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Sydowia 70 (2018) 129
DOI 10.12905/0380.sydowia70-2018-0129
Two unusual new species of Pleosporales:
Anteaglonium
rubescens
and
Atrocalyx asturiensis
Walter M. Jaklitsch1,2,*, Jacques Fournier3 & Hermann Voglmayr2
1 Institute of Forest Entomology, Forest Pathology and Forest Protection, Dept. of Forest and Soil Sciences, BOKU-University
of Natural Resources and Life Sciences, Franz Schwackhöfer Haus, Peter-Jordan-Straße 82/I, 1190 Wien, Austria
2 Division of Systematic and Evolutionary Botany, Department of Botany and Biodiversity Research, University of Vienna,
Rennweg 14, 1030 Wien, Austria
3 Las Muros, F-09420 Rimont, France
* e-mail: walter.jaklitsch@univie.ac.at
Jaklitsch W., Fournier J. & Voglmayr H. (2018): Two unusual new species of Pleosporales: Anteaglonium rubescens and Atro-
calyx asturiensis. – Sydowia 70: 129–140.
Two new species of Pleosporales, Anteaglonium rubescens (Anteagloniaceae) and Atrocalyx asturiensis (Lophiotremataceae),
are described. Phylogenetic placement was determined by combined analyses of a DNA data matrix containing ITS, LSU, SSU,
rpb2, and tef1. Anteaglonium rubescens is a stromatic fungus characterized by brown didymospores disarticulating within asci,
and by the production of a red-orange to pink pigment produced in nature and in articial culture. Atrocalyx asturiensis has
massive ascomatal crests and brown phragmospores.
Key words: Anteagloniaceae, Dothideomycetes, hysteriaceous fungi, Hysterodifractum, Lophiotremataceae, Ohleria, phylo-
genetic analysis, pyrenomycetes, Xenolophium.
The genus Anteaglonium was erected by Mugam-
bi & Huhndorf (2009a) for hysterothecial taxa with
hyaline didymospores that do phylogenetically not
belong to the Hysteriales, Mytilinidiales or Patel-
lariales, but to the Anteagloniaceae, Pleosporales.
In a comprehensive molecular study of fungi form-
ing hysterothecia, Boehm et al. (2009) reached the
same conclusions and included the four species A.
abbreviatum, A. globosum, A. latirostrum and A.
parvulum, in the genus Anteaglonium. According to
Mugambi & Huhndorf (2009a) the genus can be dis-
tinguished from Glonium by its smaller ascomata
that are either elongate or globose and its small as-
cospores that are less than 8 μm long except in A.
latirostrum. However, in the latter species the asco-
mata are globose, often with raised laterally com-
pressed apices, characters not present in Glonium.
Anteaglonium latirostrum differs from the other
species also in ascospores, which become 3–5-sep-
tate and pale brown at maturity. Another species, A.
brasiliense was added by Carneiro de Almeida et al.
(2014). They also described the new genus Hyster-
odifractum, which has ascospores that disarticulate
in two-celled part-spores, similar to Ohleria (Jak-
litsch & Voglmayr 2016). However, molecular phylo-
genetic analyses placed Hysterodifractum in Hyste-
riales (Carneiro de Almeida et al. 2014). The sixth
species of Anteaglonium, A. thailandicum, was add-
ed by Jayasiri et al. (2016). Here we describe a new
species of Anteaglonium, which morphologically
differs from other species in several respects.
Phylogenetically close to the Anteagloniaceae
are the Lophiotremataceae, which were differenti-
ated from the Lophiostomataceae by Hirayama &
Tanaka (2011) and only contained the single genus
Lophiotrema until 2016 (Jaklitsch et al. 2016a).
However, Hashimoto et al. (2017) described ve new
genera in the family including Atrocalyx and erect-
ed the three new related families Aquasubmersace-
ae, Cryptocoryneaceae, and Hermatomycetaceae.
Here we describe a new species of Atrocalyx, which
morphologically deviates considerably from other
species of the genus, notably by brown phragmos-
pores, which lack a gelatinous sheath.
Materials and methods
Isolates and specimens
All newly prepared isolates used in this study
originated from ascospores of fresh specimens.
Strain identiers including NCBI GenBank acces-
sion numbers of gene sequences used to compute
the phylogenetic trees are listed in Tab. 1. Strain ac-
Published online 4 Mai 2018
130 Sydowia 70 (2018)
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
Tab. 1. Isolates and accession numbers used in the phylogenetic analyses. Isolates/sequences in bold were isolated/sequenced in the present study.
Species Family Original
no. Specimen no.aStrain no.
GenBank Accession no
SSU ITS LSU tef1 rpb2
Amniculicola immersa
Amniculicola parva
Anteaglonium abbreviatum
Anteaglonium abbreviatum
Anteaglonium brasiliense
Anteaglonium globosum
Anteaglonium globosum
Anteaglonium latirostrum
Anteaglonium latirostrum
Anteaglonium parvulum
Anteaglonium parvulum
Anteaglonium parvulum
Anteaglonium parvulum
Anteaglonium rubescens
Anteaglonium rubescens
Anteaglonium rubescens
Anteaglonium thailandicum
Antealophiotrema brunneosporum
Aquasubmersa japonica
Aquasubmersa mircensis
Atrocalyx acutisporus
Atrocalyx asturiensis
Atrocalyx bambusae
Atrocalyx lignicola
Byssolophis sphaerioides
Crassimassarina macrospora
Cryptoclypeus oxysporus
Cryptoclypeus ryukyuensis
Cryptocoryneum akitaense
Cryptocoryneum brevicondensatum
Cryptocoryneum condensatum
Cryptocoryneum japonicum
Cryptocoryneum longicondensatum
Cryptocoryneum paracondensatum
Amniculicolaceae
Amniculicolaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
Anteagloniaceae
incertae sedis
Aquasubmersaceae
Aquasubmersaceae
Lophiotremataceae
Lophiotremataceae
Lophiotremataceae
Lophiotremataceae
incertae sedis
Lophiotremataceae
Lophiotremataceae
Lophiotremataceae
Cryptocoryneaceae
Cryptocoryneaceae
Cryptocoryneaceae
Cryptocoryneaceae
Cryptocoryneaceae
Cryptocoryneaceae
–
_
–
–
–
–
–
–
–
–
–
–
–
OR
OR1
OR2
–
–
KT 2813
KT 2436
OF
–
–
–
KT 1764
KT 2772
KT 3534
KT 3019
yone 152
KT 2892
KT 3300
KT 2913
KT 3241
CBS H-20226H
CBS H-20227H
ANM 925.1
GKM 1029
HUEFS 192250H
ANM 925.2H
SMH 5283
GKM 1119H
GKM L100Nb
MFLU 16-0473
MFLU 16-0472
MFLU 16-0474
MFLU 16-0470
WU 36962
WU 36960H
WU 36963
MFLU 16-0471H
CBS H-20222H
HHUF 30468P
MFLU 111001H
HHUF 30504H
WU 36964H
MFLU 11-0150H
CBS H-20221H
–
HHUF 29084H
HHUF 30507H
HHUF 30509H
HHUF 30477H
HHUF 30478H
HHUF 30479H
HHUF 30482H
HHUF 30486P
HHUF 30489H
CBS 123083
CBS 123092
–
–
–
–
–
–
–
MFLUCC 14-0815
MFLUCC 14-0817
MFLUCC 14-0821
MFLUCC 14-0823
–
CBS 143911
–
MFLUCC 14-0816
CBS 123095
MAFF 245218
MFLUCC 11-0401 = IFRDCC 2572
MAFF 245613 = NBRC 112316
CBS 143912
MFLUCC 10-0558
CBS 122364
IFRDCC 2053
JCM 13096 = MAFF 239606
MAFF 245614 = NBRC 112317
MAFF 245615 = NBRC 112318
MAFF 245365 = NBRC 111758
MAFF 245366 = NBRC 111759
MAFF 245367 = NBRC 111760
MAFF 245370 = NBRC 111761
MAFF 245374 = NBRC 111762
MAFF 245377 = NBRC 111763
GU456295
GU296134
–
–
–
–
–
–
–
KU922912
KU922914
KU922916
KU922918
–
–
–
KU922910
LC194298
LC061581
JX276956
LC194299
–
KX672159
LC194300
GU456296
LC194302
LC194303
LC194305
LC194306
LC194307
LC194311
LC194314
LC194318
LC194321
–
–
–
–
–
–
–
–
–
–
–
–
–
MG912909
MG912910
MG912911
–
LC194474
LC061591
JX276954
LC194475
MG912912
KX672154
LC194476
–
LC194478
LC194479
LC194481
LC096154
LC096155
LC096159
LC096162
LC096166
LC096169
FJ795498
GU301797
GQ221877
GQ221878
KF906410
GQ221879
GQ221911
GQ221874
GQ221876
KU922911
KU922913
KU922915
KU922917
MG912909
MG912910
MG912911
KU922909
LC194340
LC061586
JX276955
LC194341
MG912912
KX672162
LC194342
GU456318
LC194344
LC194345
LC194347
LC194348
LC194349
LC194353
LC194356
LC194360
LC194363
GU456273
GU349065
GQ221924
GQ221915
KF906410
GQ221925
GQ221919
GQ221937
GQ221938
KU922919
–
KU922921
KU922922
MG912913
MG912914
MG912915
KU922920
LC194382
LC194383
–
LC194386
MG912916
KX672161
LC194387
GU456263
LC194389
LC194390
LC194392
LC096136
LC096137
LC096141
LC096144
LC096148
LC096151
GU456358
–
–
–
–
–
–
–
–
–
–
–
–
MG912917
MG912918
MG912919
–
LC194419
LC194420
–
LC194423
MG912920
KX672149
LC194424
GU456348
LC194426
LC194427
LC194429
LC194430
LC194431
LC194435
LC194438
LC194442
LC194445
Sydowia 70 (2018) 131
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
Species Family Original
no. Specimen no.aStrain no.
GenBank Accession no
SSU ITS LSU tef1 rpb2
Flammeascoma bambusae
Flammeascoma lignicola
Galeaticarpa aomori
Hermatomyces iriomotens
Hermatomyces tectonae
Hermatomyces thailandica
Lepidosphaeria nicotiae
Lophiostoma arundinis
Lophiostoma crenatum
‘Lophiotrema’ boreale
Lophiotrema eburnoides
Lophiotrema fallopiae
Lophiotrema neoarundinaria
Lophiotrema neohysterioides
Lophiotrema nucula
Lophiotrema vagabundum
Polyplosphaeria fusca
Pseudoastrosphaeriella bambusae
Pseudoastrosphaeriella longicolla
Pseudoastrosphaeriella thailandensis
Pseudocryptoclypeus yakushimensis
Pseudolophiotrema elymicola
Pseudotetraploa curviappendiculata
Quadricrura septentrionalis
Tetraploa sasicola
Triplosphaeria maxima
Ulospora bilgramii
Verruculina enalia
Anteagloniaceae
Anteagloniaceae
Lophiotremataceae
Hermatomycetaceae
Hermatomycetaceae
Hermatomycetaceae
Testudinaceae
Lophiostomataceae
Lophiostomataceae
incertae sedis
Lophiotremataceae
Lophiotremataceae
Lophiotremataceae
Lophiotremataceae
Lophiotremataceae
Lophiotremataceae
Tetraplosphaeriaceae
Pseudoastrosphaeriellaceae
Pseudoastrosphaeriellaceae
Pseudoastrosphaeriellaceae
Lophiotremataceae
incertae sedis
Tetraplosphaeriaceae
Tetraplosphaeriaceae
Tetraplosphaeriaceae
Tetraplosphaeriaceae
Testudinaceae
Testudinaceae
–
–
KT 2563
KH 361
–
–
–
–
–
–
KT 1424-1
KT 274
KT 856
KH 17
–
–
–
–
–
–
KT 2186
KT 1450
–
–
–
–
–
–
MFLU 11–0143H
MFLU 10-0061H
HHUF 30505H
HHUF 30518H
MFLU 15-3437H
MFLU 15-3440H
–
–
–
–
HHUF 30079H
HHUF 30506H
HHUF 27547
HHUF 30511
–
F-634236
HHUF 29399H
MFLU 11-0155H
MFLU 11-0207H
MFLU 11-0145H
HHUF 30503H
HHUF 28984H
HHUF 28582H
HHUF 28781P
HHUF 27566H
HHUF 29390H
–
–
MFLUCC 10-0551
MFLUCC 10-0128
MAFF 245618 = NBRC 112319
MAFF 245730 = NBRC 112471
MFLUCC 14-1140
MFLUCC 14-1143
CBS 101341
CBS 621.86
CBS 629.86
CBS 114422 = JCM 14136
JCM 17826 = MAFF 242970
MAFF 245612
MAFF 239461
MAFF 245619
CBS 627.86 = JCM 14132
CBS 113975 = JCM 14138
JCM 13175 = MAFF 239685
MFLUCC 11-0205
MFLUCC 11-0171
MFLUCC 10-0553
MAFF 245622 = NBRC 112320
JCM 13090 = MAFF 239600
JCM 12852 = MAFF 239495
CBS 125429
JCM 13167 = MAFF 239677
JCM 13172 = MAFF 239682
CBS 101364
BCC 18402
KP753952
KT324584
LC194324
LC194325
KU712465
KU712468
–
DQ782383
DQ678017
LC194333
LC001706
LC149911
AB524455
LC194334
AB618703
AB618707
AB524463
KT955455
–
KT955456
LC194338
LC194339
AB524467
AB524474
AB524490
AB524496
DQ678025
GU479771
KP744440
KT324582
LC194482
LC194483
KU144917
KU144920
–
AJ496633
–
LC194491
LC001709
LC149913
AB524786
LC194493
LC194497
LC194502
AB524789
–
–
–
LC194504
LC194505
AB524792
AB524799
AB524807
AB524812
–
–
KP744485
KT324583
LC194366
LC194367
KU764695
KU764692
DQ678067
DQ782384
DQ678069
LC194375
LC001707
LC149915
AB524596
LC194376
AB619021
AB619025
AB524604
KT955475
KT955476
KT955477
LC194380
LC194381
AB524608
AB524615
AB524631
AB524637
DQ678076
GU479803
–
KT324585
LC194393
LC194394
KU872757
KU872754
DQ677910
DQ782387
DQ677912
LC194402
LC194403
LC194404
AB539109
LC194406
LC194410
LC194415
–
KT955437
KT955438
KT955439
LC194417
LC194418
–
–
–
–
DQ677921
GU479864
–
KT324586
LC194448
LC194449
KU712486
KU712488
DQ677963
DQ782386
DQ677965
LC194457
LC194458
LC194459
AB539096
LC194461
LC194465
LC194470
–
KT955414
KT955420
KT955411
LC194472
LC194473
–
–
–
–
DQ677974
GU479836
a H = holotype; P = paratype.
132 Sydowia 70 (2018)
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
ronyms other than those of ofcial culture collec-
tions are used here primarily as strain identiers
throughout the work. Representative isolates have
been deposited at the Westerdijk Fungal Biodiver-
sity Centre, Utrecht, The Netherlands (CBS culture
collection). Details of the specimens used for mor-
phological investigations are listed in the Taxonomy
section under the respective descriptions. Speci-
mens have been deposited in the Herbarium of the
Institute of Botany, University of Vienna (WU).
Culture preparation, growth rate determination
and phenotype analysis
Cultures were prepared and maintained as de-
scribed previously (Jaklitsch 2009) except that 2 %
malt extract agar (MEA; 2 % w/v malt extract, 2 %
w/v agar-agar; Merck, Darmstadt, Germany) was
used as the isolation medium. Cultures used for the
determination of growth rates and study of asexual
morph micro-morphology were grown on CMD, 2 %
MEA or potato dextrose agar (PDA, 39 g/l; Merck,
Darmstadt, Germany) at 22–25 °C in darkness. Mi-
croscopic observations were made in tap water ex-
cept where noted. Morphological analyses of micro-
scopic characters were carried out as described ear-
lier (Jaklitsch 2009). Data were gathered using a
Nikon Coolpix 995 or Coolpix 4500 or a Nikon DS-
U2 digital camera and measured with NIS-Ele-
ments D v. 3.0, or with a Zeiss Axiocam 506 colour
digital camera and measured with Zeiss ZEN Blue
Edition software. Methods of microscopy included
stereomicroscopy using an Olympus SZ 60 or Nikon
SMZ 1500 and Nomarski differential interference
contrast (DIC) using the compound microscopes
Nikon Eclipse E600 or Zeiss Axio Imager.A1. For
certain images of ascomata the stacking software
Zerene Stacker v. 1.04 (Zerene Systems LLC, Rich-
land, WA, USA) was used. Measurements are report-
ed as maximum and minimum in parentheses and
the range representing the mean plus and minus the
standard deviation of a number of measurements
given in parentheses.
DNA extraction and sequencing methods
The extraction of genomic DNA was performed
as reported previously (Voglmayr & Jaklitsch 2011,
Jaklitsch et al. 2012) using the DNeasy Plant Mini
Kit (QIAgen GmbH, Hilden, Germany). The follow-
ing loci were amplied and sequenced: the complete
internally transcribed spacer region (ITS1-5.8S-
ITS2) and a ca. 900 bp fragment of the large subunit
nuclear ribosomal DNA (nLSU rDNA), amplied
and sequenced as a single fragment with primers
V9G (de Hoog & Gerrits van den Ende 1998) and
LR5 (Vilgalys & Hester 1990); a ca. 1.1 kb fragment
of the RNA polymerase II subunit 2 (rpb2) with
primers fRPB2-5f and fRPB2-7cr (Liu et al. 1999);
and a ca. 1.3–1.5 kb fragment of the translation
elongation factor 1-alpha (tef1) with primers EF1-
728F (Carbone & Kohn 1999) and TEF1LLErev
(Jaklitsch et al. 2005) or EF1-2218R (Rehner &
Buckley 2005). PCR products were puried using an
enzymatic PCR cleanup (Werle et al. 1994) as de-
scribed in Voglmayr & Jaklitsch (2008). DNA was
cycle-sequenced using the ABI PRISM Big Dye Ter-
minator Cycle Sequencing Ready Reaction Kit v. 3.1
(Applied Biosystems, Warrington, UK) with the
same primers as in PCR; in addition, primers ITS4
(White et al. 1990), LR2R-A (Voglmayr et al. 2012)
and LR3 (Vilgalys & Hester 1990) were used for the
ITS-LSU region. In some cases the tef1 was cycle-
sequenced with internal primers TEF1_INTF (for-
ward; Jaklitsch 2009) and TEF1_INT2 (reverse;
Voglmayr & Jaklitsch 2017). Sequencing was per-
formed on an automated DNA sequencer (3730xl
Genetic Analyzer, Applied Biosystems).
Analysis of sequence data
For the phylogenetic analyses, a combined ma-
trix of ITS-LSU, SSU, rpb2 and tef1 sequences was
produced. For this, GenBank sequences of selected
families of Pleosporales were selected from Hashi-
moto et al. (2017) and supplemented with GenBank
nucleotide sequences of some additional taxa. All
alignments were produced with the server version
of MAFFT (www.ebi.ac.uk/Tools/mafft), checked
and rened using BioEdit version 7.0.9.0 (Hall
1999). Due to the lack of tef1 intron sequence data
for most species, only the exon was included for this
marker. The combined matrix contained 5054 nu-
cleotide characters, i.e. 2105 from the ITS-LSU,
1001 from the SSU, 1027 from rpb2, and 921 from
tef1.
Maximum likelihood (ML) analyses were per-
formed with RAxML (Stamatakis 2006) as imple-
mented in raxmlGUI 1.3 (Silvestro & Michalak
2012), using the ML + rapid bootstrap setting and
the GTRGAMMAI substitution model with 1000
bootstrap replicates. The matrix was partitioned for
the individual gene regions, and substitution model
parameters were calculated separately for them.
Maximum parsimony (MP) bootstrap analysis of
the combined matrix was performed with PAUP v.
4.0a159 (Swofford 2002), implementing 1000 boot-
strap replicates with 5 rounds of replicates of heu-
ristic search with random addition of sequences
Sydowia 70 (2018) 133
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
and subsequent TBR branch swapping (MULTREES
option in effect, steepest descent option not in ef-
fect) during each bootstrap replicate. In all MP
analyses molecular characters were unordered and
given equal weight; analyses were performed with
gaps treated as missing data; the COLLAPSE com-
mand was set to minbrlen.
Results
Molecular phylogeny
Of the 5054 nucleotide characters of the com-
bined matrix, 1122 are parsimony informative (367
of ITS-LSU, 46 of SSU, 443 of rpb2, and 266 of tef1).
The best tree revealed by ML analyses (-lnL =
34887.9896) is shown as phylogram in Fig. 1. Tree
topologies are similar to those obtained by Hashi-
moto et al. (2017) and are therefore not described in
detail here, except for the results relevant for the
two new species described herein. The Anteagloni-
aceae receive high support (99 % ML and 97 % MP),
containing the two monophyletic genera Anteaglo-
nium and Flammeascoma. Anteaglonium rubescens
is placed within the moderately supported Anteag-
lonium clade, but its closest relatives remain unre-
solved due to lack of signicant bootstrap support
of most nodes within Anteaglonium.
The Lophiotremataceae are highly supported
(94 % ML, 89 % MP), but the Atrocalyx clade re-
ceives only weak (69 %; ML) or no (MP) support.
Atrocalyx asturiensis is revealed as sister species to
A. lignicola with maximum (ML) or high (99 %, MP)
support.
Taxonomy
Anteagloniaceae
Anteaglonium rubescens
Jaklitsch & Voglmayr, sp.
nov. – Figs. 2, 3.
MycoBank no.: MB 824180
Etymology. – rubescens means reddening,
due to the reddening of natural and articial sub-
strates.
H o l o t y p e . – GREECE. Corfu, Kanakades, on twigs of
Pistacia lentiscus, soc. Xylaria xylarioides and a coelomycete,
20 April 2012, leg. W. Jaklitsch & H. Voglmayr (WU 36960; cul-
ture CBS 143911 = OR1).
Description. – Stromata erumpent from
wood or immersed in or erumpent from bark as ef-
fused dark brown to black zones or crusts, running
parallel to the wood surface for several cm or some-
times erumpent as stripes up to 1.8 mm long; in
places encasing one to several ascomata, often con-
siderably thickened at the sides and particularly at
the apices of the latter, sometimes absent at their
bases; parts containing ascomata forming erumpent
to supercial roundish pustules with convex or at,
rounded, angular or slightly elongate laterally com-
pressed apices, less commonly with variable black
papillae of 25–195 μm diam; in places erumpent as
hysterothecioid domes ca. 200–900 μm long and up
to ca. 150 μm thick; pseudoparenchymatous, con-
sisting of dark brown, thick-walled cells (2.8)3.2–
6(10) μm (n = 40) diam. Hysterothecioid domes dark
brown to black, smooth and with a sunken longitu-
dinal slit containing several inconspicuous ostiolar
apices; when fresh sometimes with longitudinal
crests or striae. Stromata often covered or under-
lain by brown or brightly orange or red to pink or
purple subiculum of septate, thick-walled, smooth
to warted, 3–6 μm wide hyphae encrusted with
amorphous pigment remaining unchanged in 3 %
KOH. Subiculum merging into dark reddish brown
pseudoparenchyma of thick-walled cells becoming
gradually smaller toward stroma tissue; at the stro-
ma surface sometimes becoming decomposed into
amorphous particles; subiculum often also present
between wood and bark as compressed tissue. Pig-
ment diffusing and staining wood and bark in
patches with pink to purple or violaceous tones. –
A s c o m a t a (256)265–367(425) μm diam, (230)285–
393(460) μm high (n = 13), globose to subglobose or
subconical, individually or collectively encased by
stromatic tissue. – O s t i o l e s minute, rounded in
section, openings scarcely visible. – Pe r i d i u m
10–55 μm thick, laterally sometimes up to 90 μm,
often appearing thicker due to coalescence with
stromatic tissue, pseudoparenchymatous, consist-
ing of rather thin-walled cells (3)4.5–10.5(16) μm
(n = 53) diam., red when young, turning pale to dark
brown, often surrounded by a whitish to brown,
brittle or hard cellular tissue followed by dark stro-
matic tissue. – H a m a t h e c i u m trabeculate, con-
sisting of numerous richly branched, 1–2.5(3) μm
wide pseudoparaphyses embedded in a gel matrix.
– A s c i (65)72–87(95) × (4.3)4.7–5.7(6.2) μm (n = 32),
cylindrical, bitunicate, ssitunicate, with a refrac-
tive ocular chamber, a stipe up to ca. 30 μm long, a
simple or knob-like base, containing 4–8 ascospores
in uniseriate, sometimes partly biseriate arrange-
ment. – A s c o s p o r e s 2-celled, dark brown, dark-
ening in 3% KOH, thick-walled, smooth, biconical
or dimorphic, cells disarticulating, distal cells
broader, subglobose to wedge-shaped, (4.0)4.8–
6.0(7.0) × (3.2)3.5–4.0(4.5) μm, l/w (1.2)1.3–1.6(1.9)
(n = 75), proximal cell wedge-shaped or oblong,
(4.4)5.2–6.7(7.6) × (2.7)3.0–3.6(4.0) μm, l/w (1.3)1.5–
134 Sydowia 70 (2018)
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
Fig. 1. Phylogram of the best ML tree (lnL = −34887.9896) revealed by RAxML from an analysis of the combined ITS-LSU-SSU-
rpb2-tef1 matrix of selected Pleosporales, showing the phylogenetic position of Anteaglonium rubescens and Atrocalyx asturien-
sis (in bold). ML and MP bootstrap support above 50% are given at the rst and second positions, respectively, above or below the
branches. Familial classication is according to Hashimoto et al. (2017).
Sydowia 70 (2018) 135
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
Fig. 2. Anteaglonium rubescens, sexual morph. a–g. Stromata in face view. h. Wood with purple stain. i, j. Ascomata and stro-
mata in vertical section. k. Subiculum. l. Peridial cells in vertical section. m. Stroma cells in vertical section near subiculum. n, o.
Ascus apices with ascospores. p–s. Asci. t–z. Ascospores (t. before disarticulation; v. aberrant, unicellular, non-disarticulating). m,
q–s, z. in 3 % KOH. a. WU 36962. b, g, k–m, q, t–v, z. WU 36963. c–f, h–j, n–p, w–y. WU 36960. r, s. WU 36961. b, g. photographs by
J. Martin. Scale bars: a, e–g 0.5 mm; b–d, h 1 mm; i, j 200 μm; k–m, p–s 10 μm; n, t–x 3 μm; o, y, z 5 μm.
136 Sydowia 70 (2018)
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
2.0(2.6) (n = 75), each containing 1–2 guttules; cal-
culated entire ascospore length (8.5)10–12.5(14.5)
μm, l/w (2.4)2.7–3.4(4.0) μm (n = 75).
C u l t u r e s . – Growth slow and variable. On
MEA colony radius 11–13 mm after 2 months at
22–25 °C; colony thick, surface rst whitish, turning
greyish, brown to black, often becoming zonate
with white margin, orange zones and centre covered
with rosy or reddish conidial drops, reverse black;
odour indistinct to unpleasant; after a few days pig-
ment diffusing into agar and discolouring sur-
roundings of the colony and later the whole agar
Fig. 3. Anteaglonium rubescens, asexual morph in culture at 22–25 °C. a, b. Cultures. c, d. Colonies (sections) with pycnidia and
conidial drops. e. Pycnidial wall. f. Pycnidial hyphae. g. Short conidiophores and phialides. h, i. Phialides (note thickened col-
larette in the left phialide in h). j. Prophialide. k–q. Conidia. e, f, h. in 3 % KOH. a, d–f, h, p. CBS 143911. b, c, g, i–o , q. O R. a , b,
d–f, h–j, m–p. On/from MEA. c, g, k, l, q. On/from PDA. a, d. After 60 d; b, i, j, m–o. 37 d; c, g, k, l, q. 29 d; e, f, h, p. 22 d. Scale bars:
c, d 1.5 mm; e, g, i, k, l, n 5 μm; f 10 μm; h, j, m, o–q 3 μm.
Sydowia 70 (2018) 137
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
intensely pink or red to red-orange. On CMD colony
radius up to 5–6 mm after 1 month; colony thick,
red, dark brown to black, zonate, with hyaline to
reddish conidial drops in the centre; agar turning
pink to red. On PDA colony radius ca. 10 mm after
1 month; colony surface dark brown to black, vel-
vety, reverse black; very intense pigment diffusing
into agar.
Asexual morph. – Pycnidia formed on
CMD, MEA and PDA, scattered to mostly tightly ag-
gregated and conuent in the colony centre, more or
less globose with slightly papillate central ostiole,
130–350 μm diam., sometimes with broadened base
and often collapsing apically, dark olive-brown to
black, often covered with a mat of white or reddish
aerial hyphae or by conidial drops. – P y c n i d i a l
w a l l pseudoparenchymatous, consisting of (3)4–
7(9) μm (n = 30) wide, thin-walled, inhomogenously
pigmented, dark red or dark brown cells incrusted
with red pigment, surrounded by red to brown hy-
phae. Interior lined with a palisade of hyaline to
pinkish, lageniform to ampulliform or subglobose,
less commonly cylindrical phialides (3.7)5.0–7.0(8.3)
× (2.0)2.4–3.7(5.0) μm (n = 37), usually with broad,
often thickened collarette; formed on roundish cells
and short simple cylindrical conidiophores with
roundish base cells. – C o n i d i a very variable, sub-
globose to globose or ellipsoid to oblong, (2.7)3.5–
5.5(7.0) × (2.0)2.3–3.5(4.5) μm, l/w (1.0)1.2–2.0(2.8)
(n = 86), 1-celled, with broadly rounded ends, hya-
line to pink, with 1–2(3) guttules, smooth, some-
times with 1–2 lateral protuberances.
H a b i t a t . – On wood and bark of various trees
and shrubs.
D i s t r i b u t i o n . – Southern Europe (Greece,
Spain), possibly also southern USA (Florida).
Other material examined. – SPAIN.
Canary Islands, Tenerife, Anaga mountains, Chino-
bre, pista cortada, on Laurus novocanariensis, 26
June 2002, leg. W. Jaklitsch W.J. 1908 (WU 36961);
Chinobre, La Ensillada, on Laurus novocanariensis,
soc. cf. Dothidotthia sp., Xylaria xylarioides, 16 De-
cember 2010, leg. H. Voglmayr & W. Jaklitsch (WU
36962; culture OR); Basque Country, Gipuzkoa, To-
losa, in a garden, 30TWN7577, on wood of Rhamnus
cathartica, on/soc. black stromatic pyrenomycete,
the latter also colonized by Tubeua cerea and
Bisporella sulfurina, 22 June 2016, leg. P.M. Pasa-
ban, comm. J. Martin (WU 36963; culture OR2).
N o t e s . – Ascomata may sometimes appear
larger, particularly wider, due to difcult optical
differentiation of stromatic tissue and peridium;
only the true peridium was included in measure-
ments. Red or purple colours in specimens are fad-
ing with time, particularly in bark: in the specimen
WU 36962 the bark was intensely pink coloured one
month after collection, six years later the pink col-
our was gone and had partly turned brown. A pos-
sible occurrence of A. rubescens in the USA was
deduced from personal communication of W.J. with
Margaret Barr in 2002, who reported a specimen
collected in Coconut Grove, Miami, Florida (Thax-
ter 5687, FH; not examined) with virtually identical
morphology and tentatively named it Ohleria rube-
scens.
Lophiotremataceae
Atrocalyx asturiensis
Jaklitsch, J. Fourn. & Vogl-
mayr, sp. nov. – Fig. 4.
MycoBank no.: MB 824181
Etymology. – asturiensis due to its occur-
rence in Asturias, Spain.
H o l o t y p e . – SPAIN, Asturias, Lago del Valle, elev. ca.
1550 m, on decorticated stump of Cytisus sp., soc. Coniochaeta
sp., 6 June 2017, leg. J. Fournier J.F. 17052 (WU 36964; culture
CBS 143912 = OF).
Description. – Ascomata erumpent from
bleached wood with their bases remaining more or
less immersed, loosely to most often densely aggre-
gated and in contact, rarely fusing by 2–3, then
forming a black continuous crust to 15 mm in great-
est dimension; subglobose to obpyriform, the base
rounded to attened, 320–380(430) μm high,
(250)290–380(420) μm diam (n = 30); surface black,
somewhat shiny, roughened by shallow wrinkles
and low warts, sometimes encrusted with adherent
wood bres. Apex prominent, sharply delimited
from the venter or integrated, variable in shape, ob-
tusely rounded to crest-like, typically laterally at-
tened, ellipsoid in top view, with a longitudinal slit
lined by two thick lips, rarely sub-circular in top
view with a central rounded pore or Y-shaped,
(90)120–170(210) μm high, 170–250(290) μm long,
125–210(240) μm wide (n = 30), less roughened than
the venter to almost smooth, eventually turning
shiny black. – P e r i d i u m dark brown, leathery,
(22)38–45(55) μm thick at sides and base, pseudo-
parenchymatous, of two intergrading regions: outer
region textura angularis of small, thick-walled iso-
diametric to slightly elongated cells 3–9 × 3–4.5 μm,
wall dark brown, 1–1.8 μm thick; inner layer textu-
ra prismatica of less pigmented cells 8–11 × 2.5–
5 μm, wall 0.5–0.8 μm thick; apical wall 70–80 μm
thick, blackish brown, comprised of small cells sim-
ilar to those of the outer layer but thicker-walled to
occluded, with 2.5–3.5 μm thick cell walls, internal-
ly lined by subhyaline, thin- to thick-walled angu-
138 Sydowia 70 (2018)
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
Fig. 4. Atrocalyx asturiensis (WU 36964). a–e. Ascomata in face and side views. f, g. Ascomata in vertical section. h. Peridium. i.
Ascus apex with ascospores. j–l. Asci (l. In Pelikan blue ink diluted in 1 % SDS). m–z. Ascospores (m. immature; y, z. with ver-
ruculose ornament). Scale bars: a 1 mm; b 500 μm; c–e 200 μm; f, g 100 μm; h, j–l 20 μm; i 10 μm; m–z 5 μm.
Sydowia 70 (2018) 139
Jaklitsch et al.: Anteaglonium rubescens and Atrocalyx asturiensis
lar cells merging with the periphyses. – H a m a t h e -
c i u m of narrow cellular pseudoparaphyses, ba-
sally 1.5–2 μm wide, tapering upwards to 1 μm,
ramied and anastomosed above asci, with free
ends, embedded in a gelatinous matrix. A s c i
(117)122–128(132) × 10–12(13.5) μm (n = 12), cylin-
drical, bitunicate, ssitunicate, with short stipe and
furcate base, apically rounded with an ocular cham-
ber, containing 6–8 obliquely uniseriate overlap-
ping ascospores. – A s c o s p o r e s (15.2)15.8–
18.3(19.6) × (5.8)6.2–6.9(7.1) μm, l/w = (2.2)2.4–
2.8(3.1) (n = 60), ellipsoid-fusiform with broadly
rounded ends, straight, equally 3-euseptate, slightly
constricted at the median septum, not to barely
constricted at other septa, mid cells slightly swol-
len, not disarticulating, olivaceous brown to tobac-
co brown, wall verruculose, without mucilaginous
sheath or appendages.
A s e x u a l m o r p h . – Not detected in cul-
tures.
H a b i t a t . – On wood and bark of Cytisus sp.
D i s t r i b u t i o n . – Southern Europe (Spain),
only known from the type location.
Notes. – Atrocalyx asturiensis differs from
other species of the genus by brown ascospores,
which lack a mucilaginous sheath.
Discussion
Both species described here are difcult to place
morphologically. Their phylogenetic positions are
rather unexpected. Anteaglonium rubescens differs
from all other species in the genus by brown
didymospores, which disarticulate within asci, and
by the production of a red-orange to pink pigment
formed both in nature and in articial culture. This
pigment, which does not change its appearance in
3 % KOH, adds to other interesting substances such
as anteagloniolides, which were extracted from cul-
tures of a moss endophyte identied as Anteagloni-
um sp. and characterized by Xu et al. (2015).
Anteaglonium rubescens is clearly a stromatic
fungus. As we did not study other species of the ge-
nus, this may be a special feature of this fungus or it
was not interpreted or addressed correctly with
other species earlier. In none of the earlier publica-
tions on the genus Anteaglonium the peridium was
studied in detail, but elongate ascomata were inter-
preted as true hysterothecia. In A. rubescens the
apical hysterothecioid dome often remains even
when ascomata are decomposed or eaten by insects.
A typical dome may contain 3 ascomata, which are
separated by very delicate walls. In all specimens of
A. rubescens black lines are present in the wood
caused by xylariaceous fungi. In material from Ten-
erife the xylariaceous fungus is Xylaria xylarioides;
in WU 36963 the Anteaglonium grows in part di-
rectly on stromata of an effused stromatic fungus.
These tight associations suggest that A. rubescens
may be fungicolous.
Jayasiri et al. (2016) described pycnidial asexual
morphs of two Anteaglonium spp. However, the def-
inition of the conidiogenous cells remained unclear,
as they give a size of 3–5 × 2–3 μm for both conid-
iogenous cells and conidia of both species, whereas
their illustrations rather indicate lageniform to am-
pulliform conidiogenous cells similar to those ob-
served by us.
The morphology of Atrocalyx asturiensis, par-
ticularly the massive ascomatal apices and brown
ascospores, may, e.g., suggest Lophiostoma or Xe-
nolophium (Hirayama & Tanaka 2011, Huhndorf
1993), which differ from Atrocalyx by long-stipitate
clavate asci, or Ohleria (Jaklitsch & Voglmayr 2016),
a member of Melanommataceae (Jaklitsch & Vogl-
mayr 2017, Mugambi & Huhndorf 2009b) or Tei-
chosporaceae (Jaklitsch et al. 2016b), but molecular
analyses place the fungus in Lophiotremataceae.
Hashimoto et al. (2017) described ve new genera in
the family Lophiotremataceae and erected three
new related families. Judging from poor phyloge-
netic support of several clades within Lophiotrem-
ataceae, this splitting may seem excessive, on the
other hand morphology and particularly asexual
morphs were used as arguments for taxonomic con-
clusions. Here we recognize a new species of their
genus Atrocalyx, which morphologically deviates
by brown phragmospores, which are not surround-
ed by a gelatinous sheath. On the other hand, the
well-developed crest and particularly the peridium
of A. asturiensis as used for differentiation from
Lophiotrema by Hashimoto et al. (2017) but also as-
cus morphology t well with the generic concept.
Acknowledgements
We are indebted to Pedro Pasaban and Joaquin
Martin for fresh material and images and Walter
Till and Irmgard Greilhuber for managing collec-
tions at WU. The nancial support by the Austrian
Science Fund (FWF; project P25870-B16) is grate-
fully acknowledged.
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