Endolichenic Fungi: A Promising Medicinal Microbial Resource to Discover Bioactive Natural Molecules—An Update

Lichens are some of the most unique fungi and are naturally encountered as symbiotic biological organisms that usually consist of fungal partners (mycobionts) and photosynthetic organisms (green algae and cyanobacteria). Due to their distinctive growth environments, including hot deserts, rocky coasts, Arctic tundra, toxic slag piles, etc., they produce a variety of biologically meaningful and structurally novel secondary metabolites to resist external environmental stresses. The endofungi that live in and coevolve with lichens can also generate abundant secondary metabolites with novel structures, diverse skeletons, and intriguing bioactivities due to their mutualistic symbiosis with hosts, and they have been considered as strategically significant medicinal microresources for the discovery of pharmaceutical lead compounds in the medicinal industry. They are also of great importance in the fundamental research field of natural product chemistry. In this work, we conducted a comprehensive review and systematic evaluation of the secondary metabolites of endolichenic fungi regarding their origin, distribution, structural characteristics, and biological activity, as well as recent advances in their medicinal applications, by summarizing research achievements since 2015. Moreover, the current research status and future research trends regarding their chemical components are discussed and predicted. A systematic review covering the fundamental chemical research advances and pharmaceutical potential of the secondary metabolites from endolichenic fungi is urgently required to facilitate our better understanding, and this review could also serve as a critical reference to provide valuable insights for the future research and promotion of natural products from endolichenic fungi.


Introduction
As one of the most critically important groups of unique fungi, lichens are broadly acknowledged for their unique biological characteristics, lacking any obvious differentiation of their roots, stems, and leaves.Lichens are naturally occurring, stable microorganisms, and they are mutually beneficial organic complexes with a complex composition of fungal and chlorophyll organisms [1,2].Mutualistic symbiosis is the biologically distinctive characteristic that allows lichens to be differentiated from other common plants [3].Among the symbiotic microorganisms existing in lichens, ascomycetes are the pivotal symbiotic fungi that account for their fungi diversity.Basidiomycetes are also important members of endolichenic microorganisms, and symbiotic partners are usually present as cyanobacteria or green algae [4].
Lichens can survive in a variety of extreme living environments, such as during nutrient shortages, under wet, cold, and dark conditions, in high temperatures.Notably, they often grow on the surfaces of rocks or trees, without any typical nutrient requirements, and they are widely distributed across almost all land surfaces on Earth, even when there are no suitable loamy soils for the survival of other plants or animals.It is well known that lichens can be readily encountered in nature, ranging from the North and South Poles to the equator, from mountains to plains, forests to deserts, and land to sea [5][6][7][8][9].Due to their complex environmental living conditions, they display an outstanding capacity to produce numerous intriguing bioactive secondary metabolites to resist external habitat stresses [3].
Throughout the centuries-long use of traditional medicine to treat human health conditions, lichens have been widely used in the pharmacopeias of different countries as effective remedies for various diseases; thus, they have attracted attention from both pharmaceutical and natural product chemists, who have investigated their bioactive chemical compositions in recent decades [10,11].The critical chemical constituents of lichens have been consequently demonstrated, and they are usually exemplified by depsides and their esters, depsidones, polysubstituted benzenes, anthraquinones, dibenzofurans, terpenoids, and steroids, together with lichen polysaccharides [12].These structurally diverse natural products from lichens have been illustrated to show potent enzyme-inhibitory, antibacterial, anti-fungal, anti-viral, anti-tumor, insecticidal, antioxidant, and other biological properties, which closely correspond to their traditional medicinal applications and therapeutic efficacy [4,13].Notably, numerous excellent scientific efforts to elucidate the bioactive chemical composition of lichens have disclosed that different species of lichens can produce various types of bioactive natural compounds with different structural characteristics [14].
Fungi are well known as a promising strategic pharmaceutical bioresource for the discovery of structurally intriguing and biologically significant medicinal lead compounds, and they show great advantages with the pivotal characteristics of a short growth cycle, easy fermentation, and readily operational cultivation.The number of natural products derived from fungi and the proportion of active substances in all natural sources are sharply increasing year by year, accompanied by technological innovations of natural product chemistry towards compound separation and structure identification in recent decades.Moreover, fungi play important roles in plants, terrestrial ecosystems, and other related ecosystems.Fungi found in unique habitats have shown great potential in pharmaceutical research and innovative drug development due to their unique metabolic and defense systems under the influence of complex environmental factors, and the biologically meaningful natural products derived from fungi material serve as an important source of drug lead compounds in both the medicinal and medical industries.
As a plant group closely related to fungi, lichens are well known to be composed of a variety of fungi, such as surface symbiosis fungi and endophytic fungi [15,16].Endolichenic fungi (ELF) are usually recognized as the fungi living inside healthy lichen tissues, and they do not cause any directly or indirectly noticeable negative effects on the lichen thallus [17].Conventionally, lichens supply stable living environments and suitable nutrients to meet the survival requirements of endolichenic fungi for normal growth.In turn, ELF will produce abundant secondary metabolites for lichens to resist biotic stresses due to extreme environments and accelerate their growth.ELF are similar to plant endophytic fungi; they also show a potent ability to produce pharmacologically meaningful secondary metabolites with novel structures and extensive biofunctions due to their unique living environments.They are thus becoming a new research hotspot for the discovery of new active compounds [17].
The first chemical research study on the secondary metabolite system of ELF can be traced back to 2007 [18].As a result, Paranagama successfully reported the isolation and purification of two new heptaketides from ELF, and their cytotoxicity was also evaluated.This excellent research effort was the first scientific report on the endophytic fungal metabolites of lichens, and it marked the beginning of extensive chemical research in the field of natural product chemistry using ELF.Since then, a growing number of pharmaceutical and natural product chemists have carried out fundamental studies and innovative drug developments on the metabolites of lichens and endophytic fungi.To date, thousands of secondary metabolites with different structural types, including alkaloids, steroids, xanthones, benzopyranoids, peptides, and allycylic compounds, have been discovered from ELF [19].Moreover, a huge number of these natural compounds from ELF have exhibited significant biological activity, such as antibacterial, antioxidant, cytotoxic, reversal resistance, and enzyme inhibitory activities [20].
The chemical diversity and biological activity of the secondary metabolites of ELF were comprehensively reviewed by Gao et al., covering the period up to 2015 [19].Since then, efforts towards the discovery and pharmacological investigation of the secondary metabolites of ELF have rapidly increased.According to the literature statistics from the database of Web of Science, there have been more than 90 articles on the chemistry of the secondary metabolites of endolichenic fungi, and up to 34 species of endophytic fungi in lichens have been studied since the first work on the secondary metabolites of endolichenic fungi by Paranagama et al.In the previous reports on ELF, a total of 583 natural products have been reported from endolichenic fungi.Among them, 370 compounds are novel ones, including polyketides, polyphenyls, terpenoids, steroids, alkaloids, and others.Notably, many more related studies have described new discoveries regarding the secondary metabolites of ELF in other journals on natural product chemistry, thus highlighting the relevance of ELF research.
The growing number of outstanding research works and rapid developments in the field of bioactive natural products from ELF make them increasingly important in the modern medicinal industry.Therefore, our present overview intends to provide relevant scientific information covering the recent progress in the study of bioactive natural products from ELF for readers from both the fundamental medicinal research and related industries.Previous reviews of bioactive natural products from ELF have focused on their chemical compositions and pharmacological effects, but they fail to provide an extensive discussion and analysis of endolichenic fungi metabolites in terms of their research status and future trends.In this review, the structural characteristics and composition types, biological activities, potential applications, strain distributions, and origins, as well as the research status and future trends of the bioactive secondary metabolites from ELF are comprehensively summarized, discussed, and analyzed, covering the period since 2015, in order to provide a prospective view to address the increasing demand for ELF-related fundamental research and drug innovation.

Different Types of Natural Products from Endolichenic Fungi
In recent years, chemical efforts devoted to the discovery of pharmacologically meaningful secondary metabolites of ELF have emerged and seen massive growth.Since 2015, a total of 519 natural products, excluding the repeatedly isolated ones, have been reported from ELF.Among them, 266 are novel compounds, including polyketides, polyphenyls, terpenoids, steroids, alkaloids, and others.According to a statistical analysis (Figure 1) of the structural types of natural products and their numbers from ELF, it is found that polyketides account for the largest proportion of structural types discovered from ELF each year.Overall, polyketides are the most abundant members among the secondary metabolites of ELF, followed by alkaloids, terpenes, steroids, and other types of natural products.In addition, the total number of compounds discovered each year remained stable, ranging from 40 to 80, with the exception of 2022.

Polyketides
Polyketide compounds account for the most critical family of secondary metabolites of ELF.This intriguing result might be due to their specific biosynthetic gene clusters.Polyketides are one of the most important groups of natural products, and they exhibit a variety of biologically meaningful properties.There are a variety of natural products belonging to polyketides, such as quinones, anthrones, chromogenones, isocoumarins, and other small molecules.Since the first anthraquinones from endolichenic fungi were reported by Paranagama et al. in 2007 [18], 419 members of the polyketide family have been consequently reported so far, of which 258 natural polyketides have been reported since 2015.In this review, the polyketides are further subdivided into simple aromatic polyketides, complex aromatic polyketides, and non-aromatic polyketides.Their chem-ical structures and biological activities are also extensively summarized and discussed as follows.
Figure 1.The structural types and number of natural products reported annually in ELF.

Polyketides
Polyketide compounds account for the most critical family of secondary metabolites of ELF.This intriguing result might be due to their specific biosynthetic gene clusters.Polyketides are one of the most important groups of natural products, and they exhibit a variety of biologically meaningful properties.There are a variety of natural products belonging to polyketides, such as quinones, anthrones, chromogenones, isocoumarins, and other small molecules.Since the first anthraquinones from endolichenic fungi were reported by Paranagama et al. in 2007 [18], 419 members of the polyketide family have been consequently reported so far, of which 258 natural polyketides have been reported since 2015.In this review, the polyketides are further subdivided into simple aromatic polyketides, complex aromatic polyketides, and non-aromatic polyketides.Their chemical structures and biological activities are also extensively summarized and discussed as follows.

Simple Aromatic Polyketides
Quinones are organic compounds characterized by a typical six-membered cyclic diketone structure with two double bonds, which are formulated as a highly conjugated scaffold.Among these quinone compounds, their chemical structure types can be divided

Simple Aromatic Polyketides
Quinones are organic compounds characterized by a typical six-membered cyclic diketone structure with two double bonds, which are formulated as a highly conjugated scaffold.Among these quinone compounds, their chemical structure types can be divided into benzoquinones, anthraquinones, perylene quinones, and quinone derivatives.Notably, benzoquinones are the most abundant family, and they show fascinating structural diversity.Quinones frequently occur in ELF and usually show potent biological activity.To date, 29 quinones (Figure 2) have been successfully discovered from ELF since 2015.In this part, the structural characteristics and biological activities of the quinones reported since 2015 are summarized and discussed.
According to the structural characteristics of these simple aromatic polyketide compounds, their possible biosynthetic pathways were proposed, as shown in Scheme 1 [21].The simple aromatic polyketides from ELF were considered to be genenrated from a commutual precusor, C 16 -octaketide.Firstly, C 16 -octaketide was catalyzed by non-reducing polyketide synthase to undergo aldol condensation and cyclization to yield the intermediate atrochryone carboxylic acid, which can be further oxidized and dehydrated to generate endocrocin (1) or subjected to a series of chemical transformations involving dehydration/decarboxylation/spontaneous oxidation to offer the critical compound emodin.Emodin can be transformed into intermediate a through the enzymatically catalyzed oxidative ring-opening reaction with a carbon reduction between C-4 and C-5.Moreover, emodin can also be subsequently dehydrated to generate the intermediate b.Padhi et al. reported the isolation and identification of three xanthones, funiculosone (38), mangrovamide J (39), and ravenilin (40), from a crude extract of the endolichenic fungus Talaromyces funiculosus in 2019 [26].Among them, compound 38 was reported as an undescribed substituted dihydroxanthene-1,9-dione that showed moderate antibacterial activity, with MIC values ranging from 23 to 58 µg/mL towards E. coli and Staphylococcus aureus.Moreover, 38 displayed significant inhibitory activity against Candida albicans, with an MIC value of 35 µg/mL.The daldipyrones A-C (41-43) were identified from an endolicanic fungus, Daldinia pyrenaica 047188, by Lee et al. in 2023 [27].Notably, the structures of daldipyrones A-C (41)(42)(43) were determined to share an unprecedented caged xanthone [6,6,6,6,6] polyketide future with a spiro-azaphilone unit using a spectroscopic analysis and chemical derivatization.Interestingly, the biological evaluation results illustrated that daldipyrenone A (41) displayed conspicuous antimelanogenic activity with an EC50 value lower than that of the positive control as well as moderate adiponectin-secretion-promoting activity.
According to the structural characteristics of these simple aromatic polyketide compounds, their possible biosynthetic pathways were proposed, as shown in Scheme 1 [21].The simple aromatic polyketides from ELF were considered to be genenrated from a commutual precusor, C16-octaketide.Firstly, C16-octaketide was catalyzed by nonreducing polyketide synthase to undergo aldol condensation and cyclization to yield the intermediate atrochryone carboxylic acid, which can be further oxidized and dehydrated to generate endocrocin (1) or subjected to a series of chemical transformations involving dehydration/decarboxylation/spontaneous oxidation to offer the critical compound emodin.Emodin can be transformed into intermediate a through the enzymatically catalyzed oxidative ring-opening reaction with a carbon reduction between C-4 and C-5.Moreover, emodin can also be subsequently dehydrated to generate the intermediate b.In addition, chromones and isocoumarins are also widely observed and recognized as very common secondary metabolites (Figure 4) in endolichenic fungi.Since 2015, 25 chromogenic ketones together with 22 isocoumarins have been reported.For example, Kim et al. isolated two new chromones, phomalichenones C-D (44)(45), and three known chromone derivatives (46)(47)(48) from the endolichenic fungus Phoma sp. in 2018 [28].In 2019, a phomalone derivative, (7-hydroxy-8-[2-hydroxyethyl]-5-methoxy-2-methylchroman-4-one (58), was isolated from the endolichenic fungus Cochliobolus kusanoi [29].In the same year, two other new chromone derivatives, ophiochromanone (49) and ophiolactone (50), were isolated and identified from the EtOAc extract of Ophiosphaerella korrae by Li et al. [30].Furthermore, the chromones spororrminone A (51) and 2-epi-spororrminone A (52) were also isolated as two new compounds from the crude extract of the endolichenic fungus Sporormiella irregularis; they represent the first examples of 2-(5-oxotetrahydrofuran-2-yl)chromone with a 7-carboxylic functional group [31].However, neither of these two new compounds showed obvious antimicrobial activity or cytotoxicity.
In 2023, Gamage et al. found that the strain Arthrinium sp.EL000127 could produce phthalide derivatives, such as the known compound 3-O-methylcyclopolic acid (114) and two new analogues, 3-O-phenylethylcyclopolic acid (115) and 3-O-p-hydroxyphenylethylcyclopolic acid (116) [43].The compounds 114-116 exhibited very weak cytotoxicity against HUVECs, with IC 50 values of 215.6, 43.8, and 1.8 mM, respectively.Moreover, compound 116 exhibited antiangiogenic activity by inhibiting the mRNA expression of genes to regulate epithelial cell survival and motility, suggesting that compound 116 is a potent antiangiogenic agent with promising potential as a lead compound for the development of novel cancer therapeutic agents.
Li et al. isolated and identified nine polyketide-derived compounds from the crude extract of O. korrae and assigned them with the trivial names of ophiosphaerellins A-I textls [-25](192-200), this lichen endophytic fungus was isolated from Physcia sp.[50].Ophiosphaerellins A-I (192-200) shared a novel type of unprecedented scaffold with a bicyclo[4.1.0]heptenonebackbone.In addition, the stereochemistry of C-1 in the bicyclo[4.1.0]heptenonering system of ophiosphaerellins A-I (192-200) had been innovatively determined by considering the CEs at 210-240 nm as referring to π → π* transitions.Moreover, ophiosphaerellin C (194) exhibited obvious anti-ACH activity, with minimum inhibitory quantities of 1.25 µg/mL, which was weaker than that of galantamine (the positive control, 0.006 µg/mL).
In 2018, two new macrolides, myxotrilactone A (204) and cladospolide B (205), were isolated from the extract of a solid-substrate culture of the endolichenic fungus Myxotrichum sp. by Yuan et al. [59].Compound 204 displayed potent inhibitory activity on the root elongation of Arabidopsis thaliana, with more than a 75% inhibition rate at a concentration of 32 µg/mL.Similarly, compound 205 also displayed excellent inhibitory activity on the root elongation of A. thaliana, with a 69% inhibition rate at a concentration of 32 µg/mL.The aforementioned informative results collectively indicated that the endolichenic fungus Myxotrichum sp.might contribute to the defense capability of host lichens by producing phytotoxic secondary metabolites, which might also serve as ecologically beneficial and environmentally friendly mycoherbicides.Madyranga et al. also reported the isolation of a novel microlide compound, neurosporalol 206, from the endolichenic fungus N. ugadawe in 2021, which was isolated from the lichen host G. tsunodae [44].Neurosporalol 206 showed antioxidant activity, with an IC 50 value of 5.03 ± 0.15 µg/mL, comparable to that of the positive control, BHT.

Complex Aromatic Polyketides
Complex aromatic polyketides are usually constructed from two or more aromatic polyketide fragments.These types of natural products from endolichenic fungi are exemplified by naphthol polymers, xanthone dimers, perylenequinonoids, and spiciferonederived dimers.Among them, naphthol polymers account for the largest proportion of the complex aromatic polyketides from endolichenic fungi, and the spirodioxynaphthalenes are well recognized as the typical structural types.The spirodioxynaphthalenes contain two 1,8-dihydroxynaphthalene-derived units with the formation of an interesting 6/6-6-6/6 spiroketal ring skeleton bridging through a typical spiroketal linkage, and most of them have been revealed to display a variety of biologically significant properties [60].The main types of dinaphthalene compounds reported so far include spirodi-oxynaphthalenes with two oxygen bridges, dioxynaphthalenes with one oxygen bridge, spiromonoxynaphthalenes with a furan-sipro skeleton through one oxygen bridge and one C-C bridge, and fusodioxynaphthalenes with a new fuso-nonadiene skeleton.
are well recognized as the typical structural types.The spirodioxynaphthalenes contain two 1,8-dihydroxynaphthalene-derived units with the formation of an interesting 6/6-6-6/6 spiroketal ring skeleton bridging through a typical spiroketal linkage, and most of them have been revealed to display a variety of biologically significant properties [60].The main types of dinaphthalene compounds reported so far include spirodioxynaphthalenes with two oxygen bridges, dioxynaphthalenes with one oxygen bridge, spiromonoxynaphthalenes with a furan-sipro skeleton through one oxygen bridge and one C-C bridge, and fusodioxynaphthalenes with a new fuso-nonadiene skeleton.

Polyphenyls
Polyphenyl derivatives are also commonly encountered in the secondary metabolites of endolichenic fungi, and most of them are terphenyls.Terphenyls are aromatic hydrocarbons consisting of one phenol core and two benzyl substituents.The terphenyl deriva- In 2023, six spiciferone-derived dimers with an unprecedented skeleton, named phaeosphaerones A-F (251-256), were reported and isolated from the endolichenic fungus Phaeosphaeria sp.[64], and most of them were composed of a novel 4H-chromene-4,7(8H)dione scaffold and an α-pyrone unit, forming fascinating dimeric compounds.Among them, compounds 251 and 253-256 possessed unconventional carbon skeletons featuring an ethylidene bridge, whereas compound 252 was a structurally interesting homodimer of spiciferone, which might be furnished by an unusual Rauhut-Currier reaction.Moreover, compounds 251-253 showed significant inhibitory effects towards the growth of dicot Arabidopsis thaliana at 100 µM.In addition, two biosynthetically related known secondary metabolites, lecanicillone A (257) and lecanicillolide (258), were also reported, and both showed very strong inhibitory effects on the fresh weight and root elongation of Arabidopsis thaliana, with IC 50 values of 32.04 and 26.78 µM, respectively.

Polyphenyls
Polyphenyl derivatives are also commonly encountered in the secondary metabolites of endolichenic fungi, and most of them are terphenyls.Terphenyls are aromatic hydrocarbons consisting of one phenol core and two benzyl substituents.The terphenyl derivatives include p-terphenyls, m-terphenyls, and o-terphenyls, all of which usually show a wide range of biological activities.Among them, p-terphenyl derivatives are the most common natural terphenyls, in which two terminal benzene rings connect to the phenol mother ring at the p-position.
In recent years, more and more p-terphenyls have been found from endolichenic fungi (Figure 13).274), from the extract of the endolichenic fungus Floricola striata in 2016 [65].Their biological activity assays showed that floricolins A-C (259-261) displayed moderate antifungal activity against Candida albicans, with MICs of 16, 8, and 8 µg/mL, respectively.Further antifungal mechanistic investigations revealed that the most active compound, compound 261, exerted fungicidal action through the destruction of the cell membrane.
The biosynthetic pathway of terphenyl compounds originates from L-tyrosine, which undergoes an enzymatic amino transformation reaction to provide 4-hydroxylpheylpyruvic acid.The biosynthetic pathway of terphenylquinone is related to the critical precursor 4-hydroxyphenylpyruvic acid through the Claisen-type condensation reaction, and this conclusion was experimentally confirmed by 13 C and 14 C isotope labeling experiments.The biosynthesis of atromentin further demonstrated that the L-tyrosine was deaminated to 4-hydroxyphenylpyruvic acid by the PLP-dependent transaminase AtrD, which can transform the amino group into 2-oxoglutaric acid (2-OG).The further Claisen-type condensation of two molecules of 4-hydroxyphenylpyruvic acid to atromentin was catalyzed by the quinone synthetase AtrA (Scheme 3) [67,68].
The biosynthetic pathway of terphenyl compounds originates from L-tyrosine, which undergoes an enzymatic amino transformation reaction to provide 4-hydroxylpheylpyruvic acid.The biosynthetic pathway of terphenylquinone is related to the critical precursor 4-hydroxyphenylpyruvic acid through the Claisen-type condensation reaction, and this conclusion was experimentally confirmed by 13 C and 14 C isotope labeling experiments The biosynthesis of atromentin further demonstrated that the L-tyrosine was deaminated to 4-hydroxyphenylpyruvic acid by the PLP-dependent transaminase AtrD, which can transform the amino group into 2-oxoglutaric acid (2-OG).The further Claisen-type condensation of two molecules of 4-hydroxyphenylpyruvic acid to atromentin was catalyzed by the quinone synthetase AtrA (Scheme 3) [67,68].

Terpenoids
Terpenoids are a series of structurally specific polymers constructed by isoprene units with different amounts and their structurally modified derivatives; their general formula can be theoretically considered as (C5H8)n.Terpenoids are broadly distributed in various plants, and they can also be frequently found in the secondary metabolites of microorganisms.Terpenoids are also commonly discovered in lichens' endophytic fungi as one of their most major bioactive chemical constituents, and they show outstanding structural diversity, mainly composed of sesquiterpenoids, diterpenoids, and triterpenoids.The terpenoids isolated from lichens' endophytic fungi usually exhibit a wide range of pharmaceutically significant bioactivities, with especially potent cytotoxic and antibacterial activity.Moreover, the terpenoids Scheme 3. The plausible biosynthetic pathway of terphenyl.
In 2015, Li et al. isolated the tetracyclic triterpene helvolic acid (325) from the extract of endolichenic fungus Nectria sp.[54].Moreover, six novel sesterterpenoids, asper- Xylaria hypoxylon is a lichen-associated fungus that was also found to have an outstanding capacity to produce novel secondary metabolites, and there were seven new bioactive eremophilane sesquiterpenes, eremoxylarins D-J (306-312), isolated from this fungus in 2023 [72].The antimicrobial activity of eremoxylarins D-J (306-312) was evaluated, and the biological results showed that eremoxylarins D (306), F (308), G (309), and I (311) exhibited potent antibacterial activity against a series of Gram-positive bacteria (S. aureus, methicillinresistant S. aureus (MRSA), and S. epidermidis), showing minimum inhibitory concentration (MIC) values ranging from 0.39 to 12.5 µg/mL.Among them, eremoxylarin I (311) was the most antibacterially active sesquiterpene, and it showed MIC values of 0.39-1.56µg/mL, which were much greater than or similar to those of the positive control drug.Moreover, eremoxylarin I (311) was also biologically active against HCoV-229E, with an IC 50 value of 18.1 µM, which was the concentration obtained without any noticeable toxicity to the hepatoma Huh-7 cell line.
In 2022, Varlı et al. isolated three known diterpenoids, radianspenes C-D (321-322), and dahliane D (323), from the crude extract of Nemania sp.EL006872 ELF [75], and the three diterpenoids showed promising potential for innovative drug development for immuneand immuno-tumor therapies.Moreover, a previous pharmacological study revealed that radianspene C (321) had a significant cytotoxic effect on MDA-MB-435, with an IC 50 value of 0.91 µM [76].In 2023, Libertellenone T (324) was isolated also as a novel diterpenoid from the endolichenic fungus Pseudoplectania sp.(EL000327), and it exerted strong cytotoxicity towards the human CRC cell lines of Caco2, HCT116, DLD1, and HT29, with IC 50 values of 17.5, 28, 36.6, and 28 µg/mL, respectively [77].The investigation into its pharmacological mechanism confirmed that 324 could cause G2/M phase arrest and ROS/JNK signaling activation to induce the apoptosis of Caco2 cells.Moreover, 324 exhibited perfect synergistic effects in combination with various known and novel anticancer clinical agents.

Alkaloids and Their Biological Activities
Alkaloid compounds, with various unique structures or skeletons, are a group of basic organic compounds containing nitrogen atoms that widely exist in nature.Most of these compounds possess complex fused or bridged ring scaffolds, with the nitrogen atoms generally existing in the ring system.Numerous excellent medicinal research efforts have revealed that alkaloid compounds play significant and extensive roles in various biological processes, and they are recognized as an important research focus in the natural product, synthesis, biosynthesis, and medicine biochemical fields.Since 2015, a total of 52 alkaloids with nine cyclic peptide compounds have been found in the secondary metabolites of endolichenic fungi, and many of them have shown biologically meaningful activity (Figure 16).For example, Lee et al. successfully isolated three new diketopiperazines, cyclo(L-Pro-D-trans-Hyp) (337), cyclo(L-Pro-D-Glu) (338), and cyclo(D-Pro-D-Glu) (339), together with five known diketopiperazines, cyclo(L-Pro-D-allo-Thr) (340), cyclo(L-Pro-L-Asp)(341), cyclo(D-Pro-Gly) (342), cyclo(L-Pro-L-Ala) (343), and cyclo(L-Pro-D-Ala) (344), from the endolichenic fungus Colpoma sp.CR1465A in 2016 [79].
Alkaloid compounds, with various unique structures or skeletons, are a group of basic organic compounds containing nitrogen atoms that widely exist in nature.Most of these compounds possess complex fused or bridged ring scaffolds, with the nitrogen atoms generally existing in the ring system.Numerous excellent medicinal research efforts have revealed that alkaloid compounds play significant and extensive roles in various biological processes, and they are recognized as an important research focus in the natural product, synthesis, biosynthesis, and medicine biochemical fields.Since 2015, a total of 52 alkaloids with nine cyclic peptide compounds have been found in the secondary metabolites of endolichenic fungi, and many of them have shown biologically meaningful activity (Figure 16).For example, Lee et al. successfully isolated three new diketopiperazines, cyclo(L-Pro-D-trans-Hyp) (337), cyclo(L-Pro-D-Glu) (338), and cyclo(D-Pro-D-Glu) (339), together with five known diketopiperazines, cyclo(L-Pro-D-allo-Thr) (340), cyclo(L-Pro-L-Asp)(341), cyclo(D-Pro-Gly) (342), cyclo(L-Pro-L-Ala) (343), and cyclo(L-Pro-D-Ala) (344), from the endolichenic fungus Colpoma sp.CR1465A in 2016 [79].In 2019, a chlorinated isocoumarin and indole alkaloid hybrid metabolite named isocoumarindole A (345) was obtained from the endolichenic fungus Aspergillus sp.CPCC400810 by Chen et al. [80].The novel alkaloid isocoumarindole A (345) was constructed with a chlorinated isocoumarin and an indoledione piperazine unit by the linkage of a carbon-carbon bond to form an unprecedented dimeric skeleton.The subsequent bioactivity test showed that isocoumarindole A (345) exhibited considerable cytotoxicity against the cancer cell lines of BT-549 and RKO, with IC 50 values of 1.63 and 5.53 µM, respectively.In 2020, Padhi et al. isolated and identified a novel pyro-alkaloid, pyrophen (346), from the secondary metabolites of an endolichenic fungus, Aspergillus niger [40], which showed moderate antimicrobial activity, with an IC 50 ranging from 35 to 97 mg/mL, against a broad spectrum of bacteria.

Steroids
Steroids are well known as one of the most critical chemical constituents participating in the construction of the cell membrane for various fungi, and they are also a group of natural compounds widely adopted as novel lead compounds in the medicinal industry.The structural types of steroids include phytosterols, bile acids, C21 steroids, cardiac glycosides, steroid saponins, and so on.Most steroid compounds share the basic skeletal structure of cyclopentanophenanthrene.In addition, there are usually two angularmethylgroups (C-10, C-13) and a side chain with different carbon atoms or oxygen-containing groups, such as hydroxyl and carbonyl groups (C-17), on the parent nucleus of cyclopentanophenanthrene.However, the number of steroids discovered from endolichenic fungi is very limited, as there are only 25 types of steroid compounds reported to date, with a total of 12 discovered since 2015 (Figure 18).

Steroids
Steroids are well known as one of the most critical chemical constituents participating in the construction of the cell membrane for various fungi, and they are also a group of natural compounds widely adopted as novel lead compounds in the medicinal industry The structural types of steroids include phytosterols, bile acids, C21 steroids, cardiac glycosides, steroid saponins, and so on.Most steroid compounds share the basic skeletal structure of cyclopentanophenanthrene.In addition, there are usually two angularmethylgroups (C-10, C-13) and a side chain with different carbon atoms or oxygen-containing groups, such as hydroxyl and carbonyl groups (C-17), on the parent nucleus of cyclopentanophenanthrene.However, the number of steroids discovered from endolichenic fungi is very limited, as there are only 25 types of steroid compounds reported to date, with a total of 12 discovered since 2015 (Figure 18).
For example, Yuan et al. isolated two steroids, ergone (389) and ergosterol (390), from Talaromyces sp.associated with X. angustiphylla in 2018 [57].Zhao et al. isolated eight new viridins, nodulisporiviridins A-H (371-378), from the extract of the endolichenic fungus Nodulisporium sp. in 2015 [85].Among them, compounds 371-374 are a series of novel viridins with a unique ring-opened skeleton.Furthermore, the authors disclosed that nodulisporiviridin G (377) displayed potent Aβ42 aggregation-inhibitory activity, with an IC50 value of 1.2 µM, which then led to the discovery that 377 showed an outstanding capacity to significantly improve short-term memory, via a biological assay in an Aβ transgenic drosophila model for Alzheimer's disease.For example, Yuan et al. isolated two steroids, ergone (389) and ergosterol (390), from Talaromyces sp.associated with X. angustiphylla in 2018 [57].Zhao et al. isolated eight new viridins, nodulisporiviridins A-H (371-378), from the extract of the endolichenic fungus Nodulisporium sp. in 2015 [85].Among them, compounds 371-374 are a series of novel viridins with a unique ring-opened skeleton.Furthermore, the authors disclosed that nodulisporiviridin G (377) displayed potent Aβ42 aggregation-inhibitory activity, with an IC 50 value of 1.2 µM, which then led to the discovery that 377 showed an outstanding capacity to significantly improve short-term memory, via a biological assay in an Aβ transgenic drosophila model for Alzheimer's disease.
In the same year, they found another rare class of steroids (4-methyl-pro-gesteroids) in this fungus (N.sp.) via the OSMAC (one strain, many compounds) approach [86], and they named these ten new 4-methyl-progesteroid derivatives as nodulisporisteroids C-L (379-388).Interestingly, their biogenic synthesis (Scheme 4) has also been chemically proposed.In the proposed biosynthetic pathway of compounds 379-390, all of the fungal steroids are generally derived from lanosterol [87], and the demethylation of C-14 of lanosterol results in 4a-methyl-zymosterol, which is the critical precursor of compound 388.Compound 381 can be generated by the Baeyer-Villiger oxidation of 4-methyl-8-enpregnan-3,20-dione between the C-3 and C-4 positions, whereas compounds 382-387 are considered to be transformed from the intermediates 379 or 380 via chain opening and oxidative demethylation.

The Main Research Groups Engaged in the Study of Endolichenic Fungi
During the summary of chemical composition and biological activity, a comprehen sive analysis of the research on the secondary metabolites of endolichenic fungi from 2015 to the present was conducted, as shown in Figure 20.It unambiguously shows that the number of published articles worldwide is very stable and is maintained at approximately 10 articles per year.Meanwhile, the total number of compounds reported annually fluc tuates slightly, usually maintained at a rate of between 40 and 80 compounds for each year.In particular, 20-40 new compounds have been discovered in the field of natura product chemistry every year since 2015.
Based on the research articles published in internationally renowned journals on the secondary metabolites of lichen endophytic fungi since 2015, the main research groups contributing to the natural product chemistry of lichen endophytic fungi are also prelim inarily summarized.As shown in Figure 21, it can be found that the most active research groups related to lichen endophytic fungi are those of Hongxiang Lou, Hanggun Kim Hao Gao, and Priyani A. Parargama.These four research groups have contributed more than a half of all published research articles from 2015 to the present.

The Main Research Groups Engaged in the Study of Endolichenic Fungi
During the summary of chemical composition and biological activity, a comprehensive analysis of the research on the secondary metabolites of endolichenic fungi from 2015 to the present was conducted, as shown in Figure 20.It unambiguously shows that the number of published articles worldwide is very stable and is maintained at approximately 10 articles per year.Meanwhile, the total number of compounds reported annually fluctuates slightly, usually maintained at a rate of between 40 and 80 compounds for each year.In particular, 20-40 new compounds have been discovered in the field of natural product chemistry every year since 2015.
Based on the research articles published in internationally renowned journals on the secondary metabolites of lichen endophytic fungi since 2015, the main research groups contributing to the natural product chemistry of lichen endophytic fungi are also preliminarily summarized.As shown in Figure 21, it can be found that the most active research groups related to lichen endophytic fungi are those of Hongxiang Lou, Hanggun Kim, Hao Gao, and Priyani A. Parargama.These four research groups have contributed more than a half of all published research articles from 2015 to the present.
Moreover, the total number of isolated compounds and new ones discovered from endolichenic fungi by the aforementioned main research groups are tentatively analyzed (Figure 22).Hongxiang Lou's research group has made a major contribution to the discovery of secondary metabolites from endophytic fungi in lichen.As illustrated in Figure 22, a total of 215 natural products have been reported by this group, and there are 124 natural products that have been reported as new compounds.Interestingly, the total number of natural products and new compounds reported by Hongxiang Lou's research group accounts for more than a half of all the secondary metabolites isolated from endolichenic fungi.Moreover, the total number of isolated compounds and new ones discover endolichenic fungi by the aforementioned main research groups are tentatively a (Figure 22).Hongxiang Lou's research group has made a major contribution to covery of secondary metabolites from endophytic fungi in lichen.As illustrated i 22, a total of 215 natural products have been reported by this group, and there  Moreover, the total number of isolated compounds and new ones discovered endolichenic fungi by the aforementioned main research groups are tentatively ana (Figure 22).Hongxiang Lou's research group has made a major contribution to th covery of secondary metabolites from endophytic fungi in lichen.As illustrated in F 22, a total of 215 natural products have been reported by this group, and there ar natural products that have been reported as new compounds.Interestingly, the total ber of natural products and new compounds reported by Hongxiang Lou's res At the same time, according to the research articles on the secondary metabolites of lichen endophytic fungi published in internationally renowned journals since 2015, the main research area distribution of natural products of lichen endophytic fungi is further summarized.As shown in Figure 23, it can be found that research related to endolichenic fungi is mainly distributed in Asia, with a slight level of engagement in Europe and North America.Since 2015, China has contributed more than half of the research papers on endophytic fungi in lichens, followed by South Korea with 18%.Therefore, it is not difficult to find that the research of endophytic fungi in lichen is mainly concentrated in Asia, while there is less research being conducted in other continents, which also indicates that a large number of endophytic fungi in lichen need to be explored.At the same time, according to the research articles on the secondary metabolites of lichen endophytic fungi published in internationally renowned journals since 2015, the main research area distribution of natural products of lichen endophytic fungi is further summarized.As shown in Figure 23, it can be found that research related to endolichenic fungi is mainly distributed in Asia, with a slight level of engagement in Europe and North America.Since 2015, China has contributed more than half of the research papers on endophytic fungi in lichens, followed by South Korea with 18%.Therefore, it is not difficult to find that the research of endophytic fungi in lichen is mainly concentrated in Asia, while there is less research being conducted in other continents, which also indicates that a large number of endophytic fungi in lichen need to be explored.At the same time, according to the research articles o lichen endophytic fungi published in internationally ren main research area distribution of natural products of lic summarized.As shown in Figure 23, it can be found that fungi is mainly distributed in Asia, with a slight level of en America.Since 2015, China has contributed more than ha dophytic fungi in lichens, followed by South Korea with 1 to find that the research of endophytic fungi in lichen is ma there is less research being conducted in other continents, number of endophytic fungi in lichen need to be explored

The Fungal Sources and Structural Characteristics of the Isolated Secondary Metabolites
The endolichenic fungi obtained from lichens are strategically important bioresources for the microbial and medicinal industries.Thus, the origination and species of endolichenic fungi are also comprehensively analyzed in this review, in order to provide detailed and clear information for potential readers to facilitate a better understanding.A species analysis of the endolichenic fungi reported in recent years is illustrated in Figure 24, and it can be readily found that 35 endolichenic fungi from 20 species of lichens in five districts have been investigated since 2015.Among these endolichenic fungi, Xylaria sp., Aspergillus sp., Nodulisporium sp., Daldinia sp., and Talaromyces sp.collectively represent the predominant endophytes with the largest proportions and that are the most frequently studied.
sources for the microbial and medicinal industries.Thus, the origination and species endolichenic fungi are also comprehensively analyzed in this review, in order to provi detailed and clear information for potential readers to facilitate a better understanding species analysis of the endolichenic fungi reported in recent years is illustrated in Figu 24, and it can be readily found that 35 endolichenic fungi from 20 species of lichens in fi districts have been investigated since 2015.Among these endolichenic fungi, Xylaria s Aspergillus sp., Nodulisporium sp., Daldinia sp., and Talaromyces sp.collectively represe the predominant endophytes with the largest proportions and that are the most frequen studied.With the further aim of providing an unambiguous depiction of the secondary m tabolites generated by the major strains of endolichenic fungi, a careful analysis of t critical chemical compositions of the major strains is also performed, and the informat results are depicted in Figure 25.From the statistical data, it is easily found that the s ondary metabolites from most of the endolichenic fungi are mainly polyketides, with t exception of the fungi of Xylaria sp., Aspergillus sp., and Nodulisporium sp.Moreover, t structural types of the secondary metabolites from these three strains are relatively verse, evenly distributed as polyketides, terpenoids, and alkaloids.Interestingly, a ser  With the further aim of providing an unambiguous depiction of the secondary metabolites generated by the major strains of endolichenic fungi, a careful analysis of the critical chemical compositions of the major strains is also performed, and the informative results are depicted in Figure 25.From the statistical data, it is easily found that the secondary metabolites from most of the endolichenic fungi are mainly polyketides, with the exception of the fungi of Xylaria sp., Aspergillus sp., and Nodulisporium sp.Moreover, the structural types of the secondary metabolites from these three strains are relatively diverse, evenly distributed as polyketides, terpenoids, and alkaloids.Interestingly, a series of novel compounds with unprecedented skeletons have been found in the four endolichenic fungal strains of Xylaria sp., Aspergillus sp., Daldinia sp., and Phaeosphaeria sp.
of novel compounds with unprecedented skeletons have been found in the four endolichenic fungal strains of Xylaria sp., Aspergillus sp., Daldinia sp., and Phaeosphaeria sp.

The Biological Activity of the Secondary Metabolites of Endolichenic Fungi
The biological activity of secondary metabolites is an important subject for natural product and medicinal chemists.In this review, the biological activity of the secondary metabolites discovered from endolichenic fungi since 2015 is also extensively summarized and carefully analyzed (Tables 1-5).According to the statistical data, more than 40 biologically meaningful natural products have been isolated and pharmacologically evaluated since 2015, and their biological properties include cytotoxic, antimicrobial, anti-inflammatory, antioxidative, anti-influenza A virus, and other related biological effects.The structural types of the reported compounds and their biological activity are comprehensively analyzed, and the conclusive results are shown in Figure 26.Notably, these results collectively indicate that the rich and significant biological activity of secondary metabolites from endolichenic fungi might be closely related to their structural diversity.For example, a larger number and various structural types of polyketides are included in the secondary metabolites from endolichenic fungi, and additional types of bioactive molecules remain to be discovered.

The Biological Activity of the Secondary Metabolites of Endolichenic Fungi
The biological activity of secondary metabolites is an important subject for natural product and medicinal chemists.In this review, the biological activity of the secondary metabolites discovered from endolichenic fungi since 2015 is also extensively summarized and carefully analyzed (Tables 1-5).According to the statistical data, more than 40 biologically meaningful natural products have been isolated and pharmacologically evaluated since 2015, and their biological properties include cytotoxic, antimicrobial, antiinflammatory, antioxidative, anti-influenza A virus, and other related biological effects.The structural types of the reported compounds and their biological activity are comprehensively analyzed, and the conclusive results are shown in Figure 26.Notably, these results collectively indicate that the rich and significant biological activity of secondary metabolites from endolichenic fungi might be closely related to their structural diversity.For example, a larger number and various structural types of polyketides are included in the secondary metabolites from endolichenic fungi, and additional types of bioactive molecules remain to be discovered.
Finally, the bioactivity of the secondary metabolites of the most prominent strains is statistically analyzed (Figure 27), and it is found that the bioactivity of different strains is also significantly different, such as that of the endolichenic fungi Xylaria sp.The secondary metabolites mainly reflect the cytotoxicity and antibacterial activity of the strain Aspergillus sp.It mainly shows cytotoxicity, antimicrobial activity, and anti-inflammatory effects.Meanwhile, the strain Daldinia sp.shows antioxidant and antiviral activities.Finally, the bioactivity of the secondary metabolites of the most prominent strains is statistically analyzed (Figure 27), and it is found that the bioactivity of different strains is also significantly different, such as that of the endolichenic fungi Xylaria sp.The secondary metabolites mainly reflect the cytotoxicity and antibacterial activity of the strain Aspergillus sp.It mainly shows cytotoxicity, antimicrobial activity, and anti-inflammatory effects.Meanwhile, the strain Daldinia sp.shows antioxidant and antiviral activities.

Conclusions and Outlook
Lichens are naturally occurring as stable microorganism-like plants with complex compositions of known ascomycetes, photosynthesizing partners, and specific basidiomycete yeasts.Because of their extremely unique growth environments, they are widely acknowledged as impressive microorganisms with an outstanding capacity to produce a variety of biologically significant and structurally fascinating secondary metabolites with rich structural diversities.Endophytic fungi, as the main components of lichens, can also produce secondary metabolites with novel structures and extensive bioactivities based on their mutualistic symbiosis with their hosts, which have thus made them appealing targets and attracted extensive research attention from natural product and pharmaceutical chemists.In recent years, many research groups have been motivated to conduct research on the discovery of novel secondary metabolites from endolichenic fungi.According to the number of isolated compounds and the research articles published in renowned journals in recent years, it can be readily found that research efforts and contributions with regard to the secondary metabolites of endophytic fungi in lichens have steadily increased, and they collectively demonstrate endolichenic fungi's increasing importance in the field of natural product chemistry.
According to the careful inspection and review of research efforts made since 2015, a variety of endophytic fungi were obtained from lichens by surface sterilization, and a number of species of endolichenic fungal strains with broad biological activities and high chemical abundance were selected as targeted strains for further extensive chemical studies, to obtain promising lead compounds with biologically significant activities and potential economic value.Thus, the urgent requirement for innovative drug development in the modern pharmaceutical industry can be satisfied.The specific process followed by natural product chemists in studying endolichenic fungi is introduced in Figure 28.Among past research achievements, a total of 583 compounds have been successfully isolated and identified from endolichenic fungi, of which 374 have been introduced as new compounds.Since 2015, 35 species of lichen endophytic fungi have been extensively investigated with the aim to discover biologically active or structurally novel natural products.As a result, a total of 407 compounds, with 270 new ones, have been reported, which include 16 novel compounds with unprecedented skeletons.Most importantly, more than 30 active lead compounds have been described to show significant potential in new drug development.After a comprehensive analysis of the research status of this topic since 2015, it is easily found that the main secondary metabolites in endolichenic fungi share outstanding characteristics in terms of structural diversity, and they usually contain polyketides as their major bioactive chemical components, followed by alkaloids, terpenes, steroids, and so on.The great structural diversity of the secondary metabolites in endolichenic fungi may also contribute to the diversity of their biological activity.Through careful inspection and a statistical analysis, it is found that the secondary metabolites of lichen endophytic fungi show extensive biological activities, including not only significant cytotoxic, antioxidant, and anti-inflammatory activities, but also noticeable antiviral and root-growth-inhibitory biofunctions.In summary, the numerous excellent studies on the secondary metabolites of endophytic fungi in lichens collectively indicate that they are emerging as a research hotspot for both natural product and pharmaceutical chemists.Moreover, it is believed that further scientific research on the secondary metabolites of endolichenic fungi will be beneficial to enrich the compound library of chemical molecules, as well as improve the biological activities of drug molecules.
Author Contributions: Writing-original draft preparation, writing-review and editing, W.Z.; vis- After a comprehensive analysis of the research status of this topic since 2015, it is easily found that the main secondary metabolites in endolichenic fungi share outstanding characteristics in terms of structural diversity, and they usually contain polyketides as their major bioactive chemical components, followed by alkaloids, terpenes, steroids, and so on.The great structural diversity of the secondary metabolites in endolichenic fungi may also contribute to the diversity of their biological activity.Through careful inspection and a statistical analysis, it is found that the secondary metabolites of lichen endophytic fungi show extensive biological activities, including not only significant cytotoxic, antioxidant, and anti-inflammatory activities, but also noticeable antiviral and root-growth-inhibitory biofunctions.In summary, the numerous excellent studies on the secondary metabolites of endophytic fungi in lichens collectively indicate that they are emerging as a research hotspot for both natural product and pharmaceutical chemists.Moreover, it is believed

Figure 1 .
Figure 1.The structural types and number of natural products reported annually in ELF.
Compound 31 is derived from intermediate b via methylation.Intermediate b can transform into intermediate c though decarboxylation and oxidation reactions, whereas methylation and glycosylation can attain compound 30 from intermediate c.

Figure 5 .
Figure 5.Chemical structures of polyketides 59-79 from ELF. Shevkar et al. obtained a new compound, peniazaphilin B (73), from the endolichenic fungus Talaromyces pinophilu in 2022[34].In the same year, Yuan et al. isolated two new 3,4-dihydroisocoumarin derivatives with a novel dihydrothiophene skeleton from Talaromyces sp. and named them as talarolactone A (74) and talarolactone A (74a)[35].Moreover, a plausible biosynthetic pathway for 74 has been proposed, as shown in Scheme 2. The isocoumarin skeleton is speculated to be formed through the polyketide synthetase (PKS) pathway, whereas the dihydrothiophene scaffold might be biosynthetically constructed by a cascade reaction of sulfhydrylation and cyclodehydration.In 2023, three new isocoumarin analogues, aspermarolides A-C (75-77), coupled with two known related

Figure 21 .
Figure 21.The published SCI papers since 2015 by famous research groups on the secon tabolites of endolichenic fungi.

Figure 21 .
Figure 21.The published SCI papers since 2015 by famous research groups on the secondar tabolites of endolichenic fungi.

SCIFigure 21 .
Figure 21.The published SCI papers since 2015 by famous research groups on the secondary metabolites of endolichenic fungi.

Figure 22 .
Figure 22.The numbers of compounds discovered by famous research groups.

Figure 22 .
Figure 22.The numbers of compounds discovered by famous re

Figure 23 .
Figure 23.The proportion of countries and regions contributin metabolites of endolichenic fungi since 2015.

SCIFigure 23 .
Figure 23.The proportion of countries and regions contributing to the research on the secondary metabolites of endolichenic fungi since 2015.

Figure 24 .
Figure 24.The main types of endolichenic fungi and their proportions.

Figure 24 .
Figure 24.The main types of endolichenic fungi and their proportions.

Figure 25 .
Figure 25.The structure types for secondary metabolites from endolichenic fungi.

0Figure 25 .
Figure 25.The structure types for secondary metabolites from endolichenic fungi.

Figure 26 .
Figure 26.Types of metabolites and their biological activities.

Figure 26 .
Figure 26.Types of metabolites and their biological activities.

Figure 28 .
Figure 28.The research sequence for secondary metabolites of endolichenic fungi.

Figure 28 .
Figure 28.The research sequence for secondary metabolites of endolichenic fungi.