Available via license: CC BY 4.0
Content may be subject to copyright.
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=tmyc20
Mycology
An International Journal on Fungal Biology
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tmyc20
Proposal of a new family Pseudodiploösporeaceae
fam. nov. (Hypocreales) based on phylogeny
of Diploöspora longispora and Paecilomyces
penicillatus
Jingzu Sun, Shuang Yu, Yongzhong Lu, Hongwei Liu & Xingzhong Liu
To cite this article: Jingzu Sun, Shuang Yu, Yongzhong Lu, Hongwei Liu & Xingzhong Liu
(2022): Proposal of a new family Pseudodiploösporeaceae fam. nov. (Hypocreales) based
on phylogeny of Diploöspora�longispora and Paecilomyces�penicillatus, Mycology, DOI:
10.1080/21501203.2022.2143919
To link to this article: https://doi.org/10.1080/21501203.2022.2143919
© 2022 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 24 Nov 2022.
Submit your article to this journal
View related articles
View Crossmark data
Proposal of a new family Pseudodiploösporeaceae fam. nov. (Hypocreales) based
on phylogeny of Diploöspora longispora and Paecilomyces penicillatus
Jingzu Sun
a
, Shuang Yu
a,b,c
, Yongzhong Lu
d
, Hongwei Liu
a
and Xingzhong Liu
e
a
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3 Park 1, Beichen West Road, Chaoyang
District, 100101, Beijing, China;
b
School of Medical Devices, Shenyang Pharmaceutical University, 110016, Shenyang, China;
c
School of
Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 110016, Shenyang, China;
d
School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology,
550003, Guiyang, China;
e
Department of Microbiology, College of Life Science, Nankai University, 300350, Tianjin, China
ABSTRACT
During a eld survey of cultivated Morchella mushroom diseases, Diploöspora longispora and
Paecilomyces penicillatus, causal agents of pileus rot or white mould disease were detected,
which resulted in up to 80% of yield losses. Multi-locus phylogenic analysis revealed that the
fungi were aliated in a distinct clade in Hypocreales. We further constructed a phylogenetic tree
with broader sampling in Hypocreales and estimated the divergence times. The D. longispora and
P. penicillatus clades were estimated to have diverged from Hypocreaceae around 129 MYA and
Pseudodiploösporeaceae fam. nov is herein proposed to accommodate species in this clade. Two
new genera, i.e. Pseudodiploöspora and Zelopaecilomyceswere, were introduced based on morpho-
logical characteristics and phylogenic relationships of Diploöspora longispora and Paecilomyces
penicillatus, respectively. Five new combinations – Pseudodiploöspora cubensis, P. longispora,
P. fungicola, P. zinniae, and Zelopaecilomyces penicillatus – were proposed.
ARTICLE HISTORY
Received 16 August 2022
Revised 10 October 2022
Accepted 31 October 2022
KEYWORDS
Five new combinations; two
new genera; one new family;
fungal pathogen; mushroom
disease
Introduction
True morels (Morchella, Morchellaceae, Pezizales,
Ascomycota) are one of the most popular edible
mushrooms with a long history of consumption in
Asia, Europe, and North America (Pilz et al. 2017; Liu
et al. 2018). Because of their good taste, culinary
qualities, and pharmacological performances in anti-
tumor, anti-inammatory, and antioxidant activities
(Tietel and Masaphy 2018; Zhang et al. 2019),
demands for morels have signicantly increased in
the market. In recent years, large-scale eld cultiva-
tion of Morchella was successfully achieved in China
(Liu et al. 2018). The morel cultivation area reached
approximately 12,000 ha in the production season of
2021–2020 in China, with an economic value of over
RMB 10 billion. However, with the rapid expansion of
cultivation, diseases become a bottleneck for morel
production, especially for diseases caused by fungi.
Several common fungal diseases have been identied
in the fruiting bodies of cultivated Morchella: stipe rot
disease caused by the Fusarium incarnatum–F. equiseti
species complex (Guo et al. 2016) and by
Purpureocillium lilacinum (Masaphy, 2022), cobweb
disease caused by Hypomyces/Cladobotryum species
(Lan et al. 2020), white mould disease caused by
Paecilomyces penicillatus (He et al. 2017) and pileus
rot disease caused by Diploöspora longispora (He et al.
2018; Liu et al. 2018). Previous investigations showed
that white mould diseases and pileus rot resulted in
up to 80% of morel yield losses each year, which was
attributed to a large number of conidia quickly
spreading around the cultivation areas (Wang et al.
2020). However, the fungal pathogens were mainly
identied based on sequence similarity of internal
transcribed spacer gene region (ITS) but lacked con-
vincing morphological evidence (Hyde et al. 2017,
2018; Liu et al. 2018). When did a blast of the ITS
sequence of D. longispora, Tanney et al. (2015) also
showed that D. longispora is most closely related to
P. penicillatus including its ex-type (CBS 448.69).
The genus Diploöspora was established by Grove
(1916) with Diploöspora rosea as the type species. This
genus was characterised by producing chains of hya-
line, cylindrical to fusiform, aseptate, or 1–3-septate
conidia (Tanney et al. 2015). Currently, phylogenetic
analysis of the partial sequences of small subunit
CONTACT Hongwei Liu liuhw@im.ac.cn; Xingzhong Liu liuxz@nankai.edu.cn
MYCOLOGY
https://doi.org/10.1080/21501203.2022.2143919
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Published online 24 Nov 2022
(SSU) ribosomal RNA gene, internal transcribed
spacers (ITS), and large subunit (SSU) ribosomal RNA
gene reveals that D. rosea is an onygenalean fungus
(Tanney et al. 2015). Diploöspora longispora was rstly
isolated from a dead leaf of Colocasia esculenta var.
antiquorum in Japan (Matsushima 1976). Two varieties
of D. longispora are available, namely Diploöspora
longispora var. longispora and Diploöspora longispora
var. cubensis, and the latter was originally obtained
from the fallen leaves of Leguminosae in Cuba
(Castaneda 1987). Tanney et al. (2015) presented
that D. longispora and its varieties were most closely
related to P. penicillatus belonging to the order
Hypocreales and reached anity with Hypocreaceae.
However, apart from conidial chains, there is little
morphological similarity between P. penicillatus and
D. longispora, namely the penicillate conidiophores of
P. penicillatus with their basipetal conidiogenesis ver-
sus the branched conidiophores and acropetal con-
idiogenesis of D. longispora (Tanney et al. 2015).
The genus Paecilomyces was introduced by Bainier
(1907) with Paecilomyces variotii as the type species
(Samson 1974). This genus was featured by verticillate
conidiophores with divergent whorls of phialides,
having a cylindrical or inated base tapering to
a long and distinct neck and producing typically hya-
line, one-celled conidia. Phylogenetic analysis based
on 18S rDNA demonstrates that Paecilomyces is poly-
phyletic across two classes (Luangsa-Ard et al. 2004).
The type species, P. variotii, and its thermophilic
relatives were placed in Eurotiales (Eurotiomycetes),
while mesophilic species are in Hypocreales
(Sordariomycetes) (Luangsa-Ard et al. 2004, 2005).
Paecilomyces penicillatus was introduced by Samson
in 1974, which was rst isolated from rotten mush-
rooms. Based on the morphological characteristics, it
was placed in Eurotiales (Samson 1974). Based on the
molecular phylogenetic analysis of 18S rRNA
sequences and β-tubulin, P. penicillatus was trans-
ferred to the order Hypocreales (Sordariomycetes)
and revealed an uncertain anity with
Hypocreaceae (Luangsa-ard et al. 2004, 2005); how-
ever, P. penicillatus is still placed in Paecilomyces
sensu stricto.
According to Hyde et al. (2020), this order com-
prises 14 families, including Bionectriaceae,
Calcarisporiaceae, Clavicipitaceae, Cocoonihabitaceae,
Cordycipitaceae, Flammocladiellaceae, Hypocreaceae,
Myrotheciomycetaceae, Nectriaceae, Niessliaceae,
Ophiocordycipitaceae, Sarocladiaceae,
Stachybotryaceae, and Tilachlidiaceae. Recently,
Polycephalomycetaceae was introduced for the
accommodation of the fungicolous species from
Ophiocordycipitaceae based on a concatenated matrix
of six genetic markers (LSU, ITS, SSU, TEF, RPB1, and
RPB2, personal communication). These hypocrealean
fungi are mostly found as saprobes on decaying wood
and in soil, pathogens or endophytes of plants, nema-
todes, and insects (Zhang et al. 2018, personal com-
munication), as well as parasites on other fungi and
lichens (Zhu and Zhuang 2013; Sun et al. 2019a).
Generally, hypocrealean fungicolous taxa are the
more serious, common pathogens of most cultivated
mushrooms (Sun et al. 2019b). Diploöspora longispora
and Paecilomyces penicillatus (Hypocreales) are recog-
nised as the serious pathogens of cultivated
Morchella, and previous studies indicated that their
family ranks are uncertain (Luangsa-Ard et al. 2004;
Luangsa-ard et al. 2005; Tanney et al. 2015).
Additionally, Tanney et al. (2015) and our analyses
presented that D. longispora and its variants were
most closely related to several P. penicillatus strains
(including CBS 448.69, the ex-type strain) based on an
ITS BLAST query. Currently, multi-locus phylogenetic
analyses and divergence time estimation have been
used to clarify the higher ranking of fungal taxa (Hyde
et al. 2017, 2020). This study performed phylogenetic
analysis and divergence time estimation using
a concatenated matrix of ve genetic markers (LSU,
ITS, SSU, TEF, and RPB2). Based on the results, a new
family, Pseudodiploösporeaceae, and two new genera
Pseudodiploöspora and Zelopaecilomyces, are intro-
duced to accommodate the fungal taxa misplaced
under Diploöspora and Paecilomyces, respectively.
Additionally, we introduce four combinations includ-
ing the fungal pathogens causing pileus rot disease
and white mould disease of cultivated Morchella.
Materials and methods
Specimens, isolates, and morphological
observation
Fresh specimens were collected along with the fruit-
ing bodies of cultivated Morchella mushrooms in
Kunming Yunnan province, Ankang, Shanxi province,
Baoding, Hebei, province, Ningxia Hui Autonomous
Region during the 2019 and 2021 cultivation seasons.
2J. SUN ET AL.
Fruiting bodies were examined from free-hand sec-
tions using a stereomicroscope. The conidia were
picked and streaked on potato extract agar (PDA)
and incubated at 25°C for 7 days. A Nikon Eclipse 80i
light microscope, equipped with dierential interfer-
ence contrast (DIC) optics, was used to capture digital
images. Tarosoft (R) v.0.9.7 Image Frame Work was
used to measure the morphologic structures, and
Adobe Photoshop CS6 Extended version 13.0.1 soft-
ware (Adobe Systems, USA) to edit the photographic
plates.
For observation by SEM, each patch (0.3 × 0.3 cm)
of the fresh infected and un-infected M. sextelata was
xed in 2.5% glutaraldehyde in 0.05 M phosphate-
buered saline (BPS, pH 7.2) at 4°C. After 24 hours,
the samples were washed with deionised 0.1 M PBS
for 7 min three times, then dehydrated in graded
ethanol (50%,70%, 80%, 95%) for 15 min, respectively.
Subsequently, the samples were dehydrated in 100%
ethanol for 15 min three times and dried in a fume
hood using critical point dryers (Autosamdri® 931,
Tousimis, MD, USA) with CO
2
. Finally, the samples
were sputter-coated with gold by an ion sputter
coater (ISC150, SuPro Instruments, Shenzhen, China)
with a voltage of 110 V, a frequency of 50/60 Hz, and
a current of 10 mA under vacuum of lower than 1–
2 Pa for 60 s. The samples were loaded onto the SEM
(SU8010, Hitachi, Tokyo, Japan) and observed.
DNA extraction, PCR amplication, and sequencing
Genomic DNA of each strain was extracted from fresh
mycelium grown on PDA after 7 days of growth fol-
lowing the rapid “thermolysis” method described by
Zhang et al. (2010). For the amplication of SSU, ITS,
LSU, RPB2, and TEF1-α gene fragments, the following
primer pairs: NS1/NS4 primer pair for partial small
subunit ribosomal RNA gene region (SSU), ITS4/ITS5
primer pair for internal transcribed spacer gene
region (ITS) (White et al. 1990), LROR/LR5 for partial
large subunit rRNA gene region (LSU), 983 F/2218 R
for partial translation elongation factor 1-alpha gene
region (TEF-1α) (Carbone and Kohn 1999); RPB2-5 F/
RPB2-7 R for partial RNA polymerase II largest subunit
gene region (RPB2) (Liu et al. 1999) was used. Each
PCR reaction consisted of 12.5 μl 2× Taq PCR
SuperMix (TianGen Biotech Co., Beijing, China), 1 μl
of each forward and reverse primer (10 μM), 0.5 μl
DMSO, 3 μl DNA template, and 7 μl double sterilised
water. PCR reactions were performed in a fast thermal
cycler (LongGene Co., Hangzhou, China), following
the protocols described by Gu et al. (2020). The PCR
products were sequenced by Beijing Tianyihuiyuan
Bioscience and Technology after being evaluated by
electrophoresis.
Phylogenetic analysis
SeqMan Pro v. 7.1.0 (DNASTAR Lasergene) was used
to trim the low-quality bases at both ends of the raw
forward and reverse reads and to assemble them. The
newly obtained sequences were queried against the
nuclear database of NCBI. For species delimitation,
the aligned ITS sequence matrix of 58 taxa including
our isolates, Diploöspora, and available species of
Paecilomyces and its allied fungi, as well as Alternaria
species (outgroup taxa) were used to construct the
phylogenetic tree. The SSU, ITS, LSU, RPB2, and TEF
sequences of available generic type species and
reprehensive of Hypocreales and representative spe-
cies of all accepted Hypocaceae from recent studies
(Sun et al. 2017) were employed for multi-locus phy-
logenetic analysis. Gelasinospora tetrasperma,
Neurospora crassa and Sordaria micola were chosen
as the outgroup taxa. The alignments were generated
by using MAFFT version 7.03 with the Q-INS-I strategy
(Katoh and Standley 2013). Conserved blocks were
selected from the initial alignments with Gblocks
0.91 b (Castresana 2000). The best nucleotide substi-
tution model for each gene was determined by using
jModeltest2.1.1 (Darriba et al. 2012). GTR+G + I was
estimated as the best-t model for ITS; RPB2, TN93 + G
was estimated as the best-t model for SSU; and LSU,
TN93 + G + I as the best-t model for TEF-1α under
the output strategy of BIC. The multi-locus phyloge-
netic analyses included 1403 characters for SSU, 607
characters for ITS, 893 characters for LSU, 1044 char-
acters for RPB2, and 907 characters for TEF. All char-
acters were weighted equally, and gaps were treated
as missing characters.
Maximum likelihood (ML) analyses were per-
formed by RAxML2.0 (Edler et al. 2021), using the
GTR+GAMMA+I model. The maximum likelihood
bootstrap proportions (MLBP) were determined
using 1000 replicates. Bayesian inference (BI) ana-
lyses were conducted with MrBayes v3.2.7
(Ronquist et al. 2012). Metropolis-coupled Markov
Chain Monte Carlo (MCMC) searches were
MYCOLOGY 3
calculated for 10,000,000 generations, sampling
every 100th generation with the best-t model
for each gene. Two independent analyses with six
chains each (one cold and ve heated) were car-
ried out until the average standard deviation of the
split frequencies dropped below 0.01. The initial
25% of the generations of MCMC sampling were
discarded as burn-in. The renement of the phylo-
genetic tree was used for estimating Bayesian
inference posterior probability (PP) values. The
tree was viewed in FigTree v1.4 (Rambaut 2012),
and values of maximum likelihood bootstrap pro-
portion (MLBP) greater than 50% and Bayesian
inference posterior probabilities (BIPP), greater
than 95% at the nodes, are shown along branches.
Relative divergence time estimation
Molecular dating analysis was performed using BEAST
v1.10.4 (Suchard et al. 2018). The aligned data were
partitioned for each SSU, ITS, LSU, RPB2, and TEF1 data-
set, and these were loaded to BEAUti v1.10.4. to prepare
the XML le. The data partitions were set with unlinked
substitution and clock models to independently esti-
mate each gene partition. Taxa sets were developed for
each calibration of the common ancestor nodes, asso-
ciated with the most recent common ancestor (TMRCA).
The Hypocreales crown with a normal distribution
(mean = 216, SD = 27.5, with 97.5% of CI = 269 MYA).
Calibration of the core Clavicipitaceae, using a normal
distribution (mean = 133.7, SD = 20.8, with 97.5% of
CI = 174.5 MYA). Calibration of the Ophiocordyceps
crown, using an exponential distribution (oset = 100,
mean = 27.5, with 97.5% CI of 200 MYA) (Samarakoon
et al. 2016; Hyde et al. 2017). The Yule process tree prior
was used to model the speciation of nodes in the topol-
ogy with a randomly generated starting tree. The ana-
lyses were performed for 100 million generations, with
sampling parameters every 1000 generations. The eec-
tive sample sizes were checked in Tracer v.1.7.2 and the
acceptable values are higher than 200. The rst 10,000
trees (10%) representing the burn-in phase were dis-
carded based on Tracer v.1.7.2, and 90,000 trees were
combined in LogCombiner v1.10.4. The maximum clade
credibility (MCC) tree was given by summarised data and
estimated in TreeAnnotator v1.10.4. The molecular dat-
ing tree was viewed in FigTree v1.4 (Rambaut 2012). In
the MCC tree, node bars indicate 90% condence inter-
vals for the divergence time estimates.
Results
Phylogenetic analyses
The phylogenetic trees showed that generic type
species of Diploöspora rosea (DAOM 250100) and
Paecilomyces variotii (CBS 101075) were positioned
in the class Eurotiomycetes, while isolates of
Diploöspora longispora and Paecilomyces penicillatus
were placed in the class Sordariomycetes (Figure 1).
Within Sordariomycetes, D. longispora (UAMH 340,
UAMH 6404, UAMH 6367, strain 60319, and strain
60320), D. longispora var. cubensis (CBS 727.87), and
P. penicillatus (CBS 448.69, IMI 186962) clustered
together with maximum support (MLBP/
BIBP = 100%/1.00, Figure 1). In the phylogenetic
tree, those fungi also showed anities with hypocrea-
lean fungi, especially close to Hypomyces corticiicola
(MLBP/BIBP = 100%/1.00, Figure 1).
To determine the family placement of
Diploöspora longispora and Paecilomyces variotii,
a phylogenetic tree was constructed with
a sequence matrix of ve alignment les including
SSU (1043 bp), ITS (607), LSU (884 bp), EF1-α (907
bp), and RPB2 (1044 bp) sequence data (a total of
4853 characters) from 111 taxa of Hypocreales and
three outgroup taxa (Gelasinospora tetrasperma,
Neurospora crassa, and Sordaria micola;
Figure 2). The phylogenetic tree well explained
the phylogenetic relationship within Hypocreales.
There were 15 clades formed in Hypocreales cor-
responding to the families Bionectriaceae,
Calcarisporiaceae, Clavicipitaceae, Cordycipitaceae,
Flammocladiellaceae, Cocoonihabitaceae,
Hypocreaceae, Nectriaceae, Niessliaceae,
Ophiocordycipitaceae, Polycephalomycetaceae,
Sarocladiaceae, Stachybotryaceae, and the clade
comprising Diploöspora longispora and
Paecilomyces penicillatus. Diploöspora longispora
and P. penicillatus are phylogenetically distinct
from the type species of their respective genera
and are better accommodated in as-yet unde-
scribed genera. They are hereafter referred to as
Pseudodiploöspora longispora and
Zelopaecilomyces penicillatus, respectively, and for-
mally described below. Pseudodiploöspora longis-
pora and Z. penicillatus form a strongly supported
(MLBP/BIBP = 100%/1.00; Figure 2) distinct clade sister
to Hypocreaceae with robust support (MLBP/
BIBP = 94%/1.00; Figure 2). Based on its phylogenetic
4J. SUN ET AL.
distinction from Hypocreaceae, this clade is described
below as Pseudodiploösporeaceae fam. nov.
Relative divergence time estimation
According to the divergence time estimates, the
crown age of Hypocreales is around 206 (165–246)
MYA (Figure 3). Based on our analysis,
Sarocladiaceae was the earlier diverged family in
Hypocreales, which diverged from other hypocrealean
fungi at approximately 159 MYA. Flammocladiellaceae
and Tilachlidiaceae were the youngest families within
Hypocreales, which diverged from each other about
116 MYA. In general, the divergence time for the
currently accepted 15 families is within the range of
116–159 MYA, suggesting that a family can at best be
as young as 116 MYA. In the MCC tree, our newly
generated Pseudodiploösporeaceae diverged from
Hypocreaceae at about 129 MYA, falling within the
temporal band of families.
Taxonomy
Pseudodiploösporeaceae Jing Z. Sun, X.Z. Liu & H.W.
Liu, fam. nov.
Fungal name: FN 571280
Etymology: Pseudodiploöspor-, from the genus
name Pseudodiploöspora, and -aceae is the family
sux
Type genus: Pseudodiploöspora Jing Z. Sun, X.Z.
Liu & H.W. Liu, gen. nov.
Figure 1. Phylogenetic analysis of Diploöspora longispora and Paecilomyces penicillatus based on ITS data set. The tree is rooted with
three Alternaria species (Dothideomycetes). Bootstrap values higher than 50% from RAxML (BSML) (left) are given above the nodes.
Bayesian posterior probabilities greater than 0.95 are indicated (BYPP) (right). Hyphens indicate bootstrap values less than 50% or
Bayesian posterior probability values lower than 0.90.
T
indicates the type. The type species of Diploöspora and Paecilomyces are in
blue.
MYCOLOGY 5
Figure 2. Multi-locus phylogenetic analysis of Hypocreales based on a combined SSU, ITS, LSU, TEF, and RPB2 data set. The tree
is rooted with Gelasinospora tetrasperma, Neurospora crassa, and Sordaria fimicola. Bootstrap values higher than 50% from
RAxML (BSML) (left) are given above the nodes. Bayesian posterior probabilities greater than 0.90 are indicated (BYPP) (right).
Hyphens indicate bootstrap values less than 50% or Bayesian posterior probability values lower than 0.90. The generic type
species are in blue.
6J. SUN ET AL.
Figure 3. The MCC tree of Hypocreales, including some representative strains of Sordariales, was obtained from a Bayesian approach
(BEAST). Bars correspond to the 95% highest posterior density (HPD) intervals. The fossil minimum age constraints and second
calibrations used in this study are marked with green dots. The divergence time of orders is marked in purple dots and families with
blue dots. The generic type species are in blue.
MYCOLOGY 7
Description: Saprobic or fungicolous; Sexual
morph: Undetermined. Asexual morph: Colonies on
natural substrate euse, whitish. Mycelia, supercial
or immersed; Hyphae branched, septate, hyaline.
Conidiophores micronematous to macronematous,
mononematous, penicillate. Conidiogenous cells sym-
podial, acropetal, basipetal, hyaline. Conidia cylindri-
cal, ellipsoidal, limoniform, solitary, or catenate in
simple or branched chains. Ramoconidia cylindrical
or fusiform, aseptate or septate, truncate at the
base, with terminal scars.
Note: Both phylogenetic analysis and molecular clock
evidence based on SSU, ITS, LSU, TEF, and RPB2
sequence data support Pseudodiploösporeaceae as
a sister group of Hypocreaceae. The MCC tree estimates
that Pseudodiploösporeaceae split from Hypocreaceae
around 129 MYA, falling within the temporal band of
families (50–150 MYA) (Hyde et al. 2017). Therefore,
Pseudodiploösporeaceae is introduced as a new family
within Hypocreales, to accommodate Pseudodiploöspora
and Zelopaecilomyces.
Pseudodiploöspora Jing Z. Sun, X.Z. Liu & H.W. Liu,
gen. nov.
Fungal name: FN 571281
Etymology: pseudo, in Latin, meaning “false or spur-
ious thing”, referring to members of this genus being
morphologically similar to Diploöspora but phylogen-
etically distinct to the Diploöspora species
Type species: Pseudodiploöspora longispora
(Matsush.) Jing Z. Sun, X.Z. Liu & H.W. Liu, comb. nov.
Description: Saprobic or fungicolous; Sexual
morph: Undetermined. Asexual morph: Colonies on
natural substrate euse, whitish. Mycelia, supercial
or immersed; Hyphae branched, septate, hyaline.
Conidiophores micronematous to macronematous,
aseptate or septate. Conidiogenous cells sympodial,
acropetal, hyaline. Conidia cylindrical, ellipsoidal, fusi-
form, catenate in simple or branched chains, hyaline.
Ramoconidia cylindrical or fusiform, truncate at the
base, with terminal scars, hyaline.
Note: The genus Diploöspora was established by
Grove (1916) with Diploöspora rosea as the type spe-
cies. Phylogenetic evidence supported that D. rosea is
an onygenalean fungus within Eurotiomycetes
(Tanney et al. 2015). Diploöspora longispora and its
two variants, D. longispora var. longispora and
D. longispora var. cubensis, were isolated originally
from the fallen leaves (Matsushima 1976; Castañeda
1987). Based on an ITS BLAST query, Tanney et al.
(2015) proposed that D. longispora and its varieties
belong to the order Hypocreales, and reached anity
with Hypocreaceae. While, our phylogenetic analysis
based on the ITS sequence data also showed strains of
D. longispora (UAMH 340, UAMH 6404, UAMH 6367,
strain 60,319, and strain 60,320), and D. longispora var.
cubensis (CBS 727.87, IMI 186962) grouped with
strong support (MLBP/BIBP = 100%/1.00, Figure 1) in
Sordariomyetes rather than in Eurotiomyetes. In our
multi-locus phylogenetic tree, those taxa clustered
in a distinct clade within Hypocreales but do not
belong to Hypocreaceae (Figure 2), representing
a new genus rank. Morphologically, despite those
taxa and Diploöspora producing conidial chains, they
are distinct from Diploöspora in acropetal conidiogen-
esis and the shape and size of conidia.
Pseudodiploöspora is therefore introduced herein to
accommodate those species misplaced in
Diploöspora.
Pseudodiploöspora fungicola (R.F. Castañeda) Jing
Z. Sun, X.Z. Liu & H.W. Liu, comb. nov.
Fungal name: FN 571282
Basionym: Diploöspora fungicola R.F. Castañeda,
Fungi Cubenses II: 4 (1987)
Type: INIFAT C86/132 (Holotype)
Description: See the original description in
Castañeda Ruiz, R.F. (1987), Fungi Cubenses II, p. 22
Substrate/Host: On dead basidioma of Auricularia
Distribution: Cuba
Note: There is no available sequence of Diploöspora
fungicola, and its morphological characters are highly
similar to Pseudodiploöspora longispora. Additionally,
this species colonised the basidioma of Auricularia,
which suggests a similar fungicolous ecology relating
it to Pseudodiploöspora longispora (Castañeda Ruiz, R.
F. 1987).
Pseudodiploöspora longispora (Matsush.) Jing Z. Sun,
X.Z. Liu & H.W. Liu, comb. nov. Figures 4 and 5
Fungal name: FN 571283
Basionym: Diploöspora longispora Matsush., Icones
Microfungorum a Matsushima lectorum: 61 (1975)
Figure 5
Synonym: Diploöspora longispora var. longispora
Matsush., Icones Microfungorum a Matsushima lec-
torum: 61 (1975)
Type: INIFAT C87/58 (Holotype)
8J. SUN ET AL.
Description: See the original description in
Matsushima, T. (Matsushima and Matsushima 1976),
Icones Microfungorum a Matsushima Lectorum, p. 61.
Substrate/Host: On dead leaf of Colocasia esculenta
var. antiquorum, Japan (Matsushima and Matsushima
1976). On the fruiting body of cultivated Morchella spp.,
China (CGMCC 3.23768, CGMCC 3.23769, CGMCC
3.23770, CGMCC 3.23771). Skin and foot, Canada
(UAMH 340) Canada (https://www.uamh.ca/index.html)
Distribution: Japan, China
Note: Diploöspora longispora was rst isolated from
a dead leaf of Colocasia esculenta var. antiquorum in
Japan (Matsushima and Matsushima 1976). We intro-
duce a new combination of Pseudodiploöspora long-
ispora to accommodate D. longispora. Both analyses in
Tanney et al. (2015) and this study suggested that
Pseudodiploöspora longispora is most closely related
to Paecilomyces penicillatus. However, the latter diers
from Pseudodiploöspora longispora in the penicillate
conidiophore, and basipetal conidiogenesis, as well as
the shape and size of conidia (Figures 5 and 6). We did
not treat Paecilomyces penicillatus as a synonym of
Pseudodiploöspora longispora herein because of the
great morphological dierences.
Pseudodiploöspora cubensis (R.F. Castañeda) Jing
Z. Sun, X.Z. Liu & H.W. Liu, comb. nov. Figures 4 and 5
Fungal name: FN 5712997
Synonym: Diploöspora longispora var. cubensis R.F.
Castañeda, Fungi Cubenses II: 5 (1987)
Type: CBS 727.87 (ex-type strain)
Description: See the original description in
Castañeda (1987), Fungi Cubenses II.:1-22
Figure 4. Pseudodiploöspora longispora (CGMCC 3.23768). (a, b) Pseudodiploöspora longispora and its host fungus (Morchella sextelata). (c,
d) Mycelia on a fruiting body of M. sextelata. (e–h) Conidiophores with conidia. (i) Conidiogenous cell. (j) Conidiogenous scars and
conidia. (k–m) Hypha and conidia of P. longispora (in red) across the fruiting body of M. sextelata. Scale bars: c = 200 μm; d = 100 μm;
e-g, i = 25 μm; h, j = 10 μm.
MYCOLOGY 9
Substrate/Host: On fallen leaves of Leguminosae:
Cuba (Castaneda 1987). On porcupine dung in
a cave (Including UAMH 6367, UAMH 6404) (https://
www.uamh.ca/index.html)
Distribution: Cuba
Note: Pseudodiploöspora cubensis was originally
obtained from the fallen leaves of Leguminosae in
Cuba (Castaneda 1987). The ITS sequence of CBS
727.87 is 96% similar to Pseudodiploöspora longispora
(identities, 514/537, gaps, 5/537). Additionally, regard-
ing the ellipsoidal conidia of Pseudodiploöspora
cubensis against cylindrical or ramoconidia of
P. longispora, as well as the original isolation resource
and location, we introduce a new combination of
Pseudodiploöspora cubensis to accommodate
D. longispora var. cubensis.
Pseudodiploöspora zinniae (Matsush.) Jing Z. Sun, X.Z.
Liu & H.W. Liu, comb. nov.
Fungal name: FN 571284
Basionym: Diploöspora zinniae Matsush.,
Matsushima Mycological Memoirs 2: 8 (Matsushima
1981)
Type: MBT 70959
Description: See the original description in Matsush.
(1981), Matsushima Mycological Memoirs 2, p. 8
Substrate/Host: Seed of Zinnia elegans
Distribution: Japan
Note: There is no available sequence of Diploöspora
zinnia, and we transfer this fungus to
Pseudodiploöspora based on its sympodial, acropetal
conidiogenesis, and the cylindrical-fusiform conidia
(Matsushima 1981).
Zelopaecilomyces Jing Z. Sun, X.Z. Liu & H.W. Liu,
gen. nov.
Fungal name: FN 571285
Etymology: Zelo, meaning “emulation”, refers to
members of this genus being morphologically similar
to Paecilomyces but phylogenetically distinct from the
true Paecilomyces species
Type species: Zelopaecilomyces penicillatus
(Samson) Jing Z. Sun, X.Z. Liu & H.W. Liu,
comb. nov.
Description: Saprobic or fungicolous; Sexual morph:
Undetermined. Asexual morph: Colonies on natural
substrate euse, whitish. Mycelia, supercial or
immersed; Hyphae branched, septate, hyaline.
Conidiophores mononematous, penicillate, with
whorls of phialides. Phialides cylindrical, basal portion
with distinct neck. Conidiogenous cells basipetal, hya-
line. Conidia cylindrical, ellipsoidal, solitary, or cate-
nate in simple or in chains, aseptate, truncate at the
base, with terminal scars. Chlamydospores produced
Figure 5. Pseudodiploöspora longispora (Diploöspora longispora,
INIFAT C87/58, Holotype!). (a, b) Conidiophores with conidia. ().
Conidiogenous cell. (d) Conidiogenous scars and conidia. Scale
bars = 20 μm. Redrawn from Matsushima (1975).
Figure 6. Zelopaecilomyces penicillatus (CBS 448.69, ex-type strain).
(a) Conidiophores with conidia (CBS 448.69). (b) Phialides (type
Spicaria penicillate). (c) Chlamydospores. (d) Conidia. Scale
bars = 10 μm. Redrawn from Samson (1974).
10 J. SUN ET AL.
submerged in the agar, single, ellipsoidal to pyriform,
aseptate.
Note: The genus Paecilomyces was introduced by
Bainier (1907) with Paecilomyces variotii as the type
species. The type species, P. variotii, and its thermophi-
lic relatives were placed in Eurotiales (Eurotiomycetes),
while entomopathogenic mesophilic species were
placed in Hypocreales (Sordariomycetes) under the
genus Isaria but did not include Paecilomyces penicilla-
tus (Luangsa et al. 2004; Luangsa-Ard et al. 2005). Those
taxa placed in Isaria were accepted in Samsoniella
(Hypocreales, Cordycipitaceae) (Mongkolsamrit et al.
2018). Our phylogenetic analyses showed that
Z. penicillatus (CBS 448.69, ex-type strain) was posi-
tioned in Pseudodiploösporeaceae (Figure 2).
Despite a more than 99% similarity of the SSU (iden-
tities, 1589/1590, gap, 1/1590) and ITS sequence
(identities, 502/505; gaps, 3/505 gaps) between
Z. penicillatus (CBS 448.69) and Pseudodiploöspora
loogispora, respectively. The ITS of Z. penicillatus is
4% dierent from Pseudodiploöspora cubensis (iden-
tities, 471/477; gaps, 6/477). Additionally,
Z. penicillatus diers from both P. cubensis and
P. loogispora in having penicillate conidiophores
and basipetal conidiogenesis. Herein, we introduce
Zelopaecilomyces for the accommodation of
P. penicillatus based on its morphological distinc-
tions.
Zelopaecilomyces penicillatus (Höhn.) Jing Z. Sun, X.Z.
Liu & H.W. Liu, comb. nov. Figure 6
Fungal name: FN 571286
Basionym: Paecilomyces penicillatus (Höhn.)
Samson, Studies in Mycology 6: 72 (1974)
Spicaria penicillata Höhn., Annales Mycologici 2 (1):
56 (1904)
Type: ex-type strain CBS 448.69
Description: See the original description in Samson
(1974), Paecilomyces and some allied Hyphomycetes,
Studies in Mycology, 6, p.72
Substrate/Host: Peridia of Arcyria cinerea, rotting
Agaricus bisporus mushroom
Distribution: Austria, Belgium
Note: Zelopaecilomyces penicillatus (Spicaria penicil-
lata) was introduced by Höhnel (1904) based on its
morphological characteristics. It was rst isolated
from the peridia of the myxomycete Arcyria cinerea
and later isolated from a rotten Agaricus bisporus
mushroom, with the resulting strain (CBS 448.69) trea-
ted as the ex-type strain (Samson 1974). Herein, we
introduce a new combination, Zelopaecilomyces peni-
cillatus, in consideration of the distinct phylogenetic
position and morphological features of P. penicillatus.
Discussion
A combination of phylogenetic analyses and divergence
time estimation has been widely used in solving the
classication schemes and higher ranking of taxa (Hyde
et al. 2017). According to this polyphasic approach,
a large number of taxonomic positions of fungi have
been rened (Hyde et al. 2020; He et al. 2022). Hyde
et al. (2020) gave an update of Sordariomycetes based
on phylogenetic analyses and divergence time estima-
tion. According to their results, Hypocreales contained 14
families: Bionectriaceae, Calcarisporiaceae, Clavicipitaceae,
Cocoonihabitaceae, Cordycipitaceae, Flammocladiellaceae,
Hypocreaceae, Myrotheciomycetaceae, Nectriaceae,
Niessliaceae, Ophiocordycipitaceae, Sarocladiaceae,
Stachybotryaceae, and Tilachlidiaceae. Both our multi-
locus phylogeny and divergence time evidence reveal
the proposed natural classication of Hypocreales. Multi-
locus phylogeny reals a family rank for
Pseudodiploösporeaceae because its taxa formed
a strongly supported and distinct clade sister to
Hypocreaceae. Hyde et al. (2017) introduced a temporal
banding for Ascomycota, and time ranges of 150–250
MYA and 50–150 MYA were recommended as the
boundary for orders and families, respectively. Our MCC
results presented that the crown age of Hypocreales is
around 206 (165–246) MYA (Figure 3), which concurs with
the previous results (Hyde et al. 2017, 2020). Within
Hypocreales, the divergence time for currently accepted
families is within the range of 116–159 MYA suggesting
that a family can at best be as young as 116 MYA in
Hypocreales. Divergence time showed that the family
Pseudodiploösporeaceae divorced from Hypocreaceae
about 129 MYA, falling within the temporal band of
families. Additionally, Polycephalomycetaceae was
recently introduced as a new family based on
a concatenated matrix of six genetic markers (SSU, ITS,
LSU, RPB1, RPB2, and TEF) (personal communication),
both our phylogenetic tree and MCC tree also sup-
port its family rank in Hypocreales herein. Vu et al. (Vu
et al. 2019) proposed a taxonomic threshold pre-
dicted for lamentous fungal identication, and
88.5% similarity of ITS barcodes was suggested for
MYCOLOGY 11
family rank. A BLAST querying the ITS sequence of
species from Pseudodiploösporeaceae presented less
than 89% similarity against that species from
Hypocreales, which also supported distinct family
rank for Pseudodiploösporeaceae.
The taxonomic position of Diploöspora Grove was
conrmed as a member of Eurotiomycetes by re-
examination of its generic type species D. rosea
(Tanney et al. 2015). Several species including
Pseudodiploöspora longispora (previously known as
Diploöspora longispora) and Pseudodiploöspora cuben-
sis (previously known as Diploöspora longispora var.
cubensis) placed previously in Diploöspora were
shown an anity for Hypocrealean fungi
(Sordariomycetes) based on the phylogenetic analysis
(Luangsa-Ard et al. 2004, 2005; Tanney et al. 2015).
Our phylogenetic analysis also supported that
P. longispora and P. cubensis were more closely related
to Hypocreaceae (Figure 1–2). In our multi-locus phy-
logenetic tree, those taxa clustered in a distinct clade
within Hypocreales but were outside of the core
Hypocreaceae (Figure 2), representing a new family
and subsequent genera. Pseudodiploöspora is there-
fore introduced herein to accommodate those species
misplaced in Diploöspora concerning the original
nomenclature. Pseudodiploöspora is distinct from
Diploöspora in having head-to-tail (acropetal) arrays
of conidiogenesis against the latter of tail-to-head
(basipetal) arrays of conidiogenesis. Additionally, the
conidia of Pseudodiploöspora are longer but more
slender than that of Diploöspora (Tanney et al. 2015).
We introduce P. longispora and P. cubensis for accept-
ing D. longispora and D. longispora var. cubensis
regarding the 95.66% similarity of ITS sequence
between P. cubensis (CSB 727.877) and other
P. longispora isolates. Despite lacking molecular data
on Diploöspora fungicola and Diploöspora zinnia, we
enrolled them in Pseudodiploöspora according to the
morphological features in the original description.
Diploöspora coprophilia with phialides and producing
subglobose conidia is unlikely to be related to
Diploöspora rosea (Tanney et al. 2015) and
Pseudodiploöspora longispora. Its taxonomic position
needs to be further demonstrated. It was suggested
that Diploöspora indica producing brown conidio-
phores may be better placed in Parapleurotheciopsis
but not in Diploöspora (Tanney et al. 2015). We also
excluded D. indica from Pseudodiploöspora in consid-
eration of the brown conidiophore of the fungus.
Both analyses by Tanney et al. (2015) and this study
presented that P. longispora is most closely related to
Z. penicillatus (CBS 448.86) (Figure1–2). Vu et al. (2019)
proposed a 99.6% similarity of ITS barcode for
a species taxonomic threshold. When comparing the
similarity of the ITS sequence, Z. penicillatus presented
less than 98.63% similarity to that of P. longispora, and
showed less than 94.3% similarity to that of P. cubensis
(KT279809, CBS 727.87, ex-living type, previously
known as Diploöspora longispora var. cubensis),
respectively. There were no available EF1-α and RPB2
sequences in GenBank. We did not compare the simi-
larity of EF1-α and RPB2 sequences. However,
P. penicillatus diers from the latter in having penicil-
late conidiophores and basipetal conidiogenesis.
Herein, we introduce a new genus, Zelopaecilomyces,
for the accommodation of P. penicillatus.
The taxonomic position of Paecilomyces was
revised and rened by phylogenetic analyses, habi-
tats, host range, etc. (Luangsa-Ard et al. 2004, 2005).
The entomopathogenic mesophilic species were
placed in the class Sordariomycetes belonging to
Hypocreales (Luangsa et al. 2004, 2005). Generally,
those taxa were placed in Isaria, which were accepted
by a new genus Samsoniella (Hypocreales,
Cordycipitaceae) currently (Mongkolsamrit et al.
2018). Our phylogenetic analyses presented that
Z. penicillatus (CBS 448.69, ex-type strain) was posi-
tioned in Pseudodiploösporeaceae (Figure 2), which
was owing to a higher similarity of the SSU and ITS
sequence between P. penicillatus and D. longispora.
However, previous phylogenetic studies have evi-
denced that the SSU and ITS sequence data alone
are insucient to provide good resolution in most
of the groups in Sordariomycetes(Hyde et al. 2020).
Morphologically, Z. penicillatus diers from
P. longispora by penicillate conidiophore, basipetal
conidiogenesis, and the shape and size of conidia
(Figure 5 and 6). Additionally, the divergence time
revealed that Z. penicillatus diverged from
P. longispora about 14 MYA (Figure 3). Tanney et al.
(2015) thought that P. longispora and Z. penicillatus
may be two extremes of a continuum, However, we
treat P. penicillatus as a distinct species other than
a synonym of D. longispora not only relying on mor-
phological dierences but also following the diver-
gence time.
Both P. longispora and Z. penicillatus were ori-
ginally isolated from decaying leaves and found on
12 J. SUN ET AL.
the rotten mushroom successively (Samson 1974;
Matsushima 1976; Castaneda 1987). He et al. (2017)
identied Z. penicillatus (as Paecilomyces penicilla-
tus) as the causing agent of the white mould dis-
ease of cultivated Morchella only relying on ITS
phylogenetic analysis but lacking morphological
evidence. Liu et al. (2019) reported P. longispora
(as D. longispora) infecting cultivated Morchella,
resulting in pileus rot but not oered the typical
morphological feature of P. longispora. The phylo-
genetic analyses in both Tanney et al. (2015) and
this study revealed that ITS and SSU are unable to
adequately distinguish D. longispora and
Z. penicillatus, but our study oered robust mor-
phological evidence on the taxonomy of
P. longispora and Z. penicillatus. Since
P. longispora has been reported as a serious fungal
pathogen (Hyde et al. 2017; Liu et al. 2018), reliably
taxonomic information will facilitate tracing the
origin and understanding of pathogenesis.
Acknowledgements
This research was supported by the Natural Science Foundation
of China (no. 32072645). The authors would like to thank Chun-
Li Li from the Institute of Microbiology, Chinese Academy of
Sciences, for their instructions on the sample process and
observations by SEM. The authors would like to thank Prof.
Liwei Zhou for his suggestions on phylogenetic analysis and
divergence time estimation. We also thank Prof. Rob Samson for
the permission to redraw the plates of Paecilomyces penicillatus.
Disclosure statement
No potential conict of interest was reported by the author(s).
Funding
This work was supported by the The Natural Science
Foundation of China [no. 32072645].
ORCID
Jingzu Sun http://orcid.org/0000-0003-1893-1869
References
Carbone I, Kohn LM. 1999. A method for designing primer sets
for speciation studies in lamentous ascomycetes.
Mycologia. 91(3):553–556. doi:10.1080/00275514.1999.
12061051
Castaneda R. 1987. Fungi cubenses II: instituto de investiga-
ciones fundamentales en agricultura tropical “Alejandro
Humboldt”. Cuba (Academia de Ciencas de Cuba): la Habana.
Castresana J. 2000. Selection of conserved blocks from multiple
alignments for their use in phylogenetic analysis. Mol Biol Evo.
17(4):540–552. doi:10.1093/oxfordjournals.molbev.a026334
Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2:
more models, new heuristics and parallel computing. Nat
Meth. 9(8):772. doi:10.1038/nmeth.2109
Edler D, Klein J, Antonelli A, Silvestro D. 2021. raxmlGUI 2.0:
a graphical interface and toolkit for phylogenetic analyses
using RAxML. Methods Ecol Evol. 12(2):373–377. doi:10.
1111/2041-210X.13512
Grove WB. 1916. New or noteworthy fungi. Part V. The London
J Bot. 54:220.
Guo MP, Chen K, Wang G, Bian YB. 2016. First report of stipe rot
disease on Morchella importuna caused by Fusarium
incarnatum-F. equiseti species complex in China. Plant Dis.
100:2530. doi:10.1094/PDIS-05-16-0633-PDN
Gu X, Wang R, Sun Q, Wu B, Sun JZ. 2020. Four new species of
Trichoderma in the Harzianum clade from northern China.
MycoKeys. 73:109–132. doi:10.3897/mycokeys.73.51424
He P, Li CC, Cai YL, Zhang Y, Bian YB, Liu W. 2018. First report of
pileus rot disease on cultivated Morchella importuna caused
by Diploöspora longispora in China. J Gen Plant Pathol. 84
(1):65–69. doi:10.1007/s10327-017-0754-3
He MQ, Zhao RL, Liu DM, Denchev TT, Begerow D, Yurkov A,
Kemler M, Millanes AM, Wedin M, McTaggart AR, et al. 2022.
Species diversity of Basidiomycota. Fungal Divers. 114
(1):281–325. doi:10.1007/s13225-021-00497-3
Hyde KD, Maharachchikumbura SSN, Hongsanan S,
Samarakoon MC, Lücking R, Pem D, Harishchandra D,
Jeewon R, Zhao RL, Xu JC, et al. 2017. The ranking of fungi:
a tribute to David L. Hawksworth on his 70th birthday. Fungal
Divers. 84(1):1–23. doi:10.1007/s13225-017-0383-3
Hyde KD, Norphanphoun C, Maharachchikumbura SSN, Bhat, DJ,
Jones, EBG, Bundhun, D, Chen, YJ, Bao, DF, Boonmee, S,
Calabon, MS, Chaiwan, N, Chethana, KWT, Dai, DQ,
Dayarathne, MC, Devadatha, B, Dissanayake, AJ, Dissanayake,
LS, Doilom, M, Dong, W, Fan, XL, Goonasekara, AJ, Dissanayake
LS, Doilom M, Dong W, Fan XL, Goonasekara ID, Hongsanan S,
Huang SK, Jayawardena RS, Jeewon R, Karunarathna A, Konta S,
Kumar V, Lin CG, Liu JK Liu NG, Luangsa-ard J, Lumyong S, Luo
ZL, Marasinghe DS, McKenzie EHC, Niego AGT, Niranjan M,
Perera RH, Phukhamsakda C, Rathnayaka AR, Samarakoon
MC, Samarakoon SMBC, Sarma VV, Senanayake IC, Shang QJ,
Stadler M, Tibpromma S, Wanasinghe DN, Wei DP,
Wijayawardene NN, Xiao YP, Yang J Zeng XY, Zhang SN,
Xiang MM. 2020. Rened families of Sordariomycetes.
Mycosphere. 11(1):305–1059. doi:10.5943/mycosphere/11/1/7
Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment
software version 7: improvements in performance and usability.
Mol Biol Evol. 30(4):772–780. doi:10.1093/molbev/mst010
Lan YF, Cong QQ, Wang QW, Tang LN, Li XM, Yu QW, Cui X, An XR,
Kong FH, Li XD. 2020. First Report of Cladobotryum protrusum
MYCOLOGY 13
causing cobweb disease on cultivated Morchella importuna.
Plant Dis. 104(3):977–978. doi:10.1094/PDIS-07-14-0757-PDN
Liu W, Cai YL, He PX, Ma XL, Bian YB. 2019. Occurrence and
control of pests and diseases in eld cultivation of Morchella
mushrooms. Acta Edulis Fungi. 26(2):128–134. doi:10.16488/
j.cnki.1005-9873.2019.02.018
Liu QZ, Ma HS, Zhang Y, Dong CH. 2018. Articial cultivation of true
morels: current state, issues, and perspectives. Crit Rev
Biotechnol. 38(2):259–271. doi:10.1080/07388551.2017.1333082
Liu YJ, Whelen S, Hall BD. (1999). Phylogenetic relationships
among ascomycetes: evidence from an RNA polymerse II
subunit. Mol Biol Evol.16(12), 1799–1808. 10.1093/oxford
journals.molbev.a026092
Luangsa-ard JJ, Hywel-Jones NL, Manoch L, Samson RA. 2005. On
the relationships of Paecilomyces sect Isarioidea Species. Mycol
Res. 109(Pt 5):581–589. doi:10.1017/S0953756205002741
Luangsa-ard JJ, Hywel-Jones NL, Samson RA. 2004. The poly-
phyletic nature of Paecilomyces sensu lato based on
18S-generated rDNA phylogeny. Mycologia. 96(4):773–780.
doi:10.1080/15572536.2005.11832925
Masaphy S. 2022. First report on Purpureocillium lilacinum
infection of indoor-cultivated morel primordia. Agriculture.
12(5):695. doi:10.3390/agriculture12050695
Matsushima T. 1976. Icones Microfungorum a Matsushima
Lectorum. Mycologia. 68(4):955. doi:10.2307/3758819
Mongkolsamrit S, Noisripoom W, Thanakitpipattana D, Wutikhun T,
Spatafora JW, Luangsa A. 2018. Disentangling cryptic species
with Isaria-like morphs in Cordycipitaceae. Mycologia. 110
(1):230–257. doi:10.1080/00275514.2018.1446651
Pilz D, McLain R, Alexander S, Villarreal-Ruiz L, Shannon B,
Wurtz TL, Parks CG, McFarlane E, Baker B, Molina R, et al.
2017. Ecology and management of morels harvested from
the forests of western North America. Portland (OR): U.S.
Department of Agriculture, Forest Service, Pacic
Northwest Research Station; p. 166.
Rambaut A. 2012. FigTree v1. 4. Molecular evolution, phyloge-
netics and epidemiology. Edinburgh: University of
Edinburgh, Institute of Evolutionary Biology.
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A,
Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012.
MrBayes 3.2: ecient bayesian phylogenetic inference and
model choice across a large model space. Syst Biol. 61
(3):539–542. doi:10.1093/sysbio/sys029
Samarakoon MC, Hyde KD, Promputtha I, Ariyawansa HA,
Hongsanan S. 2016. Divergence and ranking of taxa across
the kingdoms Animalia, Fungi and Plantae. Mycosphere. 7
(11):1678–1689. doi:10.5943/mycosphere/7/11/5
Samson RA. 1974. Paecilomyces and some allied Hyphomycetes.
Stud Mycol. 6:1–119.
Suchard MA, Lemey P, Baele G, Ayres DL, Drummond AJ,
Rambaut A. 2018. Bayesian phylogenetic and phylodynamic
data integration using BEAST 1.10. Virus Evol. 4(1):vey016.
doi:10.1093/ve/vey016
Sun JZ, Li YL, Lin CG, Tian Q, Zhao Q, Xiao YP, Hyde KD,
Nilthong S. 2019. Fifteen fungicolous Ascomycetes on edible
and medicinal mushrooms in China and Thailand. Asian
J Mycol. 2(1):129–169. doi:10.5943/ajom/2/1/7
Sun JZ, Liu XZ, Hyde KD, Zhao Q, Maharachchikumbura SS,
Camporesi E, Nilthong S, Lumyong S, Lumyong S. 2017.
Calcarisporium xylariicola sp. nov. and introduction of
Calcarisporiaceae fam. nov. in Hypocreales. Mycol Prog. 16
(4):433–445. doi:10.1007/s11557-017-1290-4
Sun JZ, Liu XZ, McKenzie EHC, Jeewon R, Liu JK, Zhang XL, Zhao Q,
Hyde KD. 2019a. Fungicolous fungi: terminology, diversity, dis-
tribution, evolution, and species checklist. Fungal Divers. 95
(1):337–430. doi:10.1007/s13225-019-00422-9
Tanney JB, Nguyen HDT, Pinzari F, Seifert KA. 2015. A century
later: rediscovery, culturing and phylogenetic analysis of
Diploöspora rosea, a rare onygenalean hyphomycete.
Anton Leeuw Int J G. 108(5):1023–1035. doi:10.1007/
s10482-015-0555-7
Tietel Z, Masaphy S. 2018. True morels (Morchella)-nutritional
and phytochemical composition, health benets and avor:
a review. Crit Rev Food Sci Nutr. 58(11):1888–1901. doi:10.
1080/10408398.2017.1285269
von HFXR. 1904. Mycologische fragmente. (Fortsetzung)
[XLII-LXIX]. Ann Mycol. 2(1):38–60.
Vu D, Groenewald M, de Vries M, Gehrmann T, Stielow B,
Eberhardt U, Al-Hatmi A, Groenewald JZ, Cardinali G,
Houbraken J, et al. 2019. Large-scale generation and analysis
of lamentous fungal DNA barcodes boosts coverage for
kingdom fungi and reveals thresholds for fungal species
and higher taxon delimitation. Stud Mycol. 92(1):135–154.
doi:10.1016/j.simyco.2018.05.001
Wang XX, Peng JY, Sun L, Bonito G, Guo YX, Li Y, Fu YP. 2020.
Genome Sequencing of Paecilomyces Penicillatus Provides
Insights into Its Phylogenetic Placement and Mycoparasitism
Mechanisms on Morel Mushrooms. Pathogens. 9(10):834.
doi:10.3390/pathogens9100834.
White T, Bruns T, Lee S, Taylor J. 1990. Amplication and direct
sequencing of fungal ribosomal RNA genes for phyloge-
netics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors.
PCR protocols: a guide to methods and applications. London
(UK): Academic Press; p. 315–322.
Zhang Q, Cai W, Wang T, Sun YJ, Li TT, Fan GJ. 2019.
Improvement of biological activity of morchella esculenta
protein hydrolysate by microwave-assisted selenization.
J Food Sci. 84(1):73–79. doi:10.1111/1750-3841.14411
Zhang YJ, Zhang S, Liu XZ, Wen HA, Wang M. 2010. A simple
method of genomic DNA extraction suitable for analysis of
bulk fungal strains. Lett Appl Microbiol. 51(1):114–118.
doi:10.1111/j.1472-765X.2010.02867.x
Zhang WW, Zhang XL, Li K, Wang CS, Cai L, Zhuang WY,
Xiang MC, Liu XZ. 2018. Introgression and gene family con-
traction drive the evolution of lifestyle and host shifts of
hypocrealean fungi. Mycology. 9(3):176–188. doi:10.1080/
21501203.2018.1478333
Zhu ZX, Zhuang WY. 2013. Resources of nonlichenized fungi-
colous Ascomycota from China. Mycosystema. 32
(suppl):79–88. in Chinese.
14 J. SUN ET AL.
