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Phytotaxa 400 (1): 001–013
https://www.mapress.com/j/pt/
Copyright © 2019 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Jian-Kui Liu: 8 Mar. 2019; published: 2 Apr. 2019
https://doi.org/10.11646/phytotaxa.400.1.1
1
Molecular taxonomy reveals the sexual morph of Nodulosphaeria digitalis in
Phaeosphaeriaceae from Campanula trachelium in Italy
NAPALAI CHAIWAN1, DHANUSHKA N. WANASINGHE1,2, ERIO CAMPORESI3,4,5, SAOWALUCK
TIBPROMMA1,2, SARANYAPHAT BOONMEE1, SAISAMORN LUMYONG6,7 & KEVIN D. HYDE1,2
1Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand
2Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Science, Kun-
ming 650201, Yunnan, People’s Republic of China
3A.M.B. Gruppo Micologico Forlivese ‘‘Antonio Cicognani’’, Via Roma 18, Forlì, Italy
4A.M.B. Circolo Micologico ‘‘Giovanni Carini’’, C.P. 314, Brescia, Italy
5Societa` per gli Studi Naturalistici della Romagna, C.P. 144, Bagnacavallo, RA, Italy
6Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 5200 Thailand
7Center of Excellence in Microbial Diversity and Sustainable Utilization, Faculty of Science, Chiang Mai University, Chiang Mai 50200,
Thailand
*Corresponding author: Kevin D. Hyde. email: kdhyde3@gmail.com
Abstract
The fungal genus Nodulosphaeria is one of the oldest phaeosphaeriaceous genera that comprises saprobic, endophytic and
pathogenic species associated with a wide variety of substrates. There are 71 species epithets listed under Nodulosphaeria
in Index Fungorum and most of these published records lack illustrations and descriptions, or DNA sequence data and thus
it is challenging to confirm their names or investigate taxonomic relationships. In this study, we report the sexual morph of
Nodulosphaeria digitalis, a terrestrial saprobe belonging to the genus Nodulosphaeria (Phaeosphaeriaceae, Pleosporales),
on stems of Campanula trachelium from Arezzo Province, Italy. This is the first record of a Nodulosphaeria taxon on Cam-
panulaceae. Maximum likelihood, Maximum parsimony and Bayesian inference analyses of combined ITS, LSU, SSU and
TEF1-α sequence data reveal that the novel isolate belongs to the family Phaeosphaeriaceae and nests with Nodulosphaeria
digitalis with strong bootstrap support. The detailed descriptions and illustrations of the sexual morph of N. digitalis, an
updated phylogenetic tree for the genus Nodulosphaeria are provided.
Keywords: Multi-gene, Phylogeny, Pleosporales, Saprobe, Taxonomy
Introduction
Phaeosphaeriaceae is one of the largest families in Pleosporales, containing more than 50 genera and 400 species
which are endophytic, hyperparasitic, pathogenic or saprobic on monocotyledons and some dicotyledons (Phookamsak
et al. 2014, 2017, Wijayawardene et al. 2018). Some taxa in this family i.e. Neosetophoma, Parastagonospora,
Phaeosphaeria, Phaeosphaeriopsis, Setophoma, Wojnowicia and Xenoseptoria may be pathogens and can be a risk
to agriculture (Carson 2005, Arzanlou & Crous 2006, Quaedvlieg et al. 2013, Phookamsak et al. 2014). Due to their
economic importance and diversity, there has been a great research interest on the taxa in this family (Hyde et al. 2013,
2017, Phookamsak et al. 2014, 2017, Liu et al. 2015, Mapook et al. 2016, Thambugala et al. 2017a, b, Wanasinghe et
al. 2014, 2018). This has resulted in a rapid taxonomic expansion of this family as numerous novel genera and species
have been introduced (Li et al. 2015). Even though they have provided a better taxonomic understanding, wider
taxon sampling and further taxonomic information based on morphological and phylogenetic data is needed especially
for those monospecific genera (e.g. Acericola, Camarosporioides, Equiseticola, Melnikia, Pseudophaeosphaeria,
Vrystaatia and Yunnanensis) in Phaeosphaeriaceae.
The genus Nodulosphaeria (Rabenhorst 1858) is one of the oldest phaeosphaeriaceous genera has a long history
of taxonomic debate. Nodulosphaeria species are widely distributed throughout the Northern Hemisphere and
comprise saprobic, endophytic and pathogenic species associated with a wide variety of substrates (Mapook et al.
2016, Tibpromma et al. 2017, Wijayawardene et al. 2017, Farr & Rossman 2019). A simple generic diagnosis defined
CHAIWAN ET AL.
2 • Phytotaxa 400 (1) © 2019 Magnolia Press
ascomata with brown ostiolar setae, and three-to multi-septate ascospores, with a swollen cell and with terminal
appendages (Shoemaker 1984), which is similar to some species of Leptosphaeria and Ophiobolus. This has resulted
in the genus becoming a large, heterogeneous assemblage and Index Fungorum currently lists 71 epithets (January,
2019). The exact genera generic placements of these epithets are confused due to lack of molecular data and it is
therefore necessary to recollect these taxa from type localities, isolate them in axenic culture, and analyze their DNA
sequence data to integrate them into appropriate taxonomic ranks with epitypification (Ariyawansa et al. 2014).
During our investigation on the diversity of microfungi in Italy, an isolate was recovered from
Campanula trachelium
in Arezzo Province which based on our morphology and preliminary phylogenetic analyses, was found to belong to
Nodulosphaeria. Subsequently, with morphological and molecular characterization (multigene-based phylogeny of
nuclear ribosomal and protein-coding loci), the taxon was shown to be the sexual morph of Nodulosphaeria digitalis.
Materials and methods
Isolates and morphology
Dead aerial stems of Campanula trachelium were collected from Arezzo Province in Italy and the samples were taken
to laboratory in an envelope. Specimens were observed under a light microscope (Nikon ECLIPSE 80i compound
microscope) and micro-morphological structures were photographed with a Canon EOS 600D digital camera fitted
to the microscope. All measurements were calculated using Tarosoft (R) Image Frame Work program. Figures were
processed with an Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, USA). Single spore isolations
were obtained using the methods described in Chomnunti et al. (2014). Germinated ascospores were transferred to
potato dextrose agar (PDA) plates and incubated at 16 °C. The growth rates and culture characteristics were recorded
after one week and four weeks. Isolates including accession numbers of gene sequences are listed in TABLE 1. The
cultures are deposited in Mae Fah Luang University Culture Collection (MFLUCC) and Culture collection of Kunming
Institute of Botany (KUMCC). Specimens are deposited in the herbarium of Kunming Institute of Botany Academia
Sinica (HKAS) and Mae Fah Luang University (MFLU). Faces of Fungi and Index Fungorum numbers are provided
as outlined in Jayasiri et al. (2015) and Index Fungorum (2019).
DNA extraction, PCR amplification and sequencing
DNA was extracted from mycelium of each isolate grown on PDA for 3–4 weeks at 16 °C; total genomic DNA
was extracted from approximately 150 ± 50 mg axenic mycelium scraped from the edges of the growing culture.
Mycelium was ground to a fine powder with liquid nitrogen and DNA extracted using the Biospin Fungus Genomic
DNA Extraction Kit-BSC14S1 (BioFlux, China) following the instructions of the manufacturer. DNA sequence data
was obtained from the partial sequences of four gene regions, the internal transcribed spacers region of ribosomal DNA
(ITS: ITS5/ITS4) (White et al. 1990), large subunit nuclear ribosomal DNA (LSU: LR0R/LR5) (Vilgalys & Hester
1990), small subunit nuclear ribosomal DNA (SSU: NS1/NS4) (White et al.1990) and the translation elongation factor
1-alpha gene (TEF1-α : EF1-983F/EF1-2218R) (Rehner & Buckley 2005). The polymerase chain reaction (PCR) was
carried out using the following protocol described in Tibpromma et al. (2017). The amplified PCR fragments were sent
to a commercial sequencing provider (Sangon Biotech Co., Shanghai, China). The nucleotide sequence data acquired
were deposited in GenBank (TABLE 1).
Phylogenetic analysis
Sequences generated from different primers of the four gene regions were analyzed with other sequences retrieved
from GenBank (TABLE 1). Sequences with high similarity indices were determined from a BLAST search to find
the closest matches with taxa in Phaeosphaeriaceae, and from recently published data. The multiple alignments of
all consensus sequences, as well as the reference sequences were automatically generated with MAFFT v. 7 (http://
mafft.cbrc.jp/alignment/ server/index.html; Katoh & Standley 2017), and were improved manually when necessary
using BioEdit v. 7.0.5.2 (Hall 1999). Ambiguous regions were excluded manually from the analyses and gaps were
treated as missing data. The finalized alignment and tree were deposited in TreeBASE, submission ID 22294 (Study
Accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S22294). Phylogenetic analyses of both individual
and combined aligned data were performed under maximum-likelihood, maximum parsimony and Bayesian criteria.
THE SEXUAL MORPH OF NODULOSPHAERIA DIGITALIS Phytotaxa 400 (1) © 2019 Magnolia Press • 3
TABLE 1. Taxa used in the phylogenetic analyses and their GenBank accession numbers. The newly generated sequences
are indicated in bold.
Taxa Culture accession
No.
GenBank Accession no References
ITS LSU SSU TEF1-α
Chaetosphaeronema
achilleae MFLUCC 16-0476 KX765265 KX765266 NA NA Thambugala et al.
(2014)
Chaetosphaeronema
hispidulum CBS 216.75 KF251148 KF251652 EU754045 NA Quaedvlieg et al.
(2013)
Dematiopleospora
cirsii MFLUCC 15-0615 KX274243 KX274250 NA KX284708 Phookamsak et al.
(2017)
Dematiopleospora
fusiformis MFLU 15-2133 KY239029 KY239030 KY239028 NA Huang et al. (2017)
Dematiopleospora
mariae MFLUCC 13-0612 KX274244 KJ749653 KJ749652 KJ749655 Phookamsak et al.
(2017)
Dematiopleospora
salsolae MFLUCC 17-0828 MG828889 MG829007 MG829118 MG829201 Wanasinghe et al.
(2018)
Dlhawksworthiana
alliariae MFLUCC 13-0070 KX494876 KX494877 KX494878 NA Hyde et al. (2016)
Dlhawksworthiana
clematidicola MFLUCC 14-0910 MG828901 MG829011 MG829120 MG829202 Wanasinghe et al.
(2018)
Dlhawksworthiana
lonicera MFLUCC 14-0955 MG828902 MG829012 MG829121 MG829203 Wanasinghe et al.
(2018)
Muriphaeosphaeria
ambrosiae MFLU 15-1971 KX765267 KX765264 NA NA Phukhamsakda et al.
(2015)
Muriphaeosphaeria
galatellae MFLUCC 14-0614 KT438333 KT438329 KT438331 NA Phukhamsakda et al.
(2015)
Muriphaeosphaeria
galatellae MFLUCC 15-0769 NA KT438330 KT438332 NA Phukhamsakda et al.
(2015)
Nodulosphaeria
aconiti MFLUCC 13-0728 KU708848 KU708844 KU708840 KU708852 Mapook et al. (2016)
Nodulosphaeria
derasa CBS 184.57 MH857689 MH869228 GU456299 GU456275 Mapook et al. (2016)
Nodulosphaeria
digitalis MFLUCC 17-2418 MG891749 MG891750 MH791042 MH791041 This study
Nodulosphaeria
digitalis MFLUCC 15-2716 KU058710 KU058720 NA NA Li et al. (2015)
Nodulosphaeria
guttulatum MFLUCC 15-0069 KY496746 KY496726 KY501115 KY514394 Tibpromma et al.
(2017)
Nodulosphaeria hirta MFLUCC 13-0867 KU708849 KU708845 KU708841 KU708853 Mapook et al. (2016)
Nodulosphaeria italica MFLU 16-1359 KX672153 KX672158 NA NA Phookamsak et al.
(2014)
Nodulosphaeria
modesta MFLUCC 11-0461 NA KM434285 KM434294 NA Phookamsak et al.
(2014)
Nodulosphaeria
multiseptata MFLUCC 15-0078 KY496748 KY496728 NA KY514396 Tibpromma et al.
(2017)
Nodulosphaeria
sambuci MFLUCC 15-0068 KY496742 KY496721 KY501112 NA Tibpromma et al.
(2017)
Nodulosphaeria
scabiosae MFLUCC 14-1111 KU708850 KU708846 KU708842 KU708854 Mapook et al. (2016)
......continued on the next page
CHAIWAN ET AL.
4 • Phytotaxa 400 (1) © 2019 Magnolia Press
TABLE 1 (Continued)
Taxa Culture accession
No.
GenBank Accession no References
ITS LSU SSU TEF1-α
Nodulosphaeria
spectabilis MFLUCC 14-1112 KU708851 KU708847 KU708843 KU708855 Mapook et al. (2016)
Ophiobolus artemisiae MFLU 15-1966 MG520940 MG520960 MG520978 MG520904 Phookamsak et al.
(2017)
Ophiobolus
disseminans MFLUCC 17-1787 MG520941 MG520961 MG520980 MG520906 Phookamsak et al.
(2017)
Ophiobolus rossicus MFLU 17-1639 MG520944 MG520964 MG520983 MG520909 Phookamsak et al.
(2017)
Ophiobolus rudis CBS 650.86 KY090650 GU301812 NA GU349012
Ophiosphaerella
agrostidis MFLUCC 11-0152 KM434271 KM434281 KM434290 KM434299 Phookamsak et al.
(2017)
Ophiosphaerella
agrostidis MFLUCC12-0007 KM434272 KM434282 KM434291 KM434300 Phookamsak et al.
(2017)
Ophiosphaerella
aquatica MFLUCC 14-0033 KX767088 KX767089 KX767090 MG520911 Ariyawansa et al.
(2015)
Phaeosphaeriopsis
dracaenicola MFLUCC 11-0157 KM434273 NG_059532 KM434292 KM434301 Phookamsak et al.
(2014)
Phaeosphaeriopsis
glaucopunctata MFLUCC 13-0265 KJ522473 KJ522477 KJ522483 MG520918 Thambugala et al.
(2014)
Phaeosphaeriopsis
triseptata MFLUCC 13-0271 KJ522475 KJ522479 KJ522484 MG520919 Thambugala et al.
(2014)
Pseudoophiobolus
galii MFLUCC 17-2257 MG520947 MG520967 MG520989 MG520926 Phookamsak et al.
(2017)
Pseudoophiobolus
italicus MFLUCC 17-2255 MG520948 MG520968 MG520990 MG520927 Phookamsak et al.
(2017)
Pseudoophiobolus
mathieui MFLUCC 17-1785 MG520951 MG520971 MG520992 MG520929 Phookamsak et al.
(2017)
Phaeosphaeria
chiangraina MFLUCC 13-0231 NR_155643 KM434280 KM434289 KM434298 Phookamsak et al.
(2014)
Phaeosphaeria
thysanolaenicola MFLUCC 10-0563 NR_155642 KM434276 KM434286 KM434295 Phookamsak et al.
(2014)
Phaeosphaeria musae MFLUCC 11-0151 KM434268 KM434278 KM434288 KM434297 Phookamsak et al.
(2014)
Staurosphaeria
rhamnicola MFLUCC 17-0814 MF434201 MF434289 MF434377 MF434463 Wanasinghe et al.
(2017)
Staurosphaeria
rhamnicola MFLUCC 17-0813 MF434200 MF434288 MF434376 MF434462 Wanasinghe et al.
(2017)
The parsimony analysis was carried out with the heuristic search option in PAUP (Phylogenetic Analysis Using
Parsimony) v. 4.0b10 with the following parameter settings: characters unordered with equal weight, random taxon
addition, branch swapping with tree bisection-reconnection (TBR) algorithm, branches collapsing if the maximum
branch length was zero. Alignment gaps were treated as missing characters in the analysis of the combined data
set, where they occurred in relatively conserved regions. Trees were inferred using the heuristic search option with
1000 random sequence additions, with maxtrees set at 1000. Descriptive tree statistics for parsimony; 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 (Kishino & Hasegawa
1989) were performed in order to determine whether trees were significantly different. Maximum parsimony bootstrap
values (MP) equal or greater than 70 % are given above each node (FIGURE. 1).
THE SEXUAL MORPH OF NODULOSPHAERIA DIGITALIS Phytotaxa 400 (1) © 2019 Magnolia Press • 5
The evolutionary models for Bayesian analysis and maximum-likelihood were selected independently for each
locus using MrModeltest v. 2.3 (Nylander 2004) under the Akaike Information Criterion (AIC) implemented in both
PAUP v. 4.0b10. GTR+I+G model was the best-fit model of each locus for Bayesian analysis and maximum-likelihood
as determined by AIC in MrModeltest.
Bayesian analysis was conducted with MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001) to evaluate Bayesian
posterior probabilities (BYPP) (Rannala & Yang 1996 by Markov chain Monte Carlo sampling (BMCMC). GTR+I+G
was used in the command. Six simultaneous Markov chains were run for 2000000 generations and trees were sampled
every 200th generation. The distribution of log-likelihood scores was examined to determine stationary phase for each
search and to decide if extra runs were required to achieve convergence, using the program Tracer 1.4 (Rambaut &
Drummond 2007). First 10% of generated trees were discarded and remaining 90% of trees were used to calculate
posterior probabilities of the majority rule consensus tree. BYPP greater than 0.95 are given above each node (FIGURE.
1).
FIGURE 1. Phylogram generated from RAxML based on combined ITS, LSU, SSU and TEF1-α sequence data. Bootstrap support
values for maximum parsimony (MP, left) greater than 70%, maximum likelihood (ML, middle) greater than 70% and Bayesian posterior
probabilities (PP, right) equal to or greater than 0.95 are indicated at the nodes. The ex-type strains are marked with a T and the report of
the sexual morph (Nodulosphaeria digitalis) is in white.
CHAIWAN ET AL.
6 • Phytotaxa 400 (1) © 2019 Magnolia Press
Maximum likelihood trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008,
Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using GTR+I+G model of evolution.
Maximum likelihood bootstrap values (ML) equal or greater than 70% are given above each node (FIGURE. 1).
Phylograms were visualized with FigTree v1.4.0 program (Rambaut 2012) and reorganized in Microsoft power
point (2007).
Results
Phylogenetic analyses
The combined dataset (ITS, LSU, SSU and TEF1-α loci) consisted of 44 taxa with selected genera in Phaeosphaeriaceae
including our strain and Staurosphaeria rhamnicola (MFLUCC 17-0813 and MFLUCC 17-0814) as outgroup. The
RAxML analysis of the combined dataset yielded a best scoring tree (FIGURE. 1) with a final ML optimization
likelihood value of-13845.948892. The matrix had 865 distinct alignment patterns, with 17.25% of undetermined
characters or gaps. Parameters for the GTR + I + G model of the combined ITS, LSU, SSU and TEF1-α were as
follows: Estimated base frequencies; A = 0.244869, C = 0.238356, G = 0.264219, T = 0.252556; substitution rates AC
= 0.954803, AG = 3.043523, AT = 1.950135, CG = 0.798160, CT = 7.129973, GT = 1.000000; proportion of invariable
sites I = 0.697595; gamma distribution shape parameter α = 0.629274.
The Bayesian analysis resulted in 10001 trees after 2000000 generations. The first 1000 trees, representing the
burn-in phase of the analyses, were discarded, while the remaining 9001 trees were used for calculating posterior
probabilities in the majority rule consensus tree.
The maximum parsimonious dataset consisted of 3397 characters, of which 2769 were constant, 483 parsimony-
informative and 145 parsimony-uninformative. The parsimony analysis of the data matrix resulted in 268 equally most
parsimonious trees with a length of 1770 steps (CI = 0.497, RI = 0.723, RC = 0.359, HI = 0.503) in the first tree. Tree
topologies (generated under MP and Bayesian criteria) from single gene datasets were also compared and the overall
tree topology was congruent to those obtained from the combined dataset ML tree (FIGURE 1).
The ML tree generated based on sequence analysis of the combined dataset indicated that our strain grouped with
Nodulosphaeria digitalis (MFLUCC 15-2716, asexual morph) with 75% ML and 75% MP statistical support (FIGURE
1).
Taxonomic treatment
Nodulosphaeria digitalis W.J. Li, Camporesi, D.J. Bhat & K.D. Hyde, Mycosphere 6 (6): 681–708 (2015) amend.
FIGURE 2
Index Fungorum number: IF551664; Faces of fungi number: FoF01302.
Saprobic on Campanula trachelium. Sexual morph: Ascomata 180–250 μm high × 180–400 μm diam. (
x
= 141.9 ×
154 µm, n = 5), superficial to semi-immersed, flat at the base, solitary, scattered, uniloculate globose to subglobose
dark-brown to black, smooth-wall, conspicuous on the host. Ostioles 50–60 × 45–60 μm (
x
= 53.5 × 52.5 μm, n =
5), protruding from the center of the ascomata, without setae. Peridium 40–80 μm wide, comprising several layers
brown to dark brown cells of textura angularis, outer layer darkened cells and inner layer pale brown to hyaline cells.
Hamathecium comprising numerous, filiform, septate, hyaline pseudoparaphyses. Asci 70–100 × 7–11 μm (
x
= 83
× 9 μm, n = 20), 8-spored, bitunicate, cylindric-clavate, thick walled at the apex, with minute ocular chamber, short
pedicellate or sessile with club-shaped pedicel. Ascospores 20–30 × 4.5–5.5 μm (
x
= 25 × 5 μm, n = 20), overlapping
1–2-seriate, fusiform, ellipsoidal to subcylindrical, slightly curved, upper part sorter and wider than the lower part,
4-transversely septate, constricted at the middle septum, initially hyaline, becoming yellowish-brown at maturity, ends
remaining lighter and cone-shaped, with narrowly rounded ends, wall firm, smooth-walled, guttulate, with appendages
(3–5 μm long, 3–4 μm wide) at both ends. Asexual morph: Coelomycetous, See Li et al. (2015).
Culture characteristics:—Ascospores germinating on PDA and producing germ tubes within 24 hr. Colonies
growing on PDA, circular, umbonate, rough and wrinkled at surface, edge entire, reaching 1–2 cm in 2 weeks at 25 °C,
white to brown, mycelium superficial and partially immersed, branched, septate, hyaline to pale brown, smooth.
THE SEXUAL MORPH OF NODULOSPHAERIA DIGITALIS Phytotaxa 400 (1) © 2019 Magnolia Press • 7
Specimen examined:—ITALY, Arezzo Province (AR), Stia, Montemezzano, on dead aerial stem of Campanula
trachelium L. (Campanulaceae), 14 July 2017, Erio Camporesi, IT3404 (MFLU 17-1274, HKAS 97488), living culture,
MFLUCC 17-2418.
FIGURE 2 Nodulosphaeria digitalis (MFLU 17-1274). a–c. Ascomata on a dead stem of Campanula trachelium. d. Pseudoparaphyses.
e. Section of ascoma. f–h. Ascospores. i–k. Asci. l, m. Culture on PDA (note m reverse). Scale bars: b = 100 μm, c = 500 μm, d = 10 μm,
e, i–k = 20 μm, f–h = 10 μm.
CHAIWAN ET AL.
8 • Phytotaxa 400 (1) © 2019 Magnolia Press
TABLE 2. Synopsis of the sexual morphs of Nodulosphaeria species with morphological features discussed in this study.
Species Ascomata/ ascostromata
(μm) Peridium (μm) Asci (μm) Ascospore (μm) Host Family References
Size Vertical septa Terminal appendages
N. aconiti 208–210 ×113–120 11–16 62–79×9–10 29–33 × 4–5 4 Absent Ranunculaceae Mapook et al. (2016)
N. derasa 250–350 × 250–300 35–40 90–110 ×11–14 28–55 × 5–6 8 Present (curved and cylindric) Asteraceae Shoemaker (1984)
N. digitalis 178–245 × 176–395 35–85 74–95 × 7–11 22–29 × 4.5–5.5 4 Present Campanulaceae This study
N. guttulatum 197–218 × 163–244 15–30 40–89 × 10–15 29–38 × 5–6.5 4 Present Caprifoliaceae Tibpromma et al. (2017)
N. hirta 273–277 × 220– 250 19–23 95–97 × 12–15 56–62 × 5–6 8 Absent Asteraceae Mapook et al. (2016)
N. multiseptata 232–260 × 267–272 19–27 74–127 × 17–21 56–70 × 5–6 12–14 Absent Adoxaceae Tibpromma et al. (2017)
N. sambuci 204–220 × 173–215 23–44 53–80 × 7–10 32–35 ×3–5 8 Absent Adoxaceae Tibpromma et al. (2017)
N. scabiosae 200–210 × 215–232 25–30 105– 125 × 11–13 35–40 × 3–6 4 Absent Caprifoliaceae Mapook et al. (2016)
N. spectabilis 210–290 × 250–300 20–25 100–125 × 13–16 30–42 × 5–7 4 Absent Apiaceae Mapook et al. (2016)
THE SEXUAL MORPH OF NODULOSPHAERIA DIGITALIS Phytotaxa 400 (1) © 2019 Magnolia Press • 9
Notes:—Li et al. (2015) introduced Nodulosphaeria digitalis which was collected on dead stems of Dactylis
sp. in Italy. In this study, we have acquired DNA from a sexual morph which is characterized by uniloculate,
globose to subglobose ascomata, ostioles, a peridium comprising cells of textura angularis, filiform, septate, hyaline
pseudoparaphyses, short pedicellate cylindric-clavate asci, and fusiform, ellipsoidal to subcylindrical ascospores
with appendages in agreement with the descriptions in Shoemaker (1984) for Nodulosphaeria species. In multi-gene
phylogeny our strain and Nodulosphaeria digitalis grouped in a monophyletic clade. Even though this is not strongly
supported in BI (0.91), both MP and ML acquired a good support (75%). Also, the comparison of the Nodulosphaeria
digitalis nucleotides across the ITS regions there were not differences. Therefore, report the sexual morph of
Nodulosphaeria digitalis and amended here to include sexual morph descriptions.
Acknowledgments
N. Chaiwan would like to thank the Thailand Research Fund (PHD60K0147) and Kunming Institute of Botany for
the help with molecular work. K.D. Hyde would like to thank the Thailand Research Fund (project No. RDG6130001
and DBG6080013) and Molecular Biology Experimental Center for the help on molecular work, and the Mushroom
Research Foundation (MRF), Chiang Rai, Thailand for supporting this research. Saranyaphat Boonmee thanks to
the Thailand Research Fund, project number TRG6180001. D.N. Wanasinghe would like to thank CAS President’s
International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008), the National
Science Foundation of China and the Chinese Academy of Sciences for financial support under the following grants:
41761144055, 41771063 and Y4ZK111B01.
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