Proc. Natl. Acad. Sci. USA Vol. 96, pp. 8534–8539, July 1999 Ecology Natural 13C abundance reveals trophic status of fungi and host-origin of carbon in mycorrhizal fungi in mixed forests PETER HO¨GBERG*†,AGNETA H. PLAMBOECK*, ANDREW F. S. TAYLOR‡, AND PETRA M. A. FRANSSON‡ *Section of Soil Science, Department of Forest Ecology, SLU (Swedish University of Agricultural Sciences), S-901 83 Umeå, Sweden; and ‡Department of Forest Mycology and Pathology, SLU, S-750 07 Uppsala, Sweden Communicated by Harold Alfred Mooney, Stanford University, Stanford, CA, May 24, 1999 (received for review March 4, 1999) ABSTRACT Fungi play crucial roles in the biogeochem- could differentiate between saprophytic and mycorrhizal istry of terrestrial ecosystems, most notably as saprophytes fungi. decomposing organic matter and as mycorrhizal fungi en- We also hypothesized (ii) that, because overstorey trees hancing plant nutrient uptake. However, a recurrent problem generally have a higher natural abundance of 13C than under- in fungal ecology is to establish the trophic status of species storey trees (13), their associated ectomycorrhizal fungi should in the field. Our interpretations and conclusions are too often display similar differences. The higher 13C abundance of based on extrapolations from laboratory microcosm experi- overstorey trees is the result of less pronounced discrimination 13 ments or on anecdotal field evidence. Here, we used natural against CinCO2 during photosynthesis because of higher variations in stable carbon isotope ratios (␦13C) as an ap- illumination (and, therefore, higher rates of photosynthesis) proach to distinguish between fungal decomposers and sym- and also because of more exposure to drought stress in biotic mycorrhizal fungal species in the rich sporocarp flora overstorey trees (13–15). Short plants in a closed forest may (our sample contains 135 species) of temperate forests. We obtain particularly low 13C abundance through assimilation of 13 also demonstrated that host-specific mycorrhizal fungi that C-depleted CO2 derived directly from soil respiration (13). receive C from overstorey or understorey tree species differ in We also hypothesized (iii) that most of the mycorrhizal fungi their ␦13C. The many promiscuous mycorrhizal fungi, asso- ought to receive their C from overstorey rather than from ciated with and connecting several tree hosts, were calculated understorey trees because rates of photosynthesis are higher in to receive 57–100% of their C from overstorey trees. Thus, overstorey trees. To test the three hypotheses (i–iii), we visited overstorey trees also support, partly or wholly, the nutrient- two two-storied, successional, temperate forests in Sweden and absorbing mycelia of their alleged competitors, the understo- sampled trees and a large number of fungi to analyze their 13C rey trees. abundance. In terrestrial ecosystems, fungi are integral components of METHODS most biogeochemical cycles. Decomposer fungi degrade or- ganic macromolecules whereas mycorrhizal fungi, symbionts The study was conducted at Åheden in the Svartberget Re- of plant roots, actively forage for plant nutrients (1, 2). In the search Forest, 60 km northwest of Umeå, northern Sweden complex settings of forest ecosystems, even in those with few (64°14ЈN, 19°46ЈE, 175 m above sea level) and at Stadsskogen, plant species, there is often a diverse flora of sporocarps of an urban forest in Uppsala, central Sweden (59°52ЈN, 17°13ЈE, fungal decomposers and mycorrhizal fungi (3–5). However, the 35 m above sea level). Both forests had 150-year-old Scots pine trophic status of many fungal species remains unclear: that is, (Pinus sylvestris L.) in the overstorey, but with Norway spruce whether they obtain their C by decomposing dead organic (Picea abies [L.] Karst.) approaching the role of codominant. material or from living plants by forming mycorrhizal root At Åheden, the dominant Scots pine were 25 m tall, and birch symbioses (6). The small size and cryptic growth form of most (Betula pendula Roth) was the only understorey species. At fungal mycelia effectively restricts in situ observations. Fur- Stadsskogen, the dominant Scots pine sometimes reached Ͼ30 thermore, the connection between mycorrhizal fungi and their m, and understorey deciduous tree species included aspen host trees cannot be easily established because many fungi are (Populus tremula L.), birch (B. pendula), willow (Salix caprea promiscuous and can receive C from several species of trees (2, L.), and alder (Alnus incana [L.] Moench). 7, 8). Such fungi form hyphal links transferring C and nutrients At each site, leaves and current needles were sampled (Ͼ10 between plants and can thus influence plant competition (2, g dry weight͞sample) from branches on the south side, close 7–10). Recently, it was suggested, after feeding one seedling to the top of the tree, from up to 10 specimens per tree species. 14 13 with CO2 and one with CO2, that there was a net transfer Between one and eight fruit bodies from each fungal species of C from one seedling to another via mycorrhizal connections found at each site were sampled (Table 1). Sampling was (8). However, this type of tracer approach is highly elaborate conducted on an area of 1 hectare at Åheden in August 1997 and cannot easily be used to study a greater complexity: that and at 5 plots with an area of 0.25 hectare each at Stadsskogen is, flows of C to many fungal species simultaneously. in September 1997. Specificity between tree hosts and fungal Fungal decomposers break down complex C sources like symbionts was inferred from the literature relevant to the cellulose, hemicellulose, and lignin whereas mycorrhizal fungi region (16, 17). Because associations between fungi and po- receive simpler carbohydrates directly via the plant phloem (1, tential hosts are based primarily on repeated observations of 2). These C sources may differ significantly in their natural fruit bodies and not on establishing direct contacts between abundance of the heavy stable carbon isotope 13C (11, 12). the two symbionts, the question of specificity must be treated Accordingly, we speculated (i) that analysis of 13C abundance with some caution. In this study, fungal species were only regarded as specific to a single host genus when this was clearly The publication costs of this article were defrayed in part by page charge indicated in the literature. payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: Peter. PNAS is available online at www.pnas.org. [email protected]. 8534 Downloaded by guest on September 25, 2021 Ecology: Ho¨gberg et al. Proc. Natl. Acad. Sci. USA 96 (1999) 8535 Table 1. Natural 13C abundance (␦13C) of fungal fruit bodies sampled in mixed temperate forests at two sites, Åheden and Stadsskogen, in Sweden Host n ␦13C value SD ÅÅheden site Mycorrhizal species Boletales Boletaceae Leccinum molle (Bon) Bon Betula 1 Ϫ27.6 Leccinum scabrum (Bull. Ex Fr.) S.F. Gray Betula 4 Ϫ27.5 0.4 Leccinum variicolor Watl. Betula 4 Ϫ27.6 0.8 Leccinum versipelle (Fr.) Snell Betula 4 Ϫ27.5 0.6 Suillus variegatus (Swartz ex Fr.) O. Kuntze Pinus 6 Ϫ25.0 0.4 Gomphidiaceae Chroogomphus rutilus (Schff. ex Fr.) O.K. Miller Pinus 4 Ϫ26.0 0.6 Tricholomataceae Tricholoma flavovirens (Pers. ex Fr.) Promiscuous 4 Ϫ25.4 0.8 Tricholoma virgatum (Fr.:Fr.) Kumm. Promiscuous 1 Ϫ25.2 Laccaria bicolor (Maire) Orton Promiscuous 2 Ϫ25.3 0.6 Amanitaceae Amanita muscaria (L.) Hook Promiscuous 5 Ϫ26.6 1.0 Cortinariaceae Cortinarius armeniacus (Schaeff.:Fr.) Fr. Conifers 5 Ϫ24.5 0.6 Cortinarius armillatus (Fr.) Fr. Betula 5 Ϫ26.9 1.3 Cortinarius bataillei (Moser) Hiøland Conifers 1 Ϫ25.3 Cortinarius biformis Fr. Conifers 4 Ϫ25.2 0.6 Cortinarius camphoratus (Fr.:Fr.) Fr. Conifers 1 Ϫ25.6 Cortinarius evernuis (Fr.:Fr.) Fr. Picea 1 Ϫ24.8 Cortinarius hemitrichus (Pers.:Fr.) Fr. Betula 1 Ϫ27.8 Cortinarius laniger Fr. Picea 6 Ϫ26.4 1.7 Cortinarius malachius Fr. Conifers 2 Ϫ24.8 0.2 Cortinarius pholideus (Fr.:Fr.) Fr. Betula 6 Ϫ27.6 1.0 Cortinarius scaurus (Fr.:Fr.) Fr. Promiscuous 1 Ϫ25.2 Cortinarius semisanguineus (Fr.) Gill. Conifers 3 Ϫ24.9 0.7 Cortinarius strobilaceus Mos. Conifers 1 Ϫ27.0 Cortinarius traganus Fr. (Pinus) 2 Ϫ25.2 0.5 Rozites caperata (Pers. ex Fr.) Karst. Promiscuous 4 Ϫ25.8 0.4 Russulales Russulaceae Lactarius rufus (Scop.) Fr. Promiscuous 6 Ϫ24.9 0.7 Russula paludosa Britz. Conifers 1 Ϫ25.0 Aphyllophorales Thelphoraceae Phelloden tomentosa (L.:Fr.) Baker Promiscuous 1 Ϫ24.1 Saprophytic species Boletales Boletaceae Chalciporus piperatus (Bull. ex Fr.) Bat. na 1 Ϫ22.3 Tricholomataceae Armillaria borealis Marx. & K. Korh. na 2 Ϫ23.8 0.1 Strophariaceae Stropharia hornemanii (Fr.:Fr.) Lund. na 1 Ϫ23.1 Cortinariaceae Gymnopilus penetrans (Fr.) Murr. 1 Ϫ22.4 Aphyllophorales Polyporaceae Trametes hirsuta (Wulfen:Fr.) Pil. na 1 Ϫ24.0 Stadsskogen site Mycorrhizal species Boletales Boletaceae Boletus edulis Bull. ex Fr. Promiscuous 5 Ϫ24.6 0.5 Boletus pinophilus Pil. & Dermek Pinus 1 Ϫ24.7 Leccinum aurantiacum (Bull. ex St. Am.) S.F. Gray Populus 3 Ϫ27.3 0.9 Leccinum holopus (Rostk.) Watl. Betula 1 Ϫ29.2 Leccinum scabrum (Bull. ex Fr.) S.F. Gray Betula 6 Ϫ26.4 1.9 Leccinum variicolor Watl. Betula 1 Ϫ26.1 Leccinum versipelle (Fr.) Snell Betula 2 Ϫ27.1 1.1 Leccinum vulpinum Watl. Pinus 5 Ϫ25.0 0.7 (Table continues on the next page.) Downloaded by guest on September 25, 2021 8536 Ecology: Ho¨gberg et al. Proc. Natl. Acad. Sci. USA 96 (1999) Table 1. (Continued) Host n ␦13C value SD Suillus bovinus (L.) Kuntze Pinus 6 Ϫ25.2 0.5 Suillus luteus (L. ex Fr.) S.F. Gray Pinus 6 Ϫ24.6 0.4 Suillus variegatus (Swartz ex Fr.) O.