sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ Faculty of Forestry Journal
supplemented with 5% (v/v) bovine serum albumin (BSA) as a PCR enhancer.
DNA fragments encompassing the ITS and tefa DNA regions were amplified using
the primer combinations ITS1/ITS4 and EF595F/EF1160R, respectively (White et
al., 1990; Kauserud and Schumacher, 2001). The DNA was PCR-amplified as in
previous studies (Tomšovský et al., 2006) using a Mastercycler® ep thermocycler
(Eppendorf, Hamburg, Germany). In cases when amplification of the ITS and tefa
regions was difficult, the primer pairs ITS1F/ITS4B and EF-526F/EF-1567R,
respectively, were used in nested PCR (for primer sequences, see Gardes and
Bruns, 1993; O’Donnell et al., 1998).
The amplified fragments were sequenced by the DNA Sequencing Service of
Macrogen Inc. (Seoul, Korea). We added an ITS sequence from the Porodaedalea
chrysoloma neotype (Genbank acc. no. AF123440; Larsen and Stenlid, 1999) to the
data set. ITS and tefa sequences of Onnia leporina were chosen as outgroups based
on the results of Wagner and Fischer 2002.
Sequences of each individual marker were aligned using the Clustal W
algorithm in BioEdit and adjusted manually. To determine whether the datasets
from different genetic markers were in significant conflict, partition homogeneity
tests were performed between the markers in all possible pair-wise combinations.
The tests were done in PAUP 4.0b10 using 100 replicates and the heuristic general
search option. The null hypothesis of congruence was rejected if p < 0.01
Phylogenies were generated in MrBayes version 3.1.2.The best-fit model and
parameters given by MrModeltest were used in the analyses. Markov chains were
initiated from a random tree and were run for 2,000.000 generations; the samples
were taken every 100th generation. Posterior probabilities (PP) were used as
Bayesian branch supports on the consensus trees. In addition, bootstrap branch
support values (BP) were estimated in PAUP 4.0b10 under the maximum
parsimony criterion using 1000 replicate datasets with random sequence addition
during each heuristic search.
3. RESULTS
Partition homogeneity tests showed significant conflict between the two genetic
markers used (p ≤ 0.01). This did not allow us to perform a combined analysis of
the ITS and tefa sequence data. The results of the phylogenetic analyses of the two
datasets are ambiguous. The ITS phylogram shows three main groups, while the
tefa phylogram shows four. In ITS data set, the most basal lineage within the
ingroup is composed of P. chrysoloma specimens from the Czech Republic,
Estonia, Romania, and Southern Sweden, including the P. chrysoloma neotype.
This P. chrysoloma group is also consistent in the tefa phylogram.
The second group includes P. pini specimens from the Czech Republic, Estonia,
Croatia, Lithuania, and Sweden, including a specimen growing on Larix. The
position of this group is variable; it forms a well supported terminal clade in the
ITS phylogram, but it is placed in the centre of the tefa phylogram.
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