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© 2023 Westerdijk Fungal Biodiversity Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
Studies in Mycology
Revising Clonostachys and allied genera in Bionectriaceae
L. Zhao1,2, J.Z. Groenewald1, M. Hernández-Restrepo1, H.-J. Schroers3*, P.W. Crous1,2,4*
1Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, Utrecht, 3584 CT, The Netherlands; 2Microbiology, Department of Biology, Utrecht University,
Padualaan 8, Utrecht, 3584 CH, The Netherlands; 3Plant Protection Department, Agricultural Institute of Slovenia, Hacquetova ulica 17, Ljubljana, 1000,
Slovenia; 4Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Natural and Agricultural
Sciences, University of Pretoria, Private Bag X20, Hateld, Pretoria, 0028, South Africa
*Corresponding authors: Hans-Josef Schroers, hans.schroers@kis.si, Pedro W. Crous, p.crous@wi.knaw.nl
Abstract: Clonostachys (Bionectriaceae, Hypocreales) species are common soil-borne fungi, endophytes, epiphytes, and saprotrophs. Sexual morphs of
Clonostachys spp. were placed in the genus Bionectria, which was further segregated into the six subgenera Astromata, Bionectria, Epiphloea, Myronectria,
Uniparietina, and Zebrinella. However, with the end of dual nomenclature, Clonostachys became the single depository for sexual and asexual morph-
typied species. Species of Clonostachys are typically characterised by penicillate, sporodochial, and, in many cases, dimorphic conidiophores (primary and
secondary conidiophores). Primary conidiophores are mononematous, either verticillium-like or narrowly penicillate. The secondary conidiophores generally
form imbricate conidial chains that can collapse to slimy masses, particularly on sporodochia. In the present study, we investigated the species diversity
within a collection of 420 strains of Clonostachys from the culture collection of, and personal collections at, the Westerdijk Fungal Biodiversity Institute in
Utrecht, the Netherlands. Strains were analysed based on their morphological characters and molecular phylogeny. The latter used DNA sequence data
of the nuclear ribosomal internal transcribed spacer regions and intervening 5.8S nrDNA (ITS) and partial 28S large subunit (LSU) nrDNA and partial
protein encoding genes including the RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) and β-tubulin (TUB2).
Based on these results, the subgenera Astromata, Bionectria, Myronectria and Zebrinella are supported within Clonostachys. Furthermore, the genus
Sesquicillium is resurrected to accommodate the former subgenera Epiphloea and Uniparietina. The close relationship of Clonostachys and Sesquicillium
is strongly supported as both are inferred phylogenetically as sister-genera. New taxa include 24 new species and 10 new combinations. Recognition of
Sesquicillium distinguishes species typically forming a reduced perithecial stroma supercially on plant tissue from species in Clonostachys often forming
well-developed, through bark erumpent stromata. The patterns of observed perithecial wall anatomies, perithecial wall and stroma interfaces, and asexual
morph diversications described in a previously compiled monograph are used for interpreting ancestral state reconstructions. It is inferred that the common
ancestor of Clonostachys and Sesquicillium may have formed perithecia supercially on leaves, possessed a perithecial wall consisting of a single region,
and formed intercalary phialides in penicilli of conidiophores. Character interpretation may also allow hypothesising that diversication of morphs occurred
then in the two genera independently and that the frequently stroma-linked Clonostachys morphs evolved together with the occupation of woody host niches
and mycoparasitism.
Key words: Biocontrol, Bionectriaceae, multi-locus, mycoparasitism, new taxa, phylogeny, soil-borne, taxonomy.
Taxonomic novelties: New species: Clonostachys aurantiaca L. Zhao & Crous, Clonostachys australiana L. Zhao & Crous, Clonostachys bambusae L.
Zhao & Crous, Clonostachys buxicola L. Zhao & Crous, Clonostachys cylindrica L. Zhao & Crous, Clonostachys ellipsoidea L. Zhao & Crous, Clonostachys
ava L. Zhao, Crous & Schroers, Clonostachys fujianensis L. Zhao & Crous, Clonostachys fusca L. Zhao, Crous & Schroers, Clonostachys garysamuelsii
L. Zhao & Crous, Clonostachys hongkongensis L. Zhao & Crous, Clonostachys longiphialidica L. Zhao, Crous & Schroers, Clonostachys obovatispora, L.
Zhao & Crous, Clonostachys palmae L. Zhao, Crous & Schroers, Clonostachys parasporodochialis L. Zhao & Crous, Clonostachys penicillata L. Zhao, Crous
& Schroers, Clonostachys reniformis L. Zhao & Crous, Clonostachys vacuolata L. Zhao, Crous & Schroers, Clonostachys venezuelae L. Zhao, Crous &
Schroers, Mycocitrus synnematus L. Zhao & Crous, Nectriopsis didymii L. Zhao & Crous, Sesquicillium intermediophialidicum L. Zhao & Crous, Sesquicillium
neerlandicum L. Zhao & Crous, Sesquicillium symmetricum L. Zhao & Crous. New combinations: Mycocitrus coccicola (J.A. Stev.) L. Zhao & Crous,
Mycocitrus coxeniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium essexcoheniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium lasiacidis (Samuels) L.
Zhao, Crous & Schroers, Sesquicillium phyllophilum (Schroers) L. Zhao, Crous & Schroers, Sesquicillium rossmaniae (Schroers) L. Zhao, Crous & Schroers,
Sesquicillium saulense (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium sesquicillii (Samuels) L. Zhao, Crous & Schroers, Sesquicillium spinulosisporum
(Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium tornatum (Höhn.) Schroers. New synonyms: Clonostachys aranearum W.H. Chen et al., Clonostachys
chuyangsinensis H. Yu & Y. Wang, Clonostachys eriocamporesiana R.H. Perera & K.D. Hyde, Clonostachys granuligera (Starbäck) Forin & Vizzini,
Clonostachys indica Prasher & R. Chauhan, Clonostachys spinulosa R.H. Perera et al., Clonostachys squamuligera (Sacc.) Forin & Vizzini, Clonostachys
wenpingii (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang. Epitypes (basionyms): Fusidium buxi J.C. Schmidt ex Link, Verticillium candelabrum Bonord.
Available online at www.studiesinmycology.org StudieS in Mycology 105: 205–266 (2023).
Citation: Zhao L, Groenewald JZ, Hernández-Restrepo M, Schroers H-J, Crous PW (2023). Revising Clonostachys and allied genera in Bionectriaceae.
Studies in Mycology 105: 205–266. doi: 10.3114/sim.2023.105.03
Received: 19 February 2023 ; Accepted: 30 May 2023; Effectively published online: 12 June 2023
Corresponding editor: Robert A. Samson
INTRODUCTION
The family Bionectriaceae is based on the sexual morph-typied
genus Bionectria (Spegazzini 1919). Rossman et al. (1999) included
a total of 21 perithecial and v cleistothecial genera when describing
the family. Classication of four of the cleistothecial genera
(Emericellopsis, Heleococcum, Mycoarachis, and Roumegueriella)
in the family were supported based on phylogenetic analysis of the
partial 28S large subunit (LSU) nrDNA gene, which also suggested
that the Bionectriaceae, including related asexual morphs, could be
monophyletic (Rossman et al. 2001). Phylogenetic analyses that
are based on widely available LSU rDNA sequences now suggest
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Zhao et al.
that the Bionectriaceae includes 47 genera (Wijayawardene et al.
2018, Hyde et al. 2020b).
Members of the Bionectriaceae are herbicolous, corticolous,
lichenicolous, fungicolous or coprophilous. They mostly occur in
terrestrial or freshwater habitats and are less common in marine
habitats. Ascomata occur supercially on the substratum or are
formed on a poorly or well-developed erumpent stroma and are
solitary or densely aggregated, crowded, perithecial, and rarely
cleistothecial. Their colour is pallid and ranges from white, yellow,
orange to tan or brown and does not change in KOH or lactic acid.
Asexual morphs are acremonium-, gliocladium-, gyrostroma-,
penicillium-, or verticillium-like. Conidiophores are mono- or
dimorphic, mononematous or sporodochial or synnematous and
typically hyaline and smooth, while those of a few taxa can be
subhyaline to brown or blackish brown and nely echinulated.
Conidia, produced typically by phialidic conidiogenous cells,
are unicellular to multi-septate, ellipsoid, fusoid to subfusoid,
sometimes with papillate or truncate ends, hyaline to greenish
hyaline or olivaceous grey, and smooth or striated (Rossman et al.
1999, Hyde et al. 2020b).
Spegazzini (1919) proposed the genus Bionectria based
on a single species, B. tonduzii. Samuels (1988a) combined
B. tonduzii to Nectria tonduzii and noted its similarity to N.
ochroleuca. Nectria apocyni, N. aureofulva, N. byssicola, N.
pallidula, N. ochroleuca, and N. subquaternata were classied in
the N. ochroleuca group (Samuels 1976). Schroers & Samuels
(1997) transferred the N. ochroleuca group to Bionectria based
on morphological characters.
According to dual nomenclatural concepts, the sexual morph-
typied Bionectria was selected as the generic umbrella by
Schroers (2001), while at the same time, the use of binominals
in the asexual morph-typied genus Clonostachys were equally
promoted. However, with the end of dual nomenclature,
Clonostachys was recommended for conservation over Bionectria
(Rossman et al. 2013). Clonostachys includes soil-borne species,
mycoparasites, endophytes, epiphytes or saprotrophs (Schroers
2001, Moreira et al. 2016). It was rst described by Corda (1839)
and is based on C. araucaria, which now is considered a synonym
of C. rosea (basionym Penicillium roseum), the asexual morph of
N. ochroleuca.
Schroers (2001) described 44 species of Clonostachys/
Bionectria based on morphology and molecular phylogenetic
data [the internal transcribed spacer region and intervening 5.8S
nrRNA gene (ITS) and partial β-tubulin (TUB2)]. Because of
considerable diversity of morphological patterns seen mainly in
the sexual morphs, Schroers (2001) divided the genus Bionectria
into six newly distinguished subgenera, namely Astromata,
Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella.
For example, different kinds of ascomatal wall anatomy
with either one, two or three regions, and morphologically
diverse ascospores that could be smooth, spinulose, striate
or warted and 0- or 1-septate were accepted to characterise
Clonostachys/Bionectria. Mainly elements of the asexual morph
were considered useful for linking the different subgenera into
one generic concept (Schroers 2000: g. 5). They consisted of
penicillate, frequently sporodochial, in many cases dimorphic
conidiophores (referred to here as the primary and secondary
conidiophores), and phialidic conidiogenous cells formed either
terminally or subapically by phialide supporting cells. The
primary conidiophores are mononematous, either verticillium-
like or narrowly penicillate, and form heads of watery conidial
masses. The secondary conidiophores are loosely to adpressed
penicillate, or sporodochial, forming imbricate conidial chains that
can collapse to slimy masses (Schroers 2001).
Although Clonostachys undoubtedly belongs to Bionectriaceae,
its taxonomic position in relation to other genera within the family
remains unclear (Rossman et al. 1999, Hyde et al. 2020b). In
the present study, we therefore investigated 420 additional,
preliminarily identied strains of Clonostachys and allied genera,
deposited in the CBS culture collection and the Johanna Westerdijk
(JW) citizen science collection (collected from Dutch soils by school
children; Groenewald et al. 2018, Giraldo et al. 2019, Hou et al.
2020, Crous et al. 2021) of the Westerdijk Fungal Biodiversity
Institute. We used morphological and molecular phylogenetic
analyses. The latter were based on the nuclear ribosomal internal
transcribed spacer regions and intervening 5.8S nrDNA (ITS) and
partial sequences for the 28S large subunit (LSU) nrDNA. These
were combined with partial protein-encoding genes including the
DNA-directed RNA polymerase II second largest subunit (RPB2),
translation elongation factor 1-alpha (TEF1), and β-tubulin (TUB2)
gene regions to reconstruct a phylogenetic backbone of, and
dene robust species boundaries within, the genus Clonostachys.
An additional aim of this study was to re-evaluate the taxonomic
circumscription of Clonostachys among related genera within
Bionectriaceae.
MATERIALS AND METHODS
Isolates
Fungal strains were obtained from the CBS culture collection and
Johanna Westerdijk (JW) citizen science collection of the Westerdijk
Fungal Biodiversity Institute (WI; Utrecht, the Netherlands, formerly
CBS-KNAW). New and interesting strains from JW were deposited
in the CBS fungal collection. A total of 420 isolates including taxa
preliminarily identied as Clonostachys and of allied genera were
included in this study and their morphological traits and molecular
phylogeny characterised (Supplementary Table S1).
DNA extraction, PCR amplication and sequencing
Genomic DNA was extracted from fungal colonies growing
on oatmeal agar (OA; Crous et al. 2019) for 7–14 d at room
temperature using the Wizard® Genomic DNA purication
Kit (Promega Corporation, Madison, WI, USA), following the
manufacturer’s protocol. Five loci were amplied: the ITS region
was amplied using the primer pair ITS5/ITS4 (White et al. 1990);
the LSU region was amplied using the primer pair LR0R/LR5
(Vilgalys & Hester 1990, Rehner & Samuels 1994); RPB2, TEF1
and TUB2 genes were amplied using the primer pairs RPB2-
5F2/RPB2-7CR (Liu et al. 1999, Sung et al. 2007), EF1-983F/
EF1-2218R (Rehner & Buckley 2005) and T1/T22 (O’Donnell &
Cigelnik 1997). For sequencing, the same primer sets were used
except for the partial TUB2 gene, where the additional primer
TUB4Rd (Woudenberg et al. 2009) was used. The consensus
sequences of each isolate were assembled from forward and
reverse sequences using Geneious Prime 2022 (Biomatters Inc.,
New Zealand). Sequences newly generated in this study and their
GenBank (http://www.ncbi.nlm.nih.gov) accession numbers are
shown in Supplementary Table S1.
207www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Phylogenetic analyses
Subsequent alignments for ve individual loci (ITS, LSU, RPB2,
TEF1, and TUB2) were generated with MAFFT v. 7 using the default
settings on the web server of the European Bioinformatics Institute
(EMBL-EBI) (http://www.ebi.ac.uk/Tools/msa/ mafft/) (Katoh &
Standley 2013, Li et al. 2015). These alignments were manually
edited in MEGA v. 7.0.21 when necessary (Tamura et al. 2013).
Maximum-likelihood (ML) and Bayesian analyses (BA) were used
for phylogenetic inferences of individual sequence alignments and
the concatenated (ITS, LSU, RPB2, TEF1, and TUB2) alignments.
Maximum-likelihood analyses were conducted using the CIPRES
Science Gateway portal v. 3.3 (https://www.phylo.org/; Miller et al.
2012) and RAxML-HPC2 on XSEDE v. 8.2.12 (Stamatakis 2014)
with default GTR substitution matrix and 1 000 rapid bootstrap
replications. Additional ML analyses were performed using IQ-
TREE v. 2.1.2 (Nguyen et al. 2015, Minh et al. 2020) with ultrafast
bootstrapping (UFBoot2; Hoang et al. 2018) for estimation of
branch support. The most suitable evolutionary model for each
partition was estimated using ModelFinder (Kalyaanamoorthy
et al. 2017; Minh et al. 2020) as implemented in IQ-TREE.
MrModelTest v. 2.2 (Nylander 2004) was used to determine the
optimal nucleotide substitution model for each locus. Bayesian
analyses were performed using MrBayes v. 3.2.6 (Ronquist et al.
2012). Markov Chain Monte Carlo sampling (MCMC) analyses of
four chains were started in parallel from a random tree topology.
Four simultaneous Markov chains were run for 10 M generations
and trees were sampled every 1 000 generation or until the run
was stopped automatically when the average standard deviation
of split frequencies fell below 0.01. The rst 25 % of the trees were
discarded as the burn-in phase of each analysis, and the remaining
trees were used to calculate posterior probabilities (PP). The
resulting trees were plotted using FigTree v. 1.4.2 (http://tree.bio.
ed.ac.uk/ software/gtree). Alignments and the phylogenetic trees
derived from this study were uploaded to gshare (doi: 10.6084/
m9.gshare.22894592).
Morphology
Macroscopic descriptions were made from colonies on oatmeal
agar (OA), potato dextrose agar (PDA), and synthetic nutrient-
poor agar (SNA; Nirenberg 1976, Crous et al. 2019) after 7 d in
the darkness at 25 °C. Colony diameters and characters were
measured after 7 d. Colony colours (upper surface and reverse)
were rated following the colour charts of Rayner (1970). Micro-
morphological characters were recorded mostly from 5–14-d-old
colonies on OA or SNA under near-UV light at room temperature,
using structures from relatively young parts of the colony. Clear
lactic acid was used as mounting medium for the observation of
micromorphological structures of stromata, perithecia, perithecial
walls, asci, ascospores, conidiophores and conidia (Schroers
2001). Observations of micro-morphological characteristics were
processed with a Nikon Eclipse 80i compound microscope with
differential interference contrast (DIC) optics and a Nikon AZ100
dissecting microscope. Photomicrographs and measurements
were taken with a Nikon DS-Ri2 high-denition colour digital
camera using the NIS-elements D software v. 4.50 (Nikon, Tokyo,
Japan). All descriptions, illustrations and nomenclatural data were
deposited in MycoBank (www.MycoBank.org; Crous et al. 2004),
and specimens were deposited in the CBS Fungarium.
RESULTS
Phylogenetic analyses
For inferring the phylogeny of the genus Clonostachys within the
Bionectriaceae, we analysed aligned DNA sequence data from ve
concatenated loci (ITS, LSU, RPB2, TEF1, and TUB2) in dataset 1.
To obtain a more precise phylogenetic relationship of species within
Clonostachys, more inclusive analyses based on DNA sequence
data from ve loci were carried out for the genus (dataset 2).
Dataset 1: Concatenated and aligned ITS, LSU, RPB2, TEF1, and
TUB2 sequences from 269 taxa represent several genera belonging
to the Bionectriaceae, with Tilachlidium brachiatum (CBS 363.97,
CBS 505.67), Flammocladiella anomiae (CLL 16017), F. aceris
(CBS 138906) and F. decora (CBS 142776) serving as outgroups
(Hypocreales, Tilachlidiaceae & Flammocladiellaceae; Fig. 1). The
nal alignment consisted of 4 245 characters, including alignment
gaps (gene boundaries ITS: 1–691, 691 bp; LSU: 692–1 495, 804
bp; RPB2: 1 496–2 288, 793 bp; TEF1: 2 289–3 103, 815 bp, TUB2:
3 104–4 245, 1 142 bp). Among these, 2 315 character sites were
conserved (ITS: 257, LSU: 605, RPB2: 297, TEF1: 484, TUB2:
672), 1 872 were variable (ITS: 407, LSU: 198, RPB2: 477, TEF1:
330, TUB2: 460), and 1 646 were parsimony informative characters
(ITS: 338, LSU: 167, RPB2: 453, TEF1: 281, TUB2: 407).
The phylogenetic trees based on dataset 1 were generated
with Maximum-likelihood and Bayesian analyses. According to
the result of MrModelTest, the GTR+I+G model was proposed for
all loci investigated. The Bayesian analysis of the concatenated
ve-locus alignment lasted for 10 075 000 generations and
20 152 trees were generated after the average standard deviation
of split frequencies value was below 0.01 in the BI analysis. A
total of 15 114 trees were used for calculating the posterior
probabilities (PP) after the rst 25 % of trees were discarded as
the burn-in phase. The three phylogenetic analyses (RAxML, IQ-
TREE, and MrBayes) overall displayed the same species clades
and mainly differed with regards to the backbone relationships
between species clades/lineages. The best RAxML tree based
on the combined dataset is presented here with bootstrap
support values of ML analyses (RAxML-BS / IQ-TREE-BS) and
relevant Bayesian posterior probabilities (PP) shown at the nodes
(RAxML-BS > 50 % / IQ-TREE-BS > 90 % / PP > 0.90) (Fig.
1). RAxML trees targeting each of the used partitions individually
(ITS, LSU, RPB2, TEF1) are presented as Supplementary Figs
S1–S4; TUB2 is not shown as those sequences were mainly
available only for Clonostachys.
The resulting phylogenetic tree (Fig. 1) resolved 24 well-
supported clades, representing 24 genera in the Bionectriaceae.
In our study, one clade has isolates preliminarily identied as
Sesquicillium microsporum that are here reassigned to Nectriopsis
(100 % /100 % / 1), with one new species, Nectriopsis didymii.
Two clades have isolates preliminarily identied as Clonostachys
that are here assigned to Mycocitrus (96 % / 98 % / 0.99) and
Stephanonectria (100 % / 100 % / 1). The genus Sesquicillium
(97 % / 100 % / 1) is resurrected to accommodate the subgenera
Epiphloea (except C. setosa) and Uniparietina, with three new
species and seven new combinations. The genus Clonostachys
(99 % / 100 % / 1) includes the subgenera Astromata, Bionectria,
Myronectria and Zebrinella with 49 known and 19 new species
(Figs 1, 2). However, the here presented dataset also supports that
Sesquicillium and Clonostachys derive from the same ancestor,
as their phylogenetic sister group relatedness is highly supported
(95 % / 99 % / 1).
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Zhao et al.
The individual gene trees had variable success in resolving
genus clades (Supplementary Figs S1–S4). Although they generally
resolved the same genus clades, the order and association
between the clades were not always the same due to low support
in the backbones of the trees. The ITS phylogeny (Supplementary
Fig. S1) recovered all the genus clades presented in Fig. 1, with
the exception that it intermixed species of Emericellopsis and
Stanjemonium. The LSU phylogeny (Supplementary Fig. S2)
recovered all genera presented in Fig. 1, but did not cluster species
of Lasionectria in a monophyletic clade and intermixed several
subclades of Sesquicillium and Clonostachys intermixed in a
polytomy. The RPB2 phylogeny (Supplementary Fig. S3) recovered
all genera presented in Fig. 1, with the exception that it intermixed
species of Emericellopsis and Stanjemonium. The TEF1 phylogeny
(Supplementary Fig. S4) recovered all genera presented in Fig. 1,
but did not cluster all species of Nectriopsis in a monophyletic clade
(Nectriopsis lindauiana clustered basal to Stilbocrea without any
support) while Clonostachys buxicola and Clonostachys pityrodes
formed a clade sister to the monophyletic Stephanonectria clade
but with an unsupported connecting node.
Dataset 2: This dataset consisted of 394 ingroup isolates that
formed a fully supported clade representing Clonostachys, with
the bionectriaceous Acremonium alternatum (CBS 407.66) and
A. stroudii (CBS 138820) used as outgroups (Fig. 2). The nal
alignment consisted of 4 055 characters, including alignment gaps
(gene boundaries ITS: 1–565, 565 bp; LSU: 566–1 366, 801 bp;
RPB2: 1 367–2 133, 767 bp; TEF1: 2 134–2 941, 808 bp; TUB2:
2 942–4 055, 1 114 bp). Among these, 2 654 character sites were
Emericellopsis donezkii CBS 489.71_T
Stilbocrea macrostoma CBS 114375
Stanjemonium fuscescens CBS 264.96_T
Stilbocrea colubrensis CLLM 16003_T
Acremonium egyptiacum CBS 114785_T
Stanjemonium ochroroseum CBS 656.79_T
Ovicillium oosporum CBS 110151_T
Acremonium alternatum CBS 407.66_T
Acremonium stroudii CBS 138820_T
Ovicillium subglobosum CBS 101963_T
Emericellopsis mirabilis CBS 176.53
Geosmithia lavendula CBS 344.48_T
Emericellopsis stolkiae CBS 159.71_T
Flammocladiella anomiae CLL 16017
Emericellopsis salmosynnemata CBS 182.56_T
Emericellopsis fimetaria CBS 176.60
Acremonium charticola CBS 547.86
Emericellopsis pallida CBS 624.73
Ovicillium attenuatum CBS 399.86_T
Tilachlidium brachiatum CBS 363.97
Emericellopsis glabra CBS 119.40_T
Emericellopsis pallida CBS 490.71_T
Emericellopsis humicola CBS 180.56_T
Emericellopsis maritima CBS 491.71_T
Emericellopsis maritima CBS 379.70F
Stanjemonium grisellum CBS 655.79_T
Acremonium egyptiacum CBS 124.42
Stilbocrea macrostoma CBS 141849
Emericellopsis pusilla CBS 226.62_T
Emericellopsis alkalina CBS 120049
Acremonium psychrophilum CBS 139.93
Emericellopsis robusta CBS 105.70_T
Acremonium charticola CBS 117.25
Acremonium brachypenium CBS 866.73_T
Emericellopsis terricola CBS 120.40_T
Emericellopsis alkalina CBS 127350_T
Emericellopsis minima CBS 190.55_T
Geosmithia pallidum CBS 260.33_T
Acremonium sordidulum CBS 385.73_T
Stilbocrea walteri CBS 144627_T
Emericellopsis microspora CBS 380.62_T
Flammocladiella aceris CBS 138906_T
Emericellopsis stolkiae CBS 139531
Tilachlidium brachiatum CBS 505.67
Flammocladiella decora CBS 142776
Acremonium sordidulum CBS 102413
Emericellopsis mirabilis CBS 177.53_T
85/95/- 100/100/1
92/99/1
100/100/1
96/100/1
100/100/1
92/99/1
100/100/1
69/100/1
100/100/1
100/100/1
100/100/1
88/98/1
50/-/0.94
100/100/1
100/100/1
58/95/0.99
68/93/1
100/100/1
51/-/-
100/100/1
100/100/1
100/100/1
57/94/1
100/100/1
67/99/0.95
88/100/1
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97/100/1
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100/100/1
100/100/1
99/100/1
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84/100/1
97/100/1
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97/100/1
81/100/0.99
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68/100/1
100/100/1
100/100/1
2X
2X
Stilbocrea
Ovicillium
Geosmithia
Acremonium
Stanjemonium
Emericellopsis
Fig. 1. Phylogenetic tree inferred from a Maximum Likelihood (RAxML) analysis based on aligned and concatenated ITS, LSU, RPB2, TEF1 and TUB2
sequences of 269 strains representing Bionectriaceae and outgroups. Numbers at branches indicate support values (RAxML-BS / IQ-TREE-BS / BI-PP)
above 50 % / 90 % / 0.9. New species are printed in red font, new combinations in blue font and coloured boxes highlight genera. Roman numerals indicate
subgenera as coded in the legend. “T” indicates ex-type strains. The tree is rooted to Flammocladiella aceris CBS 138906, F. decora CBS 142776, F.
anomiae CLL 16017, Tilachlidium brachiatum CBS 363.97, and T. brachiatum CBS 505.67 (Hypocreales, Flammocladiellaceae & Tilachlidiaceae). Scale bar
represents expected number of changes per site.
209www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
conserved (ITS: 330, LSU: 694, RPB2: 378, TEF1: 576, TUB2:
676), 1 366 were variable (ITS: 220, LSU: 106, RPB2: 382, TEF1:
232, TUB2: 426), and 1 185 were parsimony informative (ITS: 175,
LSU: 90, RPB2: 353, TEF1: 194, TUB2: 373).
The phylogenetic trees based on dataset 2 were generated with
Maximum-likelihood analyses and Bayesian analyses. According to
the result of MrModelTest, the SYM+I+G model was proposed for
ITS, GTR+I+G model for LSU, RPB2, and TEF1, and HKY+I+G
model for TUB2. The Bayesian analysis of the concatenated ve-
locus alignment lasted for 67 905 000 generations and 135 812
trees were generated after the average standard deviation of split
frequencies value was below 0.013 in the BI analysis. A total of
101 860 trees were used for calculating the posterior probabilities
(PP) after the rst 25 % of trees were discarded as the burn-in
phase. The three phylogenetic analyses (RAxML, IQ-TREE, and
MrBayes) overall displayed the same species clades and mainly
differed with regards to the backbone relationships between
species clades/ lineages. The best RAxML tree based on the
combined dataset is presented here with bootstrap support values
of ML analyses (RAxML-BS / IQ-TREE-BS) and relevant Bayesian
posterior probabilities (PP) shown at the nodes (RAxML-BS > 50 %
/ IQ-TREE-BS > 90 % / PP > 0.90) (Fig. 2). RAxML trees targeting
each of the used partitions individually (ITS, LSU, RPB2, TEF1) are
presented in Supplementary Figs S5–S8; TUB2 is not shown as no
sequences were available for the outgroup taxa. Well-supported
clades in the phylogenetic tree based on dataset 2 include 49
known and 19 new species of Clonostachys (Fig. 2, Supplementary
Fig. S9).
The individual gene trees had variable success in resolving
species clades (Supplementary Figs S5–S8). Although they generally
resolved the same species clades, with the exception of LSU (see
below), the order and association between the clades were not
always the same due to low support in the backbones of the trees.
The ITS phylogeny (Supplementary Fig. S5) recovered the majority
of the species clades presented in Fig. 2, with some exceptions
where species were not monophyletic (e.g., C. eriocamporesii) or
Lasionectria antillana CBS 122797_T
Hydropisphaera fungicola CBS 122304_T
Gliomastix roseogrisea CBS 134.56_T
Geonectria subalpina CBS 143540_T
Gliomastix masseei CBS 794.69_T
Lasionectria mantuana CBS 114291
Protocreopsis phormiicola CBS 567.76_T
Lasionectria mantuana A.R. 4029
Fusariella atrovirens CBS 311.73
Paracylindrocarpon pandanicola KUMCC 17-0272_T
Synnemellisia aurantia COAD 2070_T
Fusariella arenula CBS 330.77
Lasionectriopsis germanica CBS 143538_T
Lasionectria sylvana CBS 566.76
Fusariella concinna CBS 312.73
Fusariella hughesii CBS 435.70
Gliomastix masseei CBS 557.75
Fusariella sp. CBS 128364
Lasionectria antillana CBS 114748
Hydropisphaera peziza CBS 296.65
Hydropisphaera peziza CBS 139487
Gliomastix roseogrisea CBS 380.70A
Lasionectriopsis pteridii CBS 783.69
Gliomastix polychroma CBS 181.27_T
Protocreopsis caricicola CBS 140572_T
Hydropisphaera cyatheae CBS 575.76
Paracylindrocarpon nabanheensis KUMCC 16-0147_T
Fusariella curvata MFLUCC 15-0844_T
Ochronectria calami CBS 125.87
Protocreopsis pertusa CBS 568.76
Paracylindrocarpon aloicola CBS 141300_T
Paracylindrocarpon aloicola CBS 135907
Ochronectria thailandica MFLUCC 15-0140_T
Hydropisphaera suffulta CBS 122.87
Verrucostoma martinicense CBS 138731_T
Lasionectria krabiense MFLUCC 15-0673_T
Lasionectriella herbicola CBS 140156_T
Verrucostoma freycinetiae MAFF 240100_T
Gliomastix murorum CBS 154.25_T
Ochronectria calami CBS 445.96
Gliomastix murorum CBS 195.70
Gliomastix polychroma CBS 184.30
Fusariella arenula CBS 329.77
Lasionectriopsis pteridii CBS 782.69_T
Paracylindrocarpon xishuangbannaensis KUMCC 16-0144_T
Roumegueriella rufula CBS 346.85
Lasionectriella rubioi CBS 140157_T
98/100/1
100/100/1
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59/-/-
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62/100/-
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86/100/1
52/-/0.98
100/100/1
Protocreopsis
Lasionectriopsis
Lasionectriella
Ochronectria
Lasionectria
Geonectria
Hydropisphaera
Gliomastix
Synnemellisia
Roumegueriella
Verrucostoma
Paracylindrocarpon
Fusariella
Fig. 1. (Continued).
210
Zhao et al.
Mycocitrus aurantium BAFC 3843
Nectriopsis didymii CBS 326.79
Sesquicillium neerlandicum CBS 148201
Nectriopsis fuliginicola CBS 400.82_T
Sesquicillium symmetricum CBS 124.79_T
Sesquicillium neerlandicum CBS 148202
Sesquicillium symmetricum CBS 485.78
Nectriopsis microspora CBS 102560
Nectriopsis microspora CBS 933.69_T
Sesquicillium saulense BRFM 2782_T
Sesquicillium buxi JW6017
Stephanonectria keithii CBS 943.72
Sesquicillium neerlandicum CBS 148214
Sesquicillium spinulosisporum CLLG12001_T
Nectriopsis candicans CBS 440.65
Mycocitrus coxeniae BRIP 49599a_T
Nectriopsis lindauiana CBS 897.70_T
Nectriopsis didymii CBS 395.82
Stephanonectria keithii CBS 100007
Nectriopsis didymii CBS 355.70
Sesquicillium rossmaniae CBS 210.93
Mycocitrus coccicola BUcCo
Nectriopsis rexiana CBS 305.70A
Sesquicillium neerlandicum CBS 148213
Sesquicillium sesquicillii CBS 180.88_T
Nectriopsis violacea CBS 914.70_T
Stephanonectria chromolaenae MFLUCC 18-0589_T
Nectriopsis candicans CBS 701.79_T
Stephanonectria chromolaenae CBS 476.91
Stephanonectria chromolaenae CBS 475.91
Sesquicillium buxi JW94008
Sesquicillium neerlandicum CBS 148210
Mycocitrus phyllostachydis CBS 330.69
Nectriopsis microspora CBS 954.72
Stephanonectria keithii CBS 434.70
Nectriopsis microspora CBS 582.77
Sesquicillium neerlandicum CBS 148212
Sesquicillium essexcoheniae CBS 918.97
Nectriopsis didymii CBS 852.70A_T
Sesquicillium phyllophilum CBS 662.83
Sesquicillium buxi CBS 288.62
Sesquicillium phyllophilum CBS 921.97_T
Mycocitrus odorus CBS 120610
Nectriopsis microspora CBS 354.70
Mycocitrus aurantium BAFC 51693
Sesquicillium buxi JW58015
Nectriopsis rexiana CBS 305.70C
Nectriopsis didymii CBS 852.70B
Sesquicillium rossmaniae CBS 211.93_T
Sesquicillium buxi JW182006
Nectriopsis violacea CBS 849.70
Nectriopsis rexiana CBS 542.92
Mycocitrus coccicola BUcS
Sesquicillium rossmaniae CBS 221.93
Nectriopsis didymii CBS 788.85
Sesquicillium buxi JW259005
Mycocitrus synnematus CBS 126677_T
Sesquicillium intermediophialidicum CBS 685.96_T
Sesquicillium buxi JW199009
Sesquicillium neerlandicum CBS 148215
Sesquicillium buxi CBS 696.93
Sesquicillium neerlandicum CBS 148209
Sesquicillium neerlandicum CBS 148203_T
Mycocitrus odorus CBS 100104_T
Sesquicillium buxi CBS 202.69
Nectriopsis didymii CBS 652.70
Nectriopsis sporangiicola CBS 166.74_T
Sesquicillium essexcoheniae BRIP 75170a_T
61/94/0.99
52/95/1
100/100/1
90/100/1
78/99/1
97/100/1
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52/-/-
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57/98/-
99/100/1
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100/100/1
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63/-/0.92
94/100/1
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61/98/-
99/100/1
100/100/1
90/100/1
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78/99/1
55/96/0.98
59/96/0.97
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Nectriopsis
Mycocitrus
Stephanonectria
Sesquicillium
II
I
I
Fig. 1. (Continued).
211www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
indiscernible (e.g. C. rogersoniana / C. cylindrica, C. reniformis / C.
viticola, C. farinosa / C. apocyni, and numerous species intermingled
with C. rosae and C. solani). The LSU phylogeny (Supplementary
Fig. S6) recovered some of the species clades presented in Fig.
2, but the majority were poorly resolved or intermixed. The RPB2
phylogeny (Supplementary Fig. S7) recovered all species clades
presented in Fig. 2, with variable branch lengths. The TEF1
phylogeny (Supplementary Fig. S8) recovered all species clades
presented in Fig. 2, with some exceptions where species were not
monophyletic [e.g. C. farinosa, C. sporodochialis, C. rosea (one
isolate not clustering with the rest)].
Taxonomy
Based on multi-locus phylogenetic inferences, supported by
morphological observations, habitat information and geographical
comparisons, a total of 420 isolates including taxa preliminarily
identied as Clonostachys and of allied genera were examined
in this study. Cultures were shown to represent taxa belonging
to Clonostachys, Mycocitrus, Nectriopsis, Sesquicillium and
Stephanonectria. Among these, 24 new species and 10 new
combinations are proposed. Furthermore, one epitypication is
proposed, one genus resurrected, and eight species epithets
are reduced to synonymy. Two new species that proved to be
sterile are described based on DNA sequence data, following the
approach of Hou et al. (2023). Genera are arranged according to
their position on the phylogenetic tree (Fig. 1), and species are
arranged alphabetically (Figs 1, 2).
Nectriopsis Maire, Ann. Mycol. 9: 323. 1911.
Synonyms: Dasyphthora Clem., Gen. Fung. (Minneapolis): 45.
1909.
Peloronectriella Yoshim. Doi, Bull. Natl. Sci. Mus., Tokyo, N.S. 11:
179. 1968.
Type: Nectriopsis violacea (J.C. Schmidt ex Fr.) Maire
Sesquicillium candelabrum JW31018
Clonostachys australiana CBS 102423
Sesquicillium lasiacidis NL19-086015
Clonostachys australiana CBS 102421_T
Clonostachys pityrodes CBS 102033_T
Sesquicillium lasiacidis CBS 147133
Clonostachys chlorina CBS 287.90_T
Sesquicillium lasiacidis CBS 179.88_T
Clonostachys fusca CBS 101925
Clonostachys fusca CBS 207.93_T
Clonostachys vesiculosa HMAS 183151_T
Sesquicillium candelabrum JW79008
Clonostachys setosa CBS 917.97_T
Sesquicillium candelabrum YFCC 896
Sesquicillium lasiacidis CBS 190.38
Clonostachys leucaenae MFLUCC 20-0008_T
Sesquicillium candelabrum YFCC 895
Clonostachys venezuelae CBS 107.87_T
Sesquicillium lasiacidis NL19-089008
Clonostachys lucifer CBS 100008_T
Sesquicillium candelabrum CBS 513.67
Clonostachys pilosella CLLG19028_T
Sesquicillium lasiacidis CBS 504.67
Sesquicillium lasiacidis NL19-085006
Clonostachys buxicola CBS 102419_T
Sesquicillium candelabrum CBS 205.69
Sesquicillium lasiacidis JW235005
Sesquicillium lasiacidis NL19-085003
Clonostachys grammicosporopsis CBS 114.87_T
Clonostachys longiphialidica CBS 112.87_T
Sesquicillium candelabrum YHH 896
Sesquicillium candelabrum CBS 119045_T
Clonostachys levigata CBS 101916_T
Sesquicillium candelabrum CBS 194.53
Clonostachys grammicospora CBS 209.93_T
Clonostachys fusca CBS 996.97
Sesquicillium candelabrum CBS 512.67
Clonostachys lucifer CBS 126.87
Clonostachys ellipsoidea CBS 175.76_T
Clonostachys intermedia CBS 508.82_T
Clonostachys subquaternata CBS 100003_T
Clonostachys aurantiaca CBS 124757_T
Clonostachys ellipsoidea CBS 102566
Clonostachys vacuolata CBS 191.93_T
Sesquicillium candelabrum JW1015
Clonostachys flava CBS 915.97_T
Sesquicillium candelabrum CBS 204.69
Clonostachys pallens PAD S00004_T
97/99/1
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78/96/1
97/100/1
71/100/-
83/98/1
100/100/1
98/99/1
95/99/1
Clonostachys
III
IV
I
Sesquicillium
Fig. 1. (Continued).
212
Zhao et al.
Sexual morph on the natural substratum. Perithecia supercial
or immersed in substratum, rarely inconspicuously stromatic, up
to 200 μm diam, nearly white to pale yellow or orange, KOH-.
Perithecial wall up to 20 μm thick, comprising a single region of
small, thin-walled, non-descript cells; cells at surface of perithecia
wall forming a textura epidermoidea. Asci cylindrical to clavate,
apex simple or with a ring, 8-spored. Ascospores ellipsoidal,
hyaline, (0–)1-septate. Asexual morphs acremonium-, gliocladium-,
or verticillum-like (adapted from Rossman et al. 1999).
Notes: Nectriopsis was established with four species of
hypocrealean fungi having ascomata in a byssoid stroma and
considered intermediate between Nectria and Hypomyces.
Clements (1909) placed Dasyphthora in Hypocreaceae with only
0.08
Clonostachys rhizophaga CBS 202.37_T
Clonostachys compactiuscula CBS 913.97_T
Clonostachys rogersoniana CBS 920.97_T
Clonostachys sporodochialis CBS 101921_T
Clonostachys pnagiana CLLG19041_T
Clonostachys eriocamporesii CBS 148221
Clonostachys ambigua PAD S00003_T
Clonostachys krabiensis MFLUCC 16-0254_T
Clonostachys fujianensis CBS 127474_T
Clonostachys oligospora HMAS 290895_T
Clonostachys farinosa IBP2
Clonostachys hongkongensis CBS 116542
Clonostachys rosea CBS 710.86_T
Clonostachys garysamuelsii CBS 123964_T
Clonostachys apocyni CBS 130.87
Clonostachys kunmingensis CBS 101920
Clonostachys swieteniae MFLUCC 18-0572_T
Clonostachys farinosa CBS 124067
Clonostachys kunmingensis YFCC 898_T
Clonostachys rosea f. catenulata CBS 154.27_T
Clonostachys chongqingensis HMAS 290894_T
Clonostachys samuelsii CBS 699.97_T
Clonostachys leptoderma HMAS 255834_T
Clonostachys ralfsii CBS 703.97_T
Clonostachys epichloe CBS 101037_T
Clonostachys agarwalii CBS 533.81_T
Clonostachys penicillata CBS 653.70
Clonostachys pseudostriatopsis MAFF 239827_T
Clonostachys reniformis CBS 695.86_T
Clonostachys aureofulvella CBS 100980_T
Clonostachys palmae CBS 119.87_T
Clonostachys penicillata CBS 729.87_T
Clonostachys hongkongensis CBS 115291_T
Clonostachys moreaui CLL19024_T
Clonostachys eriocamporesii MFLUCC 19-0486_T
Clonostachys pseudochroleuca CBS 187.94_T
Clonostachys cylindrica CBS 101113_T
Clonostachys farinosa CBS 364.78_T
Clonostachys oblongispora CBS 100285_T
Clonostachys chloroleuca CBS 141588_T
Clonostachys farinosa MFLUCC 17-2620
Clonostachys viticola CAA 944_T
Clonostachys obovatispora CBS 118752_T
Clonostachys farinosa MFLUCC 17-0131
Clonostachys divergens CBS 967.73B_T
Clonostachys farinosa PAD S00020
Clonostachys capitata CBS 218.93_T
Clonostachys solani CBS 697.88_T
Clonostachys parasporodochialis CBS 192.93_T
Clonostachys zelandiaenovae CBS 100979_T
Clonostachys eriocamporesii CBS 647.91
Clonostachys bambusae CBS 139411_T
Clonostachys farinosa PAD S00011
Clonostachys penicillata CBS 148211
Clonostachys solani f. nigrovirens CBS 183.30_T
Clonostachys pseudostriata CBS 120.87_T
Clonostachys sp. CBS 496.90
Clonostachys miodochialis CBS 997.69_T
Clonostachys kowhai CBS 461.95_T
100/100/1
53/92/-
52/97/0.93
51/-/-
54/97/-
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56/-/0.93
66/-/-
75/91/-
87/98/-
55/98/-
Clonostachys subgenera:
I: Epiphloea
II: Uniparietina
III: Myronectria
IV: Zebrinella
V: Astromata
VI: Bionectria
-/99/-
-/92/-
V
VI
Clonostachys
Fig. 1. (Continued).
213www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
one species, D. lasioderma, that was later included in Nectriopsis
(Samuels 1988a). Samuels (1988a) presented a thorough
account of the genus, treating 43 species. The monotypic genus
Peloronectriella was introduced for a fungus on bamboo, having
elongate, tuberculate stromata with a nectria-like, but pale
yellowish, KOH- ascomata, and 1-septate ascospores (Doi 1968).
Rossman et al. (1999) regarded Peloronectriella as synonym of
Nectriopsis according to its morphological characters.
Clonostachys subquaternata CBS 133487
Clonostachys australiana CBS 102421_T
Clonostachys leucaenae MFLUCC 20-0008_T
Clonostachys chlorina CBS 287.90_T
Clonostachys levigata CBS 124005
Clonostachys setosa CBS 834.91
Clonostachys fusca CBS 996.97
Clonostachys buxicola CBS 102419_T
Clonostachys grammicosporopsis CBS 114.87_T
Clonostachys aurantiaca CBS 124757_T
Clonostachys australiana CBS 102423
Clonostachys lucifer CBS 126.87
Clonostachys setosa CBS 917.97_T
Clonostachys pityrodes CBS 322.78
Clonostachys lucifer CBS 100008_T
Clonostachys intermedia CBS 508.82_T
Clonostachys grammicosporopsis CBS 102834
Clonostachys pilosella CLLG19028_T
Clonostachys grammicosporopsis CBS 111.87
Clonostachys subquaternata CBS 108.87
Clonostachys pityrodes CBS 102035
Clonostachys levigata CBS 203.69
Clonostachys ellipsoidea CBS 102566
Clonostachys pityrodes CBS 246.78
Clonostachys levigata JW182010
Clonostachys grammicosporopsis CBS 102835
Clonostachys fusca CBS 101925
Clonostachys pityrodes CBS 249.78
Clonostachys vacuolata CBS 191.93_T
Clonostachys grammicosporopsis CBS 115.87
Clonostachys pallens PAD S00004_T
Clonostachys fusca CBS 207.93_T
Clonostachys vesiculosa HMAS 183151_T
Acremonium alternatum CBS 407.66_T
Clonostachys setosa CBS 112025
Clonostachys levigata JW199015
Clonostachys subquaternata CBS 100003_T
Clonostachys flava CBS 915.97_T
Clonostachys venezuelae CBS 107.87_T
Clonostachys grammicosporopsis CBS 102843
Clonostachys levigata JW259015
Clonostachys grammicospora CBS 209.93_T
Clonostachys levigata CBS 948.97
Clonostachys ellipsoidea CBS 175.76_T
Clonostachys levigata CBS 101916_T
Acremonium stroudii CBS 138820_T
Clonostachys longiphialidica CBS 112.87_T
Clonostachys pityrodes CBS 126394
Clonostachys pityrodes CBS 102033_T
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4X
4X
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-/91/-
Fig. 2. Phylogenetic tree inferred from a Maximum Likelihood (RAxML-ML) analysis based on aligned and concatenated ITS, LSU, RPB2, TEF1 and TUB2
sequences of 394 strains representing Clonostachys and outgroups. Numbers at branches indicate support values (RAxML-BS / IQ-TREE-BS / BI-PP)
above 50 % / 90 % / 0.9. New species are printed in red font and coloured boxes highlight species clades / lineages. “T” indicates ex-type strains. A detailed
view of the collapsed clade at the bottom of the phylogenetic tree can be found in Fig. S9. The tree is rooted to Acremonium alternatum CBS 407.66 and A.
stroudii CBS 138820 (Hypocreales, Bionectriaceae). Scale bar represents expected number of changes per site.
214
Zhao et al.
Fig. 2. (Continued).
Clonostachys compactiuscula CBS 123781
Clonostachys penicillata CBS 653.70
Clonostachys hongkongensis CBS 115291_T
Clonostachys ralfsii CBS 141088
Clonostachys compactiuscula CBS 592.93
Clonostachys penicillata CBS 729.87_T
Clonostachys compactiuscula CBS 919.97
Clonostachys compactiuscula CBS 122571
Clonostachys ralfsii CBS 129.87
Clonostachys oligospora HMAS 290895_T
Clonostachys compactiuscula CBS 913.97_T
Clonostachys eriocamporesii CBS 647.91
Clonostachys ralfsii CBS 102852
Clonostachys ralfsii CBS 102849
Clonostachys compactiuscula CBS 101923
Clonostachys samuelsii CBS 700.97
Clonostachys miodochialis CBS 997.69_T
Clonostachys divergens CBS 532.69
Clonostachys divergens CBS 229.80
Clonostachys ralfsii CBS 703.97_T
Clonostachys samuelsii CBS 198.93
Clonostachys ralfsii CBS 267.36
Clonostachys divergens JW190011
Clonostachys samuelsii CBS 188.94
Clonostachys divergens CBS 102426
Clonostachys ralfsii CBS 102850
Clonostachys divergens CBS 967.73B_T
Clonostachys eriocamporesii CBS 148221
Clonostachys ralfsii CBS 127880
Clonostachys divergens JW183010
Clonostachys obovatispora CBS 118752_T
Clonostachys ralfsii CBS 102845
Clonostachys samuelsii CBS 196.93
Clonostachys ralfsii CBS 141089
Clonostachys hongkongensis CBS 116542
Clonostachys compactiuscula CBS 123.79
Clonostachys epichloe CBS 101037_T
Clonostachys penicillata CBS 148211
Clonostachys compactiuscula CBS 123759
Clonostachys cylindrica CBS 101113_T
Clonostachys samuelsii CBS 100976
Clonostachys compactiuscula CBS 123786
Clonostachys compactiuscula CBS 122580
Clonostachys eriocamporesii MFLUCC 19-0486_T
Clonostachys divergens JW158008
Clonostachys samuelsii CBS 699.97_T
Clonostachys compactiuscula CBS 102563
Clonostachys divergens CBS 381.77
Clonostachys compactiuscula CBS 119318
Clonostachys samuelsii CBS 199.93
Clonostachys fujianensis CBS 127474_T
Clonostachys divergens CBS 120975
Clonostachys samuelsii CBS 701.97
Clonostachys ralfsii CBS 102851
Clonostachys samuelsii CBS 201.93
100/100/1
69/99/1
100/100/1
52/-/-
99/100/1
85/100/1
96/98/0.96
100/100/1
92/98/0.99
100/100/1
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100/100/1
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-/-/0.94
215www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Clonostachys aureofulvella CBS 102839
Clonostachys zelandiaenovae CBS 217.93
Clonostachys ambigua PAD S00003_T
Clonostachys zelandiaenovae CBS 234.80B
Clonostachys zelandiaenovae CBS 124343
Clonostachys palmae CBS 119.87_T
Clonostachys rogersoniana CBS 668.70
Clonostachys zelandiaenovae CBS 100978
Clonostachys rogersoniana CBS 102572
Clonostachys zelandiaenovae CBS 123949
Clonostachys rogersoniana CBS 394.85
Clonostachys zelandiaenovae CBS 102846
Clonostachys pseudostriata CBS 309.96
Clonostachys zelandiaenovae CBS 233.80
Clonostachys sp. CBS 496.90
Clonostachys viticola CAA 944_T
Clonostachys zelandiaenovae CBS 102844
Clonostachys zelandiaenovae CBS 234.80A
Clonostachys pseudostriatopsis MAFF 239829
Clonostachys aureofulvella CBS 195.93
Clonostachys agarwalii CBS 533.81_T
Clonostachys rogersoniana CBS 582.89
Clonostachys pseudostriatopsis MAFF 239841
Clonostachys pseudostriatopsis MAFF 239827_T
Clonostachys rogersoniana CBS 102564
Clonostachys pseudostriata CBS 120.87_T
Clonostachys zelandiaenovae CBS 197.93
Clonostachys rogersoniana CBS 377.65
Clonostachys zelandiaenovae CBS 234.80C
Clonostachys aureofulvella CBS 102837
Clonostachys rogersoniana CBS 139551
Clonostachys aureofulvella CBS 102836
Clonostachys krabiensis MFLUCC 16-0254_T
Clonostachys viticola CAA 946
Clonostachys zelandiaenovae CBS 102422
Clonostachys zelandiaenovae CBS 100977
Clonostachys krabiensis CBS 192.96
Clonostachys bambusae CBS 139411_T
Clonostachys reniformis CBS 695.86_T
Clonostachys aureofulvella CBS 235.80
Clonostachys rogersoniana CBS 121653
Clonostachys zelandiaenovae CBS 232.80
Clonostachys chongqingensis HMAS 290894_T
Clonostachys aureofulvella CBS 100980_T
Clonostachys rogersoniana CBS 920.97_T
Clonostachys aureofulvella CBS 200.93
Clonostachys aureofulvella CBS 102838
Clonostachys capitata CBS 218.93_T
Clonostachys rogersoniana CBS 139287
Clonostachys viticola CAA 945
Clonostachys leptoderma HMAS 255834_T
Clonostachys zelandiaenovae CBS 100979_T
Clonostachys swieteniae MFLUCC 18-0572_T
91/100/1
95/99/1
90/100/1
100/100/1
93/98/-
57/90/-
98/100/-
92/98/0.96
94/98/-
75/-/-
98/100/1
81/98/-
67/-/-
96/100/1
99/100/1
96/100/1
100/100/-
90/100/1
98/100/1
99/100/1
94/100/1
Fig. 2. (Continued).
Nectriopsis didymii L. Zhao & Crous, sp. nov. MycoBank MB
848432. Fig. 3.
Etymology: Referring to the host, Didymium melanospermum, from
which the holotype strain was collected.
Typus: Germany, Eifel, Geeser Wald near Gerolstein, on Didymium
melanospermum, Sep. 1970, W. Gams (holotype designated here: CBS
H-18226, ex-type living culture CBS 852.70A).
Sexual morph unknown. Mycelia consisting of branched, septate,
hyaline, smooth- and thin-walled hyphae, 1.5–2.8 μm diam.
216
Zhao et al.
Conidiophores hyaline, smooth, verticillately branched, lateral
branches decreasing in length towards the apex of the main axis.
Conidiogenous cells consisting of discrete phialides, terminal, in
whorls of 2–4, ask-shaped, (4.2–)6.0–12.3(–19.8) μm long, (1.0–)
1.1–1.7(–1.9) μm wide at base, 0.6–0.9(–1.0) μm wide near apex (n
= 70); intercalary phialides rare, below whorls of terminal phialides,
with up to 3 μm long lateral pegs. Conidia aseptate, hyaline, smooth-
walled, globose or subglobose, without laterally displaced hilum,
(1.8–)2.0–2.5(–2.7) × 1.6–1.9(–2.1) μm (av. = 2.2 × 1.8 μm, n = 150).
Culture characteristics: Colonies on OA reaching 30–35 mm diam
after 7 d in darkness at 25 °C, at, with entire margin, pure white,
Clonostachys solani f. nigrovirens CBS 223.72A
Clonostachys solani f. nigrovirens CBS 229.74
Clonostachys solani CBS 142837
Clonostachys pseudochroleuca CBS 192.94
Clonostachys solani f. nigrovirens CBS 144943
Clonostachys solani f. nigrovirens JW96008
Clonostachys pseudochroleuca CBS 187.94_T
Clonostachys solani f. nigrovirens CBS 147.65
Clonostachys solani JW11016
Clonostachys solani JW191026
Clonostachys solani CBS 697.88_T
Clonostachys solani CBS 142845
Clonostachys solani f. nigrovirens CBS 325.78
Clonostachys pseudochroleuca CBS 194.93
Clonostachys solani f. nigrovirens CBS 728.69
Clonostachys solani f. nigrovirens CBS 101924
Clonostachys solani CBS 102418
Clonostachys solani f. nigrovirens CBS 101919
Clonostachys solani CBS 708.86
Clonostachys solani f. nigrovirens NL19-049009
Clonostachys solani f. nigrovirens CBS 191.31
Clonostachys solani f. nigrovirens CBS 223.72B
Clonostachys solani f. nigrovirens CBS 223.72C
Clonostachys solani f. nigrovirens JW10009
Clonostachys solani f. nigrovirens CBS 125111
Clonostachys solani CBS 144941
Clonostachys solani CBS 224.72A
Clonostachys pseudochroleuca CBS 185.94
Clonostachys solani f. nigrovirens CBS 183.30_T
Clonostachys solani NL19-22014
Clonostachys solani CBS 144338
Clonostachys solani CBS 702.97
Clonostachys pseudochroleuca CBS 220.93
Clonostachys solani CBS 144944
Clonostachys solani f. nigrovirens NL19-008005
Clonostachys solani f. nigrovirens JW211002
Clonostachys solani f. nigrovirens CBS 142.91
Clonostachys solani f. nigrovirens CBS 101926
Clonostachys solani f. nigrovirens CBS 350.76
Clonostachys pseudochroleuca CBS 124610
Clonostachys solani CBS 224.72F
Clonostachys solani f. nigrovirens JW187017
Clonostachys solani BE19-005010
Clonostachys pseudochroleuca CBS 219.93
Clonostachys pseudochroleuca CBS 447.96
Clonostachys solani f. nigrovirens CBS 144349
Clonostachys solani f. nigrovirens JW1072
Clonostachys solani CBS 752.68
Clonostachys pseudochroleuca CBS 124754
Clonostachys solani CBS 102425
Clonostachys pseudochroleuca CBS 191.94
Clonostachys solani CBS 906.72D
Clonostachys solani f. nigrovirens CBS 228.74
Clonostachys pseudochroleuca CBS 186.94
98/100/1
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Fig. 2. (Continued).
217www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
aerial mycelium sparsely developed, mealy, with a thin layer of
conidiophores, distinctly zonate, reverse whitish. Colonies on PDA
reaching 27–28 mm diam, at, with entire margin, aerial mycelium
moderate, felty, pale yellow, reverse concolourous. Colonies on
SNA reaching 19–22 mm diam, at, with dendritic margin and
sparsely produced aerial mycelium, whitish, sporulation absent or
sparse, reverse concolourous.
Additional materials examined: Canada, Ontario, Petawawa, forest soil, B2
horizon, under Populus tremuloides, Aug. 1968, G.C. Bhatt, No. PET 114
(B), culture CBS 652.70; Ontario, Petawawa, forest soil under Populus
tremuloides, Oct. 1968, G.C. Bhatt, No. PET 160, specimen CBS H-18229,
culture CBS 355.70. Germany, Eifel, Pelmer Wald bei Gerolstein, on
Didymium melanospermum, 15 Sep. 1970, W. Gams, specimen CBS
H-18225, culture CBS 852.70B. Netherlands, Noord-Brabant, St. Anthonis,
on Pseudotsuga menziesii, litter, Oct. 1985, W. Gams & M. Schlag, specimen
CBS H-3999, culture CBS 788.85. Sweden, dung of isopod, date unknown,
B.E. Söderström, culture CBS 326.79. Trinidad and Tobago, Nariva
Swamp, twig, Aug. 1981, D.J. Stradling, culture CBS 395.82.
Notes: Based on our phylogenetic analysis, N. didymii (Fig. 1; 98 %
/ 99 % / 1) and N. microspora (Fig. 1; 61 % / 98 % / -) form a fully-
supported clade (Fig. 1). Morphologically, N. didymii differs from N.
microspora in shorter hyphal width (1.5–2.8 μm vs 2.5–4.0 μm), and
longer terminal phialides [(4.2–)6.0–12.3(–19.8) μm vs (6–)7–10(–
15) μm]. Furthermore, sequences clearly distinguish the ex-type
strains CBS 852.70A (N. didymii) from CBS 933.69 (N. microspora)
[ITS (99.6 % identity, with 2 bp differences), RPB2 (96.1 %, 29 bp),
Clonostachys rhizophaga CBS 229.48
Clonostachys moreaui CLL19024_T
Clonostachys rhizophaga CBS 149.72
Clonostachys rhizophaga NL19-052004
Clonostachys rhizophaga CBS 124507
Clonostachys oblongispora CBS 100285_T
Clonostachys sporodochialis CBS 205.93
Clonostachys kunmingensis YFCC 898_T
Clonostachys chloroleuca CBS 124004
Clonostachys kowhai CBS 461.95_T
Clonostachys chloroleuca CBS 141652
Clonostachys sporodochialis CBS 100975
Clonostachys apocyni CBS 360.78
Clonostachys apocyni CBS 130.87
Clonostachys rhizophaga CBS 361.77
Clonostachys chloroleuca CBS 227.48
Clonostachys rhizophaga CBS 907.72I
Clonostachys kunmingensis YFCC 892
Clonostachys chloroleuca CBS 141588_T
Clonostachys sporodochialis CBS 101921_T
Clonostachys sporodochialis CBS 133758
Clonostachys apocyni CBS 359.78
Clonostachys moreaui CBS 127881
Clonostachys apocyni CBS 956.73
Clonostachys garysamuelsii CBS 123964_T
Clonostachys chloroleuca CBS 141592
Clonostachys rhizophaga NL19-052003
Clonostachys rhizophaga CBS 124509
Clonostachys rhizophaga CBS 125416
Clonostachys rhizophaga CBS 124510
Clonostachys rhizophaga CBS 124508
Clonostachys chloroleuca CBS 532.86
Clonostachys pnagiana CLLG19041_T
Clonostachys chloroleuca CBS 141589
Clonostachys rhizophaga CBS 202.37_T
Clonostachys chloroleuca CBS 570.77
Clonostachys chloroleuca CBS 100495
Clonostachys kunmingensis CBS 101920
Clonostachys rhizophaga CBS 124511
Clonostachys rhizophaga CBS 100494
Clonostachys rhizophaga CBS 529.80
Clonostachys chloroleuca CBS 141591
Clonostachys rhizophaga CBS 906.72A
Clonostachys chloroleuca CBS 141590
Clonostachys parasporodochialis CBS 192.93_T
72/97/1
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79/99/0.96
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79/99/1
99/100/1
96/100/1
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61/95/-
55/-/-
98/100/0.97
93/100/1
99/100/1
-/-/0.92
Fig. 2. (Continued).
218
Zhao et al.
TEF1 (98.2 %, 15 bp) and TUB2 (99.1 %, 7 bp)]. Therefore, a new
species is introduced here as N. didymii. Both species have an
overall similar conidiophore morphology that differs clearly from the
frequently irregularly branched, acremonium- or, less frequently,
gliocladium-like conidiophores of other Nectriopsis species
(Samuels 1988a). Intercalary phialides are not often seen in
conidiophores in Nectriopisis, however, Samuels (1988a) illustrated
such conidiogenous cells for N. epimyces and N. microthecia.
Nectriopsis microspora (Jaap) L.W. Hou et al., Stud. Mycol. 105:
109. 2023. Fig. 4.
Basionym: Verticillium microsporum Jaap, Verh. Bot. Vereins Prov.
Brandenburg 58: 38. 1916.
Synonyms: Sesquicillium parvulum Veenb.-Rijks, Acta Bot. Neerl.
19: 323. 1970.
Sesquicillium microsporum (Jaap) Veenb.-Rijks & W. Gams, in
Gams, Cephalosporium-artige Schimmelpilze (Stuttgart): 226.
1971.
Tolypocladium microsporum (Jaap) Bissett, Canad. J. Bot. 61:
1318. 1983.
Gliocladium microsporum (Jaap) Arx, Mycotaxon 25: 157. 1986.
Typus: Netherlands, Oostelijk Flevoland, wheat eld soil, date unknown,
J.W. Veenbaas-Rijks (holotype CBS H-7751, ex-type culture CBS 933.69
= ATCC 18.932).
Fig. 2. (Continued).
0.09
Clonostachys farinosa CBS 552.84
Clonostachys farinosa CBS 124609
Clonostachys farinosa CBS 288.78
Clonostachys farinosa MFLUCC 17-0131
Clonostachys farinosa MFLUCC 17-2620
Clonostachys farinosa CBS 194.94
Clonostachys farinosa CBS 124575
Clonostachys farinosa CBS 115882
Clonostachys farinosa CBS 363.78
Clonostachys farinosa CBS 135613
Clonostachys farinosa CBS 119317
Clonostachys farinosa CBS 364.78_T
Clonostachys farinosa IBP2
Clonostachys farinosa CBS 203.93
Clonostachys farinosa CBS 113336
Clonostachys farinosa CBS 208.93
Clonostachys farinosa CBS 202.93
Clonostachys farinosa CBS 204.93
Clonostachys farinosa CBS 552.95
Clonostachys farinosa CBS 123785
Clonostachys farinosa CBS 149.52
Clonostachys farinosa CBS 324.78
Clonostachys farinosa CBS 137553
Clonostachys farinosa CBS 293.78
Clonostachys farinosa CBS 914.97
Clonostachys farinosa CBS 135759
Clonostachys farinosa PAD S00020
Clonostachys farinosa CBS 101918
Clonostachys farinosa CBS 118757
Clonostachys farinosa CBS 326.78
Clonostachys farinosa CBS 365.78
Clonostachys farinosa CBS 206.93
Clonostachys farinosa PAD S00011
Clonostachys farinosa CBS 115292
Clonostachys farinosa CBS 101917
Clonostachys farinosa CBS 101922
Clonostachys farinosa CBS 245.78
Clonostachys farinosa CBS 133759
Clonostachys farinosa CBS 124067
Clonostachys farinosa MFLUCC 18-0587
Clonostachys farinosa CBS 323.78
Clonostachys farinosa PAD S00021
Clonostachys farinosa PAD S00022
99/100/1
98/100/1
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82/-/1
99/100/0.99
99/100/1
60/-/0.96
71/99/0.96
60/98/0.96
58/98/0.93
90/100/0.96
99/100/1
58/98/-
81/98/0.96
70/-/0.93
85/-/0.95
Clonostachys rosea
Clonostachys rosea f. catenulata
219www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Descriptions and illustrations: Veenbaas-Rijks (1970), Gams
(1971).
Additional materials examined: Canada, Ontario, Aberfoyle, forest
soil under Thuja occidentalis, Sep. 1966, G.C. Bhatt, specimen CBS
H-18228, culture CBS 354.70. Japan, Kamogawa, Hachijo Island, living
Cycadaceae, Mar. 1996, T. Okuda, culture CBS 102560. Netherlands,
Utrecht, Berenkuil, soil, Sep. 1972, D. Mulder, culture CBS 954.72. USA,
Wisconsin, Madison, leaf litter of Acer saccharum, unknown date and
collector, culture CBS 582.77.
Notes: This species was originally introduced as S. parvulum
(Veenbaas-Rijks 1970). Its taxonomic history is complex.
Veenbaas-Rijks (1970) described S. parvulum as a new species
of Sesquicillium, distinguishing it from S. buxi and S. candelabrum
(Gams 1968) by the size and shape of its conidia. Gams (1971)
treated S. parvulum as a synonym of S. microsporum, with
Verticillium microsporum as basionym. Bissett (1983) transferred
S. microsporum to Tolypocladium because of the rather irregular
branching pattern of its conidiophores, false phialidic whorls,
intercalary phialides with elongated phialidic pegs, and masses
of slimy conidia. Samuels (1989) accepted classication of this
species in Sesquicillium, while noting that S. microsporum differed
from other Sesquicillium species in size ranges and shapes of
its phialides and conidia, rather dry conidial chains, and more
irregularly branched and poorly developed penicilli. Relatedness
of S. microsporum and Nectriopsis was hypothesised earlier on
the basis of the myxomyceticolous lifestyle and analyses of LSU
sequences (Rogerson & Stephenson 1993, Schroers 2001). In
the present study, according to our phylogenetic analyses, strains
identied as S. microsporum clustered in a lineage (Fig. 1; 61 % /
98 % / -) of the genus Nectriopsis and is closely related to N. didymii
(Fig. 1). For morphological comparison with the other species of
this genus, see notes under N. didymii.
Fig. 3. Nectriopsis didymii (ex-type CBS 852.70A). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Conidiophores. G, H. Conidia. Black arrows
indicate intercalary phialides. Scale bars: D = 50 μm; E–H = 10 μm.
220
Zhao et al.
Mycocitrus Möller, Bot. Mitt. Tropen. 9: 297. 1901.
Synonym: Shiraiella Hara, Bot. Mag. (Tokyo) 28: 274. 1914.
Type: Mycocitrus aurantium Möller
Sexual morph on the natural substratum. Stroma well-developed,
surface buff to rufous or light orange, KOH-, clasping and
surrounding the substratum. Ascomata perithecial, surface partially
to fully immersed, with apices barely visible, densely gregarious,
forming a single layer. Asci cylindrical, ascal apex simple, 8-spored.
Ascospores 1-septate, ellipsoid, hyaline, spinulose. Asexual
morphs commonly acremonium-like (adapted from Rossman et al.
1999).
Notes: Mycocitrus was rst described from culms of living bamboo
and Microstachys in southern Brazil (Möller 1901). The type species,
M. aurantium, is characterised by its large, eshy, orange stromata
that clasp and surround bamboo culms, with perithecial ascomata
partially to fully immersed in the upper region of the stromata.
Later, a second species, M. phyllostachydis (bas. Ustilaginoidea
phyllostachydis), was collected in Japan on Phyllostachys and
added to the genus (Doi 1967). Mycocitrus was originally placed
in the “Hypocreaceen, Didymosporae” (Möller 1901), and later
in Hypocreaceae (Doi 1967). The proposal by Rossman et al.
(1999) to include Mycocitrus in the Bionectriaceae was based on
morphology. Leite et al. (2018) obtained two bamboo-inhabiting M.
aurantium cultures in South America. Their phylogeny was based
on ITS sequences and suggested prematurely that Mycocitrus
forms an independent lineage within Hypocreales, distinct from
Bionectriaceae, Nectriaceae, Cordycipitaceae, Clavicipitaceae,
and Hypocreaceae. However, Hou et al. (2023) revealed that
Mycocitrus clusters within the Bionectriaceae based on a multi-
locus phylogenetic analysis, what is supported in the present
analysis (Fig. 1).
Mycocitrus coccicola (J.A. Stev.) L. Zhao & Crous, comb. nov.
MycoBank MB 848434.
Basionym: Tubercularia coccicola J.A. Stev., Rep. (Annual) Puerto
Rico Insular Exp. Sta., 1916–1917: 91. 1917.
Synonyms: Clonostachys coccicola (J.A. Stev.) H.T. Dao, Mycol.
Prog. 15: 6. 2016.
Nectria tuberculariae Petch, Trans. Brit. Mycol. Soc. 7: 157. 1921.
Materials examined: Australia, New South Wales, Cornwallis, armoured
scale insects, date and collector unknown, culture BucCo; New South
Wales, Somersby, armoured scale insects, date and collector unknown,
culture BucS.
Notes: Mycocitrus coccicola was originally described as
Tubercularia coccicola (Stevenson 1917) before Dao et al.
(2016) placed it in Clonostachys as C. coccicola. In our study, the
phylogenetic analysis of the combined ITS, LSU, TEF1, RPB2, and
TUB2 dataset revealed that C. coccicola clusters within the genus
Mycocitrus (Fig. 1) and a new combination is proposed here.
Fig. 4. Nectriopsis microspora (ex-type CBS 933.69). A–C. Colonies on OA, PDA, SNA after 7 d at 25 °C. D, E. Conidiophores. F. Conidia. Black arrows
indicate intercalary phialides. Scale bars = 10 μm.
221www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Mycocitrus coxeniae (Y.P. Tan et al.) L. Zhao & Crous, comb.
nov. MycoBank MB 848911.
Basionym: Clonostachys coxeniae Y.P. Tan et al., Index of
Australian Fungi 5: 3. 2023.
Description: Tan & Shivas (2023).
Notes: Tan & Shivas (2023) recently coined Clonostachys coxeniae,
but without any proper description, illustration or discussion of
morphological characters. Their deposited sequence identies this
species as a member of genus Mycocitrus (Fig. 1).
Mycocitrus odorus L.W. Hou et al., Stud. Mycol. 105: 111. 2023.
Fig. 5.
Typus: Netherlands, Amsterdam, Slotervaart Hospital, onychomycosis
(human), unknown date, W.C. van Dijk & W. Pauw (holotype specimen
CBS H-24690, ex-type living culture CBS 100104).
Description and illustration: Hou et al. (2023).
Additional materials examined: Netherlands, human skin, unknown
collection date and collector, culture CBS 120610. Sweden, Stockholm,
human skin, unknown collection date and collector, culture CBS 232.75B.
Fig. 5. Mycocitrus odorus (CBS 120610). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–K. Conidiophores. L. Conidia. Scale bars = 10 μm.
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Zhao et al.
Note: Mycocitrus odorus was recently described by Hou et al.
(2023) as a novel species in Mycocitrus (Fig. 1).
Mycocitrus phyllostachydis (Syd. & P. Syd.) Yoshim. Doi, Bull.
Natl. Sci. Mus., Tokyo, N.S. 10: 31. 1967.
Basionym: Ustilaginoidea phyllostachydis Syd. & P. Syd., Mém.
Herb. Boissier 4: 5. 1900.
Synonyms: Hypocreopsis phyllostachydis (Syd. & P. Syd.) I. Miyake
& Hara, Bot. Mag., Tokyo 24: 333. 1910.
Shiraiella phyllostachydis (Syd. & P. Syd.) Hara, Bot. Mag., Tokyo
28: 402. 1914.
Description and illustration: Doi (1967).
Material examined: Japan, on Phyllostachys sp. (Poaceae), unknown
date, W. Gams, specimen CBS H-14839, culture CBS 330.69 = IFO 8912.
Notes: Gams (1971) examined the culture of M. phyllostachydis
(CBS 330.69) isolated from Phyllostachys sp. in Japan. Based on
the phylogenetic analyses, classication of U. phyllostachydis in
Mycocitrus, as proposed by Doi (1967), is conrmed here (Fig. 1).
Fig. 6. Mycocitrus synnematus (ex-type CBS 126677). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Synnemata. G. Detail of the apical
portion of a synnema. H–K. Conidiogenous cells. L. Conidia. Scale bars: F = 100 μm; G = 50 μm; H–L = 10 μm.
223www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Mycocitrus synnematus L. Zhao & Crous, sp. nov. MycoBank
MB 848435. Fig. 6.
Etymology: Name refers to the production of synnemata.
Typus: Sri Lanka, from wood, date unknown, G.J. Samuels (holotype
designated here CBS H-25131, ex-type living culture CBS 126677).
Sexual morph unknown. Synnemata erect, golden brown,
occurring singly or in groups, abundant in culture. Conidiophores
macronematous, branched, asymmetric-biverticillate or
monoverticillate. Conidiogenous cells phialidic, cylindrical, straight
or slightly curved, slightly tapered at the apex, in terminal whorls
of 2–5, (13.2–)16.2–45.4(–59.6) × (1.2–)1.5–2.0(–2.1) μm. Conidia
aseptate, hyaline, smooth, ellipsoid, straight to slightly curved,
distally broadly rounded, without recognisable hilum, (4.3–)5.5–
7.7(–8.0) × (2.7–)3.0–3.8(–4.0) μm (av. = 6.8 × 3.5 μm, n = 150).
Culture characteristics: Colonies on OA reaching 30–35 mm
diam after 7 d in darkness at 25 °C, at, with entire margin, aerial
mycelium scanty, nely occose, whitish, reverse concolourous.
Colonies on PDA reaching 27–28 mm diam, at, with entire margin,
aerial mycelium moderately dense, occose, pale yellow, reverse
concolourous. Colonies on SNA reaching 19–22 mm diam, at, with
entire margin, aerial mycelium moderate, occose white, reverse
whitish.
Notes: Phylogenetically, M. synnematus is closely related to M.
coccicola, M. coxeniae, M. odorus, and M. phyllostachydis (Fig.
1). Morphologically, M. synnematus can be distinguished from M.
odorus and M. coccicola by its larger conidia, (4.3–)5.5–7.7(–8.0) ×
(2.7–)3.0–3.8(–4.0) μm (av. = 6.8 × 3.4 μm) in M. synnematus, 3.2–
5.5 × 2.1–3.3 (av. 4 × 2 μm) in M. coccicola and (3.4–)3.7–5.0(–
6.6) × (1.8–)2.1–2.8(–3.0) μm (av. = 4.4 × 2.5 μm) in M. odorus.
Furthermore, CBS 126677 (M. synnematus) and CBS 330.69 (M.
phyllostachydis) have clearly different ITS (93.9 % identity, with
31 bp differences), LSU (97.6 %, 18 bp), RPB2 (89.1 %, 81 bp),
and TEF1 (96.6 %, 27 bp) sequences, while M. synnematus (CBS
126677) has different ITS (95 % identity, with 24 bp differences)
sequences when compared with M. coxeniae (BRIP 49559a).
Stephanonectria Schroers & Samuels, Sydowia 51: 116. 1999.
Type: Stephanonectria keithii (Berk. & Broome) Schroers &
Samuels
Sexual morph on the natural substratum. Stroma supercial,
reduced or erumpent through bark. Perithecia brown, KOH-, smooth
to rough, minutely papillate. Ostiolum surrounded by a crown-like
arrangement of cells. Perithecial wall consisting of two regions. Cells
of the crown merging with the cells of the outer wall region, angular
to oblong, outwards toothlike. Ascospores 1-septate, covered
with short striae. Asexual morph. Conidiophores monomorphic,
sporodochial, towards the margin of the colony solitary, irregularly
penicillate, or sparsely aggregated, not showing regular patterns,
with 1–5 phialides on each supporting cell; branches overall
diverging, sometimes joined by anastomoses. Phialides cylindrical,
sometimes widening in the middle or in the upper part, typically
becoming narrower just underneath the apex, with apical periclinal
thickening visible, without collarette. Conidia aseptate, hyaline,
smooth, ellipsoidal, hilum median, slightly laterally displaced, or not
visible (adapted from Schroers et al. 1999a).
Notes: Stephanonectria was introduced as a new genus because of
characters of the ascomatal wall and aggregations of cells forming
a crown-like structure around the ostiolum. The ascospores are
covered with short striae that are more or less parallel with the
long axis of the spore. The asexual morph was identied as being
myrothecium-like (Schroers et al. 1999a) because of details seen in
the shape of phialides. Despite the more irregular branching pattern
of conidiophores, sporodochia seen in S. keithii are similar to those
formed by many Clonostachys species. The use of “myrothecium-
like” for the asexual morph in Stephanonectria (Schroers et al.
1999a) is therefore obsolete.
Stephanonectria chromolaenae R.H. Perera & K.D. Hyde, Fungal
Diversity 118: 134. 2023. Fig. 7.
Typus: Thailand, Chiang Mai Province, Mae Rim District, on dead stem of
Chromolaena odorata (Asteraceae), 18 Sep. 2017, R.H. Perera (holotype
MFLU 19-0972, ex-type living culture MFLUCC 18-0589).
Description and illustration: Perera et al. (2023).
Additional materials examined: Turkey, isolated from soil, date unknown,
G. Turhan, No. 10, culture CBS 475.91; soil, date unknown, G. Turhan, No.
13, culture CBS 476.91.
Notes: Stephanonectria chromolaenae was described from dead
stems of Chromolaena odorata. Based on our phylogenetic
analysis, S. chromolaenae and S. keithii formed a fully-supported
clade within the genus Stephanonectria (Fig. 1).
Stephanonectria keithii (Berk. & Br.) Schroers & Samuels,
Sydowia 51: 116. 1999.
Basionym: Nectria keithii Berk. & Br., Ann. Mag. Nat. Hist., Ser. 4,
27: 144. 1876.
Synonym: Nectriella keithii (Berk. & Br.) Sacc., Michelia 1: 279.
1879.
Typus: UK, Scotland, Forres, on decorticated stems of cabbage, collection
date unknown, Rev. J. Keith, specimen IMI 77877.
Description and illustration: Schroers et al. (1999a).
Additional materials examined: Netherlands, Utrecht, Berenkuil, soil,
Sep. 1972, collector unknown, culture CBS 943.72; Oostelijk Flevoland,
agricultural soil, under permanent potato, J. W. Veenbaas-Rijks, Oct.
1969, culture CBS 434.70. New Zealand, Gisborne, Lake Waikaremoana,
Ngamoko Trail, on Beilschrniedia tawa, 30 May 1983, G. J. Samuels et al.,
Samuels culture 83-165, CBS 100007 (PDD 46342; BPI 737629).
Notes: This species was originally described as Nectria keithii
(Berkeley & Broome 1876). Schroers et al. (1999a) introduced a
new genus Stephanonectria to accommodate S. keithii. The three
strains included in the phylogenetic analysis cluster in a supported
clade (Fig. 1; 87 % / 100 % / 1).
Sesquicillium W. Gams, Acta Bot. Neerl. 17. 455. 1968.
Type: Sesquicillium buxi (J.C. Schmidt ex Link) W. Gams
Sexual morph on the natural substratum. Perithecial stroma
typically formed supercially on plant tissue, reduced, supporting
solitary perithecia, typically consisting of prosenchymatous
cells not integrating with cells of major perithecial wall regions,
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Zhao et al.
sometimes erumpent through bark, supporting several perithecia
and consisting of pseudoparenchymatous cells. Perithecia
solitary, gregarious or loosely aggregated, crowded if formed on
an erumpent stroma, globose to subglobose, 200–400 µm diam,
sometimes smaller, up to 200 µm diam, pale yellow, pale to
light orange, apically or laterally pinched when dry, not papillate,
glabrous; ostiolar region somewhat sunken and slightly darker
(brownish). Perithecial wall either consisting of two or one major
wall region, sometimes with an additional outermost cell-layer
that continues into the prosenchymatous stroma. Asci narrowly to
broadly clavate, 8-spored, with at or rounded apex, with or without
visible ring. Ascospores typically 1-septate, equally 2-celled,
sometimes aseptate, hyaline, spinulose, warted, with short striae
or with warts arranged in striae, typically ellipsoidal to fusiform.
Asexual morph. Conidiophores macronematous, mononematous,
monomorphic penicillate or somewhat dimorphic, penicillate
and verticillium-like, mostly arising from the agar surface or from
sparsely formed aerial mycelium. Penicillate conidiophores bi- to
quaterverticillate; branches of the penicilli divergent or adpressed;
terminal whorls consisting of narrowly ask-shaped phialides
and/or typically one or sometimes two successive intercalary
phialides, of which the uppermost bears a solitary terminal phialide;
Fig. 7. Stephanonectria chromolaenae (CBS 475.91). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–I. Conidiophores. J. Conidia. Scale bars:
E, F = 50 μm; G–J = 10 μm.
225www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
conidiogenous pegs of intercalary phialides typically short, formed
laterally just below the upper septum. Conidia aseptate, smooth,
hyaline, obovoid, ellipsoid, or fusoid, slightly curved or straight,
generally with a slightly laterally displaced hilum and arranged in
imbricate chains forming columns or, rarely, with a centrally located
hilum and arranged in linear chains (adapted from Schroers 2001).
Notes: In this study, Bionectria subgenera Epiphloea and
Uniparietina clustered in a statistically supported clade (Fig. 1;
97 % / 99 % / 1) that is sister to clades accommodating the other
Clonostachys subgenera (Fig. 1). This separate clade includes C.
buxi and C. candelabrum classied by Gams (1968) in Sesquicillium.
The type species of the subgenus Uniparietina is Bionectria
coronata, for which S. buxi is currently in use. Furthermore, the
genus Sesquicillium, already used by Samuels (1989) for several
below considerd species, is resurrected to accommodate the
subgenera Epiphloea and Uniparietina.
Sesquicillium buxi (J.C. Schmidt ex Link) W. Gams, Acta Bot.
Neerl. 17: 455. 1968.
Basionym: Fusidium buxi J.C. Schmidt ex Link, Willdenow, Sp. pl.,
Edn 4 6(2): 97. 1825.
Synonyms: Fusisporium buxi (J.C. Schmidt ex Link) Fr., Syst.
Mycol. 3: 447. 1832.
Verticillium buxi (J.C. Schmidt ex Link) Auersw. & Fleischh.,
Hedwigia 6: 9. 1867.
Ramularia buxi (J.C. Schmidt ex Link) Fuckel, Symb. Mycol. p. 97.
1870.
Paecilomyces buxi (J.C. Schmidt ex Link) Bezerra, Acta Bot. Neerl.
12: 63. 1963.
Clonostachys buxi (J.C. Schmidt ex Link) Schroers, Stud. Mycol.
46: 193. 2001.
Nectriella coronata Juel, Arkiv før Botanik 19: 4. 1925.
Bionectria coronata (Juel) Schroers, Stud. Mycol. 46: 203. 2001.
Typus: Germany, Leipzig, Auerswald [neotype for Fusidium buxi: B,
designated by Gams (1968)].
Descriptions and illustrations: Juel (1925), Gams (1968), Rossman
et al. (1993), Schroers (2001).
Additional materials examined: France, Pyrénées Atlantiques, Île de
Sauveterre de Béarn; 400 m alt., Buxus sempervirens, leaf litter, 17 Oct
1993, F. Candoussau, BPI 802851, culture CBS 696.93. Netherlands,
Buxus sempervirens, collection date unknown, J.L. Bezerra, culture
CBS 288.62; Culemborg, soil, collection date unknown, R. Fuld, culture
JW182006; Netherlands, Utrecht, soil, collection date unknown, M.
Wickham, culture JW199009; Ermelo, soil, collection date unknown,
Marit en Mette Elmers, culture JW259005; Deurne, soil, collection date
unknown, Martina Hoeben, culture JW58015; Eemnes, soil, collection
date unknown, Herman Wim Vos, culture CBS 147861 = JW6017; Rijen,
soil, collection date unknown, Gijs & Lotte Schijvenaars, culture JW94008.
Sweden, Uppsala, Botanical Garden, on leaves of Buxus sempervirens,
10 Oct. 1924, O. Juel (type of Nectriella coronata: holotype S, isotype BPI).
UK, England, Kew Gardens, Buxus sempervirens, leaf litter, Nov. 1967, W.
Gams, CBS H-18217, culture CBS 202.69.
Notes: Sesquicillium buxi was originally described as Fusidium buxi
(Link 1825). Later, it was transferred to the genus Sesquicillium
(Gams 1968) as S. buxi (type species) with conidiophores having
sparsely branched whorls, and intercalary phialides. By accepting
a broader genus concept, Schroers (2001) combined S. buxi as
Clonostachys buxi. In the present study, the phylogenetic analysis of
the combined ITS, LSU, TEF1, RPB2 and TUB2 dataset reveals that
taxa of Sesquicillium form the phylogenetic sister of Clonostachys
sensu stricto (Fig. 1). Therefore, S. buxi is resurrected here.
Sesquicillium candelabrum (Bonord.) W. Gams, Acta Bot. Neerl.
17: 457. 1968. Fig. 8.
Basionym: Verticillium candelabrum Bonord., Handb. Allgem.
Mykol. (Stuttgart): 97. 1851.
Synonyms: Clonostachys candelabrum (Bonord.) Schroers, Stud.
Mycol. 46: 192. 2001.
Clonostachys chuyangsinensis Hong Yu bis & Yao Wang, Frontiers
Microbiol. 14: 3. 2023.
Typus: Switzerland, Kt. Bern, Büetigen, Waldhaus Hörnli, Ischlag, strongly
decayed Fomitopsis pinicola, 13 Oct. 2005, W. Gams (epitype designed
here CBS H-25178, MBT 10012943, ex-epitype culture CBS 119045);
Rabenhorst ‘Fungi europaei’ No. 2148, on leaves of Laurus nobilis, leg.
P.A. Saccardo, Selva, 1875 (B), designated by Gams (1968) (neotype for
Verticillium candelabrum).
Sexual morph unknown. Asexual morph. Conidiophores
monomorphic, scattered on the agar surface or arising from strands
of aerial hyphae, mono- to quaterverticillate, divergent or with
branches at somewhat acute angles. Conidiogenous cells phialidic
divergent or adpressed; stipes 20–90 μm long, 2.4–4.3 μm wide at
base; penicilli 30–90 μm high, up to 100 μm wide; terminal phialides
in adpressed whorls of 2–6, (6.6–)7.4–12.7(–14.4) long, (1.9–)2.0–
2.7(–3.2) wide at base, (2.8–)2.9–3.6(–3.9) at widest point, 0.8–
1.1(–1.2) wide near aperture (n = 80), ask-shaped or cylindrical,
generally with widest point in the lower third, slightly tapering
toward the apex; intercalary phialides below solitary terminal
phialides, in whorls, sometimes below whorls of terminal phialides,
(4.6–)5.4–11.0(–12.4) × (2.2–)2.5–3.5(–3.7) μm (av. 7.8 × 3.0 μm,
n = 60); subterminally formed lateral pegs 1.5–3.5 μm long (n =
60). Conidia aseptate, hyaline, smooth, ellipsoidal to subglobose,
slightly curved, sometimes with a laterally displaced hilum, broadly
rounded at the end, (3.6–)3.9–4.9(–5.5) × (2.5–)2.6–3.2(–3.7) μm
(av. = 4.3 × 2.9 μm, n = 150), arranged in imbricate chains.
Culture characteristics: Colony on OA attaining 31–32 mm after 7
d at 25 °C in darkness, at, with entire margin, aerial mycelium
moderate, nely to coarsely granular, occose to felty, whitish,
reverse concolourous. Colony on PDA attaining 24–26 mm diam,
at, with entire margin, aerial mycelium moderate, felty, whitish,
reverse concolourous. Colony on SNA attaining 24–26 mm diam,
at, with entire margin, aerial mycelium sparsely developed, nely to
coarsely granular, occose to felty, whitish, reverse concolourous.
Additional materials examined: China, Yunnan Province, Kunming City,
Wild Duck Forest Park (25°13’N, 102°87′E, 2 100 m alt.), from soil on the
forest oor, 20 Aug. 2018, Y. Wang, culture YFCC 895; Yunnan Province,
Kunming City, Songming County, Dashao Village (25°24’N, 102°55′E, 2
697 m alt.), from Ophiocordyceps highlandensis, 25 Aug. 2018, D.-X. Tang,
culture YFCC 8591. Netherlands, Limburg Province, loampit near Tegelen,
dead stem of Equisetum hyemale, Jun. 1968, W. Gams, culture CBS 205.69;
North Holland Province, Bergen, needle of Pinus pinaster, 8 Oct. 1967, W.
Gams, culture CBS 512.67; Amsterdam, soil, collection date unknown,
J. Dijk, culture JW1015. Huis ter Heide, soil, collection date unknown, L.
Grootscholten, culture JW31018; Kapel Avezaath, soil, collection date
unknown, A. Panneman, culture JW79008. UK, England, Manchester, root
of Avena sativa, Mar. 1966, G.S. Taylor, No. 2, culture CBS 513.67; soil,
1948, E.G. Jefferys, No. 650, culture CBS 194.53; Kew Gardens, leaf litter
of Buxus sempervirens, Nov. 1967, W. Gams, culture CBS 204.69. Vietnam,
Dak Lak Province, Chu Yang Sin National Park (12°29’N, 108°43′E, 1 659 m
alt.), on a spider on the underside of a leaf, Oct. 22, 2017, collected by Y.-B.
Wang, specimen YHH 896, culture YFCC 896).
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Zhao et al.
Notes: Sesquicillium candelabrum was originally described as
Verticillium candelabrum. Gams (1968) described the new genus
Sesquicillium and introduced the combination S. candelabrum.
Type material of the species described by Bonorden (1851)
has not been preserved. Therefore, well-preserved fungarium
material of Rabenhorst No. 2148 was designated as neotype for
Verticillium candelabrum (Gams 1968). Schroers (2001) placed S.
candelabrum into Clonostachys as both genera form (i) penicillate
and sometimes dimorphic conidiophores, (ii) intercalary phialides
(although less commonly in Clonostachys), and (iii) conidia with a
laterally displaced hilum and arranged in imbricate chains. In our
study, based on the phylogenetic analyses, both genera cluster in
closely related, however, separate sister clades (Fig. 1). Therefore,
S. candelabrum is resurrected here. Isolate CBS 119045, which
was collected near the type locality, is herewith designed as ex-
epitype, and the specimen CBS H-25178 as epitype of Verticillium
candelabrum. In addition, according to the phylogenetic analyses,
C. chuyangsinensis (Wang et al. 2023) is conspecic with S.
candelabrum and included as synonym of S. candelabrum.
Fig. 8. Sesquicillium candelabrum (ex-type CBS 119045). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–H. Conidiophores. I. Conidia. Scale
bars = 10 μm.
227www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Sesquicillium essexcoheniae (Y.P. Tan et al.) L. Zhao & Crous,
comb. nov. MycoBank MB 848448. Fig. 9.
Basionym: Clonostachys essexcoheniae Y.P. Tan et al., Index of
Australian Fungi 5: 3. 2023.
Typus: Australia, Queensland, Brisbane, from soil, 14 Jul. 2022, Y.P. Tan
(holotype BRIP 75170a permanently preserved in a metabolically inactive
state).
Description based on CBS 918.97: Sexual morph unknown.
Asexual morph. Conidiophores monomorphic, penicillate, rising
from the agar surface, up to quaterverticillate, branches typically
divergent, phialides divergent or adpressed; stipes 20–70 μm
long, 2–4 μm wide at base; penicilli 30–75 μm high, to 70 μm
wide; terminal phialides (5.5–)7.2–9.8(–12.3) μm long, (1.6–)2.0–
2.5(–2.9) μm wide at base, (2.5–)2.6–3.1(–3.5) μm at widest
point, 0.8–1.2 μm wide near aperture (n = 80), in whorls of up to
ve, ask-shaped, slightly tapering toward the apex; intercalary
phialides common, subapically formed lateral pegs to 3 μm long.
Conidia aseptate, hyaline, smooth, ellipsoid to subglobose,
slightly curved, with a slightly visible, typically laterally displaced
hilum, broadly rounded at the end, (3.9–)4.3–5.6(–5.8) × (2.1–)
Fig. 9. Sesquicillium essexcoheniae (CBS 918.97). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia. Scale bars
= 10 μm.
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Zhao et al.
2.3–2.8(–3.0) μm (av. = 4.8 × 2.5 μm, n = 110), arranged in
imbricate chains.
Culture characteristics: Colonies on OA reaching 27–28 mm
diam after 7 d at 25 °C in darkness, at, with entire margin, aerial
mycelium moderate, nely to coarsely granular, occose to felty,
whitish, reverse concolourous. Colonies on PDA reaching 25–29
mm, at, with crenate margin, aerial mycelium abundant, nely
granular, felty to cottony, whitish, reverse concolourous. Colonies
on SNA reaching 26–30 mm, at, with entire margin, aerial
mycelium scanty, nely granular, dirty white, reverse concolourous.
Additional material examined: USA, Puerto Rico, Caribbean National
Forest, Luquillo Mts., Big Tree Trail, from Sphaeriales, 23 Feb. 1996, G.J.
Samuels & H.J. Schroers, 101, culture CBS 918.97.
Notes: Sesquicillium essexcoheniae was described as C.
essexcoheniae based on ITS sequence data (holotype BRIP
75170a), but without proper morphological illustrations and
morphological characters (Tan & Shivas 2023). Accordingly, the
identication of strain CBS 918.97 is based on a direct comparison
of ITS sequences. Both sequences differ from each other by two
single nucleotide indels. Phylogenetically, it is a member of the
genus Sesquicillium. Multi-locus sequences deriving from CBS
918.97 places S. essexcoheniae in a statistically supported sister-
group relationship with S. rossmaniae and S. neerlandicum (Fig. 1).
Schroers (2001) included CBS 918.97 under S. candelabrum (as C.
candelabrum) although the sequences of S. essexcoheniae (CBS
918.97) and S. candelabrum (CBS 119045) differ considerably
(ITS: 91.6 % identity, with 37 bp differences; LSU: 98.6 %, 11 bp;
RPB2: 84.9 %, 112 bp; and TEF1: 95.3 %, 40 bp).
Sesquicillium intermediophialidicum L. Zhao & Crous, sp. nov.
MycoBank MB 848449. Fig. 10.
Etymology: Name refers to the whorls of intercalary phialides
regularly occurring below solitary terminal phialides.
Typus: Cuba, unknown substrate, 19 Mar. 1996, R.F. Castañeda (holotype
designated here CBS H-25133, ex-type living culture CBS 685.96).
Sexual morph unknown. Asexual morph. Conidiophores
monomorphic, penicillate, scattered on the agar surface or arising
from strands of aerial hyphae, up to ter-verticillate, branches and
phialides adpressed or somewhat divergent; stipes up to 300 μm
long, 2.0–3.5 μm wide at base; penicilli up to 80 μm high, to 40 μm
diam at widest point; terminal phialides adpressed, in whorls of up to
ve, straight to slightly curved, ask-shaped, slightly tapering in the
upper part, with or without a visible collarette, (5.8–)6.5–9.8(–11.5)
μm long, (1.8–)2.0–2.8(–3.2) μm wide at base, (2.4–)2.8–3.4(–3.9)
μm at widest point, 1.0–1.3(–1.4) μm wide near aperture (n = 70);
intercalary phialides regularly occurring below solitary terminal
phialides, in whorls, sometimes in whorls together with terminal
phialides, sometimes in chains of two, cylindrical, 5.6–11.5 × 2.2–
3.8 μm wide; lateral pegs formed subapically, to 2 μm long. Conidia
aseptate, ellipsoidal to oblong-ellipsoidal, hyaline, smooth-walled,
almost straight or one side straight and the other slightly curved,
with a rather rounded distal end, with a median or slightly laterally
displaced hilum, (5.2–)6.0–8.7(–10.0) × (1.8–)2.0–2.4(–2.5) μm
(av. = 7.3 × 2.2 μm, n = 170), arranged in imbricate chains.
Culture characteristics: Colonies on OA reaching 28–30 mm diam
after 7 d in darkness at 25 °C, at, with entire margin, membranous
without aerial mycelium, whitish, reverse concolourous. Colonies
on PDA reaching 26–29 mm diam, at, with entire margin, aerial
mycelium sparsely developed, nely felty, dirty white, reverse
whitish. Colonies on SNA reaching 16–18 mm diam, at, with
dendritic margin, membranous without aerial mycelia, white,
reverse concolourous.
Notes: Phylogenetically, S. intermediophialidicum is closely
related to S. saulense, S. spinulosisporum and S. phyllophilum
(Fig. 1). Conidial size ranges distinguish the three species from
one another. Conidia are (5.2–)6.0–8.7(–10.0) × (1.8–)2.0–2.4(–
2.5) μm in S. intermediophialidicum, 8.5–11.0(–12.0) × 3.5–4.0
μm in S. saulense (Lechat et al. 2019) and 4.5–6.5 × 3.5–4.0
μm in S. spinulosisporum (Lechat & Fournier 2018). In addition,
S. intermediophialidicum (stipes up to 300 μm long) can be
distinguished from S. saulense (15–30 μm) and S. spinulosisporum
(11–35 μm) by its longer stipes. Schroers (2001) placed the strains
of S. intermediophialidicum (CBS 685.96) under S. phyllophilum
(as C. phyllophila).
Sesquicillium lasiacidis (Samuels) L. Zhao, Crous & Schroers,
comb. nov. MycoBank MB 848454. Fig. 11.
Basionym: Nectria lasiacidis Samuels, Mem. New York Bot. Gard.
49: 273. 1989.
Synonyms: Bionectria lasiacidis (Samuels) Schroers, Stud. Mycol.
46: 187. 2001.
Clonostachys lasiacidis Schroers, Stud. Mycol. 46: 187. 2001.
Typus: French Guiana, ca. 3 h walk W of Marouini River, toward Roche
Koutou, 02°55’N, 54°03’W, 150–350 m alt., on dead culms of Lasiacis
ligulata, 18 Aug. 1987, G.J. Samuels, G.J.S. 5864; G.J.S. isolate 87-149
(isotype CBS H-7411; culture ex-type CBS 179.88).
Descriptions and illustrations: Samuels (1989), Schroers (2001).
Additional materials examined: Germany, root-associated soil using
Hordeum vulgare as bait, 22 May 2018, J.G. Maciá-Vicente, culture CBS
147133. Netherlands, Pinus nigra var. austriaca, root collar, unknown
date and collector, culture CBS 190.38; wheat eld soil, collection date
unknown, J.H. van Emden, specimen CBS H-18223, culture CBS 504.67;
Eindhoven, soil, 2017, T. Tuinier, culture JW235005; Drenthe, soil, 2019,
L. Jurjens, S. Bilstra & J. van Hoorn, culture NL19-085003; Drenthe, soil,
2019, L. Jurjens, S. Bilstra & J. van Hoorn, culture NL19-085006; Drenthe,
soil, 15 Nov. 2019, N. Schoon & S. Krol, culture NL19-086015; Drenthe,
soil, 2019, S. Schabel & M. Geerisma, culture NL19-089008.
Notes: Based on the phylogenetic analyses, S. lasiacidis has a close
phylogenetic afnity to S. candelabrum (Fig. 1). Morphologically,
S. lasiacidis differs from S. candelabrum in producing longer and
narrower conidia, (5.6–)6.4–7.6(–8.2) × (1.8–)2.2–2.8(–3.2) μm
vs (3.6–)3.9–4.9(–5.5) × (2.5–)2.6–3.2(–3.7) μm (Schroers 2001).
Although phylogenetically closely related, isolate CBS 504.67,
which produces shorter conidia, (3.2–)3.6–4.5 µm, may represent
another phylogenetic species.
Sesquicillium neerlandicum L. Zhao & Crous, sp. nov. MycoBank
MB 848455. Fig. 12.
Etymology: Named after the Netherlands where the strains of this
species were isolated in the context of a Dutch citizen science project
of the Westerdijk Institute and Utrecht University, in collaboration with
various schools, sampling garden soils in urban areas.
229www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Typus: Netherlands, Gelderland Province, Ravenswaaij, soil, Mar. 2017,
L. & N. de Klijne (holotype designated here CBS H-25136, ex-type living
culture CBS 148203 = JW 17023).
Sexual morph unknown. Asexual morph. Conidiophores
monomorphic, scattered on the agar surface or arising from strands
of aerial hyphae, penicillate, branches divergent or adpressed, bi-
to quaterverticillate; stipes up to 80 μm long, to 2.4–4.4 μm wide
at base, penicilli up to 100 μm high and 100 μm wide; terminal
phialides in adpressed whorls of up to ve, straight to slightly
curved, narrowly ask-shaped, slightly tapering in the upper part,
(5.4–)6.5–9.7(–12.7) μm long, 1.9–2.4 μm wide at base, 2.4–3.4
μm at widest point, 0.8–1.2 μm wide near apex (n = 38); intercalary
phialides common, solitary or in pairs, subterminally formed lateral
pegs up to 7 μm long. Conidia hyaline, aseptate, ellipsoidal to
cylindrical, almost straight, without a laterally displaced hilum, with
rounded distal end, (4.2–)4.5–5.6(–6.0) × (2.5–)2.7–3.2(–3.5) μm
(av. = 5.1 × 3.0 μm, n = 150), arranged in imbricate chains.
Fig. 10. Sesquicillium intermediophialidicum (ex-type CBS 685.96). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–H. Conidiophores. I, J.
Conidia. Scale bars: E = 50 μm; F–J = 10 μm.
230
Zhao et al.
Culture characteristics: Colonies on OA reaching 24–27 mm diam
after 7 d at 25 °C in darkness, at, with crenate margin, aerial
mycelium moderate, felty, nely granular, dirty white, reverse
concolourous. Colonies on PDA reaching 24–26 mm diam, at, with
crenate margin, aerial mycelium abundant, felty to cottony, nely to
coarsely granular, whitish, reverse concolourous. Colonies on SNA
reaching 23–25 mm diam, at, with crenate margin, aerial mycelium
sparse, nely to coarsely granular, whitish, reverse concolourous.
Additional materials examined: Netherlands, Gelderland Province, Kapel
Avezaath, soil, Mar. 2017, A. Panneman, CBS 148215 = JW79004;
Limburg Province, Ell, soil, Mar. 2017, K. Brennand, culture CBS 148213
= JW53028; North Holland Province, Hilversum, soil, Mar. 2017, H., J., A.
& J. Bezemer, culture CBS 148201 = JW135005; South Holland Province,
Hillegom, soil, Mar. 2017, M. & L. Fleur, culture CBS 148214 = JW71013;
Utrecht Province, Nieuwegein, soil, Mar. 2017, J. Schmidt, culture CBS
148212 = JW45022; Utrecht Province, Utrecht, soil, Mar. 2017, G.
Bleijlevens, culture CBS 148202 = JW143015; Utrecht, soil, Mar. 2017, R.
van den Brink, culture CBS 148209 = JW263008; Utrecht, soil, Mar. 2017,
R. van den Brink, culture CBS 148210 = JW263012.
Notes: Sesquicillium neerlandicum was isolated from Dutch
garden soil. Its species clade and phylogenetic relatedness to S.
Fig. 11. Sesquicillium lasiacidis (CBS 504.67). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia. Scale bars = 10
μm.
231www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
rossmaniae and S. essexcoheniae is statistically strongly supported
(Fig. 1; 100 % / 100 % / 1). It differs from S. rossmaniae in the
production of longer stipes (up to 80 μm long in S. neerlandicum vs
40 μm long in S. rossmaniae) and higher and wider penicilli (100
μm high and 100 μm wide in S. neerlandicum vs 50 μm high and
60 μm wide in S. rossmaniae). Sesquicillium neerlandicum differs
from S. essexcoheniae in higher and wider penicilli (100 μm high
and wide in S. neerlandicum, 30–75 μm high and 70 μm wide in S.
essexcoheniae).
Sesquicillium phyllophilum (Schroers) L. Zhao, Crous &
Schroers, comb. nov. MycoBank MB 848456.
Basionym: Clonostachys phyllophila Schroers, Stud. Mycol. 46:
193. 2001.
Typus: France, Forêt des buis, Coudrée (Haute Savoie), Buxus forest, on
fallen leaves of Viscum album, Sep. 1996, H.-J. Schroers & T. Gräfenhan
(holotype CBS H-7945, ex-type culture CBS 921.97).
Description and illustration: Schroers (2001).
Fig. 12. Sesquicillium neerlandicum (ex-type CBS 148203). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia.
Scale bars: E, F = 50 μm; G–I = 10 μm.
232
Zhao et al.
Additional material examined: Japan, Tokyo, Shinjuku Gyoen Garden,
Sep. 1983, W. Gams, culture CBS 662.83.
Notes: Sesquicillium phyllophilum was originally described as
Clonostachys phyllophila by Schroers (2001) from fallen leaves
of Viscum album collected in France. The present study places it
phylogenetically in the genus Sesquicillium and therefore a new
combination is proposed here (Fig. 1).
Sesquicillium rossmaniae (Schroers) L. Zhao, Crous & Schroers,
comb. nov. MycoBank MB 848457.
Basionym: Clonostachys rossmaniae Schroers, Stud. Mycol. 46:
177. 2001.
Synonym: Bionectria rossmaniae Schroers, Stud. Mycol. 46: 177.
2001.
Typus: French Guiana, Piste de Saint-Elie, km 16 on road between
Sinnamary and St. Elie, ‘Ecerex’, Orstom research area, 05°20’N,
00°53’W, on twigs of recently dead tree, Feb.–Mar. 1986, G.J. Samuels,
G.J.S. 3970, G.J.S. isolate 86-246 (isotype CBS H-7944, ex-type culture
CBS 211.93).
Description and illustration: Schroers (2001).
Additional materials examined: French Guiana, on bark of living liana,
Jan.–Mar. 1986, G.J. Samuels, culture CBS 210.93; ibid. bark of recently
dead tree, Jan.–Mar. 1986, G.J. Samuels, culture CBS 221.93.
Note: Sesquicillium rossmaniae was originally described as
Clonostachys (Bionectria) rossmaniae by Schroers (2001) and is
shown here to cluster with other species of Sesquicillium (Fig. 1).
Phylogenetically, S. rossmaniae is closely related to S. neerlandicum,
but with clearly different ITS (99.4 % identity, with 3 bp differences),
RPB2 (97.4 %, 19 bp), and TEF1 (97.5 %, 20 bp) sequences. For
morphological comparison, see notes under S. neerlandicum.
Sesquicillium saulense (Lechat & J. Fourn.) L. Zhao & Crous,
comb. nov. MycoBank MB 848458.
Basionym: Clonostachys saulensis Lechat & J. Fourn., Ascomycete.
org 11(3): 65. 2019.
Typus: French Guana, Saül, Gros Arbres trail, on dead bark of Bauhinia sp.,
22 Aug. 2018, C. Lechat, CLLG18023-A5 (holotype LIP CLLG18023-A5,
ex-type culture BRFM 2782).
Description and illustration: Lechat et al. (2019).
Notes: The culture was isolated from dead bark of Bauhinia
sp. collected from Saül in French Guiana, and described as
Clonostachys saulensis (Lechat et al. 2019). Phylogenetically, it
falls in a well-supported lineage among other species of the genus
Sesquicillium (Fig. 1).
Sesquicillium sesquicillii (Samuels) L. Zhao, Crous & Schroers,
comb. nov. MycoBank MB 848459.
Basionym: Nectria sesquicillii Samuels, Mem. New York Bot. Gard.
49: 268. 1989.
Synonyms: Bionectria sesquicillii (Samuels) Schroers, Stud. Mycol.
46: 190. 2001.
Clonostachys sesquicillii Schroers, Stud. Mycol. 46: 190. 2001.
Typus: Guyana, Cuyuni-Mazaruni Region, No. VII, Mazaruni Subregion,
No. VII-2, foothills immediately S of Mt. Ayanganna, ca. 1 km W of Pong
River, 05°28’N, 60°04’W, 550–600 m alt., on twigs and lichen, 26 Feb.
1987, G.J. Samuels, J. Pipoly & G. Gharbarran, G.J.S. 4825, G.J.S. isolate
87-23 (isotype CBS H-7413, culture ex-type CBS 180.88).
Descriptions and illustrations: Samuels (1989), Schroers (2001).
Notes: Sesquicillium sesquicillii was originally described as
Nectria sesquicillii by Samuels (1989) from twigs and a lichen.
It was subsequently transferred to Clonostachys (Schroers
2001). According to our phylogenetic inference, the ex-type of N.
sesquicillii falls in the highly supported Sesquicillium clade (Fig. 1;
97 % / 100 % / 1). Perithecia of S. sesquicillii are formed in dense
groups on erumpent stromata (Schroers 2001: g. 87a, b, d), while
the perithecia of other Sesquicillium species are formed solitarily on
supercial, thus, non-erumpent, typically reduced stromata.
Sesquicillium spinulosisporum (Lechat & J. Fourn.) L. Zhao &
Crous, comb. nov. MycoBank MB 848460.
Basionym: Clonostachys spinulosispora Lechat & J. Fourn.,
Ascomycete.org 10(4): 128. 2018.
Typus: French Guiana, Régina, nouragues natural Reserve, Inselberg
camp, primary rainforest, on aerial, dead palm leaf of Astrocaryum vulgare
(Arecaceae), 16 Jun. 2012, C. Lechat [holotype CLLG12001 (LIP), ex-
type culture CBS 133762].
Description and illustration: Lechat & Fournier (2018).
Notes: The type culture was isolated from a dead palm leaf
of Astrocaryum vulgare (Arecaceae) collected from Régina in
French Guiana and originally described as C. spinulosispora. The
present study places it phylogenetically in the genus Sesquicillium,
being closely related to S. phyllophilum (Fig. 1). Sesquicillium
spinulosisporum can be distinguished from S. phyllophilum based on
the absence of intercalary phialides and its shorter conidia (4.5–6.5
μm in S. spinulosisporum vs (5.4–)5.8–7(–8.8) μm in S. phyllophilum).
Sesquicillium symmetricum L. Zhao & Crous, sp. nov. MycoBank
MB 848461. Fig. 13.
Etymology: Name refers to the symmetrical conidia produced by
this species.
Typus: Colombia, Dep. de Meta, Municipio de Villavicencio, 25 km from
Villavicencio to Acacías, 550 m alt., agricultural soil, 18 Feb. 1978, O.
Rangel (holotype designated here CBS H-25134, ex-type living culture
CBS 124.79).
Sexual morph unknown. Asexual morph. Conidiophores monomorphic,
penicillate, arising from the agar surface or aerial mycelium; stipe 20–
130 μm long, 1.9–3.9 μm wide at base; penicilli up to 120 μm high,
90 μm wide; terminal phialides in whorls of up to six, adpressed or
divergent at acute angles, narrowly ask-shaped, (8.7–)10.4–21.6(–
22.6) μm long, (1.2–)1.6–2.5(–2.8) μm wide at base, (2.0–)2.5–3.6(–
4.0) μm at widest point, (0.8–)0.9–1.2(–1.3) μm wide near aperture (n =
60); intercalary phialides present, subterminally formed lateral pegs 3–5
μm long. Conidia hyaline, ellipsoidal to obovoid to somewhat clavate,
symmetrical, distally broadly rounded, without laterally displaced hilum,
(5.5–)5.8–8.9(–9.8) × (2.2–)2.3–3.3(–3.9) μm (av. = 7.1 × 2.7 μm, n =
100), arranged in linear chains.
Culture characteristics: Colony on OA attaining 18–20 mm after 7
d at 25 °C in darkness, at, with entire margin, aerial mycelium
sparsely developed, nely felty, dirty white, reverse whitish.
Colony on PDA attaining 20–21 mm, at, with entire margin, aerial
233www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
mycelium moderate, felty, whitish, reverse concolourous. Colony on
SNA attaining 20–21 mm, at, with entire margin, aerial mycelium
moderate, felty, dirty white, reverse concolourous.
Additional material examined: Colombia, Dep. de Meta, Municipio de
Villavicencio, 25 km from Villavicencio to Acacías, 550 m alt., maize-eld
soil, collection and isolation date unknown, O. Rangel, culture CBS 485.78.
Notes: Sesquicillium symmetricum is closely related to S.
candelabrum, S. essexcoheniae, S. lasiacidis, S. neerlandicum,
and S. rossmaniae (Fig. 1). Sesquicillium symmetricum can
be morphologically distinguished from S. candelabrum, S.
essexcoheniae, S. rossmaniae and S. neerlandicum based
on its longer conidia [(5.5–)5.8–8.9(–9.8) μm long, av. = 7.1
μm, in S. symmetricum; (3.6–)3.9–4.9(–5.5) μm av. = 4.3 μm in
S. candelabrum; (3.9–)4.3–5.6(–5.8) μm, av. = 4.84 μm in S.
essexcoheniae; (4.2–)4.6–5.4(–6.6) μm, av. = 5 μm in S. rossmaniae;
[(4.6–)5.0–6.0 μm, av. = 5.05 μm in S. neerlandicum]. Sesquicillium
symmetricum differs from S. lasiacidis in having longer phialides
[(8.7–)10.4–21.6(–22.6) μm in S. symmetricum, 8.4–13.8 μm in S.
Fig. 13. Sesquicillium symmetricum (ex-type CBS 124.79). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–H. Conidiophores. I, J. Conidia in
linear chains. Scale bars = 10 μm.
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Zhao et al.
lasiacidis]. Due to the symmetrical shape of conidia and central
placement of their hila, S. symmetricum produces not imbricate but
linear conidial chains.
Sesquicillium tornatum (Höhn.) Schroers, comb. nov. MycoBank
MB 848462.
Basionym: Pseudonectria tornata Höhn., Sitzungsber. Akad. Wiss.
Wien, Math.-Naturwiss, Kl., Abt. 1. 118: 1470. 1909.
Synonyms: Bionectria tornata (Höhn.) Schroers, Stud. Mycol. 46:
184. 2001.
Clonostachys tornata (Höhn.) Rossman et al., Stud. Mycol. 80:
242. 2015.
Sesquicillium asymmetricum Samuels, Mem. New York Bot. Gard.
49: 276. 1989.
Clonostachys asymmetrica (Samuels) Schroers, Stud. Mycol. 46:
184. 2001.
Nectria sesquiphialis Samuels, Mem. New York Bot. Gard. 49: 276.
1989.
Typus: Type for Pseudonectria tornata: Indonesia, Java, Tjibodas, on
decaying leaves of Pandanus sp. (no holotype specimen recorded). Type
for Nectria sesquiphialis and Sesquicillium asymmetricum: Venezuela,
Edo, Bolivar, 110–111 km S of El Dorado on road between El Dorado and
Sta Elena, on leaf of Zingiberaceae, 6 Aug 1972, R.F. Cain, G.J. Samuels
& C. Blanco [holotype Dumont-VE 7184 (VEN), isotype NY, dried culture
of C.T.R. isolate 72-193 (= ATCC 66892), derived from ascospores of
Dumont-VE 7184, type of N. sesquiphialis, and led with it (NY)].
Descriptions and illustrations: Von Höhnel (1909), Samuels (1989),
Schroers (2001).
Notes: Pseudonectria tornata and Nectria sesquiphialis, including
the asexual morph-typied S. asymmetricum, were synonymised
based on sexual and asexual morph characters encountered in the
type of N. sesquiphialis (Dumont-VE 7184) and P. tornata (FH no.
2899) (Schroers 2001: gs 81 vs 82) and sexual morph characters
described by von Höhnel (1909). Asexual morph characters
consisted of conidiophores showing intercalary phialides below
solitary terminal phialides and size range and shape of conidia;
sexual morph characters, the habit of supercially formed perithecia
on decaying leaves of Pandanus sp., perithecial walls composed of
two regions, and 1-septate ascospores (9–12.8 × 2–2 μm for P.
tornata; 10.8–20.8 × 2–4 μm for N. sesquiphialis) (Schroers 2001).
Accordingly, morphological character interpretations were used
for linking P. tornata and N. sesquiphialis to Bionectria subgenus
Epiphloe and combining P. tornata into Bionectria and the same
arguments are here adopted for combining the species into
Sesquicillium. Obtaining and analysing phylogenetic marker genes
from strain ATCC 66892 (= C.T.R. isolate 72-193, ex-type strain of
N. sesquiphialis) is therefore required to further support the here
suggested taxonomy.
Clonostachys Corda, Pracht-Fl. Eur. Schimmelbild.: 31. 1839.
Type: Clonostachys araucaria Corda
Sexual morph. Stromata typically present, well developed if
erumpent through bark, reduced if formed on associated fungus
hosts, typically made of pseudoparenchymatous cells, rarely
entirely supercial. Perithecia orange, yellowish orange, brownish
orange, rarely brownish, KOH-, sometimes becoming paler in lactic
acid, crowded in often large groups on stromata, rarely solitary,
globose or somewhat higher than wide, sometimes obovoid,
smooth or warted. Perithecial wall of two or three regions, rarely a
single region, texture prosenchymatous or pseudoparenchymatous.
Perithecial surface smooth, rough, or warted. Perithecial warts, if
present, strongest developed in the upper part of the perithecia,
irregularly scattered or radiating from the perithecial apex
downwards. Asci 8-spored, apically rounded or at, sometimes with
prominent edges, with subapically thickened walls and apical ring.
Ascospores typically 1-septate, coarsely or nely warted or striate,
hyaline, ellipsoidal, tapering slightly towards their ends, sometimes
with warts arranged in striae, rarely rough or smooth. Asexual
morph. Sporodochia often on erumpent stroma when formed on
the natural substratum or cushion-shaped in culture and without
stroma; synnemata present in some species. Conidiophores
frequently dimorphic (primary and secondary conidiophores),
sometimes monomorphic. Primary conidiophores mononematous,
early-formed, verticillum-like or narrowly penicillate. Secondary
conidiophores mononematous, later-formed, penicillate, often
aggregating into cushin-shaped, more or less distinct sporodochia,
less commonly synnematous; stipes typically arising from
submerged or aerial hypha or from aerial hyphal fascicles, ropes
or strands; penicilli monoverticillate or bi- to more-level verticillate).
Conidiogenous cells phialidic. Phialides on primary conidiophores
almost cylindrical, slightly and gradually tapering; phialides on
secondary conidiophores narrowly ask-shaped, widest in the
lower third or middle, and slightly and continuously tapering
toward tip or, in some species, intercalarly formed below terminal
phialides and with subapically formed conidiogenous, lateral pegs.
Conidiophores rarely with setae. Conidia aseptate, ellipsoid to
subfusoid, typically with one more attened side and a laterally
displaced hilum resulting in a somewhat kidney-like appearance,
hyaline or greenish hyaline, held in watery droplets or heads when
formed on primary conidiophores or imbricate chains when formed
on secondary conidiophores or sporodochia, conidial masses
collapsing in either off-white, orange or greenish slimy masses.
Notes: Clonostachys araucaria, the type species of Clonostachys,
was described as producing white colonies “on forest soil”,
incubated at around 17 °C, and oblong-ellipsoidal, obviously
hyaline, and imbricately arranged conidia that adhere in columns
and are formed by a penicillate conidiophore (Corda 1839).
Although type material is not available, it is clear that Corda’s
drawing illustrates a secondary conidiophore formed by many
Clonostachys species. Samuels (1988a) linked members of the
Nectria ochroleuca group to Clonostachys, and Schroers et al.
(1999b) classied Clonostachys as the asexual morph of Bionectria.
Thereafter, Clonostachys binominals were also provided for sexual
morph-typied Bionectria names to allow usage of Clonostachys
binomials for all species considered at that time (Schroers 2001),
and Clonostachys became the name selected in the single name
system (Rossman et al. 2013).
Dimorphic conidiophores, described for Clonostachys rosea
(as Gliocladidum roseum) for the rst time by Bainier (1905,
1907), i.e., primary and secondary conidiophores, slightly curved
conidia showing laterally displaced hila (i.e., a scar at the base of
a conidium), perithecial walls consisting of three regions, warted
ascospores and stromata erumpent through bark, characterise
a core group of species of subgenus Bionectria (Fig. 1 and 2,
terminal clade including C. reniformis). The group is further
subdivided morphologically based on characteristics of both types
of conidiophores, bearing either diverging or adpressed branches
or phialides, while further differentiations of species based on
morphological characters are difcult (Moreira et al. 2016). Species
235www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
of this core group show rather homogenous character patterns,
however, two of its species do not produce dimorphic conidiophores
but sporodochia only. This core group also includes C. rosea and
C. solani, for which two forms were accepted based on either light
or (partly dark) green conidial masses (Schroers 2001) and named
by using the reduced species epithets from Gliocladium catenulata
Gilman & Abbott or, respectively, G. nigrovirens van Beyma. We
recommend using the form-based system in situations where users
of names wish to emphasise the green phenotype trait of their
strains. However, the use of the binominals C. rosea or C. solani is
equally correct (Fig. 2, Supplementary Fig. S9).
Our phylogenetic concept of subgenus Bionectria presented
here mirrors the earlier provided morphocentric concept
(Schroers 2001) (Fig. 1), although Fig. 2 suggests that subgenus
Astromata may have evolved from within subgenus Bionectria.
However, the phylogenetic analysis also conrms relatedness
of morphologically diverse taxa including, e.g., C. samuelsii
forming sporodochia only and symmetrical conidia, C. ralfsii with
a differing perithecial wall anatomy, outstandingly large conidia
and synnematous conidiomata, and several others including C.
compactiuscula, C. bambusae, and C. divergens. Even after
excluding the vast majority of sesquicillium-like species, the
phylogenetic analyses furthermore support a morphologically
broad concept for Clonostachys with diversifying character
patterns. These include, e.g., (i) reduced, non-erumpent
stromata often on plant associated fungal hosts and sporodochia
forming green conidial masses (subgen. Astromata), (ii) striate
ascospores, perithecial walls consisting of two regions, with cells
not integrating with cells of stromata (subgenus Zebrinella), (iii)
penicilli with numerous intercalary phialides indistinguishable
from those seen in Sesquicillium (C. setosa, C. bambusae),
(iv) perithecia consisting of a single region only, supercially
formed not on woody substrata but on a dicotyledonous leaf [C.
vesiculosa (Luo & Zhuang 2010) that Schroers (2001) would most
likely have classied in subgen. Uniparietina, now Sesquicillium],
and (v) curved and smooth ascospores, cup-shaped conidiomata
combined with symmetrical conidia, etc. (subgenus Myronectria).
Based on phylogenetic analysis and morphological characters
employed in the present study, the genus Clonostachys has 49
known and 19 new species, distributed among four subgenera:
Astromata (six species, four known and two new species), Bionectria
(40 species, 32 known and eight new species), Myronectria (two
species, one known and one new species) and Zebrinella (18
species, 10 known and eight new species). Additionally, there
are two known species, namely C. vesiculosa (morphologically
comparable with subgenus Uniparietina, now Sesquicillium) and
C. setosa (sesquicillium-like), which do not belong to any of the
subgenera.
Clonostachys aurantiaca L. Zhao & Crous, sp. nov. MycoBank
MB 848477. Fig. 14.
Etymology: From Latin aurantiacus, meaning orange. Referring to
the production of orange ascomata.
Typus: Cameroon, Korup National Park, bark of recently fallen tree tropical
wet forest, collection and isolation date unknown, G.J. Samuels (holotype
designated here CBS H-25140, ex-type living culture CBS 124757).
Sexual morph produced in culture on OA. Stroma generally well-
developed, erumpent, bearing perithecia, cells prosenchymatous,
densely hyphal. Perithecia solitary or crowded in groups of to 10,
sometimes overgrown by mycelium, globose to subglobose, 140–
240 μm diam, yellowish to brownish orange, slightly papillate, scaly
to warty. Perithecial warts whitish to pale orange, up to 70 μm high,
cells subglobose to globose, of the same type as the cells of the
outer perithecial wall region, (6.0–)7.0–13.0(–15.0) × (4.5–)6.5–
11.5–12(–14.5) μm (n = 100), walls generally evenly thickened to 1
μm thick. Perithecial wall 25–50 μm thick composed of two regions;
outer region 12–27 μm or 1–3 cells thick, cells merging with the
cells of the warts, angular to subglobose, (5.5–)6.5–13.5(–15.0) ×
(4.0–)5.0–9.5(–12.0) μm (n = 100), with uniformly thickened walls
around 1.5 μm thick; sometimes with vacuoles; middle region
lacking; inner region 10–20 thick. Asci 8-spored, clavate, 39–60
× 7–16 μm (n = 35), apex at, edges rounded, ring clearly visible.
Ascospores 1-septate, striate, striae parallel, constriction at the
median septum frequently observed in discharged ascospores,
ellipsoid, (8.5–)9.0–12.0(–12.5) × 4.0–5.5(–6) μm (n = 80). Asexual
morph not observed.
Culture characteristics: Colonies on OA reaching 29–33 mm
diam after 7 d at 25 °C in darkness, with crenate margin,
aerial mycelium moderate, felty to cottony, pale yellow, reverse
concolourous. Colonies on PDA reaching 29–34 mm diam, with
crenate margin, aerial mycelium moderate, felty, white yellowish,
reverse pale yellow. Colonies on SNA reaching 26–31 mm diam,
with crenate margin, aerial mycelium sparse, whitish, reverse
concolourous.
Notes: Phylogenetically, Clonostachys aurantiaca is closely related
to C. fusca (Figs 1, 2), but with clearly different ITS (98.0 % identity,
with 11 bp differences), RPB2 (98.9 %, 8 bp), and TEF1 (98.2 %,
15 bp) sequences. For morphological comparison with C. fusca,
see notes under C. fusca. Observed morphological characters,
especially cell morphology of stroma not integrating with cells of
the perithecial wall, perithecial walls consisting of two regions,
perithecial warts consisting of cells with evenly thickened walls, and
striate ascospores, support its classication in subgen. Zebrinella.
Clonostachys australiana L. Zhao & Crous, sp. nov. MycoBank
MB 848478. Fig. 15.
Etymology: Named after the country where the fungus was
collected, Australia.
Typus: Australia, New South Wales, Blue Mountains Nat. Park, bark of
recently dead tree, Aug. 1999, G.J. Samuels (holotype designated here
CBS H-25138, ex-type living culture CBS 102421).
Sexual morph unknown. Asexual morph. Conidiophores dimorphic.
Primary conidiophores sparsely branched, penicillate, or
acremonium-like, arising from the agar surface or strands of aerial
mycelium; stipe 30–110 μm long, 2.6–4.7 μm wide at base; penicilli
40–70 μm high; terminal phialides in apical whorls of up to ve,
straight, almost cylindrical, slightly tapering towards the tip, without
or with a somewhat visible collarette, (12.4–) 16.6–30.8(–35.8) μm
long, (1.4–)1.5–2.7(–3.1) μm wide at base, (1.0–)1.2–2.0(–2.2) μm
wide near aperture (n = 60). Secondary conidiophores, penicillate,
mono- to terverticillate, branches divergent or adpressed; stipe 20–
60 μm long, 2.6–4.4 μm wide; penicilli 30–65 μm high, up to 65 μm
wide; terminal phialides in loose whorls of up to nine, adpressed,
straight or slightly curved, ask-shaped, widest in the lower third,
slightly tapering in the upper part towards the tip, without a collarette,
(6.5–)7.6–16.7(–23.3) μm long, (1.5–)1.6–2.7(–3.2) μm wide at
236
Zhao et al.
Fig. 14. Clonostachys aurantiaca (ex-type CBS 124757). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Perithecia. F. Section through
perithecium. G. Ostiole. H. Lateral perithecial wall showing two regions. I. Perithecial base and stroma or stroma below a perithecium. J. Immature asci with
slightly visible ring. K. Mature asci. L, M. Discharged ascospores in optical section (L) and surface view (M). Scale bars: F = 50 μm; G–M = 10 μm.
237www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
base, (2.2–)2.4–3.3(–3.9) μm at widest point, (0.9–)1.0–1.4(–1.5)
μm wide near aperture (n = 90); intercalary phialides sometimes
observed, bearing several or one terminal phialides, cylindrical,
8–14 × 2–4 μm wide, lateral conidiogenous pegs to 5.5 μm long.
Conidia aseptate, hyaline, smooth, ellipsoid, broadly rounded, with
a median or invisible hilum, (4.5–)5.4–7.7(–9.7) × (2.5–)2.7–3.6(–
4.1) μm (av. 6.3 × 3.1 μm, n = 150), arranged in imbricate chains
that may collapse into slimy masses.
Culture characteristics: Colonies on OA reaching 21–23 mm diam
after 7 d at 25 °C in darkness, with entire margin, aerial mycelium
moderate, felty to cottony, whitish, reverse concolourous. Colonies
on PDA reaching 21–24 mm diam, with entire margin, aerial
mycelium moderate, felty to cottony, whitish, reverse concolourous.
Colonies on SNA reaching 19–20 mm diam, with entire margin,
aerial mycelium moderate in the centre, sparse at periphery, felty,
whitish, reverse concolourous.
Fig. 15. Clonostachys australiana (ex-type CBS 102421). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G, H.
Secondary conidiophores. I, J. Conidia. Black arrows indicate intercalary phialides. Scale bars = 10 μm.
238
Zhao et al.
Additional material examined: Australia, New South Wales, Blue
Mountains Nat. Park, bark of recently dead tree, Aug. 1999, G.J. Samuels,
culture CBS 102423.
Notes: Phylogenetically, C. australiana is closely related to C.
grammicosporopsis (Figs 1, 2), but differs genetically in ITS
(98.1 % identity, with 9 bp differences), LSU (98.9 %, 9 bp), and
TEF1 (96.7 %, 26 bp) sequences. Morphologically, C. australiana
differs from C. grammicosporopsis in producing smaller conidia
[(4.5–)5.4–7.7(–9.7) × (2.5–)2.7–3.6(–4.1) μm vs (4.8–)6.6–8.4(–
11.6) × (2.2–)3.0–3.6(–4.6) μm] (Schroers 2001). Morphology of
conidiophores and conidia are similar to other species in subgen.
Zebrinella.
Clonostachys bambusae L. Zhao & Crous, sp. nov. MycoBank
MB 848479. Fig. 16.
Etymology: Referring to the host, bamboo, from which the type of
this species was collected.
Fig. 16. Clonostachys bambusae (ex-type CBS 139411). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–H. Conidiophores. I. Conidia. Black
arrows indicate intercalary phialides. Scale bars: D, E = 50 μm; F–I = 10 μm.
239www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Typus: Thailand, Chiang Mai Province, Mae Teng Distr. Highway 1095
at 22 km marker, 750 m alt., dead leaf of bamboo, Aug. 2014, W. Gams
(holotype designated here CBS H-25151, ex-type culture CBS 139411).
Sexual morph unknown. Asexual morph. Conidiophores
monomorphic, penicillate, scattered on the agar surface or arising
from strands of aerial hyphae, bi- to quaterverticillate, branches
somewhat divergent to adpressed; stipes 35–140 μm long, to 4.6
μm wide at base; penicilli up to 100 μm high, 90 μm wide; terminal
phialides almost adpressed in whorls of up to 5, narrowly ask-
shaped, slightly tapering in the upper part, with a minute collarette,
(7.3–)7.7–12.0(–12.6) μm long, (1.2–)1.7–2.6(–2.7) μm wide at
base, 2.3–3.0(–3.4) μm at widest point, 0.9–1.3(–1.5) μm wide
near aperture (n = 80); intercalary phialides rather frequent, formed
solitarily below whorls of terminal phialides; almost cylindrical,
6.0–10.0 × 2.0–3.2 μm, lateral conidiogenous peg 2.0–5.0 μm
long. Conidia aseptate, hyaline, smooth, oblong-ellipsoid, almost
straight, but with a laterally displaced hilum, broadly rounded at the
end, (5.6–)6.4–9.2(–10.3) × 1.6–2.0(–2.4) μm (av.= 7.7 ×1.9, n =
150), arranged in imbricate chains.
Culture characteristics: Colonies on OA reaching 32–36 mm diam
after 7 d at 25 °C in darkness, at, with crenate margin, aerial
mycelium moderate, felty, dirty white to pale yellow, with concentric
rings, reverse pale yellow. Colonies on PDA reaching 34–36 mm
diam, at, with crenate margin, aerial mycelium moderate, felty to
cottony, dirty white, with concentric rings, reverse concolourous.
Colonies on SNA reaching 28–31 mm diam, at, with entire margin,
aerial mycelium sparse, dirty white, reverse concolourous.
Notes: Phylogenetically, C. bambusae (CBS 139411) and
Clonostachys sp. CBS 496.90 (Figs 1, 2) form a sister clade to
the core group of subgenus Bionectria characterised by regularly
formed dimorphic conidiophores and slightly curved conidia
having a laterally displaced hilum. Monomorphic, penicillate
conidiophores, with somewhat divergent to adpressed branches
and frequent occurrence of intercalary phialides characterise
C. bambusae. The two strains differ genetically in ITS (99.2 %
identity, with 4 bp differences), LSU (99.6 %, 3 bp), RPB2 (94 %,
43 bp), TEF1 (98.5 %, 12 bp) and TUB2 (98 %, 16 bp) sequences.
Schroers (2001: g. 93a) illustrated CBS 496.90 to directly
compare conidiophores and intercalary phialides with those of C.
setosa, which also is characterised by a sesquicillium-like asexual
morph. Due to their phylogenetic afnity, Clonostachys sp., CBS
496.90 and C. setosa are to be classied in Clonostachys and not
Sesquicillium.
Clonostachys buxicola L. Zhao & Crous, sp. nov. MycoBank MB
848480. Fig. 17.
Etymology: Name refers to the host genus, from which this fungus
was isolated, Buxus.
Typus: France, Pyrénées Atlantiques, Isle de Sauveterre de Bearn,
on bark of dead Buxus sempervirens, 25 Oct. 1998, G.J. Samuels
& F. Candoussau (holotype designed here CBS H-25137; ex-type
culture CBS 102419).
Sexual morph unknown. Asexual morph. Conidiophores variable,
somewhat penicillate or typically with short conidiophores gradually
integrating into indistinct sporodochia. Sporodochia arising from
the aerial mycelium or from agar surface, appearing at rst as
distinct white pustules, with time coalescing, arranged in tufts
throughout the colony, covered with greenish grey to dark green
conidial masses. Penicillate mononematous conidiophores arising
from agar surface or aerial mycelium, with irregularly branched
penicilli. Sporodochial conidiophores irregularly penicillate, ter- to
quaterverticillate or more frequently branched; cells supporting
phialides frequently widening distally; terminal phialides in whorls
of 2–5, also singly, adpressed or divergent at acute angles, straight
or curved, cylindrical or somewhat narrowly ask-shaped, generally
slightly tapering in the upper part, without a visible collarette, (7.4–)
8.7–16.9(–21.0) μm long, (1.4–)1.6–2.4 μm wide at base, 0.9–1.2
μm wide near aperture; intercalary phialides rare, solitary, mostly
below a single terminal phialide, sometimes arising from ± square-
shaped cells, the conidiogenous peg of intercalary phialides
sometimes as long as terminal phialides. Conidia aseptate,
greenish hyaline, smooth, ovoid to ellipsoid, straight or minutely
curved, sometimes widest in the lower part, with a median or
slightly laterally displaced, not protruding, distinctly at or almost
invisible hilum, (4.6–)5.2–8.0(–8.6) × (2.3–)2.7–3.5(–3.8) μm (av. =
6.4 × 3.1 μm, n = 200), arranged in linear chains.
Culture characteristics: Colonies on OA reaching 24–26 mm
diam after 7 d at 25 °C in darkness, at, with entire margin, and
aerial mycelium sparsely developed, felty, dirty white, reverse
concolourous. Colonies on PDA reaching 25–28 mm diam, at,
with entire margin, aerial mycelium moderate, felty, white, reverse
concolourous. Colonies on SNA reaching 16–17 mm diam,
at, with irregular margin, nely granulose, felty, white, reverse
concolourous.
Notes: Based on phylogenetic analysis, C. buxicola and C. pityrodes,
both forming distinctly greenish pigmented conidial masses, were
included in a fully-supported clade (Figs 1, 2). Clonostachys
buxicola and C. pityrodes are morphologically similar, but have
clearly differing ITS (96.3 % identity, with 18 bp differences), LSU
(98.7 %, 10 bp), RPB2 (84.2 %, 119 bp), TEF1 (95.3 %, 38 bp),
and TUB2 (90.3 %, 104 bp) sequences. Both species form a
unique lineage near the root of the Clonostachys clade (Figs 1, 2).
Clonostachys pityrodes was placed in its own subgenus, subgenus
Myronectria, because of morphological characters deviating from
those of the other subgenera (Schroers 2001).
Clonostachys cylindrica L. Zhao & Crous, sp. nov. MycoBank
MB 848481. Fig. 18.
Etymology: Name refers to the cylindrical conidia produced by this
species.
Typus: Venezuela, Estación Biológica de Rancho Grande, Parque
Nacional Henry Pittie, Estado Aragua, 1 200 m alt., Nov. 1997, R.F.
Castañeda (holotype designated here CBS H-25150, ex-type living
culture CBS 101113).
Sexual morph unknown. Asexual morph. Conidiophores dimorphic.
Primary conidiophores verticillium-like, or acremonium-like, arising
from the agar surface or from aerial mycelium, with monoverticillate
or more-level-verticillate divergent phialides, sometimes with
side-branches; stipe up to 165 μm long, 2.3–4.5 μm wide at
base; penicilli up to 134 μm high; terminal phialides in whorls of
4, in lower levels also solitary, straight, cylindrical, but slightly and
continuously tapering towards the tip, with or without a minute
collarette, (10.4–)18.4–39.0(–41.7) μm long, (1.7–)1.8–2.6(–3.1)
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Zhao et al.
μm wide at base, (1.0–)1.2–1.6(–1.7) μm wide near aperture (n =
52), each producing a small, hyaline drop of conidia. Secondary
conidiophores penicillate, scattered on the agar surface or arising
from strands of aerial hyphae, up to terverticillate, branches
adpressed or somewhat divergent; stipes 41–152 μm long, to
3.2–5.0 μm wide at base; penicilli up to 93 μm high, to 71 μm diam
at widest point; terminal phialides adpressed, in whorls of up to
seven, straight to slightly curved, narrowly ask-shaped, slightly
tapering in the upper part, with or without a visible collarette, (7.7–
)8.2–12.8(–15.7) μm long, (1.6–)1.8–2.9(–3.3) μm wide at base,
(2.4–)3.0–4.0 μm at widest point, (0.9–)1.0–1.3(–1.4) μm wide near
aperture (n = 100); intercalary phialides (5.4–)6.7–10.2(–12.3) ×
(2.8–)3.0–4.0(–4.3) with to 4.5 μm long lateral pegs, below a whorl
of terminal phialides. Conidia aseptate, hyaline, smooth, cylindrical,
straight or slightly curved, with a laterally displaced or sometime
central hilum, (7.7–)8.6–11.9(–13.5) × (2.3–)2.5–3.3(–4.3) μm (av.
= 10.1 × 2.9 μm, n = 150), arranged in imbricate chains.
Culture characteristics: Colonies on OA reaching 32–34 mm diam
after 7 d at 25 °C in darkness, with entire margin, aerial mycelium
Fig. 17. Clonostachys buxicola (ex-type CBS 102419). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D. Sporodochia producing green conidial
masses on OA. E, F. Sporodochia. G. Phialides. H. Conidiophores. I, J. Conidia. Scale bars = 10 μm.
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Revising ClonostaChys and allied geneRa in BioneCtriaCeae
moderate, felty, whitish, reverse concolourous. Colonies on PDA
reaching 35–38 mm diam, with entire margin, aerial mycelium
moderate, felty to cottony, nely to coarsely granular, whitish,
reverse concolourous. Colonies on SNA reaching 31–36 mm
diam, with entire margin, aerial mycelium sparse, granular, whitish,
reverse concolourous.
Notes: Clonostachys cylindrica resembles C. compactiuscula and
C. penicillata, but these species can be distinguished based on
conidial size [(7.7–)8.6–11.9(–13.5) × (2.3–)2.5–3.3(–4.3) μm in C.
cylindrica) vs (3.9–)5.4–6.6–7.5(–12.4) × (1.5–)1.9–2.2–2.5(–3.2)
μm in C. compactiuscula and (4.5–)5.0–6.8(–7.6) × (1.6–)1.7–2.2(–
2.4) μm in C. penicillata. However, the morphologically differing
C. divergens and C. samuelsii (sporodochial conidiophores) and
C. hongkongensis and C. rogersoniana (broadly ellipsoidal to
oval conidia) are also members of the two clades. Clonostachys
cylindrica produces mononematous, dimorphic conidiophores, pale
conidial masses, and clearly longer conidia (av. = 10.1 μm), while
C. divergens (clostest phylogenetic sister lineage in Fig. 2) forms
greenish pigmented conidial masses, and less than 5 μm long
Fig. 18. Clonostachys cylindrica (ex-type CBS 101113). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G–I. Secondary
conidiophores. J. Conidia. Scale bars: E = 50 μm; D, F–J = 10 μm.
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Zhao et al.
conidia. Also the molecular sequences distinguish both species
clearly (ITS: 96.3 % identity, with 18 bp differences; LSU: 99.4 %, 5
bp; and TEF1: 97.9 %, 17 bp).
Clonostachys ellipsoidea L. Zhao & Crous, sp. nov. MycoBank
MB 848482. Fig. 19.
Etymology: Name refers to the broadly ellipsoidal conidia produced
by this fungus.
Typus: Indonesia, Java, Jogyakarta, date unknown, F.J.J. Jongeleen
(holotype designated here CBS H-25144, ex-type living culture CBS
175.76).
Sexual morph unknown. Asexual morph. Conidiophores
monomorphic, penicillate, arising from the sparse aerial mycelium,
branches divergent or somewhat divergent, mono- to biverticillate;
stipes 10–60 to μm long, to 4.3 μm wide at base; terminal phialides
in loose apical whorls of 2–6, adpressed or divergent at acute
angles, straight or curved, cylindrical and slightly tapering towards
Fig. 19. Clonostachys ellipsoidea (ex-type CBS 175.76). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–H. Conidiophores. I, J. Conidia. Scale
bars = 10 μm.
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Revising ClonostaChys and allied geneRa in BioneCtriaCeae
the tip or narrowly ask-shaped and slightly widening in the middle,
without visible collarette, (7.9–)8.5–18.9(–21.5) μm long, (1.4–)1.5–
2.4(–2.7) μm wide at base, (2.2–)2.4–3.5(–3.9) μm at widest point,
1.0–1.4 μm wide near aperture (n = 100); intercalary phialides rare,
conidiogenous pegs to 5 μm long, formed solitarily below whorls
of terminal phialides. Conidia aseptate, hyaline, smooth, broadly
ellipsoidal, rarely minutely curved, ends broadly rounded, hilum
laterally displaced, almost median or invisible, (4.2–)4.8–5.9(–
6.3) × (3.0–)3.2–3.7(–4.0) (av.= 5.3 × 3.5, n = 150), arranged in
imbricate chains that may collapse into slimy masses.
Culture characteristics: Colonies on OA reaching 40–42 mm
diam after 7 d at 25 °C in darkness, at, with entire margin,
aerial mycelium sparse, occose, whitish, reverse concolourous.
Colonies on PDA reaching 39–41 mm diam, with entire margin,
aerial mycelium moderate, felty to cottony, whitish, reverse
concolourous. Colonies on SNA reaching 38–40 mm diam, at, with
entire margin, aerial mycelium sparse, felty, nely granular, whitish,
reverse concolourous.
Additional material examined: India, Amarkantak, Shahdol, soil, Jan. 1993,
T. Okuda, No. TC 1304, culture CBS 102566.
Notes: Based on the phylogenetic analyses (Figs 1, 2), C.
ellipsoidea is closely related to C. leucaenae (MFLUCC 20-0008),
C. pallens (PAD S00004), C. vacuolata (CBS 191.93) and C.
venezuelae (CBS 107.87). Clonostachys ellipsoidea differs from C.
leucaenae in ITS (95.3 %, with 23 bp differences), LSU (98.3 %,
14 bp), from C. pallens in the ITS (92.5 % identity, with 14 bp
differences) sequence, and from C. venezuelae (CBS 107.87) in
ITS (95.5 % identity, with 23 bp differences), LSU (98.6 %, 11 bp),
RPB2 (96 %, 30 bp), TEF1 (96.2 %, 30 bp), and TUB2 (97.1 %, 30
bp) sequences. In addition, C. ellipsoidea differs from C. vacuolata
in producing smaller and wider conidia (av. = 5.3 × 3.5 μm vs av. =
6.0 × 2.9 μm). Morphology of conidiophores and conidia supports
classication of C. ellipsoidea in subgen. Zebrinella.
Clonostachys eriocamporesii R.H. Perera & K.D. Hyde, Fungal
Diversity 100: 199. 2020. Fig. 20.
Typus: Thailand, Southern Thailand, on dead stems of Pennisetum
polystachion (Poaceae), 11 Nov. 2017, A. Karunarathne, Bion 78 (holotype
MFLU 18-2718, ex-type culture MFLUCC 19-0486).
Description based on CBS 647.91: Sexual morph unknown.
Asexual morph. Sporodochia formed as distinct white pustules,
with time coalescing to support dark greenish conidial masses.
Conidiophores monomorphic, sporodochial, phialides straight or
curved, cylindrical and slightly tapering towards the tip or narrowly
ask-shaped and slightly widening in the middle, with or without
a visible collarette, (7.6–)9.5–17.5(–19.6) μm long, (2.0–)2.2–
3.2(–3.5) μm at base, (2.3–)2.5–3.5(–3.7) at widest point, (0.8–)
0.9–1.2(–1.3) μm wide near aperture (n = 60). Conidia aseptate,
greenish hyaline, ellipsoidal to narrowly clavate, straight or slightly
curved, with or without a slightly protruding or slightly laterally
displaced hilum and a rounded distal end, (4.4–)5.9–8.6(–12.6) ×
(2.4–)2.7–3.8(–4.2) μm (av. 7.4 × 3.2 μm, n = 150), arranged in
linear chains.
Culture characteristics: Colonies on OA reaching 30–45 mm diam
after 7 d at 25 °C in darkness, with entire margin, aerial mycelium
moderate, felty to cottony, whitish, reverse concolourous. Colonies
on PDA reaching 32–41 mm diam, with entire margin, aerial
mycelium moderate, felty to cottony, whitish, reverse concolourous.
Colonies on SNA reaching 27–32 mm diam, with entire margin,
aerial mycelium moderate in the centre, sparsely at periphery,
occose, whitish, reverse concolourous.
Additional materials studied: Germany, Berlin, unknown date and collector,
culture CBS 647.91. Netherlands, soil, Oct. 27, 2019, T. Vercruisse,
culture CBS 148221= NL19-060010.
Notes: Clonostachys eriocamporesii was rst described from dead
stems of Pennisetum polystachion (Hyde et al. 2020a). According
to our phylogenetic analyses (Figs 1, 2), C. eriocamporesii is
closely related to C. epichloe, C. fujianensis, C. miodochialis and
C. obovatispora. Morphologically, C. eriocamporesii differs from
C. epichloe, C. miodochialis and C. obovatispora, in producing
larger conidia [(4.4–)5.9–8.6(–12.6) × (2.4–)2.7–3.8(–4.2) av. 7.4
× 3.2 μm in C. eriocamporesii, (4.8–)6.0–7.0(–9.6) × (1.6–)2.2–
2.8(–3.6) av. = 6.6 × 2.6 μm in C. epichloe, (5.2–)5.8–7.2(–8.0)
× (1.8–)2.6–3.0(–3.4) av. = 6.6 × 2.8 μm in C. miodochialis, and
(4.7–)5.8–7.6(–8.2) × (1.7–)2.0–2.5(–2.9) av. = 6.74 × 2.26 μm in
C. obovatispora]. Clonostachys eriocamporesii (CBS 647.91) and
C. fujianensis (CBS 127474) are clearly different by ITS (98.2 %
identity, with 9 bp differences), LSU (99.6 %, 3 bp), TEF1 (95.8 %,
34 bp), and TUB2 (91.5 %, 90 bp) sequences.
Clonostachys farinosa (Henn.) Rossman, IMA Fungus 5: 86.
2014.
Basionym: Nectriella farinosa Henn., Hedwigia 36: 219. 1897.
Synonyms: Nectria farinosa (Henn.) Möller, in Schimper, Bot. Mitt.
Tropen 9: 296. 1901.
Nectria byssicola Berk. & Broome, J. Linn. Soc. Bot. 14: 116. 1873.
Bionectria byssicola (Berk. & Broome) Schroers & Samuels, Z.
Mykol. 63: 152. 1997.
Clonostachys byssicola Schroers, Stud. Mycol. 46: 80. 2001.
Clonostachys eriocamporesiana R.H. Perera & K.D. Hyde, Fungal
Diversity 100: 197. 2020.
Clonostachys indica Prasher & R. Chauhan [as ‘indicus’], Kavaka
48: 22. 2017.
Clonostachys wenpingii (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y.
Zhuang, Mycol. Progr. 13: 969. 2014.
Clonostachys granuligera (Starbäck) Forin & Vizzini, Persoonia 45:
240. 2020.
Clonostachys squamuligera (Sacc.) Forin & Vizzini, Persoonia 45:
245. 2020.
Clonostachys spinulosa R.H. Perera, E.B.G. Jones & K.D. Hyde,
Fungal Diversity 118: 109. 2023.
Description and illustration: Schroers (2001).
Typus: Venezuela, Edo Sucre, between Los Pocitos and Santa Isabel, on
unidentied wood, 11 Jul. 1972, G.J. Samuels & K.P. Dumont (isotype of
C. byssicola, CBS H-7918, ex-type living culture CBS 364.78 = C.T.R. 72-
123-ss7 = VE 4681).
Notes: Nectria byssicola was described by Berkeley & Broome
(1873) from the portion ‘173d’ (type specimen) as having pale
orange, scurfy perithecia and ascospores. It was transferred
to Bionectria as B. byssicola (Schroers & Samuels 1997).
Subsequently, Clonostachys byssicola was newly described
by Schroers (2001). However, with the introduction of the One
Fungus–One Name concept, B. byssicola became a synonym for
C. byssicola. Therefore, the oldest available name, N. byssicola
(1873), cannot be recombined in Clonostachys. Accordingly, the
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Zhao et al.
next available epithet, Nectriella farinosa (1897), was placed in
Clonostachys and published as a new combination, C. farinosa
(Rossman 2014). According to the multi-gene phylogenetic
inferences, the ex-type strains of C. eriocamporesiana (MFLUCC
17-2620), C. granuligera (PAD S00011), C. indica (IBP2), C.
spinulosa (MFLUCC 17-0131), C. squamuligera (PAD S00020)
and C. wenpingii (CBS 124067) cluster near the ex-type culture
of C. byssicola (CBS 364.78; Figs 1, 2) and together with several
other strains identied by Schroers (2001) as C. byssicola (Fig. 2).
Judged on the basis of gatherings of perithecia on recently dead
trees, C. farinosa is one of the more frequently occurring species
in tropical regions (Samuels 1976, Schroers 2001). A couple
of asexual morph characters were discussed that could help
identifying C. farinosa in cultures (Schroers 2001, as C. byssicola);
however, distinguishing this species from others forming dimorphic
conidiophores is difcult. It is therefore not surprising that rather
numerous synonymous species were described since 2001 on the
basis of asexual morph-typied material.
Fig. 20. Clonostachys eriocamporesii (CBS 647.91). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D. Sporodochia after 1 wk. E. Sporodochia after
2 wk. F, G. Sporodochia. H, I. Conidia. Scale bars = 10 μm.
245www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Clonostachys ava L. Zhao, Crous, & Schroers, sp. nov.
MycoBank MB 848483. Fig. 21.
Etymology: Name refers to the yellow colonies of this species (OA,
PDA, and SNA).
Typus: French Guiana, St. Laurent-Du-Marouni, Canton de Maripasoula,
S. along Route De belizon, decaying bark, 200 m alt., 14 Sep. 1994, S.M.
Huhndorf, BPI 737845, culture G.J.S. 94-75 (holotype designated here
CBS H-25139, ex-type culture CBS 915.97).
Sexual morph known from natural specimen (not shown). Asci 49–
70 × 8.5–10 μm. Ascospores 1-septate, hyaline, striate, ellipsoid,
9.7–10.8–12.8 × 3.3–4.0–4.9 μm. Asexual morph. Conidiophores
dimorphic, mononematous, scattered on the agar surface or arising
from strands of aerial hyphae. Primary conidiophores narrowly
penicillate, adpressed, mono- to terverticillate; stipe 20–120 μm long,
2.2–4.0 μm wide at base; penicilli 40–100 μm high; phialides straight
to slightly curved, almost cylindrical, slightly tapering towards the
tip, sometimes with a short collarette, (22.4–)25.9–43.2(–46.9) μm
long, (1.4–)1.6–2.4(–2.6) μm wide at base, (1.1–)1.2–1.6(–1.7) μm
Fig. 21. Clonostachys ava (ex-type CBS 915.97). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G, H. Secondary
conidiophores. I, J. Conidia. Scale bars = 10 μm.
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Zhao et al.
wide near aperture (n = 50). Secondary conidiophores penicillate,
ter- to quaterverticillate, branches adpressed or divergent, phialides
adpressed or somewhat divergent; phialides in loose whorls of 2–5,
straight or slightly curved, ask-shaped or somewhat cylindrical,
widest in the lower third, slightly tapering in the upper part towards
the tip, without a collarette, (11.4–)12.6–18.8(–20.7) μm long,
(1.2–)1.5–2.3(–2.8) μm wide at base, (1.8–)2.0–2.7(–2.8) μm at
widest point, (0.8–)0.9–1.3(–1.4) μm wide near aperture (n = 50).
Conidia aseptate, hyaline, smooth, ellipsoid to obovoid, distally
broadly rounded, apex minutely tapering, with a median, invisible,
or rarely somewhat laterally displaced hilum, (4.7–)4.9–6.5(–7.8)
× (2.4–)2.6–3.3(–3.4) μm (av. 5.7 × 3.0 μm, n = 150), arranged in
imbricate chains that may collapse into slimy masses.
Culture characteristics: Colonies on OA reaching 22–30 mm diam
after 7 d at 25 °C in the darkness, at, with crenate margin, aerial
mycelium abundant, felty to cottony, nely to coarsely granular,
yellowish white to pale yellow, reverse yellowish to pale yellow.
Colonies on PDA reaching 24–29 mm diam, at, with entire margin,
aerial mycelium abundant, felty to cottony, granular, yellowish white
to pale yellow, reverse pale yellow to pale orange. Colonies on SNA
reaching 22–24 mm diam, at, with crenate margin, aerial mycelia
sparsely developed, felty, nely granular, pale yellow, reverse
concolourous.
Notes: Based on phylogenetic analyses, C. ava and C.
grammicospora formed a well-supported clade (Figs 1, 2). Morpho-
logically, the longer phialides (both in primary and secondary
conidiophores) can be used to distinguish C. ava from C.
grammicospora [primary conidiophore: (22.4–)25.9–43.2(–46.9) μm
vs (11–)17.2–24.6(–43.4) μm; secondary conidiophore: (11.4–)12.6–
18.8(–20.7) μm vs (4.4–)9.8–12.6(–18.6) μm]. Furthermore, CBS
915.97 (C. ava) and CBS 209.93 (C. grammicospora) have clearly
different ITS (97.4 % identity, with 12 bp differences), LSU (99.5
%, 4 bp), RPB2 (96.1 %, 29 bp), TEF1 (96.9 %, 25 bp), and TUB2
(96.8 %, 30 bp) sequences. Schroers (2001) listed CBS 915.97
under C. grammicospora, characterised by an ascospore size of
(8.2–)10.6–12.6(–17.6) × (3.0–)3.8–4.6(–6.2) µm (av. 11.6 × 4.2 μm).
Accordingly, C. ava forms similarly sized ascospores as specimen
Raunkiaer 3103 (isotype of Nectria grammicospora, ascospores
10.7–13.1 × 3.8–4.9 µm) and specimen Samuels 3285 (type of C.
grammicospora, ascospores 9–13.5 × 3.6–5.4 μm) (Samuels 1988b,
Schroers 2001). Overall characters of the sexual morph of C. ava
and C. grammicospora are similar. The morphology of conidiophores
and conidia supports classication of C. ava in subgen. Zebrinella.
Clonostachys fujianensis L. Zhao & Crous, sp. nov. MycoBank
MB 848484.
Etymology: Named after the location where the fungus was
collected, Fujian, China.
Typus: China, Fujian, Bamboo stem, collection and isolation date unknown,
W.Y. Zhuang & J. Luo (holotype designated here CBS H-25146, ex-type
living culture CBS 127474).
Cultures sterile. Clonostachys fujianensis differs from its closest
phylogenetic neighbours C. eriocamporesii (Fig. 2) by unique
nucleotide substitutions and indels in the ve investigated loci
(see direct sequence comparisons deposited at doi: 10.6084/
m9.gshare.22894592): C. fujianensis and C. eriocamporesii (CBS
647.91): ITS position 116 (C), 167 (gap), 171 (T), 182 (gap), 429
(gap), 477 (A), 478 (G), 481 (C), 533 (C), 534 (T), 548 (A), 564 (A);
LSU position 1 007 (C), 1 008 (T), 1 045 (C); TEF1 position 2 150
(C), 2 180 (T), 2 186 (T), 2 198 (C), 2 270 (T), 2 306 (T), 2 339 (A),
2 360 (T), 2 361 (G), 2 369 (C), 2 384 (A), 2 414 (T), 2 429 (C), 2 469
(G), 2 471 (T), 2 504 (G), 2 513 (T), 2 522 (C), 2 525 (C), 2 556 (G),
2 621 (T), 2 643 (A), 2 690 (C), 2 702 (C), 2 705 (T), 2 748 (C), 2 759
(C), 2 819 (C), 2 831 (C), 2 849 (C), 2 885 (C); TUB2 position 2 945
(T), 2 946 (G), 2 996 (gap), 3 000–3 002 (gap), 3 006 (T), 3 007
(G), 3 009 (A), 3 011 (A), 3 012 (T), 3 013 (G), 3 014 (C), 3 015 (T,
insertion), 3 018–3 021 (gap), 3 028 (G, insertion), 3 029 (A), 3 038
(T), 3 039 (G), 3 045 (G), 3 062 (A), 3 109 (C), 3 118 (T), 3 121 (T),
3 172 (T), 3 203 (G), 3 217 (T), 3 219 (C), 3 221 (T), 3 222 (C), 3 223
(T), 3 224 (C), 3 225–3 226 (gap), 3 227 (A), 3 229 (A), 3 236 (T),
3 239 (T), 3 242 (C), 3 244 (A), 3 245 (G), 3 246 (C), 3 248 (T), 3 252
(A), 3 255 (A), 3 257 (C), 3 258 (A), 3 261 (G), 3 269 (G), 3 271 (T),
3 272 (gap), 3 287 (A), 3 292 (C), 3 294 (G), 3 295 (G), 3 308 (T),
3 311 (C), 3 365 (C), 3 371 (A), 3 377 (T), 3 398 (T), 3 410 (T), 3 416
(T), 3 461 (G), 3 468 (G), 3 470 (G), 3 473 (T), 3 482 (C), 3 497 (C),
3 542 (C), 3 569 (A), 3 635 (T), 3 684 (T), 3 705 (T), 3 714 (G), 3 744
(C), 3 768 (T), 3 780 (G), 3 786 (T), 3 789 (C), 3 813 (T), 3 834 (T),
3 864 (G), 3 873 (C), 3 888 (C), 3 930 (T), 3 960 (C), 3 972 (T), 4 002
(A), 4 032 (C), 4 047 (T).
Notes: Clonostachys fujianensis was collected from stems
of a bamboo. Unfortunately, it does not sporulate in culture.
Phylogenetically, it is distinct from all other sequenced species
(Figs 1, 2).
Clonostachys fusca L. Zhao, Crous & Schroers, sp. nov.
MycoBank MB 848485. Fig. 22.
Etymology: Epithet derived from the brown ascomata produced by
this species.
Typus: French Guiana, unknown, herbaceous stem, Jan.–Mar. 1986, G.J.
Samuels (holotype designated here CBS H-25141, ex-type living culture
CBS 207.93); also kept as G.J.S. 3731, culture G.J.S. 86-200.
Sexual morph produced in culture on OA. Stroma supercial or
erumpent, bearing perithecia, cells prosenchymatous, densely
hyphal. Perithecia crowded in groups of up to 20, globose to
subglobose, 190–300 μm diam, pale to brownish orange or reddish
brown. Perithecial warts whitish to pale orange, up to 70 μm high,
cells subglobose to globose, of the same type as the cells of the outer
perithecial wall region, (6.5–)7.5–17.0(–26.0) × (6.0–)7.0–16.0(–20.0)
μm (av. = 11.6 × 10.7 μm, n = 100). Perithecial wall 20–45 μm thick
composed of two regions; outer region 15–36 μm or 1–3 cells thick,
cells merging with the cells of the warts, cells angular to subglobose,
(5.5–)6.5–13.0(–15.0) × (3.5–)4.5–12.0(–15.5) μm (av. = 9.5 × 8.0
μm, n = 100), with evenly thickened walls around 1.5 μm thick,
sometimes with vacuoles; middle region lacking; inner region 10–25
mm thick. Asci 8-spored, clavate, 35–70 × 7.5–12.0 μm (av. = 48.1 ×
10.5 μm, n = 35), apex at or sometimes rounded, edges rounded,
ring clearly visible. Ascospores 0–1-septate, striate, striae parallel,
constriction at the median septum frequently observed in discharged
ascospores, ellipsoid, (11.5–)12.0–15.0(–16.0) × (4.0–)4.5–5.5 μm
(av. = 13.2 × 4.8 μm, n = 80). Asexual morph not observed.
Culture characteristics: Colonies on OA reaching 33–36 mm diam
after 7 d at 25 °C in darkness, with crenate margin, aerial mycelium
moderate, felty to cottony, pale yellow, reverse concolourous.
Colonies on PDA reaching 38–40 mm diam, with entire margin,
247www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Fig. 22. Clonostachys fusca (ex-type CBS 207.93). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Perithecia. F. Section through perithecium.
G. Ostiole. H. Perithecial wart. I. Lateral perithecial wall showing two regions. J, K. Asci. L, M. Discharged ascospores in optical section (L) and surface view
(M). Scale bars: F = 50 μm; G–M = 10 μm.
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Zhao et al.
aerial mycelium moderate, felty, white yellow, reverse pale yellow.
Colonies on SNA reaching 31–37 mm diam, with crenate margin,
aerial mycelium sparse, felty, whitish, reverse concolourous.
Additional materials examined: USA, Puerto Rico, Caribbean National
Forest, Luquillo Mts., Bisley Experimental Watershed, bark of recently
dead tree, 21 Feb. 1996, G.J. Samuels, H.J. Schroers 74 & D.J. Lodge,
BPI 749156, culture CBS 996.97; Puerto Rico, Caribbean National Forest,
Liquillo Mts., trail to Cocle Falls from Rt 191, 475 m alt., bark, 23 Feb. 1996,
G.J. Samuels & H.-J. Schroers, culture CBS 101925.
Notes: Based on phylogenetic analyses, C. fusca is closely related
to C. aurantiaca (Figs 1, 2). Morphologically, C. fusca differs from
C. aurantiaca in producing more crowded perithecia, and larger
ascospores [(11.5–)12.0–15.0(–16.0) × (4.0–)4.5–5.5 μm vs (8.5–)
9.0–12.0(–12.5) × 4.0–5.5(–6.0) μm]. Observed morphological
characters, especially perithecial walls consisting of two regions,
perithecial warts consisting of cells with evenly thickened walls
and the striate ascospores support their classication in subgen.
Zebrinella.
Clonostachys garysamuelsii L. Zhao & Crous, sp. nov.
MycoBank MB 848486. Fig. 23.
Etymology: Named in honour of Dr Gary J. Samuels, who collected
many sexual morphs of Clonostachys and Sesquicillium species,
isolated ascospores and deposited cultures in the CBS collection.
Typus: Venezuela, unknown, tree bark, date unknown, G.J. Samuels
(holotype designated here CBS H-25154, ex-type culture CBS 123964).
Sexual morph unknown. Asexual morph. Conidiophores dimorphic.
Primary conidiophores arising from the agar surface or strands of
aerial mycelium, mononematous, acremonium- or verticillium-like,
mono- to terverticillate; phialides adpressed or divergent; sometimes
with main side branches arising from the lower part of the stipe;
stipe varying in length, (8.0–)20.0–90.0 μm long, 2.0–3.5 μm wide
at base; penicilli 40–70 μm high; phialides in whorls of 2–5, straight,
generally slightly tapering towards the tip, with or without a visible
collarette, (15.6–)16.8–41.4(–50.0) μm long, (1.6–)1.8–2.7 μm wide
at base, (1.1–)1.2–1.8 μm wide near aperture (n = 60). Secondary
conidiophores ter- to quinquiesverticillate, densely aggregated,
formed in pustules or sporodochia on agar surface or from strands
of aerial mycelium; branches divergent; phialides in loose whorls
of 3–5, straight to slightly curved, narrowly ask-shaped, generally
with widest point in the lower third, or almost cylindrical, tapering in
the upper part, without a visible collarette, (7.8–)9.9–18.8(–20.2)
μm long, (1.5–)1.7–2.5(–2.6) μm wide at base, (2.0–)2.2–2.8(–3.0)
μm at widest point, 1.0–1.4(–1.5) μm wide near aperture (n = 50).
Conidia aseptate, hyaline, smooth, ellipsoid, straight or minutely
curved, some asymmetric with one more attened side, ends
broadly rounded, without a visible hilum, or with a hardly visible,
laterally displaced hilum, (3.5–)4.5–7.1(–8.2) × (2.0–)2.3–3.3(–3.7)
μm (av. = 5.7 × 2.7 μm, n = 150), arranged in imbricate chains that
may collapse into slimy masses.
Culture characteristics: Colonies on OA reaching 30–35 mm
diam after 7 d at 25 °C in darkness, at, with entire margin, aerial
mycelium moderate, felty, whitish, reverse concolourous. Colonies
on PDA reaching 28–31 mm diam, at, with entire margin, aerial
mycelium moderate, felty to cottony, whitish, reverse concolourous.
Colonies on SNA reaching 28–30 mm diam, at, with entire margin,
aerial mycelium sparse, felty, whitish, reverse concolourous.
Notes: According to the multi-gene phylogenetic inference, C.
garysamuelsii groups together with C. moreaui and C. oblongispora
(Fig. 2; support below threshold values) or C. parasporodochialis and
C. sporodochialis (Fig. 1, poorly to well-supported). Morphologically,
C. garysamuelsii differs from C. moreaui and C. oblongispora in
producing smaller conidia [(3.5–)4.5–7.1(–8.2) × (2.0–)2.3–3.3(–
3.7) μm vs 5.0–10.0(–12.0) × 3.5–5.0 μm and (9.0–)12.6–14.0(–
19.8) × (2.6–)3.2–3.8(–4.2) μm]. However, C. garysamuelsii
differs from C. sporodochialis in producing larger conidia [(3.5–)
4.5–7.1(–8.2) × (2.0–)2.3–3.3(–3.7) μm vs (3.2–)4.4–5.4(–6.8) ×
(1.6–)2.0–2.2(–2.6) μm]. Clonostachys garysamuelsii can also be
distinguished from C. moreaui and C. parasporodochialis by having
dimorphic conidiophores, while conidiophores of C. moreaui and C.
parasporodochialis are monomorphic (Lechat et al. 2020).
Clonostachys hongkongensis L. Zhao & Crous, sp. nov.
MycoBank MB 848487. Fig. 24.
Etymology: Named after the location where the fungus was
collected, Hong Kong, China.
Typus: China, Hong Kong, Pokfulam Reservoir, wood, 14 Jun. 2001,
unknown collector (holotype designated here CBS H-25149, ex-type living
culture CBS 115291).
Sexual morph unknown. Asexual morph. Conidiophores dimorphic,
mononematous. Primary conidiophores verticillium-like, arising
from the agar surface or from aerial mycelium, with monoverticillate
or more-level-verticillate divergent phialides, sometimes with side-
branches; stipe up to 225 μm long, 2.3–4.6 μm wide at base;
penicilli 40–130 μm high; phialides in whorls of 2–4, in lower
levels also solitary, straight, cylindrical, slightly and continuously
tapering towards the tip, with or without a minute collarette, (19.9–)
20.4–39.1(–43.4) μm long, (1.6–)1.9–2.3(–2.6) μm wide at base,
1.1–1.5(–1.7) μm wide near aperture (n = 50), each producing a
small, hyaline drop of conidia. Secondary conidiophores penicillate,
scattered on the agar surface or arising from strands of aerial
hyphae, bi- to quaterverticillate; branches adpressed or somewhat
divergent; stipes 93–135 μm long, to 5 μm wide at base; penicilli 30–
58 μm, to 55 μm diam at widest point; terminal phialides adpressed,
in whorls of up to 4–5, straight to slightly curved, narrowly ask-
shaped, slightly tapering in the upper part, with or without a visible
collarette, (9.0–)10.3–14.5(–15.9) μm long, (1.5–)1.6–2.2(–3.0) μm
wide at base, (2.2–)2.4–2.9(–3.4) μm at widest point, (0.8–)0.9–1.3
μm wide near aperture (n = 50); intercalary phialides rare, with to
3 μm long lateral pegs, below a whorl of terminal phialides (not
shown). Conidia aseptate, hyaline, smooth, ellipsoid to obovoid,
straight or slightly curved, with a laterally displaced or sometimes
median hilum, (4.1–) 4.3–5.9(–6.3) × (2.8–)2.9–3.4 μm (av. = 4.8 ×
3.1 μm, n = 70), arranged in imbricate chains.
Culture characteristics: Colonies on OA reaching 36–40 mm diam
after 7 d at 25 °C in darkness, at, with crenate margin, aerial
mycelium moderate, nely to coarsely granular, felty to cottony,
whitish to pale yellow, reverse yellowish. Colonies on PDA reaching
37–43 mm diam, at, with crenate margin, aerial mycelium
moderate, nely to coarsely granular, felty to cottony, whitish,
reverse concolourous. Colonies on SNA reaching 39–40 mm diam,
at, with crenate margin, aerial mycelium sparse, nely to coarsely
granular, whitish, reverse concolourous.
Additional material examined: China, Hong Kong, Pokfulam Reservoir,
wood, 14 Jun. 2001, unknown collector, culture CBS 116542.
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Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Notes: Based on the multi-locus phylogenetic analyses (Figs 1, 2),
C. hongkongensis has a close phylogenetic afnity to C. cylindrica,
C. divergens, C. rogersoniana and C. samuelsii. Morphologically,
C. hongkongensis differs from C. cylindrica, C. divergens, C.
rogersoniana and C. samuelsii in producing shorter conidia [(4.1–
)4.3–5.9(–6.3) μm vs (7.7–)8.6–11.9(–13.5) μm in C. cylindrica,
(4.8–)5.8–6.4(–7.4) μm in C. divergens, (4.8–)5.8–7.2(–9.6) μm in C.
rogersoniana and (4.4–)5.8–7.0(–11.6) μm in C. samuelsii]. Loosely
branched sporodochia showing diverging branches and greenish
conidial masses distinguish C. divergens from C. hongkongensis.
Clonostachys kunmingensis Hong Yu bis & Yao Wang, Frontiers
Microbiol. 14: 8. 2023. Fig. 25.
Typus: China, Yunnan Province, Kunming City, Wild Duck Forest Park
(25°13’N, 102°87′E, 2 100 m alt.), from soil on the forest oor, 10 Aug.
2019, Yao Wang (holotype YHH 898 dried specimen, ex-type culture
YFCC 898).
Description based on CBS 101920: Sexual morph from natural
specimen (not shown). Asci 49.1–59.1–66.9 × 6.7–8.4–10.0 μm (n
Fig. 23. Clonostachys garysamuelsii (ex-type CBS 123964). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Primary conidiophores. F, G.
Secondary conidiophores. H, I. Conidia. Scale bars = 10 μm.
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Zhao et al.
= 16). Ascospores 1-septate, hyaline, smooth or nely spinulose,
ellipsoid, 10.1–12.7–16.6 × 3.6–4.2–4.9 μm (n = 24). Asexual
morph. Conidiophores dimorphic, mononematous. Primary
conidiophores arising from the agar surface or from strands of
aerial mycelium, branches adpressed or somewhat divergent,
phialides adpressed; stipe 30–140 μm long, 2.8–4.8 μm wide at
base; penicilli 30–80 μm high; phialides in apical whorls of 2–5,
also solitary arising from lower levels, straight, almost cylindrical,
slightly tapering towards the tip, with a somewhat visible collarette,
(15.4–)17.6–31.2(–32.5) μm long, (1.6–)1.9–2.5(–2.7) μm wide at
base, 1.1–1.5(–1.6) μm wide near aperture (n = 80). Secondary
conidiophores penicillate, bi- to quaterverticillate, primary branches
Fig. 24. Clonostachys hongkongensis (ex-type CBS 115291). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Primary conidiophores. F, G.
Secondary conidiophores. H, I. Conidia. Scale bars: E, F = 50 μm; D, G–I = 10 μm.
251www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
typically divergent, higher level branches and phialides somewhat
divergent to almost adpressed; stipe 20–120 μm long, 2.5–4.5 μm
wide; penicilli 30–70 μm long; 25–65 μm wide; terminal phialides
in loose whorls of up to 6, straight or slightly curved, ask-shaped,
widest in the lower third, slightly tapering in the upper part towards
the tip, without or with a collarette, (9.0–)10.0–16.7(–28.1) μm
long, (1.2–)1.6–2.5(–2.8) μm wide at base, (1.9–)2.1–2.7(–2.8) μm
at widest point, (0.9–)1.0–1.3 μm wide near aperture (n = 100);
intercalary phialides sometimes observed, bearing one or several
terminal phialides, conidiogenous pegs to 5 μm long. Conidia
aseptate, hyaline, smooth, ellipsoid, commonly asymmetric with
one more attened sides, distally broadly rounded, with laterally
displaced hilum, (3.7–)4.5–6.3(–7.1) × (2.1–)2.4–3.1(–3.3) μm (av.
= 5.4 × 2.7 μm, n = 150), arranged in imbricate chains.
Culture characteristics: Colonies on OA reaching 51–53 mm
diam after 7 d at 25 °C in darkness, at, with mbriate margin,
aerial mycelium abundant, felty, whitish, reverse concolourous.
Colonies on PDA reaching 46–50 mm diam, at, with entire margin,
aerial mycelium abundant, felty, pale yellow, with greenish tinge
Fig. 25. Clonostachys kunmingensis (CBS 101920). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G–I. Secondary
conidiophores. J, K. Conidia. Scale bars: G = 50 μm; D–F, H–K = 10 μm.
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Zhao et al.
diffusing into the medium outside the colony, reverse yellowish to
light orange. Colonies on SNA reaching 38–45 mm diam, at, with
crenate margin, occose, aerial mycelium scanty, whitish, reverse
concolourous.
Material examined: China, Yunnan Province, Kunming City, Songming
County, Dashao Village (25°24’N, 102°55′E, 2 750 m alt.), from soil on the
forest oor, 24 Aug. 2019, Y. Wang, culture YFCC 892. Jamaica, Hanover
Parish, Dolphin Head Mt., vic. Askenish, wood, collection and isolation
date unknown, collected by D. Korf et al., isolated by C.T. Rogerson & G.J.
Samuels MJ 946, C.T.R. 71-116, culture CBS 101920.
Notes: Clonostachys kunmingensis was described from soil in
Kunming City, China, but its holomorph was encountered in the
neotropics. Schroers (2001) led CBS 101920 under C. solani
due to overall branching patterns of the primary and secondary
conidiophores. Based on our phylogenetic analysis, C. kunmingensis
is closely related to C. rhizophaga (Figs 1, 2). Length of phialides
in primary conidiophores distinguish both species [(15.4–)17.6–
31.2(–32.5) μm in C. kunmingensis, (15.6–)22.0–34.2(–48.2) μm
in C. rhizophaga]. Clonostachys kunmingensis (CBS 101920) has
different ITS (99.2 % identity, with 4 bp differences), LSU (99.9 %,
1 bp), RPB2 (97.5 %, 19 bp), TEF1 (99.6 %, 3 bp), and TUB2 (98.3
%, 18 bp) sequences when compared with C. rhizophaga (CBS
202.37).
Clonostachys longiphialidica L. Zhao, Crous, & Schroers, sp.
nov. MycoBank MB 848488. Fig. 26.
Etymology: Names refers to the long conidiogenous peg of
intercalary phialides produced by this species.
Typus: Venezuela, Dept. Rio Negro, Cerro de la Neblina, summit camp
5, valley at N base of Pico Phelps, 1 000–1 250 m alt., bark, cloud forest,
12 Apr 1984, G.J. Samuels, G.J.S. 1301 (holotype designated here CBS
H-25142, ex-type living culture CBS 112.87, = G.J.S. 84-330).
Sexual morph from natural specimen (not shown). Asci 46.1–66.3
× 6.6–10.3 μm (n = 11). Ascospores 1-septate, hyaline, striate,
ellipsoid, 10.4–12.7–15.8 × 3.4–4.2–5.3 μm (n = 27). Asexual
morph. Conidiophores monomorphic, aggregated in pustules,
arising from the agar surface or sparse aerial mycelium, adpressed
or with more or less divergent branches, phialides adpressed or
somewhat divergent, bi- to quaterverticillate; stipe 10–50 μm long,
1.9–3.4 μm wide at base; penicilli up to 75 μm long, 60 μm wide;
terminal phialides in whorls of 2–6, cylindrical or slightly tapering
toward the tip, or slightly widening in lower third, frequently with
a small collarette, (7.6–)9.2–16.7(–24.2) μm long, (1.5–)1.7–2.8(–
2.9) μm wide at base, (2.0–)2.3–3.3(–3.5) μm at widest point, (1.1–)
1.2–1.5(–1.6) μm wide near aperture (n = 80); intercalary phialides
rare, formed below whorls of terminal phialides, with to 8 μm long
conidiogenous pegs. Conidia aseptate, hyaline, smooth, narrowly
ellipsoid to cylindrical, straight or nearly straight, distally broadly
rounded, (6.3–)6.7–8.5(–9.3) × (2.2–)2.4–3.0(–3.4), (av. = 7.5 × 2.7
μm, n = 150), arranged in imbricate chains that may collapse into
slimy masses.
Culture characteristics: Colonies on OA reaching 40–42 mm diam
after 7 d at 25 °C in darkness, with entire margin, aerial mycelium
sparse, felty, whitish, reverse concolourous. Colonies on PDA
reaching 37–43 mm diam, with entire margin, aerial mycelium
moderate, felty to cottony, nely to coarsely granular, white
yellowish, reverse pale yellow. Colonies on SNA reaching 39–44
mm diam, with entire margin, aerial mycelium moderate, felty to
cottony, whitish, reverse concolourous.
Notes: Clonostachys longiphialidica is represented by a single
strain isolated from bark in Venezuela. It is phylogenetically different
from the closely related C. vacuolata (ITS: 96.0 % sequence
similarity; LSU: 96.0 %, RPB2: 93.6 %, and TEF1: 97.6 %). Their
morphological differences are discussed under C. vacuolata.
Specimen G.J.S. 1301 was identied as C. grammicospora on the
basis of ascospore morphology and characters of the perithecia
(Schroers 2001: g. 62a, c, e, f). However, its ascospores cover
a slightly larger length range as Raunkiaer 3103 (isotype of
Nectria grammicospora, ascospores 10.7–13.1 × 3.8–4.9 µm) and
specimen Samuels 3285 (type of C. grammicospora, ascospores
9–13.5 × 3.6–5.4 μm) (Samuels 1988b, Schroers 2001). Sexual
and asexual morphology suggest classication of C. longiphialidica
in subgen. Zebrinella.
Clonostachys obovatispora L. Zhao & Crous, sp. nov. MycoBank
MB 848489. Fig. 27.
Etymology: Names refers to the obovate conidial shown by this
species.
Typus: Germany, Frankfurt, Bergen-Enkheim, on living leaves with
Epichloe typhina, 10 Jun. 2005, R. Kirschner (holotype designated here
CBS H-25148, ex-type living culture CBS 118752).
Sexual morph unknown. Asexual morph. Conidiophores
monomorphic, sporodochial. Sporodochia appearing at rst
as distinct white pustules, with time coalescing and with green
coloured conidial masses; phialides in whorls of 2–4, ask-shaped
to cylindrical, widest near the middle, narrowing in the uppermost
part, without a visible collarette, (9.2–)9.8–16.4(–17.0) μm long,
(1.6–)1.8–2.7(–2.9) μm at base, (2.3–)2.4–3.2(–3.5) at widest
point, (0.7–)0.8–1.1(–1.2) μm wide near aperture (n = 60). Conidia
aseptate, greenish hyaline to pale green, smooth, oblong-ellipsoid
or clavate to obovoid, (4.7–)5.8–7.6(–8.2) × (1.7–)2.0–2.5(–2.9)
(av. = 6.7 × 2.3 μm, n = 100), arranged in linear chains.
Culture characteristics: Colonies on OA reaching 27–30 mm diam
after 7 d at 25 °C in darkness, with slightly lobate margin, aerial
mycelium moderate, felty, nely to coarsely granular, whitish,
reverse concolourous. Colonies on PDA reaching 26–27 mm diam,
with slightly lobate margin, aerial mycelium moderate, felty to
cottony, whitish, reverse concolourous. Colonies on SNA reaching
19–22 mm diam, at, with entire margin, membranous without
aerial mycelia, colourless, reverse colourless.
Notes: According to phylogenetic inferences in the present study
(Figs 1, 2), C. obovatispora is closely related to C. epichloe, and
C. miodochialis. However, C. obovatispora differs from C. epichloe
(CBS 101037) in ITS (98.8 % identity, with 6 bp differences), LSU
(99.3 %, 5 bp), RPB2 (91.3 %, 66 bp), TEF1 (95.8 %, 34 bp),
and TUB2 (95.1 %, 50 bp) sequences; C. obovatispora differs
from C. miodochialis (CBS 997.69) in ITS (98.4 % identity, with
8 bp differences), LSU (99.5 %, 4 bp), RPB2 (91.7 %, 62 bp),
TEF1 (96.1 %, 32 bp), and TUB2 (95.3 %, 47 bp) sequences.
Morphologically, C. obovatispora differs from C. epichloe and C.
miodochialis in producing shorter phialides [(9.2–)9.8–16.4(–17.0)
μm in C. obovatispora vs (7–)12–17(–29) μm in C. epichloe], and
narrower conidia [(1.6–)2.2–2.6–2.8 (–3.6) μm in C. obovatispora
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Revising ClonostaChys and allied geneRa in BioneCtriaCeae
vs (1.8–)2.6–3(–3.4) μm in C. miodochialis)]. However, overall
nature of morphological characters, i.e., occurrence of sporodochia,
greenish pigmentation of conidial masses and shape of conidia, is
similar in these closely related species and aligns C. obovatispora
well into the morphological concept of subgenus Astramata.
Clonostachys palmae L. Zhao, Crous & Schroers, sp. nov.
MycoBank MB 848490. Fig. 28.
Etymology: Name refers to palm, the host from which the ex-type
culture of this fungus was isolated.
Typus: Indonesia, Sulavesi, Eastern Dumoga-Bone Nat. Park, between
Maddison’s Camp, from palm leaves, 5 Oct. 1985, G.J. Samuels, 2156,
culture G.J.S. 85-155 (holotype designated here CBS H-25153, ex-type
culture CBS 119.87).
Sexual morph known from natural specimen (not shown). Asci
45–67.5 × 7.0–10.8 μm. Ascospores striate, ellipsoid with gently
Fig. 26. Clonostachys longiphialidica (ex-type CBS 112.87). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia.
Scale bars = 10 μm.
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Zhao et al.
tapering ends, 1-septate, 10.0–14.5 × 3.0–5.4 μm. Asexual morph.
Conidiophores dimorphic. Primary conidiophores mononematous,
arising from either the aerial mycelium or the agar surface,
either unbranched, acremonium-like or verticillium-like, mono- to
terverticillate, with divergent branches and phialides diverging at
more or less acute angles; stipe 40–180 μm long, 2.4–4.6 μm wide
at base; penicilli 50–100 μm high; phialides in apical whorls of 2–4,
straight, cylindrical, slightly tapering towards the tip, with a minute
visible collarette, (21.0–)22.4–42.2(–48.8) μm long, (1.6–)1.8–
2.8(–3.0) μm wide at base, 1.2–1.8(–2.0) μm wide near aperture
(n = 70). Secondary conidiophores broadly penicillate, arising from
the agar surface or aerial mycelium ter- to quinquiesverticillate,
aggregated in pustules or sporodochia; phialides slightly divergent
or adpressed, in whorls of 2–4, ask-shaped, widest in the lower
third or almost cylindrical, slightly tapering in the upper part towards
the tip, without a visible collarette, (9.2–)11.0–19.0(–21.6) μm long,
(1.5–)1.6–2.6(–2.9) μm wide at base, (2.1–)2.2–2.9(–3.1) at widest
point, (1.0–)1.1–1.7(–1.8) μm wide near aperture (n = 80). Conidia
Fig. 27. Clonostachys obovatispora (ex-type CBS 118752). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Sporodochia. F, G. Phialides. H,
I. Conidia. Scale bars = 10 μm.
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Revising ClonostaChys and allied geneRa in BioneCtriaCeae
aseptate, hyaline, smooth, ellipsoid, some slightly curved or
asymmetric with one more attened side, with a laterally displaced
hilum, (4.5–)5.3–8.2(–10.2) × (2.4–)2.7–3.2(–3.6) (av. 6.5 × 2.9 μm,
n = 150), arranged in imbricate chains.
Culture characteristics: Colonies on OA reaching 49–53 mm
diam after 7 d at 25 °C in darkness, at, with entire margin, aerial
mycelium moderate, nely to coarsely granular, felty to cottony,
whitish, reverse concolourous. Colonies on PDA reaching 54–61
mm diam, at, with entire margin, aerial mycelium abundant,
cottony, pale yellow, with greenish tinge diffusing into the medium
outside the colony, reverse yellowish. Colonies on SNA reaching
41–45 mm diam, at, with crenate margin, aerial mycelium
moderate, felty, nely granular, whitish, reverse concolourous.
Notes: Measurements provided for asci and ascospores and
description of the sexual morph are derived from observations
and notes by G. Samuels. No specimen but a dried culture was
Fig. 28. Clonostachys palmae (ex-type CBS 119.87). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G, H. Secondary
conidiophores. I, J. Conidia. Scale bars = 10 μm.
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Zhao et al.
encountered when Schroers later re-examined specimen G.J.
Samuels 2156. Schroers (2001) led this specimen under C.
pseudostriata. According to the multi-gene phylogenetic inference,
phylogenetic relatedness of C. palmae to C. pseudostriata is highly
supported (Figs 1, 2). However, C. palmae (CBS 119.87) has clearly
different ITS (98.6 % identity, with 7 bp differences), LSU (99.7 %, 2
bp), RPB2 (97.5 %, 19 bp), TEF1 (98.1 %, 15 bp), and TUB2 (97.0 %,
31 bp) sequences compared to C. pseudostriata (CBS 120.87).
Striae formed on the surface of ascospores of C. pseudostriata
consist of warts that are arranged in rows (Schroers 2001: g. 44k),
while ascosporal striae of species of subg. Zebrinella are continuous.
Clonostachys parasporodochialis L. Zhao & Crous, sp. nov.
MycoBank MB 848491. Fig. 29.
Etymology: Named after its close morphological and phylogenetic
relationship to C. sporodochialis.
Typus: Venezuela, unknown, terminal branchlet of recently dead tree,
Nov.–Dec. 1990, G.J. Samuels, 7760, culture G.J.S. 90-192 (holotype
designated here CBS H-25156, ex-type culture CBS 192.93).
Fig. 29. Clonostachys parasporodochialis (ex-type CBS 192.93). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Sporodochia. G. Phialides.
H, I. Conidia. Scale bars = 10 μm.
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Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Sexual morph known from natural specimen (not shown). Asci
57.7–84.7 × 9.2–12.6 μm (n = 16). Ascospores 1-septate, hyaline,
spinulose, ellipsoid, 8.6–13.0 × 2.6–4.4 μm. Asexual morph.
Conidiophores monomorphic, sporodochial, penicillate, ter- to
quaterverticillate, branches of conidiophores in young sporodochial
pustules divergent, in developed sporodochia adpressed; phialides
in apical whorls of 2–4, also arising from lower levels, slightly
divergent or almost adpressed, straight, hardly narrowing towards
the tip, almost cylindrical, without visible collarette, (7.5–)11.3–
24.3(–28.4) μm long, (1.5–)1.7–2.3(–2.5) μm wide at base, (0.7–)
0.8–1.1(–1.2) μm wide near aperture (n = 60). Conidia aseptate,
hyaline, smooth, oblong-ellipsoid or obovoid, some minutely
curved, asymmetric with one more attened side, distally broadly
rounded, with laterally displaced hilum, (3.5–)4.5–6.6(–8.2) ×
(1.8–)2.0–2.4(–2.7) μm (av. = 5.2 × 2.2 μm, n = 150), arranged in
imbricate chains that may collapse into slimy masses.
Culture characteristics: Colonies on OA reaching 34–52 mm
diam after 7 d at 25 °C in darkness, at, irregular margin, aerial
mycelium moderate, felty to cottony, whitish, reverse concolourous.
Colonies on PDA reaching 36–48 mm diam, with entire margin,
aerial mycelium moderate, felty, whitish, reverse concolourous.
Colonies on SNA reaching 21–24 mm diam, at, irregular margin,
aerial mycelium sparse, felty, nely granular, whitish, reverse
concolourous.
Notes: Clonostachys parasporodochialis forms a sister lineage
to C. sporodochialis (Figs 1, 2). Substitutions in sequences
distinguish both species. Clonostachys parasporodochialis
(CBS 192.93) has clearly different ITS (99.0 % identity, with 5 bp
differences), LSU (99.6 %, 3 bp), RPB2 (95.8 %, 32 bp), and TEF1
(99.3 %, 6 bp) sequences when compared with C. sporodochialis
(CBS 101921). However, both species produce morphologically
similar sporodochia in cultuers and no mononematous dimorphic
conidiophores.
Clonostachys penicillata L. Zhao, Crous & Schroers, sp. nov.
MycoBank MB 848492. Fig. 30.
Etymology: Name refers to its penicillate conidiophores.
Typus: Germany, Freiburg, forest soil, unknown date and collector
(holotype designated here CBS H-25145, ex-type living culture
CBS 729.87).
Sexual morph unknown. Asexual morph. Conidiophores mono-
morphic, mononematous, penicillate, arising from the colony
surface or sparse aerial mycelium, frequently branched, forming
independent side-branches, branches bi- to quaterverticillate,
divergent at acute angles, sometimes with setose extensions;
stipes up to 130 μm high, 4.0 μm wide at base; branching part ca.
140 μm high, penicilli of independent units around 30–100 μm high,
up to 90 μm wide; terminal phialides in adpressed whorls of up
to six, narrowly ask-shaped, (7.0–)8.0–11.0(–12.0) μm long, (1.2–
)1.6–2.3(–2.5) μm wide at base, (1.9–) 2.0–2.8 μm at widest point,
0.8–1.2(–1.3) μm wide near aperture (n = 50); intercalary phialides
formed solitarily below whorls of terminal phialides, conidiogenous
pegs to 4 μm long. Conidia aseptate, hyaline, smooth, ellipsoid to
cylindrical, almost straight, with or without laterally displaced hilum,
(4.5–)5.0–6.8(–7.6) × (1.6–)1.7–2.2(–2.4) μm (av. = 5.9 × 2.0 μm, n
= 100), arranged in imbricate chains or columns.
Culture characteristics: Colonies on OA reaching 37–41 mm diam
after 7 d at 25 °C in the darkness, with entire margin, aerial mycelium
moderate, felty to cottony, whitish, reverse concolourous. Colonies on
PDA reaching 35–38 mm diam, with crenate margin, aerial mycelium
moderate, felty to cottony, yellowish white, reverse concolourous.
Colonies on SNA reaching 29–30 mm diam, with entire margin, aerial
mycelium sparse, felty, whitish, reverse concolourous.
Additional materials examined: Netherlands, Gelderland Province,
Schovenhorst near Putten, decaying wood, unknown date and collector,
culture CBS 653.70; Utrecht Province, Bilthoven, soil, 2017, B. de Bruin,
culture CBS 148211 = JW34009.
Notes: According to the phylogenetic analyses in the current study,
C. penicillata and C. compactiuscula form a statistically supported
sister group relationship (Figs 1, 2). Clonostachys penicillata (CBS
729.87) and C. compactiuscula (CBS 913.97) share a low RPB2
(96.8 %, 24 bp) and TEF1 (98.0 %, 16 bp) sequence similarity
and a higher ITS (99.6 % identity, with 2 bp differences) and LSU
(99.5 %, 4 bp) similarity. Slightly smaller conidia in C. penicillata
may distinguish these two closely related species; av. 5.9 × 2.0
μm in C. penicillata, vs av. 6.6 × 2.2 μm in C. compactiuscula.
The two species occupy an isolated phylogenetic position (Fig.
2) as relatedness to the morphologically differing C. ralfsii is not
supported (Figs 1, 2). Clonostachys compactiuscula occurs in
temperate and tropical regions. However, its sexual morph is often
seen in North America, while sexual morphs of the majority of
Clonostachys species are typically seen in (sub)tropical regions.
Clonostachys reniformis L. Zhao & Crous, sp. nov. MycoBank
MB 848493. Fig. 31.
Etymology: Name refers to the reniform shape of its conidia.
Typus: Unknown, substrate, date and collector unknown (holotype
designated here CBS H-25152, ex-type culture CBS 695.86).
Sexual morph unknown. Asexual morph. Conidiophores dimorphic.
Primary conidiophores mononematous, formed throughout the
colony, dominating towards the margin, arising from the agar
surface or from aerial hyphae, narrowly penicillate, divergent or
adpressed, mono- to terverticillate; stipe 40–150 μm long, 2.5–4.0
μm wide at base; penicilli to 40–120 μm high; phialides in apical
whorls of to four, also solitary, straight, cylindrical, slightly tapering
towards the tip, with a visible collarette, (18.4–)19.5–35.6(–39.5)
μm long, (1.4–)1.6–2.5(–2.9) μm wide at base, (1.0–)1.2–1.6(–
1.7) μm wide near aperture (n = 60). Secondary conidiophores
broadly penicillate, mostly formed from strands of aerial mycelium,
mononematous to sporodochial, ter- to quaterverticillate or more
frequently branched, branches almost divergent or sometimes
adpressed, phialides almost adpressed; stipe 20–70 μm long,
2.0–4.5 μm wide at base; penicilli divergently branched, up to 80
μm high, 30–90 μm wide; terminal phialides in loose whorls of 2–5,
straight to slightly curved, ask-shaped, widest in the lower third or
almost cylindrical, slightly tapering in the upper part towards the tip,
without a visible collarette (10.2–)10.8–18.0(–18.6) μm long, (1.3–)
1.6–2.5(–2.7) μm wide at base, (2.1–)2.3–2.8(–3.0) μm at widest
point, 1.0–1.3 μm wide near aperture (n = 50); intercalary phialides
formed rarely, solitarily below whorls of terminal phialides. Conidia
aseptate, hyaline, smooth, reniform, curved, some asymmetric with
one more attened side with a laterally displaced hilum, distally
broadly rounded, (4.0–)4.4–6.3(–8.2) × (2.2–)2.4–3.2(–3.6) μm (av.
= 5.1 × 2.7 μm, n = 150), arranged in imbricate chains.
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Zhao et al.
Culture characteristics: Colonies on OA reaching 46–48 mm diam
after 7 d at 25 °C in darkness, at, with crenate margin, aerial
mycelium moderate, nely to coarsely granular, felty to cottony,
yellowish white to pale yellow, reverse yellowish. Colonies on PDA
reaching 46–51 mm diam, at, with crenate margin, aerial mycelium
abundant, felty, pale yellow, with greenish tinge diffusing into the
medium outside the colony, reverse yellowish. Colonies on SNA
reaching 35–37 mm diam, at, with entire margin, aerial mycelium
sparse, nely granular, felty, whitish, reverse concolourous.
Notes: According to the phylogenetic analysis in the current
study, C. reniformis groups with C. viticola and C. swieteniae
(Figs 1, 2). Clonostachys reniformis differs from C. viticola (CAA
944) in TUB2 (98.9 % identity, with 5 bp differences), and from C.
swieteniae in ITS (99.4 % identity, with 3 bp differences) and LSU
(99.5 % identity, 4 bp). Morphologically, C. reniformis differs from
C. viticola in producing longer primary conidiophores (stipe 40–
150 μm, penicilli 40–120 μm in C. reniformis vs 70.5 ± 17.9 μm
in C. viticola). Clonostachys reniformis can be distinguished from
C. swieteniae by its shorter and narrower stipes of secondary
conidiophores (20–70 × 2.0–4.5 vs 130–200 × 5.0–8.0 μm), and
curved conidia with clearly laterally displaced hila, while hila are
typically central in C. swieteniae (Perera et al. 2020).
Clonostachys rosea (Link) Schroers et al., Mycologia 91: 369.
1999.
Basionym: Penicillium roseum Link, Mag. Gesell. Naturf. Freunde,
Berlin 7: 37. 1816.
Synonyms: Gliocladium roseum Bainier, Bull. Soc. Mycol. Fr. 23:
111. 1907.
Fig. 30. Clonostachys penicillata (ex-type CBS 729.87). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Conidiophores. G, H. Conidia. Black
arrows indicate intercalary phialides. Scale bars = 10 μm.
259www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Clonostachys araucaria var. confusa Pinkerton, Ann. Mo. Bot. Gdn
23: 44. 1936.
Gliocladium aureum Rader, Phytopathology 38: 450. 1948.
Gliocladium verticillioides (G.A. Newton) Pidoplitschka, Sugar Ind.
Sci. Notes Kieff 10: 365. 1930.
Nectria aureofulva Cooke & Ellis, Grevillea 7: 8. 1878.
Bionectria aureofulva (Cooke & Ellis) Schroers & Samuels, Z.
Mykol. 63: 153. 1997.
Nectria gliocladioides Smalley & H.N. Hansen, Mycologia 49: 533.
1957.
Verticillium intertextum I. Isaac & R.R. Davies, Trans. Br. Mycol.
Soc. 38: 155. 1955.
Clonostachys araucaria Corda, Pracht-Fl. Eur. Schimmelbild.: 31.
1839.
Stachylidium araucaria (Corda) Bonord., Handb. Allgem. Mykol.
(Stuttgart): 110. 1851.
Verticillium pulverulentum Gouw., Meded. Phytopath. Labor. Willie
Commelin Scholten Baarn 8: 55. 1924.
Verticillium foexii J.F.H. Beyma, Meded. Phytopath. Labor. Willie
Commelin Scholten Baarn 12: 31. 1928.
Fig. 31. Clonostachys reniformis (ex-type CBS 695.86). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Primary conidiophores. F, G.
Secondary conidiophores. H, I. Conidia. Scale bars: D, F = 50 μm; E, G–I = 10 μm.
260
Zhao et al.
Clonostachys gneti Oudem., Verslag. Meded. K. Akad. Wetensch.,
Afd. Natuurk., ser. 3 7: 321. 1890.
Clonostachys populi Harz, Bull. Soc. Imp. Nat. Moscou 44: 116. 1871.
Clonostachyopsis populi (Harz) Höhn., Sber. Akad. Wiss. Wien,
Math.-naturw. Kl., Abt. 1 116: 149. 1907.
Nectria phyllostachydis Hara [as ‘Nectoria’], Bot. Mag., Tokyo 27:
247. 1913.
Gliocladium cholodnyi Pidopl., Fungus ora of coarse fodder: 196.
1953
Dendrodochium strictum D. Sacc., Atti Soc. Veneto-Trent. Sci. Nat.
2: 29. 1896.
Verticillium epimyces Berk. & Broome, Ann. Mag. Nat. Hist., Ser.
27: 102. 1851.
Torula rosea Preuss, in Sturm, Deutschl. Fl., 3 Abt. (Pilze Deutschl.)
6: 25. 1848.
Oospora rosea (Preuss) Sacc. & Voglino, in Saccardo, Syll. Fung.
(Abellini) 4: 18. 1886.
Alysidium roseum (Preuss) Kuntze, Revis. gen. pl. (Leipzig) 3:
442.1898.
Clonostachys populi var. aesculi Oudem., Ned. kruidk. Archf, 3 sér.
2: 1121.1904.
Clonostachys araucaria var. compacta Preuss, Linnaea 25: 727.
1853.
Verticillium stigmatellum Berk. & M.A. Curtis, in Berkeley, Grevillea
3: 110. 1875.
Dendrodochium densipes Sacc. & Ellis, in Ellis & Everhart, J.
Mycol. 4: 117. 1888.
Nectria congesta Sacc., Michelia 2: 256. 1881.
Dialonectria congesta (Sacc.) Cooke, Grevillea 12: 110. 1884.
Sphaeria ochroleuca Schwein., Trans. Am. phil. Soc., New Series
4: 204. 1832 (1834).
Nectria ochroleuca (Schwein.) Berk., Grevillea 4: 16. 1875.
Bionectria ochroleuca (Schwein.) Schroers & Samuels, Z. Mykol.
63: 151. 1997.
Clonostachys aranearum Wan H. Chen, et al., Mycosystema 35:
1063. 2016.
Description and illustration: Schroers (2001).
Typus: Netherlands, soil, on buried sclerotia of Sclerotinia minor, date
unknown, A. van Zaayen & W. Gams (neotype CBS H-7917, ex-type
culture CBS 710.86).
Additional materials studied: China, Guizhou, Qianlingshan Park, spider,
Jun. 2016, W.H. Chen, culture QLS0625clo (type of Clonostachys
aranearum). France, rotten cardboard, date unknown, G. Bainier, culture
CBS 100502 (type of Gliocladium roseum Bainier). USA, on decaying bulb
of Lilium auratum, Jan. 1955, E.B. Smalley, culture CBS 194.57 (ex-type
strains of Nectria gliocladioides Smalley & Hansen); New York, causing
lesions on stored carrot roots, date unknown, W.E. Rader, culture CBS
226.48 (ex-type strain of Gliocladium aureum).
Notes: Clonostachys rosea is the most commonly isolated species
in the genus, with complex taxonomical and nomenclatural history
(Schroers 2001). Based on our phylogenetic analyses, the recently
described C. aranearum (Chen et al. 2016) is conspecic with C.
rosea and included as synonym of C. rosea.
Clonostachys vacuolata L. Zhao, Crous & Schroers, sp. nov.
MycoBank MB 848494. Fig. 32.
Etymology: Name refers to vacuoles formed by this species inside
phialides.
Typus: Venezuela, Edo. Miranda: Parque Nacional Guatopo, trail between
Agua Blanca and La Cruceta, 10°03’N, 66°26’W N, 500–600 m alt.,
bark, 27–30 Nov. 1990, G.J. Samuels VE-7664, B. Hein & S.M. Huhndorf
(holotype designated here CBS H-25143, ex-type living culture CBS
191.93).
Sexual morph from natural specimen (not shown). Asci 48–74.4 ×
9.5–16.6 μm. Ascospores 1-septate, hyaline, striate, ellipsoid, 13.2–
14.7–17.6 × 4.4–5.4–6.3 μm (n=23). Asexual morph. Conidiophores
monomorphic, densely aggregated, conuent, formed in pustules,
not sporodochial, arising from the agar surface or sparse aerial
mycelium, branches divergent, bi- to quaterverticillate, phialides
somewhat divergent; stipe 10–55 μm long, 1.8–3.8 μm wide at
base; penicilli 20–65 μm high, up to 70 μm wide, frequently higher
than the length of the stipe; terminal phialides generally in whorls
of 2–5, straight or slightly curved, cylindrical, or ask-shaped, with
vacuoles, widest in the lower third or middle, slightly tapering in the
upper part towards the tip, collarette absent, (7.0–)9.4–21.8(–25.7)
μm long, (1.5–)1.7–2.4(–2.6) μm wide at base, (2.1–)2.2–2.9(–3.0)
μm at widest point, (1.0–)1.1–1.5(–1.6) μm wide near aperture (n
= 100); intercalary phialides rare, formed below whorls of terminal
phialides. Conidia aseptate, hyaline, smooth, ellipsoid to obovoid,
straight, or both ends broadly rounded, without visible hilum, (5.1–)
5.4–6.8(–7.7) × (2.5–)2.7–3.1(–3.3) (av. = 6.0 × 2.9 μm, n = 100),
arranged in imbricate chains that may collapse into slimy masses.
Culture characteristics: Colonies on OA reaching 37–40 mm diam
after 7 d at 25 °C in darkness, with entire margin, aerial mycelium
sparsely developed, felty, nely to coarsely granular, yellowish
white, reverse concolourous. Colonies on PDA reaching 38–45 mm
diam, with entire margin, and aerial mycelium moderate, felty, nely
to coarsely granular, pale yellow, reverse concolourous. Colonies
on SNA reaching 35–37 mm diam, with entire margin, aerial
mycelium sparse, felty, whitish, reverse concolourous.
Notes: Clonostachys vacuolata clusters together with C.
longiphialidica, C. pallens, C. venezuelae and C. ellipsoidea (Figs
1, 2). It can be morphologically distinguished from C. longiphialidica
by producing shorter conidia [(5.1–)5.4–6.8(–7.7) μm vs (6.3–)6.7–
8.5(–9.3) μm]. Clonostachys vacuolata differs from C. ellipsoidea
in producing longer and narrower conidia (av. = 6.0 × 2.9 μm vs
av. = 5.3 × 3.5 μm). A morphological comparison with C. vacuolata
and C. pallens is difcult, because C. palllens was only described
based on a sexual morph (Forin et al. 2020), while we observed
only asexual morph characters in culture although working with
the same strain. Schroers (2001) led the specimen incorrectly
under C. grammicospora, as the longer ascospores distinguish VE-
7664 from this species. Sexual and asexual morphology suggest
classication of C. vacuolata in subgen. Zebrinella.
Clonostachys venezuelae L. Zhao, Crous & Schroers, sp. nov.
MycoBank MB 848495.
Etymology: Named after the country where the fungus was
collected, Venezuela.
Typus: Venezuela, Edo Merida, 7 km NE of Merida, ca. 4 km inside
San Javier del Valle resort, 24 Jul. 1971, K.P. Dumont & G.J. Samuels
(holotype designated here CBS H-25240, ex-type living culture CBS
107.87); specimen VE 2865 (NY), culture C.T.R. 71-349.
261www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Sexual morph known from natural specimen (not shown). Asci
63.0–90.0 × 10.0–16.0 μm (n = 14). Ascospores 1-septate, hyaline,
striate, ellipsoid, (16.0–)17.9(–21.0) × 5.0–6.1–7.0 μm (n = 22).
Asexual morph (not shown), from dried CMD culture, obtained from
isolated ascospores. Conidia (8.3–)13.1(–18.8) × (3.8–)5.5(–6.8)
μm (de Schroers) or (5.0–)10.3(–18.0) × (3.0–)5.0(–7.0) μm (de
Samuels). Deposited CBS cultures not sporulating. Clonostachys
venezuelae differs from its closest phylogenetic neighbours C.
pallens and C. ellipsoidea by unique nucleotide substitutions
and indels in the ve investigated loci (see direct sequence
comparisons deposited at doi: 10.6084/m9.gshare.22894592): C.
venezuelae and C. pallens: ITS position 24 (C), 43 (C), 44 (T), 45
(A, insertion), 52 (C), 53 (gap), 67 (gap), 111 (C), 142 (G), 143 (A),
149(C), 153 (C), 157 (C), 158 (C), 162 (gap), 171 (C), 192 (C), 207
(A, insertion). C. venezuelae and C. ellipsoidea: ITS position 44 (T,
insertion), 52 (C), 67 (gap), 69 (A), 111 (C), 143 (A), 150 (T), 153
(C), 158 (C), 162 (gap), 171 (C), 172 (T), 191 (C), 200 (A), 207 (A,
insertion), 277 (T), 359 (C), 403 (T), 429 (A), 430 (G), 431 (C), 494
Fig. 32. Clonostachys vacuolata (ex-type CBS 191.93). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia. Scale
bars = 10 μm.
262
Zhao et al.
(C), 496 (A), 516 (G), 519 (C), 522 (C), 548 (G), 552 (C, insertion);
LSU position 643 (A), 704 (C), 987 (A), 990 (A), 991 (G), 1 005 (T),
1 007 (T), 1 008 (C), 1 042 (T), 1 045 (T), 1 065 (C); RPB2 position
1 398 (T), 1 407 (C), 1 422 (T), 1 493 (G), 1 599 (A), 1 614 (T),
1 620 (T), 1 650 (C), 1 701 (C), 1 710 (G), 1 719 (A), 1 743 (G),
1 779 (A), 1 782 (C), 1 788 (T), 1 803 (A), 1 804 (C), 1 809 (C),
1 812 (A), 1 833 (C), 1 862 (C), 1 870 (C), 1 900 (G), 1 916 (T),
2 002 (C), 2 011 (G), 2 047 (T), 2 105 (C), 2 126 (C); TEF1 position
2 156 (G), 2 165 (T), 2 174(C), 2 180 (C), 2 186 (T), 2 198 (C),
2 255 (T), 2 375 (T), 2 391 (A), 2 392 (A), 2 393 (G), 2 414 (C),
2 416 (G), 2 426 (T), 2 432 (C), 2 447 (T), 2 454 (T), 2 468 (T),
2 469 (C), 2 591 (C), 2 528 (C), 2 585 (T), 2 645 (C), 2 652 (C),
2 748 (T), 2 749 (T), 2 752 (C), 2 840 (C), 2 846 (T), 2 914(A); TUB2
position 3 196 (A), 3 199 (C), 3 205–3 206 (gap), 3 207 (A), 3 209
(gap), 3 219 (G), 3 223 (C), 3 225 (C), 3 236 (A), 3 241 (G), 3 246
(G), 3 249 (T), 3 250 (T), 3 252 (A), 3 260 (T), 3 268 (A), 3 282 (T),
3 356 (C), 3 416 (C), 3 620 (C), 3 684 (A), 36 93 (T), 3 705 (C),
3 744 (C), 3 816 (C).
Notes: The CBS culture of C. venezuelae does not sporulate
anymore, but the phylogenetic analyses show no similarity of
this culture with other sequenced cultures (Figs 1, 2). However,
the asexual morph is known from a dried culture led together
with specimen VE 2865 studied by G.J. Samuels (see Samuels
1988b) and Schroers (2001). Both led the specimen under C.
(Nectria) subquaternata. Sexual and asexual morphology suggest
classication of C. venezuelae in subgen. Zebrinella.
DISCUSSION
In this study, we investigated 420 strains identied as Clonostachys
and allied genera based on morphological characters and
phylogenetic analyses. The current study presents the largest
sampling of Clonostachys ever subjected to multi-locus sequencing
analyses and provides a comprehensive phylogenetic backbone
and framework for future studies of Clonostachys.
Most cultures examined previously by Schroers et al. (1999b)
and Schroers (2000, 2001) were incorporated and newly compared
with so far unstudied isolates. Based on phylogenetic analyses
of ve loci, ITS, LSU, RPB2, TEF1, TUB2, and morphological
characters, a rich set of taxa were identied as new species of
Clonostachys, Mycocitrus, Nectriopsis, and Sesquicillium (Fig. 1)
and our data support that they all belong to the Bionectriaceae.
Sesquicillium microsporum differs from other Sesquicillium species
in size ranges of phialides and conidia (Samuels 1989) and a
myxomyceticolous lifestyle (Rogerson & Stephenson 1993). Our
phylogenetic analyses (Fig. 1) support an earlier hypothesis that
S. microsporum is a species of Nectriopsis (Schroers 2001), which
accommodates also other myxomyceticolous species (Samuels
1988a).
We claried that Clonostachys accommodates the subgenera
Astromata, Bionectria, Myronectria and Zebrinella with 19 new and
49 known species, while the genus Sesquicillium, with three new
species and eight new combinations was resurrected for subgenera
Epiphloea and Uniparietina. Although the purpose of describing
subgenera in Clonostachys/Bionectria was to delineate groups of
species with similar morphological characters, Schroers (2001)
reported that some but not all subgenera are mono- or paraphyletic
based on ITS and TUB2 sequences.
In the present study, the subgenus Astromata (Clade V in Fig.
1) is comprised of six species, including known species C. epichloe,
C. eriocamporesii, C. miodochialis and C. oligospora, which agrees
with the suppositions in the previous study of Schroers (2001), and
new species C. fujianensis and C. obovatispora. Most species of
this group form perithecia either directly on their substrata such
as fruiting bodies of other fungi or on clearly reduced stromata,
sporodochial asexual morphs and greenish pigmented conidial
masses, and conidia with a somewhat laterally protruding hilum,
that results in a somewhat clavate spore shape (Schroers 2001).
Within subgenus Bionectria (Clade VI in Fig. 1), the known
species C. compactiuscula, C. divergens, C. ralfsii, C. rogersoniana
and C. samuelsii and the new species C. bambusae , C. cylindrica, C.
hongkongensis and C. penicillata accumulate numerous nucleotide
differences in sequences (Figs 1, 2) when compared to others. All
these species cluster outside a well-supported clade comprising
the above discussed core group with homogeneous species of the
subgenus Bionectria characterised by dimorphic conidiophores
(Fig. 1: 90 % / 99 % / 1; Fig. 2: 92 % / 98 % / 0.96). Also see the
discussion under the taxonomic treatment of Clonostachys above.
The subgenus Myronectria (Clade III in Fig. 1), comprising two
species, C. pityrodes and C. buxicola, is fully supported (Fig. 1:
100 % / 100 % / 1; Fig. 2: 100 % / 100 % / 1). It accommodates
an individual branch within the supported Clonostachys clade
and is unrelated to the other subgenera. Schroers (2001) erected
subgenera Myronectria and Astromata due to the circumstance
that they produce synnematous conidiophores and rather dark
greenish pigmented conidial masses. Clonostachys pityrodes is
also the only Clonostachys species forming somewhat curved,
broadly rounded, and comparatively large ascospores.
The subgenus Zebrinella forms a monophyletic lineage in
Clonostachys (Clade IV in Fig. 1: 93 % / 98 % / 1). Our phylogenetic
analyses agree well with the previous study of Schroers (2001),
who placed C. chlorina, C. grammicospora, C. grammicosporopsis,
C. intermedia, C. levigata, C. lucifer and C. subquaternata into
the subgenus Zebrinella. The subgenus, is, however, also well-
supported on the basis of morphological characters. Clearly striate
ascospores and perithecial walls showing two regions distinguish
it from species of the subgenus Bionectria typically forming warted
ascospores and perithecial walls consisting of three regions.
The subgenus Epiphloea is shown to be polyphyletic in the
present study (Clades labelled I in Fig. 1), with the majority of
species now accepted in Sesquicillium, while the sesquicillium-like
Clonostachys setosa is accepted in Clonostachys sensu stricto.
The subgenus Uniparietina (Clade II in Fig. 1) is represented by
Sesquicillium buxi in the present study.
Species of Sesquicillium are characterised by macronematous
conidiophores that form typically one, rarely two, intercalary
phialides just below a single terminal phialide in their penicilli.
They should be distinguished from intercalary phialides typically in
micronematous conidiophores that look like hyphal cells possessing
a lateral conidiogenous peg, for example, in some Acremonium
(Gams 1971) and Nectria sensu stricto species (Seifert 1985). This
revision, however, shows that high numbers of intercalary phialides
are not only formed by species of Sesquicillium but also by C.
setosa and Clonostachys sp. CBS 496.90, both classied therefore
in subgenus Epiphloea by Schroers (2001). In addition, there
are also quite many Clonostachys species producing intercalary
phialides at least sporadically [see C. australiana, C. longiphialidica,
C. vacuolata, C. ellipsoidea, C. penicillata (this study), C.
agarwalii, C. verrucispora, C. compactiuscula, C. rogersoniana,
C. grammicospora, C. levigata, and C. chlorina (Schroers 2001)].
It is thus the occurrence of intercalary phialides that link the
genera Clonostachys and Sesquicillium morphologically (see also
263www.studiesinmycology.org
Revising ClonostaChys and allied geneRa in BioneCtriaCeae
Schroers 2000: g. 5). Because Clonostachys and Sesquicillium
are phylogenetically closely related sister genera (Fig. 1) it is
not far-fetched assuming that their intercalary phialides are of
homologous nature and perhaps plesiomorphic. The ancestor of
these two genera may have possessed conidiophores forming
solitary intercalary phialides below terminal phialides.
Recognition of Sesquicillium distinguishes species typically
forming a reduced perithecial stroma supercially on plant tissue
from species in Clonostachys often forming well-developed, through
bark erumpent stromata. Also, the asexual morphs, when observed
on the natural substratum, are typically mononematous and formed
supercially in Sesquicillium, while they are often sporodochial and
formed on stromata in Clonostachys. Sesquicillium accommodates
a set of species that inhabit leaves, while this lifestyle is rarely
seen in Clonostachys, where species may occur as endophytes in
woody hosts and subcortical colonisers in recently dead trees, or on
fungal hosts associating woody plants. It is possible that the ability
to produce stromata erumpent through bark may have evolved in
woody hosts (Clonostachys), while stromata were ecologically not
required for the supercial lifestyle on plant tissues including leaves
(Sesquicillium).
However, Bionectria vesiculosa (Luo & Zhuang 2010), here
conrmed on the basis of available nrDNA sequences as a species
of Clonostachys, clearly contradicts the concept mentioned
above. The sexual morph of this species is astromatous,
formed supercially on leaves, and, remarkably, its perithecial
wall region consists of a single region only. A similar situation
is encountered in Sesquicillium buxi, whose sexual morph
consists of astromatous perithecia and a single perithecial wall
region (Schroers 2001: g. 96), nearly indistinguishable from C.
vesiculosa (Luo & Zhuang 2010: g. 1). While it was the asexual
morph linking Nectriella coronata rst to Sesquicillium (Gams
1968) and then to Clonostachys (Schroers 2000: g. 5; Schroers
2001), ITS sequences supported classication of C. vesiculosa in
Clonostachys (Luo & Zhuang 2010; Supplementary Fig. S1), while
LSU places it in an unresolved polytomy among Sesquicillium and
Clonostachys (Supplementary Fig. S2). Occurrence of supercially
formed perithecia with a simple perithecial wall in both Sesquicillium
and Clonostachys may allow hypothesising that these character
patterns are (i) plesiomorphic and that (ii) also the common ancestor
of Sesquicillium and Clonostachys formed perithecia supercially
on plant tissue, perhaps on leaves. Most importantly, however, this
concept allows hypothesising that (iii) a diversication of morphs,
including perithecial wall anatomies and perithecial wall and stroma
interfaces occurred then independently within Sesquicillium and
Clonostachys and (iv) this morphological diversication allowed
occupation of woody host-related ecological niches and perhaps
even mycoparasitism, especially in Clonostachys.
Species of Clonostachys are widely distributed all over the
world, with the highest known species diversity occurring in tropical
regions. These species are commonly found in soils, litter, and
dead plant substrata as saprotrophs. They have also been reported
as endophytes and epiphytes of living plants (Torcato et al. 2020).
Destructive mycoparasitism is especially well documented for C.
rosea and a mycophilic habit was inferred for several Clonostachys
species that formed their (a)sexual morph structures just on top of
other wood associating fungi (Samuels 1976, Schroers et al. 1999b,
Schroers 2001). However, while C. rosea has been exhaustively
studied (see below), hardly any data are available for the many
other species of Clonostachys and Sesquicillium. Sporadic
reports have also emphasised parasitic interactions of C. rosea
with myxomycetes, nematodes, ticks, molluscs, and leafhoppers
(reviewed by Schroers 2001, Toledo et al. 2006, Zhang et al. 2008).
However, these reports may rather illustrate the opportunistic
nature of this species.
Clonostachys rosea was reported as an aggressive mycoparasite
in the late 1950s (Barnett & Lilly 1962), and initial attempts to use it for
biological control of plant diseases soon followed (Shigo 1958). Since
then, there has been a wealth of new knowledge emerging concerning
the ecology, physiology and genetics of C. rosea, and its applied
use as a biological control agent (BCA) including its formulation,
application strategy, efciency and safety (Jensen et al. 2022). The
biocontrol mechanisms of C. rosea against plant pathogenic fungi
are primarily attributed to direct parasitism, secretion of fungal cell
wall degrading enzymes, production of secondary metabolites such
as antibiotics and toxins, and induction of plant resistance (Sun et al.
2020). Although most reports of biological control of plant diseases
involves the species C. rosea, there is evidence to suggest that certain
strains from other, closely related species, also possess biocontrol
properties, such as C. byssicola, C. chloroleuca, C. rhizophaga and
C. solani (García et al. 2003, Krauss et al. 2013, Sun et al. 2017,
Broberg et al. 2021).
Biological control of plant pathogens via microbial antagonists
is one promising component in future disease control strategies
(Karlsson et al. 2015). As large-scale genomic sequencing
becomes economically viable, the impact of single nucleotide
polymorphisms (SNPs) on biocontrol-associated phenotypes can
be easily studied across entire genomes of fungal populations.
Recently, genome assemblies of four C. rosea strains have been
published (Karlsson et al. 2015, Sun et al. 2015, Liu et al. 2016,
Broberg et al. 2018, Wang 2021). The available genome resources
are valuable for identifying biocontrol-related genes what will
improve our understanding of the biological control ability of C.
rosea and related species.
The present study should serve as phylogenetic backbone
for future taxonomic studies of Clonostachys. Further studies are
presently underway to generate full genome sequences of the
species studied here in an attempt to identify additional taxa that
have biocontrol properties of potential interest to industry.
ACKNOWLEDGEMENTS
We would like to thank the China Scholarship Council (CSC) for nancial
support to L.Z. (CSC student number: 202006510014) and the Slovenian
Research Agency (ARRS) for nancial support to H.-J.S. (grant no. P4-
0072; Agrobiodiversity program). P.W.C. is grateful to the European Union’s
Horizon 2020 research and innovation program (RISE) under the Marie
Skłodowska-Curie grant agreement No. 101008129, project acronym
‘Mycobiomics’, and the Dutch NWO Roadmap grant agreement No. 2020/
ENW/00901156, project ‘Netherlands Infrastructure for Ecosystem and
Biodiversity Analysis – Authoritative and Rapid Identication System for
Essential biodiversity information’ (acronym NIEBA-ARISE) for funding.
The authors are grateful to Dr Gary J. Samuels, who collected most of
the holomorphs included in this study. Without his forward sight to deposit
specimens and cultures in international biological resource centres, this
study would not have been possible.
DECLARATION ON CONFLICT OF INTEREST
The authors declare that there is no conict of interest.
264
Zhao et al.
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Supplementary Material: https://studiesinmycology.org/
Fig. S1. Phylogenetic tree of representatives of the Bionectriaceae and
outgroups resulting from a RAxML analysis of aligned ITS sequences.
Bootstrap support values > 50 % are indicated at nodes. Flammocladiella
aceris CBS 138906, F. decora CBS 142776, F. anomiae CLL 16017,
Tilachlidium brachiatum CBS 363.97, and T. brachiatum CBS 505.67 are
used as outgroup. Scale bar represents expected number of changes per
site. “T” indicates ex-type strains.
Fig. S2. Phylogenetic tree of representatives of the Bionectriaceae and
outgroups resulting from a RAxML analysis of aligned LSU sequences.
Bootstrap support values > 50 % are indicated at nodes. Flammocladiella
aceris CBS 138906, F. decora CBS 142776, F. anomiae CLL 16017,
Tilachlidium brachiatum CBS 363.97, and T. brachiatum CBS 505.67 are
used as outgroup. Scale bar represents expected number of changes per
site. “T” indicates ex-type strains.
Fig. S3. Phylogenetic tree of representatives of the Bionectriaceae and
outgroups resulting from a RAxML analysis of aligned RPB2 sequences.
Bootstrap support values > 50 % are indicated at nodes. Flammocladiella
aceris CBS 138906 and F. anomiae CLL 16017, Tilachlidium brachiatum
CBS 363.97 and T. brachiatum CBS 505.67 are used as outgroup. Scale
bar represents expected number of changes per site. “T” indicates ex-type
strains.
Fig. S4. Phylogenetic tree of representatives of the Bionectriaceae and
outgroups resulting from a RAxML analysis of aligned TEF1 sequences.
Bootstrap support values > 50 % are indicated at nodes. Flammocladiella
aceris CBS 138906 and F. anomiae CLL 16017 are used as outgroup.
Scale bar represents expected number of changes per site. “T” indicates
ex-type strains.
Fig. S5. Phylogenetic tree of Clonostachys species and outgroups
resulting from a RAxML analysis of aligned ITS sequences. Bootstrap
support values > 50 % are indicated at nodes. Acremonium alternatum
CBS 407.66 and A. stroudii CBS 138820 are used as outgroup. Scale bar
represents expected number of changes per site. “T” indicates ex-type
strains.
Fig. S6. Phylogenetic tree of Clonostachys species and outgroups
resulting from a RAxML analysis of the aligned LSU sequences. Bootstrap
support values > 50 % are indicated at nodes. Acremonium alternatum
CBS 407.66 and A. stroudii CBS 138820 are used as outgroup. Scale bar
represents expected number of changes per site. “T” indicates ex-type
strains.
Fig. S7. Phylogenetic tree of Clonostachys species and outgroups resulting
from a RAxML analysis of aligned RPB2 sequences. Bootstrap support
values (1 000 replicates, GTR-GAMMA model) > 50 % are indicated at
nodes. Acremonium alternatum CBS 407.66 and A. stroudii CBS 138820
are used as outgroup. Scale bar represents expected number of changes
per site. “T” indicates ex-type strains.
Fig. S8. Phylogenetic tree of Clonostachys species and outgroups resulting
from a RAxML analysis of aligned TEF1 sequences. Bootstrap support
values (1 000 replicates, GTR-GAMMA model) > 50 % are indicated at
nodes. Acremonium alternatum CBS 407.66 and A. stroudii CBS 138820
are used as outgroup. Scale bar represents expected number of changes
per site. “T” indicates ex-type strains.
Fig. S9. A detailed view of the collapsed clade (Clonostachys rosea and
Clonostachys rosea f. catenulata) at the bottom of the phylogenetic tree
presented in Fig. 2.
Table S1. Strains used in this study with details of their host, location, and
GenBank accessions numbers
