A New Species of Ijuhya, I. Fournieri, from French Guiana

A new species of Ijuhya, I. fournieri, from French Guiana

Christian LECHAT Summary: A detailed description of Ijuhya fournieri sp. nov. is presented based on two collections on dead Laurence LESAGE-MEESSEN palm leaves in French Guiana. The asexual state has been obtained in culture and sequenced. Molecular Anne FAVEL data indicate that it is an undescribed species of Ijuhya. A key to the species of Ijuhya with fasciculate hairs is provided. Keywords: Ascomycota, Hypocreales, Bionectriaceae, acremonium-like, taxonomy. Ascomycete.org, 7 (3) : 101-104. Juin 2015 Résumé : une description détaillée de Ijuhya fournieri sp. nov. est présentée, basée sur deux récoltes sur Mise en ligne le 13/06/2015 feuilles mortes de palmier en Guyane Française. Le stade asexué a été obtenu en culture et séquencé. Les données moléculaires indiquent qu’il s’agit d’une espèce d’Ijuhya non décrite. Une clé des espèces d’Ijuhya avec des poils fasciculés est fournie. Mots-clés : Ascomycota, Hypocreales, Bionectriaceae, type acremonium, taxinomie.

Introduction cycles at 94ºC, 54ºC and 72ºC (45, 30 and 45 s respectively) and a final 72ºC step 10 min. Chromatograms were checked searching for putative reading errors, and these were corrected. Analyses were The genus Ijuhya Starbäck is based on the lectotype Ijuhya vitrea performed online at www.phylogeny.lirmm.fr (DEREEPER et al., 2008). Starbäck (1899). The ascomata of Ijuhya are white, pale yellow, yel- Maximum likelihood phylogenetic analyses were performed with low to pale orange or brownish-orange, not changing color in 3% PhyML 3.0 aLRT (ZWICKL, 2006), using the GTR + I + Γ model of evo- KOH or lactic acid; thus the genus Ijuhya belongs to the Bionectria- lution. Branch support was assessed using the nonparametric ver- ceae as defined by ROSSMAN et al. (1999). The genus Ijuhya is distin- sion of the approximate likelihood-ratio test, implemented in PhyML guished from other genera in the Bionectriaceae by hairs arranged (SH-aLRT; ANISIMOVA & GASCUEL, 2006). in a stellate fringe around the upper margin of the perithecia, rarely without hairs. Based on pale perithecia not changing color in 3% KOH or lactic Taxonomy acid, presence of fasciculate hairs arranged in an apical crown and comparison with known species in the genus, two specimens were Ijuhya fournieri Lechat sp. nov. Plate 1 determined to represent a previously undescribed species of Ijuhya. Mycobank MB 811831 Both collections CLLG10113 and CLLG12002 were cultured from ascospores and produced an acremonium-like asexual state. Diagnosis: Differs from known species ofIjuhya by the combination of white to pale yellow ascomata with an apical crown of white, Materials and methods fasciculate hairs up to 250 μm long, asci 6–8-spored, striate ascospores (18–)22–23.5(–25) × 4.5–5(–5.5) μm and its occurrence on palm of Astrocarium sp. Specimens were examined using the method described by ROSS- Holotypus: French Guiana, Sinnamary, Piste St Elie, 22 Apr. 2010, MAN et al. (1999). Microscopic observations and measurements were on dead leaves of Astrocarium sp., coll. C. Lechat CLLG10113 (LIP); made in water and ascospore ornamentation was observed in lactic ex-type culture CBS 128283. cotton blue without heating. Cultures of the living specimen were Additional specimen examined: French Guiana, Nouragues Na- made on PDA (Potato Dextrose Agar) with 5mg/l of streptomycin in tional Natural Reserve, Inselberg camp, 16 Jun. 2012, on dead leaves Petri dishes 9 cm diam. A mass of ascospores and asci was removed of Astrocarium sp., coll. C. Lechat CLLG12002. from a perithecium with a fine needle and placed in a drop of sterile water that was stirred with a needle to distribute the elements on Etymology: The epithet “fournieri” refers to the renowned French the slide. A part of the drop containing ascospores was placed on mycologist Jacques Fournier to whom the author dedicates this spe- PDA using a sterile micropipette, then the Petri dish was incubated cies. at 25°C. DNA extraction, amplification, and sequencing were performed Ascomata solitary or crowded in groups of 2–4, superficial, non- by ALVALAB (Santander, Spain): Total DNA was extracted from dried stromatic, subglobose, (160–)180–210(–230) μm high × (170–)200– specimens blending a portion of them using a micropestle in 600 μl 220(–240) μm diam. (m = 200 × 215 μm, n = 15), white to pale CTAB buffer (CTAB 2%, NaCl 1.4 M, EDTA pH 8.0 20 mM, Tris-HCl yellow, not collapsing when dry, not changing color in 3% KOH or pH8.0 100 mM). The resulting mixture was incubated for 15 min. at lactic acid. Perithecial apex flattened with short, acute papilla, mar- 65ºC. A similar volume of chloroform: isoamylalcohol (24:1) was gin with fasciculate, thick-walled hairs. Hairs hyaline, 2–3.5 μm diam, added and carefully mixed with the samples until their emulsion. It cylindrical, flexuous, thick-walled (0.7–1 μm), rounded at tips, sep- was then centrifuged for 10 min at 13,000 g, and the DNA in the su- tate, arising from base of ascomata, covering totally the lateral as- pernatant was precipitated with a volume of isopropanol. After a comatal wall, agglutinated to form white teeth 160–250 μm long, new centrifugation of 15 min at the same speed, the pellet was wa- 20–30 μm wide at base, arranged in a stellate fringe around upper shed in cold 70% ethanol, centrifuged again for 2 min and dried. It margin of ascomata. Ascomatal wall 25–45 μm thick, composed of was finally resuspended in 200 μl ddH2O. PCR amplification was per- three regions; outer region 15–20 μm thick of thick-walled hyphae, formed with the primer LR0R and LR5 (VILGALYS & HESTER, 1990) to am- 2–3.5 μm diam, proliferating to form teeth at upper margin of peri- plify the 28S nLSU region. PCR reactions were performed under a thecium; middle region 10–15 μm wide, of globose to ellipsoidal, program consisting of a hot start at 95ºC for 5 min, followed by 35 nearly angular cells, 5–8 × 5–6 μm, with pale yellow wall 1–1.5 μm 101 Plate 1 — Ijuhya fournieri

102 Fig. 2 — Maximum likelihood phylogeny of Ijuhya fournieri based on LSU sequences.

thick; inner region 5–10 μm wide, of elongate, flattened cells, 5–11 hairs up to 300 μm long. The phylotree (fig. 2) indicates thatI. four- × 2–2.8 μm, with hyaline wall. Asci (55–)60–65(–70) × (7–)9–10 nieri is close to I. paraparilis but this one differs mainly by lacking (–12) μm (m = 63.5 × 10 μm, n=20), clavate, apices rounded, without fasciculate hairs. ML phylogeny based on ITS was not possible be- ring, with 6–8 irregularly biseriate ascospores. No interthecial ele- cause there is only one available sequence at GenBank but the maxi- ments seen. Ascospores (18–)22–23.5(–25) × 4.5–5(–5.5) μm (m = mum likelihood phylogeny based on LSU sequences (fig. 2) places 22.7 × 4.7 μm, n=30), fusiform, 1-septate, hyaline with 2–3 drops in clearly the new species in the genus Ijuhya. each cell, strongly striate with 2–3 striations visible in one plane of view, completely filling each ascus. Asexual state: acremonium-like. Acknowledgments

Cultural characteristics: Colony after two weeks on PDA, 18– The author gratefully acknowledges Dr. Amy Rossman, 1808 NW 22 mm diam, greyish white in center, white at margin, reverse pale Sunview Dr. Corvallis, OR 97330, U.S.A. and Dr. Pierre-Arthur Moreau yellow to pale yellowish brown, sporulating at margin. Conidio- (Laboratoire des sciences végétales et fongiques, Faculté des phores macronematous, mononematous, unbranched, flexuous, sciences pharmaceutiques et biologiques, Université de Lille 2, hyaline, smooth. Conidiogenous cells monophialidic, 23–45 μm France) for their advices and scientific help. long, 1.5–1.8 μm wide at apex with a slightly flared collarette, 2– 2.2 μm wide at base. Conidia solitary or grouped at tip of phialide to form a mucous head, aseptate, narrowly ellipsoidal to subcylin- References drical with rounded apex, attenuated at base with a flat abscission scar, smooth, hyaline, 6.5–9 × 2.2–3 μm (m = 7.9 × 2.8 μm, n = 30). ANISIMOVA M. & GASCUEL O. 2006. — Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Systematic Discussion Biology, 55: 539-552. DEREEPER A., GUIGNON V., BLANC G., AUDIC S., BUFFET S., CHEVENET F., DU- FAYARD J.F., GUINDON S., LEFORT V., LESCOT M., CLAVERIE J.M. & GASCUEL O. All characteristics of the species described herein fit well with the 2008. — Phylogeny.fr: robust phylogenetic analysis for the nons- genus Ijuhya as defined by ROSSMAN et al. (1999). Ijuhya fournieri is pecialist. Nucleic Acids Research, supplement (2001), 2008, 36 characterised by the combination of white to pale yellow ascomata with an apical crown of white, fasciculate hairs. The closest species (Web Server issue): W465-W469. of I. fournieri having fasciculate hairs more than 130 μm long arran- LECHAT C. & COURTECUISSE R. 2010. — A new species of Ijuhya, I. antil- ged in apical crown are Ijuhya antillana Lechat & Courtec. (2010), lana, from the French West Indies. Mycotaxon, 113: 443-447. I. dentifera (Samuels) Rossman & Samuels (1999), I. oenanthicola Le- LECHAT C. & HAIRAUD M. 2012. — A new species of Ijuhya, I. oenanthi- chat & Hairaud and I. peristomialis (Berk. & Broome) Rossman & Sa- cola. Mycotaxon, 119: 249-253. muels (1999). Ijuhya antillana differs from I. fournieri mainly in having ROSSMAN A.Y., SAMUELS G.J., ROGERSON C.T. & LOWEN R. 1999. — Genera dark orange ascomata, shorter hairs and smaller, striate ascospores; of Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, As- I. dentifera has smaller, spinulose ascospores and fasciculate hairs comycetes). Studies in Mycology, 42: 1-248. not more than 200 μm long unlike I. fournieri; I. oenanthicola differs STARBÄCK K. 1899. — Ascomyceten der ersten Regnellschen Expedi- in having hairs up to 140 μm long and smaller ascospores while I. pe- tion I. Bihang til Kongliga Svenska Vetenskaps-akademiens Hand- ristiomalis differs mainly in having larger, striate ascospores and lingar, 25, Afd. 3 (no. 1): 3-68 + pl. I-II. VILGALYS R. & HESTER M. 1990. — Rapid genetic identification and map- ping of enzymatically amplified ribosomal DNA from several Cryp- Legend of Plate 1: Ijuhya fournieri (Holotype CLLG10113). a, b: Pe- tococcus species. Journal of Bacteriology, 172: 4238-4246. rithecia on natural substratum. c: Close-up of perithecium in water. ZWICKL D.J. 2006. — Genetic algorithm approaches for the phylogenetic d: Perithecium in top view showing apical crown of fasciculate hairs. analysis of large biological sequence datasets under the maximum e: Section through the ascomatal wall. f: Ascus and ascospores (in likelihood criterion. Ph.D. Dissertation. Austin, The University of water). g: Ascospores in Cotton Blue showing ornamentation. Texas. 103 Key to species of Ijuhya with fasciculate hairs (modified from LECHAT & HAIRAUD, 2012)

1. Hairs more than 200 μm long ...... 2 1. Hairs less than 200 μm long ...... 4 2. Hairs up to 300 μm long; ascospores (24–)30–60(–110) × 4–7(–8) μm, striate; ascomata pale yellow...... I. peristomialis 2. Hairs averaging 145–240 μm long ...... 3 3. Hairs 150–200 × 3–4 μm; ascospores 6–8(–9) × 3–4 μm, spinulose; ascomata orange-yellow ...... I. dentifera 3. Hairs 160–250 × 2–3.5 μm; ascospores (18–)22–23.5(–25) × 4.5–5(–5.5) μm, striate...... I. fournieri 4. Ascospores striate ...... 5 4. Ascospores spinulose 14.5–20 × (2.5–)3–5(–5.4) μm, ascomata brownish-orange, hairs sparse, 30–50 μm long, or absent... I. parilis 5. Ascospores faintly striate (8.5–)9.5–11.5(–12.5) × 2.8–3.2(–3.5) μm; ascomata brownish-orange, hairs 28–80 μm long...... I. equiseti-hiemalis 5. Ascospores strongly striate...... 6 6. Ascospores averaging 14–28 μm long...... 7 6. Ascospores (10.5–)11–13(–14) × 2.5–3.5 μm; ascomata dark orange, hairs 100–160 μm × 2.5–3 μm...... I. antillana 7. Ascospores (12–)14–16.5(–18) × (2.5–)3–3.5(3.8) μm; ascomata pale orange to dark orange, hairs up to 140 μm long ...... I. oenanthicola 7. Ascospores (19–)21–28 × 3.5–4.5 μm; ascomata dull orange, hairs up to 100 μm long ...... I. chilensis

Christian Lechat Laurence Lesage-Meessen 64 route de Chizé INRA, UMR 1163 Biodiversité et Biotechnologie fongiques 79360 Villiers-en-Bois 13288 Marseille Cedex France France [email protected] [email protected]

Anne Favel Université d’Aix-Marseille, UMR 1163 Biodiversité et Biotechnologie fongiques 13288 Marseille Cedex France [email protected]

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