Daldinia vernicosa from the Eastern Forebalkan (Bulgaria)

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PHYTOLOGIA BALCANICA 25 (2): 153 – 155, Sofia, 2019
Daldinia vernicosa from the Eastern Forebalkan (Bulgaria)
Dimitar Y. Stoykov1 & Pablo Alvarado2
1 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences,
2 Gagarin St., Sofia 1113, Bulgaria, e-mail: stoykovdimitar@abv.bg
2 ALVALAB, La Rochela 47, E-39012, Santander, Spain,
e-mail: pablo.alvarado@gmail.com (corresponding author)
Received: May 05, 2019 ▷ Accepted: August 19, 2019
Abstract. Daldinia vernicosa was found in the Eastern Forebalkan on the bark of Carpinus orientalis, which is the first
report on this host. Identification is based on molecular analysis and morphological data. A description and
illustrations of the specimens found are provided.
Key words: Bulgarian mycota, Carpinus, Xylariaceae
Introduction
The genus Daldinia Ces. & De Not. is known to be
taxonomically difficult and, presently, seventeen
species of it are accepted in Europe (Ju & al. 1997;
Rogers & al. 1999; Johannesson & al. 2000; Stadler &
al. 2001, 2004; Wollweber & Stadler 2001; Stadler &
al. 2014). Of these, only D. concentrica (Bolton : Fr.)
Ces. & De Not., the type species, seems to have been
recorded from the countries of the Balkan Peninsu-
la, although different larger ascomycetes, including
some interesting and uncommon xylarialean fungi,
have been mentioned in a number of publications
from Bulgaria and the adjacent regions (e.g. Læssøe
1997; Zervakis & al. 1999; Karadelev & al. 2007; Sesli
& Denchev 2009; Stoykov 2011). During field work,
the authors have repeatedly collected a small species
of Daldinia, growing on dead branches of Carpinus
orientalis Mill., which is subject to morphological
and genetic studies here.
Material and methods
Air-dried specimens of the fungal stromata have
been conserved in the Mycological Collection of the
Institute of Biodiversity and Ecosystem Research
of the Bulgarian Academy of Sciences (SOMF). The
micro scopic features were studied in water, KOH and
Melzer’s reagent. Measurements of the microstructures
were always taken in water. The size of ascospores
is presented below in the following format: (min-)
mean ± 1 s (-max), n; where ‘n’ – denotes the number
of measured spores, and 's' represents the standard
deviation. Scanning electronic microphoto graphs
(SEM) were taken with a JEOL JSM-6390 device at
10 kV. Spores for SEM observations were obtained
from pieces of stromata, mounted on metal stubs with
double-sided adhesive tape and sputter-coated with
gold.
For genetic analysis, total DNA was extracted from
dry specimens employing a modified protocol based
on Murray & Thompson (1980). PCR reactions
(Mullis & Faloona 1987) included 35 cycles with
annealing temperature of 54 °C. Primers ITS1F
and ITS4 (White & al. 1990; Gardes & Bruns 1993)
were employed to amplify ITS rDNA region. PCR
products were checked in 1 % agarose gels, and
positive reactions were sequenced with one or both
PCR primers. Chromatograms were searched for
putative reading errors, and these were corrected.

154 Stoykov, D.Y. & Alvarado, P. • Daldinia vernicosa on Carpinus orientalis (Bulgaria)
Results and discussion
Daldinia vernicosa Ces. & De Not., Comment.
Soc. Crittog. Ital. 1: 198, 1863 (Figs 1-3)
Syn. D. fissa Lloyd, Mycol. Writ. 7: 1313, 1922.
Stromata turbinate, stipitate, solitary or in groups,
up to 1.2 cm in diameter and up to 1.5 cm high, vina-
ceous-brown, subsequently blackening, varnished
when old; KOH-extractable pigments livid; tissue
between perithecia greyish, pithy to woody; tissue
below the perithecial layer composed of alternat-
ing darker and lighter zones; darker zones blackish-
grey to black, pithy to woody; lighter zones whit-
ish, initially gelatinous, subsequently disintegrating
and becoming loculate. Perithecia tubulate, 530–
1050 μm high and up to 215–400 μm in diameter;
ostioles slightly papillate. Asci not seen in the Bul-
garian specimens. Ascospores blackish to dark-
brown, ellipsoid, equilateral or slightly inequilateral,
with broadly rounded ends, (11.5-) 12.2 ± 0.6 (-13.5)
× (5.5-) 6.6 ± 0.2 (-7) μm, n=100, length/width ratio
(1.6-) 1.9 ± 0.1 (-2.1), with straight germ slit spore-
length on the more convex side, perispore indehis-
cent in 10 % KOH, epispore smooth in SEM.
Specimens examined. Bulgaria: Lovech dis-
tr., Golyama Zhelyazna village (Troyan Municipali-
ty), 43°0'1.5"N, 24°29'42.2"E, alt. ca 445 m, on dead
branches of Carpinus orientalis, 15.08.2008, D. Stoyk-
ov & B. Assyov (SOMF 28163, GenBank MN535762);
idem., 13.05.2011, D. Stoykov (SOMF 28164).
Notes. So far, D. vernicosa was known only from a
single locality at the Southern Black Sea Coast, Burgas
distr., Sozopol town, on Celtis cf. australis L. (Stadler
& al. 2014: 87).
The newly-collected Bulgarian specimens match
the morphological concept of Daldinia fissa as cir-
cumscribed in Ju & al. (1997) and Wollweber &
Stadler (2001), but this species is considered an aber-
rant form of D. vernicosa on the basis of genetic evi-
dence (Stadler & al. 2014). The specimens analyzed in
the present study have an ITS rDNA 100 % identical
with GenBank NR152501, obtained from culture CBS
119316, which comes from specimen KR 0026316,
collected on the same site of the epitype (specimen KR
0026318, culture BCRC 34048, GenBank EF026146).
Some differences between NR152501 and EF026146
can be observed, but they are accepted as part of in-
traspecific variability. The size of ascospores generally
agrees with that reported by Ju & al. (1997), Wollwe-
ber and Stadler (2001) and Chlebicki (2008), although
we have recorded a somewhat lower value for the min-
imal spore width. This difference can be interpreted
as intraspecific variability until further samples from
new localities are studied, or insignificant (derived
from measurements of only two collections from sin-
gle locality). The spores of the studied specimens have
a smooth epispore under SEM (Fig. 3), thus closely re-
sembling the illustrations in Stadler & al. (2001) and
Stadler & al. (2014).
Because of its small-sized stromata and livid to
purplish extractable pigments, D. vernicosa seems
to resemble to some extent the species D. caldario-
rum Henn. and D. singularis Y.-M. Ju, Vassilyeva &
J.D. Rogers, which are present in Europe. The latter
has been reported to date only from the Russian Far
Fig. 1. Daldinia vernicosa, stromata in situ. Fig. 2. D. vernicosa, cross-section of stroma, ex situ.

155Phytol. Balcan. 25(2) • Sofia • 2019
East, where it occurs in association with Carpinus
(Stadler & al. 2014). The two species can be distin-
guished by the presence of lighter zones on the stro-
mata, and by their distinctly smaller ascospores, not
exceeding 11 μm in length and 5.5 μm in width. In
external morphology, Daldinia vernicosa can resem-
ble to some extent D. childiae J.D. Rogers & Y.-M. Ju
(Stadler & al. 2014: 74), mainly by the short stipitate
to turbinate stromata. However, it differs clearly in the
constitution of lighter zones on the stromata, greater
spore length (up to 17 μm), and transversal striations
of spores observed with SEM.
Rhoads (1918) considered D. vernicosa as a pyro-
xylo philous fungus. The size of ascospores in the col-
lections studied by Rhoads (1918: 281) usually var-
ied within 10-14.5 × 7-7.5 µm, and thus conform well
with the current concept of Stadler & al. (2014). These
authors stated that D. vernicosa was mostly found on
‘freshly felled wood or on damaged, still living trees’.
Wollweber & Stadler (2001), Stadler & al. (2001: 176)
and Stadler & al. (2014: 84) consider D. vernicosa a
pyro philic species, often found on burnt or fire-dam-
aged woody hosts. However, both Bulgarian collec-
tions studied in the present work were attached to
dead, but not burnt large branches of Carpinus orien-
talis, which seems to be also a new substrate of this
species.
Acknowledgements. This study was carried out within the
framework of the project ‘Taxonomy, conservation and sustaina-
ble use of fungi’. Dr. Boris Assyov (IBER, BAS, Sofia) is acknowl-
edged for the macrophotograph of stromata in situ.
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Fig. 3. D. vernicosa, ascospores under SEM.