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48Paxillus cuprinus (Agaricomycota, Boletales, Paxillaceae), first record of basidiomes registered in Russia
E. F. Malysheva, V. F. Malysheva
Komarov Botanical Institute of the Russian Academy of Sciences, St. Petersburg, Russia Corresponding author: E. F. Malysheva, e_malysheva@binran.ru
Abstract. Specimens of Paxillus cuprinus, a species recently described from P. involutus complex and characterized by less massive, dully coloured basidiomes with more slender stipes, as well as longer basidiospores, were collected by authors during an expedition to the Southern Urals (Republic of Bashkortostan). The studied collection represents the first record of the species in Russia based on basidiomes. The identity of the studied collection was confirmed by molecular data (nrITS and tefl sequence analyses) and morphology. A full description, illustration of specimens, and results of phylogenetic analyses are provided.
Keywords: Paxillus cuprinus, Boletales, taxonomy, Southern Urals.
Paxillus cuprinus (Agaricomycota, Boletales, Paxillaceae), первая находка плодовых тел в России
Е. Ф. Малышева, В. Ф. Малышева
Ботанический институт им. В. Л. Комарова РАН, Санкт-Петербург, Россия Автор для переписки: Е. Ф. Малышева, e_malysheva@binran.ru
Резюме. Образцы Paxillus cuprinus, вида, недавно описанного из комплекса P. involutus и характеризующегося менее массивными, тускло окрашенными базидиомами с более тонкими ножками, а также более длинными спорами, были собраны авторами во время экспедиции на Южный Урал (Республика Башкортостан). Исследованная коллекция представляет собой первую находку базидиом вида в России. Идентичность коллекции была подтверждена молекулярными данными (анализ последовательностей nrITS и tefl) и морфологией. Приводится полное описание и иллюстрации образцов, а также результаты филогенетического анализа.
Ключевые слова: Paxillus cuprinus, Boletales, таксономия, Южный Урал.
The genus Paxillus (Paxillaceae, Boletales), with P. involutus (Batsch) Fr. as a type species, is widely distributed in the Northern Hemisphere. Approximately 38 valid species of Paxillus are known (Kirk et al, 2019), and five of them (P. involutus, P. ob-scurisporus C. Hahn, P. rubicundulus P. D. Orton, P. validus C. Hahn and P. vernalis Watling) have been reported from Russia (Bolshakov et al., 2021, Svetasheva, 2021).
Currently, most representatives of the genus Paxillus are considered ectomycorrhi-zal fungi associated with various woody host plant species, including deciduous and coniferous trees.
The genus has always been the object of close attention and interest not only by taxonomists, but also by geneticists and biochemists. The genomes of P. involutus and
https://doi.org/1031111/nsnr/2022.56.2323 323
Paxillus rubicundulus strains have been fully sequenced (Martin et al., 2011; Martin, Bonito, 2012), and the secondary metabolites of P. involutus that can be harmful to human health have also been closely studied (Winkelmann et al., 1986; Anthowiak et al., 2003).
It is recognized that P. involutus in the traditional sense is a complex of species, including at least four taxa in Europe (Fries, 1985; Hahn, Agerer, 1999; Jarosch, Bre-sinsky, 1999; Bresinsky, 2006). A thorough molecular study of the P. involutus complex in Europe, recently conducted by Jargeat et al. (2014) confirmed and extended the results previously obtained by Hedh et al. (2008) and Vellinga et al. (2012). As a result, European collections sequenced from this complex have been divided into four phylogenetic lineages, each with sufficient morphological and cultural features to be recognized as distinct species (P. ammoniavirescens Contu et Dessi, P. obscurispo-rus, P. involutus, and newly described P. cuprinus).
During a taxonomic survey of agaricoid fungi collected in the Republic of Bashkortostan, we found specimens that corresponded to the description of P. cuprinus. Therefore, the collection has become the object of our close attention and study. Herein we describe and illustrate the morphological characteristics of the studied material and provide supporting evidence based on nrlTS and tef1 sequences analyses.
Here we introduce the studied collection, representing it as the first record in Russia. At the same time, we must point out that this species has previously appeared in ecological studies devoted to the analysis of the soil microbiome (Vasar et al., 2022). This very recent work was devoted to the study of soil samples using analysis of metagenome. The nrlTS nucleotide sequences of this species were detected in three soil samples from the territory of Russia. However, in such works, conclusions about the presence of a particular species in the plant community are based on molecular data only. The scarce information of such studies does not allow us to answer the question of what stage of the life cycle the taxon is (propagule, part of the hyphae, etc.) and whether it is integrated into a given community and has a functional load in it. Following Zamora et al. (2018), we believe that not only taxonomic studies, but also the study of the biodiversity of an area, whenever possible, should be supported by voucher specimens with the complete set of morphological characters available for study. Therefore, our study reliably confirms the detection of the species on the territory of Russia on the basis of the studied basidiomes.
Materials and Methods
Study site
The studied specimens were collected in the Bashkiriya National Park (53°03'N, 56°32'E). The park is located in the Southern Ural Mountains, Republic of Bashkortostan, and has an area of 820 km2. The rivers Belaya and Nugush form its natural borders. The average elevation is 500-700 m, the maximum point being 758 m a. s. l. The mountainous part of the park is mainly folded-block terrain, with steep slopes and flat peaks. The linearly elongated low mountains of the western slope of the Southern Urals mostly
represent a karst zone of the fold Urals. The climate is humid continental. This climate is characterized by large swings in temperature, both diurnally and seasonally, with mild summers and cold, snowy winters. The average annual air temperature increases from +0.1 °C to +4.5 °C as you move from the eastern to the western part of the territory. The average annual precipitation in the eastern part of the park is 582 mm, in the western part — 404 mm. A large territory of the Bashkiriya National Park is mostly covered with broad-leaved deciduous forests. Pine and pine-birch forests as well as shrub steppes are found on some slopes. In river valleys, there are alder and bird-cherry forests and wet meadows (Natsional'nyi..., 2022). The studied specimens of P. cuprinus were collected in vicinity of the Nugush Reservoir, near the Fourth Kutuk Cave, in the forest dominated by Betula pendula Roth, on soil beneath a dense thicket of ferns.
Morphological examination
Macroscopic descriptions were based on observations of fresh basidiomes from the original collection and photos taken at the site. The specimens were examined and drawn using standard microscopic techniques (Clemengon, 2009). Microscopic observations were made from dried material mounted in 5% KOH and Congo Red using an Axio Scope.Al light microscope (CarlZeiss, Germany). For statistical evaluation of ba-sidiospore dimensions, at least 30 basidiospores were measured from each basidiome; (n = 60, s = 2) indicates measurements based on 60 basidiospores from 2 basidiomes in 1 collection. Spore dimensions (without hilar appendage) are given following the form (a)b-c(d), with b-c containing at least 90% of all values and the extremes (a, d) enclosed in parentheses; xm, the arithmetic mean of spore length x width; Q, quotient of spore length x width for all measured basidiospores; Qm, the mean of Q-values. The measurements of the other morphological structures are based on at least 20 elements.
The studied collection was deposited in the Mycological Herbarium of the Koma-rov Botanical Institute (LE).
Molecular techniques
For DNA extraction, small fragments of dried basidiomes were used. The procedure of DNA extraction completely corresponded to the manufacturer's protocol of the PureLink™ Genomic DNA Mini Kit (Invitrogen, USA). The following primers were used for amplification and sequencing: ITS1F-ITS4 (White et al, 1990; Gardes, Bruns, 1993) for the internal transcribed spacer (ITS1-5.8S-ITS2); EF1-983F and EF1-1567R for part of the translation elongation factor 1-alpha (tefl) (Rehner et al., 2005). PCR products were purified applying the GeneJET Gel Extraction Kit (Thermo Scientific, Thermo Fisher Scientific Inc., MA, USA). Sequencing was performed with an ABI model 3500 Genetic Analyzer (Applied Biosystems, CA, USA). Raw data were edited and assembled in MEGA X (Kumar et al., 2018).
All microscopic and molecular studies of specimens were carried out at the Center for collective use of scientific equipment «Cellular and molecular technology of studying plants and fungi» (Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg).
Phylogenetic analyses
For this study one new nrITS and one tef1 sequences were generated. In addition, 17 nrlTS and 17 tef1 sequences, including an outgroup, were retrieved from the GenBank database (www.ncbi.nlm.nih.gov/genbank/), using the BLAST application and taxonomic considerations (Jargeat et al, 2014). The taxonomic identities of these sequences with GenBank accession numbers are given in Fig.1. Sequence alignment was performed using the Muscle tool incorporated into MEGA X separately for each genetic marker and then combined into a single dataset.
Phylogenetic reconstructions were performed with Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Before the analyses, the best-fit substitution model was estimated based on the Akaike Information Criterion (AIC) using FindModel web server (http://www.hiv.lanl.gov/content/sequence/findmodel/find-model.html). For each dataset (nrITS and tef1) the model turned out to be the same, thereby HKY+G model was chosen for concatenated dataset (nrITS+tef1) and used for further analyses. Maximum likelihood analysis was run on RAxML servers, v.1.0.0 (https://raxml-ng.vital-it.ch/#/) with one hundred rapid bootstrap replicates. BI analysis was performed with MrBayes 3.2.5 software (Ronquist et al., 2012), for two independent runs, each with 10 million generations under described model and four chains with sampling every 100 generations. To check for convergence of MCMC analyses and to get estimates of the posterior distribution of parameter values, Tracer v1.7.1 was used (Rambaut et al., 2018). We accepted the result where the ESS (Effective Sample Size) was above 200 and the PSRF (Potential Scale Reduction Factor) was close to 1.
Newly generated sequences were deposited in GenBank with corresponding accession numbers (Fig. 1).
Results
Molecular phylogeny
The final general nrITS + tef1 dataset contained 18 nrITS and 18 tef1 sequences and consisted of 1034 characters (with gaps). The overall topologies of the ML and BI trees were substantially the same.
To confirm the identity and to reveal the position of our collection among the taxa of P. involutus species complex, the nrITS and tef1 sequences of all species involved into key work of Jargeat et al. (2014) were selected for phylogenetic analysis with P. rubicundulus as an outgroup. In concordance with this paper, our nrITS+tef1 phylogeny supports four clades in the P. involutus complex (Fig. 1). These highly supported phylogenetic lineages correspond to four morphological species: P. ammoniavirescens, P. obscurisporus, P. involutus, and P. cuprinus. In the tree, P. cuprinus is sister to P. involutus, while P. ammoniavirescens is more similar to P. obscurisporus. The sequences of the Russia collection are nested in P. cuprinus clade.
100/1
95/0.99 97/1
100/1
74/0.89
P. ammoniavirescens KF261420/KF261552
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P. ammoniavirescens KF261395/KF261533 P. ammoniavirescens KF261380/KF261518 P. obscurosporus KF261386/KF261523 P. obscurosporus KF261367/KF261505 L/P obscurosporus DQ647827/DQ457629 г P involutus KF261358/KF261498 P. involutus KF261411/KF261544 P. involutus KF261363/KF261501 P. involutus KF261368/KF261507
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P. cuprinus ОР554270ЮР562164
P. cuprinus KF261389/KF261527 P. cuprinus KF261412/KF261546 P. cuprinus KF261419/KF261551 P. cuprinus KF261379/KF261517 (Holotype) P. cuprinus KF261357/KF261497 P. cuprinus KF261384/KF261521 P. rubicundulus KF261421 /KF261553
0.02
Fig. 1. Phylogenetic tree of Paxillus involutus and allies derived from the nrITS+te/1 dataset using Maximum Likelihood analysis. The bootstrap support/Bayesian PPs are shown above branches. For all taxa the GenBank accession numbers (nrITS/te/1) are presented. The specimen studied for this article is highlighted in bold. Scale bar indicates the mean number of nucleotide substitutions
per site.
Taxonomy
Paxillus cuprinus Jargeat, Gryta, J.-P. Chaumeton et Vizzini, in Jargeat, Chaumeton, Navaud, Vizzini et Gryta, Fungal Biology 118(1): 26 (2014). (Figs. 2, 3)
Basidiomes medium-sized to large, fleshy. Pileus 70-100 mm diam, with deeply depressed centre, without umbo; surface slightly felted, sticky in wet conditions, with radially arranged darker fibrils; margin undulating or lobate, not or slightly in-rolled, smooth; surface colour yellow-brown, clay-buff or reddish brown with an olivaceous shade at centre and some whitish areas. Lamellae narrow, crowded,
strongly decurrent, some anastomosing and forked towards the stipe; rusty brown or red-brown in mature basidiomes, with concolorous, slightly undulated edges. Stipe 45-55 x 0.7-15 mm, almost cylindrical or slightly tapering downwards; dry and felted, olive-brown or reddish brown; basal mycelium with bundles of pale brown rhizo-morphs.
Flesh rather thick, yellowish in the stipe and in the pileus. Smell distinct, fruity. Taste mild. The chemical reaction with ammonia solution was not observed.
Basidiospores (7.6)8.3-10.6(11.3) x 5.0-6.6(7.1) mm, meanxm = 9.4 x 6.0 mm, Q = (1.3)1.5-1.7(1.8), Qm = 1.6 (n = 60, s = 2), broadly ellipsoid infrontal view and slightly amygdaliform in side view, smooth, slightly thick-walled, brownish in KOH. Basidia (26.5)30.7-40.0 x 7.5-10.0 mm, clavate, 4-spored, rarely 2-spored, with wide sterigmata, usually with heterogeneous content, thin-walled. Lamella edge heteroge-nous. Cheilocystidia (55.0)60.0-74.0(84.0) x 7.5-14.0 mm, scattered, not forming sterile layer, most narrowly to broadly fusiform, or lageniform with subacute apex, usually shortly pedunculated, more often thin-walled, some with thickened yellowish walls, hyaline or yellowish in KOH. Pleurocystidia sparse, similar to cheilocystidia in shape and size. Pileipellis a cutis, slightly gelatinized, made of 5.5-8.0 mm wide, thin-walled hyphae, with ochraceous-brown intracellular pigment. Stipitipellis a cutis, composed of clamped, thin-walled, yellowish hyphae 5.0-10.0 mm wide. Cau-locystidia (25.0)28.0-43.5 x 6.3-8.8 mm, abundant, often in bunches, almost cylindrical, narrowly lageniform or broadly fusiform, usually thin-walled, hyaline or with light yellowish content. Clamp connections present and numerous in all tissues.
Studied collection: Russia, Republic of Bashkortostan, Bashkiriya National Park, near the Fourth Kutuk Cave, 53°00'26"N, 56°45'10"E, birch forest, on soil beneath a dense thicket of ferns, 25 VII 2022, V. Malysheva, LE 313731 (GenBank nrITS - 0P554270, tefl - OP562164).
Discussion
Although species of the genus Paxillus are ubiquitous in a wide variety of habitats, and despite of avalanche of molecular data accumulation, including molecular-ecological studies, Vellinga et al. (2012) in their introduction call the genus "a nightmare for species recognition", also emphasizing that species recognition in the P. involutus complex is still very difficult.
Certainly, without sequencing, some collections cannot be reliably identified, but species within the P. involutus complex can still be separated with some certainty based on habitat and macrofeatures. According to the descriptions provided by Jargeat et al. (2014) and Henrici and Kibby (2014), P. cuprinus is characterized by rather slender (not robust) basidiomes with a gracile stipe, growing separately, but often in groups. The species was named because a rather striking change in the pileus color with age, especially in dry weather, from rather pale olive when young to reddish brown or copper brown; also, the pale yellow flesh darkens quickly when cut or bruised. Basidiospores are longer than those of the other three species of the P. involutus complex (P. ammoniavirescens, P. obscurisporus and P. involutus); they
Fig. 2. Basidiomes of Paxillus cuprinus (LE 313731). Scale bar: 1 cm.
typically ovoid or amygdaliform with a frequent slight depression on their dorsal side near the apex and therefore showed a slight apical constriction, which is very rare in P. involutus. On the basis of all morphological characteristics, the collection studied by us rather confidently corresponds to the description of P. cuprinus (Jargeat et al., 2014).
Paxillus cuprinus is noted to occur in parks and gardens but also wood edges, river banks etc., and belongs to heliophilous (sunloving) species in strong contrast to its closest relative P. involutus which is almost entirely a woodland species, described as sciophilous (shade-loving). P. cuprinus is possibly confined to Betulaceae, recorded with Betula, Corylus, Alnus, Carpinus, while other three species of complex have a wider known host range, even including conifers. Our specimen was also found in a light birch forest, with sparsely trees, but under a dense canopy of ferns.
According to all available finds, this species is probably widespread in Europe, North America, and Iran (Henrici, Kibby, 2014; Jargeat et al., 2014). However, Paxillus cuprinus, being recently described, needs further recording to determine its frequency and distribution in the territory of Russia.
Fig. 3. Microscopic features of Paxillus cuprinus (LE 313731). A — basidia and basidiospores; B — cheilocystidia; C — pleurocystidia; D — caulocystidia.
Scale bar: 10 |im.
Acknowledgments
We express our gratitude to the director of the park, V. M. Kuznetsov, and V. G. Petrova, as well as to all the staff of the National Park for their help in conducting the expedition. The research was supported by the project 122011900033-4 of the Komarov Botanical Institute of the Russian Academy of Sciences.
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