The internal transcribed spacers, ITS1 and ITS2, and 5.8S region
of ribosomal DNA (rDNA) from 20 species of Ascosphaera were
amplified by PCR, and their sequences determined and compared. No variation
was detected in the sequences from 23 widely
distributed isolates of A. apis, in sequences from 11 widely distributed
A. atra isolates, in four A. aggregata isolates, or in
sequences
from two isolates each of A. acerosa, A. duoformis,
A. flava, A. larvis, A. pollenicola and
A. proliperda. However, the ribosomal sequences
from each of these nine species, and from another 11 species of which only
a single isolate was examined, differed from one another
by 0·18–30·9%. Thus these sequences provide a rapid
method for identifying species, and searches using them showed that the
sequence of A. apis rDNA recorded in the international databases
is,
in fact, that of A. atra.
The rDNA sequences also provided data for assessing the relationships
of
these fungi. Of the rDNA sequences in current
international databases, that of Eremascus albus was very close
to,
but distinct from, those of the Ascosphaera species. Comparisons
of
the Ascosphaera sequences showed that most formed consistent clusters
irrespective of the method of comparison used (distance
matrix and parsimony), or whether the ITS1 or ITS2 portions were used;
A. acerosa with A. asterophora, A. atra with
A. duoformis,
A. colubrina closely with A. flava, A. larvis,
A. major, A. variegata and more distantly with
A. apis and A. celerrima, and, also,
A. aggregata with A. subcuticulata, A. proliperda
and more distantly with A. solina. The apparent relationships
of these clusters were
inconsistent, depended on the alignment of several regions of ‘indels’,
and could not be resolved. The A. fusiformis, A. naganensis
and
A. osmophila sequences showed inconsistent relationships with
the others,
especially that of A. osmophila, which had an A. solina-like
ITS2 region, but an atypical ITS1 sequence with a large unique repetitive
insertion. The clusters based on gene sequence
comparisons clearly correlated with groupings based on ascospore morphology
and other characters.