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
a
Department of Evolutionary Biology, Biological Institute, University of Copenhagen, Gothersgade 140, DK-1123 Copenhagen K, Denmark
b
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 ampli-
fied and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a mem-
ber 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 et al., 1997; Roberts, 1999; Warcup, 1981; Table 1).

All orchids studied thus far have mycorrhizal associ-
ations 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 form-
ing pelotons (hyphal coils) in the cortical tissue of proto-
corms, 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: kimak@bot.ku.dk (K.A. Kristiansen).
We follow the classification by Roberts (1999) for hete-
robasidiomycetes acting as orchid endophytes.
The traditional way to identify orchid mycobionts
has been to isolate and make axenic cultures of fungi re-
covered from infected tissue for morphological compar-
ison with type cultures kept in culture collections (e.g.,
Centraalbureau voor Schimmelcultures (CBS) or Uni-
versity of Alberta Microfungus Collection & Herbarium
(UAMH)). These methods usually involve surface steri-
lization of the orchid root and dissection of the myco-
biont 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 grow-
ing hyphae onto new media, thereby obtaining a pure

0378-5955/$ - see front matter
2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2004.05.015
252 K.A. Kristiansen et al. / Molecular Phylogenetics and Evolution 33 (2004) 251–258
Table 1
Heterobasidiomycetes belonging to the complex of Rhizoctonia-like
fungi (Moore, 1987; Roberts, 1999)
Order Teleomorphic genera Anamorphic genera
Ceratobasidiales Ceratobasidium Ceratorhiza
Thanatephorus Rhizoctonia
Thanatephorus Moniliopsis
Waitea Rhizoctonia
Waitea Moniliopsis
Exidiales Ceratosebacina (Sebacina) Unnamed
Endoperplexa (Sebacina) Opadorhiza
Heteroacanthella (Sebacina) Acanthellorhiza
Oliveonia (Sebacina) Oliveorhiza
Serendipita (Sebacina) 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 fungal-
specific primers (Bidartondo and Bruns, 2001; Bidart-
ondo 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 my-
cobionts of Orchidaceae, eliminating the time-consum-
ing culture step (McKendrick et al., 2000; Taylor and
Bruns, 1997, 1999). Most previous workers have extract-
ed total DNA from sections of fungal-infected plant tis-
sue and then sequenced fungal loci and/or performed
restriction fragment length polymorphism (RFLP) anal-
ysis. 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 rela-
tionship indicated by peloton formation (Bayman et al.,
1997; Warcup, 1981). Kristiansen et al. (2001b) used dis-
section 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 identifica-
tion of sterile hyphae, such as those found in orchid my-
corrhizae, is to become possible in practice. For orchids
the relevant fungal group is Basidiomycota and numer-
ous sequences have been collected for various loci for
phylogenetic studies. Most recent phylogenetic studies
of basidiomycetes have focused on the homobasidi-
omycetes, 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–Epidendroide-
ae, 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 apostasi-
oids, this conclusion must be tentative. Although Kris-
tiansen 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 iden-
tification 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 sum-
mer 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 se-
quencing and morphological identification. Fungi
known to be orchid endophytes were obtained from cul-
ture 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 man-
ufacturerÕs 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 prim-
er 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,
 K.A. Kristiansen et al. / Molecular Phylogenetics and Evolution 33 (2004) 251–258 253

Table 2
Material of Apostasia Bl and Neuwiedia Bl
Taxon Collection site Habitat Collection number, material, and voucher
Neuwiedia Near Sin Tuong Tuong, Ranau road, c. 65 km E of Kota Kinabalu. C-668. 150899 Fixed and silicagel dried
veratrifolia c. 6–8 km W of Tambunan Steep slope facing SE, leaves and root, fixed protocorm from
dense secondary scrubby forest. seedling, C. No voucher taken since only
One adult plant and a few plantlets were seen 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 Tenom Orchid Centre,
veratrifolia Agricultural Park Sabah,
c. 145 km S of Kota Kinabalu
Spontaneous seedling among C-669. 100899 ‘‘Motherplant’’ leaf,
cultivated specimens 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 Poring Orchid Center, Spontaneous seedling among Isolate K14. 170899. Isolate taken from
veratrifolia Sabah Parks. cultivated specimens protocorm. Due to Sabah Parks rules no
Poring Hot Springs 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
? K7.1A MLIN3 C-669 Neuwiedia veratrifolia
? K7.1B MLIN3 C-669 Neuwiedia veratrifolia
? K7.4 MLIN3 C-669 Neuwiedia veratrifolia
? K10.1 MLIN3 C-669 Neuwiedia veratrifolia
? K10.2 MLIN3 C-669 Neuwiedia veratrifolia
? K11.1A MLIN3 C-668 Neuwiedia veratrifolia
? K11.1B MLIN3 C-668 Neuwiedia veratrifolia
? K11.1C MLIN3 C-668 Neuwiedia veratrifolia
? K14 MLIN3 Neuwiedia veratrifolia (see Table 2)
Thanatephorus obscurus UAMH 5443 MLIN3 Orchis rotundifolia. Mycorrhizae septum
(Ceratobasidium obscurum) structure of orchid endophytes Currah
and Sherburne (1992)
Epulorhiza repens (Rhizoctonia repens) CBS 326,47 MLIN3 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) follow-
ing 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 Ter-
minator 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 acces-
sion numbers see Table 4).
254 K.A. Kristiansen et al. / Molecular Phylogenetics and Evolution 33 (2004) 251–258

Table 4 Table 4 (continued)

Taxa included in the DNA matrix for phylogenetic analysis
Fungal taxon (reference) Source/
Fungal taxon (reference) Source/ Accession No.
Accession No. Russula brevipes Bruns et al. (1998) L46395
K-7 (this study) AF490610 Sebacina sp. Bruns et al. (1998) S:1333525
K-10-1 (this study) AF490611 Serpula incrassata Bruns et al. (1998) S:1333528
K-10-2 (this study) AF490612 Suillus tomentosus Bruns et al. (1998) S:1333534
K-11 (this study) AF490613 Tomentella atrorubra Bruns et al. (1998) S:1333538
C668 (this study) AF490608 Tricholoma flavovirens Bruns et al. (1998) S:1333542
C669 (this study) AF490609 Tricholoma moseri Horton (2000) AY323514
Thanatephorus obscurus (this study) AF490614 Tricholoma portentosum Horton (2000) AY323520
Epulorhiza repens (this study) AF490615 Tricholoma saponaceum Horton (2000) AY323519
Ceratorhiza goodyera-repentis AF345556 Tulasnella irregularis Bruns et al. (1998) S:1333546
Kristiansen et al. (2001b) Waitea circinata Bruns et al. (1998) S:1333645
DM1 Kristiansen et al. (2001b) AF345854
DM2 Kristiansen et al. (2001b) AF345855 Outgroup
DM3 Kristiansen et al. (2001b) AF345856 Schizosaccharomyces pombe Lang et al. (1987) NC_001326
Epulorhiza anaticula Kristiansen et al. (2001b) AF345559
Moniliopsis anomala Kristiansen et al. (2001b) AF345557
Sistotrema sp. Kristiansen et al. (2001b) AF345558
Tulasnella deliquescens (D47-7A) AF345852
All sequences were submitted to a Blast search in
Kristiansen et al. (2001b) GenBank as a first step to determine whether they con-
Tulasnella deliquescens (D47-7B) AF345853 tained mitochondrial ribosomal LsDNA. Sequences
Kristiansen et al. (2001b) were aligned using ClustalX (Thompson et al., 1997)
Tulasnella irregularis Kristiansen et al. (2001b) AF345560 with default parameters: opening gap = 15, gap exten-
Tulasnella pruinosa Kristiansen et al. (2001b) AF345561
Tulasnella violea Kristiansen et al. (2001b) AF345562
sion = 6.6, transition weight = 0.50, and delay diver-
Albatrellus flettii Bruns et al. (1998) S:1333189 gent = 30%. At least two taxa from each of the fungal
Albatrellus peckianus Bruns et al. (1998) S:1333202 groups recognized by Bruns et al. (1998) were included
Albatrellus skamanius Bruns et al. (1998) S:1333243 in the alignment, except for group 6 (from which only
Albatrellus syringae Bruns et al. (1998) S:1333261 Suillus tomentosus was included) and group 16 (which
Amanita calyptrata Bruns et al. (1998) S:1333278
Amanita phalloides Bruns et al. (1998) S:1333373
is not represented in this analysis). Fourteen sequences
Armillaria albolanaripes Bruns et al. (1998) S:1333375 from the updated mitochondrial ribosomal LsDNA fun-
Boletopsis subsquamosa Horton (2000) AY323508 gal database (Horton, 2000) were also included in the
Boletus satanas Bruns et al. (1998) S:1333387 data matrix, which comprised a total of 74 taxa (Table
Cantharellus-like sp1 MR14 Bruns et al. (1998) L46388 4). Schizosaccharomyces pombe (Ascomycota) was used
Cantharellus-like sp1 MR15 Bruns et al. (1998) L46389
Cantharellus tubaeformis Bruns et al. (1998) S:1333396
as the outgroup.
Clavulina cristata Horton (2000) AY323510 The matrix was analyzed with PAUP* 4.0 (Swofford,
Coniophora arida Bruns et al. (1998) S:1333400 2000) using equally weighted parsimony. Uninformative
Coniophora puteana Bruns et al. (1998) S:1333401 characters were excluded prior to searching, and all
Craterellus tubaeformis Horton (2000) AY323513 characters were treated as unordered. Tree search con-
Gyroporus cyanescens Bruns et al. (1998) S:1333416
Hebeloma crustuliniforme Bruns et al. (1998) S:1333418
sisted of 500 random addition replicates saving up to 5
Hygropgorus pudorius Bruns et al. (1998) S:1333428 trees each and using TBR branch swapping. Zero length
Hygrophorus brunnescens Horton (2000) AY323512 branches were collapsed. Jackknife branch support val-
Hygrophorus purpurescens Horton (2000) AY323511 ues (Farris et al., 1996) were obtained using PAUP*
Hygrophorus speciosus Bruns et al. (1998) S:1333488 with 37% deletion, JAC emulation, 10,000 jackknife rep-
Inocybe sororia Bruns et al. (1998) S:1333490
Inocybe sp. (OD6264) Horton (2000) AY323515
licates with one random addition replicate each, and
Inocybe sp. (OD6271) Horton (2000) AY323516 saving up to two trees per replicate.
Inocybe sp. trh415 Horton (2000) AY323518
Laccaria amethysteo-occidentalis L46394
Bruns et al. (1998) 3. Results
Laccaria laccata Bruns et al. (1998) S:1333492
Laccaria trh416 Horton (2000) AY323517
Russula xerampelina @Monotropa uniflora AY323505 The phylogenetic analysis resulted in 197 most parsi-
Horton (2000) monious trees (l = 407, CI= 0.48, RI = 0.89). The hete-
Paragyrodon sphraerosporus Bruns et al. (1998) S:1333503 robasidiomycetes are distributed in three clades (Fig.
Paxillus involutus Bruns et al. (1998) S:1333505 1, clades A–C), forming a polytomy with the remaining
Paxillus statuum Bruns et al. (1998) S:1333506
Phaeogyroporus portentosus Bruns et al. (1998) S:1333507
homobasidiomycetes. Clade A (branch support = 0.99)
Phylloporus rhodotaxus Bruns et al. (1998) S:1333508 comprises Sistotrema sp. (Aphyllophorales, Homobasid-
Pluteus cervinus Horton (2000) AY323509 iomycota; Hibbett and Thorn, 2001), Sebacina sp. (Ex-
Pseudotomentella tristis Bruns et al. (1998) S:1333512 idiales, Heterobasidiomycota) and Waitea circinata
 K.A. Kristiansen et al. / Molecular Phylogenetics and Evolution 33 (2004) 251–258 255

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, tives of K11 and one representative of K14 (all obtained
1999). Clade B (branch support = 0.99) includes the fun- from N. veratrifolia ; Table 2). Clade C (branch sup-
gal cultures Thanatephorus obscurus (Ceratobasidiales, port = 0.99) is subdivided into clades C1–C4 (Fig. 1).
Heterobasidiomycota; Roberts, 1999), three representa- Clades C1–C3 consist of pelotons from D. majalis
256 K.A. Kristiansen et al. / Molecular Phylogenetics and Evolution 33 (2004) 251–258
(Kristiansen et al., 2001b), Tulasnella teleomorphs, the
Tulasnella anamorph Epulorhiza anaticula (Tulasnella-
les), Ceratorhiza goodyera-repentis (= Ceratobasidium,
Ceratobasidiales) and Moniliopsis anomala (= Thanathe-
phorus 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 re-
solve 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 Neu-
wiedia endophyte sequences decisively with known se-
quences, given the strong branch support for these
relationships.
Neuwiedia mycobionts fall into two strongly support-
ed clades (B and C), where all terminals except for Tu-
lasnella 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 Aposta-
sia 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 or-
chid 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 sup-
port = 0.66), suggesting that Tulasnellales may be para-
phyletic. The Neuwiedia mycobionts found in C4
clearly represent T. deliquescens or a very closely relat-
ed 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 to-
gether with T. obscurus (Ceratobasidiales), suggesting
that these should be referred to Ceratobasidiales. Fur-
thermore, K11 and K14 show distinct morphological
differences from K7 and K10 (unpublished data), fur-
ther emphasizing the distinctness of these isolates. Iso-
lates K7 and K10 are from protocorms/seedlings
collected in the Tenom Orchid Center, while K11 iso-
lates are from a wild growing plantlet and K14 isolates
are from protocorms/seedlings found at the Poring Or-
chid Center. The K11 mycobionts were cultured from
the same seedling as the C668 peloton amplification,
clearly indicating the presence of more than one myco-
biont 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 infect-
ed 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 fun-
gi, 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 Te-
nom 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 mt-
LrRNA 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 brev-
ipes, 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 Pax-
illus indicated by Bruns et al. (1998) and further sup-
ported 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 homobasidi-
omycete 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 com-
prising genera with uncertain affinities.
 K.A. Kristiansen et al. / Molecular Phylogenetics and Evolution 33 (2004) 251–258
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 cur-
rently 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 endo-
phyte clades, a more variable locus such as nuclear ribo-
somal 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 addi-
tion of additional loci and sampling. Furthermore, more
intensive sampling of pelotons from individual roots
should be implemented, thereby improving our knowl-
edge 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 col-
lections during a visit in August 1999, especially Anth-
ony Lamb and the staff at Sabah Agricultural Park,
Tenom, Axel D. Poulsen, Tropical Biology and Conser-
vation Unit, Universiti Malaysia Sabah, and Harry Lo-
hok ( ), Poring Orchid Centre, Sabah Parks. This study
was partially funded by the DANCED (Danish Co-op-
eration for Environment and Development) program
‘‘Collaboration on biodiversity between Universiti Ma-
laysia 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 re-
search 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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