Major lineages of Dothideomycetes (Ascomycota) inferred from SSU and LSU rDNA sequences

Mycol. Res. 105 (8) : 901–908 (August 2001). Printed in the United Kingdom.

901

Major lineages of Dothideomycetes (Ascomycota) inferred from SSU and LSU rDNA sequences

H. Thorsten LUMBSCH* and Ralf LINDEMUTH Botanisches Institut, UniversitaW t Essen, 45117 Essen, Germany. E-mail : lumbsch!uni-essen.de Received 11 December 2000 ; accepted 14 March 2001.

The phylogeny of the Dothideomycetes was investigated using nucleotide sequences of SSU and LSU rDNA. 32 new sequences of these regions from 18 species were aligned with five sequences obtained from GenBank, including two representatives of Pezizomycetes used as outgroup. A combined data set of SSU and LSU rDNA was analysed phylogenetically using neighbour-joining and maximum likelihood. The pseudoparaphysate taxa (Pleosporales incl. Melanommatales) form a monophyletic group. A separation of Melanommatales can be rejected using parametric bootstrapping, and this supports previous results obtained from SSU rDNA studies. The aparaphysate Dothideomycetes form a sister-group to the Pleosporales, but with low bootstrap support. Within these species, two well-supported groups can be distinguished, the Dothideales s. str. and the Capnodiales with Myriangiales as sister-groups. The Myriangiales appear paraphyletic, but this has only poor bootstrap support. A monophyly of Dothideales comprising Capnodiales can be rejected with parametric bootstrapping as well as a subdivision of the loculoascomycetes based on form and opening of the ascomata and ascus. The phylogeny of the aparaphysate taxa requires further studies.

INTRODUCTION The loculoascomycetes is a group of ascomycetes including more than 6000 species (Hawksworth et al. 1995). The ascoma development is initiated with the production of an ascostroma and the asci are usually thick-walled and functionally bitunicate. Loculoascomycetes were originally proposed to be a separate group of ascomycetes on the basis of ontogenetic data (Nannfeldt 1932). Later, Luttrell (1951, 1955, 1973) put special emphasis on the bitunicate ascus-type present in this group of fungi. The loculoascomycetes are currently placed in two classes, viz. the Chaetothyriomycetes and Dothideomycetes (Eriksson & Winka 1997, Eriksson 2000). The Chaetothyriomycetes appear to be more closely related to the Lecanoromycetes (Winka, Eriksson & Ba/ ng 1998) or Eurotiomycetes (Berbee 1996, Liu, Whelen & Hall 1999, Lumbsch, Lindemuth & Schmitt 2000, Silva-Hanlin & Hanlin 1999). However, a sister-group relationship of the Chaetothyriomycetes and Dothideomycetes, and hence a monophyly of the loculoascomycetes cannot be rejected with the current molecular data (Liu et al. 1999, Lumbsch et al. 2000). The relationships of the Dothideomycetes to other classes remain uncertain, but also the subdivision of this class is currently very confusing. Some authors (von Arx & Mu$ ller 1975, Hawksworth et al. 1995) place all loculoascomycetes in one order, while Barr (1987) classifies the Dothideomycetes into seven orders. Other authors accept three * Corresponding author.

(Mu$ ller & von Arx 1950, 1962), five (Luttrell 1951, 1955, 1973), or six (Barr 1979) orders. Mu$ ller & von Arx (1950, 1962) used the form of the asci and the opening of the ascomata as main characters to distinguish groups among loculoascomycetes (Dothiorales with broad asci in non-ostiolate ascomata, Pseudosphaeriales with cylindrical-saccate asci in ostiolate ascomata, and Myriangiales with uniascal locules). The presence and type of hamathecium was used as the main character for recognizing different groups by Luttrell (1951, 1955, 1973) beside the recognition of orders with special ascoma-types, such as Hemisphaeriales with dimidiate-scutate pseudothecia or Hysteriales with apothecioid or lirelliform ascomata. The Pleosporales were defined as having pseudoparaphyses as hamathecium, while such hyphae are absent in the Dothideales. The circumscription of the Myriangiales basically remained unchanged between the different authors, although the affiliation of certain species varies. Barr (1979, 1987) based her classification on Luttrell in using hamathecial variation as the basis for the circumscription of orders in the loculoascomycetes, but recognized additional orders. These include, among others, the Melanommatales distinguished from the Pleosporales by trabeculate pseudoparaphyses, instead of cellular ones, and the Capnodiales (previously included in the Asterinales by Barr 1979) which were separated from the Dothideales on the basis of their superficial ascomata in abundant subiculum and by differing anamorphs. Given the confusing classifications based on morphology, and the unsatisfactory results of phylogenetic analyses based

Major lineages of Dothideomycetes on morphological characters due to extensive morphological homoplasy (Reynolds 1991), molecular data are urgently needed to elucidate the relationships among the loculoascomycetes (Eriksson 1994). 18S rDNA sequence data provided support for the distinction of the Pleosporales based on the presence of pseudoparaphyses as a monophyletic grouping (Berbee 1996, Liew, Aptroot & Hyde 2000, Silva-Hanlin & Hanlin 1999, Winka et al. 1998). The melanommatalean taxa clustered within the Pleosporales and hence their separation was not confirmed. The published RPB2 data do not allow any certain conclusion at the moment, although Sporormiella minima, the single Melanommatales included in that study, clustered within the Pleosporales (Liu et al. 1999). The Dothideales formed a monophyletic group in the 18S rDNA analyses by Berbee (1996) and Winka et al. (1998), but with poor bootstrap support. The relationships of other usually aparaphysate groups, such as Capnodiales and Myriangiales are currently not well understood. In the most recent classification, Eriksson (2000) accepted five orders in the Dothideomycetes, including Capnodiales, Dothideales, Myriangiales, Pleosporales, and the apothecioid Patellariales, but classified most members of the loculoascomycetes as incertae sedis. We have sequenced parts of the SSU rDNA (ca 1750 bp) and ca 1400 bp of the LSU rDNA of 18 Dothideomycetes to shed further light on the phylogeny within the Dothideomycetes. Beside the supplementation of the results of the published SSU rDNA analyses (i.e. monophyly of Pleosporales incl. Melanommatales), we were especially interested in the phylogenetic position of the Capnodiales and Myriangiales. In addition to a standard phylogenetic analysis, we have evaluated alternative topologies using parametric bootstrapping. With the help of such a statistical approach we can check the resolution ability of the data set to reject a topology not present in the most likely tree as being significantly worse or not.

902 primers.htmFLarge subunit RNA (25-28S) primer sequences) for the nuclear LSU rRNA gene. Amplifications were performed in 25 µl volumes containing a reaction mixture bead (Pharmacia Biotech Ready to Go PCR kit). 2n5 µl diluted DNA, 2n5 µl of each primer (10 µ), and 17n5 µL H O were # added. The amplification was performed in a Stratagene Robocycler using the following program : initial denaturation at 95 mC for 3 min, and 30 cycles of 95 m for 1n3 min, 45 m (or 48 m or 53 m) for 1n5 min, 73 m for 2n5 min, and a final extension at 73 m for 7 min. Sequencing Fragments were cleaned using the QIAquick PCR Purification kit (Qiagen) and sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing kit (Perkin Elmer). To obtain complete, overlapping sequences in both directions the following sequencing primers were used : (a) for the SSU rRNA gene : nu-SSU-0021-5h (Gargas & DePriest 1996), nuSSU-0402-5h, nu-SSU-0819-5h, nu-SSU-1203-5h, nu-SSU0852-3h, nu-SSU-1293-3h, nu-SSU-1750-3h (Gargas & Taylor 1992), nu-SSU-1184-3h (Gargas, DePriest & Taylor 1995), and nu-SSU-0553-3h (White et al. 1990) ; and (b) for the LSU rRNA gene : nu-LSU-0155-5h (l AL1R) (Do$ ring et al . 2000), nuLSU-0042-5h (l LR0R), nu-LSU-1050-5h (l LR17R), nu-LSU0654-5h (l LR3R), nu-LSU-0635-3h (l LR3), nu-LSU-1125-3h (l LR6), nu-LSU-1432-3h (l LR7) (Vilgalys homepage) and the newly designed primer nu-LSU-973-5h (AGGTAAAGCGAATGATTAG). Cycle sequencing was executed with the following program : 25 cycles of 95 mC for 30 s, 48 m for 15 s, 60 m for 4 min. Sequencing products were precipitated and dried before they were loaded on an ABI Prism 377 (Perkin Elmer) automatic DNA sequencer. Sequence fragments obtained were assembled with SeqMan 4n03 (DNAStar) and manually adjusted. Sequence alignment

MATERIALS AND METHODS Specimens and DNA extraction New SSU and LSU rDNA sequences were obtained from 18 specimens as listed in Table 1. The taxa were selected to include members of different orders according to varying authors, as listed in Table 2. Total DNA was extracted from fresh culture material using a modified CTAB method (Armaleo & Clerc 1995, Cubero et al. 1999). PCR Amplification Dilutions (10−") of the total DNA or undiluted DNA were used for PCR amplifications of the genes coding for the nuclear SSU and LSU rRNA. Primers (primer nomenclature follows Gargas & DePriest 1996) for amplification were : nuSSU-0021-5h (Gargas & DePriest 1996), nu-SSU-0819-5h, nuSSU-1293-3h, nu-SSU-1750-3h (Gargas & Taylor 1992) for the nuclear SSU rRNA gene, and nu-LSU-0155-5h (l AL1R) (Do$ ring et al. 2000), nu-LSU-0042-5h (l LR0R), nu-LSU1432-3h (l LR7) and nu-LSU-1125-3h (l LR6) (Vilgalys homepage : http :\\www.botany.duke.edu\fungi\mycolab\

Sequences of 18 species were aligned with sequences of three species obtained from GenBank (Table 1). Preliminary multiple alignments were generated using Clustal W (Thompson, Higgins & Gibson 1994) and manually optimized. Missing data at the 5h- and 3h-end of partial SSU and LSU rDNA sequences were coded by ‘?’. Major insertions were excluded. Portions of the alignment with ambiguous positions that may not be homologous were eliminated. The two separate data sets were combined and their congruence was examined, employing the partition homogeneity test (Farris et al. 1994) as implemented in PAUP*. Invariant characters were excluded before applying the test as recommended by Cunningham (1997). Phylogenetic analysis The alignment was analysed using the PAUP* 4n0 (Swofford 1998) and TREE-PUZZLE 5n0 (Strimmer & von Haeseler 1996) software packages. The polarity of characters was assessed using two representatives of Pezizomycetes as outgroup, which appear as a basal group to other filamentous ascomycetes in recent molecular studies (Gargas & Taylor

H. T. Lumbsch and R. Lindemuth

903

Table 1. Specimens of Dothideomycetes and Pezizomycetes used for phylogenetic analysis with GenBank accession number (newly obtained sequences in bold). GenBank accession no. Species

Collection\strain no.

SSU

LSU

Aureobasidium pullulans Bimuria novae-zelandiae Byssothecium circinans Capnodium citri Delphinella strobiligena Discosphaerina fagi Dothidea ribesia Kirschsteiniothelia aethiops Letendraea helminthicola Lojkania enalia Morchella esculenta Myriangium duriaei Phaeotrichum benjaminii Piedraia hortae Pleomassaria siparia Raciborskiomyces longisetosum* Setosphaeria monoceras Stylodothis puccinioides Trematosphaeria heterospora Westerdykella cylindrica Wilcoxina mikolae

– CBS 107n79 CBS 675n92 CBS 451n66 CBS 735n71 CBS 171n93 CBS 195n58 CBS 109n53 CBS 884n85 CBS 304n66 ESS 20437 CBS 260n36 CBS 541n72 CBS 480n64 CBS 279n74 CBS 180n53 CBS 154n26 CBS 193n58 CBS 644n86 CBS 454n72 –

M55639 AY016338 AY016339 AY016340 AY016341 AY016342 AY016343 AY016344 AY016345 AF053730 (first 543 bp)jAY016346 U42642 AY016347 AY016348 AY016349 AF164373 (first 775 bp)jAY016350 AY016351 AY016352 AY016353 AY016354 AY016355 U62014

AF050239 AY016356 AY016357 AY004337 AY016358 AY016359 AY016360 AY016361 AY016362 AY016363 U42669 (first 573 bp)jAY016364 AY016365 AY004340 AY016366 AY004341 AY016367 AY016368 AY004342 AY016369 AY004343 AF156926

* according to Barr (1997) (syn. Epipolaeum longisetosum). Table 2. Ordinal placement of Dothideomycetes according to different classifications.

Species

Mu$ ller & von Arx (1950, 1962)

Luttrell (1973)

Barr (1979)

Barr (1987)

Eriksson (2000)

Bimuria novae-zelandiae Byssothecium circinans Capnodium citri Delphinella strobiligena Discosphaerina fagi Dothidea ribesia Kirschsteiniothelia aethiops Letendraea helminthicola Lojkania enalia Myriangium duriaei Phaeotrichum benjaminii Piedraia hortae Pleomassaria siparia Raciborskiomyces longisetosum Setosphaeria monoceras Stylodothis puccinioides Trematosphaeria heterospora Westerdykella cylindrica

– Pseudosphaeriales Pseudosphaeriales Dothiorales Dothiorales Dothiorales (Pseudosphaeriales) Pseudosphaeriales Pseudosphaeriales Myriangiales Pseudosphaeriales (Myriangiales) Pseudosphaeriales – Pseudosphaeriales Dothiorales Pseudosphaeriales Pseudosphaeriales

– (Pleosporales) Dothideales Dothideales (Dothideales) Dothideales (Pleosporales) Pleosporales (Pleosporales) Myriangiales (Pleosporales) Myriangiales Pleosporales – (Pleosporales) (Dothideales) Pleosporales (Pleosporales)

– (Pleosporales) Asterinales (Dothideales) Dothideales Dothideales (Pleosporales) Pleosporales Melanommatales Myriangiales Pleosporales Myriangiales Pleosporales – Pleosporales (Dothideales) Melanommatales (Pleosporales)

Pleosporales Pleosporales Capnodiales Dothideales Dothideales Dothideales Pleosporales Pleosporales Melanommatales Myriangiales Pleosporales – Pleosporales – Pleosporales Dothideales Melanommatales Pleosporales

Pleosporales incertae sedis Capnodiales incertae sedis incertae sedis Dothideales Pleosporales incertae sedis incertae sedis Myriangiales incertae sedis incertae sedis incertae sedis incertae sedis Pleosporales Dothideales Pleosporales incertae sedis

(Names of orders are given in brackets when the genus is not mentioned expressis verbis in the cited text. Their ordinal placement is deduced from characters used by the respective authors.)

1995, Liu et al. 1999, Lumbsch et al. 2000). A maximum likelihood (ML) tree was inferred with PAUP* using the heuristic search option starting with a neighbor-joining tree with 200 simple sequence additions using the general-timereversible model with rate heterogeneity (Yang 1994). A search using random sequence addition was abandoned after ten days due to limited computer resources. Therefore the simple sequence addition modus was used for the final analysis. The model was chosen with a likelihood ratio test (Huelsenbeck & Crandall 1997) using the program MODELTEST (Posada & Crandall 1998). No bootstrap analysis was

performed on the ML tree because of the high computational requirements. As an alternative, quartet-puzzling support (QPS) values were calculated (with 10000 puzzling steps) using TREE-PUZZLE. A neighbour joining (NJ) tree was constructed using PAUP*. In the NJ analysis the LogDet transformation (Lockhart et al. 1994) was employed, which is consistent for sequences with differing nucleotide frequencies. All invariant sites were excluded as necessary for LogDet transformation (Huson 1998). Non-parametric bootstrap support (BSP) (Felsenstein 1985) for each clade was tested based on 2000 replications, using the neighbour-joining bootstrap

Major lineages of Dothideomycetes

904

Morchella esculenta Pezizomycetes

Outgroup

Wilcoxina mikolae Myriangium duriaei Myriangiales

87

Piedraia hortae 60

Capnodium citri 85

Capnodiales Raciborskiomyces longisetosum

52

Pseudoparaphyses lacking

Aureobasidium pullulans

97

Discosphaerina fagi 91

Dothideales

Delphinella strobiligena 97 100 98

Dothidea ribesia Stylodothis puccinioides Kirschsteiniothelia aethiops

98

Pleosporales

Phaeotrichum benjaminii 89

Lojkania enalia Melanommatales Trematosphaeria heterospora

100

Westerdykella cylindrica 95

Pseudoparaphyses present

Byssothecium circinans

64

Pleomassaria siparia

82

Setosphaeria monoceras Bimuria novaezelandiae

Pleosporales

Letendraea helminthicola

Fig. 1. ML tree obtained from a heuristic search using PAUP*. Quartet support values gained from a TREE-PUZZLE analysis are shown at nodes. Ordinal placement according to Barr (1979, 1987) and the presence of pseudoparaphyses is indicated at the margin.

option calculated with the LogDet transformation. Phylogenetic trees were drawn using TREEVIEW (Page 1996). Parametric bootstrapping (Goldman et al. 2000) was employed to check whether alternative topologies could be rejected as being significantly worse than the ML tree. For each test, a certain constrained ML tree was calculated using the same conditions as for the unconstrained ML search. Each constrained ML tree was used to generate 1000 simulated data sets for each actual test with the computer program SeqGen (Rambaut & Grassly 1997), using the parameters estimated from each constrained ML tree. The difference in likelihood values under the constrained and unconstrained hypothesis for each of the 1000 simulated data sets was then calculated for each test and used to generate a null distribution of likelihood differences. The null distribution was obtained from MP heuristic searches (200 random addition replicates) rather than ML searches to save computational time, as

suggested by Carlini et al. (2000). The likelihood value differences for the actual data set were then compared with the null distribution of each test to determine whether the actual difference was statistically significant. RESULTS Between 1699–2158 bp of SSU rDNA and 1396–1803 bp of LSU rDNA were obtained (including insertions). Approximately 98 % of the SSU and LSU rDNA of all species were sequenced in both directions. Major insertions were present in the genes of two species (Kirschsteiniothelia aethiops, SSU ; Dothidea ribesia, SSU and LSU) ; these were excluded from the analyses and will be discussed elsewhere. Sequences of the 21 taxa included in this study were aligned to produce a matrix of 3187 nucleotide-position characters. In the alignment, 1852 nucleotides were constant.

H. T. Lumbsch and R. Lindemuth

905

Morchella esculenta Pezizomycetes

Outgroup

Wilcoxina mikolae Myriangium duriaei Myriangiales

98

Piedraia hortae Capnodium citri

56 99

Capnodiales Raciborskiomyces longisetosum

60

Pseudoparaphyses lacking

Aureobasidium pullulans

100

Discosphaerina fagi 100

Dothideales

Delphinella strobiligena 100

100

100

Dothidea ribesia Stylodothis puccinioides Kirschsteiniothelia aethiops

83

Pleosporales

Phaeotrichum benjaminii 88

Lojkania enalia 100

Melanommatales

Westerdykella cylindrica Trematosphaeria heterospora

88

Pleosporales Melanommatales

Byssothecium circinans

Pseudoparaphyses present

95

59

Pleomassaria siparia 77

Setosphaeria monoceras 72 100

Pleosporales

Bimuria novae-zelandiae Letendraea helminthicola

Fig. 2. NJ tree obtained from a NJ-LogDet analysis using PAUP*. Bootstrap support values are shown at nodes. Ordinal placement according to Barr (1979, 1987) and the presence of pseudoparaphyses is indicated at the margin.

Twenty-two characters were excluded due to alignment ambiguities. The alignment is available in TreeBASE SN654 (http :\\herbaria.harvard.edu\treebase\). The partition homogeneity test revealed that the two data sets are congruent and can be analysed in a combined analysis. The tree obtained in a ML search (Fig. 1) has a likelihood value of k13068n883, base frequencies were estimated as follows : A l 0n26, C l 0n22, G l 0n28, and T l 0n24, the estimated value of the proportion of invariable sites was 0n552, and the gamma shape parameter was calculated as 0n542. The NJ tree (Fig. 2) has a very similar topology to the ML tree. The only difference in the topology is the sistergroup relationship of Trematosphaeria and Westerdykella in the ML tree, while Westerdykella is basal to Trematosphaeria in the NJ tree. However, in both cases there is no bootstrap support for this specific node. The ordinal placement of the species examined in this study in the various classifications is listed in Table 2. Taxa

producing pseudoparaphyses (i.e., Pleosporales sensu Luttrell 1973) form a monophyletic group with a QPS support of 89 % (88 % BSP in the NJ analysis). The two species with trabeculate pseudoparaphyses (Melanommatales sensu Barr 1979, 1987), Lojkania enalia and Trematosphaeria heterospora, cluster within the Pleosporales and a monophyly of the Melanommatales is rejected using parametric bootstrapping (Table 3). The aparaphysate species are the sister-group of the Pleosporales s. lat., but this grouping has no support (QPS : 52 %, BSP : 60 %). Within this group, two sister-groups can be distinguished : the Dothideales sensu Barr (1979, 1987) with 91 % QPS (BSP : 100 %) support, and the Myriangiales sensu Luttrell (1973) and Barr (1979) with the Capnodiales sensu Barr (1987) which have 87 % QPS (BSP : 98 %) support. The Myriangiales appear as paraphyletic and basal to the Capnodiales. However, there is only poor support (QPS : 60 %, BSP : 56 %) for a relationship of Piedraia hortae with the Capnodiales. The mitosporic fungus Aureobasidium pullulans clusters with 97 % QPS (BSP : 100 %)

Major lineages of Dothideomycetes

906

Table 3. Data of the parametric bootstrap analyses.

Constraints ML tree Monophyletic Dothideales sensu Luttrell (1973) Monophyletic Pseudosphaeriales sensu Mu$ ller & von Arx (1950, 1962) Monophyletic Melanommatales sensu Barr (1979, 1987)

Difference in likelihood value between unconstrained and constrained ML trees

Result of parametric bootstrap method (p)

Rejected?

– 29n78 109n171

– 0n001* 0n001*

Best Yes Yes

139n775

0n001*

Yes

support within the Dothideales. The monophyly of the Dothideales sensu Luttrell (1973), i.e. including the Capnodiales, is rejected by the parametric bootstrapping (Table 3). A placement of the Dothideomycetes according to the form and opening of the ascomata and asci, as proposed by Mu$ ller & von Arx (1950, 1962), into Pseudosphaeriales and Dothiorales, in addition to Myriangiales, is rejected by the parametric bootstrapping test (Table 3), in favour of a classification based on the presence of a hamathecium. DISCUSSION The results of our combined analysis of SSU and LSU rDNA sequence data revealed good bootstrap support for some clades of Dothideomycetes, while separate analyses of these data sets gave less resolution (data not shown), supporting a total evidence approach. Our results reinforce the previously published molecular studies, based on SSU rDNA data (Berbee 1996, Liew et al. 2000, Winka 2000) in supporting the monophyly of Pleosporales including the Melanommatales. In addition they allow a rejection of the monophyly of the Melanommatales based on parametric bootstrapping. These results suggest that the development of the ascomatal centrum (i.e. presence of pseudoparaphyses) is an important phylogenetic discriminator within this group of fungi, while the morphology of these hyphae appears as of minor importance. This agrees with opinions expressed, e.g. by Corlett (1975) and Eriksson (1981), who doubted the fundamental difference between cellular and trabeculate pseudoparaphyses. These pseudoparaphyses-types were used by Barr (1979), based on a concept by Groenhart (1965), as the main characters to separate Melanommatales from Pleosporales. As pointed out by Liew et al. (2000), the determination of the pseudoparaphysestype in mature ascomata may not be reliable. Detailed analyses of the early stages of development are required, however earlier studies sometimes yield differing results. For example in the type genus of the Pleosporales, Pleospora, very different observations of the hamathecial development have been published (Corlett 1975, Henssen & Thor 1994, PargueyLeduc 1966). A detailed re-evaluation of the early hamathecial development in the Pleosporales is urgently needed and may help us to understand the phylogeny within this group more clearly. The results of the molecular studies could provide a basis for the analysis of the evolution and taxonomic importance of this character set within the Pleosporales. The shape and opening of the ascomata, used by Mu$ ller & von Arx (1950, 1962) as the main character to subdivide the loculoascomycetes, also appears as of minor importance. A

grouping of Dothideomycetes according to this classification is rejected as significantly worse than the best tree, which places fungi having and lacking pseudoparaphyses together. In previous molecular studies including aparaphysate Dothideomycetes, only representatives of Dothideales sensu Barr (1979, 1987) were studied (e.g. Berbee 1996). The monophyly of this order is supported in our analyses. The circumscription of the Dothideales differs between authors, but our analyses supports a narrow concept of this order, excluding the Capnodiales. An alternative classification including Myriangiales and Capnodiales in Dothideales, however, would also be possible, since the aparaphysate taxa form a monophyletic group, although with poor bootstrap support. Such a group would somewhat resemble the suborder Dothideineae of von Arx & Mu$ ller (1975). No definite statement can be made with the data available. Additional examinations are required. The Capnodiales sensu Barr (1987) form a monophyletic group as also suggested by SSU analysis (Reynolds 1998). Using parametric bootstrapping a closer relationship to the Dothideales sensu Barr (1979, 1987) than to Myriangiales can be ruled out. This suggests that the development of the ascomatal centrum (i.e. asci growing in interthecial tissue or hamathecium disintegrating early in the development and absent in mature ascomata) used to unite these fungi in a subclass Loculoparenchemycetidae (Barr 1979) is due to convergence and hence this suborder appears polyphyletic. The Capnodiales were formerly (Barr 1979) included in the Asterinales. Since no species of this order was included in the present study, the relationships of these two orders remain uncertain. The two species of Myriangiales included in the present study appear as paraphyletic, although there is only poor support for this. Moreover, the placement of Piedraia in the Myriangiales is disputed. Von Arx (1963) excluded the genus from the order, while others placed it in the Myriangiales (Luttrell 1973, Barr 1979). Sequences of further species are needed to assess the monophyly of this order. Using molecular data, the phylogenetic position of some taxa, currently (Eriksson 2000) placed incertae sedis, could be inferred and previous classifications confirmed, such as the placement of Byssothecium, Letendraea, or Pleomassaria in the Pleosporales (Barr 1987). In most cases only single representatives of families were used and thus our sampling of taxa is insufficient to draw any conclusions at the family level. However, it is remarkable that the taxa of the two families represented by more than one species (Dothideaceae, Phaeotrichaceae) do not form monophyletic groups. Phaeotrichum and Westerdykella classified in the

H. T. Lumbsch and R. Lindemuth Phaeotrichaceae by Barr (1987) do not seem to be closely related. However, in Eriksson (2000) Westerdykella is placed in Sporormiaceae. Delphinella which was classified in the Dothioraceae by Barr (1987) appears to be more closely related to the dothideaceous Dothidea and Stylodothis than to Discosphaerina, a genus also placed in the Dothideaceae (Barr 1987). Liew et al. (2000) already pointed out that several currently accepted families in the Dothideomycetes appear to be polyphyletic and require revision. Although the monophyly of Pleosporales is supported by this combined data set, the phylogeny within this order appears poorly resolved with the current data. This could – at least partly – be due to a lack of variability in the sequence data of the nuclear SSU and LSU rDNA and we hope that a more variable gene, such as mitochondrial SSU rDNA, will be more appropriate to resolve the phylogeny within the Pleosporales. The inference of the phylogeny of the aparaphysate Dothideomycetes urgently needs the inclusion of additional taxa in the analyses and we have currently started a more thorough examination of the Dothideales and allies. A C K N O W L E D G E M E N TS Sonja Ja$ hnig is thanked for excellent technical assistance and Britta Tjaden for helpful comments on molecular techniques, Imke Schmitt (all Essen) kindly performed the parametric bootstrap analyses. Funds of this research were provided by the Deutsche Forschungsgemeinschaft for which we are appreciative.

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