Highlights of the Didymellaceae - Studies in Mycology

AveSkAMp et al.
2004), which is type species of the genus (Boerema 1964). This
clade also includes the type species of the Phoma sections Phoma,
Peyronellaea, Phyllostictoides, Sclerophomella and Macrospora.
Some phytopathologically and medically relevant species of the
section Heterospora are also associated with this clade, although
some species of this section are found in other clades, such as
Ph. samarorum (clade 6) and Ph. dimorphospora, and the sectional
type Ph. heteromorphospora (clade 7). Finally, a single species
of the setose section Paraphoma, Ph. gardeniae, is found in the
Didymellaceae. Based on the sequence data obtained in this study,
it is estimated that approximately 70 % of the species recognised by
Boerema et al. (2004) can be associated with the Didymellaceae.
Besides the many Phoma species, several other anamorph
fungi are found within this clade, including Ampelomyces quercinus,
Ascochyta fabae (teleom. Didymella fabae), Asc. hordei var.
hordei, Asc. pinodes (teleom. Didymella pinodes), Chaetasbolisia
erysiphoides, Didymella exigua, Epicoccum nigrum (synanamorph
Ph. epicoccina) and Microsphaeropsis olivacea. Of these species,
Asc. pisi, C. erysiphoides and M. olivacea are recognised as type
species for their respective genera. De Gruyter et al. (2009) already
discussed the probability of finding most of these non-Phoma taxa
in the Didymellaceae clade.
It should be noted that not all Ascochyta species are found
within this clade, indicating that this genus is also polyphyletic.
Whereas A. hordei var. hordei is found to be one of the basal
taxa of clade 8, the legume associated pathogens A. fabae, A.
pinodes and the type species A. pisi are found in close association
with several species of Phoma. This result is in congruence with
the observations in the study of Peever et al. (2007). Also the
recently described Didymella clematidis has an anamorph state in
Ascochyta and is closely related to Phoma taxa in this major clade
(Woudenberg et al. 2009). A representative strain Asc. caulina and
a new Ascochyta species that is still due to be published (G.J.M.
Verkley, pers. comm.), however, have been found to be only
distantly related and are found in clades 7 and 6, respectively.
Where a sexual state is known for the Phoma and Ascochyta
species in clade 8, it is Didymella. The type species of this teleomorph
genus, D. exigua, is also found within this clade, although it is not
associated with a Phoma anamorph state. The family Didymellaceae
was introduced for this group by De Gruyter et al. (2009). However,
type species of two other teleomorph genera have also been found
within this clade. DNA sequences of Leptosphaerulina australensis
resemble a high level of similarity with those of the various Phoma
and Didymella strains, although none are identical. Also sequences
of LSU and ITS sequence data obtained from GenBank of L.
americana, L. argentinensis, L. chartarum, L. crassiasca and L.
trifolii (GenBank accession no. AY278318, AY849949, EU272493,
U79485, AY8315585 respectively) were highly similar or even
identical to the Didymellaceae sequences obtained in the present
study (data not shown). These observations are in congruence with
the results obtained by Silva-Hanlin & Hanlin (1999), who found that
D. bryoniae (anam. Ph. cucurbitacearum) was closely related with
L. chartarum and L. crassiasca. Also Macroventuria anomochaeta,
which represents the genus Macroventuria (Van der Aa 1971)
groups in Didymellaceae. The close genetical resemblance of
Macroventuria and Leptosphaerulina found in the present study is
in congruence with the results of Kodsueb et al. (2006).
The loci employed here for phylogenetic analysis are sufficient to
identify clades at the family level, but for proper resolution at generic
level or lower, additional gene regions need to be sequenced. As
the majority of Phoma species is embedded in the Didymellaceae
clade, we will define further generic and species boundaries within
this recently established family in the subsequent part of this paper.
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Systematics of the Didymellaceae
DNA phylogenetic analysis
The alignment that was used to delineate the Didymellaceae
consisted of 274 sequences belonging to 196 species. A list of the
species names and numbers, original substrates, geographical
origins and GenBank Accession numbers of the strains used in this
study is provided in Table 3. The sequence matrix had a total length
of 2 188 characters including the alignment gaps (LSU: 1 327; ITS:
508 and TUB: 353). Of those characters, 1 788 (LSU: 1 233; ITS:
374 and TUB: 181) were constant, whereas 400 characters (LSU:
94; ITS: 144 and TUB: 192) were variable.
The analysis run of the LSU-ITS-TUB sequence matrix in
MrBayes was aborted after obtaining 20 000 trees, which was
well after stationarity in the probability of the trees was reached,
whereas the standard deviation of split frequencies was below 0.02.
From the obtained tree population, the 25 % burn-in was discarded
and the consensus tree and posterior probabilities were calculated.
The topology and support values of the BI tree were in congruence
with the optimal tree obtained in the ML analysis.
Systematics: treatment of clades
As most other anamorph genera, Phoma has largely been used as
a convenient form genus, rather than a phylogenetic entity. With
the number of Phoma species that are being analysed on DNA
sequence level rapidly increasing, the question is raised whether
form genera should be maintained or that more natural groupings,
merging both phylogeny and morphological data, should be erected.
Of course, as greater numbers of taxa are collected and analysed,
the taxonomic boundaries of more clades will be resolved. However,
for the present, only those genera that could be resolved based on
available cultures are treated. The groups mentioned below refer
to those indicated A–R in Fig. 2. The unresolved clades are left
untreated, and are thus not discussed.
The taxa in this part of the study were selected based on
genetic and/or morphological similarities with the species that were
associated with the Didymellaceae in Fig. 1. Although numerous
taxa from various genera have been associated with “Phoma”, the
number of genera that could be included in the selection for the
Didymellaceae was limited. Next to Phoma, the only species found
were those accommodated on basis of previous morphological
studies in either Ampelomyces, Ascochyta, Chaetasbolisia,
Coniothyrium, Didymella, Diplodina, Dothiorella, Epicoccum,
Leptosphaerulina, Macroventuria, or Microsphaeropsis. Of three of
these generic representatives, viz. Chaetasbolisia, Diplodina and
Dothiorella, we suspect that some cultures have been preserved
under an incorrect name. The species representing Ampelomyces,
A. quercinus, was correctly identified, but as suggested earlier,
the taxonomic placement in this genus appears to be incorrect
(Szentiványi et al. 2005).
Strains belonging to a single species proved to be genetically
identical or at least highly similar, indicating that the initial
identification of these strains had been carried out correctly.
Several well-supported clusters are recognised within this family
that are treated here as novel groups of the Didymellaceae. In
this section these separate groups are treated. However, although
multiple genes were employed in this study to generate a phylogenetic
reconstruction of the family, high levels of basal polytomy were
observed as well (Fig. 2). Application of general nrDNA loci alone did
not reduce this high level of polytomy, whilst interspecies variation in
several well-supported clades was reduced drastically.