Phytochem Rev DOI 10.1007/s11101-016-9473-1 Endolichenic fungi: a new source of rich bioactive secondary metabolites on the horizon Joshua J. Kellogg . Huzefa A. Raja Received: 3 May 2016 / Accepted: 2 July 2016 Ó Springer Science+Business Media Dordrecht 2016 Abstract Endolichenic fungi are diverse groups of predominantly filamentous fungi that reside asymptomatically in the interior of lichen thalli. Natural products from endolichenic fungi, isolated from a variety of different lichen species, have been attracting increased attention for their potential to produce bioactive metabolites possessing new structures and representing different structural classes. This is evident from the steady increase of publications devoted to endolichenic fungal metabolites over the past decade, since the first report of endolichenic secondary metabolites. The bioactive metabolites produced by endolichenic fungi originate from multiple biosynthetic pathways and occupy different chemical structure classes, including steroids, quinones, terpenoids, peptides, xanthones, sulfur-containing chromenones, etc. Endolichenic fungal metabolites possess a diverse array of bioactivities, such as anticancer, antiviral, antibacterial, antifungal, and anti-Alzheimer’s disease. This review provides the first thorough assessment of endolichenic fungi, their biodiversity, secondary metabolites, and associated bioactivity. This review will highlight the bioactive metabolites reported in recent years from endolichenic fungi, as J. J. Kellogg (&)
H. A. Raja Department of Chemistry and Biochemistry, University of North Carolina Greensboro, 435 Patricia A. Sullivan Science Building, PO Box 26170, Greensboro, NC 27402-6170, USA e-mail: jjkellog@uncg.edu well as discussing the potential of these symbiotic fungi as sources of new, diverse natural products with varying bioactivities. Keywords Bioactivity
Biodiversity
Endolichenic fungi
Lichen
Natural products Abbreviations AD Alzheimer’s disease DPPH 2,2-Diphenyl-1-picrylhydrazyl FIC Fractional inhibitory concentration GPS Global positioning system IC50 50 % inhibitory concentration ITS Internal transcribed spacer MEA Malt extract agar MIC Minimum inhibitory concentration OSMAC One-strain, many compounds PDB Potato dextrose broth Endolichenic fungi Fungal strains represent a rich source of biologically active natural product metabolites with wide-ranging biological activity. Although investigations into fungal metabolites date back to the 1870 s, the first systematic survey of fungal metabolites wasn’t initiated until after World War I by Harold Raistrick (1949). Since those initial forays into fungal secondary 123 Phytochem Rev metabolites, natural products from fungal sources have been employed as pesticides, herbicides, antibiotics, immunosuppressants, anti-infectives, and anticancer agents (Hoffmeister and Keller 2007). And yet, it is believed that only about 5 % of the global fungal species have been identified (Young 1997). Fungi occupy a wide diversity of environmental and ecological niches across the globe, including terrestrial, fresh water, and marine environments where they function as saprobes, symbionts, and pathogens. Groups of highly diverse fungi reside within the internal tissue of other organisms, living asymptomatically without any obvious sign of infection. Endophytic fungi occur within tissues of host plants and are dominated by ascomycetous fungi. Endophytic fungi have received increased attention as sources of natural products, especially after the discovery of paclitaxel (taxol) in the endophytic fungus Taxomyces andreanae, which inhabits the original source of the important anticancer drug, Taxus brevifolia (Stierle et al. 1993). Multiple reviews have highlighted the metabolite diversity and potential of endophytic fungi to produce pharmaceutically valuable natural products (Kaul et al. 2012; Nisa et al. 2015; Proksch et al. 2010; Strobel et al. 2004; Tan and Zou 2001). An analogous group of fungi inhabit the thalli of lichens in a similarly asymptomatic manner: the endolichenic fungi. Lichen thalli are an emergent property arising due to symbiotic association between a fungal organism (mycobiont) and at least one chlorophyll-containing photosynthetic organism (photobiont) such as a micro alga, a cyanobacterium, or both (Lutzoni and Miadlikowska 2009). In addition to the mycobiont of the lichen, the thallus is usually home to numerous, asymptomatic, cryptic microfungi that live in close association with the photobiont (Arnold et al. 2009). These diverse groups of fungi, which reside in the interior of a lichen thallus, have been termed as ‘endolichenic fungi’ (Arnold et al. 2009; Miadlikowska et al. 2004). Endolichenic fungi were discovered when attempts were being made to isolate the lichen forming mycobiont into pure culture (Crittenden et al. 1995; McDonald et al. 2013; Petrini et al. 1990). These fungi are similar to the endophytic fungi (sometimes also referred to as endophyte-like fungi) (Arnold et al. 2009; U’Ren et al. 2016), which reside within healthy tissues of plants and are a phylogenetically and ecologically diverse without causing any disease symptoms (Arnold 2001, 2007; Petrini 1991). 123 The endolichenic fungi, however, are distinct from mycobionts (Lutzoni and Miadlikowska 2009), which make up the lichen thallus, and from lichenicolous fungi, an ecological group of meiosporic and mitosporic fungi that can often be observed on living lichens (Arnold et al. 2009). The endolichenic fungi consist of mostly horizontally transmitted, functionally advantageous fungi, and include abundant taxa belonging to diverse classes, orders and families within the Ascomycota (Pezizomycotina) (Arnold et al. 2009; Girlanda et al. 1997; Kannangara et al. 2009; Li et al. 2007; Petrini et al. 1990; Suryanarayanan et al. 2005; Tripathi and Joshi 2015; U’Ren et al. 2010, 2012). Endolichenic fungi have, of late, become a new avenue for evaluation of bioactive secondary metabolite chemistry in natural products research. Cultures of endolichenic fungi over the last 10 years have revealed potential new structures, and interest in their production of bioactive natural products has increased substantially. Since metabolites from endolichenic fungi were first reported 9 years ago, research into endolichenic fungal natural products has steadily increased, representing a small but growing body of literature (Fig. 1). Thus, the focus of the current review is on the progress made over the last decade by natural product chemists and their mycology collaborators in isolating new secondary metabolites from endolichenic fungi and their associated bioactivity. Containing over 140 novel metabolites, this review is the first to summarize the biodiversity, metabolites, and bioactivity of natural products derived from fungi that live in symbiosis with lichens. This review covers the literature available in SCOPUS (http://www.scopus.com/) up through December 2015; included articles were found using the open text string ‘‘endolichen*’’. This review is timely given that there has been a sudden surge in the natural products literature on isolation of bioactive chemical compounds from endolichenic fungi and there is a critical need for synthesis of the literature from the numerous studies that have been published thus far. Biological survey of endolichenic fungi Distribution and biodiversity Surveys to isolate endolichenic fungi are over a decade old (Girlanda et al. 1997; Petrini et al. 1990; Phytochem Rev Fig. 1 Number of published articles as well as new isolated natural products from endolichenic fungi. Graph represents articles and compounds from the first reported metabolites in 2007 through December 2015 Suryanarayanan et al. 2005), although studies focused on isolation of secondary metabolites are more recent (see below). The first study to isolate an endolichenic fungus was undertaken in 1990 by Petrini et al. (1990), where filamentous fungi were isolated from sterilized segments of fruticose lichens belonging to genera Cladonia as well as Stereocaulon. The authors did not use chemical sterilization of lichen thallus, due to the spongy nature of the lichens, instead removing superficial contaminants from the lichen thallus with sterile tap water and sieve filters to achieve surface sterilization. Subsequently, segments of the lichen thallus were plated on 2 % malt extract, 0.4 % yeastextract, 2 % agar, amended with 50 mg L-1 chlortetracycline and 1 mg L-1 cyclosporin (Petrini et al. 1990). This study demonstrated that lichen thalli could harbor a rich diversity of filamentous fungi belonging to the Pezizomycotina, Ascomycota. A total of 506 fungal strain types were isolated; 166 of them were isolated more than once. Girlanda et al. (1997) used two foliose lichens (Parmelia taractica and Peltigera praetexta) to study the range of fungal assemblages present. The authors used four different surface sterilization techniques to isolate fungi associated with the lichens and obtained a total of 117 fungal isolates along belonging to the Pezizomycotina, Ascomycota (Girlanda et al. 1997). Suryanarayanan et al. (2005) investigated five corticolous lichens (four foliose, and one fruticose) for non-obligate microfungi residing inside the lichen thalli in India, and also used four different sterilization procedures. In addition to isolating endolichenic fungi, Suryanarayana et al. also sought to understand whether endolichenic fungi were similar to those occurring as endophytes within the bark (termed as phellophytes) and leaves of the trees; hosts on which the selected lichens were investigated. However, there was little observed overlap between endolichenic fungi and fungal endophytes of the host tissue (Suryanarayanan et al. 2005). Li et al. (2007) surveyed endolichenic fungi from five families of lichens in the Baihua mountain of Beijing, China. The authors reported 32 taxa from 488 segments of lichen thalli, with low similarity among the lichens studied; most of the endolichenic fungi belonged to the phylum Ascomycota. Tripathi and Joshi (2015)investigated the endolichenic fungi from 14 lichen species collected in the Himalayan region of India. The authors isolated 25 cultivable isolates using culture morphology; the isolates mostly belonged to Ascomycota, but also to the Basidiomycota, and Mucoromycotina/ Zygomycetes (basal fungal lineages). More recently, U’Ren et al. (2010) investigated communities of endophytic fungi in mosses and endolichenic fungi in lichens using ITS rDNA sequences obtained from cultivable fungi. The authors sought to investigate whether endolichenic fungi represent distinct ecological guilds or if they can be defined as a single group of flexible symbiotrophs capable of colonizing plants or lichens indiscriminately. Endolichenic fungal assemblages differed as a function of lichen taxonomy, rather than substrate, growth form, or photobiont. The authors found no evidence that endolichenic fungi are saprobic fungi that have been inveigled by lichen thalli; rather, their study revealed the distinctiveness of endolichenic fungal communities relative to those in living and dead plant tissues. With one notable exception, the endolichenic fungi were similar to endophytic fungi occurring in mosses (U’Ren et al. 123 Phytochem Rev 2010, 2012). The aforementioned studies suggest that like fungal endophytes of plants, endolichenic fungi are present in virtually all lichen species that have been examined to date, and represent a vital yet poorly studied characteristic of lichenology. Origins and evolution Similar to endophytic fungi, the evolutionary origins of endolichenic fungi are not well understood. Arnold et al. (2009), in a recent study on phylogenetic estimation of trophic transitions of Ascomycetes, provided some interesting insights into the origins of endophytism and the evolution of endolichenic fungi. Using ancestral state reconstruction methods, which took into account phylogenetic uncertainty; they showed that endolichenic fungi or endolichenism played a key role in evolution of endophytism within the most species rich-phylum, Ascomycota. The results of the Arnold et al. (2009) study suggest that endolichenic fungi represent a rapid evolutionary rate for fungal transitions to endophytic associations in plants. This study was also the first to document the localization of endolichenic fungi inside the lichen thalli; the authors reported that endolichenic fungi were rarely isolated from the mycobiont, but were rather preferentially associated with the green algal photobiont of the lichen thallus. The association of endolichenic fungi with an algal photobiont underscores the ecological and evolutionary similarity of endolichenic fungi with endophytic fungi, which occur in every major lineage of land plants that have been examined up to the present time (Arnold 2007; Stone et al. 2000). related to a recently described novel order of fungi, Phaeomoniellales within the Eurotiomycetes (Chen et al. 2015). U’Ren et al. utilized multigene phylogenetic analysis and reported that several isolates of endolichenic fungi and fungal endophytes obtained from the continental US might represent novel species within the Xylariaceae, which require additional study. The authors also concluded that both symbiotrophic and saprotrophic fungi reside within the Xylariaceae, which is one of the largest and most diverse families within the Pezizomycotina, Ascomycota (U’Ren et al. 2016). Results from studies that have investigated endolichenic fungi suggests that the taxonomic composition, incidence of occurrence, and diversity are a consequence of the interplay of climatic patterns, geographic separation, host type, and host lineage (U’Ren et al. 2012). A recent study by Chagnon et al. (2016) investigated the network construction that is responsible for organization of community structure within symbiotic fungi such as endophytic and endolichenic fungi. The authors found that endophytic fungi were more flexible, and less nested and connected compared to the endolichenic fungi; meaning plant hosts were more selective of their fungal partners (endophytes) than the lichens that harbor endolichenic fungi. In addition, it was noted that endolichenic fungi are host generalists with respect to the lichens in which they occur, but phylogenetically they are most closely related to the endophytic fungi compared to the saprobic fungi (Chagnon et al. 2016; U’Ren et al. 2010). Taxonomic affiliations and community structure Endolichenic fungi are taxonomically and ecologically distinct from both lichenicolous fungi as well as the about 13,500 species of mycobiont fungi that form the lichen thalli (Arnold et al. 2009; Lutzoni and Miadlikowska 2009; Lutzoni et al. 2001). Most species isolated, as endolichenic fungi, are representatives of the Pezizomycotina, with most taxa phylogenetically related to seven orders (Fig. 2) within the subclass Eurotiomycetes, Dothideomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes (Arnold et al. 2009; U’Ren et al. 2010). A recent paper found endolichenic fungi were phylogenetically 123 Fig. 2 Phylogenetic distribution (ordinal) of endolichenic fungi, which have been screened for bioactive secondary metabolites. Pleosporales was the dominant order with 34 % followed by Xylariales, and Hypocreales with 14, and 12 % respectively Phytochem Rev Isolation methods The methods of isolating endolichenic fungi are very similar to those of endophytic fungi. Briefly, according to U’Ren et al. (2012), lichen material is transported to the laboratory and processed within 24–48 h of sampling. Samples are washed thoroughly in running tap water for 30 s. Lichen thalli are cut into small pieces, surface-sterilized, and then cut under sterile conditions into 2 mm segments. Segments are surface-sterilized by agitating sequentially in 95 % ethanol for 30 s, 10 % bleach (0.5 % NaOCl) for 2 min, and 70 % ethanol for 2 min, and surface-dried under sterile conditions (Arnold and Lutzoni 2007). After surface sterilization, the segments are placed on 2 % malt extract agar (MEA) in Petri dishes with Parafilm and incubated under ambient light/dark condition at room temperature (ca. 21.5 °C) for up to 1 year (U’Ren et al. 2010, 2012). MEA needs to be amended with antibiotics, such as Penicillin G and Steptomycin sulphate (500 mg/l), to avoid isolation of bacteria (see Stone et al. 2004 for a list of antibiotics). Emergent fungi are then isolated into pure culture (U’Ren et al. 2012). Arnold et al. (2009) utilized four different types of surface sterilization procedures for endolichenic fungi. In general, the longer lichen segments were sterilized in 0.5 % NaOCl, the fewer endolichenic fungi were recovered (Arnold et al. 2009). Additional methods of surface sterilization methods employed for endolichenic fungi have been outlined previously (Girlanda et al. 1997; Li et al. 2007; Petrini et al. 1990; Suryanarayanan et al. 2005). Physiological and ecological roles The biological roles of endolichenic fungi remain to be explored, but it is hypothesized that endolichenic fungi colonize the internal tissue of lichen thalli, specifically the photobiont and get nourishment and shelter from the host. In return, they may confer a multitude of benefits to their lichen host by producing a suite of biological active functional secondary metabolites. Structural diversity and biological activities During the past decade over 30 endolichenic microorganisms have been cultured and subjected to detailed investigations leading to the chemical characterization of over 140 new natural product structures, many of which have been shown to have a variety of biological activities (Table 1). These metabolites span a diverse array of structural types, which are outlined below. Alkaloids Solid rice cultures of the endolichenic fungus Chaetomium globosum (No. 64-5-8-2), originally isolated from the lichen Everniastrum nepalense, yielded the novel cytochalasan alkaloid chaetoglobosin Y (1), which possessed a macrocyclic ring with an isoindolone moiety (Zheng et al. 2014). Rice cultures of the endolichenic fungus Tolypocladium cylindrosporum, which inhabits the lichen Lethariella zahlbruckneri yielded a new pyridine-type alkaloid tolypyridone A (2), as well as three novel tetramic acid derivatives [tolypocladenols A1, A2, and B (3–5)] (Fig. 3) (Li et al. 2015b). However, (1)–(5) did not evidence any noticeable in vitro cytotoxicity against multiple cancer cell lines (Li et al. 2015b; Zheng et al. 2014). Quinones Anthraquinones Investigations into the endolichenic fungal strain Aspergillus versicolor yielded multiple novel anthraquinone derivatives (Fig. 4). Featuring a di-furan moiety similar to versicolorin B, 8-O-methylversicolorin A (6) and 8-O-methylversicolorin B (7) were isolated, along with the alkylated anthraquinone 8-Omethylaverythin (8) (the methoxy artifact 10 -O-ethyl6,8-di-O-methylaverantin (9) was also isolated) (Fig. 4) (Dou et al. 2014). The proliferation inhibition of cancer cell lines PC-3 and H460 was evaluated against (6)–(9). Moderate cytotoxic activity was evidenced by (6) and (7), with IC50 values of 12.6 and 19.5 lM against PC-3 cells and 17.3 and 27.2 lM against H460 cells, respectively (Dou et al. 2014). The xanthoquinodins represent an unusual xanthone-anthraquinone heterdimeric skeleton that derives from two polyketide metabolites. The first polyketide forms an anthraquinone monomer via decarboxylation of the terminal chain portion, and the second a xanthone monomer by decarboxylation/ oxidization reactions, which is then fused to the anthoquinone monomer in several different configurations (Tabata et al. 1993). Investigation into the endolichenic fungus Chaetomium elatum (No. 63-10- 123 Phytochem Rev Table 1 Bioactivity of isolated endolichenic fungal metabolites Endolichenic fungal strain Lichen host Natural product(s) Biological activity Cell line/species strain References Aspergillus sp. (No. 16-20-8-1) Peltigera elisabethae var. mauritzii Lobaria retigera 9-acetyldiorcinol B (90) Ab42 aggregation – Zhao et al. (2014) 8-O-methylversicolorin A (6) 8-O-methylversicolorin B (7) Diorcinol G (87) Cytotoxic PC-3/H460 Dou et al. (2014) Cytotoxic PC-3/H460 Cytotoxic PC3/A549/A2780/MDA-MB-231/ HEPG2 Zhao et al. (2014) Xanthoquinodin A4 (10) Cytotoxic HL-60/SMMC-7721/A-549/MCF-7/ SW480 Chen et al. (2013) Xanthoquinodin A5 (11) Cytotoxic Xanthoquinodin A6 (12) Cytotoxic Xanthoquinodin B4 (13a) Cytotoxic Xanthoquinodin B5 (13b) Cytotoxic Conioxepinol B (76) Cytotoxic HL-60/SMMC-7721/A-549/MCF-7/ SW480 HL-60/SMMC-7721/A-549/MCF-7/ SW480 HL-60/SMMC-7721/A-549/MCF-7/ SW480 HL-60/SMMC-7721/A-549/MCF-7/ SW480 HeLa Conioxepinol D (78) Coniothiepinol A (80) Cytotoxic Antibacterial Coniothienol A (82) Antifungal Antibacterial Aspergillus versicolor Aspergillus versicolor (125a) Chaetomium elatum (No. 63-10-3- 1) Lobaria quercizans Everniastrum cirrhatum Coniochaeta sp. Xanthoria mandschurica Coniochaeta sp. n/a CR1546C Sticta fuliginosa Geopyxis aff. Majalis Pseudevernia intensa Myxotrichum sp. Neurospora terricola Nodulisporium sp. (No. 65-172-1) Cetraria islandica Everniastrum cirrhatum Everniastrum sp. (R)-4,6,8-Trihydroxy-3,4dihydro-1(2H)naphthalenone (38) Geopyxin A (111), acetate and diester derivatives Antifungal Cytotoxic NCI-H460/SF-268/MCF-7/PC-3M/ MDA-MB-231 Geopyxin B (112) Cytotoxic Geopyxin C (113), acetate and diester derivatives Myxodiol A (62) Cytotoxic Antifungal NCI-H460/SF-268/MCF-7/PC-3M/ MDA-MB-231 NCI-H460/SF-268/MCF-7/PC-3M/ MDA-MB-231 Candida albicans (sc5314) Myxotrichin A (64) Myxotrichin D (67) Terricollene A (93) Cytotoxic Cytotoxic Cytotoxic K562 K562 HeLa/MCF-7 Terricollene C (95) 1-O-methylterricolyne (97) Nodulisporiviridin A (122) Cytotoxic Cytotoxic Ab42 aggregation HeLa/MCF-7 HeLa/MCF-7 – Nodulisporiviridin B (123) Ab42 aggregation Ab42 aggregation – Nodulisporiviridin C (124) 123 A549/MDA-MB-231 Enterococcus faecium (CGMCC 1.2025)/E. faecalis (CGMCC 1.2535) Fusarium oxysporum (CGMCC 3.2830) Enterococcus faecium (CGMCC 1.2025)/E. faecalis (CGMCC 1.2535) Candida albicans (ATCC 10231) – Wang et al. (2010b) Wang et al. (2010a) Kim et al. (2014) Wijeratne et al. (2012) Yuan et al. (2013) Zhang et al. (2009) Zhao et al. (2015a) Phytochem Rev Table 1 continued Endolichenic fungal strain Lichen host Natural product(s) Biological activity Cell line/species strain Nodulisporiviridin D (125) Ab42 aggregation Ab42 aggregation Ab42 aggregation Ab42 aggregation Ab42 aggregation Antioxidant – Antioxidant DPPH radical scavenging Antibacterial Staphylococcus aureus (ATCC 6538) Phaeosphaerin A (27) Cytotoxic PC3/DU145/LNCaP Preussochrome C (69) Cytotoxic A549 Preussochrome A (79) 7-hydroxy-3, 5-dimethylisochromen-1-one (52) Ophiobolin P (117) Cytotoxic Antifungal A549/HCT116 Candida albicans (sc5314) antibacterial Ophiobolin T (121) Cytotoxic Antibacterial cyclo(N-methyl-L-Phe-LVal-D-Ile-L-Leu-L-Pro) (142) Antifungal synergist Bacillus subtilis/methicillin-resistant Staphylococcus aureus HepG2 Bacillus subtilis/methicillin-resistant Staphylococcus aureus/Bacille Calmette–Guerin Candida albicans (sc5314) Nodulisporiviridin E (126) Nodulisporiviridin F (127) Nodulisporiviridin G (128) Nodulisporiviridin H (129) Penicillium citrinum Parmotrema sp. Pestalotiopsis sp. Clavaroids sp. Phaeosphaeria sp. Preussia africana Heterodermia obscurata Ramalina calicaris Ulocladium sp. Everniastrum sp. Xylaria sp. Leptogium saturninum 50 -acetyl-3,5,70 -trimethoxy30 H-spiro [cyclohexa [2,4]diene-1,10 isobenzofuran]-30 ,6-dione (58) 4-acetyl-20 hydroxy-30 ,50 ,6-trimethoxy biphenyl-2-carboxylic acid (85) Ambuic acid derivative (20) References – – – – DPPH radical scavenging Samanthi et al. (2015) Ding et al. (2009) Li et al. (2012) Zhang et al. (2012) Wang et al. (2012) Wang et al. (2013b) Wu et al. (2011) Only those reported to have bioactivity during the period covered by this review are listed 3-1) revealed five novel xanthoquinodins, A4–A6 (10)–(12), B4 (13a) and B5 (13b) (Fig. 4) (Chen et al. 2013). All five compounds displayed cytotoxic activity against five cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480), with (12) possessing low-lM activities against all cell lines (IC50 ranging from 2 to 6 lM) (Chen et al. 2013). Quinones From the culture of the endolichenic fungus Pleosporales sp., six new quinone metabolites were isolated. The cyclohexenone (5R)-5-hydroxy-2,3-dimethylcyclohex-2-en-1-one (14) was identified, along with three terphenyl derivatives, cucurbitarins A (15) and B (16), as well as the glycosylated cucurbitarin C (17). Two related cucurbitarins with a cyclopentenyl core were also isolated, cucurbitarins D (18) and E (19) (Fig. 4) (Jiao et al. 2015). Ambuic acid is a highly functionalized cyclohexenone, initially isolated from rainforest endophytic fungi Pestalotiopsis sp. and Monochaetia sp. (Li et al. 2001). Ambuic acid initially evidenced antifungal activity (Li et al. 2001), but also has moderate 123 Phytochem Rev Fig. 3 Structures of new alkaloid compounds isolated from endolichenic fungi antibacterial activity and quorum-sensing inhibitory activity (Nakayama et al. 2009). Six novel derivatives of the highly bioactive ambuic acid were isolated from the crude extract of the endolichenic fungus Pestalotiopsis sp. inhabiting the lichen Clavaroids sp. (Ding et al. 2009). Compounds (20)–(25) displayed varying levels of oxidation and stereochemistry around an ambuic acid skeleton. The dimeric quinone (26), similar to torreyanic acid, was isolated from the same fungal strain (Fig. 4). Metabolites (20)–(26) were screened for antibacterial activity against a panel of Gram-positive and Gram-negative bacterial strains. Only (20) exhibited moderate inhibition of Staphylococcus aureus (IC50 of 27.8 lM, compared to 43.9 lM for ambuic acid), and none of these compounds demonstrated antifungal activity against Aspergillus fumigatus (Ding et al. 2009). Few perylenequinonoid pigments have been discovered, and those originate mostly from Ascomycete fungi (Zhou and Liu 2010). These pigments are attractive cytotoxic metabolites, as they are transformed to excited triplet states by absorption of light energy, which can react with oxygen to generate reactive oxygen species that disrupt protein kinase C activity in mammalian cells (Morgan et al. 2009). From the endolichenic fungus Phaeosphaeria sp., occurring in the lichen Heterdermia obscurata, six novel perylenequinones possessing an unusual a,bunsaturated ketone moiety were isolated, phaeosphaerins A–F (27–32) (Fig. 4) (Li et al. 2012). 123 Compounds (27)–(32) were evaluated for cytotoxicity against PC3, DU145, and LNCaP cancer cell lines, with (27) demonstrating growth inhibitions of PC3 (IC50 5.84 lM), DU145 (IC50 10.77 lM), and LNCaP (IC50 10.76 lM). Further investigations revealed that (27) accumulated in the lysosomes of tumor cells, and its inhibitory activity was potentiated using light irradiation (Li et al. 2012). Three new herbarin-derived adducts—7-desmethylherbarin (33), 8-hydroxyherbarin (34), and 1-hydroxydehydroherbarin (35)—were isolated from the endolichenic fungal strain Corynespora sp. BA-10763, which occurs in the cavern beard lichen Usnea cavernosa (Fig. 4) (Paranagama et al. 2007; Wijeratne et al. 2010). The biosynthetically related compounds corynesporol (36) (Paranagama et al. 2007) and 9-Omethylscytalol A (37) (Wijeratne et al. 2010) were also isolated. These pyranonapthoquinones were evaluated for their migration inhibitory activities of PC-3M and MDA-MD-231 cancer cell lines; none of the isolated metabolites possessed significant inhibitory activity (Paranagama et al. 2007; Wijeratne et al. 2010). Other quinone derivatives A new napthalone ((R)-4,6,8-trihydroxy-3,4-dihydro1(2H)-naphthalenone (38)) was obtained from a Costa Rican endolichenic fungus Xylariaceae sp. CR1546C from the lichen Sticta fuliginosa (Kim et al. 2014). The napthalone demonstrated weak antimicrobial activity Phytochem Rev against Bacillus subtilis (MIC 150 lg mL-1) and Candida albicans (MIC 100 lg mL-1) (Kim et al. 2014). activity against B. subtilis, S. aureus, and C. albicans; however, (39)–(44) did not show any detectable activity at the 20 lg mL-1 level (Zhang et al. 2014). Oxygen heterocycles Pyrones Furanones The new a-pyrone derivatives nodulisporipyrones A–D (45)–(47) were isolated from a rice culture of the fungal strain Nodulisporium sp. (65-12-7-1), symbiotic with the lichen Everniastrum sp.(Zhao et al. 2015b), and the novel derivatives necpyrone A, B, D, and E [(48)–(50), respectively)] have been detected in the endolichenic fungus Nectria sp., occurring in the lichen Pamelia sp. (Figure 5) (Li et al. 2015a). A dehydrogenated structure, the new metabolite necpyrone C (51), was also isolated from Nectria sp. (Li et al. 2015a). The a-pyrones (45)– (47) failed to demonstrate any activity in in vitro Seven novel furanone metabolites were isolated from the crude extract of the endolichenic fungus Peziza sp. inhabiting the lichen Xanthoparmelia sp. (Zhang et al. 2014), the pezizolides A–G (39)–(44). Compounds (39)–(41) contained a bis-furanone moiety, while (42)–(44) possessed only a single furanone ring (Fig. 5). The compounds were all tested for cytotoxicity against HeLa, A549, MCF-7, HCT116, and T24 cancer cell lines, and for potential antimicrobial Fig. 4 Structures of new quinone compounds isolated from endolichenic fungi 123 Phytochem Rev Fig. 4 continued antibacterial assays against S. aureus or E. coli (MIC [ 1000 lg mL-1), and weak activity against the fungi Aspergillus niger and C. albicans (MIC 31 and 250 lg mL-1, respectively) ((Zhao et al. 2015b). And a study against six human cancer cell lines (K562, MDA-MB-231, MCF-7, SW620, HT29, and HeLa) showed no cytotoxicity (IC50 [ 120 lM) for compounds (48)–(51) (Li et al. 2015a). Benzopyranoids Endolichenic fungi have yielded a variety of benzypyranoid and coumarin derivatives, with varying bioactivity. Two novel polyketides, 7-hydroxy-3, 5-dimethyl-isochromen-1-one (52) and 6-hydroxy-8-methoxy-3amethyl-3a,9b-dihydro-3H-furo[3,2-c]isochromene-2,5dione (53), were isolated from the endolichenic fungus Ulocladium sp. and (53) possessed a completely novel tricyclic skeleton as part of its structure (Fig. 5) (Wang et al. 2012). The coumarin derivative 6,8-dihydroxy(3R)-(2-oxopropyl)-3,4-dihydroisocoumarin (54) was 123 obtained from the Costa Rican endolichenic fungus coded CR1546C (Fig. 5) (Kim et al. 2014). The polyketide 7-hydroxy-3, 5-dimethyl-isochromen-1-one (52) evidenced mild antifungal activity against Candida albicans SC 5314, possessing an IC50 of 97.9 ± 1.1 lM (Wang et al. 2012). The ethyl acetate extracts of the endolichenic fungi Nigrospora sphaerica (No. 83-1-1-2, found in Parmelinella wallichiana), Alternaria alternata (No. 58-8-4-1, from the lichen Usnea aciculifera) and Phialophora sp. (No. 96-1-8-1, from Cetrelia braunsiana) yielded the diastereomeric pair (?)-(2S,3S,4aS)-altenuene (55a) and (-)-(2S,3S,4aR)-isoaltenuene (55b) (Fig. 5) (He et al. 2012). From a suite of eight novel metabolites from the fungus Pleosporales sp., two benzocoumarins were isolated: 3,10-dihydroxy-4,8-dimethoxy-6-methylbenzocoumarin (56) and 3,8,10-trihydroxy-4-methoxy-6methylbenzocoumarin (57),(Jiao et al. 2015) and 50 acetyl-3,5,70 -trimethoxy-30 H-spiro [cyclohexa [2,4]diene-1,10 -isobenzofuran]-30 ,6-dione (58) was obtained from Penicillium citrinum, an endolichenic fungal strain Phytochem Rev Fig. 4 continued from a Sri Lankan Parmotrema species (Samanthi et al. 2015). The coumarin 3,8-dihydroxy-4-(4-hydroxyphenyl)-6-methylcoumarin (61) was obtained from the endolichenic fungus Tolypocladium cylindrosporum, inhabiting the lichen Lethariella zahlbruckneri (Fig. 5) (Li et al. 2015b). Of the benzocoumarins from endolichenic fungi, (58) demonstrated moderate antioxidant activity, scavenging the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with an IC50 value of 159.7 ± 22.3 lg mL-1. Altenusin derivatives originate from polyketide biosynthesis pathways, generally containing seven acetate units and forming either a bicyclic or tricyclic ring skeleton, such as 6/6/6, found with altenunenes, or 6/6/5 common to the rubralactones. Two new altenusins, phialophoriol (59) and xinshengin (60), were isolated from a Phialophora spp. (No. 39-1-5-1) obtained from the lichen Cladonia ochrochlora Flörke (Fig. 5). Xinshengin (60) is composed of a unique altenuene/tetrahydrofuran-fused tetracyclic skeleton, with rings of 6/6/6/5 (Ye et al. 2013). Myxidiol A (62), a novel austdiol analog, was isolated from the endolichenic fungus Myxotrichum sp. on the lichen Cetraria islandica (Fig. 5). This compound is remarkable in that it is the first endolichenic fungal metabolite isolated containing a halogen, a chlorine atom located at position five. Myxidiol A (62) demonstrated showed minimal antifungal activity against Candida albicans (sc5314) with a MIC of 128 lg mL-1 (Yuan et al. 2013). 123 Phytochem Rev Fig. 5 Structures of new oxygen-containing herterocyclic compounds isolated from endolichenic fungi 123 Phytochem Rev Fig. 5 continued 123 Phytochem Rev Xanthones Sulfur-containing chromenones Endolichenic fungi have demonstrated a great deal of biosynthetic plasticity in the production of xanthones and xanthone derivatives. The xanthone conioxanthone A (63) was isolated from a culture of Coniochaeta sp. originating on the lichen Xanthoria mandschurica (Fig. 5) (Wang et al. 2010b). A Myxotrichum sp. living in the lichen Cetraria islandica yielded three related fulvic acid derivatives myxotrichin A–C (64)–(66), and a citromycetin analog myxotrichin D (67) (Fig. 5). Of the four xanthone derivatives from Myxotrichum sp., (64) and (67) demonstrated weak in vitro cytotoxic activity against the K562 cell line, with IC50 values 32 and 20 lM, respectively (Yuan et al. 2013). The xanthone derivative preussochromone B (68) was isolated from Preussia africana (living in the lichen Ramalina calicaris), which also yielded preussochromone C (69), with a corymbiferone skeleton (Fig. 5). Compound (69) was highly cytotoxic against A549 cells, with an IC50 of 5.8 lM, yet evidenced no activity (IC50 [ 20 lM) against MCF-7, HeLa, and HCT116 cell lines (Zhang et al. 2012). Finally, oxisterigmatocystin D (70) was isolated from Aspergillus sp. (No. 16-20-8-1) associated with the lichen Peltigera elisabethae (Zhao et al. 2014). Coniofurol A (71) was identified as a new member of the furochromenone class of oxygen heterocycles, possessing the representative ringcontracted xanthone structure with novel substituents on the aryl and furan rings (Fig. 5) (Wang et al. 2010b). Preussochromes D–F (72)–(74) were similarly ring-contracted xanthones from solid cultures of an endolichenic fungus Preussia africana (Fig. 5) (Zhang et al. 2012). And novel ringexpanded xanthones were discovered from Coniochaeta sp. Conioxepinols A–D (75)–(78) possessed the base oxepinochromenone skeleton, with differing configurations at C7 and C8 (for conioxepinol A–C) and C3 and C6 (for conioxepinol D) and altered substitution patterns on the aryl and oxepine rings (Fig. 5) (Wang et al. 2010b). Conioxepinol B (76) showed modest cytotoxicity activity against HeLa cell line, with an IC50 value of 36 lM, while conioxepinol D (78) demonstrated cytotoxcity against A549 and MDA-MB-231 cells, possessing IC50 values of 40 and 41 lM, respectively (Wang et al. 2010b). Several relatively rare sulfur-containing chromenone structures have been isolated from endolichenic fungal cultures. Preussochromone A (79), isolated from the fungus Preussia africana, was found to possess a 3,4dihydrothiopyrano[2,3-b]chromen-5(2H)-one structure, which had not previously been detected in a naturallyoccurring product, only as a synthetic derivative (Majumdar and Jana 2001; Palmisano et al. 2007; Zhang et al. 2012). Preussochromone A (79) demonstrated significant cytotoxic activity against A549 and HCT116 cell lines, with IC50 values of 8 and 11 lM, respectively (Zhang et al. 2012). Three ring-altered, sulfur-contaning xanthone derivatives were observed in cultures of the endolichenic fungus Coniochaeta sp. (Figure 5). Coniothiepinols A (80) and B (81) are the first recorded naturally-occurring thiepinols, containing the unique 4,5dihydro-2H-thiepino[2,3-b]chromen-6(3H)-one skeleton. Coniothiepinol A (80) has a distinctive C5–C8 ether linkage, resulting in a 8-oxa-2-thia-bicyclo[3.2.1]octane partial structure. Coniothienol A (82) possessed a ringcontracted 2H-thieno[2,3-b]chromen-4(3H)-one base structure (Wang et al. 2010a). When evaluated against Enterococcus faecium (CGMCC 1.2025) and E. faecalis (CGMCC 1.2535), (82) demonstrated significant activity against the two Gram-positive bacterial strains (IC50 values of 2.00 and 4.89 lg mL-1, respectively), and (80) showed modest inhibition against the Gram-positive strains (3.93 and 11.51 lg mL-1, respectively) as well as the plant pathogen Fusarium oxysporum (CGMCC 3.2830) with an IC50 of 13.12 lg mL-1 (Wang et al. 2010a). 123 Simple aromatic compounds Simple aromatic structures, or aromatic rings bound together by carbon–carbon or ether linkages represent a family of natural product skeletons observed in endolichenic fungi. Discovery efforts involving the endolichenic fungal strain Scopulariopsis sp. (occurring on Cladonia gracilis) led to the isolation of two new naphthalene derivatives, 1-(40 -hydroxy-30 ,50 dimethoxy-phenyl)-1,8-dimethoxynaphthalen-2(1H)one (83) and 1,8-dimethoxynaphthalen-2-ol (84) (Fig. 6) (Yang et al. 2012). The biphenyl compound 4-acetyl-20 -hydroxy-30 ,50 ,6-trimethoxy biphenyl-2carboxylic acid (85) was isolated from Penicillium Phytochem Rev Fig. 6 Structures of new simple aromatic compounds isolated from endolichenic fungi citrinum (Fig. 6), an endolichenic fungal strain from a Sri Lankan lichen Parmotrema sp., and demonstrated radical scavenging activity in a DPPH assay with an IC50 value of 69.6 lg mL-1 (Samanthi et al. 2015). Four biphenyl ether compounds, diorcinols F–H (86)–(88) and 3-methoxyviolaceol-II (89), were found in the endolichenic fungus Aspergillus versicolor (125a) from the lichen Lobaria quercizans (Fig. 6) (Li et al. 2015c), and another biphenyl, 9-acetyldiorcinol B (90), was isolated from Aspergillus sp. (No. 16-20-8-1), endolichenic with Peltigera elisabethae var. mauritzii (Fig. 6) (Zhao et al. 2014). Aspergillus versicolor (125a) also yielded two new tris(pyrogallol ethers), sydowiols D (91) and E (92) (Fig. 6), which featured a triphenyl structure joined by aryl-ether bridges (Li et al. 2015c). The simple phenyl ethers demonstrated a range of bioactivities; (87) exhibited moderate cytotoxicity against tested human cancer cell lines PC3, A549, A2780, MDA-MB-231, and HEPG2, with IC50 values ranging from 19.0 to 31.0 lM, yet was inactive against Candida albicans (MIC [ 64 lg mL-1) (Li et al. 2015c). The metabolite 9-acetyldiorcinol B (90) inhibited Ab42 aggregation at the 100 lM level (Zhao et al. 2014). Neurospora terricola, isolated from the lichen Everniastrum cirrhatum, has yielded several unique allenyl and alkynyl phenyl ether structures. Terricollenes A–C (93)–(95) contained a p-(buta-2,3-dienyl ether)phenyl moiety, similar to xyloallenolide A (Fig. 6) (Lin et al. 2001) and the eucalyptenes (Arnone et al. 1993), and (93) and (94) were both glycosylated with glucose and mannose, respectively (Zhang et al. 123 Phytochem Rev 2009). Three alkynyl phenyl ethers were also isolated, terricolyne (96), 1-O-methylterricolyne (97), and 1-Oacetylterricolyne (98) (Fig. 6). Compounds (93), (95), and (97) evidenced modest cytotoxic activity against HeLa cells, with IC50 values ranging from 53.3 to 92.6 lM, and (93) also displayed activity against MCF-7 cell line, with an IC50 value of 59.2 lM (Zhang et al. 2009). Terpenes Sesquiterpenoids constitute a broad structural class of natural products biosynthesized by a diverse range of Fig. 7 Structures of new terpenoid compounds isolated from endolichenic fungi 123 organisms. Eight new bisabolane sesquiterpenoids, (-)-(R)-cyclohydroxysydonic acid (99), (-)-(7S,8R)8-hydroxysydowic acid (100), (-)-(7R,10S)-10-hydroxysydowic acid (101), (-)-(7R,10R)-iso-10-hydroxysydowic acid (102), (-)-12-acetoxy-1-deoxysydonic acid (103), (-)-12-acetoxysydonic acid (104), (-)-12-hydroxysydonic acid (105), and (-)-(R)-11-dehydrosydonic acid (106), were isolated from the endolichenic fungus Aspergillus versicolor (125a) living in the lichen Lobaria quercizans (Fig. 7) (Li et al. 2015c). These compounds are formed via a series of oxidation, reduction, cyclization, and esterification reactions of the bisabolane nucleus, arising from the mevalonic acid Phytochem Rev Fig. 7 continued pathway (Li et al. 2015c). The fungal strain Periconia sp. (No. 19-4-2-1), endolichenic with Parmelia sp., yielded the novel cadinane-type sesquiterpenoid Pericoterpenoid A (107), which demonstrated moderate antifungal potential against Aspergillus niger (MIC 31 lg mL-1) (Wu et al. 2015). From a Czapek’s culture of the endolichenic fungus Ulocladium sp. (cultured from Everniastrum sp.), novel mixed terpenoids possessing a tricyclic core, the tricycloalternarenes F–H (108)–(110), were identified (Fig. 7) (Wang et al. 2013a). Ent-kaurane diterpenoids of fungal origin have been rare and mainly reported from Gibberella fujikuroi and Phaeosphaeria sp. L487 (Kawaide 2006), though this class of diterpenoids has frequently been present in several plant families (Garcia et al. 2007). Obtained from the host Pseudevernia intensa, cultures of two endolichenic fungi (Geopyxis aff. majalis, and Geopyxis sp. AZ0066) led to the isolation of new ent-kaurane diterpenes, geopyxins A–F (111)– (116) (Fig. 7). Geopyxin B (112) was the only natural geopyxin to demonstrate cytotoxicity activity in the low micromolar range against the cancer cell lines NCI-H460, SF-268, MCF-7, PC-3M, and MDA-MB231; however, the monoacetate and diacetate derivatives of (111) and the methyl esters of (111)–(113) showed low or sub micromolar activities against the cell lines, as well as activating the heat-shock response (Wijeratne et al. 2012). Ophiobolins are a family of naturally occurring sesterterpenes characterized by a unique C5–C8–C5 tricyclic ring system. Via an OSMAC (one strain, many compounds) method, wherein culture conditions are altered to prompt production of a different metabolite profile, cultures of Ulocladium sp. (endolichenic in Everniastrum sp.) grown on potato dextrose broth (PDB) produced five novel ophiobolane sesterterpenes, the ophiobolins P–T (117)–(121) (Fig. 7) (Wang et al. 2013b). Ophiobolin T (121) exhibited strong cytotoxic activities against HepG2 with an IC50 value of 0.24 lM, and (117) and (121) demonstrated moderate antibacterial activity against Bacillus subtilis and methicillin-resistant Staphylococcus aureus. Ophiobolin T (121) also presented moderate antibacterial activity against the Bacille Calmette–Guerin strain. The heightened activity of (121) compared to the other ophiobolins suggested that the furan ring on the side-chain at C-15 significantly influenced the bioactivity of ophiobolin sesterterpenes (Wang et al. 2013b). Steroids Viridins represent a fairly unique class of natural product sterol derivatives, featuring a furan ring fused to a pregnane or androstane steroid nucleus at the C-4 and C-6 position. These steroid structures have attracted interest for their potent antifungal, antibiotic, phytotoxic, and anti-inflammatory activities (Hansen 1995; Wipf and Halter 2005). Eight novel viridins, nodulisporiviridins A–H (122)–(129) (Fig. 8), were isolated from the endolichenic fungus Nodulisporium sp. (No. 65-17-2-1). The nodulisporiviridins have a 123 Phytochem Rev Fig. 8 Structures of new steroid compounds isolated from endolichenic fungi unique skeleton, featuring a cleaved A ring at either C-1 or C-10 in the 10R or 10S configuration (Zhao et al. 2015a). All nodulisporiviridins possessed inhibitory activity in an anti-Ab42 aggregation assay, with nodulisporiviridin G (128) exhibiting the most potent anti-aggregation activity (IC50 of 1.2 lM), and all improved short-term memory in a human Ab42 transgenic AD fly model (Zhao et al. 2015a). Progesteroids, C21 steroid skeletons possessing a C-2 alkyl side chain at C-17, are fairly commonly found in nature; however, the 4-methyl derivatives are only rarely detected (De Rosa et al. 1999). From the endolichenic fungus Nodulisporium sp. (No. 65-17-2-1), the first examples of 3,4-seco-4-methylpregnan steroids were isolated, nodulisporisteroid A (130) and B (132) (Fig. 8) (Zheng et al. 2013). By employing an OSMAC method, ten additional 123 4-methyl-progesteroid derivatives were obtained from the same endolichenic fungus, and were termed nodulisporisteroids C (131) and D–L (133)–(141) (Fig. 8). However, none of the compounds demonstrated cytotoxic activity against HL60, SMMC-7721, A-549, MCF-7, or SW480 human cancer cell lines (Zhao et al. 2015c). Peptides At the time of this review, only two peptides have been reported from endolichenic fungi. From the endolichenic fungus, Xylaria sp. (75-1-3-1), two prolinecontaining cyclopentapeptides. were isolated. Cyclo(-NMePhe-Pro-Leu-Ile-Val-) (142) and cyclo-(-LeuPro-Leu-Ile-Val-) (143) were tested for antifungal activity against C. albicans (Fig. 9) (SC5314). While Phytochem Rev Fig. 9 Structures of new peptide compounds isolated from endolichenic fungi Fig. 10 Structures of the new allylic compound (11S,12S,13R) 11,13-dihydroxy-12-methyltetradecanoic acid, isolated from endolichenic fungi neither (142) or (143) evidenced antifungal activity at the highest concentration tested (100 lg mL-1), (142) showed synergistic activity at concentrations of 6.3 lg mL-1 in combination with 0.004 lg mL-1 ketoconazole, yielding an FIC of \0.3125 (Wu et al. 2011). Allylic compounds The endolichenic fungus Massarina sp. yielded a single novel fatty acid, (11S,12S,13R) 11,13-dihydroxy-12-methyltetradecanoic acid (144) (Fig. 10) (Yuan et al. 2015). There have been no reported bioactivity studies or biosynthetic investigations for this metabolite. Conclusions The 144 new endolichenic fungal metabolites highlighted in this review represent only a small subfraction of endolichenic fungal chemistry. Multiple studies of endolichenic fungi have evidenced bioactivity from various fungal extracts, but have provided incomplete structural identification of the bioactive constituents. Cheon et al. (2013) examined 571 endolichenic fungi for their antifungal properties, identifying four— Stereocaulon sp. (1429), Stereocaulon sp. (1430), Cryptosporiopsis sp. (0156), and Graphis sp. (1245)— that possessed high levels of antifungal activity. While several metabolites were identified in the active fractions, none were isolated and confirmed for bioactivity. Similarly, other studies have uncovered endolichenic fungi with potent bioactivity, but complete identification of the metabolites has not yet been reported (Hwang et al. 2011; Kannangara et al. 2009; Kim et al. 2012; Padhi and Tayung 2015). The new endolichenic metabolites reviewed here, as well as previously described metabolites from these organisms, share many similar carbon skeletons with metabolites produced by endophytic fungi (Kharwar et al. 2011; Kusari et al. 2012; Stierle and Stierle 2015). The overlap between endolichenic and endophytic metabolites is consistent with their biological similarities; there exists considerable overlap in the taxa represented in endolichenic and endophytic fungal strains, and they are believed to perform similar ecological roles for the host organism (Chagnon et al. 2016; U’Ren et al. 2010). However, endolichenic fungal metabolites remain relatively distinct from the natural products produced by lichens individually (Boustie et al. 2011; Romagni and Dayan 2002; Shukla et al. 2010). And despite any similarities, this review has highlighted several instances of metabolites with novel skeletons, including the phaeosphaerins A–F (Li et al. 2012), nodulisporiviridins A–H (Zhao et al. 2015a), Pericoterpenoid A (Wu et al. 2015), Conioxepinols A–D (Wang et al. 2010b), and 6-hydroxy-8-methoxy-3a-methyl-3a,9b-dihydro-3Hfuro[3,2-c]isochromene-2,5-dione (Wang et al. 2012), and Coniothiepinols A and B (Wang et al. 2010a). Thus, while endolichenic fungal metabolites do possess some overlap with endophytic fungal natural products, they also possess novel biosynthetic pathways capable of producing novel products. Endolichenic fungi and other microbial source of natural products maintain a degree of biosynthetic plasticity in producing natural product metabolites. By applying systematic variations in the cultivation parameters (media composition and phase, aeration, pH, temperature, culture vessel, addition of enzyme inhibitors, epigenetic modifiers etc.), it is possible to increase the number of metabolites produced by a fungal (or microbial) source. Altering the culturing conditions increases the metabolomic diversity available to these organisms, and has been termed the ‘‘one strain, many compounds’’ (OSMAC) approach (Bode 123 Phytochem Rev et al. 2002). Several of the endolichenic fungi have been shown to be responsive to OSMAC approaches, biosynthesizing new metabolites as a result of the variations in culturing. The endolichenic fungal strain Nodulisporium sp. (No. 65-12-7-1) was found to produce additional 4-methyl progesteroid analogs when grown in potato-dextrose-broth (PDB) (Zhao et al. 2015c). Three new terpenoids, the tricycloalternarenes F–H were isolated from a Czapek’s culture of Ulocladium sp. (CGMCC 5507) (Wang et al. 2013a), while PDB cultures of the same endolichenic fungal strain yielded five novel ophiobolane sesterterpenes, the ophiobolins P–T. Neither of these groups of metabolites were detected in the original rice cultures (Wang et al. 2013b). Thus, OSMAC approaches to probing chemical diversity in endolichenic fungi also have the potential to maximize and further develop the natural products produced by these microorganisms. Furthermore, the 31 endolichenic fungi whose metabolites have been reported here were collected from a limited number of geographic locations (Fig. 11). It is important to note that this data is only presented for those references (33 of 39) that included either GPS coordinates or a geographical descriptor, and the inclusion of geographic data remains a key metric across all natural product discovery efforts (Leal et al. 2016; Oberlies et al. 2009). With estimates for the global number of currently recognized lichens near 20,000 species (Feuerer and Hawksworth 2007), there remains a vast reservoir for prospective endolichenic fungi that have the potential to provide bioactive natural products. Further investigations at the genetic, molecular, and population level in this field are needed for a more thorough understanding of host–endolichenic fungi interactions, as well as improved understanding of the ecological role that endolichenic fungi, and their secondary metabolites, play in the symbiosis with and protection of the lichen’s photobiont. A recent study on the evolution of fungal metabolic pathways suggests that gene duplication and horizontal gene transfer have acted together in imparting diversity to metabolic gene clusters within the Pezizomycotina. Fig. 11 Geographic locations of metabolite-producing endolichenic fungal strains identified in this review (Note this data is only presented for the references that included geographic descriptors) (Ding et al. 2009; Dou et al. 2014; He et al. 2012; Jiao et al. 2015; Kim et al. 2014; Li et al. 2012, 2015a, b, c; Paranagama et al. 2007; Samanthi et al. 2015; Wang et al. 2013a, b, 2012, 2010a, b; Wijeratne et al. 2010, 2012; Wu et al. 2011, 2015; Yang et al. 2012, 2015, 2013; Ye et al. 2013, Zhao et al. 2014, 2015a, b, c; Zheng et al. 2013, 2014) 123 Phytochem Rev These data acquired from 208 diverse fungal genomes provides further impetus for studying endolichenic fungi for secondary metabolites, since most fungi isolated as endolichenic belong to the Pezizomycotina (Wisecaver et al. 2014). More research will also aid in creating a comprehensive understanding of the evolutionary origins of endolichenic fungi, as well as the mechanisms and roles of their apparent genetic plasticity in producing secondary metabolites. Understanding the ecological and genetic roles of endolichenic fungi, will aid in identifying highly active metabolite-producing strains. As a recently discovered reservoir of fungi ‘hidden’ within host lichens, endolichenic fungi are a potentially rich source of bioactive and chemically novel compounds. While these discoveries are inspirational in uncovering new areas of bioactive natural products, challenges still exist for the future development of endolichenic fungi discovery: (1) frequent rediscovery of known natural products (El-Elimat et al. 2013) (2) technical challenges associated with their purification and structural identification; (3) traditional screening strategies at for bioactive compounds (Kellogg et al. 2016); (4) uncultivable strains as yet another unexploited source of natural products (Nichols et al. 2010). A great deal of effort remains to unearth the potential of endolichenic fungi as natural product producers. However, if the current limitations of methodologies and technologies could be overcame, a new horizon could open up for natural products from endolichenic fungi as novel compounds for the benefit of human health. Acknowledgments The authors would like to thank Drs. Nicholas H. Oberlies and Nadja B. 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