Lanspora dorisauae, a new marine fungus from rocky shores in Taiwan

Sample collection

Trapped wood was collected at a rocky shore (25°14′41.9″N 121°38′04.2″E) in Jin-Shan District, New Taipei City, Taiwan on 1 June 2022, placed on Ziplock plastic bag and transported to the laboratory at National Taiwan Ocean University, Keelung (Pang et al., 2023). The wood was cleaned briefly in running water, incubated on a tray and kept in a Ziplock plastic bag at room temperature (~25 °C) under a 12:12 light dark cycle. The wood was checked periodically for the growth of marine fungi.

Morphological characterization

The methods used in Pang et al. (2013) was followed for the morphological examination of the new fungus. For sectioning of ascomata, wood blocks with ascomata were cut out and fixed in FAA solution (5% formaldehyde and 5% glacial acetic acid in 50% ethanol) overnight at 4 °C. The wood blocks were washed three times in 50% ethanol dehydrated in a graduated t-butanol/ethanol/water series (10/40/50, 20/50/30, 35/50/15, 55/45/0, 75/25/0, 100/0/0, 100/0/0, in percentage) and embedded in paraffin. Paraffin sections were cut on a ERM_200P rotary microtome (ERMA, Saitama, Japan), floated on 42 °C water-bath and mounted on microscope slides. Dried sections were deparaffinised and rehydrated through a graded series of ethanol. The sections were stained with 1% safranin O in 50% ethanol (10 s) and 0.5% Orange G in 95% ethanol (30 s). After washing and dehydration, permanent slides were made for the stained sections in Permount (Fisher, Waltham, MA, USA).

For examination of asci and ascospores, ascomata were observed using an Olympus SZ61 stereomicroscope (Tokyo, Japan), cut opened with a razor blade, picked up with a fine forceps, and mounted on a slide in sterile seawater.

Structures of ascomata, asci and ascospores were examined using an Olympus BX51 microscope (Tokyo, Japan), with photographs taken with an Olympus DP20 Microscope Camera (Tokyo, Japan).

Single spore isolation

A spore suspension was prepared by transferring a spore mass of the new fungus to a drop of sterile natural seawater on a sterilized glass slide with a flame-sterilized forceps with mixing. This spore suspension was dropped onto the surface of cornmeal seawater agar (CMAS; 17 g cornmeal agar (Difco), 1 L natural seawater, 0.5 g/L each of Penicillin G sodium salt and streptomycin sulfate) plate. The plate was incubated at 25 °C until the spores were germinated. Single germinated spores were picked up, transferred onto fresh CMAS plates, and incubated at 25 °C. The ex-type culture was kept at Bioresource Collection and Research Center, Hsinchu, Taiwan.

Molecular analysis

Aerial mycelia of the new fungus were scraped off from the CMAS plate with a flame-sterilized spatula, and ground into powder in a mortar and pestle. Total DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Extracted DNA (1 μL) was used directly for PCR reactions with the following ingredients: 0.2 μM of each primer (18S rDNA: NS1/NS2, ITS rDNA: ITS5/ITS4 (White et al., 1990), 28S rDNA: LROR/LR6 (Vilgalys & Hester, 1990; Bunyard, Nicholson & Royse, 1994), TEF1α: EF1-983F/EF1-2218R (Rehner & Buckley, 2005), RPB2: fRPB2-5F/fRPB2-7cR (Liu, Whelen & Hall, 1999)), 0.5 volume of Gran Turismo PreMix (Ten Giga BioTech, Taiwan) and topped up to 25 μL with PCR water. The amplification cycle consisted of an initial denaturation step of 94 °C for 5 min followed by 35 cycles of (i) denaturation (94 °C for 0.5 min), (ii) annealing (55 °C for 0.5 min) and (iii) elongation (72 °C for 0.5 min) and a final 11 min elongation step at 72 °C. The PCR products were shipped to Tri-I Biotech, Inc., Taiwan, for purification and Sanger sequencing with the same primers (both strands) described above.

Returned sequences were checked for ambiguity, assembled and deposited in GenBank (accession nos. in Table 1). An initial nucleotide BLAST search suggested that the sequences were affiliated with taxa of the Phomatosporales. These sequences were aligned with the 18S, 28S, ITS, TEF1a and RPB2 genes of the taxa in the Phomatosporales and Ophiostoma piliferum and O. stenoceras (outgroup taxa) in the program MUSCLE (Edgar, 2004) in MEGA11 (Tamura, Stecher & Kumar, 2021). The final dataset contained 16 sequences and a total of 7,623 nucleotide positions. The genes were analyzed simultaneously. A maximum likelihood analysis including bootstrapping was performed in MEGA11 (Tamura, Stecher & Kumar, 2021) with the following settings: 500 bootstrap, General Time Reversible model (GTR), gamma distributed (G), number of discrete gamma categories set at 5, heuristic search with Nearest-Neigbor-Interchange, initial tree from NJ/BioNJ method, branch swapping strong. A maximum parsimony (MP) analysis including bootstrapping was also run in MEGA11 with the following settings: 500 bootstrap, Tree-Bisection-Reconnection (TBR), number of initial trees (random addition) = 5, MP search level = 1, maximum number of trees to retain = 100.

For Bayesian analysis, BEAUti v1.10.4 was used for prior settings and the dataset was analyzed in BEAST v1.10.4 (Suchard et al., 2018). The following analytical settings were used for the analysis: GTR, estimated base frequency, gamma distribution, number of gamma categories set at five, a strict clock, Coalescent: Constant Size as the speciation model, running 10 million generations with parameters and trees sampled every 1,000 generations. The first 10% of the trees were discarded as the burn-in based on the effective sample size (ESS) of the parameter statistics in Tracer v1.7.2 (Rambaut et al., 2018). A summary tree was generated in TreeAnnotator v1.10.4 (Suchard et al., 2018) and viewed and edited in FigTree v1.4.4 (available at https://github.com/rambaut/figtree/releases) (Supplemental file).

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PeerJ article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication (MB 847455) to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Taxonomy

Lanspora dorisauae K. L. Pang, Suetrong, M. W. L. Chiang and E. B. G. Jones, sp. nov. Fig. 2

Mycobank (MB#847455).

GenBank (LSU = OQ130043, ITS = OQ130045, SSU = OQ130044, TEF1α = OQ570968, RPB2 = OQ570969).

Saprobic on trapped wood on a rocky shore. Sexual morph Ascomata 148–213 μm high, 240–347 μm diam. ( $\overline{\mathrm{X}}$ = 174 × 277 μm, n = 4), immersed, subglobose to ellipsoidal in side-view, solitary to gregarious, coriaceous, brown to black, papillate (Fig. 2A). Necks short, 44–55 μm long, 80–109 μm diam. ( $\overline{\mathrm{X}}$ = 48 × 99 μm, n = 4), cylindrical, brown to black (Fig. 2A). Periphyses not observed. Peridium 13–24 μm ( $\overline{\mathrm{X}}$ = 19 μm, n = 4), equal in thickness, comprising one stratum of multilayers (over five layers) of brown cells of textura angularis with large lumina (Fig. 2B). Paraphyses 74 × 9 μm (Fig. 2C). Asci 41–51 × 16 μm ( $\overline{\mathrm{X}}$ = 44 × 16 μm, n = 4), 8-spored, unitunicate, thin-walled, clavate, pedunculated (Fig. 2D). Ascospores 8–13 × 5–7 μm ( $\overline{\mathrm{X}}$ = 11 × 6 μm, n = 35), biseriate, overlapping, unicellular, broadly ellipsoidal with one half broader than the other, with longitudinal wall striations, guttulate, appendaged (Figs. 2E and 2F). Appendages at one end of ascospores, five to seven in number, crown-like, sheet-like irregular and radiating, delicate and subgelatinous (Fig. 2G). Asexual morph Undetermined.

Culture characteristics: Colonies attained 1 cm in diameter after 1 week of incubation at 25 °C in darkness, white to pale yellow; mycelia sparse, feathery, with no pigment formed in the agar.

Holotype: TAIWAN: Jin-Shan. On a piece of unidentified trapped wood, 1 June 2022, S. Y. Guo and K. L. Pang, F26641 (National Museum of Natural Science Herbarium, Taichung, Taiwan), dried wood.

Ex-type culture: BCRC FU30316 (Bioresource Collection and Research Center, Hsinchu, Taiwan).

Other culture: NTOU3803 (National Taiwan Ocean University Culture Collection).

Etymology: In memory of Dr. Doris Wai-Ting Au, a marine zoologist/mycologist who taught me (K. L. Pang) the right way to do scientific research.

Distribution: Jin-Shan, Li-lao, Ying-Ke-Shih (New Taipei City), Shih-Ti-Ping (Hualien County) (Taiwan).