Phylogeny and Taxonomic Revision of the Family Discinaceae (Pezizales, Ascomycota).

Species of Discinaceae are common macrofungi with a worldwide distribution. Some of them are commercially consumed, while a few others are reported as poisonous. Two genera were accepted in the family: the epigeous Gyromitra with discoid, cerebriform to saddle-shaped ascomata and the hypogeous Hydnotrya with globose or tuberous ascomata. However, due to discrepancies in their ecological behaviors, a comprehensive investigation of their relationship was not thoroughly explored. In this study, phylogenies of Discinaceae were reconstructed using sequence analyses of combined and separate three gene partitions (internal transcribed spacer [ITS], large subunit ribosomal DNA [LSU], and translation elongation factor [TEF]) with a matrix containing 116 samples. As a result, the taxonomy of the family was renewed. Eight genera were recognized: two of them (Gyromitra and Hydnotrya) were retained, three (Discina, Paradiscina, and Pseudorhizina) were revived, and three (Paragyromitra, Pseudodiscina, and Pseudoverpa) were newly established. Nine new combinations were made in four genera. Two new species in Paragyromitra and Pseudodiscina and an un-named taxon of Discina were described and illustrated in detail based on the materials collected from China. Furthermore, a key to the genera of the family was also provided. IMPORTANCE Taxonomy of the fungal family Discinaceae (Pezizales, Ascomycota) was significantly renewed on the basis of sequence analyses of internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU), and translation elongation factor (TEF). Eight genera were accepted, including three new genera; two new species were described; and nine new combinations were made. A key to the accepted genera of the family is provided. The aim of this study is to deepen the understanding of the phylogenetic relationships among genera of the group, as well as the associated generic concepts.

Cordyceps guangdongensis lipid-lowering formula alleviates fat and lipid accumulation by modulating gut microbiota and short-chain fatty acids in high-fat diet mice.

Obesity has caused serious health and economic problems in the world. Cordyceps guangdongensis is a high-value macrofungus with broad application potential in the food and bio-medicine industry. This current study aimed to estimate the role of C. guangdongensis lipid-lowering compound formula (CGLC) in regulating fat and lipid accumulation, gut microbiota balance, short-chain fatty acid (SCFA) contents, and expression levels of genes involved in fat and lipid metabolism in high-fat diet (HFD) mice. The results showed that CGLC intervention markedly reduced body weights and fat accumulation in HFD mice, improved glucose tolerance and blood lipid levels, and decreased lipid droplet accumulation and fat vacuole levels in the liver. CGLC decreased the ratio of Firmicutes and Bacteroidetes and increased the relative abundances of Bacteroides (B. acidifaciens) and Bifidobacterium (B. pseudolongum). In addition, CGLC treatment significantly promoted the production of SCFAs and regulated the relative expression levels of genes involved in fat and lipid metabolism in liver. Association analysis showed that several species of Bacteroides and most of SCFAs were significantly associated with serum lipid indicators. These results suggested that CGLC is a novel candidate formulation for treating obesity and non-alcohol fatty liver by regulating gut microbiota, SCFAs, and genes involved in fat and lipid metabolism.

New insights into the genus Gyroporus (Gyroporaceae, Boletales), with establishment of four new sections and description of five new species from China.

Species of Gyroporus from southern China were studied in this study. Based on morphology and molecular phylogenetic analyses of DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS), the nuclear ribosomal large subunit (nrLSU), and the mitochondrial adenosine triphosphate ATP synthase subunit 6 (atp6), Gyroporus was divided into four main branches in the phylogenetic tree, and four sections were firstly proposed i.e. Gyroporus sect. Castaneus, G. sect. Cyanescens, G. sect. Longicystidiatus and G. sect. Pallidus. Five new species, i.e. G. alboluteus, G. atrocyanescens, G. pseudolongicystidiatus, G. pallidus and G. subcaerulescens, were revealed from China, and their phylogenetic positions were also analysed. Among them, G. alboluteus and G. pallidus were nested into the sect. Pallidus, although morphologically similar to G. castaneus; G. atrocyanescens and G. subcaerulescens, with obvious cyanescent oxidation reactions, were nested into the sect. Cyanescens; and G. pseudolongicystidiatus characterised by its long cystidia and was nested into the sect. Longicystidiatus. The new species were formally described and illustrated in the present study, and a key to the sections and species of Gyroporus in China was provided.

Performance of a Chest Radiograph AI Diagnostic Tool for COVID-19: A Prospective Observational Study.

Purpose: To conduct a prospective observational study across 12 U.S. hospitals to evaluate real-time performance of an interpretable artificial intelligence (AI) model to detect COVID-19 on chest radiographs. Materials and Methods: A total of 95 363 chest radiographs were included in model training, external validation, and real-time validation. The model was deployed as a clinical decision support system, and performance was prospectively evaluated. There were 5335 total real-time predictions and a COVID-19 prevalence of 4.8% (258 of 5335). Model performance was assessed with use of receiver operating characteristic analysis, precision-recall curves, and F1 score. Logistic regression was used to evaluate the association of race and sex with AI model diagnostic accuracy. To compare model accuracy with the performance of board-certified radiologists, a third dataset of 1638 images was read independently by two radiologists. Results: Participants positive for COVID-19 had higher COVID-19 diagnostic scores than participants negative for COVID-19 (median, 0.1 [IQR, 0.0-0.8] vs 0.0 [IQR, 0.0-0.1], respectively; P < .001). Real-time model performance was unchanged over 19 weeks of implementation (area under the receiver operating characteristic curve, 0.70; 95% CI: 0.66, 0.73). Model sensitivity was higher in men than women (P = .01), whereas model specificity was higher in women (P = .001). Sensitivity was higher for Asian (P = .002) and Black (P = .046) participants compared with White participants. The COVID-19 AI diagnostic system had worse accuracy (63.5% correct) compared with radiologist predictions (radiologist 1 = 67.8% correct, radiologist 2 = 68.6% correct; McNemar P < .001 for both). Conclusion: AI-based tools have not yet reached full diagnostic potential for COVID-19 and underperform compared with radiologist prediction.Keywords: Diagnosis, Classification, Application Domain, Infection, Lung Supplemental material is available for this article.. © RSNA, 2022.

Diversity of Cantharellus (Cantharellales, Basidiomycota) in China with Description of Some New Species and New Records.

Cantharellus is a well-known genus of edible mushrooms, belonging to the family Hydnaceae in the class Agaricomycetes. In this study, a phylogenetic overview of Cantharellus subg. Cinnabarinus and C. subg. Parvocantharellus in China is carried out with the description of four new species. Species description are based on morphological characters of basidiomata and phylogenetic analyses of multi-locus dataset of 28S + tef1 + rpb2. Among the new species, two species, C. chrysanthus and C. sinocinnabarinus, belong to C. subg. Cinnabarinus and two new species, C. convexus and C. neopersicinus, belong to C. subg. Parvocantharellus. Species delimitation characters of the new taxa are compared with closely related species. In addition, three new records of Cantharellus are reported for China: C. albovenosus and C. citrinus of subg. Cinnabarinus and C. koreanus of subg. Parvocantharellus. A key to the species of subg. Cinnabarinus in China was provided.

Emending Gymnopus sect. Gymnopus (Agaricales, Omphalotaceae) by including two new species from southern China.

Based on phylogenetic analyses, some newly studied Chinese mushroom specimens were found to represent two distinct species within the genus Gymnopus. Along with G.fusipes (sect. Gymnopus) they form a distinct clade with high support, although their macromorphological characters seem to be closer to members of Gymnopus sect. Levipedes or sect. Vestipedes (Collybiopsis). When examined in detail, their micromorphological characters, especially the type of pileipellis, support them as new members of G. sect. Gymnopus. Therefore, two new species, G.omphalinoides and G.schizophyllus, and the emended circumscription of sect. Gymnopus are proposed in this paper. Detailed morphological descriptions, colour photos, illustrations of the two new species, morphological comparisons with similar taxa and the molecular-phylogenetic analyses of the combined nrITS and nrLSU data are presented. A key to the known species of G. sect. Gymnopus is also presented.

Multigene Phylogeny and Morphology Reveal Unexpectedly High Number of New Species of Cantharellus Subgenus Parvocantharellus (Hydnaceae, Cantharellales) in China.

The genus Cantharellus, commonly known as chanterelles, has recently been divided into six subgenera; however, wider sampling approaches are needed to clarify the relationships within and between these groups. A phylogenetic overview of Cantharellus subgenus Parvocantharellus in China was inferred based on the large subunit nuclear ribosomal RNA gene (nrLSU), the DNA-directed RNA polymerase II subunit 2 (rpb2), and the transcription elongation factor 1-alpha (tef1). A total of nine species from China were assigned to the subgenus, including seven novel species, namely Cantharellusaurantinus, C. austrosinensis, C. galbanus, C. luteolus, C. luteovirens, C. minioalbus, and C. sinominior, and two known species, namely C. albus and C. zangii. The detailed descriptions and illustrations were provided based on the newly obtained data, with the comparisons to closely related species. C. zangii was restudied based on the paratype specimens and multiple new collections from the type locality. Futhermore, the Indian species C. sikkimensis was identified as a synonym of C. zangii based on the morphological and molecular analyses. A key to the Chinese species belonging to the subg. Parvocantharellus is also provided.

Biosynthetic Pathway and the Potential Role of Melatonin at Different Abiotic Stressors and Developmental Stages in Tolypocladium guangdongense.

Melatonin, a bioactive compound and an important signaling molecule produced in plants and animals, is involved in many biological processes. However, its function and synthetic pathways in fungi are poorly understood. Here, the samples from Tolypocladium guangdongense, a highly valued edible fungus with functional food properties, were collected under different experimental conditions to quantify the levels of melatonin and its intermediates. The results showed that the intracellular melatonin content was markedly improved by Congo red (CR), cold, and heat stresses; the levels of intracellular melatonin and its intermediates increased at the primordial (P) and fruiting body (FB) stages. However, the levels of most intermediates exhibited a notable decrease under CR stress. Several genes related to melatonin synthesis, excluding AADC (aromatic-L-amino-acid decarboxylase), were markedly upregulated at an early stage of CR stress but downregulated later. Compared to the mycelial stage, those genes were significantly upregulated at the P and FB stages. Additionally, exogenous melatonin promoted resistance to several abiotic stressors and P formation in T. guangdongense. This study is the first to report melatonin biosynthesis pathway in macro-fungi. Our results should help in studying the diversity of melatonin function and melatonin-synthesis pathways and provide a new viewpoint for melatonin applications in the edible-medicinal fungus.

A Prospective Observational Study to Investigate Performance of a Chest X-ray Artificial Intelligence Diagnostic Support Tool Across 12 U.S. Hospitals.

Importance: An artificial intelligence (AI)-based model to predict COVID-19 likelihood from chest x-ray (CXR) findings can serve as an important adjunct to accelerate immediate clinical decision making and improve clinical decision making. Despite significant efforts, many limitations and biases exist in previously developed AI diagnostic models for COVID-19. Utilizing a large set of local and international CXR images, we developed an AI model with high performance on temporal and external validation. Objective: Investigate real-time performance of an AI-enabled COVID-19 diagnostic support system across a 12-hospital system. Design: Prospective observational study. Setting: Labeled frontal CXR images (samples of COVID-19 and non-COVID-19) from the M Health Fairview (Minnesota, USA), Valencian Region Medical ImageBank (Spain), MIMIC-CXR, Open-I 2013 Chest X-ray Collection, GitHub COVID-19 Image Data Collection (International), Indiana University (Indiana, USA), and Emory University (Georgia, USA). Participants: Internal (training, temporal, and real-time validation): 51,592 CXRs; Public: 27,424 CXRs; External (Indiana University): 10,002 CXRs; External (Emory University): 2002 CXRs. Main Outcome and Measure: Model performance assessed via receiver operating characteristic (ROC), Precision-Recall curves, and F1 score. Results: Patients that were COVID-19 positive had significantly higher COVID-19 Diagnostic Scores (median .1 [IQR: 0.0-0.8] vs median 0.0 [IQR: 0.0-0.1], p < 0.001) than patients that were COVID-19 negative. Pre-implementation the AI-model performed well on temporal validation (AUROC 0.8) and external validation (AUROC 0.76 at Indiana U, AUROC 0.72 at Emory U). The model was noted to have unrealistic performance (AUROC > 0.95) using publicly available databases. Real-time model performance was unchanged over 19 weeks of implementation (AUROC 0.70). On subgroup analysis, the model had improved discrimination for patients with "severe" as compared to "mild or moderate" disease, p < 0.001. Model performance was highest in Asians and lowest in whites and similar between males and females. Conclusions and Relevance: AI-based diagnostic tools may serve as an adjunct, but not replacement, for clinical decision support of COVID-19 diagnosis, which largely hinges on exposure history, signs, and symptoms. While AI-based tools have not yet reached full diagnostic potential in COVID-19, they may still offer valuable information to clinicians taken into consideration along with clinical signs and symptoms.

Detection of Isocitrate Dehydrogenase Mutated Glioblastomas Through Anomaly Detection Analytics.

BACKGROUND: The rarity of Isocitrate Dehydrogenase mutated (mIDH) glioblastomas relative to wild-type IDH glioblastomas, as well as their distinct tumor physiology, effectively render them "outliers". Specialized tools are needed to identify these outliers. OBJECTIVE: To carefully craft and apply anomaly detection methods to identify mIDH glioblastoma based on radiomic features derived from magnetic resonance imaging. METHODS: T1-post gadolinium images for 188 patients and 138 patients were downloaded from The Cancer Imaging Archive's (TCIA) The Cancer Genome Atlas (TCGA) glioblastoma collection, and from the University of Minnesota Medical Center (UMMC), respectively. Anomaly detection methods were tested on glioblastoma image features for the precision of mIDH detection and compared to standard classification methods. RESULTS: Using anomaly detection training methods, we were able to detect IDH mutations from features in noncontrast-enhancing regions in glioblastoma with an average precision of 75.0%, 69.9%, and 69.8% using three different models. Anomaly detection methods consistently outperformed traditional two-class classification methods from 2 unique learning models (67.9%, 67.6%). The disparity in performances could not be overcome through newer, popular models such as neural networks (67.4%). CONCLUSION: We employed an anomaly detection strategy in the detection of IDH mutation in glioblastoma using preoperative T1 postcontrast imaging. We show these methods outperform traditional two-class classification in the setting of dataset imbalances inherent to IDH mutation prevalence in glioblastoma. We validate our results using an external dataset and highlight new possible avenues for radiogenomic rare event prediction in glioblastoma and beyond.

Identification of microRNA-like RNAs in Cordyceps guangdongensis and their expression profile under differential developmental stages.

Cordyceps guangdongensis is a well-known fungus with high nutritional and medicinal value. The metabolite profile of C. guangdongensis is similar to that of Ophiocordyceps sinensis. In plants and animals, microRNAs play important roles in regulating gene expression at the post-transcriptional level. MicroRNA-like RNAs (milRNAs) have been documented in several macro-fungi. To comprehensively investigate the milRNAs in C. guangdongensis, three small RNA libraries from the differentially developmental stages were constructed. Twenty-six conserved milRNAs were identified, and 19 novel milRNA candidates were predicted. Among them, 20 milRNAs were differentially expressed across the developmental processes, and 12 milRNAs were verified using stem-loop quantitative real-time reverse transcription polymerase chain reaction. In addition, the potential target genes of milRNA were predicted to be involved in the development of fruiting bodies and metabolite biosynthesis. This study is the first to report the milRNAs of C. guangdongensis, and provides important insights into studies of milRNA regulation pathways in ascomycete fungi.

Two new species of Phallus (Phallaceae) with a white indusium from China.

Two new Phallus species, P.cremeo-ochraceus and P.rigidiindusiatus were discovered in southwestern and southern China, respectively. Phalluscremeo-ochraceus is morphologically characterized by its cream to ochraceous receptacle, white to very slightly pinkish indusium, white to pinkish pseudostipe and white to slightly purplish pink volva. Phallusrigidiindusiatus is characterized by a white to yellowish white receptacle, a strongly rigid indusium usually without serrated margin and smaller basidiospores than those of P.serratus. Phylogenetic positions of the two species are located in two independent lineages respectively. Detailed descriptions, color photographs, illustrations and a key to the related species are presented.

Three new species from Guangdong Province of China, and a molecular assessment of Hygrocybe subsection Hygrocybe.

Blackening waxcaps (Hygrocybe subsect. Hygrocybe) are a group of colorful and attractive mushrooms. However, the species diversity of subsect. Hygrocybe in China is still poorly known due to the limited sampling. In this study, three new species of this group from Guangdong Province, China are described and illustrated based on their morphological characteristics and molecular analyses of the internal transcribed spacer and large subunit ribosomal DNA regions. Hygrocybe debilipes from grasslands of South China Sea islands is mainly characterized by its orange red to vivid red pileus, fragile stipe, and ellipsoid to oblong basidiospores; H. griseonigricans from woodlands is characterized by its whitish to dull yellow pileus, quick black discoloration and the globose, subglobose to broadly ovoid basidiospores; H. rubroconica from woodlands is characterized by the hemispheric to plano-convex pileus when mature, semitranslucent fibrose stipe, and globose to ellipsoid basidiospores.

Mitogenome of Tolypocladium guangdongense.

Tolypocladium guangdongense is a high-value edible fungus with various medicinal and food safety properties. However, its evolutionary and genetic information is still limited. Mitochondrial genomes are potential models for molecular evolution and phylogenetic studies. In this study, we sequenced the complete mitogenome of T. guangdongense, demonstrating circular sequence of 46,102 bp, containing 14 standard protein-coding genes (PCGs), 2 ribosomal RNA subunit genes, and 28 tRNA genes. Phylogenetic analysis based on mitochondrial genes indicated that T. guangdongense was clustered into the Tolypocladium genus with high support value, based on the core PCG dataset. In addition, rps3 is also a suitable marker in the phylogenetic analysis in Hypocreales. Gene rearrangement analysis indicated that the gene order of PCGs was highly consistent in Hypocreales, and tRNA rearrangement events occurred in most species of Hypocreales; however, the rearrangement rates were not taxonomically correlated. Divergence time estimation based on the old fossil record and previous reports revealed that T. guangdongense originated approximately in the middle Cenozoic (42 Mya, 95% highest posterior density interval: 43-116) with the Tolypocladium genus differentiation. Our results provided more mitogenomic information of T. guangdongense and shed new insights into evolution of the Tolypocladium genus. KEY POINTS: • The general and unique features of T. guangdongense mitogenome are firstly reported. • Phylogenetic analysis further verified the taxonomic status of T. guangdongense. • Divergence time estimation provides more evolutionary information of T. guangdongense.

Distribution, evolution and expression of GATA-TFs provide new insights into their functions in light response and fruiting body development of Tolypocladium guangdongense.

BACKGROUND: Fungal GATA-type transcription factors (GATA-TFs) are a class of transcriptional regulators involved in various biological processes. However, their functions are rarely analyzed systematically, especially in edible or medicinal fungi, such as Tolypocladium guangdongense, which has various medicinal and food safety properties with a broad range of potential applications in healthcare products and the pharmaceutical industry. METHODS: GATA-TFs in T. guangdongense (TgGATAs) were identified using InterProScan. The type, distribution, and gene structure of TgGATAs were analyzed by genome-wide analyses. A phylogenetic tree was constructed to analyze their evolutionary relationships using the neighbor-joining (NJ) method. To explore the functions of GATA-TFs, conserved domains were analyzed using MEME, and cis-elements were predicted using the PlantCARE database. In addition, the expression patterns of TgGATAs under different light conditions and developmental stages were studied using qPCR. RESULTS: Seven TgGATAs were identified. They were randomly distributed on four chromosomes and contained one to four exons. Phylogenetic analysis indicated that GATA-TFs in each subgroup are highly conserved, especially for GATA1 to GATA5. Intron distribution analyses suggested that GATA1 and GATA3 possessed the most conserved gene structures. Light treatments induced the expression levels of TgGATA1 and TgGATA5-7, but the expression levels varied depending on the duration of illumination. The predicted protein structures indicate that TgGATA1 and TgGATA2 possess typical light-responsive domains and may function as photoreceptors to regulate downstream biological processes. TgGATA3 and TgGATA5 may be involved in nitrogen metabolism and siderophore biosynthesis, respectively. TgGATA6 and TgGATA7 possess unique Zn finger loop sequences, suggesting that they may have special functions. Furthermore, gene expression analysis indicated that TgGATA1 (WC1) was notably involved in mycelial color transformation, while other genes were involved in fruiting body development to some extent. These results provide valuable information to further explore the mechanisms through which TgGATAs are regulated during fruiting body development.

Transcriptome and proteome analyses reveal the regulatory networks and metabolite biosynthesis pathways during the development of Tolypocladium guangdongense.

Tolypocladium guangdongense has a similar metabolite profile to Ophiocordyceps sinensis, a highly regarded fungus used for traditional Chinese medicine with high nutritional and medicinal value. Although the genome sequence of T. guangdongense has been reported, relatively little is known about the regulatory networks for fruiting body development and about the metabolite biosynthesis pathways. In order to address this, an analysis of transcriptome and proteome at differential developmental stages of T. guangdongense was performed. In total, 9076 genes were found to be expressed and 2040 proteins were identified. There were a large number of genes that were significantly differentially expressed between the mycelial stage and the stages. Interestingly, the correlation between the transcriptomic and proteomic data was low, suggesting the importance of the post-transcriptional processes in the growth and development of T. guangdongense. Among the genes/proteins that were both differentially expressed during the developmental process, there were numerous heat shock proteins and transcription factors. In addition, there were numerous proteins involved in terpenoid, ergosterol, adenosine and polysaccharide biosynthesis that also showed significant downregulation in their expression levels during the developmental process. Furthermore, both tryptophan and tryptamine were present at higher levels in the primordium stage. However, indole-3-acetic acid (IAA) levels continuously decreased as development proceeded, and the enzymes involved in IAA biosynthesis were also clearly differentially downregulated. These data could be meaningful in studying the molecular mechanisms of fungal development, and for the industrial and medicinal application of macro-fungi.

Hygrophorus subsection Hygrophorus (Hygrophoraceae, Agaricales) in China.

Hygrophorus subsect. Hygrophorus has been relatively well-studied in Europe and North America, but studies on the taxa in Asia, particularly in China, are still limited. In this study, phylogenetic overviews of genus Hygrophorus, based on the nuclear large subunit (LSU) ribosomal RNA gene and of subsect. Hygrophorus, based on the nuclear ribosomal internal transcribed spacer (ITS) regions were generated. Four new species, i.e. H. brunneodiscus, H. fuscopapillatus, H. glutiniceps and H. griseodiscus are described from southern China; and a rarely reported edible species H. hedrychii is described in detail, based upon the materials from north-eastern China. The main characteristics of the species under subsect. Hygrophorus worldwide are summarised in a table.

Transcriptome sequencing analysis of the MSDIN gene family encoding cyclic peptides in lethal Amanita fuligineoides.

Amanita fuligineoides, a lethal mushroom discovered in China, contains abundant cyclic peptide toxins that can cause fatal poisoning. However, the MSDIN gene family encoding for these cyclic peptides in A. fuligineoides has not been systematically studied. In this research, the transcriptome sequencing of A. fuligineoides was performed and its MSDIN family members were analyzed. A total of 4.41 Gb data containing 30833 unigenes was obtained; sequence alignments throughout several databases were done to obtain their functional annotations. Based on these annotations, MSDIN genes were found and verified by RT-PCR. A total of 29 different core peptides were obtained: 3 toxin genes, encoding ß-amanitin (ß-AMA), phalloidin (PHD), and phallacidin (PCD), and 26 genes encoding unknown cyclic peptides, 20 of which are reported for the first time and may encode for novel cyclic peptides. Analysis of the predicted precursor peptides indicated that octocyclic peptides were the main MSDIN peptides synthesized by A. fuligineoides, accounting for the 45%. A phylogenetic analysis suggested that studied precursor peptides could be clustered into 7 clades, which might represent different functionalities. Results suggested that A. fuligineoides might have a strong capacity to synthesize cyclopeptides, laying the foundation for their excavation and utilization.

Corrigendum: He XL, Horak E, Wang D, Li TH, Peng WH, Gan BC (2019) Descriptions of five new species in Entoloma subgenus Claudopus from China, with molecular phylogeny of Entoloma s.l. MycoKeys 61: 1-26. https://doi.org/10.3897/mycokeys.61.46446.

[This corrects the article DOI: 10.3897/mycokeys.61.46446.].

Expression profiling of Cordyceps DnaJ protein family in Tolypocladium guangdongense during developmental and temperature stress processes.

DnaJ is an important molecular chaperone, with significant roles in growth, development, and stress resistance. Studies on the DnaJ gene family in macro-fungi such as Cordyceps spp. s.l. is scare. In this study, 22, 20, and 24 putative DnaJ genes were identified in Tolypocladium guangdongense, Ophiocordyceps sinensis, and C. militaris, respectively. They were classified into four groups based on the presence of the J, zinc finger, and C-terminal domains. We mainly studied the T. guangdongense DnaJ genes being located in the endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus. Phylogenetic analysis revealed gene duplications during the evolutionary process. Multiple cis-elements and transcription factor binding sites were observed in the promoter, suggesting their involvement in the response to multiple stresses. qRT-PCR analysis showed that 63.63% and 45.45% of T. guangdongense DnaJ genes were differentially expressed under cold and heat stress, respectively, indicating their involvement in the response to temperature stress. Many T. guangdongense DnaJ genes in the primordium and fruiting body exhibited differential expression, in comparison to those in the mycelium, suggesting a regulatory role in its growth and development process. These findings will facilitate further functional analysis, and provide information on the classification and conservative functions of DnaJ proteins in macro-fungi.