fungal biology 115 (2011) 197e213 journal homepage: www.elsevier.com/locate/funbio From pattern to process: species and functional diversity in fungal endophytes of Abies beshanzuensis Zhi-Lin YUANa,b,*, Long-Bing RAOa, Yi-Cun CHENa, Chu-Long ZHANGb,**, You-Gui WUc aInstitute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, China bKey Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, China cFengyangshan-Baishanzu National Nature Reserve, China article info abstract Article history: The biodiversityefunctional relationship in fungal ecology was recently developed and Received 10 September 2010 debated, but has rarely been addressed in endophytes. In this study, an integrative culture Received in revised form system was designed to capture a rich fungal consortium from the conifer Abies beshanzuen- 20 November 2010 sis. Results indicate an impressive diversity of fungal lineages (a total of 84 taxa classified in Accepted 22 November 2010 Dikarya) and a relatively high proportion of hitherto unknown species (27.4 %). The laccase Available online 30 November 2010 gene was used as a functional marker due to its involvement in lignocellulose degradation. Corresponding Editor: Paola Bonfante Remarkable diversity of laccase genes was found across a wide range of taxa, with at least 35 and 19 distinct sequences in ascomycetes and basidiomycetes respectively, were revealed. Keywords: Many groups displayed variable ability to decompose needles. Furthermore, many ascomy- Antagonism cetes, including three volatile-producing Muscodor species (Xylariaceae), showed the ability to White rot fungi inhibit pathogens. Notably, most laccase-producing species showed little or no antibiosis Decomposer and vice versa. Clavicipitalean and ustilaginomycetous fungi, specifically toxic to insects, Functional diversity were inferred from taxonomic information. Intra-specific physiological variation in Pezicula Helotiales sporulosa, a second dominant species, was clearly high. We conclude that a suite of defensive Intra-specific variation characteristics in endophytes contributes to improving host fitness under various stresses Laccase genes and that a diversity of laccase genes confers an ecological advantage in competition for nutrients. Intra-specific diversity may be of great ecological significance for ecotypic adapta- tion. These findings suggest a fair degree of functional complementarity rather than redundancy among endemic symbionts of natural plant populations. ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction (Arnold 2008; Rodriguez et al. 2009). Extensive literature now exists regarding global species richness and distribution pat- Fungal endophytes constitute an important component of terns of endophytic fungi. Despite ever-increasing informa- plant-associated mycobionts. In contrast to clavicipitaceous tion on basic biology and ecology, our knowledge of endophytes in the genera Epichloe€ and Balansia that generally endophytic fungal biodiversity and its importance to ecosy- grow systemically within aboveground grass tissues (Schulze stem functioning are still limited because current approaches & Boyle 2005), non-clavicipitaceous endophytes are often as- are basically taxonomy-driven (Zak & Visser 1996). In contrast, sumed to be highly diverse in a wide range of plant lineages from a functional perspective, trait-based functional diversity * Corresponding author. Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, China. Tel.: þ86 571 63105091; Fax: þ86 571 63341304. ** Corresponding author. E-mail addresses: [email protected], [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.11.002 198 Z.-L. Yuan et al. may provide greater insights into the roles of fungi in ecolog- implications of functional traits using a polyphasic analysis ical communities (Zak et al. 1994; Schadt et al. 2003; Parrent of functional gene assay (laccase-coding genes), with quanti- et al. 2010; Saunders et al. 2010). Theoretically speaking, func- tative and qualitative in vitro experiments including antibiosis tional diversity refers to the measurement of the range of spe- bioassays, decomposition test, and evaluation of intra-specific cies traits. Miller (1995) listed many aspects of functions in physiological variation; third, to infer the ecological signifi- fungi and noted that an integration of biochemical, physiolog- cance of these based on taxonomic information. These clues ical, and molecular approaches can yield timely information will provide novel insights into the geneticefunctional diver- on the contributions of fungi to ecosystem. sity relationships in endophytic fungal communities. Assessing all functional characteristics of each fungal species (or isolate) within communities is often difficulty, whereas the Materials and methods identification of key functional traits related to different fungal groups appears feasible. For arbuscular mycorrhizal fungi Study site and sampling (AMF), a set of functional traits including fungal phosphate trans- porter genes and host productivity are often measured (Van der The study site was located in Baishanzu National Nature Heijden et al. 2004; Van der Heijden & Scheublin 2007; Gamper Reserve, Zhejiang Province, China (27440 9700-E 9700Ne119 et al. 2010). For soil saprophytes, the community level substrate 120 4700E; about 1700 m a.s.l.). Abies beshanzuensis, which grows utilization patterns using the Fungi-log study may provide a sen- in mixed forests with broadleaf trees, is endemic to China. sitive indicator of their functional diversity (Sobek & Zak 2003). Two trees were located adjacent to each other and the remain- With rapid progress in the availability of fungal genomes for ing tree was about 300 m away. Five healthy and separate comparisons, phylogenetic analysis of functional marker genes branches from each tree that included three age classes of of interest might also prove to be an ideal alternative (Torsvik needles were collected, carefully packed into a cooler box, & Ovreas 2002; Raes & Bork 2008). For example, laccase genes and transported to the laboratory within 48 h. in litter fungi and nitrate reductase-encoding genes in ectomy- corrhizal fungi have been analyzed (Luis et al. 2004; Nygren et al. 2008). The glycoside hydrolase family 32 genes (GH32), an Methods for endophytic fungi isolation and incubation important functional trait for the utilization of plant-derived sucrose, have been determined in endophytic fungi (Parrent The composition and preparation of seven different media et al. 2009). Recently, diversity of polyketide synthase genes, an used for isolation were presented in Supplementary Table 1. important enzyme participating in natural products biosynthe- All media were for 1 l. After cooling, streptomycin sulfate À1 À1 sis, was also investigated in endophytes (Lin et al. 2010). (50 mg L ) and tetracycline hydrochloride (20 mg L ) were Fungal endophytes have been suggested to provide a set of added to media for suppressing bacterial growth. Healthy nee- beneficial effects to the host (Yuan et al. 2010). A hypothetical dles and twigs were thoroughly rinsed with tap water for model of endophyte-mediated defensive mutualism was recently removing dust particles on leaf surfaces. The materials were proposed (White & Torresa 2010), indicating that the production surface-sterilized in ethanol (75 %, v/v) for 1 min and then im- of antimicrobial metabolites and free radical-scavenging sub- mersed in a 2.0 % aqueous solution of sodium hypochlorite for stances in fungi enhances host’s tolerance to biotic and abiotic 10 min and finally rinsed in sterile distilled water with three stresses. Nevertheless, growing evidence indicates that some times. Needles and twigs were cut into 0.5 cm length for incu- endophytes may switch their lifestyle from mutualism to sapro- bation. To prevent the cross-contamination of segments by phytism or parasitism and serve as corresponding decomposers fast-growing or sporulating fungi and save cost and space, 24 or potential pathogens (Carroll 1988; Muller€ et al. 2001; Koide multiple-well microplates (Corning Costar) were used. Each et al. 2005; Promputtha et al. 2007; Osono & Hirose 2009). Therefore, well contained 3 mL media. 360 needle segments and 120 one can reasonably assume the occurrence and prevalence of lig- twig segments (same number of segments from each tree) ninolytic enzymes in endophytes, which are vital for lignocellu- were placed into each media. Totally, 3360 tissue segments lose decomposition (Urairuj et al. 2003; Jordaan et al. 2006; Oses were incubated in darkness at 20 C. After 1 week, the emerging et al. 2006; Wang et al. 2006). Thus, the flexible life strategy of en- hyphae from segments were cut and transferred into Petri dish dophytes makes them an ideal model to study their functional containing potato dextrose agar (PDA) medium for purification. diversity. To our knowledge, however, the diverse functional traits of endophytes in the context of an ecological community Identification of recovered isolates: combination of traditional have received much less attention. method and multi-locus molecular generic determination Abies beshanzuensis (Pinaceae) is a critically endangered co- nifer found only in the subtropical forests of eastern China, First, all isolates were