Mycologia, 94(6), 2002, pp. 1017–1031. q 2002 by The Mycological Society of America, Lawrence, KS 66044-8897 A preliminary overview of the Diaporthales based on large subunit nuclear ribosomal DNA sequences Lisa A. Castlebury1 Amy Y. Rossman eastern North America (Anagnostakis 1987). Other diseases caused by members of this order include stem canker of soybeans (Diaporthe phaseolorum (Cooke & Ellis) Sacc. and its varieties), stem-end rot of citrus fruits (Diaporthe citri F.A. Wolf), and peach canker disease (Phomopsis amygdali (Del.) J.J. Tuset & T. Portilla) (Farr et al 1999). Some species produce secondary metabolites that result in toxicoses of animals such as lupinosis of sheep (Diaporthe toxica P.M. Will. et al) (Williamson et al 1994). A number of asexually reproducing plant pathogenic fungi also belong in the Diaporthales, such Greeneria uvicola (Berk. & Curt.) Punith., cause of bitter rot of grape, and Discula destructiva Redlin, cause of dogwood anthracnose, both of which are mitotic diaporthalean species with no known sexual state (Farr et al 2001, Zhang and Blackwell 2001). As an order the Diaporthales is well-defined morphologically based on brown to black perithecial ascomata immersed in stromata or substrata, lack of true paraphyses at maturity, and unitunicate asci that often float free within the centrum at maturity and have a refractive ring in the apex (Barr 1978, Samuels and Blackwell 2001). The known asexual states of members of the Diaporthales are generally coelomycetous bearing their phialidic, rarely annellidic, conidiogenous cells and conidia in acervuli or pycnidia with or without well-developed stromata. Molecular data have supported the Diaporthales as a distinct order within the Sordariomycetes, the class of ascomycetous fungi that generally produce their asci in perithecial ascomata (Farr et al 2001, Zhang and Blackwell 2001). Within the Diaporthales eight families have been recognized by various authors over the past 25 yr. However, no single author has ever recognized all eight and the configuration of each family has varied considerably. Most recently, Eriksson et al (2001), presenting a synthesis of data from the literature with input from the mycological community, recognize three families in the Diaporthales, namely the Melanconidaceae G. Winter, Valsaceae Tul. & C. Tul., and Vialaeaceae P.F. Cannon. The familial classifications of the Diaporthales as recognized by Barr (1978, 1990), Hawksworth et al (1995), and Wehmeyer (1975) were summarized by Zhang and Blackwell (2001). They included the Gnomoniaceae G. Winter, Systematic Botany and Mycology Laboratory, USDAARS, 10300 Baltimore Ave., Beltsville, Maryland, USA 20705 Walter J. Jaklitsch Van den langen Lüssen 31/2, A-1190 Vienna, Austria Larissa N. Vasilyeva Institute of Biology & Soil Science, Far East Branch of the Russian Academy of Sciences, Vladivostok, 690022, Russia Abstract: The ascomycete order Diaporthales includes a number of plant pathogenic fungi such as Cryphonectria parasitica, the chestnut blight fungus, as well as many asexually reproducing fungi without known sexual states. Relationships among genera in the Diaporthales were evaluated as a basis for the recognition of families and to provide a taxonomic framework for the asexually reproducing diaporthalean fungi. Phylogenetic relationships were determined based on analyses of large subunit (LSU) nuclear ribosomal DNA (nrDNA) sequences. Within the Diaporthales 82 sequences representing 69 taxa were analyzed. Results suggest the presence of at least six major lineages within the Diaporthales recognized as the Gnomoniaceae sensu stricto, Melanconidaceae sensu stricto, Schizoparme complex including the anamorph genera Coniella and Pilidiella, CryphonectriaEndothia complex, Valsaceae sensu stricto, and Diaporthaceae sensu stricto. In addition, six teleomorphic and anamorphic taxa fell within the Diaporthales but were not allied with any of the six lineages. Key Words: canker fungi, Cryphonectria, Diaporthe, Discula, phylogeny, Valsa INTRODUCTION The ascomycete order Diaporthales includes a number of plant pathogenic fungi, the most notorious of which is Cryphonectria parasitica (Murrill) Barr, the chestnut blight fungus that altered the landscape of Accepted for publication April 28, 2002. 1 Corresponding author, Email: lisa@nt.ars-grin.gov 1017 1018 MYCOLOGIA Diaporthaceae Höhn. ex Wehm. and Pseudovalsaceae M.E. Barr as well as the Melanconidaceae and Valsaceae. Two additional families have been included in the Diaporthales. The Magnaporthaceae P.F. Cannon was shown to be extralimital to the Diaporthales (Berbee 2001, Farr et al 2001, Zhang and Blackwell 2001) as had been suggested by Vasilyeva (1993). The Sydowiellaceae Lar.N. Vassiljeva was established for the genus Sydowiella Petr. (Vasilyeva 1987). Of the 98 genera of plant-associated fungi in the Diaporthales 13 to 15 genera have not been referred to families (Eriksson et al 2001, Kirk et al 2001). Generic concepts within the Diaporthales are based primarily on characteristics of the stromata, perithecia and ascospores. Stromatal characteristics used in defining genera in the Diaporthales are the extent and type of development, tissue type forming the stromata, and the relationship of the stromata to the host. The position of the perithecia relative to the host surface has been used to distinguish genera, as has the arrangement of the perithecia in the stromata and convergence or not of perithecial necks within the stromata. In addition to stromatal and perithecial characteristics, variations in the ascospore shape and septation have been used to define genera (Petrak 1966, Kobayashi 1970, Barr 1978, 1990, Monod 1983, Vasilyeva 1993). In some genera these distinctions are difficult to determine and the generic concepts have been unstable with many species transferred from one genus to another depending on the author. Approximately 60% of the described species of plant-associated fungi reproduce asexually and lack any known sexual state (Rossman 1993). Although most are mitotic ascomycetes, their relationships to teleomorph taxa are generally unknown. Because these fungi include many serious plant pathogens, knowledge of their taxonomic affinities is crucial for developing measures to control the diseases they cause. With increased use of molecular sequence data for reconstructing fungal evolutionary relationships at all levels (Kohn 1992), the affinities of mitotic fungi with their sexually reproducing relatives can be determined. Recently Greeneria uvicola, a mitotic species with no known sexual affinities, was found to belong in the Diaporthales (Farr et al 2001). Since its emergence in the late 1970s, a sexual state for Discula destructiva, the cause of dogwood anthracnose, has been sought. Redlin (1991) suggested that it might have a teleomorph belonging to Apiognomonia Höhn. or Gnomonia Ces. & De Not. Zhang and Blackwell (2001) used molecular sequence data to infer the sexual state of Discula destructiva but were unable to come to any definitive conclusion except that it belonged in the Gnomoniaceae. Knowledge of the relationships among genera within the Diaporthales is needed to serve as a basis for the recognition of families. In addition such knowledge will provide a taxonomic framework for determining relationships of teleomorph genera to asexually reproducing diaporthalean fungi. Thus, a study was undertaken to determine the major lineages within the Diaporthales based on a sequence analysis of the LSU nrDNA. MATERIALS AND METHODS Isolation, maintenance, and deposition of cultures and voucher specimens. Newly sequenced isolates used in this study are listed in TABLE I. GenBank accession numbers for previously sequenced isolates are included with the species name in FIGS. 1–3. Fresh specimens were sent as air-dried collections primarily by the third and fourth authors to the second author. Isolates obtained from these specimens were grown from single ascospores or conidia that had been plated on 1.7% Difco Corn Meal Agar (CM) supplemented with 0.2% dextrose and antibiotics. Germinated spores were transferred to both 3.9% Difco Potato Dextrose Agar (PDA) and CM plates for observation. All isolates were maintained on CM agar slants and as plugs in 20% glycerol-water at 4 C (Burdsall and Dorworth 1994). Living cultures were deposited in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, and the original specimens from which isolates were obtained were deposited in the U.S. National Fungus Collections (BPI) as listed in TABLE I. For living cultures obtained from repositories such as ATCC (American Type Culture Collection), CBS and IMI (International Mycological Institute, now CABI), dried culture specimens were deposited in BPI if the culture sporulated. Nucleic acid extraction and PCR amplification. Mycelium for DNA extraction was grown in shaker flasks at 125 rpm for 5–10 d in 100 mL liquid CYM (Raper and Raper 1972) at room temperature under ambient light conditions. Mycelium was harvested by vacuum filtration on Whatman No. 1 filter paper and freeze-dried prior to DNA extraction. Alternatively, DNA was extracted directly from actively growing surface mycelium scraped from PDA plates. DNA was extracted with the Plant DNeasy Mini kit (Qiagen Inc., Chatsworth, California, USA) according to the manufacturer’s instructions using approximately 15 mg dried tissue or 50 mg fresh mycelium. The LSU nrDNA was amplified in 50 mL reactions on a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, California, USA) under the following reaction conditions: 10–15 ng of genomic DNA, 200 mM each dNTP, 2.5 units Amplitaq Gold (Applied Biosystems, Foster City, California, USA), 25 pmoles each of primers LR0R and LR7 (Vilgalys and Hester 1990, Rehner and Samuels 1994) and the supplied 103 PCR buffer with 15 mM MgCl2. The thermal cycler program was as follows: 10 min at 95 C followed TABLE I. Newly sequenced taxa included in phylogenetic analyses Specimen No.b Russia L. Vasilyeva BPI 747935 South Africa Quercus mongolica Fisch. ex Ledeb. Leaf litter K.T. van Warmelo India Soil ?Africa Cameroon — AR 2813 (5 CBS 109747) AF408334 AF408335 BPI 748425 AR 3446 (5 CBS 109758) IMI 261318 AF408336 V.V. Bhatt BPI 841767 IMI 081599 AF408391 Hibiscus sp. R.R. Cervantes BPI 748426 AR 3534 (5 CBS 109757) AF408337 I.A.S. Gibson BPI 841768 CBS 101281 AF408338 Zaire Eucalyptus urophylla S.T. Blake Eucalyptus saligna Sm. Unknown BPI 748427 CBS 505.63 AF408339 Russia Quercus mongolica L. Vasilyeva BPI 748428 AR 3444 (5 CBS 109764) AF408340 Russia Quercus mongolica L. Vasilyeva BPI 748429 AR 3433 (5 CBS 109776) AF408341 Austria W. Jaklitsch BPI 748430 S. Redlin BPI 747916 Austria Acer pseudoplatanus L. W. Jaklitsch BPI 748431 Austria Salix sp. W. Jaklitsch BPI 747938 Austria Ulmus minor Mill. W. Jaklitsch BPI 748432 AR 3580 ex WJ 1695 (5 CBS 109765) AR 2814 (5 CBS 245.90) AR 3565 ex WJ 1491 (5 CBS 109759) AR 3455 ex WJ 1463 (5 CBS 109775) AR 3552 ex WJ 1694 AF408342 USA: Maine Aesculus hippocastanum L. Cornus alternifolia L.f. Austria W. Jaklitsch BPI 748433 Austria Ulmus minor/laevis Pall. Corylus avellana L. W. Jaklitsch BPI 747942 Austria Berberis vulgaris L. W. Jaklitsch BPI 748434 Austria Acer campestre L. W. Jaklitsch BPI 748435 AR 3566 ex WJ 1497 (5 CBS 109753) AR 3459 ex WJ 1473 (5 CBS 109772) AR 3424 ex WJ 1445 (5 CBS 109770) AR 3538 ex WJ 1643 (5 CBS 109767) AF408343 AF408344 AF408345 AF408346 AF408347 AF408348 AF408349 AF408350 1019 M. Monod GenBank No. DIAPORTHALES Fagus sylvatica L. Sourcec OF THE Switzerland Collector OVERVIEW Apiognomnia errabunda (Roberge) Höhn. (anam. Discula umbrinella (Berk. & Broome) M. Morelet) Chromendothia citrina Lar. N. Vasiljeva Coniella australiensis Petr. Coniella fragariae (Oudem.) B. Sutton Coniella musaiensis B. Sutton var. hibisci B. Sutton Cryphonectria cubensis (Bruner) Hodges Cryphonectria havanensis (Bruner) M.E. Barr Cryphonectria macrospora (Tak. Kobay. & Kaz. Itô) M.E. Barr Cryphonectria nitschkei (G.H. Otth) M.E. Barr Cryptodiaporthe aesculi (Fuckel) Petr. Cryptodiaporthe corni (Wehm.) Petr. Cryptodiaporthe hystrix (Tode) Petr. Cryptodiaporthe salicella (Fr.) Petr. Cryptosporella hypodermia (Fr.) Sacc. Cryptosporella hypodermia Diaporthe decedens (Fr.) Fuckel Diaporthe detrusa (Fr.) Fuckel Diaporthe eres Nitschke Host ET AL: Locality CASTLEBURY Speciesa 1020 TABLE I. Continued Speciesa Diaporthe fibrosa (Pers. : Fr.) Nitschke Diaporthe oncostoma (Duby) Fuckel Diaporthe padi G.H. Otth Diaporthe pardalota (Mont.) Fuckel Diaporthe perjuncta Niessl Diaporthe pustulata (Desm.) Sacc. Diaporthe pustulata Locality Host Collector Specimen No.b Rhamnus cathartica L. W. Jaklitsch BPI 747929 Russia Robinia pseudoacacia L. L. Vasilyeva BPI 747934 Austria Prunus padus L. W. Jaklitsch BPI 748436 Canada: British Columbia M. Barr BPI 747946 Austria Epilobium angustifolium L. Ulmus glabra Huds. W. Jaklitsch BPI 748437 Austria Acer pseudoplatanus W. Jaklitsch BPI 747928 Austria Acer pseudoplatanus W. Jaklitsch BPI 748438 Discula destructiva Redlin Ditopella ditopa (Fr. : Fr.) J. Schröt. Gnomonia gnomon (Tode : Fr.) J. Schröt. Gnomonia leptostyla (Fr. : Fr.) Ces. & De Not. (anam. Marssonina juglandis (Lib.) Magnus) Harknessia eucalypti Cooke Harknessia lythri D.F. Farr & Rossman Hercospora tiliae (Pers. : Fr.) Fr. Leucostoma auerswaldii Nitschke Leucostoma cincta (Fr. : Fr.) Höhn. Leucostoma nivea (Hoffm. : Fr.) Höhn. Mazzantia napelli (Ces.) Sacc. Melanconis alni Tul. USA: Washington M. Daughtrey BPI 1107757 W. Jaklitsch BPI 748439 Italy Cornus nuttallii Audubon Alnus glutinosa (L.) Gaertn. Corylus avellana M. Ribaldi — USA: Illinois Juglans nigra L. D. Neely BPI 747976 Australia Eucalyptus regnans F. Muell. Lythrum salicaria L. Z-q. Yuan — Melanconis desmazierii Petr. Austria Austria USA: Minnesota Austria E. Katovich BPI 747560 W. Jaklitsch BPI 748440 Austria Tilia tomentosa Moench Frangula alnus Mill. W. Jaklitsch BPI 748456 Russia Padus maackii Rupr. L. Vasilyeva BPI 748441 Austria Salix purpurea L. W. Jaklitsch BPI 748442 Austria Aconitum vulparia Rchb. Alnus viridis (Vill.) Lam. & DC. Tilia sp. W. Jaklitsch BPI 748443 W. Jaklitsch BPI 748444 W. Jaklitsch BPI 748445 Austria GenBank Number AR 3425 ex WJ 1417 (5 CBS 109751) AR 3445 (5 CBS 109741) AR 3419 ex WJ 1458 (5 CBS 109784) AR 3478 ex MBB 10220 (5 CBS 109768) AR 3461 ex WJ 1480 (5 CBS 109745) AR 3430 ex WJ 1428 (5 CBS 109742) AR 3535 ex WJ 1628 (5 CBS 109760) AR 2596 (5 CBS 109771) AR 3423 ex WJ 1443 (5 CBS 109748) CBS 199.53 AF408351 AF408361 FAU 543 AF408362 CBS 342.97 AF408363 AR 3383 (5 ATCC PTA-2756) AR 3526 ex WJ 1600 (5 CBS 109746) AR 3428 ex WJ 1424 (5 CBS 109774) AR 3415 (5 CBS 109766) AR 3512 ex WJ 1555 (5 CBS 109743) AR 3498 ex WJ 1531 (5 CBS 109769) AR 3500 ex WJ 1542 (5 CBS 109773) AR 3525 ex WJ 1588 (5 CBS 109780) AF408364 AF408353 AF408354 AF408355 AF408356 AF408357 AF408358 AF408359 AF408360 AF408365 AF408384 AF408366 AF408367 AF408368 AF408371 AF408372 MYCOLOGIA Austria Sourcec TABLE I. Continued Speciesa Locality Host Collector Specimen No.b Sourcec Canada: British Columbia Alnus rubra Bong. M. Barr BPI 748446 Melanconis stilbostoma (Fr.) Tul. Ophiovalsa betulae (Tul. & C. Tul.) Petr. (anam. Disculina betulina (Sacc.) Hohn.) Ophiovalsa suffusa (Fr.) Petr. (anam. Disculina vulgaris (Fr.) B. Sutton) Phragmoporthe conformis (Berk. & Broome) Petr. Pilidiella castaneicola (Ellis & Everh.) Arx Pilidiella granati (Sacc.) Aa Pilidiella granati Plagiostoma conradii (Ellis) M.E. Barr Plagiostoma euphorbiae (Fuckel) Fuckel Schizoparme botrytidis Samuels Valsa cenisia De Not. Austria Betula pendula Roth W. Jaklitsch BPI 748447 Austria Betula pendula W. Jaklitsch BPI 748448 Austria Alnus incana (L.) Moench W. Jaklitsch BPI 748449 AR 3496 ex WJ 1556 (5 CBS 109750) AF408376 Canada: British Columbia Alnus rubra M. Barr BPI 748450 AR 3632 ex MBB 10338 (5 CBS 109783) AF408377 ET AL: Korea Unknown K. S. Bae BPI 748451 CBS 143.97 AF408378 Cyprus Punica granatum L. R.M. Nattrass BPI 748452 CBS 152.33 AF408379 Turkey USA: New Jersey N. Kaskalöglu G. Bills BPI 748453 BPI 746482 Unknown CBS 814.71 AR 3488 (5 CBS 109761) CBS 340.78 AF408380 AF408381 Netherlands Punica granatum Hudsonia tomentosa Nutt. Euphorbia palustris L. OVERVIEW AF408373 Melanconis marginalis (Peck) Wehm. Puerto Rico Dead wood S. Huhndorf BPI 748454 Austria Juniperus communis L. W. Jaklitsch BPI 748457 Valsa ceratosperma (Tode : Fr.) Maire Valsa ceratosperma Russia Quercus mongolica L. Vasilyeva BPI 748458 Austria Quercus robur L. W. Jaklitsch BPI 748459 Valsella adherens Fuckel Russia Betula sp. L. Vasilyeva BPI 748460 Valsella salicis Fuckel Italy Salix fragilis L. W. Jaklitsch BPI 748461 Wuestneia molokaiensis Crous & J.D. Rogers USA: Hawaii Eucalyptus robusta Sm. J. Rogers BPI 748462 AF408375 AF408382 AF408385 AF408386 DIAPORTHALES AF408383 OF THE SMH 1354 (5 AR 3504) AR 3522 ex WJ 1583 (5 CBS 109752) AR 3416 (5 CBS 109756) AR 3426 ex WJ 1425 (5 CBS 109777) AR 3549 (5 CBS 109782) AR 3514 ex WJ 1580 (5 CBS 109754) AR 3578 (5 CBS 109779) AF408374 CASTLEBURY — AR 3442 ex MBB 1021A (5 CBS 109744) AR 3501 ex WJ 1543 (5 CBS 109778) AR 3524 ex WJ 1610 (5 CBS 109763) GenBank No. AF408387 AF408388 AF408389 AF408390 a 1021 Type species of the genus in bold. BPI 5 U.S. National Fungus Collections. c AR 5 Amy Rossman, second author; ATCC 5 American Type Culture Collection; CBS 5 Centraalbureau voor Schimmelcultures; FAU 5 maintained by second author; IMI 5 International Mycological Institute, now CABI, Inc.; MBB 5 Margaret Barr Bigelow, Sidney, British Columbia; SMH 5 Sabine M. Huhndorf, Field Museum, Chicago, IL; WJ 5 Walter Jaklitsch, third author. b 1022 MYCOLOGIA by 35 cycles of 30 s at 94 C, 30 s at 55 C, 1 min at 72 C, with a final extension period of 10 min at 72 C. Following amplification, the PCR products were purified with QIAQuick columns (Qiagen Inc., Chatsworth, California, USA) according to the manufacturer’s instructions. Amplified products were sequenced with the BigDye dye terminator kit (Applied Biosystems, Foster City, California, USA) on an ABI 310 or ABI 377 automated DNA sequencer using the following primers: LR0R, LR3R, LR5R, LR7, LR5, LR3 (Vilgalys and Hester 1990, Rehner and Samuels 1994). Sequence analysis. Raw sequences were edited using Sequencher version 4.05 for Windows (Gene Codes Corporation, Ann Arbor, Michigan, USA). Alignments were manually adjusted using GeneDoc 2.6.001 (http://www.psc. edu/biomed/genedoc/). Two alignments were generated. Alignment 1 included sequences from 55 newly sequenced diaporthalean taxa, 27 diaporthalean sequences from GenBank for which only approximately 600 bp are available for some taxa with Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa and Gauemannomyces graminis (Sacc.) Arx & D. Olivier from the Magnaporthaceae as outgroup taxa. Alignment 1 was truncated to 650 aligned positions to minimize the effects of large amounts of missing data for some of the taxa in the analyses. Alignment 2 included only the 55 taxa newly sequenced for this study as well as 16 sequences recently reported in Farr et al (2001) for which approximately 1350 bp of the 59 end of the LSU nrDNA were sequenced. The sequence alignments were deposited in TreeBASE as S815. For both alignments, trees were inferred by the neighborjoining (NJ) method (Kimura 2-parameter distance calculation) and by maximum parsimony (MP) using the heuristic search option with the random addition sequence (1000 replications) and the branch swapping (tree bisection-reconnection, TBR) option of PAUP* 4.0b8 (Swofford 1998). For both types of analyses, ambiguously aligned positions were excluded. All characters were unordered and given equal weight during the analysis. Gaps were treated as missing data in the parsimony analysis and the neighbor joining analysis; missing or ambiguous sites were ignored for affected pairwise comparisons. Heuristic searches for most parsimonious trees (MPT) with the MULTREES option in effect resulted in large numbers of trees and did not search to completion. Maximum likelihood analyses were not attempted due to the length of time required for a data set of this size. All resulting MPT were compared using the ShimodairaHasegawa (S-H) test (Shimodaira and Hasegawa 1999) as implemented in PAUP* 4.0b8. The likelihood model was determined by Modeltest version 3.06 (Posada and Crandall 1998). Relative support for branches was estimated with 1000 bootstrap replications (Felsenstein 1985) with MULTREES and TBR off and 10 random sequence additions for the MP bootstraps. Phylogenetic trees were also inferred for alignment 2 using Bayesian inference as implemented in MrBayes (http:// morphbank.ebc.uu.se/mrbayes/) with the following commands: (i) exclude positions 75, 76, 116, 117, 475–488, 504, 505, 862–1227; (ii) likelihood settings (lset) number of sub- stitution types (nst) 5 6, a proportion of sites invariable and the rest drawn from the gamma distribution (rate 5 invgamma), base frequencies 5 estimate, rate matrix 5 (1.7034, 6.7182, 3.8375, 0.9578, 15.0004, 1.0000); (iii) number of generations 5 500 000, sample frequency 5 100, number of chains 5 4, temperature 5 0.5, save branch lengths 5 yes, starting tree 5 random. The first 100 000 generations were discarded as the chains were converging (burnin). Likelihood model assumptions were as determined with Modeltest version 3.06 (Posada and Crandall 1998): base frequencies A 5 0.2708, C 5 0.2196, G 5 0.2847, T 5 0.2249; number substitution types 5 6; proportion of invariable sites 5 0.719; gamma shape parameter 5 0.6023, number rate categories 5 4, mean average rate; rate matrix 5 1.7034, 6.7182, 3.8375, 0.9578, 15.0004, 1.0000. Four independent analyses, each starting from a random tree, were run under the same conditions. Phylogenetic trees corresponding to the most recent classification schemes of Barr (1990) and Eriksson et al (2001) were constructed by using taxa contained within each accepted family as a separate monophyletic constraint in MP analyses of alignment 2 using the heuristic search option (1000 random sequence additions, TBR and MULTREES off). The tree with the best 2ln likelihood score resulting from each constrained analysis and all trees resulting from the unconstrained analysis were compared by the S-H test as described above (TABLE II), including all characters in the analysis except for ambiguously aligned positions and intron sequences. The following topologies were tested with the 372 equally parsimonious trees resulting from the unconstrained analysis: (i) Barr (1990) Gnomoniaceae, (ii) Barr (1990) Melanconidaceae, (iii) Barr (1990) Valsaceae, (iv) Eriksson et al (2001) Melanconidaceae, (v) Eriksson et al (2001) Valsaceae, and (vi) Bayesian topology. The range of 2ln likelihood scores of trees from each constraint topology is shown. However, only the topology with the best 2ln likelihood score from each constraint was tested against the unconstrained trees. RESULTS Sequence alignments. Alignment 1 consisted of 650 total characters of which 20 ambiguously aligned positions were excluded. Of the remaining 630 characters, 132 were parsimony informative. Alignment 2 consisted of 1650 bases of which 366 positions were excluded due to the presence of introns in two of the sequences (Cryptodiaporthe corni AR 2814, Ditopella ditopa AR 3423) and 20 positions were excluded because of potentially ambiguous alignments, leaving 1264 positions of which 189 were parsimony informative. Sequence analyses. For MP analyses, heuristic searches resulted in excess of 5000 trees. When the MULTREES option was turned off, 210 and 372 equally parsimonious trees were generated for alignment 1 and alignment 2, respectively. A strict consensus of CASTLEBURY ET AL: OVERVIEW trees generated with MULTREES on (MAXTREES 5 5000) was identical to the strict consensus of trees generated from analyses with MULTREES off for both analyses (trees not shown). Parsimony tree scores for alignment 1 were CI 5 0.428, RI 5 0.879, RC 5 0.376, and length 5 428. For alignment 2, tree scores were CI 5 0.487, RI 5 0.893, RC 5 0.435 and length 5 503. The MPT with the best 2ln likelihood score for alignment 1 is shown in FIG. 1 and one of two MPT with the best 2ln likelihood score is shown for the alignment 2 (FIG. 2), although neither tree was significantly better than others generated in each analysis (P 5 0.05). Bootstrap values greater than 70 percent are indicated on FIGS. 1 and 2 above (MP) and below (NJ) the respective branches. To determine if trees resulting from MP analyses with the MULTREES option 5 off, in general, reflected a good approximation of relationships within this group, Bayesian phylogenetic inference was used to construct a tree and determine the probabilities of a particular group existing in that tree (given the observed data). Bayesian analysis using Markov chain Monte Carlo algorithms is computationally more practical than bootstrapping and maximum likelihood. In addition, heuristic searches are not guaranteed to converge to the optimal tree (Larget and Simon 1999), whereas the Markov chain explores possible tree topologies and dimensions of other parameters of trees in proportion to their posterior probabilities (Lewis 2001). [For detailed explanations of this method, see Larget and Simon 1999, Huelsenbeck et al 2000, and Lewis 2001.] FIGURE 3 shows the tree resulting from the Bayesian analysis with the highest 2ln likelihood score with the numbers above the branches reflecting the probability that each group exists expressed as a percentage. Four independent analyses were run with each starting from a random tree and probabilities and topologies were similar in all analyses. Topologies from the four analyses differed only in the placement of the Valsaceae and Diaporthaceae as sister taxa in two of the four analyses. However, posterior probabilities of that placement were not particularly high (0.58 and 0.69). Likelihood ratio tests. Phylogenetic analysis of the diaporthalean taxa available for this study indicates the presence of at least six lineages within the Diaporthales. Although elements of many of the previously described families are present, these lineages do not entirely conform to taxonomic schemes that have been proposed. The S-H test results for constrained tree topologies corresponding to recently recognized families are presented in TABLE II. OF THE DIAPORTHALES 1023 Shimodaira-Hasegawa test results show that when analyses are constrained to conform to the placement of genera within the three families as recognized by Barr (1990) and Eriksson et al (2001), resulting trees from all of the constraints except for the Valsaceae as recognized by Barr (1990) are significantly worse than FIG. 2. Trees resulting from analyses constraining Valsa Fr., Leucostoma (Nitschke) Höhn., Valsella Fuckel, Endothia Fr. and Chromendothia Lar.N. Vassiljeva to be a monophyletic group could not be rejected as significantly worse explanations of the data than FIG. 2. These results suggest that two of the three major families in the Diaporthales as currently circumscribed (Eriksson et al 2001) are not monophyletic and that a greater number of families should be recognized. DISCUSSION Phylogenetic analyses. Phylogenetic analysis of LSU nrDNA sequences for available taxa within the Diaporthales shows the presence of at least six lineages within this order. Likelihood ratio tests (Shimodaira and Hasegawa 1999) comparing the topologies obtained by constraining monophyletic groups that correspond to recent classifications schemes do not recognize these families as equally good explanations of the data when compared with FIG. 2, the result of an unconstrained analysis, except in the case of Barr’s (1990) Valsaceae. Recent papers have pointed out that the Kishino-Hasegawa (K-H) test (Kishino and Hasegawa 1989) is not appropriate for making multiple comparisons or for trees resulting from the analysis of the data set and designated a posteriori (Shimodaira and Hasegawa 1999, Goldman et al 2000, Whelan et al 2001). However, there is some doubt about whether or not it is appropriate to use the SH test for questions of monophyly (Goldman et al 2000, Whelan et al 2001). In light of these concerns, although the S-H test was performed, it may be more appropriate to compare only the 2ln likelihood scores of each topology and not to evaluate their statistical significance. The results of maximum parsimony, neighbor joining, and Bayesian inference analyses were all similar in topology as well as levels of support. Bootstrap support for groups was generally higher in NJ analyses than for MP analyses and support was higher in alignment 2 (1264 bp) than alignment 1 (630 bp). In order to determine if this observation might be a result of either the more limited taxon sampling or the larger number of analyzed characters in alignment 2, the first 650 bp, excluding 20 ambiguously aligned positions, of this alignment was analyzed by both MP and NJ bootstrap analyses as described in the mate- 1024 MYCOLOGIA FIG. 1. One of 210 equally parsimonious trees based on analysis of 630 bp of the 59 end of the LSU nrDNA (2ln likelihood 5 3328.6773, CI 5 0.428, RI 5 0.879, RC 5 0.376, length 5 428 steps) for 82 diaporthalean sequences. Bootstrap values greater than 70% are shown above (MP) and below (NJ) each branch. Taxa in bold represent type species of their respective genera. Thickened lines indicate that branch appeared in the strict consensus of the 210 trees. CASTLEBURY ET AL: OVERVIEW OF THE DIAPORTHALES 1025 FIG. 2. One of two trees with the best 2ln likelihood score of 372 equally parsimonious trees based on analysis of 1264 bp of the 59 end of LSU nrDNA (2ln likelihood 5 4681.5015, CI 5 0.487, RI 5 0.893, RC 5 0.435, length 5 503 steps) for 71 diaporthalean taxa. Bootstrap values greater than 70% are shown above (MP) and below (NJ) each branch. Taxa in bold represent type species of their respective genera. Thickened lines indicate that branch appeared in the strict consensus of the 372 trees. 1026 MYCOLOGIA FIG. 3. Phylogenetic tree resulting from Bayesian analysis of 1264 bp of the LSU nrDNA (2ln likelihood 5 4693.39844) for 71 diaporthalean taxa. Numbers on branches indicate the posterior probability given the observed data that the group exists, expressed as a percentage. Taxa in bold represent type species of their respective genera. CASTLEBURY TABLE II. ET AL: OVERVIEW OF THE DIAPORTHALES 1027 Shimodaira-Hasegawa likelihood test results Topology Trees Length 2ln likelihood Pa Unconstrained Barr 1990 Gnomoniaceae Barr 1990 Melanconidaceae Barr 1990 Valsaceae Eriksson 2001 Melanconidaceae Eriksson 2001 Valsaceae Bayesian analysis 372 193b 118b 216b 117b 674b 4b 503 558 549 518 549 548 523–531 4681.50151–4697.26700 4783.96828–4801.5197 4815.91753–4834.40237 4709.20630–4728.20071 4825.80000–4837.33077 4825.65341–4846.68496 4693.39844–4711.49428 — 0.000* 0.000* 0.409 0.000* 0.000* 0.784 a P-values only reported for the tree with best 2ln likelihood score. Only the tree with the best 2ln likelihood score was tested. * Indicates significant at P , 0.05 in a one-tailed test under the null hypothesis that all trees are equally good explanations of the data. b rials and methods section. In both analyses, bootstrap values were equivalent or slightly lower than those from alignment 1 (trees not shown). This would indicate that better measures of support for groups within this order are obtained when more than the 59 650 bp of the LSU nrDNA are sequenced. However, for determining basic affinities, 650 bp may be sufficient. The Bayesian topology was similar to the MP analysis and the probabilities obtained were similar to bootstrap values for both NJ and MP analyses with the exception of better support for the Diaporthaceae. When the Bayesian topology was compared to the unconstrained MP topology, the S-H test found the Bayesian tree not to be a significantly worse explanation of the data. The 2ln likelihood score of the Bayesian topology shown in FIG. 3 is contained within the range of scores for the 372 equally parsimonious trees obtained in the MP search. Order and families of the Diaporthales. The Diaporthales has been recognized as a distinct order within the perithecial ascomycetes for about half a century following the description of the Diaporthe -type centrum by Luttrell (1951). With a few minor exceptions the order has been well defined by previous mycologists as listed by Barr (1978, 1990) and Samuels and Blackwell (2001). Analyses of molecular data have confirmed the Diaporthales as a well-supported order (Farr et al 2001, Zhang and Blackwell 2001). Thus, the morphological characteristics used to define the Diaporthales are considered reliable indicators of the order. The families of the Diaporthales as circumscribed by Eriksson et al (2001) were not supported by this study. Genera placed in the Melanconidaceae by Eriksson et al (2001) and included in this study are Allantoporthe Petr. as Diaporthe decedens, Ditopella De Not., Hercospora Fr., Melanconis Tul. & C. Tul., Phragmoporthe Petr., Schizoparme Shear, and Wuestneia Auersw. In this study these genera group into several different lineages within the order and do not constitute one or even several monophyletic groups (FIGS. 1–3). Genera placed in the Valsaceae by Eriksson et al (2001) and included in this study are Apioplagiostoma M.E. Barr, Cryphonectria (Sacc.) Sacc. & D. Sacc., Cryptodiaporthe Petr., Diaporthe, Endothia, Gnomonia, Gnomoniella Sacc., Leucostoma, Linospora Fuckel, Mazzantia, Ophiovalsa Petr. (listed as Winterella (Sacc.) Kuntze in Eriksson et al 2001), Plagiostoma Fuckel, Pleuroceras Riess, Valsa, and Valsella. Similarly, these genera group in different lineages within the Diaporthales. Representatives of the Vialaeaceae could not be located for this study. All three cultures deposited as Vialaea insculpta (Fr. : Fr.) Sacc. at CBS were sequenced, but none of these proved to belong in the Sordariomycetidae. No cultures of the genus Sydowiella were available to represent the Sydowiellaceae, but based on the morphology of the type species, S. fenestrans (Duby) Petr., it seems likely that this family is a later synonym of the Gnomoniaceae. Gnomoniaceae. One major lineage within the Diaporthales includes the genus Gnomonia and thus should be regarded as the family Gnomoniaceae (Hawksworth and Eriksson 1988). Twelve teleomorph genera representing 18 species and the anamorph taxa Discula campestris, D. destructiva, D. fraxinea, and D. quercina form this lineage that groups together with 85% or greater support in all analyses (FIGS. 1–3). Teleomorph genera in the Gnomoniaceae represented here include Apiognomonia Höhn, Apioplagiostoma, Cryptodiaporthe, Cryptosporella Sacc., Ditopella, Gnomonia, Gnomoniella, Linospora, Ophiovalsa, Phragmoporthe Petr., Plagiostoma, and Pleuroceras. No well-supported subdivisions were found in this group, although a number of taxa consistently grouped together in the analyses. These results generally agree with the concept of the Gnomoniaceae as recognized by Monod (1983) 1028 MYCOLOGIA and include taxa characterized by ascomata that are immersed, solitary, without stromata or aggregated in reduced, prosenchymatous stromata in herbaceous plant material, especially in leaves or stems, but also in wood. The ascomata are generally soft-textured, thin-walled, and prosenchymatous with either central or lateral beaks. The asci may or may not have a distinct ring and the ascospores are generally small, less than 25 mm long, and range in septation from nonseptate, one-septate (median or eccentric) to multiseptate. The anamorphs of members of the Gnomoniaceae are acervular or pycnidial, often with a broad opening, and phialidic, with pallid, non- or one-septate conidia. One genus, Mazzantia, placed in the Gnomoniaceae by Monod (1983) and included in this study, does not belong in the Gnomoniaceae, rather it falls within the Diaporthaceae (see below). Another genus placed in the Gnomoniaceae by Monod (1983), Gaeumannomyces Arx & D.L. Olivier, has been shown to belong in the Magnaporthaceae outside of the Diaporthales (Berbee 2001, Farr et al 2001, Zhang and Blackwell 2001). Melanconidaceae. Unlike the Melanconidaceae sensu Eriksson et al (2001) the results of this study suggest that all genera except the type genus Melanconis should be excluded from the family. The type species of Melanconis, M. stilbostoma, and two additional species, M. alni and M. marginalis, form a well-supported group with greater than 98% support in all analyses (FIGS. 1–3). These three species of Melanconis produce well-developed stromata having a light-colored ectostromatic disc and a concolorous central column with circinately arranged, immersed ascomata; hyaline, one-septate ascospores; and anamorphic states placed in Melanconium Link. Pycnidia develop in the stromata prior to formation of the ascomata and produce unicellular, dark-brown conidia. One species, Melanconis desmazierii, falls outside the Melanconidaceae. This species is a distantly related diaporthalean taxon that is allied with Hercospora tiliae in this study (.90%) in all analyses (FIGS. 1–3). The Melanconidaceae, herein restricted to Melanconis sensu stricto, groups with the Gnomoniaceae with support of 99% or greater in all analyses (FIGS. 1–3) and these two families could be combined and regarded as the Gnomoniaceae. This result is somewhat surprising and suggests that morphological characteristics like thickness of ascospore wall and stromatal development are of less importance than suggested previously (Barr 1978, 1990). Cryphonectria-Endothia complex. Representatives of three genera, namely Chromendothia, Cryphonectria, and Endothia, and one additional species, Cryptodia- porthe corni, grouped together at greater than 80% in all analyses except MP analysis of alignment 1 (FIGS. 1–3). Morphologically these taxa are united by ascomata immersed in well-developed, yellow to orange or orange-red stromata. The pigments within the stromatal wall turn purple in 3% KOH (KOH1) and yellow in lactic acid and are produced in culture. The ascomatal wall of members of the CryphonectriaEndothia complex is dark brown to black, often evident as darkened ostiolar papillae extending beyond the stromata. Although a similar KOH reaction is also characteristic of the Nectriaceae, Hypocreales, the pigments occur in cell walls of the ascomatal wall and are not often produced in culture. Anamorphs in the Cryphonectria-Endothia complex produce small, hyaline, one-celled conidia enteroblastically in multiloculate pycnidia in well-developed stromata similar in appearance to those producing ascomata. The close relationship of these three genera was recognized by Vasilyeva (1998) who placed them in tribe Endothiae M.E. Barr. Cryptodiaporthe corni, a species having the typical KOH1 purple color reaction characteristic of this group and occurring on the temperate host, Cornus alternifolia (Redlin and Rossman 1991) falls in this group rather than in the Gnomoniaceae with the type species of Cryptodiaporthe, C. aesculi (FIGS. 1–3). This species undoubtedly belongs in either Cryphonectria or Endothia. Three of the five species of Cryphonectria, namely C. macrospora, C. nitschkei, and C. parasitica, and Chromendothia citrina and Endothiella gyrosa occur primarily on temperate hardwood trees. Two additional species of Cryphonectria, C. cubensis and C. havanensis, appear to be more closely related to each other than to the other three species of Cryphonectria. Venter et al (2001) have recently suggested that the latter two species belong in a separate genus. Schizoparme complex. Two species of Schizoparme including the type species, S. straminea, and S. botrytidis grouped with seven strains of Coniella Höhn. and Pilidiella Petr. & Syd. at 97% or greater in all analyses (FIGS. 1–3). Although placed by Samuels et al (1993) in the Melanconidaceae, the genus Schizoparme and its allied taxa do not fall within any established family in the Diaporthales and may eventually be recognized as its own family. Unlike most members of the Diaporthales, species of Schizoparme are often erumpent through the host epidermis, becoming superficial. The ascomatal wall often includes ‘‘an epistromatic region of small, pale-colored cells around the ostiolar opening’’ (Samuels et al 1993). A similar, thickened outer wall was observed on species of Coniella and Pilidiella in culture. The ana- CASTLEBURY ET AL: OVERVIEW morph of S. straminea is P. castaneicola (Samuels et al 1993 as C. castaneicola) while that of S. botrytidis also belongs in Pilidiella (pers obs). Although Coniella is generally recognized in the broad sense to include the genus Pilidiella (Sutton 1980, Nag Raj 1993), the well-supported separation of these taxa in all analyses suggests that Pilidiella may be distinct from Coniella. These genera can be distinguished by conidial pigmentation which in Coniella fragariae, type of Coniella, and C. australiensis are dark brown while in Pilidiella castaneicola, type of Pilidiella, and related taxa including C. musaiensis, the conidia tend to be pale brown. Valsaceae. The genus Valsa and representatives of two related genera, Leucostoma and Valsella, group together with 98% or greater bootstrap values (FIGS. 1–3) in what is considered the family Valsaceae sensu stricto. The close relationship of species of Valsa to Leucostoma and Valsella has been recognized by a number of authors (Spielman 1985, Vasilyeva 1993) and is confirmed here. Members of the Valsaceae occur on woody angiosperms in temperate regions throughout the world (Barr 1978, Spielman 1985). The ascomata are aggregated in well-developed entostromata with beaks emerging centrally through a white to black stromatic disc. In Leucostoma and Valsella the entostromata are delimited basally by a black stromatic zone, while in Valsa such a zone is lacking. The genus Leucostoma has eight-spored asci while Valsella is characterized by multisporous asci. Although the type species of Leucostoma, L. massariana Höhn., was not available for this study, three species of Leucostoma were included, namely L. nivea, L. cincta and L. auerswaldii. The type of the genus Valsella, V. salicis, was included along with a second species, V. adherens. Neither the species of Leucostoma nor those of Valsella grouped together, exclusive of other taxa. Rather they were interspersed with each other and Valsa ceratosperma suggesting that neither the black stromatic disc nor polysporous asci unite related species. Within the Valsaceae, there were three well-supported subgroups in all analyses. The three species of Leucostoma with Valsella salicis and Valsa ceratosperma represent a subdivision although there is not obvious correlation with any morphological or biological features. The genus Valsa is represented in this study by the type species, V. ambiens, which grouped closely with Valsa germanica and V. cenisia. Valsa mali was basal to the V. ambiens group and the Leucostoma/Valsella group in all analyses. Diaporthaceae. This lineage consists of eleven species of Diaporthe including representatives of the type species, D. eres, and Mazzantia napelli which grouped OF THE DIAPORTHALES 1029 together at 75% or greater bootstrap support in all analyses except MP analysis of alignment 1 (FIGS. 1– 3). The family Diaporthaceae is delimited here in a much more restricted sense than by previous authors. This family was established by Höhnel (1917) who recognized this and only one other family, the Gnomoniaceae, in the Diaporthales. Wehmeyer (1975) had a somewhat narrower concept of the Diaporthaceae, including Diaporthe and Mazzantia as well as many more genera, some of which are included in this study and excluded from this lineage. The Diaporthaceae was considered a synonym of the Valsaceae by Barr (1978) and other workers since then. Based on the results presented here, the Diaporthaceae sensu stricto includes only Diaporthe and Mazzantia. All species of Diaporthe included in this study formed a well-supported group. Diaporthe is a large genus that is well-defined morphologically and includes several hundred described species, many of which have anamorphs belonging to the genus Phomopsis. Species occur on a wide range of substrates ranging from woody dicotyledonous plants to herbaceous monocots (Wehmeyer 1933). In Diaporthe each stroma covers and subtends several ascomata usually forming a black line in the hardened host tissue. The ascospores of species of Diaporthe are oneseptate, hyaline, and usually ellipsoidal. The Phomopsis anamorphic states are even more ubiquitous forming uni- or multiloculate, pycnidial stromata in which are produced hyaline, usually non-septate, primary conidia on elongate, phialidic conidiogenous cells, and often producing filiform beta conidia. The other member of the Diaporthaceae in this study is the genus Mazzantia. Although not the type species of the genus Mazzantia, M. napelli is similar to the type, M. galii (Fr.) Mont., in stromatal and ascomatal morphology, anamorph, and occurrence on dicotyledonous herbaceous hosts. Diaporthe and Mazzantia are similar in producing well-developed stromata immersed in relatively newly killed wood or stems. In Mazzantia the stromata are well-developed covering only one or a few, immersed ascomata. Although placed in the genus Mazzantiella Höhn., the anamorph of Mazzantia is similar to Phomopsis in producing hyaline, one-celled, elongate conidia on filiform, phialidic conidiogenous cells in a pycnidial locule (Wehmeyer 1975). Other taxa. The anamorph species, Greeneria uvicola, the cause of bitter rot of grapes, was recently determined to belong in the Diaporthales (Farr et al 2001). Despite its inclusion in this expanded account of the Diaporthales, G. uvicola is not closely affiliated with any of the taxa included in this study (FIGS. 1– 1030 MYCOLOGIA 3) and thus it is not possible to determine what its teleomorph may be, if one exists. One species of the genus Wuestneia having a Harknessia Cooke anamorph and the type species of Harknessia, H. eucalypti, grouped together in all analyses (FIGS. 1–3). The connection between Wuestneia and its anamorph Harknessia was established by Reid and Booth (1989) and is confirmed by these data. Harknessia lythri did not group with Harknessia and Wuestneia. This species is unusual in having longitudinal striations on the conidia (Farr and Rossman 2001) and may not belong in that genus. Many additional species of Harknessia have been described and most of them lack a known sexual state. Two species, Hercospora tiliae and Melanconis desmazierii, grouped together and may represent the Pseudovalsaceae (FIGS. 1–3). Melanconis desmazierii forms rudimentary stromata and produces a typical Melanconium anamorph. The well-developed stromata of Hercospora tiliae developed in culture producing a distinctive Rabenhorstia anamoph (Sutton 1980). Both species occur on species of Tilia in temperate regions. Our study suggests that four of the five major families previously established for members of the Diaporthales should be recognized, albeit circumscribed differently, and that two additional major lineages exist within the order. The lineages discussed in this paper were defined using a relatively high number of diaporthalean taxa, certainly the greatest number to date. Emphasis was placed on obtaining type species of key genera including those of anamorph genera where possible. Additionally, an attempt was made to locate fresh, accurately identified material from which single ascospore cultures could be isolated. Despite considerable effort, less than half of the type species of the 98 genera in the Diaporthales were obtained. With an increased number of taxa, especially those representing type species, it is likely that additional lineages will be defined. It is also expected that sequencing additional genes for taxa in this order will make it possible to identify relationships of genera within families and to determine morphological characters for reliable generic identifications. ACKNOWLEDGMENTS The authors express sincere appreciation to Margaret Barr Bigelow, Sidney, British Columbia, for providing fresh specimens of diaporthalean fungi from which cultures were isolated as well as general expertise and advice on working with the Diaporthales. In addition we thank Sabine Huhndorf and Fernando Fernandez, Field Museum, Chicago, for sending the sequence and culture of Schizoparme botrytidis obtained while working under NSF-PEET grant #DEB9521926. The following persons also sent isolates: Gerald Bills, Merck Research, Plagiostoma conradii; Scott Redlin, Animal and Plant Health Inspection Service, Apiognomnia errabunda and Cryptodiaporthe corni; and Jack Rogers, Washington State University, Pullman, Wuestneia molokaiensis. Finally, the skilled technical expertise of Douglas Linn, John McKemy, Brenda Paul, Frank Washington and Janelle Wood is acknowledged for handling the cultures and sequencing the newly obtained fungi. LITERATURE CITED Anagnostakis SL. 1987. 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