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Two new species of nematode-trapping fungi:
relationships inferred from morphology, rDNA
and protein gene sequence analyses
Yan LI
a
, Rajesh JEEWON
b
, Kevin D. HYDE
b
, Ming-He MO
a
, Ke-Qin ZHANG
a,
*
a
Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming 650091, Yunnan Province, PR China
b
Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road,
Hong Kong SAR, PR China
article info
Article history:
Received 10 August 2005
Received in revised form
4 April 2006
Accepted 10 April 2006
Corresponding Editor:
Richard A. Humber
Keywords:
Dactylellina
Fungus-invertebrate interactions
Orbiliaceae
Phylogeny
abstract
Two new nematode-trapping fungi, Dactylellina sichuanensis and D. varietas from China, which
capture nematodes by both adhesive knobs and non-constricting rings, are described and il-
lustrated. D. sichuanensis is characterized by both adhesive knobs and non-constricting rings,
solitary conidiophores and 3-(4)-6 septate conidia, as compared with species such as D. appen-
diculata,D. candida,D. leptospora and D. lysipaga. Although D. sichuanensis shares similar types
of trapping devices, the presence of simple conidiophores and spindle-shaped conidia with
these species, it can be distinguished by its larger conidia and presence of more than four
septa. In D. sichuanensis, a single conidium is born at the tip of conidiophore, while in D.
candida, 3–10 conidia are born near the apex of conidiophore in a capitate arrangement. It pro-
duces spindle-shaped conidia with 3–6 septa, whereas D. leptospora produces cylindrical-
shaped conidia with 5–15 septa. D. appendiculata captures nematodes by adhesive knobs
whereas D. sichuanensis captures nematodes by both adhesive knobs and non-constricting
rings. They also differ in conidial size (35–82.5 mminD. sichuanensis as compared with 57–
108 mminD. appendiculata). D. varietas is characterized by conidiophores that are branched
at right-angles, and elongate to fusoid conidia, with 7–8 septa (more than 25% of which are
curved). D. varietas resembles D. asthenopaga,Dactylella oxyspora and Monacrosporium multisep-
tatum, but has elongate-fusoid conidia, whereas D. asthenopaga possesses obconical or clavate
conidia. D. varietas forms both adhesive knobs and non-constricting rings whereas Dactylella
oxyspora does not produce any trapping device. M. multiseptatum differs from D. varietas in
having larger conidia with an inflated middle cell. Phylogenetic analyses based on nuclear
and protein coding DNA sequences (18 S, and a combined 28 S þ5.8 S þb-tubulin dataset) in-
dicate that these two taxa should be assigned to the family Orbiliaceae.
ª2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
Nematode-trapping fungi have been studied worldwide for
their potential as biocontrol agents and because of their
unique predatory habits (Pandey 1973; Nansen et al. 1988;
Waller & Faedo 1993; Bird & Herd 1995; Dong et al. 2003).
Most of them can capture nematodes and other microscopic
animals by forming various trapping-devices, which include
* Corresponding author.
E-mail address: kqzhang111@yahoo.com.cn.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycres
mycological research 110 (2006) 790–800
0953-7562/$ – see front matter ª2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2006.04.011
simple adhesive branches, unstalked adhesive knobs, stalked
adhesive knobs, non-constricting rings, constricting rings,
two-dimensional networks, and three-dimensional networks
(Duddington 1951; Barron 1977; Ahre
´net al. 1998; Scholler
et al. 1999). Traditionally, these fungi have been assigned to
three genera as circumscribed by Subramanian (1963):Arthro-
botrys (Corda 1839), Dactylella (Grove 1884) and Monacrosporium
(Oudemans 1885). The nematode-trapping fungi of these gen-
era are conidial anamorphs in the Orbiliales (Orbiliomycetes). In
Arthrobotrys, conidiophores have nodes, and conidia are usu-
ally characterized with a single septum but some species
may have no septum or several septa. In Dactylella, conidio-
phores are simple, unbranched or occasionally branched. Sin-
gle conidia are produced at tips of simple conidiophores and
are elongate spindle-shaped to cylindrical without a central
inflated cell. In Monacrosporium, the conidiophores are simple
or form a few short branches near the apex; conidia are spin-
dle-shaped and have an inflated central cell (longest and wid-
est) and more than two septa. These taxonomic concepts are
mainly based on the morphology of conidia (shape, septa
and size) and conidiophores (branching, modifications of the
apex). This taxonomic system has been widely used (Cooke
& Dickinson 1965; Schenck et al. 1977; Van Oorschot 1985;
Liu & Zhang 1994).
However, recent molecular studies using ITS and 18 S
rDNA sequences have indicated that trapping devices are
more informative than other morphological structures in
delimiting genera (Liou & Tzean 1997; Pfister 1997; Ahre
´n
et al. 1998; Scholler et al.1999). Based on these studies, a new
classification scheme with four genera was proposed by
Scholler et al. (1999), which were Arthrobotrys,Dactylellina,
Drechslerella and Gamsylella. Based on rDNA and b-tubulin
phylogenies, trapping devices have been shown to be more
important than conidial morphology in the systematics and
taxonomy of nematode-trapping fungi (Li et al. 2005). This
has lead to a rearrangement of the classification of these fungi
where the generic concept was redefined as follows: Arthrobo-
trys, characterized by three-dimensional adhesive networks,
Dactylellina by adhesive knobs, and Drechslerella by constrict-
ing-rings. Phylogenetic placement of taxa characterized by
stalked adhesive knobs and non-constricting rings is also con-
firmed in Dactylellina. Species that produce unstalked adhe-
sive knobs that grow out to form loops are transferred from
Gamsylella to Dactylellina, and those that produce unstalked
adhesive knobs that grow out to form networks are trans-
ferred from Gamsylella to Arthrobotrys.Gamsylella as currently
circumscribed cannot be treated as a valid genus (Li et al.
2005).
During a survey of nematode-trapping fungi in China in
2002, two new predacious species forming both adhesive
knobs and non-constricting rings were collected and are de-
scribed in this study based on a combination of morphological
and molecular characters. We follow the classification of
nematode-trapping fungi based on trapping devices as pro-
posed by Li et al. (2005) and classify our new taxa in Dactylellina.
Morphological distinctions between these new species and
similar species are discussed. Their phylogenetic affinities
with closely related genera and species are also discussed
based on rDNA sequences from (18 S, 28 S and 5.8 S) and pro-
tein coding sequences (partial b-tubulin).
Materials and methods
Morphological examination
Soil samples (30 g) were collected from the forest near Emei
Mountain, Sichuan Province, China. Each sample was placed
in zip-locked plastic bags and labelled. Samples of 0.5–1 g
soil were sprinkled onto corn meal agar (CMA) inoculated
with Paragrellus redivius (free-living nematode) and incubated
at 25 C, following methods as described by Duddington
(1955) and Wyborn et al. (1969). Samples of P.redivius were pro-
vided by Yunnan University (China) and were cultured in oat
media (10 g oats is put in 50 ml conical flask with 6 ml distilled
water and autoclaved at 121 C, 30 min) for two weeks before
use. Nematodes were then isolated from media following
Baerman’s funnel technique (Barron 1977), suspended in
sterile distilled water, and then used as bait.
After one month, samples were examined using a dissect-
ing microscope. Single spores were isolated with a sterilized
toothpick and were cultivated on CMA at 25 C, maintained
on the same medium as agar slants and stored at 4 C.
After one week the conidial characters and conidiophores
of the isolated cultures were examined. To verify the morphol-
ogy of the trapping devices, cultures were cut into small cubes
and inoculated with Paragrellus redivius at 25 C for 3–5 d
(Duddington 1955; Wyborn et al. 1969). The growth test was
performed under different temperatures and different media.
All morphological characters were measured using an
Olympus CH20 light microscope, and all photographs were
taken using an Olympus BX51 microscope. Dimensions of
the individual morphological characters were based on the
mean of 50 measurements.
DNA extraction
For each strain, single spores were cultured on potato dex-
trose agar (PDA) and incubated at 28 C for 7–10 d. DNA was
extracted using a modified Cetyltrimethyl Ammonium Bro-
mide (CTAB) method as outlined by Jeewon et al. (2002, 2004)
and Cai, (2005). Lyophilized mycelium was pulverized in
a 1.5 ml microcentrifuge tube with 200 mg sterilized quartz
sand and 600 ml2CTAB extraction buffer (2% w/v CTAB,
100 mMTris–HCl, 1.4 M NaCl, 20 mMEDTA, pH 8).
The entire contents were incubated at 65 C in a water-bath
for 60 min with occasional swirling. The solution wasthen spun
two or three times with an equal volume of phenol chloroform
(1:1) at 14 000 gfor 25 min. The upper aqueous phase containing
the DNA was transferred to microcentrifuge tubes precipitated
by addition of 800 ml cold absolute ethanol and kept at 20 C
overnight. Contents were centrifuged at 11 000 g(4 C) for
40 min, the supernatant was discarded and the DNA pellet
gently washed twice with 75% ethanol, dried under vacuum,
and finally suspended in Tris-acetate-EDTA (TE) buffer (1 mM
EDTA, 10 mMTris–HCl, pH 8) with RNase (1 mgml
1
).
Amplification and sequencing of genomic DNA
DNA amplification was performed using PCR (PTC-100
thermal Controller, MJ Research, MA). Primer pairs NS1 and
New nematode-trapping species of Dactylellina 791
NS4 (White et al. 1990), LROR and LR5 (Vilgalys & Hester 1990),
ITS5 and ITS4 (White et al. 1990), Bt1A and Bt1B and Bt2A and
Bt2B (Glass & Donaldson 1995) were used to amplify the SSu
(18 S), LSu (28 S), ITS and b-tubulin, respectively.
PCR reactions were performed using 3 ml genomic DNA
template in a 50 ml PCR mixture (1.5 units of Taq DNA polymer-
ase, 10buffer, 1.5 mMMgCl
2
, 2.5 mMdNTPs, 1.5 mMprimers)
under the following thermal conditions: 3 min at 95 C, 30
cycles of 1 min at 94 C, 50 s at 52 C, 1 min at 72 C and a final
extension step of 10 min at 72 C. Amplified products were
electrophoresed on 1% agarose gels, stained with 0.5 mg l
1
ethidium bromide for 1 h in 1Tris-acetate acid EDTA buffer
and visualized under UV light to check for size and purity.
Size estimates were made using a 100 bp DNA ladder. PCR
products were purified with a DNA and Gel Band Purification
Kit (Amersham Biosciences 27-9602-01; Buckinghamshire, UK).
The purified PCR products were directly sequenced on both
strands with the same primers that were used for amplifica-
tion. Reactions were performed in an Applied Biosystem
(Foster City, California, USA) 3730 DNA Analyser at the
Genome Research Centre (The University of Hong Kong).
rDNA sequences of the new taxa and others used in the anal-
yses are listed in Table 1.
Phylogenetic analysis
DNA sequences were aligned with additional sequences
obtained from GenBank (Table 1) using BioEdit (Hall 1999)
and ClustalX 1.83 (Thompson et al. 1997) and were manually
aligned by inserting gaps when necessary. Phylogenetic
analyses were conducted using PAUP version 4.0b10 (Swof-
ford 2002). Trees were inferred using the heuristic search op-
tion with random sequence additions. Gaps were treated as
missing data and as fifth characters to increase the probabil-
ity of finding all the most-parsimonious trees and to com-
pare tree topologies. Clade stability was assessed using
Bootstrap (BS) analysis with 1000 replicates, random se-
quence additions with MAXTREES set to 5000 as imple-
mented in PAUP. Further details are outlined in
Promputtha et al. (2005) Descriptive tree stastistics tree
length (TL), CI, RI, rescaled consistency index (RC), homo-
plasy index (HI) and log likelihood (–Ln L) were calculated
for all trees generated under different parameters. Kishino–
Hasegawa tests (Kishino & Hasegawa 1989) and Templeton
tests (Templeton 1983), as implemented in PAUP, were per-
formed for all trees to determine whether trees generated
under different parameters were significantly different.
Trees were viewed in Treeview (Page 1996).
Taxonomy
Dactylellina varietas Y. Li, K.D. Hyde & K.Q. Zhang, sp. nov.
(Fig 1)
MycoBank No. 510030
Etym.: Latin; varietas ¼variety; referring to the various
shapes of the conidia.
Coloniae in agaro CMA albidis, post 10 dies 25 3.5 cm diam. My-
celium sparsum, hyphis hyalinis, septatis, ramosis. Conidiophora
hyalinis, erecta, septata, simplicia vel ramosa, 51–123 mm longis,
basi 2.5–5 mm latis, sursum leniter attenuata, apice 1–2.5 mm latis,
primum saepe duo longis ramosae, 60–160 mm longis, deinde apice
saepe parce ramosae denique 2–3 conidia ferentes. Macroconidiis
hyalinis, elongatus-fusiformis, rectis vel leniter curvatis, basi
truncatis, 25–61.3 mm longis, 6.3–10 mm latis, 1–9-septatis, praeci-
pue 7–8 septatis.
Typus:China:Sichuan: YMF 1.00118A, Emei Mountain, Sichuan,
China, 7 Aug. 2002, L. Cao (YMF*1.00118A–holotypus; YMF1.00118–
cultura viva).
Colonies on PDA villiform, whitish reached 4 cm diam at 25 C
and 28 C and did not grow at 4Cor35C within 7 d. The
reverse side of the media was yellowish to reddish orange.
Colonies on CMA whitish, slow growing, and up to a diam
of 3.5 cm at 25 C, 5 cm at 28 C and did not grow at 4C
or 35 C within 10 d. The reverse side of the media was of
the same colour on CMA. Mycelium spreading, scanty, hya-
line, septate, branched. Conidiophores hyaline, erect, septate,
branched, 51–123 mm(
x¼76:5mm) high, 2.5–5 mm
(x¼3:5mm) wide at the base, gradually tapering upward to
a width of 1–2.5 mm at the apex, initially forming two long
branches at right angles with a single terminal spore,
60–160 mm(
x¼98 mm) long; later often producing a few
short branches near the apex of each long branch, and bear-
ing 1–5 conidia, commonly producing 2–3 conidia. Conidia
colourless, elongate fusoid, straight or curved, 23.5% some-
times curved, truncate at the base, 25–61.5 6.5–10 mm
(x¼46:59mm), (1–) 7–8 (–9) septa; proportion of conidia
with 6, 7 or 8 septa are 12%, 40% and 40%, respectively. Trap-
ping devices are of two types: stalked adhesives knobs uni-
cellular, subspherical or prolate ellipsoidal, 6.5–9 5–7.5 mm
(x¼7:57mm), stalks 2.5–40 mm(x¼20 mm) long and
1.5–2.5 mm(
x¼2mm) wide, curved; non-constricting rings
approximately circular, inner diameter 6.5–15.5 mm, outer
diameter 8–18 mm, composed of three subequal arcuate cells,
stalks 12–46.5 mm(
x¼23:5mm) long. Chlamydospores were not
observed in culture.
Dactylellina sichuanensis Y. Li, K.D. Hyde & K.Q. Zhang,
sp. nov.
(Fig 2)
MycoBank No. 510031
Etym.:Sichuanensis, was named after Sichuan Province
where the soil samples were collected.
Coloniae in agaro CMA albidis, post 10 dies 25 C 4 cm diam.
Mycelium sparsum, hyphis hyalinis, septatis, ramosis, 2.5–5 mm
latis. Conidiophora hyalinis, erecta, septata, simplicia, 139–
198.5 mm alta, basi 2–2.5 mm crassa, sursum leniter attenuata,
apice 0.5–1 mm crassa, ibi umum conidium ferentes. Conidiis hya-
linis, fusiformis, rectis vel leniter curvatis, basi truncatis, 35–
82.5 mm longis, 7.5–17.5 mm latis, 3–6 septatis, praecipue 4 septatis.
Typus:China:Sichuan: Emei Mountain, Sichuan, China, 7 Aug.
2002, L. Cao (YMF1.000234–holotypus; YMF1.00023–culture viva).
Colonies on PDA villiform, whitish, extending to 5 cm diam
at 25 C, 7 cm at 28 C and did not grow at 4Cor35C
within 7 d. The reverse side of the media was of the same col-
our. Colonies on CMA whitish, slow-growing, and extending to
a diam of 4 cm at 25 C, 6 cm at 28 C and did not grow at
* Laboratory for Conservation and Utilization of Bioresources,
Yunnan University, Kunming, PR China.
792 Y. Li et al.
4Cor35 C within 10 d. Mycelium spreading, scanty, vegeta-
tive hyphae hyaline, septate, branched, mostly 2.5–5 mm wide.
Conidiophores hyaline, erect, simple, septate, 139–198.5 mm
(x¼168:5mm) high, 2–2.5 mm wide at the base, gradually
tapering upwards to a width of 0.5–1 mm at the apex, bearing
one single conidium. Conidia 35–82.5 (x¼53:5mm) 7.5–
17.5 mm(
x¼13 mm), hyaline, spindle-shaped, straight or
sometimes slightly curved, acutely narrowing towards and
truncate at the base, the middle cell the largest, (3–)4(–6)-sep-
tate; proportion of conidia with 3, 4, 5 and 6 septa are 14%,
76%, 9% and 1%, respectively. Two types of trapping devices
were observed in culture: stalked adhesive knobs, 20–50 mm
long, unicellular, subspherical or prolate ellipsoidal; non-con-
stricting rings approximately circular, composed of three sub-
equal arcuate cells. Chlamydospores were not observed in
culture.
Table 1 – List of taxa and GenBank accession numbers of DNA sequences used in this study
Species 18 S rDNA 28 S rDNA ITS B-tubulin
b-1A1B b-2A2B
Ingroups
Arthrobotrys arcuata AY261129 AF106527 AY965790 AY965820
A. conoides AJ001983
A. musiformis AJ001985
A. gephyropaga AJ001996
A. psychrophila AJ001998
A. robusta AJ001988
A. superba AJ001989
A. thaumasia AY261137 U51972 AY965791 AY965819
Dactylella atractoides AY902796
D. oxyspora AJ001993 AY965767 AF106537 AY965787 AY965816
D. rhombospora AY902797
D. rhopalota AJ001992
Dactylellina appendiculata AY902792 AF106531 AY965793 AY965822
D. asthenopaga AY965770 U51962 AY965796 AY965825
D. candida AJ001990 AY902801 AY965749 AY965778 AY965808
D. drechsleri AY965765 AY695063 AY965784 AY965813
Drechslerella doedycoides AJ001994
Dactylellina ellipsospora AJ001995 AY261157 AY965759 AY965803 AY965832
D. entomopaga AY965774 AY965774 AY965802 AY965831
D. leptospora AY965763 AY965750 AY965783 AY965809
D. lysipaga AY261165 AY695067 AY965782 AY965812
D. mammillata AY902802 AY902794 AY965795 AY965824
D. parvicolle AY965761 AY965748 AY965777 AY965807
D. phymatopaga AY261156 U51970 AY965798 AY965827
D. sichuanensis AY902788 AY902803 AY902795 AY965776 AY965806
D. varietas AY902798 AY902800 AY902805 AY965780 AY965810
Drechslerella anchonia AY902799 AY965753 AY965786 AY965815
D. brochopaga AY261176 U72609 AY965794 AY965823
D. dactyloides AJ001997
Monacrosporium multiseptatum AY965764 AY965751 AY965781 AY965811
Orbilia auricolor U72598
O. delicatula U72603
O. fimicola AF006307
Ascozonus woolhopensis AF010590
Barssia oregonensis U42657
Cystotheca wrightii AB120747
Helvella lacunosa U42654
Leveillula taurica AB033479
Microstoma floccosum AF006313
Morchella cf. elata AY544709
M. cf. esculenta AY544708
Phillipsia domingensis AF006315
Pseudopithyella minuscula AF006317
Thelebolus stercoreus U49936
Verpa conica AY544710
Wynnella silvicola U42655
Outgroup 18 S
Chromocleista cinnabarina AB006747
Trichophyton tonsurans AY083229
Outgroup 28 S D5.8 S Db-tubulin
Neurospora crassa AY681158 AY681193 AY974799 AY681226
Sordaria fimicola AY681160 AY681188 AY974800 AY681228
New nematode-trapping species of Dactylellina 793
Fig 1 – Dactylellina varietas. A, C. Conidiophores with two long branches at right angle. B. Simple conidiophore. D–I. Conidia
with septa from 2 to 9 from immature to mature. J–O. Curved conidia with 3–6 septa. P. Germinating conidium with an
immature conidium with a mature conidium. Q. Adhesive knob. R. Non-constricting ring. S. Adhesive knob and
non-constricting ring. All images at the same magnification. Bars [20 mm.
794 Y. Li et al.
Fig 2 – Dactylellina sichuanensis. A. Conidiophore with a immature conidium. B. Conidium on conidiophore. C–D, M–O. Mature
conidia. E–J. The process of conidia growth. K–L. Abnormal conidia. P. Adhesive knobs and non-constricting ring.
Q. Germinated conidium. All images at the same magnification. Bars [20 mm.
New nematode-trapping species of Dactylellina 795
Results
Phylogenetic analysis
Small subunit (18 S) dataset
This DNA matrix consisted of 34 taxa with Chromocleista cinna-
barina and Trichophyton tonsurans as outgroups. The dataset
was aligned without ambiguous regions and consisted of
1091 characters, of which 796 were constant and 191 were par-
simony-informative. Unweighted parsimony analysis, treat-
ing gaps as missing data, generated eight trees. Weighted
parsimony with a transition transversion ratio (TTr) of 1.5:1
resulted in three trees, whereas a TTr of 2:1 resulted in six
trees. All 17 trees generated under different assumptions
were similar in topology and not significantly different based
on the KH and Templeton tests. Phylogeny generated by treat-
ing gaps as missing data and under a TTr of 1.5:1 was identi-
fied as the best tree (Fig 3). Phylogenies indicate that all
nematode trapping fungi, including D. sichuanensis and
D. varietas, are closely related to each other and are in the
family Orbiliaceae. Treating gaps as a fifth character under dif-
ferent TTr yielded trees that were identical in topology.
Combined 28 S þ5.8 S þb-tubulin dataset
The combined 28 S þ5.8 S þb-tubulin dataset comprised 21
taxa and 2249 characters, of which 567 characters that were
ambiguously aligned were excluded in the analysis, and 387
were phylogenetically informative. Neurospora crassa and Sor-
daria fimicola were used as outgroups. Unweighted parsimony
analysis treating gaps as missing data resulted in one tree
(Fig 4). Similar results were obtained under a TTr of 1.5:1. Phy-
logenies derived from the 28 S þ5.8 S þb-tubulin sequences
provided a better taxonomic resolution of D. sichuanensis and
D. varietas (Fig 4). D. sichuanensis clustered with other Dactylel-
lina species characterized by adhesive knobs (some also have
non-constricting rings; Fig 4). D. sichuanensis appears to be
phylogenetically related to D. leptospora,D. lysipaga,D. appendi-
culata, and this clade received 83% BS support. The phyloge-
netics of D. varietas with Dactylella oxyspora and
Orbilia fimicola
Arthrobotrys superba
Arthrobotrys robusta
Dactylellina sichuanensis
Orbilia auricolor
Arthrobotrys conoides
Arthrobotrys musiformis
Arthrobotyrs gephyropaga
Dactylellina ellipsospora
Dactylellina candida
Arthrobotrys psychrophila
Dactylella rhopalota
Drechslerella dactyloides
Drechslerella doedycoides
Dactylella rhombospora
Dactylella oxyspora
Dactylella atractoides
Dactylellina varietas
Orbilia delicatula
Pseudopithyella minuscula
Phillipsia domingensis
Microstoma floccosum
Morchella cf. elata
Morchella cf. esculenta
Verpa conica
Wynnella silvicola
Helvella lacunosa
Barssia oregonensis
Ascozonus woolhopensis
Thelebolus stercoreus
Cystotheca wrightii
Leveillula taurica
Chromocleista cinnabarina
Trichophyton tonsurans
100
100
99
100
100
100
94
77
98
72
99
76
58
100
69
71 62
63
67
92
Eurotiomycetes
Erysiphaceae
Thelebolaceae
Leotiomycetes
Helvellaceae
Sarcoscyphaceae Pezizomycete
s
Sarcoscyphaceae
Orbiliomycetes
(Orbiliaceae)
Fig 3 – Phylogenetic tree based on partial 18 S rDNA sequences. The tree was rooted with Chromocleista cinnabarina and
Trichophyton tonsurans and constructed under the MP criterion with a TTR of 1.5:1. The number at each branch point
represents percentage Bootstrap support calculated from 1000 replicates (TL [715.5, CI [0.674, RI [0.778. RC [0.524,
HI [0.326, LLn L [5148.02358).
796 Y. Li et al.
Monacrosporium multiseptatum is also well defined (94% BS
support).
Discussion
Based on recent molecular data regarding the systematics and
evolution of nematode-trapping fungi, a modified classifica-
tion, based on trapping devices was presented, including
Arthrobotrys,Dactylellina,Drechslerella. And Dactylella were
redefined as non-tapping fungi (Li et al. 2005). Dactylellina
was accepted by Scholler et al. (1999) for species characterized
by stalked adhesive knobs and included those species produc-
ing non-constricting rings. Based on previously published
phylogenies (Li et al. 2005) and coupled with sequence analy-
ses in this study we assign the two new taxa to Dactylellina.
Sequence data based on rDNA and protein genes confirmed
that our new taxon, D. sichuanensis,isaDactylellina species
because it associates with other Dactylellina taxa (Fig 4). Phylo-
genetic analyses also indicated that it is closely related to
D. appendiculata,D. leptospora and D. lysipaga (85% BS support).
However, D. sichuanensis is more closely related to D. lysipaga
(94% BS support, Fig 4). D. sichuanensis is also unique in that
the 28 S, 5.8 S and b-tubulin sequences differ from D. appendi-
culata,D. leptospora and D. lysipaga by more than 10%. These
species could also be distinguished from each other based
on morphological characteristics.
D. sichuanensis is characterized by adhesive knobs and
non-constricting rings, which is characteristic of Dactylellina.
It is distinct in having two types of trapping devices and
larger conidia from similar species within the same genus.
D. sichuanensis is morphologically similar to D. lysipaga
(Scholler et al. 1999), which also possesses two types of
Dactylellina drechsleri
Dactylellina mammillata
Dactylellina entomopaga
Dactylellina parvicolle
Dactylellina ellipsospora
Dactylellina phymatopaga
Dactylellina candida
Dactylellina lysipaga
Dactylellina sichuanensis
Dactylellina appendiculata
Dactylellina leptospora
Drechslerella anchonia
Drechslerella brochopaga
Arthrobotrys thaumasia
Arthrobotrys arcuata
Dactylellina asthenopaga
Dactylella oxyspora
Monacrosporium multiseptatum
Dactylellina varietas
Neurospora crassa
Sordaria fimicola
100
92
54
100
55
87
85
94
68
99
76
Fig 4 – One most-parsimonous tree generated from combined 28 S rDNA D5.8 S Db-tubulin sequences. Designated
outgroups are Neurospora crassa and Sordaria fimicola. Bootstrap values obtained from 1000 replicates are shown above
respective nodes. BS values less 50 % are not shown. Gaps were treated as missing data (TL [1305, LLn L [9272.58652).
New nematode-trapping species of Dactylellina 797
trapping devices, simple conidiophores and spindle-shaped
conidia. However, the main differences between these two
are the size of the conidia and the number of conidial septa.
D. sichuanensis produces larger conidia than D. lysipaga (Table
2). Only 2–4 septa are present in D. lysipaga whereas D. sichua-
nensis is usually characterized by more than four septa.
Another species that shares close morphological affinities
is D. candida (Table 2,Li et al. 2005). Despite having the same
number of conidial septa (3–(4)–5) and conidial shape (spindle
shape), D. sichuanensis has larger conidia than D. candida, and
the conidiophores of these two species have different branch-
ing patterns (Table 2).
Molecular analyses also indicated that D. sichuanensis is
closely related to D. appendiculata and D. leptospora, but
D. sichuanensis is distinguished by its different trapping struc-
ture and conidial characteristics. D. sichuanense resembles
D. leptospora (Morelet 1968) in its trapping devices. However,
D. sichuanensis produces spindle-shaped conidia with 3–6
septa whereas D. leptospora produces cylindrical-shaped coni-
dia with 5–15 septa (Table 2). D. appendiculata (Scholler et al.
1999) captures nematodes by means of adhesive knobs but
D. sichuanensis captures nematodes by means of both adhesive
knobs and non-constricting rings. They also differ in conidial
size (35–82.5 mminD. sichuanensis but 57–108 mminD. appendi-
culata) but characterized by spindle-shaped conidia and
simple conidiophores. A summary of some important mor-
phological characters differentiating these species is provided
in Table 2.
D. varietas clustered with Dactylella oxyspora and Monacro-
sporium multiseptatum with 92% BS support (Fig 4). However,
D. varietas appears to be phylogenetically distinct and is sister
to other Dactylellina species and can be distinguished from
others based on morphological characters. All these species
share at least one morphological character.
D. varietas resembles other known Dactylella species in hav-
ing elongate fusoid conidia and multi-septate but differ from
them in presence of trapping devices. D. varietas produces
both adhesive knobs and non-constricting rings whereas Dac-
tylella is restricted to non-trapping fungi. It is unique in that it
has branched conidiophores at right-angles (Fig 1A, C) and at
least 25% of conidia are curved (Fig 1J–L, O–P). Both D. varietas
and Dactylella oxyspora share (Matsushima 1971) shaped conid-
ial similar and have the same number of septa. However,
D. varietas is characterized by smaller conidia and the pres-
ence of trapping devices as compared with Dactylella oxyspora.
The conidia of D. varietas are 25–61.5 6.5–10 mm
(x¼46:59mm), whereas the average conidia size of D. oxy-
spora is 60 10.5 mm(Table 3). However, the presence of trap-
ping devices (non-constricting rings and adhesive knobs) and
smaller conidial size and more than 25% curved conidia of
D. varietas make it morphologically distinguishable from
D. oxyspora.D. varietas is also unique in that the 28 S, 5.8 S
and b-tubulin sequences differ from Dactylella oxyspora by 11%.
D. varietas resembles M. multiseptatum (Su et al. 2005) in pro-
ducing trapping devices, elongate-fusoid conidia and 7–8
septa. However, D. varietas produces adhesive knobs with
non-constricting rings and conidia without middle-inflated
cells, whereas M. multiseptatum produces adhesives knobs
only and conidia with middle-inflated cells. A summary of
some important morphological characters differentiating
these species is provided in Table 3.
D. varietas has elongate-fusoid conidia, whereas D. astheno-
paga possesses obconical or clavate conidia. The former also is
distinct from the latter in trapping devices and conidial size
Table 2 – Morphological comparison of Dactylellina appendiculata,D. candida,D. lysipaga,D. leptospora and D. sichuanensis
Dactylellina
species
Shape of
conidia
Size of conidia (mm) Conidia
septation
Conidiophores Trapping devices
D. appendiculata Spindle 57–108 9.3–14.5 4–7 Simple Adhesive knobs
D. candida Spindle 26–52 5.5–11.5
(x¼30 45 710)
2–(4)–6 Forming 3–10 short
branches near the apex
Adhesive knobs and
non-constricting rings
D. leptospora Elongate fusoid
to cylindrical
40–105 4–5.8 5–15 Branched Adhesive knobs and
non-constricting rings
D. lysipaga Spindle 28–55 9–14
(x¼41 11:5)
2–4 (4) Simple, branched
occasionally
Adhesive knobs and
non-constricting rings
D. sichuanensis Spindle 35–82.5 7.5–17.5
(x¼53:513)
3–(4)–6 Simple Adhesive knobs and
non-constricting rings
Table 3 – Morphological comparison of Dactylella oxyspora,Dactylellina asthenopaga,D. varietas and Monacrosporium
multiseptatum
Dactylellina
species
Shape of conidia Size of conidia (mm) No. of conidia
septa
Conidiophores Trapping devices
Dactylella oxyspora Spindle 45–100 9–13 6–12 Branched None
D. asthenopaga Obconical or clavate 20–46 6.5–9.5
(x¼31:58:2)
3 Simple Adhesive knobs
D. varietas Elongate fusoid 25–61.5 6.5–10
(x¼46:59)
(1–) 7–8 (–9) Form two long
branches at the
right angle
Adhesive knobs and
non-constricting rings
Monacrosporium
multiseptatum
Elongate fusiform
with middle-inflated cell
67.5–132.5 3.8–17.5mm
(x¼91:615:5)
(4–) 6–7 (–9) Simple Adhesive knobs
798 Y. Li et al.
and septation. The nucleotide difference (28 S þ5.8 S þb-
tubulin) between D. varietas and D. ashenopaga was 14%.
Acknowledgements
This research was supported by the 973 program (No.
2003CB415102), National Natural Science Foundation of China
(No. 30230020) and the Department of Science and Technology
of Yunnan Province (No. 2005 NG 05). Molecular work and
preparation of manuscript were done at The University of
Hong Kong (CRCG Grant 2004 1115 9091, awarded to Dr Rajesh
Jeewon and Dr Kevin D. Hyde). Yunnan University (China) is
acknowledged for providing a MSc scholarship to Yan Li Lei
Cai, Dhanasekaran Vijaykrishna, Alvin M. C. Tang and Jing
Luo are thanked for assistance and advice. Heidi Kong and
Helen Leung are thanked for their technical help.
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