Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/1 Studies of coprophilous ascomycetes in Kenya – Ascobolus species from wildlife dung Mungai PG1,2,3, Njogu JG3, Chukeatirote E1,2 and Hyde KD1,2* 1Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand 2School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand 3Biodiversity Research and Monitoring Division, Kenya Wildlife Service, P.O. Box 40241 00100 Nairobi, Kenya Mungai PG, Njogu JG, Chukeatirote E, Hyde KD 2012 – Studies of coprophilous ascomycetes in Kenya – Ascobolus species from wildlife dung. Current Research in Environmental & Applied Mycology 2(1), 1-16, Doi 10.5943/cream/2/1/1 Species of coprophilous Ascobolus were examined in a study of coprophilous fungi in different habitats and wildlife dung types from National Parks in Kenya. Dung samples were collected in the field and returned to the laboratory where they were incubated in moist chamber culture. Coprophilous Ascobolus were isolated from giraffe, impala, common zebra, African elephant dung, Cape buffalo, dikdik, hippopotamus, black rhinoceros and waterbuck dung. Six species, Ascobolus amoenus, A. bistisii, A. calesco, A. immersus, A. nairobiensis and A. tsavoensis are identified and described. Ascobolus calesco, A. amoenus and A. bistisii were the most common. Two new species, Ascobolus nairobiensis and A. tsavoensis are introduced in this paper. In addition, two others, Ascobolus bistisii and A. calesco are new records in Kenya and are described and illustrated. The diversity of coprophilous Ascobolus from wildlife dung in Kenya as deduced from this study is very high. Key words – Ascobolus amoenus – A. nairobiensis – A. tsavoensis – elephant – moist chambers – national park – zebra Article Information Received 9 February 2012 Accepted 10 February 2012 Published online 26 February 2012 *Corresponding author: Paul G. Mungai – e-mail – [email protected] Introduction and incubated at room temperature. Ascobolus Fungal inventories are important prere- species that sporulated were examined under a quisites for biodiversity conservation. In this stereo microscope. This survey sought to identify study the taxonomy and biodiversity of coprophi- and classify Ascobolus Pers. found on various lous Ascobolus Pers. from wild herbivores in dung types in Kenyan wildlife and develop a Kenya, a neglected area of ecology and conserva- checklist for Ascobolus species associated with tion, was carried out. Previous studies on different dung types in wildlife. coprophilous fungi in Kenya include studies by Ascobolus belongs to the family Minoura (1969), Khan et al. (1972), Carter et al. Ascobolaceae. Ascobolus comprises 60 mostly (1982), Krug et al. (1987), Krug et al. (1989) and cosmopolitan and coprophilous species that Caretta et al. (1998). These studies show that begin to sporulate on dung within the first week coprophilous ascomycetes species richness and of incubation (Bell 2005, Kirk et al. 2008, diversity in Kenya is very high and similar to that www.indexfungorum.org, 2012). The pale- found in other tropical areas of the world yellow luteous apothecioid ascomata are superfi- (Richardson 2001). cial, gregarious, and sessile (Brummelen van Dung from wild herbivores was collected 1967, Doveri 2004, Bell 2005), but sometimes 1 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/1 with a short stalk up to 30 mm diameter. device. The criteria for sampling and site Paraphyses are filiform, cylindrical and often selection were habitat type, dung type, topogra- embedded in mucus. Asci are usually unitunicate, phy and vegetation type. Parameters such as operculate, saccate-clavate or cylindric-clavate sample number, moisture condition of dung, (Doveri 2004, Bell 2005), with rounded or dome- animal species voiding the dung, vegetation type, shaped apex, protruding above surface of location, weather details, collector’s name and hymenium when mature, amyloid in some collection date were recorded in a field datasheet. species and usually 4 or 8-spored. Asci that Over-dried dung was moistened with sterile protrude way beyond the hymenium at maturity, water taking care not to waterlog it because too contain thick-walled ascospores when young, a much water is known to inhibit fungal characteristic pore plugging mechanism of sporulation (Bell 2005). Fresh dung was taken to septum structure and similar ascus apical the laboratory and placed in moist chambers chamber and operculum (Brummelen van 1967, (moist filter paper in Petri dishes). Each dung Aas 1992, Dissing et al. 1994). Ascospores are sample was examined for signs of sporulation single-celled, subglobose to elliptical or oval, prior to incubation. The incubation was done at thick-walled, sometimes with a gelatinous room temperature under natural light to induce sheath, ornamented with pigment which is sporulation of coprophilous Ascobolus (Cain deposited externally; they can be smooth or 1934, Brummelen van 1967, Lundqvist 1972, variously roughened, 2-3-seriate in ascus, ejected Bell 2005). singly and are purple or brown (Bell 2005). These species are often saprobic and Coprophilous fungi examination coprophilous (Bell 2005). From the third day of incubation, the Globally, species of the genus Ascobolus moist chambers were examined for fungi have been recorded in Europe (Brummelen van sporulation under a stereoscopic microscope. 1967, Mireille et al. 2002, Doveri 2004), the Thereafter, the dung was examined on a regular Middle East (Abdullah et al. 1993), Australia and basis to monitor fungal sporulation and the New Zealand (Bell 1983, 2005), Asia (Liou S-C development of Ascobolus fruiting. Thorough 1977, Wang 2000a), Russia (Prokhorov 1991) examinations commenced immediately after the and the Americas (Richardson 2008). first Ascobolus fruiting body was observed and In Africa, A. albidus P. Crouan & H. continued on a regular basis thereby ensuring Crouan, A. amoenus Oudem., A. degluptus that fungal sporulation and the development of Brumm., A. hawaiiensis Brumm., A. immersus Ascobolus fruiting was well monitored (Doveri Pers. and A. stictoideus Speg. have been recorded 2004, Bell 2005). in Uganda, South Africa and Egypt (Minoura The examination of coprophilous 1969, Ebersohn et al. 1992, 1997, Abdel-Azeem Ascobolus was carried out by picking individual et al. 2005). Ascobolus americanus (Cooke & fruiting bodies with a pair of very fine forceps or Ellis) Seaver, A. immersus Pers., A. perplexans a sharp needle and transferring them directly Massee & Salmon, A. viridulus W. Phillips & onto a drop of sterile water placed on a glass Plowr. and Ascobolus sp. have been recorded in slide and covered with a cover slip. Gentle Kenya in previous studies (Minoura 1969, pressure was applied on the mount using a rubber Caretta et al. 1998). of a pencil to extrude the asci, ascospores and In this study six Ascobolus species are paraphyses for microscopic examination examined and described. (Brummelen van 1967, Bell 2005). Standard stains were employed to help examine important Materials and Methods diagnostic fungal structures. Each species was examined in detail and its sample number, dung Sample collection and incubation type, date of incubation, date of examination, Wildlife dung was collected from tentative name, notes on reaction with stains, National Parks and Reserves in a coastal forest, habit and color, photograph number and the age savannah, wet montane forest and a dry highland of dung recorded in a laboratory data sheet. forest in Kenya. The collection sites were Small pieces of dung were placed in envelopes mapped using a Global Positioning System and cryotubes for herbarium deposition. 2 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/1 Fig. 1 – Ascobolus amoenus (KWSTE003B-2009). A Ascomata on substrate. B Ascoma squashed in water. C Detail of hymenium with 8-spored mature asci (black arrow) and immature asci (red arrow). D- E Details of ectal excipulum. Note irregular shape (black arrow) and isodiametric cells (white arrow). F Paraphyses. G Dotted-granular ascospores. H Paraphyses (white arrow) and details of asci, note an ascus apex (black arrow), a short stipitate ascus (red arrow) and the greenish yellow mucus. I Mature ascospores, note the unilateral gelatinous sheath (arrow) and greenish mucus. J Open operculum (arrow). Scale bars: A-B = 500 µm, C = 200, D, H = 50 µm, E-G, J = 20 µm. 3 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/1 Dung fungi specimens were described rounded and curved above, wall bluing in using features observed under a stereoscopic and Meltzer’s reagent, operculum 16–20 µm wide, compound microscope. Staining of squash short-stipitate. Ascospores 31–34 × 16–18 µm, mounts was done to enable better observation of initially uniseriate, finally biseriate, ellipsoidal or micro-structures. Monographs, dichotomous and elongated ellipsoidal, hyaline at first, then violet, synoptic keys (Brummelen van 1969, Korf 1972, finally brownish, smooth to finely punctate or Richardson et al. 1997, Hansen et al. 1998, Bell densely granular, with no cracks in the epispo- 1983, 2005, Doveri 2004) comprising morpholo- rium, surrounded by a hyaline gelatinous enve- gical characters such as mycelium, ascomata, lope, thinner on the poles, thicker on the sides. vestiture, asci, ascospore, colour, shape, size and Material examined – KENYA, Tsavo East formation were extensively used. Comparisons National Park