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Systematic Botany and Mycology Laboratory, USDA-ARS, 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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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, Cryphonectria-Endothia 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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). 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, 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). 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.
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) 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 µL 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 10x PCR buffer with 15 mM MgCl2. The thermal cycler program was as follows: 10 min at 95 C followed 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 5' end of the LSU nrDNA were sequenced. The sequence alignments were deposited in TreeBASE as S815.
For both alignments, trees were inferred by the neighbor-joining (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 Shimodaira-Hasegawa (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 substitution types (nst) = 6, a proportion of sites invariable and the rest drawn from the gamma distribution (rate = invgamma), base frequencies = estimate, rate matrix = (1.7034, 6.7182, 3.8375, 0.9578, 15.0004, 1.0000); (iii) number of generations = 500 000, sample frequency = 100, number of chains = 4, temperature = 0.5, save branch lengths = yes, starting tree = 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 = 0.2708, C = 0.2196, G = 0.2847, T = 0.2249; number substitution types = 6; proportion of invariable sites = 0.719; gamma shape parameter = 0.6023, number rate categories = 4, mean average rate; rate matrix = 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 -ln 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 -ln likelihood scores of trees from each constraint topology is shown. However, only the topology with the best -ln likelihood score from each constraint was tested against the unconstrained trees.
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RESULTS |
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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 trees generated with MULTREES on (MAXTREES = 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 = 0.428, RI = 0.879, RC = 0.376, and length = 428. For alignment 2, tree scores were CI = 0.487, RI = 0.893, RC = 0.435 and length = 503. The MPT with the best -ln likelihood score for alignment 1 is shown in Fig. 1 and one of two MPT with the best -ln likelihood score is shown for the alignment 2 (Fig. 2), although neither tree was significantly better than others generated in each analysis (P = 0.05). Bootstrap values greater than 70 percent are indicated on Figs. 1 and 2 above (MP) and below (NJ) the respective branches.
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), 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 -ln 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).
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) 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 S-H 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 -ln 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 materials 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 5' 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 -ln 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-><011>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) 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 µm long, and range in septation from non-septate, 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, Cryptodiaporthe 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 (KOH+) and yellow in lactic acid and are produced in culture. The ascomatal wall of members of the Cryphonectria-Endothia 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 KOH+ 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 anamorph 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 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 one-septate, 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. 1–3) 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication April 28, 2002.
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LITERATURE CITED |
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