Wojnowicia viburni, sp. nov., from China and its phylogenetic placement

Abstract

A new species of Wojnowiciawas isolated from leaves of Viburnum utile in Guizhou
Province, China. The new species, Wojnowicia viburni, is described, illustrated and compared with similar species. It differs from other Wojnowicia species in its 6–8 euseptate,
smaller conidia and lack of setae. Phylogenetic analysis of LSU rDNA sequence data shows
that the species groups with W. hirtaand Ophiosphaerella herpotricha in the family Phaeosphaeriaceae.The genus Wojnowiciais emended to include species without setae. This is
the first record of the genus from China as well as from Asia. A key is provided for the four
species accepted in Wojnowicia

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* e-mail: yongwangbis@yahoo.cn (corresponding author)
Wojnowicia viburni
, sp. nov., from China and its
phylogenetic placement
Nalin N. Wijayawardene
1,2,3
, Yu Song
1
, D. Jayarama Bhat
2,4
,
Eric H. C. McKenzie
5
, Ekachai Chukeatirote
3
, Yong Wang
1
* and
Kevin D. Hyde
2,3
1
Department of Plant Pathology, Agriculture College, Guizhou University, 550025,
People’s Republic of China
2
Institute of Excellence in Fungal Research, Mae Fah Luang University,
Chiang Rai 57100, Thailand
3
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
4
Formerly, Department of Botany, Goa University, Goa 403 206, India
5
Manaaki Whenua Landcare Research, Private Bag 92170, Auckland, New Zealand
Wijayawardene N. N., Song Y., Bhat D. J., McKenzie E. H. C., Chukeatirote E., Wang Y.
& Hyde K. D. (2013) Wojnowicia viburni, sp. nov., from China and its phylogenetic place-
ment. – Sydowia 65 (1): 129–138.
A new species of Wojnowicia was isolated from leaves of Viburnum utile in Guizhou
Province, China. The new species, Wojnowicia viburni, is described, illustrated and com-
pared with similar species. It differs from other Wojnowicia species in its 6–8 euseptate,
smaller conidia and lack of setae. Phylogenetic analysis of LSU rDNA sequence data shows
that the species groups with W. hirta and Ophiosphaerella herpotricha in the family Phae-
osphaeriaceae. The genus Wojnowicia is emended to include species without setae. This is
the rst record of the genus from China as well as from Asia. A key is provided for the four
species accepted in Wojnowicia.
Keywords: asexual fungi, coelomycetes, molecular phylogeny, taxonomy.
Coelomycetes are a group of asexual fungi belonging to many families
within the Ascomycota, although a few genera (e.g. Ellula and Fibulocoela)
are Basidiomycota (Wijayawardene et al. 2012 a). Many coelomycetous asex-
ual states belong to the class Dothideomycetes (Chomnunti et al. 2011, Crous
et al. 2009, Liu et al. 2012, Zhang et al. 2012) but, in general, their taxonom-
ic placement is poorly established. Approximately 37 % of coelomycete gen-
era are linked to sexual states or can be accommodated in ascomycete fami-
lies (Wijayawardene et al. 2012 b) however, many cannot be accommodated
in the Ascomycota taxonomic framework because they lack molecular data.
Wijayawardene et al. (2012 c) emphasized the need for recollecting, isolating

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and sequencing these orphan coelomycete genera so that they can be placed
in natural taxonomic position.
Wojnowicia Sacc. was introduced by Saccardo (1892) with W. hirta
(J. Schröt.) Sacc. (≡ Hendersonia hirta J. Schröt.) as the type species. The ge-
nus was revised by Sutton (1980) and Farr & Bills (1995) accepted three spe-
cies. However, this genus was not addressed by molecular studies except de
Gruyter et al. (2009) who have shown it is belonging to Phaeosphaeriaceae.
In this paper we describe a new Wojnowicia species and discuss its taxo-
nomic placement in Phaeosphaeriaceae.
Materials and methods
Collection and isolation
Plant pathogenic and saprobic coelomycetes were collected during a
eld survey in Guizhou Province, China. Leaves of Viburnum utile with dis-
ease symptoms were returned to the laboratory where they were observed
under a stereo microscope. The fungus was isolated by the single spore isola-
tion method as described in Chomnunti et al. (2011). Germinating spores
were transferred aseptically to potato dextrose agar (PDA) plates and grown
at 25 °C. Colony colour and morphological characteristics were assessed af-
ter 2, 4 and 6 weeks. The holotype specimen is deposited in the Herbarium of
the Department of Plant Pathology, Agricultural College, Guizhou Univer-
sity (HGUP) (HGUP500 holotype) and the isotype is deposited in Mae Fah
Luang University (MFLU) Herbarium, Chiang Rai, Thailand (isotype
MFLU12–2221). Living cultures are deposited at the Culture Collection at
Mae Fah Luang University (MFLUCC), culture collection at Department of
Plant Pathology, Agricultural College, Guizhou University (HGUPCC) and
at Landcare Research, Private Bag 92170, Auckland, New Zealand (ICMP).
Genomic DNA was extracted from fresh mycelia, following the speci-
cation of Biomiga Fungus Genomic DNA Extraction Kit (GD2416). The
primers ITS5 and ITS4, NS1 and NS4 (White et al. 1990) and LROR and LR5
(Vilgalys & Hester 1990) were used to amplify the regions internal tran-
scribed spacers (ITS), small subunit rDNA (SSU) and large subunit rDNA
(LSU), respectively. Polymerase chain reaction (PCR) amplication was car-
ried out the method described in Phillips et al. (2008). LSU sequence of W.
viburni and sequences downloaded from GenBank were aligned using Bi-
oedit (Hall 2004) and ClustalX (Kohli & Bachhawat 2003). Alignments were
checked and manual adjustments were made wherever necessary. Phyloge-
netic analyses were performed by using MEGA 5 (Tamura et al. 2011) for
maximum likelihood (ML).
For phylogenetic analyses, DNA sequences of the LSU region of W. vi-
burni together with reference taxa of different families of Pleosporales ob-
tained from GenBank (Tab. 1) were aligned using Bioedit (Hall 2004). A blast
search was carried out to nd the closest matches with taxa in the family
Phaeosphaeriaceae. The whole ambiguously aligned regions within each

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Tab. 1. Sequences representing the closest taxa to Wojnowicia available in GenBank.
Taxon Accession number
GenBank number
LSU SSU
Ampelomyces quisqualis
Chaetosphaeronema hispidulum
Cochliobolus heterostrophus
Cochliobolus sativus
Coniothyrium palmarum
Coniothyrium palmarum
Cucurbitaria berberidis
Didymella exigua
Didymella pisi
Dothidotthia aspera
Dothidotthia symphoricarpi
Leptosphaeria doliolum
Leptosphaeria doliolum
Leptosphaeria doliolum
Leptosphaeria slovacica
Leptosphaerulina australis
Ophiosphaerella herpotricha
Ophiosphaerella herpotricha
Phaeosphaeria avenaria
Phaeosphaeria eustoma
Phaeosphaeriopsis musae
Phaeosphaeria nodorum
Phaeosphaeria oryzae
Phoma caloplacae
Phoma cladoniicola
Phoma cladoniicola
Phoma foliaceiphila
Phoma herbarum
Paraphoma radicina
Phoma zeae-maydis
Pleospora calvescens
Pleospora herbarum
Preussia minima
Pyrenochaeta acicula
Pyrenochaeta nobilis
Pyrenochaeta quercina
Pyrenochaetopsis decipiens
Pyrenochaetopsis leptospora
Stagonospora foliicola
Pyrenophora phaeocomes
Wojnowicia hirta
Wojnowicia hirta
Wojnowicia viburni
CBS 129.79
CBS 216.75
AFTOL–ID 54
AFTOL–ID
CBS 400.71
CBS 758.73
CBS 394.84
CBS 183.55
CBS 126.54
CPC 12933
CBS119687
CBS 541.66
CBS 155.94
CBS 125979
CBS 389.80
CBS 317.83
AFTOL–ID 1595
CBS 620.86
DAOM 226215
CBS 573.86
CBS 120026
CBS 110109
CBS 110110
CBS 129338
CBS 128027
CBS 128026
CBS 129141
CBS 615.75
CBS 102875
CBS 588.69
CBS 246.79
CBS 191.86
AFTOL–ID 1256
CBS 122789
CBS 407.76
CBS 115095
CBS 343.85
CBS 101635
CBS 343.86
AFTOL–ID 283
CBS 160.73
CBS 295.69
MFLUCC 120733
EU754128
AY544645
DQ678045
EU754153
EU754154
GQ387605
EU754155
GU237968
JF740284
JF740282
JF740283
JF740315
GU301830
DQ767656
EU754175
GQ387591
JQ238643
JQ238631
JQ238628
JQ238640
EU754186
EU754192
EU754131
GU238160
DQ678056
EU754204
EU754206
GQ387619
GQ387624
GQ387627
DQ499596
EU754222
EU754223
KC594287
EU754029
EU754041
EU673228
EU673224
DQ767650
DQ678010
AY544725
DQ678011
GU296186
EU754091
EU754118
EU754123
EU754124
KC594288

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dataset were excluded from the analyses (Begoude et al. 2010). In the analy-
ses, gaps were treated as missing data, and all characters were unordered
and of equal weight (Liu et al. 2011, 2012). Maximum-likelihood analyses
were performed using the heuristic search option with 1000 random taxa ad-
dition and tree bisection and reconnection (TBR) as the branch-swapping
algorithm. All characters were unordered and of equal weight and gaps were
treated as missing data. Branches of zero length were collapsed and all mul-
tiple, equally parsimonious trees were saved. The robustness of the most par-
simonious trees was evaluated from 1000 bootstrap replications (Hillis &
Bull 1993).
Taxonomy
Wojnowicia
Sacc., Syll. fung. (Abellini) 10: 328 (1892) emended
H a b i t : Associated with leaf spots of Viburnum utile, conifer litter, on
culms of Triticum spp. Sexual state: Ophiosphaerella-like. Asexual
s t a t e : C o n i d i o m a t a pycnidial, at rst immersed, later appearing super-
cial by decay of host tissues, separate, globose, often markedly papillate or
non papillate, dark brown; walls thick, composed of dark brown, thick-
walled textura angularis becoming hyaline and thin-walled towards the in-
ner conidiogenous region. O s t i o l e central or displaced to one side, papil-
late, circular. S e t a e absent or presence, when present formed around the
ostiole or from the lateral pycnidial walls, straight or exuous, unbranched,
brown, septate, smooth. Conidiophores absent. Conidiogenous cells
enteroblastic, phialidic, determinate, discrete, doliiform to ampulliform, hy-
aline, smooth, channel and collarette minute, formed from the inner pyc-
nidial wall cells. C o n i d i a pale brown, with several transverse eusepta, con-
tinuous, straight or curved, fusiform or cylindrical, apex and base obtuse,
thin-walled, smooth, guttulate.
Wojnowicia viburni
D. N. N. Wijayawardene, Yong Wang bis & K. D. Hyde,
sp. nov.
MycoBank no.: MB 803464
Etymology. – Named after the host genus on which the fungus occurs.
Type. – CHINA, Guizhou Province, Kaiyang, Longguang, on Viburnum utile Hemsl.
leaves, 3 June 2012, leg. D. N. N. Wijayawardene G0603–5 (HGUP500 holotype; MFLU12–
2221 isotype), ex-type living culture at MFLUCC 120733 = ICMP 19778= HGUPCC N28.
Description. – Associated with leaf spots of Viburnum utile, on both
sides, irregular, brown, leaf spots surrounded by dark brown border. S e x u a l
state not observed. Asexual state: Conidiomata pycnidial, 175–200
µm diam., 190–220 µm high, abundant on upper surface, partly immersed in
the host tissue, scattered, solitary, non-papillate, brown. P y c n i d i a l wall
17–28 µm thick, thick-walled textura angularis with pigmented outer cell
layer and colourless inner cell layer. C o n i d i o p h o r e s absent. C o n i d i o g -
e n o u s c e l l s formed on the inner layer of conidiomata, ampulliform, en-
teroblastic, phialidic, smooth, pale brown. C o n i d i a 18–25 × 4–5 µm (x¯ =

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Figs. 1–16. Wojnowicia viburni (holotype). 1. Irregular leaf spot. 2. Immersed conidiomata
3, 6. Longitudinal sections of conidiomata. 4, 5. Conidiomata wall. 7, 10–14. Conidia. 8–9.
Developing young conidia and mature conidium attached to conidiogenous cell. 15. Germi-
nating conidia. 16. Culture on PDA. Scale bars: 3, 6 100 µm, 4, 5, 7–9 25 µm, 10–14 20 µm.
20.2 × 4.3 µm, n=20), cylindrical, straight to slightly curved, gradually taper-
ing towards the rounded apex, with rounded apex, initially hyaline to pale
brown, after maturity pale golden brown, 6–8-euseptate, thin-walled.
C o l o n i e s on PDA olive brown to greyish brown, zonate, slow growing,
attaining a diam. of 5–6 cm after 14 days at 20–25 °C, later with dense myce-
lium, circular to irregular, with uneven margins, later comprising dense, my-

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celium, after 4 weeks, greyish brown, zonate, attaining 7–8 cm diam., with
thin mycelium, with conidiomata formed on the surface. Colonies after 6
weeks greyish (Fig. 3) and slightly raised.
Phylogenetic analyses
Partial nucleotide sequences of LSU ribosomal DNA (720 bp) were ob-
tained from the isolate. The other sequences used in the analysis were ob-
tained from GenBank (Tab. 1). The large subunit rDNA (LSU) data com-
prised 35 sequences of 27 taxa including outgroup taxa. New sequences in-
cluding SSU, ITS and LSU are deposited in GenBank.
Figs. 17–18. Cultural characteristics of Wojnowicia viburni colony on PDA after six weeks.
17. Colony from upside. 18. Colony from downside.
Wojnowicia viburni was aligned with a set of sequences obtained from
GenBank (Tab. 1) representing the closest taxa in Phaeosphaeriaceae follow-
ing a blast search. The LSU alignment contained 798 characters including
coded alignment gaps. Of the remaining 752, 542 were constant, while 210
were variable. Maximum likelihood analysis was carried out by MEGA 5 and
the resulting tree is shown in Fig. 19.
In the analysis of SSU and LSU, data set consists of 14 taxa with Doth-
idotthia aspera and D. symphoricarpi as the outgroup taxa. Both sets of se-
quences were rst analysed separately and then the individual datasets were
concatenated into a combined dataset. The dataset consists of 1849 charac-
ters including coded alignment gaps. Figure 20 shows the tree generated of
maximum likelihood analysis of combined gene from MEGA 5 (Tamura et al.
2011).
The phylogenetic tree obtained from maximum likelihood analysis
showed that the new isolate grouped in Phaeosphaeriaceae with Wojnowicia

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hirta, the generic type, and Ophiosphaerella herpotricha (Fr.) J. Walker. Wo-
jnowicia viburni and W. hirta cluster in the same clade but are distinct. Anal-
ysis of SSU did not show this relationship clearly (data not shown). Analysis
of combined LSU and SSU sequence data also clearly distinguish the spe-
cies. However, the bootstrap value is slightly lower than in the LSU analysis.
We have not carried out the combined gene analysis including ITS (i.e. ITS
and LSU and ITS and SSU) as the sequences of W. hirta are not available in
GenBank.
Fig. 19. Phylogenetic tree generated from maximum likelihood analysis from LSU rDNA
sequences by using MEGA 5. Bootstrap support values >50 % from 1000 replicates are
shown at nodes. The tree is rooted to Preussia minima. All type strains are in bold.
Discussion
Saccardo (1892) established Wojnowicia, with W. hirta as the type spe-
cies. The genus was characterized by Sutton (1975) as having black, setose,
pycnidial conidiomata which are often papillate, with well-dened ostioles,
and having enteroblastic and phialidic conidiogenous cells and brown,
transversely euseptate conidia. Sutton (1975, 1980) accepted only two spe-

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cies, W. hirta and W. ephedrae Hollós. Wojnowicia tenella Pat. and W. graminis
(McAlpine) Sacc. & D. Sacc. were considered synonyms of W. hirta by Sutton
(1980). Farr & Bills (1995) followed Sutton (1975, 1980) in accepting two spe-
cies and also described W. colluvium D. F. Farr & Bills. Wojnowicia buxi Ber-
tault & Malençon, described by Malençon & Bertault (1976), was considered
a younger synonym of W. ephedrae by Farr & Bills (1995). Index Fungorum
(2013) lists W. bryophila Racov. and W. exilis (Corda) Sacc. & Traverso as
epithets of Wojnowicia. However Sutton (1980) and Farr & Bills (1995) have
not accepted these two epithets in Wojnowicia.
Sutton (1980) listed the genus from Australia, Canada, France, Hungary,
Ireland, Italy, Tunisia, Turkey and the United Kingdom. Farr & Bills (1995)
described their species from the United States of America. Therefore, this is
the rst record of the genus from China and Asia.
Fig. 20. Phylogenetic tree generated from maximum likelihood analysis of combined data-
set of LSU and SSU sequences by using MEGA 5. Bootstrap support values for maximum
likelihood >50 % from 1000 replicates are shown at nodes. The tree is rooted to Dothidot-
thia aspera and D. symphoricarpi.
The 5–8-euseptate conidia found in W. hirta are quite similar to those in
W. viburni, however, conidia of W. hirta are longer (35–45 µm). The conidio-
mata of both species are subepidermal to epidermal though in W. viburni
conidiomata are not papillate. The most characteristic difference is the lack
of setae in W. viburni; setae are found in all other species of Wojnowicia
(Figs. 1–16).
Besides the study of de Gruyter et al. (2009), Wojnowicia has not been
included in any molecular study. De Gruyter et al. (2009) showed that W.
hirta grouped in Phaeosphaeriaceae with Ophiosphaerella herpotricha and
this is conrmed in our study. Zhang et al. (2012) also placed Wojnowicia in
Phaeosphaeriaceae (Pleosporales). This indicates that Wojnowicia may be

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the asexual state of Ophiosphaerella, however, O. herpotricha is not the type
species of Ophiosphaerella (Spegazzini 1909). Therefore, before Ophio-
sphaerella is synonymized under the older name Wojnowicia, it is necessary
to conrm that O. herpotricha and O. graminicola are congeneric using cul-
tural techniques or molecular data.
However we can conclude that Wojnowicia has Ophiosphaerella-like
sexual morphs, as Wojnowicia hirta, the generic type, groups with Ophio-
sphaerella herpotricha.
Key to species of
Wojnowicia
1. Conidia 5–8 euseptate ................................................................................... 2
1. Conidia 1–4 euseptate ................................................................................... 3
2. Conidia 5–7 euseptate, 35–45 µm long, pycnidia supercial at maturity
............................................................................................................. W. hirta
2. Conidia 5–8 euseptate, 17–28 µm, pycnidia immersed at maturity ............
.........................................................................................................W. viburni
3. Conidia primarily 3 euseptate, 19–36 µm long, pycnidia supercial, papil-
late .............................................................................................. W. colluvium
3. Conidia primarily 2 euseptate 20–29 µm long, pycnidia immersed, non-
papillate .......................................................................................W. ephedrae
Acknowledgements
This research was funded by Guizhou Province to tackle hard-nut prob-
lems in agricultural science and technology (No. 20113045).
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(Manuscript accepted 28 Apr 2013; Corresponding Editor: Kevin D. Hyde)