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ORIGINAL ARTICLE
Helicocentralis hyalina gen. et sp. nov., an aero-aquatic
helicosporous fungus (Leotiomycetes, Ascomycota) in Thailand
Veera Sri-indrasutdhi
1
&Clement K. M. Tsui
2
&Charuwan Chuaseeharonnachai
1
&
Kaoru Yamaguchi
3
&Satinee Suetrong
1
&Izumi Okane
4
&Akira Nakagiri
5
&
Nattawut Boonyuen
1
Received: 22 April 2015/Revised: 9 August 2015 /Accepted: 17 August 2015
#German Mycological Society and Springer-Verlag Berlin Heidelberg 2015
Abstract During a survey of aero-aquatic fungi in Thailand,
an undescribed helicosporous fungus was discovered. It is
characterized by the presence of branched and septate hyphae
with hyaline conidiophores that are micronematous or semi-
macronematous, acrogenous and holoblastic. Conidia are hy-
aline and centrifugally coiled (excentric), helicoid or circinate,
coiled 2–3 times clockwise or counterclockwise. After com-
parison of morphological and molecular characters with other
aero-aquatic, helicosporous fungi, introduction of a new genus
and species is proposed, Helicocentralis hyalina gen. et sp.
nov. Phylogenetic analyses based on the combined sequence
data from the small and large nuclear subunit ribosomal DNA
(SSU and LSU), as well as internal transcribed spacer (ITS)
rDNA sequence data, the fungus groups within the
Leotiomycetes class with strong statistical support. The new
genus is not related to other helicosporous hyphomycete
genera (Helicoma,Helicomyces,Helicoön,and
Helicosporium), which belong in the Tubeufiaceae,
Dothideomycetes. Within the Leotiomycetes, our new fungus
is distantly related to Helicodendron paradoxum (the type
species). The new fungus is also compared morphologically
to six similar helicosporous genera from Dothideomycetes
and Leotiomycetes.
Keywords Aero-aquatic fungi .Freshwater .
Dothideomycetes .Helicocentralis hyalina .Helicosporous
hyphomycetes .Leotiomycetes .Molecular phylogeny .
Wood .Thailand
Introduction
Aero-aquatic fungi are often found on submerged leaves and
woody debris in freshwater habitats, i.e. slowly moving
streams, wetlands, swamps and ponds. They are widely dis-
tributed in both temperate and tropical locations (van
Beverwijk 1951; Gönczöl and Révay 2003; Yamaguchi et al.
2012; Chuaseeharonnachai et al. 2013) and survive in low
oxygen concentrations that might be adaptive to freshwater
environments (Bärlocher et al. 1978; Fisher and Webster
1979,1981; Abdullah and Webster 1980; Field and Webster
1983). Of these, the aero-aquatic helicosporous hyphomycetes
are a large group, for example, the genera Helicomyces,
Helicosporium,Helicoma,Helicodendron and Helicoön
(Markovskaja 2012). Their common features are helicoid co-
nidia, which have the axis curved through more than 180°
(Tsui and Berbee 2006;Hawksworthetal.1995;Zhaoetal.
2007).
During an ongoing ecological study of aero-aquatic fungi
in north and northeast Thailand on their species abundance,
diversity and frequency of occurrence (Chuaseeharonnachai
Section Editor: Roland Kirschner
*Veera Sri-indrasutdhi
Veera@biotec.or.th
1
Fungal Biodiversity Laboratory, BIOTEC, National Science and
Technology Development Agency (NSTDA), 113 Thailand Science
Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe
Khlong Luang, Pathum Thani 12120, Thailand
2
Department of Pathology and Laboratory Medicine, University of
British Columbia, Vancouver, BC V6T 1Z4, Canada
3
Biological Resource Center (NBRC), National Institute of
Technology and Evaluation (NITE), 2-5-8 Kazusakamatari,
Kisarazu, Chiba 292-0818, Japan
4
Faculty of Life and Environmental Sciences, University of Tsukuba,
1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
5
Fungus/Mushroom Resource and Research Center, Faculty of
Agriculture, Tottori University, 4-101 Koyama-Minami,
Tottori, Tottori 680-8553, Japan
Mycol Progress (2015) 14:81
DOI 10.1007/s11557-015-1103-6
et al. 2013), an unknown aero-aquatic helicosporous hypho-
mycete was found on submerged wood. Based on its morpho-
logical characters, this fungus could not unequivocally be
assigned to any of the existing genera such as Helicosporium,
Helicoma and Helicomyces. We considered that the fungus
might represent an undescribed species belonging to one of
the existing genera or to a new genus. The objectives of this
study are: (1) to determine the position and the phylogenetic
relationship of the novel taxon based on partial sequences of
the combined nuc18S (SSU) and nuc28S rDNA (LSU); (2) to
compare it with other aero-aquatic helicosporous hyphomy-
cetes taxa currently included in the Leotiomycetes and
Dothideomycetes; and (3) to describe, illustrate and assign
the new taxon to a higher order based on morphological and
molecular data.
Materials and methods
Morphological study
Semi-submerged wood was collected from a peat swamp at
Ang Ka Nature Trail, Doi Inthanon National Park (2545 m
above sea level), Chiang Mai Province, Northern Thailand (N
18°35’19^, E 098°28’57^) following the procedures de-
scribed in Chuaseeharonnachai et al. (2013,2014). Samples
were returned to the laboratory and placed in plastic boxes,
lined with moist tissue paper (25×10×10 cm), with a layer of
water over the wood surface and incubated at 20 °C. Samples
were observed under a stereomicroscope for conidia/
propagules and re-examined at 14–day intervals for 90 days.
Single spores were isolated into pure culture, grown on
potato-carrot agar (PCA, extracted from 20 g/L potato, 20 g/
L carrot, 2 % agar), supplemented with two antibiotics (pen-
icillin G, 0.5 g/L from Bio Basic Canada Inc.; and streptomy-
cin, 0.5 g/L from Bio Basic Canada Inc.; Gams et al. 1998).
Germinated spores were then transferred to potato dextrose
agar plates (PDA, Criterion™dehydrated culture media, Har-
dy Diagnostics, Santa Maria, California) and coded as SS (a
culture collection of Somsak Sivichai housed at the National
Center for Genetic Engineering and Biotechnology
(BIOTEC) culture collection).
To examine and observe the fine structure of sporulation
and conidial development, a scanning electron microscope
(SEM) was performed as outlined in Yamaguchi et al.
(2012). Fungi were cultured on PCA medium and fixed with
1 % osmium tetroxide (O
s
O
4
) at 4 °C overnight or at 25 °C for
2 h. Subsequently, the specimens were dehydrated in an eth-
anol series and substituted with isoamyl acetate. After critical
point drying and coating with platinum-palladium, the fungal
specimens were examined with a JSM-6060 SEM (JEOL,
Tokyo, Japan) operated at 15 kV.
Micro-morphological structures were observed by squash-
mounted material in sterile distilled water. Some specimens
were taken from fresh material and mounted in lactophenol-
cotton blue solution and photographed using a Nomarski dif-
ferential interference contrast microscope. Permanent slides
are deposited at the BIOTEC Bangkok Herbarium (BBH,
Thailand; http://www1a.biotec.or.th/bbh/)andNITE
Biological Resource Center collection (NBRC, Japan; http://
www.nbrc.nite.go.jp/e/). Holotype axenic cultures are kept
either in the BIOTEC culture collection (BCC, Thailand;
http://www1a.biotec.or.th/BCC/) or the NBRC, Japan.
Molecular study
DNA extraction, polymerase chain reaction (PCR)
amplification, and sequencing
Helicosporous aero-aquatic fungi were grown on PDA for 4–
16 weeks at 25 °C. Actively growing mycelium was scraped
off the surface of a culture and transferred to 2 ml Eppendorf
micro-centrifuge tubes. The biomass was lyophilized at
−80 °C for 24 h. Fungal extraction buffer (1 % CTAB,
0.7 M NaCl, 50 mM Tris–HCl, 10 mM EDTA, pH 8) was
prepared and added to fungal samples. Total genomic DNA
was extracted according to the procedures in Boonyuen et al.
(2011), and the purity was assessed on a 1 % Tris-borate-
ethylenediaminetetraacetic acid (TBE) agarose gel stained
with ethidium-bromide. Primers used for PCR amplification
and sequencing of nuc18S rDNA, nuc28S rDNA and ITS
rDNA were NS1-NS4, NS3-NS6, ITS1-ITS4 and ITS5-
ITS4, respectively (White et al. 1990). Detailed protocols
and PCR conditions for the amplification were fully described
in Boonyuen et al. (2011). PCR products were purified using a
NucleoSpin® extract kit (Macherey-Nagel, Germany) to re-
move non-specific amplicons, excess primers, and
deoxynucleotide triphosphates (dNTPs). The PCR products
were sequenced by Macrogen Inc., Republic of Korea, with
the same PCR primers. The sequences obtained have been
deposited in the National Center for Biotechnology Informa-
tion (NCBI) and the accession numbers are listed in Table 1.
Sequence alignment and phylogenetic analysis
DNA sequences were edited and assembled with BioEdit
6.0.7 (Hall 2004) and homologous sequences of other fungal
sequences were retrieved using the basic local alignment
search tool (BLAST) at NCBI for phylogenetic analyses
(http://blast.ncbi.nlm.nih.gov). Alignments were manually
adjusted with other sequences obtained from the GenBank
nucleotide database. Multiple sequence alignments were
generated with other sequences obtained from GenBank by
Clustal W 1.6 (Thompson et al. 1994) and refined manually in
81 Page 2 of 12 Mycol Progress (2015) 14:81
BioEdit 6.0.7 (Hall 2004). Manual gap adjustments were done
to improve the alignment and ambiguously aligned regions
were also excluded. Portions of the 5′- and 3′-ends of the
nuclear small and large subunits ribosomal DNA (nuc18S
and nuc28S) and ITS DNA sequence were excluded from all
analyses and coded by a question mark (?). The final align-
ment was optimized by visual examination and manually
corrected using the Se-Al v. 2.0a8 program (Rambaut 1996).
The alignment of combined nuc18S and nuc28S DNA se-
quences and ITS DNA sequences is available in TreeBASE
No.17503 (http://www.treebase.org/treebase–web/home.
html).
The phylogenetic analysis was performed through PAUP*
4.0b10 software (Swofford 2002) on a Mac OS X 10.4.10.
Phylogenetic trees were visualized using the program
TreeView (Page 1996). The phylogenetic analyses of different
datasets were performed with maximum parsimony (MP),
maximum likelihood (ML) and Bayesian algorithms (BY) as
described in Suetrong et al. (2011). MP analyses were per-
formed using PAUP software (Swofford 2002) and all charac-
ters were equally weighted, followed by a heuristic search
with a stepwise starting tree, a random stepwise addition of
100 replicates and a tree-bisection-reconnection (TBR)
branch-swapping algorithm, withgaps treated as missing data.
Branch support for all parsimony analyses was calculated by
using 1000 bootstrap replicates (Felsenstein 1985)withthe
full heuristic searches on stepwise addition of sequences, 10
replicates of random addition of taxa and followed by the TBR
branch swapping algorithm. Three indices (consistency indi-
ces (CI), retention indices (RI) and re-scaled consistency indi-
ces (RC) were calculated. ML analyses were performed using
RAxML v. 7.2.6 (Stamatakis 2006). The partitioned dataset
and default settings for the remaining parameters according to
each gene and separated codons (eight partitions) were used.
A general time reversible (GTR) model with a discrete gamma
distribution and four rate classes was applied to each partition
and a tree was obtained by simultaneously running a fast
bootstrap search of 1000 pseudoreplicates followed by a
search for the most likely tree under functional setting Ba^.
MrModeltest 2.2 (Nylander 2004) was used to determine the
best-fit model of evolution for each data set of Bayesian anal-
yses. Bayesian analyses employing Markov chain Monte
Carlo (MCMC) methods was performed with MrBayes
3.0b4 (Huelsenbeck and Ronquist 2001). GTR + I + G model,
Iset nst = 6 rates invgamma; prset statefreqpr = dirichlet (1, 1,
1, 1), was implemented and four chains were run from random
starting tree for 2,000,000 generations and sampled every 100
generations. The first 2000 trees, which extend beyond the
burn-in phase in each analysis, were discarded and the remain-
ing 18,000 trees were used to calculate posterior probabilities
(BYPP) in the consensus tree. Posterior probabilities were
determined from a consensus tree for each clade. Confident
branch support is defined as Bayesian posterior probabilities ≥
0.95 as significant and strongly supported.
MP (BSMP, left) and ML (BSML, right) bootstrap support
values≥50 % are shown above the node and Bayesian poste-
rior probabilities≥0.95 are given below each branch (BYPP).
The internodes that are highly supported by all bootstrap pro-
portions (100 %) and posterior probabilities (1.00) are given
as a thicker line. The rDNA sequences, composed of nuc18S,
nuc28S and ITS, were submitted to the NCBI database and the
novel sequences generated for this work are provided in
Table 1. The GenBank accession numbers of sequences used
in the phylogenetic analysis are shown in Table 2included
with the selected aero-aquatic helicosporous hyphomycetes
(Helicoma,Helicomyces,Helicoön,Helicosporium,and
Helicodendron). Taphrina deformans and T. wi es ne ri
(Taphrinomycetes) were used as the outgroup taxa, while
two members of the Dothideomycetes (Helicomyces roseus
and H. muelleri)wereusedtoroottheITSphylogenetictree.
Results
Taxo no my
Helicocentralis Sri-indrasutdhi, Chuaseeharonnachai,
Boonyuen, Yamaguchi, Suetrong & C.K.M. Tsui, gen. nov.
Fig. 1
Table 1 Sequences used in this study
Taxon Source GenBank accession numbers
nuc18S rDNA nuc28S rDNA ITS rDNA
Helicodendron microsporum CBS100149 KR078445 KR078441 KR078437
Helicodendron paradoxum CBS300.50 N/A N/A KR078436
Helicocentralis hyalina SS04733.01/BCC36782 KR078442 KR078438 KR078433
Helicocentralis hyalina SS04733.02/BCC36783 KR078443 KR078439 KR078434
Helicocentralis hyalina SS04733.03/BCC36784 KR078444 KR078440 KR078435
Helicocentralis hyalina AF0314/BCC72621 KR152642 KR152641 KR152640
Mycol Progress (2015) 14:81 Page 3 of 12 81
Table 2 Sequences of fungal taxa obtained from GenBank, with their GenBank accession numbers
Taxon Source GenBank accession numbers
nuc18S rDNA nuc28S rDNA ITS rDNA
Acanthostigma chiangmaiensis MFLUCC 10-0125 JN865185 JN865197 N/A
Alatospora pulchella CCM F-502 N/A N/A KC834039
Anguillospora furtiva CCM –F - 20483 AY357262 N/A N/A
Annulatascus aquatorba SS 2424 JN226106 JN226107 N/A
Annulatascus velatisporus HKUCC 3701 N/A AF132320 N/A
Apiognomonia errabunda AFTOL 2120 DQ862045 AF408334 N/A
Arbusculina fragmentans CCM F-13486 N/A N/A KC834042
Arthonia caesia AFTOL 775 N/A FJ469668 N/A
Arthrobotrys elegans AFTOL 1252 FJ176810 FJ176864 N/A
Articulospora atra CCM F- 00684 N/A N/A FJ000402
Ascitendus austriacus A44 - 28A N/A AY590292 N/A
Ascocalyx abietina cf870061 N/A N/A U72259
Ascocoryne cylichnium PDD75671 N/A N/A AY789395
Ascocoryne sarcoides AFTOL 1834 FJ176830 FJ176886 N/A
Ascotaiwania mitriformis HKUCC 3706 N/A AF132324 N/A
Ascothailandia grenadoidia SS 3615 GQ390252 GQ390267 N/A
Botryotinia fuckeliana AFTOL 59 AY544695 AY544651 N/A
Bulgaria inquinans ZW-Geo52-Clark N/A N/A AY789345
Cadophora luteo-olivacea Strain 18 N/A N/A DQ404348
Caliciopsis orientalis AFTOL 1911 DQ471039 DQ470987 N/A
Calonectria colombiensis CBS 112221 N/A GQ280689 N/A
Caloscypha fulgens DJ 053103-2 DQ247807 DQ247799 N/A
Camarops microspora AFTOL 1361 DQ471036 AY083821 N/A
Camarops ustulinoides AFTOL 72 DQ470989 DQ470941 N/A
Capnodium coffeae CBS 147.52 DQ247808 DQ247800 N/A
Chaetosphaeria innumera SMH 2748 N/A AY017375 N/A
Chlamydotubeufia huaikangplaensis MFLUCC 10-0926 JN865186 JN865198 N/A
Chlorociboria aeruginosa AFTOL 151 AY544713 AY544669 N/A
Chloroscypha chloromela 66490 N/A N/A U92311
Cladochasiella divergens CCM F-13489 N/A N/A KC834043
Cochliobolus heterostrophus AFTOL 54 AY544727 AY544645 N/A
Coniochaeta ligniaria F 3331 N/A AF353583 N/A
Cordyceps militaris NRRL 28021 AF049146 AF327374 N/A
Cryptosporiopsis rhizophila CBS110609 N/A N/A AY176758
Cudonia lutea wz164 N/A N/A AF433149
Cudoniella clavus AFTOL 166 DQ470992 DQ470944 N/A
Cudoniella indica CBS 430.94 N/A N/A DQ202513
Dactylaria dimorphospora CBS 256.70 N/A N/A U51980
Daldinia concentrica ATCC 36659 U32402 U47828 N/A
Dermea acerina CBS 161.38 DQ247809 DQ247801 N/A
Diaporthe eres AFTOL 935 DQ471015 AF408350 N/A
Dimorphospora foliicola CBS 221.59 N/A N/A DQ202518
Disciotis venosa AFTOL 179 AY544711 AY544667 N/A
Dothidea sambuci AFTOL 274 AY544722 AY544681 N/A
Elsinoe veneta AFTOL 1360 DQ678007 DQ678060 N/A
Eupenici llium limosum AFTOL 2014 EF411061 EF411064 N/A
Filosporella exilis CCM F-13097 N/A N/A KC834046
Filosporella fistucella CCM F-13091 N/A N/A KC834047
Filosporella versimorpha CCM F-11194 N/A N/A KC834054
Flagellospora fusarioides CCM F-14583 N/A N/A KC834048
Fontanospora fusiramosa UMB −075.01 N/A N/A GQ411267
Gelasinospora tetrasperma AFTOL 1287 DQ471032 DQ470980 N/A
Geniculospora grandis UMB −176.01 N/A N/A GQ411354
Geoglossum glabrum OSC 60610 AY789316 AY789317 N/A
Geoglossum nigritum AFTOL 56 AY544694 AY544650 N/A
Gorgomyces honrubiae CCM F-12003 N/A N/A KC834057
81 Page 4 of 12 Mycol Progress (2015) 14:81
Table 2 (continued)
Taxon Source GenBank accession numbers
nuc18S rDNA nuc28S rDNA ITS rDNA
Gremmeniella abietina cf 870061 N/A N/A U72259
Gyoerffyella entomobryoides CBS268.63 N/A N/A KC834056
Gyoerffyella rotula 130-1090 N/A N/A AY729937
Gyoerffyella sp. B 54A4 N/A N/A EF093184
Helicodendron conglomeratum UAMH 8472 AY856943 AY856900 N/A
Helicodendron luteoalbum ICMP 15655 N/A N/A EF029237
Helicodendron paradoxum UAMH 8758 AY856945 N/A N/A
Helicod endron triglit ziense ICMP 15653 N/A N/A EF029236
Helicodendron tubulosum UAMH 8567 AY856944 N/A N/A
Helicodendron websteri ICMP15521 N/A N/A EF029229
Helicodendron westerdijkae ICMP 14616 N/A N/A EF029196
Helicoma chlamydosporum CBS 160.69 AY856923 AY856875 N/A
Helicoma muelleri CBS 964.69 N/A N/A AY916453
Helicoma vaccinii CBS 216.90 AY856926 AY856879 N/A
Helicomyces lilliputeus NBRC 32664 AY856942 AY856899 N/A
Helicomyces roseus CBS 283.51 N/A N/A AY916464
Helicoon gigantisporum BCC 3550 AY856948 AY856904 N/A
Helicoon richonis CBS 282.54 AY856952 N/A N/A
Helicosporium guianense CBS 269.52 AY856938 AY856893 N/A
Hemiphacidium longisporum ATCC26761 N/A N/A AY645899
Heyderia abietis OSC60392 N/A N/A AY789290
Hyalodendriella betulae CBS261.82 N/A N/A EU040232
Hyaloscypha vitreola M39 N/A N/A EU940231
Hydrocina chaetoc ladia CCM F-10890 N/A N/A KC834062
Hymenoscyphus ericae UAMH 8873 AY524847 N/A N/A
Hymenoscyphus varicosporoides FC-2038 N/A N/A AB481291
Hyphodiscus hymeniophilus MUCL9042 N/A N/A DQ227259
Kirschs teiniothelia e laterascus A 22-5A AF053727 N/A N/A
Kohlmeyeriella tubulata PP 1105 AY878998 AF491265 N/A
Koralionastes ellipticus JK 5771 EU863580 EU863583 N/A
Lachnum bicolor 47829 N/A N/A U59005
Lachnum virgineum AFTOL 49 N/A N/A DQ491485
Lecythophora lignicola CBS 267.33 AJ496246 AF353601 N/A
Lemonniera centrosphaera CCM F-149 N/A N/A KC834063
Leotia lubrica ZW-Geo59-Clark N/A N/A AY789360
Letendraea helminthicola CBS 884.85 AY016345 AY016362 N/A
Lindra thalassiae JK 5090 AF195634 AF195635 N/A
Lophiostoma compressum IFRD 2014 GU296164 GU301834 N/A
Lulworthia grandispora JK 4686 DQ522855 DQ522856 N/A
Menispora tortuosa AFTOL 278 AY544723 AY544682 N/A
Miniancora allisoniensis CCM F-30487 N/A N/A KC834064
Mitrula brevispora ZW 02-012 N/A N/A AY789294
Mitrula elegans ZW-Geo45-Clark N/A N/A AY789331
Mollisia cinerea AFTOL 76 DQ470990 DQ470942 DQ491498
Monascus purpureus AFTOL 426 DQ782881 DQ782908 N/A
Monilinia laxa AFTOL 169 AY544714 AY544670 N/A
Morakotiella salina BCC 12781 N/A HQ111039 N/A
Morchell a esculenta AFTOL 60 AY544708 AY544664 N/A
Mycoarthris corallinus 120162 N/A N/A AF128440
Mycosphaerella punctiformis AFTOL 942 DQ471017 DQ470968 N/A
Nectria cinn abarina GJS 89.107 U32412 U00748 N/A
Neobulgaria pura AFTOL 1259 N/A FJ176865 N/A
Neofabraea al ba MM159 N/A N/A AY359236
Neofabraea mal icorticis AFTOL 149 AY544706 AY544662 N/A
Neurospora crassa CBS 709.71 X04971 AF286411 N/A
Nimbospora effusa JK 5104A U46877 U46892 N/A
Mycol Progress (2015) 14:81 Page 5 of 12 81
Etymology: Bhelico^referring to the spiral or helical shape
and Bcentralis^referring to the origin center in conidial rotation.
MycoBank MB811729
Index fungorum IF551100
Colonies on natural substrate effuse, dispersed, hairy and
colourless. Mycelium partly superficial and partly immersed, hy-
aline, composed of branched, septate, smooth, and creeping hy-
phae. Conidiophores short, micronematous or semi-
macronematous, mononematous, solitary or sometimes forming
3–4 in a group, arising laterally from the creeping mycelium,
unbranched or rarely branched, tapering at the apex, septate,
smooth, and hyaline. Conidiogenous cells monoblastic,
integrated, terminal, determinate, conical to cylindrical. Conidia
holoblastic, hyaline, solitary, dry, acrogenous, simple, circinate,
and centrifugally coiled to clockwise or counterclockwise. Co-
nidial filament coiled in three-dimensional plane, non-hygro-
scopic, hyaline, septate, and smooth walled.
Type species: Helicocentralis hyalina
Helicocentralis hyalina Sri-indrasutdhi, Chuaseeharonnachai,
Boonyuen, Yamaguchi, Suetrong & C.K.M. Tsui, sp. nov.
(Fig. 1).
Etymology: Referring to the colourless conidia.
MycoBank MB811730
Index fungorum IF551101
Table 2 (continued)
Taxon Source GenBank accession numbers
nuc18S rDNA nuc28S rDNA ITS rDNA
Nohea umiumi JK 5103 F U46878 U46893 N/A
Oculimacula acuformis RAC 44 N/A N/A AY266146
Orbilia vinosa AFTOL 905 DQ471000 DQ470952 N/A
Ophiostoma piliferum AFTOL 910 DQ471003 DQ470955 N/A
Ophiostoma ulmi CBS 298.87 M83261 DQ368627 N/A
Penici llium freii DAMO 216705 AY640998 AY640958 N/A
Phialocephala helvetica D–ZB - 40 N/A N/A AY347413
Pleospora herbarum CBS 191.86 GU238232 GU238160 N/A
Pontogeneia microdictyi JK 5748 EU863582 N/A N/A
Rhexocercosporidium sp. G 14 N/A N/A DQ275614
Rhynchosporium orthosporum CBS 698.79 N/A N/A AY140669
Roccellographa cretacea AFTOL 93 DQ883705 DQ883696 N/A
Savoryella lignicola NTOU 791 HQ446299 HQ446377 N/A
Schismatomma decolorans DUKE 47570 NG_013155 NG_027622 N/A
Sclerotinia sclerotiorum WZ 0067 AY789346 AY789347 N/A
Sclerotinia sclerotiorum wb197 N/A N/A AF455526
Sordaria macrospora ATCC 36709 AY641007 AY346301 N/A
Taphrina deformans AFTOL 1234 DQ471024 DQ470973 N/A
Taphrina wiesneri IFO 7776 D12531 NG_027620 N/A
Tetracladium apiense CCM F-23299 N/A N/A EU883422
Tetracladium breve CCM F-12505 N/A N/A FJ000405
Tetracladium palmatum CCM F-10001 N/A N/A FJ000372
Tetracladium setigerum CCM F-10186 N/A N/A FJ000374
Thaxteriella inthanonensis MFLUCC 11-0003 JN865187 JN865199 N/A
Trichoglossum hirsutum AFTOL 64 AY544697 AY544653 N/A
Tricladium alaskense VG -2012a N/A N/A JQ417290
Tricladium angulatum CCM F-10200 N/A N/A AY204609
Tricladium kelleri VG -2012b N/A N/A JQ417288
Tricladium obesum CCM F-14598 N/A N/A KC834068
Tricladium terrestre CBS 697.73 N/A N/A DQ202519
Tricladium minutum CCM F-10203 N/A N/A JQ412863
Tubeufia cylindrothecia BCC 3559 N/A AY849965 N/A
Varicosporium delicatum CCM F-19494 N/A N/A JQ412864
Variocladium giganteum CBS 508.71 N/A N/A DQ202520
Vibrissea albofusca PDD 75692 N/A N/A AY789384
Vibrissea flavovirens MBH 39316 N/A N/A AY789427
Vibrissea truncorum AFTOL 1322 FJ176818 FJ176874 N/A
Xylaria acuta AFTOL 63 AY544719 AY544676 N/A
Xylaria hypoxylon AFTOL 51 AY544692 AY544648 N/A
Zalerion varium ATCC 28878 N/A N/A AF169303
81 Page 6 of 12 Mycol Progress (2015) 14:81
Colonies on natural substrate effuse, dispersed, hairy, and
colourless. Mycelium hyaline, partly superficial and partly im-
mersed, composed of branched, septate, smooth, creeping,
thick-walled cells, 2–2.5 μmthick.Conidiophores short,
micronematous or semi-macronematous, mononematous, sol-
itary or sometimes forming 3–4 in a group, arising laterally
from the creeping mycelium, unbranched or rarely branched,
tapering at the apex, 2.5 μm in the broadest part, 17.5–
22.5 μm long, 1–3 septate, smooth-walled, hyaline.
Conidiogenous cells monoblastic, integrated, terminal, deter-
minate, conical to cylindrical, 2–2.5 μmthick.Conidia holo-
blastic, solitary, dry, acrogenous, simple, circinate, centrifu-
gally coiled with rounded apical and basal cells and tightly
coiled 2–3 times, centrifugally coiled to clockwise or counter-
clockwise, hyaline, 17.5–25 μmindiam.Conidial filament
coiled in two- or three-dimensional plane, non-hygroscopic,
3.75–5μmthick,with14–21 septa, smooth-walled, hyaline
(Fig. 1).
Specimen examined: Thailand, Ang Ka Nature Trail, Doi
Inthanon National Park, Chiang Mai province, on semi-
submerged wood, collected by V. Sri-indrasutdhi, C.
Chuaseeharonnachai, N. Boonyuen and K. Yamaguchi, 12
February 2009 and isolated by C. Chuaseeharonnachai, 20
March 2009. Holotype (BBH27889), Isotypes (BBH39849
and BBH39850), ex-holotype culture BCC36782 (=
SS04733.01).
Gene sequence ex-holotype: KR078442 (nuc18S rDNA),
KR078438 (nuc28S rDNA), KR078433 (ITS rDNA)
Molecular phylogeny
Phylogenetic analyses of combined partial nuclear ribosomal
28S large subunit and complete internal transcribed spacer
DNA sequence data suggested that H. hyalina belongs in the
Leotiomycetes, and not Dothideomycetes.
The combined nuc18S and 28S phylogeny
The combinednuc18S-nuc28S alignment comprised a total of
3100 characters for 91 taxa, of which 1117 were parsimony-
informative, 576 were parsimony-uninformative and 1407
were constant. Tree length (TL) was 6592, CI=0.395, RI=
0.643, RC=0.254 and HI=0.605. The MP analysis resulted
in seven most parsimonious trees (MPTs). RAxML yielded a
best scoring tree with log likelihood −37095.730180, with:
alpha = 0.515038, invar = 0.201872 and TL = 5.509281 (data
not shown). Bayesian phylogenetic analysis was performed
using a uniform GTR + I + G model, as selected by hLRT in
MrModeltest 2.2: [GTR + I + G] Prset statefreqpr = dirichlet
(1, 1, 1, 1), Lset nst=6 rates=invgamma. There were no sig-
nificant conflicts between the tree topologies and well-
Fig. 1 Micrographs of
Helicocentralis hyalina.aYoung
conidia with conidiophores
arising from septate hypha. band
cCharacteristic features of
conidiophores and conidia stained
with lactophenol cotton blue. d–g
Different conidia showing the
number of rotations. Bars=
20 μm. h–jSEM micrographs h
Mature conidium breaking down
from the end of conidiogenous
locus. iSolitary and aggregate
conidia. jConidium in side view
showing thickness and
characteristic of rotation
Mycol Progress (2015) 14:81 Page 7 of 12 81
supported clades in the separate nuc18S and nuc28S analyses
(data not shown).
A tree from combined nuc18S-nuc28S data consisted of
eight major classes (Leotiomycetes, Dothideomycetes,
Arthoniomycetes, Eurotiomycetes, Geoglossomycetes,
Pezizomycetes, Taphrinomycetes and Orbiliomycetes and 12
major orders in Sordariomycetes (e.g. Savoryellales,
Microascales, Hypocreales and Sordariales).
In this tree, the new genus (four isolates of BCC36782,
BCC36783, BCC36784 and BCC72621) grouped together
in the Leotiomycetes with BSMP = 84 %, BSML = 98 %)
and BYPP = 1.00. This monophyletic group formed a close
relationship to several genera, which are grouped in this class,
i.e. several species of Helicodendron,Lachnum,
Hymenoscyphus,Dermea and Neofabraea,whileMonilinia
laxa,Sclerotinia sclerotiorum and Botryotinia fuckeliana
formed a basal clade to the other species and the new genus
(Fig. 2). Therefore, analysis of partial 28S and 18S nuc rDNA
sequences placed the new taxon in the Leotiomycetes.
The ITS rDNA phylogeny
The ITS matrix consisted of 72 taxa, with Helicomyces roseus
and H. muelleri as outgroup taxa. The phylogenetic analyses
of ITS DNA sequence data were performed with various taxa
in the Leotiomycetes from the GenBank. Fungal sequences
were aligned and analyzed separately by MP, ML and BY.
The MP dataset consists of 470 characters: 195 characters
were constant, 210 characters were parsimony-informative
and 65 characters were parsimony-uninformative. The MP
resulted in one MPT in a length of 1852 steps (CI=0.294,
RI=0.607, RC=0.178, HI=0.706). Independent Bayesian
phylogenetic analysis was performed using a uniform GTR
+ I + G model, as selected by hLRT in MrModeltest 2.2: [GTR
+ I + G] Prset statefreqpr = dirichlet (1, 1, 1, 1), Lset nst = 6
rates = invgamma. One hundred successive searches using a
rapid hill–climbing algorithm from distinct randomised
starting trees in RAxML yielded a best scoring tree with a
log likelihood = −8463.030414, with alpha = 0.956559, invar
= 0.347445, tree-length = 11.185004, rate A < −>C:
1.906065, rate A < −> G: 2.699106, rate A < −>T:
2.268633, rate C < −> G: 1.356516, rate C < −>T:
4.419870, rate G < −> T: 1.000000, freq pi (A): 0.222876,
freq pi (C): 0.247703, freq pi (G): 0.256064 and freq pi (T):
0.273357 (data not shown).
In Fig. 3,Helicocentralis hyalina is phylogenetically
placed in the Leotiomycetes with strong statistical support
(93 % BSMP, 98 % BSML, and 0.98 BYPP). It is distantly
related to other representative freshwater fungi, e.g.
Helicodendron species, Tricladium species, and
Dimorphospora foliicola that are grouped in the same class
(Leotiomycetes). Additionally, Helicodendron paradoxum
(CBS300.50, the type species of genus), Dimorphospora
foliicola (CBS221.59) and H. microsporum (CBS100149)
grouped together with high support values (100 % BSMP,
98 % BSML and 1.00 BYPP). Four strains of Helicocentralis
hyalina are grouped with Cudonia lutea (Peck) Sacc. wz164
(= Vibrissea lutea Peck 1873) and Tricladium angulatum
CCMF-1200 (the latter belonging in Helotiaceae, Helotiales),
in a poorly supported sister clade. In addition, in a further
analysis (ITS, not figured), with more members of the order
Rhytismatales, Cudonia lutea grouped together with Rhytisma
salicinum and Spathularia flavida,awayfromHelicocentralis
hyalina (data not shown).
Fig. 2 One of seven MPTs inferred from combined analyses of nuc18S
and nuc28S rDNA sequences. BSMP, BSML bootstrap support values≥
50 % and BYPPs≥0.95 are given the at left above the node, right above
the node and below the node, respectively. The original code numbers of
these sequences selected for phylogenetic analyses are given in the tree
after the taxon names
81 Page 8 of 12 Mycol Progress (2015) 14:81
Discussion
To determine the phylogenetic placement of the new aero-aquatic
hyphomycete, four isolates were analyzed with selected aquatic
hyphomycetes distributed throughout the Leotiomycetes
(Baschien et al. 2013). Based on the clustering pattern in
Baschien et al. (2013), Helicocentralis hyalina did not group
with the Hymenoscyphus-Cudoniella clade, Ascocoryne-
Hydrocina clade or Mitrula clade. It was related to Cudonia
lutea wz164 (= Vibrissea lutea Peck 1873; Helotiales,
Leotiomycetidae, Leotiomycetes) and Tricladium angulatum
CCMF-1200 (Helotiaceae, Helotiales), but the placement did
not receive strong support.
Traditionally, the delineation of helicosporous genera is based
on conidiophore and conidiogenous cell development on aerial
hyphae and whether conidia are two- or three-dimensional, hya-
line or coloured, degree of coiling and septation (Holubová-
Jechová 1991). However, these characters are proved to be evo-
lutionary uninformative (Tsui and Berbee 2006). In this study,
Helicocentralis is not closely related to morphologically similar
helicosporous hyphomycetes (e.g. Helicoma,Helicomyces,
Helicoön,Helicosporium,Helicodendron and Helicogoosia)in
Tubeufiaceae (Dothidiomycetes). Instead, molecular data sup-
ports the placement of H. hyalina in the Leotiomycetes.
Helicocentralis is unique.
Table 3summarizes the similarities and differences be-
tween the different genera when we compare the morphology
of selected genera from the Leotiomycetes and
Dothideomycetes (Zhao et al. 2007; Sánchez and Bianchinotti
2010; Sánchez et al. 2012; Boonmee et al. 2011; Tsui and
Berbee 2006). In the phylogenetic tree of ITS, Helicocentralis
within the Leotiomycetes is distant from the genus
Helicodendron (H. paradoxum as the type species,
H. microsporum); although it shares some morphological
characteristics, such as conidiophores micronematous or
semi-macronematous, and mononematous, and
conidiogenous cells monoblastic, integrated, terminal, deter-
minate, cylindrical and conidia three-dimensionally coiled
(Fisher and Webster 1983; Table 3). Although Helicocentralis
resembles Helicoma,Helicoma was not monophyletic and
most species clustered with Helicomyces and Helicosporium
in the Tubeufiaceae (Tubeufiales) based on molecular data
(Tsui and Berbee 2006,2010;Tsuietal.2006; Boonmee
et al. 2011,2014). Helicocentralis differs from other
Helicoma species by having smooth-walled conidia, hyaline
conidiophores and conidia that are circinate, tightly coiled,
centrifugally coiled and hyaline (Ellis 1971; Goos 1986;
Zhao et al. 2007; Table 3).
Helicocentralis is morphologically similar to the genus
Helicomyces by having solitary, dry, centrifugally coiled,
acrogenous, and hyaline conidia. However, the conidial fila-
ment in Helicomyces is usually centripetally coiled and coiled
in a two-dimensional plane, but sometimes tend to be three-
dimensional, hygroscopic, septate, conidiophores lacking or
sometimes short (Goos 1986;Zhaoetal.2007; Table 3).
Helicocentralis resembles Helicosporium by possessing
micronematous, mononematous, unbranched conidiophores,
but Helicosporium differs from Helicocentralis by having
pleurogenous or acropleurogenous, simple, helicoid to some-
what cochleate, centric coiled conidia, colourless or brightly
coloured in mass (Tsui et al. 2006; Ellis 1971; Zhao et al.
2007; Table 3). As shown in Table 3,Helicocentralis shows
some similarities to the genus Helicoön by having
mononematous, unbranched and hyaline conidia. However,
the conidia ofthe latter are acropleurogenous or pleurogenous,
ellipsoidal or sometimes cylindrical, barrel-shaped, colourless
Fig. 3 The best MPT obtained from the ITS rDNA dataset. Bootstrap
resampling values ≥50 % are given above the branches, and BYPPs
≥0.95 are indicated below the nodes. The original code numbers of
these sequences selected for phylogenetic analyses are given in the tree
after the taxon names
Mycol Progress (2015) 14:81 Page 9 of 12 81
Tab le 3 Morphological features of the genus Helicocentralis compared with the selected helicosporous hyphomycete genera
Character Helicoma Corda Helicomyces Link Helicoön Morgan Helicosporium Nees Helicodendron
Peyronel
Helicogoosia Hol.-Jech. Helicocentralis
gen. nov.
H. muelleri
Corda, 1837
H. roseus Link,
1809
H. sessile Morgan,
1892
H. vegetum
Nees, 1816
H. paradoxum
Peyr., 1918
H. paradoxa
Hol.-Jech., 1991
H. hyalina
Colonies on
substrate Effuse, hairy or
velvety, yellowish,
grayish brown,
brown or olivaceous
Effuse to arachnoid
or tuberculate,
white to pinkish or
brownish in age
Effuse, velvety
or loosely cottony,
yellow, grey,
olivaceous or brown
Effuse, dispersed,
hairy or cottony,
brightly coloured
or fuscous
Effuse, velvety or
loosely cottony,
white, shining,
greenish to olive,
grey or brown
Effuse, loosely cottony
or tomentose, brown Effuse, dispersed, hairy,
bright coloured,
hyaline
Mycelium on
substrate N/A Branched, septate,
hyaline to dilute
fuscous
N/A Branched, septate Branched, septate Branched, septate, smooth,
pale brown, occasionally
anastomosing
Branched, septate,
smooth, hyaline
Conidiophores Macronematous,
mononematous,
or lacking, branched or
unbranched; sometimes
apex setiform, smooth,
pale to dark brown or
olivaceous brown
Lacking or formed
as short Macro- or micronematous,
mononematous,
unbranched or branched,
smooth or verrucose,
hyaline to brown
Macronematous,
mononematous,
unbranched or loosely
branched; branches
sometimes anatomosing,
apex often setiform
Micronematous or
semi-
macronematous,
mononematous,
branched, smooth,
colourless to brown
or olivaceous
Semi-macronematous,
mononematous,
unbranched
or sometimes loosely
branched,
occasionally proliferating,
smooth, pale brown to
brown
Short, micronematous
or semi-
macronematous,
mononematous,
unbranched or rarely
branched, smooth,
hyaline
Conidiogenous
cells Mono- or polyblastic,
integrated, terminal and/or
intercalary, sympodial or
determinate, cylindrical,
denticulate; denticles cy-
lindrical
Mono- or polyblastic,
integrated, producing
conidia from the apex,
or synchronously and/
or successively from
short denticles
Mono- or polyblastic,
integrated, terminal and
intercalary, sympodial or
determinate, denticulate;
denticles cylindrical
Mono- or polyblastic,
integrated, intercalary
and sometimes terminal,
sympodial or determinate,
cylindrical, denticulate;
denticles cylindrical
Mono- or polyblastic,
integrated, terminal
and intercalary,
determinate,
cylindrical
Monoblastic, integrated,
terminal or lateral,
determinate or
proliferating, lanceolate,
becoming easily detached;
after secession leaving
clear minute pores in the
wall of conidiophores
Monoblastic,
integrated, terminal,
determinate, conical
to cylindrical
Conidia Solitary, dry,
acropleurogenous or
pleurogenous, simple,
helicoid or circinate,
centripetally coiled,
colourless to brown or
olivaceous brown
Solitary, dry, acrogenous
or acropleurogenous,
simple, helicoid or
formed in disk-like
spore body, basal cell
attached centripetally
coiled, hyaline
Solitary, dry,
acropleurogenous or
pleurogenous, simple,
ellipsoidial or sometimes
cylindrical spore body,
barrel-shaped, colourless to
brown
Solitary, dry, pleurogenous or
acropleurogenous, simple,
helicoid to somewhat
cochleate, centripetally coiled
conidium, colourless or
brightly coloured in mass
Catenate, dry, acropleu-
rogenous, simple,
cylindrical, ovoid
or ellipsoidal,
colourless to
green, olivaceous
or brown
Solitary, dry, acrogenous,
simple, circinate, tightly
coiled in centrifugally
coiled conidium, brown
Solitary, dry,
holoblastic,
acrogenous, simple,
circinate, tightly
coiled, centrifugally
coiled (excentric),
hyaline
Conidial
filament Coiled, non-hygroscopic,
septate, smooth Coiled usually in a two-
dimensional plane but
sometimes three-
dimensional,
hygroscopic, septate
Coiled three-dimensionally
to form hollow spore
bodies, septate, smooth.
Coiled, hygroscopic,
septate, smooth Coiled three-
dimensionally
to form a cylindrical,
ovoid or ellipsoidal
spore body or with
only a few loose
coils,
septate, smooth,
colourless to green,
olivaceous or brown.
Coiled in two-dimensional
plane, non-hygroscopic,
septate, smooth, brown
Coiledintwo-
dimensional planes,
but sometimes tend
to three-dimensional,
non-hygroscopic,
sepate, smooth,
hyaline
Teleomorph of
type species Thaxteriella pezizula
(Berk. & M.A. Curtis) Tubeufia cylindrothecia
(Seaver) Orbilia luteorubella
(Nyl.) Tubeufia cerea
(Berk. &
M.A. Curtis)
Hymenoscyphus
paradoxus (P.J.
Fisher & J. Webster)
unknown unknown
References Ellis (1971); Goos
(1986); Zhao et al. (2007)Goos (1985);
Zhao et al. (2007)Ellis (1971); Goos
(1986); Zhao et al.
(2007)
Ellis (1971);
Zhao et al.
(2007)
Ellis (1971); Goos
(1985);
Zhao et al. (2007)
Holubová-Jechová
(1991)This study
81 Page 10 of 12 Mycol Progress (2015) 14:81
to brown (Ellis 1971;Goos1986;Zhaoetal.2007). In the
Index of Fungi (www.indexfungorum.org), the current name
of H. sessile is Orbilia luteorubella (Orbiliomycetes).
Additionally, Helicoön sessile is the type species of the
genus; the remaining species of Helicoön are not
orbiliaceous (Orbiliomycetes), i.e. H. gigantisporum and H.
richonis (Dothideomycetes) as shown in the combined
nuc18S-nuc28S phylogeny.
Helicogoosia shares some morphological features with
Helicocentralis with conidiogenous cells being monoblastic,
integrated, terminal, determinate and sharing centrifugally
coiled conidia, and non-hygroscopic, and smooth-walled, sep-
tate conidia. However, Helicogoosia has proliferating, lance-
olate, conidiogenous cells, which become easily detached af-
ter secession leaving clear minute pores in the wall of conid-
iophores (Holubová-Jechová 1991; Table 3). In addition, no
cultures of Helicogoosia were available for sequencing.
Helicoid conidial forms have evolved a number of times in
saprobic fungi in streams or in aero-aquatic species on leaf
litter (Nawawi 1985) or on wood (Tsui and Berbee 2006).
Asexual fungi with helicoid conidia are widely distributed
and nest within a number of fungal classes: Dothideomycetes
(e.g. Helicoma,Thaxteriella,Helicangiospora,Helicomyces,
Neocanthostigma), Leotiomycetes (e.g. Halenospora,
Helicodendron) and Sordariomycetes (e.g. Zalerion,
Cumulospora), occurring on a variety of substrata (leaf litter,
wood) and in terrestrial, freshwater and marine habitats. Sex-
ual morphs of Tubeufiales (Dothideomycetes) include many
genera with helicoid conidia: Acanthohelicospora,
Chlamydotubeufia,Neoacanthostigma,andThaxteriella
(Boonmee et al. 2014). Likewise, Leotiomycetes includes taxa
with helicoid conidia and sexual morphs in Mollisia and
Hymenoscyphus (Helicodendron giganteum and
H. paradoxum, respectively; Fisher and Webster 1983).
Acknowledgements This research was conducted under collaboration
between the National Center for Genetic Engineering and Biotechnology
(BIOTEC, Thailand) and the NITE Biological Resource Center (NBRC,
Japan). Financial support by the Bioresources Research Network (BRN,
P-00-20194) is gratefully acknowledged. Dr. Nattawut Boonyuen wishes
to thank Dr. Somsak Sivichai who initiated the project of aero-aquatic
fungi as a Thai freshwater fungi at BIOTEC. We gratefully thank Dr. Lily
Eurwilaichitr, Dr. Kanyawim Kirtikara from BIOTEC, Prof. Dr. Morakot
Tanticharoen from King Mongkut’s University of Technology Thonburi,
Dr. Ken-ichiro Suzuki and Dr. Hiroko Kawasaki from NITE Biological
Resource Center for their interest in aero-aquatic fungi under the collab-
orative project between BIOTEC and NITE. Panthita Ruangareerate from
Genome Technology Research Unit, BIOTEC is thanked for RAxML.
We also acknowledge Prof. Dr. E.B. Gareth Jones, from King Saud Uni-
versity, Saudi Arabia, for his pre-submission comments.
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