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Fungi
Journal of
Article
Molecular Phylogeny and Morphology of
Amphisphaeria (=Lepteutypa) (Amphisphaeriaceae)
Milan C. Samarakoon 1,2,3,4,5 , Sajeewa S. N. Maharachchikumbura 4, Jian-Kui (Jack) Liu 4,
Kevin D. Hyde 3,6, Itthayakorn Promputtha 1, 2, * and Marc Stadler 7, *
1Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand;
samarakoon_m@cmu.ac.th
2Research Center in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University,
Chiang Mai 50200, Thailand
3Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand;
coe-fungal@mfu.ac.th
4School of Life Science and Technology, University of Electronic Science and Technology of China,
Chengdu 611731, China; sajeewa83@yahoo.com (S.S.N.M.); liujiankui@uestc.edu.cn (J.-K.L.)
5Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand
6Innovative Institute of Plant Health, Zhongkai University of Agriculture and Engineering, Haizhu District,
Guangzhou 510225, China
7Department of Microbial Drugs, Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstrasse 7,
38124 Braunschweig, Germany
*Correspondence: itthayakorn.p@cmu.ac.th (I.P.); marc.stadler@helmholtz-hzi.de (M.S.);
Tel.: +66-833-3443-92 (I.P.); +49-531-6181-4240 (M.S.)
Received: 24 August 2020; Accepted: 14 September 2020; Published: 17 September 2020
Abstract:
Amphisphaeriaceous taxa (fungi) are saprobes on decaying wood in terrestrial, mangrove,
and freshwater habitats. The generic boundaries of the family have traditionally been based on
morphology, and the delimitation of genera has always been challenging. Amphisphaeria species have
clypeate ascomata and 1-septate ascospores and a coelomycetous asexual morph. Lepteutypa is different
from Amphisphaeria in having eutypoid stromata and more than 1-septate ascospores. These main
characters have been used for segregation of Lepteutypa from Amphisphaeria for a long time. However,
the above characters are overlapping among Amphisphaeria and Lepteutypa species. Therefore, here we
synonymized Lepteutypa under Amphisphaeria based on holomorphic morphology and multigene
phylogeny. Further, our cluster analysis reveals the relationship between seven morphological
traits among Amphisphaeria/Lepteutypa species and suggests those morphologies are not specific to
either genus. Three new species (i.e., Amphisphaeria camelliae,A. curvaticonidia, and A. micheliae) are
introduced based on morphology and LSU-ITS-RPB2-TUB2 phylogenies. Furthermore, the monotypic
genus Trochilispora, which had been accepted in Amphisphaeriaceae, is revisited and synonymized
under Hymenopleella and placed in Sporocadaceae.
Keywords: 3 new taxa; 6 new combinations; asexual morph; Sporocadaceae; taxonomy
1. Introduction
Amphisphaeria, the type genus of Amphisphaeriaceae, was introduced by Cesati and De Notaris [
1
].
Amphisphaeria has immersed, clypeate, globose, periphysate ostiolate ascomata; visible as raised,
blackened, circular dots on the host surface; and several peridial layers with inner hyaline and
outer brown cells; filamentous, septate, flexuous paraphyses; 8-spored, cylindrical asci with J+or
J
−
, discoid, tubular or wedge-shaped apical ring; and 1-septate, ellipsoidal, brown ascospores [
1
,
2
].
The coelomycetous asexual morph has solitary or aggregated, globose, dark brown conidiomata with
J. Fungi 2020,6, 174; doi:10.3390/jof6030174 www.mdpi.com/journal/jof
J. Fungi 2020,6, 174 2 of 27
a thick-walled peridium, septate, branched, hyaline conidiophores, elongated, conical, thin-walled,
septate, hyaline, annellidic conidiogenous cells and hyaline, elongate-fusiform, 1-celled, smooth-walled
conidia [
3
]. Wang et al. [
2
] revised Amphisphaeria based on herbarium specimens and accepted 12 species.
Following consecutive studies, 19 Amphisphaeria species have been accepted [4–9].
Lepteutypa was introduced by Petrak [
10
] with its type L. fuckelii (
≡
Massaria fuckelii) which was
collected from Germany. Lepteutypa is characterized by scattered, weakly developed eutypoid stroma
with a single opening; immersed to semi-immersed, single or clustered perithecia, papillate or
short conical ostioles; a cellular peridium; broad, easily broken and numerous paraphyses;
8-spored, cylindrical asci; and oblong, multicellular, pigmented ascospores with thick epispore [
10
].
Fifteen Lepteutypa species have been published based on above morphology or morpho-phylogenetic
studies [
9
,
11
–
17
]. Jaklitsch et al. [
16
] proposed a neotype for L. fuckelii on Tilia cordata from Belgium,
which has aggregated perithecial colonies and 5-septate ascospores. However, in the original generic
description, L. fuckelii is characterized by eutypoid stroma as a key character in Lepteutypa [
10
].
Nevertheless, the aggregated perithecia in the generic description by Petrak [
10
] were considered to be
an eutypoid stroma [16].
Petrak [
18
] introduced Lepteutypella, which is typified by L. allospora (
≡
Cladosphaeria allospora).
The genus has poorly developed, small, eutypoid, spotty stromata, thin, sub-hyaline, fibrous tissue layer
around the ostiole as a distinct clypeus, perithecia with a conical ostiole, broad, simple, very delicate
paraphyses (metaphyses), 8-spored, delicate, cylindrical asci and broadly ellipsoidal, multicellular,
dark coloured, thick episporous ascospores. However, Lepteutypella has hitherto been regarded as a
synonym of Lepteutypa [19].
The presence of multiseptate ascospores in Lepteutypa is a key indicator to differentiate Lepteutypa
from Amphisphaeria species. However, some Lepteutypa species, such as L. uniseptate, have 1-septate
ascospores, which are morphologically similar to Amphisphaeria, but were phylogenetically related to
Lepteutypa in previous studies [
9
,
16
,
17
]. Thus, the ascospore septation is not a specific characteristic
for the generic delimitation of two genera. The correlations between concepts based on morphology
vs. phylogeny of Amphisphaeria and Lepteutypa make taxonomic instability of generic delimitation,
which needs further studies.
In this study, we evaluate the morphology and phylogeny of accepted species in
Amphisphaeria and Lepteutypa, and synonymize Lepteutypa under Amphisphaeria. Furthermore, as part
of our continuous studies on inconspicuous xylariaceous taxa from China and Thailand, we introduce
three novel taxa which are associated with senescent plant substrates.
V.P. Abreu, A.W.C. Rosado & O.L. Pereira introduced Trochilispora in Hyde et al. [
20
], typified by
T. schefflerae from Brazil. The ITS-LSU phylogenies in Hyde et al. [
20
] showed that T. schefflerae has an
affinity to Hymenopleella hippophaeicola. However, Trochilispora was introduced in Amphisphaeriaceae
with an uncertainly of the phylogenetic position. Here, we reconstruct the phylogeny including
selected taxa and provide evidence for the phylogenetic placement of T. schefflerae.
2. Materials and Methods
2.1. Sample Collection, Isolation and Morphological Studies
We have been focusing on collecting microfungi from senescent twigs, branches, and culms in
both mono- and dicotyledonous plants with emphasis on xylarialean species. Surveys from 2017
−
2019
investigated several interesting inconspicuous xylariaceous species from northern Thailand [
7
,
21
].
In this study, we collected samples from northern Thailand and Sichuan Province, China. Specimens
were placed in paper bags and dried at room temperature. External and internal macro-micro structural
observations were made as described in Samarakoon et al. [7].
Single spore isolations were carried out as detailed in Chomnunti et al. [
22
] and germinating
spores were transferred aseptically to potato dextrose agar (PDA). The cultures were incubated at
25–30
◦
C for 4–6 weeks and colonies observed frequently. The type specimens were deposited in the
J. Fungi 2020,6, 174 3 of 27
Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand and the Cryptogamic Herbarium,
Kunming Institute of Botany, Academia Sinica (HKAS), Kunming, China. Ex-type living cultures
were deposited in the Culture Collection at Mae Fah Luang University (MFLUCC) and International
Collection of Microorganisms from Plants (ICMP), New Zealand. Facesoffungi and MycoBank numbers
are provided as explained in Jayasiri et al. [23] and MycoBank (http://www.MycoBank.org).
2.2. DNA Extraction, PCR Amplification and Sequencing
Fresh mycelia were scraped from two-week-old cultures on PDA using sterilized scalpels.
Cleaned ascomata were picked up from specimens using sterilized needles. Genomic DNA was
extracted using the Ezup DNA Extraction Kit (Sangon Biotech, Shanghai, China) according to the
manufacturer’s protocol. The internal transcribed spacer (ITS) and partial 28S large subunit rDNA (LSU)
nuclear ribosomal DNA were amplified using ITS5/ITS4 [
24
] and LR0R/LR5 [
25
] primers respectively
following 94
◦
C/30 s, 55
◦
C/50 s, 72
◦
C/60 s protocol. Partial RNA polymerase II second largest subunit
(RPB2) and
β
-tubulin (TUB2) were amplified using fRPB2-5f/fRPB2-7cR [
26
] and T1/T22 [
27
] primers
following 95
◦
C/45 s, 52
◦
C/50 s, 72
◦
C/60 s and 95
◦
C/60 s, 54.5
◦
C/50 s, 72
◦
C/90 s protocols, respectively.
All the PCR protocols were followed by 35 cycles including 94
◦
C/5 min initial denaturation and
72 ◦C/10 min final extension.
The 25
µ
L total volume of PCR mixture contained 9.5
µ
L of ddH
2
O, 12.5
µ
L of 2X PCR Master Mix
(TIANGEN Co., China), 1
µ
L of DNA template, and 1
µ
L of forward and reverse primers (10
µ
M each)
in each reaction. PCR amplified products were checked on 1% agarose electrophoresis gels stained
with GoldView I nuclear staining dye (1
µ
L/10 mL of agarose). Purification and sequencing of PCR
products were done by Invitrogen Biotechnology Co. Ltd., Beijing, China. A consensus sequence for
each gene region was assembled in SeqMan (DNAStar, Inc., Madison, WI, USA).
2.3. Phylogenetic Analyses
Generated ITS, LSU, RPB2, and TUB2 sequences were subjected to BLASTn searches (https://blast.
ncbi.nlm.nih.gov) and related sequences were downloaded from GenBank
®
(Table 1). The individual
gene matrix was aligned using MAFFT v7 (https://mafft.cbrc.jp/alignment/server/large.html; [
28
]) with
E-INS-i and L-INS-i Iterative refinement methods, respectively, and improved when necessary in
BioEdit v. 7.0 [
29
]. All absent sequences were coded as missing data and characters were assessed to
be unordered and equally weighted.
Evolutionary models were estimated by using MrModeltest 2.2 [
30
] and model parameters
were selected independently for different gene regions under the Akaike information criterion (AIC)
implemented in PAUP v. 4.0b10. The GTR+G+I model was the best-fit model for all loci.
Single (LSU, ITS, RPB2, TUB2) and combined (LSU-ITS, LSU-ITS-RPB2-TUB2) matrices were used
for phylogenetic analyses in order to compare the topology with previous studies which only used
LSU-ITS matrices. Maximum parsimony (MP) analyses were carried out using PAUP v.4.0b 10 [
31
].
The parameters were set up with tree bisection-reconnection (TBR). Trees were inferred using the
heuristic search option with 1000 random sequence additions, with maxtrees set at 1000. Tree length (TL),
consistency index (CI), retention index (RI), relative consistency index (RC), and homoplasy index
(HI) were calculated for trees generated under different optimality criteria. The Kishino-Hasegawa
tests [32] were performed to determine whether trees were significantly different.
Maximum likelihood (ML) analyses were performed at the CIPRES webportal [
33
] using
RAxML-HPC2 on XSEDE (v 8.2.8) with GTR+G+I model and default parameters, and bootstrapping
with 1000 replicates [34].
The Bayesian inference (BI) analysis was generated by using Markov Chain Monte Carlo sampling
in MrBayes v3.1.2 [
35
,
36
] for 3,000,000 generations using four chains with 100 sample frequencies which
products 30,000 trees. The first 3000 (10% from total) trees were the burn-in phase and were discarded.
The remaining 27,000 trees were used to calculate the posterior probability (PP). The final alignment
and tree were registered in TreeBASE under the submission ID: 26768 (http://www.treebase.org).
J. Fungi 2020,6, 174 4 of 27
The resulting trees were viewed in FigTree v.1.4.0 [
37
] and the final layout was done with Adobe
Illustrator®CS5 (Adobe Systems, San Jose, CA, USA).
Table 1.
List of taxa used in the current phylogenetic study and GenBank acc. nos of DNA sequences.
Strains and corresponding sequences that were newly obtained are printed in bold.
Species Code GenBank Accession Numbers References
LSU ITS RPB2 TUB2
Achaetomium
macrosporum CBS 532.94 KX976699 KX976574 KX976797 KX976915 [38]
Amphisphaeria
acericola MFLU 16-2479 MK640424 MK640423 - - [8]
Amphisphaeria
acericola MFLUCC 14-0842 * MF614131 MF614128 - - [8]
Amphisphaeria
camelliae HKAS 107021 *,a MT756615 MT756621 MT789850 MT774368 This study
Amphisphaeria
camelliae MFLUCC 20-0122 bMT756616 MT756622 MT789851 MT774369 This study
Amphisphaeria
curvaticonidia HKAS 102288 aMT756618 MT756624 MT789853 - This study
Amphisphaeria
curvaticonidia MFLUCC 18-0620 *,b MT756617 MT756623 MT789852 - This study
Amphisphaeria
flava MFLUCC 18-0361 * MH971234 MH971224 - MK033638 [7]
Amphisphaeria
fuckelii WU 33555 KT949903 KT949903 - - [16]
Amphisphaeria
fuckelii CBS 140409 * KT949902 KT949902 MH554918 MH554677 [16,39]
Amphisphaeria
mangrovei NFCCI-4247 * MG844275 MG844283 - - [6]
Amphisphaeria
micheliae HKAS 107012 *,a MT756619 MT756625 MT789854 MT774370 This study
Amphisphaeria
micheliae MFLUCC 20-0121bMT756620 MT756626 MT789855 MT774371 This study
Amphisphaeria
neoaquatica MFLUCC 14-0045 * MK835805 MK828607 - - [17]
Amphisphaeria
qujingensis KUMCC 19-0186 MN707567 MN707568 - - [9]
Amphisphaeria
qujingensis KUMCC 19-0187 * MN556316 MN477033 - - [9]
Amphisphaeria
sambuci CBS 131707 * KT949904 KT949904 MH554911 MH704632 [16,39]
Amphisphaeria
sambuci WU 33557 KT949905 KT949905 - - [16]
Amphisphaeria
sambuci WU 33558 KT949906 KT949906 - - [16]
Amphisphaeria
sorbi MFLUCC 13-0721 * KP744475 KR092797 - - [5]
Amphisphaeria
thailandica MFLU 18-0794 * MH971235 MH971225 MK033640 MK033639 [7]
Amphisphaeria
umbrina AFTOL-ID 1229 FJ176863 FJ176809 - - [40]
Amphisphaeria
uniseptata CBS 114967 * MH554197 - MH554878 MH554638 [39]
Amphisphaeria
yunnanensis KUMCC 19-0188 * MN556306 MN477177 - - [9]
Amphisphaeria
yunnanensis KUMCC 19-0189 MN550992 MN550997 - - [9]
Anungitea
eucalyptorum CBS 137967 KJ869176 KJ869118 - - [41]
J. Fungi 2020,6, 174 5 of 27
Table 1. Cont.
Species Code GenBank Accession Numbers References
LSU ITS RPB2 TUB2
Anungitea
grevilleae CBS 141282 KX228304 KX228252 - - [42]
Bagadiella
lunata CBS 124762 * GQ303300 GQ303269 - - [43]
Bartalinia pini CBS 143891 * MH554330 MH554125 MH555033 MH554797 [39]
Bartalinia
pondoensis CBS 125525 * MH875078 MH863602 MH554904 MH554663 [39,40]
Beltrania
pseudorhombica CBS 138003 * KJ869215 KJ869158 MH555032 - [39,41]
Beltrania
rhombica CBS 123.58 MH869260 MH857718 MH554899 MH704631 [39,44]
Beltraniella
endiandrae CBS 137976 * KJ869185 KJ869128 - - [41]
Beltraniopsis
neolitseae CBS 137974 * KJ869183 KJ869126 - - [41]
Castanediella
acaciae CBS 139896 * KR476763 KR476728 - - [45]
Castanediella
brevis KUMCC 18-0132 * MH806358 MH806361 - - [46]
Castanediella
eucalypti CBS 139897 * KR476758 KR476723 - - [45]
Castanediella
eucalypticola CBS 141317 * KX228317 KX228266 - KX228382 [42]
Chaetomium
elatum CBS 374.66 MH870466 KC109758 KF001820 KC109776 [38,44]
Clypeophysalospora
latitans CBS 141463 * KX820261 KX820250 - - [47]
Cylindrium
corymbiae CBS 146087 * MT223887 MT223792 MT223679 MT223732 [48]
Cylindrium
grande CPC 35403 * MK876425 MK876384 MK876481 MK876502 [49]
Heterotruncatella
acacigena CBS 143880 * MH554295 MH554084 MH554996 MH554756 [39]
Heterotruncatella
vinaceobubalina CBS 143897 MH554341 MH554139 MH555045 MH554812 [39]
Hymenopleella
hippophaeicola CBS 140410 * KT949901 KT949901 MH554919 MH554678 [16,39]
Hymenopleella
polyseptata CBS 143887 * MH554321 MH554116 MH555024 MH554789 [39]
Hymenopleella
schefflerae COAD 2371 * MH084761 MH128360 - MH231215 [20]
Hymenopleella
austroafricana CBS 143886 * MH554320 MH554115 MH555023 MH554788 [39]
Neophysalospora
eucalypti CBS 138864 * KP004490 KP004462 - - [50]
Phlogicylindrium
uniforme CBS 131312 * JQ044445 JQ044426 MH554910 MH704634 [39,51]
Phlogicylindrium
eucalyptorum CBS 111680 KF251707 KF251204 KF252209 KF252698 [52]
Plectosphaera
eucalypti CBS 120063 * DQ923538 DQ923538 - - [53]
Robillarda
roystoneae CBS 115445 * MH874545 KR873254 MH554880 KR873317 [39,44,54]
J. Fungi 2020,6, 174 6 of 27
Table 1. Cont.
Species Code GenBank Accession Numbers References
LSU ITS RPB2 TUB2
Seimatosporium
rosae MFLUCC 14-0621 * MH823070 LT853105 LT853153 LT853253 [3,55]
Seiridium
marginatum CBS 140403 * MH878679 KT949914 LT853149 LT853249 [16,44,55]
Sordaria
fimicola CBS 723.96 MH874231 - DQ368647 - [44,56]
Types and authentic strains are indicated with (*). Codes with
a
and
b
are denoted holotypes and ex-type
cultures, respectively. Missing sequences are indicated by (-). Newly generated sequences are in bold.
Abbreviations: AFTOL-ID—Assembling the Fungal Tree of Life; CBS—Centraalbureau voor Schimmelcultures,
Utrecht, The Netherlands; COAD—Culture Collection of the Universidade Federal de Viçosa, Brazil; CPC—Culture
collection of Pedro Crous, housed at CBS; HKAS—The Herbarium of Cryptogams Kunming Institute of Botany
Academia Sinica, Kunming, China; KUMCC—Kunming Institute of Botany Culture Collection, Kunming, China;
MFLU—Mae Fah Luang University Herbarium, Chiang Rai, Thailand; MFLUCC—Mae Fah Luang University
Culture Collection, Chiang Rai, Thailand; NFCCI—National Fungal Culture Collection of India, Agharkar Research
Institute, India. WU—Herbarium of the Institute of Botany, University of Vienna, Austria.
2.4. Cluster Analysis
A cluster analysis was performed to assess morphological similarities of Amphisphaeria and
Lepteutypa taxa. The presence of poorly- (0) or well- (1) developed clypeus; solitary (0) or
solitary/aggregated (0.5) or aggregated (1) nature of ascomata; l/w ratio of asci
≤
15 (0) or >15
(1); J
−
(0) or J+(1) apical ring; l/w ratio of ascospores
≤
2.8 (0) or >2.8 (1); one septaum (0) or more than
one septum (1) in ascospores; and the absence (0) or presence (1) of a mucilaginous sheath around
ascospores of Amphisphaeria and Lepteutypa species were used for the analysis.
The analysis was conducted using Python 3.8 coupled with SciPy 1.5.1 package. We obtained a
dendrogram using the unweighted pair group method with arithmetic mean (UPGMA) algorithm and
also obtained the Average distance measure. The dendrogram of obtained results was plotted using
Matplotlib 3.3.0 package. The heatmap was obtained in a similar way. Plotting was performed by
using Seaborn 0.10.1 (https://docs.scipy.org).
3. Results
3.1. Topology of Phylogenetic Analyses
The LSU-ITS alignment comprised 1374 characters (LSU 1–786, ITS 787–1374) while the
LSU-ITS-RPB2-TUB2 combined alignment comprised 2515 characters (LSU 1–786, ITS 787–1377,
RPB2 1378–2212, TUB2 2213–2515) including 56 strains.
We follow LSU-ITS-RPB2-TUB2 to show the relationship among related taxa here (Figure 1).
The best scoring RAxML tree with a final likelihood value of—1224.694904 is presented. The matrix
had 1227 distinct alignment patterns, with 28.04% of undetermined characters or gaps. Estimated base
frequencies were as follows; A =0.247366, C =0.234669, G =0.265996, T =0.251970; substitution rates
AC =1.117478, AG =3.507552, AT =1.477282, CG =1.121674, CT =6.681156, GT =1.000000;
gamma distribution shape parameter
α
=0.485915. The ML, MP, and BY phylogenetic trees resulting
from analyses of the alignment of LSU-ITS were different among taxa within the Amphisphaeriaceae
(Figure S1). ML and BY results using LSU-ITS-RPB2-TUB2 combined matrix were similar in topologies,
and Amphisphaeriaceae was comprised of two well-supported clades containing Amphisphaeria
species (clade Y) and species that had previously been classified under the genus Lepteutypa (clade X).
Amphisphaeria curvaticonidia (HKAS 102288, MFLUCC 18-0620) formed a basal lineage to all the other
amphisphaeriaceous taxa in only MP analysis by using the LSU-ITS-RPB2-TUB2 combined matrix.
J. Fungi 2020,6, 174 7 of 27
J. Fungi 2020, 6, x FOR PEER REVIEW 6 of 27
Figure 1. ML tree revealed by RAxML from an analysis of the LSU-ITS-RPB2-TUB2 matrix of the
Amphisphaeriaceae and selected families of Amphisphaeriales. Bootstrap supports (≥50%) of MP and
ML and the posterior probability values (≥0.9) of BI analyses are indicated above or below the
respective branches. Newly generated sequences are in blue and type strains are in bold. The tree is
rooted to Achaetomium macrosporum, Chaetomium elatum, and Sordaria fimicola (Sordariales). The scale
bar represents the expected number of nucleotide substitutions per site. Ascospore septation among
taxa are shown in white (one septum) and black (more than one septum) circles. Taxon in red denotes
topological conflict with previous phylogenies. Previous taxa are in “•”.
The maximum parsimony dataset consisted of 2515 characters, of which 1430 were constant, 881
parsimony-informative (35.03%), and 204 parsimony-uninformative. The parsimony analysis of the
data matrix resulted in 1000 equally most parsimonious trees with a length of 3898 steps (CI = 0.445,
RI = 0.671, RC = 0.299, HI = 0.555) in the first tree.
Amphisphaeria curvaticonidia (HKAS 102288, MFLUCC 18-0620; 100%/100%/1.00 PP) formed a
clade with poor statistical support. Amphisphaeria camelliae (HKAS 107021, MFLUCC 20-0122;
100%/100%/1.00 PP) is sister to “Lepteutypa uniseptata” CBS 114967 (91%/100%/1.00 PP). Amphisphaeria
micheliae (HKAS 107012, MFLUCC 20-0121; 100%/100%/1.00 PP) formed a clade sister to “L. sambuci”
(CBS 131707, WU 33557 and WU 33558) with strong statistical support (100%/100%/1.00 PP). The
Figure 1.
ML tree revealed by RAxML from an analysis of the LSU-ITS-RPB2-TUB2 matrix of the
Amphisphaeriaceae and selected families of Amphisphaeriales. Bootstrap supports (
≥
50%) of MP
and ML and the posterior probability values (
≥
0.9) of BI analyses are indicated above or below the
respective branches. Newly generated sequences are in blue and type strains are in bold. The tree is
rooted to Achaetomium macrosporum,Chaetomium elatum, and Sordaria fimicola (Sordariales). The scale
bar represents the expected number of nucleotide substitutions per site. Ascospore septation among
taxa are shown in white (one septum) and black (more than one septum) circles. Taxon in red denotes
topological conflict with previous phylogenies. Previous taxa are in “•”.
The maximum parsimony dataset consisted of 2515 characters, of which 1430 were constant,
881 parsimony-informative (35.03%), and 204 parsimony-uninformative. The parsimony analysis of
the data matrix resulted in 1000 equally most parsimonious trees with a length of 3898 steps (CI =0.445,
RI =0.671, RC =0.299, HI =0.555) in the first tree.
Amphisphaeria curvaticonidia (HKAS 102288, MFLUCC 18-0620; 100%/100%/1.00 PP) formed
a clade with poor statistical support. Amphisphaeria camelliae (HKAS 107021, MFLUCC 20-0122;
100%/100%/1.00 PP) is sister to “Lepteutypa uniseptata” CBS 114967 (91%/100%/1.00 PP). Amphisphaeria
J. Fungi 2020,6, 174 8 of 27
micheliae (HKAS 107012, MFLUCC 20-0121; 100%/100%/1.00 PP) formed a clade sister to “L. sambuci”
(CBS 131707, WU 33557 and WU 33558) with strong statistical support (100%/100%/1.00 PP). The strain
“Trochilispora schefflerae” (COAD 2371) clustered with Hymenopleella austroafricana (CBS 143886) with
high statistical support (100%/100%/1.00 PP) in Sporocadaceae.
The ITS sequence of “Lepteutypa uniseptata”; CBS 114967 (MH553979) is dubious because it
appeared highly similar to Robillarda species in a BLASTn search and clustered in Sporocadaceae in our
ITS-based phylogenies, which is why it has been excluded from our final analyses.
3.2. Cluster Analysis
The cluster analysis was comprised of 37 species with seven characters (Figure 2). There are three
clusters (A, B and C). The type of species of Amphisphaeria;A. umbrina and the type of “Lepteutypa”;
“L. fuckelii” cluster with clusters B and C, respectively. Species in cluster A shared asci l/w greater
than 15. Clusters B and C were mainly distinguished by having 1-septate (B) and multiseptate (C)
ascospores. All other characteristics were distributed among the species without a unique characteristic
for each genus.
J. Fungi 2020, 6, x FOR PEER REVIEW 7 of 27
strain “Trochilispora schefflerae” (COAD 2371) clustered with Hymenopleella austroafricana (CBS 143886)
with high statistical support (100%/100%/1.00 PP) in Sporocadaceae.
The ITS sequence of “Lepteutypa uniseptata”; CBS 114967 (MH553979) is dubious because it
appeared highly similar to Robillarda species in a BLASTn search and clustered in Sporocadaceae in
our ITS-based phylogenies, which is why it has been excluded from our final analyses.
3.2. Cluster Analysis
The cluster analysis was comprised of 37 species with seven characters (Figure 2). There are three
clusters (A, B and C). The type of species of Amphisphaeria; A. umbrina and the type of “Lepteutypa”;
“L. fuckelii” cluster with clusters B and C, respectively. Species in cluster A shared asci l/w greater
than 15. Clusters B and C were mainly distinguished by having 1-septate (B) and multiseptate (C)
ascospores. All other characteristics were distributed among the species without a unique
characteristic for each genus.
Figure 2. Cluster analysis dendrogram showing similarity among Amphisphaeria and Lepteutypa
species (linkage algorithm paired group UPGMA; Average Distance measure). Characteristics: (1)
nature of the clypeus; (2) nature of the ascomata; (3) l/w ratio of asci; (4) apical ring; (5) l/w ratio of
ascospores; (6) ascospore septation; and (7) mucilaginous sheath around ascospores. “X” denotes
missing data. Newly introduced species are in bold. Taxa which needed to be revised are in blue.
Holomorphic taxa are denoted as “##”.
3.3. Taxonomy
3.3.1. Amphisphaeria
Amphisphaeria Ces. & De Not., Comm. Soc. crittog. Ital. 1(fasc. 4): 223 (1863), emend.
MycoBank: MB173; Facesoffungi number: FoF02099
= Poikiloderma Füisting, Bot. Ztg. 26: 369 (1868)
= Massariopsis Niessl, Verh. nat. Ver. Brünn 14: 199 (1876)
= Phorcys Niessl, Verh. nat. Ver. Brünn 14: 200 (1876)
= Conisphaeria Cooke, Grevillea 7(no. 43): 86 (1879)
Figure 2.
Cluster analysis dendrogram showing similarity among Amphisphaeria and Lepteutypa species
(linkage algorithm paired group UPGMA; Average Distance measure). Characteristics: (1) nature of
the clypeus; (2) nature of the ascomata; (3) l/w ratio of asci; (4) apical ring; (5) l/w ratio of ascospores;
(6) ascospore septation; and (7) mucilaginous sheath around ascospores. “X” denotes missing data.
Newly introduced species are in bold. Taxa which needed to be revised are in blue. Holomorphic taxa
are denoted as “##”.
3.3. Taxonomy
3.3.1. Amphisphaeria
Amphisphaeria Ces. & De Not., Comm. Soc. crittog. Ital. 1(fasc. 4): 223 (1863), emend.
MycoBank: MB173; Facesoffungi number: FoF02099
=Poikiloderma Füisting, Bot. Ztg. 26: 369 (1868)
J. Fungi 2020,6, 174 9 of 27
=Massariopsis Niessl, Verh. nat. Ver. Brünn 14: 199 (1876)
=Phorcys Niessl, Verh. nat. Ver. Brünn 14: 200 (1876)
=Conisphaeria Cooke, Grevillea 7(no. 43): 86 (1879)
=Massariella Speg., Anal. Soc. cient. argent. 9(4): in tabula [facing p. 192) (1880)
=Lepteutypa Petr., Annls mycol. 21(3/4): 276 (1923)
=Rhynchostomopsis Petr. & Syd., Annls mycol. 21(5/6): 377 (1923)
=Lepteutypella Petr., Annls mycol. 23(1/2): 98 (1925)
=Macrothelia M. Choisy, Bull. mens. Soc. linn. Soc. Bot. Lyon 18: 107 (1949)
Saprobic on woody branches, twigs and culms in terrestrial, freshwater, and mangrove habitats.
Sexual morph: appearing as slightly raised, black dots on host surface, often surrounded by a dark or
light-coloured, halo-like area. Pseudostromata, when present, are made up of host cells and brown to
black fungal hyphae. The Clypeus is often poorly developed. Ascomata: perithecial, scattered, solitary or
clustered, immersed, erumpent or rarely superficial, globose, subglobose or ellipsoidal, coriaceous,
dark brown, papillate ostiole. Papilla: narrow, conical, periphysate, often umbilicate. Periphyses: hyaline,
filamentous. Peridium: two-layered, with an outer layer comprising dark brown cells arranged in
atextura angularis and inner layer comprising thin-walled, hyaline cells. Hamathecium: comprising
numerous, filamentous, septate, slightly tapering paraphyses. Asci: 8-spored, unitunicate, cylindrical,
indistinctly pedicellate, apex rounded, with J+or J
−
apical ring. Ascospores: uniseriate to overlapping
uniseriate, light brown to dark brown, ellipsoid to fusiform, rarely curved, 1–3-septate, not or
slightly constricted at the septum, rarely 2–4(6)-distoseptate, smooth-walled, some guttulate, with or
without a mucilaginous sheath. Asexual morph: Coelomycetous. Conidiomata: solitary or aggregated,
globose to sub-globose, dark brown. Peridium: comprised of thick-walled, septate, brown mycelium.
Conidiophores: septate, branched, thick-walled, hyaline to light brown. Conidiogenous cells: elongated,
thin-walled, septate, hyaline, annellidic. Conidia: hyaline, elongate-fusiform, 1-celled with or without
appendage derived from the middle of the conidia, smooth-walled.
Type species: Amphisphaeria umbrina (Fr.) De Not., Sfer. Ital.: 69 (1863)
MycoBank: MB222981; Facesoffungi number: FoF08735
≡Sphaeria umbrina Fr., Syst. mycol. (Lundae) 2(2): 461 (1823) nom. sanct.
=Sphaeria mammillaris Schumach., Enum. pl. (Kjbenhavn) 2: 157 (1803)
=Sphaeropsis conica L
é
v., in Demidov, Voyage dans la Russie Meridionale et la Crime
é
, par la
Hongrie, la Valachie et la Moldavie 2: 112 (1842)
=Diplodia conica (Lév.) Lév., Annls Sci. Nat., Bot., sér. 3 9: 258 (1848)
=Amphisphaeria conica (Lév.) Ces. & De Not., Comm. Soc. crittog. Ital. 1(fasc. 4): 224 (1863)
=Hypocrea gelatinosa var. umbrina (Fr.) Sacc., Syll. fung. (Abellini) 2: 524 (1883)
=Kirschsteiniella conica (Lév.) Petr., Sydowia 7(1-4): 57 (1953)
Typus: Italy, Flaventino, on trunk of Ulmus sp. (Ulmaceae), Nov. 1860, L. Caldesi, Rabenhorst Fungi
Europaei 327 (RO, epitype).
Notes: Fries [
57
] described Sphaeria umbrina on the wood of Alnus (Betulaceae) from Sweden.
Cesati and de Notaris [
1
] introduced Amphisphaeria without a type species. Petrak [
10
] revisited
the morphology of the Amphisphaeria umbrina on the bark of Ulmus (Ulmaceae) from Italy and
proposed it as the type of species of Amphisphaeria. Consecutive studies from Clements and Shear [
58
],
Müller and Arx [
59
], and Korf [
60
] accepted A. umbrina as the type species of Amphisphaeria represented
by Sphaeria umbrina. Hyde et al. [
61
] re-examined a specimen of S. umbrina from the collection of
Cesati and de Notaris [
1
], also which was used by Petrak [
10
] for description. The specimen was
proposed as the epitype for A. umbrina. Hyde et al. [
61
] provided another A. umbrina specimen
from Austria on Quercus petraea (Fagaceae) (AFTOL-ID 1229; CBS 172.96). Amphisphaeria umbrina
is distributed in temperate habitats on Quercus (Fagaceae), Salix (Salicaceae), Tilia (Malvaceae) and
Ulmus (Ulmaceae) [
61
]. In addition, Amphisphaeria has been described from Alnus (Betulaceae),
Fraxinus (Oleaceae), Quercus (Fagaceae), Salix (Salicaceae) and Ulmus (Ulmaceae) [62].
J. Fungi 2020,6, 174 10 of 27
Petrak [
10
] introduced Lepteutypa to accommodate species with eutypoid stroma and multicellular
ascospores different from Amphisphaeria.Lepteutypa species do not possess eutypoid stroma in their
morphological descriptions, figures, and illustrations except aggregated ascomata. Jaklitsch et al. [
16
]
proposed a neotype for Lepteutypa fuckelii, the type species of the genus, which has scattered or
aggregated ascomata on the host but not eutypoid stroma. Jaklitsch et al. [
16
] further noted that
Petrak [
10
] interpreted those aggregated perithecial colonies as eutypoid stromata. Among previously
known Lepteutypa species, L. alpestris,L. cisticola,L. fusispora,L. hederae and L. tropicalis also possess
aggregated perithecia, while L. hexagonalis possesses two perithecia under a single clypeus rarely.
The aggregated ascomata also can be observed in A. bertiana and A. seriata.
Amphisphaeria and Lepteutypa were previously separated based on stromatic nature and
ascospore septation. However, with the introduction of Amphisphaeria camelliae and A. micheliae,
which are typical of Amphisphaeria and not “Lepteutypa” where they cluster, we conclude the
Amphisphaeria and “Lepteutypa” are congeneric. There is no distinct characteristic used to separate
Amphisphaeria (clade Y) and “Lepteutypa” (clade X) clusters in Figure 1. Both clusters have 2-celled
and multicelled ascospores. No single characteristic exists in one clade. The predicament here is
whether to follow the molecular data, which indicates there are two distinct genera (Amphisphaeria
and Lepteutypa) or the morphological data which indicates the group is one genus (Amphisphaeria).
We adopt a single genus, Amphisphaeria, due to the morphological similarity. However, further studies
with fresh collections may resolve the taxonomic relationships in Amphisphaeriaceae and sexual
asexual connections.
The coelomycetous asexual morph of Amphisphaeria sorbi is the only asexual record in
Amphisphaeriaceae. Several morphological records of pestaloid-like asexual morphs for “Lepteutypa”
have been suggested, but not confirmed [
63
]. In our study, we obtained two asexual morphs which are
not pestaloid-like, but similar to the coelomycetous asexual morph of A sorbi. Since there is lack of
sequence data for most of the “Lepteutypa” species, here we provided five taxonomic combinations
of “Lepteutypa” species which have molecular data. However, the remaining “Lepteutypa” species
are treated as ambiguous taxa and need to be revisited with molecular and morphology data in
further studies.
3.3.2. Additional Accepted Species in Amphisphaeria
Amphisphaeria acericola Senan., Camporesi & K.D. Hyde, Phytotaxa 403(4): 285–292 (2019)
MycoBank: MB553774; Facesoffungi number: FoF03594
Typus: Italy, Province of Forl
ì
-Cesena, Galeata, Strada San Zeno, on a branch of Acer campestre
(Sapindaceae), 26 Mar 2014, E. Camporesi, IT 1779 (MFLU 16-2479, holotype); ex-type living culture
MFLUCC 14-0842.
Notes: Amphisphaeria acericola is similar to A. pseudoumbrina in having immersed, oblate ascomata,
J+, a discoid apical ring, cylindrical asci with rugose-walled, brown, uniseptate ascospores, but different
in having non-clypeate ascomata, peridium with textura angularis cells, and aseptate, cellular paraphyses.
The LSU-ITS phylogeny of A. acericola forms a basal clade distinct from other Amphisphaeria species.
Amphisphaeria bertiana Fairm., Proc. Rochester Acad. Sci. 4: 217 (1906)
MycoBank: MB190709; Facesoffungi number: FoF08738
Typus: USA, New York, Lyndonville, in cavities at the end of a rotting log, Oct. 1905,
Fairman (CUP, holotype).
Notes: Wang et al. [
2
] re-examined the holotype. Amphisphaeria bertiana has erumpent or superficial
ascomata on a subiculum and J−apical ring.
Amphisphaeria camelliae Samarak., Jian K. Liu & K.D. Hyde, sp. nov. Figures 3and 4
J. Fungi 2020,6, 174 11 of 27
J. Fungi 2020, 6, x FOR PEER REVIEW 10 of 27
Figure 3. The sexual morph of Amphisphaeria camelliae (HKAS 107021, holotype). (a,b) Host Camellia
japonica; (c) substrate; (d–f) ascomata on the substrate; (g) vertical section of ascoma; (h) peridium; (i)
ostiole; (j) paraphyses; (k–n) asci; (o,p) apical ring bluing in Melzer’s reagent; (q) ascospore top view;
(r–w) ascospores (v in Congo Red). Scale bars are set at (c) 1 cm; (f) 500 μm; (d,e) 200 μm; (g) 100 μm;
(h,i,k–n) 20 μm; (o,p,r–w) 10 μm; (q) 5 μm.
Figure 3.
The sexual morph of Amphisphaeria camelliae (HKAS 107021, holotype). (
a
,
b
) Host Camellia
japonica; (
c
) substrate; (
d
–
f
) ascomata on the substrate; (
g
) vertical section of ascoma; (
h
) peridium;
(
i
) ostiole; (
j
) paraphyses; (
k
–
n
) asci; (
o
,
p
) apical ring bluing in Melzer’s reagent; (
q
) ascospore top view;
(
r
–
w
) ascospores (v in Congo Red). Scale bars are set at (
c
) 1 cm; (
f
) 500
µ
m; (
d
,
e
) 200
µ
m; (
g
) 100
µ
m;
(h,i,k–n) 20 µm; (o,p,r–w) 10 µm; (q) 5 µm.
J. Fungi 2020,6, 174 12 of 27
J. Fungi 2020, 6, x FOR PEER REVIEW 11 of 27
Figure 4. The asexual morph of Amphisphaeria camelliae (MFLUCC 20-0122, ex-type living culture). (a)
Germinating ascospore; (b) upper view, (c) reverse view of the 2 weeks old colony on PDA; (d)
conidiomata in the culture; (e) setae; (f–h) conidiophores, conidiogenous cells and conidiogenesis; (i–
n) conidia. Scale bars are set at (e) 50 μm; (a,f–h) 10 μm; (i–n) 5 μm.
Saprobic on the dead wood of Camellia japonica (Theaceae). Sexual morph: Ascomata: 300–480
μm high × 160–310 μm diameter (M = 410 × 260 μm, n = 5), immersed, visible as black spots covered
with pale brown and blackish area, solitary or aggregated, scattered, globose to subglobose, papillate
ostiole 80–150 μm high × 50–85 μm diameter (M = 110 × 60 μm, n = 5), centric. Periphyses: 1–2 μm wide
(M = 1.5 μm, n = 20), hyaline, short. Peridium: two-layered; outer layer 5–7 μm (M = 5.5 μm, n =10),
dense, reddish-brown cells of textura angularis 8–15 × 1–2 μm (M = 10.8 × 1.6 μm, n = 15), thick-walled.
Inner layer: 7–12 μm (M = 9.5 μm, n =10), loosely arranged, hyaline cells of textura angularis 9.5–18 ×
1–3 μm (M = 12 × 1.5 μm, n = 15), thin-walled, loosely arranged. Paraphyses: 2–4 μm wide (M = 3 μm,
n = 20), hyaline, highly delicate, cellular, constricted septate, guttulate; 1–2 μm wide (M = 1.5 μm, n =
20), hyaline, filiform, longer than asci, blunt end, cellular, guttulate, embedded in a gelatinous matrix.
Asci: 85–130 × 5–8 μm (M = 110 × 6.5 μm, n = 25), 8-spored, unitunicate, cylindrical, thin-walled, short-
pedunculate, apically rounded, with a J+, discoid apical ring. Ascospores: 12–17.5 × 4–5.5 μm (M = 15
× 5 μm, n = 40), l/w 3.1, uniseriate, oblong or narrowly fusiform, first hyaline, guttulate, turning
yellow to yellowish-brown, with a median septum, slightly constricted at the septum, straight to
slightly curved, smooth-walled. Asexual morph: Coelomycetous. Conidiomata: superficial on PDA,
solitary or aggregated, globose, dark brown. Conidiophores: 24–40 × 1–3 μm (M = 31 × 2 μm, n = 15),
arising from peridium, septate, branched, thick-walled, light brown to hyaline. Setae: 60–92 × 3.5–5
μm (M = 76 × 4.5 μm, n = 5), septate, thick-walled, blunt end, brown to light brown. Conidiogenous
cells: 7.5–14.5 × 1.5–2.5 μm (M = 11.5 × 2 μm, n = 15), elongated conical, thin-walled, hyaline, annellidic,
guttulate. Conidia: 14.5–18 × 1.5–2.5 μm (M = 16 × 2 μm, n = 25), elongate-fusiform, curved, smooth-
walled, hyaline, guttulate.
Figure 4.
The asexual morph of Amphisphaeria camelliae (MFLUCC 20-0122, ex-type living culture).
(
a
) Germinating ascospore; (
b
) upper view, (
c
) reverse view of the 2 weeks old colony on PDA;
(
d
) conidiomata in the culture; (
e
) setae; (
f
–
h
) conidiophores, conidiogenous cells and conidiogenesis;
(i–n) conidia. Scale bars are set at (e) 50 µm; (a,f–h) 10 µm; (i–n) 5 µm.
MycoBank: MB836110; Facesoffungi number: FoF08740
Etymology: The specific epithet reflects the host genus Camellia.
Holotype: HKAS 107021
Saprobic on the dead wood of Camellia japonica (Theaceae).
Sexual morph:
Ascomata: 300–480
µ
m
high
×
160–310
µ
m diameter (M =410
×
260
µ
m, n=5), immersed, visible as black spots covered with
pale brown and blackish area, solitary or aggregated, scattered, globose to subglobose, papillate ostiole
80–150
µ
m high
×
50–85
µ
m diameter (M =110
×
60
µ
m, n=5), centric. Periphyses: 1–2
µ
m wide
(M =1.5
µ
m, n=20), hyaline, short. Peridium: two-layered; outer layer 5–7
µ
m (M =5.5
µ
m,
n=10), dense, reddish-brown cells of textura angularis 8–15
×
1–2
µ
m (M =10.8
×
1.6
µ
m,
n=15), thick-walled. Inner layer: 7–12
µ
m (M =9.5
µ
m, n=10), loosely arranged, hyaline cells
of textura angularis 9.5–18
×
1–3
µ
m (M =12
×
1.5
µ
m, n=15), thin-walled, loosely arranged.
Paraphyses: 2–4
µ
m wide (M =3
µ
m, n=20), hyaline, highly delicate, cellular, constricted septate,
guttulate; 1–2
µ
m wide (M =1.5
µ
m, n=20), hyaline, filiform, longer than asci, blunt end, cellular,
guttulate, embedded in a gelatinous matrix. Asci: 85–130
×
5–8
µ
m (M =110
×
6.5
µ
m, n=25),
8-spored, unitunicate, cylindrical, thin-walled, short-pedunculate, apically rounded, with a J+,
discoid apical ring. Ascospores: 12–17.5
×
4–5.5
µ
m (M =15
×
5
µ
m, n=40), l/w 3.1, uniseriate,
oblong or narrowly fusiform, first hyaline, guttulate, turning yellow to yellowish-brown, with a
median septum, slightly constricted at the septum, straight to slightly curved, smooth-walled.
Asexual morph:
Coelomycetous. Conidiomata: superficial on PDA, solitary or aggregated, globose,
dark brown. Conidiophores: 24–40
×
1–3
µ
m (M =31
×
2
µ
m, n=15), arising from peridium,
septate, branched, thick-walled, light brown to hyaline. Setae: 60–92
×
3.5–5
µ
m (M =76
×
4.5
µ
m,
J. Fungi 2020,6, 174 13 of 27
n=5), septate, thick-walled, blunt end, brown to light brown. Conidiogenous cells: 7.5–14.5
×
1.5–2.5
µ
m (M =11.5
×
2
µ
m, n=15), elongated conical, thin-walled, hyaline, annellidic, guttulate.
Conidia: 14.5–18
×
1.5–2.5
µ
m (M =16
×
2
µ
m, n=25), elongate-fusiform, curved, smooth-walled,
hyaline, guttulate.
Culture characteristics: colonies on PDA, reaching 16–17 mm diameter after one week at 25
◦
C,
the colonies are flat, circular, dense, with a smooth surface, entire margin, and white to light brown.
Media become pale brown; reverse light brown at center and dirty white edges.
Material examined: China, Sichuan Province, Chengdu, University of Electronic Science and
Technology of China (UESTC) campus, on the wood of Camellia japonica (Theaceae), 30 September 2019,
M.C. Samarakoon, SAMC254 (HKAS 107021, holotype; MFLU 20-0504, isotype); ex-type living culture
MFLUCC 20-0122.
Notes: Our specimens have solitary and aggregated, immersed ascomata with two-layered
peridium, unitunicate asci with J+, a discoid apical ring, and brown ascospores similar to
amphisphaeriaceous species. Amphisphaeria camelliae possesses 1-septate ascospores similar to
A. uniseptata, but differs in having single or aggregated, globose to subglobose (vs. single, subglobose
or applanate) ascomata, thin paraphyses (3.1 vs. 5
µ
m) and large ascospores (l/w 3.1 vs. l/w 2.7).
In the phylogenetic analyses, our collection also clusters with A. uniseptata. Based on morphology and
phylogeny, our collection is introduced as a novel species A. camelliae.
Amphisphaeria curvaticonidia Samarak., Promp. & K.D. Hyde, sp. nov. Figures 5and 6
J. Fungi 2020, 6, x FOR PEER REVIEW 13 of 27
our strains with J− and an apical ring in asci. In addition, some of the immature ascospores are
covered with a thin mucilaginous sheath as in A. flava, A. lusitanica, A. seriata, A. sorbi and A. vibratilis.
In addition, our collection has 1 median, slightly constricted at the euseptum, and two distosepta
which are characteristic to A. depressa. Wang et al. [2] re-examined the herbarium specimen of A.
depressa and noted that this unusual distoseptate ascospores characteristic is not matched with the
generic description. However, based on unituniate asci with J+, apical ring, Wang et al. [2] accepted
this species in Amphisphaeriaceae. The asexual coelomycetous from the culture of our strain is
similar to the only known asexual morph of A. sorbi by having septate, branched, hyaline
conidiophores, elongated conical, thin-walled, septate, hyaline, annellidic conidiogenous cells, and
elongate-fusiform, 1-celled, hyaline conidia. The asexual morph of our strain is characterized with
curved appendage derived from the middle of the conidia cell. Here we introduce our new collection
as A. curvaticonidia.
Figure 5. Sexual morph of Amphisphaeria curvaticonidia (MFLU18-0789, holotype). (a–c) Ascomata on
the substrate; (d) vertical section of ascoma; (e) ostiole; (f) peridium; (g,h) paraphyses; (i) apical ring
(in water); (j) apical ring bluing in Melzer’s reagent; (k) ascospore (top view); (l–p) asci; (q–v)
ascospores (v in Indian Ink). Scale bars are set at, (d) 100 μm; (e) 50 μm; (f,l–p) 20 μm; (i,j,q–v) 10 μm;
(g,h,k) 5 μm.
Figure 5.
Sexual morph of Amphisphaeria curvaticonidia (MFLU18-0789, holotype). (
a
–
c
) Ascomata on the
substrate; (
d
) vertical section of ascoma; (
e
) ostiole; (
f
) peridium; (
g
,
h
) paraphyses; (
i
) apical ring (in water);
(
j
) apical ring bluing in Melzer’s reagent; (
k
) ascospore (top view); (
l
–
p
) asci; (
q
–
v
) ascospores (v in
Indian Ink). Scale bars are set at, (d) 100 µm; (e) 50 µm; (f,l–p) 20 µm; (i,j,q–v) 10 µm; (g,h,k) 5 µm.
J. Fungi 2020,6, 174 14 of 27
J. Fungi 2020, 6, x FOR PEER REVIEW 14 of 27
Figure 6. Asexual morph of Amphisphaeria curvaticonidia (MFLUCC 18-0620, ex-type living culture).
(a) Germinating ascospore; (b) upper view, (c) reverse view of the 2 weeks old colony on PDA; (d)
upper view, (e) reverse view of the 6 weeks old colony on PDA; (f,g) conidiomata in the culture; (h)
vertical section of conidiomata in dried PDA; (i–k) conidiophores, conidiogenous cells, and
conidiogenesis (white arrows show conidia attachment); (l–p) conidia. Scale bars are set at (h) 200
μm; (a) 20 μm; (i–p) 10 μm.
Amphisphaeria depressa Petr., Sydowia 7(5–6): 381 (1953)
MycoBank: MB292502; Facesoffungi number: FoF08743
Typus: USA, Hawaii, Kaihea, Oahu, on Cassia bicapsularis (Fabaceae), 24 Feb. 1928, Shear (W
11997, holotype; BPI 618577, isotype).
Notes: Wang et al. [2] re-examined the holotype and remarked the distoseptate ascospores are
an unusual character of this species and accommodated in Amphisphaeria until further collections are
made.
Amphisphaeria doidgeae Marinc., M.J. Wingf. & Crous, in Marincowitz et al., CBS Diversity Ser.
(Utrecht) 7: 20 (2008)
MycoBank: MB506197; Facesoffungi number: FoF08744
Typus: South Africa, Western Cape Province, Kleinmond, on twig litter of Leucadendron sp.
(Proteaceae), 11 July 2000, S. Marincowitz, S.L. 485 (PREM 59556, holotype).
Notes: Amphisphaeria doidgeae is characterized by having broader peridium (59–72 μm), septate
and branched paraphyses, asci with a J– apical ring and broadly fusiform with a base often projected
and truncate ascospores.
Amphisphaeria fallax De Not., Hedwigia 4: 21 (1865)
MycoBank: MB187770; Facesoffungi number: FoF08745
Typus: Czech Republic, Weiβkirchen, on Quercus robur (Fagaceae), Apr. 1936, F. Petrak (DAOM
148328, neotype).
Notes: Wang et al. [2] designated a neotype by observing a slide collection from Quercus robur
deposited by Petrak in 1936 with similar morphology.
Amphisphaeria flava Samarak. & K.D. Hyde, Phytotaxa 391 (3): 210–211 (2019)
Figure 6.
Asexual morph of Amphisphaeria curvaticonidia (MFLUCC 18-0620, ex-type living culture).
(
a
) Germinating ascospore; (
b
) upper view, (
c
) reverse view of the 2 weeks old colony on PDA;
(
d
) upper view, (
e
) reverse view of the 6 weeks old colony on PDA; (
f
,
g
) conidiomata in the culture;
(
h
) vertical section of conidiomata in dried PDA; (
i
–
k
) conidiophores, conidiogenous cells, and
conidiogenesis (white arrows show conidia attachment); (
l
–
p
) conidia. Scale bars are set at (
h
) 200
µ
m;
(a) 20 µm; (i–p) 10 µm.
MycoBank: MB836111; Facesoffungi number: FoF08742
Etymology: The specific epithet curvaticonidia reflects the curved conidia.
Holotype: MFLU 18-0789
Saprobic on a dead branch.
Sexual morph:
Ascomata: 320–390
µ
m high
×
360 410
µ
m diameter,
(M =350
×
385
µ
m, n=8), immersed, visible as black spots, solitary, scattered, globose to subglobose or
ovoid, papillate ostiole 75–140
µ
m wide (M =110
µ
m, n=5), yellowish-brown, centric. Periphyses: 1.5–3
µ
m
wide (M =2.5
µ
m, n=20), hyaline, short. Peridium: two-layered; outer layer: 18–26
µ
m (M =21
µ
m,
n=15), reddish-brown cells of textura angularis 7–21
×
1.5–4
µ
m (M =12
×
2.5
µ
m, n=20), thick-walled.
Inner layer: 5.5–11
µ
m (M =8
µ
m, n=15), hyaline cells of textura angularis 8–20
×
1–2.5
µ
m (M =
12
×
2
µ
m, n=20), thin-walled, loosely arranged. Paraphyses: 3.5–5
µ
m wide (M =4.5
µ
m, n=20),
hyaline, highly delicate, cellular, constricted septate, guttulate; 2–3.5
µ
m wide (M =3
µ
m, n=20),
hyaline, filiform, longer than asci, blunt end, cellular, guttulate, embedded in a gelatinous matrix.
Asci: 121–162
×
10.5–17.5
µ
m (M =135
×
12.5
µ
m, n=25), 8-spored, unitunicate, cylindrical, thin-walled,
short-pedunculate, apically rounded, with a J+, discoid apical ring. Ascospores: 17–23
×
6–9
µ
m (M =
20.5
×
7.5
µ
m, n=50), l/w 2.7, uniseriate, oblong or narrowly fusiform, first hyaline with a thin mucous
sheath, turning yellow to yellow-brown, with 1 median, slightly constricted euseptum and two distosepta,
straight to slightly curved, smooth-walled, with 8 longitudinal ridges which render the ascospore to
appear octagonal in transverse section.
Asexual morph:
Coelomycetous. Conidiomata: 590–640
µ
m high
×
370–490
µ
m diam. (M =615
×
425
µ
m, n=3), immersed in PDA, solitary or aggregated, globose to
sub-globose, dark brown. Peridium: comprised of thick-walled, septate, brown mycelium. Conidiophores:
11–17
×
1.5–2.5
µ
m (M =14
×
2
µ
m, n=15), septate, branched, thick-walled, hyaline to light brown.
Conidiogenous cells: 7–17
×
2–2.5
µ
m (M =12.5
×
2.2
µ
m, n=15), elongate, thin-walled, hyaline, annellidic,
guttulate. Conidia: 23.5–30
×
1–2
µ
m (M =26.5
×
1.5
µ
m, n=25), hyaline, cylindrical, curved, smooth-walled,
J. Fungi 2020,6, 174 15 of 27
appendage derived from the middle of the conidia cell, 6–10.5
µ
m long (M =8.5
µ
m, n=20), curved. Culture
characteristics: Colonies on PDA, reaching 9–10 mm diameter after 2 weeks at 25
◦
C; colonies are flat,
irregular, and dense, with immersed and superficial mycelia, with a rough surface, fimbriate margin,
white to light brown becoming orange to dark brown, media becoming brown; reverse light brown at
center with dirty white edges, later becoming yellowish-brown.
Material examined: Thailand, Chiang Rai, Thoeng, Ban Mae Loi Rai, on a dead branch,
19
◦
54
0
N 100
◦
06
0
E, 350 m msl, 11 September 2017, M.C. Samarakoon, SAMC040 (MFLU 18-0789,
holotype; HKAS 102288, isotype); ex-type living culture MFLUCC 18-0620.
Notes: Our collection shares immersed, clypeate, globose to subglobose ascomata with inner
hyaline and outer brown peridium, sepatate, flexuose paraphyses and ellipsoidal, brown ascospores
typical for Amphisphaeria. The J+discoid apical ring is similar to several other species, including
A. flava described from Thailand. However, A. flava differs from our new collection in having a halo
on the host surface around the ostiole and 1-septate ascospores. Amphisphaeria mangrovei differs from
our strains with J
−
and an apical ring in asci. In addition, some of the immature ascospores are
covered with a thin mucilaginous sheath as in A. flava,A. lusitanica,A. seriata,A. sorbi and A. vibratilis.
In addition, our collection has 1 median, slightly constricted at the euseptum, and two distosepta which
are characteristic to A. depressa. Wang et al. [
2
] re-examined the herbarium specimen of A. depressa
and noted that this unusual distoseptate ascospores characteristic is not matched with the generic
description. However, based on unituniate asci with J+, apical ring, Wang et al. [
2
] accepted this
species in Amphisphaeriaceae. The asexual coelomycetous from the culture of our strain is similar
to the only known asexual morph of A. sorbi by having septate, branched, hyaline conidiophores,
elongated conical, thin-walled, septate, hyaline, annellidic conidiogenous cells, and elongate-fusiform,
1-celled, hyaline conidia. The asexual morph of our strain is characterized with curved appendage
derived from the middle of the conidia cell. Here we introduce our new collection as A. curvaticonidia.
Amphisphaeria depressa Petr., Sydowia 7(5–6): 381 (1953)
MycoBank: MB292502; Facesoffungi number: FoF08743
Typus: USA, Hawaii, Kaihea, Oahu, on Cassia bicapsularis (Fabaceae), 24 Feb. 1928, Shear (W 11997,
holotype; BPI 618577, isotype).
Notes: Wang et al. [
2
] re-examined the holotype and remarked the distoseptate ascospores are
an unusual character of this species and accommodated in Amphisphaeria until further collections
are made.
Amphisphaeria doidgeae Marinc., M.J. Wingf. & Crous, in Marincowitz et al., CBS Diversity Ser.
(Utrecht) 7: 20 (2008)
MycoBank: MB506197; Facesoffungi number: FoF08744
Typus: South Africa, Western Cape Province, Kleinmond, on twig litter of Leucadendron sp.
(Proteaceae), 11 July 2000, S. Marincowitz, S.L. 485 (PREM 59556, holotype).
Notes: Amphisphaeria doidgeae is characterized by having broader peridium (59–72
µ
m), septate and
branched paraphyses, asci with a J– apical ring and broadly fusiform with a base often projected and
truncate ascospores.
Amphisphaeria fallax De Not., Hedwigia 4: 21 (1865)
MycoBank: MB187770; Facesoffungi number: FoF08745
Typus: Czech Republic, Wei
β
kirchen, on Quercus robur (Fagaceae), Apr. 1936, F. Petrak (DAOM
148328, neotype).
Notes: Wang et al. [
2
] designated a neotype by observing a slide collection from Quercus robur
deposited by Petrak in 1936 with similar morphology.
Amphisphaeria flava Samarak. & K.D. Hyde, Phytotaxa 391 (3): 210–211 (2019)
MycoBank: MB555396; Facesoffungi number: FoF04976
Typus: Thailand, Chiang Mai, Chang Wat, Amphoe Mae Taeng, Tambon Pa Pae, on a
senescent branch, 01 September 2017, M.C. Samarakoon, SAMC019 (MFLU 18-0102, holotype;
HKAS 102301, isotype); ex-type living culture MFLUCC 18-0361.
J. Fungi 2020,6, 174 16 of 27
Notes: Samarakoon et al. [
7
] introduced A. flava from Thailand which is easy to observe on the
host with its round, pale yellow appearance around the ostiole.
Amphisphaeria fuckelii (G.H. Otth) Samarak., Maharachch. & K.D. Hyde, comb. nov.
MycoBank: MB836119; Facesoffungi number: FoF08746
≡Massaria fuckelii G.H. Otth, Mitth. Naturf. Ges. Bern Nr. 654–683: 50 (1868)
=Massaria fuckelii Fuckel, Jahrb. Nassauischen Vereins Naturk. 23–24: 155. 1870. [1869–70]
=Lepteutypa fuckelii (G.H. Otth) Petr., Ann. Mycol. 21: 276 (1923)
Typus: Belgium, Leuven, Heferlee, Heferleebos, on Tilia cordata (Malvaceae), 31 May 2012, P.
Bormans (WU 33554, neotype); ex-type living culture CBS 140409.
Notes: Jaklitsch et al. [
16
] proposed a neotype for Lepteutypa fuckelii on Tilia cordata from Belgium
(see the generic amendment above).
Amphisphaeria gaubae (Petr.) Y. Z. Wang, Aptroot & K.D. Hyde, Fungal Diversity Res. Ser. 13:
13 (2004)
MycoBank: MB373366; Facesoffungi number: FoF08748
≡Didymosphaeria gaubae Petr., Sydowia 8: 195. (1954)
Typus: Australia, Australian Capital District, Jervis Bay, on dead leaves of Lambertia formosa
(Proteaceae), 14 July 1950, Gauba (H, holotype).
Notes: Aptroot [
64
] transferred Didymosphaeria gaubae to Amphisphaeria and Wang et al. [
2
]
re-examined the holotype and accepted the transfer to Amphisphaeria due to the combination of clypeate
perithecia, unitunicate asci with J+apical ring and 1-septate, slightly curved ascospores.
Amphisphaeria lusitanica (Niessl) Y. Z. Wang, Aptroot & K.D. Hyde, Fungal Diversity Res. Ser. 13:
16 (2004)
MycoBank: MB373365; Facesoffungi number: FoF08751
≡Phorcys lusitanica Niessl, Inst. Coimbra 31: 15 (1883)
=Didymosphaeria lusitanica (Niessl) Berl. & Voglino, in Saccardo, Syll. fung., Addit. I-IV (Abellini):
115 (1886)
=Microthelia lusitanica (Niessl) Kuntze, Revis. gen. pl. (Leipzig) 3(3): 498 (1898)
Typus: Portugal, Figueira da Foz, on Arundo donax (Poaceae), June 1880, Moller, 986 (M, holotype).
Notes: The wedge-shaped apical ring and ascospores strongly constricted at the septum have a
close affinity to Arecophila in Cainiaceae [2].
Amphisphaeria mangrovei Devadatha & V.V. Sarma, in Phookamsak et al.,
Fungal Diversity 95: 161 (2019)
MycoBank: MB554279; Facesoffungi number: FoF04273
Typus: India, Tamil Nadu, Tiruvarur, Muthupet mangroves, on intertidal branches and twigs of
Suaeda monoica (Chenopodiaceae), 29 October 2016, B. Devadatha (AMH-9948 holotype); ex-type living
culture NFCCI-4247.
Notes: Phookamsak et al. [
6
] introduced Amphisphaeria mangrovei on intertidal branches and
twigs as the first report of Amphisphaeria species associated with mangrove habitats. LSU-SSU-ITS
phylogenies revealed that A. mangrovei is a sister to A. sorbi.
Amphisphaeria micheliae Samarak., Jian K. Liu & K.D. Hyde, sp. nov. Figure 7
J. Fungi 2020,6, 174 17 of 27
J. Fungi 2020, 6, x FOR PEER REVIEW 16 of 27
Figure 7. Sexual morph of Amphisphaeria micheliae (HKAS 107012, holotype). (a,b) Host Michelia alba;
(c) substrate; (d,e) ascomata on the substrate; (f) paraphyses; (g) vertical section of ascoma; (h) apical
ring bluing in Melzer’s reagent; (i) ostiole; (j) peridium; (k–m) asci; (n–v) ascospores (v in Melzer’s
reagent); (w) upper view, (x) reverse view of the 2 weeks old colony on PDA. Scale bars are set at (c)
1 cm; (d,e) 500 μm; (g) 200 μm; (i,k–m) 50 μm; (j) 20 μm; (h,n–v) 10 μm.
Saprobic on a dead branch of Michelia alba. Sexual morph: Ascomata: 180–210 μm high × 225–370
μm diameter (M = 190 × 300 μm, n = 8), immersed, visible as black spots in light coloured area on the
host, solitary, scattered, subglobose to oblate, papillate ostiole 60–76 μm high × 34–50 μm diam. (M =
68 × 42 μm, n = 8), centric. Periphyses: 1–2 μm wide (M = 1.5 μm, n = 20), hyaline, short. Peridium: two-
layered; outer layer: 6.5–11.5 μm (M = 8.5 μm, n =10), dense, reddish brown cells of textura angularis
Figure 7.
Sexual morph of Amphisphaeria micheliae (HKAS 107012, holotype). (
a
,
b
) Host Michelia alba;
(
c
) substrate; (
d
,
e
) ascomata on the substrate; (
f
) paraphyses; (
g
) vertical section of ascoma; (
h
) apical
ring bluing in Melzer’s reagent; (
i
) ostiole; (
j
) peridium; (
k
–
m
) asci; (
n
–
v
) ascospores (v in Melzer’s
reagent); (
w
) upper view, (
x
) reverse view of the 2 weeks old colony on PDA. Scale bars are set at (
c
) 1
cm; (d,e) 500 µm; (g) 200 µm; (i,k–m) 50 µm; (j) 20 µm; (h,n–v) 10 µm.
MycoBank: MB836112; Facesoffungi number: FoF08752
Etymology: The specific epithet reflects the host genus Michelia.
Holotype: HKAS 107012
J. Fungi 2020,6, 174 18 of 27
Saprobic on a dead branch of Michelia alba. Sexual morph: Ascomata: 180–210
µ
m high
×
225–370
µ
m
diameter (M =190
×
300
µ
m, n=8), immersed, visible as black spots in light coloured area on the
host, solitary, scattered, subglobose to oblate, papillate ostiole 60–76
µ
m high
×
34–50
µ
m diam.
(M =68
×
42
µ
m, n=8), centric. Periphyses: 1–2
µ
m wide (M =1.5
µ
m, n=20), hyaline, short.
Peridium: two-layered; outer layer: 6.5–11.5
µ
m (M =8.5
µ
m, n=10), dense, reddish brown cells of textura
angularis 6.5–12.5
×
1–2.5
µ
m (M =9
×
1.5
µ
m, n=15), thick-walled. Inner layer: 4–8
µ
m (M =6
µ
m,
n=10), loosely arranged, hyaline cells of textura angularis 10.5–17.5
×
2.5–4
µ
m (M =14.5
×
3
µ
m, n=15),
thin-walled, loosely arranged. Paraphyses: 3.5–4.5
µ
m wide (M =4
µ
m, n=20), hyaline, highly delicate,
cellular, constricted septate, guttulate, embedded in a gelatinous matrix. Asci: 92–135
×
7–10.5
µ
m
(M =115
×
8.5
µ
m, n=25), 8-spored, unitunicate, cylindrical, thin-walled, short-pedunculate,
apically rounded, with a J+, discoid apical ring. Ascospores: 15.5–21
×
6–7.5
µ
m (M =18
×
6.5
µ
m,
n=40), l/w 2.7, uniseriate, oblong or narrowly fusiform, first hyaline, guttulate, turning yellow to
yellow-brown, 1-septate, slightly constricted at septum, straight to slightly curved, smooth-walled.
Asexual morph: Undetermined.
Culture characteristics: colonies on PDA, reaching 20–22 mm diameter after one week at 25
◦
C;
colonies are flat, circular, and dense, with a smooth surface, entire margin, concentrically zonate,
white to light brown, media becoming pale brown; reverse yellowish brown at center and light
brown edges.
Material examined: China, Sichuan Province, Chengdu, University of Electronic Science and
Technology of China (UESTC) campus, on a dead branch of Michelia alba (Magnoliaceae), 30 September
2019, M.C. Samarakoon, SAMC244 (HKAS 107012, holotype; MFLU 20-0503, isotype); ex-type living
culture MFLUCC 20-0121.
Notes: Two of our collections have solitary, immersed ascomata with two-layered peridium,
unitunicate asci with J+, discoid apical ring, and brown ascospores. This collection also has 1-septate
ascospores similar to Amphisphaeria unisepta and A. camelliae. Compared to those two similar species,
this collection has subglobose to oblate ascomata and oblong or narrowly fusiform ascospores.
The phylogenetic analyses show MFLUCC 20-0121 and HKAS 107012 are closely related to A. sambuci,
isolated from partly decorticated branches of Sambucus nigra.Amphisphaeria sambuci is different from our
new collection in having large, depressed globose ascomata and oblong-ellipsoid, straight, rarely curved,
2–4(–6)-distoseptate ascospores with a thick mucilaginous sheath. Based on the morphology and
phylogeny, here we introduce it as the novel species A. micheliae.
Amphisphaeria multipunctata (Fuckel) Petr., Annls mycol. 21(3/4): 329 (1923) (as “Amphisphaeria
millepunctata”)
MycoBank: MB271193; Facesoffungi number: FoF08753
≡Diaporthe multipunctata Fuckel, Jb. nassau. Ver. Naturk. 27–28: 37 (1874) [1873–74]
=Sphaeria acerina (Rehm) Cooke & Plowr. (1833)
=Didymosphaeria acerina Rehm, Ascomyceten: no. 2237 (1874)
=Didymosphaeria acerina var. fraxini G. Winter ex Sacc., Syll. fung. (Abellini) 1: 714 (1882)
=Massariopsis acerina (Rehm) Kirschst., Annls mycol. 33(3/4): 218 (1935)
Typus: Switzerland, Neuch
â
tel, Ca. Neuchatel, on dry branches of Corylus avellana (Betulaceae),
in spring, Morthier, Fuckel, Fungi Rhen. Exs. nr. 2661 (holotype?)
Notes: Aptroot [
64
] synonymized several Didymodphaeria species under Amphisphaeria
multipunctata. Samuels et al. [
65
] noted that Petrak [
10
] has spelled as “Diaporthe millepunctata”
erroneously and the Amphisphaeria multipunctata is the correct name. Aptroot [
64
] documented the host
association of A. multipunctata as Acer campestre (Aceraceae), Carpinus betulus,Quercus robur (Fagaceae),
Fraxinus excelsior,Syringa vulgaris (Oleaceae), Prunus spinosa,Sorbus aucuparia (Rosaceae), Rhus glabra
(Anacardiaceae) and Viburnum opulus (Caprifoliaceae).
Amphisphaeria neoaquatica Samarak., Maharachch. & K.D. Hyde, nom. nov.
MycoBank: MB836114; Facesoffungi number: FoF08737
J. Fungi 2020,6, 174 19 of 27
≡
Lepteutypa aquatica Z.L. Luo, K.D. Hyde & H.Y. Su, in Luo et al., Fungal Diversity: 99:629
(2019) non-Amphisphaeria aquatica (Ellis & Everh.) Berl. & Voglino, in Saccardo, Syll. fung., Addit.
I–IV (Abellini): 125 (1886); Amphisphaeria aquatica Plöttn. & Kirschst., in Kirschstner, Verh. bot. Ver.
Prov. Brandenb. 48: 52 (1906) [1907]
Typus: Thailand, Chiang Rai Province, on submerged decaying wood in a freshwater stream,
Nov. 2013, K.D. Hyde, ZL-23 (MFLU 15-0077, holotype); ex-type living culture MFLUCC 14-0045.
Notes: Luo et al. [
17
] introduced Amphisphaeria aquatica (later re-named A. neoaquatica) which is
similar to A. uniseptata by having subglobose, black, immersed ascomata and long cylindrical asci.
ITS-LSU phylogeny shows that A. neoaquatica is basal to amphisphaeriaceous species [17].
Amphisphaeria paedida (Berk. & Broome) Sacc., Syll. fung. (Abellini) 1: 724 (1882)
MycoBank: MB208832; Facesoffungi number: FoF08754
≡Sphaeria paedida Berk. & Broome, Ann. Mag. nat. Hist., Ser. 4 11: 348 (1873)
=Conisphaeria paedida (Berk. & Broome) Cooke, in Cooke & Plowright, Grevillea 7(no. 43): 86 (1878)
=Melanomma paedida (Berk. & Broome) Cooke (?)
Typus: United Kingdom, on Fagus sylvatica (Fagaceae), Apr. 1859 (K(M), holotype).
Notes: Wang et al. [
2
] re-examined a specimen of A. paedida and noted that the superficial
ascomata, J
−
apical ring and ascospores with thickened septa are characteristic of the species. However,
fresh collection and taxonomic revision is needed.
Amphisphaeria pakistanae E. Müll. & S. Ahmad, Biologia, Lahore 3: 10 (1957)
MycoBank: MB292509; Facesoffungi number: FoF08755
Typus: Pakistan. Swat, Kalam, on dead branches of Indigofera sp. (Fabaceae), 18 Aug. 1952, S.
Ahmad (Z+ZT 9032, holotype).
Notes: Amphisphaeria pakistanae has large ascomata and relatively small, light brown ascospores [
2
].
Amphisphaeria pseudoumbrina Sacc., Atti Soc. Veneto-Trent. Sci. Nat. 2(1): 112 (1873)
MycoBank: MB225907; Facesoffungi number: FoF08757
Typus: Italy, on the bark of Acer campestre (Sapindaceae) (VER, isotype).
Notes: Saccardo [
66
] described A. pseudoumbrina which has oblate ascomata, smaller asci and
relatively broader ascospores compared to A. umbrina. Wang et al. [
2
] re-examined the isotype,
which was found on Acer campestre in Italy, and accepted in Amphisphaeria.
Amphisphaeria qujingensis (Dissan., J.C. Kang & K.D. Hyde) Samarak., Maharachch. & K.D. Hyde,
comb. nov.
MycoBank: MB836127; Facesoffungi number: FoF06506
≡Lepteutypa qujingensis Dissan., J.C. Kang & K.D. Hyde, Phytotaxa 446 (3): 150 (2020)
Typus: China, Yunnan Province, Qujing, on a senescent branch of an unknown host, 06 May 2019,
L.S. Dissanayake, DW1137-045 (HMAS 290478, holotype; HKAS 107065, isotype); ex-type living culture
KUMCC 19-0187.
Notes: Amphisphaeria qujingensis is similar in morphology to A. fuckelii in having immersed
ascomata, J+apical ring, and multiguttulate, hyaline to brown ascospores. LSU-ITS phylogenies also
showed that A. qujingensis is sister to A. fuckelii with strong statistical support.
Amphisphaeria sambuci (Jaklitsch & Voglmayr) Samarak., Maharachch. & K.D. Hyde, comb. nov.
MycoBank: MB836129; Facesoffungi number: FoF08759
≡Lepteutypa sambuci Jaklitsch & Voglmayr, Persoonia 37: 88 (2016a)
Typus: England, Yorkshire, Worksop, Rotherham, Anston, Anstonstones Wood, on partly
decorticated branches of Sambucus nigra (Adoxaceae), 16 May 2011, T. Læssøe et al. (WU 33556,
holotype); ex-type living culture CBS 131707.
Notes: Jaklitsch et al. [
16
] introduced Amphisphaeria sambuci on mostly decorticated branches
of Sambucus nigra lying on the ground and sometimes submerged in aquatic habitats from Europe.
Amphisphaeria sambuci has immersed ascomata, asci with J+, apical ring and ascospores with a central
scarcely constricted euseptum and 2–4(–6)-distoseptate.
Amphisphaeria seriata M.E. Barr & A.W. Ramaley, Mycotaxon 58: 350 (1996)
J. Fungi 2020,6, 174 20 of 27
MycoBank: MB414608; Facesoffungi number: FoF08760
Typus: USA, Texas, Enchanted rock state park, Gillespie Co., on leaf of Nolina sp. (Asparagaceae),
23 October 1993, A.W. Ramaley (BPI 802953, holotype).
Notes: The finely foveolate ascospores are reminiscent of Arecophila foveata, but this species differs
by having a discoid apical ring [
2
]. Furthermore, Arecophila species are mainly from monocots and
A. seriata needs to be re-collected and taxonomically revised.
Amphisphaeria sorbi Senan. & K.D. Hyde, in Liu et al., Fungal Divers 71: 10 (2015)
MycoBank: MB550904; Facesoffungi number: FoF00414
Typus: Italy, Trento [TN], Dimaro, Folgarida, on the branch of Sorbus aucuparia (Rosaceae),
02 August 2013, E. Camporesi, IT 1400 (MFLU 14-0797, holotype); ex-type living culture
MFLUCC 13-0721.
Notes: Liu et al. [
5
] introduced Amphisphaeria sorbi with its holomorph and LSU phylogeny from
Italy. Amphisphaeria sorbi is highly similar to A. vibratilis but differs in having small perithecia, wide,
non-flexuose paraphyses and smooth-walled ascospores without deeply pigmented septa.
Amphisphaeria thailandica Samarak. & K.D. Hyde, Phytotaxa 391 (3): 210–211 (2019)
MycoBank: MB555397; Facesoffungi number: FoF04977
Typus: Thailand, Phayao, Phu Sang, Doi Phu Nang, on a dead branch, 20 July 2017, M.C.
Samarakoon, SAMC097 (MFLU 18-0794, holotype; HKAS 102290, isotype).
Notes: Amphisphaeria thailandica is distinguished from other Amphisphaeria species by having
subglobose to oval, hyaline bi-guttulate immature and light brown to greyish bi-guttulate ascospores,
lacking a mucilaginous sheath.
Amphisphaeria uniseptata (C.K.M. Tsui, K.D. Hyde & Hodgkiss) Samarak., Maharachch. & K.D.
Hyde, comb. nov.
MycoBank: MB836132; Facesoffungi number: FoF08763
≡Clypeosphaeria uniseptata C.K.M. Tsui, K.D. Hyde & Hodgkiss, Mycologia 93(5): 1004 (2001)
=Lepteutypa uniseptata (C.K.M. Tsui, K.D. Hyde & Hodgkiss) Jaklitsch & Voglmayr, Persoonia 37:
88 (2016a)
Typus: Hong Kong, Tai Po, Lam Tsuen River, on submerged wood, Sept. 1997, R.M. Tsui, EjM 247
(HKU(M) 8095, holotype); ex-type living culture HKUCC 6579.
Notes: Tsui et al. [
67
] introduced Amphisphaeria uniseptata on submerged wood from Hong Kong
and noted that it is similar to A. pakistanae in having ellipsoidal, brown, 1-sepatate but differs from
having thick-walled ascospores. Jaklitsch et al. [
16
] placed this species in Lepteutypa emphasizing
the presence of clypeus, long cylindrical asci with the J+apical ring and uniseriate, ellipsoidal,
brown ascospores.
Amphisphaeria vibratilis (Fuckel) E. Müll., in Müller & von Arx, Beitr. Kryptfl. Schweiz 11(no. 2):
695 (1962)
MycoBank: MB326176; Facesoffungi number: FoF08764
≡Massaria vibratilis Fuckel, Jb. nassau. Ver. Naturk. 23–24: 154 (1870) [1869–70]
=Massariella vibratilis (Fuckel) Sacc., Syll. fung. (Abellini) 1: 716 (1882)
=Phorcys vibratilis (Fuckel) J. Schröt., Kryptogamische Flora Schlesiens 3: 381 (1897)
=Massariella vibratilis var. mespili Pass., in Brunaud, Ann. Soc. Sci. nat. Char.-Marit. 25: 29 (1888)
Typus: Canada, British Columbia, on the stem of Prunus sp. (Rosaceae), June 1915, J. Macoun
(DAOM, isotype).
Notes: Wang et al. [
2
] re-examined the isotype. Amphisphaeria vibratilis has asci with the J
−
apical
ring and verrucose ascospores with a mucilaginous sheath.
Amphisphaeria yunnanensis Dissan., J.C. Kang & K.D. Hyde, in Dissanayake et al. Phytotaxa 446
(3): 144–158 (2020)
MycoBank: MB556876; Facesoffungi number: FoF06505
J. Fungi 2020,6, 174 21 of 27
Typus: China, Yunnan Province, Qujing, on a dead branch of an unknown host, 06 May 2019, L.S.
Dissanayake, DW1137-048 (HMAS 290476, holotype; HKAS 107066, isotype); ex-type living culture
KUMCC 19-0188.
Notes: Amphisphaeria yunnanensis has ascomata with narrow and long ostioles, asci with the
J–apical ring and ascospores without a gelatinous sheath.
3.3.3. Taxa Needing Further Revisions
Ten species of Lepteutypa lack molecular data and asexual morphs. There are some “Lepteutypa”
species that have been considered with pestaloid-like asexual morphs as mentioned in the relevant
notes below and it is not possible to place them in Amphisphaeria. Therefore, these “lepteutypa-like”
taxa need to be recollected, and sexual–asexual connections, molecular data, and generic affiliations
established. We therefore do not treat them in Amphisphaeria sensu stricto.
Lepteutypa alpestris (Ellis & Everh.) M.E. Barr, Mycotaxon 46: 56 (1993)
=Melanomma alpestre Ellis & Everh., Proc. Acad. nat. Sci. Philad. 46(3): 328 (1894)
Typus: USA, Washington, Mt. Paddo, on dead twigs of Arctostaphylos nevadensis (Ericaceae),
July 1886, W.N. Suksdorf, 268 (NY, holotype; Washington 342, isotype).
Notes: Ellis and Everhart [
68
] introduced Lepteutypa alpestris with the sexual morph. However,
they noted that one perithecium contained stromatic nature. Barr [
14
] revisited the species and accepted
it in “Lepteutypa” which is closely related to L. cupressi by having 3-septate ascospores covered with
mucilaginous sheath and their sizes. However, L. cupressi has been accepted as Seiridium cupressi [
16
,
69
].
Lepteutypa biseptata Petr., Sydowia 8 (1–6): 197 (1954)
Typus: Australia, New South Wales, on thin branches of Daviesia latifolia (Fabaceae),
16 February 1951, E. Gauba (holotype).
Notes: Petrak [
12
] introduced Lepteutypa biseptata from Australia which has clypeate like
greyish-brown discoloration, perithecia single or aggregated (two perithecia), and 1–3-septate
ascospores with a mucilaginous sheath.
Lepteutypa cisticola Ade, Bot. Jb. 142: 109 (1928)
Typus: Spain, Canary Island, Tenerif, Barranco Anadigo, on Cistus monspeliensis (Cistaceae),
27 May 1926, A. Ade (W, Lectotype).
Notes: Lepteutypa cisticola has 3–5 gregarious ascomata, asci with J+apical ring and euseptate,
with 3-septate (or occasionally two transverse and one oblique) ascospores [
70
]. Petrak [
71
] introduced
Adea canariensis while suggesting it was possibly the anamorph of L. cisticola. However, Nag Raj and
Kendrick [70] re-described Seiridium canariense (=Adea canariensis).
Lepteutypa fusispora Petr., Sydowia 7(5–6): 387 (1953)
Typus: USA, Hawaii, on thin branches of Wisteria sp. (Fabaceae), 14 January 1928, Volcano, Nr.
1131a (holotype).
Notes: Lepteutypa fusispora has more or less often aggregated (often paired) perithecia and 1-septate,
straight or curved ascospores [
11
]. The author has noted that the specimen was young, and asci and
ascospores often shrink, and Diaporthe seposita is often found on the same collection. Nag Raj and
Kendrick [
70
] re-examined the type specimen and remarked that “L. fusispora” is not congeneric with
A fuckelii based on the J+asci and ascospores.
Lepteutypa hederae (Fuckel) Rappaz, Mycol. helv. 7(1): 160 (1995)
≡Amphisphaeria hederae Fuckel, Jb. nassau. Ver. Naturk. 25-26: 304 (1871)
=Anthostoma hederae (Fuckel) Sacc., Syll. fung. (Abellini) 1: 301 (1882)
Typus: Switzerland, near Neuchatel, on dead, corticated branches of Hedera helix (Araliaceae),
by xylotomy probably Viburnum sp. (Adoxaceae), Mar. 1867, P. Morthier (G00111726!; Herbier Fuckel
1894 ex Herb, holotype).
Notes: Rappaz [
72
] accommodated the species in Lepteutypa. Jaklitsch et al. [
16
] re-examined
the holotype, which was reported in the protologue to grow on corticated branches of Hedera helix
J. Fungi 2020,6, 174 22 of 27
(but as revealed by xylotomy, the host is probably Viburnum sp.) from Switzerland. Lepteutypa hederae
possesses consistently 3-septate ascospores, lacking a median euseptum and asci with a J
−
apical ring.
Lepteutypa hexagonalis Goh & K.D. Hyde, Mycol. Res. 101(1): 85 (1997)
Typus: Ecuador, Oriente, Napo Province, Rio Cuyabeno, Cuyabeno rainforest, on dead trunk of
Pinanga sp. (Arecaceae), August 1993, K. D. Hyde (BRIP 23007, holotype).
Notes: Goh and Hyde [
15
] introduced Lepteutypa hexagonalis which is characterized by having
single or in groups of two, deeply immersed ascomata with a distinct ostiole, cylindrical asci with a J+
apical ring and brown, 3-septate, ascospores with 6–7 longitudinal ridges.
Lepteutypa podocarpi (Butin) Aa, Sydowia 39: 1 (1987) [1986]
=Keissleriella podocarpi Butin, Sydowia 27(1–6): 273 (1975) [1973–1974]
Typus: Chile, La Uni
ó
n, leaves of Podocarpus nubigenus (Podocarpaceae), 29 August 1968,
H. Butin (holotype).
Notes: Lepteutypa podocarpi was introduced by Butin [
73
] from Podocarpus nubigenus in Chile.
Based on similar ascospore septation and a pestaloid asexual morph, van der Aa [
74
] accepted this
taxon in Lepteutypa.
Lepteutypa sabalicola (Ellis & G. Martin) M.E. Barr, Mycotaxon 46: 57 (1993)
=Sphaeria sabalicola Ellis & G. Martin, Am. Nat. 16: 810 (1882)
=Leptosphaeria sabalicola (Ellis & G. Martin) Sacc., Syll. fung. (Abellini) 2: LVII (1883)
=Heptameria sabalicola (Ellis & G. Martin) Cooke, Grevillea 18(no. 86): 32 (1889)
Typus: USA, Florida, on petioles of Serenoa serrulata (=Sabal serrulata, Arecaceae), Feb. 1881,
Martin (holotype).
Notes: Barr [
14
] re-examined the type of the specimen on Serenoa serrulata from Florida. Ascomata
are usually gregarious beneath a conspicuous blackened clypeus. Ascospores are verruculose under
the narrow hyaline coating. Another collection from Florida on Aralia spinosa is identical in asci and
ascospores, but the ascomata form beaks that protrude 330–449 beyond the blackened clypeus.
Lepteutypa tropicalis Dulym., Sivan., P.F. Cannon & Peerally, in Dulymamode, Cannon & Sivanesan,
Mycol. Res. 105(2): 249 (2001)
Typus: Mauritius, Petrin, on dead basal leaves of Pandanus rigidifolius (Pandanaceae), 30 April 1996,
R. Dulymamode, P52 (mycol. herb. Univ. Mauritius, holotype; IMI 376738, isotype).
Notes: Dulymamode et al. [
75
] introduced Lepteutypa tropicalis on the adaxial surface of dead
fallen leaf bases of Pandanus rigidifolius and P. palustris from Mauritius.
Lepteutypa ulmicola (Ellis & Everh.) M.E. Barr, Mycotaxon 46: 57 (1993)
=Clypeosphaeria ulmicola Ellis & Everh., Proc. Acad. nat. Sci. Philad. 45: 138 (1893)
Typus: Canada, Ontario, on dead branches of Ulmus sp. (Ulmaceae), April 1892, Dearness,
1776 (NY, holotype).
Notes: Ascospores of Lepteutypa ulmicola have one median septum and two distosepta with
strongly pigmented and irregularly roughened walls.
3.3.4. Hymenopleella
Hymenopleella Munk, Dansk Bot. Ark. 15(no. 2): 89 (1953)
MycoBank: MB2416; Facesoffungi number: FoF08765
=Dyrithiopsis L. Cai et al., Mycologia 95: 912 (2003)
=Neotruncatella Hyang B. Lee & T.T.T. Nguyen, Fungal Diversity 80: 198 (2016)
=Trochilispora V.P. Abreu, A.W.C. Rosado & O.L. Pereira, Fungal Diversity: 96, 169 (2019)
Type species: Hymenopleella hippophaëicola Jaklitsch & Voglmayr, Persoonia 37: 96 (2016)
MycoBank: MB814829; Facesoffungi number: FoF08766
Typus: Austria, Niederösterreich, Gerasdorf, Marchfeldkanalweg, on twigs ofHippophaë rhamnoides
(Elaeagnaceae), 12 August 2012, W. Jaklitsch (WU 32027, epitype); ex-type living culture CBS 140410.
Notes: Hymenopleella is a sexual morph genus and sexual-asexual connection was confirmed
through phylogeny by Liu et al. [
39
] while synonymizing Dyrithiopsis and Neotruncatella under
J. Fungi 2020,6, 174 23 of 27
Hymenopleella. Jaklitsch et al. [
16
] epitypified the type species, Hymenopleella hippophaëicola,
while Liu et al. [39] emended the generic description and provided updated phylogeny.
Hymenopleella schefflerae (V.P. Abreu, A.W.C. Rosado & O.L. Pereira) Samarak., Maharachch. &
K.D. Hyde, comb. nov.
MycoBank: MB836133; Facesoffungi number: FoF08767
≡
Trochilispora schefflerae V.P. Abreu, A.W.C. Rosado & O.L. Pereira, in Hyde et al., Fungal Divers
96: 171–173 (2019)
Typus: Brazil, Minas Gerais, Paraopeba, Floresta Nacional de Paraopeba (FLONA-Paraopeba),
on leaves of Schefflera morototoni (Araliaceae), 30 January 2016, V.P. Abreu & O.L. Pereira (VIC 44384,
holotype); ex-type living culture COAD 2371.
For description, see Hyde et al., Fungal Divers 96: 1–242 (2019)
Notes: A BLASTn search of ITS and LSU sequences of Trochilispora schefflerae (COAD 2371) are
most similar to Hymenopleella (=Dyrithiopsis) (Sporocadaceae). The LSU-ITS-RPB2 phylogeny in this
study also revealed that T. schefflerae clustered with Hymenopleella austroafricana (CPC 21940) with
high statistical support. When the genus was introduced in Amphisphaeriaceae, the phylogeny
lacked sufficient taxa selection, i.e., they used taxa in Bartaliniaceae, Discosiaceae, Pestalotiopsidaceae,
and Robillardaceae which are now synonymized under Sporocadaceae [
16
]. In addition, the genera used
in Amphisphaeriaceae are now accepted in Sporocadaceae (Monochaetia,Morinia) and Sordariomycetes
genera incertae sedis (Ellurema) [
39
]. The phylogenetic study in Hyde et al. [
20
] showed that
T. schefflerae is closely related to Hymenopleella hippophaeicola (CBS 140410). However, Trochilispora
was introduced as a new genus with an uncertain phylogenetic position in Amphisphaeriaceae [
20
].
Trochilispora schefflerae was introduced based on acervular conidiomata, a 3–5-celled, thickened,
brown peridium, small conidiophores, discrete, annellidic conidiogenous cells and fusiform, straight
or slightly curved, 3–4-septate conidia with medium brown central cells and hyaline to subhyaline end
cells with an apical cell with a tubular, filiform, single, eccentric, unbranched, aseptate appendage and
basal cell without a basal appendage. However, the photo plate shows a coelomycetous conidiomata
and similar morphology to Hymenopleella (see Hyde et al. [
20
], Liu et al. [
39
]). Trochilispora schefflerae
and H. austroafricana possess discrete, annellidic conidiogenous cells (3.5–11.5
×
1.5–3
µ
m vs. 4–11.5
×
1.5–3
µ
m) and septate conidia (3–4-septate, 13–21
×
3.5–5
µ
m vs. 3–5 mostly 4-septate, 15.5–
×
4–5
µ
m).
Conidia of Trochilispora schefflerae have longer apical appendages (2.5–7.5
µ
m) compared to those of
H. austroafricana (1.5–4.5
µ
m). Moreover, the spores of T. schefflerae lack basal appendages, which may
be present in H. austroafricana. Based on morphology and phylogeny, here we treat Trochilispora as a
synonym of Hymenopleella and accordingly recombine T. schefflerae in Hymenopleella.
4. Discussion
Jaklitsch et al. [
16
] concluded that the taxonomy of Lepteutypa and related genera is still unclear.
Our morpho-molecular study corroborated this fact and revealed that Lepteutypa is an ill-defined
genus and cannot be clearly segregated from Amphisphaeria. Barr [
76
] provided a key for genera in
Amphisphaeriaceae. Amphisphaeria and Lepteutypa are placed together with brown ascospores (end cells
hyaline or lightly pigmented at times) and single or few ascomata beneath the clypeus. According to
her concept, taxa with 1-septate, smooth-walled or ornamented ascospore bearing species were placed
in Amphisphaeria, while 3-septate ascospores with brown terminal cells, lacking elongate appendages,
distoseptate at times, a wall usually ornamented and surrounded by narrow mucilaginous sheath
were placed in Lepteutypa. This means that the ascospore septation played a key role in distinguishing
the two genera. Jaklitsch et al. [
16
] combined Clypeosphaeria unisepta with L. unisepta addressing the
ascospores septation as an insignificant characteristic for generic delimitation. Samuels et al. [
65
] had
previously mentioned that the number of septa in the ascospores is not taxonomically significant at the
generic level.
Several other studies also show that intraspecific morphological variations are not sufficient
for generic delimitation (e.g., Hirayama and Tanaka [
77
]; Jaklitsch et al. [
78
]; Hyde et al. [
79
];
J. Fungi 2020,6, 174 24 of 27
Wijayawardene et al. [
80
]). Amphisphaeria and Lepteutypa species differ in eutypoid stromata and
septation of ascospores in the protologues. However, we consider that these morphologies are not
sufficient to distinguish these genera. Here, we synonymize Lepteutypa under Amphisphaeria.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2309-608X/6/3/174/s1.
Figure S1: ML tree revealed by RAxML from an analysis of the LSU-ITS matrix of the Amphisphaeriaceae and
selected families of Amphisphaeriales.
Author Contributions:
Conceptualization, M.C.S., S.S.N.M., K.D.H. and M.S.; methodology, M.C.S., S.S.N.M.
and K.D.H.; resources, K.D.H., J.-K.L. and I.P.; writing—original draft preparation, M.C.S.; writing—review and
editing, S.S.N.M., K.D.H., M.S., J.-K.L. and I.P.; supervision, I.P. and K.D.H.; funding acquisition, K.D.H., J.-K.L.
and I.P. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by Thailand Research Fund (TRF), grant number RDG6130001 (Impact of
climate change on fungal diversity and biogeography in the Greater Mekong Sub region) and High-end Foreign
Expert Introduction Plan to Kunming Institute of Botany, granted by the Ministry of Science and Technology of
the People’s Republic of China, grant number G20190139006.
Acknowledgments:
M.C.S. and I.P. acknowledge the Chiang Mai University and Graduate School of Chiang
Mai University for support of this research work. M.C.S. thanks the Mushroom Research Foundation (MRF),
Chiang Rai, Thailand and the international exchange program between the Chiang Mai University and the
University of Electronic Science and Technology of China (UESTC). M.S. and K.D.H. would like to thank the Royal
Golden Jubilee PhD Program under Thailand Research Fund (RGJ) and the German Academic Exchange Service
(DAAD) for a joint TRF-DAAD (PPP 2017–2018) academic exchange Grant. Authors would like to thank Pawel
Dzialak, Konstanze Bensch, Thilini Chethana Kandawatthe and Shaun Pennycook for their advice and supports
during this study.
Conflicts of Interest: The authors declare no conflict of interest.
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