Clonostachys Moreaui (Hypocreales, Bionectriaceae), a New Species from the Island of Madeira (Portugal)

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Clonostachys moreaui (Hypocreales, Bionectriaceae), a new species from the island of Madeira (Portugal)

Christian LECHAT Abstract: Clonostachys moreaui sp. nov. is described and illustrated based on a collection on dead corticated Jacques FOURNIER branches of Laurus novocanariensis in Madeira (Portugal). This species is placed in Clonostachys based on its Antonia GASCH clonostachys-like asexual morph, ascomata not changing colour in 3% koh or lactic acid and phylogenetic comparison of its LSU sequence with those of 17 known species of Clonostachys. Clonostachys moreaui is primarily characterized by smooth to slightly roughened, orange, globose, bionectria-like ascomata with Ascomycete.org, 12 (2) : 35–38 wall cells containing numerous orange oily droplets, crowded on an erumpent pseudoparenchymatous Mise en ligne le 19/04/2020 stroma and faintly warted ascospores (14–)15–18(–20) × 6–7 µm. Based on the comparison of morphological 10.25664/ART-0295 characteristics of sexual-asexual morphs and molecular data with known species, C. moreaui is proposed as a new species. Keywords: Ascomycota, Laurus novocanariensis, Macaronesia, ribosomal DnA, taxonomy.

Résumé : Clonostachys moreaui sp. nov. est décrite et illustrée d’après une récolte eﬀectuée sur écorce de branches mortes de Laurus novocanariensis à Madère (Portugal). Cette espèce est placée dans le genre Clo- nostachys d’après sa forme asexuée de type clonostachys, les ascomes ne changeant pas de couleur dans koh à 3 % ou dans l’acide lactique et la comparaison phylogénétique de la séquence LSU avec celles de 17 espèces connues de Clonostachys. Clonostachys moreaui est principalement caractérisée par des ascomes globuleux, oranges, lisses à faiblement rugueux, rassemblés sur un stroma pseudoparenchymateux érom- pant et des ascospores faiblement verruqueuses, (14–)15–18(–20) × 6–7 µm. en se fondant sur la comparaison des caractères morphologiques et des données moléculaires avec les espèces connues, C. moreaui est proposée comme une nouvelle espèce. Mots-clés : ADn ribosomal, Ascomycota, Laurus novocanariensis, Macaronésie, taxinomie.

Introduction nLSU region. PCr reactions were performed under a program con- sisting of a hot start at 95ºC for 5 min, followed by 35 cycles at 94ºC, 54°C and 72ºC (45, 30 and 45 s respectively) and a final 72ºC step During a foray in Levada dos Cedros, Madeira (Portugal) in July 10 min. Chromatograms were checked searching for putative read- 2019, an hypocrealean fungus morphologically similar to Clonos- ing errors, and these were corrected. tachys rosea (Link) Schroers, Samuels, Seifert & W. Gams (SChroerS, Analyses were performed online at www.phylogeny.lirmm.fr 2001) was collected on dead corticated branches of Laurus novoca- (DereePer et al., 2008). Maximum likelihood phylogenetic analyses nariensis, an endemic tree of Canary Islands and Madeira, typical of were performed with PhyML 3.0 aLrT (ZWICkL, 2006), using the GTr laurisilva forests. This fungus was successfully cultured and, as ex- + I + Γ model of evolution. Branch support was assessed using the pected, yielded a clonostachys-like asexual morph, characterized by non-parametric version of the approximate likelihood-ratio test, im- ChroerS a distinctive penicillate conidiogenous structure (S , 2001). plemented in PhyML Sh-aLrT (AnISIMoVA & GASCUeL, 2006). nomen- We report here our observations showing the morphological differ- clature follows MycoBank (CBS-knAW Fungal Biodiversity Center, ences of both its sexual and asexual morphs with those of typical Utrecht, The netherlands). C. rosea. A LSU sequence was obtained from the culture and com- pared with those of 17 Clonostachys species, confirming its affiliation to Clonostachys Corda and its affinities with C. rosea. Its differences Taxonomy with the widespread C. rosea are discussed and shown to support the status of the collection from Madeira as a distinct species for Clonostachys moreaui Lechat, J. Fourn. & Gasch, sp. nov. – MB which we propose the name C. moreaui. 834843 – Fig. 2

Diagnosis: Most similar to Clonostachys rosea but different by Materials and methods larger ascospores (14–)15–18(–20) × 6–7 µm vs. (7.4–)9.4–10.8(– 14.4) × (2.2–)3–3.6(–4.8) µm in C. rosea and wider ellipsoidal conidia Dry specimens were rehydrated and examined using the method without hilum 5–10(–12) × 3.5–5 µm vs. 7–9(–15.4) × 2.8–3.4 µm. described by roSSMAn et al. (1999). Microscopic observations and Holotype: PorTUGAL, Madeira, Levada dos Cedros, on dead corti- measurements were made in water. The holotype specimen was de- cated branches of Laurus novocanariensis rivas Mart., Lousã, Fern. posited in LIP herbarium (University of Lille). Cultures of the living Prieto, e. Dias, J.C. Costa & C. Aguiar (Lauraceae), 10 oct. 2019, leg. specimen were made on PDA (Potato Dextrose Agar) with 5 mg/L A. Gasch, CLL19024 (LIP), LSU GenBank sequence MT160524. of streptomycin in Petri dishes 5 cm diam., incubated at 25°C. DnA Etymology: named in honour of our dear friend and colleague extraction, amplification, and sequencing were performed by AL- Dr. Pierre-Arthur Moreau. VALAB (oviedo, Spain): Total DnA was extracted from pure cultures blending a portion of them using a micropestle in 600 µL CTAB Ascomata solitary to gregarious, in groups of 3–40, crowded on buffer (CTAB 2%, naCl 1.4 M, eDTA ph 8.0 20 mM, Tris-hCl ph 8.0 an erumpent, pseudoparenchymatous stroma, globose, 330– 100 mM). The resulting mixture was incubated for 15 min at 65ºC. A 540 µm diam., smooth to faintly roughened, laterally pinched or not similar volume of chloroform: isoamylalcohol (24:1) was added and when dry, orange to bright orange, with a slightly darker ostiolar re- carefully mixed with the samples until their emulsion. It was then gion, not changing colour in 3% koh or lactic acid. Ascomatal apex centrifuged for 10 min at 13.000 g, and the DnA in the supernatant rounded to flat around ostiole, minutely papillate, composed of was precipitated with a volume of isopropanol. After a new centrifu- cylindrical to subglogose cells 8–12 × 2–4 µm, with pale orange wall gation of 15 min at the same speed, the pellet was washed in 70% 1.5 µm thick. Ascomatal wall 50–60 µm thick, of two regions; outer cold ethanol, centrifuged again for 2 min and dried. It was finally re- region 30–35 µm thick, composed of subglobose to ellipsoidal cells suspended in 200 µL ddh2o. PCr amplification was performed with 7–15 × 5–8 µm, with pale yellow walls 1–1.5 µm thick, containing the primers Lr0r and Lr5 (VILGALyS & heSTer, 1990) to amplify the 28S numerous pale orange oily droplets; inner region 20–25 µm thick,

35 Fig. 1 – Maximum likelihood phylogeny (-lnL = 2070.86989) of Clonostachys moreaui inferred by PhyML 3.0, model hky85 from a 860 bp matrix of 28S rrnA sequences, rooted with Dialonectria diatrypicola.

composed of subglobose to elongate cells 7–16 × 2.5–3.5 µm with tria ochroleuca (Schwein.) Schroers & Samuels (SChroerS et al., 1999). hyaline wall 1.5–2 µm thick. Asci evanescent, unitunicate, clavate, Clonostachys has now priority over the former sexual morph genus short-stipitate (80–)85–90(–95) × 12–15 µm (Me = 88 × 13.5 µm, Bionectria (roSSMAn et al., 2013). n=30), with 8 ascospores biseriate or irregularly disposed in upper Clonostachys sexual morphs are characterized by white, yellow to part and uniseriate in lower part, apex simple. evanescent, monili- orange or brown, usually crowded and roughened ascomata, not form paraphyses inserted between asci, up to 12 µm diam. at base. changing colour in 3% koh or lactic acid, which places them in the Ascospores (14–)16–18(–20) × (4.5–)5.5–6.5(–7) µm (Me = 17 × Bionectriaceae as defined by roSSMAn et al. (1999) and SChroerS 6 µm, n = 50), ellipsoidal, equally 1-septate, slightly constricted or (2001). The new species described above matches well this set of not at septum, faintly warted, hyaline. characters, which is supported by our phylogenetic analysis of LSU Cultural characteristics: After two weeks on PDA at 25°C, colony sequences (Fig. 1) placing this fungus in Clonostachys. Clonostachys 30–40 mm diam., aerial mycelium white to pinkish, salmon at point of inoculation, off-white at margin, without colouration in medium. moreaui is primarily characterized by smooth to faintly warted, or- Mycelium composed of septate, hyaline, smooth hyphae 2–3 µm ange to bright orange globose ascomata, crowded on an erumpent diam. Conidiophores monomorphic, penicillate, arising from aerial pseudoparenchymatous stroma, not changing colour in 3% koh or hyphae, macronematous, flexuous, hyaline, stipe and lateral lactic acid, with wall cells containing numerous pale orange oily branches 25–40 µm long, 3(–3.5) µm diam., apically bearing subu- droplets, a feature encountered in many Clonostachys species. our late conidiogenous cells 18–34(–40) µm long, 2.5–3 µm diam. at phylogenetic analysis (Fig. 1) showed that C. moreaui is nested on a base. Conidia hyaline, aseptate, ellipsoidal with rounded apex or sister branch of C. rosea, both species having 99% similarity of their rounded at both ends, or attenuated towards base with or without LSU sequences. however, C. rosea primarily differs in having signifi- apiculate hilum, smooth-walled, 5–10(–12) µm long, 3.5–5 µm wide cantly smaller ascospores 9.4–10.8 × 3–3.6 µm vs. 15–18 × 6–7 µm in widest part. in C. moreaui, and smaller conidia. A further different characteristic regarding the asci is the presence of a conspicuous apical ring ap- Results and discussion pearing triangular in optical section (SChroerS, 2001) that was not detected in C. moreaui. Comparison of morphological characteristics Clonostachys was introduced to accommodate the type species of sexual-asexual morphs of our fungi with known Clonostachys C. araucaria Corda, an asexual morph synonym of Clonostachys species, as well as molecular analysis, lead us to recognise C. moreaui rosea, which was shown to represent the asexual morph of Bionec- as an undescribed species.

36 Ascomycete.org Fig. 2 – a–h: Clonostachys moreaui (holotype CLL19024). a: habit of ascomata on bark; b: Vertical section through an ascoma and basal stroma; c: Ascomatal wall in vertical section; d: Asci and ascospores in water; e: Ascospores in lactic cotton blue; f: Paraphyses inserted between asci, in water; g: Culture at two weeks; h: Conidiophores and conidia from culture, in lactic acid. Scale bars: a = 300 µm; b = 30 µm; c = 20 µm; d-f, h = 10 µm.

Ascomycete.org 37 Acknowledgements D.M., ZhUAnG W.y., hIrookA y., herrerA C., SALGADo-SALAZAr C. & ChAVerrI P. 2013. — Genera of Bionectriaceae, Hypocreaceae and Dr Amy rossman (oregon State University, Corvallis, U.S.A.) is Nectriaceae (Hypocreales) proposed for acceptance or rejection. warmly thanked for her advice and scientiﬁc help and for her pre- IMA Fungus, 4 (1): 41–51. doi: 10.5598/imafungus.2013.04.01.05 submission review. SChroerS h.-J. 2001. — A monograph of Bionectria (Ascomycota, Hypocreales, Bionectriaceae) and its Clonostachys anamorphs. References Studies in Mycology, 46: 1–214. SChroerS h.-J., SAMUeLS G.J., SeIFerT k.A. & GAMS W. 1999. — Classiﬁca-

AnISIMoVA M. & GASCUeL o. 2006. — Approximate likelihood-ratio test tion of the mycoparasite Gliocladium roseum in Clonostachys as for branches: A fast, accurate, and powerful alternative. Systematic C. rosea, its relationship to Bionectria ochroleuca, and notes on Biology, 55 (4): 539–552. doi: 10.1080/10635150600755453 other Gliocladium-like fungi. Mycologia, 91 (2): 365–385. doi: DereePer A., GUIGnon V., BLAnC G., AUDIC S., BUFFeT S., CheVeneT F., DUFA- 10.1080/00275514.1999.12061028 yArD J.F., GUInDon S., LeForT V., LeSCoT M., CLAVerIe J. M. & GASCUeL o. VILGALyS r. & heSTer M. 1990. — rapid genetic identiﬁcation and map- 2008. — Phylogeny.fr: robust phylogenetic analysis for the non- ping of enzymatically ampliﬁed ribosomal DnA from several Cryp- specialist. Nucleic Acids Research, 2008 Jul 1; 36 (Web Server issue): tococcus species. Journal of Bacteriology, 172 (2): 4238–4246. doi: W465–469. doi: 10.1093/nar/gkn180 10.1128/jb.172.8.4238-4246.1990 roSSMAn A.y., SAMUeLS G.J., roGerSon C.T. & LoWen r. 1999. — Genera of Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, As- ZWICkL D.J. 2006. — Genetic algorithm approaches for the phylogenetic comycetes). Studies in Mycology, 42: 1–248. analysis of large biological sequence datasets under the maximum roSSMAn A.y., SeIFerT k.A, SAMUeLS G.J., MInnIS A.M., SChroerS h.-J., LoM- likelihood criterion. Ph.D. Dissertation. Austin, The University of BArD L., CroUS P.W., PõLDMAA k., CAnnon P.F., SUMMerBeLL r.C., GeISer Texas. ef

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1: C. Lechat – 64 route de Chizé, 79360 Villiers-en-Bois, France – [email protected] 2: J. Fournier – Las Muros, 09420 Rimont, France – [email protected] 3: A. Gasch Illescas – Av. Italia, 9D, 41012 Sevilla, Spain – [email protected]

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