office Excel 2007, (AIC), implemented in jModelTest 2.1.1 (Guindon and with outliers shown in parentheses. A line drawing was made Gascuel 2003; Darriba et al. 2012) with default parameters, using a drawing tube (Carl Zeiss, Oberkochen, Germany). A was used to select the best model of evolution, which was culture plate inoculated with the fungus was sent to the determined to be the GTR+I+G model for each region. Advanced Microscopy Facility of the University of Victoria, Maximum likelihood (ML) analyses using PhyML (Guindon British Columbia, Canada for scanning electron microscopy and Gascuel 2003) in Seaview 4.2 were conducted under the (SEM), where the sample was processed following a standard GTR substitution model with six rate classes and invariable microwave fixation protocol and dried with sites optimized. An unrooted BioNJ starting tree was con- Hexamethyldisilazane (E. Humphrey, personal communica- structed and the best of nearest neighbor interchange (NNI) tion), sputter-coated with gold-palladium, and examined using and subtree pruning and regrafting (SPR) tree improvement a Hitachi S-4800 field emission scanning electron microscope was implemented during the heuristic search. Nonparametric at 0.5–1.0 kV. Selected images of light and SEM microscopy bootstrap support (BS) (Felsenstein 1985) was determined were edited in Adobe Photoshop Elements 7.0. The type spec- with 100 replicates. Clades were considered significant and imens, in the form of dried cultures and semi-permanent highly supported when BS≥70 % (Hillis and Bull 1993). slides, are deposited in the Herbarium of the US National Bayesian inference employing a Markov Chain Monte Fungus Collections (BPI) and the Illinois Natural History Carlo (MCMC) algorithm was performed using MrBayes v. Survey Fungarium (ILLS). A living culture is also deposited 3.1.2 (Huelsenbeck and Ronquist 2001)ontheCIPRES in the Centraalbureau voor Schimmelcultures (CBS). Science Gateway Teragrid (Miller et al. 2010)asanadditional means of branch support. The GTR+I+G model with six rate DNA extraction, PCR amplification, and sequencing classes was employed. Four independent chains of MCMC were run for 100 million generations with every 1000th tree Detailed protocols for DNA extraction, PCR amplification, sampled to ensure that trees were not trapped in local optima. andSangersequencingaredescribedinPromputthaand Effective sample size (ESS) was estimated using Tracer v. 1.5 Miller (2010). A DNeasy® Mini Plant extraction kit (Qiagen (Rambaut and Drummond 2009), which suggested removing Inc., Valencia, CA) was used to extract DNA from the culture the first 10,000 trees as burn-in. Posterior probabilities were following the manufacturer’s protocols, except tissues were determined from a 95 % consensus tree generated in PAUP not ground in liquid nitrogen and final elution volume was 4.0b10 (Swofford 2002) with the remaining 90,000 trees. 25 μL. The complete ITS region and partial LSU of nrDNA Clades with Bayesian posterior probability (BPP)≥95 % were was amplified using two overlapping primer sets: ITS1F–LR3 considered significant and highly supported (Alfaro et al. and LROR–LR6 (Gardes and Bruns 1993; Rehner and 2003). Samuels 1995; Vilgalys and Hester 1990; White et al. 1990) and sequenced with the PCR primers along with ITS4 and LR3R on an Applied Biosystems 3730XL high-throughput Results capillary sequencer (Applied Biosystems, Foster City, CA) at the W. M. Keck Center at the University of Illinois A single sequence (1,666 bp) containing both the LSU and the Urbana-Champaign. ITS (KM503089) was obtained and deposited in GenBank. Phylogenetic analyses show that Curviclavula belongs within Phylogenetic analyses the Helotiales (Leotiomycetes) with the ML analysis of the LSU sequences yielding a single most-likely tree (Fig. 1). Two independent datasets were compiled and analyzed, a Curviclavula groups in a single branch with Hyalodendriella LSU dataset 903 bp in length consisting of 31 taxa betulae Crous with poor bootstrap support value (50 %) but representing nine families in the Helotiales based on high posterior probability (>95 %) and forms an unsupported Baschien et al. (2013), Hyde et al. (2011), and Réblová and sister clade with members of Hyaloscyphaceae sensu lato that Gams (2011), and a more exclusive ITS dataset 595 bp in includes Cistella spicicola Huhtinen & Söderh, Hyphodiscus Mycol Progress (2015) 14: 3 Page 3 of 7 3

Fig. 1 Phylogenetic tree generated after ML analysis of the LSU nrDNA sequence data showing the position of Curviclavula among Helotiales. Numbers above or below branches are bootstrap values≥ 50 % and thickened branches are Bayesian posterior probabilities≥ 95 %

hymeniophilus (P. Karst.) Baral and Catenulifera anamorphs. macronematous, mononematous, repeatedly dichotomously The ML analysis of the ITS data also generated a single tree branched few times, septate, brown. Conidiogenous cells (not shown) where Curviclavula clustered most closely with monoblastic or polyblastic, integrated, terminal or intercalary, Chalara species belonging to Helotiales and again to indeterminate, sympodial, cylindrical or geniculate; H. betulae. conidiogenous loci flattened, inconspicuous. Conidial seces- sion schizolytic. Conidia acropleurogenous, complanate, pale brown, smooth, variable in shape, didymosporous, Taxonomy phragmosporous, minutely cheiroid at first or eventually subglobose to broadly clavate, the apical cell curving and Curviclavula G. Delgado, F.A. Fernández & A.N. Mill., gen. fusing with other conidial cells once in contact forming a nov. closely appressed conidium; basal cell with truncate, darkened MycoBank MB 809670 hilum and minute marginal frill. Etymology: from the Latin curvus=curved, bent and Type species: Curviclavula anemophila G. Delgado, F.A. clavula=small club, referring to the conidial shape. Fernández & A.N. Mill. Colonies on MEA moderately slow growing, velvety, dark Curviclavula anemophila G. Delgado, F.A. Fernández & gray. Mycelium immersed and superficial. Conidiophores A.N. Mill., sp. nov. (Figs. 2 and 3) 3 Page 4 of 7 Mycol Progress (2015) 14: 3

subglobose, oblong or broadly obovoid, but usually minutely cheiroid at first and consisting of a subcylindrical to obconical, often inflated, straight or curved basal cell bearing a single, subglobose lateral cell and a short row of 2–4cells, the apical cell of the developing conidium curves and fuses with the lateral and remaining cells once in contact eventually forming a closely appressed, subglobose to broadly clavate conidium, (7) 8–13 (15)×(4) 6–9 (10) μm, 4–6 μm thick; basal cell with a truncate, darkened hilum, 2–2.5 μm wide, often showing a minute marginal frill. Teleomorph unknown. Specimen examined: UNITED STATES. New Mexico, Los Alamos Co., Los Alamos, 35°53′16″ N, 106°18′56″ W, 2,200 m asl., from outdoor air, isol. F.A. Fernández, 24.1.2013. Holotype (BPI 892883), Isotype (ILLS 71919), ex-holotype culture (CBS 138123).

Discussion

Certain aspects of conidial morphology and development Fig. 2 Curviclavula anemophila (from holotype). Line drawings of a branched conidiophore, conidiogenous cells and conidia. Scale bar= make Curviclavula unique among previously described gen- 15 μm era of hyphomycetes (Seifert et al. 2011). The most significant developmental feature involves the curving and sequential fusing of the apical cell with other elements of the conidium, MycoBank MB 809671 first with the lateral cell and later with the remaining conidial Etymology: from the Greek ánemos=wind and philein=to cells during maturation. Initially, the conidial filament elon- love, referring to the air, where the fungus was isolated. gates while the tip bends to one side, and the apical cell is then Colonies on MEA moderately slow growing, reaching 23– delimited by a thin septum. The bottom and now basal cell 28 mm diam. after 21 days at 25 °C, velvety, dark gray or produces a small bud laterally somewhat below the septum, blackish gray (22 F1/22G1), becoming dark brown (6 F7) in which also elongates forming a lateral cell after being age, with cottony, light gray (22D1) aerial mycelium in the delimited by a second septum. At this point the conidium center, margin narrow, entire or slightly undulate, reverse pale initial shows a resemblance to a tiny hand in side view. yellow (2A3), often with concentric rings, sporulation abun- Meanwhile, a further septum is formed at the conidial filament dant. Mycelium immersed and superficial, composed of delimiting a subapical cell. More or less synchronously with branched, septate, smooth or finely rough, hyaline to light the formation and delimitation of the lateral cell, the apex of brown hyphae, 1–2 μm wide. Conidiophores arising interca- the conidial filament curves and grows towards it. Once both lary or terminally from the aerial hyphae, macronematous, cell tips are in contact, they press together or even fuse but mononematous, erect, flexuous, smooth, septate, slightly at- remain delimited by the cell walls as a new, additional septum. tenuated at the septa, repeatedly dichotomously branched 1–5 Subsequent swelling of the filament causes the walls of the times, brown, length indeterminate, 2–3 μm wide; branches lateral and subapical cell to also fuse delimiting another new usually formed below a septum, flexuous, somewhat genicu- septum. The final conidium is composed of four cells and five late, brown, paler toward the apex, up to 120 μm long. septa, one longitudinal and four oblique, and is more or less Conidiogenous cells monoblastic or polyblastic, integrated, broadly clavate in shape, often showing a tiny space in the terminal or intercalary, indeterminate, irregularly sympodial, center as a result of incomplete fusing. cylindrical, subcylindrical or sometimes geniculate, with 1–2 The pattern described above is the most common, but there conidiogenous loci per cell; conidiogenous loci flattened, in- are variations among cheiroid conidia (Figs. 2 and 3d). conspicuous, apical, lateral or on shoulders formed by sym- Occasionally only the lateral walls of the apical, subapical, podial proliferation, non-protuberant, neither thickened nor and lateral cells press together while the cell tips remain free. darkened. Conidial secession schizolytic. Conidia holoblastic, Some conidia have only four cells tightly appressed and ar- solitary, dry, acropleurogenous, complanate, pale brown, ranged side by side forming a cross. Sometimes, it is the smooth, composed of 2–5 (6) cells, each individual cell 3– suprabasal cell of the row and not the basal cell that elongates 7×3–6 μm, septate, constricted at the septa, variable in shape, forming a lateral cell after being delimited by a septum. The occasionally simple, didymosporous or phragmosporous and final conidium is then composed of five cells and six septa Mycol Progress (2015) 14: 3 Page 5 of 7 3

Fig. 3 Curviclavula anemophila (from holotype) (a)ColonyonMEA indicates the irregular marginal frill left after schizolytic secession (d) after 21 days incubation at 25 °C. Conidiophores, conidiogenous cells light microscopy. Scale bars a=1 cm, b-c, e-f=3 μm, g-h=5 μm, d= and conidia (b–c), (e–h)SEM(b) Seceding conidium showing irregular 10 μm tear of the outer conidiogenous cell wall (e) single conidium. Arrow once the apex curves and fuses. Often, the basal cell is inflated apical cell curves, it then contacts this second upper lateral and no delimiting septum is laid down. A lateral cell is, there- cell, which is located in the middle between both the apical fore, not formed and the apical cell fuses with one side of the and the bottom lateral cell. The final conidium possesses six basal cell forming a subglobose conidium made of three tight- cells and up to nine septa with different longitudinal, trans- ly appressed cells and three septa more or less arranged in a verse, or oblique orientation. Another variation occurs when BY^. A variation of this pattern occurs when the inflated basal the suprabasal cell enlarges laterally but no septum is laid cell enlarges laterally without forming a delimiting septum. down to form a second lateral cell. That enlargement is then The enlarged side then contacts the tip or the lateral wall of surrounded by the apical cell, which curves to contact the tip the apical cell and the resulting conidium is made up also of of the lateral cell, resulting in a five-celled conidium with three cells and three septa arranged in a BY^, often with a tiny seven septa also in different orientations. space in the center. A similar variation occurs when the This peculiar conidial morphology and development is in suprabasal cell is the one that is inflated or enlarges laterally, combination with other features such as repeatedly dichoto- and the conidium then possesses four cells, a subcylindrical or mously branched conidiophores and terminal or intercalary, obconical basal cell and three distal cells disposed in the same irregularly sympodial conidiogenous cells bearing one or BY^ septal arrangement. Rarely, the basal cell produces a lat- two loci per cell. Conidial secession is schizolytic, but the eral cell delimited by a septum, but also the suprabasal cell conidial basal cells often bear minute, hardly visible, marginal enlarges laterally forming a second lateral cell. When the frills under the light microscope. SEM microscopy revealed 3 Page 6 of 7 Mycol Progress (2015) 14: 3 that the lytic zone between the wall of the conidiogenous cell or in freshwater habitats as aero-aquatic or aquatic (Hyde et al. and the conidium basal septum is relatively broad and often 2011). The relationship of Curviclavula with thickened and the seceding conidium tears the surface of the Hyaloscyphaceae could be further supported by its morpho- supporting conidiogenous cell rather irregularly (Fig. 3b, f). logical similarity with Cheiromycella, particularly Once released, the conidium carries around the base a small Ch. microscopica (Braun et al. 2009; Ellis 1971; Sutton portion of the ruptured outer wall as a frill of irregular margin 1985). They share the presence of macronematous, repeatedly and leaves a more or less circular scar on the conidiogenous branching conidiophores with monoblastic or polyblastic cell. An ambiguous conidial feature was wall ornamentation, conidiogenous cells and occasionally simple, didymosporous smooth under the light microscope but finely rough to wrin- or phragmosporous but also branched cheiroid conidia that are kled under SEM microscopy, and possibly the result of sample variable in shape and usually with two arms arising from a preparation artifacts. basal cell and constricted at the septa. Matsushima (1981) Phylogenetic analyses suggest that Curviclavula illustrated a specimen of Ch. microscopica isolated on culture anemophila belongs to the Helotiales with strong affinity to media from the inside of dead bark of Fagus crenata Blume in the family Hyaloscyphaceae. The LSU phylogeny shows a Japan with conidia very similar to those in Curviclavula. relationship supported only in Bayesian analysis with Conidiophores in Cheiromycella, however, are short and ir- Hyalodendriella betulae, a morphologically unrelated regularly branched, conidiogenous cells are swollen, determi- helotialean anamorph characterized by limoniform to ellip- nate, clavate, doliiform, sphaerical or subsphaerical in shape, soid, aseptate conidia in short chains of 2–3 and dimorphic sometimes connected to one another by narrow isthmi, and conidiophores with conidiogenous cells bearing a single or conidia are aggregated in firm slimy masses, secede numerous, aggregated conidiogenous loci (Crous et al. schizolytically without bearing a basal marginal frill, and have 2007). Helotiales is a large and polyphyletic order in the class divergent and flexuous arms with a greater number of cells Leotiomycetes that includes several anamorphic genera dis- that never fuse (Ellis 1971). tributed within eleven teleomorphic families, but phylogenetic relationships at familial level are still unresolved for many Acknowledgments Special thanks are due to Drs. Ondřej Koukol for other genera like Hyalodendriella (Hyde et al. 2011). ITS serving as a presubmission reviewer and his many valuable comments on the manuscript, Walter Gams, Keith Seifert, and Guozhu Zhao for their sequence data also showed a relationship with H. betulae opinions on the identity of the fungus and aspects of conidial secession and with species of the morphologically dissimilar, phialidic and to Seppo Huhtinen for providing relevant literature on Hyaloscypha. genus Chalara belonging to Helotiales such as C. recta G.D. gratefully acknowledges Kamash Ramanathan, Joshua Cox, Shan- Koukol, C. longipes (Preuss) Cooke, and C. piceae-abietis non Nast, Josef Juhas, and Christopher Weedon (EMLab P&K) for pro- vision of laboratory facilities and financial or other supports. Hol.-Jech. (Koukol 2011). 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