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Mycologia, 95(2), 2003, pp. 340-346.
© 2003 by The Mycological Society of America

Lignicolous freshwater Ascomycota from Thailand: Hymenoscyphus varicosporoides and its Tricladium anamorph


S. Sivichai 1
E. B. G. Jones
N. L. Hywel-Jones

     BIOTEC-Mycology, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Science Park, 113 Pahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
 GROWTH OF TELEOMORPH AND...
 DISCUSSION
 LITERATURE CITED
 

A Tricladium anamorph for the discomycete Hymenoscyphus varicosporoides was established in culture from both conidia and ascospores collected in KhaoYai National Park, Thailand, and is compared with Tricladium indicum and T. marylandicum. Hymenoscyphus varicosporoides is compared with Cudoniella indica.

Key words: anamorph/teleomorph connections, Freshwater ascomycete fungi, systematics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 TAXONOMY
 GROWTH OF TELEOMORPH AND...
 DISCUSSION
 LITERATURE CITED
 
In a survey of lignicolous freshwater fungi of Thailand (Sivichai et al 2000aCitation, bCitation), a discomycete referable to Hymenoscyphus was collected on test blocks exposed in Khao Yai National Park. This species is similar to the description given by Tubaki (1966)Citation for Hymenoscyphus varicosporoides Tubaki. Sporulating structures of the teleomorph and an anamorph were observed on test blocks and on agar media in the laboratory. Tubaki (1966)Citation also encountered an anamorph state that he ascribed to Varicosporium but also said it equally well could be referred to Polycladium or Tricladium. He did not formally describe this anamorph.

The anamorph collected in this study matches that described by Tubaki (1966)Citation but, in our opinion, the conidia do not conform to those characteristic of Varicosporium. Recently, Sati and Tiwari (1992)Citation described a new species—Tricladium indicum—from India. Furthermore Webster et al (1995)Citation showed that Tricladium indicum was the anamorph of a new species, Cudoniella indica. However, they did not refer to Hymenoscyphus varicosporoides, a species almost identical to C. indica. Because of the confusion over the assignment of this fungus we describe and illustrate the Thai material and then discuss the taxonomic implications.

These taxonomic issues need to be resolved: 1) Is the anamorph described for Hymenoscyphus varicosporoides best placed in Varicosporium, as originally proposed by Tubaki (1966)Citation, or better placed in Tricladium? 2) Is Tricladium indicum the anamorph of Cudoniella indica? 3) Are Hymenoscyphus varicosporoides and Cudoniella indica conspecific?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
TAXONOMY
 GROWTH OF TELEOMORPH AND...
 DISCUSSION
 LITERATURE CITED
 
The methods used have been explained previously (Sivichai et al 1998Citation). Test blocks of Alstonia scholaris and Anisoptera oblonga were submerged in streams at Tad Tha Phu and km 29.2 on Sept. 13, 1996. Xylia dolabriformis and Dipterocarpus alatus test blocks were exposed in the same streams and locations on Aug. 12, 1997. These two sets were recovered each month and incubated, as previously described.

Specimens were dried and deposited in the Bangkok BIOTEC Herbarium (BBH); isolates are maintained in the BIOTEC Culture Collection (BCC).


   TAXONOMY
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
GROWTH OF TELEOMORPH AND...
 DISCUSSION
 LITERATURE CITED
 

Hymenoscyphus varicosporoides Tubaki (Trans Br Mycol Soc 49, 345–349, 1966) Figs. 2–9



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  		 FIGS. 1–9. Hymenoscyphus varicosporoides. 1. Colony of the anamorph Tricladium varicosporoides growing on wood. 2. Hymenoscyphus varicosporoides arising from the substratum with a colony of the anamorph stage. 3. The last stage of the colony with only Hymenoscyphus varicosporoides on wood. 4. Squash of asci and paraphyses. Figs. 5–8. Asci at different stages of development. Fig. 9. Ascospores. Bars (1–3) = 1 mm, (4–9) = 10 µm

 
Apothecia generally scattered or occasionally aggregated, superficial, disk and stalk originally white, becoming pale brown when mature and dry, disks circular, slightly convex, and up to 380 µm diam (Figs. 2, 3). Receptacle smooth, soft, white, stalk cylindrical, length variable (up to 1400 µm long), and pale brown at the base. Flesh thin walled, compact with slightly interwoven, hyaline hyphae becoming parallel toward the excipulum. Asci persistent, cylindric-clavate, 70–100 x 10–12.5 µm, 8-spored, apex round, pedicellate, unitunicate and J+ (Figs. 4–8). Ascospores 14–20 x 5–7.5 µm, hyaline, biseriate or partly biseriate, elliptic-fusiform, equally or unequally 1-septate, with ends rounded, sometimes continuous, and often slightly curved (Fig. 9). Paraphyses numerous, unbranched, filamentous, non-septate, with rounded apex 1.2 µm wide and similar in length to the asci (Fig. 4). The anamorph appeared on test blocks after seven days of incubation, while apothecia appeared after 6–10 days, and were mixed with the anamorph.

Tricladium anamorph. FIGS. 1, 10–15



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  		 FIGS. 10–17. Tricladium varicosporoides on CMA. 10–12. Conidia of Tricladium varicosporoides. 13. Septa of conidia. 14. Germination of conidium on CMA after two days. 15. Colony of Hymenoscyphus varicosporoides and Tricladium varicosporoides on CMA in water. 16–17. Conidia with lateral branches. Arrow indicates isthmus-like attachment to main axis. Bars (10–12) = 20 µm, (13, 16, 17) = 10 µm, (14) = 100 µm, (15) = 1 mm

 
Colonies up to 20 mm long, and 6 mm wide (Fig. 1), appeared on the wood after 6–10 days of incubation and were originally white, becoming pale brown when mature and dry. Conidiophores were simple, bearing branched conidia. Conidia were hyaline, with a main axis, 300–600 µm long, and 5–7.5 µm wide, with one to three laterals of the same width, usually developing on one or both sides of the main axis, up to 220–500 µm long (Figs. 10–14). Conidia typically had two lateral branches, which were positioned on the same side of the main axis and in the same plane. Occasionally, the two laterals were on opposite sides of the conidial axis. Rarely, three branches were seen, which either could be all on the same side or on opposite sides. In the typical form, the distance between the laterals was consistently between 60–80 µm. No tertiary branches were observed either from material in culture or from field material. The main axis and laterals are multiseptate, each cell ca 10–20 µm (Figs. 13, 14) with the lateral arms joined to the main axis by a narrow isthmus (Figs. 16, 17).

Specimens examined. THAILAND. NAKORN RATCHASSIMA PROV: all specimens were from Khao Yai National Park, a stream at Tad Tha Phu. A. On trapped river hardwood, 2 Dec. 1996, S. Sivichai BBH SS76; B. On exposed test blocks: exposure I: Anisoptera oblonga test blocks submerged 7 months, 8 May 1997, S. Sivichai SS335 (anamorph), BBH SS336 (teleomorph); Anisoptera oblonga test blocks submerged 12 months, 17 Sep. 1997, S. Sivichai SS367 (anamorph). Xylia dolabriformis submerged for 11 months, 15 July 1998, S. Sivichai BBH SS585 (teleomorph). Exposure II: Dipterocarpus alatus test blocks submerged 4 months, 23 Dec. 1997, S. Sivichai SS451 (anamorph). D. alatus test blocks submerged 6 months, 20 Feb. 1998, S. Sivichai SS472 (anamorph). X. dolabriformis test blocks submerged 11 months, 15 July 1998, S. Sivichai SS586(anamorph). D. alatus submerged 12 months, 24 Aug. 1998, S. Sivichai SS618(anamorph).

Known distribution. Teleomorph: AUSTRALIA, JAPAN, HONG KONG, and THAILAND.

Spatial and temporal distribution. Hymenoscyphus varicosporoides and its anamorph were found only in Thailand's Tad Tha Phu in Khao Yai National Park. It was recovered from a single sample of unidentified wood and from three species of timber that had been immersed in the stream in a colonization study (Sivichai et al 2000bCitation).

For Anisoptera/Alstonia timbers (first exposure trial) only the presence/absence of a species was recorded. Pooling the data with the Dipterocarpus/Xylia data, a presence–absence contingency table for Hymenoscyphus/Tricladium produced a Yule's Q of association of 1. A {chi}2 of 29.97, and was significant at P < 0.001, indicating a strong association between the presence/absence of the anamorph/teleomorph. Between October (1996) and September (1997) Hymenoscyphus/Tricladium was recorded only from Anisoptera test blocks collected in May (1997) and October (1996). It was not recorded at all from the Alstonia test blocks. In contrast, the second experiment recorded Hymenoscyphus/Tricladium from November (1996) to August (1997). Thus, September was the only month that the Hymenoscyphus/Tricladium combination was not recorded in the two years of this study.

Sporulation of the anamorph/teleomorph was generally within one week of the wood being removed from the river and being placed in incubation chambers (Table I). The Tricladium anamorph appeared in the first week for 10 of the 12 Dipterocarpus alatus test blocks recovered, compared with five for Xylia dolabriformis. The teleomorph sporulated on one and five of the test blocks of X. dolabriformis and D. alatus, respectively, at Week 1. No records were found after three months' incubation on both timbers. At Month 2, a single specimen of Hymenoscyphus was reported from D. alatus while a single specimen of Tricladium was recorded from X. dolabriformis.


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  		TABLE I. Sporulation of Hymenoscyphus/Tricladium on incubated test blocks of Dipterocarpus alatus and Xylia dolabriformis

 

   GROWTH OF TELEOMORPH AND ANAMORPH IN CULTURE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
 GROWTH OF TELEOMORPH AND...
DISCUSSION
 LITERATURE CITED
 
Ascospores germinated on cornmeal agar (CMA) within 24–36 hours when incubated in a cool, white fluorescent light at 20 C. Germ tubes were produced from both end cells. Conidia germinated on CMA within 24–36 hours, under similar conditions, and produced germ tubes from the main axis or lateral branches. Colonies of H. varicosporoides and T. indicum on CMA were identical, growing rapidly (10 mm in 20 days), with the superficial mycelium white, fluffy, septate, ca 3 µm wide; immersed mycelium light brown, becoming brown (15–20 mm in 20 days). The anamorph sporulated on the surface of the agar and mainly at the edge of the colony. Agar disks placed in water produced both the anamorph and teleomorph (Fig. 15).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
 GROWTH OF TELEOMORPH AND...
 DISCUSSION
LITERATURE CITED
 
Both anamorph and teleomorph were collected on test blocks of three timbers (A. oblonga, D. alatus and X. dolabriformis) at Tad Tha Phu, but the fungus was not collected at the km 29.2 site in the same park. It is important to note that collections of river wood from many streams throughout Thailand have yielded only a single other record, also from Tad Tha Phu. The fact that it was present throughout the year (absence in September is considered an anomaly) at Tad Tha Phu suggests that absence of records from other rivers is further evidence of its limited spacial distribution (as opposed to temporal distribution). Also, its occurrence on three of four exposed test blocks further hints that the timber species might not be the limiting substratum. Both anamorph and teleomorph were not sporulating when test blocks were recovered from the stream and appeared only after incubation in the laboratory.

The Thai collection of Hymenoscyphus agrees with the description given by Tubaki (1966)Citation, but the apothecia of the Japanese collection were larger (Table II). Ho et al (2001)Citation also reported a discomycete on river wood in Hong Kong and referred the collection to Hymenoscyphus sp. but did not report an anamorph. Cribb (1991)Citation also reported this species from Australia. Hymenoscyphus varicosporoides differs from other aquatic Hymenoscyphus species described by Abdullah et al (1981)Citation in ascospore dimensions, which are generally smaller, and in having anamorph connections with other Ingoldian hyphomycete genera.


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  		TABLE II. Comparison of the measurements of various collections of Hymenoscyphus varicosporoides and Cudoniella indica measurement (µm)

 
Various anamorphs have been described for Hymenoscyphus species: Anguillospora, Articulospora, Dimorphospora, Geniculospora, Tricladium, Varicosporium in Ingoldian hyphomycetes and Helicodendron in aero-aquatic fungi (Tubaki 1966Citation, Abdullah et al 1981Citation, Fisher and Webster 1983Citation, Webster 1992Citation). These are all genera with very different conidial morphologies. It is important to note that Tricladium also has been shown to have different teleomorphs: T. splendens with Hymenoscyphus splendens (Leotiaceae); T. chaetocladium with Hydrocina chaetocladia (Hyaloscyphaceae) (Abdullah et al 1981Citation, Webster et al 1990Citation); while Webster et al (1995)Citation described Cudoniella indica as the teleomorph of T. indicum.

Tubaki (1966)Citation referred the anamorph of H. varicosporoides to Varicosporium, although noting that it equally well could be assigned to the genera Tricladium or Polycladium. The conidia derived from the Japanese culture had a main axis 300–500 µm long with 1–3 laterals, with tertiary laterals (not illustrated), a characteristic of the genus Varicosporium. Species recently placed in the genus Varicosporium are characterized by conidia that have increasingly branched spores.

Other genera considered by Tubaki (1966)Citation were Polycladium, Tricladium and Tricladiomyces. Polycladium is ruled out, because the number of lateral branches is greater with tertiary laterals. Roldán et al (1987)Citation, in describing Dendrospora polymorpha, noted that the genera Varicosporium, Tricladium and the Dendrospora-Dendrosporomyces complex "have become increasingly ill-defined in recent years." Although the genus Tricladium is considered to have only primary branching (Roldán et al 1987Citation), there "are examples of frequent secondary branching in several Tricladium species" (Table III), although "this is definitely not a characteristic of T. splendens," the type species. Tricladiomyces conidia are U- or S-shaped, with doliporus septa, characteristics not exhibited by the Thai collection. We therefore prefer to assign our collection to Tricladium, which is already an accepted anamorph for Hymenoscyphus species (Abdullah et al 1981Citation).


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  		TABLE III. Tricladium species with the main conidial axis greater than 100 µm (species listed in decreasing order of the length of the main axis of the conidium)

 
The second issue concerns the identity of T. indicum, described by Webster et al (1995)Citation, and the specific assignment of our collection. The South African and Indian collections of T. indicum have similar conidial measurements (450–640 x 12 µm and 150–580 x 10–12 µm respectively) and number of laterals (1–3 and 1–4-(5) respectively). They differ in that the South African conidia have a main axis up to 1800 µm, considerably longer than that of the Indian collection. Webster et al (1995)Citation reported that colonies have gray mycelium with sparse aerial growth, while colonies of the Indian material are dark gray to black (Sati and Tawari 1992Citation). Further studies are required to determine if these two collections are of the same species.

The Thai material differs from the above collections in having a conidial main axis (300 x 600 µm) which is narrower (5–7.5 µm), and longer laterals (220–500 µm), again narrower, while the colonies in culture are pale brown, never gray or black. We consider these differences sufficient to distinguish the Thai collection from C. indica/T. indicum. The differences in the teleomorphs revolve around the staining of the asci: in C. indica asci are J- but J+ in H. varicosporoides. However, the staining of the ascus tip in Hymenoscyphus is considered variable (Spooner pers comm). Ascospores of C. indica are said to be constricted at the septum, while in the Thai collection they are not. The stalk of C. indica is considerably longer at 6 mm than those reported for the H. varicosporoides collections (up to 2 mm). One other detail is that in C. indica, cylindrical or tapering, thin-walled, septate, colorless hairs 3–4 µm in diam are reported by Webster et al (1995)Citation on the stipe, whereas these were not observed in H. varicosporoides. For now, we refer the Thai collection to Hymenoscyphus until there is a more detailed assessment of the genus, including its relationship to Cudoniella.

Tubaki (1966Citation, Fig. 1c) provided a single illustration of a conidium, which had laterals about 60 µm apart. This is similar to the separations reported in our study (60–80 µm). In contrast, the illustration for T. indicum (Webster et al 1995Citation, Fig. 1) suggests a separation of about 10–25 µm. Therefore, the Thai material bears closer comparison to Tubaki's material than to the T. indicum material of Webster et al (1995)Citation. It is possible, then, that the separation of the laterals in Tricladium might be a consistent and a useful character for determining species.

Webster (1992)Citation lists 27 teleomorph connections for Ingoldian hyphomycetes and notes that certain genera, such as Nectria, Massarina and Hymenoscyphus, have several Ingoldian anamorphs. He speculates as to the reason for this: selection pressure, which has conserved a rather uniform ascomatal and ascospore morphology and has led to the evolution of such different kinds of conidia, presumably with different strategies for dispersal, settlement and attachment (Webster 1992Citation). Discomycetes must form their apothecia out of water if the ascospores are to be effectively discharged and dispersed. Consequently, ascospores often are small for dispersal by air, while conidia are more elaborate for entrapment during attachment to suitable substrata (Jones 1994Citation). Read et al (1992)Citation and Au et al (1996)Citation have shown that the larger the conidia, the more branches to the conidia, the greater surface area there is for attachment to a surface. Therefore the anamorph has undergone greater adaptation to environmental conditions, while the teleomorph has had little need for such adaptation.

Hyde et al (1997)Citation drew attention to the fact that few discomycetes are reported in wood in tropical streams, while Shearer (1993)Citation reported 112 species from temperate regions. During the course of our study of lignicolous freshwater fungi of Thailand, we have collected several discomycetes. All have developed after an extended period, often several months, of incubation in the laboratory. The use of different techniques to survey for tropical freshwater discomycetes might yield a wider range of species.

We posed three questions in the introduction and conclude that the anamorph of Hymenoscyphus varicosporoides is best referred to Tricladium. The remaining issues cannot be resolved because cultures of Tricladium indicum (type material from India) and Cudoniella indica are not available for molecular study.


   ACKNOWLEDGMENTS
 
This work was supported by the Biodiversity Research and Training Programme (BRT Thailand) with co-financing by the National Center for Genetic Engineering and Biotechnology (BIOTEC) and the Thailand Research Fund (TRF) grants No. BRT 143016 and No. BRT 145006. We thank the Royal Forest Department for permission to study in Khao Yai National Park and Professors John Webster, David Hawksworth and Dr. Enrique Descals for their invaluable comments.


   FOOTNOTES
 
1 Corresponding author, sivichai{at}biotec.or.th Back

Accepted for publication August 27, 2002.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
 GROWTH OF TELEOMORPH AND...
 DISCUSSION
 LITERATURE CITED
 
Abdullah SK, Descals E, Webster J., 1981 Teleomorphs of three aquatic hyphomycetes. Trans Br Mycol Soc 77:475-483

Au DWT, Jones EBG, Moss ST., 1996 Spore attachment and extracellular mucilage of aquatic Hyphomycetes. Biofouling 10:123-140

Cribb AB., 1991 The aquatic Discomycete Hymenoscyphus varicosporoides in Queensland. Queensland Naturalist 31:26-28

Fisher PJ, Webster J., 1983 The teleomorphs of Helicodendron giganteum and H. paradoxum. Trans Br Mycol Soc 81:646-659

Ho WH, Hyde KD, Hodgkiss IJ, Yanna, 2001 Fungal communities on submerged wood from streams in Brunei, Hong Kong and Malaysia. Mycol Res 105:1492-1501

Hyde KD, Wong SW, Jones EBG., 1997 Freshwater ascomycetes. In: Hyde KD, ed. Biodiversity of tropical microfungi. Hong Kong: Hong Kong Univ. Press. p 179–187

Jones EBG., 1994 Fungal adhesion. Mycol Res 98:961-981

Read SJ, Moss ST, Jones EBG., 1992 Germination and development of attachment structures by conidia of aquatic Hyphomycetes, a scanning electron microscope study. Can J Bot 70:838-845

Roldán A, Descals E, Honrubioa M., 1987 Dendrospora polymorpha sp. nov. a new hyphomycete from Spanish streams. Mycotaxon 29:21-27

Sati SC, Tiwari N., 1992 A new species of Tricladium from Kumaum Himalaya, India. Mycol Res 96:229-232

Shearer CA., 1993 The freshwater Ascomycetes. Nova Hedwigia 56:1-33

Sivichai S, Hywel-Jones NL, Somrithipol S., 2000a Lignicolous freshwater Ascomycota from Thailand: Melanochaeta and Sporoschisma anamorphs. Mycol Res 104:478-485

———, Jones EBG, Hywel-Jones NL., 2000b Fungal colonization of wood in a freshwater stream at Khao Yai National Park, Thailand. Fung Divers 5:71-88

———, Goh TK, Hyde KD, Hywel-Jones NL., 1998 The genus Brachydesmiella from submerged wood in the tropics, including a new species and a new combination. Mycoscience 39:239-247

Tubaki K., 1966 An undescribed species of Hymenoscyphus, a perfect stage of Varicosporium. Trans Brit Mycol Soc 49:345-349

Webster J., 1992 Anamorph-teleomorph relationships. In: Barlocher F, ed. The ecology of aquatic hyphomycetes. Springer-Verlag, Berlin Press. p 99–117

———, Scheuer C, Om Khaltoum Khattab S., 1990 Hydrocina chaetocladia gen. et spec. nov., the teleomorph of Tricladium chaetocladium. Nova Hedwigia 52:69-72

———, Eicker A, Spooner BM., 1995 Cudoniella indica sp. nov. (Ascomycetes, Leotiales), the teleomorph of Tricladium indicum, an aquatic fungus isolated from a South African river. Nova Hedwigia 60:493-498




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