Morphological characteristics and molecular phylogeny of Disciseda hyalothrix (Gasteromycetes) from Altai Mountains, a new record to Northern Asia

Abstract

Vlasenko VA, Rebriev YuA, Asbaganov SV, Dejidmaa T, Vlasenko AV 2020-Morphological and molecular characteristics of Disciseda hyalothrix (Gasteromycetes) from Altai Mountains. Abstract Morphological characteristics and molecular phylogeny of the gasteroid fungus, Disciseda hyalothrix, and data on its localities, habitat and distribution are provided. This rare species of D. hyalothrix was found in the protection zone of the Tigirek Reserve, Altai Territory, Russia (Altai Mountains, Western Siberia, Northern Asia). A fruiting body of D. hyalothrix was found in "Dragunskaya" cave. Detailed descriptions, illustrations of basidiocarp and basidiospores are given. The main diagnostic features of Disciseda species are the size of the spore and the nature of the ornamentation. Spores of D. hyalothrix are globose, brown, grossly verrucose, (6) 6.5-7.5 μm in diameter, without ornamentations, without pedicels or rarely with colorless pedicels 4-6 (up to 10) μm. In the SEM, ornamentation in the form of powerful pyramidal tufts consisting of thin spines pressed together to 1-1.3 μm in height. We present a scanning electron micrograph study and morphological characteristic comparison of D. hyalothrix and other Disciseda species found in Eurasia, which currently includes 5 taxa. We first generated new sequences for rDNA (ITS1-5.8S-ITS2 region and partly for LSU) of D. hyalothrix (NSK 1014099). The new record of D. hyalothrix broadens information on the ecology of the rare gasteromycete species, which was growing in the conditions of stony steppe communities. New sequence data for studied loci of rDNA will help clarify the phylogenetic relationships of species from the genera Disciseda and Bovista.

Full text

Submitted 11 August 2019, Accepted 16 December 2019, Published 31 January 2020
Corresponding Author: Vyacheslav A. Vlasenko – e-mail – vlasenkomyces@mail.ru 34
Morphological characteristics and molecular phylogeny of Disciseda
hyalothrix (Gasteromycetes) from Altai Mountains, a new record to
Northern Asia
Vlasenko VA1*, Rebriev YuA2, Asbaganov SV1, Dejidmaa T3, Vlasenko AV 1
1 Central Siberian Botanical Garden, Siberian branch, Russian Academy of Sciences, Zolotodolinskaya, 101,
Novosibirsk, 630090, Russia
2 South Science Center RAS, Chehova, 41, Rostov-on-Don, 344006, Russia
3 Plant Protection Research Institute of Mongolia, Khoroo 11, Ulaanbaatar, 17024, Mongolia
Vlasenko VA, Rebriev YuA, Asbaganov SV, Dejidmaa T, Vlasenko AV 2020 – Morphological and
molecular characteristics of Disciseda hyalothrix (Gasteromycetes) from Altai Mountains. Current
Research in Environmental & Applied Mycology (Journal of Fungal Biology) 10(1), 34–41,
Doi 10.5943/cream/10/1/4
Abstract
Morphological characteristics and molecular phylogeny of the gasteroid fungus, Disciseda
hyalothrix, and data on its localities, habitat and distribution are provided. This rare species of D.
hyalothrix was found in the protection zone of the Tigirek Reserve, Altai Territory, Russia (Altai
Mountains, Western Siberia, Northern Asia). A fruiting body of D. hyalothrix was found in
“Dragunskaya” cave. Detailed descriptions, illustrations of basidiocarp and basidiospores are given.
The main diagnostic features of Disciseda species are the size of the spore and the nature of the
ornamentation. Spores of D. hyalothrix are globose, brown, grossly verrucose, (6) 6.5–7.5 μm in
diameter, without ornamentations, without pedicels or rarely with colorless pedicels 4–6 (up to 10)
μm. In the SEM, ornamentation in the form of powerful pyramidal tufts consisting of thin spines
pressed together to 1–1.3 μm in height. We present a scanning electron micrograph study and
morphological characteristic comparison of D. hyalothrix and other Disciseda species found in
Eurasia, which currently includes 5 taxa. We first generated new sequences for rDNA (ITS1-5.8S-
ITS2 region and partly for LSU) of D. hyalothrix (NSK 1014099). The new record of D. hyalothrix
broadens information on the ecology of the rare gasteromycete species, which was growing in the
conditions of stony steppe communities. New sequence data for studied loci of rDNA will help
clarify the phylogenetic relationships of species from the genera Disciseda and Bovista.
Key words – gasteroid fungi – molecular data – morphology – puffballs – new sequences
Introduction
The genus Disciseda was described by B. Chernyaev in 1845. It was later described by other
authors as Catastoma Morgan and Bovistina Long et Stouffer, in 1982 and 1941 respectively
(references are needed). In nature, this fungus is found in arid habitats such as deserts, sandy and
stony steppes, and also in dry low-grass meadows, less often in drying light coniferous forests
(Rebriev 2009). When totally mature, the exoperidium incrusted by the substratum breaks up in the
lower part. The basidiocarp detaches from mycelium and turns over. The upper part of the
exoperidium that is firmly attached at the apex, remains a cup-shaped structure. A small basal
Current Research in Environmental & Applied Mycology (Journal of Fungal Biology)
10(1): 34–41 (2020) ISSN 2229-2225
www.creamjournal.org Article
Doi 10.5943/cream/10/1/4

35
rupture appears, through which spores can escape. The cup-like residue of the exoperidium
encrusted with the substrate, in which the fruiting bodies is located, is also observed in some
representatives of Bovista genus, but the original development of fruiting bodies with inversion is
observed only in the species of Disciseda.
Index Fungotum gives 46 names of Disciseda records (Index Fungorum 2019), a lot of them
seem to be synonyms. Four species are found in Russia. Species of the genus Disciseda in Russia
are most often found in arid areas, in steppes and deserts. The most widespread and common
species among the genus are D. bovista (Klotzsch) Henn. and D. candida (Schwein.) Lloyd. The
remaining species are of rare occurrence. For Disciseda cervina (Berk.) G. Cunn. about 20 records
are known. Rare species of D. hyalothrix (Cooke et Massee) Hollós, in Russia, was previously
recorded only for European part of the country (Rostov Region).
New findings and herbarium materials are important, as at present, due to the lack of modern
monographic treatments and abundance of synonyms, it is difficult to determine the number of
species in the genus Disciseda. The literature on the genus morphology is controversial. For
example, presence of a parenchymal layer of exoperidia is indicated not only in D. candida
(Moravec 1954), but also in D. bovista (Kers 1975, Jeppson 1997, Rebriev 2009). Some
morphological differences in D. candida and D. cervina were revealed with SEM (Kers 1975,
Rebriev 2009). SEM is currently the main method used to reliably differentiate species of the genus
Disciseda, as well as to reveal the variability range for diagnostically significant morphological
characteristics.
Phylogenetic studies of species of the genus Disciseda are poorly represented (Larsson &
Jeppson 2008, Bates et al. 2009), data on the nucleotide sequences for ITS region was available for
only two species before starts our study, and they were D. candida and D. bovista. New sequence
data for studied loci of rDNA generated by us, can be used as barcodes to identify D. hyalothrix.
Materials & Methods
Field studies
A rare species of D. hyalothrix was found in the Altai Territory, in the protection zone of the
Tigirek Reserve, in the vicinity of the Tigirek village (Fig. 1). This is the first finding of this
species in the Northern Asia.
Fig. 1 – Map of the Altai Territory with the locality details of Disciseda hyalothrix (color drawing
Google Earth Pro V 7.3 2019).
The Tigirek State Nature Reserve was established in 1999 to preserve the biodiversity of the
natural complexes in the middle mountains of Western Altai. The reserve is located in the south-

36
western part of the Altai Territory, i.e., in the western part of the Altai Mountains on the left bank
of the Upper Charysh basin. The total area of the reserve is 40,693 hectares, the conservation zone
of the reserve in total covers 26,257 hectares (Davydov et al. 2011).
A basidiocarp of D. hyalothrix was found in “Dragunskaya” cave (or “Pasechnaya”). It was
described by P.S. Pallas during an expedition in 1771. The cave is located in the Krasnoschekovsky
district of the Altai Territory, at Altai northern foothills, 3 km west of the Tigirek village, on the
eastern edge of “Dragunsky” ravine, which starts in the vicinity of “Shlyapnaya” Mountain. It is a
small erosive limestone cave shaped as a horizontal gallery, with a flat bottom, vertical walls, and a
lancet arch. The cave has a small slope in the direction from the end towards the entrance. The
bottom of the cave is soil, on which 1 old fruiting bodies of D. hyalothrix was found.
We do not have reliable data to show the fruiting bodies were growing on soil in a cave. In
addition to soil and seeping rainwater, the gallery of the cave is partially illuminated by sunlight.
The aggregate factors were conducive for the appearance and growth of the fungus. Possibility that
the fruiting bodies of D. hyalothrix could be deposited into the cave by wind, thawing or rainwater
from the surrounding area of the cave cannot be excluded.
The terrain in the fungus area is typical for low mountains and is located in the mountain-
forest-steppe belt, with a combination of grass, mixed grass petrophytic and meadow steppes
(Fig. 2). Shrub communities are well developed. They are widely represented in the steppes, and
form a separate belt with thickets of Sibiraea laevigata, Caragana arborescens, Lonicera tatarica,
Crataegus sanguinea, Sambucus sibirica along the pristine slopes and logs. Forest vegetation is
represented by larch, birch-larch or pine forests on the tops of the northern slopes of the hills.
Fig. 2 – Habitats of Disciseda hyalothrix. A Dragunsky ravine. B Rocky (petrophytic) steppe on a
hillside above the cave. Photos by V. Vlasenko.
Since D. hyalothrix is a species growing in arid habitats, its growth is possible in habitats
entering the petrophytic steppe plant communities on the hilltops above the cave, which are formed
by Sedum hybridum, Thymus petraeus, Aster alpinus, Dianthus versicolor, Bupleurum multinerve,
Caragana pygmaea, Spiraea trilobata, Juniperus sabina. Habitats of D. hyalothrix in the Tigirek
Reserve will be preserved, which in turn contributes to conservation of the species in nature, like
many other species (Davydov et al. 2018).
Morphological examination
Initial morphological examination was performed using a Carl Zeiss Stemi DV4
stereomicroscope, a Carl Zeiss Axiolab E re light microscope and a Carl Zeiss Axioskop-40 light
microscope. The examination of microstructures under the light microscope was made after boiling
the preparation for a short time in polyvinyl lactophenol cotton blue. Specimens were prepared for

37
scanning electron microscopy using traditional SEM techniques, summarized as follows. The
specimens were viewed and photographed using a Carl Zeiss EVO MA 10 scanning electron
microscope. For photographing fruiting bodies in transmitted light, we used a Panasonic-Lumix
DFC-XZ7 camera. Voucher specimens of the species are stored in the MG Popopv Herbarium
(NSK), Novosibirsk, Russia. The species description is based on the literature data (Jeppson 2018,
Moreno et al. 2003, 2007, Silva & Baseia 2014) and original observations by Yu. Rebriev.
DNA extraction and sequencing
Specimens of D. hyalothrix (NSK 1014099) was used for molecular analyses. A piece of
fungal fruiting body (50 μg) was homogenized in 300 μl lysis buffer and extract the DNA with
NucleoSpin Plant II kit was used. The ITS1-5.8S-ITS2 region of the rDNA were amplified by PCR
with the primers ITS1F and ITS4B. For PCR, HS Taq DNA Polymerase (Evrogen, Moscow) was
used. PCR reactions were performed in an C1000 Thermal Cycler (Bio-Rad, USA). PCR results
were checked at Gel Doc XR+ Imager (Bio-Rad, USA). DAN amplicons sequencing performed in
SB RAS Genomics Core Facilities (Novosibirsk, Russia).
Phylogenetic analyses
Additional 6 ITS sequences of other Disciseda and Bovista species based on BLAST results
and 3 ITS sequences of other species were retrieved from GenBank
(http://www.ncbi.nlm.nih.gov/Genbank/). Mycenastrum corium was used as an outgroup (Larsson
& Jeppson 2008). We first generated a new sequence for ITS1-5.8S-ITS2 region and partly for
large subunit rDNA (28S rDNA) for D. hyalothrix (GenBank No: MN151399). The final dataset
consisted of 10 ITS sequences. An overview of all taxa and on sequences used for tree
reconstruction, shows the species names, herbarium vouchers/strain and Genbank accession
numbers given in Table 1.
Table 1 Sequences used in the phylogenetic analyses.
Species
Herbarium voucher/strain
Genbank accession number
Bovista aestivalis
MJ1122
DQ112620
Bovista pusilliformis
CBS 397.74
MH860864
Bovista promontorii
MJ7070
DQ112621
Bovista polymorpha
DA-29
AJ237613
Disciseda bovista
MJ5078
DQ112627
Disciseda candida
STB304
EU833654
Disciseda hyalothrix
NSK 1014099
MN151399
Lycoperdon perlatum
MJ4684
DQ112630
Mycenastrum corium
KM162954
GQ981488
Vascellum pratense
MJ4864
DQ112554
Sequences were align using ClustalW methods (Thompson et al. 1994). The ITS sequences
were aligned in MEGA 7 (Kumar et al. 2016). The evolutionary history was inferred using the
Neighbor-Joining method (Saitou & Nei 1987). The optimal tree with the sum of branch length =
0,016 is shown. The percentage of replicate trees in which the associated taxa clustered together in
the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein 1985). The tree is
drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to
infer the phylogenetic tree. The evolutionary distances were computed using the Maximum
Composite Likelihood method (Tamura et al. 2004) and are in the units of the number of base
substitutions per site. The differences in the composition bias among sequences were considered in
evolutionary comparisons (Tamura & Kumar 2002). The analysis involved 10 nucleotide

38
sequences. All positions containing gaps and missing data were eliminated. There were a total of
630 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.
Results
Disciseda hyalothrix (Cooke et Massee) Hollós, 1902, Növényt. Közlem. 1: 107. Fig. 3
Description – Basidiocarps subglobose to depressed-globose, 1–2.8 cm in diameter.
Exoperidium thin, whitish to grayish-brown, fragile, often completely falling away or remaining in
the form of a shell-encrusted “case” encrusted with remnants of the substrate with the
endoperidium lying in it. Endoperidium is coriaceous, hard, gray to dark brown, smooth, with a
fibrillose pore of irregular shape. Gleba is brown when maturing. Threads of capillitium rarely
branched, smooth, curved, light brown, easily breakable into short fragments, 2.3–2.7 μm. Spores
globose, grossly verrucose, brown, (6) 6.5–7.5 μm in diameter without ornamentation, without
pedicels or rarely with a colorless pedicels 4–6 (up to 10) μm. In the electron microscope,
ornamentation in the form of powerful pyramidal tufts to 1–1.3 μm consisting of thin spines
pressed together.
Habitat – steppes and deserts.
Known distribution – Europe, Asia, North and South America, Australia. In Russia, currently
known only from Rostov Region (Rebriev 2009).
Fig. 3 – Disciseda hyalothrix. A Fruiting body in the collection site. B Gleba (color in RL).
C Spores (SEM). D Spores (in TL, polyvinyl lactophenol). E Spore (SEM). Scale bars: A = 1 cm.
B = 1 mm. C = 2 µm. D = 10 µm. E = 2 µm. Photos by: A, B, D – V. Vlasenko. C, E – A.
Vlasenko.

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Material examined – Russia, Altay Territory, Krasnoschekovsky district, Tigirek Reserve, 3
km west of the Tigirek village, on the eastern edge of “Dragunsky” ravine, “Dragunskaya” cave, on
soil in a cave, 51° 09' 195" N, 82° 58' 798" E, 05 July 2018, V.A. Vlasenko, NSK 1014099.
Discussion
The main diagnostic feature of Disciseda species are the size of the spores and the nature of
the ornamentation (Table 2).
Table 2 Morphological comparison of D. hyalothrix and other species found in Eurasia.
D. cervina
D. candida
D. bovista
D. verrucosa
D. hyalothrix
Spore size (with
ornamentation), µm
4–5.5
3.5–5.5
5.5–7 (8)
8–12
8.5–13
Spore ornamentation
smooth to
asperulate
finely
verrucose
verrucose
strongly
verrucose
strongly
verrucose*
*Spores often with a pedicel 4–6 (up to 15) μm.
The studied specimen is characterized by relatively small spores, 6.5–7.5 μm. Another
significant difference from the typical description is absence of pedicels in most spores, if any,
small sizes of 4–6 μm. This was noted in specimens from Mexico, which can be explained by the
degree of maturity of the fruiting bodies, its preservation and/or growth conditions (Moreno et al.
2007).
Small smooth or verrucose spores up to 5.5 μm in diameter are found in two species in Russia
– D. cervina and D. candida. In the remaining species, spores are large, more than 5.5 μm in
diameter, with well-developed ornamentation. Several species of this group are found in Eurasia.
Disciseda bovista is the most common form with well-ornamented spores. It is distinguished by a
smaller size of spores. The spores are large verrucose 5.5–7 (8) μm in diameter with ornamentation.
Pedicels up to 3.5 μm are present or absent. Ornamentation of the spores of D. verrucosa G. Cunn.
(=D. arida Velen.) – finger-shaped spines often up to 2 μm high. The spores are 8–12 μm in
diameter, taking into account the ornamentation.
Another species described from Europe, D. nigra, has verrucose spores 7.5–8.5 μm in
diameter with ornamentation about 1.8 μm (Dörfeld & Nowak 2002). The species description is
invalid, the type is not specified. According to the obtained molecular data, the species should be
classified as Geastrum genus (http://www.indexfungorum.org).
Disciseda ochrochalcea Kreisel was described from Nepal, the Himalayas Mountains, where
it was collected in a dry Alpine meadow at an altitude of 4,700–4,800 m (Kreisel 1976), and is
nowhere to be found. It is characterized by grossly verrucose olive-brown to almost black spores of
8.2–10.5 μm in diameter, taking into account the ornamentation. Pedicels up to 4.7 μm. In the
description of the species only the drawings are presented and there is no detailed information on
the ornamentation of the spores. Of all the listed species, D. hyalothrix is clearly distinguished by
the ornamentation of its spores in the form of pinned thin spines, assembled into powerful
pyramidal tufts.
The molecular phylogenetic analyses placed the specimens of Disciseda hyalothrix (NSK
1014099) from Northern Asia close to Disciseda and Bovista genera. Genetic distance 0.024 with
bootstrap to support D. hyalothrix branch 70% (Fig. 4). Phylogenetic studies of species of
Disciseda genus are poorly represented (Larsson & Jeppson 2008, Bates et al. 2009), data on the
nucleotide sequences of ITS1-5.8S-ITS2 region rDNA are available for Disciseda bovista and D.
candida. New sequence data for studied loci of rDNA received by us, can be used as barcodes to
identify the D. hyalothrix. Classification of Disciseda genus will be updated with the advent of new
sequences for members of the genus.

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Fig. 4 – Neighbor Joining (NJ) tree showing phylogenetic relationships between the D. hyalothrix
(NSK 1014099) from Northern Asia and other related species of Ggasteromycetes, based on the
ITS rDNA sequences. Mycenastrum corium (GQ981488) was used as the outgroup taxon. Values
on the branches represent the percentage of 1000 bootstrap replicates and bootstrap values over
75% are shown in the tree.
Acknowledgements
The work of V.A. Vlasenko, A.V. Vlasenko, T. Dejidmaa and S.V. Asbaganov was funded
by RFBR and MCESSM according to the research project 19-54-44002 Mong_T. The work of Yu.
Rebriev was carried out within frame of the government assignment for South Science Center RAS
(project AAAA-A18-118011990300-9). Herbarium specimens from MG Popov Herbarium (NSK),
Novosibirsk, were used.
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