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12Two species of Amanita (Basidiomycota) new to Russia from the South Siberia
N. V. Filippova1, I. A. Gorbunova2
'Yugra State University, Khanty-Mansiysk, Russia 2Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Sciences,
Novosibirsk, Russia Corresponding author. N. V. Filippova, filippova.courlee.nina@gmail.com
Abstract. The paper provides data on records of two new to Russia agaricoid species. Amanita betulae from the Krasnoyarsk Territory (Eastern Sayan) and A. coryli from the Republic of Altai. Species identity was confirmed by molecular phylogeny based on nrlTS sequences. Detailed morphological descriptions, data on ecology and distribution of these species are provided. The morphological features matched the descriptions of type specimens, while the ecology of the specimens of Amanita coryli and A. betulae from South Siberia differed from the European collections.
Keywords: Agaricales, Amanita betulae, Amanita coryli, Vaginatae, taxonomy, mycobiota, Siberia.
Два новых для России вида Amanita из Южной Сибири Н. В. Филиппова1, И. А. Горбунова2
'Югорский государственный университет, Ханты-Мансийск, Россия 2Центральный стбирский ботанический сад СО РАН, Новосибирск, Россия Автор для переписки. Н. В. Филиппова, filippova.courlee.nina@gmail.com
Резюме. В статье представлены морфологические описания, фотографии плодовых тел и микроструктур новых для России видов Amanita betulae из Красноярского края (Западный Саян) и A. coryli из Республики Алтай. Приводятся особенности экологии и данные о местонахождениях. Для подтверждения идентификации видов грибов проведен филогенетический анализ на основе маркера рибосомальной ядерной ДНК (ITS регион). Сравнительный анализ морфологических особенностей показал сходство образцов Amanita betulae и А. coryli, произрастающих на юге Сибири, с описаниями типовых образцов из Европы, тогда как экология сибирских образцов отличалась от европейских.
Ключевые слова: Agaricales, Amanita betulae, Amanita coryli, Vaginatae, таксономия, мико-биота, Сибирь.
The genus Amanita Pers. belongs to the family Amanitaceae and comprises 95% of all its species. It is one of the most recognizable and widespread genera among the agaricoid fungi (Agaricales) worldwide. According to recent studies, Amanita is divided into three subgenera and eleven sections. The subgenus Amanita includes the sections: Amanita Pers., Amarrendiae (Bougher et T. Lebel) Zhu L. et al., Caesareae Singer, and Vaginatae Quel. (Cui et al., 2018). The Vaginatae section, comprising species with stipe lacking a ring, presents the greatest taxonomical complexity.
https://doi.org/10.31111/nsnr/2024.58.1.F37 Received: 26 February 2024 Accepted: 29 March 2024
Published: 1 April 2024
In Russia, about 57 species of Amanita are known from literature review up to present (Bolshakov et al, 2021), with Amanita vaginata s. l. (Bull.) Lam. being the most commonly found species across the country. Some researchers have already shown the complexity of A. vaginata and new species were described for Russian territory (Maly-sheva et al, 2014; Malysheva, Kovalenko, 2015).
The Vaginatae section have been extensively studied by the European scientists (Neville, Poumarat, 2009; Vizzini et al., 2016; Bozok et al., 2023; etc.), as well as researchers from Asian countries (Kim et al., 2013) during recent years. All studies indicate hidden diversity within the group, with the number of newly described species increasing and old taxa revised with new molecular phylogeny methods.
The aim of this article was to expand the knowledge on the distribution, morphological, and ecological features of Amanita betulae and A. coryli based on collections made significantly farther east from the previously known species range.
Materials and Methods
Amanita betulae was collected in July 2010 in the dryad and grass-lichens tundra in Eastern Sayan, Krasnoyarsk Territory. Amanita coryli was gathered in July 2013 in the dryad tundras of the Republic of Altai.
Fresh collections were photographed and described in the field. Specimens were dried using standard methods (Clemengon, 2009). The morphological description of the basidiomata is based on the examination of fresh and dried material. A standard set of reagents (5-10% KOH, 1% Congo Red solution, Melzer's reagent), and a Zeiss AxioStar microscope with a digital camera AxioCam ERc5s. Spore dimensions were calculated for 20 basidiospores and are given as minimum minus maximum of length x minimum minus maximum of width (average values). Q (the ration of length to width) is given as minimum minus maximum of ratio length/width (average values). Microscopic measurements were made using the Zeiss AxioVision 4.8.2 software.
The specimens are stored in the M. G. Popov Herbarium of the Central Siberian Botanical Garden, Siberian Branch of the Russian Academy of Sciences (NSK), and the Yugra State University Biological Collection (YSU-F). Information about the specimens has been entered into the Specify collection information system (http://specify.ugrasu. ru/). Collection data is available for searching and downloading, including specimen photographs and obtained sequences, through GBIF (Gorbunova, Filippova, 2024).
Molecular techniques. The PCR was made using the TransDirect® Plant Tissue PCR Kit without DNA extraction. PCR reactions were performed in 20 |iL of reaction mixtures containing 4 |iL of ScreenMix (Evrogen), 0.2 |iL of each PCR primer, 14 |iL of deionized H2O, and 1.6 |iL of template DNA. For amplification of the ITS region the primers ITS1-F (Gardes, Bruns, 1993) and ITS4 (White et al, 1990) were used. PCR cycle parameters were as follows: initial denaturation for 5 min at 95°C, 30 cycles (denaturation for 20 sec at 95°C, primers annealing for 30 sec at 54°C, extend DNA for 60 sec at 72°C), final extension for 7 min at 72°C. PCR and sequence reaction products were purified using CleanMag DNA (Evrogen) and Dynabeads™ Sequencing Clean-Up
kits. Sequencing was performed with BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (NimaGen) using Applied Biosystems® Sanger Sequencing 3500 Series Genetic Analyzer. Newly generated sequences were deposited in the GenBank.
Phylogenetic analyses. Raw data were edited and assembled using MEGA11 software (Koichiro et al, 2021). Additionally, 18 ITS sequences were selected from GenBank following the previous publication (Bozok et al, 2023). Sequences were aligned using MAFFT version 7 web tool (Kazutaka et al, 2019) using the E-INS-1 option. The resulting alignment was manually processed in MEGA11 and trimmed for maximum overlap of the ITS1 and ITS2 regions.
The best-fit substitution model (HKY+F+G4) was estimated based on the Baye-sian Information Criterion (BIC) using ModelFinder (Kalyaanamoorthy et al, 2017). Maximum likelihood (ML) phylogenetic reconstruction was performed in IQTREE 1.6.12 (Nguyen et al, 2015). The support was tested by 1000 ultrafast bootstraps (BS) (Hoang et al., 2017), as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test (Guindon et al., 2010). BS consensus tree was constructed from 1000 bootstrap trees (Nguyen et al, 2015); the final technical formatting of the resulting tree was made in InkScape software (https://inkscape.org). Similarity percentage of the sequences was calculated in Basic Local Alignment Search Tool (BLAST) (Altschul et al, 1990).
Results
Molecular phylogeny. The final nrITS dataset contained 20 sequences of 563 total sites. Molecular phylogeny revealed that the sequences of our specimens were nested in Amanita betulae and A. coryli clades respectively (Fig. 1).
PP277295 Amanita coryli Russia, Republic of Altai
MT980921 Amanita coryli Norway ON115219 Amanita coryli Turkey " — KM658298 Amanita vaginata Russian Far East — MN490667 Amanita coryli HOLOTYPE Norway MT980919 Amanita coryli Germany
MT229849 Amanita betulae Norway MT229878 Amanita betulae Norway MW013157 Amanita betulae Sweden — MN490668 Amanita betulae holotype PP277327 Amanita betulae Russia, Krasnoyarsk Territory -KU248104 Amanita friabitis
MN490695 Amanita mairei
-KX834245 Amanita lividopaiiescens
-MN490649 Amanita aiseides
-NR154690 Amanita protects
-NR154689 Amanita popuiiphiia
-KX834255 Amanita simulans
- MN490686 Amanita beckeri
KM658295 Amanita submembranacea
Fig. 1. Maximum Likelihood best tree of selected ITS sequences of Amanita section Vaginatae
(following Bozok et al., 2023), obtained by analysis IQTREE 1.6.12. The newly generated sequences are highlighted in bold font, and bootstrap percentage with > 70% values is provided above the branches. Two key species (Amanita betulae, A. coryli) highlighted in gray.
Table 1
Comparison of the obtained sequences of Amanita coryli and A. betulae with sequences of these species, including holotypes, from GenBank
Amanita coryli PP277295.1 (Russia, Republic of Altai) Amanita betulae
Sequence PP277327.1 (Russia, Krasnoyarsk Territory)
Amanita coryli ON115219 (Turkey) 99.8% (1 base del) -
A. coryli MT980919 (Germany) 99.6% (1 base del, 1 base substit) -
A. coryli MT980921.1 (Norway) 100% -
A. vaginata KM658298.1 (Russia, Far East) 99.4% (1 base insert, 1 base subst) -
A. coryli MN490667.1 (Norway, holotype) 99.1% (2 bases insert, 2 bases del, 1 base subst) -
A. betulae MT229878.1 (Norway) - 99.6% (1 base del, 1 base subst)
A. betulae MW013157.1 (Sweden) - 99.6% (1 base insert, 1 base subst)
A. betulae MT229849.1 (Norway) - 99.6% (1 base del, 1 base subst)
A. betulae MN490668.1 (Norway, holotype) - 99.5% (1 base del, 2 bases subst)
Note: The sequences generated in this work are shown in boldface.
Amanita betulae was nested in the clade with other 5 selected sequence of this species, including holotype specimen. The difference with holotype specimen was two bases deletion and 1 base substitution (99.5% percentage similarity) (Table 1). Am-anita coryli was nested in the clade with other 5 selected sequence of this species, including holotype specimen. The difference with holotype specimen was 2 bases insertion, 2 bases deletion, and 2 bases substitution (99.1%), while the percentage similarity was up to 100% with other sequences.
Taxonomy
Amanita betulae Neville et Poumarat, 2009, Fungi Non Delineati, Raro vel Haud Perspecte et Explorate Descripti aut Definite Picti 51-52: 27. (Fig. 2A, B)
Pileus 50-70 mm in diam., from campanulate to plano-convex, with convex, low broad umbo, edge smooth, dry, glabrous, subviscid when wet, beige to dark gray with age in central part, whitish to beige toward margin, without velar remnants; margin deeply striate on 1/4 of the pileus radius. Lamellae free, non-crowded, wide, white or cream, edge concolorous. Stipe 70-100 x 6-9 mm, up to 15 mm broad at base, subcylindric, slightly broadened towards base, without basal bulb, whitish to light
Fig. 2. Macro- and microscopical features of studied specimens. A, B — Amanita betulae (NSK 1013056): A — photo of basidiocarps in situ; B —basidiospores and basidia; C, D — A. coryli (NSK 1013054): C — photo of basidiocarps in situ; D — basidiospores and basidia. Scale bars: 10 |im.
beige, covered with white flaked squamules. Volva membranous, outer surface velvety, both surfaces white, sometimes with a few ochre spots on the outer surface. Context white. Odour indistinguishable. Taste not recorded. Spore print white.
Basidiospores 10-15 x 9-13 (13.2 x 11.2) |m, Q = 1.05-1.17 (1.1), globose, sub-globose, hyaline, inamyloid, smooth, thin-walled. Basidia 45.5-62 x 12.0-15.8 |m, 4-spored, but 2- or 1-spored also present, clavate to elongate clavate, thin-walled; sterigmata 4-6 |m long, without basal clamps. Lamellar edge sterile, composed of numerous broadly utriform, pyriform cells with grainy content. Pileipellis ixocutis,
made of filamentose hyphae up to 7 |im broad with yellow-brown pigment in KOH. Basal volva made up of loosely interwoven, thin-walled, hyaline hyphae, with very rare sphaerocysts. Clamp connections not seen in all tissues.
Specimen examined: Russia, Krasnoyarsk Territory (Eastern Sayan), Ermakovsky District, Ergaki Natural Park, upper reaches of the Bolshaya Baklanikha river, 52°42'96"N, 93°17'94"E, 1787 m a. s. l., grass-lichens and dryad tundras, on soil, 30 VII 2010, Gorbunova, det. Filippova, Gorbunova, NSK 1013056, YSU-F-13932 (GenBank PP277327.1).
Amanita coryli Neville et Poumarat, 2009, Fungi Non Delineati, Raro vel Haud Pers-pecte et Explorate Descripti aut Definite Picti 51-52: 34. (Fig. 2C, D)
Pileus 20-80 mm diam., from campanulate to plano-convex, umbonate, margin striate to 1/5 of the pileus radius, smooth, dry to slightly sticky in wet weather, from pale clay to nut-colored at margin to dark gray-brown at center, with or without numerous velar remnants, which are initially white, later grayish-reddish. Lamellae free, crowded, with rare lamellulae, from white to whitish-cream, turning pink when dry, becoming yellow-brown in dry basidiomata, edge smooth, concolorous. Stipe 70-120 x 5-15 mm, about twice longer than pileus diameter, thin, cylindrical, slightly widened at base, without bulb, whitish to light gray, longitudinally striate, with grayish, small, sparse flakes (universal veil remnants) on the stem surface, sometimes almost smooth, color not changing when touched. Volva membranous, tightly adjacent at the base, free at the edge, sometimes 2- or 3-lobed, up to 25 mm high, whitish on the outside, pale cream, grayish-cream inside, sometimes spotted yellowish-ocher on the outer side. Context white. Odor and taste not recorded. Spore print white.
Basidiospores 10.0-11.0 x 9.0-9.8 (10.2 x 9.7) jm, Q = 1.0-1.1 (1.05), globose, subglobose, hyaline, inamyloid, smooth, thin-walled, with small apiculus. Basidia 31.5-40.1 x 13.0-15.8 jim, clavate, hyaline, thin-walled, 4-spored, sterigmata 4-5 |im long, without basal clamps. Lamellar edge sterile, composed of numerous clavate or ovoid elements. Pileipellis ixocutis, made of filamentous hyphae up to 5 |im broad. Volva composed predominantly of filamentous hyphae 4-5 |im broad and very rare spherical or ovoid elements 15-20 |im broad, with yellowish-brown content in KOH. Universal veil remnants on pileus of similar architecture as volva. Clamp connections not seen in all tissues.
Specimen examined: Russia, Republic of Altai, Ulagan district, near village Aktash, 50°27'20.5"N, 87°40'28.8"E, 1957 m a. s. l., dryad tundras, on soil, solitary or scattered, 28 VII 2013, Gorbunova, det. Gorbunova, Filippova, NSK 1013054, YSU-F-13816 (GenBank PP277295).
Discussion
Amanita betulae was described from France (Neville, Poumarat, 2009). It is characterized as a medium-sized mushroom from the Vaginatae section, growing in coniferous-deciduous forests, with a hazel-colored pileus that becomes grayish with
age, white gills, a white stipe with zebroid bands becoming same color as pileus with age, white volva, and predominantly globose large spores. The morphological features of A. betulae specimen found in the Southeastern Siberia (Western Sayan) match the original description (Table 2). The holotype of A. betulae was found under birch and willow trees. In the Western Sayan, basidiomata were collected in tundra communities at an altitude of 1787 m a. s. l., among Dryas sp., Salix sp., and Vaccinium sp., indicating a broader ecological flexibility and new mycorrhizal symbionts for Amanita betulae.
Table 2
Comparison of micromorphological features and mycorrhizal symbionts of studied collections of Amanita coryli and A. betulae with published descriptions of these species
Species Basidiospores Basidia, |m Mycorrhizal symbiont
L x W, |im Q
Amanita coryli (Russia, Republic of Altai) 10-11 x 9-9.8 (10.2 x 9.7) 1.0-1.1 (1.05) 31.5-40.1 x 13.015.8, 4-spored Dryas oxyodonta Juz., Salix spp.
A. coryli [France (Neville, Poumarat, 2009)] 9.5-13 x 9.5-11 (10.4 x 10.1) 0.94-1.26 (1.29) 35-70 x 12-17, 4-spored Corylus avellana
A. betulae (Russia, Krasnoyarsk Territory) 10-15x9-13 (13.2 x 11.2) 1.05-1.17 (1.1) 45.5-62 x 12-15.8, 4-spored, rare 1- and 2-spored Dryas oxyodonta, Salix spp.
A. betulae [France (Neville, Poumarat, 2009)] 10-13 x 9-14.5 (11.2-11.1) 1.01-1.03 (0.97-1.04) 42-75 x 12-18, 4-, 1- and 2-spored Betula pendula Roth, Salix cap-rea L., Picea abies (L.) H. Karst.
Note: The specimens examined are in boldface.
The previous observations of Amanita betulae were reported from most of the central and northern European countries. There are about 120 occurrences according to GBIF download (GBIF, 2024a), about two thirds of them verified by voucher specimens with sequences or represented by environmental DNA sequences. The new finding of this species in Western Sayan marks considerable extension of our knowledge of its range to the east into the Asian part of Russia (Fig. 3).
The main morphological features of Amanita coryli described by Neville and Pou-marat (2009) was slender basidiomata and an unusual habitat — growing under Co-rylus avellana (L.) H. Karst., which influenced the choice of the specific epithet. The pileus surface was described as smooth, greyish-beige, with prominent umbo. The stipe slender, two to three times longer than the pileus diameter, with a white membranous or slightly ochre-stained volva. Spores range from globose to broadly ellipsoid [Q = 0.94-1.26 (1.29)].
The morphological features of the basidiomata of Amanita coryli discovered in the studied collection from the Republic of Altai are mostly correspond to the protologue
(Table 2). The only differences noted were the absence of sharp umbo and the ratio pileus to stipe is smaller (basidiomata less stout). The ecology of specimens from the Southwest Siberia was completely different: they were found in tundra communities and formed mycorrhizal associations with Dryas sp. and Salix sp. Other researchers from Southeastern Europe have also noted that Amanita coryli were not restricted to the presumed host-trees of the genus Corylus L. and may also occur with some Faga-ceae (Bozok et al, 2023).
The previously known distribution of Amanita coryli was restricted to Western (France) and Southeastern (Bulgaria and Turkey) Europe (Bozok et al., 2023). According to GBIF download (GBIF, 2024b) there is a number of collections documented from Scandinavian countries (Norway, Finland, Denmark) and one from Canada, with most findings validated by voucher specimens and sequences. The new finding of this species in the Republic of Altai marks considerable extension of our knowledge of its range to the east into the Asian part of Russia (Fig. 3).
Fig. 3. Distribution maps of Amanita betulae (A) and A. coryli (B) based on GBIF download, new to Russia records marked by larger red hexagons.
Acknowledgments
The study was carried out within the framework of the State Assignment of the Central Siberian Botanical Garden SB RAS (AAAA-A21-121011290024-5). The article used material from the Bioresourse collection of the Central Siberian Botanical Garden of the Russian Academy of Sciences "Herbarium of Higher Vascular Plants, Lichen and Fungi (NS, NSK)", No. USU 440537. Nina Filippova was supported by a grant for organization of a new laboratory for young researchers at the Yugra State University as part of implementation of the National Project "Science and Universities".
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