New Species of Entoloma Subgenera Cubospora and Leptonia (Agaricales, Basidiomycota) from Central Vietnam

Four new species of Entoloma from Kon Chu Rang Nature Reserve and Ta Dung National Park were discovered during an investigation of the diversity of the mycobiota of Central Vietnam and are described here on the base of the molecular and morphological data. Phylogenetic analysis was based on nrITS1-5.8S-ITS2, nrLSU and tef1α regions. Illustrated descriptions of their macro- and microscopic features and discussion on similar taxa are given. Entoloma cycneum and E. peristerinum belong to the subgenus Cubospora. They are morphologically similar species and are characterized by white or whitish basidiomata with yellowish or beige tinges and with mainly smooth, glabrous, and hygrophanous pileus, longitudinally fibrillose or fibrillose-scaly white stipe, cuboid spores, and more or less cylindrical cheilocystidia, arising from hymenophoral trama. Entoloma peristerinum posseses initially more coloured beige conical pileus, discolouring to white with age and drying. The pileus of E. cycneum is initially white, hemisphaerical to convex, usually with thin pubescence near the margin. The species can be recognized also by the cheilocystidia form: serrulatum-type in E. cycneum vs. porphyrogriseum-type in E. peristerinum. Another two species belong to the subgenus Leptonia. Entoloma tadungense is close to E. percoelestinum from which it differs by smaller spores with pronounced angles, presence of the cheilocystidia, and the lilac discolouration of the stipe. E. dichroides is named after its similarity to E. dichroum, a dark blue coloured species with pronouncedly angled basidiospores. It is distinguished by the basidiospores form—irregularly 5(–6) angled with elongated apiculus, as well as by absence of the cheilocystidia and darker basidiomata with conical pileus. The article also describes the history of the study of the genus Entoloma in Vietnam with a list of 29 species mentioned in the publications for this country.


Introduction
According to estimates for 2018 [1], 21 species of the genus Entoloma (Fr.) P. Kumm. were known from the Central Vietnam, including unpublished data of the authors. In subsequent years, five more new species were described from this territory [2][3][4]. The full history of study of the genus Entoloma in Vietnam with a list of 29 species mentioned in the publications for this country is described in the discussion part. However, the real diversity of the genus is much higher, and many species are still waiting to be described.
As proven by recent molecular genetic studies, the shape of the basidiospores is a key feature in understanding evolution and relatedness in Entolomataceae [5][6][7].
The cuboid and cuboid-like form of spores is remarkable and interesting from a phylogenetic point of view in terms of the multiplicity of its occurrence in the course of evolution. The molecular genetic data [7] supports a clear segregation of clades with cuboid spores from cuboid-like ones (pentagonal, prismatic, pseudocuboid, squamiferum). The

Collecting and Site Description
The material for this study was collected during the expeditions of the Joint Vietnam-Russia Tropical Science and Technology Research Centre (VRTC) to the Central Highlands of Vietnam.
The Kon Chu Rang Nature Reserve is located in the north-eastern part of Gia Lai Province of Vietnam (Son Lang Commune, K'Bang District), between 14.50 • N-14.58 • N and 108.5 • E-108.65 • E. The area of the reserve is 15,446 hectares, of which 99% are primary and intact forests, which is the highest forest coverage in the country. The relief is hilly to mountainous in the northern part, with heights from 800 to 1452 m a. s. l. (Kon Chu Rang Mt). The average annual temperature is about 21 • C (from 28 • C in May to 12 • C in January). The average annual precipitation is about 1900-2000 mm with the peak in September (340 mm). The dry season is from January to April [40,41]. The main forest type in the reserve is middle-mountain evergreen broad-leaved and mixed forest dominated by Fagaceae (Lithocarpus, Quercus, Castanopsis), Lauraceae, Fabaceae, Clusiaceae, Myrtaceae, Ericaceae, Burseraceae, and Magnoliaceae, mixed with gymnosperms (Dacrycarpus imbricatus, Dacrydium elatum), distributed at elevations between 900 and 1500 m in the north-west of the nature reserve. The first data on the mycobiota of Kon Chu Rang Nature Reserve were published only recently, including those on several subgenera of Entoloma, and on the Boletaceae [3,4,42,43].
Ta Dung National Park is located in the Central Highlands of Vietnam within the administrative boundary of Dak Som commune, Dak G'long district, Dak Nong province, 50 km northeast of Gia Nghia commune's exam centre. It extends between 11.79 • N-11.99 • N and 107.89 • E-108.11 • E, occupying a total area of 20,973.7 hectares. The National Park is located on the Dak Nong Plateau and part of the Di Linh Plateau. Dak Nong Plateau is an arched elevation with an average height of 600-1200 m and has many mountain ranges with an average height of 1200-1500 m, with Ta Dung peak being 1982 m high, the lowest elevation being the land. Ta Dung National Park is situated in an area with a tropical highland monsoon climate regime with two distinct seasons, the rainy season lasts from April to October, the dry season-from November to March. The average annual temperature is 22.0 • C. The total average annual rainfall is 2339 mm; precipitation falls mainly from May to October. Ta Dung National Park is characterized by subtropical humid evergreen closed forests and mixed broadleaf and coniferous closed forests [44]. The mycobiota of the National Park previously was not studied.
Specimens were photographed in the field, and their macromorphological characters, such as size, colour, shape, and surface of all parts of the basidiomata as well as odour, were documented before drying. Colour codes refer to Kornerup & Wanscher [45]. GPS coordinates of collection site, habitat, and substrate type were also documented for each collection. Specimens were then dried either in airtight plastic containers with silica gel, or with an electric dryer at a temperature ca. 50 • C, placed on a piece of absorbent paper and packed in plastic Ziploc bags with small amounts of silica gel.

Morphological Study
Microscopic measurements and drawings were made with an AxioScope A1 light microscope equipped with Zeiss AxioCam 1Cc3 digital camera with AxioVisionRel.4.6 software (CarlZeiss, Jena, Saxe-Weimar-Eisenach, Germany). Spores, basidia, and cystidia were observed in squash preparations of small parts of the lamellae in 5% KOH or 1% Congo Red in concentrated NH4OH. The pileipellis was examined from a radial section of the pileus in 5% KOH. Basidiospore dimensions were based on 20 spores; cystidia and basidia dimensions on at least 10 structures per collection. Basidia were measured without sterigmata, and the spores without apiculus. Spore length to width ratios were reported as Q. When studying the specimens and compiling morphological descriptions, we followed the recommendations and terminology of [8]. The dried specimens were deposited in the Mycological Herbarium of the Komarov Botanical Institute RAS (LE) and in the Herbarium of the Joint Vietnam-Russia Tropical Science and Technology Research Centre, Hanoi (VRTC).

DNA Extraction, Amplification, and Sequencing
PCR products were obtained without DNA purification step using the Thermo Scientific Phire Tissue Direct PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) standard protocol. The ribosomal ITS1-5.8S-ITS2 region was amplified with the fungal specific primers ITS1F and ITS4B [46]; http://www.biology.duke.edu/fungi/mycolab/ primers.htm, accessed on 1 March 2017. Sequences of nrLSU-rDNA were generated using primers LR0R and LR5 [47]. Primers EF1-983F and EF1-1567R were used to amplify approximately 500 bp of tef1 [48]. For ITS, PCR was carried out under the following cycling parameters: initial denaturation: 98 • C for 4 min; followed by 35 cycles: 98 • C for 1 min, 52 • C for 1 min, and 72 • C for 1 min, and final extension at 72 • C for 3 min. For nrLSU: initial denaturing at 98 • C for 5 min; then 12 cycles of denaturing at 98 • C for 5 s, annealing at 67 • C for 1 min, extension at 72 • C for 1.5 min; then 35 cycles of denaturing at 98 • C for 5 s, annealing at 56 • C for 1 min, extension at 72 • C for 1.5 min; and a final extension step of 72 • C for 10 min. For tef1-a: initial denaturing at 98 • C for 5 min; then 8 cycles of denaturing at 98 • C for 5 s, annealing at 60 • C for 40 s, extension at 72 • C for 2 min; then 36 cycles of denaturing at 98 • C for 5 s, annealing at 53 • C for 1.5 min, extension at 72 • C for 2 min; and a final extension step of 72 • C for 10 min.
PCR products were visualized using agarose gel electrophoresis and Gel Red staining, and subsequently purified with the Fermentas Genomic DNA Purification Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequencing was performed with an ABI model 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
This work was carried out using equipment of the Core Facility Centre 'Cell and Molecular Technologies in Plant Science' of the Komarov Botanical Institute. Raw data were edited and assembled in MEGA X [49]. Newly generated sequences have been deposited in the GenBank.

Alignment and Phylogenetic Analyses
For this study, 12 nrITS and 9 tef1α, and 10 nrLSU sequences were newly generated. In addition, 38 nrITS and 20 tef1α, and 43 nrLSU sequences, including outgroups, were retrieved from the GenBank database (www.ncbi.nlm.nih.gov/genbank, accessed on 15 March 2023), using the BLASTn application (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 March 2023). The information on all these sequences is presented the Table 1.  Three datasets were analysed: nrITS, tef1α, and nrLSU. DNA sequences were aligned with the MAFFT v.7.110 web tool [56] using the G-INS-i option, and then manually modified where necessary in MEGA X [49]. To determine the phylogenetic positions of the studied collections, both datasets were analysed using Bayesian Analysis (BA). BA was performed using MrBayes 3.2.1 [57], under a GTR model. The analyses were run with two parallel searches: four chains for 5 million generations for ITS and LSU and for 1 million generations for tef1α, starting with a random tree. The trees were sampled every 100 generations. To check for convergence of MCMC analyses and to obtain estimates of the posterior distribution of parameter values, Tracer v1.7.2 was used [58]. The phylogenetic trees were edited in Adobe Illustrator CS4. Posterior probability (PP) values ≥ 0.95 are considered significant.

Phylogenetic Analysis
The full nrITS dataset contained 50 sequences with 1059 characters (gaps included). The/Entocybe clade was selected as outgroup due to its basal position in the Entoloma phylogeny [6]. Besides our specimens, the tree includes 7 more sequences of the subgenus Cubospora retrieved from the GenBank NCBI data base, 16 representatives of the subgenus Leptonia, and 1-3 representatives of the other main subdivisions of the Entoloma s.l.
Since in the GenBank the ITS data are absent for many species of the subgenus Cubospora, a tree was constructed for this subgenus based on the tef1α as well. For the subgenus Leptonia, such information is insufficient for analysis. The full tef1α dataset contained 26 sequences with 535 characters (gaps included). It included Entoloma prunuloides as an outgroup due to its basal position in the Entoloma phylogeny [53,54], and E. luteolamellatum, the type-species of the subgenus Cubospora. In addition to our specimens, 11 more representatives of this subgenus and 1-3 representatives of the other main subgenera of the Entoloma s.l. were included in the analysis.
The full nrLSU dataset contained 52 sequences with 781 characters (gaps included). Clitopilus prunulus and Clitopilopsis hirneola were chosen as an outgroup because of their basal position in the Entolomataceae phylogeny [5]. In addition to new species specimens, 8 representatives of the subgenus Cubospora and 15 sequences of the subgenus Leptonia, as well as 1-3 sequences of the other main subgenera of the Entoloma s.l., were added to the analysis.
The results of the phylogenetic analysis are presented in the Figure 1 (nrITS), Figure 2 (tef1α), and Figure 3 (nrLSU). Entoloma cycneum and E. peristerinum form highly supported branches within the/Cubospora clade in all trees. E. tadungense and E. dichroides clearly nest within the/Leptonia clade in the ITS tree. They are not represented on the tef1α tree. Generally, in the LSU tree, the topology mostly is not or hardly resolved due to small differences between the sequences in the subgenera. At the same time, the subgenus Cubospora forms a highly supported clade in which two new species (E. cycneum and E. peristerinum) nest.
Subgenus Leptonia does not form a separate clade in the LSU tree, but it is represented by several small clades and singletons. However, it is clearly seen here that Entoloma tadungense clusters together with E. percoelestinum and E. coelestinum with high support. Entoloma dichroides distinctly nests in the/dichroum clade, but it differs from the known Leptonia species.
Basidiospores  Notes-Entoloma cycneum and E. peristerinum are morphologically very similar species characterized by white or whitish with yellowish or beige tinged basidiomata with mainly smooth, glabrous, and hygrophanous pileus, longitudinally fibrillose or fibrillosescaly white stipe, cuboid spores and more or less cylindrical cheilocystidia, arising from hymenophoral trama. Entoloma peristerinum posseses initially more coloured beige conical pileus, discolouring to white with age and drying. The pileus of E. cycneum is initially Diagnosis. Entoloma peristerinum is characterized by initially cream, pale beige to beige or greyish-beige becoming white basidiomata, with smooth, glabrous, hygrophanous pileus, longitudinally fibrillose or fibrillose-scaly stipe, cuboid spores and cheilocystidia mostly narrowly clavate or tapering towards the apex-of porphyrogriseum-type.
Basidiospores Notes-Entoloma cycneum and E. peristerinum are morphologically very similar species characterized by white or whitish with yellowish or beige tinged basidiomata with mainly smooth, glabrous, and hygrophanous pileus, longitudinally fibrillose or fibrillose-scaly white stipe, cuboid spores and more or less cylindrical cheilocystidia, arising from hymenophoral trama. Entoloma peristerinum posseses initially more coloured beige conical pileus, discolouring to white with age and drying. The pileus of E. cycneum is initially white, hemisphaerical to convex, usually with thin pubescence near the margin. E. peristerinum differs from E. cycneum also by more differentiated porphyrogriseum-type [8] cheilocystidia, vs. serrulatum-type in E. cycneum. Yellow tinge can present in old or damaged basidiomata of both species.
Phylogenetically both new species are also close to each other but rather distant from other known species in the subgenus Cubospora [7]. Entoloma cervinum (Karstedt & Capelari) Blanco-Dios and E. acutipallidum E. Horak & Cheype from the South America are the closest species according to the phylogenetical analysis. Morphologically they differ from our species by darker, distinctly coloured pileus [28].
Several species with cuboid spores and predominantly white or whitish basidiomata have been described at different times from different regions of the Earth.
The pileus of Entoloma alboumbonatum Hesler from North America is darker coloured, and only umbo is white. This species is characterized by the clavate or capitate cheilocystidia, presence of the pleurocystidia, and absence of the clamp connections [31].
E. albidoquadratum Manim. & Noordel., described from India, is characterized by non hygrophaneous, non-translucently striate pileus, presence of pleurocystidia, and large spores [20]. E. minutoalbum E. Horak is a species of Southern Hemisphere, a common fungus of the subantarctic Nothofagus forests of Tierra del Fuego and New Zealand. Mor-phologically it differs from new species by small basidiomata (with the pileus less than 10 mm diam.), smaller spores, and absence of the cheilocystidia [13]. E. albogracile E. Horak is also species from the Southern Hemisphere-Papua New Guinea. It is characterized by the small size, pileus covered with minute scales and fibrils, and by the absence of clamp-connections [13]. E. laccaroides T.H. Li, E. Horak & Xiao Lan He is recognized by the umbilicate pileus, and numerous conspicuous broadly fusoid to utriform pleurocystidia [12].
Due to the discolouration new species would be compared with cuboid-spored lightyellow species. Entoloma dennisii from Trinidad is a rather robust species with pileus up to 5 cm broad, deep to pale yellow colour, and small spores. E. pallidoflavum differs by the predominance of light-yellow colour in the pileus, yellow content of the oleiferous hyphae, and smaller spores [13]. Diagnosis. Entoloma tadungense is distinguished among the other Leptonia species by the tiny dark blue basidiomata with discolouring to lilac of the stipe, and by the small spores with rather pronounced angles.
Habitat and distribution-In small groups on soil in middle-mountain evergreen mixed forest. Known from Vietnam.  Notes-Entoloma tadungense is a species of the subgenus Leptonia due to presence of clamp connections, absence of brilliant granules, longitudinally fibrillose stipe sur-face and plagiotrichoderm to trichoderm pileipellis. It resembles Entoloma percoelestinum O.V. Morozova, Noordel., Vila & Bulyonk. by the small-sized dark blue mycenoid basidiomata [39]. Microscopically the absence of cheilocystidia and small spores also make them similar. However, E. tadungense can be recognized by smaller spores with pronounced angles, as well as the lilac discolouration of the stipe. Molecular data support their differences (p-distance from the closest species E. percoelestinum (ITS1-5.8S-ITS2 region)-4.9%). The similar non tropical species with dark blue colour and small size of the basidiomata distinguish from the new species in the following: E. coelestinum (Fr.) Hesler-by the smooth stipe and slightly larger spores, E. lepidissimum (Svrček) Noordel. and E. venustum-by the coloured lamellae, distinctly larger spores, and presence of the cheilocystidia, E. chytrophilum possesses large nodulose spores [39]. The American species E. subcoelestinum (Largent) Blanco-Dios is characterized by the initially coloured lamellae, moniliform cells in the pileipellis, and larger indistinctly angular spores [32]. Diagnosis. Entoloma dichroides is a species of subgenus Leptonia, characterized by the dark blue basidiomata with squamulose surface of the pileus and stipe, initially white lamellae, absence of the cheilocystidia, and spores with 5(-6) pronounced angles and elongated apiculus.
Notes-Entoloma dichroides is similar to European E. dichroum and Australian E. panniculus due to dark blue basidiomata with squamulose surface of the pileus and stipe, initially white lamellae, and spores with pronounced angles [26]. New species differs from them by darker basidiomata with conical pileus, basidiospores with elongated apiculus, and absence of the cheilocystidia. The Eastern species E. eugenei Noordel. & O.V. Morozova is also close. It is recognized by the more robust basidiomata and presence of the cheilocystidia [21]. The p-distance (ITS1-5.8S-ITS2 region) of the new species from E. dichroum-6.9%, from E. eugenei-10.4%.

Discussion
The genus Entoloma is the second largest genus in the order Agaricales with over 1000 species worldwide [59]. However, studies devoted to it in Vietnam have not yet been carried out enough.
The first most important contribution to the study of Vietnamese mycobiota was made by N. Patouillard, who described many new species from Northern and Central

Discussion
The genus Entoloma is the second largest genus in the order Agaricales with over 1000 species worldwide [59]. However, studies devoted to it in Vietnam have not yet been carried out enough.
The first most important contribution to the study of Vietnamese mycobiota was made by N. Patouillard, who described many new species from Northern and Central Vietnam based on collections by V. Demange, L. Duport, P. A. Eberhardt, E. Poilane, and others. First records of the genus Entoloma (Rhodophyllus clypeatus, Rh. sericeus) were also published by him [60,61], including originally described from Vietnam Rh. submurinus [61]. Heim and Malençon [62] published information on E. madidum. The first checklist summarizing all the data on the species composition of fungi and slime-molds in Vietnam was published in 1998 and included 829 species, of which only 6 belonged to the genus Entoloma [63]. The same species are presented in the list of plant species of Vietnam [64]. In 2003, Le Ba Dung listed 300 species of macromycetes for the Central Highlands, with only two Entoloma species: E. lividum and E. madidum [65]. Later, Ngô Anh and Nguyễn Thị Kim Cúc recorded two more species of the genus in Thua Thien Hue Province-E. prunuloides in Bach Ma National Park [66] and E. abortivum in Phong Dien Nature Reserve [67]. The data on Vietnamese mycobiota have been summarized in a monography published in 2011-2012, which provides information on the ecology and distribution of more than 900 species of macromycetes [68,69] with only 3 Entoloma species.
More intensive studies of the genus Entoloma in Vietnam began in the frame of the work of the Joint Vietnam-Russia Tropical Science and Technology Research Centre in collaboration with the Komarov Botanical Institute RAS. In 2012, Morozova et al. [11] reported 12 species of the genus Entoloma (Agaricales, Basidiomycota) for the Bidoup-Nui Ba (Lam Dong Province) and Cat Tien (Dong Nai Province) National Parks, of which 11 species were recorded for Vietnam for the first time, and a new species for science, E. myriadophyllum O.V. Morozova, was described.
The above information was based only on morphological data. In connection with the revision of the system of the genus Entoloma [8], these data are not entirely credible and require confirmation. In cases where herbarium material is absent or old and destroyed, this is not possible. However, sequences were later obtained for some of these records. Some of the identifications were revised, and some of these findings were described as species new to science-E. daphnis and E. bidupense [4,42]. Our specimens with cuboid spores were used in the work of Karstedt and colleagues [7], where additional data on tef1α, mtSSU, rpb2, and LSU markers were obtained for them.
As a result, the data on 29 species of Entoloma have been published for Vietnam so far. Only for 14 of them the molecular data have been published and submitted into the GenBank. The resulting list is presented here.
List of the species of the genus Entoloma mentioned in the publications for Vietnam, 1910-2022 (species, supported for the molecular data are marked by the asterisk (*)): Entoloma Four more species are described in the presented article. For E. pallidoflavum, the information on ITS sequence is published here.
In conclusion, it is important to note that the area of tropical forests has been drastically reduced in recent years, including in Vietnam. Along with them, the species confined to them disappear, often without even being assigned a name. Nature reserves and national parks serve to save biologically valuable forest areas. However, even here the species are vulnerable. It sometimes happens that type localities are destroyed during road reconstructions (e.g., E. atricolor, E. arion). It remains to be hoped that they will continue to be found in the adjacent forests. In central Vietnam, the greatest diversity of fungi of the genus Entoloma was observed in middle-mountain evergreen broad-leaved forests. They require special attention, study, and careful treatment.
The study of the fungal diversity of typical tropical forests complements the information on the biota of macromycetes in Vietnam and may be useful in the development of measures for the conservation of these valuable nature areas and the species inhabiting them.  Data Availability Statement: The DNA sequence data obtained from this study have been deposited in GenBank NCBI (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 13 April 2023).