Phylogenetic Significance of the Pseudoparaphyses in Loculoascomycete Taxonomy

Molecular Phylogenetics and Evolution Vol. 16, No. 3, September, pp. 392– 402, 2000 doi:10.1006/mpev.2000.0801, available online at http://www.idealibrary.com on

Phylogenetic Significance of the Pseudoparaphyses in Loculoascomycete Taxonomy Edward C. Y. Liew,* ,1 Andre´ Aptroot,† and Kevin D. Hyde* *Centre for Research in Fungal Diversity, The University of Hong Kong, Pokfulam Road, Hong Kong; and †Centraalbureau voor Schimmelcultures, P.O. Box 273, NL-3740 AG Baarn, The Netherlands Received September 22, 1999; revised March 9, 2000

The ontogeny of the ascostroma, in particular the centrum structures, has always been regarded as an important criterion in the subdivision of the Loculoascomycetideae (ascomycetous fungi). However, the use of pseudoparaphysis type, cellular or trabeculate, to classify taxa at the ordinal level has been contentious due to the lack of information about their evolution. To determine the phylogenetic significance of the pseudoparaphysis and its variants, DNA sequences of the 18S nuclear rRNA genes from representatives of the orders Pleosporales and Melanommatales were obtained and analyzed. Species with pseudoparaphyses formed a monophyletic group with high statistical confidence. The monophyly of a distinct lineage of species with cellular pseudoparaphyses (the order Pleosporales) is rejected. Likewise, monophyly of a distinct lineage of species with trabeculate pseudoparaphyses (the order Melanommatales) is rejected also. The Pleosporales and Melanommatales are, therefore, not natural orders. The Lophiostomataceae, Phaeosphaeriaceae, and Melanommataceae are most probably polyphyletic, as is the genus Massarina. © 2000 Academic Press

Key Words: pseudoparaphysis; pleosporales; melanommatales; loculoascomycete; bitunicate ascomycete; molecular phylogenetics.

INTRODUCTION The recognition of a distinct series of ascomycetes in which the asci are produced in locules in an ascostroma began by Fuckel (1870) with the establishment of the Dothideaceae (Luttrell, 1951). Since then various names have been applied to this ascostromatic group of ascomycetes, viz. Dothideales (Ho¨hnel, 1907, 1909), Dothidiineae (Theissen and Sydow, 1918), Ascoloculares (Nannfeldt, 1932), and Bitunicatae (Luttrell, 1 To whom correspondence should be addressed at Department of Ecology and Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong. Fax: (852) 2559 5984. E-mail: ecyliew@hkucc. hku.hk.

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1951). Luttrell (1955) proposed a formal taxonomic status for this group, referring to it as the Loculoascomycetes, a subclass to the ascomycetes, characterized by “ascis bitunicatis, in ascostromate evolutis.” To conform to nomenclatural conventions, this name was later modified to Loculoascomycetidae (Luttrell, 1981). Since Nannfeldt (1932) defined the ascostromatic ascomycetes as distinct from the Ascohymeniales (the Euascomycetes), the taxonomic subdivision within this group of ascomycetes has undergone a considerable amount of revision. The taxonomic confusion has been primarily due to the differing emphasis placed on various characters and interpretation of different aspects of the ascoma ontogeny. The Loculoascomycetidae have been arranged into 4 orders (Nannfeldt, 1932), 3 orders (Mu¨ller and Arx, 1950, 1962), 5 orders (Miller, 1949; Luttrell, 1951, 1955, 1973; Wehmeyer, 1975; Dennis, 1981), 1 order (Arx and Mu¨ller, 1975), 8 orders (Barr, 1976, 1979), or 10 orders (Barr, 1987b), with various infraordinal organizations. Most recently, Hawksworth et al. (1995) placed all of these ascostromatic ascomycetes into 1 order, the Dothideales, subdivided into 58 families. The recognition of the ascostroma (Ho¨hnel, 1907, 1909; Theissen and Sydow, 1918; Nannfeldt, 1932) as the essential character uniting all members of the Loculoascomycetidae focused research on patterns in the ontogeny of the ascostromatic ascoma. Although Miller (1949) had earlier described and included centrum characteristics as important criteria for his ordinal arrangement, Luttrell (1951, 1955) was the first to define the different loculoascocarp developmental types: monascous locules characterizing the Myriangiales, represented by Elsinoe¨ Racib.; fascicles of aparaphysate asci characterizing the Dothideales, represented by Dothidea Fr.; and the pseudoparaphysate centrum characterizing the Pleosporales, represented by Pleospora Rabenh. The main diagnostic character in the Pleosporales is the pseudoparaphysis, which has been the cause of much confusion and discussion. Luttrell (1951, 1955) defined pseudoparaphyses as distinct, vertical, paraph-

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ysis-like hyphae, which occupy the locule prior to the formation of asci. They are usually observed to grow downward from the upper portion of the ascoma and are ultimately attached at both ends of the hyphae. These pseudoparaphyses had previously been thought to be remnants of the stromal tissue persisting as interthecial strands (threads) as the asci develop (Ho¨hnel, 1907; Theissen and Sydow, 1918; Nannfeldt, 1932). Subsequent workers (Miller, 1949; Luttrell, 1955; Wehmeyer, 1955) noted this to be a misconception, which contributed greatly to the confusion in the ordinal delimitation. If these pseudoparaphyses were remnants of the disintegrating stromal tissue, then species in which the interthecial tissue remains between the mature asci (Myriangiales) and species in which no interthecial tissue is present (Dothideales) would be developmentally the same as those with pseudoparaphyses, differing only in degrees to which the interthecial tissue is compressed or has disintegrated (Luttrell, 1955). Groenhart (1965) pointed out that the term “developmental type” should indicate a uniformity of ascoma development in all species of a particular type. He further noted that ascomata of several species regarded as belonging to the Pleospora-type develop very differently. Wehmeyer (1954) observed that the ascoma development of Pyrenophora trichostoma (Fr.) Fuckel [as Pleospora trichostoma (Fr.) Ces. & de Not.] was largely of the Pleospora-type but, rather than being vertically arranged in the locule, the pseudoparaphyses grew out from the individual stromal cells with a roughly radial orientation. They branched and twined about each other and were not attached at both ends. Similarly, in Pleospora armeriae (Corda) Ces. & De Not., the pseudoparaphyses were observed to grow in an indefinite radial fashion initially, and only later were they arranged vertically, especially in the upper part of the ascostroma (Wehmeyer, 1955). Kerr (1961) compared the ascoma development of Pleospora herbarum (Pers.) Rabenh. and several species of Venturia Sacc. and noted that whereas the species of Venturia and P. herbarum had the same developmental type, the downward growth of pseudoparaphyses with free ends was not observed in either genus. Rather, the pseudoparaphyses appeared to be always attached at both ends. Moreover, there was no evidence of radially ingrowing hyphae. Kerr (1961) concluded that the variations described by various workers were largely due to the differences in interpretation of sections. The intercalary growth of pseudoparaphyses with no free ends had been described earlier by Chesters (1938) in Melanomma pulvis-pyrius (Pers.) Fuckel, M. fuscidulum Sacc., and Thyridaria rubro-notata (Berh. & Broome) Sacc., in which the central cells of the developing ascoma separated into strands which remained attached to the outer cells at the apex and base. Chesters (1938) named these strands, which were visual-

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ized as branched and anastomosing, trabeculae. More recently, similar structures were described in several species of Lophiostoma Ces. & De Not. (Chesters and Bell, 1970). Kowalski (1965) observed similar pseudoparaphyses which were attached to the top and bottom of the ascoma from the start of development in Preussia typharum (Sacc.) Cain, but named them paraphysoids. Groenhart (1965) introduced the term tinophyses to characterize the pseudoparaphyses in P. herbarum as interpreted by Kerr (1961) and the trabeculae in M. pulvis-pyrius, M. fuscidulum, and T. rubro-notata described by Chesters (1938) and in species of Lophiostoma by Chesters and Bell (1970), as well as the paraphysoids in Preussia typharum described by Kowalski (1965). In contrast, cataphyses were introduced to characterize pseudoparaphyses which grow downward from the roof of the locule as those described in Sporormia leporina Niessl. by Arnold (1928) and those of Luttrell’s earlier descriptions of the Pleospora-type development (Luttrell, 1951, 1955). In view of the variations reported by various workers and the resulting confusion, Corlett (1973) made detailed studies on the ascoma development of P. herbarum and noted that pseudoparaphyses appeared to grow in essentially the same manner as vegetative hyphae, i.e., by apical expansion. In addition, certain intercalary cells were observed to undergo elongation. As the pseudoparaphyses grew downward, the free lower ends generally became attached at the base of the centrum. They intertwined and anastomosed with ascogenous hyphae and basal pseudoparenchyma, prior to the development of the asci. Corlett’s observations and interpretations were generally in accordance with Luttrell’s original descriptions (Luttrell, 1951, 1955). In addition, Corlett (1973) collated and reviewed the data of all previously studied Pleosporales species in terms of their centrum development. He maintained that the Pleospora-type was remarkably uniform and variations, in particular the trabeculae (paraphysoids, tinophyses), were a result of differences in interpretation because earlier stages of the ascoma development were not observed. In revising the classification of the Loculoascomycetidae, Barr (1976, 1979, 1987b) emphasized the variations of the pseudoparaphysis appearance, rather than the origin. On this basis, she separated species having the Pleospora-type development into two orders, viz. the Pleosporales, characterized by the presence of cellular pseudoparaphyses, and the Melanommatales, characterized by the presence of trabeculate pseudoparaphyses. According to Barr’s (1979) definitions, cellular pseudoparaphyses are septate, branched or unbranched, sometimes anastomosing above the asci, and at times deliquescing at maturity (as in P. herbarum), whereas trabeculate pseudoparaphyses are narrow, threadlike, apparently nonseptate, and branched and

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anastomosing or unbranched above the asci and embedded in a gelatinous matrix (as in Melanomma, Thyridaria, and Lophiostoma). However, her comparison of trabeculate pseudoparaphyses as being equivalent to Groenhart’s (1965) cataphyses and cellular pseudoparaphyses to Groenhart’s tinophyses is somewhat confusing and contradictory. Eriksson (1981) also pointed out this confusion and emphasized the importance of studying the early stages of ascoma development. Although the ordinal separation of the Pleosporales and Melanommatales (Barr, 1976, 1979, 1987b) has been widely accepted, the phylogenetic significance of this classification has never been tested by a phylogenetic analysis using molecular data. However, a phylogenetic analysis using morphological characters was performed by Reynolds (1991), who could not find much support for the monophyly of these taxa. Recently Berbee (1996), based on the analysis of ribosomal DNA (rDNA) sequences, showed convincingly that the Pleosporales form a monophyletic group. This monophyletic group, however, included one species, Sporormia lignicola Phill. & Plowr., with trabeculate pseudoparaphyses (Berbee, 1996). Silva-Hanlin and Hanlin (1999), using the same DNA region, examined the phylogenetic relationships of S. lignicola and Sporormiella australis Speg. and found that both these genera clustered within the Pleosporales with high bootstrap support. The current investigation aims to assess the phylogenetic significance of the pseudoparaphysis, as well as the pseudoparaphysis types, by examining the relationships of representative species of the Pleosporales and Melanommatales. DNA sequence data from the 18S small subunit (SSU) of the rDNA were used for the analysis. MATERIALS AND METHODS Cultures and DNA Sequences Fungal strains and GenBank sequences used in the study are listed in Table 1. All cultures were obtained from the Centraalbureau voor Schimmelcultures (CBS) and Hong Kong University Culture Collection (HKUCC). Cultures from CBS were purified by subculturing from a single hyphal tip onto the culture medium recommended by CBS. The HKUCC cultures were all isolated from single spores. All cultures were subcultured onto V8 agar (40% Campbell V8 Juice) 4 to 8 days prior to DNA extraction for rapid growth. DNA Extraction Actively growing mycelia were directly scraped off culture plates and transferred into 1.5-ml centrifuge tubes. DNA extraction followed a modified protocol of Doyle and Doyle (1987). Approximately 0.05 g of my-

celium was mixed with ca. 0.3 g of white quartz sand (Sigma) in warm (ca. 60°C) 2⫻ CTAB buffer [2% (w/v) CTAB; 100 mM Tris–HCl; 1.4 M NaCl; 20 mM EDTA, pH 8.0]. Mycelium–sand mixture was ground with a glass pestle and incubated at 60°C for 1 h before being subjected to multiple phenol:chloroform (1:1) extractions. DNA was precipitated from the purified aqueous extraction layer by ethanol precipitation. The DNA pellet was washed (70% ethanol), dried (vacuum centrifuge), and resuspended in 100 ␮l TE buffer. DNA samples were checked for purity and integrity by gel electrophoresis before storing at 4°C. DNA Fragment Amplification The 18S rDNA was partially amplified using primers NS1 and NS4 (White et al., 1990). Two to 5 ␮l of suspended DNA was used for each polymerase chain reaction (PCR) with 1.5 mM MgCl 2, 0.2 mM each dNTP, 0.3 ␮M each primer, and 2.0 U of Taq DNA polymerase in a 50-␮l reaction volume. The thermal cycling program was as outlined in White et al. (1990), with primer annealing at 52°C for 50 s. The size of each amplified fragment was verified by gel electrophoresis with EtBr staining of a 2-␮l product sample and visualized over a uv transilluminator. PCR products were purified using the Wizard PCR Preps DNA Purification System (Promega) and the purified products were further assessed for purity and sufficient concentration by gel electrophoresis (using 0.2 ␮l). Where necessary, fragment suspensions were further concentrated in a vacuum centrifuge and stored for not more than 1 week before DNA sequencing. DNA Sequencing The amplified 18S DNA fragments were directly sequenced using the ALFexpress Automated DNA Sequencer AM V 3.0 (Pharmacia Biotech). Sequencing reactions were conducted in a thermal cycler using the Auto Cycle 200 Sequencing Kit (Pharmacia) according to the manufacturer’s recommendations. CY-5-labeled primers NS1, NS2, NS3, and NS4 (White et al., 1990) were used, which allowed for the determination of both DNA strands. The optimal annealing temperature for each primer used in the sequencing reaction was empirically determined, taking into consideration the calculated temperature based on the manufacturer’s instructions for the Sequencing Kit. Sequence Alignment For each fungal strain, four separate sequences obtained for the respective primers were manually aligned to obtain a consensus sequence using the biosequence editor SeqPup v0.8 (Gilbert, 1998). During this process of alignment, individual bases were verified by comparison with the fluorescence signal printout for each sequence. Consensus sequences for each strain, together with the sequences obtained from Gen-

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TABLE 1 Fungal Taxa and Their 18S rDNA GenBank Accession Nos. Species

Source of culture a

GenBank Accession No.

Order sensu Hawksworth et al. (1995)

Order sensu Barr (1987b)

Botryosphaeria quercuum (Schw.:Fr.) Sacc. B. rhodina (Berk. & M.A.Curti.) Arx B. ribis Grossenb. & Duggar Cucurbitaria berberidis (Pers.:Fr.) Gray Dangeardiella macrospora (Schr.) Sacc. & Syd. Delitschia winteri Phill. & Plowr. Didymella exigua (Niessl.) Sacc. Dothidea insculpta Wallr. Entodesmium rude Riess. Herpotrichia diffusa (Schw.) Ellis & Everh. Hypocrea schweinitzii (Fr.) Sacc. b Hysterium pulicare Pers.:Fr. Hysteropatella clavispora (Peck) Seaver Keissleriella cladophila (Niessl.) Corb. Leptosphaeria doliolum (Pers.:Fr.) Ces. & De Not Leptospora rubella (Pers.:Fr.) Rabenh. Lophiostoma caulium (Fr.) Ces. & De Not b Massaria platani Ces. Massarina australiensis K.D.Hyde b Massarina bipolaris K.D.Hyde b Massarina eburnea (1) (Tul. & Tul.) Sacc. b Massarina eburnea (2) (Tul. & Tul.) Sacc. Massariosphaeria phaeospora (Mu¨ll.) Criv. Melanomma pulvis-pyrius (Pers.:Fr.) Fuckel Montagnula opulenta (De Not) Aptroot b Phaeodothis winteri (Niessl.) Aptroot b Phaeosphaeria nodorum (Mu¨ll.) Hedj. Pleomassaria siparia (Berk. & Broome) Sacc. Pleospora herbarum (Fr.) Rabenh. ex Ces. & De Not Pseudotrichia aurata (Rehm) Wehm. Pyrenophora trichostoma (Fr.:Fr.) Fuckel Rhytidhysteron rufulum (Spreng.) Speg. Sordaria fimicola Ces. & De Not Taphrina deformans (Berk.) Tul. Trematosphaeria hydrela (Rehm) Sacc. Xylaria hypoxylon (Linn.:Fr.) Grev.

CBS 177.89 — — — CBS 179.58 CBS 139.83 CBS 183.55 — CBS 650.86 — CBS 836.91 CBS 239.34 CBS 247.34 CBS 104.55 — CBS 132.80 CBS 624.86 CBS 222.37 HKUCC189 HKUCC1053 HKUCC4054 CBS 473.64 CBS 611.86 CBS 109.77 CBS 168.34 CBS 182.58 CBS 438.87 CBS 279.74 — CBS 143.41 — CBS 306.38 — — CBS 880.70 —

AF164352 U42476 U42477 U42481 AF164353 AF164354 AF164355 U42474 AF164356 U42484 AF164357 AF164358 AF164359 AF164360 U04205 AF164361 AF164362 AF164363 AF164364 AF164365 AF164366 AF164367 AF164368 AF164369 AF164370 AF164371 AF164372 AF164373 U05201 AF164374 U43459 AF164375 X69851 U00971 AF164376 U20378

Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Hyocreales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Pyrenulales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Dothideales Patellariales Sordariales Taphrinales Dothideales Xylariales

Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Melanommatales Pleosporales Dothideales Pleosporales Pleosporales — Pleosporales Patellariales Melanommatales Pleosporales Pleosporales Pleosporales Melanommatales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Melanommatales Pleosporales — Pleosporales Pleosporales Pleosporales Melanommatales Pleosporales Patellariales — — Melanommatales —

a Cultures were obtained for new sequences only. CBS, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; HKUCC, The University of Hong Kong Culture Collection, The University of Hong Kong, Hong Kong. b Identification of cultures verified by morphological structures or comparison of sequence similarity of more than one strain/accession.

Bank, were aligned using Clustal W (Thompson et al., 1994). The result was further adjusted manually to allow for maximum alignment. Phylogenetic Analysis The data were subjected to two methods of phylogenetic analysis: maximum-parsimony (MP) and maximum-likelihood (ML). Searches for most-parsimonious trees were conducted using the heuristic maximumparsimony algorithms with random stepwise sequence addition and tree bisection–reconnection branch swapping in PAUP 3.1.1 (Swofford, 1993). For each search, 1000 replications were performed. Gaps (insertions and deletions) of more than one nucleotide in length were not observed in the alignment data and all sites were subsequently included in the phylogenetic analy-

ses. Gaps were treated as fifth character state and were given equal weight as the others. Character states were all treated as unordered. A symmetric stepmatrix as an assumption block in the analysis input was used assigning a transition:transversion ratio of 2. Rooting of trees was determined by the inclusion of three unitunicate ascomycetes and a basal ascomycete in the data set, as well as the specific assignment of the basal ascomycete (Taphrina deformans) as the outgroup when setting the heuristic search parameters. Statistical support for the internal branches was estimated by bootstrap analysis with 1000 replications. Analysis using maximum-likelihood algorithms (Felsenstein, 1981) was performed using the DNAML program from the PHYLIP v.3.5c computer package

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FIG. 1. The 50% majority-rule consensus cladogram generated from 16 equally most-parsimonious trees containing 36 taxa. The designated outgroup is Taphrina deformans. Values at branch nodes indicate percentage of trees (of the 16) in which a particular branch node appears.

(Felsenstein, 1995). The resulting expected ratio of transition:transversion was set at 1.0 or 2.0. One category of substitution rates for all characters was used. Input order of sequences was randomized once with global rearrangements. T. deformans was used as the outgroup to root the tree. RESULTS Approximately 1100 bp of the 5⬘ end of the SSU rDNA for each strain yielded 1690 aligned nucleotide positions, of which 149 were phylogenetically informa-

tive. Heuristic search for most-parsimonious trees with 1000 replications and random addition of taxa yielded 16 equally most-parsimonious trees. A 50% majorityrule consensus tree was generated (Fig. 1). The branch support of the MP tree was evaluated using bootstrap analysis with 1000 replications (Fig. 2). The monophyly of loculoascomycete species with pseudoparaphysate centrum is highly supported with a bootstrap confidence of 91% (Figs. 1 and 2). There is, however, no evidence for separate monophyletic lineages of species with cellular pseudoparaphyses and species with trabeculate pseudoparaphyses.

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FIG. 2. Phylogram of single most-parsimonious tree containing 36 taxa (length ⫽ 553 steps, consistency index ⫽ 0.649, and retention index ⫽ 0.724). Bootstrap values ⬎50% from 1000 bootstrap replicates are shown at the nodes. The designated outgroup is Taphrina deformans. Branch lengths are proportional to nucleotide substitutions.

Within the pseudoparaphysis clade, there appear to be several subclades (sister groups) with high bootstrap support (78 –99%), although the basal branches (between these subclades) did not receive high bootstrap support (Fig. 2). Three of these subclades contain species with cellular pseudoparaphysis, as well as species with the trabeculate type. Pseudotrichia aurata, with trabeculate pseudoparaphyses, clustered within the subclade comprising cellular pseudoparaphysate species, Pleospora herbarum, Pyrenophora trichostoma, Leptosphaeria doliolum, etc. (subclade A); three trabeculate pseudoparaphysate species, Didymosphaeria astragalina, Massaria platani, and Keissleriella cladophila, formed a cluster with cellular pseudoparaphysate species, Montagnula opulenta, Massarina eburnea, and Massariosphaeria phaeospora (subclade B); and similarly Melanomma pulvis-pyrius clustered together with Dangeardiella macrophila, Herpotrichia diffusa, and Pleomassaria siparia (subclade D). Another group within the pseudoparaphysis lineage com-

prises Lophiostoma caulium, Massarina australiensis, and Massarina bipolaris (subclade C). L. caulium clustered with the two Massarina species with high bootstrap support (83%). The Lophiostomataceae, Phaeosphaeriaceae, and Melanommataceae (sensu Hawksworth et al., 1995 or Barr, 1987) do not appear to be monophyletic, whereas the two Pleosporaceae representatives formed a clade with strong bootstrap support (100%) (Fig. 2). Although the two strains of Massarina eburnea clustered together with 100% bootstrap support (subclade B), M. australiensis and M. bipolaris appear to be phylogentically distinct from M. eburnea, clustering together with Lophiostoma caulium instead (subclade C) (Fig. 2). The three Botryosphaeria species clustered together in the Dothideales clade, but the clustering with Dothidea received low bootstrap support (Fig. 2). Hysteropatella clavispora [Patellariaceae sensu Barr (1987b)] and Hysterium pulicare (Hysteriaceae), which clus-

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FIG. 3. Cladogram with the highest likelihood with 36 taxa generated from a maximum-likelihood search, with the expected transition:transversion ratio set at 2.0. Taphrina deformans is the designated outgroup.

tered together in 98% of the bootstrap replicates, also clustered within this Dothideales clade with low bootstrap support. For the maximum-likelihood analysis of the same alignment data with the transition:transversion ratio set at 2.0, a ML tree of log likelihood ⫺6224.48 was obtained (Fig. 3). Nucleotide frequencies were estimated from the data set to be A ⫽ 0.27, C ⫽ 0.20, G ⫽ 0.26, and T ⫽ 0.27. When the transition:transversion ratio was set at 1.0, a ML tree with a similar topology was obtained but with a lower log likelihood (⫺6253.49) (results not shown). A comparison between trees obtained from the two different algorithms shows that the MP and ML trees are nearly identical in their topologies (Figs. 2 and 3). The trees differ only slightly in the branching pattern within the subclades and in the relationships between the subclades C and D. DISCUSSION Monophyly of the Pseudoparaphyses The current investigation supports the monophyly of loculoascomycete species with pseudoparaphysate cen-

trum. Based on 18S rDNA sequence data, 24 species disposed in either the Pleosporales or the Melanommatales (sensu Barr, 1979, 1987b) clustered together, forming a monophyletic clade with high bootstrap support (91%) in the maximum-parsimony analysis. The clustering of this clade was corroborated by the analysis based on the maximum-likelihood algorithm. Although Hysterium pulicare and the three Botryosphaeria species clustered with Dothidea, this phylogenetic affinity is not resolved due to the absence of bootstrap support. Moreover, these two genera, placed within the Pleosporales by Barr (1979, 1987b), did not consistently cluster within the Dothideales clade when different parameters were employed or when different combinations of taxa were included in the analyses (results not shown). The monophyly of pseudoparaphysate species based on rDNA sequence data has previously been shown by Berbee (1996) and Winka et al. (1998), but with smaller samples of species. Berbee (1996) suggested that the presence of pseudoparaphyses is a good monophyletic character to delimit pleosporaceous taxa. Silva-Hanlin and Hanlin (1999), however, disagreed with this viewpoint, because of two Leptosphaerulina species (nonpseudoparaphysate) clustering within the Pleoporales clade with high bootstrap confidence in their study. The current work involves a more representative sample of taxa, compared to these previous studies, with the inclusion of more melanommataceous species (sensu Barr, 1979, 1987b) and more representative families. The Hysteriaceae, although often linked with the Patellariaceae based on the apothecioid ascoma with an elongated opening and on the structure of the peridium (Luttrell, 1974; Arx and Mu¨ller, 1975; Barr, 1987b), is commonly regarded as having the Pleosporatype (cellular) pseudoparaphyses (Miller, 1949; Luttrell, 1973; Barr, 1987b) or paraphysoids (trabeculae) (see Eriksson, 1981; Belleme`re, 1971). Zogg (1962), in his monograph of the family, noted that the pseudoparaphyses (as paraphysoids) were a result of the stretching of the parenchymatous ascomal wall. However, Luttrell (1953) had already observed that the pseudoparaphyses of Glonium stellatum Muhl.: Fr., another representative of the Hysteriaceae, grow downward from the cavity roof. This appeared to be representative of the Pleospora-type, although the typical vertical arrangement of these interascal hyphae at early stages was not observed. Luttrell (1953) also noted several other differences and suggested that the Hysteriaceae and Pleosporaceae should be placed in separate orders. The ontogeny of the pseudoparaphyses in Hysterium pulicare, which could be more closely related to that in the Patellariales, has not been studied. The monophyletic nature of taxa with pseudoparaphysate centrum could be further verified by re-

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solving the phylogeny of Hysterium pulicare and Botryosphaeria species. A wider sampling of representatives from the Hysteriaceae, Botryosphaeriaceae, and taxa having the Dothidea-type centrum would be required. Neither Berbee (1996) nor SilvaHanlin and Hanlin (1999) were able to determine conclusively the ordinal placement of Botryosphaeria. Berbee (1996) suggested that Botryosphaeria could very well have affinities to both the Pleosporales and the Dothideales, which would concur with it having a combination of both pleosporareous and dothideaceous morphological features. A curious point is that the pseudoparaphyses in Botryosphaeria are not persistent in mature ascomata and hence resemble the aparaphysate Dothidea-type ascomata. Evolution of Cellular and Trabeculate Pseudoparaphyses The hypothesis of separate evolutionary lineages of species with cellular pseudoparaphyses and species with trabeculate pseudoparaphyses, respectively belonging to the Pleosporales and the Melanommatales (sensu Barr, 1983, 1987b), is rejected in the current study. Species from these two groups did not cluster into two distinct clades in any of our phylogenetic analyses. Moreover, combinations of both types of species grouped together in several subclades with high bootstrap values. Berbee (1996) showed that Sporormia lignicola Phill. & Plowr. [Sporormiella lignicola sensu Ahmed and Cain, (1972)], the only species with trabeculate pseudoparaphyses used in her analysis, nested confidently within her Pleosporales clade, and Silva-Hanlin and Hanlin (1999) obtained similar results with Sporormia lignicola and Sporormiella australis Speg. The differences between these two types of pseudoparaphysis, regardless of the definitions followed (Groenhart, 1965; Eriksson, 1981; Barr, 1983, 1987b), appear to be straightforward. However, in reality, this character is often determined without thorough examination of the early developmental stages. The determination of the pseudoparaphysis type present (if at all present) in the mature ascoma is often subject to personal interpretation and hence may not be reliable (Corlett, 1975; Eriksson, 1981; Janex-Fauvre, 1988; Eriksson and Hawksworth, 1992). Whether the pseudoparaphyses are attached to the ascomal wall at both ends is frequently anomalous in developmental studies conducted. Corlett (1973) maintained that the lack of examination of very early stages could be the reason for not observing the pseudoparaphysis free-ends, corresponding to the development of trabeculae. If indeed these pseudoparaphysis types are not uniform in their development, then the different types would have evolved separately several times after the divergence of the general pseudoparaphysis lineage. Furthering this line of argument, the determination of

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the ancestral type, and the number of times a particular type appeared or was lost, would require as prerequisites the unequivocal establishment of the developmental type for each taxa within this lineage. The low resolution (low bootstrap confidence) at the basal nodes of the pseudoparaphysis clade observed in this study does not allow any inferences as to the ancestral type of these pseudoparaphyses. Phylogeny and Taxonomy among the Pseudoparaphysate Taxa The acceptance of the pseudoparaphysis type as a primary criterion for taxonomic divisions within the Loculoascomycetidae has not been unanimous. The species within the pseudoparaphysis lineage defined in this study have undergone numerous taxonomic revisions. These species have been placed in 13 families in two orders (Barr, 1979, 1987b) and 10 families in one order (the Dothideales; Hawksworth et al., 1995). Based primarily on ascal and ascospore morphology, Arx and Mu¨ller (1975) included all species used in the present study in the Pleosporaceae, except for Pyrenophora trichostma, Lophiostoma caulium, and Delitschia winteri, placed, respectively, in the Pseudosphaeriaceae, Lophiostomataceae, and Sporormiaceae. Relationships among the taxa within the pseudoparaphysis clade are largely unresolved in this study due to the relatively low bootstrap support obtained. For some cases, e.g., taxa within subclade A (Fig. 2), this low resolution is probably due to the presence of short branches indicating insufficient nucleotide substitutions for unambiguous lineage separation (Berbee and Taylor, 1995). Nevertheless, several monophyletic subclades within this pseudoparaphysis lineage are statistically well supported. Subclade A contains the Pleosporaceae, the type family for the Pleosporales, represented here by Pleospora herbarum and Pyrenophora trichostoma (sensu Hawksworth et al., 1995). This subclade, in addition, contains representatives from the Cucurbitariaceae, Leptosphaeriaceae, Phaeosphaeriaceae, and Melanommataceae (sensu Hawksworth et al., 1995). Barr (1987b) placed Pleospora and Pyrenophora into separate families based on their habit, anamorphic states, and ascospore morphology. The Pleosporaceae, Leptosphaeriaceae, and Phaeosphaeriaceae are morphologically closely related. Barr (1987a) separated the Leptosphaeriaceae from the Pleosporaceae “because of the coelomycetous rather than hyphomycetous anamorphs as well as the narrower and thinner-walled asci.” She separated the Leptosphaeriaceae from the Phaeosphaeriaceae “primarily on the shape of the ascomata and large scleroplectenchymatous or thick-walled cells of the peridium.” The current study does not support the monophyly of a combination of these three families.

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The Pleosporaceae and Phaeosphaeriaceae have recently been shown to be of distinct and separate lineages, whereas the Leptosphaeriaceae, sensu lato, is polyphyletic (Khashnobish and Shearer, 1996; Dong et al., 1998). Entodesmium rude clustered within subclade A. The position of this genus has always been uncertain. Although it is currently placed in the Lophiostomataceae, it has been suggested that it is related to Leptosphaeria (Mu¨ller and Arx, 1950) and was previously assigned to the Pleosporaceae (Arx and Mu¨ller, 1975) and Phaeosphaeriaceae (Eriksson and Hawksworth, 1991). Subclades B and C (Fig. 2) consist largely of representatives from the Lophiostomataceae, the others belonging to Phaeosphaeriaceae and Massariaceae. On the basis of morphological characteristics, Phaeodothis was recently reinstated to accommodate several species excluded from Didymosphaeria (Didymosphaeriaceae) (Aptroot, 1995a,b). This appears to be justified here, as P. winteri is shown to have more phylogenetic affinity to Montagnula, also in the Phaeosphaeriaceae. The Massariaceae, represented by Massaria platani here, may have a close relationship with the Phaeosphaeriaceae and, as emphasized by Eriksson (1981), is not closely related to Massarina as claimed by some previous authors (e.g., Wehmeyer, 1975). Phaeodothis winteri and Montagnula opulenta, however, do not form a monophyletic group with Phaeosphaeria nodorum, the representative of the type genus of the family Phaeosphaeriaceae sensu Barr (1987b). This family, shown here to be polyphyletic, certainly needs further investigation. The genus Massarina appears to be polyphyletic, with M. eburnea clustering away from M. australiensis and M. bipolaris. Hyde (1995) illustrated M. eburnea and Aptroot (1998) recently revised this genus based on morphological characters. The latter commented that the accepted 43 species may not be monophyletic on the grounds that some species of Massarina appear to be very similar to species of other genera, differing only in very few characters, in particular the pseudoparaphysis type. The analysis here indicates that the delineation of this genus, along with other genera and families within the pseudoparaphysis lineage, will need to be revised, assigning greater weighting to some characters other than the pseudoparaphysis type. Referring to the terminal clade of the two Massarina eburnea strains, it could be argued that the relatively long branch (Fig. 2), which is indicative of a rapidly evolving lineage, may be phylogenetically misleading. As pointed out by Berbee and Taylor (1995), such lineages have the tendency to cluster together with other rapidly evolving lineages, away from the true closest relatives. This aberration can be ruled out here, as the M. eburnea clade does not exhibit this phenomenon of long branch attraction toward other long branches within the trees.

Some species of Massarina are morphologically very similar to Lophiostoma, which differs mainly by the consistently laterally compressed slot-like ostioles (Hyde, 1995; Aptroot, 1998; Hyde and Aptroot, 1998). Barr (1987b) included Massarina in the Lophiostomataceae, concurring with the opinions of previous authors (e.g., Eriksson, 1981). However, it was also suggested that the Lophiostomataceae, sensu stricto, primarily circumscribed by the presence of the compressed slot-like ostiole, is most probably heterogeneous and should be disassociated due to the unstable and highly adaptive ostiolar character (Holm and Holm, 1988). Results from the current phylogenetic analyses do not support the monophyly of the Lophiostomataceae. Subclade D contains Melanomma pulvis-pyrius, the type of the Melanommatales, as well as Dangeardiella macrophila, Herpotrichia diffusa, and Pleomassaria siparia. D. macrophila has the lophiostomataceous slotlike ostiole and has been placed in the Lophiostomataceae accordingly (Barr, 1987b). The taxonomy of Herpotrichia has been contentious (Bose, 1961; Sivanesan, 1971; Arx and Mu¨ller, 1984; Barr, 1987), and the genus was noted to be related to Melanomma. The current study does not support the monophyly of Melanommataceae as delimited by either Barr (1987b, 1990) or Hawksworth et al. (1995). Further work is definitely required to resolve the taxonomy of the taxa within this subclade. It is worth noting that the interpretation of polyphyly of families from our data set depends on identification of cultures which could not always be verified. Patellariales Taxa Two recent studies have shown Rhytidhysteron rufulum to be basal to the Pleosporales lineage based on the 18S rDNA sequences (Winka et al., 1998; SilvaHanlin and Hanlin, 1999). This concurs with the results obtained in this study using a newly obtained sequence of the same region; R. rufulum clusters at the base of the pseudoparaphysis lineage. Winka et al. (1998) noted that the absence of other representatives from the Patellariales in their sample did not allow a precise inference regarding the relationships of this order with the Pleosporales and the Dothideales. The taxa used here include Hysteropatella clavispora, which clustered together with Hysterium pulicare with high bootstrap confidence. The former, previously placed in the Hysteriaceae (Eriksson and Hawksworth, 1986) was included by Barr (1987b) in the Patellariaceae while acknowledging the heterogeneity of this family. Kutorga and Hawksworth (1997), in their comprehensive revision of the Patellariaceae, did not include H. clavispora in the family. On the basis of morphological characters and the current DNA data, this species should be redisposed into the Hysteriaceae. As

PHYLOGENETIC SIGNIFICANCE OF THE PSEUDOPARAPHYSES

mentioned earlier, these two families are similar in several aspects. The pseudoparaphyses in the Patellariales are similar to the trabeculate type, but develop further at the top, regenerating apical branches. The tips are slightly swollen, agglutinated, and pigmented, forming an epithecium (Kutorga and Hawksworth, 1997). This type of development is related to that of the pseudoparaphyses, but they are certainly not the same. Several genera within the Hysteriaceae have this epithecium at maturity. CONCLUSION Loculoascomycete taxa with pseudoparaphyses within their ascostroma evolved as a monophyletic lineage. There is no evidence for separate lineages of cellular and trabeculate pseudoparaphyses. The Pleosporales and Melanommatales are not natural orders. It is probably more appropriate to classify these groups of loculoascomycetes together as a single taxon, e.g., the suborder Pleosporineae, as proposed by Eriksson and Winka (1997). These results have important implications to the taxonomy of taxa within the pseudoparaphysis lineage. Several currently accepted families within the lineage appear to be polyphyletic and hence require revision. ACKNOWLEDGMENTS This research was funded by The Hong Kong Research Grants Council. E. C. Y. Liew thanks The University of Hong Kong for a Post-Doctoral Research Fellowship. Dr Edwin Abeln is thanked for comments on the manuscript.

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