Phylogeny and comparative analysis of mitochondrial genomes of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes), with descriptions of G. matijun J.W. Liu & F.Q. Yu and G. bijiensis sp. nov.

357 Phylogeny and comparative analysis of mitochondrial genomes of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes), with descriptions of G. matijun J.W. Liu & F.Q. Yu and G. bijiensis sp. nov. Xianyi Wang1,2,3*, Zhongyao Guo1,2*, Jiawei Tao1, Yunhan Guo1,2, Guoyu Wang1, Guangyin Xu2, Qirui Li4, Hongmei Liu2,3 1 Engineering Research Center of Health Medicine, biotechnology of Institution of higher education of Guizhou Province, Guizhou Medical University, Guiyang, China 2 Engineering Research Center of Medical Biotechnology, School of Biology and Engineering, Guizhou Medical University, Guiyang, China 3 Laboratory Animal Center of Guizhou Medical University, Guiyang, China 4 TheHighEfficacyApplicationofNaturalMedicinalResourcesEngineeringCenterofGuizhouProvince(TheKeyLaboratoryofOptimalUtilizationofNatural Medicine Resources), School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang, China Correspondingauthors:QiruiLi([email protected]);HongmeiLiu([email protected]) Copyright: © Xianyi Wang et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The genus Gomphus Pers. presents persistent taxonomic challenges due to its morphological similarities with related genera. In this study, we collected two specimens of Gomphus from Guizhou, China; one specimen is described as a new species, Gomphus bijiensis sp. nov. and the other is identified as G. matijun J.W. Liu & F.Q. Yu based on morphological traits and phylogenetic analyses of the nuclear rDNA internal transcribed spacer (ITS) and nuclear rDNA large subunit (LSU). To resolve their evolutionary relationships we assembled and annotated the mitochondrial genomes of both species using next-generation sequencing. Comparative analyses revealed codon usage strongly biased toward Aor U-ending codons, consistent with the low GC content typical of fungal mitochondria. Variation in protein-coding gene lengths and base composition suggests that diverse evolutionary pressures have shaped these genomes. Divergence time estimation indicates that morphological diversity within Gomphus and related macrofungi has largely resulted from convergent evolution. Phylogenetic reconstruction places G. bijiensis and G. matijun within a distinct clade, supporting their close evolutionary affinity and the coexistence of ancestral and derived traits. This study provides the first comprehensive mitochondrial genomic data for Gomphus, offering new insights into its taxonomy, phylogeny, and evolutionary dynamics, and establishing a framework for future studies within the Gomphaceae. Key words: Clavarioid fungi, comparative genomics, evolutionary adaptation, fungal diversity, Gomphaceae Introduction The genus Gomphus Pers. (family Gomphaceae) represents a taxonomically challenging group due to its pronounced morphological overlap with related genera. First established by Persoon in 1797 with G. clavatus designated as the type species, the genus has since undergone repeated reassessment. Academic editor: Samantha C. Karunarathna Received: 19 May 2025 Accepted: 6 October 2025 Published: 13 November 2025 Citation: Wang X, Guo Z, Tao J, Guo Y, Wang G, Xu G, Li Q, Liu H (2025) Phylogeny and comparative analysis of mitochondrial genomes of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes), with descriptions of G. matijun J.W. Liu & F.Q. Yu and G. bijiensis sp. nov. MycoKeys 124: 357–381. https://doi.org/10.3897/ mycokeys.124.158670 MycoKeys 124: 357–381 (2025) DOI: 10.3897/mycokeys.124.158670 This article is part of: Exploring the Hidden Fungal Diversity: Biodiversity, Taxonomy, and Phylogeny of Saprobic Fungi Edited by Samantha C. Karunarathna, Danushka Sandaruwan Tennakoon, Ajay Kumar Gautam * These authors contributed equally to this work. 358 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Morphologically, Gomphus species are typically fan-shaped to funnel-shaped, display distinctive coloration, and form ectomycorrhizal associations with members of the Fagaceae, Myrtaceae, and Pinaceae (Giachini 2004). This variation in macromorphology has frequently obscured taxonomic boundaries, contributing to the reassignment of several species into allied genera such as Gloeocantharellus Singer, Phaeoclavulina (Corner) Brinkmann, and Turbinellus Earle (Giachini et al. 2010). Molecular phylogenetic analyses further complicate the picture, with earlier studies suggesting paraphyly within the Gomphaceae (Miller 2002). On the basis of Giachini’s taxonomic framework and subsequent molecular evidence, Gomphus sensu stricto, once comprising about 35 species, has now been reduced to 16 accepted species (Index Fungorum, accessed August 2025). Gomphus is primarily distributed in subtropical and temperate regions of Asia, Europe, and North America. Within the PR of China, eight species are recognized, including G. clavatus, G. roseus R.H. Petersen, G. matijun, and the newly described taxon presented in this study (Liu et al. 2022). This update highlights the increasing recognition of Gomphus diversity, particularly in Asia, where ongoing molecular and morphological studies continue to refine its taxonomy (Singh et al. 2024). The morphological variability within the genus includes traits such as the wrinkled to nearly poroid structure of the hymenium and the warty (verrucose) nature of basidiospores, once considered distinctive to Gomphus, which have now been observed in other genera within Gomphaceae (Humpert et al. 2001). This morphological convergence has caused confusion and debate among mycologists, complicating the accurate identification and systematic placement of Gomphus species (Scambler et al. 2018). In addition to morphological complexities, the ecological roles of Gomphus species underscore their importance in forest ecosystems. As ectomycorrhizal fungi, they form symbiotic relationships with key forest trees, facilitating the exchange of nutrients and promoting the growth and health of their hosts (Fan et al. 2023). This ecological significance requires an understanding of Gomphus taxonomy to inform conservation efforts and protect forest biodiversity (Ye et al. 2020). Molecular studies have further highlighted the complexity of Gomphus classification. Earlier molecular phylogenetic analyses revealed that Gomphus sensu lato—which includes genera such as Gloeocantharellus Singer, Phaeoclavulina (Corner) Brinkmann, and Turbinellus Earle, as well as Ramaria sensu lato—are paraphyletic within the Gomphaceae (Giachini et al. 2011). These findings demonstrate the complex evolutionary relationships within the family and the need for taxonomic refinement. In response, several taxonomic rearrangements have occurred over the past few decades (Liu et al. 2022), resulting in the reclassification of some species previously placed in Gomphus into other genera (Petersen 1973; Villegas et al. 1999). Nouhra highlighted that several species within Gomphus sensu lato lacked clear diagnostic traits, creating persistent uncertainty regarding their taxonomic placement (Nouhra et al. 2005). Building on these earlier observations, Giachini and Castellano (2011) further refined the genus by demonstrating that Gomphus sensu stricto can be distinguished by specific features, including violet to lavender-brown or milky coffee-colored hymenia, recurrent clamp connections, and verrucose spores. These refinements 359 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions helped clarify the boundaries of Gomphus s. str. within the Gomphaceae, although some taxonomic ambiguities remain. In recent years, the study of mitochondrial genomes (mitogenomes) have gained attention as a powerful tool for phylogenetic analyses. The mitogenome, maternally inherited and relatively small, offers a valuable source of genetic information for reconstructing evolutionary relationships among species (Sun et al. 2022). Its high mutation rate and frequent recombination make it particularly useful for distinguishing closely related species and tracing their evolutionary history (Luo et al. 2024). This genetic approach has enabled researchers to uncover evolutionary lineages with greater precision, allowing for the identification of species that were previously overlooked due to morphological similarities (Li et al. 2023). Moreover, mitogenome analysis provides insights into the timing of evolutionary events, allowing researchers to estimate species divergence times and construct more accurate phylogenetic trees. This method is especially useful in cases where fossil records are sparse or incomplete, offering a genetic perspective on the evolutionary history of organisms (Arcones et al.2021). The increasing availability of mitogenome data, combined with advanced computational tools, has revolutionized our understanding of fungal evolution and diversification. As more mitogenomes are sequenced, new insights into the processes shaping species boundaries and evolutionary trajectories are expected. In this study, we investigate the mitogenomes of two Gomphus species collected from Guizhou, China, including one previously undescribed taxon. Alongside comparative mitogenomic analyses, we formally identify and described this new species, G. bijiensis sp. nov., integrating morphological and molecular evidence. The objectives of our study are: (1) to formally describe G. bijiensis as a distinct lineage within the genus; (2) to elucidate the structural and compositional features of the two Gomphus mitogenomes; (3) to identify variation and conservativeness between Gomphus and other Gomphaceae mitogenomes through comparative analyses; (4) to determine the phylogenetic position of Gomphus within the Basidiomycota using mitochondrial gene datasets; and (5) to examine the evolutionary significance of Gomphus morphology through divergence-time estimation. This work represents the first publication of complete mitogenomes in Gomphus, while simultaneously advancing its taxonomy and deepening our understanding of the evolutionary dynamics of the Gomphaceae. Methods Specimen collection and preservation Specimens were collected and catalogued from distinct locations in Guizhou Province, PR of China (Rathnayaka et al. 2025). The specimen of G. matijun was collected from the Qiannan Buyi and Miao Autonomous Prefecture, while G. bijiensis was collected from Niubang Township in Weining County, Bijie City. Following collection, specimens were identified morphologically and their habitats were photographed. The samples were then air-dried naturally and deposited at the Engineering Research Center of Medical Biotechnology, Guizhou Medical University, with specimen identifiers MY1010 and MY449. 360 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Morphological identification To identify the species, detailed morphological examinations were conducted on both fresh and dried specimens. Macroscopic features, such as overall shape, size, coloration, surface texture, and hymenophore arrangement, were described using the terminology outlined in Giachini (Giachini et al. 2012). Macroscopic features were examined and photographed using the Nikon SMZ-745T stereomicroscope (Nikon, Japan). Spore ornamentation was further analyzed using the Phenom LE scanning electron microscope (Phenom, Netherlands). Measurements were based on at least 30 spores per specimen, excluding abnormally shaped or immature spores. Morphological descriptions were compared with taxonomic treatments and regional keys in Flora Fungorum Sinicorum (Liu et al. 2022) and other relevant monographs to ensure accurate identification and comparison with previously described Gomphus taxa. DNA extraction Genomic DNA from two Gomphus species were extracted using the DNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s protocols, with modifications to optimize DNA yield and purity for downstream genomic analysis. Freshly collected or herbarium-preserved specimens were used for DNA extraction. To minimize contamination and ensure DNA quality, small pieces of inner context tissue were carefully excised from the fruiting bodies. For fresh collections, tissue was taken immediately after field sampling while for preserved material dried specimens deposited in the herbarium were used. Uncontaminated internal tissue was selected to obtain reliable molecular data. Approximately 50 mg of tissue was excised from the pileus (cap) region of the fruiting bodies, ensuring a representative sample for DNA extraction. The tissue samples were initially ground into a fine powder using a sterile mortar and pestle under liquid nitrogen to break down the cell walls and facilitate the release of cellular contents. The powdered tissue was then transferred into a 1.5 mL microcentrifuge tube, to which 400 µL of the provided lysis buffer (AP1) was added. The samples were incubated at 65 °C for 10 minutes, allowing for cell lysis. During this time, proteins, lipids, and other cellular materials were solubilized, making the DNA more accessible for extraction. Following incubation, the samples were briefly centrifuged to remove any large particulate matter, and an equal volume of buffer AP2 was added to neutralize the lysate. The mixture was then vortexed and centrifuged at 13,000 rpm for 5 minutes to separate the debris from the supernatant, which contained the genomic DNA. The supernatant was transferred to a new tube, and the DNA was precipitated by adding an equal volume of 100% ethanol. After mixing, the sample was centrifuged at 13,000 rpm for 10 minutes to pellet the DNA. The DNA pellet was washed twice with 70% ethanol to remove any residual impurities. Following air drying, the DNA pellet was resuspended in 50 µL of sterile, deionized water. The quality and concentration of the extracted DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific) by measuring the absorbance at 260 nm (A260) and 280 nm (A280). The purity of the DNA was evaluated by calculating the A260/A280 ratio, which typically ranged from 1.8 361 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions to 2.0, indicating high-quality DNA suitable for downstream applications such as PCR amplification, sequencing, and genome assembly. For long-term storage, the extracted DNA was kept at -20 °C for future analysis. The final DNA samples were ready for PCR amplification, sequencing, and other molecular analyses aimed at identifying species-specific markers and constructing the complete mitogenomes of the two Gomphus species. Molecular identification Molecular identification of the novel Gomphus species was performed using DNA barcoding techniques. PCR was conducted using universal primers for the nuclear ribosomal internal transcribed spacer (ITS) region and the large subunit rDNA (LSU) gene (Xue et al. 2019), both of which are used in species identification. PCR reactions were performed in a 25 µL volume containing extracted DNA, primers, dNTPs, Taq DNA polymerase, and buffer. The thermocycler protocol consisted of an initial denaturation step followed by cycles of denaturation, annealing, and extension, culminating in a final extension step. The internal transcribed spacer (ITS) and nuclear large subunit (nLSU) regions were amplified using primer pairs ITS1/ITS4 and LR0R/LR5, respectively (Xue et al. 2019). PCR reactions were performed in 25 μL volumes containing 1 × reaction buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.4 μM of each primer, 1 μL Taq DNA polymerase (Takara, Dalian, PR of China), and approximately 30 ng of template DNA. The thermocycler program consisted of an initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 40 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. PCR products were purified using a Gel Extraction Kit (Omega Bio-Tek, USA) and sequenced bi-directionally at Sangon Biotech (Shanghai). The resulting sequences were edited and assembled using Geneious Prime 2023.2.1 software (https://www.geneious.com). Consensus sequences were compared against reference sequences in the NCBI GenBank (Suppl. material 1: table S1) database via BLAST to confirm species identity. Phylogenetic analyses were performed using PhyloSuite v1.2.3 (Zhang et al. 2020), an integrated platform for phylogenetic reconstruction that incorporates maximum likelihood (ML) methods. For ML analysis, the optimal evolutionary model was selected using ModelFinder within PhyloSuite, and the tree was constructed with IQ-TREE (1,000 bootstrap replicates) to assess branch support. Mitochondrial genome sequencing, assembly, and annotation The mitogenomes were sequenced using next-generation sequencing technology. Raw sequence data were assembled using Geneious Prime 2023.2.1 (Wang et al. 2025a) and aligned with the mitochondrial reference sequence of Turbinellus floccosus (Schwein.) Giachini, Osmundson, A. Sánchez-García & Spatafora (NC045218) and Ramaria flavescens (Schaeff.) Quél. (PP847337). Genome annotation was conducted using MFannot (Valach et al. 2014) and MITOS (Bernt et al. 2013) based on the mitochondrial genetic code 4 to identify protein-coding genes (PCGs), open reading frames (ORFs), rRNAs, tRNAs, and introns in the two Gomphus mitogenomes. ORFs exceeding 100 amino acids were further analyzed through BLASTP searches against the NCBI Open 362 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Reading Frame Finder and the NCBI non-redundant protein sequence database. The annotated tRNA genes were validated using tRNAscan-SE v1.3.1 software (Chan et al. 2021), and physical maps of the two Gomphus mitogenomes were constructed using OGDraw v1.2 (Greiner et al. 2019). Base composition and strand asymmetry analysis The base composition of the mitogenomes of Gomphus and other Gomphaceae species were determined using DNASTAR Lasergene v7.1 (http://www.dnastar. com/). Strand asymmetry was calculated using the formulas: GC skew = [G − C] / [G + C] and AT skew = [A − T] / [A + T]. Codon usage and substitution rate analysis Codon usage in the Gomphus mitogenomes were analyzed using Sequence Manipulation Suite (Stothard 2000) with genetic code 4. The nonsynonymous (Ka) and synonymous (Ks) substitution rates of protein-coding genes (PCGs) in the mitogenomes of Gomphus and other Phallomycetidae were calculated using DnaSP v6.10.01 (Rozas et al. 2017). Genetic distance and genome homology The genetic distance between each pair of the 15 major PCGs was determined using the Kimura-2 parameter (K2P) substitution model in MEGA v6.06. Genome-wide homologies among the seven chimeric species were assessed using AliTV (https://alitvteam.github.io/AliTV/d3/AliTV.html), with all mitochondrial genomes aligned starting from the cox1 gene. Phylogenetic analysis A comprehensive molecular approach was used to construct a phylogenetic tree of Basidiomycetes based on mitochondrial genes. Two datasets were created from 59 Basidiomycetes: (1) PCG, consisting of the concatenated sequences of 15 conserved PCGs, and (2) PCG12, comprising the first and second codon positions of these 15 conserved PCGs. Tremella fuciformis Berk. (Ascomycota) was used as the outgroup. Individual mitochondrial genes were aligned using MAFFT v7.037. The aligned genes were then concatenated into a single mitochondrial gene set using PhyloSuite v1.2.3 (Zhang et al. 2020). A preliminary partition homogeneity test was conducted to assess phylogenetic inconsistencies among genes. The best-fit evolutionary model and partitioning scheme were determined using PartitionFinder 2.1.1 (Lanfear et al. 2017). Phylogenetic trees were constructed using Bayesian inference (BI) with MrBayes v3.2.6 (Ronquist et al. 2012) and maximum likelihood (ML) with IQ-tree v1.6.3 (Nguyen et al. 2015). Phylogenetic analysis and time-divergence estimation A time-divergence phylogenetic tree was constructed using 59 mitogenomes. The complete mitogenomes were aligned using the MAFFT v7.037 software to ensure accurate alignment of homologous regions. Poorly aligned 363 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions regions were identified and removed using Gblocks v0.91b to enhance the reliability of the alignment. Bayesian inference (BI) analyses were performed using MrBayes v3.2.7 (Ronquist et al. 2012) with two independent runs, each comprising four chains, for 10 million generations, sampling every 1,000 generations. The average standard deviation of split frequencies fell below 0.01, indicating convergence between runs. The first 25% of sampled trees (2,500 trees) were discarded as burn-in, and the remaining trees were used to construct a majority-rule consensus tree. Posterior probabilities were calculated from the post-burn-in trees to assess branch support. Convergence and effective sample size (ESS > 200) were further confirmed using Tracer v1.7 (Drummond et al. 2012). For time-divergence estimation, a molecular clock model was implemented using BEAST v2.6.3. The alignment was analyzed under an uncorrelated log-normal relaxed clock model, which accounts for rate variation among lineages. Calibration points, based on fossil records and previously published divergence times, were applied to estimate the divergence times of key nodes (Wang et al. 2025b). The Markov chain Monte Carlo (MCMC) analysis was run for a sufficient number of generations, with parameters sampled at regular intervals. The resulting phylogenetic tree, annotated with divergence times, was initially visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). For further refinement of the visual presentation, we utilized Adobe Illustrator to optimize the tree’s graphical elements. Referencing the taxonomic hierarchy and divergence time frameworks from TimeTree (http://timetree.org/), adjustments were made to enhance clarity, including refining branch lines and standardizing node labeling. Results Identification of a new Gomphus species To confirm the novelty of the new species, BLAST analyses of its ITS and nLSU sequences were performed against the NCBI nucleotide database (nr/nt) using BLASTn. For the ITS sequence, the top hit was G. clavatus (PQ652817), with 85.78% identity, 94% query coverage, an E-value of 0, and a max score of 891. The second closest match was G. ludovicianus R.H. Petersen & Justice, N. Amer (NR169660) with 85.19% identity, 91% coverage, an E-value of 0, and a max score of 873. For the nLSU sequence, the highest similarity was observed with G. matijun (NG228916) (96.03% identity, 91% coverage, E-value=0, max score=1539), followed by G. ludovicianus (NG241961) (95.51% identity, 97% coverage, E-value=0, max score=1,519). The relatively low sequence identities (<98%) with known species in both ITS and nLSU regions further support the recognition of this taxon as a new species. The maximum likelihood (ML) phylogenetic tree inferred from ITS sequences (Fig. 1) provides strong molecular evidence in support of recognizing Gomphus bijiensis as a distinct species. In this tree, G. bijiensis forms a well-supported monophyletic clade with a bootstrap value of 100, clearly separated from other congeners. In addition, the phylogenetic tree inferred from LSU sequences (Suppl. material 2) revealed that G. bijiensis was distinctly separated from other congeneric species within Gomphus, which is congruent with the ITS-based inference in reflecting its affinity to these taxa while still maintaining genetic distinctiveness. 364 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Collectively, both molecular datasets strongly support the designation of G. bijiensis as a phylogenetically independent species within Gomphus. Taxonomy Gomphus bijiensis Wang, Guo & Liu, sp. nov. Fungal Names: FN 572719 Fig. 2a–c Diagnosis. Gomphus bijiensis is phylogenetically distinct from G. indicus K. Das, Hembrom & R. Kujur and G. matijun (bootstrap support = 100) and differs Figure 1. Maximum likelihood (ML) phylogeny of 49 fungal species based on ITS sequences. Bootstrap support values (≥65%) are indicated at the nodes. The newly described species G. bijiensis is highlighted in red, while G. matijun and other ingroup taxa are shown in black. Outgroup taxa are indicated with a lighter background. 365 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions morphologically in having (1) smaller basidiocarps (6–11 cm vs. up to 15 cm in G. indicus), (2) a darker violet to purplish-brown pileus (6F6–7E8 vs. lighter brownish-orange in G. matijun), and (3) smaller, more prominently warted basidiospores [(7.5–)8.0–9.8(–10.5) × (4.5–)5.0–6.0(–6.5) μm vs. larger and less ornamented spores in G. indicus]. Etymology. The epithet bijiensis refers to the type location, Guizhou Province, PR of China. Holotype. PR of China • Guizhou Province, Bijie City, collected on soil under a Pinus forest, August 2023, coll. MY1010 (Herbarium of Guizhou Medical University). Description. Basidiocarps are robust, funnel-shaped to irregularly lobed, pileus 6–11 cm diam., violet to purplish-brown (Munsell: 6F6–7E8), surface rugulose to wrinkled. Hymenophore with blunt, forked folds, lacking lamellae, concolorous with pileus but paler at maturity. Stipe 4–9 × 1.5– 2.5 cm, solid, tapering toward base, surface whitish to cream (4A2–4A3) with brown fibrils. Flesh white, unchanging. Basidiospores (7.5–)8.0–9.8(– 10.5) × (4.5–)5.0–6.0(–6.5) μm, Q = 1.45–1.72, Qm = 1.58 ± 0.06 (n = 50), ellipsoid to broadly ellipsoid, hyaline, thick-walled, with irregular warted ornamentation. Basidia 35–42 × 7–9 μm, clavate, 4-spored, with basal clamp connections. Hyphae in trama hyaline, 3–6 μm wide, with irregularly thickened walls, clamp connections present. Habit, habitat, and distribution. Growing gregariously on soil in mixed Pinus forests. Known only from the type locality in Bijie City, Guizhou Province, PR of China (approx. 27°05'10"N, 103°48'50"E). Notes. Gomphus bijiensis is phylogenetically closely related to G. indicus and G. matijun, but both molecular and morphological data support its recognition as a distinct species. It differs from G. indicus in its smaller basidiocarps, darker pileus coloration, and more prominently ornamented spores, while it differs from G. matijun in pileus colour (deep violet to purplish-brown Figure 2. Gomphus bijiensis, a. Fresh samples; b. SEM images of basidiospores showing warted ornamentation; c. SEM images of mycelial structure. 372 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Time divergence analysis The phylogenetic analysis and timeline reveal that macrofungi with agaricoid (Fig. 9), coral-shaped, and spike-shaped morphologies have evolved independently across multiple lineages. Agarics, such as Agaricus bisporus (J.E. Figure 8. Phylogenetic analysis of 59 species (Suppl. material 1: table S4) using Bayesian inference (BI), based on 15 PCGs and 2 rRNA sequences. Species included in this study are marked with purple text. 373 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Lange) Imbach and Amanita muscaria (L.) Lam., emerged more recently during the Cenozoic era, likely in response to changing ecological conditions. Coral-shaped fungi, represented by Clavaria fumosa Pers. and R. rubella (Schaeff.) Quél., have a broader evolutionary timeframe, with origins dating back to the Jurassic period, suggesting early adaptation to diverse environments. Spikeshaped fungi, including Hericium erinaceus (Bull.) Pers. and Auricularia heimuer J.Z. Ying, exhibit a wide temporal range, indicating that this morphology has been a successful strategy across various ecological contexts. Discussion Macrofungi mitochondrial genomes represent a valuable source of information for understanding the evolutionary history, adaptation strategies, and taxonomic relationships of diverse lineages. Patterns of nucleotide Figure 9. Phylogenetic analysis of basidiomycetes based on temporal analysis. 374 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions composition, codon usage bias, gene content, and genome organization often reflect both conserved functional constraints and lineage-specific evolutionary pressures. Comparative mitochondrial studies have demonstrated that closely related species can share highly conserved genomic features while simultaneously exhibiting distinct adaptations linked to ecology, metabolism, or life history strategies (Wang et al. 2025c). In this context, analyses of newly sequenced mitogenomes not only provide insights into species-level taxonomy but also contribute to broader understanding of fungal genome evolution, phylogeny, and ecological diversification. The analyses of codon usage biases in the mitogenomes of G. bijiensis and G. matijun reveal several important insights into the genetic architecture and evolutionary dynamics of these species. The observed codon preferences, particularly the significant bias towards UUA (Leu) and AGA (Ser) codons, reflect a selective pressure towards optimizing translational efficiency in the mitochondrial environment. This pattern aligns with findings in other fungal species (Tao et al. 2024), where codon usage bias is commonly influenced by the combined effects of mutational pressures and natural selection. A strong preference for codons ending in A or U in both species is indicative of a low GC content, a common feature in many mitochondrial genomes. This bias may influence the structural stability of the mitochondrial RNA and could be an adaptation to the specific metabolic demands of these organisms. The conserved usage patterns of certain codons between G. bijiensis and G. matijun further suggest that these codons may play a crucial role in the functional optimization of mitochondrial genes, particularly those involved in critical cellular processes such as oxidative phosphorylation. Overall, the codon usage bias observed in these two Gomphus species underscores the importance of nucleotide composition and codon preference in shaping mitochondrial genome evolution. These findings provide a foundation for further comparative studies across other related species, which may shed light on the evolutionary pressures driving codon usage and genome organization in fungal mitochondria. The observed variations in genomic parameters provide valuable insights into the evolutionary dynamics and genomic architecture of the studied species. The significant differences in PCG lengths among the species may reflect diverse evolutionary pressures and functional requirements. Longer PCGs could be associated with more complex protein functions or regulatory mechanisms, while shorter PCGs might indicate streamlined genetic coding for essential functions. The relatively stable GC content within each species implies that GC content is a conserved feature, potentially associated with genome stability and replication fidelity. Species exhibiting elevated GC content may have developed particular adaptations that provide advantages in their surroundings, such as enhanced thermal stability of DNA. The variations in AT and GC skew across the species highlight the asymmetric nature of nucleotide composition in their genomes. Positive AT or GC skews could indicate strand-specific mutational biases or selective pressures favoring certain nucleotide compositions. These skews may also be related to replication and transcription processes, influencing gene expression and genome organization. Overall, these findings underscore the complexity of genomic evolution and the diverse strategies employed by different species to adapt to their environments. Further investigation into the functional 375 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions implications of these genomic features could provide deeper insights into the molecular mechanisms driving evolutionary change. The patterns identified in our collinearity research offer significant insights into the evolutionary dynamics of these species’ groups. The extensive synteny shared by certain pairs, such as G. bijiensis and R. bruneilacinia Schild & T. Ohenoja, could imply recent common ancestry or strong selective pressures maintaining gene order across these genomes. This high degree of conservation may be indicative of essential biological functions that have been preserved over evolutionary time. Conversely, the scattered nature of syntenic blocks in other comparisons, such as between R. rubripiremans Marr & D.E. Stuntz and R. ichunensis Y.C. Dai & F. Wu, may reflect either ancient divergence events leading to significant genome reshuffling or adaptive responses to disparate ecological niches driving genome evolution independently within each lineage. These findings underscore the complexity inherent in genome evolution processes, while also highlighting potential avenues for further investigation. They pinpoint specific genes within highly conserved regions that may be critical for shared biological functions among these species. The time divergence analysis provides significant insights into the evolutionary history of the studied species. The high bootstrap support for various clades suggests robust evolutionary relationships, consistent with previous studies indicating close genetic and morphological similarities among these species. G. bijiensis and G. matijun form a distinct clade, indicating a close evolutionary relationship. This clade’s placement within the larger Gomphus group suggests that these species have retained several ancestral traits while also developing unique characteristics, potentially driven by specific ecological niches or adaptive pressures unique to their environments. The clear delineation of various clades, including the Gomphus and Ramaria clades, has important taxonomic implications, supporting the classification of these species within their respective genera and highlighting the need for further taxonomic revisions to accurately reflect their evolutionary relationships. From a conservation perspective, understanding the evolutionary relationships and genetic diversity within these genera is crucial. Conservation strategies should consider the unique evolutionary history of species like G. bijiensis and G. matijun to ensure the preservation of their genetic diversity and ecological roles. The morphological analysis of G. bijiensis spores, conducted under both optical and electron microscopes, has provided critical insights into its taxonomic classification within the Gomphus genus. The ellipsoidal to fusiform shape of the spores and their smooth surface, observed under optical microscopy, are characteristic of the genus. However, the distinct size range and surface microstructure identified through SEM analysis set G. bijiensis apart from closely related species. The SEM imagery, which revealed a finely textured surface with intricate ridges, suggests a potential adaptation to its ecological niche, possibly aiding in spore dispersal or environmental adherence. The morphological features of the fresh basidiocarp also contribute to the identification of this new species. The robust, fleshy structure and distinctive capstipe differentiation, combined with the pigmentation patterns, suggest that G. bijiensis occupies a unique position within the genus. The observed characteristics suggest a potential adaptation to specific environmental conditions, which may involve interactions with soil microorganisms or symbiotic relationships with 376 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions surrounding flora. These morphological distinctions provide a strong foundation for classifying G. bijiensis as a novel species and open up avenues for further ecological and phylogenetic studies to understand its role in its native habitat. The discovery and characterization of G. bijiensis adds to the biodiversity of the Gomphus genus and highlights the importance of microscopic analysis in fungal taxonomy. Future research should aim to explore the ecological roles and potential applications of G. bijiensis, including its interactions within its ecosystem and possible contributions to biotechnological fields. The results indicate that the morphological diversity in large fungi is largely driven by convergent evolution, with similar forms evolving independently in response to similar ecological pressures. The umbrella shape likely evolved during the Cenozoic to enhance spore dispersal in forested environments, while the coral shape, with its much older origins, suggests an adaptation to humid or aquatic ecosystems. Spike-shaped fungi exhibit broad evolutionary success across various periods, underscoring the adaptive advantages of this morphology. Overall, the study highlights the importance of morphological adaptations in the evolutionary diversification of fungi and their ecological success in diverse niches. Conclusions The complete mitogenomes of G. bijiensis and G. matijun were successfully assembled, measuring 85,105 bp and 71,976 bp, respectively. The GC content was 24.7% for G. matijun and 24.8% for G. bijiensis. Strand asymmetry analysis revealed negative AT skews and positive GC skews in both mitogenomes, consistent with patterns in other fungal mitogenomes. Each contained a complete set of protein-coding genes (PCGs), including atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3. Analysis of genomic parameters across eight species demonstrated significant variations, with PCG lengths reaching up to 2,500 base pairs, indicating differences in gene structure and function. The GC content of PCGs ranged from 10% to 45%, with most exhibiting stability, while some showed higher GC content linked to evolutionary adaptations. The AT skew varied between -0.15 and 0.15, revealing species-specific abundances of adenine and thymine, while the GC skew showed similar ranges, indicating varied guanine and cytosine content. Divergence time analyses suggest that morphological diversity in macrofungi, including Gomphus species, has been shaped by convergent evolution. The phylogenetic tree places G. bijiensis and G. matijun in a distinct clade, indicating a close evolutionary relationship characterized by both the retention of ancestral traits and the emergence of unique adaptations. This study describes a new species and provides the first comprehensive mitochondrial genomic data for Gomphus, significantly advancing our understanding of its evolutionary relationships and divergence timelines. This, in turn, enhances Gomphus taxonomy and lays a foundation for future research on the evolutionary dynamics within the Gomphaceae family. Acknowledgements We thank Junhong Li and Jiayu Li for providing the specimens. 377 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement All fungal materials were collected with the consent of local authorities and in compliance with institutional and national regulations governing the collection of biological specimens. No endangered or legally protected species were collected. This research adhered to ethical standards for biodiversity research and specimen handling. Use of AI No use of AI was reported. Funding This study was supported by the Science and Technology Program of Guizhou Province (Qian Ke He Foundation-ZK [2024] General 128; Qian Ke He Foundation-ZK [2022] General 364); the High-Level Talent Initiation Project of Guizhou Medical University (J [2022] 052); the Project of the State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University (FAMP2021Z1-1); the Guizhou Provincial Education Department Scientific Research Program (Qian Jiao He KY Zi [2018] 088; Qian Jiao He KY Zi [2020] 021); the Guizhou Provincial Education Department Engineering Research Center Program (Qian Jiao Ji [2023] 036); and the Guizhou Medical University National Natural Science Foundation Cultivation Project (20NSP055). Author contributions Xianyi Wang and Zhongyao Guo contributed equally to this work. Xianyi Wang designed and conducted the experiments, analyzed the data, and wrote the manuscript. Zhongyao Guo assisted with the experimental design, data analysis, and manuscript revision. Jiawei Tao contributed to the data analysis and figure preparation. Yun Han Guo provided expertise in bioinformatics and phylogenetic analysis. Guoyu Wang supported the collection and preservation of samples. Guangyin Xu participated in the experimental design and data interpretation. Qirui Li supervised the research, contributed to the study design, and revised the manuscript. Hongmei Liu led the project, guided the overall study, and provided critical revisions to the manuscript. All authors read and approved the final manuscript. Author ORCIDs Qirui Li https://orcid.org/0000-0001-8735-2890 Data availability All data generated or analyzed during this study are included in this published article and its additional files. The complete mitogenomes of G. bijiensis and G. matijun were deposited in the GenBank database under the accession numbers PX373692 and PX373691, respectively. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.158670.suppl1 381 MycoKeys 124: 357–381 (2025), DOI: 10.3897/mycokeys.124.158670 Xianyi Wang et al.: Phylogeny of Gomphus spp. Pers. (Basidiomycota, Agaricomycetes) mitochondrial genomes with new species descriptions Supplementary material 2 Supplementary charts Author: Zhongyao Guo Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.158670.suppl2