Can ascospore ultrastructure differentiate between the genera

Linocarpon and Neolinocarpon and species therein?

YANNA, Wai Hong HO and Kevin D. HYDE*

Department of Ecology & Biodiversity, Centre for Research in Fungal Diversity, The University of Hong Kong,Pokfulam Road, Hong Kong SAR, People’s Republic of China.E-mail : kdhyde@hkucc.hku.hk

Received 24 April 2002; accepted 12 August 2003.

Ascospore appendages of two species of Linocarpon and three species of Neolinocarpon are illustrated using electron

microscopy. The structure of these appendages can be used to distinguish between species. The ascospore appendages ofspecies in both genera, however, were found to have similar structure. Thus, species of Linocarpon and Neolinocarponcould not be delineated at the generic level based on ascospore appendage structure.

INTRODUCTION

Linocarpon (Hyponectriaceae) was introduced based onL. pandani (syn. Linospora pandani) (Sydow & Sydow1917). Neolinocarpon was introduced to accommodateLinocarpon-like species with deeply immersed ascomataforming below a slightly raised or flattened clypeus,and with a refractive globose body below the apicalascus ring (Hyde 1992a). Linocarpon was restricted tospecies with superficial or slightly immersed ascomata,and the lack of a refractive globose body (Hyde 1992b,Hyde, Taylor & Frohlich 1998). Species of Linocarponand Neolinocarpon are very similar in producing8-spored, cylindrical asci with a non-amyloid cylindri-cal apical ring, and filiform, hyaline, usually unicellular,curved ascospores with refringent septum-like bands(Hyde 1988, 1992a, b, 1997, Dulymamode, Cannon& Peerally 1998, Hyde, Taylor & Frohlich 1998).Linocarpon presently comprises 35 species, while Neo-linocarpon comprises eight species (Hyde 1988, 1992a,1997, Dulymamode et al. 1998, Hyde & Alias 1999,Thongkantha et al. 2003). Both genera are currentlyplaced in the Hyponectriaceae, although Wang & Hyde(1999) suggest their inclusion is unlikely.

Species of Linocarpon and Neolinocarpon are ident-ified based on the size of the asci and ascospores, theshape of ascospores, and the presence of ascosporeappendages. Ascospores of these two genera are verythin, ranging from 2 to 6 mm. It is therefore difficult toexa-

mine the ascospore poles under the light microscope.Poonyth et al. (2000) examined Linocarponappendiculatum and L. nipae at the ultrastructural leveland showed that the morphology of the appendageswas a useful character in the delineation of species inLinocarpon. Therefore, to establish further criteria todelineate species of Linocarpon and Neolinocarpon,ascospore appendages of selected species were exam-ined at the electron microscope level.

The presence of a refractive globose body below theascus apical ring in Neolinocarpon appears to be in-consistent, as in species of Lasiosphaeria (Hyde, Taylor& Frohlich 2000). Refractive globose bodies were re-corded in the asci of Neolinocarpon globosicarpon (typespecies), N. australiense,N. calamae, N. eutypoides, andN. nonappendiculatus (Hyde 1992a, Hyde et al. 1998),but were absent in N. enshiensis, N. inconspicuus andN. nypicola (Hyde 1992a, 1997, Hyde et al. 1998, Hyde& Alias 1999). Therefore, delineation of Linocarponand Neolinocarpon based on the presence and absenceof the refractive globose bodies is unreliable, althoughno species of Linocarpon has asci with subapical re-fractive globose bodies.

Linocarpon and Neolinocarpon species are thereforesolely delineated based on the position of the ascomata,i.e. deeply immersed vs superficial. Frohlich & Hyde(2000) have discussed the need to establish fur-ther criteria to delineate species of Linocarpon andNeolinocarpon at the generic level. Ascospore ap-pendage structure of selected species is, therefore,examined at the ultrastructural level. The terminologyof Kirk (1966) is used to define the wall layers of* Corresponding author.

Mycol. Res. 107 (11): 1305–1313 (November 2003). f The British Mycological Society 1305

DOI: 10.1017/S095375620300861X Printed in the United Kingdom.

ascospores : mesosporium (the innermost layer) ; epi-sporium (the middle layer, except where the exosporiumis absent and generally the first formed layer) ; andexosporium (the outer layer). The term appendageis used to describe structures at the ends of theascospores. Ultrastructural work was carried out with

the following objectives : (1) to determine and com-pare the ultrastructure of ascospore appendages ofselected taxa in the two genera; and (2) to determineif species in both genera can be delineated at the gen-eric level by the ultrastructure of ascospore append-ages.

Figs 1–9. Light, SEM and TEM micrographs of ascospores of Linocarpon species. Fig. 1. L. aquaticum. Note thebipolar appendages (arrowed). Fig. 2. L. livistonae. In this species appendages are basal (arrowed). Figs 3–4. L. aquaticum.The appendages are bipolar (arrowed in 3). At one end the appendage has spread and is attached to the polycarbonate

membrane. Figs 5–7. L. livistonae. The apex in rounded while the base has a mucilaginous appendage (arrowed in 5).Figs 8–9. L. livistonae. The ascospore wall comprised an electron-dense episporium (E) and a bilamellate mesosporium(M1, M2). The basal appendage comprised electron-dense fibrillar material (Fm) that was derived from the episporium.

Bars: 1–2=25 mm; 3, 5=10 mm; 4, 6–7=1 mm; 8=500 nm; 9=100 nm.

Ultrastructure of Linocarpon and Neolinocarpon 1306

MATERIAL AND METHODS

Light microscopy (LM)

Freshly collected decaying palm samples were in-cubated separately in plastic bags lined with moistpaper towels and examined within one month. Fungalfruit bodies were mounted on slides in water or lacto-phenol for microscopic examination. The collectiondetails of material examined are listed in Table 1.

Scanning electron microscopy (SEM)

Fresh samples were fixed with 2% (w/v) osmiumtetroxide at 4 xC overnight. Samples were then washedwith distilled water, dehydrated through a gradedalcohol series to absolute alcohol, followed by criticalpoint drying, sputter-coating with gold/palladium for40 s, and examined using a Leica Cambridge Stereo-scan 440 scanning electron microscope operating at20 kV.

Transmission electron microscopy (TEM)

Fresh samples were embedded in 2% (w/v) ion agar,fixed with 4% (v/v) glutaraldehyde at room tempera-ture for 4 h, and post-fixed with 2% (w/v) osmiumtetroxide at 4 x overnight. Fixed samples were washedwith distilled water and dehydrated through a gradedalcohol series and acetone series. Dehydrated sampleswere embedded in Mollenhauer’s resin (Mollenhauer1964) through a series : 25, 50, 75 and 90% resin inacetone for 36 h for each concentration. Samples werethen transferred to 100% resin and infiltrated in avacuum chamber at 20 cmHg for 4 h, followed bypolymerization at 60 x for 3 d. Ultra-thin sections werestained with lead citrate for 15 min and post-stained

Table 1. Collection details of material examined.

Fungus Site Substratum Date

Herbarium

assession no.

Linocarpon aquaticum Australia, North Queensland,

Atherton Tablelands

Decaying rachis of

Oraniopsis appendiculata

May 1998 YAN M6

(HKU(M) 3358)

L. livistonae Brunei Darussalam, Temburong,

Batu Apoi Forest Reserve, The

University of Brunei Darussalam

Kuala Belalong Field Studies

Centre (KBFSC), Baki Tributary

Decaying rachis of

Oncosperma horridum

Feb. 1999 YAN 92 On

(HKU(M) 13181)

Neolinocarpon australiense Brunei Darussalam, Temburong, Batu

Apoi Forest Reserve, The University

of Brunei Darussalam Kuala Belalong

Field Studies Centre (KBFSC),

Baki Tributary

Decaying rachis of

Oncosperma horridum

Feb. 1999 YAN 93 On

(HKU(M) 13210)

N. globosicarpon Brunei Darussalam, Temburong,

mangrove

Decaying rachis of

Nypa fruticans

April 1998 YAN M3

(HKU(M) 3359)

N. nonappendiculatus Brunei Darussalam, Temburong, Batu

Apoi Forest Reserve, The University

of Brunei Darussalam Kuala Belalong

Field Studies Centre (KBFSC),

Baki Tributary

Decaying rachis of

Oncosperma horridum

Dec. 1997 YAN 108 On

(HKU(M) 10153)

Figs 10–16. Light and SEM micrographs of ascospores ofNeolinocarpon australiense. Figs 10–11. Note the roundedapex and basal truncate appendage (arrowed in 11). Fig. 12.

Rounded apex. Figs 13–16. The truncate base of each asco-spore is surrounded by a membranous collar with enclosedgranular material. Bars: 10=25 mm; 11=10 mm; 12–16=1 mm.

Yanna, W. H. Ho and K. D. Hyde 1307

with uranyl acetate for 40 min and examined using aJEOL 100S transmission electron microscope operat-ing at 80 kV.

RESULTS

Linocarpon

Linocarpon aquaticum

The ascospores comprise round appendages of uniformwidth at both ends (Figs 1, 3). Usually one of the asco-spore appendages was attached to the polycarbonatemembrane, while the other was unattached (Fig. 3).Unattached ends were thick, pad-shaped, with smoothirregular edges, which may have folded. Attached ap-pendages had slightly spread out forming thin layers ofmucilage which adhered to the polycarbonate mem-brane (Fig. 4).

Linocarpon livistonae

The apex of each ascospore was rounded and lackedappendages, while the base was covered with muci-laginous material (Figs 2, 5–7). The ascospore wallcomprised an electron-dense episporium, 10.5–14 nmthick and a bilamellate mesosporium, 40–60 nm thick(Figs 8–9). The outer mesosporium (11–15 nm thick)was more electron-dense than the inner one (34.5–40 nm thick) (Figs 8–9). The basal appendages com-prised electron-dense fibrillar material that was derivedfrom the episporium (Figs 8–9).

Neolinocarpon

Neolinocarpon australiense

The apex of each ascospore was rounded and lackedappendages (Figs 10–12), while the base was sur-rounded by a membranous collar with enclosed

Figs 17–20. TEM micrographs of ascospores of Neolinocarpon australiense. Fig. 17. Longitudinal section of the basal

region of an ascospore. The ascospore (A) incorporates compact uniseriate lipid globules (G). The base of the ascosporeis surrounded by a collar-like (C) membranous structure. Figs 18–19. Longitudinal sections of ascospores illustrating theappendages that are made up of a collar-like structure (C) containing amorphic and dense fibrillar material (Fm). Fig. 20.Longitudinal section of ascospore (A). The wall comprises an episporium (E) and a trilamellate mesosporium, including an

outer (M1), middle (M2) and inner mesosporium (M3). Bars: 17=1 mm; 18=500 nm; 19–20=200 nm.

Ultrastructure of Linocarpon and Neolinocarpon 1308

granular material (Figs 13–16). The edge of the mem-brane collar was irregular and usually spread outwards(Figs 14–16). When attached, the membranous ma-terial of the collar may break open, releasing thegranular material that sticks to the polycarbonatemembrane.

The ascospore wall comprised a 3.6–4 nm thickelectron-dense episporium and a 48–52 nm thick, tri-lamellate mesosporium (outer : 15–16.5 nm thick,middle: 9–10 nm thick, inner: 22–28 nm thick) (Fig.20). The middle layer of the mesosporium was the mostelectron-dense and the inner layer of the mesosporiumwas least electron-dense (Fig. 20). The basal end com-prised a trilamellate collar which was not continuousfrom the ascospore wall (Figs 17–19). The collar was50–58 nm thick, comprising a thin electron-dense layer(7.5–11 nm thick) which was sandwiched between twoless electron-dense layers (21–25 nm thick). The asco-spore wall surrounded by the collar was covered withamorphic material and electron-dense fibrillar materialwhich extended from the middle layer of the meso-sporium (Figs 17–19).

Neolinocarpon globosicarpon

The apex of each ascospore was rounded and lackingappendages, while the base of each ascospore was tan-gentially flattened with a collar-like swelling, internallycovered with granular material, bearing brush-likestructures adhering to the surface of the polycarbonatemembrane (Figs 21–24).

Neolinocarpon nonappendiculatus

The ascospores were rounded and lacked appendagesat the apex, but were tangentially flattened 18–20 mmfrom the base, tapering to a narrow rounded end(Figs 25–30). The flattened surface was covered withgranular material that adhered to the surface of thepolycarbonate membrane (Figs 28–30).

The ascospore wall is comprised of an electron-dense episporium (24–26 nm thick) and a bilamellatemesosporium (53.5–59 nm thick). The outer layerof mesosporium (23–26 nm thick) was slightly moreelectron-dense than the inner layer (30–34 nm thick)(Figs 31–32). The flattened surface of the ascosporeswas covered with granular, electron-dense materialthat was derived from the episporium (Fig. 33).

DISCUSSION

Characterisation of species of Linocarponand Neolinocarpon

The morphology, texture and origin of the ascosporeends and ascospore wall layers in Linocarpon andNeolinocarpon species are compared in Table 2. Linedrawings illustrating the spore ends following obser-vation under light and electron microscopy are pro-vided in Figs 34–39. Observation of ascospore endsat the SEM level clearly distinguished all the speciesexamined. For instance, the pad-like structures ofL. aquaticum spread out and became attached to the

Figs 21–24. Light and SEM micrographs of ascospores of Neolinocarpon globosicarpon. Fig. 21. Note the basal appendage(arrowed). Fig. 22. The apex is rounded while the base has a tangentially flattened end. Fig. 23. Rounded apex. Fig. 24. Thebase of each ascospore is tangentially flattened and contains granular material. Bars: 21=25 mm; 22=10 mm; 23, 24=1 mm.

Yanna, W. H. Ho and K. D. Hyde 1309

Figs 25–33. Light, SEM and TEM micrographs of ascospores of Neolinocarpon nonappendiculatus. Figs 25–26. In this species theapex is rounded while the base is narrowly attenuated (arrowed). Fig. 27. Rounded apex. Figs 28–30. The attenuated surface has atone side a needle-shaped area of granular material (arrowed in Fig. 28). Figs 31–33. Longitudinal sections of ascospores (A). Theascospore wall comprised an electron-dense episporium (E) and a bilamellate mesosporium, comprising outer (M1) and inner (M2)mesosporial layers. The attenuated end of ascospores were covered with granular, electron-dense material (Gm) that was derived fromthe episporium. Bars: 25=25 mm; 26=10 mm; 27–28=2 mm; 29–30=1 mm; 31=500 nm; 32=200 nm; 33=100 nm.

Ultrastructure of Linocarpon and Neolinocarpon 1310

Figs 34–39. Diagrammatic representation of ascospore ends of Linocarpon and Neolinocarpon species. Fig. 34. L. appen-diculatum (redrawn from Poonyth et al. 2000). A membranous collar enclosing granular material (Gm) enclosing tufts offibrillar material (Fm) covered by a membrane (Mb). The ascospore wall comprised an electron-dense episporium (E) anda bilamellate mesosporium, including a less electron-dense outer mesosporium (M1) and an electron-dense inner

mesosporium (M2). Fig. 35. L. aquaticum. A flared attached appendage under SEM. Fig. 36. L. livistonae. The appendagecomprised fibrillar material (Fm). The ascospore wall comprised an electron-dense episporium (E) and a bilamellate

(continued on p. 1312)

Yanna, W. H. Ho and K. D. Hyde 1311

polycarbonate membrane (Fig. 4). L. livistonae asco-spore ends were similar to L. aquaticum in producingmucilaginous structures, but only at the bases (Fig. 5).N. australiense had a distinct collar-like end containinga fibrous structure (Figs 13–16). Neolinocarpon non-appendiculatus produced a layer of mucilage liningthe tapering region towards the basal tip (Fig. 28).Although L. appendiculatum, L. nipae and N. globosi-carpon produced brush-like structures at the SEM level,the structural details of each were distinct. One sporeend of L. appendiculatum comprised a collar which wasfilled with short fibrils (Poonyth et al. 2000). The sporeends of L. nipae were made up of long fibrils (Poonythet al. 2000), whereas those of N. globosicarpon com-prised granular material (Fig. 24).

Generic delineation of Linocarpon and Neolinocarpon

Ascospore appendage ontogeny at the ultrastructurallevel has been widely used in the delineation of fungalgenera. For instance, Moss (1990) and Jones (1995)have demonstrated delineation in genera of Halo-sphaeriaceae based on ascospore appendage ontogeny.Annulatascus, Cataractispora, Diluvicola, Fluminicola,Pseudoproboscispora and Rivulicola described in theAnnulatascaceae were also delineated based on theirdifference in ascospore appendage ontogeny (Ho, Hyde& Hodgkiss 1999, Wong, Hyde & Jones 1999, Ho &Hyde 2000).

Ascospore end structures of all examined Linocarponand Neolinocarpon species originated as a direct out-growth of the episporium (L. livistonae, L. nipae, and

N. nonappendiculatus), mesosporium (N. australiense),and episporium and mesosporium (L. appendiculatum).In a review of generic delineation in Halosphaeriaceae,Jones (1995) recognised ten ascospore appendageontogeny types, including four which do not have anexosporium: (1) appendage formed as a direct out-growth from one or more of the spore wall layers ; (2)appendage exuded through a pore or pores in the as-cospore wall ; (3) ascospores with end chambers formedas outgrowths of the mesosporium and episporium, thechamber containing mucilage ; and (4) ascospores withpolar mucilaginous appendages. In Linocarpon andNeolinocarpon species, the ascospore ends are of similarontogeny, as a direct outgrowth of one or more of thespore wall layers. It is therefore not possible to delin-eate species of Linocarpon and Neolinocarpon at thegeneric level based on ascospore appendage ontogeny.The distinction between species based on ascospore endstructure is also confused.

The position of ascomata therefore remains the maincriterion to delineate Linocarpon and Neolinocarponspecies. However, using the position of ascomata todelineate the two genera is not straightforward. Theascomata of Linocarpon should be superficial and thoseof Neolinocarpon should be deeply immersed (Hyde1992a, Hyde et al. 1998). Unfortunately, L. aquaticumand L. palmetto produce immersed ascomata (Hyde1992b). It is therefore arguable, that these last twospecies should be placed in Neolinocarpon. With theconfusion in delineation of Linocarpon and Neolino-carpon species now demonstrated, the genera need to becompared using molecular techniques.

Table 2. Comparison of ultrastructural morphologies of some Linocarpon and Neolinocarpon species.

Texture Morphology Origin of appendages Ascospore wall layer

Linocarpon appendiculatum

(Poonyth et al. 2000)

Collar with fibrils

(brush-like)

Fibrillar Episporium and

mesosporium

1 episporium

2 mesosporium

L. aquaticum Pad-like and

mucilaginous

– – –

L. livistonae Mucilaginous Fibrillar Episporium 1 episporium

2 mesosporium

L. nipae (Poonyth et al. 2000) Brush-like Fibrillar Episporium 1 episporium

1 mesosporium

Neolinocarpon australiense Collar Fibrillar Mesosporium 2 1 episporium

3 mesosporium

N. globosicarpon Brush-like – – –

N. nonappendiculatus Mucilaginous Granular Episporium 1 episporium

2 mesosporium

mesosporium, including an electron-dense outer mesosporium (M1) and an less electron-dense inner mesosporium (M2).Fig. 37. L. nipae. The appendage comprised fibrillar material (Fm). Also note the remnants of the membrane (Mb) whichpartially covers the fibrillar material and the ascospore wall adjacent to the appendage. The ascospore wall comprised

an electron-dense episporium (E) and a less electron-dense mesosporium (M). Fig. 38. N. australiense. A membranous collarcomprising amorphous material (Am) and fibrillar material (Fm) under TEM. The appendage comprised fibrillar material(Fm). The ascospore wall comprised an electron-dense episporium (E) and a trilamellate mesosporium, including an outer

mesosporium (M1), middle (M2) and inner mesosporium (M3). Fig. 39. N. nonappendiculatus. The ascospore wall comprisedan electron-dense episporium (E) and a bilamellate mesosporium (M1, M2). Note the granular material (Gm) which isderived from the episporium. This is a needle-shaped area of granular material as seen under SEM.

(continued )

Ultrastructure of Linocarpon and Neolinocarpon 1312

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