Mycologia, 95(3), 2003, pp. 426–433. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897
Saccharicola, a new genus for two Leptosphaeria species on sugar cane
Ove E. Eriksson1
onstrated by single ascospore cultures to have an anamorph that the authors referred to as Stagonospora. The genus Phaeosphaeria Miyake, a segregate from Leptosphaeria (cf. Holm 1957, Shoemaker and Babcock 1989), includes many species with a Stagonospora anamorph (Leuchtmann 1984). However, analyses of SSU rDNA sequences did not support such a classification (Morales et al 1995). The relationships and position of the species have remained uncertain, although it has been included in some recent molecular studies. Dong et al (1998) found that L. bicolor is not closely related to Leptosphaeria, nor to Phaeosphaeria and assumed that a new genus might have to be described after further studies. An SSU rDNA study by Olivier et al (2000) suggested a close relationship among L. bicolor and two Helminthosporium species but not with any teleomorphs. Caˆmara et al (2002) reported that Leptosphaeria bicolor is most closely related to the morphologically and biologically similar Leptosphaeria taiwanensis W. Y. Yen et C. C. Chi. We re-examined original dried reference material and found that L. bicolor is more similar to Massarina than to Phaeosphaeria or any other genus in Dothideomycetes. In an SSU rDNA study by Liew, Aptroot and Hyde (2000), three Massarina species, but not L. bicolor, were among the 31 ‘‘loculoascomycetes’’ included. Their study indicated that Massarina was polyphyletic. The purpose of our study was to find answers to three questions: (i) Is Leptosphaeria bicolor closely related to the type species of Massarina, M. eburnea (Tul. et C. Tul.) Sacc.? (ii) If so, should it be transferred to Massarina or be accommodated in a separate genus? (iii) To which family does it belong? To answer these questions we re-examined herbarium material of L. bicolor and M. eburnea and performed a maximum-parsimony analysis of all SSU rDNA sequences from teleomorphs (63) and some anamorphs in class Dothideomycetes that were available from GenBank in April 2001.
Department of Ecology & Environmental Science, Umea˚ University, SE-90187 Umea˚, Sweden
David L. Hawksworth Departamento de Biologı´a Vegetal II, Facultad de Farmacia, Universidad Complutense, Plaza de Ramo´n y Cajal, Ciudad Universitaria, E-28040 Madrid, Spain
Abstract: Leptosphaeria bicolor, causal agent of a leaf scorch disease of sugar cane, is referred to the new genus Saccharicola. The ascospores are 1–3 transseptate and hyaline at first but become melanized and rough after release, as is the case in some members of Massarina and Lophiostoma. SSU rDNA data indicate that it is closely related to M. eburnea but is biotrophic in leaves of sugar cane and not corticolous, the ascomata are less melanized, and it has Stagonospora- and Phoma-like synanamorphs, not a Ceratophoma-like anamorph. A second species, Leptosphaeria taiwanensis, is transferred to Saccharicola. It differs in slightly larger, normally 1-septate, hyaline spores with more attenuated ends. The family Massarinaceae is resurrected to accommodate Massarina s. str., Keissleriella, Saccharicola and Helminthosporium. These genera formed a clade with 100% bootstrap support in a parsimony analysis of SSU rDNA sequences from 38 ascomycetes, 30 of them members of Pleosporales (including Melanommatales). Key words: ascomycetes, Leptosphaeria bicolor, Massarinaceae, phylogeny, Pleosporales, SSU rDNA, morphology
INTRODUCTION
Leptosphaeria bicolor D. Hawksw., W. Kaiser et Ndimande (Kaiser et al 1979) was described to accommodate the causal agent of a leaf scorch disease of sugar cane (Saccharum officinarum L.) in Kenya. It was included in Leptosphaeria on the basis of the small, innate perithecioid ascomata, bitunicate asci separated by branched, anastomosed, septate pseudoparaphyses, and 3-septate ascospores. It was dem-
MATERIALS AND METHODS
The holotype (IMI 215888) and an authenticated collection (IMI 214025) of Leptosphaeria bicolor, one collection of Massarina eburnea (Lundell and Nannfeldt, Fungi Exs. Suec. 775, UPS), and isotype material of Leptosphaeria taiwanensis (BPI 621856) were examined under the light microscope.
Accepted for publication September 9, 2002. 1 Corresponding author. E-mail:
[email protected]
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Taxa included in parsimony analysis and their SSU rDNA GenBank accession numbers
Capnodium dematum (V.A.M. Mill. et Bonar) D.R. Reynolds Capronia pilosella (P. Karst.) E. Mu¨ll. Ceramothyrium linnaeae (Dearn.) S. Hughes Coccodinium bartschii A. Massal. Cochliobolus sativus (Ito et Kurib.) Drechsler ex Dastur Cucurbidothis pityophila ( J.C. Schmidt et Kunze) Petr. Cucurbitaria berberidis (Pers.) Gray Dothidea hippophae¨s (Pass.) Fuckel Dothidea insculpta Wallr. Helicascus kanaloanus Kohlm. Helminthosporium solani Dur. et Mont. Helminthosporium velutinum Link Herpotrichia nigra Hartig (‘‘Herpotrichia juniperi’’) Keissleriella cladophila (Niessl) Corbaz Kirschsteiniothelia elaterascus Shearer Leptosphaeria bicolor D. Hawksw., W. Kaiser et Ndimande Leptosphaeria doliolum (Pers.) Ces. et De Not. Leptosphaeria maculans (Desm.) Ces. et De Not. Lophiostoma crenatum (Pers.) Fuckel Massaria platani Ces. Massarina australiensis K.D. Hyde Massarina bipolaris K.D. Hyde Massarina eburnea (Tul. et C. Tul.) Sacc. Massariosphaeria phaeospora (E. Mu¨ll.) Criv. Melanomma pulvis-pyrius (Pers.) Fuckel Montagnula opulenta (De Not.) Aptroot Neurospora crassa Shear et B.O. Dodge Paraphaeosphaeria filamentosa (Ellis et Everh.) M.E. Barr Paraphaeosphaeria glauco-punctata (Grev.) Shoemaker et C.E. Babcock Paraphaeosphaeria michotii (Westend.) O.E. Erikss. Paraphaeosphaeria obtusispora (Speg.) O.E. Erikss. Paraphaeosphaeria pilleata Kohlm., Volkm.-Kohlm. et O.E. Erikss. Phaeodothis winteri (Niessl) Aptroot Phaeosphaeria microscopica (P. karst.) O.E. Erikss. Pleospora herbarum (Pers.) Rabenh. Pyrenophora trichostoma (Fr.) Fuckel Venturia liriodendri R.T. Hanlin
All SSU rDNA sequences in GenBank from teleomorphs (and some anamorphs) of the Dothideomycetes, together with two members of the Chaetothyriomycetes and one of the Sordariomycetes (TABLE I), were aligned manually with Se-Al v.1 (Rambaut 1996). The latter (Neurospora crassa) was used as outgroup in maximum-parsimony analyses performed with PAUP 4.0b7 (Swofford 2001). Gaps were coded as missing data. Trees were obtained by heuristic search (100 replicates, random addition sequence and TBR branch swapping algorithms). Bootstrap values for nodes were computed from 500 replicates. The TreeBase study accession number is S865; and the matrix accession number is M1402.
RESULTS
Morphological data. We re-examined the original material of Leptosphaeria bicolor and found that the descriptions of the species provided by Kaiser et al
AF006724 U42473 AF022715 U77668 U42479 U42480 U42481 U42475 U42474 AF053729 AF120253 AF120254 U42483 AF164360 AF053728 U04202 U04205 U04238 U42485 AF164363 AF164364 AF164365 AF164367 AF164368 AF164369 AF164370 X04971 AF250825 AF250819 AF250817 AF250822 AF250821 AF164371 U04235 U43458 U43459 U79489
(1979), Punithalingam (1983), Sivanesan (1984), and Sivanesan and Waller (1986) agreed in most respects with our observations. However, we did not see any fully melanized spores inside the asci nor were any illustrated in any of these publications. The ascospores appeared to be released in a hyaline or very pale brown stage, with 1–3 transsepta. The upper central cell in the 3-septate spores of this species is slightly inflated and can become dark brown to almost black at maturity. We found such spores on the surface of the host near the ostioles of the fungus and free inside some ascomatal cavities. Some spores were arranged in groups of eight, but there were no remnants of ascus walls. However, in many free spores all four cells were melanized, sometimes with the upper central cell much darker than the other three cells. We also found melanized 1-septate and 3-sep-
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FIG. 2. Saccharicola taiwanensis. Ascospores. Bar: 30 mm. From isotype.
FIG. 1. Saccharicola bicolor. Ascospores from the host surface in different stages of development, from almost hyaline to spores with all cells strongly melanized. Two spores with a collapsed cell. Bar: 30 mm. From holotype.
tate spores with two dark cells. A distinct pore often was clearly visible in the center of older, melanized septa. In some spores of L. bicolor, we observed groups of small pores in the walls, but the origin and nature of these pores could not be determined. The hyaline spores always were smooth, but the melanized ones had an ornamentation of fine warts, as observed by Kaiser et al (1979). The variation in ascospore shape, color and septation is shown in FIG. 1. While the asci are clearly bitunicate in structure, no fissitunicate discharge was seen. There are two possible explanations for these observations. The spores are forcibly ejected while hyaline and become melanized only on the leaf surface, brown spores in the cavity being over-ripe and abortive, or the spores are released by deliquescence of the ascus walls within the cavity, become melanized and more inflated, then ooze out of the ostiole. Studies of fresh material are needed to reveal which of these two alternatives is correct. Molecular data. We aligned all SSU rDNA sequences from teleomorphs and some anamorphs in the Dothideomycetes available in GenBank, using Se-Al. In a first preliminary parsimony analysis (not shown), with two outgroup taxa in Chaetothyriomycetes and one in Sordariomycetes, we found that Leptosphaeria bicolor appeared with a strong bootstrap (BT) support in a clade of perithecioid taxa, most of which currently are
accommodated in Pleosporales (including Melanommatales), Dothideales and Capnodiales. Taxa in Pleosporales formed one subclade, those in Dothideales and Capnodiales another subclade, both with strong BT support. We performed a BLAST search for the sequences that were most similar to the SSU rDNA of L. bicolor. Almost all taxa from that search were members of the Pleosporales clade. The hyphomycetes Helminthosporium solani and H. velutinum had the most similar sequences, as already reported by Olivier et al (2000). In a second parsimony search we excluded Dothideomycetes taxa that, based on the first parsimony analysis, obviously were not related closely to L. bicolor, viz. members of Myriangiales, Patellariales and some genera with uncertain relationships, e.g., Acrospermum (Winka and Eriksson 2000) and Aliquandostipite (Inderbitzin et al 2001). Excluded also were many taxa in genera with several closely related species that differed very little in their SSU rDNA sequences. Many of the sequences in the alignment were about 1700 bp, and only a short part at the ends of each gene was lacking. However, some sequences were little more than 1000 bp (from near the 59 end of the gene). This second heuristic analysis (100 repl.) of 1708 characters (225 phylogenetically informative) in 37 taxa resulted in 12 equally most-parsimonious trees (MPTs). The consensus tree (FIG. 3) resembled that from the first broader analysis but with slightly different BT values. All members of the Dothideomycetes clustered in one clade with 97% BT support. The two members of Chaetothyriomycetes formed a separate clade with 100% BT support. The Dothideomycetes clade again consisted of two subclades, both with 99% BT support, viz. one with all members of Pleosporales, the other with members of Dothideales and Capnodiales. The Pleosporales clade of 30 taxa was
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FIG. 3. A maximum-parsimony consensus tree from 12 equally most-parsimonious trees in an analysis of SSU rDNA sequences from ascomycetes. The numbers represent the bootstrap percentages from 500 replicates. Orders: 1. Pleosporales. 2. Dothideales. 3. Capnodiales. 4. Hypocreales. 5. Chaetothyriales. Clade A: Massarinaceae. Clade B: Montagnulaceae.
not very well resolved. Almost all of the differences between the 12 MPTs were in this clade, and six branches collapsed in the consensus tree. It also contains genera, such as Melanomma and Massaria, which have been placed in the orders Melanommatales and Pyrenulales, respectively. Inclusion of more taxa representing these two orders might be useful in resolving the phylogeny of the clade. However, some subclades received a strong BT support, six of
them with 95–100% support. One of the subclades that received 100% BT support consisted of Leptosphaeria bicolor, Massarina eburnea and the two Helminthosporium species. The position of Leptosphaeria bicolor and the type species of Leptosphaeria, L. doliolum (Pers.) Ces. et De Not., is of particular interest because we were able to confirm the results of some previous studies that indicated that Leptosphaeria bicolor is not closely related to the generic type Leptos-
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phaeria doliolum (Morales et al 1995, Dong et al 1998, Olivier et al 2000, Tehler et al 2000). DISCUSSION
Morphological data. Ascospores of the L. bicolor type, which become melanized at a late stage, are common in Lophiostoma and Massarina s. auct. (currently in the Lophiostomataceae) but not in Leptosphaeria and related genera. Leptosphaeria, as now conceived, is restricted to species with scleroplectenchymatous ascomatal walls (Holm 1957). Indeed, Kaiser et al (1979: 490) had observed that L. bicolor occupied a somewhat isolated position among the Leptosphaeria species known from Poaceae generally. Leptosphaeria bicolor does not fit in Leptosphaeria but agrees in several respects with Massarina. The latter genus was monographed by Aptroot (1998), who accepted 43 species, but many more still are hidden in Leptosphaeria, Metasphaeria, etc. A recent molecular paper by Liew et al (2000) indicated that Massarina might be polyphyletic. The type species, M. eburnea, clustered with 83% BT support with four species in other genera (Keissleriella, Massaria, Montagnula, Phaeodothis), whereas two other Massarina species (M. australiensis K.D. Hyde and M. bipolaris K.D. Hyde) formed a clade with 89% BT support with Lophiostoma caulium (Fr.) Ces. et De Not. As we obtained similar results in our analysis, and, moreover, found that L. bicolor clustered with M. eburnea with very high BT support in all of our parsimony analyses with different constellations of taxa, we re-examined microscopic characters of L. bicolor and M. eburnea. Massarina eburnea has been examined by several mycologists (e.g., Bose 1961, Eriksson 1981, Aptroot 1998, Hyde 1995), all with slightly different observations. Key observations in our morphological studies of L. bicolor and M. eburnea are: (i) M. eburnea, as most other species in Massarina s. lat., occurs in bark or on wood of shrubs and trees. A few Massarina species are saprotrophs on other substrates, e.g., grass culms, canes of Rubus and leaves of Lycopodium, but it is uncertain now whether these species are closely related to M. eburnea. Leptosphaeria bicolor is a perthotroph that occurs only in discolored dead areas in living leaves of Saccharum officinarum. (ii) The wall of the ascomata usually is melanized irregularly in Massarina and opaque black near the ostiole. The ascomatal cell walls are dark brownish in L. bicolor, but the ostiolar region is not opaque black. (iii) A ring is visible in the apex of M. eburnea asci in mounts stained with Cotton blue (Eriksson 1981, Fig. 103B). No ring was seen in the ascal apex of L. bicolor mounted in Cotton blue or Congo red. (iv) The as-
cospore wall is about 1–1.5 mm in M. eburnea but is somewhat thicker at the septa, which are thick in the peripheral parts and gradually thinner toward the central pore (Eriksson 1981, Fig. 103C). Thus, the locules are lenticular. In L. bicolor the spore wall and septa are of equal and even thickness, c. 1 mm. (v) The ascospores are consistently hyaline in M. eburnea. We have searched for melanized spores on the bark near the ostiole in this species but have found none. In old ascospores of L. bicolor, the septa also are melanized and the central pore is visible in the each septum. That feature recalls a septal pore in Pyrenidium actinellum Nyl. (Dacampiaceae, Hawksworth 1980, 1983) and Navicella pileata (Tode) Fabre (Massariaceae; Eriksson 1981, Fig. 99H). However, pores can be demonstrated also in species with hyaline spores if a stain is added to the mount, e.g., in Clathrospora heterospora (De Not.) Wehm. (Eriksson 1967, Fig. 2m). A central pore probably always is present in spore septa and thus not a phylogenetically important character. (vi) The perispore is thick in M. eburnea and divided at the primary septum (Eriksson 1981, Fig. 103C). No perispore was seen in L. bicolor, even in young ascospores. (vii) M. eburnea, has a Ceratophoma-like anamorph (Bose 1981). The anamorph of L. bicolor is Stagonospora-like, but a Phoma-like anamorph also has been reported (Venkatasubbaiah et al 1988). Many species of Phaeosphaeria have a Stagonospora anamorph, but the conidia are more numerous and smaller in comparison to the size of the pycnidia than are those of the anamorph of L. bicolor. Stagonospora conidia are comparatively much fewer and larger in L. bicolor (see Kaiser et al 1979, Fig. 5E). Also, Caˆmara et al (2002) have argued that the ‘‘Stagonospora’’ anamorphs of Phaeosphaeria are different from that of L. bicolor. One of the 43 Massarina species, accepted by Aptroot (1998), M. arundinacea (Sow.) Leuchtm., has been reported to have a Stagonospora anamorph (S. elegans (Berk.) Sacc.), but the connection seems never to have been proven, and the conidia of this fungus are very large (52–84 3 8.5–14 mm, Sutton 1980: 108) and it is highly improbable that the presence of a Stagonospora-like fungus in L. bicolor and M. arundinacea (unproven) is a synapomorphy. The latter species has ascospores that are similar to immature Lophiostoma spores, and it might be related to that genus. Molecular data. As mentioned above, our morphological studies indicate that L. bicolor is closely related to Massarina. In a molecular study of ascostromatic taxa, Liew et al (2000) found that Massarina might be a polyphyletic genus. They did not include L. bicolor in their study. In our analysis of a broader set of taxa, we obtained a phylogram similar to that in Oliv-
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ier et al: two strongly supported subclades (A and B) that formed a clade that was the sister clade of a large group containing Leptosphaeria s. str., Phaeosphaeria, Pleospora, etc. Some other taxa were located close to, but not in, these clades and will require further studies (Massariosphaeria phaeospora, etc.), but adding more taxa to the analysis probably will not influence the stability of Clade A. Clade A (100%) contained L. bicolor and the two Helminthosporium species mentioned above and the generic type Massarina eburnea. This clade might be referred to as Massarinaceae Munk, a family name that has been treated as a synonym of Lophiostomataceae (e.g., Eriksson and Yue 1986, Eriksson 1999). Clade B (96%) contained six taxa, Cucurbidothis pityophila and Venturia liriodendrii (as in Olivier et al), but also two Paraphaeosphaeria species (P. michotii and P. pilleata), Phaeodothis winteri and Montagnula opulenta (syn. Didymosphaerella opulenta, Barr 2001). The two latter species were included by Barr (2001) in her new family Montagnulaceae. Whether the other species in Clade B have any specific morphological traits in common with the Montagnulaceae is as yet unclear. Paraphaeosphaeria michotii is the type species of Paraphaeosphaeria, and, as pointed out by Caˆmara et al (2001), the genus might be polyphyletic. Paraphaeosphaeria pilleata is closely related. Both species have small, 2-septate ascospores. Other species in the genus clustered with Leptosphaeria, Phaeosphaeria and Cucurbitaria and might require a new generic name. Molecular characters indicate that Leptosphaeria bicolor is closely related to Massarina eburnea, the type species of Massarina, but there are several morphological differences between the two species. They are best accommodated in separate genera, hence, L. bicolor is transferred to the new genus Saccharicola. L. taiwanensis is closely related and also is accommodated in Saccharicola. Saccharicola D. Hawksw. et O.E. Erikss. gen. nov. Ascomata pseudothecia dispersa, singularia, immersa, subglobosa, nigra, cum ostiolis papillatibus; muri e cellularum atrobrunneum et pseudoparenchymaticarum. Pseudoparaphyses filiformes, ramosae, rarissime anastomosantes. Asci subcylindrices vel elongato-clavati, fissitunicati, 8-spori. Ascosporae distichae, late fusiformes, inequilateralae, 1–3 septatae, hyalinae vel pallide brunneae in ascis, brunneae et verruculosissimae ex ascis, cellula juxta cellulam apicis inflata et interdum atrobrunneae. Conidiomata pycnidia, Stagonospora similia.
Ascomata pseudothecia, scattered, arising singly in dead spots of leaves, immersed, subglobose, black, ostiole papillate; walls composed of a few layers of dark brown pseudoparenchymatous cells. Hamathecium of filiform, branched, rarely anastomosing pseudoparaphyses. Asci subcylindrical or elongate-clavate, bi-
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tunicate, 8-spored. Ascospores distichously arranged in the asci, broadly fusiform, inequilateral, 1–3-septate, hyaline to pale brown in the asci, brownish and usually finely warted after release, penultimate cell inflated, often darker than the other cells and sometimes dark brown. Conidiomata pycnidia, similar to Stagonospora. Typus: Saccharicola bicolor (D. Hawksw., W. Kaiser et Ndimande) D. Hawksw. et O.E. Erikss. (HOLOTY PUS). Saccharicola bicolor (D. Hawksw., W. Kaiser et Ndimande) D. Hawksw. et O.E. Erikss. comb. nov. FIG. 1 Basionym: Leptosphaeria bicolor D. Hawksw., W. Kaiser et Ndimande, in Kaiser et al, Mycologia 71: 483 (1979). Type: KENYA: near Nairobi, on leaves of Saccharum officinarum, 24.viii.1977, W.J. Kaiser (HOLOTY PE IMI 215888; ISOTY PES BPI, DAOM, H). Other collection studied: KENYA: Mumias (W. Kenya), Stagonospora sp. isolated from Saccharum officinarum, 3.vi.1977, W.J. Kaiser (IMI 214025). Descriptions: Kaiser et al (1979), Punithalingam (1983), Sivanesan (1984), Sivanesan and Waller (1986). Saccharicola taiwanensis (W.Y. Yen et C.C. Chi) O.E. Erikss. et D. Hawksw. comb. nov. FIG. 2 Basionym: Leptosphaeria taiwanensis W.Y. Yen et C.C. Chi, Journal of Sugar cane Research Taiwan 6: 213 (1952). Type: TAIWAN: Taichung, on leaves of Saccharum officinarum, 4.ii.1953, W.Y. Yen (ISOTY PE BPI 621856). Descriptions: Yen and Chi (1952), Shoemaker and Babcock (1989). Camara et al (2002) found in their ITS analyses that L. bicolor is very closely related to L. taiwanensis J.M. Yen et C.C. Chi, another species on Saccharum, and another unnamed species on Miscanthus, a grass genus related to Saccharum. There were no differences between the sequences from different isolates of L. taiwanensis, but the sequence from L. bicolor differed from these in 26 positions. Shoemaker and Babcock (1989) examined type material of L. taiwanenis and said the ascospores were hyaline and 1-septate, although some older spores appeared ‘‘3-septate from separation of protoplasts.’’ Therefore, they transferred the species to Didymella. From their excellent description and illustrations and our studies of type material, it is clear that the fungus is more similar to young specimens of Saccharicola than to Didymella, which have larger peridial cells and no tendency to phragmoseptation. The two Saccharicola species are found in dead spots on leaves of Sacchar-
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um. They can be identified only under the microscope. S. taiwanensis has 1-septate hyaline ascospores that are more attenuated toward the ends than the spores of S. bicolor, which, moreover, are smaller. Sivanesan (1984) said the spores were ‘‘hyaline and brown’’ and 40–46 3 6.5–12.5 mm, whereas Shoemaker and Babcock (1989) gave the spore size of L. taiwanensis as 44–56 3 11–13 mm, and whose observations are in agreement with our studies. The spores of L. bicolor measure 29–42 3 8–11 mm. There has been a controversy about the identity of the anamorph of L. taiwanenis, either Cercospora taiwanensis T. Matsumoto et W. Yamam. or Stagonospora tainanensis Hsieh (see Shoemaker and Babcock 1989). Further collecting and cultural studies are needed to confirm Hsiehs (1979) studies that demonstrated Phoma and Stagonospora synanamorphs. Our results support those of previous studies suggesting: (1) the genus Massarina is heterogenous (Liew et al 2000). The type species can be accommodated in the family Massarinaceae (Pleosporales). Among the other species is the type of the genus Bertiella (Sacc.) Sacc et Sydow (Eriksson and Yue 1986, Aptroot 1998), which probably is closely related to the Lophiostomataceae. (2) The genus Paraphaeosphaeria is heterogeneous (Caˆmara et al 2001). One fraction seems to be related to the Montagnulaceae; the other is closest to Leptosphaeria or Phaeosphaeria. (3) Kirschsteiniothelia might be heterogenous. Material identified as the type species, Kirschsteiniothelia aethiops (Berk. et Curtis) D. Hawksw. (Hawksworth 1985), clustered with unitunicate pyrenomycetes, and a BLAST search listed only members of the Sordariomycetes. More information on this sequence and the voucher material must be examined and the misidentification excluded before any changes are made to the generic concept. K. elaterascens might be closely related to Helicascus kanaloanus, and these two species will require restudy and closer morphological comparison. K. maritima does not seem to be related to any of the two other species discussed above. ACKNOWLEDGMENTS
We thank K. Winka for useful comments on the manuscript. We are grateful for the loan of material of Leptosphaeria bicolor from IMI (CABI Bioscience, Egham, U.K.), of Massarina eburnea from UPS, and of Leptosphaeria taiwanensis from BPI. The authors acknowledge support by a grant from the Swedish Natural Science Research Council. D.L.H. was financed by the Ministerio de Ciencia y Technologı´a de Espan ˜ a (Project PO 98–0774) and was in receipt of an award under the Programa Ramo´n y Cajal from the same ministry while this study was completed.
LITERATURE CITED
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