Ascus and ascospore appendage structures can be important characters in delineation of genera. We have therefore examined the ascus and ascospore ultrastructure of Massarina ingoldiana and M. purpurascens, as Massarina is thought to be a heterogeneous genus. The ascus wall of M. ingoldiana is two layered and comparable to M. thalassiae. Ascospores of both species comprise an electron-transparent mesosporium, an electron-dense episporium and an exosporial mucilaginous sheath. In this respect they are similar to the ultrastructure of other Massarina species, and should be maintained in Massarina.published_or_final_versio

Hodgkiss, IJ

Hyde, KD

Tsui, KM

Wong, SW

Mycologia

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Title Ultrastructural studies of Massarina ingoldiana and M.purpurascensAuthor(s) Tsui, KM; Wong, SW; Hyde, KD; Hodgkiss, IJCitation Mycologia, 1999, v. 90 n. 5, p. 721-726Issued Date 1999URL http://hdl.handle.net/10722/53362Rights Creative Commons: Attribution 3.0 Hong Kong LicenseJl~rolo ,q fc ,9 l (.i).199'1. PP. 721 -726. 'C 1999 h\ The  hlvcological Society of .lmerica. I.awrence, IS 660448897 Issued I5 Septeliiher- 19I)Il Ultrastructural studies of Massarina ingoldiana and M. puqburascens K. M. Tsuil S. W. Wong' K. D. Hyde I. J. Hodgkiss Fttngal Dzverszty Research Project, Llepartment of bcolog? and B~odzverszty, The Crnzvmszt> of Hong Kong Pokftfam Road, Hong Kong Abstract: Ascus and ascospore appendage structures can be important characters in delineation of genera. We have therefore examined the ascus and ascospore ultrastructure of ;Classarina ingoldiana and 121. pur- purascens, as Massarirza is thought to be a heteroge- neous genus. The ascus wall of 1M. ingoldiana is two layered and comparable to lM. thalassiae. Ascospores of both species comprise an electron-transparent me- sospoi-ium, an electron-dense episporium and an ex- osporial mucilaginous sheath. In this respect they are similar to the ultrastructure of other Massarina spe-cies, and should be maintained in iMassavina. Key Words: aquatic fungi, ascomycetes, fine struc- ture, electron microscopy INTRODY( TIOS The genus iVassarina Sacc. comprises 43 species of which eight are known from freshwater habitats (Ap- troot 1998) and is thought to be heterogeneous. Most aquatic species have ascospores with polar ap- pendages, e.g., M. bipolaris K. D. Hyde (Hyde 1995a), AM. fronsisubrn~rsa K. D.  Hyde (Hyde 1994), or they are surrounded by a mucilaginous sheath, e.g., :M. ingoldiana Shearer & K. D. Hyde (Shearer and Hyde 1997) and AM. pz~rpura.scens K. D. Hyde & Aptroot (Hyde and Aptroot 1998). Since the ascospore ap- pendage is significant in the delineation of genera (-Tones 1995), this study was initiated to establish if there are any major differences between species at the ultrastructural level, and whether the differences Accepted for p~tblicatiori April 26. 19919. ' Email: 1i93081~i7@I~husua.liku.lih 'Present hdtiress: Sitlgnpore E\e Research Institute Laborator!, \ID0 Phvsioloqy Building, #01-15. Faculty of hledicine, Sntional L'ni\rrcit~ of Sillgapore, 10 Kent Ridgr Crescr~i t ,  Singapore 1 l<I?IiO. can be used to place the Massarina species into dif- ferent genera. Investigation on the fine structure of Massanna species has been restricted to marine species, includ- ing 121. thalasszae Kohlm. & \lolkm.-Kohlm. and M. armatis@oraK. D.  Hyde et al (Read et a1 1994). Mas-sanna purpurascens and M.  zngoldzana are particular- ly common in streams in Hong Kong, and we have therefore examined the fine structure of the asco-spores of these species at the ultrastructural level. The results are also compared with ultrastructural data from other bitunicate species. Submerged wood was collected from rivers and reservoirs in the New Territories. Hong k n g ,  and in Brunei and re- turned to the laboratory in plastic bags, where they were incubated for 2-3 wk on moist paper in sterile plastic boxes. Material was periodically examined under a dissecting mi- croscope and fungal fruiting bodies present identified. For scanning electron microscopy (SEM) and transmis- sion electron microscopy (TEM), an ascospore suspension was prepared in distilled water using the ascomata content, and fresh asconlata were removed from wood. They were fixed and prepared following the methods described by Ho et a1 (1999). For cryoscanning electron microscopy (cryo- SEM), the polycarbonate membrane with specimens were air-dried for 5 mi11 at room temperatrlre mounted on a h -  lniniuln stubs using carbon cement. The material was then frozen in an external chamber (Polaron LT7400 (kyopep) which was connected to the main chamber of Leica Ster- eoscan 440 SEM. The material was lowered to -150 (: fol-lowed by coating with gold for 10 s using a built-in sputter coater. Subsequently. the specimen was transferred under vacuum into the main chamber and placed on the cryostage for low temperature (-150 C) observation using acceler- ating voltages of 10 kkT. iMassarirza purpurascens produces fusiform, two- celled ascospores that are constricted at the septum, and surrounded by a mucilaginous sheath (F~c,s.1, 2) ,  although the appearance of the sheath depends on the condition of material. In a fresh specimen from Brunei, the sheath had an outer thin, undulat- ing membrane and was examined in this study (FIGS. 3, 4).  In dried specimens the sheath is often incon- spicuous or lacking. FIGS. 1, 2. Massarina purpurascas, SEM of ascospore surrounded with a characteristic sheath. Scale bars: 5 pm. Mature ascospores of M. pLrpLrascens were sur- rounded by a thick electron-transparent mucilagi- nous sheath (0.86-1.35 pm thick) with an outer thin electrondense layer (ca 90 nm thick) (FIGS. 3, 4). This outer layer was electron dense, undulating and composed of compacted fibrillar material. Remnants of the membrane complex were also present (FIG. 4). In addition, the outer layer was connected to the as- cospore wall at some regions e.g., ascospore apex (not illustrated here) and appeared to agree with the characteristic morphology of the ascospore sheath at the SEM level (FIGS. 1, 2). The thick mucilaginous sheath comprised diffuse material (FIG. 4) and some electron dense inclusions (FIG. 4). Both the mucilag- inous sheath and the outer layer were exosporial. The mature ascospore wall comprised an outer elec- tron dense amorphous episporium (ca 30 nm) and an inner electron-transparent mesosporium (ca 130 nm) (FIG. 4). Ascospores of Massanna ingoldiana were also two- celled, fusiform, slightly constricted at midseptum, hyaline, guttulate and surrounded by an inconspic- uous mucilaginous sheath that could only be ob- served following the addition of India ink (Shearer and Hyde 1997). When viewed by scanning and cryoscanning elec- tron microscopy, mature ascospores were fusiform and surrounded by wide elongate mucilaginous sheath (FIGS. 5, 6). The drying process in conven- tional SEM dehydrated the mucilaginous sheath (FIG. 5), whereas the mucilaginous sheath was in a con- densed form when examined by cryo-SEM (FIG. 6). This sheath was closely adpressed to the polycarbon- ate membrane and may aid in ascospore attachment. When viewed with transmission electron micros- copy the bitunicate ascus of Massarina ingoldiana in- cluded an outer thin electrondense ectoascus (ca 300 nm thick) and an inner electron-transparent en- doascus (ca 770 nm thick) (FIGS. 7, 8). The walls of mature ascospores comprised an out- er, thin, electrondense episporium (ca 12 nm wide) and an inner thick electron-transparent mesospor- ium (ca 100 nm wide) (FIGS. 10, 12). Some electron- dense inclusions occurred within the mesosporium (FIG. 10). Condensed mucilaginous material aggre- gated on the outside of the ascospore wall, near to the septum (FIG. 11). In the ascus, ascospores were surrounded by a thick, dense exosporial fibrillar layer (300-470 nm thick) with an outer discontinuous membrane (pos- sibly the membrane complex) (25-55 nm) (FIG. 8). This membrane was irregular (FIG. 9) and in released ascospores, it coiled up at the edges where discontin- uous (FIG. 8). The dense mucilaginous sheath (ca 1.85 pm thick) of released ascospores expanded (FIG. 7), which may have disrupted the outer membrane. The fibrillar component of the sheath became more diffuse and spread in water via the discontinuities of the outer membrane (FIGS. 9, 10). The outer mem- brane appeared to slough off or disintegrate, and the ascospore was then surrounded by a diffuse and ex- panded fibrillar sheath (RGs. 11, 12). DISCUSSION Ascus waU.-The ascus wall in M. ingoldiana shares structural similarities to M. thalassiae (Read et a1 1994) in having an outer narrow electrondense ec- toascus and an inner thick electron-transparent en- doascus surrounded by mucilage. It is, however, dif- ferent from that in Pleospora gaudeJi-9i Pat., which is characterized by a much narrower ectoascus and a TSUI ET AL: ULTRASTRUCTURE OF MSSARNA SPP. 723 .- - - --- , r- bipartite endoascus (Yusoff et a1 1994). No partition of the endoascus has been observed in Massarina species. I The separation of the ascus wall into an inelastic ectoascus and an elongate elastic endoascus is a unique character of bitunicate fungi (Reynolds 1989). This separation occurs irrespective of the number of sublayers within the ectoascus and en- doascus. The asci of Massarina ingoldiana and M. pu@rascens are bitunicate, and only have two sub- layers. Ascospore wall and appendage.-The ascospore wall of M. thalassiae (Read et a1 1994), M. ingoldiana and M. purplrascens include an electron-transparent me- sosporium, an electrondense episporium and an ex- 2 b osporial mucilaginous sheath with an outer electron- - 0/1 dense layer. Massarina purplrascens differs slightly 24  from the other two species in having electrondense inclusions within the mesosporium. The outer mem- brane of the mesosporial sheath is undulate in M. purplrascens and M. ingoldiana, whereas in M. thal- assiae it is linear. The mucilaginous sheath of M. thaL assiae and M. purplrascens is constricted in some re- gions, and this results in the characteristic shape of the sheath. In M. ingoldiana, the outer membrane is discontinuous, probably due to swelling of the exos- porial sheath. Some aquatic Loculoascomycetes also possess similar sheaths. In M. ramunculicola K. D. Hyde, ascospores are at first surrounded by a thin membrane, and this subsequently breaks at the poles \ d to form pads. The membrane then disintegrates and L-L a wide mucilaginous sheath is formed (Hyde 1991). - In M. ricifma K. D. Hyde the sheath is two-layered and is only visible in India ink (Kohlmeyer et a1 1995). The inner layer is thought to rupture the out- er membrane. In Trematosphaeria c m . s a  K. D. Hyde and Vaginatispora aquatica K. D. Hyde, the sheath is rf. 7 formed by rupture of an outer membrane (Hyde '&#b,g 1995a, b). The outer membranes in ascospores of M. thalassiae and M. puqburascens are persistent even when the ascospore sheath has expanded in water, I \ t m-. IEIGS. 3, 4.  Massarina purpurascens, TEM. 3. Oblique longitudinal section illustrating the whole ascospore sur- rounded by an outer irregular electrondense layer (OM) and inner electron-transparent layer (TL). 4. Higher mag- nification of the ascospore sheath illustrating the outer lay- er (OM) composed of compact fibrillar material. Remnants of the discontinuous membrane complex (MC) are present to the outside undulating layer (OM). Longitudinal sections of ascospore wall also illustrating the outer electrondense episporium (E) and the inner electron-transparent meso- sporium. Scale bars: 3 = 5 pm, 4 = 0.5 pm. r EcA f FIGS. 5-10. Massanna ingoldiana. 5.  Ascospore surrounded by a wide elongated sheath, SEM. 6. Condensed form of mucilaginous sheath surrounding the ascospore, crydEM. 7,8. Longitudinal sections of mature asci illustrating the ectoascus (EcA) and endoacus (EnA), TEM. Some mucilaginous material (Mu) occurs on the ascus wall. Ascospores in the ascus are surrounded by a mucilaginous sheath (MuS) with an outer membrane (OM). 9. Oblique longitudinal sections illustrating released ascospores surrounded by a mucilaginous sheath (MuS) with outer membrane (OM), TEM. Note the discontinuities in the membrane (arrowed). 10. Longitudinal section illustrating the outer membrane (OM) coiled at the discontinued edge, TEM. The sheath comprises fibrillar material, which remains condensed against the episporium (E). Some electron- dense inclusions (DI) occur within the mesosporium (M). Scale bars: 5 = 20 pm, 6 = 10 pm, 7, 8, 10 = 0.5 pm, 9 = 0.1 w .  TSUI ET AL: ULTRASTRUCTURE OF MASSARINA SPP. 725 FIGS. 1 1, 12. Massarina ingoldiana, TEM. 1 1. Longitudi- nal sections of ascospores illustrating the lack of outer mem- brane and dense mucilaginous sheath surrounding the as- cospores (As), which is more d i i s e .  12. Higher magnifica- tions of the ascospore wall illustrating condensed mucilagi- nous material (CMu) which has accumulated on the ascospore wall at the septum (Sp). Electrondense fibrillar material (DF) appears to be associated with this condensed mucilaginous material (CMu) and the episporium (E) of the released ascospore. Scale bars: 11 = 5 pm, 12 = 0.1 pm. although a discontinuity may occur occasionally at the ascospore tip in M. thahsiae (Read et a1 1994). Electrondense granular inclusions occur in the ex- osporial sheath of immature ascospores of M. thal- assiae and M. armatispora (Read et a1 1994). These inclusions may be condensed mucilaginous material, which absorbs water to form the extensive sheath. We have only examined mature ascospores of M. ingo& diana and M. purplrascens, however, electrondense inclusions have been found in the exosporial sheath of M. plrplrascens. Episporial polar chambers con- taining concentrated fibrillar material occur at the apices of ascospores of M. thahsiae, whereas no chambers have been observed in other Massarina species. Although both Pleospora and Massarina are placed in the Pleosporales (sensu Barr 1987), their asco- spore walls differ. In Pleospora gaudefi-oyi the asco- spore wall comprises an electron-transparent mesos- porium (Wl) enclosing each cell (Yusoff et a1 1994). All cells are embedded in a common layer with elec- trondense inclusions (WZ), and the whole ascospore is surrounded by a layer containing compact melanin bodies (W3) with an outer thin episporium (W4). No partition of mesosporium has been observed in Mas- sarina species. In addition, although the episporium of ascospores of Massarina species are thick and elec- tron dense, no melaninlike bodies have been ob- served, although they may be present in older asco- spores. The data presented here indicated that ascospores in both M. ingoldiana and M. plrpurascens have sim- ilar wall structure, including a mesosporium, an e p  isporium and an exsosporial sheath with an outer lay- er. These characters are similar to those found in as- cospores of M. thahsiae (Read et a1 1994), and the similarities imply they have a similar appendage on- togeny. As the type species M. eburnea has not yet been studied, it is premature to separate Massarina species into further genera. ACKNOWLEDGMENTS We acknowledge the staff in the electron microscopy unit, Queen Mary Hospital, Hong Kong, and Mr. Lee for tech- nical help. K. M. Tsui and S. W. Wong thank The University of Hong Kong for the award of Postgraduate Studentship. LITERATURE CITED Aptroot A. 1998. A world revision of Massarina (Ascomy- cota) . Nova Hedwigia 66:89-162. Barr ME. 1987. Prodromus to class Loculoascomycetes. Am- herst, Massachusetts: University of Massachusetts. 168 p. Ho WH, Hyde KD, Hodgkiss IJ. 1999. Ultrastructure of An- nulatascus aquaticus sp. nov., a freshwater ascomycete on  submerged wood from Hong Kong. Fung Divers 2: 119-128. Hyde KD. 1991. Ma~srcrina vrlntospora and a llew ntangrove- inhabiting species, IM.mmztnn~licolcisp. nov. Mycologia 83:839-845. . 1994. Aquatic fungi on rachides of I,ivistontl in the Western Province of Papua New Guinea. Mycol Res 98: 719-725. . 199ia. Tropical Australian fi-eshwater fungi. \?I. New genera and species of i\scomycetes. Nova Hedwig- ia 61:119-140. . 199513. Tropical Austl-alian freshwater fungi. IX. Lh-ginnti,spora nqzinticcl gen. et sp. nov. Nova Hedwigia 61: 233-241. , Xptroot A. 1998. Tropical freshwater species of the genera Lophiosto~rrnand ,\.lnssnrina. Nova Hedwigia 66: 489-<502. Jones EBG. 1995. IJltrastructure in taxonomy of the iiqllatic ascomycetous order Halosphaeriales. (:an ,I Ro t  73: 790-801. Kohlmeyer ,J, L'olkmann-Kohlme~rB, Eriksson (1)E. 1995. Fungi on ,/z~ncli.r 7-or)r~r.riarz 1. 7i-ir.12orladizi n~tdlil-113. tn kcerr sp. nov. hlycotaon 53:349-3.53. Read SJ. Jones EBG, hloss ST. 1994. Ultrastructure of asci and ascospore sheath of' ~Mns.rnrinn thflla~iae (I>ocl~-loascomycetes. Ascorn!-cotina). Bot Mar 37:547-55.7. Reynolds DR. 1989. The hitunicate ascus paradigm. Bot Rev 55:l-52. Shearer (:A. Hyde KD. 1997. ,Mrcssnrinn ingolrlicinrc, a new ascornycete from fi-eshwater habitats. Slycologia 89: 114-119. Yusoff M,Jones EBG, Moss ST. 1994. Ultrastructt~ral of .in-tr?ino,spor(e ~snlinn comb. nov. hlycol Kes 98:997-1003. 

Ultrastructural studies of Massarina ingoldiana and M. purpurascens

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Ultrastructural studies of Massarina ingoldiana and M. purpurascens

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