Exploring macrofungal diversity hotspot of the Qinghai–Xizang Plateau revealed by integrative taxonomy with 31 new species

Xixi Han; Ruilin Zhao; Bin Cao; Jiaxin Li; Rui Xing; Maoqiang He; Haiying Feng; Shihui Wang; Kevin D. Hyde; Naritsada Thongklang; Dorji Phurbu; Xixi Han; Ruilin Zhao; Bin Cao; Jiaxin Li; Rui Xing; Maoqiang He; Haiying Feng; Shihui Wang; Kevin D. Hyde; Naritsada Thongklang; Dorji Phurbu

doi:10.48130/mycosphere-0026-0017

Macrofungi are key components of terrestrial ecosystems, yet their diversity remains poorly documented in high-elevation regions. The Qinghai–Xizang Plateau, characterized by strong environmental gradients and extreme conditions, provides an ideal system for investigating fungal diversity and adaptation. In this study, extensive field surveys conducted between 2015 and 2024 across multiple vegetation types were combined with morphological and multilocus phylogenetic analyses to assess macrofungal diversity. We describe 31 previously unknown species belonging to 10 genera of Agaricales, spanning multiple phylogenetic lineages and ecological guilds. Saprotrophic taxa dominate the assemblage, with ectomycorrhizal species also being well-represented. These findings indicate that macrofungal diversity in high-elevation ecosystems remains substantially underestimated and highlight the importance of integrative taxonomy for resolving species diversity. A subset of species exhibits a recurrent morphological pattern, characterized by robust basidiomata with relatively small, thick pileus and short, stout stipes, along with the occasional co-occurrence of two-spored and four-spored basidia. Based on these observations, we propose a preliminary hypothesis that coordinated shifts in structural and reproductive traits may occur in some macrofungi inhabiting high-elevation environments. Overall, this study expands current knowledge of macrofungal diversity on the Qinghai–Xizang Plateau and emphasizes the importance of mountain systems for understanding fungal diversification and ecological adaptation.

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Introduction

Macrofungi constitute a fundamental yet still profoundly underexplored component of global terrestrial biodiversity^[1−4]. As dominant decomposers of lignocellulosic biomass, they play indispensable roles in organic matter turnover, nutrient mobilization, and soil formation across a wide range of ecosystems^[5−10]. In addition, numerous macrofungal taxa establish ectomycorrhizal symbioses with woody plants, thereby influencing plant productivity, vegetation dynamics, and ecosystem resilience^[11−14], and are important in other biotechnological applications including compound discovery^[15−17]. Through these ecological functions, macrofungi regulate carbon fluxes, mediate nutrient cycling, and contribute to the maintenance of ecosystem stability^[18−21]. Despite their ecological prominence and deep evolutionary history, global estimates indicate that only a small fraction of macrofungal diversity has been formally documented, with vast numbers of species remaining undescribed^[22−24]. This taxonomic knowledge gap is particularly pronounced in mountainous regions such as the Qinghai–Xizang Plateau, where pronounced environmental heterogeneity, arising from rugged topography, steep climatic gradients, and geographic isolation, is hypothesized to drive lineage diversification and endemic radiations^[25−27].

Traditionally, macrofungal taxonomy has relied primarily on phenotypic characterization, including macromorphological features of basidiomata and micromorphological traits such as basidiospore ornamentation, hymenophoral configuration, and hyphal structure^[28−30]. However, pervasive morphological plasticity across many fungal lineages, frequently driven by environmental factors such as substrate composition, moisture availability, temperature regimes, and altitude, can obscure species boundaries^[31−34]. Convergent evolution and parallel morphological adaptations further contribute to taxonomic frameworks that do not accurately reflect phylogenetic relationships^{[24,31,35,36]}. As a result, numerous historically described taxa circumscribed solely on morphological evidence lack phylogenetic support, leading to uncertain systematic placement and complicating the delimitation and recognition of newly discovered species^[37−39]. Such limitations may result in taxa being recovered as nested within established clades, generating paraphyly and persistent taxonomic instability^[40]. Over the past two decades, the rapid maturation of molecular systematics and the paradigm shift toward integrative taxonomy, which synthesizes phenotypic characters, ecological niches, and robust multilocus phylogenetic inference, have revolutionized fungal systematics and revealed extensive cryptic diversity across major macrofungal lineages^[34,37,41].

The Qinghai–Xizang Plateau represents the largest and highest alpine ecosystem on Earth, providing an exceptional setting for studies of biodiversity and ecological processes^[42−44]. Shaped by a complex geological history and marked environmental gradients, the Plateau supports diverse alpine and montane vegetation types that create a wide range of fungal habitats^[45−47]. The extreme conditions of high-elevation environments, including low temperatures, intense ultraviolet radiation, fluctuating moisture regimes, and reduced oxygen availability, impose strong selective pressures that may drive adaptive divergence and lineage diversification in fungal populations^[48−53]. Owing to steep environmental gradients and topographic complexity, mountain systems function as major engines of diversification, promoting speciation through geographic isolation, ecological niche differentiation, and climatically driven range dynamics^[54−58]. Under these conditions, substantial fungal diversity, including cryptic taxa, may remain overlooked when relying solely on morphology-based identification. Consequently, integrative taxonomic investigations remain essential for revealing fungal diversity and improving our understanding of evolutionary processes in high-elevation ecosystems.

Although fungal diversity across parts of the Qinghai–Xizang Plateau and adjacent regions has received increasing attention in recent years^[59−63], current knowledge remains taxonomically fragmented and geographically uneven. Existing studies have largely focused on individual genera, specific ecological guilds, or restricted sampling areas^[64−67], resulting in an incomplete understanding of diversity patterns, lineage diversification, and systematic placement, particularly for taxa lacking multilocus phylogenetic assessment. Moreover, the scarcity of integrative studies spanning multiple phylogenetic lineages across vegetation belts and elevational gradients continues to constrain a comprehensive understanding of macrofungal diversity and diversification in high-elevation ecosystems^{[25,26,68,69]}. Here, we address these gaps through extensive plateau-wide field surveys and an integrative systematic investigation of 10 focal fungal lineages. By combining morphological, ecological, and multilocus phylogenetic evidence, this study provides a more robust evolutionary framework for species delimitation and taxonomic placement, thereby improving our understanding of macrofungal diversity and diversification on the Qinghai–Xizang Plateau. These findings also contribute to a broader understanding of how environmental heterogeneity and elevational gradients shape fungal diversification on the plateau.

Based on field collections made between 2015 and 2024 across multiple high-elevation habitats of the Qinghai–Xizang Plateau, this study uses an integrative taxonomic approach that combines morphological examination, ecological observations, and multilocus phylogenetic analyses to investigate macrofungal diversity. We describe 31 previously undocumented species belonging to 10 genera across 10 families of Agaricales, with specimens collected from multiple vegetation types, including coniferous forests, broad-leaved forests, mixed forests, alpine meadows, and shrublands. Although not intended as a comprehensive regional inventory, these findings highlight the presence of substantial, previously unrecognized taxonomic diversity across diverse high-elevation habitats. This work also provides a baseline framework for future studies addressing fungal diversity patterns, ecological associations, and evolutionary processes in mountain ecosystems.

Materials and methods

Sample collection and herbarium specimen preparation

Fresh basidiomata were collected during extensive field surveys conducted from 2015 to 2024 across the Qinghai–Xizang Plateau and adjacent regions, including Xizang Autonomous Region, Qinghai, Sichuan, Gansu, and Yunnan Provinces. Collections were obtained from five major vegetation types, namely shrublands, alpine meadows, coniferous forests, broad-leaved forests, and mixed conifer and broad-leaved forests. Macromorphological features were documented from fresh specimens in the field and photographed before preservation. All collections were dehydrated at 50 °C, sealed, and deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS) and the Alpine Fungarium (AF).

Morphological studies
Morphological investigations were conducted using both fresh and dried materials. Macroscopic characters were recorded from mature basidiomata in the field, whereas microscopic structures were examined from rehydrated specimens^[66]. Colour descriptions followed the Methuen Handbook of Colour^[70]. Sections of pileus, lamellae, and stipe tissues were mounted in 5%–10% KOH and, when necessary, stained with Congo red. Microscopic observations were performed using an Olympus CX31 microscope following the procedures outlined by Largent et al.^[30]. Basidia, basidiospores, cystidia, and pileipellis structures were examined and measured, with a minimum of 50 measurements obtained for each character. Spore dimensions are expressed as (a)b–c(d), where b–c encompasses at least 90% of the observations. The notation n/m/p refers to the number of spores measured, the number of basidiomata examined, and the number of collections studied, respectively. Q represents the spore length-to-width ratio and Qm its mean value ± standard deviation. Basidiospore ornamentation and other ultrastructural features were further examined using scanning electron microscopy (JEM-1400)^[66,71,72].

DNA extraction, amplification, and sequencing
Total genomic DNAs were isolated from fresh or dried basidiomata using a Broad-spectrum Plant Rapid Genomic DNA Kit (Beijing Biomed Gene Technology Co., Ltd., China), following the manufacturer's protocol with slight modifications. DNA extracts were stored at −20 °C before PCR amplification. Four loci widely used in fungal phylogenetic studies include the internal transcribed spacer region (ITS1–5.8S–ITS2), the nuclear large subunit ribosomal DNA (LSU), partial sequences of the second largest subunit of RNA polymerase II (rpb2), and the translation elongation factor 1-α gene (tef1α). The ITS region was amplified using ITS1-F (ITS5 or ITS1) and ITS4^[73,74]; LSU using LR0R and LR5 (or LR7)^[75,76]; rpb2 using brpb2-6F (or frpb2-5F) and brpb2-7.1R^[77]; and tef1α using 983F and 1567R (or 2218R)^[78]. For samples that failed to amplify with universal primers, specific or internal primers were used, following previous studies^[79,80].

PCR amplification was performed in a T30 Multi-Block Gradient PCR Thermocycler (Hangzhou LongGene Scientific Instruments Co., Ltd., China) using a total reaction volume of 25 µL containing 2 µL genomic DNA, 1 µL of each primer, 9 µL double-distilled water, and 12 µL 2× Es Taq MasterMix (Dye) (Beijing ComWin Biotech Co., Ltd., China). PCR amplifications were carried out using the following thermal cycling programme: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 48–55 °C for 1 min, and extension at 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. For samples that failed to amplify under these conditions, gradient PCR was conducted using the same thermocycler with annealing temperatures ranging from 50 to 60 °C. PCR products were examined on 1.4% agarose gels stained with GoldenView. Successfully amplified products were purified and sequenced using the same primers at BGI Genomics Co., Ltd. (Beijing, China). In a few cases when direct sequencing failed, PCR products were cloned into the pMD18-T vector (Takara Biomedical Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions, then transformed into Escherichia coli DH5-α competent cells, and sequenced using M13F/M13R primers^[81]. Sequences were assembled and edited using SeqMan implemented in the DNASTAR software package (DNASTAR Inc., Madison, USA). All newly generated sequences were deposited in GenBank and are listed under 'Specimens examined' for each species in the Taxonomy. For new species described from single collections, PCR amplification and sequencing were independently repeated to minimize potential sequencing errors.

Phylogenetic analyses
Newly generated sequences were initially subjected to blastn searches in the GenBank database to identify closely related taxa. Sequences with query coverage > 90% and a percentage identity of 96% were selected, and one to three representative sequences per species were included for subsequent analyses. In addition, published sequences of morphologically similar taxa were incorporated into the final dataset. Individual loci were aligned separately using the online MAFFT v7 server (https://mafft.cbrc.jp/alignment/server)^[82] with the default strategy, and manually adjusted in BioEdit v7.1.3.0^[83]. The aligned datasets were subsequently concatenated using PhyloSuite v1.2.3^[84], with missing data and ambiguous regions coded as 'N'.

Phylogenetic relationships were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML analyses were conducted on the CIPRES Science Gateway (www.phylo.org)^[85] using RAxML-HPC BlackBox v8.2.12^[86], and branch support was estimated based on 1,000 nonparametric bootstrap replicates. The best partitioning scheme and substitution models for BI analyses were determined using PartitionFinder2 v2.1.1^[87], implemented in PhyloSuite, applying the greedy algorithm and the AICc criterion. Bayesian analyses were performed on the CIPRES Science Gateway using MrBayes on ACCESS v3.2.7a^[88], with two independent runs and four Markov chains. For each group, the DNA loci used, analytical method(s), partitioning strategy, and outgroup(s) setting were described in the figure legend of the phylogenetic tree. The resulting phylogenetic trees were visualized using the tvBOT online platform (www.chiplot.online/tvbot.html)^[89] and further edited in Adobe Illustrator 2024.

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Exploring macrofungal diversity hotspot of the Qinghai–Xizang Plateau revealed by integrative taxonomy with 31 new species

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