Mycosphere 9(3): 565–582 (2018) www.mycosphere.org
ISSN 2077 7019
Article
Doi 10.5943/mycosphere/9/3/8
Copyright © Guizhou Academy of Agricultural Sciences
Deniquelata vittalii sp. nov., a novel Indian saprobic marine fungus on
Suaeda monoica and two new records of marine fungi from Muthupet
mangroves, East coast of India
Devadatha B1, Sarma VV1*, Ariyawansa HA2 and Gareth Jones EB3,4
1
Fungal Biotechnology Lab, Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet,
Pondicherry-605014, India
2
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
3
Division of Plant Pathology, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai
University, Chiang Mai 50200, Thailand
4
Nantgaredig, 33B St. Edwards Road, Southsea, Hants., PO5 3DH, UK
Devadatha B, Sarma VV, Ariyawansa HA, Gareth Jones EB 2018 – Deniquelata vittalii sp.nov., a
novel Indian saprobic marine fungus on Suaeda monoica and two new records of marine fungi from
Muthupet mangroves, East coast of India. Mycosphere 9(3), 565–582, Doi
10.5943/mycosphere/9/3/8
Abstract
Deniquelata vittalii, a novel species of marine fungi in the genus Deniquelata, found saprobic
on a decaying woody stem of the halophyte Suaeda monoica, collected from Muthupet mangroves,
southeast coast of India is described and illustrated in this paper. Morphologically D. vittalii
resembles D. barringtoniae, but it is distinct in having larger ascomata, asci and golden yellow to
dark brown ascospores with 3–6 transverse septa. Phylogenetic analyses inferred from combined
LSU, SSU and ITS datasets indicate that D. vittalii shares a sister relationship with D.
barringtoniae with high statistical support and forms a strongly supported monophyletic clade.
Both morphological differences and DNA based sequence data strongly support the establishment
of the new taxon. New records of Farasanispora avicenniae and Hysterium rhizophorae are also
reported in this paper supplemented with molecular sequence data.
Key words – Dothideomycetes – Didymosphaeriaceae – Pleosporales – Mangrove Fungi –
Molecular phylogeny
Introduction
Didymosphaeriaceae was introduced by Munk (1953), which is typified by Didymosphaeria
Fuckel with D. epidermidis as the type species. This family is characterized by brown, thickwalled, 1-septate ascospores and trabeculate pseudoparaphyses, which anastomose above the asci in
a gelatinous matrix and includes 25 accepted genera (Aptroot 1995, Hyde et al. 2013, Ariyawansa
et al. 2014a, b, Wijayawardene et al. 2017). However, very few genera were reported in this family
from marine based habitats (Jones et al. 2015).
The monotypic genus Deniquelata Ariyawansa & K.D. Hyde was established by Ariyawansa
et al. (2013) to accommodate Deniquelata barringtoniae Ariyawansa & K.D. Hyde, found as a
pathogen on living leaves of Barringtonia asiatica (Lecythidaceae). Both morphology and
phylogenetic analyses of a concatenated dataset of the SSU and LSU rDNA supported Deniquelata
Submitted 6 March 2018, Accepted 18 April 2018, Published 15 June 2018
Corresponding Author: Venkateswara Sarma V – e-mail – sarmavv@yahoo.com
565
as a new genus within Montagnulaceae, which was later synonymized and transferred to
Didymosphaeriaceae based on its sister group relationship to Bimuria D. Hawksw., Chea &
Sheridan and Tremateia Kohlm., Volkm-Kohlm. & OE. Erikss. (Ariyawansa et al. 2013, 2014b).
This genus is characterized by ascomata that are immersed, globose to sub-globose, dark brown to
black, asci bitunicate, clavate to broadly-clavate with a short furcate pedicel. Ascospores oblong to
narrowly oblong, reddish brown to dark yellowish brown, muriform with three transverse septa and
1–2 vertical septa, verruculose and lacking a sheath (Ariyawansa et al. 2013).
Farasanispora Abdel-Wahab, Bahkali & E.B.G. Jones is a monotypic genus established by
Li et al. (2016) to accommodate Farasanispora avicenniae Abdel-Wahab, Bahkali & E.B.G. Jones
recorded on decaying wood of Avicennia marina from Farasan Island. Phylogenetic analysis of a
concatenated dataset of the SSU and LSU rDNA supported Farasanispora as a new genus within
Pleosporales and related to well established families such as Trematosphaeriaceae,
Ascocylindricaceae and Morosphaeriaceae that are known to harbour marine fungi. However, any
close affinities to other genera or an accurate position within a particular family remained
unresolved (Li et al. 2016). Currently the genus is monotypic with Farasanispora avicenniae.
The genus Hysterium was circumscribed by morphological characteristics such as
hysterothecial, pigmented, carbonaceous ascomata and ascospores that are 3 or more transverselyseptate (Bisby 1923). The recent molecular studies showed that the species of Hysterium are
polyphyletic (Schoch et al. 2009). Hyde et al. (2017) reported Hysterium rhizophorae, a new
species, from Rhizophora apiculata based on the differences in the asci and ascospore dimensions
in contrast with H. angustatum, which also formed a distinct lineage in the phylogenetic analyses.
We have reported some new species from our ongoing studies on biodiversity of marine fungi
from Muthupet mangroves, Tamil Nadu, south east coast of India (Devadatha et al. 2017, 2018,
Devadatha & Sarma 2018). In the present study, we introduce a new species Deniquelata vittalii in
Deniquelata based on morphological characters and phylogenetic analyses. Further, two new
records of marine fungi to India, viz., Farasanispora avicenniae and Hysterium rhizophorae are
also reported in this paper supplemented with molecular sequence data.
Materials & Methods
Sample collection and morphological studies
Decaying mangrove woody stem pieces of the halophyte Suaeda monoica Forssk. ex J.F.
Gmel and Aegiceras corniculatum (L.) Blanco were collected from Muthupet mangroves (10.4 °N,
79.5 °E), Kaveri River Delta, Tamil Nadu, southeast coast of India as detailed in Devadatha et al.
(2017). Specimens were incubated in moist chambers and examined under an Optika stereo zoom
SZM-LED1 microscope. Hand sections of the ascomata were taken, where necessary, and the spore
mass contents were scooped out with the help of forceps or a needle and mounted in sterile sea
water and/or Lactophenol to observe the microscopic characters. Images were captured using Nikon
ECLIPSE TiU upright microscope with DIC objectives connected to Nikon DS-Fi2 digital camera.
Single spore isolation was performed as described in Chomnunti et al (2014) with the
modifications outlined in Devadatha et al. (2017). The herbarium specimens and the type cultures
were deposited in the Ajrekar Mycological Herbarium (AMH) and National Fungal culture
collection of India (NFCCI), Agharkar Research Institute (ARI), Pune, India. Facesofffungi and
MycoBank numbers are provided (Jayasiri et al. 2015, MycoBank 2017).
DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from the axenic cultures grown on Malt Extract Agar (MEA)
medium by using the DNeasy plant DNA extraction kit (QIAGEN, Germany) following the
manufacturer’s protocol. In this study, several loci were amplified using known universal primer
pairs: ITS1 and ITS4 to amplify ITS region and nuclear small subunit rDNA region (SSU) was
amplified using NS1 and NS4 (White et al. 1990). Nuclear large subunit rDNA (LSU) was
amplified using LR0R and LR5 (Vilgalys & Hester 1990). The translation elongation factor 1-alpha
566
gene (TEF-1α) was amplified using primers EF1–983F and EF1–2218R (Rehner & Buckley 2005).
The RNA polymerase II second largest subunit (RPB2) gene was amplified using the fRPB2-5F
and fRPB2-7cR primer (Liu et al. 1999).
The amplifications were performed employing 50µL of Polymerase chain reaction (PCR)
mixtures containing 5 µL of 10X Ex Taq buffer, 4 µL of deoxy nucleotide triphosphate mixture
(2.5 mM of each dNTP), 1 µL of each primer (10 µM), 2µL of DNA template, 0.25 µL of Takara
EX Taq polymerase and 36.75 µL of Nuclease free water. The PCR amplification conditions and
amplified PCR amplicons were purified as reported in Devadatha et al. (2018). The purified PCR
products were sequenced at Macrogen Inc. (Seoul, Korea).
Phylogenetic analyses
Taxa used in the phylogenetic analyses were obtained based on the BLAST search similarity
resulted from LSU and ITS regions and through published literature (Ariyawansa et al. 2013,
2014a, b) for the taxonomic placement of Deniquelata vittalii. Multi-gene phylogenetic analyses of
combined LSU, SSU, ITS, TEF-1α, RPB2 sequence data were performed for Farasanispora
avicenniae (Li et al. 2016) and Hysterium rhizophorae (Hyde et al. 2017), based on the published
data. Multiple sequence alignments for individual regions were generated online at MAFFT server
(http://mafft.cbrc.jp/alignment/server/) (Katoh & Standley 2013) and alignments were improved
manually using BioEdit, where necessary. The individual sequence datasets (LSU, SSU, ITS, TEF1α, RPB2) were combined using BioEdit v.7.0.5.2 (Hall 1999). Three different datasets were
prepared for the multigene phylogenetic analyses.
Maximum-parsimony analysis was performed by using PAUP v.4.0b10 (Swofford 2002)
software to generate the most parsimonious trees. 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 for Figure 1 and equal
or greater than 75 % for Figs 2, 3.
The evolutionary models for phylogenetic analyses were selected for each gene using
MrModeltest v. 2.3 (Nylander 2004) under the Akaike Information Criterion (AIC) as implemented
in both PAUP v. 4.0b10 and Mr. Bayes v.3.1.2. GTR+I+G model was selected as the most
appropriate in each locus for Bayesian analysis and maximum-likelihood by AIC in MrModeltest as
the best-fit model.
Bayesian analysis was performed with MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001) to
evaluate Bayesian posterior probabilities (BYPP) (Rannala &Yang 1996, Zhaxybayeva & Gogarten
2002) by Markov Chain Monte Carlo sampling (BMCMC). GTR+I+G was used in the command.
Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every
100th generation (resulting in 10001trees). 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 20% of generated
trees were discarded and remaining 80% trees were used to calculate posterior probabilities in the
majority rule consensus tree. BYPP greater than 0.95 are given above each node (Figs. 1, 2, 3).
RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008, Stamatakis 2014) in the CIPRES
Science Gateway platform (Miller et al. 2010) was used to construct a maximum likelihood (ML)
tree. Maximum Likelihood bootstrap values greater than 70% were given above each node for (Fig.
1) and equal or greater than 75 % for (Figs. 2, 3).
The Phylogenetic trees were viewed in FigTree v1.4.0 program (Rambaut 2012) and
reorganized in Microsoft power point (2016) and Adobe Illustrator® CS5 (Version 15.0.0, Adobe®,
San Jose, CA). Novel sequences generated in the current study were deposited in GenBank (Tables
567
1, 2, 3) and the finalized alignment and tree of Deniquelata vittalii were deposited in TreeBASE,
submission ID: 22390 (http://www.treebase.org/).
Results
Phylogenetic analyses
The first phylogenetic analyses were carried out with 24 sequences with our new taxon and
strains from Didymosphaeriaceae, with Stemphylium vesicarium and Stemphylium botryosum as the
outgroup taxa using combined LSU, SSU and ITS gene datasets (Table 1). Three different
alignments corresponding to each individual gene and a combined alignment of the three genes
were analyzed. The maximum parsimony dataset consists of 2332 characters with 1848 characters
as constant, 311 characters were counted as parsimony-informative and 173 characters as
parsimony-uninformative. The parsimony analyses resulted in two equal parsimonious trees with a
tree length of 974 steps, CI = 0.633, RI = 0.661, RC = 0.419, HI = 0.367 values. RAxML analysis
yielded a best scoring tree (Fig. 1) with a final ML optimization likelihood value of -8221.245191.
The matrix had 586 distinct alignment patterns, with 16.69% of undetermined characters or gaps.
Estimated base frequencies were as follows; A = 0.247437, C = 0.227513, G = 0.277892, T =
0.247158; substitution rates AC =1.689545, AG = 2.522196, AT = 1.252478, CG = 0.689774, CT =
6.758270, GT = 1.000; proportion of invariable sites I = 0.576761; gamma distribution shape
parameter α = 0.582903. The Bayesian analyses resulted in 10001 trees after 1,000,000 generations.
The first 2000 trees, representing the burn-in phase of the analyses, were discarded, while the
remaining 8001 trees were used for calculating posterior probabilities in the majority consensus tree
for all the phylogenetic analyses (Figs 1, 2, 3). Phylogenetic trees obtained from ML, MP and
Bayesian analyses yielded trees with similar topology and congruent with previous work-based on
MP, ML and Bayesian analyses (Ariyawansa et al. 2013, 2014b). MP tree is shown with the
bootstrap support (BS) values of MP and ML (≥70 %) and Bayesian posterior probabilities (BYPP)
greater than 0.95 are shown above the internal branches respectively (Fig. 1).
Table 1 GenBank and culture collection accession numbers of species included in the phylogenetic
study of Deniquelata vittalii. Sequences generated in this study are in blue.
Taxon
Voucher/ Culture
Alloconiothyrium aptrootii
Bimuria novae-zelandiae
Deniquelata barringtoniae
Deniquelata barringtoniae
Deniquealta vittalii
Didymocrea sadasivanii
Didymosphaeria rubi-ulmifolii
Kalmusia spartii
Karstenula rhodostoma
Laburnicola muriformis
Letendraea cordylinicola
Leptosphaerulina australis
Montagnula cirsii
Neokalmusia brevispora
Paraphaeosphaeria michotii
Paraconiothyrium hawaiiense
Phaeodothis winteri
Pseudopithomyces chartarum
Stemphylium vesicarium
Stemphylium botryosum
Pseudocamarosporium corni
Spegazzinia tessarthra
Tremateia arundicola
Xenocamarosporium acaciae
CBS 980.95
CBS 107.79
MFLUCC 11-0257
MFLUCC 11-0422
NFCCI4249
CBS 438.65
MFLUCC 14-0023
MFLUCC 14-0560
CBS 691.94
MFLUCC 16-0290
MFLUCC 11-0150
CBS 317.83
MFLUCC 13-0680
KT 2313
MFLUCC 13-0349
CBS 120025
CBS 182.58
UTHSC 04-678
CBS 191.86
CBS 714.68
MFLUCC 13-0541
Yone 211
MFLUCC 16-1275
CPC 247.55
ITS
JX496121
KM213997
JX254654
MF406218
KP744441
KU743197
KM213996
GU237829
KX274242
LC014574
KJ939279
JX496027
HG518060
KC584239
KC584238
KJ747048
JQ673429
KX274241
KR476724
GenBank Accessions
LSU
SSU
JX496234
AY016356
AY016338
KM214000
KM214003
JX254655
JX254656
MF182395
MF622059
DQ384103
DQ384066
KJ436586
KJ436588
KP744487
KP753953
AB807531
AB797241
KU743198
KU743199
KM213999
KM214002
FJ795500
GU296160
KX274249
KX274255
AB524601
AB524460
KJ939282
KJ939285
JX496140
EU295655
DQ678073
DQ678021
HG518065
GU238160
GU238232
KC584345
KC584603
KJ813279
AB807582
AB797294
KX274248
KX274254
KR476759
-
568
Table 2 GenBank and culture collection accession numbers of species included in the phylogenetic
study of Farasanispora avicenniae. Sequences generated in this study are in blue.
Taxon
Aegeanispora elanii
Aquilomyces patris
Ascocylindrica marina
Ascocylindrica marina
Camarographium koreanum
Clypeoloculus akatensis
Halomassarina thalassiae
Halomassarina thalassiae
Helicascus elaterascus
Helicascus nypae
Falciformispora lignalitilis
Falciformispora senegalensis
Falciformispora tompkinsii
Farasanispora avicenniae
Farasanispora avicenniae
Macrodiplodiopsis desmazieri
Massarina eburnea
Massarina ignaria
Medicopsis romeroi
Morosphaeria ramunculicola
Morosphaeria velatispora
Lentithecium fulviatile
Lentithecium arundinaceum
Pseudochaetosphaeronema
larense
Trematosphaeria hydrela
Trematosphaeria pertusa
Ulospora bilgrami
Verriculina enalia
Voucher/
Culture
MAW-2017
CBS135661
MD6011
MD6012
CBS 117159
KT788
BCC 17054
BCC 17055
KT2673
BCC36751
BCC21118
CBS19679
CBS20079
MF1207
NFCCI-4220
CBS123812
H3953
CBS 122784
BCC18404
BCC17058
CBS 123090
CBS123131
LSU
KY026052
KP184041
KT252906
JQ044451
AB807543
GQ925849
GQ925850
AB807533
GU479788
GU371827
KF015631
KF015625
KT950962
MG844277
KR873269
AB521735
DQ810223
EU754208
GQ925853
GQ925851
FJ795450
GU456320
GenBank Accessions
SSU
ITS
TEF1α
KY026051
KP184077
NR137961
KT252907
JQ044432
AB797253
AB809631
AB808519
GQ925842
GQ925843
AB797243
AB809626
AB808508
GU479754
GU479854
GU371835
KF432943
GU371820
KF015636
KF015673
KF015687
KF015639
NR132041
KF015685
KT950961
MG844281 MG844285 MG948548
KR873234
AF164367
AB808517
DQ813511
EU754109
KF366447
KF015679
GU479760
GQ925840
FJ795493
GU456298
GU456281
CBS639.94
CBS88070
CBS122368
AFTOLD1598
BCC18402
KF015610
KF314116
FJ201990
DQ678076
GU479803
KF015651
DQ678025
GU479771
KF015655.
NR132040
-
KF015683
KF314136
DQ677921
GU479864
RPB2
GU479826
KF015717
KF015719
MG973031
KF015707
FJ795467
FJ795476
DQ677974
Table 3 GenBank and culture collection accession numbers of species included in the phylogenetic
study of Hysterium rhizophorae. Sequences generated in this study are in blue.
Taxon
Voucher/ Culture
Hysterium angustatum
Hysterium angustatum
Hysterium angustatum
Hysterium angustatum
Hysterium angustatum
Hysterium angustatum
Hysterium hyalinum
Hysterium barrianum
Hysterium barrianum
Hysterium pulicare
Hysterium pulicare
Hysterium pulicare
Hysterium pulicare
Hysterium pulicare
Hysterium vermiforme
Hysterium rhizophorae
Hysterium rhizophorae
Psiloglonium clavisporum
Psiloglonium clavisporum
CBS 236.34
CBS123334
GKM5211
SMH5216
GKM243a
CMW:20409
CBS:237.34
ANM1495
ANM1442
ANM85
EB 0238
CBS:119331
AFTOL-ID 1254
ANM1455
GKM1234
MFLU 16-1179
NFCCI-4250
CBS:123338
CBS:123340
LSU
FJ161180
FJ161207
GQ221906
GQ221908
GQ221899
FJ161194
FJ161181
GQ221885
GQ221884
GQ221898
FJ161201
DQ678055
GQ221904
GQ221897
KX611364
MG844276
FJ161197
FJ161205
GenBank Accessions
SSU
ITS
GU397359.
FJ161180
FJ161153.
FJ161141
FJ161161
EU552137
DQ678002
KX611365
KX611363
MG844280
MG844284
-
RPB2
FJ161117
GU566751
FJ161127
FJ238433
MG968956
-
The phylogenetic analyses show that our new taxon Deniquelata vittalii clustered together
with the strains of D. barringtoniae with a strong statistical support in a monophyletic clade (100%
569
ML, 100%MP, 1.00 BYPP) in the family Didymosphaeriaceae. Deniquelata vittalii shares a sister
relationship with D. barringtoniae with significant statistical support (88% ML, 89% MP, 1.00
BYPP, Fig. 1).
The second multigene phylogenetic analyses include 27 in-group taxa from different genera
from Pleosporales, including our taxon, while Verruculina enalia served as the outgroup taxon
(based on LSU, SSU, TEF-1α, ITS and RPB2 sequence data, Table 2). RAxML analysis yielded a
best scoring tree (Fig. 2) with a final ML optimization likelihood value of -22392.941365. The
matrix had 1629 distinct alignment patterns, with 42.78% of undetermined characters or gaps.
Estimated base frequencies were as follows; A =0.249321, C = 0.235804, G = 0.273367, T =
0.241508; substitution rates AC = 1.425242, AG =3.166282, AT = 1.605461, CG = 1.085865, CT =
7.895025, GT = 1.000000; proportion of invariable sites I = 0.436939; gamma distribution shape
parameter α =0. 442521. The maximum parsimony dataset consisted of 4613 characters with 3128
characters as constant, 1009 characters were counted as parsimony-informative and 476 characters
as parsimony-uninformative. The parsimony analyses resulted in one equal parsimonious tree with
a tree length of 3657 steps, CI = 0.596, RI = 0.484, RC = 0.288, HI = 0.404 values. Phylogenetic
analyses indicate that both strains of Farasanispora avicenniae cluster together with high bootstrap
support (100% ML, 100% MP, 1.00 BYPP, Fig. 2). Our phylogenies generated herein, under
different criteria, yielded similar results as previously reported in connection to the uncertain
familial placement of Farasanispora avicenniae (Li et al. 2016).
The phylogenetic analyses inferred from the third dataset include 19 taxa from Hysterium
including our strain, while two strains of Psiloglonium clavisporum served as outgroup taxa (Table
3). RAxML analysis yielded a best scoring tree (Fig. 3) with a final ML optimization likelihood
value of -9131.172652. The matrix had 297distinct alignment patterns, with 50.91% of
undetermined characters or gaps. Estimated base frequencies were as follows; A = 0.253481, C =
0.222684, G = 0.285514, T = 0.238321; substitution rates AC = 0.906683, AG =1.276344, AT =
0.828696, CG = 0.793370, CT = 2.357133, GT = 1.000000; proportion of invariable sites I =
0.492153; gamma distribution shape parameter α = 586.774160. The maximum parsimonious
dataset consists of 19 taxa with 3632 characters, of which 2500 were constant, 312 were
parsimony-informative and 820 parsimony-uninformative. The parsimony analysis of the data
matrix resulted in one hundred and ninety-eight equally parsimonious trees with a length of 1523
steps (CI = 0.922, RI = 0.711, RC = 0.656, HI = 0.078). The molecular phylogeny indicates that
both the strains of H. rhizophorae (MFLUCC161179 and NFCCI4250) nested together with
significant bootstrap support of 77% in MP, 0.96% in BYPP and moderate support of 64% in ML
(Fig. 3). Phylogenetic trees resulted from ML, MP, BYPP analyses were in congruent to the earlier
study (Hyde et al. 2017).
Taxonomy
Deniquelata vittalii Devadatha, V.V Sarma, E.B.G Jones, sp. nov.
Fig. 4
MycoBank number: MB820842; Facesoffungi number: FoF04375
Etymology – In honour of Professor B.P.R. Vittal, formerly Professor in the University of
Madras, India for his contributions to Indian mycology
Holotype – AMH-9888
Saprobic on decaying woody stem of the halophyte Suaeda monoica. Sexual morph:
Ascomata 95–360 μm high, 75–350 μm in diameter ( x = 225 × 212 µm, n = 10), immersed to semiimmersed, erumpent, globose to sub-globose, dark brown to black, aggregated to solitary,
obpyriform, coriaceous, fused with the host tissues, with a papillate to depressed ostiole. Peridium
10–40 μm ( x = 23 μm, n = 10) wide at the base, 10–55 μm ( x = 30 μm, n = 10) at the sides,
comprising 3–5 layers of thin-walled hyaline to pale brown cells inwardly and thick-walled pale
brown to dark brown cells of textura angularis outwardly fused with the host tissue. Hamathecium
composed of 1.5–3 μm wide ( x = 2 μm, n = 10), cellular, profusely branched, hyaline, septate
570
Figure 1 – Phylogram generated from maximum parsimony analysis based on a combined dataset
of LSU, SSU and ITS. Bootstrap support values for maximum likelihood (ML, green), maximum
parsimony (MP, blue) equal to or greater than 70 % and the values of Bayesian posterior
probabilities (BYPP, purple) equal to or greater than 0.95 are given above each branch,
respectively. The new species is in purple colour. The tree is rooted with Stemphylium vesicarium
and Stemphylium botryosum.
571
Figure 2 – Phylogram generated from maximum parsimony analysis based on combined LSU,
SSU, TEF1, ITS and RPB2 sequence data of Farsanispora avicenniae NFCCI-4220 and other
related taxa in Pleosporales. Verruculina enalia is the outgroup taxon. Bootstrap support values for
maximum likelihood (ML, green), maximum parsimony (MP, blue) equal to or greater than 75 %
and the values of Bayesian posterior probabilities (BYPP, purple) equal to or greater than 0.95 are
given above each branch, respectively. The new record is in purple colour.
572
Figure 3 – Phylogram generated from maximum parsimony analysis based on combined LSU, SSU
and RPB2 sequence data of selected taxa. Bootstrap support values for maximum likelihood (ML,
green), maximum parsimony (MP, blue) equal to or greater than 75 % and the values of Bayesian
posterior probabilities (BYPP, purple) equal to or greater than 0.95 are given above each branch,
respectively. The new record is in purple. The tree is rooted with Psiloglonium clavisporum.
pseudoparaphyses, anastomosing above the asci, enclosed in a gelatinous matrix. Asci 75–135 ×
10–19 μm ( x = 98 × 15 μm, n = 30), 8-spored, bitunicate, fissitunicate, cylindric-clavate to clavate,
apically rounded with an ocular chamber, with a 7–15 μm long and broad furcate pedicel.
Ascospores 18–26 × 7.5–13.5 μm ( x = 22.5 × 10.7 μm, n = 50), 1–2 seriate, muriform, hyaline
when young, developing into golden yellow to dark brown at maturity, rarely guttulate, ellipsoidal
to broadly oblong, planate, flat, verrucose, deeply constricted in the middle, slightly curved to
straight, apically conical to elliptical, with 1–2 longitudinal septa in each cell, 3–6 transverse
septate, lacking mucilaginous sheath. Asexual morph: Undetermined.
Culture characteristics – Ascospores germinating on 50% sea water agar producing germ
tubes at both ends of the ascospores within 24 hours. Colonies on MEA showed moderate growth
attaining 20–24 mm in diameter within a week and reached 35–50 mm after 20 days of incubation
at 25°C, front pale ochraceous salmon, reverse capucine buff, with pale yellow diffusible pigments,
margin filamentous, pulvinate, circular, cottony and fluffy.
Material examined – INDIA, Tamil Nadu, Tiruvarur, Muthupet mangroves (10.4°N 79.5°E),
on decaying woody stem of the halophyte Suaeda monoica (Amaranthaceae) 15 August 2015, B.
Devadatha (AMH-9888, holotype), ex-type living culture NFCCI-4249.
Notes – Multigene analyses of LSU, SSU and ITS sequence data show that our new taxon
belongs to the family Didymosphaeriaceae and is closely related to Deniquelata barringtoniae with
a strongly-supported monophyletic clade (Fig. 1). Morphologically Deniquelata vittalii resembles
the generic type Deniquelata barringtoniae in having sub-globose to globose ascomata, shorter
573
papilla with a depressed ostiole; asci with short furcate pedicel and ascospores that are oblong,
straight or slightly curved, muriform with 1–2 vertical septa, verruculose (Ariyawansa et al. 2013,
2014b). However, Deniquelata vittalii can be easily distinguished from D. barringtoniae in having
larger ascomata, asci and ascospore dimensions (Fig. 4, Table 4). Deniquelata barringtoniae is
distinct in having smaller, reddish-brown ascospores (13–16 × 5–7), with 3-transverse septa
whereas D. vittalii has larger, brown ascospores (17.5–25 × 7.5–13.5) with 3–6 transverse septa
(Fig. 4, o–s). Further, D. barringtoniae is pathogenic on living leaves of Barringtonia asiatica from
terrestrial environments (Ariyawansa et al. 2013), while D. vittalii is saprobic on decaying woody
stem of the halophyte Suaeda monoica from marine environments. Distinct nucleotide differences
between Deniquelata vittalii and D. barringtoniae were noted across various gene regions analyzed
[35 within ITS; 34 within LSU; 12 within SSU; 46] which comply with recommendations outlined
by Jeewon & Hyde (2016) to establish new species based on DNA sequence data. Hence the new
species D. vittalii has been proposed to be accommodated in the genus Deniquelata.
Farasanispora avicenniae Abdel-Wahab, Bahkali & E.B.G. Jones, Fungal Diversity. 78: 63 (2016)
Fig. 4
Facesoffungi number: FoF01635
Saprobic on decaying woody stem of the halophyte Suaeda monoica. Sexual morph:
Ascomata 170–330 µm high, 165–345 µm diam. ( x = 239 × 240 µm, n = 10), immersed to
erumpent, sub globose, solitary to gregarious, coriaceous, dark brown to black, ostiolate. Peridium
unequal in thickness, 18–40 µm ( x = 28 µm, n = 10) wide at the sides comprising two different cell
layers, outer layer of brown to hyaline polygonal cells fused with host tissue and thick inner layer
composed of several hyaline flattened cells of textura angularis. Peridium less developed at the
base 15–30 µm ( x = 22.5 µm, n = 10) wide, hyaline to light brown cells fused with the host tissue.
Hamathecium composed of 1.5–2.5 µm ( x = 2.1 µm, n= 20) wide, numerous, septate, branched,
filamentous pseudoparaphyses resembling hyphae embedded in a gelatinous matrix, anastomosing
above the asci. Asci 70–135 × 20–32 µm ( x = 105 × 26 µm, n = 40), 8-spored, bitunicate,
fissitunicate, cylindrical to clavate, short pedicellate, apically rounded and thickened with an ocular
chamber. Ascospores 30–37 × 7–15 µm ( x = 34 × 11 µm, n = 50), biseriately arranged, constricted
at the septa, rugose, hyaline, fusiform,1–3 septate, the septum is sub-median, upper cell longer and
wider, slightly curved, guttulate, lacking a mucilaginous sheath. Senescent ascospores are larger
35–42 × 10–15 µm ( x = 37 × 12 µm, n = 10), light brown, flattened, distinctly constricted at the
middle septum, striate, verruculose, 2–3 septate.
Asexual morph: After 25 days of fermentation in Czapek-Dox broth, oval to ellipsoidal
chlamydospores were found, 8–25 × 10–20 µm ( x = 15 × 12.5 µm, n = 10), hyaline to purple
colour filamentous hyphae, pinkish diffusible pigments produced as extracellular metabolites (Fig.
6).
Culture characteristics – Ascospores germinating on seawater agar within 24 hours, germ
tubes arising from terminal ends of the ascospores. Colonies on malt extract agar moderately
growing, reaching 45–60 mm diameter after 25 days of incubation at room temperature, initially
white to grey becoming dark grey to brown in older cultures, flexuous on surface, zonate, undulate,
effuse, flattened, medium dense, reverse pink, producing diffusible pigments into media.
Material examined – INDIA, Tamil Nadu, Tiruvarur, Muthupet mangroves, decaying woody
stem of the halophyte Suaeda monoica Lam. (Amaranthaceae), (10.4°N 79.5°E), 29 October 2016,
B. Devadatha, (AMH-9911) living culture (NFCCI-4220).
Notes – Multigene phylogenetic analyses show that Farasanispora avicenniae (NFCCI-4220)
belongs to the order Pleosporales sharing sister relation to Trematosphaeriaceae and
Morosphaeriaceae and distantly related to Massarinaceae (Fig. 2). However, it did not group with
any known family and formed a distinct monophyletic clade with the existing Farasanispora
avicenniae (MF1207) with a high bootstrap support from (100% ML, 100%MP, 1.00 BYPP, Fig. 2)
and shares a sister group relation to Aegeanispora elanii with a lower bootstrap support.
Morphological characteristics of Farasanispora avicenniae occurring on the decaying woody stems
574
Figure 4 – Deniquelata vittalii (AMH-9888, holotype). a Ascomata semi-immersed in the decaying
woody stem of the halophyte Suaeda monoica. b–c Longitudinal sections of ascomata d Section of
peridium. e Cellular and hyaline pseudoparaphyses. f-k Immature and mature asci. l-s Ascospores. t
Germinating ascospores. Scale bars: b = 100 μm, c = 50 μm, d-t = 10 μm.
575
Table 4 Synopsis of morphological differences between Deniquelata vittalii and D. barringtoniae
Species
Deniquelata barringtoniae
Deniquelata vittalii
Host
Barringtonia asiatica
Suaeda monoica
Life mode
Parasitic on leaves
Saprobic on decaying woody stems
Habitat
Terrestrial
Marine
Ascomata (μm)
Peridium
thickness (μm)
Asci (μm)
150–180 × 164–189
100–350 × 75–350
9–17
10–40
67–78 × 10–15
13–16 × 5–7, oblong, reddish brown to dark
yellowish brown, 3-transverse septa and 1–2
longitudinal septa
Ariyawansa et al. (2013)
70–140 × 10–20
17.5–25 × 7.5–13.5, golden yellow to dark brown,
deeply constricted in the middle, 3–5 transverse
septate, 1–2 longitudinal septa in each cell
This study
Ascospores (μm)
References
of halophyte Suaeda monoica, reported in the present study, are similar to Farasanispora
avicenniae reported from Avicennia marina (Li et al. 2016). However, its occurrence on Suaeda
monoica, in India, constitute new host and geographic records. Farasanispora avicenniae shares
similar morphological characters with Halomassarina thalassiae (Kolhm. & Volkm -Kohlm.)
Suetrong et al. (Suetrong et al. 2009). However, it differs from Halomassarina thalassiae (Suetrong
et al. 2009, Li et al. 2016) in having smaller ascomata, without a clypeus or papillae and in the
absence of periphyses in the ostiolar canal, lacking senescent ascospores and a prominent
gelatinous sheath (Fig. 5) (Kohlmeyer & Kohlmeyer 1987). Prior to the present study, only LSU
and SSU gene sequence data of F. avicenniae were available (Li et al. 2016). This study provided
ITS, TEF-1α and RPB2 in addition to LSU and SSU (Table 2).
Hysterium rhizophorae Dayarathne & K. D. Hyde, in Fungal Diversity 87:42 (2017)
Fig. 7
Facesoffungi number: FoF02911
Saprobic on decaying wood of Aegiceras corniculatum. Sexual morph Ascomata
hysterothecial, 650–2100 μm long ( x = 1060 µm, n = 5), 100–400 μm high, 170–200 μm wide ( x
= 243 × 199 µm, n = 5), erumpent to superficial with base immersed, solitary to gregarious, straight
to flexuous, ellipsoid or elongate, with pointed ends, opening by a depressed longitudinal slit, in
vertical section sub-globose to globose, carbonaceous, black. Peridium 25–75 ( x = 50 µm, n = 5)
μm wide, carbonaceous, comprising an outer layer of dark brown cells of textura globosa and an
inner layer of hyaline to pale brown cells of textura globosa. Pseudoparaphyses 1–2 µm (n = 30)
wide, cellular, septate, flexuous, branched. Asci 40–65 × 6–12 µm (x̅ = 48 × 10 μm, n = 20), 8spored, bitunicate, cylindric to claviform, short pedicellate. Ascospores 10–17 × 3–5 µm (x̅ = 14 ×
4 μm, n = 30), overlapping biseriate, light brown, ellipsoidal, straight to slightly curved, with 3transverse septa, often slightly constricted at the median septum, with or without guttules. Asexual
morph: Undetermined.
Culture characteristics – Ascospores germinated on sea water agar within 24 hours, germ
tubes arisen from terminal ends of the ascospore. Colonies on malt extract agar reaching 30–75 mm
diameter after 25 days of incubation at room temperature, initially hyaline becoming ash grey in
older cultures and reverse light brown, irregular, undulate, floccose.
Material examined – INDIA, Tamil Nadu, Tiruvarur, Muthupet mangroves, on intertidal
wood of Aegiceras corniculatum (Primulaceae), (10.4°N 79.5°E), 29 October 2016, B. Devadatha
(AMH-9947), living culture (NFCCI-4250).
Notes – Combined phylogenetic analyses of LSU, SSU, ITS and RPB2 sequence data placed
our taxon (Hysterium rhizophorae NFCCI4250) in the family Hysteriaceae and clustered together
with Hysterium rhizophorae (MFLUCC161179) in a monophyletic clade with a significant
bootstrap support from MP 77%, BYPP 0.96% and moderate support in ML 64% (Fig. 3). This
Hysterium species was found on the decaying wood of Aegiceras corniculatum having overlapping
576
Figure 5 – Farasanispora avicenniae (AMH-9911). a Ascomata erumpent on the decaying wood
of Suaeda monoica. b-c Longitudinal sections of ascomata d Section of peridium e filamentous
pseudoparaphyses. f-j Immature and mature asci. k-m Hyaline ascospores. n Mature senescent
ascospore. o Germ tubes developed from terminal ends of ascospore p Culture. Scale bars:
b–c = 100 μm, d–o = 10 μm.
577
Figure 6 – Farasanispora avicenniae (NFCCI-4220). a Fermented culture in Czapek-Dox broth.
b, d hyphae showing development of chlamydospores and pink pigments. c, e-f chlamydospores.
Scale bars: b–c = 100 μm, d–o = 10 μm.
morphological characters with Hysterium rhizophorae reported from Rhizophora apiculata (Hyde
et al. 2017). The occurrence of H. rhizophorae on a new host i.e. A. corniculatum increases its host
range and also this is the first report of this fungus from India thus expanding its geographical
range. ITS pairwise alignment resulted in a very low (3) base pair difference with ITS sequence
data of the two strains of H. rhizophorae (Jeewon & Hyde 2016). Previously only ITS, LSU, SSU
and TEF-1α gene sequences were carried out (Hyde et al. 2017). The present study provides
additional information on RPB2 sequence in the GenBank (RPB2: MG968956) in addition to the
ITS, LSU and SSU gene sequence data for the new record.
Discussion
The monotypic genus Deniquelata typified by D. barringtoniae was established by
Ariyawansa et al. (2013) who reported this fungus as a pathogen on living leaves of Barringtonia
asiatica with brown spots and fruiting bodies scattered in the necrotic tissues (Ariyawansa et al.
2013, 2014b). The pathogenic nature of Deniquelata barringtoniae was proved by in vitro
pathogenicity testing of healthy leaves of Barringtonia asiatica (Ariyawansa et al. 2013).
Multi-gene analyses showed that Deniquelata vittalii nested together with D. barringtoniae
with significant bootstrap support (88% ML, 89% MP, 1.00 BYPP, Fig. 1) and separated from
Bimuria novae-zelandiae and Tremateia arundicola sister groups. Deniquelata and Bimuria share
similarities in having scattered, semi-immersed, sub-globose ascomata with muriform ascospores
and are saprobic (D. vittalii and B. novae-zelandiae). Deniquelata species distinct from Bimuria
typified by B. novae-zelandiae, in having shorter papilla with a depressed ostiole; 8-spored asci
with short furcate pedicel and ascospores that are oblong, straight or slightly curved, muriform with
578
Figure 7 – Hysterium rhizophorae (AMH-9947) a Appearance of hysterothecia on host. b Vertical
section through hysterothecium. c, d Peridium. e Pseudoparaphyses. f–h Asci. i, k–l Ascospores.
j Germinating ascospores. Scale bars: b = 100 μm, c-k = 10 μm.
579
3–6 transverse septa, 1–2 vertical septa, verruculose. Bimuria novae-zelandiae has 2–3-spored asci
with short and small knob-like pedicel and comparatively larger ascospores with 5–7 transverse
septa, without vertical septa, verrucose and by occurring in terrestrial habitat (Hawksworth 1979,
Ariyawansa et al. 2013). The species in Tremateia can be clearly distinguished from Deniquelata
species in having clavate to broadly clavate, short pedicellate, long asci and ellipsoid ascospores
with 3–6 transverse septa and 1 vertical septum in each row (Kohlmeyer et al 1995, Hyde et al
2016).
This study provides sequence data of protein coding genes RPB2 and TEF1α (MF168942,
MF182398) in GenBank for Deniquelata vittalii whereas we lack these sequences data of D.
barringtoniae for a comparison. This is the first report of a Deniquelata species from marine
habitats (Jones et al. 2015). Recently (Devadatha & Sarma 2018) reported a new species,
Pontoporeia mangrovei from decaying woody stem of the halophyte Suaeda monoica. By adding
the present new taxon, Suaeda monoica could be considered as a host that supports several novel
marine fungi.
Farasanispora avicenniae and Hysterium rhizophorae were recorded for the first time from
India and hence constitute new geographic records. Also, their occurrence on new hosts extend
their host range. The present study provides new sequences data for these two-known species.
Acknowledgements
We would like to thank the Ministry of Earth sciences, Govt. of India for funding a project to
carry out studies on Marine fungi from Muthupet mangroves (Sanction order:
MOES/36/OO1S/Extra/40/2014/PC-IV dt.14.1.2015). We thank the District Forest Office,
Tiruvarur, Tamil Nadu and PCCF (Head of Forest Force), Chennai, Tamil Nadu Forest Department,
India for providing permission to collect samples from Muthupet mangroves. Department of
Biotechnology, Pondicherry University is thanked for providing the facilities. B. Devadatha would
like to thank the Ministry of Earth Sciences, Govt. of India for providing a fellowship and also
grateful to Dhanushka Nadeeshan of Mae Fah Luang University, Thailand for his valuable
suggestions and help. H.A. Ariyawansa thanks the Ministry of Science and Technology, Taiwan
(MOST project ID: 106-2621-B-002-005-MY2) for a partial funding of this work. We thank the
anonymous reviewers for their suggestions to improve the manuscript.
References
Aptroot A. 1995 – A monograph of Didymosphaeria. Studies in Mycology 37, 1–160.
Ariyawansa HA, Maharachchikumbura SS, Karunarathne SC, Chukeatirote E et al. 2013 –
Deniquelata barringtoniae gen. et sp. nov., associated with leaf spots of Barringtonia
asiatica. Phytotaxa. 105, 11–20.
Ariyawansa HA, Camporesi E, Thambugala KM, Mapook A et al. 2014a – Confusion surrounding
Didymosphaeria phylogenetic and morphological evidence suggest Didymosphaeriaceae is
not a distinct family. Phytotaxa 176, 102–119.
Ariyawansa HA, Tanaka K, Thambugala KM, Phookamsak R et al. 2014b – A molecular
phylogenetic reappraisal of the Didymosphaeriaceae (= Montagnulaceae). Fungal Diversity
68, 69–104.
Bisby GR. 1923 – The literature on the classification of the Hysteriales. Transactions of the British
Mycological Society 8, 176–189.
Chomnunti P, Hongsanan S, Aguirre-Hudson B, Tian Q et al. 2014 – The sooty moulds. Fungal
Diversity 66, 1–36.
Devadatha B, Sarma VV. 2018 – Pontoporeia mangrovei sp.nov., a new marine fungus from an
Indian mangrove along with a new geographical and host record of Falciformispora
lignalitis. Current Research in Environmental & Applied Mycology 8, 238–246.
580
Devadatha B, Sarma VV, Wanasinghe DN, Hyde KD, Jones EBG 2017 – Introducing the new
Indian mangrove species, Vaginatispora microarmatispora (Lophiostomataceae) based on
morphology and multigene phylogenetic analysis. Phytotaxa 329, 139–149.
Devadatha B, Sarma VV, Jeewon R, Wanasinghe DN et al. 2018 – Thyridareilla a novel marine
fungal genus from India: morphological characterization and phylogeny inferred from
multigene
DNA
sequence
analyses.
Mycological
Progress
1–14.
https://doi.org/10.1007/s11557-018-1387-4
Hall TA. 1999 – BioEdit: a user- friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95–98.
Hawksworth DL, Yen CC, Edmund J. 1979 – Bimuria novae-zelandiae gen. et sp. nov., a
remarkable ascomycete isolated from a New Zealand barley field. New Zealand journal of
botany 17, 267–273.
Huelsenbeck JP, Ronquist F. 2001 – MRBAYES: Bayesian inference of phylogenetic trees.
Bioinformatics 17, 754–755.
Hyde KD, Jones EBG, Liu JK, Ariyawansa H et al. 2013 – Families of Dothideomycetes. Fungal
Diversity 63, 1–313.
Hyde KD, Hongsanan S, Jeewon R, Bhat DJ et al. 2016 – Fungal diversity notes 367–490:
taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 80, 1–270.
Hyde KD, Norphanphoun C, Abreu VP, Bazzicalupo A et al. 2017 – Fungal diversity notes 603–
708: taxonomic and phylogenetic notes on genera and species. Fungal Diversity 87, 1–235.
Kohlmeyer J, Volkman-Kohlmeyer B. 1987 – Marine fungi of Aldabra, the Galapagos, and other
tropical islands. Canadian Journal of Botany 65, 571–582
Kohlmeyer J, Volkmann-Kohlmeyer B, Eriksson OE. 1995 – Fungi on Juncus roemerianus 2. New
dictyosporous ascomycetes. Botanica Marina. 38, 165–174.
Jayasiri CS, Hyde KD, Ariyawansa HA, Bhat DJ et al. 2015 – The Faces of Fungi database: fungal
names linked with morphology, phylogeny and human impacts. Fungal Diversity 74, 3–18.
Jeewon R, Hyde KD. 2016 – Establishing species boundaries and new taxa among fungi:
recommendations to resolve taxonomic ambiguities. Mycosphere 7, 1669–1677.
Jones EBG, Suetrong S, Sakayaroj J, Bahkali AH et al. – 2015 – Classification of marine
Ascomycota, Basidiomycota, Blastocladiomycota and Chytridiomycota. Fungal Diversity 73,
1–72.
Katoh K, Standley K. 2013 – MAFFT Multiple Sequence Alignment Software Version 7:
Improvements in Performance and Usability. Molecular Biology & Evolution 30, 772–780.
Kishino H, Hasegawa M. 1989 – Evaluation of the maximum likelihood estimate of the
evolutionary tree topologies from DNA – sequence data, and the branching order in
Hominoidea. Journal of Molecular Evolution 29, 170–179.
Li GJ, Hyde KD, Zhao RN, Hongsanan S et al. 2016 – Fungal diversity notes 253–366: taxonomic
and phylogenetic contributions to fungal taxa. Fungal Diversity 78, 1–237.
Liu YJ, Whelen S, Hall BD. 1999 – Phylogenetic relationships among ascomycetes: evidence from
an RNA polymerase II subunit. Molecular Biology and Evolution 16, 1799–1808.
Miller MA, Pfeiffer W, Schwartz T. 2010 – Creating the CIPRES science gateway for inference of
large phylogenetic trees. In Proceedings of the Gateway Computing Environments Workshop
(GCE). San Diego Supercomput. Center, New Orleans, 1–8.
Munk A. 1953 – The system of the pyrenomycetes. A contribution to a natural classification of the
group Sphaeriales sensu Lindau. Dansk Botanisk Arkiv 15, 1–163.
MycoBank. 2017 – available from: http://www.MycoBank .org/- (accessed: October 2017).
Nylander JAA. 2004 – MrModeltest v2 Program distributed by the author. Evolutionary Biology
Centre, Uppsala University, Uppsala.
Rambaut A. 2012 – FigTree v. 1.4.0. http://tree.bio.ed.ac.uk/software/figtree/
Rambaut A, Drummond AJ. 2007 – Tracer ver. 1.4. Available at http://tree. bio. ed. ac.
uk/software/tracer.
581
Rannala B, Yang Z. 1996 – Probability distribution of molecular evolutionary trees: a new method
of phylogenetic inference. Journal of Molecular Evolution 43, 304–311.
Rehner SA, Buckley E. 2005 – A Beauveria phylogeny inferred from nuclear ITS and EF1-α
sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs.
Mycologia 97, 84–98
Schoch CL, Crous PW, Groenewald JZ, Boehm EWA et al. 2009 – A class– wide phylogenetic
assessment of Dothideomycetes. Studies in Mycology 64, 1–15.
Stamatakis A, Hoover P, Rougemont J. 2008 – A rapid bootstrap algorithm for the RAxML webservers. Systematic Biology 57, 758–771.
Stamatakis A. 2014 – RAxML version 8: a tool for phylogenetic analysis and post-analysis of large
phylogenies. Bioinformatics. 30, 1312–1313.
Suetrong S, Schoch CL, Spatafora JW, Kohlmeyer J et al. 2009 – Molecular systematics of the
marine Dothideomycetes. Studies in Mycology 64, 155–173.
Swofford DL. 2002 – PAUP*: Phylogenetic analysis using parsimony, Version 4.0b10. Sinauer
Associates, Suderland.
Vilgalys R, Hester M. 1990 – Rapid genetic identification and mapping of enzymatically amplified
ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172, 4238–
4246.
White T, Bruns T, Lee S, Taylor J. 1990 – Amplification and direct sequencing of fungal ribosomal
RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications 18,
315–322.
Wijayawardene NN, Hyde KD, Rajesh Kumar KC et al. 2017 – Notes for genera: Ascomycota.
Fungal Diversity 86, 1–594.
Zhaxybayeva O, Gogarten JP. 2002 – Bootstrap, Bayesian probability and maximum likelihood
mapping: exploring new tools for comparative genome analyses. BMC genomics 3, 4.
582