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Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae)
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 33 (2004) 251–258 www.elsevier.com/locate/ympev
Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae) Kim A. Kristiansena,*, John V. Freudensteinb, Finn N. Rasmussena, Hanne N. Rasmussenc b
a Department of Evolutionary Biology, Biological Institute, University of Copenhagen, Gothersgade 140, DK-1123 Copenhagen K, Denmark Herbarium, Department of Evolution, Ecology and Organismal Biology, Ohio State University, 1315 Kinnear Road, Columbus, OH 43212-1157, USA c Danish Forest and Landscape Research Institute, Hørsholm Kongevej 11, DK-2970 Hørsholm, Denmark
Received 8 March 2002; received in revised form 24 February 2004 Available online 30 July 2004
Abstract We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant. 2004 Elsevier Inc. All rights reserved. Keywords: Basidiomycota; Cladistics; Mitochondrial ribosomal LsDNA; Molecular identification; Orchidaceae; Mycorrhizal fungi
1. Introduction All orchids studied thus far have mycorrhizal associations for at least part of their life (Rasmussen, 1995). The orchid mycorrhiza has been recognized as a distinct type (Smith and Read, 1997), with the mycobiont forming pelotons (hyphal coils) in the cortical tissue of protocorms, roots, tubers, and rhizomes (Rasmussen, 1995). These pelotons are at some stage digested by the orchid. Most orchid mycobionts isolated from photosynthetic orchids have been identified as Rhizoctonia fungi, which are anamorphic states of heterobasidiomycetes (Currah *
Corresponding author. Fax: +45-3532-2154. E-mail address: [email protected] (K.A. Kristiansen).
0378-5955/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.05.015
et al., 1997; Roberts, 1999; Warcup, 1981; Table 1). We follow the classification by Roberts (1999) for heterobasidiomycetes acting as orchid endophytes. The traditional way to identify orchid mycobionts has been to isolate and make axenic cultures of fungi recovered from infected tissue for morphological comparison with type cultures kept in culture collections (e.g., Centraalbureau voor Schimmelcultures (CBS) or University of Alberta Microfungus Collection & Herbarium (UAMH)). These methods usually involve surface sterilization of the orchid root and dissection of the mycobiont from the infected tissue, followed by a series of rinsings in sterile water before transfer to suitable growth media. This is followed by subculturing of growing hyphae onto new media, thereby obtaining a pure
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Table 1 Heterobasidiomycetes belonging to the complex of Rhizoctonia-like fungi (Moore, 1987; Roberts, 1999) Order
Teleomorphic genera
Anamorphic genera
Ceratobasidiales
Ceratobasidium Thanatephorus Thanatephorus Waitea Waitea
Ceratorhiza Rhizoctonia Moniliopsis Rhizoctonia Moniliopsis
Exidiales
Ceratosebacina (Sebacina) Endoperplexa (Sebacina) Heteroacanthella (Sebacina) Oliveonia (Sebacina) Serendipita (Sebacina)
Unnamed Opadorhiza Acanthellorhiza Oliveorhiza Unnamed
Tulasnellales
Tulasnella
Epulorhiza
culture (for more detailed descriptions of methods see Clements et al., 1986; Rasmussen et al., 1990; Zelmer et al., 1996). Unfortunately, not all isolated fungi grow willingly, and cessation of growth after some time often makes the culture useless. Recently, molecular methods have been employed for direct identification of endophytes in roots using fungalspecific primers (Bidartondo and Bruns, 2001; Bidartondo et al., 2000; Bruns et al., 1998; Cullings et al., 1996; Gardes and Bruns, 1993; Kretzer et al., 2000). Such methods have been used to characterize mycobionts of Orchidaceae, eliminating the time-consuming culture step (McKendrick et al., 2000; Taylor and Bruns, 1997, 1999). Most previous workers have extracted total DNA from sections of fungal-infected plant tissue and then sequenced fungal loci and/or performed restriction fragment length polymorphism (RFLP) analysis. The nuclear ribosomal internal transcribed spacer (ITS) and the mitochondrial large subunit ribosomal RNA (mt-LsDNA) are the loci most often examined. Many orchids harbor a number of fungi in their tissues, but not all are necessarily involved in the parasitic relationship indicated by peloton formation (Bayman et al., 1997; Warcup, 1981). Kristiansen et al. (2001b) used dissection and sequencing of single pelotons from the root cortex of Dactylorhiza majalis to focus on fungi involved in this particular interaction, and found that even single pelotons could contain more than one mycobiont. A database of sequences that represent the diversity of the fungal group is necessary if molecular identification of sterile hyphae, such as those found in orchid mycorrhizae, is to become possible in practice. For orchids the relevant fungal group is Basidiomycota and numerous sequences have been collected for various loci for phylogenetic studies. Most recent phylogenetic studies of basidiomycetes have focused on the homobasidiomycetes, using either the nuclear or mitochondrial rDNA subunits as the object for sequencing (Binder and Hibbett, 2002; Bruns et al., 1998; Hibbett et al.,
1997, 2000; Jarosch, 2001; Moncalvo et al., 2000, 2002; Tehler et al., 2000), but a significant number of heterobasidiomycete sequences are also available. Previous studies of orchid mycobionts have focused on the larger subfamilies of Orchidaceae–Epidendroideae, Orchidoideae and Cypripedioideae (following the classification of Chase et al., 2003). Neuwiedia is one of two small genera of Apostasioideae; this subfamily is significant because it is sister to the remainder of the Orchidaceae (Cameron et al., 1999; Freudenstein and Rasmussen, 1999). The orchid endophyte relationship is believed to be a synapomorphy for Orchidaceae, but until details are known of this relationship for apostasioids, this conclusion must be tentative. Although Kristiansen et al. (2001a) showed that Neuwiedia has typical orchid protocorms that contain peloton-forming fungi, nothing is yet known of the identity of these fungi. The aim of this study was to apply the peloton identification method of Kristiansen et al. (2001b) to the endophytes of Neuwiedia to determine if they fall into the same group of heterobasidiomycete families known to be associated with other photosynthetic orchids.
2. Materials and methods Neuwiedia veratrifolia was collected during the summer of 1999 in Sabah, Borneo, East-Malaysia (Table 2). Roots, tubers, and protocorms were rinsed in water and dried in silica gel for later DNA analysis. Pelotons from N. veratrifolia were isolated (Rasmussen et al., 1990) and cultured on potato-dextrose agar for both sequencing and morphological identification. Fungi known to be orchid endophytes were obtained from culture collections (Table 3). Pelotons were dissected from the cortex of the roots or tubers following Kristiansen et al. (2001b) with some modifications: the pelotons were rinsed twice in double distilled water, recovered in 1 ll water and transferred to 0.5 ml Eppendorf-tubes containing 42.4 ll ddH2O and 5 ll 10· PCR buffer (see below). The mix was heated to 95 C for 10 min in a heat block to lyse the hyphae. DNA from fungal reference cultures was extracted using the DNeasy Plant Mini Kit (Qiagen) following the manufacturerÕs instructions. The 50 ll PCR mix used for each peloton contained 5 ll 10· PCR buffer [100 mM Tris–HCl (pH 8.8), 25 mM MgCl2, and 250 mM KCl], fungal DNA (in 1 ll H2O), 0.2 mmol each primer, 0.2 mM equimolar dNTP mix, and 1 U Taq polymerase. The forward primer MLIN3 (Kristiansen et al., 2001b) and the reverse primer ML6 (White et al., 1990) were used to amplify ca. 500 bp of the mt-LrRNA gene. The PCR parameters were as described by Gardes and Bruns (1993). The products were run on a 1.4% agarose gel to separate the bands; in cases where several bands were observed,
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Table 2 Material of Apostasia Bl and Neuwiedia Bl Taxon
Collection site
Habitat
Collection number, material, and voucher
Neuwiedia veratrifolia
Near Sin Tuong Tuong, c. 6–8 km W of Tambunan
Ranau road, c. 65 km E of Kota Kinabalu. Steep slope facing SE, dense secondary scrubby forest. One adult plant and a few plantlets were seen
C-668. 150899 Fixed and silicagel dried leaves and root, fixed protocorm from seedling, C. No voucher taken since only few plants were at location. Photographs of infructescence of adult plant, 26 Feb 1997 at Z (A. Kocyan Photo # 11215/ 11217, copies at C). Isolates K11A, K11B, and K11C taken from protocorms and stored at Danish Forest and Landscape Research Institute
Neuwiedia veratrifolia
Tenom Orchid Centre, Agricultural Park Sabah, c. 145 km S of Kota Kinabalu
Spontaneous seedling among cultivated specimens
C-669. 100899 ‘‘Motherplant’’ leaf, seedling leaf, and root dried. Seedling roots and protocorm FAA at C. Live culture at Tenom Orchid Centre. Pressed shoot from this culture at Z (AK971129/ 1/03). Isolates K7-1A and K7-1B, K7-4, K10-1, and K10-2, taken from protocorms and stored at Danish Forest and Landscape Research Institute
Neuwiedia veratrifolia
Poring Orchid Center, Sabah Parks. Poring Hot Springs
Spontaneous seedling among cultivated specimens
Isolate K14. 170899. Isolate taken from protocorm. Due to Sabah Parks rules no voucher was taken. Isolate K14 taken from protocorms and stored at Danish Forest and Landscape Research Institute
Herbarium abbreviation according to Holmgren et al. (1990).
Table 3 Cultivated orchid endophytes used in this study Fungal taxon
Source/accession
Forward primer
Orchid source and reference to fungal description
? ? ? ? ? ? ? ? ? Thanatephorus obscurus (Ceratobasidium obscurum)
K7.1A K7.1B K7.4 K10.1 K10.2 K11.1A K11.1B K11.1C K14 UAMH 5443
MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3
Epulorhiza repens (Rhizoctonia repens)
CBS 326,47
MLIN3
C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-668 Neuwiedia veratrifolia C-668 Neuwiedia veratrifolia C-668 Neuwiedia veratrifolia Neuwiedia veratrifolia (see Table 2) Orchis rotundifolia. Mycorrhizae septum structure of orchid endophytes Currah and Sherburne (1992) Orchis morio Bernard Saksena and Vaartaja (1961)
CBS is Centraalbureau voor Schimmelcultures Baarn and Delft, The Netherlands. UAMH is University of Alberta Microfungus Collection and Herbarium, Canada.
individual bands were cut out and purified from the gel using the Qiaquick Gel Extraction Kit (Qiagen) following the manufacturerÕs instructions. In cases where a clean sequence was not readily obtained, PCR products were cloned using the PCR-Script Amp Cloning Kit (Stratagene, CA). All PCR products were purified prior to sequencing using Wizard PCR Preps (Promega, WI) following the
manufacturerÕs instructions. The DNA was eluted in 20 ll sterile water and cycle sequenced using the MLIN3/ML6 primers and the ABI Prism BigDye Terminator Ready Reaction kit. Sequences were run on a PE Biosystems 377 sequencer at the DNA Sequencing Center, Brigham Young University, Provo, Utah. Sequences have been deposited in GenBank (for accession numbers see Table 4).
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Table 4 Taxa included in the DNA matrix for phylogenetic analysis Fungal taxon (reference)
Source/ Accession No.
K-7 (this study) K-10-1 (this study) K-10-2 (this study) K-11 (this study) C668 (this study) C669 (this study) Thanatephorus obscurus (this study) Epulorhiza repens (this study) Ceratorhiza goodyera-repentis Kristiansen et al. (2001b) DM1 Kristiansen et al. (2001b) DM2 Kristiansen et al. (2001b) DM3 Kristiansen et al. (2001b) Epulorhiza anaticula Kristiansen et al. (2001b) Moniliopsis anomala Kristiansen et al. (2001b) Sistotrema sp. Kristiansen et al. (2001b) Tulasnella deliquescens (D47-7A) Kristiansen et al. (2001b) Tulasnella deliquescens (D47-7B) Kristiansen et al. (2001b) Tulasnella irregularis Kristiansen et al. (2001b) Tulasnella pruinosa Kristiansen et al. (2001b) Tulasnella violea Kristiansen et al. (2001b) Albatrellus flettii Bruns et al. (1998) Albatrellus peckianus Bruns et al. (1998) Albatrellus skamanius Bruns et al. (1998) Albatrellus syringae Bruns et al. (1998) Amanita calyptrata Bruns et al. (1998) Amanita phalloides Bruns et al. (1998) Armillaria albolanaripes Bruns et al. (1998) Boletopsis subsquamosa Horton (2000) Boletus satanas Bruns et al. (1998) Cantharellus-like sp1 MR14 Bruns et al. (1998) Cantharellus-like sp1 MR15 Bruns et al. (1998) Cantharellus tubaeformis Bruns et al. (1998) Clavulina cristata Horton (2000) Coniophora arida Bruns et al. (1998) Coniophora puteana Bruns et al. (1998) Craterellus tubaeformis Horton (2000) Gyroporus cyanescens Bruns et al. (1998) Hebeloma crustuliniforme Bruns et al. (1998) Hygropgorus pudorius Bruns et al. (1998) Hygrophorus brunnescens Horton (2000) Hygrophorus purpurescens Horton (2000) Hygrophorus speciosus Bruns et al. (1998) Inocybe sororia Bruns et al. (1998) Inocybe sp. (OD6264) Horton (2000) Inocybe sp. (OD6271) Horton (2000) Inocybe sp. trh415 Horton (2000) Laccaria amethysteo-occidentalis Bruns et al. (1998) Laccaria laccata Bruns et al. (1998) Laccaria trh416 Horton (2000) Russula xerampelina @Monotropa uniflora Horton (2000) Paragyrodon sphraerosporus Bruns et al. (1998) Paxillus involutus Bruns et al. (1998) Paxillus statuum Bruns et al. (1998) Phaeogyroporus portentosus Bruns et al. (1998) Phylloporus rhodotaxus Bruns et al. (1998) Pluteus cervinus Horton (2000) Pseudotomentella tristis Bruns et al. (1998)
AF490610 AF490611 AF490612 AF490613 AF490608 AF490609 AF490614 AF490615 AF345556 AF345854 AF345855 AF345856 AF345559 AF345557 AF345558 AF345852 AF345853 AF345560 AF345561 AF345562 S:1333189 S:1333202 S:1333243 S:1333261 S:1333278 S:1333373 S:1333375 AY323508 S:1333387 L46388 L46389 S:1333396 AY323510 S:1333400 S:1333401 AY323513 S:1333416 S:1333418 S:1333428 AY323512 AY323511 S:1333488 S:1333490 AY323515 AY323516 AY323518 L46394 S:1333492 AY323517 AY323505 S:1333503 S:1333505 S:1333506 S:1333507 S:1333508 AY323509 S:1333512
Table 4 (continued) Fungal taxon (reference)
Source/ Accession No.
Russula brevipes Bruns et al. (1998) Sebacina sp. Bruns et al. (1998) Serpula incrassata Bruns et al. (1998) Suillus tomentosus Bruns et al. (1998) Tomentella atrorubra Bruns et al. (1998) Tricholoma flavovirens Bruns et al. (1998) Tricholoma moseri Horton (2000) Tricholoma portentosum Horton (2000) Tricholoma saponaceum Horton (2000) Tulasnella irregularis Bruns et al. (1998) Waitea circinata Bruns et al. (1998)
L46395 S:1333525 S:1333528 S:1333534 S:1333538 S:1333542 AY323514 AY323520 AY323519 S:1333546 S:1333645
Outgroup Schizosaccharomyces pombe Lang et al. (1987)
NC_001326
All sequences were submitted to a Blast search in GenBank as a first step to determine whether they contained mitochondrial ribosomal LsDNA. Sequences were aligned using ClustalX (Thompson et al., 1997) with default parameters: opening gap = 15, gap extension = 6.6, transition weight = 0.50, and delay divergent = 30%. At least two taxa from each of the fungal groups recognized by Bruns et al. (1998) were included in the alignment, except for group 6 (from which only Suillus tomentosus was included) and group 16 (which is not represented in this analysis). Fourteen sequences from the updated mitochondrial ribosomal LsDNA fungal database (Horton, 2000) were also included in the data matrix, which comprised a total of 74 taxa (Table 4). Schizosaccharomyces pombe (Ascomycota) was used as the outgroup. The matrix was analyzed with PAUP* 4.0 (Swofford, 2000) using equally weighted parsimony. Uninformative characters were excluded prior to searching, and all characters were treated as unordered. Tree search consisted of 500 random addition replicates saving up to 5 trees each and using TBR branch swapping. Zero length branches were collapsed. Jackknife branch support values (Farris et al., 1996) were obtained using PAUP* with 37% deletion, JAC emulation, 10,000 jackknife replicates with one random addition replicate each, and saving up to two trees per replicate.
3. Results The phylogenetic analysis resulted in 197 most parsimonious trees (l = 407, CI= 0.48, RI = 0.89). The heterobasidiomycetes are distributed in three clades (Fig. 1, clades A–C), forming a polytomy with the remaining homobasidiomycetes. Clade A (branch support = 0.99) comprises Sistotrema sp. (Aphyllophorales, Homobasidiomycota; Hibbett and Thorn, 2001), Sebacina sp. (Exidiales, Heterobasidiomycota) and Waitea circinata
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Fig. 1. Strict consensus of 197 equally most parsimonious trees with a length of 407 steps. Terminals marked with @ are unknown fungi isolated from the plants named. Jackknife support values are shown above branches.
(Ceratobasidiales, Heterobasidiomycota; Roberts, 1999). Clade B (branch support = 0.99) includes the fungal cultures Thanatephorus obscurus (Ceratobasidiales, Heterobasidiomycota; Roberts, 1999), three representa-
tives of K11 and one representative of K14 (all obtained from N. veratrifolia ; Table 2). Clade C (branch support = 0.99) is subdivided into clades C1–C4 (Fig. 1). Clades C1–C3 consist of pelotons from D. majalis
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(Kristiansen et al., 2001b), Tulasnella teleomorphs, the Tulasnella anamorph Epulorhiza anaticula (Tulasnellales), Ceratorhiza goodyera-repentis (= Ceratobasidium, Ceratobasidiales) and Moniliopsis anomala (= Thanathephorus anomala?, Ceratobasidiales; Roberts, 1999). Subclade C4 comprises cultured fungi or single pelotons from N. veratrifolia together with Tulasnella deliquescens teleomorphs and an anamorph (Epulorhiza).
4. Discussion The cladistic analysis presented here is in accordance with the groupings reported in the neighbour-joining tree by Bruns et al. (1998; Fig. 1). The tree does not resolve relationships among many of the smaller clades; although a high degree of resolution is always desirable in a cladistic analysis, the purpose of this study was not to study the detailed relationships of basidiomycetes but rather to identify fungi associated with Neuwiedia using phylogenetic methods. The analysis did place the Neuwiedia endophyte sequences decisively with known sequences, given the strong branch support for these relationships. Neuwiedia mycobionts fall into two strongly supported clades (B and C), where all terminals except for Tulasnella pruinosa and Tulasnella violea were amplified either directly from pelotons or from fungal isolates from N. veratrifolia or higher orchids (cf. Tables 1 and 3 in Kristiansen et al., 2001b). Although nothing is yet known about the fungal associates of Apostasia, it is clear that Apostasioideae as represented by Neuwiedia have the same mycorrhizal associates that are present in other orchids; we predict that examination of Apostasia will also reveal Rhizoctonia-type mycobionts. Hence, it appears that presence of this type of fungus, and the orchid mycorrhizal type in general, is a synapomorphy for the entire Orchidaceae. Most of the mycobionts fall in subclade C4, with representatives of Tulasnellales also recovered from orchid roots. The Neuwiedia mycobionts C668 and C669 were amplified from pelotons from seedling roots, while K7-1A, K7-1B, K7-4, K10-1, and K10-2 are from cultured material, derived from pelotons isolated from fresh seedlings (Tables 2 and 3). Sister to these is a clade (C3) comprising members of Tulasnellales, but also representatives of Ceratobasidiales (Moniliopsis and Ceratorhiza). In subclade C3 the Ceratobasidiales members come out with Tulasnella species (branch support = 0.66), suggesting that Tulasnellales may be paraphyletic. The Neuwiedia mycobionts found in C4 clearly represent T. deliquescens or a very closely related species. The remaining Neuwiedia mycobionts, also derived from pelotons isolated from fresh seedlings, (K11-1A to K11-1C and K14) are placed in clade B together with T. obscurus (Ceratobasidiales), suggesting
that these should be referred to Ceratobasidiales. Furthermore, K11 and K14 show distinct morphological differences from K7 and K10 (unpublished data), further emphasizing the distinctness of these isolates. Isolates K7 and K10 are from protocorms/seedlings collected in the Tenom Orchid Center, while K11 isolates are from a wild growing plantlet and K14 isolates are from protocorms/seedlings found at the Poring Orchid Center. The K11 mycobionts were cultured from the same seedling as the C668 peloton amplification, clearly indicating the presence of more than one mycobiont in this individual. This finding supports previous reports of multiple active mycorrhizal fungi in the same plant (Kristiansen et al., 2001b; Warcup, 1981) and might also indicate a pattern of varying host utilization with respect to area. The Tenom seedlings were infected only with T. deliquescens-like fungi, C669, K7, and K10. The Sin Tuong Tuong seedling was infected with both T. deliquescens-like fungi and T. obscurus-like fungi, C668 and K11, whereas the Poring population was infected with only T. obscurus-like fungi, K14. This may indicate a change in mycorrhizal partner from Tenom in the south to Poring in the north, but could also be due to chance amplification and culturing success. Further study of endophytes within species and within individuals is needed to more clearly establish patterns of interaction at those levels. The taxa from HortonÕs (2000) update of the mtLrRNA sequence database are behaving as one would have predicted, though some are worth mentioning. The Russula xerampelina isolate from the leafless ericad Monotropa uniflora in clade 8 was sister to Russula brevipes, perhaps not surprising in that the latter species was also isolated from M. uniflora by Bidartondo and Bruns (2001). This analysis confirms previous indications of paraphyly for Albatrellus (Fig. 1, clades 7 and 9; Bruns et al., 1998; Hibbett et al., 2000). The paraphyly of Paxillus indicated by Bruns et al. (1998) and further supported by the phylogenetic work on Boletales by Jarosch (2001; Fig. 1 clades 2 and 3) is also supported by this analysis. Ceratobasidiales appear as polyphyletic on the tree, falling in clades A, B, and C (Fig. 1). This result is not consistent with results presented by Roberts (1999), which indicate Ceratobasidiales to be monophyletic and sister to Exidiales (paraphyletic) and Tulasnellales (monophyletic). RobertsÕ cladistic analyses were based on morphological and molecular data, and included 23 species belonging to Ceratobasidiales, but only two species of Tulasnella. Furthermore, the homobasidiomycete Sistotrema poses an interesting problem by falling among the heterobasidiomycetes Sebacina and Waitea (A), but Sistotrema is classed as belonging to Aphyllophorales (Hibbett and Thorn, 2001) which is a troublesome ‘‘lower’’ homobasidiomycete order comprising genera with uncertain affinities.
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As new taxa and additional accessions derived from carefully identified reference collections are added, the mt-LrRNA database becomes increasingly useful for identification of orchid mycobionts. The data set is currently insufficient by itself to provide strong support for structure at the base of the Basidiomycota, but is much more useful on a clade-by-clade level, as was shown here by placement of peloton and sterile fungal isolates. For detailed study of relationships within the orchid endophyte clades, a more variable locus such as nuclear ribosomal ITS needs to be examined in addition. The key to molecular identification of orchid mycobionts currently lies in better resolving the phylogeny of basidiomycetes, in particular the heterobasidiomycetes, through the addition of additional loci and sampling. Furthermore, more intensive sampling of pelotons from individual roots should be implemented, thereby improving our knowledge about patterns of fungi relationships within species.
Acknowledgments The authors thank friends and colleagues in Sabah for help with locations, logistics, and access to live collections during a visit in August 1999, especially Anthony Lamb and the staff at Sabah Agricultural Park, Tenom, Axel D. Poulsen, Tropical Biology and Conservation Unit, Universiti Malaysia Sabah, and Harry Lohok ( ), Poring Orchid Centre, Sabah Parks. This study was partially funded by the DANCED (Danish Co-operation for Environment and Development) program ‘‘Collaboration on biodiversity between Universiti Malaysia Sabah and Danish Universities’’ and by an Ohio State University Office of Research Seed Grant to J.V.F. The authors thank the Economic Planning Unit, Government of Malaysia, for permission to conduct research in Sabah (ref.: UPE: 40/200/19SJ.736) and Drs. Thomas Læssøe, Conny Asmussen, and Bo Johansen for valuable suggestions on the manuscript.
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Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae) Kim A. Kristiansena,*, John V. Freudensteinb, Finn N. Rasmussena, Hanne N. Rasmussenc b
a Department of Evolutionary Biology, Biological Institute, University of Copenhagen, Gothersgade 140, DK-1123 Copenhagen K, Denmark Herbarium, Department of Evolution, Ecology and Organismal Biology, Ohio State University, 1315 Kinnear Road, Columbus, OH 43212-1157, USA c Danish Forest and Landscape Research Institute, Hørsholm Kongevej 11, DK-2970 Hørsholm, Denmark
Received 8 March 2002; received in revised form 24 February 2004 Available online 30 July 2004
Abstract We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant. 2004 Elsevier Inc. All rights reserved. Keywords: Basidiomycota; Cladistics; Mitochondrial ribosomal LsDNA; Molecular identification; Orchidaceae; Mycorrhizal fungi
1. Introduction All orchids studied thus far have mycorrhizal associations for at least part of their life (Rasmussen, 1995). The orchid mycorrhiza has been recognized as a distinct type (Smith and Read, 1997), with the mycobiont forming pelotons (hyphal coils) in the cortical tissue of protocorms, roots, tubers, and rhizomes (Rasmussen, 1995). These pelotons are at some stage digested by the orchid. Most orchid mycobionts isolated from photosynthetic orchids have been identified as Rhizoctonia fungi, which are anamorphic states of heterobasidiomycetes (Currah *
Corresponding author. Fax: +45-3532-2154. E-mail address: [email protected] (K.A. Kristiansen).
0378-5955/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.05.015
et al., 1997; Roberts, 1999; Warcup, 1981; Table 1). We follow the classification by Roberts (1999) for heterobasidiomycetes acting as orchid endophytes. The traditional way to identify orchid mycobionts has been to isolate and make axenic cultures of fungi recovered from infected tissue for morphological comparison with type cultures kept in culture collections (e.g., Centraalbureau voor Schimmelcultures (CBS) or University of Alberta Microfungus Collection & Herbarium (UAMH)). These methods usually involve surface sterilization of the orchid root and dissection of the mycobiont from the infected tissue, followed by a series of rinsings in sterile water before transfer to suitable growth media. This is followed by subculturing of growing hyphae onto new media, thereby obtaining a pure
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Table 1 Heterobasidiomycetes belonging to the complex of Rhizoctonia-like fungi (Moore, 1987; Roberts, 1999) Order
Teleomorphic genera
Anamorphic genera
Ceratobasidiales
Ceratobasidium Thanatephorus Thanatephorus Waitea Waitea
Ceratorhiza Rhizoctonia Moniliopsis Rhizoctonia Moniliopsis
Exidiales
Ceratosebacina (Sebacina) Endoperplexa (Sebacina) Heteroacanthella (Sebacina) Oliveonia (Sebacina) Serendipita (Sebacina)
Unnamed Opadorhiza Acanthellorhiza Oliveorhiza Unnamed
Tulasnellales
Tulasnella
Epulorhiza
culture (for more detailed descriptions of methods see Clements et al., 1986; Rasmussen et al., 1990; Zelmer et al., 1996). Unfortunately, not all isolated fungi grow willingly, and cessation of growth after some time often makes the culture useless. Recently, molecular methods have been employed for direct identification of endophytes in roots using fungalspecific primers (Bidartondo and Bruns, 2001; Bidartondo et al., 2000; Bruns et al., 1998; Cullings et al., 1996; Gardes and Bruns, 1993; Kretzer et al., 2000). Such methods have been used to characterize mycobionts of Orchidaceae, eliminating the time-consuming culture step (McKendrick et al., 2000; Taylor and Bruns, 1997, 1999). Most previous workers have extracted total DNA from sections of fungal-infected plant tissue and then sequenced fungal loci and/or performed restriction fragment length polymorphism (RFLP) analysis. The nuclear ribosomal internal transcribed spacer (ITS) and the mitochondrial large subunit ribosomal RNA (mt-LsDNA) are the loci most often examined. Many orchids harbor a number of fungi in their tissues, but not all are necessarily involved in the parasitic relationship indicated by peloton formation (Bayman et al., 1997; Warcup, 1981). Kristiansen et al. (2001b) used dissection and sequencing of single pelotons from the root cortex of Dactylorhiza majalis to focus on fungi involved in this particular interaction, and found that even single pelotons could contain more than one mycobiont. A database of sequences that represent the diversity of the fungal group is necessary if molecular identification of sterile hyphae, such as those found in orchid mycorrhizae, is to become possible in practice. For orchids the relevant fungal group is Basidiomycota and numerous sequences have been collected for various loci for phylogenetic studies. Most recent phylogenetic studies of basidiomycetes have focused on the homobasidiomycetes, using either the nuclear or mitochondrial rDNA subunits as the object for sequencing (Binder and Hibbett, 2002; Bruns et al., 1998; Hibbett et al.,
1997, 2000; Jarosch, 2001; Moncalvo et al., 2000, 2002; Tehler et al., 2000), but a significant number of heterobasidiomycete sequences are also available. Previous studies of orchid mycobionts have focused on the larger subfamilies of Orchidaceae–Epidendroideae, Orchidoideae and Cypripedioideae (following the classification of Chase et al., 2003). Neuwiedia is one of two small genera of Apostasioideae; this subfamily is significant because it is sister to the remainder of the Orchidaceae (Cameron et al., 1999; Freudenstein and Rasmussen, 1999). The orchid endophyte relationship is believed to be a synapomorphy for Orchidaceae, but until details are known of this relationship for apostasioids, this conclusion must be tentative. Although Kristiansen et al. (2001a) showed that Neuwiedia has typical orchid protocorms that contain peloton-forming fungi, nothing is yet known of the identity of these fungi. The aim of this study was to apply the peloton identification method of Kristiansen et al. (2001b) to the endophytes of Neuwiedia to determine if they fall into the same group of heterobasidiomycete families known to be associated with other photosynthetic orchids.
2. Materials and methods Neuwiedia veratrifolia was collected during the summer of 1999 in Sabah, Borneo, East-Malaysia (Table 2). Roots, tubers, and protocorms were rinsed in water and dried in silica gel for later DNA analysis. Pelotons from N. veratrifolia were isolated (Rasmussen et al., 1990) and cultured on potato-dextrose agar for both sequencing and morphological identification. Fungi known to be orchid endophytes were obtained from culture collections (Table 3). Pelotons were dissected from the cortex of the roots or tubers following Kristiansen et al. (2001b) with some modifications: the pelotons were rinsed twice in double distilled water, recovered in 1 ll water and transferred to 0.5 ml Eppendorf-tubes containing 42.4 ll ddH2O and 5 ll 10· PCR buffer (see below). The mix was heated to 95 C for 10 min in a heat block to lyse the hyphae. DNA from fungal reference cultures was extracted using the DNeasy Plant Mini Kit (Qiagen) following the manufacturerÕs instructions. The 50 ll PCR mix used for each peloton contained 5 ll 10· PCR buffer [100 mM Tris–HCl (pH 8.8), 25 mM MgCl2, and 250 mM KCl], fungal DNA (in 1 ll H2O), 0.2 mmol each primer, 0.2 mM equimolar dNTP mix, and 1 U Taq polymerase. The forward primer MLIN3 (Kristiansen et al., 2001b) and the reverse primer ML6 (White et al., 1990) were used to amplify ca. 500 bp of the mt-LrRNA gene. The PCR parameters were as described by Gardes and Bruns (1993). The products were run on a 1.4% agarose gel to separate the bands; in cases where several bands were observed,
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Table 2 Material of Apostasia Bl and Neuwiedia Bl Taxon
Collection site
Habitat
Collection number, material, and voucher
Neuwiedia veratrifolia
Near Sin Tuong Tuong, c. 6–8 km W of Tambunan
Ranau road, c. 65 km E of Kota Kinabalu. Steep slope facing SE, dense secondary scrubby forest. One adult plant and a few plantlets were seen
C-668. 150899 Fixed and silicagel dried leaves and root, fixed protocorm from seedling, C. No voucher taken since only few plants were at location. Photographs of infructescence of adult plant, 26 Feb 1997 at Z (A. Kocyan Photo # 11215/ 11217, copies at C). Isolates K11A, K11B, and K11C taken from protocorms and stored at Danish Forest and Landscape Research Institute
Neuwiedia veratrifolia
Tenom Orchid Centre, Agricultural Park Sabah, c. 145 km S of Kota Kinabalu
Spontaneous seedling among cultivated specimens
C-669. 100899 ‘‘Motherplant’’ leaf, seedling leaf, and root dried. Seedling roots and protocorm FAA at C. Live culture at Tenom Orchid Centre. Pressed shoot from this culture at Z (AK971129/ 1/03). Isolates K7-1A and K7-1B, K7-4, K10-1, and K10-2, taken from protocorms and stored at Danish Forest and Landscape Research Institute
Neuwiedia veratrifolia
Poring Orchid Center, Sabah Parks. Poring Hot Springs
Spontaneous seedling among cultivated specimens
Isolate K14. 170899. Isolate taken from protocorm. Due to Sabah Parks rules no voucher was taken. Isolate K14 taken from protocorms and stored at Danish Forest and Landscape Research Institute
Herbarium abbreviation according to Holmgren et al. (1990).
Table 3 Cultivated orchid endophytes used in this study Fungal taxon
Source/accession
Forward primer
Orchid source and reference to fungal description
? ? ? ? ? ? ? ? ? Thanatephorus obscurus (Ceratobasidium obscurum)
K7.1A K7.1B K7.4 K10.1 K10.2 K11.1A K11.1B K11.1C K14 UAMH 5443
MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3 MLIN3
Epulorhiza repens (Rhizoctonia repens)
CBS 326,47
MLIN3
C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-669 Neuwiedia veratrifolia C-668 Neuwiedia veratrifolia C-668 Neuwiedia veratrifolia C-668 Neuwiedia veratrifolia Neuwiedia veratrifolia (see Table 2) Orchis rotundifolia. Mycorrhizae septum structure of orchid endophytes Currah and Sherburne (1992) Orchis morio Bernard Saksena and Vaartaja (1961)
CBS is Centraalbureau voor Schimmelcultures Baarn and Delft, The Netherlands. UAMH is University of Alberta Microfungus Collection and Herbarium, Canada.
individual bands were cut out and purified from the gel using the Qiaquick Gel Extraction Kit (Qiagen) following the manufacturerÕs instructions. In cases where a clean sequence was not readily obtained, PCR products were cloned using the PCR-Script Amp Cloning Kit (Stratagene, CA). All PCR products were purified prior to sequencing using Wizard PCR Preps (Promega, WI) following the
manufacturerÕs instructions. The DNA was eluted in 20 ll sterile water and cycle sequenced using the MLIN3/ML6 primers and the ABI Prism BigDye Terminator Ready Reaction kit. Sequences were run on a PE Biosystems 377 sequencer at the DNA Sequencing Center, Brigham Young University, Provo, Utah. Sequences have been deposited in GenBank (for accession numbers see Table 4).
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Table 4 Taxa included in the DNA matrix for phylogenetic analysis Fungal taxon (reference)
Source/ Accession No.
K-7 (this study) K-10-1 (this study) K-10-2 (this study) K-11 (this study) C668 (this study) C669 (this study) Thanatephorus obscurus (this study) Epulorhiza repens (this study) Ceratorhiza goodyera-repentis Kristiansen et al. (2001b) DM1 Kristiansen et al. (2001b) DM2 Kristiansen et al. (2001b) DM3 Kristiansen et al. (2001b) Epulorhiza anaticula Kristiansen et al. (2001b) Moniliopsis anomala Kristiansen et al. (2001b) Sistotrema sp. Kristiansen et al. (2001b) Tulasnella deliquescens (D47-7A) Kristiansen et al. (2001b) Tulasnella deliquescens (D47-7B) Kristiansen et al. (2001b) Tulasnella irregularis Kristiansen et al. (2001b) Tulasnella pruinosa Kristiansen et al. (2001b) Tulasnella violea Kristiansen et al. (2001b) Albatrellus flettii Bruns et al. (1998) Albatrellus peckianus Bruns et al. (1998) Albatrellus skamanius Bruns et al. (1998) Albatrellus syringae Bruns et al. (1998) Amanita calyptrata Bruns et al. (1998) Amanita phalloides Bruns et al. (1998) Armillaria albolanaripes Bruns et al. (1998) Boletopsis subsquamosa Horton (2000) Boletus satanas Bruns et al. (1998) Cantharellus-like sp1 MR14 Bruns et al. (1998) Cantharellus-like sp1 MR15 Bruns et al. (1998) Cantharellus tubaeformis Bruns et al. (1998) Clavulina cristata Horton (2000) Coniophora arida Bruns et al. (1998) Coniophora puteana Bruns et al. (1998) Craterellus tubaeformis Horton (2000) Gyroporus cyanescens Bruns et al. (1998) Hebeloma crustuliniforme Bruns et al. (1998) Hygropgorus pudorius Bruns et al. (1998) Hygrophorus brunnescens Horton (2000) Hygrophorus purpurescens Horton (2000) Hygrophorus speciosus Bruns et al. (1998) Inocybe sororia Bruns et al. (1998) Inocybe sp. (OD6264) Horton (2000) Inocybe sp. (OD6271) Horton (2000) Inocybe sp. trh415 Horton (2000) Laccaria amethysteo-occidentalis Bruns et al. (1998) Laccaria laccata Bruns et al. (1998) Laccaria trh416 Horton (2000) Russula xerampelina @Monotropa uniflora Horton (2000) Paragyrodon sphraerosporus Bruns et al. (1998) Paxillus involutus Bruns et al. (1998) Paxillus statuum Bruns et al. (1998) Phaeogyroporus portentosus Bruns et al. (1998) Phylloporus rhodotaxus Bruns et al. (1998) Pluteus cervinus Horton (2000) Pseudotomentella tristis Bruns et al. (1998)
AF490610 AF490611 AF490612 AF490613 AF490608 AF490609 AF490614 AF490615 AF345556 AF345854 AF345855 AF345856 AF345559 AF345557 AF345558 AF345852 AF345853 AF345560 AF345561 AF345562 S:1333189 S:1333202 S:1333243 S:1333261 S:1333278 S:1333373 S:1333375 AY323508 S:1333387 L46388 L46389 S:1333396 AY323510 S:1333400 S:1333401 AY323513 S:1333416 S:1333418 S:1333428 AY323512 AY323511 S:1333488 S:1333490 AY323515 AY323516 AY323518 L46394 S:1333492 AY323517 AY323505 S:1333503 S:1333505 S:1333506 S:1333507 S:1333508 AY323509 S:1333512
Table 4 (continued) Fungal taxon (reference)
Source/ Accession No.
Russula brevipes Bruns et al. (1998) Sebacina sp. Bruns et al. (1998) Serpula incrassata Bruns et al. (1998) Suillus tomentosus Bruns et al. (1998) Tomentella atrorubra Bruns et al. (1998) Tricholoma flavovirens Bruns et al. (1998) Tricholoma moseri Horton (2000) Tricholoma portentosum Horton (2000) Tricholoma saponaceum Horton (2000) Tulasnella irregularis Bruns et al. (1998) Waitea circinata Bruns et al. (1998)
L46395 S:1333525 S:1333528 S:1333534 S:1333538 S:1333542 AY323514 AY323520 AY323519 S:1333546 S:1333645
Outgroup Schizosaccharomyces pombe Lang et al. (1987)
NC_001326
All sequences were submitted to a Blast search in GenBank as a first step to determine whether they contained mitochondrial ribosomal LsDNA. Sequences were aligned using ClustalX (Thompson et al., 1997) with default parameters: opening gap = 15, gap extension = 6.6, transition weight = 0.50, and delay divergent = 30%. At least two taxa from each of the fungal groups recognized by Bruns et al. (1998) were included in the alignment, except for group 6 (from which only Suillus tomentosus was included) and group 16 (which is not represented in this analysis). Fourteen sequences from the updated mitochondrial ribosomal LsDNA fungal database (Horton, 2000) were also included in the data matrix, which comprised a total of 74 taxa (Table 4). Schizosaccharomyces pombe (Ascomycota) was used as the outgroup. The matrix was analyzed with PAUP* 4.0 (Swofford, 2000) using equally weighted parsimony. Uninformative characters were excluded prior to searching, and all characters were treated as unordered. Tree search consisted of 500 random addition replicates saving up to 5 trees each and using TBR branch swapping. Zero length branches were collapsed. Jackknife branch support values (Farris et al., 1996) were obtained using PAUP* with 37% deletion, JAC emulation, 10,000 jackknife replicates with one random addition replicate each, and saving up to two trees per replicate.
3. Results The phylogenetic analysis resulted in 197 most parsimonious trees (l = 407, CI= 0.48, RI = 0.89). The heterobasidiomycetes are distributed in three clades (Fig. 1, clades A–C), forming a polytomy with the remaining homobasidiomycetes. Clade A (branch support = 0.99) comprises Sistotrema sp. (Aphyllophorales, Homobasidiomycota; Hibbett and Thorn, 2001), Sebacina sp. (Exidiales, Heterobasidiomycota) and Waitea circinata
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Fig. 1. Strict consensus of 197 equally most parsimonious trees with a length of 407 steps. Terminals marked with @ are unknown fungi isolated from the plants named. Jackknife support values are shown above branches.
(Ceratobasidiales, Heterobasidiomycota; Roberts, 1999). Clade B (branch support = 0.99) includes the fungal cultures Thanatephorus obscurus (Ceratobasidiales, Heterobasidiomycota; Roberts, 1999), three representa-
tives of K11 and one representative of K14 (all obtained from N. veratrifolia ; Table 2). Clade C (branch support = 0.99) is subdivided into clades C1–C4 (Fig. 1). Clades C1–C3 consist of pelotons from D. majalis
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(Kristiansen et al., 2001b), Tulasnella teleomorphs, the Tulasnella anamorph Epulorhiza anaticula (Tulasnellales), Ceratorhiza goodyera-repentis (= Ceratobasidium, Ceratobasidiales) and Moniliopsis anomala (= Thanathephorus anomala?, Ceratobasidiales; Roberts, 1999). Subclade C4 comprises cultured fungi or single pelotons from N. veratrifolia together with Tulasnella deliquescens teleomorphs and an anamorph (Epulorhiza).
4. Discussion The cladistic analysis presented here is in accordance with the groupings reported in the neighbour-joining tree by Bruns et al. (1998; Fig. 1). The tree does not resolve relationships among many of the smaller clades; although a high degree of resolution is always desirable in a cladistic analysis, the purpose of this study was not to study the detailed relationships of basidiomycetes but rather to identify fungi associated with Neuwiedia using phylogenetic methods. The analysis did place the Neuwiedia endophyte sequences decisively with known sequences, given the strong branch support for these relationships. Neuwiedia mycobionts fall into two strongly supported clades (B and C), where all terminals except for Tulasnella pruinosa and Tulasnella violea were amplified either directly from pelotons or from fungal isolates from N. veratrifolia or higher orchids (cf. Tables 1 and 3 in Kristiansen et al., 2001b). Although nothing is yet known about the fungal associates of Apostasia, it is clear that Apostasioideae as represented by Neuwiedia have the same mycorrhizal associates that are present in other orchids; we predict that examination of Apostasia will also reveal Rhizoctonia-type mycobionts. Hence, it appears that presence of this type of fungus, and the orchid mycorrhizal type in general, is a synapomorphy for the entire Orchidaceae. Most of the mycobionts fall in subclade C4, with representatives of Tulasnellales also recovered from orchid roots. The Neuwiedia mycobionts C668 and C669 were amplified from pelotons from seedling roots, while K7-1A, K7-1B, K7-4, K10-1, and K10-2 are from cultured material, derived from pelotons isolated from fresh seedlings (Tables 2 and 3). Sister to these is a clade (C3) comprising members of Tulasnellales, but also representatives of Ceratobasidiales (Moniliopsis and Ceratorhiza). In subclade C3 the Ceratobasidiales members come out with Tulasnella species (branch support = 0.66), suggesting that Tulasnellales may be paraphyletic. The Neuwiedia mycobionts found in C4 clearly represent T. deliquescens or a very closely related species. The remaining Neuwiedia mycobionts, also derived from pelotons isolated from fresh seedlings, (K11-1A to K11-1C and K14) are placed in clade B together with T. obscurus (Ceratobasidiales), suggesting
that these should be referred to Ceratobasidiales. Furthermore, K11 and K14 show distinct morphological differences from K7 and K10 (unpublished data), further emphasizing the distinctness of these isolates. Isolates K7 and K10 are from protocorms/seedlings collected in the Tenom Orchid Center, while K11 isolates are from a wild growing plantlet and K14 isolates are from protocorms/seedlings found at the Poring Orchid Center. The K11 mycobionts were cultured from the same seedling as the C668 peloton amplification, clearly indicating the presence of more than one mycobiont in this individual. This finding supports previous reports of multiple active mycorrhizal fungi in the same plant (Kristiansen et al., 2001b; Warcup, 1981) and might also indicate a pattern of varying host utilization with respect to area. The Tenom seedlings were infected only with T. deliquescens-like fungi, C669, K7, and K10. The Sin Tuong Tuong seedling was infected with both T. deliquescens-like fungi and T. obscurus-like fungi, C668 and K11, whereas the Poring population was infected with only T. obscurus-like fungi, K14. This may indicate a change in mycorrhizal partner from Tenom in the south to Poring in the north, but could also be due to chance amplification and culturing success. Further study of endophytes within species and within individuals is needed to more clearly establish patterns of interaction at those levels. The taxa from HortonÕs (2000) update of the mtLrRNA sequence database are behaving as one would have predicted, though some are worth mentioning. The Russula xerampelina isolate from the leafless ericad Monotropa uniflora in clade 8 was sister to Russula brevipes, perhaps not surprising in that the latter species was also isolated from M. uniflora by Bidartondo and Bruns (2001). This analysis confirms previous indications of paraphyly for Albatrellus (Fig. 1, clades 7 and 9; Bruns et al., 1998; Hibbett et al., 2000). The paraphyly of Paxillus indicated by Bruns et al. (1998) and further supported by the phylogenetic work on Boletales by Jarosch (2001; Fig. 1 clades 2 and 3) is also supported by this analysis. Ceratobasidiales appear as polyphyletic on the tree, falling in clades A, B, and C (Fig. 1). This result is not consistent with results presented by Roberts (1999), which indicate Ceratobasidiales to be monophyletic and sister to Exidiales (paraphyletic) and Tulasnellales (monophyletic). RobertsÕ cladistic analyses were based on morphological and molecular data, and included 23 species belonging to Ceratobasidiales, but only two species of Tulasnella. Furthermore, the homobasidiomycete Sistotrema poses an interesting problem by falling among the heterobasidiomycetes Sebacina and Waitea (A), but Sistotrema is classed as belonging to Aphyllophorales (Hibbett and Thorn, 2001) which is a troublesome ‘‘lower’’ homobasidiomycete order comprising genera with uncertain affinities.
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As new taxa and additional accessions derived from carefully identified reference collections are added, the mt-LrRNA database becomes increasingly useful for identification of orchid mycobionts. The data set is currently insufficient by itself to provide strong support for structure at the base of the Basidiomycota, but is much more useful on a clade-by-clade level, as was shown here by placement of peloton and sterile fungal isolates. For detailed study of relationships within the orchid endophyte clades, a more variable locus such as nuclear ribosomal ITS needs to be examined in addition. The key to molecular identification of orchid mycobionts currently lies in better resolving the phylogeny of basidiomycetes, in particular the heterobasidiomycetes, through the addition of additional loci and sampling. Furthermore, more intensive sampling of pelotons from individual roots should be implemented, thereby improving our knowledge about patterns of fungi relationships within species.
Acknowledgments The authors thank friends and colleagues in Sabah for help with locations, logistics, and access to live collections during a visit in August 1999, especially Anthony Lamb and the staff at Sabah Agricultural Park, Tenom, Axel D. Poulsen, Tropical Biology and Conservation Unit, Universiti Malaysia Sabah, and Harry Lohok ( ), Poring Orchid Centre, Sabah Parks. This study was partially funded by the DANCED (Danish Co-operation for Environment and Development) program ‘‘Collaboration on biodiversity between Universiti Malaysia Sabah and Danish Universities’’ and by an Ohio State University Office of Research Seed Grant to J.V.F. The authors thank the Economic Planning Unit, Government of Malaysia, for permission to conduct research in Sabah (ref.: UPE: 40/200/19SJ.736) and Drs. Thomas Læssøe, Conny Asmussen, and Bo Johansen for valuable suggestions on the manuscript.
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