Ophiobolus hydei sp. nov. (Phaeosphaeriaceae, Ascomycota) from Cirsium and Phlomoides in Uzbekistan

Abstract

We introduce a new fungal species, Ophiobolus hydei, from dead stems of Cirsium alatum (Compositae) and Phlomoides brachystegia (Lamiaceae), based on morphological and phylogenetic evidence. The species was collected from the Mountains of Western Tien Shan and southwestern Hissar in Uzbekistan. Ophiobolus hydei is characterized by globose to subglobose ascomata with short to long papilla, cylindrical to subcylindric-clavate asci, broad pseudoparaphyses, and scolecosporous, yellowish-brown to brown, filiform, multiseptate ascospores that can split into several part-spores at the septa. Multigene phylogenetic analyses using a combined gene analysis of ITS, LSU, SSU, and TEF1- indicated that the new species has a close affinity to Ophiobolus ponticus, but differs from that species in the micromorphological characteristics of the ascomata, asci, and ascospores, as well as biogeographic distribution. A distribution map, morphological descriptions, and illustrations with colour photographs of the novel species are provided.

Full text

ARTICLE
Ophiobolus hydei sp. nov. (Phaeosphaeriaceae, Ascomycota)
from Cirsium and Phlomoides in Uzbekistan
Yusufjon Gafforov, Rungtiwa Phookamsak, Hong-Bo Jiang, Dhanushka N. Wanasinghe,
and Mukhiddin Juliev
Abstract: We introduce a new fungal species, Ophiobolus hydei, from dead stems of Cirsium alatum (Compositae) and
Phlomoides brachystegia (Lamiaceae), based on morphological and phylogenetic evidence. The species was collected from
the Mountains of Western Tien Shan and southwestern Hissar in Uzbekistan. Ophiobolus hydei is characterized by
globose to subglobose ascomata with short to long papilla, cylindrical to subcylindric-clavate asci, broad pseudopara-
physes, and scolecosporous, yellowish-brown to brown, filiform, multiseptate ascospores that can split into several
part-spores at the septa. Multigene phylogenetic analyses using a combined gene analysis of ITS, LSU, SSU, and TEF1-␣
indicated that the new species has a close affinity to Ophiobolus ponticus, but differs from that species in the micromor-
phological characteristics of the ascomata, asci, and ascospores, as well as biogeographic distribution. A distribution
map, morphological descriptions, and illustrations with colour photographs of the novel species are provided.
Key words: new species, ascomycetous microfungi, Central Asia, Compositae, GIS, multigene, Lamiaceae,Pleosporales,
phylogeny.
Résumé : Les auteurs présentent une nouvelle espèce, Ophiobolus hydei, isolée de tiges mortes de Cirsium alatum
(Compositae) et de Phlomoides brachystegia (Lamiaceae), identifiée sur la base de données morphologiques et phylo-
génétiques. L’espèce a été récoltée dans les montagnes du Tien Shan de l’Ouest et des monts Hissar du Sud-ouest,
en Ouzbékistan. Ophiobolus hydei est caractérisé par des ascomes globuleux à subglobuleux avec des papilles courtes
à longues, des asques cylindriques à subcylindriques à clavés, de larges pseudoparaphyses et des ascospores
scolécosporeuses, de couleur brune jaunâtre à brune, filiformes, multiseptées qui peuvent se fragmenter en
plusieurs spores au septum. Les analyses phylogénétiques multigéniques réalisées par la combinaison de l’analyse
des gènes de l’ITS, de la LSU, de la SSU et de TEF1-␣ont indiqué que cette nouvelle espèce présente une affinité
étroite avec Ophiobolus ponticus mais qu’elle s’en distingue par les caractéristiques micromorphologiques des
ascomes, des asques, et des ascospores, de même que par sa distribution biogéographique. Une carte de la
distribution, des descriptions et des illustrations morphologiques et des photographies en couleur de la nouvelle
espèce sont présentées. [Traduit par la Rédaction]
Mots-clés : une nouvelle espèce, microchampignon ascomycète, Asie centrale, Compositae, GIS, multigénique, Lamiaceae,
Pleosporales, phylogénie.
Introduction
Phaeosphaeriaceae is one of the largest families in
Pleosporales, comprising over 60 genera and more than
400 species (Phookamsak et al. 2014,2017,2019;Wanasinghe
et al. 2018a;Wijayawardene et al. 2018;Yang et al. 2019;
Bakhshi et al. 2019;Maharachchikumbura et al. 2019;
Marin-Felix et al. 2019). Species of this family are sapro-
trophs, nectrotrophs, or endophytes. Some species, espe-
Received 12 June 2019. Accepted 24 September 2019.
Y. Gafforov.* Laboratory of Mycology, Institute of Botany, Academy of Sciences of the Republic of Uzbekistan, 32 Durmon Yuli Street,
Tashkent 100125, Uzbekistan; Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China; Department of
Ecology, University of Kassel, Heinrich-Plett-Strasse, 40, DE-34132 Kassel, Germany.
R. Phookamsak and D.N. Wanasinghe. Key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming
Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, China.
H.-B. Jiang. Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand.
M. Juliev. Institute of Mountain Risk Engineering, University of Natural Resources and Life Sciences, Vienna 1190, Austria; Tashkent
Institute of Irrigation and Agricultural Mechanization Engineers, Qori Niyoziy Street 39, Tashkent 100000, Uzbekistan.
Corresponding author: Yusufjon Gafforov (email: yugafforov@yahoo.com).
*Present address: Laboratory of Mycology, Institute of Botany, Academy of Sciences of the Republic of Uzbekistan.
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.
A correction was made to the e-First version of this paper on 26 November 2019 prior to the final issue publication. The current online
and print versions are identical and both contain the correction.
671
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cially in their asexual life stages, are reported as important
plant pathogens that have been introduced worldwide
(Arzanlou and Crous 2006;Schoch et al. 2009;Zhang et al.
2012;Phookamsak et al. 2014;Farr and Rossman 2019).
The genus Ophiobolus was established by Riess based on
the type species Ophiobolus disseminans, which was found
on Cirsium arvense in 1854 (Reiss 1854), and this genus
belongs to the order Pleosporales (Phookamsak et al.
2014,2017;Ariyawansa et al. 2015). Ophiobolus is one of the
most species-rich genera of ascomycetous fungi. They are
saprotrophic on dead branches and stems of herbaceous
flowering plants; their ecological function is that they
degrade dead plants. Most species of Ophiobolus have been
described in the Northern Hemisphere (Holm 1948;
Shoemaker 1976,1984;Barr 1979;Walker 1980;Shoemaker
and Babcock 1989;Winter 1886). Recently, some new spe-
cies of Ophiobolus have been described from Italy and Rus-
sia (Ariyawansa et al. 2015;Phookamsak et al. 2017,2019;
Tibpromma et al. 2017;Wanasinghe et al. 2018a). Cur-
rently, 287 species are recognised in worldwide (Index
Fungorum 2019).
Species in Ophiobolus are characterized by their large or
small size ascomata, which are scattered or clustered,
semi-immersed, or erumpent through the host tissue,
globose with a long cylindrical erumpent beak lined
with hyaline periphyses. The asci are numerous, short or
long-stalked, cylindrical to cylindric-clavate, pedicellate,
eight-spored, usually in linear fascicles, sometimes tetra-
seriate, and thin or thick-walled at the apex. The asco-
spores are variously shaped, parallel or spirally arranged
in asci, multiseptate, phragmosporous to scolecosporous,
elliptical to fusiform, sometimes bearing globose ap-
pendages at each end, sometimes with bandlike or
cushion-shaped appendages near the first-formed sep-
tum, without a refractive sphere near each end, lacking
detectable polar germ pores. The ascospores can be sep-
arated into part-spores, or may not break into several
spores; light brown to dark brown or reddish brown
(Holm 1948;Shoemaker 1976;Phookamsak et al. 2017).
Recently, Phookamsak et al. (2017) revisited Ophiobolus
and demonstrated that Ophiobolus-like fungi are polyphyletic
within Phaeosphaeriaceae. The type, Ophiobolus disseminans,
had a close alliance with species of Entodesmium and
Premilcurensis, and therefore those species have been syn-
onymized under Ophiobolus (Phookamsak et al. 2017). In
addition, Ophiobolopsis,Paraophiobolus, and Pseudoophiobolus
were introduced to accommodate other remaining
Ophiobolus-like taxa within Phaeosphaeriaceae (Phookamsak
et al. 2017).
The diversity of fungi in Uzbekistan, especially asco-
mycetous microfungi, has received little attention. Our
aim is to improve the knowledge of the Uzbekistan my-
cota, especially Ascomycetes, and provide a detailed de-
scription and illustrations, as well as information on the
molecular phylogeny of this group of fungi. Additionally,
we aim to revise the taxonomic limits, and obtain more
specific information on the species ecology and geographic
distribution. Recently, several new genera, species, and
new records of ascomycetous microfungi were reported
from Uzbekistan (Solieva and Gafforov 2001,2002;Gafforov
2002,2015,2016a,2016b,2017;Gafforov and Rakhimov
2017;Wanasinghe et al. 2017,2018a,2018b;Samarakoon
et al. 2018;Pem et al. 2018,2019a,2019b;Hyde et al. 2019).
Materials and methods
Study site
The Tien Shan and Hissar mountain systems occupy
15% of the territory of Uzbekistan. The unique diversity
of flora and landscapes makes this mountainous area
one of the most biologically interesting places in Central
Asia, with a wide range of habitats from xeric and moist
forests, to alpine meadows and semi-arid grasslands. These
Central Asian Mountains lie at a biological crossroads at the
most westerly part of the Himalayan range, and support
both Palearctic species and others representative of more
southerly subtropical latitudes. The peculiarity of biota is
due to its mixed character: Indo-Himalayan, Mongolian,
Eurasian, and Mediterranean species are represented, as
well as local endemics. In any geographic region, species
richness and endemism are always higher in the moun-
tains than on the surrounding plains, first, because moun-
tains serve as refugia during unfavorable climatic epochs,
and second, because of greater habitat diversity.
Collection and preservation of samples
During a survey of ascomycetous microfungi in
Uzbekistan, several undescribed Ophiobolus specimens
were collected from dead stems and leaves of Cirsium and
Phlomoides from Compositae and Lamiaceae in the
Surkhandaryo and Tashkent provinces of Uzbekistan.
The studied specimens were deposited in the Tashkent
Mycological Herbarium (TASM; Institute of Botany, Acad-
emy of Sciences of Uzbekistan). Herbarium acronyms
used in the paper are from Index Herbariorum (Thiers
2019, continuously updated).
Morphological examination
Dried specimens were studied following the methods
described in Phookamsak et al. (2017). The specimens
were brought to the laboratory and examined under an
Olympus SZ61 stereomicroscope (Model SZ2-ILST; Olym-
pus, Tokyo, Japan). Micromorphological characters were
examined under a Nikon ECLIPSE 80i (Nikon, Tokyo,
Japan) compound microscope, and images were cap-
tured using a Nikon ECLIPSE 80i compound microscope
with a Canon 550D (Tokyo, Japan) digital camera and
illustrated using DIC microscopy. All morphological
character measurements were made with Tarosoft Im-
age Frame Work (version 0.9.7), and the images used for
figures were processed using Adobe Photoshop CS6 Ex-
tended (version 10.0; Adobe Systems). Faces of Fungi and
MycoBank numbers are provided as outlined in Jayasiri
672 Botany Vol. 97, 2019
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et al. (2015). The new species is justified based on the rec-
ommendations as outlined by Jeewon and Hyde (2016).
DNA extraction, PCR, and sequencing
The genomic DNA was extracted directly from dried
ascomata using a DNA extraction kit (E.Z.N.A Fungal
DNA Mini Kit, D3390-02; Omega Bio-Tek) following the
manufacturer’s protocol. The DNA product was kept at
4 °C for DNA amplification and maintained at −20 °C for
long term storage. DNA was amplified by polymerase
chain reaction (PCR) for four gene regions, namely the
28S large ribosomal subunit (LSU), the 18S small subunit
nuclear rDNA, the internal transcribed spacers (ITS1–
5.8S–ITS2) and the translation elongation factor 1-alpha
(TEF1-␣). The LSU gene region was amplified using the
primers LR0R and LR5 (Vilgalys and Hester 1990;Rehner
and Samuels 1994); the SSU gene region was amplified
using the primers NS1 and NS4 (White et al. 1990); nu-
clear ITS was amplified using the primers ITS5 and ITS4
(White et al. 1990). The TEF1-␣gene region was amplified
using primers EF1-983F and EF1-2218R (Rehner and
Buckley 2005). PCR was carried out in a volume of 25 ␮L
that contained 8.5 ␮L of sterilized water, 12.5 ␮Lof2×
Power Taq PCR MasterMix, 1 ␮L each of forward and re-
verse primers (0.2 ␮mol/L) and 2 ␮L of DNA template
(50 ␮g). The PCR thermal cycle program for ITS, LSU, SSU,
and TEF1-␣gene regions were as detailed by Phookamsak
et al. (2017). PCR amplification was confirmed on 1% aga-
rose electrophoresis gels containing Safeview DNA stain.
The amplified PCR fragments were sent to a commer-
cial sequencing provider (Tsingke Biological Technol-
ogy Co.).
Sequence alignment and phylogenetic analyses
Consensus sequences were assembled in BioEdit
version 7.0.5.2 (Hall 1999) and additional reference se-
quences were obtained from GenBank (Table 1). Subse-
quent alignments for each locus (LSU, SSU, TEF1-␣, and
ITS) were generated with MAFFT version 7 (http://mafft.
cbrc.jp/alignment/server/index.html;Kuraku et al. 2013;
Katoh et al. 2017), and manually corrected when neces-
sary in BioEdit version 7.0.9 (Hall 1999). Each locus and
the concatenated aligned dataset were analysed sepa-
rately using Maximum Likelihood (ML) and Bayesian In-
ference (BI). The best-fit models of evolution for the four
loci tested (GTR+I+G for all gene regions) were estimated
using MrModeltest version 2.3 (Nylander 2004).
BI analyses were performed using MrBayes version
3.2.1 (Ronquist et al. 2012) based on the models selected
by the MrModeltest. The Markov Chain Monte Carlo
(MCMC) algorithm of six chains was initiated for2×10
6
generations in parallel from a random tree topology. The
trees were sampled every 200th generation. The distribu-
tion of log-likelihood scores was examined to determine
the stationary phase for each search and to decide
whether extra runs were required to achieve conver-
gence, using the program Tracer version 1.5 (Rambaut
and Drummond 2007). All sampled topologies beneath
the asymptote (10%) were discarded as part of a burn-in
procedure; the remaining trees were used for calculating
PP in the majority rule consensus tree. Posterior proba-
bilities values of the BI analyses (BYPP) over 0.95 were
considered statistically significant. The ML analyses were
conducted with RAxML-HPC BlackBox (version 8.2.8)
(Stamatakis et al. 2008;Stamatakis 2014) in the CIPRES
Science Gateway platform (Miller et al. 2010) using a
GTR+I+G substitution model with 1000 bootstrap repli-
cates. The robustness of the analyses was evaluated by
bootstrap support (MLBS).
Phylograms were visualized using FigTree version 1.4.0
program (Rambaut 2012) and a phylogenetic tree was
edited in Microsoft Office PowerPoint 2007.
Thirty-six taxa are used as ingroup taxa (including our
newly generated sequences) and Phaeosphaeria chiangraina
(MFLUCC 13-0231), P. oryzae (CBS 110110), and P. thysanolaenicola
(MFLUCC 10-0563) were selected as the outgroup taxa in the
final dataset. Sequences generated in this study were depos-
ited in GenBank (Table 1) and the final matrices and trees in
TreeBASE (accession number: 24465; Study Accession URL:
http://purl.org/phylo/treebase/phylows/study/24465).
Reviewer access URL: http://purl.org/phylo/treebase/
phylows/study/TB2:S24465?x-access-code=7ee4c3843699
05d924a11df642a16d50&format=html.
Data compilation for the GIS map
A point distribution map of new species Ophiobolus
hydei (Fig. 1) was produced using ArcGIS 10.3 desktop
software. A global navigation system (GPS) navigation
device and Google Earth software were used for georef-
erencing recently collected samples in study sites for
Ophiobolus specimens. A WGS84 geographic coordinate
system was used as a reference datum. Information for
Ophiobolus hydei such as collection date, location includ-
ing latitude and longitude, elevation, and host plants are
provided. Landsat satellite data was utilized for the Nor-
malized Difference Vegetation Index (NDVI 2019) and the
Advanced Spaceborne Thermal Emission and Reflection
Radiometer Digital Elevation Model (ASTER DEM 2019)to
prepare the elevation map. Using open source datasets is
a way to upgrade remote sensing and GIS skills, espe-
cially for researchers in developing countries. Landsat
optical satellite images and ASTER DEM are frequently
used for different research areas (Lu et al. 2004;Juliev
et al. 2019).
Results
Phylogenetic inferences
Topologies of trees (under ML and BI criteria) recovered
for each gene dataset were visually compared, and the overall
tree topology was congruent to those obtained from the com-
bined dataset. The RAxML analysis of the combined dataset
yielded a best scoring tree (Fig. 2) with a final ML optimization
likelihood value of −11460.866279. The matrix had 636 dis-
tinct alignment patterns, with 17.17% proportion of gaps
Gafforov et al. 673
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Table 1. A list of species, specimens, strains, and GenBank accession numbers for the sequences used in the phylogenetic analysis.
Species Host/Family Strain/voucher No.
GenBank accession numbers
ITS LSU SSU TEF1-␣Reference Origin
Chaetosphaeronema achilleae Achillea nobilis/Compositae MFLUCC 16-0476 KX765265 KX765266 KX765265 n/a Hyde et al. 2016 Russia
C. hispidulum Anthyllis vulneraria/Fabaceae CBS 216.75 KF251148 EU754144 KF251148 KF253108 Quaedvlieg et al. 2013 Germany
Dematiopleospora cirsii Cirsium sp./Compositae MFLUCC 15-0615 KX274243 KX274250 KX274243 KX284708 Hyde et al. 2016 Italy
D. fusiformis Achillea millefolium/Compositae MFLU 15-2133 KY239029 KY239030 KY239029 n/a Huang et al. 2017 Russia
Dermatiopleospora mariae Ononis spinosa/Fabaceae MFLUCC 13-0612 KX274244 KJ749653 KJ749652 KJ749655 Wanasinghe et al. 2014 Italy
Dlhawksworthia alliariae Alliaria petiolata/Brassicaceae MFLUCC 13-0070 KX494876 KX494877 KX494876 n/a Hyde et al. 2016,
Wanasinghe et al. 2018a
Italy
D. clematidicola Clematis vitalba/Ranunculaceae MFLUCC 14-0910 MG828901 MG829011 MG828901 MG829202 Wanasinghe et al. 2018aRussia
D. lonicera Lonicera sp./Caprifoliaceae MFLUCC 14-0955 MG828902 MG829012 MG828902 MG829203 Wanasinghe et al. 2018aItaly
Muriphaeosphaeria ambrosiae Ambrosia artemisiifolia/Compositae MFLU 15-1971 KX765267 KX765264 KX765267 n/a Hyde et al. 2016 Russia
M. galatellae Galatella villosa/Compositae MFLUCC 14-0614 KT438333 KT438329 KT438331 MG520900 Phukhamsakda et al. 2015 Russia
M. galatellae Galatella villosa/Compositae MFLUCC 15-0769 n/a KT438330 KT438332 n/a Phukhamsakda et al. 2015 Russia
Nodulosphaeria guttulatum Scabiosa sp./Caprifoliaceae MFLUCC 15-0069 KY496746 KY496726 KY501115 KY514394 Tibpromma et al. 2017 Italy
N. multiseptate Sambucus ebulus/Adoxaceae MFLUCC 15-0078 KY496748 KY496728 KY501116 KY514396 Tibpromma et al. 2017 Italy
N. scabiosae Scabiosa sp./Caprifoliaceae MFLUCC 14-1111 KU708850 KU708846 KU708842 KU708854 Mapook et al. 2016 Italy
Ophiobolopsis italica Ononis spinosa/Fabaceae MFLUCC 17-1791 MG520939 MG520959 MG520977 MG520903 Phookamsak et al. 2017 Italy
Ophiobolus artemisiae Artemisia austriaca/Compositae MFLU 15-1966 MG520940 MG520960 MG520978 MG520904 Phookamsak et al. 2017 Russia
O. artemisiae Artemisia campestris/Compositae MFLUCC 14-1156 KT315508 KT315509 MG520979 MG520905 Ariyawansa et al. 2015,
Phookamsak et al. 2017
Russia
O. artemisiicola Artemisia austriaca/Compositae MFLUCC 15-2137 MG828930 MG829039 MG829145 MG829220 Wanasinghe et al. 2018aRussia
O. artemisiicola Artemisia santonicum/Compositae MFLUCC 15-2140 MG828931 MG829040 MG829146 MG829221 Wanasinghe et al. 2018aRussia
O. disseminans Cirsium arvense/Compositae MFLUCC 17-1787 MG520941 MG520961 MG520980 MG520906 Phookamsak et al. 2017 Italy
O. italicus Lathyrus sp./Fabaceae MFLUCC 14-0526 KY496747 KY496727 n/a KY514395 Tibpromma et al. 2017 Italy
O. hydei Cirsium alatum/Compositae TASM 6143 MK981301 MK981305 MK981303 MK993651 This study Uzbekistan
O. hydei Cirsium alatum/Compositae TASM 6144 MK981300 MK981304 MK981302 MK993650 This study Uzbekistan
O. malleolus Cirsium arvense/Compositae MFLUCC 15-1077 MH399730 MH399731 MH399729 n/a Phookamsak et al. 2019 Russia
O. ponticus Centaurea sp./Compositae MFLUCC 17-2273 MG520943 MG520963 MG520982 MG520908 Phookamsak et al. 2017 Italy
O. rossicus Medicago falcata subsp. romanica/
Fabaceae
MFLU 17-1639 MG520944 MG520964 MG520983 MG520909 Phookamsak et al. 2017 Russia
O. rudis none CBS 650.86 KY090650 GU301812 n/a GU349012 Schoch et al. 2009 n/a
O. senecionis Senecio sp./Caprifoliaceae MFLUCC 13-0575 KT728365 KT728366 n/a n/a Tibpromma et al. 2015 Italy
Paraophiobolus arundinis Arundo pliniana/Poaceae MFLUCC 17-1789 MG520945 MG520965 MG520984 MG520912 Phookamsak et al. 2017 Italy
P. plantaginis Plantago sp./Plantaginaceae MFLUCC 17-0245 KY797641 KY815010 KY815012 MG520913 Phookamsak et al. 2017 Italy
Pseudoophiobolus achilleae Achillea millefolium/Compositae MFLU 17-0925 MG520946 MG520966 n/a n/a Phookamsak et al. 2017 Italy
P. galii Galium sp./Rubiaceae MFLUCC 17-2257 MG520947 MG520967 MG520989 MG520926 Phookamsak et al. 2017 Italy
P. italicus Onobrychis viciifolia/Fabaceae MFLUCC 17-2255 MG520948 MG520968 MG520990 MG520927 Phookamsak et al. 2017 Italy
P. mathieui Salvia sp./Lamiaceae MFLUCC 17-1785 MG520951 MG520971 MG520991 MG520929 Phookamsak et al. 2017 Italy
Phaeosphaeria chiangraina Oryza sativa/Poaceae MFLUCC 13-0231 KM434270 KM434280 KM434289 KM434298 Phookamsak et al. 2014 Thailand
P. oryzae Oryza sativa/Poaceae CBS 110110 KF251186 KF251689 n/a n/a Quaedvlieg et al. 2013 South Korea
P. thysanolaenicola Thysanolaena latifolia/Poaceae MFLUCC 10-0563 KM434266 KM434276 KM434286 KM434295 Phookamsak et al. 2014 Thailand
Note: Sequences generated in this study are in bold font.
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and completely undetermined characters in this align-
ment. Parameters for the GTR+I+G model of the com-
bined LSU, SSU, TEF1-␣, and ITS were as follows:
estimated base frequencies: A = 0.243022, C = 0.238618,
G = 0.264759, T = 0.253601; substitution rates: AC =
1.084693, AG = 3.131132, AT = 1.890296, CG = 0.922679,
CT = 8.054753, GT = 1.000; proportion of invariable sites:
I = 0.707451; gamma distribution shape parameter: ␣=
0.582839. The Bayesian analysis resulted in 10001 trees
after 2 M generations. Therefore, the first 1000 trees,
representing the burn-in phase of the analyses, were dis-
carded, while the remaining 9001 trees were used for
calculating posterior probabilities in the majority rule
consensus tree.
Fig. 1. Distribution of Ophiobolus hydei sp. nov. in Uzbekistan. [Colour online.]
Gafforov et al. 675
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All available sequence data from Ophiobolus species and
representative taxa in Chaetosphaeronema,Dematiopleospora,
Dlhawksworthia,Muriphaeosphaeria,Nodulosphaeria,Ophio-
bolopsis,Paraophiobolus, and Pseudoophiobolus were included
in our final dataset. All these genera received high statisti-
cal support for their phylogenetic lineages (Fig. 2). Species
of Ophiobolus are monophyletic with good statistical sup-
port, and Ophiobolus hydei sp. nov. showed a close phyloge-
Fig. 2. Phylogram generated from maximum likelihood analysis based on the combined LSU, SSU, ITS, and TEF1-␣sequence data
for Ophiobolus species and several related genera in Phaeosphaeriaceae. Bootstrap values for maximum likelihood (ML) equal to or
greater than 70% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are listed above the branches, respectively.
The newly generated sequences are indicated in blue bold font. Ex-type strains are indicated in black bold font. The tree is rooted
with Phaeosphaeria chiangraina (MFLUCC 13-0231), P. oryzae (CBS 110110), and P. thysanolaenicola (MFLUCC 10-0563). [Colour online.]
0.0080
Phaeosphaeria oryzae CBS 110110
Muriphaeosphaeria ambrosiae MFLU 15-1971
Dematiopleospora cirsii MFLUCC 15-0615
Ophiobolus ponticus MFLUCC 17-2273
Ophiobolus italica MFLUCC 14-0526
Ophiobolopsis italica MFLUCC 17-1791
Phaeosphaeria chiangraina MFLUCC 13-0231
Ophiobolus artemisiicola MFLU 15-2137
Dlhawksworthia alliariae MFLUCC 13-0070
Chaetosphaeronema achilleae MFLUCC 16-0476
Ophiobolus rossicus MFLU 17-1639
Dematiopleospora fusiformis MFLU 15-2133
Pseudoophiobolus achilleae MFLU 17-0925
Ophiobolus artemisiae MFLUCC 14-1156
Ophiobolus hydei TASM 6144 (paratype)
Dematiopleospora mariae MFLUCC 13-0612
Dlhawksworthia clematidicola MFLUCC 17-0693
Pseudoophiobolus italicus MFLUCC 17-2255
Muriphaeosphaeria galatellae MFLUCC 15-0769
Pseudoophiobolus galii MFLUCC 17-2257
Nodulosphaeria multiseptata MFLUCC 15-0078
Paraophiobolus plantaginis MFLUCC 17-0245
Ophiobolus hydei TASM 6143 (holotype)
Ophiobolus artemisiae MFLU 15-1966
Dlhawksworthia lonicera MFLUCC 14-0955
Paraophiobolus arundinis MFLUCC 17-1789
Pseudoophiobolus mathieui MFLUCC 17-1785
Muriphaeosphaeria galatellae MFLUCC 14-0614
Phaeosphaeria thysanolaenicola MFLUCC 10-0563
Chaetosphaeronema hispidulum CBS 216.75
Ophiobolus disseminans MFLUCC 17-1787
Ophiobolus malleolus MFLUCC 15-1077
Ophiobolus rudis CBS 650.86
Ophiobolus senecionis MFLUCC 13-0575
Nodulosphaeria scabiosae MFLUCC 14-1111
Ophiobolus artemisiicola MFLU 15-2140
Nodulosphaeria guttulatum MFLUCC 15-0069
73/1.00
100/1.00
84/-
100/1.00
100/1.00
94/1.00
100/1.00
99/0.99
100/1.00
100/1.00
100/1.00
94/1.00
100/1.00
100/1.00
87/-
78/1.00
100/1.00
95/1.00
100/1.00
100/1.00
100/1.00
100/1.00
77/0.99
88/1.00
-/1.00
-/0.99
676 Botany Vol. 97, 2019
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netic affinity to O. disseminans (MFLUCC 17-1787), O. malleolus
(MFLUCC 15-1077), and O. ponticus (MFLUCC 17-2273).
Taxonomy
Ophiobolus hydei Gafforov, Phookamsak & Wanas, sp.
nov. (Fig. 3)
MYCOBANK:MB831142.
ETYMOLOGY:hydei (Lat.): in honour of the international
mycologist Kevin D. Hyde for his contribution to the
taxonomy and phylogeny of ascomycetous fungi.
HOLOTYPE:UZBEKISTAN, Surkhandaryo province, Boysun
district, Qizilnaur village, right slope of the Baysuntau
Mountains, on a dead stem of Cirsium alatum (S.G.Gmel.)
Bobrov (Compositae), coll. 14 May 2016, Y. Gafforov, YG-
Fig. 3. Ophiobolus hydei sp. nov. (TASM 6143; holotype). (a) Appearance of ascomata on the host surface. (b) Vertical section
through ascoma. (c) Section through peridium of textura angularis to textura globulosa. (d) Ostiolar canal filled with periphyses.
(e) Pseudoparaphyses embedded in a hyaline gelatinous matrix (stained with Indian ink). (fand g) Asci (g: stained with Indian ink).
(h–j) Ascospores (j: stained with congo red). (k) Apical ascus showing fissitunicate and ocular chamber (stained with Congo red).
(land m) Ascospores split into part-spores. Scale bars: b= 100 ␮m; c=50␮m; d–jand l=20␮m; kand m=10␮m. [Colour online.]
Gafforov et al. 677
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S82-2 (holotype TASM 6143); Surkhandaryo province,
Qizilnaur village, in southwestern spurs of the Hissar
Range, on dead stem and leaves of Cirsium alatum, coll.
14 May 2016, Y. Gafforov, YG-S81-1 (paratype TASM 6144).
DESCRIPTION:Saprobic on dead stems of Cirsium and
Phlomoides plant species from Compositae and Lami-
aceae, respectively. Sexual morph:Ascomata 270–450 ␮m
long (excluding papilla), 290–345 ␮m in diameter, scat-
tered or often grouped under the host tissues, solitary,
immersed in host tissue with long neck, visible as black
dots when immature, in groups on the host surface, dark
brown to black, globose to subglobose, ostiole lined with
hyaline periphyses, coriaceous, papillate. Papilla 125–
160 ␮m long, 70–160 ␮m in diameter, with rounded to
truncate apex, dark brown to black. Peridium 18–33 ␮m
wide, thick-walled cells, pseudoparenchymatous cells,
arranged in textura angularis to textura globules, 8.5–
10 ␮m in diameter, comprising 3–5 layers to multilay-
ered, of thin, flattened, dark-brown cells. Hamathecium
comprising numerous, long, anastomosed pseudopara-
physes embedded in a hyaline gelatinous matrix, sep-
tate, 1.2–2.8 ␮m wide, filamentous, embedded in
mucilage. Asci (123−)162–258(−290) ␮m × (7.3−)8.3–
12.5 ␮m(xˉ = 210 × 10.2 ␮m, n= 20), numerous in a broad
hymenium, 8-spored, sometimes ascospores overlapping
in asci, bitunicate, fissitunicate, cylindrical to subcylindrical-
clavate, pedicellate, apically rounded with a well-developed,
apical ascus. Ascospores fasciculate, scolecosporous, fil-
iform, (110.5–)162–258(–264.2) ␮m × (1.7–)1.9–2.8(–3.2) ␮m
(xˉ = 137 × 2.5 ␮m, n= 20), hyaline when immature, then
becoming slightly pale brown, and becoming brown to
yellowish brown at full maturity, multiseptate up to
30-septate, split into part-spores at the septa, with
rounded ends, smooth-walled, without sheath, without
appendages. Asexual morph: Undetermined.
OTHER SPECIMENS EXAMINED:UZBEKISTAN, Tashkent Prov-
ince, Bustonliq District, Ugam-Chatkal State National Na-
ture Park, Beldersay, the Greater Chimgan Mountain,
Chatkal Range of Western Tien Shan Mountains, on dead
stems of Phlomoides brachystegia (Bunge) Adylov, Kamelin
et Makhm. (Lamiaceae), 7 May 2016, Y. Gafforov, YG-B9-2
(TASM 6145).
KNOWN DISTRIBUTION AND ECOLOGY:northeast and southern
Uzbekistan (Tashkent and Surkhandaryo Province). The
new species was collected in two different localities in
the mountain systems of Western Tien Shan and Pamir-
Alay, and we provided a distribution map for new species
in Uzbekistan (Fig. 1). The new species found on two host
plant species belonging to two families and two genera.
It can be found on other dead plant species from Com-
positae and Lamiaceae in mountains of Central Asia.
Discussion
Ophiobolus hydei differs from O. ponticus by its larger
ascomata, with long papilla (125–160 ␮m long 70 –160 ␮m
in diameter) and with rarely truncate apex on host plants
surface; larger asci [(123–)162–258(–290) ␮m × (7.3–)8.28–
12.5 ␮m] and larger ascospores [(110.5–)162–258(–264.2) ␮m×
(1.7–)1.9–2.8(–3.2) ␮m], and the ascospores can be up to
30-septate and split into several part-spores. Ophiobolus
ponticus has smaller ascospores [(65–)70–85(–94) ␮m×
(2.5–)3.0–4.5 ␮m] and the ascospores have 10–12 septa,
are swollen at the fourth triangular cell, and do not sep-
arate into part-spores (Phookamsak et al. 2017).
Ophiobolus hydei morphologically resembles O. rossicus
Wanas., Bulgakov., E.B.G. Jones & K.D. Hyde, O. disseminans
Riess, and O. rudis (Riess) Rehm by having a similar shape
of ascospores that are broken into pieces (spores) at the
septa (up to 30 part-spores). However, these three species
differ from O. hydei mainly in their smaller ascomata with
shorter necks, and smaller asci and ascospores. Multigene
phylogenetic analyses of combined LSU, SSU, ITS, and
TEF1-␣sequence data showed that O. hydei clusters with
O. ponticus and shares a close relationship to O. disseminans
with support (94% ML and 1.00 PP). However, O. rudis and
O. rossicus are phylogenetically far from O. hydei.
Ophiobolus artemisiae (S. Konta, Bulgakov and K.D. Hyde)
Wanas., Phookamsak and K.D. Hyde resembles O. hydei
in morphology, but O. artemisiae has smaller ascospores
(80–140 ␮m×3–5␮m) that do not break into part-spores,
and lack a swollen cell; it is saprobic on Artemisia species
(Phookamsak et al. 2017). In addition, O. hydei and O. artemisiae
are not closely related in the phylogenetic analysis (Fig. 2).
Ophiobolus was previously known from Europe and
North America. Regarding the ecological aspects, species
of Ophiobolus have been reported on various representa-
tives of Caprifoliaceae, Compositae, Fabaceae, and Lami-
aceae (Table 1): species of Artemisia,Astragalus,Cirsium,
Coronilla,Lathyrus,Medicago,Senecio, and Vicia (Holm 1948;
Shoemaker 1976,1984;Ariyawansa et al. 2015;Phookamsak
et al. 2017,2019;Tibpromma et al. 2017;Wanasinghe et al.
2018a;Farr and Rossman 2019). However, we found
Ophiobolus hydei on Cirsium alatum and Phlomoides brachystegia
from Compositae and Lamiaceae, respectively, in the
Uzbekistan. These two host plants are new substrates for
species of the genus Ophiobolus. Therefore our new spe-
cies from Uzbekistan represents a significant addition to
both the biogeography and host range of Ophiobolus species. A
systematic survey of ascomycetes in Central Asia is
needed. Given the special geographic position of this
central region of Eurasia into consideration, this kind of
survey would greatly improve our knowledge on the
worldwide diversity of ascomycetous microfungi.
Acknowledgements
This work was supported by Ministry of Innovative De-
velopment of the Republic of Uzbekistan (Project No. P3-
2014-0830174425 and PÇ-20170921183), CAS President’s
International Fellowship Initiative (PIFI) for Visiting Scien-
tist (Grant No.: 2018VBB0021) and German Academic Ex-
change Service (DAAD) for a Visiting Fellowship (Grant No.:
57314018). Dhanushka Wanasinghe would like to thank the
CAS President’s International Fellowship Initiative (PIFI) for
funding his postdoctoral research (Grant No.: 2019PC0008),
the National Science Foundation of China, and the Chinese
Academy of Sciences for financial support under the fol-
lowing grants: 41761144055, 41771063, and Y4ZK111B01.
678 Botany Vol. 97, 2019
Published by NRC Research Press
Botany 2019.97:671-680.
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Rungtiwa Phookamsak thanks the CAS President’s Interna-
tional Fellowship Initiative (PIFI) for young staff (Grant No.:
2019FYC0003), the Research Fund from the China Postdoc-
toral Science Foundation (Grant No.: Y71B283261), the Yun-
nan Provincial Department of Human Resources and Social
Security (Grant No.: Y836181261), and National Science Foun-
dation of China (NSFC) project code 31850410489 for financial
research support. We are thankful to Dr. Barbara Wilson (Cor-
vallis, Oregon) for reviewing the manuscript, and for valuable
comments on an earlier version of the manuscript.
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