Wawelia amyloasca, sp. nov., – the only species of Wawelia (Xylariaceae) discovered in the field

Abstract

The new species Wawelia amyloasca is described from the dung of Siberian roe deer in the Russian Far East. This is the first
published record of Wawelia for more than 20 years, the only record outside Europe, and the only discovery made in the field, even
though the genus was described in 1908. The new taxon is characterized by the longest stromata in the genus reaching 120 mm,
ascospores with polar pad-like appendages, and asci with a well-noticeable amyloid apical apparatus, which is unique within the
genus. Wawelia is accommodated within the family Xylariaceae together with other coprophilous genera, which was proved by
the phylogenetic analysis using four markers, ITS, LSU, BenA, and RPB2, the latter two being obtained for the first time for the
genus. A complete bibliography is given and a comparative table including all known species of Wawelia is presented.

Full text

Sydowia 76 (2024) 45
DOI 10.12905/0380.sydowia76-2024-0045 Published online 18 October 2023
Wawelia amyloasca,
sp. nov., – the only species of
Wawelia
(Xylariaceae) discovered in the eld
Fedor M. Bortnikov1,*, Nadezhda A. Bortnikova2 & Evgeny A. Antonov1, 3
1 Lomonosov Moscow State University, Faculty of Biology, Leninskie Gory 1–12, Moscow 119234, Russia
2 Komarov Botanical Institute of the Russian Academy of Sciences, Laboratory of Systematics and Geography of Fungi,
Prof. Popov Street 2, St. Petersburg, 197376, Russia
3 Kurchatov Institute National Research Center, Institute of Information Technology, Kurchatov Square 1,
Moscow 123098, Russia
* e-mail: f.m.bortnikov@gmail.com
Bortnikov F.M., Bortnikova N.A. & Antonov E.A. (2023) Wawelia amyloasca, sp. nov., – the only species of Wawelia (Xylari-
aceae) discovered in the eld. – Sydowia 76: 45–61.
The new species Wawelia amyloasca is described from the dung of Siberian roe deer in the Russian Far East. This is the rst
published record of Wawelia for more than 20 years, the only record outside Europe, and the only discovery made in the eld, even
though the genus was described in 1908. The new taxon is characterized by the longest stromata in the genus reaching 120 mm,
ascospores with polar pad-like appendages, and asci with a well-noticeable amyloid apical apparatus, which is unique within the
genus. Wawelia is accommodated within the family Xylariaceae together with other coprophilous genera, which was proved by
the phylogenetic analysis using four markers, ITS, LSU, BenA, and RPB2, the latter two being obtained for the rst time for the
genus. A complete bibliography is given and a comparative table including all known species of Wawelia is presented.
Key words: apical apparatus, Ascomycota, coprophilous fungi, molecular phylogeny, rare species. – 1 new species.
In 1908, B. Namysłowski cultivated coprophilous
fungi on rabbit dung using moist chamber cultures
in the Botanic Garden of the Jagiellonian Univer-
sity (Kraków, Poland). He was supplied with this
substrate by one of the garden workers, who lived
on the Wawel hill, where the Royal Castle was lo-
cated, and raised rabbits there. Namysłowski found
a curious fungus from a previously unknown genus
and described it as Wawelia regia Namysł.
(Namysłowski 1908). The scientist included the ge-
nus in the subfamily “Waweliaceae” in the family
“Hypocreales”. Later F. Vincens studied the type
material of W. regia and supposed that this genus
was related to Xylariaceae rather than Hypocreace-
ae, which was based on the presence of an ascospore
germ slit, a feature not mentioned by Namysłowski
(Vincens 1918). Since then, almost 50 years passed
without any records of Wawelia in the world. In
1956, W. regia was rediscovered by B. Gumin´ska,
while she was incubating rabbit droppings in the
same botanical garden in Kraków (Gumin´ska 1957).
Her material was sent to G. Doguet and E. Müller,
who examined it (Müller 1959; Doguet 1960, 1961a,
b, 1963) and concluded that the genus Wawelia be-
longed to the family Melanosporaceae because of
the features of asci: they were thin-walled and
lacked an iodine-positive apical apparatus, which is
present in typical xylariaceous species (Müller
1959). Doguet followed the perithecia development
of W. regia during the experiment and pointed out
that this species should be either classied as a
member of the family Melanosporaceae or main-
tained in the initially suggested family of its own,
Waweliaceae (Doguet 1961b).
For a long time, the genus Wawelia remained
monotypic. W. Wojewoda speculated that the in-
credible rarity of this fungus could be explained by
the fact that it originated from tropical or subtropi-
cal regions and spores or whole fragments of myce-
lium were accidentally brought to the Kraków Bo-
tanic Garden with the introduction of exotic plants
(Wojewoda 1983). However, the same year, a new
species, W. octospora Minter & J. Webster, was de-
scribed from England, but it was characterized by
8-spored instead of 4-spored asci as in the case of
W. regia (Minter & Webster 1983). Subsequently,
three more species of Wawelia were introduced:
W. effusa N. Lundq. from Sweden and Hungary
(Lundqvist 1992), W. argentea J. Webster, and W. m i -
crospora J. Webster, both from England (Webster et
al. 1999). As such, the genus Wawelia currently
comprises ve species, all described from Europe.

46 Sydowia 76 (2024)
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Although the genus Wawelia is quite small and
there are not many studies focusing on it, some of
them still appear little known and are rarely or nev-
er cited. Therefore, we provide full bibliography
considering this taxon (Tab. 1).
Despite a number of mycological studies and
specic interest of some researchers in coprophilous
fungi in Europe, Wawelia has never been observed
in the eld before (Gumin´ska 2000). All the known
specimens (less than 15–20 in the world) were ob-
tained from the substrate incubated in moist cham-
bers. Moreover, only W. regia has been recorded by
someone else besides its discoverer (Gumin´ska
1957).
In July 2020, we were lucky enough to encounter
extremely long liform stromata of a mature but
unknown coprophilous ascomycete during a myxo-
mycete inventory in Kedrovaya Pad Nature Reserve
in the south-west of Primorsky Krai (Russia)
(Fig. 1). Further research proved that this specimen
represents an undescribed species of the genus
Wawelia, because it possesses several unique fea-
tures, distinguishing it from other taxa.
Materials and methods
Field and morphological study
Stromata were collected in the eld and then
fully dried out at room temperature. Microscopic
morphological features were studied using tempo-
rary slides prepared with water using a Micromed 3,
Var. 3 LED M microscope (Shenzhen, China). The
amyloid reaction of apical apparatus was revealed
via Lugol’s iodine and Melzer’s solution. Ascospore
appendages and ascus walls were observed in Indi-
an ink. All measurements were performed in water.
Measurements are given as follows: (minimum)
mean-standard deviation – mean + standard devia-
tion (maximum). Some images were obtained using
a CamScan S2 scanning electron microscope (Cam-
bridge Instruments Co Ltd, Cambridge, UK). Speci-
mens were preliminary sputter coated with gold-
palladium alloy. Tissue types of stromata and peri-
thecia were interpreted following Hengstmengel
(2020).
DNA extraction, amplication, and sequencing
Total genomic DNA was extracted from 1 g of
dry stroma using the cetyltrimethyl ammonium
bromide (CTAB) method. Samples were grounded
in liquid nitrogen and incubated in eppendorfs for
60 min at 65 °C with 700 µl extraction buffer (2 %
CTAB, 1.4 M NaCI, 20 mM EDTA, 100 mM Tris-HCI
pH 8). After 5 min cooling, 500 µl chloroform were
added. Samples were vortexed and then centrifuged
at 13000 g for 10 min. This aqueous phase was
transferred to a new 1.5 ml tube and 400 µl cold
isopropanol + 70 µl 5M CH3COOK were added.
Samples were vortexed and then centrifuged at
13000 g for 10 min. Supernatant was removed and
the DNA was washed with 70 % ethanol, then cen-
trifuged at 13000 g for 5 min. The step was repeated
twice. The DNA pellet was dried thoroughly and
dissolved in TE buffer. The DNA samples were
stored at -20 °C until use.
PCR was performed with the use of ScreenMix
supermix (Evrogen JSC, Moscow, Russia) in T100
Thermal Cycler (Bio-Rad, CA, USA). Primer pairs
used were as follows: ITS1/ITS4 for ITS (White et
al. 1990), LROR/LR5 for LSU (Vilgalys & Hester
1990), Bt2a/Bt2b for benA β-tubulin locus (Glass &
Donaldson 1995), and fRPB2-5F/fRPB2-7R for
RPB2 (Liu et al. 1999).
Amplication products were cut out from the gel
and puried on the silica spin-columns using a
Cleanup Standard kit (Evrogen JSC, Moscow, Rus-
sia). Sequencing reactions were performed by Evro-
gen JSC (Moscow, Russia) following the BigDye ter-
minator protocol on the Applied Biosystems 3730xl
automatic sequencer (Applied Biosystems, Inc, CA,
USA) with both forward and reverse primers. New-
ly generated sequences were deposited in the NCBI
(GenBank) nucleotide database.
Phylogenetic analyses
Two datasets were analyzed, ITS+LSU and
BenA+ITS+RPB2. Sequences were aligned using
MUSCLE implemented in UNIPRO Ugene v46.0
(Okonechnikov et al. 2012) and then optimized with
TrimAl v1.3 (Capella-Gutierrez et al. 2009) with au-
tomated 1 option using webserver version imple-
mented in Phylemon 2.0. (Sánchez e al. 2011) in the
rst case and aligned via MAFFT version 7 (Katoh
et al. 2019) using the auto strategy and then manu-
ally optimized using MEGA X (Kumar et al. 2018)
in the second case. The phylogenetic sampling was
based on taxonomic studies of Xylariaceae
(Daranagama et al. 2015, 2018), coprophilous Xy-
lariaceae (Becker et al. 2020), and polyphyletic gen-
era of Xylariaceae (Konta et al. 2020). Graphostro-
ma platystomum CBS 270.87 and Biscogniauxia
nummularia MUCL 51395 (Graphostromataceae)
were chosen as the outgroup in both cases.
The ITS+LSU dataset comprised 36 sequences
concatenated in SequenceMatrix (Vaidya et al.
2011) with a total length of 1854 characters includ-

Sydowia 76 (2024) 47
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Tab. 1. Bibliography of the genus Wawelia Namysł.
Literary source Language Content of the article regarding Wawelia Figures and tables
Namysłowski (1908) French Description of the genus Wawelia and
the type species, W. regia; commentary
on cultivation, development, and
taxonomic position of the new genus and
species
Five hand drawings of stromata, perithecia,
asci, paraphyses, and anamorphic stage; one
black-and-white photograph of W. regia type
specimen
Vincens (1918) French Short commentary on the ascospore mor-
phology of W. regia and its taxonomic
position within the family Xylariaceae
One hand drawing of ascospores of the W. regia
type specimen
Gumin´ ska (1957) English Report of the second record of W. regia
on terra typica (Krakow, Poland).
Morphological description of the new
specimen
Six black-and-white photographs of the
second W. regia specimen: stromata (3), asci
and paraphyses (2), and ascospores (1)
Müller (1959) German Description of W. regia morphology
(Gumin´ ska’s material), commentary on
the genus taxonomic position
Four hand drawings of the second W. regia
specimen: stromata, perithecium (general view
and section), and ascus with spores
Doguet (1960) French Detailed description of different ways of
treating and cultivating W. regia
(Gumin´ ska’s material and fresh cultures
isolated from it) and their inuence on
ascospore germination and development
of fertile stromata with perithecia
Three tables
Doguet (1961a) French Detailed cytological report on W. regia
asci development (Gumin´ ska’s material
and fresh cultures isolated from it) and
nuclei distribution in ascospores
Forty-two hand drawings of different asci
development stages and normal and abnormal
ways of nuclei distribution in ascospores
Doguet (1961b) French Detailed description of W. regia mor-
phology (Gumin´ ska’s material and fresh
cultures isolated from it) and perithe-
cium development. Commentary on
taxonomic position of the genus based
on the observations provided
Twenty hand drawings: ascogenous hyphae,
paraphyses, asci, ascospores, anamorph (6),
and perithecium development stages (14);
14 tables
Doguet (1963) French Detailed description of the ascospore
treatment required for their activation
(dormancy breaking)
Seven graphs and 13 tables
Gumin´ ska (1963) Polish Short review of four out of ve articles
of Müller and Doguet considering W.
regia
–
Rogers (1981) English Short commentary on the issue that
Wawelia should not be considered
synonymous to Sarcoxylon (based on the
study of W. regia material in Royal
Botanic Gardens, Kew)
–
Wojewoda (1983) Polish Brief information on the history of W.
regia discovery, different opinions on its
taxonomic position, and assumption
about the native range of this fungus
–
Minter & Webster
(1983)
English Description of the second Wawelia
species (W. octospora). Commentary on
its taxonomic position, a way of spore
release, and preferred conditions for the
development of fertile stromata
Five hand drawings of stromata, perithecia,
asci, paraphyses, and anamorph of W. octos-
pora
Gumin´ ska (1987) Polish Review of the Minter & Webster (1983)
article with a W. octospora description
and additional commentary
Two schematic hand drawings, comparing
morphology of stromata, perithecia, and asci of
W. regia and W. octospora

48 Sydowia 76 (2024)
Bortnikov et al.: Wawelia amyloasca, sp. nov.
ing gaps (ITS, 1–486, GTR+G model; LSU, 487–1770,
GTR+G) (Tab. 2). The best-t models were estimated
based on the Akaike Information Criterion (AIC)
using FindModel web server (http://www.hiv.lanl.
gov/content/sequence/ndmodel/ndmodel.html).
Maximum likelihood (ML) analysis of the ITS+LSU
dataset with two partitions (Chernomor et al. 2016)
was run in IQ-TREE v1.6.12 (Nguyen et al. 2015);
ultrafast bootstrap was performed with 100000
samples (Hoang et al. 2018). Bayesian inference (BI)
analysis was performed with MrBayes v. 3.2.6 (Ron-
quist et al. 2012); two sets of four chains were run
for 8 million generations, trees were sampled every
100th generation. The convergence of MCMC chains
Literary source Language Content of the article regarding Wawelia Figures and tables
Lundqvist (1992) English Description of the third Wawelia species
(W. effusa). Commentary on the perithe-
cia development and taxonomic position
of the genus
Eight hand-drawn illustrations (Fig. 1 A-H) of
stroma, perithecium (general view and section),
asci, and ascospores of W. effusa; ve black-
and-white photos of stromata, perithecia, and
the outer layer of W. effusa
Webster et al. (1999) English Description of the fourth and fth
Wawelia species (W. argentea and W.
microspora). Identication key of species
known at that time. Commentary on
preferred conditions for the development
of fertile stromata
Eleven black-and-white photographs of W.
argentea (6) and W. microspora (5)
Gumin´ ska (2000) Polish Overview of the history of the genus
research
Five schematic hand drawings, comparing
stromata and asci morphology of different
Wawelia species
Wojewoda &
Karasin´ ski (2010)
English Short review of the history of W. regia
discovery
–
Daranagama et al.
(2018)
English Description of the genus morphology
and W. octospora holotype. NB: there are
a few inaccuracies about Wawelia
Nine colored photographs of W. octospora
herbarium specimen and morphological
features; three black-and-white reprinted
illustrations of W. octospora from Minter &
Webster (1983)
Fig. 1. Type locality of Wawelia amyloasca.

Sydowia 76 (2024) 49
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Tab. 2. Specimens and GenBank accession numbers of DNA sequences used in this study.
Species Strain /
voucher
GenBank accession numbers
References
ITS LSU RPB2 BenA
Arthroxylaria elegans CBS 537.79 AF432179 – – – Seifert et al. (2002)
Biscogniauxia nummu-
laria
MUCL
51395
KY610382 KY610427 KY624236 KX271241 Wendt et al. (2018)
Brunneiperidium
gracilentum
MFLUCC
14-0011
KP297400 – KP340528 KP406611 Daranagama et al. (2015)
B .involucratum MFLUCC
14-0009
KP297399 – KP340527 KP406610 Daranagama et al. (2015)
Daldinia concentrica CBS
113277
AY616683 KY610434 – – ITS: Triebel et al. (2005), LSU:
Wendt et al. (2018)
D. dennisii CBS
114741
JX658477 KY610435 – – ITS: Stadler et al. (2014), LSU:
Wendt et al. (2018)
D. loculatoides CBS
113279
MH862918 – KX271246 KY624247 ITS: Vu et al. (2019), RPB, BenA:
Wendt et al. (2018)
D. macaronesica CBS
113040
KY610398 – KY624294 KX271266 Wendt et al. (2018)
Dematophora bunodes CBS
123597
MN984619 MN984625 – – Wittstein et al. (2020)
D. buxi JDR 99 GU300070 – GQ844780 GQ470228 Hsieh et al. (2010)
D.necatrix CBS 349.36 AY909001 – KY624275 KY624310 Wendt et al. (2018)
Graphostroma
platystoma
CBS 270.87 JX658535 DQ836906 KY624296 HG934108 ITS: Stadler et al. (2014), LSU:
Zhang et al. (2006), RPB2:
Koukol et al. (2015), BenA:
Wendt et al. (2018)
Hypocopra anomala TTI-000339 — – MT901033 MT901030 Becker et al. (2020)
H. rostrata TTI-000009 MT896134 MT903246 MT901034 MT901031 Becker et al. (2020)
Hypoxylon fragiforme MUCL
51264
KC477229 KM186295 KM186296 KX271282 ITS: Stadler et al. (2013), LSU,
RBP2: Daranagama et al. (2015),
BenA: Wendt et al. (2018)
H. monticulosum MUCL
54604
KY610404 KY610487 KY624305 KX271273 Wendt et al. (2018)
Kretzschmaria deusta CBS 163.93 KC477237 – KY624227 KX271251 Stadler et al. (2013)
K. deusta CBS 288.30 MH855142 MH866592 – – Vu et al. (2019)
K. guyanensis HAST
89062903
GU300079 – GQ478214 GQ844792 Hsieh et al. (2010)
Nemania beaumontii HAST 405 GU292819 – GQ844772 GQ470222 Hsieh et al. (2010)
N. serpens CBS 679.86 KU683765 – KU684284 KU684188 U’Ren et al. (2016)
N. serpens AT-114 DQ631942 DQ840075 – – Tang et al. (2007)
Podosordaria mexicana WSP 176 GU324762 – GQ853039 GQ844840 Hsieh et al. (2010)
P. muli WSP 167 GU324761 – GQ853038 GQ844839 Hsieh et al. (2010)
Poronia erici DSM
107106
MN954396 MN954397 MN956523 MN956524 Peric & Wendt (2017)
P. pileiformis WSP
88113001
GU324760 – GQ853037 GQ502720 Hsieh et al. (2010)
P. punctata CBS 656.78 KT281904 KY610496 KY624278 KX271281 Wendt et al. (2018)
Rhopalostroma
angolense
CBS
126414
KY610420 KY610459 – – Wendt et al. (2018)

50 Sydowia 76 (2024)
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Species Strain /
voucher
GenBank accession numbers
References
ITS LSU RPB2 BenA
Rh. indicum CBS
113035
MH862909 MH874483 – – Vu et al. (2019)
Rosellinia aquila MUCL
51703
KY610392 – KY624285 KX271253 Wendt et al. (2018)
R. australiensis CBS
142160
KY979742 KY979797 – – Crous et al. (2017)
R. corticium MUCL
51693
KY610393 – KY624229 KX271254 Wendt et al. (2018)
R. necatrix CBS 349.36 AY909001 KF719204 – – Peláez et al. (2008)
Sarcoxylon compunc-
tum
CBS 359.61 KT281903 KY610462 KY624230 KX271255 Wendt et al. (2018)
Stromatoneurospora
phoenix
BCC 82040 MT703666 MT735133 MT742605 MT700438 Becker et al. (2020)
Thamnomyces
dendroideus
CBS
123578
FN428831 KY610467 KY624232 KY624313 ITS: Stadler et al. (2010), LSU,
RPB2 and BenA: Wendt et al.
(2018)
Wawelia amyloasca
LE
F-334908
OP687954 OP687953 OP700449 OP700450 Present study
W. regia CBS 110.10 MH854595 MH866123 – – Vu et al. (2019)
Xylaria adscendens JDR 865 GU322432 – GQ844818 GQ487709 Hsieh et al. (2010)
X. arbuscula CBS
126415
MH864101 KY610463 KY624287 KX271257 ITS: Vu et al. (2019), LSU, RPB2
and BenA: Wendt et al. (2018)
X. bambusicola JDR 162 GU300088 – GQ844801 GQ478223 Hsieh et al. (2010)
X. bambusicola BCC22739 MT710944 MT735135 – – Becker et al. (2020)
X. brunneovinosa isolate ZE OL656078 OL704809 – – Direct submission
X. carpophila isolate
Z191
MZ621171 MZ703409 – – Direct submission
X. crozonensis HAST 398 GU324748 – GQ848361 GQ502697 Hsieh et al. (2010)
Xylaria cubensis BCC20646 MT703672 MT735142 – – Becker et al. (2020)
X. curta HAST 494 GU322444 – GQ844831 GQ495937 Hsieh et al. (2010)
X. discolor Y.M.J 1280 JQ087405 – JQ087411 JQ087414 Ju et al. (2012)
X. grammica HAST 479 GU300097 – GQ844813 GQ487704 Hsieh et al. (2010)
X. grammica BCC20655 MT703670 MT735138 – – Becker et al. (2020)
X. hypoxylon CBS
122620
KY610407 KY610495 KY624231 KX271279 Wendt et al. (2018)
X. ianthinovelutina HAST 553 GU322441 – GQ844828 GQ495934 Hsieh et al. (2010)
X. ianthinovelutina isolate 246 MZ620706 MZ703179 – – Direct submission
X. multiplex HAST 580 GU300098 – GQ844814 GQ487705 Hsieh et al. (2010)
X. nigripes isolate ZK OL656080 OL704811 – – Direct submission
X. polymorpha MUCL
49884
KY610408 KY610464 KY624288 KX271280 Wendt et al. (2018)
X. telfairii HAST 421 GU324737 – GQ848350 GQ502686 Hsieh et al. (2010)
X. telfairii BCC23019 MT703674 MT735139 – – Becker et al. (2020)

Sydowia 76 (2024) 51
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Fig. 2. Wawelia amyloasca (holotype): A. Type locality of Wawelia amyloasca. B. Stromata in the eld. C. Herbarium specimen
(holotype). D. Part of stroma. E. Immature asci with distinctive J+ apical apparatuses. F. Immature and mature ascospores.
G, H. Conidiogenous cells fragments (SEM). I. Perithecium (SEM). J. Perithecium viewed with transmitted light; strongly mela-
nized neck is well-noticeable. K, L. Anamorphic structures on the surface of stroma: conidiophores with conidia (c) and conidial
secession scars. Scale bars: C = 50 mm; D = 200 µm; E, F = 10 µm; G, H = 1 µm; I = 100 µm; J = 200 µm; K = 5 µm; L = 1 µm.

52 Sydowia 76 (2024)
Bortnikov et al.: Wawelia amyloasca, sp. nov.
was estimated using Tracer 1.7.2 (Rambaut et al.
2018). The result with the minimum ESS (effective
sample size) above 5400 and the PSRF (potential
scale reduction factor) equal to 1 was accepted.
The BenA+ITS+RPB2 dataset included 40 con-
catenated sequences with a total length of 1963
characters with gaps: BenA (1–419, K80+I+G mod-
el), ITS (420–1157, SYM+G model), RPB2 (1158–
1963, GTR+I+G model). The most suitable models
were selected based on the Bayesian Information
Criterion (BIC), calculated in jModelTest v2.1.10
(Darriba et al. 2012). ML analysis with three parti-
tions was run in IQ-TREE v2.1.2 (Minh et al. 2020);
bootstrapping was performed using standard boot-
strap with 200 bootstrap replicates. BI was per-
formed in MrBayes v. 3.2.6; two sets of four chains
were run for 2 million generations, trees were sam-
pled every 100th generation. All chains were con-
verged to <0.005 average standard deviation of split
frequencies, and a burn-in of 25 % was used.
Bayesian posterior probabilities (BPP) were ex-
ported to the ML-tree. Branch support values are
shown only for clades with ML bootstrap higher
than 60 % and BPP above 0.6. Clades with supports
higher than 90 % ML and 0.98 BPP simultaneously
are marked with thicker lines. Trees were visualized
in FigTree v1.4.4 and then prepared and edited
using Inkscape 1.2.
Results
Taxonomy
Wawelia amyloasca
Bortnikov, Bortnikova & An-
tonov, sp. nov. – Figs. 2–4.
MycoBank: MB 841454
E t y m o l o g y. – The name refers to the amyloid apical ap-
paratus, which is a unique feature within the genus Wawelia.
H ol ot yp us . – Russian Federation, Primorsky Krai, Ke-
drovaya Pad Nature Reserve, N 43.158293° E 131.473090°, 97
± 5 m a.s.l. (Fig. 1), oodplain forest with a predominance of
Chosenia arbutifolia (Pall.) A.K. Skvortsov and Phellodendron
amurense Rupr., on dung of the Siberian roe deer (Capreolus
pygargus Pallas, 1771), 8 Jul. 2020, leg. F.M. Bortnikov & N.A.
Bortnikova, (LE F-334908). GenBank ITS: OP687954; LSU:
OP687953; BenA: OP700450; RPB2: OP700449.
Fig. 3. Wawelia amyloasca (holotype): A. Asci in Indian ink. B. Amyloid apical apparatuses in Melzer’s solution. C, D. Ascospores
with non-cellular appendages in Indian ink (C) and water (D). E. Conidia. Scale bars: A = 20 µm, B–E = 5 µm.

Sydowia 76 (2024) 53
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Diagnosis. – Wawelia amyloasca differs from other
species of Wawelia by its stromata up to 120 mm long, asci
with a distinctive amyloid apical apparatus, and ascospores
with polar pad-like appendages.
Description. – Stromata liform, exu-
ous, unbranched, (30)40–120 mm (on average 50–
70 mm) long, 0.1–0.3 mm wide, tapering toward the
apex, cylindrical or sometimes slightly attened,
exible when fresh; initially whitish grey, due to co-
nidiogenesis on the surface, blackish brown at ma-
turity; with yellow hairs in the lower part. Ecto-
stroma melanized, rather soft, never carbonaceous
or brittle, made up of 2–3 layers of textura porrecta
composed of closely packed cells about 3.4–4.4 µm
wide and 25–90 µm long; entostroma hyaline, com-
posed of somewhat sinuous and loosely assembled
Fig. 4. Wawelia amyloasca (holotype): A. Stromata on Siberian roe deer droppings. B. Schematic illustration of an immature
perithecium, developing under a stromal layer. C. Conidiogenous cells. D. Conidia. E. Ascus and paraphyses, with a more detailed
illustration of the apical apparatus (dashed lines indicate ascus walls which are almost invisible in water, but well-contrasted in
Indian ink). F. Ascospores. G. Structure of the perithecial wall from the outer layer to the inner one. Scale bars: A = 20 mm, B =
100 µm, C–G = 20 µm.

54 Sydowia 76 (2024)
Bortnikov et al.: Wawelia amyloasca, sp. nov.
cells similar to the cells of ectostroma. – Pe r i t h e -
c i a evenly distributed or sometimes aggregated in
the upper 3/4 of the stroma, individual or rarely in
twos or threes under the common outer layer, stro-
mal, with fully exposed contours; dark greyish
brown to black, globose, slightly asymmetrical,
(295)310–420 (460) µm long and (230)290–380(435)
µm wide, with opaque black, eminent, conical osti-
oles, glabrous, yellowish brown in transmitted light;
tissue surrounding perithecia composed of four lay-
ers, with the outer two being stromal and the inner
two being perithecial. The outermost layer com-
posed of large, melanized, thick-walled textura
prismatica cells, 10–20 (up to 30) µm wide, which
are presumably the overgrown cells of ectostroma –
textura porrecta (Fig. 4b). The following layer made
up of loose cells, mainly lined along the stroma,
though often twisted and curved (textura intricata),
that previously composed the peripheral layer of
entostroma. The third layer consists of small, very
densely packed cells of textura epidermoidea. The
innermost layer lining the lumen of perithecium
composed of large, at cells of textura epider-
moidea, which are bigger and have a more isodia-
metrical form than those of the layer above. – Asci
cylindrical, unitunicate, containing eight uniseri-
ately arranged ascospores, thin-walled, 100–150 ×
5.0–6.1 µm in total length, with the ascospore-bear-
ing part 70–80 µm long and the stipe up to 60(70)
µm long; apical apparatus bluing in Lugol’s and
Melzer’s solutions with or without KOH pre-treat-
ment, tubular to urn-shaped, 1.6–1.9 × 1.6–2.0 µm.
– A s c o s p o r e s unicellular, broadly ellipsoid in
front view, inequilateral in side view, smooth,
(6.9)7.6–8.5(9.8) µm long, (3.1)3.5–3.9(4.2) µm wide,
8.0 × 3.7 µm on average (n = 150), dark olive-brown
to dark greyish brown, mostly biguttulate, with
conspicuous bright longitudinal germ slit along the
entire length on the ventral side, bearing non-cellu-
lar, pad-like or irregular appendages 3.5–4 µm long
at both ends. – Pa r a p h y se s thin-walled, septate,
1.5–2 times longer than asci, composed of sac-like
cells 4.7–6.7(7.7) µm wide, slightly tapering toward
the tips. – Anamorph Geniculosporium-type, de-
taching from the surface of stroma, varying in
length, 20–40 µm (rarely to 150 µm), straight or ex-
uous, sometimes zigzag-like, almost hyaline, with
slightly darker conidial secession scars and thicker
walls near detachment point (better viewed by
SEM). – C o n i d i a hyaline, oblong ellipsoid,
(5.1)7.0–9.2(10.6) × (1.7)2.1–2.7(3.1) µm, 8.1 × 2.4 µm
on average (n = 100), with rounded or slightly nar-
rowed tips, sometimes with prominent, darker,
truncated base and secession scar.
Phylogeny
Phylogenetic analyses resulted in two phyloge-
netic trees with three congruent major clades, cor-
responding to the families Xylariaceae, Hypoxy-
laceae, and Graphostromataceae (genera of this
family were used as the outgroup). All major clad-
es are characterized by maximum support ML
bootstrap values and BPP. Division of the Xylari-
aceae s.s. clade into two subclades is maximally
supported in the ITS+BenA+RPB2 phylogeny.
However, another situation is observed on the
ITS+LSU tree: two subclades are also present, but
poorly supported. We suppose this can be explained
by the unexpected clustering of Kretzschmaria de-
usta (Hoffm.) P.M.D. Martin with the “coprophil-
ous” clade. Wawelia amyloasca clusters with Hy-
pocopra spp., Podosordaria spp., Poronia spp.,
Stromatoneurospora phoenix (Kunze ex Fr.) S.C.
Jong & E.E. Davis and Sarcoxylon compunctum
(Jungh.) Cooke with 69 % ML, 1.00 BPP (Fig. 5)
and with the same taxa plus Arthroxylaria elegans
Seifert & W. Gams with 98 % ML and 1.00 BPP
(Fig. 6). Hypocopra (Fr.) J. Kickx f., Podosordaria
Ellis & Holw., Poronia Willd, and Arthroxylaria el-
egans are coprophilous fungi and Stromatoneuros-
pora phoenix is a pyrophilous one. However, the
latter has recently been proven to be closely re-
lated to coprophilous xylarialean fungi by a com-
bination of morphological, chemotaxonomic, and
molecular data (Becker et al. 2020). Sarcoxylon
compunctum is a rather rare tropical species, lack-
ing detailed studies on its morphology, ecology, and
phylogeny. Nevertheless, despite typically inhabit-
ing wood, Sarcoxylon Cooke clusters together with
coprophilous and pyrophilous genera of Xylari-
aceae according to the published data and the pre-
sent study (Becker et al. 2020, Konta et al. 2020,
Voglmayr & Beenken 2020, Xie et al. 2020, Pi et al.
2021).
Wawelia amyloasca forms a separate clade with
the maximum support values (100 % ML, 1.00 BPP)
together with W. regia, the type species of the genus
and the only one currently available for intragener-
ic comparison. There are two partial sequences pre-
sent in GenBank, ITS and LSU. Phylogeny obtained
using ITS+BenA+RPB2 might be confusing since
ITS is the only marker present for both species of
Wawelia and branch lengths are practically equal
(Fig. 5). However, the p-distance between ITS se-
quences of W. amyloasca and W. regia is 5 %, and
zooming-in Fig. 5 allows to see their difference.
Also, the ITS+LSU tree (Fig. 6) clearly shows dis-
similarity of branch lengths. These sequences were

Sydowia 76 (2024) 55
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Fig. 5. Three-locus (ITS+BenA+RPB2) phylogenetic tree was constructed by BI analysis. Support values are given only for branch-
es with maximum likelihood bootstrap > 60 % and Bayesian posterior probabilities > 0.60. Statistically signicant branches (ML
> 90 % and BPP > 0.98) are marked with thicker lines, the bar indicates the number of substitutions per site. New taxon is high-
lighted in bold.
probably obtained from the holotype since it was
cultured by B. Namysłowski himself (as indicated
on the CBS website) and can be considered an ex-
holotype.
A sister taxon to the Wawelia-clade is Arthroxy-
laria elegans, which was the second result of the
NCBI BLAST search of W. amyloasca ITS. Only one
marker (ITS) is available for this monotypic genus.

56 Sydowia 76 (2024)
Bortnikov et al.: Wawelia amyloasca, sp. nov.
ITS+LSU phylogeny shows rather high support
(91 % ML, 0.96 BPP) for the bigger clade of Wawe-
lias and A. elegans.
It is worth mentioning that obtaining partial se-
quences of benA and RPB2 for W. amyloasca was
not an easy task: all efforts to sequence the former
locus with reverse Bt2b primer and the latter – with
forward RPB2-5f primer failed, so only one-way
reads of these loci were used.
Fig. 6. Two-locus (ITS+LSU) phylogenetic tree was constructed by ML analysis. Support values are given only for branches with
maximum likelihood bootstrap > 60 % and Bayesian posterior probabilities > 0.60. Statistically signicant branches (ML > 90 %
and BPP > 0.98) are marked with thicker lines, the bar indicates the number of substitutions per site. New taxon is highlighted
in bold.

Sydowia 76 (2024) 57
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Discussion
Wawelia amyloasca displays typical characteris-
tics for the genus Wawelia, such as a) liform, exu-
ous stromata (similar to those of W. argentea, W. mi-
crospora, and W. octospora), forming on the surface
of herbivorous animal droppings; b) perithecia de-
veloping under the outer layer of stroma and later
erupting through it, thus making the stromal layer a
part of the perithecial wall (typical for all the spe-
cies); c) cylindrical, thin-walled asci and dark
smooth ascospores with a longitudinal germ slit; d)
non-palisadic anamorph of Geniculosporium-type,
developing on the surface of stroma.
Besides Wawelia spp., there are other species
with liform stromata in Xylariaceae s.l., such as
Xylaria liformis (Alb. & Schwein.) Fr., X. hip-
potrichoides (Sowerby) Sacc., Thamnomyces spp.
However, they all noticeably differ from Wawelia
amyloasca morphologically. Xylaria liformis is
characterized by liform stromata up to 72 mm
long, but ascospores are much larger (13–16 ×
5–7 µm vs. 8.0 × 3.7 µm of W. amyloasca) and con-
idiophores form a compact palisadic layer along the
stromata (Rogers & Samuels 1986, Hashemi et al.
2014). Stromata of Xylaria hippotrichoides are also
liform, although usually branched. Ascospores of
this species possess similar appendages, which are
much bigger – 14–20 µm long (Sowerby 1799, Den-
nis 1981, Minter & Webster 1983). All species of the
genus Thamnomyces Ehrenb. have been recorded
only from Africa and the Neotropics and are char-
acterized by brittle, strongly melanized stromata
that are often branched and contain KOH-extract-
able pigments. The anamorph of Thamnomyces is
Nodulisporium-type, while Wawelia has a Genicu-
losporium-type anamorph. Molecular data show
that Thamnomyces is placed within the family Hy-
poxylaceae, which has recently been segregated
from Xylariaceae (Stadler et al. 2010, Daranagama
et al. 2018). Despite all of the aforementioned simi-
larities, these species inhabit plant debris and never
animal dung.
A number of genera and particular species be-
longing to Xylariaceae s.l. are coprophilous, but
only some of them are attributed to Xylaria (San
Martin et al. 1998a). Nevertheless, even then it was
questioned, whether they belong to the genus Xy-
laria, and assumptions about their position within
the coprophilous genus Podosordaria were made
(San Martin et al. 1998b). Xylaria pileiformis (Berk.)
Curr. is currently regarded as Poronia pileiformis
(Berk.) Fr. (Ju & Rogers 2001, Hsieh et al. 2010); be-
sides, its stroma has a long stalk and a hemispheri-
cal fertile apex, which makes this species different
from all Wawelia members (San Martin et al. 1998a,
Deepna Latha & Manimohan 2012). Xylaria equina
San Martin & Guervara can be easily distinguished
from W. amyloasca by massive stromata, smaller as-
cospores surrounded by a hyaline mucilaginous
sheath, and a different shape of apical apparatus
(San Martin et al. 1998a). Xylaria coprophila Wehm.
differs by non-liform stromata of smaller size (up
to 1.5 mm long) and larger ascospores (9–10.5 × 3.5–
4.5 µm vs. 7.6–8.5 × 3.5–3.9 µm of W. amyloasca)
(Wehmeyer 1942). Xylaria guepini (Fr.) Ces. has
slightly smaller ascospores (5–7.5 × 2.7–4.4 µm vs.
7.6–8.5 × 3.5–3.9 µm of W. amyloasca). This species
was previously regarded along with coprophilous
Xylarias (San Martin et al. 1998a), but even then, its
preference for soil rich in organic matter was ac-
centuated. Recently, X. guepini has been thoroughly
studied, and its ecology has been nally settled
(Hsieh et al. 2022). Taking all the above-stated mor-
phological and ecological features into account,
placing the new species in the coprophilous genus
Wawelia is justied.
Wawelia amyloasca is characterized by several
unique features, distinguishing it from the species
of the genus described before (Tab. 3). Firstly, this
species was found on dung of the Siberian roe deer
(Capreolus pygargus), while all other species of
Wawelia were described from leporoid dung, al-
though it has to be mentioned that W. effusa has
also been recorded from the European roe deer
droppings (C. capreolus Linnaeus, 1758) besides
rabbit ones (Lundqvist 1992). Secondly, stromata of
W. amyloasca reach up to 120 mm in length, while
stromata of other species do not exceed 30 mm.
However, the fact that stromata of the new species
have developed under natural conditions and not in
a limited space of moist chamber has to be consid-
ered. It might be assumed that the comparison of
two specimens collected in the eld might show less
difference in the length of stromata. Thirdly, peri-
thecia of W. amyloasca do not bear any hairs on the
surface, while the other species are characterized
by more or less hairy perithecia and tend to have
even longer hairs around ostioles. In particular,
W. octospora is characterized by such long hairs
that they might tangle in a ball up to 1000 µm in
diameter (as in Chaetomium Kunze). Fourthly, as-
cospores of W. amyloasca bear appendages, which
are sometimes poorly-visible due to their weak re-
fraction (Figs. 3C–D, 4F). Pad-like appendages are
common for other coprophilous members of Xylari-
aceae, for instance, Poronia australiensis (Læssøe,
C.A. Pearce & K.D. Hyde) J.D. Rogers, Y.M. Ju &

58 Sydowia 76 (2024)
Bortnikov et al.: Wawelia amyloasca, sp. nov.
Tab. 3. Morphological comparison of Wawelia spp.
W. amyloasca W. microspora W. octospora W. argentea W. regia W. effusa
Literary
source Present study Webster et al.
(1999)
Minter &
Webster (1983)
Webster et al.
(1999)
Namysłowski
(1908)
Gumin´ska
(1957) Müller (1959) Doguet
(1961b)
Lundqvist
(1992)
Shape of
stroma
Filiform,
straight or
exuous,
usually
unbranched,
sometimes
slightly
attened
Filiform,
unbranched
near the base,
cylindrical,
then curved
and
branched;
attened or
with
longitudinal
ridges
Filiform,
exuous,
unbranched
or rarely
branched
Filiform,
branched or
unbranched,
wavy and
curved,
cylindrical or
slightly
attened
Half-cylin-
drical,
half-conical,
sometimes
more or less
attened,
sometimes
furcate
Conical, 1-2
times
branched,
sometimes
quite
attened
Varies greatly.
Besides
individual
bulbous
structures,
there are
branched and
quite delicate
ones
Shape and
size vary.
Sometimes
simple,
conical;
sometimes
branched in
every
dimension;
sometimes
spherical;
sometimes
attened
Prostrate on the
substrate
Length of
stroma, mm (30–) 40–120 20–30 2–25 < 30 5–12 < 10 < 5 5–10
Width of
stroma, mm 0.1–0.3 0.1–0.3 0.1–0.3 0.1–0.5 1–2 mm NA NA NA
Size of
perithecia,
µm
310–420 ×
290–380 250 × 230–360 350–500 < 400 × 400
240 (on
average) (up
to 300)
NA 300–400 250–400 650–960 ×
615–720
Asci 8-spored 8-spored 8-spored 4-spored 4-spored 8-spored
Size of asci,
µm
100–150 ×
5.0–6.1 75–88 × 5 70–90 × 6–8
87–93 × 10–12
(holotype),
50–67 × 10–11
(another
specimen)
60–80 × 8 52.9–71.3 × 8
(on average) 55–65 × 7-8 55–80 × 7–8 110–140 × 13–15
Paraphyses
1.5–2 longer
than asci (on
average),
4.7–6.7 (–7.7)
µm wide
Longer than
asci, up to 8
µm wide
Slightly
longer than
asci
Much longer
than asci, up
to 12 µm wide
120 × 6 µm 105.8–142.6 ×
6 µm NA 100–120 ×
<10 µm
Longer than
asci, 5–12 µm
wide
Ascospores,
µm
7.6–8.5 × 3.5–3.9
7.5–8 × 3–4 9–12 × 6–8
15–18 × 9–12
(holotype),
12–15 ×
9.5–12.5
(another
specimen)
8 × 6 (on
average) 9.2–11.5 × 6 9–11 × 5.5–7 8–11 (on
average 9.3) 15–19 × 9–10
Conidia, µm
7.0–9.2 × 2.1–2.7
3–4 × 2 8–12 × 2–4 3–4 × 2–2.5 4–6 × 2 NA NA 5–6 × 3 NA
Country of
record and
dung type
Russia:
Siberian roe
deer
(Capreolus
pygargus)
England:
rabbit
(Oryctola-
gus), hare
(Lepus)
England:
European
rabbit
(Oryctolagus
cuniculus)
England:
rabbit
(Oryctolagus)
Poland:
European
rabbit
(Oryctolagus
cuniculus)
Poland:
European
rabbit
(Oryctolagus
cuniculus)
Gumin´ska’s material
Sweden: hare
(Lepus),
Hungary: roe
deer (Capreolus
capreolus)
Conditions
of record In the eld
After
incubation for
several weeks
under limited
water access
conditions at
about 20–25 °C
Under low
humidity
conditions (90
and 95 % RH)
and at room
temperature
(around 20 °C)
After
incubation for
several weeks
under limited
water access
conditions at
about 20–25 °C
Not stated
explicitly;
presumably
similar to those
of Gumin´ ska
In closed glass
dishes, under
high humidity
conditions at
room
temperature
(around 20 °C)
After incubation
in moist
chambers, on
almost dried-out
substrate

Sydowia 76 (2024) 59
Bortnikov et al.: Wawelia amyloasca, sp. nov.
F. San Martin and Podosordaria elephantis J.D.
Rogers & Y.M. Ju (Hyde et al. 1996, Deepna Latha &
Manimohan 2012), although they have never been
recorded for any Wawelia species. Fifthly, the com-
bination of measurements of ascospores and conid-
ia is unique within the genus.
Finally, the most important and truly distinctive
feature of Wawelia amyloasca is the presence of an
amyloid apical apparatus. Doguet stated that the
apical apparatus of W. regia had neither a ring nor
a nasse; moreover, it was reduced to a circular thick-
ening of the ascus inner layer which formed a rather
well-developed apical dome, bluing slightly in Cot-
ton Blue (Doguet 1961b). All other species also do
not have a noticeably amyloid reaction, and that
was one of the main arguments that E. Müller and
G. Doguet presented to exclude Wawelia from the
family Xylariaceae (Müller 1959, Doguet 1961b).
The presence of the J+ apical apparatus in W. a m -
yloasca clearly suggests positioning of the genus
Wawelia in Xylariaceae.
Furthermore, biology of W. amyloasca is of great
interest. Four out of ve species of Wawelia (except
for W. regia) are apparently xerophilous, since sub-
strates for incubation were collected on the windy
seashore cliffs or sand dunes with scarce vegetation,
and fertile stromata were cultivated under condi-
tions of low humidity or on almost dry substrate
(Tab. 3). On the contrary, W. amyloasca does not
seem to be xerophilous. Since the specimen was
found in the eld, we cannot be certain under which
conditions stromata and perithecia were formed.
Nevertheless, the specimen was encountered not on
the dry south slopes, but in a oodplain forest less
than 50 m from a stream (Fig. 2A), where humidity
is generally higher. In addition, according to the
weather station 4.3 km from the type locality, there
were 210 mm of rainfall during two months before
the record (58.3 mm between May 8 and June 7 and
151.6 mm between June 8 and July 7). All these facts
allow us to consider that W. amyloasca is not a xe-
rophilous species as W. argentea, W. effusa, W. mi-
crospora, and W. octospora.
The reasons for the apparent extreme rarity of
Wawelia species are still unknown due to the lack of
data on their distribution and physiology. It is
known that ascospores of Wawelia can be dormant
and high temperature is required to trigger their
activation. Germination also depends on other fac-
tors, such as substrate composition and the pres-
ence of metabolites from other organisms (Doguet
1960). It can be suggested that under natural condi-
tions ascospores, activated after passing through
the animal digesting system, do not remain in this
state for a long time, and if the environment is not
suitable for the formation of stromata, fungus may
die before reproduction. We believe that new Wawe-
lia discoveries might shed light on true reasons of
its extreme rarity in the world.
Acknowledgements
The work of F.M. Bortnikov and E.A. Antonov
was carried out within the state assignment of the
Ministry of Science and Higher Education of the
Russian Federation (№ 075-15-2021-1396) and the
work of N.A. Bortnikova was supported by the Min-
istry of Science and Higher Education of the Rus-
sian Federation (agreement № 075-15-2021-1056)
and the state task “Biodiversity, ecology, structural
and functional features of fungi and fungus-like
protists” (BIN RAS, 122011900033-4).
We are grateful to the staff of the Land of the
Leopard National Park that helped to organize the
eld work. Aleksandra I. Golubeva (University of
Szczecin, Poland), Ilya A. Viner (University of Hel-
sinki, Finland), and the staff of the Central Univer-
sity Library of Cluj-Napoca (Romania) provided
some rare publications on Wawelia. Moreover, we
thank Alina V. Alexandrova (Lomonosov Moscow
State University, Russia) and Evgeniy S. Popov
(Komarov Botanical Institute RAS, Russia) for the
specimen initial identication to the genus level.
Without their help and advice this work could not
have been fullled. We also thank the Interdepart-
mental Laboratory of Electron Microscopy (ILEM)
at the Faculty of Biology of Moscow State Univer-
sity for their assistance in obtaining SEM micro-
graphs.
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(Manuscript accepted 29 March 2023; Corresponding Editor:
I. Krisai-Greilhuber)