Mycologia, 98(4), 2006, pp. 571–583. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897 The family Pleosporaceae: intergeneric relationships and phylogenetic perspectives based on sequence analyses of partial 28S rDNA Rampai Kodsueb niothelia, which is probably polyphyletic. Anamorphic Department of Biology, Faculty of Science, Chiang Mai characters appear to be significant (especially in University, Chiang Mai, Thailand Cochliobolus) while ascospore morphologies, such as Vijaykrishna Dhanasekaran shape and color and substrate occurrence are poor Centre for Research in Fungal Diversity, Department of indicators of phylogenetic relationships among these Ecology & Biodiversity, The University of Hong Kong, loculoascomycetes. Pokfulam Road, Hong Kong Key words: anamorphs, ascospore morphology, Andre´ Aptroot Loculoascomycetes, phylogeny, Pleospora, polyphy- Centraalbureau voor Schimmelcultures, P.O. Box letic, ribosomal DNA 85167, 3508 AD Utrecht, The Netherlands Saisamorn Lumyong INTRODUCTION Department of Biology, Faculty of Science, Chiang Mai The largest family within the Pleosporales, Pleospor- University, Chiang Mai, Thailand aceae, comprises 17 genera and 111 species (Kirk et al Eric H.C. McKenzie 2001). Species are parasites or saprobes on wood and Landcare Research, Private Bag 92170, Auckland, dead herbaceous stems or leaves (Sivanesan 1984). New Zealand The classification in the Pleosporaceae has been Kevin D. Hyde based primarily on the Pleospora type of centrum Rajesh Jeewon1 development (Dong et al 1998) and asci that are Centre for Research in Fungal Diversity, Department of interspersed with pseudoparaphyses in the asco- Ecology & Biodiversity, The University of Hong Kong, stroma. These pseudoparaphyses originate above the Pokfulam Road, Hong Kong hymenial layer and grow downward among the asci to fuse at the base of the locule (Wehmeyer 1975). Ascomata are perithecial, initially immersed and Abstract: The Pleosporaceae is an important locu- become erumpent and are usually black and some- loascomycete family. There has been disagreement, times hairy or setose. Asci are fissitunicate, cylindrical, however, regarding the taxonomic placement of with an ocular chamber and pseudoparaphyses are many genera within this family. This study investigates cellular. Ascospores are usually brown and phragmo- phylogenetic relationships among the genera Cochlio- sporous or dictyosporous (Dong et al 1998, Kirk et al bolus, Kirschsteiniothelia, Leptosphaerulina, Macroven- 2001). Many pleosporaceous taxa are important turia, Pleospora, Pyrenophora, and Wettsteinina. Partial plant pathogens. For instance Cochliobolus heterostro- 28S rDNA sequences from taxa within these genera phus causes southern corn leaf blight and Pyrenophora were analyzed with maximum parsimony, likelihood graminea is the causal agent of barley leaf stripe and Bayesian methods. Cochliobolus can be segregated (Agrios 2005). Their anamorphs are usually hypho- broadly into two groups as previously proposed. mycetes and also have been reported to cause plant Pleospora is polyphyletic in its current sense. Taxa disease in cereals (e.g. Bipolaris maydis, Exserohilum with Stemphylium anamorphs are closely related to turcicum and Helminthosporium oryzae) (Farr et al Cochliobolus and fit within the Pleosporaceae, whereas 1989, Berbee 1996, Krupinsky et al 2004, Agrios the affinities of Pleospora herbarum and P. ambigua are 2005). still ambiguous. Pyrenophora constitutes a monophy- The Pleosporaceae historically was placed in the letic group within the Pleosporaceae, whereas Lepto- Sphaeriales (e.g. Winter 1887, Ellis and Everhart sphaerulina and Macroventuria appear to share 1892, Lindau 1897) based on immersed perithecia phylogenetic affinities with the Leptosphaeriaceae containing paraphyses (Wehmeyer 1975). The family and Phaeosphaeriaceae. Phylogenies indicate that then was transferred to the Pseudosphaeriaceae, Wettsteinina should be excluded from the Pleospor- which was later raised to ordinal rank as the Pseudo- aceae. Similar findings are reported for Kirschstei- sphaeriales (Theissen and Sydow 1917, Wehmeyer 1975). Luttrell (1955) treated the name Pseudo- Accepted for publication 30 May 2006. 1 Corresponding author. E-mail: [email protected], rjeewon@ sphaeriales as a synonym of the Dothideales and graduate.hku.hk suggested that pseudoparaphyses were important in 571 572 MYCOLOGIA their taxonomy. He considered the Pseudosphaer- Lophiostoma caulium, Massarina ramunculicola, Phaeo- iales as synonym of the Pleosporales and assigned sphaeria vegans and Venturia carpophila) were selected the Pleosporaceae, Venturiaceae and Lophiostomata- for this study. Species names and accession numbers of ceae, to the Pleosporales. Such a scheme largely was the isolates in this study are listed (TABLE II). For each isolate, pure cultures were plated on potato dextrose accepted by other mycologists (e.g. Wehmeyer 1975). agar and incubated at 25 C 10–20 d before DNA Wehmeyer (1975) pointed out that the family extraction. Genomic DNA was extracted from fresh Pleosporaceae has never been clearly delimited fungal mycelia following a protocol as outlined by following Luttrell’s concept and as a result taxa with Jeewon et al (2002, 2003, 2004) and Cai et al (2005). ascostromata of many different types, which pre- Briefly, mycelia were scraped off from the surface of viously were placed in other families, were placed the plate. The mycelia were ground with 200 mg of arbitrarily in the Pleosporaceae. sterilized quartz sand and 600 mLof23 CTAB The family Pleosporaceae is a heterogeneous extraction buffer (2% w/v CTAB, 100 mM Tris-HCL, group of bitunicate ascomycetes with genera pri- 1.4 M NaCl, 20 mM EDTA, pH 8) in a 1.5 mL Eppen- dorf tube. The contents were incubated at 60 C in marily included based on ascospore characteristics, a water bath 40 min with gentle swirling every 10 min. including shape, color, septation, pigmentation and The solution was extracted three times with an equal presence or absence of mucilaginous sheaths (Lut- volume of phenol:chloroform (1:1) at 13 000 g 30 min trell 1955, 1973; Wehmeyer 1961, 1975; Eriksson until no interface was visible. The upper aqueous phase 1981; Sivanesan 1984; Barr 1987b; Abler 2003). Other containing the DNA was precipitated by addition of families, such as the Leptosphaeriaceae, Melanom- 2.5 volumes of absolute ethanol and kept at 220 C mataceae, Phaeosphaeriaceae and Sporormiaceae, overnight. The precipitated DNA was washed two times however, also possess morphological characters sim- with 70% ethanol, dried under vacuum and suspended ilar to those of the Pleosporaceae, and this has in TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) resulted in considerable ambiguity and confusion and treated with RNase (1 mg/mL) before DNA amplification. in intergeneric and familial classification (Luttrell 1955, 1973; Wehmeyer 1961, 1975; von Arx and DNA amplification and sequencing of 28S rDNA.—Approx- Mu¨ller 1975; Sivanesan 1984; Barr 1987a, b; Eriksson imately 900 nucleotides at the 59 end of the 28S rDNA and Hawksworth 1986, 1991). Most genera in these region were amplified by primer pairs LROR/LRO5 families have morphological characters that overlap (Vilgalys and Hester 1990). PCR was carried out in m m in many respects and the taxonomic organization 50 L reaction volume containing 31.7 L sterile water, 5 mLof103 Mg free PCR buffer, 3 mLof25mMMgCl , among them has been modified on several occasions 2 4 mL of 2.5 mM deoxyribonucleotide triphosphate (TABLE I). Barr (1987b) redefined the Pleosporaceae (dNTPs), 1.5 mL of each 10 mM primers (LROR and to include Clathrospora (5Comoclathris), Kirschstei- LRO5), 3 mL of DNA template, 0.3 mL of 2.5 units of niothelia, Lewia and Pleospora and grouped Cochliobo- Taq DNA polymerase (Promega, Madison, Wisconsin). lus, Pyrenophora and Setosphaeria into the family Typical amplification parameters were: initial denatur- Pyrenophoraceae. Berbee (1996) disagreed, suggest- ation of 95 C for 3 min; 35 cycles of denaturation at ing that all those genera belong to the Pleosporaceae. 95 C 1 min, annealing at 52 C 50 s and extension at Given the considerable taxonomic confusion, this 72 C 1 min and final extension of 72 C 10 min. Double- TM study, based on phylogenetic analyses of rDNA stranded DNA products were purified with GFX PCR DNA and Gel Band Purification Kit (Amersham sequence data, was undertaken to (i) verify the Biosciences, catalogue No. 27-9602-01) following man- familial placement of these genera and assess whether ufacturer’s protocol. Sequencing reactions were per- they represent natural groups, (ii) determine which formed and sequences determined automatically in an morphological characters are phylogenetically signi- Applied Biosystem 3730 Genetic Analyzer/Sequencer ficant and therefore are useful for generic delinea- (Genome Research Center, The University of Hong tion and (iii) assess whether phylogenies based on Kong) using PCR primers mentioned above. molecular characters are concordant with any of the Phylogenetic analyses.—Fifty-four taxa from different traditional morphology-based classification schemes. fungal families were aligned initially with the computer program Bioedit (Hall 1999) and Clustal X (Thompson MATERIALS AND METHODS et al 1997) with default parameter settings, and alignments were manually edited by inserting gaps for DNA extraction.—Twenty isolates of Pleosporaceae (Co- optimization using Se-Al (Rambaut 1996). Phylogenetic chliobolus [two species], Kirschsteiniothelia