As a result of molecular phylogenetic analyses and morphological studies, two new species of the genus Pseudosperma, namely P. fulvidiscum and P. singulare were discovered in Wuzhishan Nature Reserve of Hainan Province of China. The two new species are distinct from other known Pseudosperma species in the phylogenetic polygram inferred from a combined nuclear ribosomal internal transcribed spacer region (ITS), the nuclear ribosomal large subunit (nrLSU), and the nuclear second-largest subunit of RNA polymerase II (RPB2) sequence data. Muscarine contents of the two new species were detected using ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach. In addition, a new combination was proposed based on phylogenetic evidences.
Field sampling and morphological studiesFresh materials were collected in the Wuzhishan and Yinggeling substations of Hainan Tropical Rainforest National Park and Fujian Province in China. Firstly, basidiomata were photographed using a digital camera in the eld. Secondly, fresh specimens were recorded and described macroscopically later. Then, the specimens were dried overnight at 45 ℃ with an electronic drier, and the dried specimens were packed in sealed plastic bags with silica gel (Yu et al. 2020;Deng et al. 2021a). Color codes follow Kornerup and Wanscher (1978). Microscopical characteristics are observed using an optical microscope (Olympus CX23, Olympus Industrial limited company, Guangzhou, China) in the laboratory. Mushroom tissues from the pileus, the lamellae, and the stipes were cut into thin sections by freehand with the aid of a stereoscope (AV100-240V). Dried materials were rehydrated in KOH (5%) and stained with Congo Red (1%) when necessary. Microscopic structures, including basidiospores, basidia, cheilocystidia, hymenophoral trama, pileipellis, and stipipellis were examined and measured. Methods of measurements follow Fan and Bau (2013). At least 100 basidiospores for each species were randomly measured from different specimens. The basidiospore measurements are expressed with the [n/m/p], which indicates "n" basidiospores measured from "m" basidiomata of "p" specimens. In addition, the basidiospore size is given with the (a) b-e-c (d) formula, where "b-c" contains a minimum of 90% of the measured values of the spores, "e" represents the average values of the spores, and "a" and "d" represents the minimum and maximum values of the spores (Ge et al. 2021). Q is the ratio length/width of individual spores (Q value); Q m is the average Q of all basidiospores ± sample standard deviation (Na et al. 2022;Jean et al. 2022).After the study, examined specimens were deposited in the Herbarium of Changbai Mountain Natural Reserve (ANTU) with FCAS numbers and the Fungal Herbarium of Hainan Medical University (FHMU).
DNA extraction, PCR, and sequencingIn this study, the ITS, nrLSU, and RPB2 gene fragments were analyzed. Genomic DNA was extracted using the NuClean Plant Genomic DNA kit (ComWin Biotech, Beijing), and the extracted DNA products were gen...