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Havispora longyearbyenensis gen. et sp. nov.: an arctic marine fungus from Svalbard, Norway

     Department of Biology & Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR, and Institute of Marine Biology, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 20224, Taiwan (ROC)

Michael W.L. Chiang
Lilian L.P. Vrijmoed ¹

     Department of Biology & Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR

	ABSTRACT

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Information on the diversity and ecology of arctic marine fungi is lacking. During a short visit to Longyearbyen (78°13'N 15°33'E), Svalbard, Norway, a new marine fungus growing on driftwood collected at the shore was encountered. This taxon belongs to the Halosphaeriales (Ascomycota), a fungal order of mostly marine species. Havispora longyearbyenensis gen. et sp. nov. is morphologically similar to Nautosphaeria and Nereiospora, all with tufts of appendages at polar and equatorial positions of the ascospore but differing in color and septation of the ascospore and morphology and ontogeny of the ascospore appendage.

Key words: Ascomycota, driftwood, Halosphaeriaceae, taxonomy

	INTRODUCTION

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Information on the diversity and ecology of marine fungi in the arctic is lacking. Recent studies have provided insights on the diversity of fungi in the arctic, including substrates such as subglacial ice (Sonjak et al 2006) and cryopegs (Gilichinsky et al 2005). Mitosporic fungi are common in these studies, including many Penicillium species, while members of the Halosphaeriales were not documented (Gilichinsky et al 2005, Sonjak et al 2006). During a short visit to Longyearbyen (78°13'N 15°33'E), Svalbard, Norway, a few bags of washed-up wood were collected. On return to the laboratory, periodic examination of this wood over 3 mo revealed the presence of two new species of marine fungi, one of which is described here.

	MATERIALS AND METHODS

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Collection of materials and isolation.— – Driftwood was collected on the bouldery shore at Longyearbyen, Svalbard, Norway on 10 Aug 2006 and 12 Aug 2006. Wood samples were placed in Zip-lock plastic bags and transported to the laboratory in Hong Kong. Wood pieces were incubated in plastic boxes lined with moist tissue paper, observed for sporulating structures for 3 mo and isolated as pure cultures. Herbarium materials were deposited at BIOTEC Bangkok Herbarium (BBH), Thailand.

Light Microscopy.— – Two wood pieces (2 x 1 x 1 cm³) with ascomata, cut from a larger piece of wood, were fixed by immersion in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.05% CaCl₂ overnight at 4 C. The fixed samples were rinsed three times in the same buffer, followed by three rinses in distilled water. The samples were dehydrated in a graduated ethanol series (30%, 50%, 70%, 90%, 95% and 100%), cleared with xylene, and infiltrated gradually and embedded in paraffin (Paraplast X-tra, Kendall, USA). Paraffin sections (7 µm) were cut on RM2125RT rotary microtome (Leica, Germany), floated on 42 C water-bath to relax compression and mounted on Superfrost Plus microscope slides (Menzelgläser, Germany). Dried sections were deparaffinized and rehydrated through a graded series of ethanol. The sections were stained with 0.1% safranin O in 50% ethanol and 0.5% methyl green (each for 30 min). After washing and dehydration each stained section was mounted permanently with a cover slip and Permount (Fisher, USA). Specimens were observed on an Axioplan 2 imaging microscope (Carl-Zeiss, Germany) and light micrographs were acquired by a ColorView 12 CCD camera (Soft Imaging System, Germany) using analysis (version 3.2) software.

Scanning microscopy.— – Ascospore suspension was filtered through a 0.2 µm isopore membrane filter (Millipore, Ireland). Specimens were fixed immediately by immersion in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.05% CaCl₂ for 2 h at room temperature. The specimens were rinsed three times with 0.1 M cacodylate buffer followed by postfixation in 1% osmium tetroxide in the same buffer for 1.5 h at room temperature. After rinsing with the same buffer and distilled water three times for each treatment, the samples were dehydrated in a graduated ethanol series (10%, 30%, 50%, 70%, 80%, 90%, 95% and 100%) and finally in 100% acetone (15 min each). The dehydrated materials were critical point dried in a CO₂ atmosphere (BAL-TEC CPD 030 Critical Point Dryer, Liechtenstein) and mounted on aluminum stubs. Mounted specimens were sputter coated with gold in a BAL-TEC SCD 005 Sputter Coater (Liechten-stein) and examined under a FEI/Philips XL30 Esem-FEG scanning electron microscope (Netherlands) operated at 10 kV.

	RESULTS

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A total of 27 pieces of driftwood was collected at Longyearbyen. Seven pure isolates, belonging to six species of marine fungi, were cultured. No sporulating structures were produced in all cultures. Two taxa are new to science, including a new genus, Havispora longyearbyenensis, illustrated in this paper. A new Remispora species will be described in a subsequent publication. Havispora longyearbyenensis was recorded on two pieces of the collected wood.

	TAXONOMY

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Havispora K.L. Pang & Vrijmoed, gen. nov.

Ascomata nigera, ellipsoidea vel subglobosa, immersa, coriacea. Periphyses absens. Peridium fuscum, bistratum, exterior stratum ex cellularum ex textura angularis, interior stratum ex cellularum elongatum. Asci clavati, leptodermi, unitunicati, octospori, persistentes, ex pulvino cellularum pseudoparenchymatarum ad basim ascomati orientes. Catenophyses praesens. Ascosporae ellipsoideae, leptodermae, appendiculatae. Appendices funiculus simile, ad polum et aequatorem quatuor.

Typus generis. – Havispora longyearbyenensis K.L. Pang & Vrijmoed

Ascomata black, ellipsoidal or subglobose, coriaceous. Periphyses absent. Peridium dark-colored, 2-layered, outer stratum of cells ex textura angularis, inner stratum of elongated cells. Periphyses absent. Asci clavate, thin-walled, unitunicate, 8-spored, persistent, developing at the base of ascoma venter. Catenophyses present. Ascospores ellipsoidal, thin-walled, with one appendage at each pole and four equatorial appendages of equal dimension. Appendages one tuft at polar and four tufts at equatorial positions, string-like composed of intertwining strands which split in seawater.

Etymology. – "Hav" meaning "sea" in Norwegian.

Havispora longyearbyenensis K.L. Pang & Vrijmoed, sp. nov. FIGS. 1–8

View larger version (218K): [in this window] [in a new window]   FIGS. 1–8. Havispora longyearbyenensis gen. et sp. nov. (HOLOTYPE, 1–6 light micrographs, 7–8 scanning electron micrographs). 1. Black ascomata on wood. 2. Peridium composed of two cell types, with an outer textura angularis layer and an inner layer of elongated cells. 3. Subglobose ascoma with a short neck without periphyses. 4. Persistent asci of different developmental stages. 5. Catenophyses. 6. Hyaline, 3-septate ascospore with polar and equatorial appendages. 7. A smooth-walled ascospore with one tuft of appendages at polar and four tufts at equatorial positions. 8. String-like appendage, composed of intertwining strands that split in seawater. Bars: 1 = 500 µm, 2, 4 = 20 µm, 3 = 100 µm, 5, 6 = 10 µm, 7 = 5 µm, 8 = 2 µm.

Ascomata 442–(616)–787 x 306–(422)–607 µm (n = 15), solitary, black, ellipsoidal to subglobose, immersed, coriaceous. Peridium 29–(37)–44 µm (n = 7), dark-colored, 2-layered, outer stratum with 5–7 rows of cells of textura angularis, inner stratum with 3–4 rows of elongated cells. Necks 43–(61)–80 x 33–(69)–88 µm (n = 2), periphyses absent. Asci 88–(103)–114 x 20–(25)–33 µm (n = 23), clavate, pedunculate, thin-walled, unitunicate, 8-spored, persistent, developing at the base of the ascoma venter. Catenophyses present, 105–(132)–165 x 5–(6)–8 µm (n = 6). Ascospores 24–(30)–36 x 8–(11)–14 µm (n = 52), ellipsoidal, hyaline, thin-walled, 3-septate with constriction, with tufts of appendage. Appendages 4–(10)–14 µm long (n = 50), one tuft at polar and four tufts at equatorial positions, string-like composed of intertwining strands that split in seawater.

Etymology. – In reference to the place of discovery, Longyearbyen, Svalbard.

Specimens examined. – NORWAY. SVALBARD: Longyearbyen. On unidentified driftwood growing along with a Remispora sp., 12 Aug 2006, K.L. Pang. (HOLOTYPE: BIOTEC Bangkok Herbarium), BBH 18329, dried wood.

Known geographical distribution. – Svalbard, Norway. Substrata. Driftwood.

	DISCUSSION

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Diversity of arctic marine lignicolous fungi is largely unknown. Plentiful driftwood, originating from the Siberian and North American boreal forests, washes up at Spitsbergen, Svalbard, (Alix 2005) and is a suitable substratum for supporting growth of marine fungi. Marine fungi can produce powerful wood-degrading enzymes to mineralize the wood for nutrition (Pointing and Hyde 2000). One of two new marine lignicolous fungus is reported here, Havispora longyearbyenensis, discovered from a collection of 27 pieces of driftwood during a short trip to Longyearbyen, Svalbard, Norway. Whether this fungus is endemic to Spitsbergen requires further collections in the area and associated islands.

Havispora longyearbyenensis belongs to the Halo-sphaeriales (Sordariomycetes, Ascomycota), an order of fungi mostly isolated from marine habitats, with its saprobic nature, presence of catenophyses, persistent asci with no apical structure and hyaline ascospores with appendages (Kohlmeyer and Kohlmeyer 1979). Ascospore appendage morphology and ontogeny are the fundamental characters for the delineation of taxa in the Halosphaeriales. Havispora longyearbye-nensis possesses unique ascospore appendage morphology and other characters that differ from other taxa in the order.

Among the genera in the Halosphaeriales, polar and equatorial appendages are also present in the following genera: Corollospora, Halosphaeria, Halo-sphaeriopsis, Marinospora, Nautosphaeria, Nereiospora, Ocostaspora and Sablecola. Ascospore appendages of Havispora are string-like, composed of intertwining strands that split in seawater. These appendages differ significantly from the thorn-like spines and equatorial and polar exosporic appendages in Corollospora ( Jones et al 1983), the deeply spoon-shaped appendages in Halosphaeria ( Jones et al 1984), the polar cap-like and equatorial crescent-shaped appendages in Halosphaeriopsis ( Jones et al 1984), the obclavate to subcylindrical and tapering appendages in Marinospora ( Johnson et al 1984) and the awl-shaped and tapering appendages in Ocostaspora ( Jones et al 1984).

Havispora longyearbyenensis closely resembles Nautosphaeria, Nereiospora and Sablecola in terms of the position and gross morphology of the ascospore appendages (Hyde and Jones 1989, Pang et al 2004). Nautosphaeria differs from Havispora in having colorless, membranous ascomata, deliquescing asci and 1-celled, gray-fuscous ascospores. Information on the ascospore appendage of N. cristaminuta is insufficient to have an adequate comparison with H. longyearbyenensis ( Jones 1964). Nereiospora has carbonaceous ascomata in which the peridium is one-layered, the asci are deliquescing, the central asco-spores cells are fuscous with apical hyaline cells, and the appendages are more delicate and fibrillar and do not intertwine ( Jones and Moss 1978, Jones et al 1983). Transmission electron micrographs showed that appendages of N. comata are attached to the spore wall by a pad, which is not present in Havispora ( Jones and Moss 1980). In Sablecola ascomata are light-colored, ascospores are uniseptated, and asco-spore appendages are flattened, attenuate, strap-like and have parallel striations (Pang et al 2004). Nautosphaeria, Nereiospora and Sablecola lack cateno-physes. These morphological differences justify the establishment of a new genus to accommodate the new fungus.

	ACKNOWLEDGMENTS

 
We thank the governor of Svalbard for letting us collect in Svalbard and Mr Ian Gertz for the swift process of our application of permit; Dr Steve Coulson (UNIS, Svalbard) for the provision of laboratory space and equipment; Pen Chan, Eric Wong and Daniel Tsang for help during collection.

	FOOTNOTES

 
Accepted for publication December 26, 2007.

¹ Corresponding author. E-mail: bhlilian{at}cityu.edu.hk

	LITERATURE CITED

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Alix C. 2005. Deciphering the impact of change on the driftwood cycle: contribution to the study of human use of wood in the Arctic. Glob Planet Change 47:83–98.[CrossRef]

Gilichinsky D, Rivkina E, Bakermans C, Shcherbakova V, Petrovskaya L, Ozerskaya S, Ivanushkina N, Kochkina G, Laurinavichuis K, Pecheritsina S, Fattakhova R, Tiedje JM. 2005. Biodiversity of cryopegs in permafrost. FEMS Microbiol Ecol 53:117–128.[CrossRef][Medline]

Hyde KD, Jones EBG. 1989. Observation on ascospore morphology in marine fungi and their attachment to surfaces. Bot Mar 32:205–218.

Johnson RG, Jones EBG, Moss ST. 1984. Taxonomic studies of the Halosphaeriaceae: Remispora Linder, Marinospora Cavaliere and Carbosphaerella Schmidt. Bot Mar 27:557–566.

Jones EBG. 1964. Nautosphaeria cristaminuta gen. et sp. nov., a marine pyrenomycete on submerged wood. Trans Brit Mycol Soc 47:97–101.

———, Moss ST. 1978. Ascospore appendages of marine ascomycetes: an evaluation of appendages as taxonomic criteria. Mar Biol 49:11–26.[CrossRef]

———, ———. 1980. Further observation on the taxonomy of the Halosphaeriaceae. Bot Mar 23:483–500.

———, Johnson RG, Moss ST. 1983. Taxonomic studies of the Halosphaeriaceae: Corollospora Werdermann. Bot J Linn Soc 87:193–212.[CrossRef]

———, ———, ———. 1984. Taxonomic studies of the Halosphaeriaceae: Halosphaeria Linder. Bot Mar 27: 129–143.

Kohlmeyer J, Kohlmeyer B. 1979. Marine mycology: the higher fungi. New York: Academic Press.

Pang KL, Jones EBG, Vrijmoed LLP. 2004. Two new marine fungi from China and Singapore, with the description of a new genus, Sablecola (Halosphaeriales, Ascomycota). Can J Bot 82:485–490.

Pointing SB, Hyde KD. 2000. Lignocellulose-degrading marine fungi. Biofouling 15:221–229.

Sonjak S, Frisvad JC, Gunde-Cimerman N. 2006. Penicillium mycobiota in arctic subglacial ice. Microb Ecol 52:207–216.[CrossRef][Medline]

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