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DNA barcoding of fungi: A case study using ITS sequences for identifying
aquatic hyphomycete species

Article  in  Fungal Diversity · October 2010

DOI: 10.1007/s13225-010-0056-y

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Fungal Diversity
DOI 10.1007/s13225-010-0056-y
DNA barcoding of fungi: a case study using ITS sequences
for identifying aquatic hyphomycete species
Sahadevan Seena & Cláudia Pascoal &
Ludmila Marvanová & Fernanda Cássio
Received: 24 May 2010 / Accepted: 3 August 2010
# Kevin D. Hyde 2010
Abstract Aquatic hyphomycetes are a polyphyletic group
of fungi that play a crucial role in organic matter turnover in
streams. They have been traditionally identified based on
the morphology of conidia collected in stream water or
obtained from leaves colonized in nature upon aeration in
the laboratory. Therefore, species identification is limited
by our ability to induce conidium production and to
establish pure cultures. Conidial shapes are believed to be
the result of convergent evolution, so similar conidia may
be produced by different conidiogenesis processes, which
may prevent unambiguous identification. Currently, a great
effort in fungal taxonomy is being made at introducing a set
of criteria based on comparisons of selected nucleotide
sequences instead of or in addition to phenotypic charac-
ters. We examined the suitability of ITS1-5.8S-ITS2 rRNA
gene region or its subregions (ITS1 and ITS2) to identify
aquatic hyphomycetes, by sequencing and comparing these
regions in 94 fungal isolates belonging to 19 species
collected in Portuguese streams with different environmen-
tal conditions during 8 years. Sequences of ITS1, ITS2 and
ITS1-5.8S-ITS2 rRNA genes of the Portuguese isolates of
aquatic hyphomycetes and those from the GenBank
exhibited taxonomic cohesiveness, the isolates grouped
with their respective species but all Tricladium species did
S. Seena (*) : C. Pascoal : F. Cássio
CBMA (Centre of Molecular and Environmental Biology),
Department of Biology, University of Minho,
Campus de Gualtar,
4710-057 Braga, Portugal
e-mail: ssahadevan@bio.uminho.pt
L. Marvanová
Czech Collection of Microorganisms, Faculty of Science,
Masaryk University,
Tvrdého 14,
602 00 Brno, Czech Republic
not group within the Tricladium genus. Cohesiveness was
not observed between isolates with respect to location,
condition of stream or date of collection. Evolutionary
divergences (ITS1-5.8S-ITS2 sequences; Kimura 2-
parameter distance) between con-specific isolates were
shallow and a deep divergence between species was
generally observed. The NJ trees based on ITS1 or ITS2
rRNA gene sequences had lower statistical support for
some internal nodes, we therefore propose ITS1-5.8S-ITS2
rRNA gene as barcode for identifying species of aquatic
hyphomycetes.
Keywords Aquatic hyphomycetes . ITS . Barcode .
Evolutionary divergence
Introduction
Aquatic hyphomycetes were first reported by Ingold in
1942, but it was over 30 years ago that stream ecologists
realized their importance (Bärlocher and Kendrick 1974;
Suberkropp and Klug 1976). This polyphyletic group of
fungi play a crucial role in organic matter turnover in
streams, acting as intermediaries between plant litter and
invertebrate shredders and thereby contributing to the
functioning of freshwater ecosystems (Bärlocher 2005;
Gessner et al. 2007). More than 300 species of aquatic
hyphomycetes are currently known with a worldwide
distribution (Descals 1997; Shearer et al. 2007), mainly in
lotic habitats from the equator to the Arctic (Shearer et al.
2007) and to the Tierra del Fuego (Godeas 1985), although
some species are likely to be limited to certain latitudes and
altitudes (Bärlocher 2007). Meiospores may be more apt
than the asexual spores (conidia) for ensuring the cosmo-
politan distribution of aquatic hyphomycetes (Bärlocher

2009). However, to date, the sexual state has been reported
in only ca 10% of the aquatic hyphomycetes (Bärlocher
2007; Marvanová 2007), and the majority of anamorph/
teleomorph associations are still a mystery. Undoubtedly,
various kinds of vegetative resting structures (chlamydo-
spores, microsclerotia), known to be part of the life cycle in
many species, contribute to the long-distance dispersal.
The traditional aquatic hyphomycete classification is
based on morphology of conidia, i.e. their shape and
ontogeny (Alexopoulos et al. 1996; Marvanová 1997).
However, practicing identification based on these criteria is
often limited by our ability to induce conidium production
from colonized plant debris and to establish pure cultures.
The common approach in stream ecology is to identify
aquatic hyphomycetes on the basis of detached conidia
collected in water or obtained from leaves colonized in
nature through aeration methods in laboratory (Descals
2005). Both approaches have some pitfalls: the most
common conidium morphologies include sigmoid (worm
shaped, scolecoform) and multiradiate (stauroform, often
tetraradiate) forms that are believed to have evolved
convergently as independent adaptations to similar envi-
ronmental pressures. Hence, similar conidia may be
produced by different conidiogenous processes and ignor-
ing this may lead to false identification. Conidium
production induced by aeration may be more advantageous
for rheophilic species adapted to quick conidium release
than for those that sporulate later or prefer slowly moving
water. Thus, some species may remain undetected, when
the laboratory conditions for sporulation do not meet their
requirements in nature. Moreover, the few published keys
comprise mainly the most common species and the
dispersion of formal taxonomic descriptions make reliable
species identification difficult.
Currently, a great effort in fungal taxonomy is being
made at introducing a set of criteria based on comparisons
of nucleotide sequences of selected genes instead of or in
addition to phenotypic characters (Webster and Weber
2007). The relevance of DNA sequence information in
contemporary mycology is exemplified by the recent
concept of fungal barcoding, which seeks species identifi-
cation through the use of standardized DNA sequences
from annotated reference specimens (Hebert et al. 2003;
Hajibabaei et al. 2006, Seifert et al. 2007; Nilsson et al.
2008). In the All Fungi Barcoding meeting held in 2007 the
internal transcribed spacer (ITS) region of nuclear rRNA
gene was proposed as the most appropriate candidate for
barcoding fungi (Rossman 2007). Certain merits of this
region are that it is very easy to sequence with specific
primers and it also has high capacity to evolve rapidly.
However, it was admitted that this region does not provide
precise species recognition for some fungal groups, such as
for yeasts, members of the Glomeromycota and plant
Fungal Diversity
pathogenic ascomycetes (Rossman 2007). The fungal
kingdom is estimated to comprise 1.5 million species out
of which only less than 1% is sequenced for the ITS region
(Hawksworth 2001; Nilsson et al. 2006). Nilsson et al.
(2008) have shown that on average the variability of ITS1
region exceeds that of ITS2; however, they found greater
variability in ITS2 than ITS1 in 34% of the studied fungal
species. It was also concluded that these two spacer regions
are not subjected to independent evolution because the
variation of both regions are highly correlated. The 5.8S
rRNA gene region is well conserved within all fungal
species (Nilsson et al. 2008).
The problem of misidentification of aquatic hyphomy-
cetes when relying on conidial shape could be overcome by
barcodes. Bärlocher et al. (2010) successfully extracted
DNA from single conidia and amplified partial ITS region.
Recently, it has been demonstrated that ITS sequences of
aquatic hyphomycetes provided the most appealing clado-
gram along with optimal threshold factor of 10 between the
interspecific and intraspecific variability for the Tetracla-
dium spp. (Letourneau et al. 2010). In January 2010,
species level records on Barcode of Life Data Systems
(BOLD) based on cytochrome c oxidase I (CO1) gene
sequences were 759,950 and those of ITS rRNA gene
sequences were very few; so most queries with ITS
sequence data are not likely to yield a successful match
(http://www.boldsystems.org). The other major hurdle is
that there are a low number of ITS sequences of aquatic
hyphomycetes in the GenBank, so there is a demand to
scale up sequencing of aquatic hyphomycete isolates and
thereby enhance data for generating barcodes. Inopportune-
ly, the type strains of many aquatic hyphomycetes do not
exist in any registered culture collection (http://wdcm.nig.
ac.jp/simple_search.html). In such cases, the isolates whose
identity has been guaranteed by a specialist would serve as
a reference material for generating barcodes (Letourneau et
al. 2010; http://www.cbs.knaw.nl/research/collection.aspx).
In an attempt to examine the suitability of ITS sequences to
identify aquatic hyphomycetes, this rRNA gene region was
sequenced and compared in 94 isolates belonging to 19
species. We used the ITS1-5.8S-ITS2, ITS1 and ITS2
rRNA gene sequences to assess the feasibility of using the
whole ITS region or one of these subregions as DNA
barcodes in aquatic hyphomycetes. The fungi employed in
this study were isolated from Portuguese streams with
different environmental conditions between the years of
1999 and 2007, and include cosmopolitan and rare species
of aquatic hyphomycetes (Marvanová et al. 2003; Pascoal
et al. 2005). Conidium morphology of the selected species
is well documented elsewhere (novel and rare species from
Portugal: Collembolispora barbata, Flagellospora penicil-
lioides originally identified as F. curta, Geniculospora
grandis, Heliscus submersus, Tricladium terrestre—
Fungal Diversity
Marvanová et al. 2003; common species in temperate
regions: Articulospora tetracladia, Anguillospora filifor-
mis, Clavariopsis aquatica, Dimorphospora foliicola,
Fontanospora fusiramosa, Heliscus lugdunensis, Lunulo-
spora curvula, Tetracladium marchalianum Tricladium
chaetocladium, T. splendens, Varicosporium elodeae and
Ypsilina graminea—Gulis et al. 2005; and other species,
namely Tetracladium breve—Letourneau et al. 2010, and
Tricladium indicum—Sati and Tiwari 1992). We also
attempted to visualize the phylogenetic clustering of these
species using published ITS sequences at the National
Center for Biotechnology Information (NCBI) (http://
www.ncbi.nlm.nih.gov). This study is expected to lead to
an assembly of reference libraries of barcode sequences
for aquatic hyphomycete species in order to develop an
identification tool for biodiversity assessment with direct
applications in ecological studies.
Materials and methods
The fungal isolates used in this study are monoconidial and
are maintained in the culture collection of the Centre of
Molecular and Environmental Biology (CBMA), Depart-
ment of Biology of the University of Minho. A total of 19
species were used, namely, Articulospora tetracladia (10
isolates), Anguillospora filiformis (5 isolates), Clavariopsis
aquatica (4 isolates), Collembolispora barbata (2 isolates),
Dimorphospora foliicola (5 isolates), Flagellospora pen-
icillioides (4 isolates), Fontanospora fusiramosa (4 iso-
lates), Geniculospora grandis (2 isolates), Heliscus
lugdunensis (11 isolates), H. submersus (2 isolates),
Lunulospora curvula (3 isolates), Tetracladium breve (1
isolate) T. marchalianum (5 isolates), Tricladium chaeto-
cladium (4 isolates), T. cf. indicum (2 isolates), T. splendens
(7 isolates), T. terrestre (4 isolates), Varicosporium elodeae
(16 isolates), Ypsilina graminea (3 isolates). The origin of
fungal isolates and the substrate sampled (stream water,
foam, leaves or twigs) are outlined in Table 1. All cultures
were grown at room temperature on 1.5% malt extract agar.
After 2 weeks, the fungal DNA was extracted using MoBio
Ultraclean Soil DNA Isolation Kit according to manufac-
turer’s instruction to obtain maximum yield and stored at
−20°C. Fungal DNA (2 μl) was mixed with 2 μl of ITS1
and 2 μl of ITS4 primers (White et al. 1990), 14 μl of
GoTaq® Green Master Mix (Promega) and 5 μl water
supplied with the GoTaq® Green Master Mix in 0.2 ml strip
tubes. The DNA amplification was done in a BIO RAD
MyCycler™ Thermal Cycler as follows: 1. initial denatur-
ation (94°C, 2 min); 2. denaturation (94°C, 45 sec); 3.
annealing (55°C, 45 sec); 4. extension (73°C, 1.30 min); 5.
repeat steps 1 to 4 (38 cycles); 6. final extension (70°C,
10 min); 7. pause and incubation until retrieved at 4°C. The
PCR products were run on a 1.3% agarose gel at 90 V to
check the presence of the desired band. The PCR products
were then cleaned using GenElute™ PCR Clean-Up Kit
(Sigma) according to manufacturer’s protocols and its
concentration was confirmed using nanodrop (Spectropho-
tometer ND-1000). The amplicons were sequenced using
ITS1, ITS2 and ITS3 primers (White et al. 1990). Cycle
sequencing was performed using Big Dye Terminator V3.1
Kit (Applied Biosystems) according to the manufacturer.
Excess dyes were removed using NucleoSEQ kit
(Macherey-Nagel). Purified sample was denaturated with
formamide during 5 min at 95°C and then run on an ABI
310 Genetic analyzer (Applied Biosystems) using POP4
polymer.
Consensus sequences of ITS1-5.8S-ITS2 region were
obtained using CodonCode Aligner 2.0.6 (CodonCode Co.,
USA) and aligned using MEGA 4 (Tamura et al. 2007), the
alignment was further refined with BioEdit 7.0.9 (Hall
1999). Sequence divergence was analyzed by using Kimura
2-parameter (K2P) distance (Kimura 1980) and the dendro-
grams for ITS1- 5.8S-ITS2, ITS1 and ITS2 regions were
generated with neighbour-joining (NJ) method (Saitou and
Nei 1987) using MEGA 4 (Tamura et al. 2007), after testing
the suitability of data for estimating NJ trees by analyzing
the average Jukes-Cantor (JC) distance (Hall 2008). All
positions containing alignment gaps and missing data were
eliminated only in pairwise sequence comparisons. Branch
support was assessed with bootstrap analysis (1000 repli-
cates, Felsenstein 1985). The sequence of Cudonia lutea
(AF433151) from GenBank was used to root the trees.
Sequence data obtained from the Portuguese isolates were
deposited in GenBank (Table 1) and alignments in
TreeBASE (http://purl.org/phylo/treebase/phylows/study/
TB2:S10536). Sequences of the same gene regions of
anamorph/teleomorph when available from GenBank were
included in this study (Table 2). The known anamorph/
teleomorph connections are outlined in Table 3.
Results
All 19 aquatic hyphomycete species had different ITS1-
5.8S-ITS2, ITS1 and ITS2 sequences. The average se-
quence divergence employing JC distance was < 1 for
ITS1-5.8S-ITS2, ITS1 and ITS2 sequences and hence these
were used to construct NJ trees with K2P distances. The
ITS1-5.8S-ITS2 region of all species consisted of 436–
583 bp. The 5.8S rDNA sequences were 158 bp long and
were similar among isolates of the same species.
The ITS1-5.8S-ITS2 sequences of the Portuguese iso-
lates and those from the GenBank exhibited taxonomic
cohesiveness and all the isolates grouped within their
respective species, but Tricladium species appeared in two
 Fungal Diversity

Table 1 Aquatic hyphomycete taxa, isolate reference, stream loca- River sites (E1, E5), Laja Stream (La), Lamas (Lm), Maceira River
tion, sampled substrate and GenBank accession number of sequenced (Ma), Patanha Stream (Pa), Pelhe River (Pe), Peliteira Stream (Pi),
isolates. The sampling sites are: Alhões Stream (Al), Ave River sites Preguiça Stream (Pr), São João do Campo Stream (Sj) and Tinhela
(L1, L2, L6 and L7), Botão Stream (Bo), Cávado River (Ca), Este Stream (Ti)

Taxon Isolate Stream location Sampled GenBank accession

reference substrate number

Articulospora tetracladia Ingold UMB-014.00 L6 (41°21′N 8°25′W) SW GQ411288

UMB-087.01 Ma (41°46'N 8º08'W) F GQ411289
UMB-122.01 Sj (41°45′N 8°11′W) F GQ411284
UMB-320.07 Al (40º30'N 7º52'W) L GQ411292
UMB-323.07 Pa (40º59'N 8º00'W) L GQ411286
UMB-328.07 Pa (40º59'N 8º00'W) L GQ411287
UMB-332.07 Al (40º30'N 7º52'W) L GQ411291
UMB-333.07 Al (40º30'N 7º52'W) L GQ411290
UMB-343.07 Pa (40º59'N 8º00'W) L GQ411293
UMB-376.07 Pi (41°26′N 7°33′W) L GQ411285
Anguillospora filiformis Greath. UMB-015.00 L7 (41°20′N 8°31′W) SW GQ411263
UMB-102.01 Pe (41°24′N 8°30′W) L GQ411261
UMB-117.01 E5 (41°30′N 8°27′W) L GQ411262
UMB-148.01 E5 (41°30′N 8°27′W) L GQ411260
UMB-232.02 E1 (41°34′N 8°19′W) L GQ411259
Clavariopsis aquatica De Wild. UMB-019.99 L2 (41°32′N 8°15′W) L GQ411316
UMB-110.01 Pe (41°24′N 8°30′W) L GQ411318
UMB-156.01 E5 (41°30′N 8°27′W) L GQ411319
UMB-195.01 E5 (41°30′N 8°27′W) L GQ411317
Collembolispora barbata Marvanová, UMB-048.01 Sj (41°45′N 8°11′W) F GQ411302
Pascoal and Cássio UMB-088.01 Sj (41°45′N 8°11′W) F GQ411303
Dimorphospora foliicola Tubaki UMB-030.01 E5 (41°30′N 8°27′W) L GQ411314
UMB-031.01 E5 (41°30′N 8°27′W) L GQ411311
UMB-034.01 E5 (41°30′N 8°27′W) L GQ411313
UMB-172.01 E5 (41°30′N 8°27′W) L GQ411312
UMB-218.00 E5 (41°30′N 8°27′W) L GQ411315
Flagellospora penicillioides Ingold UMB-300.05 E1 (41°34′N 8°19′W) SW GQ411324
UMB-302.05 E1 (41°34′N 8°19′W) SW GQ411326
UMB-304.05 E1 (41°34′N 8°19′W) SW GQ411325
UMB-350.07 Bo (40°18′N 8°23′W) L GQ411323
Fontanospora fusiramosa Marvanová, P.J. UMB-075.01 Ma (41°46'N 8º08'W) F GQ411267
Fisher, Descals and Bärlocher UMB-085.01 Ma (41°46'N 8º08'W) F GQ411264
UMB-167.01 La (41°44′N 8°09′W) T GQ411265
UMB-208.01 Sj (41°45′N 8°11′W) F GQ411266
Geniculospora grandis (Greath.) Nolan UMB-176.01 Lm (41°30′N 8°25′W) F GQ411354
UMB-198.01 E1 (41°34′N 8°19′W) F GQ411353
Heliscus lugdunensis Sacc. and Thérry UMB-003.00 L6 (41°21′N 8°25′W) SW GQ411335
UMB-044.01 Sj (41°45′N 8°11′W) F GQ411329
UMB-079.01 La (41°44′N 8°09′W) T GQ411338
UMB-160.01 La (41°44′N 8°09′W) T GQ411333
UMB-164.01 Ma (41°46'N 8º08'W) F GQ411334
UMB-311.06 Ca (41°38′N 8°19′W) F GQ411336
UMB-362.07 Ti (41°26′N 7°32′W) F GQ411331
UMB-364.07 Pi (41°26′N 7°33′W) L GQ411330
UMB-369.07 Ti (41°26′N 7°32′W) T GQ411332
UMB-378.07 Pi (41°26′N 7°33′W) T GQ411337
UMB-385.07 Pi (41°26′N 7°33′W) T GQ411339
Fungal Diversity
Table 1 (continued)
Taxon Isolate Stream location
reference
Heliscus submersus H.J. Huds. UMB-001.99 L1 (41°33′N 8°14′W)
UMB-135.01 E5 (41°30′N 8°27′W)
Lunulospora curvula Ingold UMB-108.01 E5 (41°30′N 8°27′W)
UMB-115.01 E5 (41°30′N 8°27′W)
UMB-305.05 E1 (41°34′N 8°19′W)
Tetracladium breve A. Roldán UMB-046.01 Sj (41°45′N 8°11′W)
Tetracladium marchalianum De Wild. UMB-064.01 Pe (41°24′N 8°30′W)
UMB-094.01 E5 (41°30′N 8°27′W)
UMB-095.01 E5 (41°30′N 8°27′W)
UMB-118.01 E1 (41°34′N 8°19′W)
UMB-239.02 Pe (41°24′N 8°30′W)
Tricladium chaetocladium Ingold UMB-013.99 L1 (41°33′N 8°14′W)
UMB-035.01 E5 (41°30′N 8°27′W)
UMB-062.01 E5 (41°30′N 8°27′W)
UMB-065.01 Pe (41°24′N 8°30′W)
Tricladium indicum Sati and N. Tiwari UMB-026.01 Lm (41°30′N 8°25′W)
UMB-241.02 E1 (41°34′N 8°19′W)
Tricladium splendens Ingold UMB-068.01 Sj (41°45′N 8°11′W)
UMB-100.01 E5 (41°30′N 8°27′W)
UMB-330.07 Pa (40º59'N 8º00'W)
UMB-347.07 Al (40º30'N 7º52'W)
UMB-352.07 Pa (40º59'N 8º00'W)
UMB-356.07 Pa (40º59'N 8º00'W)
UMB-372.07 Pi (41°26′N 7°33′W)
Tricladium terrestre D. Park UMB-179.01 E1 (41°34′N 8°19′W)
UMB-181.01 E1 (41°34′N 8°19′W)
UMB-083.01 Lm (41°30′N 8°25′W)
UMB-089.01 Lm (41°30′N 8°25′W)
Varicosporium elodeae W. Kegel UMB-005.00 Ca (41°38′N 8°19′W)
UMB-055.01 E1 (41°34′N 8°19′W)
UMB-096.01 Ma (41°46'N 8º08'W)
UMB-101.01 Sj (41°45′N 8°11′W)
UMB-171.01 E1 (41°34′N 8°19′W)
UMB-213.02 Sj (41°45′N 8°11′W)
UMB-249.02 E1 (41°34′N 8°19′W)
UMB-310.06 Ca (41°38′N 8°19′W)
UMB-314.06 Pi (41°26′N 7°33′W)
UMB-319.06 Pi (41°26′N 7°33′W)
UMB-331.07 Pa (40º59'N 8º00'W)
UMB-340.07 Al (40º30'N 7º52'W)
UMB-345.07 Al (40º30'N 7º52'W)
UMB-366.07 Pi (41°26′N 7°33′W)
UMB-382.07 Pi (41°26′N 7°33′W)
UMB-384.07 Pi (41°26′N 7°33′W)
Ypsilina graminea (Ingold, P.J. McDougall UMB-098.01 E5 (41°30′N 8°27′W)
and Dann) Descals, J. Webster and Marvanová UMB-111.01 E5 (41°30′N 8°27′W)
UMB-354.07 Al (40º30'N 7º52'W)
L leaves; F foam; SW stream water; T twigs
Sampled GenBank accession
substrate number
L GQ411327
L GQ411328
L GQ411321
L GQ411322
SW GQ411320
F GQ411301
L GQ411294
L GQ411298
L GQ411296
F GQ411295
L GQ411299
L GQ411309
L GQ411310
L GQ411308
L GQ411307
F GQ411341
F GQ411340
F GQ411349
L GQ411347
L GQ411348
L GQ411346
L GQ411351
L GQ411352
L GQ411350
F GQ411344
F GQ411345
F GQ411342
F GQ411343
F GQ411279
F GQ411272
F GQ411274
F GQ411275
F GQ411276
F GQ411282
F GQ411270
L GQ411283
F GQ411273
F GQ411281
L GQ411277
F GQ411269
F GQ411271
L GQ411278
L GQ411268
L GQ411280
F GQ411304
F GQ411306
L GQ411305
 Fungal Diversity

Table 2 GenBank accession number for ITS region sequences for Table 3 Teleomorph/anamorph connections in aquatic hyphomycete
aquatic hyphomycetes species used in this study

Species Accession Origin of isolate Anamorph Order Teleomorph

nº
Articulospora tetracladia Helotiales Ombrophila tetracladia
Articulospora tetracladia EU998929 CCM F-11101 Anguillospora filiformis Unknown Unknown
Anguillospora filiformis AY148104 CCM F-20687 Clavariopsis aquatica Pleosporales Massarina sp.
Clavariopsis aquatica GQ152143 Unknown Collembolispora barbata Unknown Unknown
Cudoniella indica DQ202513 CBS 430.94 = CCM Dimorphospora foliicola Helotiales Hymenoscyphus
F-100 93a foliicola
Cudonia lutea AF433151 Unknown Fontanospora fusiramosa Unknown Unknown
Dimorphospora foliicola DQ202518 Unknown Flagellospora Hypocreales Nectria penicillioides
Hymenoscyphus DQ202514 CBS 651.66 = IFO penicillioides
varicosporoides 8268a Geniculospora grandis Helotiales Hymenoscyphus
Nectria lugdunensis DQ247778 CCM F-245 africanus
Tetracladium breve FJ000405 CCM F-12505 Heliscus lugdunensis Hypocreales Nectria lugdunensis
Tetracladium marchalianum FJ000370 CCM F-19399 Heliscus submersus Unknown Unknown
Tricladium chaetocladium DQ202515 CBS 249.90 = HME Lunulospora curvula Sordariales Unknown
4375a Tricladium Helotiales Hydrocina chaetocladia
Tricladium splendens DQ202510 SS 2274 chaetocladium
Tricladium indicum Helotiales Cudoniella indica
Tricladium terrestre DQ202519 CBS 697.73a
Tricladium splendens Helotiales Hymenoscyphus
Varicosporium elodeae DQ202517 CBS 541.92 = CCM
splendens
F-04276
Tricladium terrestre Helotiales Unknown
a
ex-type or ex-isotype Tetracladium breve Unknown Unknown
Tetracladium Unknown Unknown
marchalianum
rather distant groups (Fig. 1). The average overall evolu- Varicosporium elodeae Helotiales Unknown
tionary divergence (i.e., number of base substitutions per Ypsilina graminea Unknown Unknown
site from averaging over all sequence pairs) for ITS1-5.8S-
ITS2 sequences was 23%. The maximum evolutionary
divergence was observed within T. indicum, Varicosporium sequences of isolates of the same species exhibited very
elodeae, Articulospora tetracladia, Anguillospora filiformis shallow divergences.
and Ypsilina graminea but never exceeded 2%. All the The evolutionary divergences among species based on
Portuguese A. tetracladia isolates diverged by 1.7–1.9% ITS1-5.8S-ITS2 sequences are given in Table 4. The
from EU998929 (sequence originating from CCM sequences exhibited deep divergences between the species
F-11101). One of the isolates of T. cf. indicum (UMB- with some exceptions. The T. chaetocladium diverged by
026.01) differed by 1.0–1.2% from Hymenoscyphus vari- 23.8–24.8% from the other Tricladium species (T. terrestre,
cosporoides IFO 8268, Cudoniella indica DQ202513 T. splendens and T. indicum); however, the evolutionary
(sequence from CCM F-10093, the ex-type culture of T. divergence among T. terrestre, T. splendens and T. indicum
indicum) and T. cf. indicum UMB 241.02. The 16 isolates was only 2.9–3.4%. Geniculospora grandis diverged from
of V. elodeae from Portugal formed two groups each T. indicum by only 5% and Heliscus submersus diverged
consisting of 9 and 7 isolates (Fig. 1) diverging by 1.8%. only by 2.2% from Flagellospora penicillioides but by
The colonies of isolates included in the same group of V. 7.4% from H. lugdunensis. Among the other species,
elodeae DQ202517 (sequence from CCM F-04276, a namely V. elodeae, A. tetracladia, A. filiformis and
typical V. elodeae isolate producing green pigment) were Fontanospora fusiramosa, an evolutionary divergence of
pigmented (green or yellow) while the others were not. The 2.6–7.4% was found. For instance, F. fusiramosa showed
Portuguese isolates of A. filiformis diverged by 1.5% from an evolutionary divergence from V. elodeae, A. tetracladia
the Canadian isolate of A. filiformis AY148104 (sequences and A. filiformis by 2.6, 3.6 and 6.2%, respectively. In
from CCM F-20687). Ypsilina graminea UMB-098.01 addition, Y. graminea diverged from Collembolispora
differed from the two other Portuguese isolates of this barbata by 8.7%. The divergence rate between other
species by 1%. Dimorphospora foliicola DQ202518 species ranged between 16.3–46.5%.
exhibited 0.9% divergence from the five Portuguese The ITS1 and ITS2 regions of the fungal isolates used in
isolates. All the other taxa or groups included in this study this work consisted of 132–252 bp and 146–173 bp,
did not exhibit any internal divergence. On an overall basis, respectively. The ITS1 sequences varied in length among
Fungal Diversity

ƒFig. 1 Neighbour joining tree based on ITS1-5.8S-ITS2 sequences

using Kimura 2-parameter distances. Bootstrap values above 95%,
calculated from 1000 full heuristic replicates are shown at the nodes.
Scale bar indicates one base change per 100 nucleotide positions

the species by 120 bp, whereas ITS2 only by 27 bp. The

average overall divergence for ITS1 and ITS2 region in all
sequences was found to be 42% and 38%, respectively;
however, for some sequence pairs of ITS1 the evolutionary
divergence was >100% (not shown). The NJ trees based on
ITS1 and ITS2 sequences exhibited taxonomic cohesive-
ness, grouped the anamorphs with their respective tele-
omorphs and T. chaetocladium formed a separate clade
from other Tricladium species (Figs. 2 and 3), similar to NJ
tree based on ITS1-5.8S-ITS2 sequences (Fig. 1). However,
V. elodeae isolates formed two clear separate groups only in
NJ tree based on ITS2 sequences with a bootstrap support
of 98 and 100% (Fig. 3). The NJ trees obtained with the
ITS1, ITS2 and ITS-5.8S-ITS2 sequences did not share
similar topology and the bootstrap support was higher for
ITS1-5.8S-ITS2 rRNA gene sequences followed by ITS2
and ITS1 rRNA gene sequences (Figs. 1, 2 and 3).

Discussion

There is an ever increasing amount of pure cultures and

environmental sequences being accumulated, especially
from ecological surveys, waiting for a rapid and reliable
method of identification. To date there are very few DNA
sequences, including those for ITS-5.8S-ITS2 region, of
aquatic hyphomycetes available in the NCBI for compar-
ison. Moreover, the anamorph-teleomorph connections and
order classifications of aquatic hyphomycetes included in
our study are scanty (Table 3). Although there are many
methods for estimating the base pair distances between
DNA sequences, the K2P distance was used in our study as
it is the most widely employed in barcoding studies (Ward
2009). The ITS1-5.8S-ITS2, ITS1 and ITS2 sequences
generated were species/genus specific, but the NJ trees
obtained with these sequences did not share similar
topology (Figs. 1, 2 and 3). Moreover, the ITS1-5.8S-
ITS2 sequences of Lunulospora curvula available in the
NCBI (FJ000404 and AY729938) did not exhibit taxonom-
ic cohesiveness and formed two separate sister taxa with
Collembolispora barbata and Ypsilina graminea (not
shown), and hence were not used further in our analyses.
However, all the Portuguese isolates of L. curvula formed a
single group. Our current knowledge about the L. curvula is
scarce and further studies are yet to be conducted with
regard to its phylogeny.
Since the divergence rate of ITS1 region was 4% higher
than that of ITS2, there are chances that the evolution of
most of the aquatic hyphomycetes in our study may result
Table 4 Evolutionary divergence (%) between aquatic hyphomycete species based on ITS-5.8S-ITS2 sequences. The number of base substitutions per site from averaging overall sequence pairs
between the species is shown. Analyses were done with Kimura 2-parameter method

Aquatic hyphomycetes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Tricladium chaetocladium (1) 0

Tricladium splendens (2) 23.9
Tricladium terrestre (3) 24.8 3.0
Tricladium indicum (4) 23.8 2.9 3.4
Geniculospora grandis (5) 25.6 6.3 6.2 5.0
Varicosporium elodeae (6) 23.3 22.2 23.0 20.9 20.7
Articulospora tetracladia (7) 23.2 23.0 22.5 20.9 22.3 4.7
Anguillospora filiformis (8) 24.7 22.8 23.7 21.4 21.0 6.8 7.4
Fontanospora fusiramosa (9) 22.5 20.4 20.5 18.9 19.9 2.6 3.6 6.2
Tetracladium marchalianum (10) 25.0 24.3 23.7 23.7 23.4 17.3 17.4 21.5 16.9
Tetracladium breve (11) 25.4 25.6 24.1 24.2 22.5 17.4 17.1 20.3 16.3 2.6
Dimorphospora foliicola (12) 27.5 22.4 22.4 22.7 22.5 22.0 23.8 23.5 21.9 25.1 25.4
Collembolispora barbata (13) 30.2 25.5 25.2 25.1 24.2 22.2 20.9 23.6 22.3 24.8 24.8 36.7
Ypsilina graminea (14) 29.0 23.9 23.6 23.9 23.9 21.9 20.1 24.0 21.2 24.2 24.2 36.0 8.7
Clavariopsis aquatica (15) 37.8 35.7 35.2 34.3 35.3 37.1 34.6 36.1 35.8 38.7 39.1 42.4 33.2 33.1
Lunulospora curvula (16) 38.0 37.7 37.9 39.4 38.7 36.2 35.6 38.7 34.2 37.8 36.5 40.4 35.2 34.2 46.5
Flagellospora penicillioides (17) 31.1 32.1 33.8 32.4 32.7 28.6 29.6 30.0 28.4 31.2 30.8 33.1 28.4 30.2 42.5
Heliscus submersus (18) 31.5 32.3 34.0 32.6 33.2 30.0 31.0 31.4 29.8 33.0 32.6 34.3 29.0 30.7 43.7 26.0 2.2
Heliscus lugdunensis (19) 30.7 31.8 33.1 32.9 33.9 29.1 30.8 30.5 28.9 31.0 30.3 33.6 27.0 28.5 41.7 23.0 8.0 7.4 0

Numbers in parentheses denote each aquatic hyphomycete species

Fungal Diversity
Fungal Diversity

ƒFig. 2 Neighbour joining tree based on ITS1 sequences using Kimura

2-parameter distances. Bootstrap values above 95%, calculated from
1000 full heuristic replicates are shown at the nodes. Scale bar
indicates one base change per 100 nucleotide positions

from variations in the ITS1 region. However, our results

suggest that the vulnerability of ITS1 region to variations is
not specific because the ITS2 region in Fontanospora
fusiramosa, Varicosporium elodeae, Articulospora tetracla-
dia and Anguillospora filiformis appears to be more
diverged than ITS1 region (not shown). These findings
agree with those of Nilsson et al. (2006) pointing to a
greater variability in the ITS1 region than in the ITS2
region (see above). Since the evolutionary distances for the
overall divergence rate of ITS1 and ITS2 almost doubled
compared to ITS1-5.8S-ITS2 sequences (not shown) and
divergence was > 100% for some pairs of ITS1 sequences
(probably due to large standard errors of distances and
sequence alignment difficulties), the NJ tree based on ITS1
sequences cannot be considered reliable. However, we were
able to employ 132 to 252 bp (ITS1 and ITS2) to
successfully group the strains to the respective species. A
minimalist DNA barcodes finds its use in identifying a
partially degraded specimen (Hajibabaei et al. 2006). From
our study, it is evident that very short sequences of ITS1 or
ITS2 can become handy when the DNA template is
degraded or often when encountering difficulties with the
DNA polymerase reaction.
The aquatic hyphomycetes are known to be polyphy-
letic. Sixteen species of Tricladium were placed into 6
clades rather distant from each other (Campbell et al.
2009). Three Tricladium species are associated with
Cudoniella, Hydrocina and Hymenoscyphus belonging to
Helotiales (Table 3), but the relationships differ at the
family level: Cudoniella and Hymenoscyphus are members
of Helotiaceae, whereas Hydrocina is classified in Hyme-
noscyphaceae. This may probably explain the evolution-
ary distance of Tricladium chaetocladium from T. indicum,
T. splendens and T. terrestre. The interspecific differences
of an ideal barcode are expected to exceed intraspecific
distances (Hebert et al. 2003). In our study, the majority of
sequences exhibited deep evolutionary divergences be-
tween the species but the differences in rate of divergence
between some species assigned to Tricladium genus were
huge. So it becomes difficult to assign a reliable threshold
factor to define species due to the polyphyletic nature of
most aquatic hyphomycetes. The assignment of a thresh-
old value to differentiate species may also lead to some
erroneous results due to the presence of young taxa (Ward
2009). In monophyletic species like those of Tetracladium
genus, the ITS sequences exhibited a threshold factor of
10 in sequence variations between and within species
(Letourneau et al. 2010).
 Fungal Diversity

ƒFig. 3 Neighbour joining tree based on ITS2 sequences using Kimura

2-parameter distances. Bootstrap values above 95%, calculated from
1000 full heuristic replicates are shown at the nodes. Scale bar
indicates one base change per 100 nucleotide positions

Articulospora tetracladia teleomorph was classified in

Ombrophila (Baral and Krieglsteiner 1985). In our study,
the divergence rate between A. tetracladia, V. elodeae, A.
filiformis and F. fusiramosa were only ≤ 7.4% (Table 4);
hybridization may be a possible explanation for sequence
sharing between these species. In the NJ tree based on SSU
rDNA, A. tetracladia is a sister taxon of A. filiformis
(Belliveau and Bärlocher 2005) and these two species are
sister taxa of V. elodeae (Baschien et al. 2006). Anguillo-
spora filiformis is not closely associated with A. longissima,
A. crassa and A. furtiva (Baschien et al. 2006), however the
teleomorphs of A. filiformis and V. elodeae are unknown
making it difficult to clarify the phylogenetic relationships
between these species.
There are controversies associated with the usage of ITS
region as barcodes. Studies on Fusarium suggest that ITS
region is not an universal species level marker (Lieckfeldt
and Seifert 2000), however Baschien et al. (2006) were able
to successfully separate Tetracladium spp., Alatospora
acuminata, Tricladium spp. and Anguillospora spp. based
on ITS sequences. In our study, we could successfully
distinguish 19 species and we were able to group the
conspecific strains using ITS1, ITS2 and ITS1-5.8S-ITS2
sequences (Figs.1, 2 and 3). Cohesiveness was not
observed between isolates with respect to location, condi-
tion of stream or date of collection. For instance, between
the two groups of V. elodeae only São João do Campo
appears to be the common location (41°45′N 8°11′W).
Further studies have to be undertaken to better understand
the cohesiveness of strains with respect to geographic
location.
Although our study has included 19 species of aquatic
hyphomycetes, representing 6.3% of the total described
species in this group of fungi, it was confined to Portuguese
isolates except for sequences retrieved from NCBI that are
likely to be from different geographic regions. Thus, further
studies conducted with isolates spanning various parts of
the world are expected to improve our knowledge on
aquatic hyphomycete classification, but this requires
experts in taxonomy and it is constrained by the establish-
ment of pure cultures, which is a time consuming task.
The DNA barcoding is reported to be cost effective in
bioinventory and biomonitoring studies. It is estimated that
a DNA barcode could be generated with $5 when compared
to $50 to $100 per specimen if a team of experts is targeting
a huge number of species in a specific geographic area
(Smith 2005; Hebert and Gregory 2005). In this study,
taxonomic cohesiveness of most aquatic hyphomycete
Fungal Diversity
species/genus or isolates was maintained in the three types
of trees generated; however, NJ trees based on ITS1 or
ITS2 sequences had lower statistical support for some
internal nodes, so we propose mainly the usage of entire
ITS1-5.8S-ITS2 rRNA sequences as the barcodes for
identifying species of aquatic hyphomycetes.
Acknowledgement L. Marvanová was supported by the project
MSM0021622416.
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