Geastrumsuae sp. nov. (Geastraceae, Basidiomycota) a new species from Yunnan Province, China

Abstract Background Geastrum is the largest genus of Geastraceae and is widely distributed all over the world. Four specimens which belong to Geastrum were collected during our scientific expedition to Cangshan Mountain, Yunnan, China. Based on morphological characteristics and phylogenetic analysis, a new species was introduced. New information Geastrumsuae is characterised by its large basidiomata (height 35–70 mm, diameter 18–37 mm) with long stipe (height 10–45 mm), smooth pink exoperidium and sessile globose endoperidial body. Phylogenetic analysis has been carried out, based on the internal transcribed spacer (ITS) and large subunit ribosomal ribonucleic acid (nrLSU) sequence data. The illustration and description for the new taxa are provided.


Introduction
Geastrum Pers. is the largest genus of Geastraceae and was established by Persoon (1794).Geastrum is commonly known as the earthstars with worldwide distribution and the most species-diverse in the family Geastraceae.Up to now, there are 140 valid species in this genus (Wijayawardene et al. 2022, Zhou et al. 2022, Cabral et al. 2022, Wang and Bau 2023).Geastrum clearly differs from Myriostoma by a single endodermal stoma (Sousa et al. 2014).Due to the non-splitting ectoderm and the poorly-developed endoperidium being different from Geastrum, researchers thought that Radiigera is one of the genera closely related to Geastrum (Sunhede 1989, de Toledo andCastellano 1996).Later, some studies have found that specimens in Radiigera are nested in Geastrum (Hosaka et al. 2006, Hosaka and Castellano 2008, da Silva et al. 2013), but the relationship between these two genera has not been studied in depth until Jeppson et al. (2013) classified the species of Radiigera into the genus Geastrum.Species of this genus are distributed globally, especially in temperate and tropical regions, such as Brazil-Amazon and Europe (de Leόn 1968, da Silva et al. 2013, Jeppson et al. 2013, Zamora et al. 2014, Cabral et al. 2014a, Cabral et al. 2014b, Sousa et al. 2015, Crous et al. 2016, Cabral et al. 2017, Crous et al. 2017, Sousa et al. 2017, Crous et al. 2018a, Crous et al. 2018b, Assis et al. 2019, Finy et al. 2021, Rodrigues et al. 2021).However, the taxonomic relationship under the genus was chaotic (Zamora et al. 2013) until Zamora et al. (2014) divided it into 14 Sections using polygenic analysis, viz.Sect.Campestria, Corollina, Elegantly, Exareolata, Fimbriata, Fornicata, Geastrum, Hariotia, Hieronymia, Myceliostroma, Papillata, Pseudoilmbata, Schmidelia and Trichaster.In China, the early systematic report of Geastrum can be found in "Fungi in China" (Deng 1963) and "The Confluence of Chinese Fungi" (Dai 1979).Zhou et al. (2007) detailed descriptions of 16 species of Geastrum in China in "Flora Fungorum Sinicorum-Geastraceae and Nidulariaceae".Later, three new records and nine new species were reported (Han and Bau 2016, Zhou et al. 2022, Wang and Bau 2023).
Four specimens which belong to Geastrum were collected during our scientific expedition to Cangshan Mountain, Yunnan, China.Morphological and phylogenetic analysis revealed that these specimens are the same species and are different from other species in Geastrum.Therefore, we introduced it as a new species and provided the detailed description and illustration.

Morphological description
Macro-morphological descriptions were based on fresh specimens, which were photographed in the field with notes and laboratory supplemental measurements.The colour is compared with the standard colours in the colorhexa website (https:// www.colorhexa.com).Micro-morphological data were obtained from the fresh specimens and observed by using a light microscope, following Accioly et al. (2019).Sections were studied at magnification of up to 1000× using a NiKon eclipse Ni microscope and phase contrast illumination and scanning electron microscope (SEM) analysis was done under a Shimadzu SSX-550.Preparation of the material examined under SEM followed da Silva et al. (2011).Microscopic features and measurements were made from slide preparations stained with 5% potassium hydroxide (KOH).Basidiospore features, hyphal system, colour, sizes and shapes were recorded and photographed.Measurements were made using the Image Framework v.0.9.7 to represent variation in the size of basidiospores, 5% of measurements were excluded from each end of the range and extreme values are given in parentheses.
The abbreviation for spore measurements (n/m/p) denote "n" spores measured from "m" basidiocarps of "p" specimens.Basidiospore dimensions (and "Q" values) are given as (a) b-av-c (d), where "a" represents the minimum, "d' the largest, "av" the average "b" and "c" covers a minimum of 90% of the values."Q" is the length/width ratio of a spore inside view and "Qm" for the average of all spores ± standard deviation.Voucher specimens are deposited in the Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (KUN-HKAS).

DNA extraction, PCR amplification and sequencing
The DNA extractions were performed from a small piece of the dried basidioma by using Trelief Plant Genomic DNA Kit from Tsingke Biotechnology Co., Ltd (Beijing, China).Two DNA regions were amplified: the internal transcribed spacer nuclear ribosomal DNA (ITS), nuclear ribosomal large subunit (nrLSU) with the primer pairs ITS1F/ITS4 and LR0R/ LR5, respectively (Table 1).

Sequence alignment
Sequence data of two partial loci, internal transcribed spacer region (ITS) and the large subunit ribosomal RNA gene (nrLSU) were analysed.All the sequences, except those which were obtained from this study, were selected from GenBank for phylogenetic analyses (Table 2).Sequences were aligned using the online version of MAFFT v.7 (http:// mafft.cbrc.jp/alignment/server/)(Katoh and Standley 2013) and adjusted using BioEdit v. 7.0.9(Hall 1999) by hand to allow maximum alignment and minimise gaps.Ambiguous regions were excluded from the analyses and gaps were treated as missing data.AliView 1.19-beta was used to convert the alignment fasta file to Phylip and Nexus format for phylogenetic analysis.Phylogenetic analyses were obtained from Maximum Likelihood (ML) and Bayesian Inference (BI).

Molecular phylogenetic analyses
The Maximum Likelihood (ML) and Bayesian Inference (BI) methods were used to analyse the combined dataset of ITS and nrLSU sequences.ML analysis was conducted with RAxML-HPC2 on the CIPRES Science Gateway (Miller et al. 2010), involving 100 ML searches; all model parameters were estimated by the programme.The ML bootstrap values (ML-BS) were obtained with 1000 rapid bootstrapping replicates.
Bayesian analysis was performed with MrBayes v.3.2 (Ronquist et al. 2012), with the bestfit model of sequence evolution estimated with MrModelTest 2.3 (Nylander et al. 2008) to evaluate posterior probabilities (PP) (Rannala and Yang 1996, Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo (MCMC) sampling.Six simultaneous Markov chains were run for 100,000,000 generations, trees were sampled every 500 generation and 200,000 trees were obtained.The first 50,000 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 150,000 trees were used for calculating posterior probabilities in the majority rule consensus tree (the critical value for the topological convergence diagnostic is 0.01).
The phylogenetic tree was visualised with FigTree version 1.4.0 (Rambaut 2012) and made in Adobe Illustrator CS5 (Adobe Systems Inc., USA).Sequences derived in this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov).

Diagnosis
Geastrum suae is characterised by long stipes and larger basidiomata; Pseudoparenchymatous layer is pink, smooth; globose endoperidial body, grey; the ends of eucapillitium hyphae taper and are bluntly rounded; and they live in groups.

Etymology
The species is named suae (Lat.), in memory of the Chinese mycologist Prof. Hong-Yan Su, who kindly helped the authors in many ways and sadly passed away on 3 May 2022 during the preparation of the current paper.

Habit
It grows in groups on the ground in mixed coniferous and broad-leaved forests where there are Alnus nepalensis and Pinus yunnanensis, with thick humus.Currently, it is known only from Cangshan Mountain.

Analysis Phylogenetic analysis
Firstly, we constructed the ML tree of Geastrum genus, based on ITS (1-540 bp) and nrLSU (541-1498 bp) genes and found that G. suae is in Sect.Mycelioatroma.The Maximum Likelihood bootstrap values (ML) equal to or greater than 70% are given above each node (Fig. 2), with the Final ML Optimisation Likelihood: -24127.230142.The aligned matrix had 856 distinct alignment patterns, with 6.78% completely undetermined characters or gaps.The base frequency and rate are as follows: A = 0.274187, C = 0.208839, G = 0.265219, T = 0.251755; rate AC = 1.202699,AG = 3.054698, AT = 1.472914,CG = 0.671195, CT = 5.726232, GT = 1.000000; gamma distribution shape: α = 0.269052.Therefore, we constructed the ML tree and Bayesian tree of Sect.Mycelioatroma, based on ITS and nrLSU genes and clarified the position of G. suae in this Section.The dataset is composed of ITS and nrLSU genes, comprising a total of 1478 characters including gaps, ITS (1-591 bp) and nrLSU (592-1478 bp), including 35 taxa with Myriostoma coliforme (MA-Fungi 83759) as the outgroup taxon (Fig. 3).The best fit model for the combined 2-gene dataset estimated and applied in the Bayesian analysis was GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1).The phylogenetic analysis of ML and BI produces similar topology.The combined dataset analysis of RAxML generates a best-scoring tree (Fig. 3  Phylogenetic analysis showed that four new collections of G. suae clustered together with high bootstrap support and are sister to G. rubellum with good bootstrap support (74% ML/ 1 PP Fig. 3).

Discussion
Geastrum suae can be easily recognised by the basidiomata with pink neat, smooth 6lobed ectoderm, globose sessile endoperidium and longer prosthecae.
In the phylogenetic inferences, Geastrum suae is sister to G. rubellum, which is known from the biome Tropical and Subtropical Moist Broadleaf Forests in Brazil (Accioly et al. 2019) (Fig. 3).Morphologically, both species share similar characteristics of the mesopodal basidiomata, but G. rubellum has reddish to brownish exoperidium with longer exoperidium hairs.G. suae hardly has such hairs and the reddish pseudoparenchymatous layer in G. rubellum also clearly differentiates G. suae.Not only that, but G. rubellum also has reddish to brownish exoperidium with a verrucose to hairy mycelial layer, while the exoperidium of G. suae is almost smooth.Their size is different, the expanded basidiomata saccate of G. rubellum being 10 mm high × 8.5-30 mm wide, while G. suae is 35-70 mm high × 18-37 mm wide.The warts on the basisiospore of G. suae are shorter than those of G. rubellum.
The pseudoparenchymatous layer of G. rubellum is pure (or mostly pure) pink (#fa007d) when fresh, brownish-grey when dried, but is very pale red (#ffccd5) for G. suae.

Figure 2 .
Figure 2. Phylogenetic tree of Geastrum species and related taxa, based on ITS and nrLSU sequence data.

Figure 3 .
Figure 3.Phylogenetic tree of the new Geastrum species and related taxa which belong to sect.Myceliostroma, based on ITS and nrLSU sequence data.Branches are labelled with bootstrap values (ML) higher than 70% and posterior probabilities (PP) higher than 0.95.The new species are shown in red bold.

Table 1 .
Amplification primers information used in this study. TM

Table 2 .
Species, specimens, Collection locality and GenBank accession numbers of sequences used in this study (newly-generated sequences are indicated in bold).