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The polyphyletic nature of Pleosporales: an example from Massariosphaeria based on rDNA and RBP2 gene phylogenies

The polyphyletic nature of Pleosporales: an example from Massariosphaeria based on rDNA and RBP2 gene phylogenies

mycological research 111 (2007) 1268–1276 journal homepage: www.elsevier.com/locate/mycres The polyphyletic nature of Pleosporales: an example from ...

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mycological research 111 (2007) 1268–1276

journal homepage: www.elsevier.com/locate/mycres

The polyphyletic nature of Pleosporales: an example from Massariosphaeria based on rDNA and RBP2 gene phylogenies Hong-Kai WANGa, Andre APTROOTb, Pedro W CROUSc, Kevin D HYDEd, Rajesh JEEWONd,* a

Biotechnology Institute, Zhejiang University, 310029, PR China Adviesbureau voor Bryologie en Lichenologie, ABL Herbarium, G.v.d.Veenstraat 107, NL-3762 XK Soest, The Netherlands c Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands d Division of Microbiology, School of Biological Sciences, Room 2S-17, Kadoorie Biological Sciences Building, University of Hong Kong, Pokfulam Road, Hong Kong, P.R. China b

article info

abstract

Article history:

Massariosphaeria is a loculoascomycetous fungus currently accommodated within the

Received 2 February 2007

Pleosporales. However, based on morphology alone, it has been difficult to assess its familial

Received in revised form

position and its affinities to other fungi with bitunicate asci. In order to establish its

8 June 2007

evolutionary relationships, two regions of the rDNA (18S and 28S) and two regions of the

Accepted 22 August 2007

RPB2 protein-coding gene were sequenced and analysed phylogenetically. Multigene phy-

Published online 1 September 2007

logenies revealed that Massariosphaeria is not monophyletic and results are in disagree-

Corresponding Editor:

ment with existing morphological-based classification schemes. Characters, such as

H. Thorsten Lumbsch

ascomatal shape and ascospore morphology, have evolved more than once within the Pleo-

Keywords:

which could be confidently assigned to the Lophiostomaceae. M. typhicola is closely related

sporales. The familial placement of several species is still obscure, except M. grandispora, Ascospore evolution

to Trematosphaeria hydrela (Melanommataceae), whereas M. triseptata is related to Melanomma

Dothideomycetes

radicans but shares close affinities to the Sporormiaceae. The placement of M. roumeguerei is

Loculoascomycetes

still unresolved, and it does not appear to have any close evolutionary relationship to any

Multigene phylogeny

known melanommataceous or pleosporaceous genera. Our molecular data also refute the

Pleosporaceae

monophyly of Kirschsteiniothelia, Massarina, Melanomma, and Pleospora, and support previous

Systematics

phylogenetic hypotheses that Melanommataceae is polyphyletic. There is a need for more phylogenetic (and taxonomic) studies within the Pleosporales, especially incorporation of more anamorphic taxa and type species. ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction The genus Massariosphaeria (Pleosporales, Dothideomycetes) is characterised by a typical pleosporaceous morphology (Leuchtmann 1987), which include: black, bean-shaped or elongated,

immersed (sometimes semi-immersed) ascomata; black and papillate ostioles; a thin-walled, smooth peridium (textura prismatica) with black to grayish cells; abundant, hyaline, septate hamathecium filaments; and bitunicate, cylindric-clavate asci. It is morphologically a relatively well-characterised genus,

* Corresponding author. E-mail addresses: [email protected]; [email protected] 0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2007.08.014

Polyphyletic nature of Massariosphaeria

easily recognisable by the fusiform, multiseptate ascospores with relatively large thick walls, yellow to brown colour, some with transverse septum only, with the cell above the septum the largest, and surrounded by a prominent mucilaginous sheath (Tanaka & Harada 2004; Van 2005). All species have a tendency to form red pigments, on the host and especially in culture (Van 2005). The genus was first established as a section of Leptosphaeria (Mu¨ller 1950), but was later given generic status. This taxonomic arrangement has largely been accepted (Leuchtmann 1984; Huhndorf et al. 1990), although Barr (1989) included Massariosphaeria in Chaetomastia. Currently, there are about 28 accepted species Index Fungorum, June 2006 and both phragmosporous and dictyosporous taxa are included (Leuchtmann 1987; Shoemaker & Babcock 1989; Tanaka & Harada 2004). Lophiostoma, Leptosphaeria, Melanomma, and Trematosphaeria are pleosporaceous genera with species that are morphologically similar to various Massariosphaeria species. This has resulted in taxonomic uncertainty with species being transferred from one genus to another (e.g. Shoemaker & Babcock 1989; Huhndorf et al. 1990; Tanaka & Harada 2003, 2004). The familial position of Massariosphaeria is still not well defined. This is partly because the circumscription of other families within the order Pleosporales is still unclear. Massariosphaeria is generally accepted to belong to the family Lophiostomaceae (Eriksson & Hawksworth 1991; Kirk et al. 2001). However, it has also been referred to the family Dacampiaceae by Barr (1992), no doubt because she included some species of Massariosphaeria in Chaetomastia (Barr 1989). Most of the known anamorphic Massariosphaeria species are aposphaeria-like, which is also the anamorph produced by Melanomma (Melanommataceae) (Kirk et al. 2001; Tanaka & Harada 2004). On morphological grounds, it has been very difficult to predict the familial placement, and to date, only one species of Massariosphaeria (M. phaeosphaeria, the type species), has had its 18S rDNA partially analysed (Liew et al. 2000). It is thus unknown whether the genus is mono- or polyphyletic. This issue needs to be addressed as there have been a few reports on polyphyly of some pleosporaceous genera. (e.g. Kodsueb et al. 2006a; Liew et al. 2002). This paper is a continuity of several taxonomic studies on the Pleosporales where we have targeted important dothideomycetous genera, such as Leptosphaerulina, Letendraea, Pleospora, Tubeufia, Pyrenophora, and Wettsteinina (Kodsueb et al. 2006a,b; Pinnoi et al. 2007). The present work had three objectives: (1) to determine whether Massariosphaeria represents a natural group; (2) to verify the familial placement of Massariosphaeria; and (3) to discuss phylogenetic findings with respect to morphological-based classification schemes.

Materials and methods DNA extraction, amplification and sequencing Cultures of fungi used in this study were obtained from the Centraalbureau voor Schimmelcultures (Massariosphaeria grandispora ¼ CBS613.86; M. roumeguerei ¼ CBS612.86; M. triseptata ¼ CBS614.86; M. typhicola ¼ CBS609.86). Isolates were grown on potato-dextrose agar (PDA) and malt-extract agar (MEA) for two to four weeks and total genomic DNA was

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extracted from mycelia following the protocols as outlined by (Cai et al. 2005, 2006a,b). GenBank accession numbers are as follows and shown on the trees: 18S rDNA (Massariosphaeria grandispora ¼ EF165038; M. roumeguerei ¼ EF165035; M. triseptata ¼ EF165036; M. typhicola ¼ EF165037); 28S rDNA (Massariosphaeria grandispora ¼ EF165034; M. roumeguerei ¼ EF165032; M. triseptata ¼ EF165031; M. typhicola ¼ EF165033); RPB2 (Massariosphaeria grandispora ¼ EF165034/EF165042; M. roumeguerei ¼ EF165032/EF165039; M. triseptata ¼ EF165031/EF165040; M. typhicola ¼ EF165033/EF165041). DNA amplification was performed by PCR. For partial 18S and 28S rDNA amplification, NS1 & NS4 and LROR & LR5 primers (White et al. 1990; Vilgalys & Hester 1990) were used. Two separate regions of the RPB2 gene were amplified with primer pairs RPB2-5f & RPB2-7cr and RPB2-7f & RPB2-11ar (Liu et al. 1999). All amplification reactions were performed in a 50 ml reaction volume as follows: 1  PCR buffer, 0.2 mM dNTP, 0.3 mM of each primer; 1.5 mM MgCl2, 0.8 units Taq Polymerase and 5–10 ng DNA. PCR thermal cycle parameters for partial 18S and 28S rDNA amplification was as follows: 94  C for 3 min, followed by 34 cycles of denaturation at 94  C for 1 min, annealing at 52  C for 30 s and elongation at 72  C for 1 min, with a final extension step of 72  C for 10 min. The parameters for the PCR thermal cycle of the partial RPB2 gene amplification consisted of 95  C for 5 min, followed by 35 cycles of denaturation at 95  C for 1 min, annealing at 55  C for 2 min and elongation at 72  C for 1.5 min, with a final extension step of 72  C for 10 min (Liu et al. 1999). PCR products were purified using minicolumns, purification resin, and buffer according to the manufacturer’s protocols (Amersham, Foster, CA). DNA sequencing was performed using the primers mentioned above in an Applied Biosystem 3730 DNA analyser.

Sequence alignment and phylogenetic analyses Sequences were aligned in Clustal X (Thompson et al. 1997) and Bioedit (Hall 1999). Four single gene datasets were analysed (18S rDNA, 28S rDNA, RPB2 5f-7cr, RPB2 7f-11ar) and two combined ones (RPB2 5f-7cr þ RPB2 7f-11ar and RPB2 5f-7cr þ 28S rDNA). Phylogenetic analyses were performed in PAUP 4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded from all analyses. Unweighted parsimony (UP) and weighted parsimony (WP) analyses were performed. Gaps were treated as missing data and fifth character to increase the probability of finding the most parsimonious tree/s but only gapmode ¼ missing was used in the final analyses. WP analyses were also performed using a symmetric step matrix generated with the program STMatrix v2.2, by which the relative frequencies of nucleotide substitutions were calculated and converted into costs of changes (Francois Lutzoni & Stefan Zoller, Department of Biology, Duke University, Durham, NC). Trees were inferred using the heuristic search option with 1 K random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed, and all multiple parsimonious trees were saved. Descriptive tree statistics [Tree Length (TL), CI, RI, RC, HI] were calculated for trees generated under different optimality criteria. Clade stability was assessed in BS analyses with 1 K replicates, each with ten replicates of random stepwise addition of taxa. Kishino–Hasegawa (KH) tests (Kishino & Hasewaga 1989) were performed in order to determine

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whether trees were significantly different. Trees were figured in TreeView (Page 1996). Other details are outlined in Jeewon et al. (2002, 2003). ML and Bayesian analyses (using models of nucleotide substitution derived from Model test) were also performed as outlined by Shenoy et al. (2006).

Results Ribosomal DNA phylogenies The 18S rDNA dataset comprises 60 taxa with 1022 characters (16 % parsimony informative). WP analyses yielded two trees that were identical in topology and not statistically different, based on KH tests. One of them is shown in Fig 1 (TL ¼ 957.95, CI ¼ 0.568, RI ¼ 0.756, RC ¼ 0.430, HI ¼ 0.432). The trees generated in UP analyses were identical to those from WP but had slightly less nodal support. Phylogenies obtained show that Massariosphaeria spp. do not constitute a monophyletic lineage. M. roumeguerei constitutes an unresolved clade basal to Lepidosphaeria nicotia, Neotestudina rosatii and Arthopyrenia salicis. M. grandispora is a sister taxon to Lophiostoma caulium, Trematosphaeria heterospora and Massarina bipolaris with adequate support. Massariosphaeria phaeospora, the type species is basal to a clade consisting of Kirschsteiniothelia elaterascus and Helicascus kanaloanus with 65 % BS support (BS). T. hydrela and M. typhicola cluster together with high statistical confidence, but their relationship to other members of the Pleosporales was not statistically supported. The 28S rDNA sequence data consisted of 70 taxa with 890 characters (27 % parsimony-informative characters). WP analyses yielded two trees (not significantly different), one of which is shown in Fig 2 [TL ¼ 2412.07, CI ¼ 0.359, RI ¼ 0.655, RC ¼ 0.235, HI ¼ 0. 641]. Similar phylogenies were recovered with those from the 18S rDNA sequence data except for M. triseptata. This taxon clusters with Melanomma radicans and these two are nested in between the Sporormiaceae and Lophiostomaceae (with no BS). In contrast, in the 18S rDNA phylogenies, M. triseptata is nested between members of the Sporormiaceae, but this relationship was not resolved (all clades comprising Eremodothis, Sporormia, Preussia, and Westerdykella collapsed). Massariosphaeria typhicola is basal to Lepidosphaeria, Lojkania, Neostestudina, Pleospora rubicunda, and Verruculina, but this sister relationship did not receive any statistical confidence. A combined 28S and 18S rDNA dataset was also analysed to check for any topological incongruence. This dataset consisted of 39 taxa and 1913 characters (18.3 % parsimonyinformative) and our taxon sampling also included some newly released loculoascomycetous DNA sequences as used by Kruys et al. (2006) in their combined 28S and 18S rDNA dataset. Phylogenies obtained were almost identical to those obtained from those recovered from the 28S rDNA sequence data (results not shown).

RPB2 DNA phylogenies The polyphyletic nature of Massariosphaeria was also strongly supported in the RPB2 sequence data analyses. Two separate datasets (A and B) were analysed for DNA sequences generated from the RPB2 gene region. Dataset A includes the first

H.-K. Wang et al.

1130 nucleotides of the RPB2 region amplified and sequenced with primer pairs RPB2-5f and RPB2-7cr, whereas dataset B (920 sites) includes another region of the same gene amplified and sequenced with primer pairs RPB2-7f and RPB2-11ar. WP analyses of dataset A, as well as dataset B, resulted in a single tree [TL ¼ 8387.20, CI ¼ 0.241, RI ¼ 0.525, RC ¼ 0.126, HI ¼ 0.759 (dataset A); TL ¼ 8888.0, CI ¼ 0.275, RI ¼ 0.541, RC ¼ 0.148, HI ¼ 0.725 (dataset B)] and depicted similar topologies with respects to the in-groups under study. Phylogenies from dataset A were essentially similar to those obtained from single gene rDNA datasets (results not shown). It should be noted that any slight difference in placement was only due to the unavailability of more sequence data from the Pleosporales. Both RPB2 gene regions were also combined but results are not discussed, as there were not enough taxa available to properly assess the relationship of Massariosphaeria species. A dataset of 43 taxa combining the 28S rDNA þ RPB2 (dataset A) was also informative. This dataset consisted of 2038 characters, but 412 had to be excluded (ambiguous alignment). WP, with 45 % parsimony-informative characters, yielded one tree [TL ¼ 8558.09, CI ¼ 0.338, RI ¼ 0.529, RC ¼ 0.179, HI ¼ 0.662; Fig 3]. Clade support was moderate and results are generally concordant with phylogenetic placement of Massariosphaeria as determined by 28S rDNA. Massariosphaeria species fall into four distinct lineages. M. triseptata clusters with Melanomma radicans whereas M. grandispora fits within the Lophiostomaceae with strong statistical support. Conversely, a proper phylogenetic connection of M. roumeguerei and M. typhicola is still obscure as they constituent independent lineages without support. For all gene datasets, ML, NJ, and Bayesian analyses were also performed. ML and Bayesian based phylogenies were identical to those of MP and are not discussed here. Clades from NJ trees were not statistically strong although phylogenies were essentially similar (results not shown).

Discussion The objective of this study was to assess the phylogenetic relationships of Massariosphaeria and establish its familial placement within the Pleosporales. Based on 18S rDNA phylogenies, Liew et al. (2000) found that M. phaeospora (the type species) was basal to other members of the Massarinaceae (as circumscribed by Eriksson & Hawksworth 2003). Inderbitzin et al. (2002) could not confidently assess the proper affinities of M. phaeospora as its phylogenetic placement was largely dependent upon the analytical methods used. They found that it formed an unresolved sister taxon relationship to other members of the Pleosporales. Phylogenies obtained here somewhat corroborate those of Liew et al. (2000) who suggested a close affinity to the Massarinaceae. Using a different taxon sampling, we found that the type species is basal to Kirschsteiniothelia elaterascus and Helicascus kanaloanus with moderate support. Although these two taxa have been referred to the Pleosporales, there is no agreement and conclusive evidence to justify an appropriate familial placement. In addition, M. phaeospora also appears to be closely related to other members of the Massarinaceae and other genera such as Bimuria, Letendraea, Cucurbidothis, and Didymocrea. These latter taxa have been referred to the Melanommataceae, Cucurbitariaceae, and Zopfiaceae,

Polyphyletic nature of Massariosphaeria

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60 90 83

65

64

Didymocrea sadasivanii DQ384066 Cucurbidothis pityophila U42480

Letendraea helminthicola AY016345 Bimuria novae-zelandiae AY016338 Helminthosporium velutinum AF120254 96 100 Massarina eburnean AF164366 Leptosphaeria bicolor U04202

59

Melanommataceae Massarinaceae

97

Helicascus kanaloanus AF053729 Kirschsteiniothelia elaterascus AF053727 Massariosphaeria phaeospora AF164368 Cochliobolus heterostrophus AY544727 100 Pleosporaceae Pleospora herbarum U43448 Pyrenophora tritici-repentis U42486 53 Leptosphaeria doliolum U43447 Phaeosphaeria avenaria AY544725 Melanomma sanguinarium AF242265 Melanommataceae 90 Pseudotrichia aurata AF164374 Didymella cucurbitacearum AY293779

Byssothecium circinans AY016339 Pleomassaria siparia AF164373 100 Pleomassaria siparia DQ678027 Melanomma pulvis-pyrius AF164369 71 Trematosphaeria pertusa DQ678020 Melanommataceae Trematosphaeria hydrela AF164376 91 Massariosphaeria typhicola EF165037 Massarina australiensis AF164364 Lophiostoma caulium AF164362 51 Trematosphaeria heterospora AY016354 64 Lophiostomaceae Massariosphaeria grandispora EF165038 Lophiostoma crenatum U42485 89 Massarina bipolaris AF164365 Westerdykella dispersa U42488 50 Westerdykella cylindrica AY016355 Sporormiaceae Eremodothis angulata DQ384067 82

80

Massariosphaeria triseptata EF165036 90 Berkleasmium nigroapicale DQ280269 Berkleasmium micronesicum DQ280268 Melanomma radicans U43461 Melanommataceae Sporormia lignicola U42478 Sporormiaceae 89 50 Preussia terricola AY544726 Verruculina enalia AF053730 Neotestudina rosatii DQ384069 Arthopyrenia salicis AY538333 Lepidosphaeria nicotiae DQ384068 Massariosphaeria roumeguerei EF165035 Botryosphaeria dothidea AF27112 Botryosphaeria ribis U42477 68 Spilocaea oleaginea AF338393 100 Fusicladium effusum AY251125

93

Phaeoramularia hachijoensis AY251123 Phaeotrichum benjaminii AY016348 Trichodelitschia munkii DQ384070 92 Dothidea sambuci AY544722 100 Stylodothis puccinioides AY016353 Sydowia polyspora AY544718 79 Delphinella strobiligena AY016341 Eurotium herbariorum AB008402 Spiromastix warcupii AB015768 Xylaria hypoxylon U20378 100

10

Fig 1 – Phylogram of one of two most parsimonious tree generated based on the 18S rDNA sequences. Data were analysed with random addition sequence, WP and treating gaps as missing characters. Values above the branches are parsimony BS (equal or above 50 %). The tree is rooted with Xylaria hypoxylon.

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H.-K. Wang et al.

85 Pleospora tomatonis AY849959 Pleospora halophila AY849955 89 94 Curvularia brachyspora AF279380 98 Pleosporaceae Cochliobolus lunatus AF163988 84 60 Pyrenophora dictyoides AY849960 Pyrenophora triticirepentis DQ384097 Pleospora ambigua AY787987 Pleospora bjoerlingii AY849954 Phaeosphaeria avenaria AY544684 90 78 68 Phaeosphaeriopsis musae DQ885894 Leptospora rubella DQ195792 Leptosphaeria macrospora DQ384092 100 Leptosphaerulina argentinensis AY849947 Macroventuria anomochaeta AY787936 85 Massarina ramunculicola DQ528762 Kirschsteiniothelia elaterascus AY787934 80 Montagnula opulenta DQ677984 54 Dacampiaceae Munkovalsaria appendiculata AY772016 55 Bimuria novae-zelandiae AY016356 Melanommataceae 73 Letendraea padouk AY849951 Letendraea helminthicola AY016362 86 Karstenula rhodostoma AY787933 Melanommataceae Leptosphaeria coniothyrium AY849945 Aquaticheirospora sp. AY736378 Massarinaceae Massarina phragmaticola Massariosphaeria roumeguerei EF165032 100 Arthopyrenia salicis AY538339 Pleospora leptosphaerulinoides AY849956 84 Lophiostoma arundinis DQ782384 86 Massariosphaeria grandispora EF165034 92 Lophiostoma caulium DQ528723 100 Lophiostomaceae Lophiostoma crenatum DQ678069 88 Trematosphaeria heterospora AY016369 91 Lophiostoma fuckelii DQ399531 Berkleasmium nigroapicale DQ280273 Massariosphaeria triseptata EF165031 Melanomma radicans U43479 Melanommataceae 53 Westerdykella dispersa DQ384099 94 Westerdykella ornata AY853401 85 Eremodothis angulata DQ384105 Westerdykella cylindrica AY004343 Sporormiaceae 88 Preussia minima EF100938 Sporormia lignicola DQ384098 Preussia terricola AY544686 Pleospora iqbalii AY787931 71 Melanomma pulvis-pyrius DQ384095 Melanommataceae Trematosphaeria pertusa DQ678072 62 Wettsteinina pachyasca AY849970 100 Wettsteinina macrotheca AY849969 Lophiostoma macrostomum DQ384094 60 Pleomassaria siparia AY004341 Lepidosphaeria nicotiae DQ384106 Neotestudina rosatii DQ384107 100 Verruculina enalia DQ678079 Lojkania enalia AY016363 Pleospora rubicunda AY849957 67 Massariosphaeria typhicola EF165033 100 Berkleasmium sp DQ280271 65 Berkleasmium typhae DQ280275 67 Mycopepon smithii AF279400 Delitschia didyma DQ384090 67 66 Delitschia winteri DQ678077 52 Zopfia rhizophila DQ384104 Kirschsteiniothelia aethiops AY016361 78 100 Helicomyces roseus AY787932 Tubeufia cylindrothecia DQ341103 100 Kirschsteiniothelia maritima AY849943 100 Spilocaea oleaginea AF338398 Venturia carpophila AY849967 Cladonia rangiferina AY300832 10

Fig 2 – Phylogram of one two most parsimonious tree based on 28S rDNA sequences. Data were analysed with random addition sequence, WP and treating gaps as missing state. Values above the branches are parsimony BS (equal or above 50 %). The tree is rooted with Cladonia rangiferina. Genera that are underlined are those that have been shown to be polyphyletic.

Polyphyletic nature of Massariosphaeria

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Pleospora herbarum DQ247804/DQ247794

98 100

Curvularia brachyspora AF279380/AF107803

100

Pleosporaceae

Alternaria alternata DQ678082/DQ677980 Leptosphaeria maculans DQ470946/DQ470894 92

70

Phaeosphaeria avenaria AY544684/ DQ499802 99

99

99

Melanommataceae

Bimuria novae-zelandiae AY016356/ DQ470917 Sporidesmiella fusiformis DQ408577/DQ435079 Montagnula opulenta DQ678086/DQ677984

Byssothecium circinans AY016357/DQ767646 Massariosphaeria triseptata EF165031/EF165040 Melanomma radicans U43479/AY485625 Preussia terricola AY544686/DQ470895 84 75 99

Preussia minima DQ678056/DQ677950

Melanommataceae

Sporormiaceae

Trematosphaeria pertusa DQ678072/ DQ677969 Pleomassaria siparia AY004341/ DQ677976 Herpotrichia diffusa DQ678071/DQ677968

Massariosphaeria roumeguerei EF165032/EF165039 100 63 100 54

Lophiostoma arundinis DQ782384/ DQ782386 Massariosphaeria grandispora EF165034/EF165042 Lophiostoma crenatum U43469/ DQ677965

Lophiostomataceae

Trematosphaeria heterospora AY016369/ DQ497615 Sporidesmium australiense DQ408554/DQ435080 78

86

Ulospora bilgramii DQ384108/DQ677974 Verruculina enalia DQ678079/DQ677977

99

Lepidosphaeria nicotiae DQ384106/ DQ677963 63

Massariosphaeria typhicola EF165033/EF165041 Delitschia winteri DQ678077/DQ677975 Anisomeridium polypori DQ782906/DQ782864 100

100

Sydowia polyspora AY544675/DQ677953 Delphinella strobiligena AY016341/DQ677951

75 100

Dothidea insculpta AY640949/AF107800 Dothiora cannabinae DQ470984/DQ470936

89 84 100

Capnodium coffeae DQ247800/DQ247788 Scorias spongiosa AF338398/DQ677973 Mycosphaerella punctiformis AY490776/AY485626

100 99 99 100

Botryosphaeria stevensii DQ377864/DQ677960 Botryosphaeria tsugae DQ377867/DQ767644 Botryosphaeria rhodina DQ377858/AF107802

Botryosphaeria viticola DQ377873/DQ677985

100

100

Pyrgillus javanicus DQ823103/DQ842009 Pyrenula cruenta AF279407/AY641067 Pyrenula pseudobufonia AY640962/AY641068

Dibaeis baeomyces AF279385/AY485622 100

Fig 3 – Phylogram of the most parsimonious tree based on combined 28S nrDNA and RPB2 gene sequences. Data were analysed with random addition sequence,WP and treating gaps as missing state. Values above the branches are parsimony BS (equal or above 50 %). The tree is rooted with Dibaeis baeomyces.

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respectively (e.g Lumbsch & Lindemuth 2001; Kruys et al. 2006), except Letendraea, which has been excluded from the Tubeufiaceae (Kodsueb et al. 2006b). Despite this, and given that the systematics of related genera are still in a transitional stage, it would be inappropriate to confidently accommodate the type species of Massariosphaeria in a particular loculoascomyceteous family. Barr (1989, 1992) considered that Massariosphaeria (and its type M. phaeospora transferred to Chaetomastia) should be classified in the Dacampiaceae. The type of Chaetomastia has tetrasporous asci and dark-brown 3-septate ascospores and differs in form from the species Crivelli (1983) placed in Massariosphaeria. Phylogenetic data in this study do not support this classification and no close affinities were found with taxa of the Dacampiaceae such as Munkovalsaria and Polycoccum (Fig 2). Polycoccum was not included in this study as we found that it was not related to any Pleosporales or Dothideales. However, the reliability of the 28S rDNA sequence of P. vermicularium deposited in GenBank (AY961601), needs to be verified. Parsimony analyses indicate that Munkovalsaria appendiculata is more related to Montagnula opulenta (Montagnulaceae) and Bimuria novae-zelandiae (Melanommataceae) and not Massariosphaeria species. The genus Munkovalsaria, with M. donacina as the type species, was established by Aptroot (1995) for bitunicate fungi with a black stroma that forms a clypeus around the ostiole; cylindrical asci with eight ascospores in one row (IKI negative); reddish to deep brown ascospores that are 1euseptate, surrounded by a thin gelatinous sheath but without germ pore or slit. The Dacampiaceae are not very strongly characterized morphologically anyway, the main characters being the multilayered, rather soft ascoma walls and the relatively wide ostioles. It may turn out to be polyphyletic, but few sequences are available for any phylogenetic comparison. Our studies provide some insights into the relationships of M. typhicola. However, in most phylogenies (except 18S rDNA), the association of this species with other taxa was not statistically supported and there are uncertainties with respects to its proper phylogenetic connections. 18S rDNA phylogeny (Fig 1) reveals a close relationship between M. typhicola and Trematosphaeria hydrela (Melanommataceae) with high statistical confidence. Although the latter is currently a melanommataceous loculoascomycete, its affinities with known bitunicate genera remains unclear, and any sister relationship lacks support. Similar findings were reported by Liew et al. (2000) and Inderbitzin et al. (2002). A close affinity between M. typhicola and Trematosphaeria hydrela is not surprising. It is probably significant that the ascomatal wall surface of M. typhicola described by Shoemaker & Babcock (1989) as deviating, viz. with radiating cells of textura prismatica. Similar structures can also be observed in Trematosphaeria hydrela. Due to unavailability of other DNA sequences from T. hydrela other gene phylogenies (in this case, 28S rDNA and RPB2) indicate that M. typhicola is an independent lineage basal to other fungi with bitunicate asci whose familial placement is still not fully resolved (Kruys et al. 2006). M. typhicola was considered to be a species of Chaetomastia (Dacampiaceae) by Barr (1989) but this familial placement could not be confirmed here. Another particular feature in our gene phylogenies is the evolutionary relationships of M. triseptata. This taxon is nested in between the Sporormiaceae (Fig 1) and constitutes an unsupported sister relationship to Berkleasmium and Melanomma radicans. In

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contrast, 28S rDNA and combined 28S þ RPB2 gene phylogenies (Figs 2 and 3, respectively) indicate a close association to Melanomma radicans. Although weakly supported, the later would make sense in light of morphological similarities, as the ascospores are not typical for Massariosphaeria, for instance because they are only three-septate. Although the 18S rDNA gene tree resulted in a slightly different topological arrangement, incongruence is not statistically supported. We believe that given the number of available taxa analysed and number of parsimony informative characters from the datasets, the 28S rDNA (Fig 2) and the combined 28S þ RPB2 phylogenies (Fig 3) is more likely to represent the true phylogeny. Molecular data provide evidence to support that M. roumeguerei is not phylogenetically related to other melanommataceous genera. There were no topological conflicts, but phylogenies are incongruent with morphologicalbased classification schemes. We are unable to fully resolve these relationships, and the results obtained do not provide any insights into current species separation based on morphologies. All gene phylogenies differ largely from the conventional classification system of Massariosphaeria, which recognizes that the genus belongs to the Lophiostomaceae (Eriksson & Hawksworth 1991; Kirk et al. 2001). Surprisingly only one species, M. grandispora, could be confidently assigned to the Lophiostomaceae. There is a close phylogenetic relationship between M. grandispora, Lophiostoma caulium, L. crenatum, Massarina australianensis, M. bipolaris, and Trematosphaeria heterospora (Fig 1). It should be pointed out here that T. heterospora has been referred to the Melanommataceae (Lumbsch & Lindemuth 2001) but in other papers in preparation, we found that it would be more appropriate to accommodate this taxon in the Lophiostomaceae, which also makes sense morphologically as it has the sideways compressed ostioles characteristic of most Lophiostoma species. Morphologically, M. grandispora possesses cellular pseudoparaphyses, ascospores that are 9–12-septate and hyaline with a wide sheath. It also has the sideways compressed ostioles characteristic of most Lophiostoma species. It has generally been classified as Lophiotrema grandispora (Shoemaker & Babcock 1989) but was reassigned to Massariosphaeria based on the presence of multitransseptate ascospores and ascomata that are composed of thin-walled pale brown cells around the ostiole (Tanaka & Harada 2003). Our results support the classification of Massariosphaeria grandispora in a genus of the Lophiostomaceae, possibly in Lophiotrema. Of special interest was the sister group relationship of M. triseptata and Melanomma radicans to Berkleasmium micronesmium and B. nigroapicale (Figs 1 and 2). The latter are hyphomycetous species, which have been shown to belong to the Pleosporales, in particular to the family Sporormiaceae (Pinnoi et al. 2007). Despite a close relationship between these taxa, there is little chance for a close genetic connection between them, as M. triseptata is known to produce mostly Aposphaeria-like anamorphs (Leuchtmann 1984; Tanaka & Harada 2004). Berkleasmium is a rather small group of hyphomycetes that includes fungi characterised by macronematous conidiophores that are mostly unbranched and closely packed in a sporodochium, while spores are solitary, brown, muriform, clavate or oblong, with rounded ends or irregular, and often have a protruding hilum (Bussaban et al. 2003;Ellis 1971; Zhao & Liu, in press). No teleomorphs are known for Berkleasmium, but phylogenies have indicated a close affinity of

Polyphyletic nature of Massariosphaeria

B. nigroapicale to Westerdykella cylindrica and Sporormia australis, which are both currently in the family Sporormiaceae (Pinnoi et al. 2007). Similar results are obtained here but a close relationship between Berkleasmium, Melanomma radicans, and M. triseptata is unexpected. However, any relationship between them is possibly largely dependent upon taxon sampling. There are simply inadequate DNA sequences available from known databases to further elucidate the phylogeny of M. triseptata. Conversely, Aposphaeria is a coelomycetous genus with globose conidomata, conidiophores that are cylindrical to lageniform and formed all around the locular cavity and conidia that are ellipsoid, hyaline, and aseptate (Leuchtmann 1984). The latter has been suggested to represent anamorphs of Melanomma, but in this study, no phylogenetic associations were observed between Massariosphaeria species and Melanomma species such as Melanomma pulvispyrius and Melanomma sanguinarium (Fig 2). Therefore, any proper phylogenetic conclusions regarding relationships of Aposphaeria, Massariosphaeria and Melanomma should be interpreted with caution and future studies should incorporate more taxa, including the anamorphic types. We would also advocate the use of alternative phlylogenetic markers, if possible, as either rDNA or RPB2 DNA sequence data reported lack of phylogenetic signal to fully resolve evolutionary relatedness. Molecular data indicate that the morphologically wellcharacterised genus Massariosphaeria is polyphyletic. Further members of the Melanommataceae are interspersed in different clades among the Pleosporales. Classification among many loculoascomycete taxa has always been subjected to controversy because of overlapping morphological characters used to delimit genera and species. The polyphyletic nature of Massariosphaeria supports previous speculation that ascomatal and ascospore morphologies have undergone convergent evolution among the Pleosporales (e.g. Berbee 1996; Kodsueb et al. 2007; Lumbsch & Lindemuth 2001; Schoch et al. 2006). A number of loculoascomycetes genera such as Kirschsteiniothelia, Massarina, Melanomma, Pleospora, and Trematosphaeria included in this study are shown to be polyphyletic. Therefore, a similar evolutionary scenario for Massariosphaeria is not surprising. The familial placement and phylogenetic relationships of Massariosphaeria taxa studied were also not fully resolved, but there is sufficient evidence to justify its placement within the Pleosporales. However, its familial position is still partially resolved and precarious because of: (1) inadequate taxa available for analytical comparison; (2) its affinities to other taxa whose placement are obscure and presumably polyphyletic; and (3) lack of statistical support to conclusively determine any appropriate phylogenetic connection.

Acknowledgements The University of Hong Kong (RGC 7322/04M-awarded to R.J. and K.D.H.) is thanked for funding this research and providing H.K.W. with a training stipend to visit Hong Kong. This study was also partially supported by the National Science foundation of China (No. 30300229).

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