Phylogenetic relationships of Astrocystis eleiodoxae sp. nov. (Xylariaceae)

Abstract

An ascomycete with morphological similarities to Astrocysti (Xylariaceae) was collected from the peat swamp palms Eleiodoxa conferta and Licuala longicalycata in southern Thailand and is introduced here. The new taxon is characterized by carbonaceous black stromata on persistent white hyphae, and brown ascospores surrounded by a thin mucilaginous sheath and with a longitudinal germ slit. No anamorph was observed in nature or in culture. Phylogenetic relationships were investigated based on ITS1-5.8S-ITS2, partial LSU and SSU rDNA sequences using maximum parsimony and Bayesian analyses. Phylogenetic analyses of the ITS regions places the taxon in Xylariaceae, in a clade comprising Astrocystis eleiodoxae and Stilbohypoxylon elaeicola with good support and a sister group to Astrocystis, Kretzschmaria, Rosellinia and Xylaria species, with moderate support. LSU and SSU rDNA data places A. eleiodoxae in a clade with A. cocoes, Rosellinia necatrix and Stilbohypoxylon elaeicola in the Xylariaceae. The data indicates a relationship between A. eleiodoxae and Stilbohypoxylon elaeicola. There are no other Astrocystis sequence data available in GenBank, and based on molecular data shown here and morphological data we described Astrocystis eleiodoxae as a new species.

Full text

Mycosphere
1
Phylogenetic relationships of Astrocystis eleiodoxae sp. nov. (Xylariaceae)
Pinnoi A1,3, Phongpaichit P1, Jeewon R2, Tang AMC2, Hyde KD4 and Jones EBG3*
1Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkla, Thailand 90112 –
aom5736@gmail.com
2School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China –
rajeshjeewon@yahoo.com
3BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science
Park, Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani, Thailand 12120 – corresponding author Gareth Jones
– remispora@googlemail.com
4School of Science, Mae Fah Luang University, Chiang Rai, Thailand – kdhyde3@gmail.com
Pinnoi A, Phongpaichit P, Jeewon R, Tang AMC, Hyde KD, Jones EBG. 2010 – Phylogenetic
relationships of Astrocystis eleiodoxae sp. nov. (Xylariaceae). Mycosphere 1, 1–9.
An ascomycete with morphological similarities to Astrocystis (Xylariaceae) was collected from the
peat swamp palms Eleiodoxa conferta and Licuala longicalycata in southern Thailand and is
introduced here. The new taxon is characterized by carbonaceous black stromata on persistent white
hyphae, and brown ascospores surrounded by a thin mucilaginous sheath and with a longitudinal
germ slit. No anamorph was observed in nature or in culture. Phylogenetic relationships were
investigated based on ITS1-5.8S-ITS2, partial LSU and SSU rDNA sequences using maximum
parsimony and Bayesian analyses. Phylogenetic analyses of the ITS regions places the taxon in
Xylariaceae, in a clade comprising Astrocystis eleiodoxae and Stilbohypoxylon elaeicola with good
support and a sister group to Astrocystis, Kretzschmaria, Rosellinia and Xylaria species, with
moderate support. LSU and SSU rDNA data places A. eleiodoxae in a clade with A. cocoës,
Rosellinia necatrix and Stilbohypoxylon elaeicola in the Xylariaceae. The data indicates a
relationship between A. eleiodoxae and Stilbohypoxylon elaeicola. There are no other Astrocystis
sequence data available in GenBank, and based on molecular data shown here and morphological
data we described Astrocystis eleiodoxae as a new species.
Key words – molecular phylogeny – new species – palm fungi – Stilbohypoxylon
Article Information
Received 12 December 2009
Accepted 8 January 2010
Published online 14 January 2010
*Corresponding author – remispora@googlemail.com
Introduction
A study of saprobic fungi on the peat
swamp palms Eleiodoxa conferta and Licuala
longicalycata (Pinnoi et al. 2006; Pinruan et al.
2007) yielded a new xylariaceous ascomycete
with morphological similarities to Astrocystis,
Nemania and Stilbohypoxylon. Taxa from
palms within these and related genera have
been reviewed by Smith et al. (2001) and based
on morphological characteristics our taxon
most closely resembles Astrocystis.
Astrocystis is a genus mostly confined to
monocotyledons and has uni- rarely multi-
peritheciate stromata, which may develop
beneath the host cuticle and appear superficial.
The asci have a relatively short stipe and the
ascal ring is relatively small, amyloid and
stopper-shaped (Smith et al. 2001). A key to
six accepted species was provided by Smith et
al. (2001).
This study introduces a new species,
Astrocystis eleiodoxae and explores the phylo-

2
genetic relationships of this and related species
based on rDNA sequences of the ITS1-5.8S-
ITS2 and partial LSU and SSU genes.
Methods
Sample collection, fungal isolation
Decaying petioles of the palm Eleiodoxa
conferta were collected from Sirindhorn Peat
Swamp Forest, Thailand. Palm material was
placed in sterile plastic bags, returned to the
laboratory and incubated in moist plastic boxes
at 25°C. Fungi were observed under a
stereomicroscope, and then measured and
illustrated under a compound microscope. All
morphological measurements were carried out
in sterile water, with a mean from 25
measurements for most characters. Melzer’s
reagent was used to test the amyloidity of the
apical ring and 10% KOH for testing the
dehiscence of the perispore. A single spore
technique was used for isolation of the species.
Axenic cultures were grown on potato dextrose
agar medium (PDA) for 2−3 weeks and used
for the molecular study. Herbarium specimens
and living cultures are deposited in the
BIOTEC Herbarium (BBH) and Culture
Collection (BCC), respectively.
DNA extraction, amplification and
sequencing
DNA extraction was performed by
following a modified protocol as defined and
outlined previously (Jeewon et al. 2004, Wang
et al. 2005, Pinruan et al. 2007, Promputtha et
al. 2007). Three different regions of the rDNA
gene (characterised by different rates of
evolution) were amplified. Primer pairs NS1
(5′−GTAGTCATATGCTTGTCTC−3′) and
NS4 (5′−CTTCCGTCAATTCCTTTAAG−3′)
primer pairs were used for the small 18S
subunit (White et al. 1990). LROR
(5′−ACCCGCTGAACTTAAGC−3′) and LR5
(5′−TCCTGAGGGAAACTTCG−3′) primer
pairs were used to amplify a segment of the
large 28S subunit (about 900 nucleotides)
(Vilgalys & Hester 1990). ITS4 (5′−TCCTCC
GCTTATTGATATGC−3′) and ITS5 (5′−GGA
AGTAAAAGTCGTAACAAGG−3′) as def-
ined by White et al. (1990) were used to
generate about 550 nucleotides from the
complete ITS including 5.8S regions. The
amplification conditions were performed in a
50 μL reaction volume as follows: 1 × PCR
buffer, 0.2 mM each dNTP, 0.3 M of each
primer, 1.5 mM MgCl2, 0.8 units Taq
Polymerase and 10 ng DNA. PCR parameters
for all the regions were as follows: initial
denaturation 94ºC for 3 minutes, 35 cycles of
94ºC for 1 minute, 52ºC for 50 seconds, 72ºC
for 1 minute, final extension of 72ºC for 10
minutes. Characterization of PCR products was
done via agarose gel electrophoresis on a 1%
agarose gel containing ethidium bromide as the
staining agent. DNA sequencing was perf-
ormed using primers as mentioned above in an
Applied Biosystem 3730 DNA Analyzer at the
Genome Research Centre (University of Hong
Kong).
Phylogenetic analysis
DNA sequences were aligned using
BioEdit (Hall 2005) and Clustal X 1.83
(Thompson et al. 1997) with other sequences
obtained from GenBank. A blast search was
performed to find the possible sister groups of
the newly sequenced taxa. In addition, fungal
members from different families of the order
Xylariales and related orders were also
included in the analyses. Phylogenetic analyses
were performed using PAUP* version 4.0b10
(Swofford 2002). Prior to phylogenetic analy-
sis, ambiguous sequences at the start and the
end were deleted and gaps manually adjusted
to optimize alignment. Maximum parsimony
analyses were conducted using heuristic
searches as implemented in PAUP, with the
default options method. Analyses were done
under different parameters including
unweighted parsimony, and weighted
parsimony criteria. Clade stability was assessed
in a bootstrap analysis with 1000 replicates,
random sequence additions with maxtrees set
to 1000 and other default parameters as
implemented in PAUP*. Kishino-Hasegawa
(KH) tests (Kishino & Hasegawa 1989) and
Templeton tests (Templeton 1983), were
performed in order to determine whether trees
inferred from the different tree building
methods were significantly different. The
Bayesian analyses were conducted with the
Markov chains run for 1000000 generations.

Mycosphere
3
Trees were viewed in Treeview (Page 1996).
The nucleotide sequences reported in this paper
have been deposited in GenBank.
Results
Astrocystis eleiodoxae A. Pinnoi, E.B.G. Jones
& K.D. Hyde, sp. nov. Figs 1–14
MycoBank 513077.
Etymology – eleiodoxae refers to the
palm host.
Stromata perithecilis similia, superficialis,
solitaria vel gregaria, atra, carbonacea, 825–
1375 µm diam. × 250–375 µm alta, subglobosa.
Peridio 22.5–62.5 µm latus, aliquot strata ex
compressus cellular, atro. Paraphysa 2 µm latus,
filamentosae, septatae, numerosa. Asci 107.5–
155 × 6.2–10 µm, 8-spori, cylindrici,
pedicellati, unitunicati, truncata ad apicem, 5 ×
2.5 µm, annulo apicali in liquore iodato
Melzeri cyanescente. Ascosporae brunnae,
inequilaterallis ellipsoidae, 17.5–23 × 4.5–6.2
µm, exiguus curvata, unicellulae, leavia piries.
Holotypus – BBH9822.
Stromata semi-superficial, solitary or
gregarious, black, shiny, carbonaceous, without
apparent KOH-extractable pigments, synne-
mata absent; covering 1–3 perithecia; in
vertical section 825–1375 µm diam., 250–375
µm high, subglobose (Figs 1–3). Peridium
22.5–62.5 µm (
x
= 32 µm, n = 15) wide,
comprising several layers of compressed cells,
black (Fig. 3). Paraphyses 2 µm wide,
filamentous, septate, numerous and embedded
in a gelatinous matrix (Figs 2, 4). Asci 107.5–
155 × 6.2–10 µm (
x
= 131 × 8.5 µm, n = 25),
8−spored, cylindrical, relatively short, apically
truncate with a 5 × 2.5 µm wedge-shaped, J+,
subapical ring (Figs 5–8). Ascospores 17.5–23
× 4.5–6.2 µm (
x
= 19 × 5 µm, n = 25),
uniseriate, brown, inequilaterally ellipsoidal,
slightly curved, unicellular, smooth-walled,
germ slit full-length and with a thin
mucilaginous sheath (Figs 9–14).
Colonies of A. eleiodoxae on PDA at
25ºC are relatively fast growing, white, effuse,
producing globose structure, black,
carbonaceous in the center within 1 month but
not sporulating.
Material examined – Thailand, Narathi-
wat, Sirindhorn Peat Swamp Forest, on
submerged petiole of Eleiodoxa conferta, 12
May 2001, A. Pinnoi, BBH 9822 (holotype) –
ex-type cultures BCC 12874 and BCC 12875;
ibid., on submerged petiole of Eleiodoxa
conferta, 22 June 2001, A. Pinnoi, BBH 9825 –
cultures BCC 12512.
SSU based phylogenies
The 18S dataset contained 25 taxa
including 830 characters with 114 parsimony
informative sites, 62 parsimony uninformative
sites and 654 constant characters, with
Dothidea sambuci as the outer group (not
shown). Unweighted parsimony analysis (with
gap treated as missing character), which
yielded 6 parsimonious trees of 903.7 steps
with CI, RI, RC and HI of 0.667, 0.771, 0.514
and 0.333 respectively. Bootstrap values
(generated from 1000 replicates) and Bayesian
posterior probabilities were generated from
1000000 generations. Astrocystis eleiodoxae is
a member of the family Xylariaceae, order
Xylariales and clustered with Stilbohypoxylon
elaeicola, Rosellinia necatrix and Astrocystis
cocoës with high support, with Xylaria
hypoxylon as a sister group. The Astrocystis
species are in a monophyletic sub-clade
(Xylariaceae) with Amphisphaeriaceae as a
sister group in the Xylariales clade.
Phylogenetically the Xylariales are distinct
from other orders included in the analysis, with
high support (92%).
LSU based phylogenies
The 28S DNA matrix consisted of 33
taxa with Dothidea sambuci as an outgroup.
The aligned dataset was 835 characters, out of
which 207 were parsimony informative, 77
parsimony uninformative and 551 constant
characters. The tree shows 737 steps with CI,
RI, RC and HI of 0.541, 0.713, 0.386 and
0.459, respectively (not shown). Bootstrap
values (generated from 1000 replicates) and
Bayesian posterior probabilities were generated
from 1000000 generations. Astrocystis
eleiodoxae and S. elaeicola grouped with
Rosellinia necatrix and Astrocystis cocoës with
high bootstrap support and Bayesian posterior
probabilities support of 100%. Astrocystis
eleiodoxae and S. elaeicola clustered together
with weak parsimony bootstrap support and
Bayesian posterior probabilities. Stilbohypo-
xylon quisquiliarum was distantly placed from

4
Figs 1–14 – Light micrographs of Astrocystis eleiodoxae (from holotype). 1 Stroma on natural
substrata. 2 Squash mount illustrating asci and paraphyses. 3 Section of stroma with ascomata. 4
Paraphyses. 5–6 Asci with relatively short stipe. 7–8 Asci with J+ stopper-shaped apical ring
(arrowed). 9–14 Ascospores. Bars 1 = 1 mm, 2 = 100 µm, 3 = 1 mm, 4 = 5 µm, 5–8 = 25 µm, 9–14
= 10 µm.

Mycosphere
5
Astrocystis eleiodoxae and Stilbohypoxylon
elaeicola and grouped with Xylaria and
Nemania species in a poorly supported
subclade.
ITS based phylogenies
The ITS data consisted of 36 taxa with
Diatrype disciformis as an outgroup. The
aligned dataset was 772 characters, out of
which 330 were parsimony informative, 114
parsimony uninformative and 328 constant
characters (Fig. 15). Clade A comprises two
Xylaria, two Kretzschmaria species and
Stilbohypoxylon quisquiliarum. Clade B
consists of Astrocystis and Rosellinia species
and Halorosellinia oceanica. Clade C includes
two strains of Astrocystis eleiodoxae that are
monophyletic and Stilbohypoxylon elaeicola
with high bootstrap support. Clade D is a well
supported Nemania group, while clade E
constitutes Nemania maritima, N. confluens,
Rosellinia aquila, R. pepo and Astrocystis
cocoës. Clade F (Daldinia species) and G
(Hypoxylon and Annulohypoxylon species) are
basal to the family (Fig. 15).
Combined 28S and ITS1-5.8S-ITS2 based
phylogenies
The combined dataset consisted of 27
taxa with Pestalotiopsis versicolor as an
outgroup. The aligned dataset was 1746
characters. The tree obtained was 737 steps
with CI, RI, RC and HI of 0.583, 0.510, 0.297
and 0.417, respectively. Six Xylariaceae clades
are identified, all with high bootstrap support
(Fig. 16). Clade F is basal to the family and
comprises Annulohypoxylon and Hypoxylon
species in subclades, while clade E consists of
Biscogniauxia species. Clade A has high
support with Rosellinia and Astrocystis species.
Clade B comprises Astrocystis eleiodoxae
which clusters with S. elaeicola with weak
support. Halorosellinia oceanica and
Rosellinia necatrix form a sister group. Clade
D supports Stilbohypoxylon quisquiliarum,
Kretzschmaria deusta and Xylaria grammica.
Three Nemania species constitute a well-
supported monophyletic Clade C that is
phylogenetically distinct from the other
xylariaceous genera.
Discussion
Astrocystis eleiodoxae possesses several
morphological characters which it shares with
the other six Astrocystis species. These include
a raised stroma under which the ascomata
develop, an ascus with a relatively short stipe, a
J+ wedge-shaped subapical ring and brown
ascospores with a germ slit. It is however
different from any of the accepted species in
the monograph of the genus by Smith et al.
(2001) and is thus described here as a new
species.
Sequences data from 18S rDNA and 28S
rDNA confirm the monophyly of the two
Astrocystis eleiodoxae strains isolated on
different occasions, and are well positioned in
the Xylariaceae. The species clustered with
Astrocystis cocoës and Rosellinia necatrix with
high Bayesian posterior probabilities support
99% (data not shown).
Tang et al. (2007, 2009) have used
various genes to test their use in separating taxa
in the Xylariaceae. The phylogeny of several
xylariaceous genera has also been evaluated
using protein-coding genes, such as β-tubulin
and α-actin genes (Hsieh et al. 2005). These
gene regions may be particularly useful since
limited success has been achieved in
delineating genera and resolving generic
relationships based on ribosomal DNA genes
(Sánchez-Ballesteros et al. 2000, Smith et al.
2003, Triebel et al. 2005, Peláez et al. 2008).
Unfortunately this study could not utilize these
genes as there are no sequences in GenBank
representing the genes of genera discussed here.
The phylogenetic results therefore provide little
indication of the genera to which this new
taxon belongs, especially as the identification
of those sequences used from GenBank in this
study could not be verified (Zhang et al. 2008).
Petrini (2004) undertook a revision of
Stilbohypoxylon accepting ten species and
providing a key to the taxa. In her paper an
earlier epithet ‘elaeicola” in Rosellinia
elaeicola Henn. was used to represent S.
moelleri Henn and Astrocystis cocoës was
considered a synonym. Stilbohypoxylon
stromata are characterized by spine-like
synnemata covered with yellow granules
(Fröhlich & Hyde 2000). As stromata mature

6
Fig. 15 Phylogenetic tree based on ITS1-5.8S-ITS2 sequences. The tree is rooted with Diatrype
disciformis and constructed under un-weighted maximum parsimony criterion. The number at each
branch point represents percentage bootstrap support calculated from 1000 replicates and Bayesian
posterior probabilities (thickened branches). Branch lengths are proportional to the numbers of
nucleotide substitutions and are measured by scale bar (Bar; 10 % sequence divergence).
these may be lost and this may lead to
confusion in identification. Petrini (2004)
found these spines to be present in some
collections but not others while in the material
examined by Smith et al. (2001) spines were
lacking.
In the case of the new species described
here, there were no spines or yellow granules at
any stage of development and for this reason
the taxon is best placed in Astrocystis. Whether
Astrocystis cocoës belongs in Astrocystis or
Stilbohypoxylon has yet to be resolved.
Furthermore, whether Stilbohypoxylon species
should all be transferred to Astrocystis needs
consideration following re-examination of the
type of Astrocystis, A. mirabilis Berk. &
Broome. What is clear is that the phylogeny of
these tropical xylariaceous species is a long
way from being resolved and requires chemical
as well as molecular data.
10
X
ylaria
sp. AJ309350
X
ylaria
mali AF163040
K
retzschmaria
clavus AJ390434
K
retzschmaria
deusta AJ390435
Stilbohypoxylon
quisquiliarum DQ631937
A
strocystis
bambusae AY862573
A
strocystis
mirabilis AY862572
H
alorosellinia oceanica
R
osellinia
necatrix AY909001
R
osellinia
arcuata AB017660
A
strocystis eleiodoxae
A
strocystis eleiodoxae
Stilbohypoxylon elaeicola
N
emania
aenea AJ390427
N
emania
aenea
var. aureolateum AJ390428
N
emania
serpens AJ390431
N
emania
chestersii AJ390430
N
emania
serpens DQ631942
N
emania
aenea AJ390426
N
emania
maritima
N
emania confluens
R
osellinia
aquila
AY881727
R
osellinia
pepo AB017659
A
strocystis
cocoës AY862571
M
uscodo
r
vitigenus AY100022
D
aldinia
concentrica AF163021
D
aldinia
fissa AF176976
D
aldinia
petriniae AF176970
D
aldinia
loculatoides AF176982
D
aldinia
loculata AF176959
D
aldinia
concentrica AY616681
A
nnulohypoxylon
atroroseum AJ390397
A
nnulohypoxylon
stygium AJ390409
A
nnulohypoxylon
annulatum AJ390395
H
ypoxylon
fendleri AJ390400
D
iatrype
disciformis AJ390410
100
99
100
76
60
100
75
100
73
73
88
100
67
98
100 100
100
88
100
100
99
100
100
99
87
100
100
80
100
100
Clade A
Clade B
Clade C
Clade F
Clade G
Clade D
Clade E

Mycosphere
7
Fig 16. Phylogenetic tree based on combined 28S and ITS1-5.8S-ITS2 sequences. The tree is rooted
with Pestalotiopsis versicolor and constructed under un-weighted maximum parsimony criterion.
The number at each branch point represents percentage bootstrap support calculated from 1000
replicates and Bayesian posterior probabilities (thickened branches). Branch lengths are
proportional to the numbers of nucleotide substitutions and are measured by scale bar (Bar; 10 %
sequence divergence).
Petrini (2004) reduced Astrocystis cocoës
to synonymy with S. elaeicola, but molecular
data presented here does not support this
taxonomic assignment. Ju & Rogers (2002)
have suggested that N. maritima and N.
confluens are not well placed in the genus
Nemania and in the ITS data set they group
with Rosellinia aquila, R. pepo and Astrocystis
cocoës. The phylogenetic relationships of both
these groups warrant further study.
Acknowledgements
We thank the Biodiversity Research and
Training Program (BRT R 148008) and
Graduate School of Prince of Songkla
University for financial support, Manetr
Boonyanant and his staff for research facilities
at the Sirindhorn Nature Study and Research
Center, Narathiwat, Thailand. Aom Pinnoi
thanks Thailand Graduate Institute of Science
and Technology (TG-22-18-84OD) for the
10
R
osellinia arcuata
R
osellinia pepo
A
strocystis cocoës
A
strocystis bambusae
A
strocystis mirabilis
A
strocystis eleiodoxae
Stilbohypoxylon elaeicola
H
alorosellinia oceanica
R
osellinia necatrix
N
emania aenea
N
emania serpens
N
emania chesterii
Stilbohypoxylon quisquiliarum
K
retzschmaria deusta
X
ylaria grammica
B
iscogniauxia capnodes
B
iscogniauxia sp.
A
nnulohypoxylon sp. TH
A
nnulohypoxylon sp. GZ
H
ypoxylon fragiforme
H
ypoxylon monticulosum GZ
H
ypoxylon fendleri
A
nnulohypoxylon nitens
A
nnulohypoxylon stygium
X
ylariaceae sp.
P
estalotiopsis versicolor
A
nnulohypoxylon atroroseum
Clade A
Clade C
Clade E
Clade D
Clade F
1
2
76
66
100
100
70
86
100
79
100
100
100
100
99
99
100
100
99
100
94 Clade B

8
award of a Ph.D Scholarship. The University of
Hong Kong (RGC HKU 7322/04M) is thanked
for providing funds to support the molecular
study, provide Aom Pinnoi with a student-
training stipend, and Rajesh Jeewon with a Post
Doctoral Fellow to enable manuscript pre-
paration. Alvin Tang thanks The University of
Hong Kong for supporting a postgraduate
studentship. Prasert Srikitikulchai, Jariya
Sakayaroj, Rattaket Choeyklin and Umpava
Pinruan are acknowledged for their assistance
with the field work.
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