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Revision of the genus Thyronectria (Hypocreales) from China
Z.Q. Zeng
W.Y. Zhuang
1
State Key Laboratory of Mycology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101,
People’s Republic of China
Abstract: Recent collections and herbarium speci-
mens of Thyronectria from different regions in China
were examined. Using combined analyses of morpholo-
gy and molecular data, we recognized eight species.
Among them, Thyronectria atrobrunnea,T. orientalis,
and T. sinensis are described and illustrated as new spe-
cies. Thyronectria atrobrunnea is characterized by black-
ish brown perithecia that become cupulate when dry,
and 8-spored asci containing ellipsoidal to broadly
fusiform or subcylindrical ascospores that bud to form
bacillar to subellipsoidal ascoconidia within the asci.
Thyronectria orientalis can be easily recognized by stro-
mata that are erumpent through the epidermis of
the host, immersed or semi-immersed perithecia
covered with yellowish green scurf, and ellipsoidal to sub-
fusiform, muriform ascospores. Thyronectria sinensis on
Pinus features solitary ascomata that are rarely aggre-
gated, and 8-spored asci giving rise to subcylindrical
to vermiform, multiseptate ascospores that form bacil-
lar to allantoid ascoconidia that fill the asci. The new
species and their close relatives are compared and dif-
ferences between them are discussed. Thyronectria strobi
is reported for the first time in China. Name changes
for the previously recorded species are noted. Phyloge-
netic analyses inferred from 28S, ITS, RPB1, TEF 1, and
TUB2 hint that phenotypic characters, viz. stromata,
ascospores, appendage of perithecial wall, and host
specificity may carry phylogenetic information as previ-
ous papers discussed.
Key words: morphology, multigene analyses,
Nectriaceae, taxonomy
INTRODUCTION
Thyronectria Sacc., typified by Thyronectria patavina Sacc.,
was established by Saccardo (1875) to include nectria-
like fungi having immersed ascomata and muriform
ascospores. A year later, Pleonectria Sacc., typified by
P. lamyi (Desm.) Sacc., was published to accommodate
nectriaceous fungi with discrete or caespitose perithe-
cia seated on stromata and with muriform ascospores
that produce ascoconidia in the asci (Saccardo 1876).
Considering that perithecia of T. patavina were not
immersed in stromata, Seaver (1909) and Seeler
(1940) concluded that it was unjustified to separate
them as two genera, and treated Pleonectria as a syno-
nym of Thyronectria. However, Rossman et al. (1999)
detected distinct pseudoparaphyses in the holotype of
T. patavina and concluded that the fungus does not
belong to Hypocreales. Subsequently, Thyronectria was
regarded a nomen dubium (Kirk et al. 2008). In their
comprehensive study, Hirooka et al. (2012) proposed
that Pleonectria was the oldest available name for this
group of fungi and accepted 26 species. Recent work
based on type examinations by Jaklitsch and Voglmayr
(2014) proved again that Pleonectria and Thyronectria
are congeneric, and that the type species of Mattirolia
Berl. & Bres. and Thyronectrioidea also belong to Thyro-
nectria. They interpreted the pseudoparaphyses
observed by Rossman et al. (1999) as apical paraphyses
that originate at the top of a perithecium and continue
down to the ascal base. The generic name Thyronectria
was thus retained and 29 species were accepted.
Recently, two new additional species of the genus
were added from Spain (Chaca et al. 2015).
Thyronectria is characterized by: (i) well-developed
erumpent stromata that are often covered with yellow-
green amorphous scurf; (ii) densely aggregated peri-
thecia that are superficial or immersed in a stroma
and that often become cupulate when dry; (iii) ascos-
pores that sometimes bud in the ascus to produce
ascoconidia; and (iv) zythiostroma-like asexual morphs
(Jaklitsch and Voglmayr 2014, Lombard et al. 2015).
Many members of the genus are distributed worldwide
mainly in temperate and subtropical regions and occur
on dead corticated twigs or branches of woody plants
in association with or directly on other fungi (Hirooka
et al. 2012, Jaklitsch and Voglmayr 2014), indicating
that at least they may be fungicolous. Otherwise,
they are mostly saprobic, while a few species are plant
pathogens (Hirooka et al. 2012). For example, T. aus-
troamericana is a facultative parasite causing canker of
honey locust in Alabama, Colorado, Kansas, Mississippi,
Massachusetts, Oklahoma, and Tennessee, USA (Crowe
et al. 1982, Conway and Morrison 1983, Jacobi and Riffle
1989, Hirooka et al. 2012). Because of the importance
of the genus, several studies focused on its taxonomy,
biology, and pathogenicity (Crowe et al. 1982, Bedker
and Wingfield 1983, Jacobi and Riffle 1989, Hirooka
et al. 2012).
Research on Thyronectria in China was started by
Tai (1979) when T. rosellinii (Carestia) Jaklitsch &
Voglmayr (as Ophionectria cylindrospora [Sollm.] Berl. &
Voglino) was first reported on fallen twigs of Larix sp.
Submitted 12 Jan 2016; accepted for publication 25 Aug 2016.
1
Corresponding author. E-mail: zhuangwy@im.ac.cn
Mycologia, 108(6), 2016, pp. 1130–1140. DOI: 10.3852/16-004
#2016 by The Mycological Society of America, Lawrence, KS 66044-8897
1130
from Heilongjiang Province. Thyronectria balsamea (Cooke
& Peck) Seeler (as Nectria balsamea Cooke & Peck) on
rotten twigs from Liaoning Province (Zhang and Zhuang
2003, Zhuang 2013), and T. pinicola (Kirschst.) Jaklitsch &
Voglmayr (as N. balsamea)onPinus taiwanensis from
Taiwan Province (Guu et al. 2007, Hirooka et al. 2012)
were later added. The genus appears to inhabit northern
and central China. In connection with our current work
on the Chinese fungus flora, recent collections and
herbarium specimens of the genus were examined. On
the basis of morphology and combined phylogenetic
analyses of the nuc 28S rDNA D1-D2 domains (28S)
and nuc rDNA region encompassing the internal
transcribed spacers 1 and 2, along with the 5.8S rDNA
(ITS), partial sequences of genes for RNA polymerase II
largest (RPB1), translation elongation factor 1-agene
(TEF1) and b-tubulin (TUB2), eight taxa were identified,
including three new species. The new species are com-
pared with their close relatives, their phylogenetic posi-
tions are revealed, and infraspecific variation of a few
other species is discussed.
MATERIALS AND METHODS
Specimens and strains.—Specimens were collected from Beijing
and Heilongjiang, Henan, Hubei, Liaoning, and Sichuan
provinces, and are deposited in Herbarium Mycologicum
Academiae Sinicae (HMAS). Cultures were obtained by single
ascospore isolation from fresh ascomata, and the strains
are deposited in the China General Microbiological Culture
Collection Center (CGMCC).
Morphological observations.—The methods of Luo and Zhuang
(2010) and Hirooka et al. (2012) were generally followed
for morphological studies. 3% KOH and 100% lactic acid
(LA) were used to test ascomatal wall reactions. Longitudinal
sections through ascomata were made with a freezing micro-
tome (YD-1508-III, Jinhua, China) at a thickness of 6–8mm.
Microscopic examinations and measurements were taken
from longitudinal sections and squash mounts in LA solution
using an Olympus BH-2 Microscope (Tokyo, Japan). Descrip-
tive statistics of ascospores and conidia (minimum, maxi-
mum, mean and standard deviation) were calculated
following the methods of Hirooka et al. (2012). Continuous
measurements were based on 30 individuals, except as other-
wise noted. Photographs were taken with a Canon G5 Digital
Camera (Tokyo, Japan) connected to a Zeiss Axioskop 2 Plus
Microscope (Göttingen, Germany). Characteristics of colo-
nies were recorded from cultures on potato dextrose agar
(PDA) and synthetic low nutrient agar (SNA) (Nirenberg
1976) at 25 C for 1 wk.
DNA extraction, amplification, and sequencing.—Genomic DNA
was extracted from fresh mycelium from the colony surface
by the methods of Wang and Zhuang (2004). For PCR ampli-
fications of phylogenetic markers, five different primer pairs
were used: (i) LR0R and LR5 for 28S (Vilgalys and Hester
1990, Rehner and Samuels 1994); (ii) ITS5 and ITS4 for ITS
(White et al. 1990); (iii) crpb1a and rpb1c for RPB1 (Castlebury
et al. 2004); (iv) 728F and 1567R for TEF1 (Carbone and Kohn
1999, Rehner 2001); and (v) T1 and T2 for TUB2(O’Donnell
and Cigelnik 1997). PCR reactions were performed on an
ABI 2720 Thermal Cycler (Applied Biosciences, Foster City,
California) with a 25 mL reaction volume consisting of 12.5
mL Taq MasterMix, 1 mL each primer (10 mM), 1 mL template
DNA, and 9.5 mL purified water. PCR conditions generally
followed the procedures of Hirooka et al. (2012). DNA sequenc-
ing was carried out in both directions with an ABI 3730XL
DNA Sequencer (Applied Biosciences, Foster City, California).
GenBank accession numbers for sequences used in the phylo-
genetic analyses are listed (SUPPLEMENTARY TABLE I).
Phylogenetic analyses.—Sequences were assembled, aligned,
and manually edited with BioEdit 7.0.5 (Hall 1999) and
converted to NEXUS files by ClustalX 1.8 (Thompson et al.
1997). Hydropisphaera fungicola and Verrucostoma freycinetiae
were selected as outgroup taxa. To determine the phylogenet-
ic positions of the new species, sequences of 28S, ITS, RPB1,
TEF1, and TUB2 regions were combined and analyzed with
maximum parsimony (MP) and Bayesian inference (BI) anal-
yses. The MP analysis was performed with PAUP 4.0b10 (Swof-
ford 2002) using 1000 replicates of heuristic search with
random addition of sequences and subsequent TBR (tree
bisection and reconnection) branch swapping. Topological
confidence of the resulting was tested by maximum parsimo-
ny bootstrap proportion (MPBP) with 1000 replications,
each with 10 replicates of random addition of taxa. The BI
analysis was conducted by MrBayes 3.1.2 (Ronquist and Huel-
senbeck 2003) using a Markov chain Monte Carlo (MCMC)
algorithm. Nucleotide substitution models were determined
by MrModeltest 2.3 (Nylander 2004). The nucleotide substitu-
tion model for each locus was analyzed. GTR+I+G was shown
to be the best-fit model for all these regions as well as for com-
bined sequences. Four Markov chains were run simultaneous-
ly for 1 000 000 generations with the trees sampled every 100
generations. The first 2500 trees were excluded from further
analysis and Bayesian inference posterior probability (BIPP)
was determined from the remaining trees. Trees were exam-
ined in TreeView 1.6.6 (Page 1996), with BIPP .90% and
MPBP .50% shown at the nodes.
RESULTS
The partition homogeneity test (P 50.01) indicated
that the individual partitions were not highly incongru-
ent (Cunningham 1997), and thus the five loci were
combined for sequence analyses. The combined data-
sets include 3556 characters, of which 2060 were con-
stant, 257 were variable and parsimony-uninformative,
and 1239 were parsimony-informative. The final matrix
was deposited in TreeBASE with accession No. S18072.
In the MP analysis, the trees reconstructed were 7806
steps long with CI 50.3509, HI 50.6491, RI 50.5871,
and RCI 50.2060. The BI tree generated is shown
(FIG. 1). The MPBP are also shown at the nodes.
The 44 investigated species belonging to Allantonec-
tria,Nectria, and Thyronectria grouped together (BIPP/
ZENG AND ZHUANG:THYRONECTRIA FROM CHINA 1131
FIG. 1. A Bayesian Inference trees inferred from the combined 28S, ITS, RPB1, TEF 1, and TUB2 sequence datasets. BIPP
.90% (left) and MPBP .50% (right) are indicated at nodes. New species proposed here are shown in boldface. The final
matrix was deposited in TreeBASE with accession No. S18072.
1132 MYCOLOGIA
MPBP 5100%/100%), and segregated into two major
clades (FIG. 1). All species of Nectria constituted one
clade (BIPP/MPBP 5100%/100%). Members of
Allantonectria and Thyronectria formed another (BIPP/
MPBP 5100%/99%), which was further divided into
seven subclades (I–VII). Subclades I–VI includes Thyro-
nectria species, while subclade VII contains only the two
known species of Allantonectria with high bootstrap sup-
port (BIPP/MPBP 5100%/99%).
The three new species were all placed within Thyro-
nectria.Thyronectria sinensis clustered with T. balsamea,
T. cucurbitula,T. pinicola,T. rosellinii, and T. strobi, which
occur only on conifers and produce budding ascoconi-
dia. These six species constituted the subclade I (BIPP/
MPBP 5100%/99%). In subclade II (BIPP/MPBP 5
100%/99%), T. boothii on Picea abies did not group
with species on conifers but appeared as a sister of
T. coryli. The perithecial walls of the fungi in this
subclade are three-layered, and the perithecia are
rounded at apical portion (Hirooka et al. 2012). Thyro-
nectria lamyi and T. caudata formed subclade III (BIPP/
MPBP 5100%/100%). They both live on the plant
genus Berberis.Thyronectria atrobrunnea was associated
with, but clearly separated from, T. sinopica and T. oki-
nawensis in subclade IV (BIPP/MPBP 5100%/98%).
Species of this subclade are common in perithecia
superficial on well-developed stromata and are charac-
terized by one-septate ellipsoidal to fusiform ascos-
pores. Thyronectria orientalis was in subclade V (BIPP/
MPBP 5100%/95%), which was a sister of T. austroa-
mericana (BIPP/MPBP 5100%/100%). They further
grouped with T. rhodochlora,T. virens, and T. zanthoxyli.
All members of this subclade have muriform ascos-
pores that do not produce ascoconidia within the asci.
Thyronectria asturiensis,T. giennensis,T. obscura,T. pista-
ciae, and T. roseovirens were located in subclade VI
(BIPP/MPBP 5100%/100%).
TAXONOMY
Thyronectria atrobrunnea Z.Q. Zeng & W.Y. Zhuang,
sp. nov. FIG.2
MycoBank MB814050
Typification: CHINA, HEILONGJIANG, Wuyin, 425
m, on rotten twigs of Eleutherococcus senticosus, 26 Aug
2014, Z.Q. Zeng, H.D. Zheng &W.T. Qin 9206 (holotype
HMAS 271280). Ex-type culture CGMCC 3.11739. Ex-
type sequences: 28S: KT423108; ITS: KT423111; RPB1:
KT423102; TEF1: KX372534; TUB2: KT423105.
Etymology: ater (Latin), dull black, and brunnea (Latin),
brown; referring to the blackish brown perithecia.
Mycelium not visible around ascomata or on host.
Stromata erumpent through epidermis, bay, turning
dark red in KOH and yellow in LA, pseudoparenchy-
matous, cells forming a textura angularis, intergrading
with ascomatal wall. Ascomata superficial on well-
developed stromata, aggregated in a group of 4–17,
subglobose to globose, cupulate upon drying, blackish
brown, no color change in KOH or LA, 295–370 6
305–410 mm(n540). Perithecial surface covered
with concolorous warts, 5–30 mm thick, cells forming
textura angularis or textura prismatica, 5–15 63–6mm,
walls 1–1.5 mm thick. Perithecial wall of two regions, 38–
63 mm thick, outer region of textura angularis, intergrad-
ing with stroma, 23–50 mm thick, cells 5–15 63–5mm,
walls 1–1.5 mm thick; inner region of textura prismatica,
8–13 mm thick, cells 5–15 62–3mm, walls 1–1.5 mm
thick. Apical paraphyses sparse, 2–4mm wide, septate
and branched. Asci clavate, with a simple apex,
8-spored, increasing in size as ascospores mature, 63–
103 65–7.5 mm(n550). Ascospores ellipsoidal
to broadly fusiform or subcylindrical, straight, 1-septate,
hyaline, smooth, mainly uniseriate or biseriate above in
the asci, 6–10 62–3mm(7.3¡1.1 62.6¡0.4 mm), l/w
2–4(n560), budding to produce hyaline, thin-walled,
bacillar to subellipsoidal ascoconidia, 1.8–3.2 60.8–1.5
mm(2.5¡0.5 61.2¡0.2 mm), l/w 2.1–4(n540).
In culture, colony on PDA growing fast, 78 mm diam
after 1 wk at 25 C, surface cottony, aerial mycelium
white, producing pale yellow pigment in the medium.
Colony on SNA 14 mm diam after one wk at 25 C,
surface slightly floccose, with sparse, whitish aerial myce-
lium. Conidiophores in aerial mycelium unbranched or
with 1–2 branches, 10–40 mm long, 1.5–3mm wide at
base. Conidiogenous cells in aerial mycelium mono-
phialidic, cylindrical, slightly tapering toward the tip
with widest point in the middle, 5–8mm long, 1.5–
2.5 mm wide at base. Sporodochial conidiophores
densely branched, 12.5–33 mm long, 1.5–2.5 mm wide
at base. Sporodochial conidiogenous cells monophiali-
dic, cylindrical, slightly tapering toward the tip or nar-
rowly flask-shaped with the widest point in the middle,
5–12.5 mm long, 1–2mm wide at base. Conidia ellipsoi-
dal, allantoid to rod-shaped, straight or slightly curved,
rounded at both ends, non-septate, hyaline, smooth,
2.5–13(–15) 61–3.5 mm (7.5¡462.2 ¡0.5 mm),
l/w 3.2–7(n540). Chlamydospores not observed.
Other specimen examined: CHINA, HEILONGJIANG, Wuyin,
425 m, on rotten twigs of Eleutherococcus senticosus, 26 Aug
2014, Z.Q. Zeng, H.D. Zheng &W.T. Qin 9207 (HMAS
252895).
Notes: The perithecial wall reactions to 3% KOH and
100% LA are negative, which is different from most
species of the genus. However, the major taxonomic
characters of the fungus, such as well-developed stro-
mata on natural substrates, ascospores budding to pro-
duce ascoconidia inside the asci, non-septate conidia,
and the sequence data, provide strong evidence for
placement of this species in Thyronectria.
ZENG AND ZHUANG:THYRONECTRIA FROM CHINA 1133
Among the known species of the genus, T. atrobrun-
nea resembles T. coryli in ascomatal gross morphology,
clavate asci, and ascospores forming ascoconidia. But
T. coryli differs from T. atrobrunnea in having wider
asci (40–115 65–15 mm), and larger ascospores (8.5–
15.5 62–5.5 mm) and ascoconidia (2–11 61–3.5 mm)
(Hirooka et al. 2012). The 5-locus phylogeny supports
recognition of T. atrobrunnea as a well-separated taxon.
It is related to, but clearly distinct from, T. sinopica.
The two species are easily distinguishable by gross mor-
phology of perithecia, shape and size of asci, and bud-
ding ascospores.
Thyronectria orientalis Z.Q. Zeng & W.Y. Zhuang,
sp. nov. FIG.3
MycoBank MB814058
Typification: CHINA, HENAN, Jiaozuo, Yuntaishan,
1000 m, on twigs of ?Pinus sp. (according to the ITS
sequence), 25 Sep 2013, H.D. Zheng, Z.Q. Zeng & Z.X.
Zhu 8912 (holotype HMAS 252896). Ex-type culture
CGMCC 3.11741. Ex-type sequences: 28S: KT423107,
ITS: KT423110, RPB1: KT423101, TEF1: KX372535,
TUB2: KT423104.
Etymology: orientalis (Latin), eastern; referring to the type
locality, eastern Asia.
Mycelium not visible around ascomata or on host.
Stromata erumpent through epidermis, pale white to
pale brown, not changing color in KOH or LA, of pro-
senchymatous cells. Ascomata semi-immersed in stro-
mata or immersed only at base, aggregated in groups
of 2–40, subglobose to globose, not becoming cupulate
FIG.2. Thyronectria atrobrunnea (HMAS 271280, holotype). a–c. Ascomata on natural substrate. d. Median section of an ascoma.
e, f. Asci with ascoconidia. g, h. Asci with ascospores. i, j. Budding ascospores. k, l. Ascospores. m. Colony on PDA. n–q.
Conidiophores and conidia. r, s. Conidia. Scale bars: a, b 51mm;c50.5 mm; d 550 mm; e–h510 mm; i–l, n–s55mm; m 51cm.
1134 MYCOLOGIA
upon drying, often firmly attached, brown to dark
brown, apical region slightly darker, turning brownish
black in KOH, with yellow substance effused from the
tissues, becoming slightly yellow in LA, 216–314 6
265–333 mm(n527). Perithecial surface covered with
abundant yellowish green to greenish yellow scurf, 25–
125 mm thick, cells forming a textura globulosa to tex-
tura angularis, cells 3.5–23 63–20 mm, walls 0.5–1mm
thick. Perithecial wall of two regions, 15–50 mmthick,
outer region of textura angularis to textura prismatica,
7.5–15 mmthick,cells5–12.5 62–3.5 mm, walls about
1mm thick; inner region of textura prismatica, 7.5–15
mmthick,cells10–17.5 62–3.5 mm, walls 0.5–1mm
thick. Apical paraphyses not observed. Asci clavate,
with a simple apex, (4–)8-spored, 53–100 67.5–12.5
mm(n542). Ascospores ellipsoidal with narrow ends
to subfusiform with blunt ends, muriform, with 4–6
transverse septa and one longitudinal septum, rarely
with up to three transverse septa, hyaline, smooth,
biseriate above and uniseriate below, (10–)11–20(–21)
65–7mm[15.3¡2.6 66¡1] mm, l/w 2–2.8 (n 568).
Colony on PDA growing fast, 70 mm diam after 1 wk
at 25 C, surface velvety, aerial mycelium white, produc-
ing light purple pigment in medium. Colony on SNA
FIG.3. Thyronectria orientalis (HMAS 252896, holotype). a–c. Ascomata on natural substrate. d. Colony on PDA. e. Median
section of ascomata. f. Median section of an ascoma. g, h. Asci with ascospores. i–l. Ascospores. m, o, p. Conidiophores and
conidia. n. Conidiophores. q. Chlamydospores. r. Conidia. Scale bars: a–c51 mm; d 51 cm; e, f 550 mm; g–r510 mm.
ZENG AND ZHUANG:THYRONECTRIA FROM CHINA 1135
10 mm diam after 1 wk at 25 C, surface radial, slightly
floccose, with sparse whitish aerial mycelium. Conidio-
phores with short simple branches, 5.5–19.5 61.5–2.5
mm. Conidiogenous cells monophialidic, cylindrical,
slightly tapering toward the tip, 3–661.5–2mm.
Conidia ellipsoidal, allantoid to rod-shaped, hyaline,
aseptate, 2–561–2mm [3.4 ¡161.4 ¡0.3 mm],
l/w 2–5(n560). Chlamydospores intercalary, globose
to subglobose, smooth, 3–7.5 62.5–7.5 mm.
Other specimen examined: CHINA, HENAN, Jiaozuo, 500 m,
on twigs of ?Pinus sp., 6 Sep 2013, Z.Q. Zeng, H.D. Zheng &
Z.X. Zhu 8941 (HMAS 271398).
Notes: Morphologically T. orientalis resembles T. aus-
troamericana in having stromata that are not immersed
in substrate, and muriform ascospores that do not
bud inside or outside the asci (Hirooka et al. 2012).
The latter differs significantly by its yellowish brown,
reddish gray to nearly black ascomata aggregated in
groups of up to hundreds, and subglobose to ellipsoidal
ascospores that are shorter (9.5–15 65–10 mm) and
with fewer transverse septa (1–2 septa) (Hirooka et al.
2012). The new species also resembles T. virens in
having immersed or semi-immersed perithecia, and
ellipsoidal muriform ascospores that do not bud inside
or outside the asci (Hirooka et al. 2012). However,
T. virens differs in its ascomatal surface covered with
dark green scurf, larger perithecia (270–410 6210–
400 mm), thicker perithecial wall (20–70 mm thick),
shorter and wider asci (55–80 610–20 mm), wider
ascospores (13–23 65.5–9.5 mm), and formation of
white to whitish yellow mycelium on PDA (Hirooka et al.
2012). Phylogenetically T. orientalis appears to be close-
ly related to T. austroamericana with strong statistical
support (BIPP/MPBP 5100%/100%), while morphol-
ogy distinguishes them.
Thyronectria sinensis Z.Q. Zeng & W.Y. Zhuang, sp.
nov. FIG.4
MycoBank MB814057
Typification: CHINA, SICHUAN, Jiuzhaigou, 2500 m,
on twigs of Pinus sp., 4 Aug 2013, Z.Q. Zeng, Z.X. Zhu &
F. Ren 8614 (holotype HMAS 271282). Ex-type culture
CGMCC 3.11740. Ex-type sequences: 28S: KT423106,
ITS: KT423109, RPB1: KT423100, TEF 1: KX372533,
TUB2: KT423103.
Etymology: sinensis (Latin), coming from China; referring to
the type locality, China.
Mycelium not visible around ascomata or on natu‐
ral substrates. Stromata erumpent through epidermis,
orange, turning orange red in KOH and yellow in LA,
of prosenchymatous cells. Ascomata superficial on well-
developed stromata, scattered to aggregated in group
of 3–4, subglobose to globose, cupulate upon drying,
orange red, becoming dark red in KOH and yellow in
LA, 304–372 6204–420 mm(n528). Perithecial sur-
face often covered with yellow scurf, 8–30 mmthick,cells
forming textura globulosa or textura angularis, 5–10 6
2–4mm, walls 1–1.5 mm thick. Perithecial wall of two
regions, 30–60 mm thick, outer region of textura globu-
losa or textura angularis, 20–42 mm thick, intergrading
with stroma, cells 6–13 62–8mm, walls about 1 mm
thick; inner region of textura prismatica, 10–20 mm
thick, cells elongate, 5–13 62–4mm, walls 0.5–
1mm thick. Apical paraphyses sparse, 2–3.5 mmwide,
septate and branched. Asci clavate to broadly clavate,
with a simple apex, 8-spored, increasing in size as ascos-
pores mature, 50–105 65–13 mm(n536). Ascospores
subcylindrical rounded ends to vermiform, with 11–27
septa, hyaline, smooth, (28–)33–63(–70) 62.5–3.5 mm
(49.8¡9.5 62.8n ¡0.3 mm), l/w 8–18 (n 540), bud-
ding to produce hyaline, thin-walled, bacillar to allan-
toid ascoconidia with a tapering apex, 2–3.5 60.8–1.2
mm(2.6¡0.4 61.1¡0.1 mm,) l/w 2.9–4.4 (n 550).
In culture, colony on PDA growing moderately slow,
14 mm diam after 1 wk at 25 C, surface velvety, aerial
mycelium white, producing pale yellow pigment in
the medium. Colony on SNA 21 mm diam after 1 wk
under 25 C, surface slightly floccose, with sparse, whit-
ish aerial mycelium.
Other specimen examined: CHINA, HUBEI, Shennongjia,
Dalongtan, 2000 m, on twigs of Pinus sp., 13 Sep 2014, Z.Q.
Zeng, H.D. Zheng, W.T. Qin & K. Chen 9477 (HMAS 271399).
Notes: Morphologically, T. sinensis is similar to T. cucur-
bitula and T. strobi in having multiseptate ascospores bud-
ding within the asci, and its occurrence on species of
Pinus (Hirooka et al. 2012). However, T. cucurbitula dif-
fers by having ascospores with more septa (15–39 septa)
and wider ascoconidia (1.9–4.5 6,1mm). Thyronectria
strobi can be distinguished by its smaller perithecia
(174–302 6210–340 mm), ascospores having more
septa (12–44 septa), and wider ascoconidia (2.5–46
1–2.5 mm). The sequence comparisons reveal that there
are 14 bp and 17 bp divergences in the ITS, and 9 bp and
16 bp differences in the 28S between T. sinensis and the
other two species. Thyronectria sinensis shares only 84%
and 85% sequence similarities with T. cucurbitula and
T. strobi for TUB2, and has 50 bp and 49 bp sequence
divergences in RPB1 region.
In our phylogenetic tree (FIG.1),T. sinensis is closely
related to T. rosellinii (BIPP/MPBP 5100%/100%)
from Japan and USA, while the latter has smaller perithe-
cia (215–350 6200–315 mm), shorter ascospores (22.5–
60 61.5–4mm) with 8–31 septa, wider ascoconidia
(2–4.5 61–3mm), and occurs on Abies instead of Pinus
(Hirooka et al. 2012).
Other accepted species.
Thyronectria balsamea (Cooke & Peck) Seeler, J. Arnold
Arbor. 21: 442. 1940.
Specimen examined: CHINA, LIAONING, Caohekou, on
rotten twigs, May 1964, L.P. Shao, HMAS 33788.
1136 MYCOLOGIA
Notes: The fungus was previously recorded as Nectria
balsamea (Zhang and Zhuang 2003). When compared
with the North American collections (Hirooka et al.
2012), the Chinese material fits well the species con-
cept except for the smaller asci (55–89 65.5–13.5
mmvs.58–139 66.5–17.5 mm), which is treated as
infraspecific variation.
Thyronectria cucurbitula (Tode) Jaklitsch & Voglmayr,
Persoonia, Mol. Phyl. Evol. Fungi 33: 201. 2014.
Specimen examined: CHINA, HEILONGJIANG, Tangyuan,
Yaoyin Forest Farm, on twigs of Pinus sp., 29 Aug 2014, Z.Q.
Zeng, H.D. Zheng &W.T. Qin 9416 (HMAS 252897).
Notes: Thyronectria cucurbitula remained as a species
complex for a long time until Hirooka et al. (2012)
separated it into three species, T. cucurbitula (s.s.) (as
P. cucurbitula), T. rosellinii (as P. rosellinii) and T. strobi
(as P.strobi). A recent collection from Heilongjiang
Province (HMAS 252897) agrees well with the Hiroo-
ka’s concept of T. cucurbitula s.s.
Another collection (HMAS 33641) from Heilong-
jiang Province, formerly filed under Ophionectria cylin-
drospora (Tai 1979) and later treated as N. cucurbitula
(Zhang and Zhuang 2003), was re-examined. Our
observations reveal that its perithecial surface is scaly
instead of scurfy, and its ascospores are much shorter
than those of the typical T. cucurbitula [(18–)23.5–
41.5(–50) mm vs. 32.5–74.5 mm long]. The correct iden-
tification of this collection should be T. rosellinii (see
below).
FIG.4. Thyronectria sinensis.a–c. Ascomata on natural substrate. d. Colony on PDA. e. Median section of ascomata. f. Median
section of an ascoma. g, h. Asci with budding ascospores. i, j. Asci with ascoconidia. k, l. Budding ascospores. m–o.
Conidiophores and conidia in pycnidium. p, q. Conidia in pycnidium. Scale bars: a 51 mm; b, c 50.5 mm; d 51 cm; e, f 550
mm; g–q510 mm. a–l from holotype, m–q from HMAS 271399.
ZENG AND ZHUANG:THYRONECTRIA FROM CHINA 1137
Thyronectria pinicola (Kirschst.) Jaklitsch & Voglmayr,
Persoonia, Mol. Phyl. Evol. Fungi 33: 203. 2014.
Specimen examined: CHINA, BEIJING, Yudushan, on twigs
of Pinus sp., 27 Jul 2015, X.C. Wang,Z.Q. Zeng,H.D. Zheng,
W.T. Qin &K. Chen 10098 (HMAS 252898).
Notes: In the past, many specimens of T. pinicola were
erroneously identified as Nectria balsamea. According to
the recent study by Hirooka et al. (2012), the synonymy
of these names is incorrect. Thyronectria pinicola is a
distinct species, which differs from T. balsamea in size
and number of transverse septa of ascospores, presence
of long sterile hyphae on natural substrates, and its
occurrence on Pinus.
Thyronectria rosellinii (Carestia) Jaklitsch & Voglmayr,
Persoonia, Mol. Phyl. Evol. Fungi 33: 204. 2014.
Specimens examined: CHINA, HEILONGJIANG, Dailing,
Liangshui Forest Farm, on bark of Larix gmelinii, 1 Jul 1963,
X.R. Pan HMAS 33641. SICHUAN, Ruoergai, 3500 m, on
twig of Abies sp., 23 Jul 2013, L. Wang, Z.Q. Zeng, Z.X. Zhu &
F. Ren 8309, 8311 (HMAS 252899, 252900); Malcolm,
on twig of Abies sp., 30 Jul 2013, Z.Q. Zeng, Z.X. Zhu &F. Ren
8491 (HMAS 252901). HUBEI, Shennongjia, Xiaolongtan,
2100 m, on twigs of Abies sp., 13 Sep 2014, Z.Q. Zeng,
H.D. Zheng, W.T. Qin &K. Chen 9466,9467,9494-2 (HMAS
271263, 252902, 271400); Shennongjia, Jinhouling, 2500 m,
on twigs of Abies sp., 14 Sep 2014, Z.Q. Zeng, H.D. Zheng,W.T.
Qin &K. Chen 9576 (HMAS 252903).
Notes: As mentioned by Hirooka et al. (2012), T. rosel-
linii can be divided into two groups with high
phylogenetic support. Our collections seem to be closer
to the Japanese material but cannot be separated
conclusively from the American ones. The two groups
should be considered conspecific, and there are very
few sequence differences.
This species is relatively common in China. Although
the ascomata of HMAS 33641 grow on Larix sp. instead
of Abies spp., it fits well in the concept of T. rosellinii
(Hirooka et al. 2012).
Thyronectria strobi (Hirooka, Rossman & P. Chaverri)
Jaklitsch & Voglmayr, Persoonia 33: 207. 2014.
Specimen examined: CHINA, HUBEI, Shennongjia, Tian-
ziya, 2000 m, on twigs of Pinus armandii, 16 Sep 2014,
Z.Q. Zeng, W.T. Qin, K. Chen & H.D. Zheng 9772 (HMAS
252904).
Notes: Thyronectria strobi was originally described by
Hirooka et al. (2012) in Pleonectria and transferred to
Thyronectria by Jaklitsch and Voglmayr (2014). The fun-
gus was previously known only from Europe and North
America (Hirooka et al. 2012). The Chinese collection
extends its distribution to Asia. Compared with the
original description, the Chinese collection has slightly
larger perithecia (294–392 6343–402 mm vs. 174–302
6210–340 mm) and shorter ascospores [(23–)27–41
(–45) 62.5–3mm vs. 22–64 62.5–3mm], which are
treated as infraspecific variation.
DISCUSSION
Phylogenetic overviews of Thyronectria based on multilo-
cus sequence analyses by Hirooka et al. (2012) and
Jaklitsch and Voglmayr (2014) showed that the genus
is monophyletic. Our analyses inferred from sequences
of 28S, ITS, RPB1, TEF1, and TUB2 including our three
new taxa revealed a similar tree topology (FIG. 1) to that
given by Jaklitsch and Voglmayr (2014). In Thyronectria,
six subclades were recognized along with a few taxa
(T. aurigera,T. berolinensis, and T. quercicola) forming
isolated lineages. Both morphological and molecular
data support the recognition of T. atrobrunnea,T. orien-
talis, and T. sinensis as new taxa, and their placement
in different subclades of Thyronectria.Ourresults
also suggest that phenotypic characters of stromata,
ascospores, appendage of perithecial wall, and host
specificity, carry phylogenetic information, already stat-
ed in the previous studies (Hirooka et al. 2012, Jaklitsch
and Voglmayr 2014).
Thyronectria sinensis belongs to subclade I (BIPP/MPBP
5100%/99%), along with T. balsamea,T. cucurbitula,
T. pinicola,T. rosellinii,andT. strobi. These species have
been found only on conifers, and produce budding asco-
conidia within the asci.
Thyronectria atrobrunnea together with T. okinawensis
and T. sinopica comprise subclade IV (BIPP/MPBP 5
100%/98%). They all have superficial perithecia on
well-developed stromata, and ellipsoidal to fusiform
ascospores that are uniseptate.
Thyronectria orientalis grouped with T. austroamericana,
T. rhodochlora,T. virens and T. zanthoxyli in subclade V
(BIPP/MPBP 5100%/95%). They share common mor-
phological characteristics, such as perithecia immersed
in stromata and covered with abundant bright yellow
scurf, and muriform ascospores that do not bud in
the asci.
The significance of differences in substrates has
been considered in the taxonomy of some nectriaceous
fungi. Species of Nectria often occur on decaying wood
and tend to have a broad host range (Rossman et al.
1999, Hirooka et al. 2012). In contrast, species of Thyr-
onectria mostly grow on recently killed woody plants,
and show a degree of host-specificity. For example,
T. aurigera and T. austroamericana are known only
from Oleaceae and Fabaceae, respectively. Thyronectria
aquifolii and T. ilicicola are restricted to Ilex aquifolium
(Hirooka et al. 2012). Thyronectria berolinensis is restrict-
ed to Ribes, while T. lamyi and T. caudata are on Berberis
(Jaklitsch and Voglmayr 2014). Some species of the
genus are fungicolous, and their presumed plant hosts
are actually fungi parasitic on the plants. For example,
T.rhodochlora is associated with or grows directly on
Diplodia spp., and T.roseovirens colonizes Cucurbitaria
1138 MYCOLOGIA
laburni and its closely related species (Jaklitsch and
Voglmayr 2014).
The Chinese collections of T. cucurbitula and T. pinicola
occur on Pinus spp., which agrees with the results of Guu
et al. (2007) and Hirooka et al. (2012). However, the
occurrence of T. rosellinii (HMAS 33641) on Larix gmelinii
seems inconsistent with the observation that this species
exclusively occurs on Abies (Hirooka et al. 2012). Similar-
ly, T. rhodochlora grows not only on Acer campestre but
also on other woody plants as well as fungi (Jaklitsch
and Voglmayr 2014). Additional collections of fungi in
the group may reveal their selection of hosts or substrates.
In the taxonomy of hypocrealean fungi, the reaction
of the perithecial wall to KOH is considered as an
important character (Rossman et al. 1999, Zeng
and Zhuang 2014). Exceptions were observed in Thyro-
nectria. Most species of Thyronectria have perithecial
color turning darker to blood-red or purple in KOH.
However, T. austroamericana,T. chlorinella,T. lonicerae,
T. rhodochlora,T. sphaerospora,T. virens, and T. zanthoxyli
display a weak or negative reaction to KOH, which
might be influenced by the presence of scurf covering
perithecia or their dark-colored ascomata (Hirooka
et al. 2012). In our study, the dark perithecial walls
of T. atrobrunnea do not change color in KOH but the
major features, such as well-developed stromata, ascos-
pores budding to produce ascoconidia in the asci, and
a zythiostroma-like asexual morph, as well as the molec-
ular data provide strong evidence that it belongs to
Thyronectria.
ACKNOWLEDGMENTS
The authors would like to thank all collectors of the speci-
mens for this study, and Drs A.Y. Rossman, G.J. Samuels,
and Y. Hirooka for sharing their expertise. This project was
supported by the National Natural Science Foundation of
China (Nos. 31270073, 31400020, 31570018) and Ministry of
Science and Technology of China for Fundamental Research
(Nos. 2013FY110400, 2014FY210400).
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