~\.l~rolo,~~,94(5), 2002, pp 803-813 O 2002 b\ The M\cological Socieh of Ame~ica,La~rence, KS 660448895 An evaluation of the monophyly of Massarina based on ribosomal DNA sequences Edward C. Y. Liew cies were included in Massam'na, the first being Mas- Centre for Research in Fungal Diversity, Department of sam'na eburnea (Tul. & C. Tul.) Sacc., but a type was Ecology & Biodiversity, The CTniversityof Hong Kong, not indicated, nor was the genus placed in a family. Pokfulam Road, Hong Kong SAR, PR China Massam'na eburnea was later selected as lectotype of Andre Aptroot this genus by Clements and Shear (1957) and was Centraalbureau voor Schimmelcultures, P 0. Box redescribed and illustrated by Hyde (1995a). 85167, NL-3508 AD Utrecht, The Netherlnnds A new family, the Massarinaceae Munk, was intro- Kevin D. Hydel duced by Munk (1956) to accommodate Massam'na. This family was accepted by various authors (Eriksson Centle for Research zn Fungal Dzverszty, Department of Ecolqg & Bzodzversztj, The Unzverszty of Hong Kong 1981, 1982, Eriksson and Hawksworth 1985). Bose Pokfulam Road, Hong Kong SAR, PR Chzna (1961), in his review of Massam'na, noted that doubts had previously been expressed several times about whether the introduction of a new family was justified Abstract: The monophyletic status of the genus (Holm et a1 1957, Scheinpflug 1958).He subsequent- Massam'na was evaluated on the basis of phylogenetic ly placed Massam'na in the existing Pleosporaceae, analysis of the partial small subunit gene (SSU), in- due to the lack of distinctive characters distinguish- ternal transcribed spacers (ITS 1 & 2), and 5.8s gene ing the two families. This placement was supported sequences of the ribosomal DNA. Species of Massar- by some authors (Miiller and von Arx 1962, Luttrell ina used in the study clustered into two distinct 1973, von Arx and Miiller 1975), but Massam'na was clades with high bootstrap support in trees generated eventually disposed within the Lophiostomataceae from maximum parsimony, weighted parsimony, Sacc. in the Pleosporales (Eriksson and Yue 1986, maximum likelihood, and neighbor-joining analyses. Barr 1987, 1992). The hypothesis that ~Wussam'naspecies belong to a Since Saccardo's initial inclusion of 11 species in phylogenetically monophyletic group is rejected. Spe- Massarina, numerous species have been described in cies with narrowly fusiform ascospores form a mono- or transferred to this genus. Bose (1961) discussed phyletic clade with Lophiostoma, a genus highly sim- 19 species in his review, which covered mostly the ilar in morphology. The five species currently ac- European species. Srinivasulu and Sathe (1974) cov- cepted in Massarina with such spore morphology are ered nine species from India, while Barr (1992) treat- here transferred into the genus Lophiostoma. Massar- ed the North American members of the genus. None ina species with broadly fusiform to ellipsoidal asco- of these revisions were exhaustive. A list of 132 spe- spores are retained as ~Vassam'nas. str., lectotypified cies described in Massam'na was annotated by Hyde by M. ehurnea. 1Zlassam'na walkem' is presently exclud- (1995a). More recently, many newTspecies have been ed from both ~Mccssam'naand Lophiostoma. The trans- described: five from marine or mangrove intertidal fer of M. papulosa to a new genus Olethem'o.~trigulais environments (Hyde and Borse 1986, Kohlmeyer and verified. Volkmann-Kohlmeyer 1987, Hyde 1989, 1991, Hyde Kq Words: ascomycetes, fungi, Lophiostoma, Lo- et a1 1992), eight from the freshwater environment phiostomataceae (Hyde 1992, 1994, 1995b, Shearer and Hvde 1997, Hyde and Aptroot 1998, Tsui et a1 1999a), and eight from terrestrial environments (Shoemaker et a1 1991, ISTRODUCTION Hyde and Aptroot 1997, Poonvth et a1 1999, Aptroot Massam'na Sacc. was introduced by Saccardo (1883) et a1 2000). for species of pyrenocarpous ascomycetes that had In response to an obvious need to revise all previ- previously been placed in Massaria De Not. but typ- ously described species including those from the ically had hyaline ascospores (Bose 1961).Eleven spe- tropics, Aptroot (1998) published a world revision of the genus Massam'na and accepted 43 species. All of Accepted for publication February 20, 2002. these species are characterized by cellular pseudo- ' Correspondirlg author, Email: [email protected] paraphyses, hyaline, septate, fusiform to long-ellip- soidal ascospores and bttunlcnte nsc~.,bZa~snnnn rhfil- ~IATERIu 4 Ah11 hIkTHO1)4 nea has an immersed, poorly developed ascoma wall Cultuws and DA\t4 r~q~~ellr~.~.-Thesollrces of fi~ngalc~il- and broadly fusiforni, 3-septate ascospores ~vitha tures and GenBank sequences used in the stud! are listed thick gelatinous sheath. The majority of the species in T-\RI.FI. Identification of furlgal speci~nens\vas \.rrifirtl differ from the type species in various characters, es- based on morphological characters before a si~iglcspore pecially in regard to the ascospore shape, which is culture ~vasobtained and cl~lturedon corr~rnealagar. (Chl.4 often narrowly fusiform, and to the shape and exten- [Difco]). Cultures from the Ce~ltraalbure;~~~of Scliimmrl- sion of the gelatinous sheath arouild the ascospores. ctlltwes (CBS, Utrecht) \\ere purifietl by sul~cultl~ririgfr-on1 Aptroot (1998) commented that the accepted spe- a single hyphal tip onto the culturc medilmi rcconlnlentleti cies of ~Vlussam'nnprobably do not form a monophy- for the respective strain h! CBS. All c~~ltures\\.ere sr~bc.ul- 1'8 letic group. Some species are similar to other genera, tured onto 1'8 agar (40C4, Campbell ,Juice) f01- ~.;~pi(l growth 1-2 I\. prior to DN.4 extl-xction. differing in very few characters, and some of the re- ported anamorphs suggest very different groups. DDy4 ~~t~o~tZ'o~z.-;\cti\-elygro~vingm>celia \vrrtx directly Subdivisions within this genus were ho~vever not scraped off frorn culture plates and tra~lsferreclinto l..? 1111. made, mainly because of the uncertainty in selecting centrifuge tllbes. DNA4extraction follo~veda modifietl pro- the appropriate delineating morphological charac- tocol of 1)oyle and Doyle (1987). Approxirnatel! 0.0-5 g of' ni)celi~~rn\\.as rnixed ~iithca 0.3 g of tvhitc cjual.tz sarltl (Sig- ters. Aptroot (1998) poirlted out the need. for ex- ma) in warm (ca 60 (;) 2 X (:T.4B hllffel- [P!; (\t.iv) (:T.-\R; amining characters of different levels, ultrastructural 100 mhf Tris-HC1; 1.3 hl NaCl: 20 1nh1 EDT-4. pH X.OJ. 111- and molecular. Several reports of ultrastructural ex- celium-sand n~isrl~re\\.as groluntl \vith a glass pc.\tle ant1 in- amination of ~Vlc~ssam'nnspecies have not only con- cubated at 60 C; for 1 Ilr hefore heilig slil?jected to multiple firmed and clarified observatio~isbased on light mi- ~11~enol:chloroform( 1:1) estractioni;. DN.4 \$-asprecipitated croscopy, but have revealed structures that could not from the purifietl aqueous extracrio~llayer by ethanol prc- be resolved by light microscopy (Read et a1 1994, cipitatio~i. The DSA4 pellrt \fils ~rashed (70% ctlia~lol). 1997, Tsui et a1 199913). Lrltrastructural examinations dried (vacutum centl-ifi~gr)and rrs~~spendctlin 100 FL. Tt: generally reveal consistent structures in all the ,W(is- buffer containing 0.8 ~g/mLof RNase X (Sigma). DS.\ sam'na species examined, except for the occasional samples were checked for purity ancl inttgt-ity gel elcc- appearance of ascospore polar chambers formed tl-ophoresis befi~restori~~g at 3 C:. fro111 the episporial cell wall layer, and of lateral, fi- DA\:4,f'ropnrrrt an~plijccrlion.TheITS 1. 5%gene and ITS brillar appendage-like structures. Polar chambers 2 of the rDSA was amplified i~singprimers ITS4 allti ITS3 have been observed in taxonomically distinct groups (Tl'hite et al 1990). T\\.o to 3 FL of suspended 13S.4 tvcrc. of marine ascomycetes and seem to have developed used for each polvrnerasc chain 1.ractior1 (PCR) ~vitli 1.3 independently in several evolutionary lineages (Read ruM hfgCl,, 0.hihI cof each tlNTP, 0.3 ~11of each pri~iicl-. and 2.0 U of Taq DNA polymerase in a .iO ~1.reaction et a1 1997). Such convergent characters are not con- T-olume.The therrrlal c>cling program \vas as o~~tlinctlill sidered to be of primary taxonomic importance. Uhite et al (1990), rvith prinler a~lnealirlgat 32 C. fior 50 s. I11 evaluating the phylogenetic significat~ceof pseu- The size of each amplified fragrncnt was \,erifietl h! grl rlec- doparaphvses in the loculoascomycetes, Lien. et al trophoresis with ethidi~~rnbrornide staining of a 2 ~1.prod- (2000) included three ~bZus.snrinaspecies in their 18s uct sample and visualiretl over an ultraviolet tl-ansilllimi- subunit ribosomal DNA analysis. These species did nator. P(:R products xvere p~~rifiedusing tllc IYira~.tlP(:K not form a mollophyletic group. ,\.lassarinn nustml- Preps DNA Plirification Systern (Promega) ;III(~tlir pu~-ified iensis and IW. hipolaris clustered together with I,o- products were filrthcr assessccl fbr purity ant1 s~if'ficienc\.in phiostomn cnuli~rm,away from 1bZ. eburnra. Morpho- corlccntration hv gel electr-ophoresis (tising 0.3 ~1.).\Vhcrc logically, some 1Mnssarina species are remarkably sim- necesyary, fragment susperlsio~lq\wre fur the^- concr~>~~-;~tetl ilar to Lophiostoma, which differs in its laterally com- in a vacuum centrifi~ge.ancl stored tor not no re tll,ir~1 nk before DNA sequelicing. pressed slot-like ascomatal ostioles (Eriksson and Yue 1986, Hyde 1995a, Aptroot 1998, Hyde and Aptroot ULY4.crt-jtrrnrir~g.-The arnplifiecl DNA fragments \\.ere di- 1998). rectly sequenced 11sing the LiLFexprrssLiutomated DNA Sr- The current research aimed to assess the mono- quelicer Ahl V 3.0 (Pharmacia Biotech). Sequelicing I.cac- phyly of the genus Mc~ssnrinaby phylogenetic analysis tions were condtictcd in a ther~nalc!cler using thr Auto of the ribosomal DNA.