Mycologia, 104(3), 2012, pp. 000–000. DOI: 10.3852/11-238
# 2012 by The Mycological Society of America, Lawrence, KS 66044-8897
Annulatascus aquatorba sp. nov., a lignicolous freshwater ascomycete from
Sirindhorn Peat Swamp Forest, Narathiwat, Thailand
Nattawut Boonyuen1
species were Acrogenospora sphaerocephala, Annulatascus sp.1, A. velatisporus, Cancellidium applanatum,
Chaetopsina sp.2, Cryptophialoidea unilateralis, Dictyochaeta sp.3, Helicomyces roseus, Hypocrea sp.1, Jahnula
appendiculata, Monodictys sp.1, Monodictys sp.2, Trichoderma sp.1, Verticillium sp.1 and Xylomyces chlamydosporis. The site is situated in an area subject to monsoonal seasonality, with high relative humidity and
rainfall nearly year round (2560 mm on average). It
offers an unusual habitat for tropical forests with its
acidic water (with pH approximately 4.5–6.0) and
relative humidity, low input of nutrients and lack of
soil or firm ground for the growth of plants (Anon
1994, 1996). Test blocks were recovered at intervals over
an 8 y period and examined for fungi that resulted in
the discovery of this new taxon. Numerous new species
have been described from this site (Pinnoi et al. 2003a,
b; Pinruan et al. 2004a, b; Sivichai and Boonyuen 2010).
Hyde (1992) described the genus Annulatascus (type
species Annulatascus velatispora) from submerged
dead wood collected in northern Queensland, Australia. The genus is characterized by perithecioid, immersed or superficial, black ascomata with long necks;
wide, tapering paraphyses; cylindrical, unitunicate asci
with a relatively large, refractive apical ring; fusiform,
septate, hyaline ascospores with appendages or sheaths.
Currently 16 Annulatascus species (Annulatascaceae,
Sordariomycetidae, Sordariomycetes, Pezizomycotina,
Ascomycota) have been described on submerged substrata (decaying wood, stems of Phragmites australis,
dead petiole of Licuala ramsayi, bamboo) from
freshwater habitats (TABLE I). Annulatascus is a cosmopolitan genus with four species described from Queensland, Australia and nine species reported from tropical
southeastern Asia (TABLE I). Two species have been described from Africa, whereas only one species has been
reported from South America (TABLE I). Tsui et al.
(2002) provided a key to species and a synoptic table of
12 Annulatascus species based on morphological characteristics, while A. apiculatus (Barbosa et al. 2008), A.
bipolaris (Hyde 1992) and A. liputii (Cai et al. 2002)
were described subsequently.
At the molecular level, based on 28S rDNA sequences, Abdel-Wahab et al. (2011) showed that A. nilensis
grouped with A. velatisporus and A. hongkongensis in
the same clade, but A. biatriisporus did not group with
them, suggesting Annulatascus might be polyphyletic.
In this paper we describe and illustrate a new
Annulatascus species with morphological and molecular
Mycology Laboratory (BMYC), Bioresources Technology
Unit (BTU), National Center for Genetic Engineering
and Biotechnology (BIOTEC), 113 Thailand Science
Park, Phaholyothin Road, Khlong 1, Khlong Luang,
Pathumthani 12120, Thailand, and
Department of Plant Pathology, Faculty of Agriculture,
Kasetsart University, 50 Phaholyothin Road,
Chatuchak, Bangkok 10900, Thailand
Veera Sri-indrasutdhi
Satinee Suetrong
Somsak Sivichai
E.B. Gareth Jones
Mycology Laboratory (BMYC), Bioresources Technology
Unit (BTU), National Center for Genetic Engineering
and Biotechnology (BIOTEC), 113 Thailand Science
Park, Phaholyothin Road, Khlong 1, Khlong Luang,
Pathumthani 12120, Thailand
Abstract: As part of a long term study of fungi colonizing submerged wood in freshwater streams a new
Annulatascus species, A. aquatorba, is described and
illustrated from Erythrophleum teysmannii test blocks
from Sirindhorn Peat Swamp Forest, southern Thailand. It differs from other species in the genus in
ascospore measurements, thickness of the cell wall, 1–
3-septate, fusoid to lunate shape, with central brown
cells and subhyaline end cells and without a mucilaginous sheath. Asci are cylindrical, pedicellate, with a
distinct, wedge-shaped and non-amyloid apical ring.
Phylogenetic relationships of this species, based on
the combined partial 18S and 28S rDNA, place it
in the same clade as A. velatisporus (type species), A.
hongkongensis and A. nilensis.
Key words: Annulatascaceae, freshwater, peat
swamp, phylogeny, taxonomy
INTRODUCTION
In an ongoing long-term survey of fungi colonizing
nine timber species submerged in aquatic habitats in
Thailand a fungus referable to the genus Annulatascus K.D. Hyde was collected on Erythrophleum
teysmannii Craib test blocks in Sirindhorn Peat
Swamp Forest, Narathiwat Province. This fungus was
collected along with 22 other species (17 mitosporic
fungi and five ascomycetes). The most common
Submitted 17 Jul 2011; accepted for publication 26 Oct 2011.
1
Corresponding author. E-mail: [email protected]
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MYCOLOGIA
TABLE I. Comparative table of the morphological features of Annulatascus species modified from Tsui et al. 2002 (all species
listed alphabetically and measured in micrometers)
Apical ring
(high 3 diam)
Ascomata (high 3 diam)
Neck
Asci
A. aquatorba
310–350 3 390–500
100–120 3 400–480
102.5–152.5 3 6.25–8.75
A. apiculatus
400–5503 240–410
100–250 3 100–180
175–250 3 10–13
6–7.2 3 1.8–2.4
A. aquaticus
220–600 3 240–600
400–800 3 60–80
150–175 3 10–12
3.5–4 3 4.5–5
A. biatriisporus
195–325 3 390–520
No data
210–260 3 12–17
No data
155–235 diam
No data
174–205 3 9–10.5
224–310 3 350–485
No data
137.5–178.8 3 10–12
170–220 diam
ca. 210–230 in high
141–235 3 7.5–10.5
A. hongkongensis
210–250 3 250–280
140–150 3 35–40
250–275 3 25–30
3.5–4.5 3 4.7–6.0
A. joannae
180–200 3 150–200
150–200 3 40–60
150–200 3 10–13
2.5–4 3 5–6.5
A. lacteus
140–200 3 100–130
50 3 40–50
130–170 3 9–10.5
3–4 3 4–5
A. licualae
77.5–95 3 202.5–460
A. liputii
200–300 3 180–260
250–400 3 45–70
130–187.5 3 8.5–10
2 3 3.5
A. nilensis
400–600 3 220–280
240–360 3 96–112
260–400 3 12–14
3–4 3 5–6
150–440 diam
200 3 70
250–260 3 250–270
100–50 3 30–50
190–255 3 12–18
3–5 3 3–5
384 3 140
220–290 3 12–18
7–8 3 4–5
110 in high
140–218 3 9–14
Fungi
A. bipolaris
A. citriosporus
A. fusiformis
A. palmietensis
A. tropicalis
56–82 diam
450 3 260–410
A. velatisporus
Annulusmagnus
(Annulatascus)
triseptatus
222–353 diam
evidence (two loci of 18S and 28S rDNA sequences)
from submerged E. teysmannii test blocks in the peat
swamp forest.
MATERIALS AND METHODS
Specimen collection, fungal isolates, identification and growth.—
Nine timber species (Azadirachta indica var. siamensis
Mycologia myco-104-03-13.3d 25/4/12 17:04:23
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88.8–125 3 5.8–7.8
98–142 3 7–10.5
5.0–7.5 3 2.5–3
436
2.75–3.25 35–5.5
4–5 3 3–4
1.5–2 3 2.5–2.75
3–4 3 4–4.5
3 3 5.5
Valeton, Erythrophleum teysmannii Craib, Melaleuca cajuputi
Powell, Shorea obtusa Wall, S. roxburghii G. Don, S. siamensis
Miq, Wrightia tomentosa Roem. & Schult, Xylia xylocarpa
(Roxb.) W. Theob, and Zollingeria dongnaiensis Pierre)
were submerged in Sirindhorn Peat Swamp Forest, Sungai
Kolok, in Narathiwat, Thailand (6.00295N, 101.9571889E).
After 2 y (22 Feb 2003) four sets of nine timber species
(15 3 2.5 3 2.5 cm3) were recovered and returned to the
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BOONYUEN ET AL.: ANNULATASCUS AQUATORBA
TABLE I.
0
Extended
Ascospores
Septate/aseptate
15–22.5 3 5–7.5, fusoid to lunate, lacking
appendages or a sheath
(1–)3, constricted
at the septa
23–36.5 3 8.8–10, uniseriate, fusiform,
with a narrow mucilaginous sheath
0–3-septate, not
constricted
at septum
Aseptate
19–24 3 6–7, uniseriate, hyaline, ellipsoidal,
with a mucilaginous sheath
40–58 3 8–10, fusiform, with a mucilaginous
sheath
21–30 3 6.5–8.5, uniseriate, hyaline,
fusiform, with a polar appendage and a
mucilaginous sheath
22.5–30.5 3 6.4–8.5, fusiform, with a thin
mucilaginous sheath
16.5–25.5 3 6–9, uniseriate, hyaline,
fusiform, with a bipolar mucilaginous
pad–like appendage
35–37.5 3 12.5–15, uniseriate, hyaline,
ellipsoidal, with a mucilaginous sheath
20–28 3 9–12, uniseriate, hyaline,
ellipsoidal to fusiform, with a thin
mucilaginous sheath
24–28 3 6–8, uniseriate, hyaline, fusiform,
lacking appendages or a sheath
15–17.8 3 3.8–5, overlapping –uniseriate,
fusiform–rhomboid, with a thin
mucilaginous sheath
15–22.5 3 6.5–7.5, , overlapping –
uniseriate, hyaline, fusiform, with a thin
mucilaginous sheath
32–52 3 7–10, uniseriate –overlapping
uniseriate, hyaline, fusoid, with bipolar
mucilaginous pad–like appendage and
sheath
20–26 3 6–7, seriate, hyaline fusiform with
blunt ends, lacking a mucilaginous sheath
42.5–52.5 3 7.5–10, uniseriate, hyaline,
fusiform, lacking appendages or a sheath
26–42 3 9–12, fusiform, with mucilaginous
sheath
18–33 3 6–12, uniseriate, hyaline, fusiform,
with a thin mucilaginous sheath
Aseptate
Aseptate
Aseptate
1–5-septate
Three-septate
Aseptate
Aseptate
Aseptate
Two-septate
5–11-septate,
constricted
at the septa
Three-septate
1–3-septate
Aseptate
Three-septate,
constricted
at the septa
laboratory. Each set of test blocks were separated and
placed in pre-sterilized plastic boxes with tissue papers
layered at the base. They were incubated at 20 C in a
cabinet with cool white fluorescent light and examined at
intervals of 1 wk, 1, 2 and 3 mo for the presence of sporulating fungi.
Assessment procedures were as described by Sivichai
et al. (2002). Single-ascospore isolations were made on
Mycologia myco-104-03-13.3d 25/4/12 17:04:24
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Substrate
Known
distribution
Reference
Narathiwat,
Thailand
This study
Brazil
Barbosa et al.
(2008
Submerged
wood
Submerged
wood
Submerged
wood
Hong Kong,
China
Queensland,
Australia
Queensland,
Australia
Ho et al. (1999a)
Dead petiole of
Licuala sp.
(plam)
Submerged
wood
Brunei
Darussalam
Fröhlich and
Hyde (2000)
The Philippines
Hyde and Wong
(2000)
Hong Kong,
China
Hong Kong,
China
Ho et al. (1999b)
Erythrophleum
teysmannii
(test blocks)
Submerged
wood
Submerged
wood
Submerged
wood
Hyde (1995)
Hyde (1992)
Tsui et al. (2002)
Submerged
Hong Kong,
wood
China
Dead petiole of
Queensland,
Licuala ramsayi
Australia
(plam)
Submerged
The Philippines
bamboo
Islands
Tsui et al. (2002)
Submerged
stems of
Phragmites
australis
Submerged
wood
Submerged
wood
Submerged
wood
Submerged
wood
Sohag, Egypt
Abdel-Wahab
et al. (2011)
KwaZulu–Natal,
South Africa
Hong Kong,
China
Queensland,
Australia
Brunei
Darussalam
Hyde et al. (1998)
Fröhlich and
Hyde (2000)
Cai et al. (2002)
Tsui et al. (2002)
Hyde (1992)
Campbell and
Shearer (2004);
Wong et al. (1999)
cornmeal agar (CMA). Plates were incubated at 20 C in a
cabinet with cool, white fluorescent light and examined
daily for germinating ascospores. Six to eight germinated
ascospores were transferred to new plates and incubated in
the same cabinet. A culture was deposited in the BIOTEC
culture collection (BCC) as BCC16735 and dried material
in the BIOTEC Bangkok Herbarium (BBH), as BBH
29936.
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MYCOLOGIA
DNA extraction, polymerase chain reactions (PCR), sequencing
and phylogenetic analyses.—DNA was extracted from fresh
mycelium as described by Sri-indrasutdhi et al. (2010) with
extraction buffer. To amplify the partial 18S ribosomal DNA
(18S rDNA) the sequencing primers are NS1 and NS4
(White et al. 1990). To amplify the partial 28S ribosomal
DNA (28S rDNA) the sequencing primers are LROR and
LR7 (Bunyard et al. 1994, Landvik 1996). PCR reactions and
amplification cycles were carried out according to Boonyuen et al. (2011). Purified PCR products were sent to
Macrogen Inc. (Korea) with the above mentioned primers
for DNA sequencing. Newly generated sequences from
isolates were blasted in GenBank to exclude the possibility
of contamination, assembled in Bioedit 7.5.03 (Hall 2006)
to get consensus sequence and deposited in GenBank
(TABLE II). A phylogenetic tree was generated.
The assembled sequences (18S rDNA and 28S rDNA)
were aligned with related sequences obtained from GenBank (Campbell and Shearer 2004, Zhang et al. 2006,
Abdel-Wahab et al. 2011, Boonyuen et al. 2011, Sakayaroj
et al. 2011) with Clustal W 1.6 (Thompson et al. 1994)
incorporated in Bioedit 7.5.03 (Hall 2006) and improved in
MUSCLE 3.6 (Edgar 2004) with ambiguously aligned
regions excluded from phylogenetic analyses. Missing data
at the 59– and 39– ends of the partial sequences were
excluded from the analysis. Representatives of the order
Pezizales were selected as an outgroup.
Maximum parsimony (MP) analyses were performed with
PAUP 4.0b10 (Swofford 2002) with Windows and Home
Mac OS 9.2 (Apple Studio, Mac OS X 10.4.10) as heuristic
searches with 1000 random addition replicates and tree
bisection-reconnection (TBR) branch swapping to obtain the
most parsimonious trees. Gaps were given equal weight and
treated as missing data. To estimate the best tree topology
the Kishino-Hasegawa test was used (Kishino and Hasegawa
1989). The final alignments (18S rDNA and 28S rDNA
dataset) and the trees obtained were deposited in TreeBASE
(http://www.treebase.org) as accession number 11739.
Clade stability for the branching topologies was evaluated
by bootstrap analysis (BSMP) derived from 1000 replicates
with 10 random addition replicates each of a full heuristic
search (Felsenstein 1985). Descriptive tree statistics tree
length (TL), consistency index (CI), retention index (RI),
rescaled consistency index (RC) and homoplasy index (HI)
were calculated for each tree and phylogenetic trees were
displayed with Treeview (Page 1996).
Bayesian phylogenetic analysis was performed in MrBayes
3.0b4 with a uniform GRT + I + G model as the best
nucleotide substitution model (Huelsenbeck and Ronquist
2001). This model was chosen with MrModeltest 2.2
(Nylander 2004). Chains were analyzed with random starting
trees for 2 000 000 generations and sampled every 100
generations. Trees collected before the stable likelihood
value points were discarded as burn-in. The Metropoliscoupled Markov chain Monte Carlo (MCMC) sampling
approach after the exclusion of the initial set of 2000 burnin trees were used to calculate posterior probabilities (BYPP).
RAxML bootstrap values (BSML) were conducted in
RAxML 7.0.3 with the same models in MrBayes 3.0b4 (GRT
+ I + G model of nucleotide substitution) and data
Mycologia myco-104-03-13.3d 25/4/12 17:04:24
4
partitions as in the searches for the optimal trees using fast
bootstrapping 1000 pseudoreplicates (Stamatakis et al.
2006).
RESULTS
Molecular phylogeny of combined 18S and 28S
rDNA sequence.—The combined 18S rDNA and 28S
rDNA dataset including sequences from the new
species (highlighted in FIG. 1 and TABLE II) was aligned
with 84 representative taxa from the subclass Hypocreomycetidae (Savoryellales, Microascales, Hypocreales and Hypocreomycetidae Incertae sedis), Sordariomycetidae (Coniochaetales, Chaetosphaeriales,
Boliniales, Diaporthales, Ophiostomatales, Sordariales,
Sordariomycetidae incertae sedis), Trichosphaeriales
and Xylariales (TABLE II). Two representative taxa of
the order Pezizales (Morchella escuenta and Disciotis
venosa) were used as outgroup taxa.
The final aligned dataset comprised 2317 characters,
out of which 762 were parsimony informative, 302
parsimony uninformative and 1253 constant characters.
The result of Kishino-Hasegawa maximum likelihood
tests of the combined 18S rDNA and 28S rDNA from
unweight parsimony analysis yielded three most parsimonious trees with a tree length of 4030 (CI 5 0.405; RI
5 0.687; RC 5 0.278; HI 5 0.595; 2ln likelihood 5
23 519.13633; Difference ln L 5 the best tree and a
significant difference at P , 0.05). One of the three
most parsimonious trees, inferred from the combined
two-locus dataset, was the best tree (FIG. 1). Three trees
in the interordinal relationship within the subclasses
Hypocreomycetidae and Sordariomycetidae formed a
stable branching pattern but with minor branch
swapping of Annulatascus biatriisporus and branch
length in Cyanoannulus petersenii (results not shown).
A comparison of the combined 18S rDNA and 28S
rDNA dataset with the individual 28S rDNA dataset
using Kishino-Hasegawa maximum likelihood tests
(tree not shown) showed the topology of the
combined two-locus of 18S rDNA and 28S rDNA
dataset was almost identical to that of the individual
28S rDNA dataset (TL 5 3166, CI 5 0.365; RI 5
0.650; RC 5 0.237; HI 5 0.635, total character 5
1149; constant character 5 447; parsimony informative character 5 486 and parsimony uninformative
variable characters 5 216) (data not shown).
Bayesian analysis provided a topology similar to
the combined 18S rDNA and 28S rDNA analyses (data
not shown). Although minor differences in the
position of Papulosa amerospora, Ascobrunneispora
aquatica and Mirannulata samuelsii were noted, this
does not affect the overall topology of the tree in
maximum parsimony analyses and the conclusions
drawn.
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TABLE II. DNA sequences in Sordariomycetes used in the phylogenetic analyses, their specimen voucher and GenBank
accession numbers
GenBank accession numbers
Classification
Original codes
Annulatascaceae incertae sedis (Sordariomycetidae)
SS 2424
- Annulatascus aquatorba*
- Annulatascus nilensis*
MF 808
- Annulatascus hongkongensis*
HKUCC 3702
- Annulatascus velatisporus
A 70–18a
- Annulatascus velatisporus
R 047a
- Annulatascus velatisporus*
HKUCC3701
- Annulatascus biatriisporus
A 464–3a
- Pseudoproboscispora caudae–suis
A 336–2Db
- Submersisphaeria aquatica
A 95–1Bb
- Submersisphaeria aquatica
A 354–14b
- Annulusmagnus triseptatus
A 325–1Db
- Annulusmagnus triseptatus
A 353–1Fb
- Annulusmagnus triseptatus
A 413–6b
- Annulusmagnus triseptatus
A 54–10Ab
- Annulusmagnus triseptatus
A 54-10Eb
- Ascitendus austriacus*
A 44–28Ab
- Ascitendus austriacus*
A 324–1Bb
- Ascitendus austriacus*
A 324–1Fb
- Ascitendus austriacus
CBS 102665
Sordariomycetidae Incertae sedis
- Aquaticola hyalomura
- Aquaticola ellipsoidea
- Aquaticola hongkongenis*
- Cyanoannulus petersenii
- Fusoidispora aquatica*
- Ascocollumdensa aquatica*
- Vertexicola caudatus*
- Cataractispora receptaculorum
- Rhamphoria delicatula
R 038b
A 411–3a
HKUCC 3703
R 044aa
HKU(M) 17484c
HKUCC 3707
HKUCC 3715
HKUCC 3710
MR 1396/98
Papulosaceae Incertae Sedis (Sordariomycetidae)
- Papulosa amerospora
AFTOL 748
- Ascobrunneispora aquatica
HKUCC 3708
Order Ophiostomatales
- Endomyces scopularum
SSU
LSU
JN226106
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
JN226107
HQ616536
AF132319
AY316354
AY316355
AF132320
AY316352
AY094192
AY094193
AY094194
AY590288
AY590289
AY590285
AY590286
AY590287
AY590292
AY590293
AY590294
AF261067
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
AY590291
AY316356
AF132321
AY316358
AY780365
AF132325
AF132331
AF132327
AF261068
DQ470998
N/A
DQ470950
AF132326
NRRL Y–17633d (CBS
131.86)
AFTOL 910
AFTOL 1038
ATCC 32437
N/A
AF268488
DQ471003
DQ836897
M83261
DQ470955
DQ836904
DQ368627
Order Boliniales
- Camarops microspora
- Camarops ustulinoides
- Cornipulvina ellipsoides*
AFTOL 1361
AFTOL 72
SMH 1378
DQ471036
DQ470989
N/A
AY083821
DQ470941
DQ231441
Order Chaetosphaeriales
- Chaetosphaeria innumera
- Menispora tortuosa
- Striatosphaeria codinaeophora
SMH 2748
AFTOL 278
SMH 1524
N/A
AY544723
N/A
AY017375
AY544682
AF466088
Order Coniochaetales
- Barrina polyspora
- Coniochaeta ligniaria
AWR 9560A
F 3331e
N/A
N/A
AY346261
AF353583
- Ophiostoma piliferum
- Ophiostoma stenoceras
- Ophiostoma ulmi*
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TABLE II.
Continued
GenBank accession numbers
SSU
LSU
CBS 267.33T
SANK 12878
AJ496246
N/A
AF353601
AY346297
AFTOL
AFTOL
AFTOL
AFTOL
AFTOL
AFTOL
AFTOL
DQ862045
DQ471015
DQ471019
DQ862053
DQ862054
DQ862056
DQ862050
AF408334
AF408350
AF408361
AF408373
AF408374
AF362564
AF362558
AFTOL 1287
CBS 606.72
AFTOL 17
CBS 709.71
Carolina Biological Supply
Company 15–6291
ATCC 36709/Buck s.n.
DQ471032
DQ368659
DQ836894
X04971
AY545724
DQ470980
AY999113
AY436413
AF286411
AY545728
AY641007
AY346301
Order Savoryellales
- Ascotaiwania lignicola
- Ascotaiwania mitriformis*
- Ascotaiwania sawadae
- Ascothailandia grenadoidia*
- Canalisporium caribense
- Savoryella lignicola
NIL 00006
HKUCC 3706
SS 00051
SS 03615
SS 03839
NTOU 791
HQ446285
N/A
HQ446283
GQ390252
GQ390253
HQ446299
HQ446365
AF132324
HQ446363
GQ390267
GQ390268
HQ446377
Sordariomycetidae Incertae sedis
- Conioscyphascus varius*
- Carpoligna pleurothecii
CBS 113653
AFTOL 281
AY484511
AY544689
AY484512
AY544685
Order Microascales
- Kochiella crispa*
- Morakotiella salina
- Nimbospora effusa
- Nohea umiumi
- Ocostaspora apilongissima
- Remispora maritima
- Sablecola chinensis
BCC 33502
BCC 12781
JK 5104A
JK 5103F
LP 53
BBH 28309
BCC 22809
N/A
N/A
U46877
U46878
N/A
HQ111002
N/A
HQ111020
AY864844
U46892
U46893
HQ111005
HQ111012
HQ111024
Order Hypocreales
- Calonectria colombiensis*
- Cordyceps militaris
- Cylindrocladium floridanum
- Melanopsamma pomiformis*
- Nectria cinnabarina
- Sedecimiella taiwanensis*
- Stachybotrys chartarum
CBS 112221
NRRL 28021
ATCC 22677
ATCC 18873
GJS 89–107
CY 5100
ATCC 66238
N/A
AF049146
N/A
AY489677
U32412
HM451495
AY489680
GQ280689
AF327374
U17408
AY489709
U00748
HM451496
AY489712
Sordariomycetes Incertae sedis
- Mirannulata samuelsii*
smh 1880
N/A
AY578353
Order Trichosphaeriales
- Trichosphaeria pilosa
089319bb
N/A
AY590297
Classification
- Lecythophora lignicola*
- Poroconiochaeta discoidea
Order Diaporthales
- Apiognomonia errabunda
- Diaporthe eres
- Gnomonia gnomon
- Melanconis marginalis
- Melanconis stilbostoma
- Valsa ambiens
- Valsa nivea
Order Sordariales
- Gelasinospora tetrasperma
- Immersiella caudata
- Lasiosphaeria ovina
- Neurospora crassa*
- Sordaria fimicola
- Sordaria macrospora
Original codes
2120
935
952
2128
2129
2131
2125
Order Xylariales
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TABLE II.
0
Continued
GenBank accession numbers
SSU
LSU
ATCC 36659
AFTOL 63
AFTOL 51
U32402
AY544719
NG013136
U47828
AY544676
NG027599
AFTOL 179
AFTOL 60
AY544711
AY544708
AY544667
AY544664
Classification
- Daldinia concentrica*
- Xylaria acuta
- Xylaria hypoxylon
Order Pezizales
- Disciotis venosa
- Morchella esculenta
Original codes
Bold: new sequence generated for this study; Not applicable with the prefix N/A, information in a certainfield on a table is
not provided; Asterisks: ex-holotype strains; AFTOL: Assembling the Fungal Tree of Life; ATCC: The American Type Culture
Collection, Manassas, VA; BCC: BIOTEC Culture Collection, Thailand; BBH: BIOTEC Bangkok Herbarium, Thailand; CBS:
Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; CY: City University of Hong Kong Culture Collection, Hong
Kong; HKUCC: University of Hong Kong Culture Collection, Hong Kong; MF: Marine-Earth Science and Technology
(JAMSTEC), Japan; NRRL: Northern Regional Research Center (NRRL), USDA, Peoria, IL, USA;
Culture and specimen abbreviations:
a
H.A. Raja, J. Campbell and C.A. Shearer;
b
J. Campbell and C.A. Shearer;
c
D. Vijaykrishna, R. Jeewon, and K.D. Hyde;
d
S.-O. Suh and M, Blackwell;
e
E. Weber;
AWR: A.W. Ramaley; GJS: G.J. Samuels; LP: J.Sakayaroj; JK: J. Kohlmeyer; MR: M. Reblovi; NIL; K-L Pang; SANK: J. C. Krug;
SMH, smh: S.M. Huhndorf; SS: S. Sivichai.
A. aquatorba (FIG. 1) placed in the Annulatascaceae
clade with a BSMP of 66%, BSML of 59% and a BYPP
of 0.98. It forms a sister clade to A. velatisporus, A.
hongkongensis and A. nilensis that are grouped in the
same clade but without significant support (BSML of
55%). Other taxa in the Annulatascaceae clade included the freshwater genera Annulusmagnus J. Campbell &
Shearer, Ascitendus J. Campbell & Shearer, Pseudoproboscispora Punith and Submersisphaeria K.D. Hyde.
Annulatascus biatriisporus grouped in a distal clade
with Fusoidispora aquatica and Ascocollumdensa aquatica (Sordariomycetidae Incertae sedis) with low
bootstrap support (BYPP 0.99) and showed no affinities with other Annulatascus species (A. velatisporus, A. aquatorba, A. hongkongensis and A. nilensis).
TAXONOMY
Annulatascus aquatorba Boonyuen & Sri-indrasutdhi
sp. nov.
FIGS. 2–15
MycoBank MB519473
Anamorph. Not observed.
Ascomata 390–500 mm alta (
x 5 445 mm, n 5 4),
310–350 mm (
x 5 312 mm, n 5 4) diam, semi-immersa
vel superficialia, globosa, brunea usque atra, ostiolata,
periphysis, solitaria. Collo 400–480 3 100–120 mm (
x
5 450 3 110 mm, n 5 8), cylindriculus, flexus perpendicular versus erectusescens. Peridium crassus
paries, brunea, pseudoparenchymatous cella in exterior stratum et compressus. Hamathecium:
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Paraphysis hyalinus, septa, filiformis, simplex
versus rariflorus ramus, usque ad 2.5 mm latus. Asci
102.5–152.5 3 6.25–8.75 mm (
x 5 114 mm, n 5 30)
cylindriculus, 8-spora, pedicellus, unitunicatata, distinctus, cuneatus, non–amyloideus annulusio apicalis, 5.0–7.5 mm latus. Ascospora 15–22.5 3 5–7.5 mm
(
x 5 19.2 3 6.5 mm, n 5 30), uniseriata versus
biseriata imbricate, in ascus, fusoidus, 3-septata,
aliquando leviter constrictus, strictus versus aliquantum curvus, laevis, centrale cella brunnea, extremum
cella rotundus cum subhyalinus. Ascospora destitutus
appendiculatus vel vagina.
Holotype: BBH 29936.
Etymology: aqua: water and torba: peat swamp,
belonging to the aquatic habitat of the fungus.
Ascomata 390–500 mm high (
x 5 445 mm, n 5 4),
310–350 mm (
x 5 312 mm, n 5 4) diam, semi–
immersed or superficial, globose, brown to dark
brown, ostiolate, periphysate, solitary (FIG. 2). Neck
400–480 3 100–120 mm (
x 5 450 3 110 mm, n 5 8),
cylindrical, bending perpendicularly or becoming
erect. Peridium thick-walled, brown, pseudoparenchymatous cells in the outer layers and inner layer
compressed (FIG. 3). Hamathecium: Paraphyses hyaline, septate, filiform, simple to rarely branched, up to
2.5 mm wide (FIG. 4). Asci 102.5–152.5 3 6.25–8.75 mm
(
x 5 114 mm, n 5 30) cylindrical, eight-spored,
pedicellate, unitunicate with a distinct, wedge-shaped,
nonamyloid apical ring, 5.0–7.5 mm wide (FIGS. 5–10).
Ascospores 15–22.5 3 5–7.5 mm (
x 5 19.2 3 6.5 mm, n
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MYCOLOGIA
FIG. 1. Phylogenetic analyses of Annulatascus aquatorba and related species based on combined partial 18S and 28S rDNA
sequences. Bootstrap values higher than 50% from maximum parsimony analysis (BSMP) (top left) and from RAxML (BSML)
(top right) are given above the nodes respectively. Bayesian posterior probabilities greater than 0.95 are indicated below the
nodes (BYPP). The internodes that are highly supported by all bootstrap proportions (100%) and posterior probabilities
(1.00) are shown as a thicker line. Asterisks are indicated as ex-holotype.
5 30), uniseriate to overlapping biseriate in the ascus,
fusoid to lunate, straight to somewhat curved, threeseptate, slightly constricted at the septa, smoothwalled, central cells brown, end cells with rounded
Mycologia myco-104-03-13.3d 25/4/12 17:04:25
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apices, subhyaline (FIGS. 11–15). Ascospores lack
appendages or a sheath.
Culture characteristics: Colonies diffuse, no aerial
mycelium, hyaline, becoming pale brown to dark
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FIGS. 2–15. Annulatascus aquatorba. (Holotype SS2424.01). Light microscope micrographs. 2. Ascoma with long neck on
the test blocks (arrowed). 3. Section of ascomata with thick peridial wall. 4. Asci and hyaline paraphyses. 5. Apical ring
(arrowed). 6–10. Asci at various stages of development with ascospores uniseriate to overlapping biseriate. 11–15. Ascospores
three-septate, fusoid to lunate, end cells subhyaline. Bars: 2 5 1000 mm, 3 5 150 mm, 4 5 30 mm, 8–15 5 15 mm, 5–7 5 10 mm.
Mycologia myco-104-03-13.3d 25/4/12 17:04:38
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MYCOLOGIA
brown. Cultures grew moderately well, reaching 16 mm
in 24 d at 25–28 C in normal daylight conditions.
Annulatascus aquatorba was isolated on CMA, with
ascospores germinating within 24–48 h on CMA.
Appressorial-like pegs developed from each terminal
cell and usually in synchrony (BCC16735).
Specimen examined: Thailand, Narathiwat Province, Sirindhorn Peat Swamp Forest on submerged wood test block
of Erythrophleum teysmannii, 22 Feb 2003, collected by N.
Boonyuen and V. Sri-indrasutdhi (BBH 29936).
Habitat: On submerged test blocks of Erythrophleum
teysmannii, in a peat swamp forest.
Mode of life: Saprobic.
Distribution: Thailand.
DISCUSSION
Based on morphological features (FIGS. 2–15), A.
aquatorba is referred to the genus Annulatascus
because it is characterized by dark, immersed to
superficial ascomata with tapering paraphyses. Asci
are cylindrical with a relatively large refractive apical
nonamyloid apical ring with a plug to the ascus and
versicolorous, thick-walled ascospores, slightly constricted at the septa (Hyde 1992, Tsui et al. 2002).
Campbell and Shearer (2004) opined that taxa
assigned to Annulatascus were not monophyletic as
currently circumscribed based on molecular data, and
this is confirmed by Abdel-Wahab et al. (2011) and
this study. Annulatascus species differ in morphology,
dimensions of the ascomata, asci and ascospores and
the substrata they naturally colonize. The most
pronounced differences are in size of the ascus and
apical ring, ascospore septation and the presence of a
mucilaginous sheath (Tsui et al. 2002). Eight
Annulatascus species (TABLE I) have ascospores that
are aseptate (A. aquaticus, A. biatriisporus, A. bipolaris,
A. citriosporus, A. joannae, A. lacteus, A. licualae and A.
velatispora), while the remainder are (1–)-3 septate (A.
aquatorba, A. apiculatus, A. hongkongensis, A. palmietensis, A. triseptatus and A. tropicalis), 1–5-septate (A.
fusiformis), 5–11-septate (A. nilensis) and two-septate
(A. liputii).
Annulatascus apiculatus, A. hongkongensis and Annulusmagnus triseptatus have ascospores that are larger
than A. aquatorba and have a mucilaginous sheath
(TABLE I). Annulatascus fusiformis has ascospores of a
similar size to A. aquatorba but differs in having bipolar
mucilaginous pad–like appendages (TABLE I).
Annulatascus aquatorba differs from A. palmietensis
and A. tropicalis because A. palmietensis has larger
ascomata and ascospores (TABLE I), while A. tropicalis
has larger asci and larger ascospores (TABLE I).
Annulatascus aquatorba resembles two other freshwater ascomycetes: Ascitendus J. Campbell & Shearer
Mycologia myco-104-03-13.3d 25/4/12 17:04:45
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and Submersisphaeria aquatica K.D. Hyde (Campbell
et al. 2003, Campbell and Shearer 2004). However
Submersisphaeria aquatica differs from A. aquatorba in
having 1–2-septate ascospores with cap-like appendages while Ascitendus austriacus differs from A.
aquatorba in having ascospores not constricted at
the septa and a roughened spore wall.
Taxa grouping in the Annulatascaceae include
Annulatascus, Annulusmagnus, Ascitendus, Pseudoproboscispora and Submersisphaeria, all freshwater ascomycetes growing on submerged wood. Within the
Annulatascaceae clade three subclades are discerned
(FIG. 1). Subclade A includes the type species A.
velatisporus, which forms a sister group to A. hongkongensis, with A. nilensis and A. aquatorba as basal taxa
with low bootstrap support. Subclade B includes
various strains of Annulusmagnus triseptatus and
Ascitendus austriacus. Two strains of Submersisphaeria
aquatica group in subclade C with high bootstrap
support. Pseudoproboscispora forms a basal subclade
with moderate support (FIG. 1). Annulatascus aquatorba therefore can be distinguished from other taxa in
the Annulatascaceae based on morphological and
molecular evidence.
Annulatascus species have been reported from
various substrata and geographical locations, with
most growing on submerged wood in freshwater
streams. Annulatascus aquatorba was collected on a
submerged test block of Erythrophleum teysmannii in
a peat swamp, while the remaining species (n 5 13)
are reported from unknown submerged timbers,
dead palm petioles and stems of Phragmites
australis.
Annulatascus aquatorba occurred only once on the
test block that had been exposed 2 y and was not
found on the other eight timber species submerged at
the peat swamp (on Azadirachta indica var. siamensis,
Melaleuca cajuputi, Shorea obtusa, S. roxburghii, S.
siamensis, Wrightia tomentosa, Xylia xylocarpa and
Zollingeria dongnaiensis). In addition it has not been
collected on natural submerged wood and test blocks
at other test sites in Thailand (e.g. Doi Inthanon
National Park, Khao Yai National Park, Kaeng
Krachang National Park, Mo Ko Chang National
Park, Tai Rom Yen National Park, Ton Nga Chang
Wildlife Sanctuary and Hala Bala Wildlife Sanctuary)
in the past 8 y, indicating it might be a rare species
and adapted to the acidic waters of the peat swamp
forest.
ACKNOWLEDGMENTS
This study was supported by the TRF/BIOTEC special
Program for Biodiversity Research and Training grants BRT
R_145006, BRT R_252057 and R_251009. Prof Morakot
Cust # 11-238R1
BOONYUEN ET AL.: ANNULATASCUS AQUATORBA
Tanticheroen, Drs Kanyawim Kirtikara and Lily Eurwilaichitr at BIOTEC are thanked for their continued support of
the freshwater work in Thailand. Our thanks to Sunthorn
Taodam for support with the field work at Sirindhorn Peat
Swamp, Jureerat Ueapattanakit for DNA extraction and
PCR of Annulatascus aquatorba, Juntima Chanprasert for
running the RAxML analysis, Prof Mohamed A. AbdelWahab for the sequence of A. nilensis and Dr Clement K.M.
Tsui for invaluable comments on Annulatascus taxonomy.
We are also grateful for the help of two anonymous
reviewers who provided critical comments that improved
the manuscript.
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