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Botanica Marina 53 (2010): 493–498 2010 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2010.061
Sedecimiella taiwanensis gen. et sp. nov., a marine mangrove
fungus in the Hypocreales (Hypocreomycetidae, Ascomycota)
Ka-Lai Pang1,*, Siti A. Alias2, Michael W.L.
Chiang3, Lilian L.P. Vrijmoed3 and E.B. Gareth
Jones4
1
Institute of Marine Biology, National Taiwan Ocean
University, 2 Pei-Ning Road, Keelung 20224, Taiwan
(R.O.C.), e-mail: [email protected]
2
Institute of Biological Sciences, University of Malaya,
50603 Kuala Lumpur, Malaysia
3
Department of Biology and Chemistry, City University
of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong,
Hong Kong
4
BIOTEC Bioresource and Technology Unit, National
Centre for Genetic Engineering and Biotechnology, 113
Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani
12120, Thailand
* Corresponding author
Abstract
In an ongoing survey of marine mangrove fungi in Taiwan,
a fungus with light-coloured ascomata, resembling members
of the Hypocreales, was found on twigs of Kandelia obovata.
Based on morphological characteristics and partial nuclear
small and large subunit ribosomal DNA data, the fungus is
described as a new genus and species, Sedecimiella taiwanensis, and placed in the Hypocreales (Hypocreomycetidae,
Ascomycota). Phylogenetic analyses based on LSU/SSU
sequences did not resolve the familial placement of S. tawianensis within the Hypcreales, but it may be related to the
Niessliaceae. The characteristic features of S. tawianensis are
orange to dark-brown colour, soft-texture and perithecial
ascomata with the inner wall layer of the neck extending into
the upper part of the centrum; cylindrical, 16-spored, unitunicate asci without an apical pore; and hyaline, smoothwalled and globose ascospores.
Keywords: aquatic fungi; Kandelia obovata;
Sordariomycetes.
Introduction
During ongoing studies of mangrove fungi in coastal waters
of Taiwan, a new unitunicate ascomycete with 16-spored asci
was found on twigs of Kandelia obovata Sheue, Liu et Yong
(Rhizophoraceae). Morphological characters of this fungus,
such as orange to dark-brown globose, subglobose, erumpent
ascomata, periphysate, cylindrical asci, and hyaline, thin-
walled, round, unicellular ascospores are also features of the
Hypocreales. The goals of this study were to: (1) characterise
and describe this novel fungus from a mangrove in Taiwan,
and (2) analyse partial nuclear small and large subunit ribosomal DNA (SSU, LSU rDNA) data to determine the phylogeny of the new taxon.
Materials and methods
Morphological study
Drift and attached mangrove wood subject to seawater
immersion was collected from Chunan, Taiwan, on 12
August 2008 (holotype), from Futian Nature Reserve, Shenzhen, China on 14 March 2006 (isotype) and from Kukup
Island, Malaysia on 5 August 2009. Wood samples were
transferred to the laboratory and incubated on a tray lined
with moist tissue paper for up to one month. Fruiting bodies
were observed using an Olympus SZ61 stereomicroscope
(Tokyo, Japan), sectioned with a razor blade, grasped with
fine forceps, and mounted on a slide in sterile seawater. Morphology of the asci and ascospores was observed using an
Olympus BX51 microscope (Tokyo, Japan). Photographs
were taken with an Olympus DP20 Microscope Camera
(Tokyo, Japan).
Two wood pieces (2=1=1 cm3) with ascomata, cut out
from a larger piece of collected wood, were fixed by immersion in FAA (50% ethanol, 5% glacial acetic acid and 4%
paraformaldehyde) overnight at 48C. The fixed samples were
rinsed three times in the same buffer, followed by three rinses in distilled water. The samples were then dehydrated in a
graduated ethanol and 1-butanol series, infiltrated gradually
and embedded in paraffin (Paraplast X-tra, Kendall, Mansfield, USA). Paraffin sections (8–10 mm) were cut on
RM2125RT rotary microtome (Leica, Nussloch, Germany),
floated over a water-bath at 428C to relax sections, and then
mounted on Superfrost Plus microscope slides (Menzel-Gläser, Braunschweig, Germany). Dried sections were deparaffinised and rehydrated through a graded series of ethanol.
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 permanently mounted
with a cover slip and Permount (Fisher, Fair Lawn, USA).
Specimens were observed on an Axioplan 2 Imaging microscope (Carl Zeiss, Göttingen, Germany) and light micrographs were acquired using a ColorView 12 CCD camera
(Soft Imaging System, Münster, Germany) using analySIS
(version 3.2) software.
2010/032
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494 K.-L. Pang et al.: A new mangrove fungus
Figures 1–4 Sedecimiella taiwanensis gen. et sp. nov. (1) Orange to brown ascomata on wood. (2) Globose ascoma with a thickened neck
embedded in wood. (3) Base of neck protruding into the upper part of the ascomal centrum. (4) Peridium composed of an outer stratum of
cells forming textura angularis and inner stratum of elongated cells. Scale bars: 1s200 mm, 2–3s20 mm, 4s10 mm.
Molecular analysis
A mycelial culture (CY5100) from materials collected at
Futian, Shenzhen, China (see Figure 7) was used for a
molecular study as ascospores from Chunan did not germinate. The isolate was grown in GYPS (glucose 4 g l-1, yeast
extract 4 g l-1 and peptone 2 g l-1 in full strength natural sea
water). Mycelial pellets were washed with sterile water, blotted dry and ground into powder in a mortar and pestle,
pre-cooled in a -808C freezer overnight. Fungi Genomic
DNA Purification Kit (Catalogue no. E070, BioMi,
Taichung, Taiwan) was used for extraction according to the
manufacturer’s instructions. Extracted DNA was used directly for PCR reactions with the following ingredients: 0.2 mM
of each primer (NS1/NS4: White et al. 1990, LROR/LR7
Bunyard et al. 1994), 0.2 mM of each dNTP, 1.5 mM MgCl2
and 1 U of Taq Polymerase (Catalogue no. F530-S, FINNZYMES, Espoo, Finland). The amplification cycle consisted
of an initial denaturation step of 948C for 5 min followed by
35 cycles of (i) denaturation (948C for 0.5 min), (ii) annealing (558C for 0.5 min) and (iii) elongation (728C for 0.5 min)
and a final 11 min elongation step at 728C. The PCR prod-
ucts were analysed by agarose gel electrophoresis and purified using a PCR Purification Kit (Catalogue no. K310001,
Invitrogen, Carlsbad, USA) according to the manufacturer’s
instructions. Purified PCR product was shipped to Tri-I Biotech (Taipei, Taiwan) for direct sequencing with the primers
described above.
Returned sequences were checked for ambiguity, assembled and deposited in GenBank (accession numbers in
Figures 9 and 10). Sequences were compared with available
sequences using nucleotide blast search in GenBank. These
and GenBank sequences were manually adjusted in Se-Al
v1.0a1 (Rambaut 1999). Both nuclear SSU and LSU rDNA
datasets were entered into PAUP* 4.0b10 for maximum parsimony analyses (Swofford 2002). Heuristic searches were
run for both datasets with the following settings: gaps treated
as missing data, starting tree(s) obtained via stepwise addition, random sequence addition of 10,000 replicas, a treebisection-reconnection (TBR) branch-swapping algorithm,
MULTREES on. A thousand parsimony bootstrap analyses
were used to reflect the support of the clades with the same
settings, except that 10 replicates of random sequence addition were used.
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K.-L. Pang et al.: A new mangrove fungus 495
ceous, ostiolate, smooth, not collapsing upon drying, KOH-,
lactic acid-. Neck thick, inner wall extends into upper part
of ascoma centrum. Periphyses present, short, non-septate.
Peridium orange to dark brown, two-layered, outer stratum
of thick-walled cells forming textura angularis, inner stratum
of elongated, hyaline thin-walled cells. Asci unitunicate,
cylindrical, short pedunculate, thin-walled, no apical pore,
16-spored, persistent, developing from inner wall of ascoma
base. Paraphyses present, irregular, often tapering towards
apex, branched, septate. Ascospores globose, one-celled,
hyaline, smooth, thin-walled, without appendage or sheath.
Etymology
‘‘Sedecim’’ meaning 16 in Latin.
Anamorph
Unknown.
Sedecimiella taiwanensis K.L. Pang, Alias et E.B.G.
Jones, sp. nov. (Figures 1–8)
Figures 5–8 Sedecimiella taiwanensis gen. et sp. nov. (5) Thinwalled, cylindrical and unitunicate asci. (6) Ascus with 16 unicellular ascospores (collection from Taiwan). (7) Morphology of asci
and ascospores in the collection from Shenzhen, China. (8) Remnants of paraphyses. Scale bars: 5–8s5 mm.
Results and discussion
Sedecimiella K.L. Pang, Alias et E.B.G. Jones,
gen. nov.
(Mycobank MB518530). Ascomata flavida, brunnea vel
atra, globosa vel subglobosa, pyriforma, immersa, subimmersa vel exposita, coriacea, ostiolata, teres. Colla profunda. Periphyses praesentes. Peridium flavidum, brunneum vel
atrum, bistratum, exterior stratum ex cellularum ex textura
angularis, interior stratum ex cellularum elongatum. Asci
unitunicati, cylindrici, leptodermi, sedecim ascospori, persistentes, ex pulvino cellularum pseudoparenchymatarum ad
basim ascomati orientes. Paraphyses praesentes. Ascosporae
globosae, hyalinae, leptodermae, sine appendiculatae.
Sedecimiella taiwanensis K.L. Pang,
Alias et E.B.G. Jones.
Ascomata orange to dark brown, pyriform with globose to
subglobose venter, immersed, erumpent or exposed, coria-
Typus generis
(Mycobank MB518531). Ascomata 63-(110)-145=65-(100)140 mm (ns20), pyriforma, solitaria, flavida, brunnea vel
atra, globosa vel subglobosa, immersa, subimmersa vel
exposita, coriacea, ostiolata, teres. Colla 30-(63)-101=42(66)-93 mm (ns20), cum periphysibus. Peridium 14-(17)25 mm (ns20), flavidum, brunneum vel atrum, bistratum,
exterior stratum ex cellularum ex textura angularis, interior
stratum ex cellularum elongatum. Asci 25.8-(30.3)-35.3=
3.5-(4.3)-4.7 mm (ns31), unitunicati, cylindrici, pedicellati,
leptodermi, sedecim ascospori, persistentes, ex pulvino
cellularum pseudoparenchymatarum ad basim ascomati
orientes. Paraphyses praesens, 13.5-(25.3)-37.1=1.8(3.4)-7.6 mm (ns10). Ascosporae 2.3-(2.8)-3.5 mm (ns50),
globosae, sine septatae, hyalinae, leptodermae, sine
appendiculatae.
Ascomata 63-(110)-145=65-(100)-140 mm (ns20), pyriform with globose to subglobose venter, solitary, orange to
dark brown, globose to subglobose, immersed, erumpent or
exposed, coriaceous, ostiolate, smooth, not collapsing upon
drying, KOH-, lactic acid-. Neck 30-(63)-101=42-(66)93 mm (ns20), thick, inner wall layer extends into upper
part of centrum, periphyses present. Peridium 14-(17)-25 mm
in diameter (ns20), orange to dark brown, two-layered, outer stratum with 3–6 rows of thick-walled cells of textura
angularis, 2.5-(3.5)-4.5=2.0-(2.5)-3.0 mm; inner stratum
with 3–6 rows of elongated, hyaline thin-walled cells, 5.5(7.0)-9.0=0.5-(1.0)-2.0 mm. Asci 26.0-(30.0)-35.5=3.5(4.0)-4.5 mm (ns31), unitunicate, cylindrical, short
pedunculate, thin-walled, no apical pore, 16-spored, persistent, developing from inner wall of ascoma base. Paraphyses
13.5-(25.5)-37.0=2.0-(3.5)-7.5 mm (ns10), irregular, often
tapering towards apex, branched, septate. Ascospores 2.5(3.0)-3.5 mm (ns50), globose, one-celled, hyaline, smooth,
thin-walled, without appendage or sheath.
In reference to the place of discovery of
holotype, Taiwan.
Etymology
Anamorph
Unknown.
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496 K.-L. Pang et al.: A new mangrove fungus
Holotype
Taiwan: Chunan. On a twig of Kandelia obovata, 12 August 2008, K.L. Pang, BBH26390 (BIOTEC
Bangkok Herbarium), dried wood.
Isotype
China: Shenzhen: Futian Nature Reserve. On a
twig of unidentified mangrove wood, 14 March 2006, K.L.
Pang, CY5100-CY5101 (City University of Hong Kong,
China), fungal culture, HM451495(SSU)-HM451496(LSU),
GenBank sequences.
Malaysia: Kukup Island.
On a twig of unidentified mangrove wood, 5 August 2009,
K.L. Pang.
Other specimens examined
Chunan, Taiwan;
Shenzhen, China; Kukup Island, Malaysia.
Known geographical distribution
Substrata
grove wood.
Drift and dead intertidal branches of man-
Decaying mangrove substrates (wood, leaves and fruits)
support the growth of many marine mangrove fungi. Taiwan
has 22 mangrove forests and four true mangrove species
including Avicennia marina (Forssk.) Vierh., Kandelia obovata, Lumnitzera racemosa Willd. and Rhizophora stylosa
Griff. The only report of marine fungi for Taiwan is by Hsieh
et al. (2002) who documented 59 species, of which 32 were
from mangroves. Thus our knowledge of mangrove fungi for
Taiwan is fragmentary.
Thirty-one species of marine mangrove fungi were identified in the current study from 111 pieces of wood collected
at Chunan (results not shown). The dominant fungal species
were Saagaromyces abonnis (Kohlm.) K.L. Pang et E.B.G.
Jones (37 collections), Lignincola laevis Höhnk (28 collections) and Halosarpheia fibrosa Kohlm. et E. Kohlm. (26
collections). Sedecimiella taiwanensis was discovered on
only two pieces of mangrove wood, indicating that it is not
a common species in that area. Abundant ascomata, however,
were produced on the wood (Figure 1).
Figure 9 A single most parsimonious tree (C.I.s0.553, R.I.s0.707) of 1310 steps obtained from maximum parsimony analysis of the
nuclear small subunit ribosomal DNA. Maximum parsimony bootstrap values are also shown.
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K.-L. Pang et al.: A new mangrove fungus 497
Figure 10 Consensus tree of 19 most parsimonious trees (C.I.s0.452, R.I.s0.650) of 926 steps obtained from maximum parsimony
analysis of the nuclear large subunit ribosomal DNA. Maximum parsimony bootstrap values are also shown.
Sedecimiella taiwanensis was tentatively identified as a
member of the Hypocreales based on its light-coloured, softtextured, perithecial ascomata, and unitunicate asci (Rossman
et al. 1999). However, its affinity within the Hypocreales is
unknown. Seven marine genera of the Hypocreales have
been reported: Emericellopsis, Halonectria, Heleococcum,
Kallichroma, Neocosmospora, Payosphaeria and Pronectria
(Jones et al. 2009). Sedecimiella taiwanensis most closely
resembles Neocosmospora (Nectriaceae) and Payosphaeria
(Hypocreales incertae sedis) (Leong et al. 1990, Rossman et
al. 1999). In Neocosmospora, the ascomatal wall is composed of two regions, asci are cylindrical, ascospores are
yellow to yellow-brown or reddish brown, unicellular, globose and ornamented, but lack paraphyses (Rossman et al.
1999). Sedecimiella taiwanensis differs from Neocosmospora in having branched paraphyses, asci with 16 hyaline, and
round ascospores without any ornamentation. Both Sedeci-
miella and Payosphaeria have light-coloured ascomata,
branched paraphyses, cylindrical asci and hyaline, globose
ascospores, but the ascomatal wall of Sedecimiella is composed of two regions instead of one as in Payosphaeria. Only
eight ascospores are present in Payosphaeria. Sedecimiella
differs from Neocosmospora and Payosphaeria in that the
inner wall layer of the neck extends into the upper part of
the centrum (Figures 2 and 3), an anatomical feature that
appears to be previously unknown in the Hypocreales. A
similar structure has been observed in Rostrupiella danica J.
Koch, K.L. Pang et E.B.G. Jones (Lulworthiales), a Lulworthia-like marine fungus (Koch et al. 2007). Asci with 16
ascospores are also reported for many Hypocrea species, but
generally are the result of disarticulation of 1-septate ascospores (Rossman et al. 1999). Globose ascospores have also
been reported in Roumegueriella Speg. and Trichosphaerella
E. Bommer, M. Rousseau et Sacc. (Samuels and Barr 1997,
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498 K.-L. Pang et al.: A new mangrove fungus
Rossman et al. 1999). Roumegueriella differs from Sedecimiella in its saccate asci and ornamented ascospores (Rossman et al. 1999), while ascomata of Trichosphaerella bear
short, simple, dark setae that are absent in Sedecimiella, and
the 16 ascospores in the asci are the result of disarticulation
from oblong, uniseptate ascospores (Samuels and Barr 1997).
Ascospores from the holotype did not germinate, but those
from the isotype collected in China did, and these were used
for the molecular study. The isotype was morphologically
identical to the holotype and the asci and ascospores are
illustrated in Figure 7. However, no herbarium material was
kept for the isotype. In view of this, a piece of wood with
abundant ascomata from the collection in Taiwan was deposited as the holotype.
Maximum parsimony analysis of the partial nuclear SSU
rRNA gene confirmed the placement of Sedecimiella taiwanensis in the Hypocreales, Hypocreomycetidae (Figure 9).
In order to determine the familial placement of S. taiwanensis, the partial nuclear LSU rRNA gene was sequenced and
analysed with maximum parsimony (Figure 10). S. taiwanensis consistently grouped with Niesslia exilis (Alb. et
Schwein.) G. Winter (Niessliaceae), although without bootstrap support. This result is in agreement with the blast
search results of the LSU gene (results not shown). Only
three taxa from the Niessliaceae wMelanopsamma pomiformis
(Pers.:Fr.) Sacc., N. exilis and Valetoniellopsis laxa Samuels
et M.E. Barrx are available in the GenBank for comparison,
and they were not monophyletic in the LSU analysis in this
study. Members of the Niessliaceae are saprotrophic and characterised by small, dark-coloured, ascomata on a subiculum,
narrow peridium of textura epidermoidea, hamathecium of
periphysoids, thin-walled asci with a refractive, non-amyloid
ring and hyaline or light-coloured, 1-septate and disarticulating ascospores (Samuels and Barr 1997). S. taiwanensis
differs from the Niessliaceae in its light-coloured ascomata
without subicula, lack of periphysoids, asci without apical
ring and 16 unicellular ascospores. A wider range of taxa in
the Niessliaceae should be sequenced to determine whether
S. taiwanensis truly belongs in this family.
In total, there are nine marine species in seven genera of
the Hypocreales (Jones et al. 2009). However, the taxonomic
positions for many of these are unresolved, e.g., Halonectria
milfordensis E.B.G. Jones, although its coloured ascomata,
its long neck and immersed ascomata are not general features
of the group. Although generally not well documented in
marine habitats, members of the group are present and may
occupy specific habitats we.g., Cosmospora cf. diminuta
found on the petiole base of the brackish water palm Nypa
fruticans (Thun.) Wurmb.x (E.B.G. Jones and A. Loilong,
unpublished data). Further studies may therefore yield a wider range of hypocreacous marine fungi from such habitats.
Acknowledgements
K.L. Pang would like to thank National Science Council of Taiwan
for research grants (NSC-97-2621-B-019-002 and NSC-98-2621-B019-002-MY3) and Centre of Marine Bioenvironment and Biotechnology (CMBB), National Taiwan Ocean University for providing
financial support; Kuan-Hsiang Huang for technical support. E.B.G.
Jones acknowledges support from the TRF/BIOTEC Special Programme for Biodiversity and Training grant BRT R._251006.
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Received 29 March, 2010; accepted 13 July, 2010; online first 3
November, 2010
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