Fungal Diversity Associated with Thirty-Eight Lichen Species Revealed a New Genus of Endolichenic Fungi, Intumescentia gen. nov. (Teratosphaeriaceae)

Fungi from the Teratosphaeriaceae (Mycosphaerellales; Dothideomycetes; Ascomycota) have a wide range of lifestyles. Among these are a few species that are endolichenic fungi. However, the known diversity of endolichenic fungi from Teratosphaeriaceae is far less understood compared to other lineages of Ascomycota. We conducted five surveys from 2020 to 2021 in Yunnan Province of China, to explore the biodiversity of endolichenic fungi. During these surveys, we collected multiple samples of 38 lichen species. We recovered a total of 205 fungal isolates representing 127 species from the medullary tissues of these lichens. Most of these isolates were from Ascomycota (118 species), and the remaining were from Basidiomycota (8 species) and Mucoromycota (1 species). These endolichenic fungi represented a wide variety of guilds, including saprophytes, plant pathogens, human pathogens, as well as entomopathogenic, endolichenic, and symbiotic fungi. Morphological and molecular data indicated that 16 of the 206 fungal isolates belonged to the family Teratosphaeriaceae. Among these were six isolates that had a low sequence similarity with any of the previously described species of Teratosphaeriaceae. For these six isolates, we amplified additional gene regions and conducted phylogenetic analyses. In both single gene and multi-gene phylogenetic analyses using ITS, LSU, SSU, RPB2, TEF1, ACT, and CAL data, these six isolates emerged as a monophyletic lineage within the family Teratosphaeriaceae and sister to a clade that included fungi from the genera Acidiella and Xenopenidiella. The analyses also indicated that these six isolates represented four species. Therefore, we established a new genus, Intumescentia gen. nov., to describe these species as Intumescentia ceratinae, I. tinctorum, I. pseudolivetorum, and I. vitii. These four species are the first endolichenic fungi representing Teratosphaeriaceae from China.

Lichen is a symbiotic relationship between fungi and algae or cyanobacteria, resulting in the formation of thalli [14][15][16]. The fungal diversity associated with lichens includes symbiotic, lichenicolous and endolichenic fungi [16]. Endolichenic fungi secrete a variety of secondary metabolites, some of which are beneficial to the survival of lichen in severe environments [17,18]. Endolichenic and lichenicolous fungi include species of the phyla Ascomycota, Basidiomycota, and Mucoromycota [16,17,19].
Globally, studies involving endolichenic fungi led to the identification and discovery of various new and previously identified fungal taxa representing various lineages of Ascomycota, such as Pezizomycetes, Dothideomycetes, Eurotiomycetes, Leotiomycetes, and Sordariomycetes, among others [20][21][22][23]. Echoing the global trend, several novel endolichenic fungal taxa were also identified in China [24][25][26][27][28]. In a recent fungal biodiversity study by Xu et al. (2022), the authors detected a substantial diversity of endolichenic fungi in the family Teratosphaeriaceae from Heterodermia obscurata, and proposed that Teratosphaeriaceae is likely to be one of the core endophytic fungal families associated with this lichen [13]. The authors used a high-throughput sequencing platform; hence, no isolates could be retrieved.
In this study, we aimed to catalogue the fungal diversity associated with lichen species from Yunnan Province of China. We selected this province as our sampling site because this region is considered a biodiversity hotspot due to its unique climatic conditions that support a diverse vegetation and, consequently, the microbes that are associated with it [29]. We hypothesized that the diversity of fungi that are associated with each lichen species will differ; however, there will be some overlap. This fungal diversity will include endolichenic fungi as well as other fungi, representing a wide range of ecological guilds. Therefore, we aimed to detect the diversity of fungal guilds within lichens.

Collection and Identification of Lichen Samples
Five surveys were conducted in Yunnan Province of China from 2020 to 2021. During these surveys, we collected 115 lichen samples representing different growth forms, such as crustose, foliose, and fruticose (Figures 1 and 2, Table S1). We collected at least two thalli for each lichen. One of them was dried in a cabinet at 35 • C. This dried sample was intended for lichen photography and identification. The morphological identification of the lichen samples were carried out using the monographs published by Wang (2012) and Wang and Qian (2012) [30,31]. Following taxonomic identification of the lichens, all dried samples were discarded. A fresh lichen thallus was used for fungal isolation.

Isolation of Fungi from Lichen Thalli
All of the lichen samples collected during the surveys were asymptomatic. Hence, fungal isolation was conducted randomly from various parts of a thallus. The lichen samples were individually rinsed under running tap water and then with deionized sterile distilled water. Multiple 2 × 2 cm 2 pieces of each lichen thallus were dissected under a Leica Zoom 2000 stereomicroscope. The upper and lower cortices of the thallus tissue were scraped off using a sterile blade and a pair of tweezers. The medullary layer was carefully removed and rinsed repeatedly with sterile deionized water. These medulla tissues from each lichen sample were then placed onto the surface of 2% potato dextrose agar medium (PDA, Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) amended with 0.05% streptomycin. All of the Petri plates were incubated at 25 • C in darkness. Hyphal tips of mycelia emerging from the medullary tissues were sub-cultured onto fresh PDA plates.

DNA Extraction, PCR Amplification, Sequencing, and Preliminary Identification
Total genomic DNA was extracted from 15-day old fungal cultures grown on PDA using a modified CTAB protocol [32]. For the purpose of the initial screening of all isolates, the complete internal transcribed spacer (ITS) and the partial nuclear large subunit ribosomal DNA (LSU) regions were amplified using primers ITS1/ITS4 [33] and LR0R/LR5 [33,34], respectively.   Fungi isolated from 38 lichen species sampled in this study from Yunnan Province, China, between 2020 and 2021. Fungal species are listed according to their abundance in lichens. Three most frequently isolated fungal species were Anteaglonium gordoniae (11 isolates), Cladophialophora nyingchiensis (11 isolates), and Capronia rubiginosa (nine isolates). Highest number of fungal isolates were recovered from the lichens Usnea aciculifera (26 isolates), Parmotrema reticulatum (15 isolates), Parmelinella wallichiana (14 isolates), and Usnea ceratina (13 isolates). The numbers within the barplot represent the fungal species.
For all gene regions, each 25 µL of PCR reaction included 9.5 µL of water, 12.5 µL of 1-5 TM 2 × High-Fidelity Master Mix (buffer, MgCl 2 , dNTPs and Taq; Tsingke Co., China), 1 µL each of forward and reverse primers, and 1 µL DNA template. For all gene regions, PCR amplifications were conducted with an initial denaturation at 94 • C for 5 min, followed by 35 cycles of 94 • C for 30 s, 56 • C for 1 min, 72 • C for 90 s, and a final extension at 72 • C for 10 min. Positive amplification of the gene regions was determined using agarose gel electrophoresis. Samples were stained using Spark GoldView (SparkJade, China) and visualized under UV light.
All PCR products were sequenced by the Qingdao Sangong Biotechnology Co., LTD. The resulting forward and reverse sequences were assembled using Geneious v.10.2.2 (Biomatters, Auckland, New Zealand). Preliminary identification of the fungal isolates was carried out using the BLAST algorithm [39] available through the NCBI GenBank. Sequences from possibly novel fungus species were included in the phylogenetic analyses and were also submitted to GenBank (Table S2).

Guilds of Fungal Species Isolated from Lichens
Ecological guilds of the fungal species isolated from 38 lichen species were determined using the FungalTraits database [40]. Due to a paucity of data, the specific guild could not be identified for some fungal species. In those cases, we inserted an inquiry mark alongside the guild.

Phylogenetic Analyses
During preliminary identification of the fungal isolates using BLAST, six isolates emerged as potentially new fungal species from the family Teratosphaeriaceae. For the phylogenetic identification of these six isolates, separate datasets were prepared for all six gene regions: ITS (59 taxa), LSU (82 taxa), RPB2 (57 taxa), TEF1 (37 taxa), ACT (26 taxa), and CAL (29 taxa) ( Table S2). The datasets include sequences generated in this study, as well as those from the ex-type isolates retrieved from GenBank and from a previous study by Wanasinghe et al. (2018) [3]. Irrespective of the datasets, Staninwardia suttonii served as an outgroup. All sequence datasets were aligned using MAFFT v.7 [41] and if needed, they were manually adjusted using MEGA v.7 [42].
All single genes and concatenated datasets were analyzed using the maximum likelihood (ML), Bayesian inference (BI), and maximum parsimony (MP) approaches. ML and BI were analyzed using the CIPRES Scientific Gateway platform [43]. Irrespective of the phylogenetic approaches, the appropriate nucleotide substitution model was determined using jModelTest v.2.1.6 [44]. The ML analysis was performed using RAxML v.8.2.12 with 1000 bootstrap replicates using GTR + Gamma as the substitution model [45]. For the BI analyses, MrBayes v.3.2.7 [46] ran 5 million generations from a random start tree with four MCMC chains using the substitution model GTR+I+G for ITS, GTR+G for LSU, TPM3uf+I+G for TEF, TIM3+I+G for ACT and RPB2, TIM1+I+G for CAL, and GTR+I+G for the concatenated dataset. For all analyses, the stop value was set to 0.01 and a temperature was set to 0.2, and the trees were sampled after every 100 generations. A quarter of the sampled trees were discarded during burn-in. The remaining trees were used for constructing consensus trees. MP analysis was performed using MEGA v.10.2.0 with 1000 bootstrapping replicates and gap as the fifth character state. The resulting trees from the ML, MP, and BI analyses were viewed using FigTree v.1.4 [47]. The alignments and trees were deposited in TreeBASE (Study ID29830).

Morphology and Growth Studies
Multiple approaches were used to induce sporulation in the novel fungal isolates. All isolates were sub-cultured on four microbial culture media. These were PDA, malt extract agar medium (MEA, Qingdao Hope Bio-technology, Qingdao, China), oat agar medium (OA, oats 20 g; agar, 20 g from Qingdao Hope Bio-technology, Qingdao, China; distilled water 1000 mL), and synthetic nutrient-poor agar medium (SNA; KH 2 PO 4 1 g; KNO 3 1 g; MgSO 4 7H 2 O 0.5 g; KCl 0.5 g; glucose 0.2 g from Qingdao Hope Bio-technology, Qingdao, China; sucrose 0.2 g; agar, 20 g from Qingdao Hope Bio-technology, Qingdao, China; distilled water 1000 mL) [48]. If any of the isolates did not sporulate on the abovementioned media, then they were sub-cultured onto PDA, MEA, OA, and SNA amended with sterilized pine needles [49,50] and dried lichen powder (0.2 g/100 mL).
The micro-morphology of the potential new species was photographed using a Leica DM6 compound microscope attached to a Zeiss Axio Imager Z2 camera. Image J [51] was used to measure at least 50 readings for each taxonomically important attribute from all isolates.
For the growth study, all isolates from four potentially new species were sub-cultured onto PDA. All of the Petri plates were incubated at 25 • C for 15 days. Then, agar blocks measuring 5 mm in diameter were placed in the center of a 90 mm Petri dish. The isolates were incubated in three replicates at five different temperatures: 5, 10, 15, 20, 25, 30, and 35 • C (±0.5 • C). Colony diameter of each isolate was measured every two days until day 30.
Ex-holotype cultures of undescribed species were deposited at the Chinese General Library of Microbial Cultures (CGMCC), Beijing, China. Holotypic specimens were preserved in the culture collection of the Institute of Microbiology (HMAS), Beijing, China.

Collection and Identification of the Lichen Samples
One hundred and fifteen lichens were classified into 38 species based on morphological identification using monographs. The most commonly collected lichen in this study was Usnea aciculifera, which is followed by Flavoparmelia caperata and Usnea ceratina (Table S1).

Phylogenetic Analyses
The  Figure 3). This clade of six isolates emerged as the sister to a clade that encompasses fungi from the genera Acidiella and Xenopenidiella, but this relationship did not receive significant branch support ( Figure 3). In the phylogenetic analyses using single gene datasets, these six isolates formed a monophyletic clade in the ITS, LSU, ACT, and TEF trees ( Figures S1-S6). However, this relationship was only significant in the TEF tree ( Figure S3).  Within the clade that included the six previously undescribed species, CGMCC3.23633, CGMCC3.23634, and CGMCC3.23636 formed a monophyletic clade that received significant statistical support (ML/MP/PP; 100/99/1; Figure 3). The sequences for all amplified gene regions were identical between CGMCC3.23633 and CGMCC3.23634. However, when compared to CGMCC3.23636, it had two nucleotide differences in ITS and RPB2 and a single nucleotide difference in ACT, whereas there were no differences in LSU, CAL, and TEF1.
Isolate CGMCC3.23741 appeared as the sister to the clade that included CGMCC3.23633, CGMCC3.23634, and CGMCC3.23636. This relationship received high branch support (ML/MP/PP; 99/98/1; Figure 3). In addition to this, there were substantial differences in all gene regions between the two groups. With significant statistical support (ML/MP/PP; 99/99/1) and differences in the amplified gene regions, CGMCC3.23635 emerged as the sister to this clade ( Figure 3). CGMCC3.23630 emerged as the basal diverging taxa within this clade (Figure 3). The isolate CGMCC3.23630 s phylogenetic position was a wobble in the single-gene trees. On the TEF tree, this isolate appeared as the sister taxon of a clade that included three Baudoinia species, but had no substantial branch support. Similarly, CGMCC3.23630 is either clustered with Austroafricana associata or appeared as an orphan in the CAL and RPB2 trees, respectively.

Taxonomy
Intumescentia H. L. Si, R. L. Chang, T. Bose and Y. C. Wang, gen. nov. MycoBank No: 844851 Etymology: The name refers to the typical hyphal swellings that occur in this group of fungi.
Description: The slow growing colonies on PDA are black-brown in color (top and reverse), compact, superficial, with usually gray or greenish black, tomentose, the margins may be entire or finely serrated and irregularly lobed. Hyphae are asperulous or smooth, brown in color, septate, multi-guttulate, and branched; their compartments are variable in size, usually with hyphal swelling that are apical or intercalary in position, with lateral branching usually arising from the swollen compartments. Conidial cells catenulate, three to eight or more in a chain, they are intercalary or apical in position, and are caducous. The conidia are columnar to doliiform in shape, dark brown in color, and basal and intercalary conidia have flat apices, and apical conidia with an acute apex are multi-guttulate. No sexual structures were observed.
Type species: Intumescentia tinctorum H. L. Si, R. L. Chang, T. Bose and Y. C. Wang Notes: The genus Intumescentia displays a significant morphological variance with closely related genera Acidiella, Araucasphaeria and Xenopenidiella. In comparison to Intumescentia, Acidiella, and Xenopenidiella, they yield morphologically distinct mitospores [4,52]. Acidiella produces puffed and truncated arthroconidia, while Xenopenidiella produces branched chains of verruculose conidia that are ellipsoid to cylindrical-oblong in shape and brown in color. Araucasphaeria is known to produce sexual spores [53]. Fungi from the genera Acidiella, Araucasphaeria, and Xenopenidiella have a faster growth rate than Intumescentia [49,52,53]. Etymology: Named after its host lichen species, Parmotrema tinctorum. Description: Hyphae are smooth, light brown in color, septate, multi-guttulate, and branched; compartments are variable in size and are often distorted, measuring 2.01-5.05 µm (x = 3.23 µm, n = 50), usually with hyphal swelling that is globose to subglobose in shape, and apical or intercalary in position, and lateral branching usually arises from swollen or distorted compartments; juvenile hyphae are usually slightly curved. Asexual and sexual structures were not observed.
Colony morphology and growth: Colony on PDA after 30 days at 20 • C is black-brown in color (top and reverse), compact, with a surface that is greenish gray in color, minutely tomentose, margins are entire and irregularly lobed. Optimal growth temperature is 20 • C (0.31 mm/day). Growth was observed at 5 • C (0.06 mm/day), whereas no growth was detected at 35 • C.
Additional Notes: In the phylogenetic analyses of single genes and concatenated datasets, the three isolates of I. tinctorum emerged as a monophyletic lineage (Figure 3). This fungal species shares both congruent and distinct morphological characteristics with closely related species (Table 1). Additionally, I. tinctorum has the lowest optimal growth temperature compared to other species in this genus ( Table 1).
J. Fungi 2023, 9, x FOR PEER REVIEW 11 of 20 related species (Table 1). Additionally, I. tinctorum has the lowest optimal growth temperature compared to other species in this genus ( Table 1).    Etymology: Named after its host lichen species, Physcia vitii. Description: Hyphae are smooth, dark brown in color, branched, septate, with constricted septa; the compartment is often peanut-shaped, multi-guttulate with guttles small in size; the compartment is variable in size and often distorted, measuring 2.64-7.31 µm (x = 4.09 µm, n = 50), usually with irregular globose hyphal swellings that are intercalary in position, with lateral branching usually arising below the septa. Juvenile hyphae and hyphal apices are thin walled and hyaline to light brown in color; matured hyphae have thick walls and are dark brown in color. Asexual and sexual structures were not observed.
Colony morphology and growth: Colony on PDA after 30 days at 25 • C is blackish brown in color (top and reverse), compact, superficial, tomentose, and margins are entire and irregularly lobed. Optimal growth temperature is 25 • C (0.18 mm/day). Growth was observed at 5 • C (0.06 mm/day), whereas no growth was detected at 35 • C.
Notes: Intumescentia vitii appeared as the sister of I. tinctorum in the ML (Figure 3), MP, and BI trees. These two species exhibit characteristics that are both overlapping and distinctive (Table 1). In addition to the DNA data, these two species can be differentiated through colony morphologies, hyphal dimensions, and optimal growth temperatures.
Colony morphology and growth: Colony on PDA after 30 days at 25 • C is blackish brown in color (top and reverse), compact, superficial, tomentose, the margins are entire and irregularly lobed. Optimal growth temperature is 25 • C (0.27 mm/day). Growth was observed at 5 • C (0.06 mm/day), whereas no growth was detected at 35 • C.
Colony morphology and growth: Colony on PDA after 30 days at 25 • C is blackish brown in color (top and reverse), compact, superficial, slightly raised in the center and villose, with margins that are finely serrated and irregularly lobed. Optimal growth temperature is 25 • C (0.4 mm/day). Growth was observed at 5 • C (0.06 mm/day), whereas no growth was detected at 35 • C.
Notes: Intumescentia ceratinae has both unique and overlapping morphological characteristics with other Intumescentia species described in this study. The colony morphology, hyphal morphology, and conidia measurements distinguish this species from others, whereas the dimensions of the hyphal compartments, shape of the hyphal swellings, and optimal growth temperature of I. ceratinae are comparable to those of other Intumescentia species. In the phylogenetic analyses, I. ceratinae appeared as basal to other species in this family. However, this relationship did not receive significant branch support. Nonetheless, we placed this species in the genus Intumescentia. In the future, when more species from this genus are recovered, the taxonomic placement of this fungus can be re-evaluated.

Discussion
For the present study, we conducted field surveys in Yunnan Province of China between 2020 and 2021. During these surveys, we collected specimens of 38 lichen species. From these lichens, we recovered 205 fungal isolates representing 127 fungal species from the phyla Ascomycota, Basidiomycota, and Zygomycota. This diversity included fungi from a wide variety of ecological guilds. Additionally, the fungal diversity overlapped between the lichen species and also included unique species. Among the fungi recovered in this study, there were six fungal isolates from the family Teratosphaeriaceae that had low morphological and genetic similarities with previously described species from this family. Multi-gene phylogenies indicated that these six isolates represented a novel clade within Teratosphaeriaceae. We erected a new genus Intumescentia to describe these six isolates as four new species: I. ceratinae, I. vitii, I. wallichianae, I. tinctorum, and I. pseudolivetorum.
In this study, 205 fungal isolates were identified from the phyla Ascomycota, Basidiomycota, and Mucoromycota. Among them, Dothideomycetes was the most dominant class (62 species), followed by Sordariomycetes (40 species), and Eurotiomycetes (13 species). The remaining species were from Agaricomycetes, Arthoniomycetes, Cystobasidiomycetes, Exobasidiomycetes, Leotiomycetes, Tremellomycetes, and Umbelopsidomycetes. Fungi from most of these classes have been previously reported from lichens [16]. However, the abundance of these classes substantially varies between the studies. For example, among the Antarctic lichens, Park et al. (2015) reported Arthoniomycetes, Eurotiomycetes, and Lecanoromycetes as the most common classes [54], whereas in a later study, Yu et al. (2018) found Leotiomycetes, Sordariomycetes, and Dothideomycetes to be abundant [22]. Similarly, Muggia et al. (2016) found that Chaetothyriomycetes and Dothideomycetes were the major fungal classes among rock-inhabiting lichens in the alpine area, whereas Rajulu et al. (2019) found Sordariomycetes, Dothideomycetes, and Eurotiomycetes to be prevalent among lichens in the Western Ghats, India [55,56]. The findings of these studies show that the environment, growth substrate, and a variety of other abiotic variables influence the fungal diversity associated with lichens. Simultaneously, the isolation and identification strategies used in many studies, including ours, can have an impact on the documented diversity.
In the present study, we recovered an assortment of fungi representing to a wide range of guilds, including saprophytes, plant pathogens, human pathogens, as well as entomopathogenic, endolichenic, and symbiotic fungi. This study supports the prior findings that fungal diversity associated with the lichen thallus is not limited to lichenicolous, endolichenic, and symbiotic fungi [16,57,58]. These lichen thalli also yielded human and entomopathogenic fungal species. However, we are uncertain of the specific ecological roles of these fungi in lichens. It is probable that these fungi perform additional roles in the environment than are currently understood. Alternatively, these fungi might have been contaminants during the isolation procedure. This hypothesis may be correct for fungi for which only a single isolate was recovered, but not for all.
Fungi from Teratosphaeriaceae are widely distributed. Several fungal species from this family have been identified from unique habitats. However, as of now, a few endolichenic fungi from Teratosphaeriaceae have been isolated from lichens [59], such as those from the genera Xanthoriicola and Austrostigmidium [12]. Contrarily, in a recent diversity study using a high throughput sequencing platform showed that Teratosphaeriaceae is the most abundant endolichenic fungal taxa associated with Heterodermia obscurata [13]. This contradiction is likely because a majority of endolichenic fungi are slow-growing, and in this regard, the isolates in this study are not an exception. Hence, it is difficult to isolate these fungi from a lichen thallus that houses a plethora of fungi. Still, it is important to conduct culture-based studies, such as this. Isolates recovered from this study and from other similar studies [26,27,60] will allow us to thoroughly investigate the biology of these fungi. In addition to this, some endolichenic fungi can also be of biotechnological importance, such as oleaginous yeasts [24].
For Teratosphaeriaceae, LSU is frequently used as the marker gene for identifying new fungal species [4,12,[61][62][63]. This is because Crous et al. (2007) suggested LSU as a suitable marker for differentiating species within Teratosphaeriaceae [1]. However, there were insufficient variations in the LSU sequences between the six Teratosphaeriaceae fungal isolates recovered in this study. Nevertheless, amplifying the partial RPB2 gene allowed us to tease out the species. In the future, it is worth considering RPB2 along with LSU as markers for species delineation in Teratosphaeriaceae. The current study exemplifies the importance of culture-based fungal diversity studies from the lichen thallus and various other environments. In this era, a majority of the microbial diversity studies use one of the many available high-throughput sequencing platforms. This strategy is useful because it allows to identify a greater number of fungal OTUs along with many other advantages [64]. However, in most of the studies, the novel fungal species detected could not be resolved taxonomically. Additionally, now there is also provision for using sequences for describing novel fungi. However, this strategy should be used with caution, especially when using short-read sequencing platforms [65]. Even though culture-based diversity studies are currently rare, they allow for the recovery of fungal isolates that can feed into many downstream studies, such as taxonomy, genetics, and biotechnology. In the future, a hybrid approach using both of these strategies would be useful, such as those conducted for fungus-like organisms [66,67]. The data generated by these studies will be of interest to a broader scientific community and will not be limited to microbial diversity research groups.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof9040423/s1, Figure S1: Maximum likelihood phylogeny using ITS dataset for Teratosphaeriaceae. The bootstrap support value ≥75% and posterior probability ≥0.95 displayed above the node are ML/MP/PP. The isolates of Intumescentia gen. nov. obtained in this study are shown in bold. T = ex-type isolates; Figure S2: Maximum likelihood phylogeny using LSU dataset for Teratosphaeriaceae. The bootstrap support value ≥75% and posterior probability ≥0.95 displayed above the node are ML/MP/PP. The isolates of Intumescentia gen. nov. obtained in this study are shown in bold. T = ex-type isolates; Figure S3: Maximum likelihood phylogeny using TEF dataset for Teratosphaeriaceae. The bootstrap support value ≥75% and posterior probability ≥0.95 displayed above the node are ML/MP/PP. The isolates of Intumescentia gen. nov. obtained in this study are shown in bold. T = ex-type isolates; Figure S4: Maximum likelihood phylogeny using CAL dataset for Teratosphaeriaceae. The bootstrap support value ≥75% and posterior probability ≥0.95 displayed above the node are ML/MP/PP. The isolates of Intumescentia gen. nov. obtained in this study are shown in bold. T = ex-type isolates; Figure S5: Maximum likelihood phylogeny using RPB2 dataset for Teratosphaeriaceae. The bootstrap support value ≥75% and posterior probability ≥0.95 displayed above the node are ML/MP/PP. The isolates of Intumescentia gen. nov. obtained in this study are shown in bold. T = ex-type isolates; Figure S6: Maximum likelihood phylogeny using ACT dataset for Teratosphaeriaceae. The bootstrap support value ≥75% and posterior probability ≥0.95 displayed above the node are ML/MP/PP. The isolates of Intumescentia gen. nov. obtained in this study are shown in bold. T = ex-type isolates. Table S1: The lichen species sampled in this study, along with their sample numbers, habitat, distribution, and growth type; Table S2: Taxa used in the phylogenetic analyses and their corresponding GenBank numbers. T = ex-type isolates; Table S3: List of fungal species isolated from 38 lichen species sampled in this study and their respective guilds. Numbers in the cells indicate the number of isolates.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.