First records of rare epiphytic species Physarum lakhanpalii and Ph. lenticulare for Russia

Epiphytic species are still the least studied group of myxomycetes, because of their life cycle, substrate specialization and method of search using “moist chambers”. The Russian’s first records of the Physarum lakhanpalii Nann.-Bremek. et Y. Yamam. and Ph. lenticulare Nann.-Bremek. et Y. Yamam. with data on its localities, habitat, and distribution are provided. Ph. lakhanpalii is characterized by light yellow sporocarps, 0.5 mm wide and up to 10 mm long, two-layer peridium and spores, 11–12.5 μm, violaceous brown, adhering in clusters of mostly 4–6. Ph. lenticulare is characterized by gregarious sporocarps, total height 1 mm, sporotheca discoid to lenticular, black stalk, narrowed at the base, sometimes the stalks merge into 2–3 pieces, spores (9)10–11(14) μm, brown, warted and sometimes with clusters of more prominent warts. The revised description of Ph. lakhanpalii and Ph. lenticulare is given in the paper. We present micrographs in SEM for studied species, for Ph. lenticulare they presented for the first time. Ph. lenticulare is one of the rarest species of myxomycetes in the world, previously it was found only in Belgium, France, Netherlands and Japan. Most of the Physarum species in Asian part of Russia are recorded in Novosibirsk Region and currently the genus includes 29 species.


Introduction
Myxomycetes are phagotrophic eukaryotes that feed on various microorganisms, and occur in terrestrial environments as haploid, uninucleated myxamoebae and swarm cells or as diploid multinucleated plasmodium that produce funguslike fruiting bodies (Martin, Alexopoulos, 1969).
The most characteristic stage of myxomycete is the assimilative plasmodium. The plasmodium is derived from a zygote after fusion of two haploid cells carrying different mating types, or from apogamic diploid cell directly. Plasmodium engulf bacteria, yeast and other organic matter, which surrounded and digested in their food vacuoles. Under adverse conditions the plasmodium can form a resistant sclerotium which can continue growth when conditions improve. Sporulation with the production of spores triggered by the mature plasmodium undergoing starvation in the presence of light (Rollins, Stephenson, 2011;Clark, Haskins, 2015).
These organisms constitute a well-defined and homogenous group of approximately 900 species (Lado, 2005(Lado, -2019. Many species of myxomycetes have worldwide distribution, but some restricted only to the tropics or only temperate regions. Traditionally, it has been advanced that the majority of myxomycetes had cosmopolitan distribution (Rojas et al., 2014;Novozhilov et al., 2017). This idea is consistent with the "ubiquity hypothesis" (Finlay, 2002;Fenchel, Finlay, 2004). More recent work has revealed that some species of myxomycetes exhibit clear biogeographical patterns (Stephenson et al., 2008), thus supporting the "moderate endemicity hypothesis" (Foissner, 2006), which suggests that eukaryotic microorganisms do exhibit distributional patterns (Rollins, Stephenson, 2011).
Studies of soil microbiota showed the importance of this group of organisms to the ecosystems where they occur, serving as regulators of microbial populations of bacteria, yeasts and filamentous fungi and in nutrient cycling and mineralization (Keller, Everhart, 2010;Stephenson, Feest, 2012).
Physarum was described by Persoon in 1794, with Ph. aureum Pers. as a type species. The genus known as the largest genus in Myxomycetes as well comprises about 146 species worldwide (Lado, 2005-2019), 62 species recorded in Russia, 40 species recorded in Asian part of Russia, 35 species recorded in Western Siberia, 33 species recorded in south of Western Siberia. Most of the Physarum species in the Western Siberia are found in the Novosibirsk Region -27 species, in the Altai Territory -20 species, in the Republic of Altai -17 species (Novozhilov et al., 2009(Novozhilov et al., , 2010Vlasenko, Novozhilov, 2011;Vlasenko, 2013;Vlasenko et al., 2013aVlasenko et al., , b, 2016. The genus Physarum is characterized by limy fruiting bodies, non-crystalline lime granules, darkcolored spore mass and calcareous nodes-bearing capillitium (physaroid type).
Identification of Physarum species is carried out by a complex of morphological features -shape and colour of fruiting bodies, nodes of capillitium. Scanning electron microscopy, with which you can search the ornamentation of the surface of the spores is important for the diagnosis.
Species of the genus commonly inhabit bark folds of living trees, bark and wood of dead trees, dung, grass litter and other vegetable of substrates.
Samples with large sporocarps can be collected in the field. To search species with a small sporocarps, a commonly we used moist-chamber method, which is a highly efficient technique to search a hidden diversity of epiphytic myxomycetes.
By using the moist-chamber culture method reported by Gilbert and Martin (1933), many new or rare corticolous myxomycetes were discovered.
The paper also presents morphological, ecological and geographical data on Ph. lakhanpalii and Ph. lenticulare. The two species are described and illustrated below.

Myxomycetes isolation
Ph. lakhanpalii was found in natural conditions on the bark of Populus tremula during a survey of urban plantings in Novosibirsk.
Ph. lenticulare was obtained using the moistchamber method. Sporocarps of Ph. lenticulare were found in Petri dishes while examining a bark collection obtained from living Salix pentandra, growing in the arboretum in the Central Siberian Botanical Garden of the Siberian Branch of the Russian academy of sciences, (Novosibirsk). Bark pieces cut from a living tree at a height of 5 meters were placed into Petri dishes on filter paper in one layer. Traditional methods call for the substrate to the soaked 24 h, with incubation at room temperature after the excess water is poured off (Härkönen, 1977). No excess water was poured off in our experiment; the lid Vlasenko A. V. et al. First records of Physarum lakhanpalii and Ph. lenticulare for Russia of the wet chamber was opened for 4-5 h, awaiting removal of the excess water. We did not drain the water, so that there would be no removal of cysts of myxomycetes from the substrates; we used natural ventilation to evaporate the excess water. The cultures were exposed to light in the natural lighting for at least 8 hours a day. The first sporocarps of Ph.
lenticulare were obtained on the 36 day of cultivation.

Morphological examination
Microscopic examination was carried out in fresh preparations in 3 % KOH and distilled wa-  ter. For microscopy, sporocarps were preserved as permanent slides in polyvinyl lactophenol. A Carl Zeiss Stemi DV4 stereomicroscope and a Carl Zeiss Axiolab E re light microscope were used for morphological analysis. A detailed study of the morphological features was performed on a Carl Zeiss EVO MA 10 scanning electron microscope. Specimens for scanning electron microscopy were prepared as follows. Pieces of substratum bearing sporocarps were mounted on aluminum stubs using conductive adhesive tape. A gold coating was applied to the stubs in a vacuum evaporator. The specimens with metal coating were viewed and photographed using a scanning electron microscope. The nomenclature of myxomycetes follows C. Lado (2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019).
Description: Predominantly plasmodiocarpous, sessile on a narrow base, simple or sparingly branched or, often, subglobose-confluent, rough, white at the base and yellow at the top, 0.5 mm wide and up to 10 mm long. Peridium consists of two closely adherent layers, with the inner membranous, colourless layer and the outer containing lime agglomerations forming numerous scaly, yellow, irregularly confluent protruberances. Dehiscence irregular, rare dehiscence with apical longitudinal crack. Capillitium a dense, colourless net, with numerous, rounded or spiky, irregularly elongate, in part confluent (badhamioid) yellow lime nodes. Spore-mass dark brown. Spores violaceous brown, adhering in clusters of mostly 4-6 (in clusters of sometimes more than 6), subglobose, 11-12.5 µm diam., densely, rather evenly, minutely warted. The warts of the closest spores merge with each other and thereby keep the spores in clusters.
Ecology and habitat: Epiphyte. On living trees. Earlier in other regions of the world identified on Ravenala madagascarensis, Liquidambar formosana, Hedera helix, Thespesia sp., Platanus sp. On mosses growing on living trees. This species found in the Novosibirsk Region on mosses growing on a living tree. Such microhabitats are characterized by high moisture, as mosses strongly retain water.
Comments: Ph. lakhanpalii differs from related species of this genus two-layer peridium and spores, adhering in clusters (Tab. 1). Morphological difference between the type specimen described of Nannenga-Bremekamp and Yamamoto (1987) and our examined specimen is absent.
Comments: Ph. lenticulare differs from related species of this genus in the structure of capillitium threads, the shape of capillitium nodes, shape, structure and colour of the stalk (Tab. 2). Ph. lenticulare is characterized by black stalk narrowed at the base, sometimes the stalks merge into 2-3 pieces. The stalk of Ph. album subulate, rusty brown at the base and white at the top. The stalk of Ph. javanicum long, slender, narrowing above, greyish-white, darker below. The stalk of Ph. pezizoideum slender, striate, reddish brown. Morphological difference between the type specimen described of Nannenga-Bremekamp et Yamamoto (1987) and our examined specimen is the size of a spores. The spores of the samples studied by us are slightly larger in diameter, up to 14 microns, in the type specimen -up to 12 microns.