Two rare ophiocordycipitaceous fungi newly recorded in Taiwan

Ophiocordycipitaceae is a highly diverse fungal family parasitizing a wide range of arthropods and hypogeous fungi. We collected two ophiocordycipitaceous species previously unknown in Taiwan: one emerged from hypogeous fruiting bodies of an Elaphomyces fungus and the other was associated with dragonflies. Based on gross morphology, microscopic features, ITS sequences, and hosts, the two ophiocordycipitaceous fungi were identified as Tolypocladium japonicum and Ophiocordyceps odonatae. We isolated axenic cultures of these two fungi, and their anamorphs were obtained. The simplicillium-like anamorph of T. japonicum is described herein for the first time. The anamorph of O. odonatae produce conidia holoblastically in sympodial sequence and is assignable to Hymenostilbe. A dichotomous key to the species of Ophiocordycipitaceae reported in Taiwan is provided. A thorough literature study indicates that the two fungi reported herein have rarely been collected. Our identifications of T. japonicum and O. odonatae agree well with descriptions in the literature and are highly supported by DNA sequence analysis.


Background
Ophiocordycipitaceous fungi are parasites of more than ten orders of Arthropoda and one fungal genus Elaphomyces (Kobayasi 1941(Kobayasi , 1981(Kobayasi , 1982Kobayasi and Shimizu 1960;Mains 1957Mains , 1958Spatafora et al. 2007). Taxa of Ophiocordycipitaceae G. H. Sung et al. were included in Clavicipitaceae (Lindau) Earle ex Rogerson sensu lato primarily because they have fleshy stromata, filiform ascospores, and unitunicate asci with a cap-like thickening on top (Kobayasi 1941(Kobayasi , 1981(Kobayasi , 1982Kobayasi and Shimizu 1960;Mains 1957Mains , 1958. Molecular phylogenetic studies (Sung et al. 2007a, b;Spatafora et al. 2007) suggested that Clavicipitaceae sensu lato can be segregated into three families: Cordycipitaceae Kreisel ex G. H. Sung et al., Ophiocordycipitaceae, and Clavicipitaceae sensu stricto. Ophiocordycipitaceae differs from the Cordycipitaceae as circumscribed by Sung et al. (2007a) mainly in having dark, tough, fibrous to pliant stromata at maturity. In accordance with the changes in Art. 59 of ICN, Quandt et al. (2014) proposed that Ophiocordycipitaceae contains the following six genera:  (Lo et al. 2013), and Tolypocladium inflatum W. Gams, the origin of cyclosporin (Henry 1998). Ophiocordyceps and Harposporium are the two genera of Ophiocordycipitaceae that have been reported in Taiwan (Wang et al. 1999). Most records of Ophiocordyceps reported in Taiwan were from ants; others were from wasps, grasshoppers, flies, stink bugs or rove beetles , and see Key to genera of Ophiocordycipitaceae in Taiwan herein). Harposporium species reported in Taiwan thus far were exclusively nematode parasites Kuo et al. 2008).
In this study, we add to the mycobiota of Taiwan two ophiocordycipitaceous species associated with interesting hosts: Ophiocordyceps odonatae (

Morphological observation
Specimens were air-dried and stored in the herbarium of Biodiversity Research Center, Academia Sinica, Taipei (HAST). Freehand thin sections were mounted in 5 % KOH or distilled water. Microscopic features were observed with a Leica DM2500 microscope equipped with differential interference contrast. Cultures were initiated from stromatal tissue inoculated on scratch malt extract agar (MEA) (Kenerley and Rogers 1976). Descriptions of cultures and anamorphs were made based on their morphology on potato dextrose agar (PDA) at 25 C. Fungal cultures were deposited at BCRC (the Bioresource Collection and Research Center, Hsin-chu, Taiwan).

DNA extraction, amplification, sequencing and sequence analysis
Fungal cultures were grown in malt extract broth (2 g malt extract in 100 ml water). Total DNA was extracted from freeze-dried mycelia by automated magnetic bead operating platform Smart LabAssist (Taiwan Advanced Nanotec Inc., Taoyuan, Taiwan) with nucleic acid extraction kit TANBead ® Fungi DNA Auto tube (Taiwan Advanced Nanotec Inc., Taoyuan, Taiwan). PCR amplification of the internal transcribed spacers of rDNA (ITS) was described in Hsieh et al. (2009). PCR products were cleaned with DNA Advanced ™ DNA Clean Up System (Viogene BioTek Corp., Sijhih, Taiwan) following the manufacturer's protocol. Sequencing methods were as in Hsieh et al. (2009).
Specimens examined. New Taipei  Notes. Records of T. japonicam are fairly rare even in Japan (Kobayasi and Shimizu 1960) and appear sporadic in other countries, including Austria (Mains 1957;Kobayasi and Shimizu 1960) and China (Liang et al. 2003). Reflected in studies of recent years is the paucity of available material of this fungus: the DNA sequences deposited at GenBank resulting from two Japanese sources only, a specimen OSC 110991 and a culture IFO 9647. Among the three ITS sequences of T. japonicum cited below, AB027366 and EU039882 are from IFO 9647, while JN049824 is from OSC 110991.
The Taiwan collections fit well the descriptions of T. japonicum given by Kobayasi and Shimizu (1960 This is the first report describing cultures and anamorph of T. japonicum. The conidiogenous cells of T. japonicum are long, slender, lacking an inflated base. Tolypocladium, typified by T. inflatum W. Gams, was originally characterized by conidiogenous cells with an inflated base and a narrow neck. However, the genus is currently circumscribed on the basis of molecular phylogeny rather than morphology (Quandt et al. 2014). ≡ Cordyceps odonatae Kobayasi, Bull. Nat. Sci. Mus. Tokyo, Ser. B, 7: 6. 1981. = Hymenostilbe odonatae Kobayasi, Sci. Rep. Tokyo Bunrika Daig., Sect. B 5: 223.1941.
Cultures and anamorph. Colonies on PDA at 25 C attaining 1 cm in 3 wk, white, overlain with short dense aerial hyphae, diffuse at margins. Sporulation absent. Notes. Ophiocordyceps odonatae is the only species in the genus known to parasitize dragonflies. The two studied Taiwan collections were made from the dragonfly genus Planaeschna, from which the type specimen of Hymenostilbe odonatae Kobayasi was also collected (Kobayasi 1941(Kobayasi , 1981. Only the anamorph was present in the Taiwan collections. It should be noted that the basionym of O. odonatae is the teleomorph-typified binomial Cordyceps odonatae Kobayasi, which is predated by the anamorph-typified binomial Hymenostilbe odonatae. Conidiogenous cells and conidia in Taiwan collections are slightly larger than those documented in the protologue of H. odonatae (Kobayasi 1941). Also, unlike the more or less curved conidia described in Kobayasi (1941), conidia in Taiwan collections are not curved or only slightly curved. Modes of conidiogenesis were considered an important character in separating Hymenostilbe from Akanthomyces Lebert by Samson and Evans (1975), with the former producing conidia holoblastically in sympodial sequence and the latter enteroblastically. Samson and Evans (1975) related H. odonatae to Akanthomyces with reference to the description in Kobayasi (1941). Our study clearly shows that the anamorph of O. odonatae produces conidia holoblastically in sympodial sequence and can be accommodated in Hymenostilbe.