The isolation protocol of Lee & Taylor
(1990 ) was used to extract
genomic DNA from fungal mycelia grown on MEA. The primers ITS1
(White et al. 1990 )
and LR5 (Vilgalys & Hester
1990) were used to amplify part of the nuclear rRNA operon using
the PCR conditions recommended by the authors and spanning the 3' end of the
18S rRNA gene, the internal spacers, the 5.8S rRNA gene and a part of the 5'
end of the 28S rRNA gene. PCR products were separated by electrophoresis at 80
V for 1 h in a 0.8 % (w/v) agarose gel in 0.5× TAE running buffer (0.4
m Tris, 0.05 m NaAc, and 0.01 m EDTA, pH
7.85) and visualised under UV light using a GeneGenius Gel Documentation and
Analysis System (Syngene, Cambridge, U.K.) following ethidium bromide
staining. The amplification products were purified using a GFX PCR DNA and Gel
Band Purification Kit (Amersham Pharmacia Biotech Europe GmbH, Germany). The
purified products were sequenced in both directions using an ABI PRISM Big Dye
Terminator v. 3.1 Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster
City, CA) containing AmpliTaq DNA Polymerase as recommended by the
manufacturer. The primers LR0R (Rehner
& Samuels 1994), LR3R
(http://www.biology.duke.edu/fungi/mycolab/primers.htm),
LR16 (Moncalvo et al. 1993), and LR5 (Vilgalys &
Hester 1990) were used to ensure good quality sequences over the
entire length of the amplicon. The resulting fragments were analysed on an ABI
Prism 3100 DNA Sequencer (Perkin-Elmer, Norwalk, CN).
DNA sequences were assembled and added to the outgroups and additional
GenBank sequences using Sequence Alignment Editor v. 2.0a11
(Rambaut 2002 ), and manual
adjustments for improvement were made by eye where necessary. The phylogenetic
analyses of sequence data were done in PAUP (Phylogenetic Analysis Using
Parsimony) version 4.0b10 (Swofford
2002) and consisted of neighbour-joining analysis with the
uncorrected (“p”), the Kimura 2-parameter and the HKY85
substitution model in PAUP. Alignment gaps were treated as missing data and
all characters were unordered and of equal weight. Any ties were broken
randomly when encountered. For parsimony analysis, alignment gaps were treated
as both missing and as a fifth character state and all characters were
unordered and of equal weight. Maximum parsimony analysis was performed using
the heuristic search option with simple taxa additions and tree bisection and
reconstruction (TBR) as the branch-swapping algorithm. Branches of zero length
were collapsed and all multiple, equally parsimonious trees were saved. The
robustness of the trees obtained was evaluated by 1000 bootstrap replications
(Hillis & Bull 1993 ). Tree
length (TL), consistency index (CI), retention index (RI) and rescaled
consistency index (RC) were calculated and the resulting trees were printed
with TreeView v. 1.6.6 (Page
1996).
Bayesian analysis was conducted on the same aligned LSU dataset as the
distance analysis. First MrModeltest v. 2.2
(Nylander 2004 ) was used to
determine the best nucleotide substitution model. Phylogenetic analyses were
performed with MrBayes v. 3 (Ronquist
& Huelsenbeck 2003) applying a general time-reversible (GTR)
substitution model with gamma (G) and proportion of invariable site (I)
parameters to accommodate variable rates across sites. The Markov Chain Monte
Carlo (MCMC) analysis of 4 chains started from random tree topology and lasted
10 000 000 generations. Trees were saved each 100 000 generations, resulting
in 1000 saved trees. Burn-in was set at 500 000 generations after which the
likelihood values were stationary, leaving 950 trees from which the consensus
trees and posterior probabilities were calculated. PAUP 4.0b10 was used to
reconstruct the consensus tree, and maximum posterior probabilities were
assigned to branches after a 50 % majority rule consensus tree was constructed
from the 950 sampled trees.