385 The ectomycorrhizal status of Calostoma cinnabarinum determined using isotopic, molecular, and morphological methods Andrew W. Wilson, Erik A. Hobbie, and David S. Hibbett Abstract: Calostoma cinnabarinum Corda belongs to the suborder Sclerodermatineae (Boletales), which includes many well-known ectomycorrhizal basidiomycetes, but the genus Calostoma has been described as saprotrophic. This study com- bines isotopic, molecular, and morphological techniques to determine the mode of nutrition of C. cinnabarinum. d13C and d15N measurements were compared among co-occurring C. cinnabarinum, ectomycorrhizal fungi, saprotrophic fungi, and ectomycorrhizal plants. Isotopic profiles of C. cinnabarinum resembled those of ectomycorrhizal fungi but not those of saprotrophic fungi. For molecular analyses, ectomycorrhizal root tips were extracted from soil cores collected beneath C. cinnabarinum fruit bodies. Nuclear ribosomal internal transcribed spacer (nrITS) sequences were obtained from ectomy- corrhizal root tips using fungal-specific primers and screened against C. cinnabarinum nrITS sequences. Ectomycorrhizal root tips had nrITS sequences that matched C. cinnabarinum fruiting bodies. Root tips colonized by C. cinnabarinum were also described morphologically. Several morphological characters were shared between fruiting bodies and ectomycorrhizal root tips of C. cinnabarinum. Results of isotopic, molecular, and morphological analyses indicate that C. cinnabarinum is ectomycorrhizal. Molecular analysis and observations of plant associations suggest that C. cinnabarinum forms ectomycor- rhizae with Quercus. Key words: Calostoma, nrITS, ectomycorrhizae, saprotrophic, isotope, molecular ecology, fungal ecology. Re´sume´ : Le Calostoma cinnabarinum Corda appartient au sous-ordre des Sclerodermatinae (Bole´tales), qui inclut plu- sieurs espe`ces de basidiomyce`tes bien reconnues, mais on a de´crit le genre Calostoma comme saprophyte. Les auteurs ont de´termine´ le mode de nutrition du C. cinnabarinum a` l’aide de techniques isotopiques, mole´culaires et morphologiques. Ils ont compare´ les mesures des d13Cetd15N entre le C. cinnabarinum, des champignons ectomycorhiziens, des champignons saprophytes et des plantes ectomycorhiziennes, poussant sur un meˆme site. Les patrons isotopiques du C. cinnabarinum ressemblent a` ceux des champignons ectomycorhiziens, mais non a` ceux de champignons saprophytes. Afin de conduire des analyses mole´culaires, des apex ectomycorhiziens ont e´te´ extraits de carottes de sol re´colte´es sous des fructifications de C. cinnabarinum. En utilisant des amorces fongiques spe´cifiques on a obtenu des se´quences nucle´aire ribosomal espa- ceur transcrit interne (nrITS), a` partir des apex ectomycorhiziens, et on les a compare´es avec les se´quences nrITS du C. cinnabarinum. Ces se´quences nrITS des apex correspondent a` celles de fructifications du C. cinnabarinum. On pre´sente e´galement une description des apex racinaires colonise´s par le C. cinnabarinum. Les re´sultats des analyses isotopiques, mole´culaires et morphologiques indiquent que le C. cinnabarinum est ectomycorhizien. Les analyses mole´culaires et l’ob- servation des associations ve´ge´tales sugge`rent que le C. cinnabarinum forme des ectomycorhizes avec des Quercus. Mots-cle´s : Calostoma, nrITS, ectomycorhizes, saprophytes, isotope, e´cologie mole´culaire, e´cologie fongique. [Traduit par la Re´daction] Introduction indexfungorum.org/). Three of these species occur in North- ern and Central America, and the rest are found in Asian The genus Calostoma consists of morphologically unusual and Australian regions. Recent molecular studies have species of basidiomycetes that fruit on the ground in temper- placed this genus in the Sclerodermatineae, a suborder of ate and tropical forests composed of Fagaceae, Nothofaga- the Boletales (Hughey et al. 2000; Binder and Bresinsky ceae, Myrtaceae, and Dipterocarpaceae. Calostoma species 2002). The Boletales are mostly ectomycorrhizal with some form gastroid fruiting bodies often with brightly colored saprotrophic members, while all core Sclerodermatineae peristomes (ridged ostioles) and gelatinized tissues. There genera — Scleroderma (Godbout and Fortin 1983; Bue´eet are 26 species of Calostoma in the CABI database (www. al. 2004), Pisolithus (Moyersoen and Beever 2004), Astreus (Danielson 1984), Gyroporus (Agerer 2002) — are consid- Received 15 February 2007. Published on the NRC Research Press Web site at canjbot.nrc.ca on 14 June 2007. ered ectomycorrhizal. Hughey et al. (2000) described Calostoma cinnabarinum Corda as a litter decomposer after A.W. Wilson1 and D.S. Hibbett. Department of Biology, Clark Miller and Miller (1988), but this assumption is most likely University, Worcester, MA 01610, USA. based on previous taxonomic comparisons with the saprotro- E.A. Hobbie. Complex Systems Research Center, Morse Hall, phic Tulostoma (Burnap 1897) and with Geastrum (Massee University of New Hampshire, Durham, NH 03824, USA. 1888), which have been previously described as saprotrophic 1Corresponding author (e-mail: [email protected]). (Kreisel 1969; Miller and Miller 1988; Sunhede 1989), but Can. J. Bot. 85: 385–393 (2007) doi:10.1139/B07-026 # 2007 NRC Canada 386 Can. J. Bot. Vol. 85, 2007 more recently described as ectomycorrhizal (Agerer and Materials and methods Beenken 1998). However, Hughey et al. (2000) acknowl- edged that the classification of Calostoma as saprotrophic Sampling was probably based on previous taxonomic placement and Calostoma cinnabarinum fruiting bodies and soil cores suggested that the ecological role of this fungus should be were collected from four locations in Massachusetts, USA further investigated. (Table 1). Samples of fruiting bodies and plant foliage used Saprotrophic and ectomycorrhizal fungi often acquire car- in the isotopic analysis were collected from the Upton site. bon and nitrogen from different sources. This difference in The samples consisted of three fruiting bodies of Calostoma; nutrient acquisition is observed in studies of stable carbon six fruiting bodies of the ectomycorrhizal genera Boletinel- (13C) isotope abundances in fruiting bodies of known sapro- lus, Cortinarius, Gyroporus, Lactarius, and Scleroderma; trophic and mycorrhizal fungi, with saprotrophic fungi being and four fruiting bodies of the saprotrophic genera Armil- several parts per mille (%) enriched in 13C relative to my- laria, Lycoperdon, and Trametes. In addition, a total of five corrhizal fungi (Ho¨gberg et al. 1999; Hobbie et al. 1999, foliage samples were collected from three ectomycorrhizal 2001; Kohzu et al. 1999; Henn and Chapela 2001; Taylor et plant genera (Quercus, Carya, Pinus) for isotopic analysis. al. 2003; Trudell et al. 2004; Hobbie 2005; Hart et al. 2006). The fruiting bodies were dried and stored as vouchers. Soil In addition, because the creation of transfer compounds such cores of approximately 500–1000 mL were extracted from as amino acids by mycorrhizal fungi results in the transfer beneath fruiting bodies to about 15 cm below the surface. of 15N-depleted nitrogen to host plants and the retention of Ectomycorrhizal root tips were separated from roots using a dissecting microscope. Root tips extracted from soil cores 15 N-enriched nitrogen in mycorrhizal fungi, ectomycorrhizal were pooled based on morphology, stored in Eppendorf fungi that supply nitrogen to their host plants can be en- tubes in 1Â Tris–EDTA buffer, and refrigerated. Samples riched in 15N relative to co-occurring saprotrophic fungi for molecular and isotopic analyses were collected from Au- (Hobbie et al. 1999, 2001, 2005; Kohzu et al. 1999; Henn gust through October of 2003. and Chapela 2001; Taylor et al. 2003; Trudell et al. 2004; Hobbie 2005). The source of nitrogen in the environment Isotopic analysis and sample preparation may also affect 15N enrichment in ectomycorrhizal fungi 13 15 Isotopic analyses in this study follow the methods de- (Gebaur and Taylor 1999). Although values of C and N scribed in Hobbie et al. 1999 and 2001 and Taylor et al. content vary between sites and across species of fungi, 2003. For the analyses, samples of ectomycorrhizal fungi, Hobbie et al. (2001) and Taylor et al. (2003) used measure- saprotrophic fungi, and foliage from ectomycorrhizal plants ments of isotopic values from known ectomycorrhizal and were identified to genus. Samples were dried at saprotrophic fungi to infer nutrient acquisition in fungi of 13 50 8C. Approximately 250 mg of each sample was ground uncertain ecological role. These studies suggest that C and to a fine powder using a mortar and pestle with liquid nitro- 15N measurements could be used to infer whether Calostoma gen. At least half of the sampled fruiting body or plant ma- resembles ectomycorrhizal fungi or saprotrophic fungi in its terial was kept as vouchers. Up to two replicates per sample nutrient acquisition strategy. were analyzed for d13C and d15N at the University of Vir- Molecular methods have been used in many studies to de- ginia on a Carlo Erba elemental analyzer coupled to a Mi- termine the identity of mycorrhizal fungi colonizing the root cromass Optima mass spectrometer (Fisons/VG/Micromass, tips of plants (Gardes and Bruns 1993, 1996; Cullings et al. Manchester, UK). The internal standards for isotopic and 1996; Dahlburg et al. 1997; Chambers et al. 1998; concentration measurements were acetanilide and apple McKendrick et al. 2000; Bidartondo et al. 2002). The nu- leaves (NIST 1515). Stable isotope abundances are reported