Unearthing a fungal giant: Dianjunaceae fam. nov., a novel Paleocene lineage of Xylariales harboring Dianjunus rex gen. et sp. nov

1 Introduction

The order Xylariales Nannf. comprises a morphologically diverse group of ascomycetous fungi, typically characterized by carbonaceous, darkly pigmented stromata. Taxa within this order occupy varied ecological niches, functioning as endophytes, pathogens, or saprobes (Senanayake et al., 2015). Saprobic members, often wood-associated, are key decomposers of dead plant materials, particularly lignified tissues, and contribute significantly to carbon cycling in forest ecosystems (Ma et al., 2022). Endophytic xylarialean species reside asymptomatically within healthy plant tissues, sometimes shifting to saprobic life mode when host tissues senesce, while others may act as opportunistic plant pathogens under host stress (Cedeño-Sanchez et al., 2023).

Xylariales typically produce perithecial ascomata embedded within stromatic tissues. They are defined by unitunicate asci, frequently possessing a characteristic amyloid or inamyloid apical apparatus, and brown to dark ascospores that often exhibit a germ-slit (Stadler, 2011). While traditional taxonomy has relied on stromatal morphology, perithecial arrangement, and spore characteristics, these traits are often subject to convergence. Contemporary systematics of Xylariales increasingly integrates multi-locus phylogenetic and genome-scale data, leading to substantial revisions of familial and generic boundaries (Wendt et al., 2018; Chen et al., 2023). Several studies indicated that anamorph morphology in Xylariales fungi served as a better indicator of phylogenetic relationships than teleomorph morphology (Jaklitsch and Voglmayr, 2012; Jaklitsch et al., 2016). The order currently encompasses 16 recognized families (Samarakoon et al., 2016) and allied lineages reflecting considerable lifestyle diversity. Beyond their ecological relevance, Xylariales are notable for their prolific production of secondary metabolites, including pigments and bioactive compounds, and their enzymatic capacity to degrade complex plant polymers. These attributes render them of significant interest for biotechnology, natural product discovery, and studies of plant–microbe interaction (Becker and Stadler, 2021; Franco et al., 2025).

Stromatal morphology within the order varies, including both upright and penzigioid forms (Fournier et al., 2020). The order Xylariales encompasses few species capable of producing large stromata. A paramount example is Engleromyces goetzei P. Henn., which forms large, subglobose stromata on bamboo culms (Whalley et al., 2010). A documented specimen from Kenya comprised two lobes measuring 30 cm in diameter with a mass of approximately 3.2 kg, ranking among the most voluminous individual ascomata recorded in Ascomycota (Gibson and Kimeria, 1962; Whalley et al., 2010). In contrast, species such as Xylaria polymorpha (Pers.) Grev. and X. longipes Nitschke produce clustered, clavate stromata where aggregations, though comprised of modest individual units (5–8 cm height), can exceed 20 cm in total breadth (Rogers, 1979). Squamotubera le-ratii Henn., the only species of the genus, has been reported with stromata around 7 cm height and 5 cm width (Hennings, 1903; Rogers, 1981).

During fungal diversity surveys in Yunnan Province, China, several specimens producing exceptionally large, upright stromata were collected from a tropical rainforest in Xishuangbanna Dai Autonomous Prefecture and from temperate coniferous forests in Kunming and Lufeng Cities. Through integrative approaches combining detailed morphological examination with multilocus phylogenetic reconstruction, taxonomic placement of all specimens was determined.

2 Materials and methods

2.1 Sample collection and morphological examination

Specimens were collected during the following periods and in the following locations: August to September 2024 in Damaidi Village, Tuanjie Town, Luquan County, Kunming, China; July 2025 in Nanbang Village, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China; and July 2025 in Yipinglang Town, Lufeng City, Chuxiong Yi Autonomous Prefecture, Yunnan Province, China. Detailed site data, including elevation, climatic conditions, and geographical features, were recorded in situ. Photographs of fungal specimens in their natural habitat were taken using a Canon G15 camera (Canon Inc., Tokyo, Japan). Collected materials were wrapped in waterproof foil and transported to the laboratory for subsequent analysis. Macroscopic characteristics were examined using an Olympus SZ61 stereomicroscope. Micromorphological structures, including ascospores, asci, and ascomata, were photographed using a Canon 700D digital camera mounted on a compound microscope. Ascospores were cultured on Potato Dextrose Agar (PDA), and pure cultures were obtained following the methodology of Senanayake et al. (2020b). Fungal hyphae were grown on Malt extract agar (MEA), Oat Meal Agar (OMA), and PDA to examine the colony morphology. All microscopic measurements were performed using the Tarosoft Image Frame Work (v. 0.9.0.7). Image composites were processed and adjusted with Adobe Photoshop CS6 software (version 10.0, Adobe Systems, USA). Voucher specimens were deposited at the fungarium of the Kunming Institute of Botany, Chinese Academy of Sciences (HKAS). Index Fungorum numbers (http://www.indexfungorum.org) were registered for all novel taxa.

2.2 DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from the fresh stromata tissue using the Biospin DNA Extraction Kit (Hangzhou Bioer Technology Co., Ltd., Hangzhou, China) according to the manufacturer’s protocol. Extracted DNA was stored at -20 °C before amplification. Polymerase chain reaction (PCR) amplification and sequencing were performed for four genetic loci: the partial 28S Large Subunit ribosomal DNA (LSU) using primers LR0R and LR5 (Vilgalys and Hester, 1990); the complete Internal Transcribed Spacer (ITS) region using primers ITS1 and ITS4 (White et al., 1990); the beta-tubulin (TUB2) gene using primers T1 and T22 (O'Donnell and Cigelnik, 1997); and the RNA polymerase II subunit 2 (RPB2) gene using primers fRPB2-5F and fRPB2-7cR (Liu et al., 1999). PCR reactions were conducted following the protocol described in Senanayake et al. (2020b). The total volume of the PCR reaction was 25 µL, containing 1 µL of DNA template, 1 µL of each forward and reverse primer, 12.5 µL of 2 × PCR Master Mix, and 9.5 µL of sterile double-distilled water (ddH₂O). Amplification was performed for 35 cycles under the thermocycling conditions detailed in Table 1. PCR products were visualized via electrophoresis on 1% agarose gels stained with ethidium bromide. Products were purified and sequenced commercially (Sunbiotech Company, Beijing, China). Raw sequences were assembled, edited, and quality-checked using DNASTAR Lasergene v. 7.1. Newly generated sequences were deposited in the National Center for Biotechnology Information (NCBI)GenBank database; corresponding accession numbers are provided in Table 2.

Table 1. Primers and optimized PCR protocols used in this study.

Table 2. Details of the isolates used in the phylogenetic tree.

Newly generated sequences are bold.

AFTOL-ID, Assembling the Fungal Tree of Life; ATCC, The American Type Culture Collection; BAFC, Faculty of Exact and Natural Sciences, University of Buenos Aires; BCC, Biotec Culture Collection; BCMX, Baja California Mexico; BISH, Herbarium Pacificum; CBS, Westerdijk Fungal Biodiversity Institute; CPC, Culture Collection of Pedro Crous; DAOM, Canadian National Mycological Herbarium; FMR, Facultad de Medicina, Spain; GBMC, Global Microbiome Conservancy; HAST, Biodiversity Research Museum, Herbarium Academia Sinica; HKAS, Cryptogamic Herbarium of Kunming Institute of Botany; HKU-M, Hong Kong University Herbarium; HKUCC, Hong Kong University Culture Collection; HUEFS, Herbarium of the University of Helsinki; ICMP, International Collection of Microorganisms from Plants; JCM, Japan Collection of Microorganisms; KNU, Kyungpook National University, Korea; MFLU, Mae Fah Luang University Herbarium; MFLUCC, Mae Fah Luang University Culture Collection; MUCL, Agro-food & Environmental Fungal Collection; NBRC, National Institute of Technology and Evaluation; NRRL, Agricultural Research Service Culture Collection; PDD, New Zealand Fungarium; PUFD, Culture Collection at Pondicherry University; PUFNI, University of Pondicherry; SMH, National Museum of Natural History; SQUCC, Sultan Qaboos University Culture Collection; STMA, Deutsche Sammlung von Mikroorganismen und Zellkulturen microbial culture collection; TASM, Tasmanian Fungal Culture Collection; UCD, The U.C. Davis Viticulture and Enology Department Culture Collection; WA, The Western Australian Microbial Culture Collection; WSP, Herbarium at Washington State University; WU, Herbarium of University of Vienna; YMJ, The Japan Collection of Microorganisms.

2.3 Phylogenetic analyses

Newly generated sequences were subjected to BLASTn searches against the GenBank database (http://www.ncbi.nlm.nih.gov/) to determine preliminary taxonomic affiliations. Additional sequences were obtained from GenBank based on Basic Local Alignment Search Tool (BLAST) results and previous studies. A concatenated dataset of LSU, ITS, RPB2, and TUB2 sequences was assembled, comprising 202 strains. Leptosillia cordylines (MFLU 20-0056) and Delonicicola siamense (MFLUCC 15-0670) were selected as the outgroup.

Individual gene alignments were generated using the online version of Multiple Alignment using Fast Fourier Transform (MAFFT) v. 7.0362 (Katoh et al., 2019) with default parameters and manually refined in BioEdit 7.1.3 (Hall, 1999) where necessary. Maximum Likelihood (ML) analysis was performed with Randomized Axelerated Maximum Likelihood (RAxML) (Stamatakis and Alachiotis, 2010) implemented in raxmlGUI v.1.5 (Silvestro and Michalak, 2012) using the ML + rapid bootstrap setting and the general time-reversible (GTR) + I + G model of nucleotide substitution with 1,000 rapid bootstrap replicates. For Bayesian inference (BI), the optimal substitution model (GTR+G+I) was determined using MrModeltest software v. 2.2 (Nylander, 2004). BI analysis was conducted in MrBayes v. 3.2.6 (Ronquist et al., 2012) with four simultaneous Markov chain Monte Carlo chains from random trees over 3 million generations (standard deviation of split frequencies < 0.01), sampling every 500^th generation. The distribution of log-likelihood scores was detected to check whether sampling was in the stationary phase, and Tracer v1.5 was used to check if further runs were required to reach convergence (Rambaut and Drummond, 2007). The first 20% of sampled trees were discarded as burn-in before calculating a consensus tree and posterior probabilities. Resultant phylogenetic trees were visualized using FigTree v.1.4 (Rambaut, 2012).

2.4 De novo genome assembly and genome sequencing of Dianjunus rex

Whole-genome and transcriptome sequencing were performed on three stromata of Dianjunus rex collected from the wild. Internal tissue was excised from each stroma. High-quality genomic DNA was extracted from each sample using the cetyltrimethylammonium bromide (CTAB)method (Porebski et al., 1997). Total RNA was extracted with TRIzol^® Reagent (Invitrogen) extraction kit, followed by purification using the Plant RNA Purification Reagent (Invitrogen) purification kit. Short DNA and RNA reads were generated for each sample on the Illumina HiSeq 4000 platform (San Diego, CA, USA). These reads were then trimmed and filtered using fastp (v0.23.2) (Chen et al., 2018) with parameters ‘-f 10 and -F 10’ to remove adapter sequences and low-quality reads. For paired-end DNA sequencing data, afterthe clean data converted were into a FASTA file, 10,000 reads were randomly selected from each file and aligned against the NCBI Nucleotide (NT) database, indicating no significant contamination. Finally, in total, we obtained 49 Gb of clean data for DNA and 51 Gb for RNA, respectively. For genome assembly, the filtered DNA reads from each sample were assembled independently using SPAdes (v4.2.0) (Bankevich et al., 2012). Genome completeness was assessed by Benchmarking Universal Single-Copy Orthologs (BUSCO, v5.2.2) (Simão et al., 2015) with the ascomycota_odb10 orthologous gene set.

2.5 Annotation of repetitive sequences and protein-coding genes

RepeatModeler (v2.0.2) (Flynn et al., 2020) was used to build the de novo repeat libraries and then sequences from the assembly were aligned to the de novo repeat libraries and known repeat databases to identify and mask repetitive elements, using RepeatMasker (v4.1.2) (Chen, 2004). Protein-coding genes in each genome were predicted using the Genome-wide Electronic Tool for Annotation (GETA, v2.6.1) pipeline (https://github.com/chenlianfu/geta), integrating evidence from homology-based, ab initio, and RNA-seq-based prediction approaches. For homology-based prediction, protein-coding sequences of Biscogniauxia marginata, Annulohypoxylon maeteangense, Daldinia caldariorum, Hypoxylon argillaceum, Astrocystis sublimbata, Durotheca rogersii, Kretzschmaria deusta, Nemania abortiva, Poronia punctata, and Xylaria curta were downloaded from the Joint Genome Institute (JGI) (Franco et al., 2022) and were used for gene prediction using GeneWise (v2.4.1) (Birney et al., 2004). The ab initio prediction was conducted using AUGUSTUS (v3.4.0) (Stanke et al., 2006). RNA-seq reads were aligned to the genome assembly using hierarchical indexing for spliced alignment of transcripts 2 (HISAT2, v2.1.0) (Kim et al., 2015), and protein-coding regions were predicted using TransDecoder (v5.5.0) (https://github.com/TransDecoder/TransDecoder). The predictions from the three methods were then integrated and filtered against the Pfam database.

2.6 Phylogenetic tree construction and divergence time estimation

Protein sequences of 63 species in Pezizomycotina were downloaded from JGI Mycocosm database and combined with sequences from three samples generated in this study, resulting in 66 samples for phylogenetic analysis. Single-copy orthologous proteins conserved across all 66 samples were retrieved from the results of BUSCO assessment. A total of 302 single-copy orthologs were then aligned using MAFFT (v7.407) (Katoh et al., 2002) with default parameters, and the alignments were concatenated sequentially to construct a super matrix. The phylogenetic tree was constructed from this super matrix using IQ-TREE 3 (v3.0.1) (Wong et al., 2026), with Aspergillus niger and A. tetrazonus serving as the outgroup. Branch supports were assessed using a bootstrap approach with 1,000 replicates. The species tree was visualized using the web application Tree Visualization By One Table (tvBOT) (Xie et al., 2023).

To further cross-verify the topological stability across different ortholog selection workflows, an alternative dataset was constructed. Orthologous groups across the same 66 samples were clustered using OrthoFinder (v3.1.3) with the DIAMOND alignment mode (-S diamond) and MAFFT for sequence alignment (-M msa -A mafft), yielding 345 single-copy orthologous gene sets. The amino acid sequences of each orthogroup were aligned using MAFFT (v7.407) with automatic parameter selection (--auto). To eliminate poorly aligned or highly divergent regions, the alignments were systematically trimmed using trimAl (v1.4.rev15) with a gap threshold of 0.3 (-gt 0.3), ensuring that sites with more than 70% gaps were removed. Individual maximum-likelihood gene trees were then inferred from the trimmed alignments using IQ-TREE 3 (v3.0.1) under the best-fit evolutionary models automatically selected using ModelFinder Plus (MFP) with 1,000 ultrafast bootstrap replicates, utilizing the same outgroup (Aspergillus niger and A. tetrazonus). Finally, these 345 gene trees were used as input for Accurate Species TRee ALgorithm (ASTRAL, v5.7.1) to reconstruct the alternative coalescent-based species tree.

The MCMCtree module of the Phylogenetic Analysis by Maximum Likelihood (PAML)package (v4.9i) (Yang, 2007) was used to estimate each speciation time for these 66 Pezizomycotina samples. The phylogenetic tree constructed by IQ-TREE 3 served as a guide tree, with the divergence times from TimeTree (Kumar et al., 2017) used as calibration points: Aspergillus tetrazonus and Pyricularia oryzae (233.8~367.0 million years ago [MYA]), Pseudomassariella vexata and Microdochium trichocladiopsis (143.3~164.1 MYA), Microdochium bolleyi and Hypoxylon fuscum (103.4~318.2 MYA), and Colletorichum cereale and Verticillium longisporum (85.0~139.4 MYA).

2.7 Identification and analysis of carbohydrate-active enzymes

Carbohydrate-active enzymes (CAZymes) profiles were identified in the proteomes of all 66 Pezizomycotina samples. BLAST and hidden Markov model (HMM) searches were performed simultaneously using the proteomes as queries to detect putative CAZymes. For the BLAST searches, query protein sequences were used as inputs in the BLASTp tool against the database for automated CAZyme annotation (dbCAN, v11) (Yin et al., 2012). The resulting hits were filtered by e-value (1e-5) and identity threshold (20%). For the HMM search, the hmmscan program in the HMMER package was used to match the family-specific HMM profiles of CAZymes within dbCAN database (v11). The candidate genes obtained from both the BLAST and HMM searches were merged and only the overlapping hits were retained. The raw sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) under the BioProject accession number of PRJNA1265937. All genome assemblies and gene annotation results have been archived in the figshare database and can be accessed at https://doi.org/10.6084/m9.figshare.29153135.v2. The multilocus alignment of this study can be accessed via https://doi.org/10.6084/m9.figshare.32611812.

3 Results

3.1 Phylogeny

All individual trees generated under different criteria and from single-gene datasets were essentially similar in topology and not significantly different from the tree generated from the concatenated dataset (not discussed herein). Maximum likelihood analysis of new strains generated in this study and other related taxa in this study with 1,000 bootstrap replicates yielded the best ML tree (Figure 1) with the likelihood value of ln: –6728.286175 and the following model parameters: alpha: 0.191444; Π(A): 0.244188, Π(C): 0.230054, Π(G): 0.301322, and Π(T): 0.224436. The substitution rates are as follows: AC: 0.763664, AG: 2.779076, AT: 1.349374, CG: 0.639806, CT: 6.128235, GT: 1.0. The Bayesian analysis resulted in 10,000 trees after 5,000,000 generations, and the first 20% of the trees were discarded. The remaining trees were used for calculating posterior probabilities in the majority-rule consensus tree. Maximum likelihood bootstrap values ≥50% and Bayesian inference (BI) ≥0.9 are given at each node. Tree topologies of the ML and Bayesian analyses were similar to each other and there were no significant differences. Both topologies showed 15 well-supported major clades representing families in Xylariales. These new fungal collections grouped as a sister clade to Graphostromataceae with ML/BI = 100%/1.00 support.

Phylogenomic analysis confirmed that Dianjunus rex represents a distinct lineage within Dianjunaceae (Figure 2). Comparative CAZyme annotation revealed an exceptionally compact and highly conserved enzyme repository across the three sequenced D. rex strains (HKAS151626, HKAS148941, and HKAS150823), encoding 220, 224, and 219 total CAZyme genes, respectively.

Across the broad phylogenomic tree of Sordariomycetes and related Xylariales (Figure 2), D. rex exhibits the most severe species-wide CAZyme contraction observed. Compared to typical saprobic and wood-decaying lineages such as Xylaria (535–635 genes), Daldinia (458–496 genes), and Hypoxylon (423–577 genes), the total CAZyme burden of D. rex is reduced by more than half. Specifically, Glycoside Hydrolases (GHs: 100–103) and Carbohydrate Esterases (CEs: 9–11) are markedly diminished, and Pectin Lyases (PLs) are virtually depleted (PL = 0-1; completely absent in strain HKAS148941) compared to 7–19 PL genes in closely related Xylariales lineages (Figure 2). Conversely, Glycosyltransferases (GTs: 57–58) represent a disproportionately high fraction (~25.9%–26.0%) of its overall enzymatic repertoire.

3.2 Phylogenomic tree construction and divergence time estimation

The genome assembly sizes of samples HKAS148941, HKAS150823, and HKAS151626 are 31.09 Mb, 32.01 Mb, and 30.48 Mb with contig N50 values of 47.63 kb, 42.61 kb, and 76.62 kb respectively (Table 3). Genome completeness assessment revealed that at least 96.19%, 95.96%, and 96.19% of single-copy BUSCO genes were identified in the assembly (Figure 3, Table 3).

Table 3. Genome assembly statistics of Dianjunus rex.

3.3 Taxonomy

Dianjunaceae F.Q. Yu, Pérez-Moreno & Senan., fam. nov.

Index Fungorum number: IF904757

Etymology: Based on the type genus Dianjunus.

Diagnosis: Dianjunaceae is distinguished from other families in Xylariales by having large stromata with persistent powdery-fibrous surface, unique S-shaped germ slit in ascospores, the inverted-trapezoid, amyloid apical ring in Melzer’s reagent, and nodulisporium-like anamorph with periconiella-like branching patterns produce distantly-packed branchlets bearing cluster of cylindrical, conidiogenous cells.

Type genus: Dianjunus F.Q. Yu, Pérez-Moreno & Senan.

Teleomorph: Stromata upright, fleshy, variable in size, shape ranging from cylindrical, fusiform, turbinate to palm-shaped, terete to flattened, simple to branched from the base, sometimes arising from long rooting stipes, with flattened sterile apices. Outer crust coriaceous, immersed part in soil black, aerial part fertile, surface covered with a long persistent, hairy, whitish yellow fibrous substance, whitish in young stromata, turning progressively dark yellow at maturity, ostiole openings appear as black dots. Immersed part sterile, consisting of stipes, cylindrical, black, smooth. Internal tissues solid, homogeneous to fibrous, spongy, off-white to yellow, with a slightly darker core in aged specimens. Perithecia aggregated, immersed in stromata, subglobose, coriaceous, papillate, ostiolate. Ostioles raised-discoid, black, with a cylindrical papilla at center. Paraphyses sparse, hypha-like, narrowing toward the ends, hyaline, septate, slightly constricted at the septa, unbranched. Asci (6–)8-spored, unitunicate, cylindrical, long-stipitate, total length, with apical apparatus, appearing bilobed, bluing in Melzer’s reagent, reddish brown in Lugol’s solution, turning blue in Lugol’s solution after treatment with 3% (w/v) KOH. Ascospores overlapping uniseriate in the ascus, globose to oval or fusiform with acute ends, hyaline when young, becoming brown at maturity, with single, central guttule or with a large central and two small polar guttules, smooth, thick-walled, with a conspicuous, S-shaped germ slit one-half to four-fifths spore length on the flattened side. Anamorph: nodulisporium-like anamorph with periconiella-like branching patterns.

Notes: The multilocus phylogeny of ITS, LSU, TUB2, and RPB2 sequences showed that our new fungal collections clustered together forming a well-supported, distinct sister clade to Graphostromataceae in Xylariales. However, morphological characteristics of our collections are significantly different from those of species in this family. Further, only few genera in Graphostromataceae, such as Camillea and Biscogniauxia, produce upright stromata. The phylogenomic analyses also confirmed the placement of these collections as sister to Graphostromataceae in Xylariales. Therefore, we introduce this unidentified clade as a new family Dianjunaceae.

Dianjunus F.Q. Yu, Pérez-Moreno & Senan., gen. nov.

Index Fungorum number: IF904733

Etymology: The generic name is derived from the “Dian Kingdom”, a historical society situated in the region of present-day Yunnan Province in southwestern China where the samples were collected. The epithet is combined with the Chinese word “jūn”, meaning ‘mushroom’ or ‘fungus’.

Type species: Dianjunus rex F.Q. Yu, Pérez-Moreno & Senan.

Arise from roots of dying host plants. Teleomorph: Stromata upright, fleshy, variable size and shape, ranging from cylindrical, fusiform, turbinate to palm-shaped, terete to flattened, simple to branched from the base, or at the top, arising from long rooting stipes, with flattened sterile apices. Outer crust coriaceous, black, ostiole openings appear as black dots. Aerial part fertile, surface covered with a long persistent, powdery, whitish yellow fibrous substance, whitish in young stromata, turning progressively dark yellow at maturity. Immersed part sterile, consisting of stipes, cylindrical, black, no powdery substance, smooth. Interior tissues solid, homogeneous to fibrous, spongy, off-white to yellow, with a slightly darker core in aged specimens, absence of discolouration with 5% (w/v) KOH. Ascomata perithecial, aggregated, scattered, immersed in stromata, subglobose, coriaceous, papillate, ostiolate. Ostioles raised-discoid, black, with a central, cylindrical papilla. Peridium dark brown, turning subhyaline inwardly, containing cells of textura porrecta mixed with textura angularis, evenly thickened. Paraphyses sparse, hypha-like, narrowing toward the ends, hyaline, septate, slightly constricted at the septa, unbranched, longer than asci, embedded in mucilaginous matrix. Asci (6–)8-spored, unitunicate, cylindrical, long-stipitate, total length, with apical apparatus, appearing bilobed, amyloid, cuboid in Melzer’s reagent, reddish brown in Lugol’s solution, turning blue in Lugol’s solution after treatment with 3% (w/v) KOH, inverted trapezoid. Ascospores overlapping uniseriate in the ascus, globose to oval or fusiform with acute ends, hyaline when young, becoming brown at maturity, with single, central guttule or with a large central and two small polar guttules, smooth, thick-walled, with a conspicuous, S-shaped germ slit one-half to four-fifths spore length on the flattened side. Anamorph: nodulisporium-like with periconiella-like branching patterns.

Notes: The phylogenetic analyses of combined ITS, LSU, TUB2, and RPB2 loci and phylogenomic analyses showed that these five collections (HKAS150824, HKAS151626, HKAS150823, HKAS148941, HKAS148942) clustered forming a well-supported clade (Figure 1). Morphological characters of collections in this clade are significantly different from those of sister clades. Therefore, we establish this clade as Dianjunus gen. nov.

Dianjunus rex F.Q. Yu, Pérez-Moreno & Senan., sp. nov. Figures 4–6

Index Fungorum number: IF904732

Etymology: The species epithet rex (Latin = king) refers to the large size and majestic appearance of the stromata.

Type: China, Yunnan Province, Kunming City, Luquan Yi and Miao Autonomous County, Tuanjie Town, Damaidi, associated with roots of Quercus variabilis Blume (Fagaceae), 5 August 2024, Jing Song, (HKAS148941, holotype), ex-type culture GBWSCC202508860, HKAS151626 (dried culture); ibid. 6 September 2024, Senanayake IC (HKAS148942, Paratype).

From roots of dying host plants and arise from rhizosphere soil. Teleomorph: Stromata upright, slender to plump fleshy, 20–50 cm total height, with fertile parts 10–20 cm high, 7–10(–15) cm diam., 250–600 g weight, variable size and shape, ranging from cylindrical, fusiform, turbinate to palm-shaped, terete to flattened, simple to branched from the base, or at the top, arising from long rooting stipes, with flattened sterile apices. Outer crust 200–300 μm thick, coriaceous, black, ostiole openings appear as black dots, ostiolar papillae are distributed in this layer. Aerial part fertile, surface covered with a long persistent, powdery, whitish yellow fibrous substance, whitish in young stromata, turning progressively dark yellow at maturity. Immersed part 20–30 cm high, 3–6 cm wide, sterile, consisting of stipes, cylindrical, black, no powdery substance, smooth. Interior tissues solid, homogeneous to fibrous, spongy, off-white to yellow, with a slightly darker core in aged specimens, absence of discoloration with 5% (w/v) KOH. Ascomata 400–750 μm diam., 1200–1500 μm high, perithecial, aggregated, scattered, immersed in stromata, subglobose, coriaceous, centrally papillate, ostiolate. Ostioles 150–280 μm diam., raised-discoid, black, with a cylindrical papilla at center. Paraphyses 4–7 μm broad (x̄ = 5 μm), sparse, hyphal, thin-walled, narrowing toward the ends, hyaline, septate, slightly constricted at the septa, unbranched, longer than asci, embedded in mucilaginous matrix. Peridium 70–100 µm (x̄ = 80 μm), thick, dark brown, turning subhyaline inwardly, containing cells of textura porrecta mixed with textura angularis, evenly ticked. Asci (6–)8-spored, unitunicate, cylindrical, long-stipitate, 115–160 × 8–10 µm (x̄ = 2.5 × 3 μm) total length, the spore-bearing parts 90–100 × 8–10 µm, with bilobed, apical apparatus, 2–3 µm high, 3–5 µm wide (x̄ = 3 × 4 μm) in water; cuboid, amyloid, 2–3 µm high, 2.8–3.2 µm wide (x̄ = 2.5 × 3 μm), in Melzer’s reagent; inverted trapezoid, reddish brown in Lugol’s solution, 2.5–3 µm high, 3–4.5 µm wide (x̄ = 3 × 4 μm), turning blue in Lugol’s solution after treatment with 3% (w/v) KOH. Ascospores 12–17 × 5–7 µm, overlapping uniseriate in the ascus, globose to oval or fusiform with acute ends, hyaline when young, becoming brown at maturity, with single, central guttule or with a large central and two small polar guttules, smooth, thick-walled, with a conspicuous, S-shaped germ slit one-half to four-fifths spore length on the flattened side. Anamorph: nodulisporium-like anamorph with periconiella-like branching patterns. Mycelium consisting of septate, branched, smooth-walled hyphae, hyaline to pale brown. Conidiophores 17–25 µm length, 6–8 µm width (x̄ = 20 × 7 µm, n = 10), macronematous, mononematous, erect, septate, smooth, pale to dark brown, arising singly from hyphae; branching profuse and arborescent (periconiella-like), with primary and secondary branches producing numerous short terminal branchlets. Conidiogenous cells 7–10 µm length, 3–6 µm width (x̄ = 9 × 5 µm, n = 20), terminal, polyblastic, sympodially proliferating (nodulisporium-like), denticulate; conidiogenous loci minute, with small truncate scars. Conidia 6–9 µm length, 3–5 µm width (x̄ = 8 × 4 µm, n = 25), produced singly on denticles, ellipsoid to ovoid, hyaline, smooth, unicellular.

Culture characteristics: Colonies after 7 days of incubation at 20 °C in dark; on PDA reaching 5 cm diam., circular, smooth margin, flat, bearing abundant aerial mycelium, initially white, turning to yellowish-white with time, lack of pigment production, heavily sporulated with black, globose conidiomata; on MEA reaching 4.5 cm, circular, thin, arachnoid, with abundant, initially white, later turning to blackish brown, radiating, dendritic hyphae; center compact, slightly olivaceous to grayish; margin even to diffuse, reverse view blackish, radiating, dendritic hyphae start from the center and spread toward the margin, lack of pigment production, fairly sporulated with black, globose conidiomata; on OMA reaching 5 cm, circular, white, dense, cottony to floccose, with abundant aerial mycelium, center compact and grayish, surface faintly radially striate with weak zonation, margin entire to diffuse, reverse view with circular, dark olivaceous to olive-brown pigmentation; central region buff to cream, sporulation did not occur.

Additional material examined: China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Mengla County, Mengla Town, attached to trunk and root of decaying unidentified tree, 14 July 2025, Xiao Aka, (HKAS150824); ibid., Chuxiong Yi Autonomous Prefecture, Lufeng City, Yipinglang Town, from roots of Quercus sp., 10 July 2025, Xie Tao, (HKAS150823).

Notes: In the combined multilocus phylogeny analysis of ITS, LSU, TUB2, and RPB2, the collections of Dianjunus species clustered together forming a distinct clade with support value of ML/BI = 100%/1.00 (Figure 1). The bootstrap value in phylogenomic analyses was also >90% (Figure 2). All the collections of Dianjunus rex are morphologically similar, and stromata of tropical collections are larger than those of subtropical collections (Figure 4). The young stromata mostly have bulb-like apex and they form finger-like branches when mature. The fresh stromata are very fleshy and dehydrate rapidly after being detached. There is no strong smell or taste from stromata. Therefore, based on morphological characters and phylogeny, these collections are introduced as a new species, Dianjunus rex. This anamorph described above is derived from pure cultures obtained from single-ascospore isolation. Both cultures obtained from single-spore isolation and conidia suspension of sporulated culture were used for molecular sequence comparison. The sequences of the ITS locus of both cultures are identical to each other.

Our collections morphologically resemble the genus Squamotubera P. Henn. Hennings (1903) erected the genus Squamotubera for the single species, S. le-ratii P. Henn., which was collected from Noumea, New Caledonia (island between Australia and New Zealand) from soil. Lloyd (1917) noted that Squamotubera le-ratii is similar to Sarcoxylon compunctum (Jungh.) Cooke except for its horizontal or upright stromatal development from bark of living plants. Therefore, it was synonymized as Sarcoxylon le-ratii (P. Henn.) Lloyd. Rogers (1981) also argued that Squamotubera might be congeneric with Sarcoxylon. However, considering the upright stromatal development of Squamotubera from subterranean sclerotia, Rogers (1981) conserved Squamotubera as a distinct genus and mentioned the requirement of further studies to clarify it. The genus Squamotubera is only known from its short Latin protologue by Hennings (1903) with no illustrations. Rogers (1981) noted that the holotype specimen of Squamotubera le-ratii is not available in specimen repositories (Farlow Herbarium, FH).

In the original description, Hennings (1903) described the stromata of Squamotubera le-ratii as “tuberiform, round depressed with squamose external surface”. Rogers (1981) amended the description and mentioned, “the suspicion that stromata arise from subterranean sclerotia” and “the exterior is convoluted with smaller wrinkles overall”. However, our collections do not fit any of these two characteristics. It is neither “tuberiform” nor “squamose or with wrinkles overall”. Apart from these diagnostic characteristics, there are other important taxonomic traits that do not fit with the description provided by Hennings (1903) or Rogers (1981) such as “interior pallid subcarnose” or “tan, solid with irregular lacunae”, while in our fungus, the inner context was “spongy, off-white to mostly yellow”.

Rogers (1981) described “Capitate apices quickly become soft with gelatinous areas beneath the perithecial layer” as a characteristic never recorded in our specimens, whose context always remains fibrous, spongy. Additionally, Rogers (1981) described “Parasite sur les vielles souches de Casuarina” [sic] in French, which means “Parasite on the old stumps of Casuarina sp.”, a host plant not recorded for our fungus. Germ-slits in ascospores were not reported from S. le-ratii by Hennings (1903) and Rogers (1981) while our collections have S-shaped germ-slits. The stromata of Squamotubera are 7 cm high and 5 cm wide, while stromata in our collections are 20–50 cm high and 7–10 (-15) cm diam. Therefore, our collections are significantly different from Squamotubera le-ratii (Table 4).

Table 4. Morphological character comparison of collections of Squamotubera and Dianjunus.

4 Discussion

Yunnan Province, situated in southwestern China, is recognized for its exceptional fungal diversity (Feng and Yang, 2018). Its varied topography, encompassing subtropical forests, alpine meadows, grasslands, and wetlands, creates a spectrum of ecological niches and microhabitats that support a rich mycota (Sun et al., 2017). Driven by complex landscapes, diverse floristic communities hosting numerous endemic species, and predominantly tropical to subtropical climates, Yunnan constitutes a biodiversity hotspot for fungi. While over 6,000 species have been documented, total fungal richness is estimated to potentially exceed 100,000 species, with frequent discoveries of novel taxa and genera continuing to highlight the region’s underexplored status (Senanayake et al., 2022). Research has extensively documented major phyla such as Ascomycota and Basidiomycota.

The Province hosts a significant diversity of ectomycorrhizal fungi, which form symbiotic associations with tree roots, particularly in temperate and subtropical forests (Liu et al., 2009; Dong et al., 2024; Yang et al., 2025; Wang et al., 2026). Commonly encountered genera include Boletus, Ganoderma, Russula, Tricholoma, Tuber (truffles), and Xylaria. These fungi play critical roles in nutrient cycling and forest ecosystem functioning. Yunnan is also notable for its edible fungi, especially Morchella species and truffles (genus Tuber), which hold substantial culinary and economic value both locally and globally. The utilization of wild edible mushrooms represents an important economic activity though it raises concurrent concerns regarding sustainable harvest and ecological impact. Thus, the diversity of edible fungi is a major focus of both ecological and economic research.

Up to now, very few species in Xylariales have been reported to produce massive stromata. The size of the stromata in Xylariales depends on the species, mode of life, and geographical location of collections. Engleromyces goetzei P.Henn. represents the largest species reported within the order Xylariales to date. Engleromyces goetzei produces subglobose, two-lobed stromata that are seated on and partially enveloping bamboo culms (Whalley et al., 2010). Gibson and Kimeria (1962) reported Engleromyces goetzei from North Kenya, where it forms subglobose stromata growing toward the tops of bamboo stems, about 30–40 ft. from the ground, varying considerably in size, from small growths weighing a few ounces to the largest with a diameter of nearly 30 cm and weighing around 3.2 kg. Meanwhile, Engleromyces sinensis M.A. Whalley, A. Khalil, T.Z. Wei, Y.J. Yao & Whalley, sampled in Yulong County in Yunnan Province, China, presented much smaller stromata that enveloped the bamboo culms and formed two lobes; the stromata were globose to subglobose, with sizes of 4.3–4.9 × 4–5.5 cm in width and 1.6–4 cm in height (Whalley et al., 2010). Most other species in Xylariales that morphologically resemble Dianjunus rex are Daldinia concentrica (Bolton) Cesati & de Notaris, Xylaria hypoxylon (L.) Grev., X. cubensis (Mont.) Fr., and X. longipes Nitschke. However, the majority of them produce black stromata. However, Dianjunus rex differs, apart from its larger size, in having less branched, pale to dark yellow stromata with blunt apex (Phillips et al., 2005).

Beyond its morphological distinctiveness, Dianjunus rex is phylogenetically isolated from all other known taxa in Xylariales. Divergence time estimates by Samarakoon et al. (2022) place the split between Amphisphaeriales and Xylariales at 154 (117–190) million years ago (MYA), with crown ages of 127 (92–165) MYA and 147 (111–184) MYA, respectively. Our analysis estimates the family Dianjunaceae diverged at approximately 65 (50–80) MYA (Figure 2), a timeframe consistent with the common family-level divergence trend of 50–150 MYA reported for fungi (Hyde et al., 2017). The phylogenomic topologies generated by the concatenation and summary-coalescent (ASTRAL) approaches were largely congruent, with one notable exception in a non-target lineage. Specifically, the phylogenetic position of Stanjemonium grisellum exhibited topological discordance between the concatenation tree and the two ASTRAL species trees (which were perfectly consistent with each other) (Supplementary Data). In the ASTRAL analyses, the local posterior probability (LPP) for this discordant node was 0.84 for the BUSCO dataset and dropped to 0.62 for the OrthoFinder dataset (Figure 7). This moderate-to-low support indicates historical gene tree conflicts, likely arising from widespread incomplete lineage sorting (ILS) or historical hybridization events within this non-target group. Crucially, despite this isolated background conflict, our core target topology was flawlessly and robustly preserved across all three distinct workflows. Dianjunus was consistently resolved as a sister group to the Graphostromataceae with maximum support (Bootstrap = 100 in the concatenation tree; LPP = 1.0 in both ASTRAL trees). This definitive consistency across diverse datasets and evolutionary models demonstrates that the taxonomic and evolutionary placement of our target family and genus is unequivocally stable (Figure 7). There are 432 monotypic fungal families, representing 35.4% of total known families in Kingdom Fungi (Hyde et al., 2024).

Species in Graphostromataceae show wide variations in anamorph morphology mainly described from sporulate cultures by nodulisporium-like conidial formation. However, sometimes other types of conidial formation have also been observed, such as sporothrix-like, rhinocladiella-like, virgariella-like, xylocladium-like, and periconiella-like (Ju and Rogers, 1996; Wendt et al., 2018). Our collections form a nodulisporium-like anamorph with periconiella-like branching patterns. The conidiophores are 15–25 µm long, with distantly packed branchlets bearing clusters of cylindrical, conidiogenous cells that are distinct from those of species in Graphostromataceae.

Comparative genomics revealed a conserved contraction of CAZyme genes (~220 genes) across all three D. rex strains compared to related saprobic Xylariales (>350 genes; Miyauchi et al., 2020). The most striking feature is the near-total depletion of pectin lyases (PL = 0-1, vs. 7–19 in related saprobes; Figure 2). In plant–fungus interactions, pectinases play a pivotal role in the initial breakdown of host plant cell walls, and their cleavage products can act as damage-associated molecular patterns (DAMPs) that trigger host immune responses (De Lorenzo et al., 2019). The loss of PL genes indicates a reduced genomic potential for pectin degradation, a feature consistent with genomic signatures of certain biotrophic or latent fungal endophytes (Mesny et al., 2021).

Concurrently, glycosyltransferases are proportionally enriched (GTs: ~26% of total CAZymes; Figure 2), which are primarily responsible for fungal cell wall biosynthesis. Because fruiting bodies of D. rex were observed exclusively post-host mortality, these combined traits support a working hypothesis of an endophyte-to-saprotroph lifestyle transition (Selosse et al., 2018). In this scenario, low metabolic maintenance costs of extracellular degradative enzymes, paired with sustained allocation to cell wall synthesis, may facilitate rapid carbon channeling into fungal biomass to form its massive stromata (Figure 5a). Such genomic adaptations accord with the dual ecological strategies widely observed in xylarialean endophytes, where genomic streamlining aligns with metabolic trade-offs between symbiotic latency and saprotrophic competence (Ren et al., 2022).

5 Conclusion

This study elucidates the taxonomic identity and phylogenetic relationships of an exceptional macromycete discovered in the subtropical and tropical forests of Yunnan Province, China. Through an integrative taxonomic approach combining detailed morphological examination, multi-locus phylogenetic analysis (ITS, LSU, RPB2, and TUB2), and phylogenomic data, the fungus is formally described as Dianjunus rex gen. et sp. nov. Phylogenetic reconstructions robustly place this novel taxon within the order Xylariales, where it forms a distinct, well-supported lineage sister to the family Graphostromataceae. This phylogenetic isolation, coupled with a unique suite of morphological characters, including massive, upright, fleshy stromata, a persistent powdery-fibrous surface, and a nodulisporium-like anamorph, is proposed to establish the new family Dianjunaceae. Divergence time estimation, calibrated using a genome-scale dataset, indicates that the Dianjunaceae lineage diverged during the early Paleocene, approximately 65 (50–80) million years ago. This timing coincides with the Cretaceous–Paleogene (K–Pg) boundary and the subsequent period of significant planetary ecological reorganization. The emergence of this morphologically distinct lineage in the aftermath of a major extinction event may reflect an adaptive radiation or the exploitation of newly available ecological niches during forest ecosystem recovery. The stromata of D. rex, reaching dimensions of up to 50 cm in height and 2.2 kg in weight, represent the largest stromata documented within the Ascomycota. This discovery significantly expands the known morphological disparity of the Xylariales, demonstrating that the evolution of gigantism in ascomycete fructifications has occurred independently in multiple lineages, including the stromata-forming Xylariales and the apothecia- or truffle-forming Pezizales. The discovery of Dianjunus rex and the Dianjunaceae underscores the profound and largely unexplored fungal diversity within East Asian forests, particularly in the biodiverse regions of southwestern China. It affirms the critical importance of continued mycological exploration integrated with modern molecular systematics, which remains essential for revealing hidden lineages, understanding the evolutionary trajectories of fungal morphology, and elucidating the complex history of Earth’s ecosystems.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ffunb.2026.1940464/full#supplementary-material

Supplementary material Supplementary material at the source

Footnotes

Edited by: Rajesh Jeewon, University of Mauritius, Mauritius

Reviewed by: Rui-Lin Zhao, Chinese Academy of Sciences (CAS), China

Changlin Zhao, Southwest Forestry University, China