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Ultrastructure of Antennospora salina comb. nov.
Mycol. Res. 98 (9): 997-1004 (1994)
997
Printed in Great Britain
Ultrastructure of Antennospora salina comb. nov.
M. YUSOFF"", S. J. READ, E. B. G. JONES AND S. T. MOSS School of Biological Sciences. University of Portsmouth. Portsmouth POt 2DY. u.K.
The ultrastructure of the ascospore and appendages of Halosphaeria salina was studied. Ascospore walls comprise a two-zoned mesosporium. the outer more electron-dense. and an electron-dense episporium enclosed by a layer of mucilaginous material. Ascospores have 3-5 subpolar appendages at each pole that appear as electron-dense striations and an amorphous core. Striations arise as outgrowths of the outer zone of the mesosporium. which is thickened at the point of attachment of the appendage to the spore wall. Halosphaeria salina is compared to H. appendiculata and Antennospora quadricornuta. It has more features in common with A. quadricornuta. particularly the ontogeny of the spore appendage. and is therefore assigned to the genus Antennospora.
Halosphaeria Linder was initially monotypic. with H. appendiculata Linder the type species (Barghoom & Linder. 1944). Subsequently. a number of new species were described. while Kohlmeyer (1972) transferred all species of Remispora to Halosphaeria. a total of 13 species then being assigned to the genus. Revisions of genera of the Halosphaeriaceae by Jones. Johnson & Moss (1984. 1986) and Jones & Moss (1987) reduced the species assigned to Halosphaeria to three (H. appendiculata. H. cucullata (Kohlm.) Kohlm.. H. salina (Meyers) Kohlm.). The remaining species were assigned to Remispora (R. galerita Tubaki. R. maritima Linder. R. pilleata Kohlm.. R. quadriremis (Hohnk) Kohlm.• R. stellata Kohlm.). Halosphaeriopsis (H. mediosetigera (Cribb & J. W. Cribb) T. W. Johnson). Halosarpheia (H. hamata (tentative assignment). H. trullifera (Kohlm.) E. B. G. Jones. S. T. Moss & Cuomo). Ondiniella (0. torquata (Kohlm.) E. B. G. Jones. R. G. Johnson & S. T. Moss) and Antennospora (A. quadricornuta (Cribb & J. W. Cribb) T. W.Johnson) (Jones. Moss & Cuomo. 1983; Johnson. Jones & Moss. 1984; Jones. Johnson & Moss. 1984; Kohlmeyer and Volkmann-Kohlmeyer. 1991). While all the above taxonomic recommendations have been accepted by Kohlmeyer (1986) and Kirk (1986). Kohlmeyer & Volkmann-Kohlmeyer (1987. 1991) have not accepted the transfer of H. quadricornuta to Antennospora as .the appendage in both genera is connected by an isthmus of electron-dense material to the mesosporium. and an episporium is absent at the region of attachment· (Kohlmeyer. 1984). However. we maintain that the two genera are quite distinct. In Halosphaeria. ascospores have polar and equatorial appendages that arise as outgrowths of the mesosporium and are initially wrapped around the spore. but separate and extend to form spoonshaped structures. The substructure of the appendage • Now at: Institute of Advanced Studies. University of Malaya, 59100 Kuala Lumpur, Malaysia.
comprises a reticulum of electron-dense strands within a less electron-dense amorphous matrix. while in Antennospora appendages are subterminal and the episporium is absent at the point of appendage attachment. There are no equatorial appendages. The substructure of the appendage comprises a thin outer electron-dense layer. an inner layer of less electrondense material with electron-dense fibrils that run the length of the appendage and arise from the mesosporium. and an electron-transparent core (Jones & Moss. 1987). There is a fundamental difference in the ultrastructure of the appendages of these two genera. Halosphaeria cucullata undoubtedly does not belong in Halosphaeria as it has a single. cap-like. subglobose. polar. deciduous appendage and bears no resemblance to the type species (H. appendiculata). Studies at the transmission electron microscope level (TIM) are required to determine ascospore appendage ontogeny. However. the species has not been widely collected. and material has been unavailable for study by us. Halosphaeria salina is a widely collected species on wood in tropical and subtropical waters. It was first described by Meyers (1957) as Arenariomyces salina Meyers. then reduced to synonymy with Remispora quadriremis by Johnson & Sparrow (1961) and Kohlmeyer (1961). Later. Kohlmeyer (1968) recognized that the two species were distinct. based on the different position of ascospore appendages: terminal in R. quadriremis and subterminal in A. salina; and their geographic distribution: R. quadriremis temperate. A. salina tropical to subtropical. Nevertheless. A. salina was retained in Remispora because of similarities in their ascomatal structure. However. Kohlmeyer (1972) later transferred all Remispora species to Halosphaeria as described above. Thus. two species are left in Halosphaeria: H. appendiculata and H. salina. Ascomata are smaller in H. salina (336--392 X 266--308 I-lm) than in H. appendiculata (425-500 x
Ultrastructure of Antennospora salina comb. nov.
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Figs 1-4. Antennospora salina. Scanning electron micrographs of ascospores and ascospore appendages. Scale bars, 2 ~m. Figs 1-3. Subpolar attachment of three (Fig. 1) and four (Figs 2, 3) appendages (A) by which the spore adheres to the substratum. Note the sheath of fibrous material on the ascospore. Fig. 4. Spore appendage showing the 'scar' of detachment (S) from the ascospore. Note the linear markings (arrowed) on the appendage.
In the former the ascomata are globose to subglobose, immersed, membranous and coriaceous, with short necks, while in the laUer they are globose to elongate, immersed to superficial, membranous to coriaceous, with long necks (up to 750 11m) with apical hyphae. They also differ in ascospore appendage morphology. In H. appendiculata there is a single appendage at each pole and 3-4 equatorial appendages, all 'spoon-shaped' at maturity. In H. salina there • are 3-4 subpolar, 'claw-like' appendages and no equatorial appendage. The appendages have been reported to be striated by Kohlmeyer (1968), Kohlmeyer & Kohlmeyer (1971,1979) and Zainal & Jones (1974). 317-330 11m).
MATERIALS AND METHODS Material of H. salina was obtained from driftwood collected at Kuala Selangor and Morib mangroves, Malaysia and returned to Portsmouth for fixation.
A fresh spore suspension was embedded in agar, fixed in 1 % (w!v) aqueous potassium permanganate and dehydrated
through a graded ethanol series, transferred to acetone and embedded in Mollenhauer's resin (Mollenhauer, 1964). Ultrathin sections were stained with lead citrate, post-stained with uranyl acetate and examined at 80 kV in a JEOL 1005 TEM.
RESULTS Fully developed ascospores were hyaline, ovoid to ellipsoidaL 17'5-23 x 8'5-10 11m, uniseptate, slightly constricted at the septum, with rounded poles, and 4-5 subterminal appendages (Figs 1-3, 5, 7). Each appendage was characterized by striations along its length and with its terminal third tapering to a fine point from which mucilage may be released (Figs 1, 4). Figure 4 shows a scar at the base of a detached appendage indicating the narrow zone of attachment to the spore walL
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5
6
Figs 5-8. Anfennosporasalina. Transmission electron micrographs of an ascospore and ascospore appendages. Fig. 5. Oblique longitudinal section of a released ascospore showing attachment of one of the subpolar appendages (A). The spore wall comprises an outer electron-dense episporium (E) and an inner less electron - dense mesosporium (M) that also forms the cross wall. Fig. 6. Appendages with peripheral striations and an amorphous core. Note the wide appendage base (arrowed). Figs 7, 8. Transverse sections at the attachment region of the appendage showing absence of episporium (E) and a thickened outer mesosporium (M) in this zone Scale bars: Fig. 5, 2'0 ~; Fig. 6. 0'1 ~m; Figs 7. 8, 0'5 ~m.
The ascospore wall comprised an outer tripartite episporium and an inner mesosporium (Fig. 7). The episporium consisted of two electron-dense layers separated by an electrontransparent layer and with a total thickness of 15-20 nm. The
250-450 nm thick mesosporium was less electron-dense than
the episporium. An exosporium was not observed in this species, but fibrous, mUcilaginous deposits occurred on the outer surface of the episporium (Figs 1-3, 11, 12).
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Ultrastructure of Antennospora salina comb. nov.
~.
....~-M
12 Figs 9-12. Antennospora salina. Transmission e1edron micrographs of ascospores and ascospore appendages. Fig. 9. Longitudinal sedion of a released ascospore with striated appendages (A). Note the fibrous material (arrowed) on the outside of the spore wall. Fig. 10. Higher magnification of the spore walls showing the fibrous material. Figs 11, 12. Appendage attachment regions showing the eledrondense striations (arrowed) of the appendage which are continuous with the isthmus formed by the thickened mesosporium (M). Scale bars: Fig. 9, 4 \.lm; Figs 10, 11, 0'25 \.lm; Fig. 12, 1 \.lm.
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17
..
Figs 13-17. Antennospora salina. Transmission electron micrographs of ascospore appendages. Fig. 13. Transverse section of an appendage bounded by an electron-dense layer and with an electron-transparent core. Note the peripheral zone of amorphous material within which occur the striations (arrowed). Fig. 14 shows the absence of the episporium (E) at the appendage attachment region. Figs 15, 16. Longitudinal sections showing the flattened end-face, an electron-dense outer boundary and an amorphous core. Fig. 17. Oblique transverse section showing two of the subpolar appendages. Note the wide appendage base (arrowed). Scale bars: Figs 13, 14, 0·5 ~m; Figs 15, 16, 0·8 ~m; Fig. 17, 2 ~m.
The appendages had an outer electron-dense boundary which enclosed a peripheral region of longitudinal electrondense striations which surrounded a core of amorphous, mucilaginous material (Figs 6, 9, 10, 12, 15-17). A section through or near the appendage periphery showed continuous striations (Figs 6, 10, 12), but these were discontinuous in oblique sections (Fig. 6). At the base of the appendage, the electron-dense striations were continuous with the outer electron-dense zone of the mesosporium (Figs 7, 8, 10, 12, 14).
Localized electron-dense regions, continuous with the outer boundary layers, were also observed at the appendage periphery, although they were more prominent on one side of the appendage (Fig. 13). These were considered to be the sections through the striations. The amorphous material within the appendage seemed to concentrate at the peripheral region (Fig. 13) and towards the appendage tip (Figs 5,15,16). The appendages were sharply attenuated at the tip (Fig. 2) with a flattened end-face and an outer electron-dense boundary
Ultrastructure of Antennospora salina comb. nov.
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Table 1. Comparison of ascoma. ascus and ascospore characters of Halosphaeria appemliculata. AnlemlOspora salina and A. quadricornula H. appmdiculata
Ascomata Solitary/gregarious Globose/ subglobose Immersed Ostiolate Periphyses absent Membranous Dark brown/black 4--6 layers of thick-walled cells with large lumina Pseudoparenchyma cells, thin-walled, large, polygonal, deliquescing 425-500 x 3 I 7-330 ~m Asci 8-spored Clavate/ subfusoid Pedunculate Unitunicate Deliquescing early Develop at the base of ascoma from a pad of ascogenous hyphae Ascospores Ellipsoidal Uniseptate Slightly constricted at septum Hyaline 18-29 x 8-12 ~m Appendaged Membrane complex present Appendages Polar (1) Equatorial (2-4) Obclavate with deep spoon-shaped structure at attachment region II-15 ~m Appendage ontogeny Mesosporium, episporium, no exosporium Appendage = outgrowth of spore
Attached to mesosporium via an isthmus of electron-dense material No episporium at attachment region Spoon-shape near base Appendage initially wrapped around spore. folded substructure becoming electron-dense. strands with less electron-dense component. Mature appendage reticulate in appearance.
A. salina
A. quadricornuta
Solitary/gregarious Globose/ elongate Immersed/superficial Ostiolate
Solitary/ gregarious Subglobose/ellipsoidal Immersed/superficial Ostiolate Periphyses absent Subcarbonaceous Dark brown/black 3-4 layers of irregular polygonal thick-walled cells Pseudoparenchyma cells, thin-walled, large and deliquescing 210--240 Ilm in diameter
Membranous to subcarbonaceous Dark brown
266-308 x
33~392 ~m
8-spored Elongate / clavate
8-spored Clavate
Unitunicate Deliquescing early On pad at base of ascoma
Unitunicate Deliquescing early Formed from a pad of ascogenous hyphae
Ellipsoidal Uniseptate Slightly/ not constricted at septum Hyaline 17'5-28 x 8-14 ~m Appendaged Membrane complex not observed
Ellipsoidal Uniseptate Slightly constricted at septum Hyaline 20--35 x 8-12 ~m Appendaged Membrane complex present
Subpolar (3-4) Equatorial (none) Obclavate, acuminate. attenuate, clawlike/curved 10--19 Ilm
Subpolar (2-3) Equatorial (none) Cylindrical, attenuate, non-gelatinous
Mesosporium, episporium, no exosporium Appendage = outgrowth of spore Attached to mesosporium via an isthmus of electron-dense material No episporium at attachment region Bulbous base Outer electron-dense boundary enclosing peripheral longitudinal electron-dense striations. with amorphous core. Striations arise from the electron-dense layer of the mesosporium.
Mesosporium, episporium, no exosporium Appendage = outgrowth of spore Attached to mesosporium via an isthmus of electron-dense material No episporium at attachment region Bulbous base Outer electron-dense boundary enclosing peripheral longitudinal electron-dense striations. with amorphous core. Striations arise from the electron-dense layer of the mesosporium.
(Figs IS, 16). At the attachment region of the appendage to the spore, the episporium was absent and the outer electrondense zone of the mesosporium was thickened to form an isthmus (Figs 8, 10, 12). Occasionally, the appendages appeared to have a wide base (Figs 6, 17).
DISCUSSION Ascospore morphology and dimensions of H. salina recorded in this study concur with the type description of Arenariomyces salina (Meyers, 1957). The thickness of the mesosporium is within the range recorded for H. appendiculata, Le. 200-500 nm Oohnson, 1982). However, the episporium is thinner in H.
20--33 Ilm
salina than in H. appendiculata (50-70 nm). In contrast to H. appendiculata, the episporium in H. salina is tripartite, while an exosporium is absent in both species. Fibrous material envelops the entire spore of H. salina, and this probably aids in spore attachment to surfaces. This observation, particularly at the scanning electron microscope leveL has been reported several times by other workers (Zainal & Jones, 1984; Hyde & Jones, 1989). In H. appendiculata, the ascospore lacks mucilage when it is released from the ascoma, but a mucilaginous envelope forms around the spore once it has made contact with a surface (Hyde, Moss & Jones, 1994). In H. salina, the mucilage on the spore wall is present prior to ascospore release.
M. Yusoff and others Striations along the length of the appendages in H. salina have also been recorded previously (Kohlmeyer, 1968; Zainal & Jones, 1984); however, these are not visible in older spores owing to shrinkage of the appendages (Kohlmeyer & Kohlmeyer, 1971). These appendages have also been described, using light microscopy, as being channelled on the outside (Kohlmeyer, 1984), although this may be an artefact of the striations. Appendage shape, Le. a broad base tapering towards a pointed tip, agrees with the description by Kohlmeyer (1984) and Zainal & Jones (1984). Appendages are attached to the spore by a small pad or an isthmus of material which fits into a subpolar socket in the spore (Zainal & Jones, 1984). These were not observed in the present study. Zainal & Jones (1984) observed release of mucilage from the tips of the appendages. In this study, the appendages were shown to contain an amorphous core which may represent the unreleased mucilage observed in the previous study. It is assumed that the mucilage may play a role in ascospore attachment. The number and position of ascospore appendages of H. salina agree with those previously described for this species (Kohlmeyer, 1968, 1984; Kohlmeyer & Kohlmeyer, 1971, 1979; Zainal & Jones, 1984; Borse, 1987). Ascospores with 6-8 appendages have been recorded by Booth (1983) and these appendages were significantly longer in ascospores from buried driftwood than from exposed driftwood. It is unclear whether Booth's observations represent a recognizable trend in this species or are merely a coincidence. Kohlmeyer & Volkmann-Kohlmeyer (1991) noted that ascomata which developed on incubated wood often contain ascospores with abnormal shapes and appendages. Kirk (1986) suggested that the production (development?) of abnormal appendages may help to understand the origin of normal ones, or even indicate phylogenetic relationships. However, we caution the use of such criteria in the delineation of genera and species (Kohlmeyer & Volkmann-Kohlmeyer, 1987).
TAXONOMIC ASSIGNMENT Antennospora quadricornuta is distinguished from species of Halosphaeria by the absence of equatorial appendages and the presence of subpolar, rigid and non-gelatinous appendages with a bulbous base and an amorphous core. Electron-dense striations within the appendages arise from the electron-dense zone of the mesosporium, and the episporium is absent at the point of attachment to the spore wall (Table 1). All these characteristics are also present in H. salina. In H. appendiculata the appendage is reticulate and lacks electron-dense striations but arises, as an outgrowth of the spore wall, on a slight swelling with connecting fibrils. In addition to ascospore and appendage characteristics, ascoma and ascus characters of the two species should also be compared. Gross ascoma morphology of Halosphaeria salina resembles more closely that of Antennospora quadricornuta than H. appendiculata. All three species do not differ greatly in ascus and ascospore wall characters. However, ascospore appendages appear to be the key criteria in separating them, namely the position and ultrastructure of the appendages. Consequently the following nomenclatural changes are proposed.
1003
Antennospora Meyers, Mycologia 49: 503 (1957) Type Antennospora quadricornuta (Cribb & J. W. Cribb) T. W. Johnson,]. Elisha Mitchell Sci. Soc. 74: 46-47 (1958). Synonyms Halosphaeria quadricornuta Cribb & J. W. Cribb, Univ. Queensland Papers, Dept. Botany 3: 99 (1956). Antennospora caribbea Meyers, Mycologia 49: 503 (1957). Antennospora salina (Meyers) Yusoff, E. B. G. Jones & S. T. Moss, comb. nov. Synonyms Arenariomyces salina Meyers, Mycologia 49: 505 (1957). Remispora salina (Meyers) Kohlm., Mycologia 60: 262 (1968).
Halosphaeria salina (Meyers) Kohlm., Can.]. Bot. 50: 1957 (1972).
Halosphaeria Linder, Farlowia 1: 412 (1944) Type Halosphaeria appendiculata Linder, Farlowia 1: 412 (1944). Synonym Remispora ornata T. W. Johnson & A. R. CavaL Nova Hedwigia 6: 188 (1963). Species requiring further study Halosphaeria cucullata (Kohlm.) Kohlm., Can.]. Bot. 50: 1956 (1972).
Synonym Remispora cucullata Kohlm., Mycologia 56: 770 (1964). A comparison of ascoma, ascus and ascospore characters of H. appendiculata, A. salina and A. quadricornuta is presented in Table 1. Dr Yusoff is grateful to the Public Services Department of Malaysia for the award of a scholarship and to the University of Malaya for granting leave of absence. Professor E. B. G. Jones is grateful to the British Council and the University of Malaya for travelling funds for the collection of material. We thank Mr C. Derrick for photographic assistance.
REFERENCES Barghoom. E. S. & Linder. D. H. (1944). Marine fungi: their taxonomy and biology. Farlowia 1, 395-467. Booth. T. (1983). Lignicolous marine fungi from Sao Paulo. Brazil. Canadian Journal of Botany 61. 488-506. Borse, B. D. (1987). Marine fungi from India. IV. Transactions of the Mycological Society of Japan 18. 55-61. Hyde. K. D. & Jones. E. B. G. (1989). Observations on ascospore morphology in marine fungi and their attachment to surfaces. Botanica Marina 31. 205-218. Hyde, K. D., Moss. S. T. & Jones. E. B. G. (1994). A SI06 pore ultrastructure of Halosphaeria appencilulata (Halosphaeriaceae), Botanica Marina 31, 51-56. lohnson. R. G. (1982). Ultrastructure and histochemistry of the ontogeny of ascospores and their appendages in marine Ascomycetes. PhD. Thesis. CN.A.A., Porstmouth Polytechnic. Johnson, R. G., Jones. E. B. G. & Moss, S. T. (1984). Taxonomic studies of the Halosphaeriaceae: Remispora Linder. Marinospora Cavaliere and Carbosphaere/la Schmidt. Botanica Marina 11, 557-566. Johnson. T. W. & Sparrow. F. K. (1961). FUIlgi in Oceans and Estuaries, 668 pp. J. Cramer: Weinheim.
Ultrastructure of Antennospora salina comb. nov. Jones, E. B. G., Johnson, R. G. & Moss, S. T. (1984). Taxonomic studies of the Halosphaeriaceae: Halosphaeria Linder. Botanica Marina 27, 129-143. Jones, E. B. G.. Johnson, R. G. & Moss, S. T. (1986). Taxonomic studies of the Halosphaeriaceae. Philosophy and rationale for the selection of charaders in the delineation of genera. In The Biology of Marine Fungi (ed. S. T. Moss), pp. 211-229. Cambridge University Press: Cambridge, U.K. Jones, E. B. G. & Moss, S. T. (1987). Key and notes on genera of the Halosphaeriaceae examined at the ultrastructural level. Systema Ascomycetum 6, 179-200. Jones, E. B. G., Moss, S. T. & Cuomo, V. (1983). Spore appendage development in the lignicolous marine Pyrenomycetes Chaetosphaeria chaetosa and
Halosphaeria trullifera. Transactions of the British Mycological Society 80, 193-200. Kirk, P. W. (1986). Evolutionary trends within the Halosphaeriaceae. In The Biology of Marine Fungi (ed. S. T. Moss), pp. 263-273. Cambridge University Press: Cambridge, U.K. Kohlmeyer, J. (1961). Synoptic plates for quick determination of marine Deuteromycetes and Ascomycetes. Nova Hedwigia 3, 383-398. Kohlmeyer, ). (1968). Marine fungi from the tropics. Mycologia 60, 252-270. Kohlmeyer, J. (1972). A revision of Halosphaeriaceae. Canadian Journal of Botany 50, 1951-1963.
(Accepted 8 February 1994)
1004 Kohlmeyer, J. (1984). Tropical marine fungi. P.S.2-N.l.: Marine Ecology 5, 329-378. Kohlmeyer, J. (1986). Taxonomic studies of the marine Ascomycotina.ln The Biology ofMarine Fungi (ed. S. T. Moss), pp. 199-210. Cambridge University Press: Cambridge, U.K. Kohlmeyer, J. & Kohlmeyer, E. (1971). Synoptic Plates of Higher Marine Fungi. An ldmtification Guide for the Marine Environment. r. Cramer: Lehre. Kohlmeyer, J. & Kohlmeyer, E. (1979). Marine Mycology, The Higher Fungi, 690 pp. Academic Press: New York arid London. Kohlmeyer, J. & Volkmann-Kohlmeyer, B. (1987). Marine fungi from Aldabra, the Galapagos, and other tropical islands. Canadian Journal of Botany 65, 571-582. Kohlmeyer, J. & Volkmann-Kohlmeyer, B. (1991). lllustrated key to the filamentous higher marine fungi. Botanica Marina 34, 1-61. Meyers, S. P. (1957). Taxonomy of marine Pyrenomycetes. Mycologia 49, 475-528. Mollenhauer, H. H. (1964). Plastic embedding mixtures for use in eleelron microscopy. Stain Technology 39, 111-114. Zainal. A. & Jones, E. B. G. (1984). Observations on some lignicolous marine fungi from Kuwait. Nova Hedwigia 39, 569-583.
997
Printed in Great Britain
Ultrastructure of Antennospora salina comb. nov.
M. YUSOFF"", S. J. READ, E. B. G. JONES AND S. T. MOSS School of Biological Sciences. University of Portsmouth. Portsmouth POt 2DY. u.K.
The ultrastructure of the ascospore and appendages of Halosphaeria salina was studied. Ascospore walls comprise a two-zoned mesosporium. the outer more electron-dense. and an electron-dense episporium enclosed by a layer of mucilaginous material. Ascospores have 3-5 subpolar appendages at each pole that appear as electron-dense striations and an amorphous core. Striations arise as outgrowths of the outer zone of the mesosporium. which is thickened at the point of attachment of the appendage to the spore wall. Halosphaeria salina is compared to H. appendiculata and Antennospora quadricornuta. It has more features in common with A. quadricornuta. particularly the ontogeny of the spore appendage. and is therefore assigned to the genus Antennospora.
Halosphaeria Linder was initially monotypic. with H. appendiculata Linder the type species (Barghoom & Linder. 1944). Subsequently. a number of new species were described. while Kohlmeyer (1972) transferred all species of Remispora to Halosphaeria. a total of 13 species then being assigned to the genus. Revisions of genera of the Halosphaeriaceae by Jones. Johnson & Moss (1984. 1986) and Jones & Moss (1987) reduced the species assigned to Halosphaeria to three (H. appendiculata. H. cucullata (Kohlm.) Kohlm.. H. salina (Meyers) Kohlm.). The remaining species were assigned to Remispora (R. galerita Tubaki. R. maritima Linder. R. pilleata Kohlm.. R. quadriremis (Hohnk) Kohlm.• R. stellata Kohlm.). Halosphaeriopsis (H. mediosetigera (Cribb & J. W. Cribb) T. W. Johnson). Halosarpheia (H. hamata (tentative assignment). H. trullifera (Kohlm.) E. B. G. Jones. S. T. Moss & Cuomo). Ondiniella (0. torquata (Kohlm.) E. B. G. Jones. R. G. Johnson & S. T. Moss) and Antennospora (A. quadricornuta (Cribb & J. W. Cribb) T. W.Johnson) (Jones. Moss & Cuomo. 1983; Johnson. Jones & Moss. 1984; Jones. Johnson & Moss. 1984; Kohlmeyer and Volkmann-Kohlmeyer. 1991). While all the above taxonomic recommendations have been accepted by Kohlmeyer (1986) and Kirk (1986). Kohlmeyer & Volkmann-Kohlmeyer (1987. 1991) have not accepted the transfer of H. quadricornuta to Antennospora as .the appendage in both genera is connected by an isthmus of electron-dense material to the mesosporium. and an episporium is absent at the region of attachment· (Kohlmeyer. 1984). However. we maintain that the two genera are quite distinct. In Halosphaeria. ascospores have polar and equatorial appendages that arise as outgrowths of the mesosporium and are initially wrapped around the spore. but separate and extend to form spoonshaped structures. The substructure of the appendage • Now at: Institute of Advanced Studies. University of Malaya, 59100 Kuala Lumpur, Malaysia.
comprises a reticulum of electron-dense strands within a less electron-dense amorphous matrix. while in Antennospora appendages are subterminal and the episporium is absent at the point of appendage attachment. There are no equatorial appendages. The substructure of the appendage comprises a thin outer electron-dense layer. an inner layer of less electrondense material with electron-dense fibrils that run the length of the appendage and arise from the mesosporium. and an electron-transparent core (Jones & Moss. 1987). There is a fundamental difference in the ultrastructure of the appendages of these two genera. Halosphaeria cucullata undoubtedly does not belong in Halosphaeria as it has a single. cap-like. subglobose. polar. deciduous appendage and bears no resemblance to the type species (H. appendiculata). Studies at the transmission electron microscope level (TIM) are required to determine ascospore appendage ontogeny. However. the species has not been widely collected. and material has been unavailable for study by us. Halosphaeria salina is a widely collected species on wood in tropical and subtropical waters. It was first described by Meyers (1957) as Arenariomyces salina Meyers. then reduced to synonymy with Remispora quadriremis by Johnson & Sparrow (1961) and Kohlmeyer (1961). Later. Kohlmeyer (1968) recognized that the two species were distinct. based on the different position of ascospore appendages: terminal in R. quadriremis and subterminal in A. salina; and their geographic distribution: R. quadriremis temperate. A. salina tropical to subtropical. Nevertheless. A. salina was retained in Remispora because of similarities in their ascomatal structure. However. Kohlmeyer (1972) later transferred all Remispora species to Halosphaeria as described above. Thus. two species are left in Halosphaeria: H. appendiculata and H. salina. Ascomata are smaller in H. salina (336--392 X 266--308 I-lm) than in H. appendiculata (425-500 x
Ultrastructure of Antennospora salina comb. nov.
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Figs 1-4. Antennospora salina. Scanning electron micrographs of ascospores and ascospore appendages. Scale bars, 2 ~m. Figs 1-3. Subpolar attachment of three (Fig. 1) and four (Figs 2, 3) appendages (A) by which the spore adheres to the substratum. Note the sheath of fibrous material on the ascospore. Fig. 4. Spore appendage showing the 'scar' of detachment (S) from the ascospore. Note the linear markings (arrowed) on the appendage.
In the former the ascomata are globose to subglobose, immersed, membranous and coriaceous, with short necks, while in the laUer they are globose to elongate, immersed to superficial, membranous to coriaceous, with long necks (up to 750 11m) with apical hyphae. They also differ in ascospore appendage morphology. In H. appendiculata there is a single appendage at each pole and 3-4 equatorial appendages, all 'spoon-shaped' at maturity. In H. salina there • are 3-4 subpolar, 'claw-like' appendages and no equatorial appendage. The appendages have been reported to be striated by Kohlmeyer (1968), Kohlmeyer & Kohlmeyer (1971,1979) and Zainal & Jones (1974). 317-330 11m).
MATERIALS AND METHODS Material of H. salina was obtained from driftwood collected at Kuala Selangor and Morib mangroves, Malaysia and returned to Portsmouth for fixation.
A fresh spore suspension was embedded in agar, fixed in 1 % (w!v) aqueous potassium permanganate and dehydrated
through a graded ethanol series, transferred to acetone and embedded in Mollenhauer's resin (Mollenhauer, 1964). Ultrathin sections were stained with lead citrate, post-stained with uranyl acetate and examined at 80 kV in a JEOL 1005 TEM.
RESULTS Fully developed ascospores were hyaline, ovoid to ellipsoidaL 17'5-23 x 8'5-10 11m, uniseptate, slightly constricted at the septum, with rounded poles, and 4-5 subterminal appendages (Figs 1-3, 5, 7). Each appendage was characterized by striations along its length and with its terminal third tapering to a fine point from which mucilage may be released (Figs 1, 4). Figure 4 shows a scar at the base of a detached appendage indicating the narrow zone of attachment to the spore walL
M. Yusoff and others
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5
6
Figs 5-8. Anfennosporasalina. Transmission electron micrographs of an ascospore and ascospore appendages. Fig. 5. Oblique longitudinal section of a released ascospore showing attachment of one of the subpolar appendages (A). The spore wall comprises an outer electron-dense episporium (E) and an inner less electron - dense mesosporium (M) that also forms the cross wall. Fig. 6. Appendages with peripheral striations and an amorphous core. Note the wide appendage base (arrowed). Figs 7, 8. Transverse sections at the attachment region of the appendage showing absence of episporium (E) and a thickened outer mesosporium (M) in this zone Scale bars: Fig. 5, 2'0 ~; Fig. 6. 0'1 ~m; Figs 7. 8, 0'5 ~m.
The ascospore wall comprised an outer tripartite episporium and an inner mesosporium (Fig. 7). The episporium consisted of two electron-dense layers separated by an electrontransparent layer and with a total thickness of 15-20 nm. The
250-450 nm thick mesosporium was less electron-dense than
the episporium. An exosporium was not observed in this species, but fibrous, mUcilaginous deposits occurred on the outer surface of the episporium (Figs 1-3, 11, 12).
1000
Ultrastructure of Antennospora salina comb. nov.
~.
....~-M
12 Figs 9-12. Antennospora salina. Transmission e1edron micrographs of ascospores and ascospore appendages. Fig. 9. Longitudinal sedion of a released ascospore with striated appendages (A). Note the fibrous material (arrowed) on the outside of the spore wall. Fig. 10. Higher magnification of the spore walls showing the fibrous material. Figs 11, 12. Appendage attachment regions showing the eledrondense striations (arrowed) of the appendage which are continuous with the isthmus formed by the thickened mesosporium (M). Scale bars: Fig. 9, 4 \.lm; Figs 10, 11, 0'25 \.lm; Fig. 12, 1 \.lm.
M. Yusoff and others
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17
..
Figs 13-17. Antennospora salina. Transmission electron micrographs of ascospore appendages. Fig. 13. Transverse section of an appendage bounded by an electron-dense layer and with an electron-transparent core. Note the peripheral zone of amorphous material within which occur the striations (arrowed). Fig. 14 shows the absence of the episporium (E) at the appendage attachment region. Figs 15, 16. Longitudinal sections showing the flattened end-face, an electron-dense outer boundary and an amorphous core. Fig. 17. Oblique transverse section showing two of the subpolar appendages. Note the wide appendage base (arrowed). Scale bars: Figs 13, 14, 0·5 ~m; Figs 15, 16, 0·8 ~m; Fig. 17, 2 ~m.
The appendages had an outer electron-dense boundary which enclosed a peripheral region of longitudinal electrondense striations which surrounded a core of amorphous, mucilaginous material (Figs 6, 9, 10, 12, 15-17). A section through or near the appendage periphery showed continuous striations (Figs 6, 10, 12), but these were discontinuous in oblique sections (Fig. 6). At the base of the appendage, the electron-dense striations were continuous with the outer electron-dense zone of the mesosporium (Figs 7, 8, 10, 12, 14).
Localized electron-dense regions, continuous with the outer boundary layers, were also observed at the appendage periphery, although they were more prominent on one side of the appendage (Fig. 13). These were considered to be the sections through the striations. The amorphous material within the appendage seemed to concentrate at the peripheral region (Fig. 13) and towards the appendage tip (Figs 5,15,16). The appendages were sharply attenuated at the tip (Fig. 2) with a flattened end-face and an outer electron-dense boundary
Ultrastructure of Antennospora salina comb. nov.
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Table 1. Comparison of ascoma. ascus and ascospore characters of Halosphaeria appemliculata. AnlemlOspora salina and A. quadricornula H. appmdiculata
Ascomata Solitary/gregarious Globose/ subglobose Immersed Ostiolate Periphyses absent Membranous Dark brown/black 4--6 layers of thick-walled cells with large lumina Pseudoparenchyma cells, thin-walled, large, polygonal, deliquescing 425-500 x 3 I 7-330 ~m Asci 8-spored Clavate/ subfusoid Pedunculate Unitunicate Deliquescing early Develop at the base of ascoma from a pad of ascogenous hyphae Ascospores Ellipsoidal Uniseptate Slightly constricted at septum Hyaline 18-29 x 8-12 ~m Appendaged Membrane complex present Appendages Polar (1) Equatorial (2-4) Obclavate with deep spoon-shaped structure at attachment region II-15 ~m Appendage ontogeny Mesosporium, episporium, no exosporium Appendage = outgrowth of spore
Attached to mesosporium via an isthmus of electron-dense material No episporium at attachment region Spoon-shape near base Appendage initially wrapped around spore. folded substructure becoming electron-dense. strands with less electron-dense component. Mature appendage reticulate in appearance.
A. salina
A. quadricornuta
Solitary/gregarious Globose/ elongate Immersed/superficial Ostiolate
Solitary/ gregarious Subglobose/ellipsoidal Immersed/superficial Ostiolate Periphyses absent Subcarbonaceous Dark brown/black 3-4 layers of irregular polygonal thick-walled cells Pseudoparenchyma cells, thin-walled, large and deliquescing 210--240 Ilm in diameter
Membranous to subcarbonaceous Dark brown
266-308 x
33~392 ~m
8-spored Elongate / clavate
8-spored Clavate
Unitunicate Deliquescing early On pad at base of ascoma
Unitunicate Deliquescing early Formed from a pad of ascogenous hyphae
Ellipsoidal Uniseptate Slightly/ not constricted at septum Hyaline 17'5-28 x 8-14 ~m Appendaged Membrane complex not observed
Ellipsoidal Uniseptate Slightly constricted at septum Hyaline 20--35 x 8-12 ~m Appendaged Membrane complex present
Subpolar (3-4) Equatorial (none) Obclavate, acuminate. attenuate, clawlike/curved 10--19 Ilm
Subpolar (2-3) Equatorial (none) Cylindrical, attenuate, non-gelatinous
Mesosporium, episporium, no exosporium Appendage = outgrowth of spore Attached to mesosporium via an isthmus of electron-dense material No episporium at attachment region Bulbous base Outer electron-dense boundary enclosing peripheral longitudinal electron-dense striations. with amorphous core. Striations arise from the electron-dense layer of the mesosporium.
Mesosporium, episporium, no exosporium Appendage = outgrowth of spore Attached to mesosporium via an isthmus of electron-dense material No episporium at attachment region Bulbous base Outer electron-dense boundary enclosing peripheral longitudinal electron-dense striations. with amorphous core. Striations arise from the electron-dense layer of the mesosporium.
(Figs IS, 16). At the attachment region of the appendage to the spore, the episporium was absent and the outer electrondense zone of the mesosporium was thickened to form an isthmus (Figs 8, 10, 12). Occasionally, the appendages appeared to have a wide base (Figs 6, 17).
DISCUSSION Ascospore morphology and dimensions of H. salina recorded in this study concur with the type description of Arenariomyces salina (Meyers, 1957). The thickness of the mesosporium is within the range recorded for H. appendiculata, Le. 200-500 nm Oohnson, 1982). However, the episporium is thinner in H.
20--33 Ilm
salina than in H. appendiculata (50-70 nm). In contrast to H. appendiculata, the episporium in H. salina is tripartite, while an exosporium is absent in both species. Fibrous material envelops the entire spore of H. salina, and this probably aids in spore attachment to surfaces. This observation, particularly at the scanning electron microscope leveL has been reported several times by other workers (Zainal & Jones, 1984; Hyde & Jones, 1989). In H. appendiculata, the ascospore lacks mucilage when it is released from the ascoma, but a mucilaginous envelope forms around the spore once it has made contact with a surface (Hyde, Moss & Jones, 1994). In H. salina, the mucilage on the spore wall is present prior to ascospore release.
M. Yusoff and others Striations along the length of the appendages in H. salina have also been recorded previously (Kohlmeyer, 1968; Zainal & Jones, 1984); however, these are not visible in older spores owing to shrinkage of the appendages (Kohlmeyer & Kohlmeyer, 1971). These appendages have also been described, using light microscopy, as being channelled on the outside (Kohlmeyer, 1984), although this may be an artefact of the striations. Appendage shape, Le. a broad base tapering towards a pointed tip, agrees with the description by Kohlmeyer (1984) and Zainal & Jones (1984). Appendages are attached to the spore by a small pad or an isthmus of material which fits into a subpolar socket in the spore (Zainal & Jones, 1984). These were not observed in the present study. Zainal & Jones (1984) observed release of mucilage from the tips of the appendages. In this study, the appendages were shown to contain an amorphous core which may represent the unreleased mucilage observed in the previous study. It is assumed that the mucilage may play a role in ascospore attachment. The number and position of ascospore appendages of H. salina agree with those previously described for this species (Kohlmeyer, 1968, 1984; Kohlmeyer & Kohlmeyer, 1971, 1979; Zainal & Jones, 1984; Borse, 1987). Ascospores with 6-8 appendages have been recorded by Booth (1983) and these appendages were significantly longer in ascospores from buried driftwood than from exposed driftwood. It is unclear whether Booth's observations represent a recognizable trend in this species or are merely a coincidence. Kohlmeyer & Volkmann-Kohlmeyer (1991) noted that ascomata which developed on incubated wood often contain ascospores with abnormal shapes and appendages. Kirk (1986) suggested that the production (development?) of abnormal appendages may help to understand the origin of normal ones, or even indicate phylogenetic relationships. However, we caution the use of such criteria in the delineation of genera and species (Kohlmeyer & Volkmann-Kohlmeyer, 1987).
TAXONOMIC ASSIGNMENT Antennospora quadricornuta is distinguished from species of Halosphaeria by the absence of equatorial appendages and the presence of subpolar, rigid and non-gelatinous appendages with a bulbous base and an amorphous core. Electron-dense striations within the appendages arise from the electron-dense zone of the mesosporium, and the episporium is absent at the point of attachment to the spore wall (Table 1). All these characteristics are also present in H. salina. In H. appendiculata the appendage is reticulate and lacks electron-dense striations but arises, as an outgrowth of the spore wall, on a slight swelling with connecting fibrils. In addition to ascospore and appendage characteristics, ascoma and ascus characters of the two species should also be compared. Gross ascoma morphology of Halosphaeria salina resembles more closely that of Antennospora quadricornuta than H. appendiculata. All three species do not differ greatly in ascus and ascospore wall characters. However, ascospore appendages appear to be the key criteria in separating them, namely the position and ultrastructure of the appendages. Consequently the following nomenclatural changes are proposed.
1003
Antennospora Meyers, Mycologia 49: 503 (1957) Type Antennospora quadricornuta (Cribb & J. W. Cribb) T. W. Johnson,]. Elisha Mitchell Sci. Soc. 74: 46-47 (1958). Synonyms Halosphaeria quadricornuta Cribb & J. W. Cribb, Univ. Queensland Papers, Dept. Botany 3: 99 (1956). Antennospora caribbea Meyers, Mycologia 49: 503 (1957). Antennospora salina (Meyers) Yusoff, E. B. G. Jones & S. T. Moss, comb. nov. Synonyms Arenariomyces salina Meyers, Mycologia 49: 505 (1957). Remispora salina (Meyers) Kohlm., Mycologia 60: 262 (1968).
Halosphaeria salina (Meyers) Kohlm., Can.]. Bot. 50: 1957 (1972).
Halosphaeria Linder, Farlowia 1: 412 (1944) Type Halosphaeria appendiculata Linder, Farlowia 1: 412 (1944). Synonym Remispora ornata T. W. Johnson & A. R. CavaL Nova Hedwigia 6: 188 (1963). Species requiring further study Halosphaeria cucullata (Kohlm.) Kohlm., Can.]. Bot. 50: 1956 (1972).
Synonym Remispora cucullata Kohlm., Mycologia 56: 770 (1964). A comparison of ascoma, ascus and ascospore characters of H. appendiculata, A. salina and A. quadricornuta is presented in Table 1. Dr Yusoff is grateful to the Public Services Department of Malaysia for the award of a scholarship and to the University of Malaya for granting leave of absence. Professor E. B. G. Jones is grateful to the British Council and the University of Malaya for travelling funds for the collection of material. We thank Mr C. Derrick for photographic assistance.
REFERENCES Barghoom. E. S. & Linder. D. H. (1944). Marine fungi: their taxonomy and biology. Farlowia 1, 395-467. Booth. T. (1983). Lignicolous marine fungi from Sao Paulo. Brazil. Canadian Journal of Botany 61. 488-506. Borse, B. D. (1987). Marine fungi from India. IV. Transactions of the Mycological Society of Japan 18. 55-61. Hyde. K. D. & Jones. E. B. G. (1989). Observations on ascospore morphology in marine fungi and their attachment to surfaces. Botanica Marina 31. 205-218. Hyde, K. D., Moss. S. T. & Jones. E. B. G. (1994). A SI06 pore ultrastructure of Halosphaeria appencilulata (Halosphaeriaceae), Botanica Marina 31, 51-56. lohnson. R. G. (1982). Ultrastructure and histochemistry of the ontogeny of ascospores and their appendages in marine Ascomycetes. PhD. Thesis. CN.A.A., Porstmouth Polytechnic. Johnson, R. G., Jones. E. B. G. & Moss, S. T. (1984). Taxonomic studies of the Halosphaeriaceae: Remispora Linder. Marinospora Cavaliere and Carbosphaere/la Schmidt. Botanica Marina 11, 557-566. Johnson. T. W. & Sparrow. F. K. (1961). FUIlgi in Oceans and Estuaries, 668 pp. J. Cramer: Weinheim.
Ultrastructure of Antennospora salina comb. nov. Jones, E. B. G., Johnson, R. G. & Moss, S. T. (1984). Taxonomic studies of the Halosphaeriaceae: Halosphaeria Linder. Botanica Marina 27, 129-143. Jones, E. B. G.. Johnson, R. G. & Moss, S. T. (1986). Taxonomic studies of the Halosphaeriaceae. Philosophy and rationale for the selection of charaders in the delineation of genera. In The Biology of Marine Fungi (ed. S. T. Moss), pp. 211-229. Cambridge University Press: Cambridge, U.K. Jones, E. B. G. & Moss, S. T. (1987). Key and notes on genera of the Halosphaeriaceae examined at the ultrastructural level. Systema Ascomycetum 6, 179-200. Jones, E. B. G., Moss, S. T. & Cuomo, V. (1983). Spore appendage development in the lignicolous marine Pyrenomycetes Chaetosphaeria chaetosa and
Halosphaeria trullifera. Transactions of the British Mycological Society 80, 193-200. Kirk, P. W. (1986). Evolutionary trends within the Halosphaeriaceae. In The Biology of Marine Fungi (ed. S. T. Moss), pp. 263-273. Cambridge University Press: Cambridge, U.K. Kohlmeyer, J. (1961). Synoptic plates for quick determination of marine Deuteromycetes and Ascomycetes. Nova Hedwigia 3, 383-398. Kohlmeyer, ). (1968). Marine fungi from the tropics. Mycologia 60, 252-270. Kohlmeyer, J. (1972). A revision of Halosphaeriaceae. Canadian Journal of Botany 50, 1951-1963.
(Accepted 8 February 1994)
1004 Kohlmeyer, J. (1984). Tropical marine fungi. P.S.2-N.l.: Marine Ecology 5, 329-378. Kohlmeyer, J. (1986). Taxonomic studies of the marine Ascomycotina.ln The Biology ofMarine Fungi (ed. S. T. Moss), pp. 199-210. Cambridge University Press: Cambridge, U.K. Kohlmeyer, J. & Kohlmeyer, E. (1971). Synoptic Plates of Higher Marine Fungi. An ldmtification Guide for the Marine Environment. r. Cramer: Lehre. Kohlmeyer, J. & Kohlmeyer, E. (1979). Marine Mycology, The Higher Fungi, 690 pp. Academic Press: New York arid London. Kohlmeyer, J. & Volkmann-Kohlmeyer, B. (1987). Marine fungi from Aldabra, the Galapagos, and other tropical islands. Canadian Journal of Botany 65, 571-582. Kohlmeyer, J. & Volkmann-Kohlmeyer, B. (1991). lllustrated key to the filamentous higher marine fungi. Botanica Marina 34, 1-61. Meyers, S. P. (1957). Taxonomy of marine Pyrenomycetes. Mycologia 49, 475-528. Mollenhauer, H. H. (1964). Plastic embedding mixtures for use in eleelron microscopy. Stain Technology 39, 111-114. Zainal. A. & Jones, E. B. G. (1984). Observations on some lignicolous marine fungi from Kuwait. Nova Hedwigia 39, 569-583.