Symbiosis (2020) 82:79–93 https://doi.org/10.1007/s13199-020-00719-3

Axenic culture and biosynthesis of secondary compounds in symbiotic fungi, the

E. M. Díaz1 & J. C. Zamora2 & C. Ruibal2 & P. K. Divakar2 & N. González-Benítez1 & F. Le Devehat3 & M. Chollet3 & S. Ferron3 & A. Sauvager3 & J. Boustie3 & A. Crespo2 & M. C. Molina1

Received: 16 June 2020 /Accepted: 13 September 2020 / Published online: 22 October 2020 # Springer Nature B.V. 2020

Abstract produce unique secondary metabolites with a rich potential as bioactive compounds. In many cases, the use of these molecules is limited by the low concentration of these compounds in thalli, low growth rate in culture, and changes in chemical patterns between thalli and aposymbiotic culture. In addition, the massive collection of some species of industrial interest can cause damage to lichen diversity and the associated environment. Six lichenized fungi ( centrifuga, Parmelia saxatilis, tiliacea, Platismatia glauca, tinctina, and Usnea ghattensis) with biotechnological interest and belonging to Parmeliaceae have been cultured in order to test culture conditions and obtain enough biomass for further studies. In addition, we analyzed the compounds synthetized in axenic conditions and they were compared with chemosyndromes identified in complete thalli. , P. saxatilis, P. tiliacea and X. tinctina were success- fully cultivated while for P. glauca and U. ghattensis we only obtained sporulation and germination of the spores. The chemical pattern of the compounds secreted into the culture media varied significantly from the chemosyndrome of the whole thallus. Phenolic compounds of pharmacological and industrial interest (usnic acid, aspicilin, α-alectoronic acid, physodic acid, lobaric acid and nordivaricatic acid) and a wide variety of potentially bioactive compounds were obtained during the culture process.

Keywords Axenic culture . Mycobiont . Phenolic compounds . Bioactive molecules

A lichen is an intimate and long-term symbiosis consisting of terpenoids and phenolic compounds, which are deposited in a complex interaction between, at least, a (mycobiont), either the cortex (external phenols) or the medulla (internal one or more photoautotrophic partners (photobionts) and as- phenols). Although the molecular mechanisms of interaction sociated bacteria (Meessen and Ott 2013). Many yeast of bionts are still unclear (Legaz et al. 2003; Fontaniella et al. (Spribille et al. 2016) and other fungi (Muggia et al. 2017) 2004;Rikkinen2013), throughout the interactions of different have also been involved in the symbiotic nature as well. The symbiotic partners whole thalli produce secondary metabo- implications of these partners (bacteria and additional fungi) lites under a multiple interaction of environmental factors for symbiosis are being investigated (Aschenbrenner et al. (Brunauer et al. 2007). Secondary metabolites have a rich 2016). Lichen are able to produce secondary metabolites such potential as bioactive compounds such as anti-microbial, an- ti-bacteria, anti-viral, antioxidant activity and anti-cancer (e.g.Ranković 2015; Solárová et al. 2020). They could be used Electronic supplementary material The online version of this article – (https://doi.org/10.1007/s13199-020-00719-3) contains supplementary in therapy of oxidative stress related diseases (Sahin et al. material, which is available to authorized users. 2019). Among the most studied secondary compounds are phenols (see Ranković 2015; Shukla et al. 2010;Yamamoto * M. C. Molina et al. 2015;Xuetal.2016; Fernández-Moriano et al. 2016; [email protected] Gómez-Serranillos et al. 2014; Zambare and Christopher 2012; Sieteiglesias et al. 2019). Terpenoids, molecules of sec- 1 Department of Biology, Geology, Physical and Inorganic Chemistry, ondary metabolism from mevalonic acid, can also be bioactive Rey Juan Carlos University, 28933 Madrid, Spain molecules, although there is a scarcer body of evidence 2 Department Pharmacology, Pharmacognosy and Botany, (see Ranković 2015;Rehmanetal.2018;Bateetal.2018). Complutense University of Madrid, 28040 Madrid, Spain Secondary compounds have also been used since ancient 3 University Rennes, CNRS, ISCR (Institut des Sciences Chimiques de times as sources of color dyes and perfume stabilizers Rennes) - UMR 6226, F-35000 Rennes, France 80 Díaz E.M. et al.

(Shukla and Upreti 2015; Salgado et al. 2018; Shaheen et al. need to be overcome. For example, in mycobiont culture, an 2019;Calcheraetal.2019). inverse relationships between growth and the production of Unfortunately, a key characteristic of lichen-forming fungi secondary metabolites has been established (Timsina et al. is their very low growth rates (Salgado et al. 2018)compared 2013), modulated by biotic parameters, such as the presence to most other filamentous fungi under natural conditions. of the photobiont (Shanmugam et al. 2016), and abiotic pa- Although the causes of this slow growth rate are unknown, rameters, such as pH, temperature, nutrients, carbon source it appears that environmental factors (Gaio-Oliveira et al. (Deduke and Piercey-Normore 2015; Timsina et al. 2013; 2004) and biotics, such as the hormonal system Shanmugam et al. 2016). This is a major problem since we (Farkhutdinov et al. 2018) could be involved. Lichen collec- cannot achieve a greater number of phenols by increasing tion in field for biomedical or industrial use, especially in its biomass. It is also frequent that between 100 and 300 days use as perfume stabilisers and as dyes (Joulain and Tabacchi of culture the mycobiont stops growing until it is dehydrated 2009; Shaheen et al. 2019), has caused serious problems in the (Molina et al. 2013; Timsina et al. 2013;Molinaetal.2015). conservation of these organisms (Upreti et al. 2005; Shukla Moreover, in many cases, the mycobiont culture in and Upreti 2015). The collection and extraction of pigments, aposymbiotic conditions alters the phenolic pattern synthe- especially in countries such as India and Pakistan, continue in sized (e.g. Molina et al. 2003) and it is unknown to what the actuality (Shaheen et al. 2019; Upreti et al. 2010). For extent it modifies the ontogenetic development. The large example, Everniastrum nepalense, frequently picked up by number of apparently latent PKS genes present in many lichen Indian traders (Upreti et al. 2005) and consumed by ethnic fungi (Bertrand and Sorensen 2018;Calcheraetal.2019)may groups (Devkota et al. 2017), is found on the IUCN red list be responsible for these differences, when activated in (Devkota and Weerakoon 2017). The rare endemic lichen aposymbiotic condition. In this scenario, it is necessary to Xanthoparmelia beccae, listed as “vulnerable” is also fre- continue with the improvement of axenic cultivation tech- quently collected (Aptroot and Perez-Ortega 2018). niques to increase mycobiont or whole thalli growth and the Different alternatives have been investigated for produc- production of lichen compounds of interest in a standardized tion of these lichen secondary compounds: chemical synthesis and large-scale way (Shanmugam et al. 2016). (e.g. Schaubach et al. 2017), biosynthesis from mycobiont or Parmeliaceae (, ) is one of the whole thalli culture on laboratory conditions (e.g. Molina et al. largest and most diverse families among lichenized fungi, 2015), transgenic approaches (Chooi et al. 2008), and inocu- comprising more than 2700 of mainly fruticulose and foli- lation of lichen diaspores on cover slips (Anstett et al. 2014). aceous species, grouped in about 80 genera (Thell et al. However, Legaz et al. (2011) indicated that the chemical syn- 2012). This family is a group of choice for the current theses of lichen compounds are tedious and energy expensive study given that certain species have demonstrated to pro- and the polyketide synthesis through transgenic approaches is duce biologically active compounds in aposymbiotic con- in its infancy (Miao et al. 2001). Therefore, mycobiont and ditions (Gulluce et al. 2006;Manojlović et al. 2012; whole thalli culture in laboratory have been proposed as the Ranković 2015). In addition, this is one of the most best option to obtain biomass and bio-compounds, conserving researched families for phylogenetic studies and providing the diversity of the natural populations of these organisms, a set of successful conditions for axenic growth may be of also sensitive to climate change (Horák et al. 2019) and loss interest to generate enough biomass for DNA isolation and or deterioration habitat (Pykälä 2019). sequencing of the fungal partner (Alors et al. 2017). The first attempts at mycobiont cultivation date back Parmelia saxatilis and Usnea ghattensis can be taken as 70 years (Castle and Kubsch 1949). Ahmadjian and an example of lichens with demonstrated antimicrobial and Reynolds (1961) already underlined the difficulties of antioxidant properties (Kosanić et al. 2012;Srivastava cultivation and the importance of certain compounds as et al. 2013; Behera et al. 2005), but the lack of known active molecules. Oliver et al. (1989) and Crittenden and successful in vitro conditions for growth and productions Porter (1991), aware of the interest of these compounds, treat- of bioactive compounds are hampering their use (Behera ed the cultivation of many lichen fungi with debatable suc- et al. 2009). The same phenomenon has been observed cesses. Improvements in production have been achieved with Xanthoparmelia tinctina, with high antiviral potential through the use of specific culture media and new protocols (Karagoz and Aslan 2005). Antifungal activities have been (McDonald et al. 2013;Molinaetal.2015; Rafat et al. 2015), reported in this family. Due to their phenolic content, many control of abiotic parameters and the use of electronically crude extracts have been also screened as antioxidant adjustable culture chambers that can simulate variations in agents (Gulluce et al. 2006; De Paz et al. 2010; Ranković culture conditions (Stocker-Wörgötter et al. 2013). Special 2015; Fernández-Moriano et al. 2016;Thadhaniand interest is having the genetic and functional characterization Karunaratne 2017). Anticancer activity of secondary me- of polyketide synthases (PKS) (e.g. Bertrand and Sorensen tabolites from Parmeliaceae lichens has also been explored 2019). Despite the progress, there are many pitfalls that still (Yamamoto et al. 1995; Stanojković 2015;Ebrahimetal. Axenic culture and biosynthesis of secondary compounds in lichen symbiotic fungi, the Parmeliaceae 81

2017). For example, usnic acid, a characteristic metabolite 1.2 Isolation and culture of Usnea and (Abdel-Hameed et al. 2016), but also found in the Parmelia, is the most studied Multi-ascospore isolations were obtained from apothecia of among lichen substances as candidate for novel cancer the lichen-forming fungi, according to the discharged spore therapy and other pharmacological uses (Cocchietto et al. method of Ahmadjian (1993). Three samples from each spe- 2002). Preliminary analysis of Xanthoparmelia tinctina ac- cies where used to select the apothecia. They had a size of 2– etone extracts showed the ability to inhibit maturation of 7 mm to ensure sexual maturity (Molina et al. 1997; Alors biofilms of the opportunistic pathogen Candida albicans, et al. 2019) and were carefully cleaned following the protocol where complex phenolic compounds seem to be the main described by Molina and Crespo (2000). Twenty apothecia responsible for this activity (Millot et al. 2017). As far as per species (6 or 7 by thalli or sample) were used. The clean we know and, according to the precise revision apothecia were separately attached to the lid of an inverted of Ranković (2015), A. centrifuga phenolic compounds Petri dish with petroleum jelly. The bottom part of the Petri have not been described as molecular bioactive. dishes contained inorganic Basal Bold Medium [BBM; Considering the ontogenetic, phylogenetic, metabolic and Deason and Bold 1960]. Inverted Petri dishes were incubated biotechnological interest of this family, we selected Parmelia in a growth chamber at 20 °C in the dark. During the first 24– saxatilis, Usnea ghattensis, Xanthoparmelia tinctina, 48 h, the apothecia were allowed to discharge ascospores up- Platismatia glauca, Parmelina tiliacea, and Arctoparmelia wards onto the medium; after this time, they were removed to centrifuga in order to: 1) provide new media and culture reduce contaminations by bacteria or other fungi, and new knowledge (germination and development) that may favor sterile lids were placed on. One to two weeks after first ger- the production of active biomolecules without prejudice to mination was noted, we randomly selected 25 isolates by thalli lichen diversity, 2) identify the secondary metabolites by the and media. These agar pieces containing several germinated mycobiont in aposymbiotic conditions and 3) perform a com- ascospores from each species. Ideally, pieces containing a parative study between phenolic patterns from whole thalli vs. single group of germinating ascospores from a ascus, but mycobiont in aposymbiotic condition (cultures), on different sometimes several groups of ascospores were carried on a organic media. single agar fragment (no more than 24 spores by agar piece). For long-term growth and measurements, those pieces, were excised, transported and sub-cultured on different organic me- 1 Material and methods dia. The media chosen were usually successful in other cul- tures of lichen forming fungi (e.g. Molina et al. 2013;Molina 1.1 Sampling et al. 2015). They were: 0.2% glucose malt-yeast extract (w/v) [0.2%G-MY; according to Molina et al. 2013], 2% glucose Details of the selected specimens are showed in Table 1. BBM (w/v) [2%G-BBM; Behera and Makhija 2001]; Lilly Herbarium specimens are deposited in MAF-Lich and S and Barnet medium enriched with 3% glucose (w/v) [3%G- (Thiers, continuously updated). External morphology of her- LBM; according to Lilly and Barnett 1951 as modified by barium specimens was examined using an Olympus SZX16 or Lallemant 1985] and Corn Meal Agar (CMA) following man- ’ a Leica MZ 7.5 dissecting microscope. Thalli were air-dried ufacturer s instructions (Difco, Detroit, MI, USA). All proce- and stored in the dark either at 4 °C and no longer than two dures were carried out under sterile conditions in a laminar weeks (Arctoparmelia centrifuga and Parmelia saxatilis), or flow chamber. Cultures were incubated at 20 ± 5 °C in the at room temperature and from two weeks to up to four months dark and observed and measured periodically until 200 days. (Parmelina tiliacea, Platismatia glauca, Usnea ghattensis, Mycobionts were examined using an Olympus CX40 micro- and Xanthoparmelia tinctina). scope and a magnifier glass (Nikon SMZ800, Tokyo, Japan).

Table 1 Lichen-forming fungi used to isolate and cultivate the mycobiont, under axenic conditions, and its herbarium code

Species Herbarium code Location

Arctoparmelia centrifuga (L.) Hale 1986 S-9639 Sweden, Hälsingland (61°56′25”N, 15°36′36″E) Parmelia saxatilis (L.) Ach. 1803 S-9551 Sweden, Hälsingland (61°27′47,8”N, 17°05′40,3″E) Parmelina tiliacea (Hoff.) Hale 1974 MAF-Lich Spain, Ávila, (N 40 18′ 28.4” W500′ 39.0″) Platismatia glauca (L.) W.L. Culb. & C.F. Culb. 1968 S-9565 Sweden, Jämtland (62°19′26,0”N, 14°25′46,3″E) Usnea ghattensis G. Awashti 1986 MAF-Lich India, Maharashtra (N17 55.19″ E7344,04″) Xanthoparmelia tinctina (Maheu & A. Gillet) Hale 1974 MAF-Lich Spain, Madrid, San Lorenzo de El Escorial, Silla de Felipe II 82 Díaz E.M. et al.

For photography, an automatic ring flash system was attached were recorded each second. MS1 low resolution data were to the camera lens (CANON 450-D, Tokyo, Japan). The ob- obtained in the ESI negative mode using the following param- servations were carried out using white light and Nomarsky eters: capillary temperature 250 °C, capillary voltage 180 V, interference contrast. source voltage offset 20 V, source voltage span 20 V, source Different parameters were calculated to assess the gas temperature 50 °C, ion spray voltage 3,5 kV. Advion data mycobiont ontogenetic development: mean apothecia size, express software was used for data evaluation. Identification ascospore ejection capacity (after 48 h); capacity of ejection of compounds was done by comparison of their retention time and germination of plurisporic packs, mean by the number of and MS1 data with standards under the same chromatographic colony-forming units (CFU) per apothecia; growth capacity; conditions (Gadea et al. 2018). and secondary metabolic production. The growth was ana- All spectra were analyzed using an automatic and standard- lyzed with the program ImageJ (http://imagej.nih.gov/ij/) ized identification method to offer a better comparison be- with images taken under binocular (Olympus SZ30, Japan). tween metabolites found in each media for each mycobiont. We quantified the relative culture area growth rate (Evans Each .datx file was exported as *.cdf with ADVION 1972) according to the formula: DataExpress software in order to be read by MZmine 2.52 2 Relative culture area growth rate (RCAGR, mm freeware (http://mzmine.sourceforge.net/). Mass data were cm−2 day−1) = ln. The apothecia size was calculated as the detected with a noise level of 2E6. Chromatograms were largest diameter of the mycobiont culture in cm when the built with ADAP Chromatogram builder with a minimal aggregates were collected to isolate the phenolic compounds. number of scans of 4; group intensity threshold and min highest intensity of 2E6 and m/z tolerance of 0.5 m/z (Myers 1.3 Metabolite composition analysis et al. 2017). Chromatograms were deconvoluted with ADAP Wavelets algorithm. Dereplication was then achieved against 1.3.1 Phenols and terpenoids extraction two in-house .csv database files. The first file had been built with m/z and RT values of standards and led us to identify Samples of 300 mg of dried intact thalli and 30 mg of the dried fairly well these metabolites whereas the other one contains fungal culture (at the end of the growth period) were used to only calculated m/z values of most known lichen metabolites extract internal and superficial secondary compounds. First, in order to get a possible match. Dereplicated feature lists were they were washed off with 15 ml acetone at room temperature exported as .csv files for further treatment. for 5 min. The residue was also macerated with 15 ml of acetone for 5 min. Consequently, both solutions were gathered 1.3.3 Statistical analysis and filtered through a 45 μm Ico plus 3 filter (TeknoKroma, Spain) and then dried in extraction hood. The filtrate was In order to compare if culture growth depends on media and collected in an HPLC vial after the acetone was completely time, and if underlying errors are all uncorrelated with homo- evaporated. Lichen and culture extracts were solubilized in geneous variances, we run a General Linear Model (GLM) tetrahydrofuran (THF) in order to have a final concentration with culture growth as a response variable and media and time at 1 mg/mL, solutions were then filtered (0.45 μm) before as predictors. Multiple Comparisons after Post-Hoc Pairwaise TLC and HPLC analysis. Seven lichen compounds (purity test were used for checking differences between culture >95%) described in the studied lichens and referenced in the growth and data were considered significantly different when REN-LICPD library were used as standards. p < 0.05. Statistical analyses were performed with software R (R Development Core Team 2016). We used the package 1.3.2 HPLC-DAD-MS analysis ‘glm2’ for general linear models. The graphics showed the 95% confidence interval, median average and outliers. Analyses were performed on a Prominence Shimadzu LC- 20 AD system (Marne La Vallée, France) interfaced with a mass spectrometer ADVION expression CMS. A kinetex C18 2 Results column (2.6 μm, 100 × 4.6 mm, Phenomenex) was used as a stationary phase at t° = 40 °C. The mobile phase consisted of 2.1 Cultivation of mycobionts

H2O + 0.1% HCOOH as solvent A and ACN + 0.1% HCOOH as solvent B with gradient: 20% of B during The 20 selected apothecia of all the six species ejected muti- 5min,20%–80% of B during 25 min, 80%–100% of B during sporic packs and spores isolated in the first 24 h. Ascospores 5 min, 100% of B during 7 min, 100%–20% of B during produced septate hyphae with short cells in BBM in the ma- 3 min, 20% of B during 3 min. The flow rate was 0.5 mL/ jority of cases (Fig. 1). The number of multi-sporic group per min and 10 μL of each sample were injected. DAD data were apothecia ejected and able to product CFUs varied consider- recorded at 254 nm and absorption spectra (210–400 nm) ably between and within the species. The most important Axenic culture and biosynthesis of secondary compounds in lichen symbiotic fungi, the Parmeliaceae 83

Fig. 1 Ascospores germination after 15 days on BBM from a Arctoparmelia centrifuga, b Parmelina tiliacea, c Parmelia saxatilis, d Platismatia glauca, e Xanthoparmelia tinctina, f Usnea ghattensis. Scale = 10 μm differences in ejection and germination were between CMA media showed orange pink-colored (5YR; 7/8 Munsell P. glauca and X. tinctina, with a CFU average of 13.65 and colour system, Munsell 1912). In media 3%G-LBM and 1440 in BBM media, respectively (Table 2). When subcul- 0.2G.MY colonies were pink (2.5YR, 7/4) and rosewood tures where transferred to different enrichment media, inter- (10YR, 9/4) colour, respectively, with white aerial hyphae on specific and intraspecific variation of development were ob- the surfaces (Fig. 4). This fungus did not grow on 2%GBBM. served (Figs. 2 and 3). Parmelina tiliacea ejected naked and bipolar ascospores Arctoparmelia centrifuga produced a large amount of ellip- (Fig. 1b), generally in groups of eight, and we detected differ- soid ascospores with thickened walls (Fig. 1a), which are often in ent growth depending on media. The mycelial colony was groups of four, but isolated ascospores can be seen on the medi- significantly larger (p ≤ 0.001) in 2%G-BBM and 3%G- um as well. The initial superficial mycelium was observed with LBM than that observed for 0.2G-MY and CMA media intersepta along the hypha. This species showed significant dif- (Fig. 2e, f). The cultures presented a significant increase in ferences (p ≤ 0.001) among media, with the highest and lowest their growth from 2 months, then subsequently stabilized from growth values at 3%G-LBM and 2%G-BBM, respectively (Fig. 100 days until analysis had been completed. An interaction 2,Table3). After 40 days, the axenic cultures experienced sig- between media and time has not been observed in none of the nificant progressive growth (P ≤ 0.001) until 200 days, but no cases (p ≥ 0.001, Table 3). Parmelina tiliacea showed the interaction between media and time (P ≥ 0.001) was observed largest macroscopic development after six months of growth, 2 −2 −1 (Fig. 2). In 3%G-LBM the RCAGR for 200 days was 1.406 ± with 3.05 ± 0.11 mm cm day RCAGR on 3%G-LBM 0.052 mm2 cm−2 day−1 (Table 4), followed by 0.2G-MY (1.37 ± (Table 4). Compact growth was observed in every media 0.042 mm2 cm− 2 day− 1 ) and CMA (1.34 ± analysed with brownish coloration (Fig. 4) after 60 days, less 0.040 mm2 cm−2 day-1). Morphological appearance was changed 2%G-BBM colonies with orange pink colour (5YR: 7/8). along the experiment in media where growth was observed. Development on 0.2G-MY media gave rise to brown- Coloration of colonies not varied overtime. Mycobionts of coloured (7.5YR: 4/2) cultures with white aerial hyphae with

Table 2 Apothecia diameter size of twenty sample (three thalli or Species Apothecia size (mm) Spore ejection Productivity (CFU) samples by species), spore ejection in inorganic Basal Bold Arctoparmelia centrifuga 5.42 ± 1.44 after 24 h 834.62 ± 539.24 Medium (BBM) and productivity Parmelia saxatilis 3.07 ± 1.81 after 24 h 534.71 ± 453.50 per species. (CFU = colony- Parmelina tiliacea 6.15 ± 2.18 after 24 h 383.53 ± 420.10 forming units in BBM) Platismatia glauca 3.85 ± 0.89 after 24 h 13.65 ± 15.58 Usnea ghattensis 3.45 ± 0.48 after 24 h 676.85 ± 718.23 Xanthoparmelia tinctina 3.33 ± 0.61 after 24 h 1440 ± 968.11 84 Díaz E.M. et al.

Fig. 2 Mycobiont size (larger diameter) of a, b Arctoparmelia centrifuga, c, d Parmelia saxatilis and e, f Parmelina tiliacea, considering organic media (0.2- MY, 2%G-BBM, 3%G-LBG, CMA), incubation time (0 to 200 days) and the interaction between the two factors. Lower case letter denotes significantly different mean values (p <0.05; Multiple Comparisons after Post- Hoc Pairwaise test). Box extents indicate 95% confidence interval, center is mean average. All assays were done for twenty sub- cultured samples

numerous dark brown (7.5YR 2/2) tips and brown (7.5YR showed by colonies growing on 0.2G-MY (7.5YR 4/2) or 4/2) liquid released by the colonies. Same coloration and mor- their light brown (10YR: 5/10) liquid secretion. phological structure was observed in mycobiont grew on Development on 2%G-BBM media showed brown- CMA media but without presence of liquid secretion. A colored colonies (10YR: 4/4) with abundant white aerial brown (5YR: 3/2) three-dimensional growth was observed hyphae, with presence of a light brown (10YR: 5/10) liquid on 3%G-LBM cultures with many folds and with aerial hy- secretion tip. On the other hand, a compact growth was phae on the surface. observed when P. saxatilis colonies grew on 3%G-LBM Parmelia saxatilis had naked, globular and thin-walled (5YR: 4/2) with very limited presence of white aerial ascospores, packed together in groups of four or eight (Fig. hyphae. 1c). In 3%G-LBM, it was observed a significant increase Platismatia glauca ejected naked and bipolar ascospores (p ≤ 0.001) in their growth after 100 days (Fig. 2c, d). The generally in groups of eight (Fig. 1d). The growth success interaction between media and time on the growth of the for this species was the lowest over the 200 days of experi- mycobionts was not found in none of the analysed cases ment, scarcely developing in every cultured media, no more

(p ≥ 0.001, Table 3)andtheRCAGR on 3%G-LBM for than a few cells divisions in any case analysed (Fig. 3a, b). 200 days was 1.45 ± 0.05 mm2 cm−2 day−1 (Table 4). There were no significant differences (p ≤ 0.001, Table 3)on Morphologicalaspect(Fig.4e–h)ofCMAand0.2%G- media, time or the interaction between media and tie on the MY colonies was very similar along the experiment includ- growth of the mycobionts colonies. The development on all ing presence of white aerial hyphae, but colonies growing media used was poor and no change in coloration was ob- on CMA (2.5YR: 8/2) not presented the brown coloration served (data not shown). Axenic culture and biosynthesis of secondary compounds in lichen symbiotic fungi, the Parmeliaceae 85

Fig. 3 Mybobiont size of a, b Platismatia glauca, c, d Xanthoparmelia tinctina y e, f Usnea ghattensis considering organic media (0.2-MY, 2%G- BBM, 3%G-LBG, CMA), incubation time (200 days) and the interaction between the two factors. Lowercaseletterdenotes significantly different mean values (p ≤ 0.05; Multiple Comparisons after Post-Hoc Pairwaise test). Box extents indi- cate 95% confidence interval, center is mean average. All assays were done for twenty sub- cultured samples

Xanthoparmelia tinctina dispersed naked and bipolar (Fig. 1e). The cultured showed significant differences ascospores in groups of eight, where the first hyphae among treatments (p ≤ 0.001, Fig. 4c, d), with up to sometimes appeared on both sides of the ascospores 0.164 ± 0.075 cm2 generated in 0.2G-MY media. The

Table 3 Summary of statistic parameters for growth rate of Arctoparmelia centrifuga Parmelia saxatilis Parmelina tiliacea Arctoparmelia centrifuga, df F p df F p df F p Parmelia saxatilis, Parmelina Media 1 79.253 <0.001 1 73.205 <0.001 1 124.313 <0.001 tiliacea, Platismatia glauca, Xanthoparmelia tinctina, Usnea Time 3 6.098 <0.001 3 36.012 0.013 3 3.330 <0.001 ghattensis (considering organic Media*Time 3 1.345 0.275 3 0.751 0.522 3 0.538 0.656 media (0.2%G-MY, 2%G-BBM, D2 76 87 57 3%G-LBG, CMA), incubation Platismatia glauca Usnea ghattensis Xanthoparmelia tinctina time (0 to 200 days) and the interaction between the two df F p df F p df F p factors Media 1 0.555 0.357 1 2.313 0.121 1 25.850 <0.001 Time 3 0.533 0.659 3 8.809 <0.001 3 7.977 <0.001 Media*Time 3 0.469 0.704 3 0.061 0.980 3 0.011 0.998 D2 37 68 57 86 Díaz E.M. et al.

Table 4 Relative culture area 2 −1 −1 growth rate (RCAGR) of Species Experiment Growth rate (mm cm day ) Arctoparmelia centrifuga, Parmelia saxatilis, Parmelina Media Time (days) tiliacea, Usnea ghattensis and Xanthoparmelia tinctina,atdif- Arctoparmelia centrifuga 0.2%G-MY 0–200 1.369 ± 0.042 ferent media 3%-LBG 0–200 1.406 ± 0.052 2%G-BBM 0–200 0.988 ± 0.082 CMA 0–200 1.345 ± 0.040 Parmelia saxatilis 0.2%G-MY 0–200 3.140 ± 0.072 3%-LBG 0–200 1.453 ± 0.054 2%G-BBM 0–200 1.168 ± 0.063 CMA 0–200 1.350 ± 0.016 Parmelina tiliacea 0.2%G-MY 0–100 1.380 ± 0.050 3%-LBG 0–100 3.054 ± 0.107 2%G-BBM 0–100 2.996 ± 0.089 CMA 0–100 1.340 ± 0.190 Usnea ghattensis 0.2%G-MY 0–200 1.060 ± 0.017 3%-LBG 0–200 1.074 ± 0.071 2%G-BBM 0–200 0 CMA 0–200 1.079 ± 0.190 Xanthoparmelia tinctina 0.2%G-MY 0–200 1.363 ± 0.067 3%-LBG 0–200 1.194 ± 0.114 2%G-BBM 0–200 1.495 ± 0.136 CMA 0–200 1.264 ± 0.098 growth has increased significantly (p ≤ 0.001) from the grown on (Fig. 4n,p,q). Every mycelium analysed beginning to achieve the maximum at 200 days. showed brown pigmentation (5 yr: 4/2) since early Morphological appearance of the colonies showed were states of growth with white and long aerial hyphae similar and not varied with the type of media it had forming a dense layer on the surfaces.

Fig 4 Ontogenetic development of mycobiont cultures on four enriched media after 200 days. a, b, c, d Arctoparmelia centrifuga. e, f, g, h Parmelia saxatilis i, j, k, l Parmelina tiliacea, m, o, p, q Xanthoparmelia tinctina. Scale = 6mm Axenic culture and biosynthesis of secondary compounds in lichen symbiotic fungi, the Parmeliaceae 87

Usnea ghattensis produced naked and globular ascospores protocetraric acid derivatives, the chromone roccellin and ste- with thickened walls (Fig. 1f) and frequently dispersed as roids are also suspected to be produced in this medium. single or double-packed ascospores. These cultures achieved In P. tiliacea thalli, four compounds were detected and the a maximum area of 0.009 cm2 after 6 months of incubation, depsides lecanoric acid, atranorin and chloroatranorin (only reaching the maximum of growing at 20 days. There were detectable by UV detection) were found to be the major com- significant differences among media (p ≤ 0.001, Fig. 3e, f), pounds produced (Fig. 7;Table7, supplementary data). with no development of germinated spores in 2%G-BBM me- Cultivation of the mycobionts in 2% G-BBM medium dia. During this time, any colonies showed visible coloration allowed the detection of eight compounds while 0.2% G- for different media (data not shown). MY, 3% G-LBM, CMA media produced respectively six, five and two detectable compounds. Interestingly, α-andß- alectoronic acids and usnic acid were produced in the 3% G- 2.2 Chemical analysis by HPLC-DAD-MS LBM medium. One compound was shared between CMA, 3% G-LBM, 2%G-BBM and 0.2% G-MY media (m/z Qualitative detection of compounds based on UV, m/z and Rt 265.2/ Rt = 25.0 min). In the productive 2% G-BBM, several features showed a high tentative diversity of secondary com- putative compounds were detected such as stictic acid deriv- pounds by species and medium (Supp. Tables 5–8). When we atives but also nitrogen compounds (scabrosin related-com- analyzed the quantitative production of compounds in the four pound), the cycloaliphatic aspicilin, the quinone solorinic acid culture media and lichen thalli of five species by HPLC-MS and the pulvinic acid derivative epanorin. (Figs. 5, 6, 7, 8), important differences in chemosyndromes Three depsidones (protocetraric, norstictic, psoromic acids) were observed between mycobiont and thalli except for and three putative stictic acid derivatives were detected as A. centrifuga, where a-alectoronic acid, isolated from natural major compounds in X. tinctina thalli (Fig. 8;Table8, stems, was also found in G-LBM. P. tiliacea and X. tinctina, supplementary data). Cultivation of the mycobionts in 2% despite belonging to different genera, synthesized aspicilin, G-BBM media allowed the detection of seventeen compounds usnic acid and α-andβ-alectoronic acid. None of these phe- while in 3% G-LBM eleven compounds were detected, and nols was detected in natural thalli of these species. seven in 0.2% G-MY and CMA. The depside α-alectoronic In A. centrifuga, seven compounds including α-alectoronic acid was also detected in the four media while its related acid, α-collatolic acid atranorin and usnic acid were found to diphenylether α-alectoronic acid was detected in three media correspond to the compounds detected in natural thalli (Fig. 5; except in the less productive CMA medium. The dibenzofuran Table 5 supplementary data). Mycobionts grown in 3% G- usnic acid was synthesized in 0.2%-MY and CMA media LBM synthetized five compounds in this most inductive me- though its isomer isousnic acid was likely identified in the dium followed by the 0.2% G-MY (three compounds) and the crude thalli. An additional aromatic depsidone with alkyl media 2% G-BBM and CMA. The depsidone α-alectoronic chains physodic acid was also produced in the 3% G-LBM acid but also its corresponding diphenylether α-alectoronic medium. Aspicilin was also detected in 3% G-LBM medium acid were detected in 0.2% G-MY, 3% G-LBM media. One like for Psaxatilis. All extracts of mycobionts grown in the putative compound (m/z 265.1 / Rt 24.9 min) was shared for four media exhibited the same compound suspected to be 2,4- CMA and 2% G-BBM while another one (m/z 279.9 / Rt di-O-methylolivetonic acid (m/z 265.1 / Rt 25.7 min). Several 19.5 min) was shared between 0.2% G-MY and 2% G-BBM terpenoids were also suspected to be formed in 2% G-BBM (Table 5, supplementary data). medium along with several monoaromatic phenols. The depsidones salazinic and lobaric acids and the depsides Chromatographic analyses showed that atranorin, atranorin and chloroatranorin (only detectable by UV detec- divaricatic and caperatic acids were detected in P. glauca thal- tion) are found in P. saxatilis thallus as the major compounds li while usnic (as major compound), psoromic and evernic along with nordivaricatic acid (Fig. 6;Table6,supplementary acids were the only metabolites detected in U. ghattensis thalli data). Twelve and nine compounds were detected in 3% G- (data not shown). LBM and 2% G-BBM media respectively while five com- pounds were detected in 0.2% G-MY and only one in CMA. Three compounds were common between the most productive 3 Discussion media (0.2% G-MY, 3% G-LBM, 2% G-BBM). Two are suspected to be aromatic compounds belonging to quinone All species analysed showed ability to produce and eject as- family (solorinic acid), monoaromatic phenol’s (2,4-di-O- cospores (Fig. 1).Thedispersionofascosporeswereasgroups methylolivetonic acid) and one is unknown from our database. containing 4–8 ascospores (except U. gatthensis). The tempo- The most productive medium 3% G-LBM revealed the pres- ral viability of meiotically produced fungal spores is not well ence of the cycloaliphatic compound aspicilin, the aromatic documented (Leavitt and Lumbsch 2016). However, results compounds physodic acid. Some compounds like stictic, showed that, at least in P. saxatilis, P. tiliacea, and X. tinctina, 88 Díaz E.M. et al.

Fig. 5 HPLC-MS detected metabolites and identification of metabolites intensity of peak detection for shared compounds. Each frame which according to their retention time (Rt) combined to their molecular mass in surrounds the identity of the metabolites corresponds to a structural fam- negative mode ESI (reported m/z) for the mycobionts of A. centrifuga ily : dibenzofuran, : depsidone, : cultivated in the four media (CMA, 0.2%G-MY, 3% G-LBM, 2%G- diphenylether BBM) and the natural thalli. Part size in pie-charts is according to the

most of the spores from asci germinate and develop myceli- influence the production of later phenols, but it is possible that um. It is unknown how this reproductive strategy, in which the the great diversity of PKs genes detected in lichens (Calchera spores remain united to generate a new mycelium, could et al. 2019) could be related to the diversity of products

Fig. 6 HPLC-MS detected metabolites and identification of metabolites and the natural thalli. Part size in pie-charts is according to the intensity of according to their retention time (Rt) combined to their molecular mass in peak detection for shared compounds. Each frame which surrounds the negative mode ESI (reported m/z) for the mycobionts of P.saxatilis cul- identity of the metabolites corresponds to a structural family : tivated in the four media (CMA, 0.2%G-MY, 3% G-LBM, 2%G-BBM) depsidone, :depside, : cycloaliphatic compound Axenic culture and biosynthesis of secondary compounds in lichen symbiotic fungi, the Parmeliaceae 89

Fig. 7 HPLC-MS detected metabolites and identification of metabolites peak detection for shared compounds. Each frame which surrounds the according to their retention time (Rt) combined to their molecular mass in identity of the metabolites corresponds to a structural family: negative mode ESI (reported m/z) for the mycobionts of P. tiliacea cul- : dibenzofuran, : depsidone, : diphenylether, tivated in the four media (CMA, 0.2%G-MY, 3% G-LBM, 2%G-BBM) :depside; : cycloaliphatic compound and the natural thalli. Part size in pie-charts is according to the intensity of meiotic involved in the formation of multi-ascospore cultures depending on media (Molina et al. 2015; Shanmugam et al. (in aposymbiotic conditions) and in natural thalli. This hy- 2016) and time (Molina et al. 2015), but no interaction be- pothesis should be tested with new experimental designs. tween them in any of the analyzed cases (Table 3), with a The sizes of the analysed mycelial colonies were consider- notable exception of P. glauca with no more than a few cells ably different, both within and among species along the de- divisions. All mycobiont grown on CMA show a shortage of velopment (Figs. 2 and 3). Variations were detected secondary compounds that could be explained by the absence

Fig. 8 HPLC-MS detected metabolites and identification of metabolites peak detection for shared compounds. Each frame which surrounds the according to their retention time (Rt) combined to their molecular mass in identity of the metabolites corresponds to a structural family: negative mode ESI (reported m/z) for the mycobionts of X. tinctina cul- : dibenzofuran, : depsidone, : tivated in the four media (CMA, 0.2%G-MY, 3% G-LBM, 2%G-BBM) diphenylether, :depside; : cycloaliphatic and the natural thalli. Part size in pie-charts is according to the intensity of compound 90 Díaz E.M. et al. of an additional carbon source. Corn meal infusion provides conditions such as temperature, humidity or photoperiod that nitrogen, vitamins, minerals and amino acids essential for have been shared by all the species could be determining. growth, without glucose, while in the other glucose enriched Usnic acid identified in P. tiliacea and X. tinctina axenic media, a greater number of secondary compounds were culture has been used in creams, toothpastes, mouthwashes, detected. Shanmugam et al. (2016) proposed a range of opti- deodorants and sunscreen products (Ingolfsdottir 2002). The mal low sucrose concentrations for growth and a range of high enantiomers of this compound have a wide spectrum of med- concentrations for the synthesis of phenols. However, ical properties such as antiangiogenic, anti-inflammatory, an- Valarmathi et al. (2009) have reported the downregulation of timicrobial, antioxidant, antiproliferative, antitumor and cyto- the PKS genes and lower number of compounds produced toxic activity against cancer cell lines (see Machado et al. upon increasing sucrose concentration. Mycobiont colonies 2019). Some of the genera that produce this compound con- or aggregated growing on 0.2%-MY, 3%G-BBM and 2%G- tain species listed in the red list such as Cladonia perforata, LBM showed a wide range of metabolites belonging to sev- Cladonia appalachensis (Yahr 2003; Lendemer et al. 2020). eral families (depsides, depsidones, dibenzofuranes, The α-alectoronic acid (Culberson 1969; Brodo et al. 2001) diphenylethers, quinones, pulvinic acid derivatives...) was found in several cultivated thalli with or without its rela- (Table 5–8 supplementary material and Figs. 5, 6, 7, 8) when tive diphenylether: 3% G-LBM for A. centrifuga and colonies stopped growing (about 200 days). These results are P.tiliacea, 0.2% G-MY for A. centrifuga and all media for consistent with the theory that the mycobiont would use the P. tinctina. The presence of the diphenylether is usually re- carbon source to grow until one or several fundamentals nu- ported as an artefact formed during extraction or by degrada- trients were missing in the media. From then on, the carbon tion, but it could be also a pathway for its biogenetic synthesis source could be used to massively synthesize phenolic com- (Millot et al. 2008; Ismed et al. 2012). The presence of α- pounds (Bu'lock 1961; Timsina et al. 2013). Detoxification of alectoronic acid is interesting given that this secondary com- primary metabolites is another hypothesis that has been pro- pound has cytotoxic activity (Millot et al. 2008) and original posed to explain the production of secondary metabolites. If antibacterial activity acting as an enzyme inhibitor on polo- the growth of the fungus slows down (for reasons unknown like kinase1 (Williams et al. 2011). Some lichen-forming fun- until now), but the metabolism is still very active, toxic prod- gi that produce α-alectoronic acid are Ochrolechia parella ucts of the primary metabolism can accumulate. The transfor- (Millot et al. 2007), physodes (Latkowska et al. mation of these into secondary metabolites can be a method to 2019). Aspicilin is a macrolide with antibiotic potential prevent the toxic accumulation of by-products (Deduke et al. (Schmidt et al. 2017). Isolated from different lichens such as 2012). Furthermore, an astonishing diversity of PKS Aspicilia contorna (Paukov et al. 2015) it has been commonly paralogous genes (Calchera et al. 2019) has been detected in synthesized by complex physicochemical processes lichens, which, modulated by abiotic factors, can amplify the (Schaubach et al. 2017; Saidhareddy et al. 2014). We propose synthesis response qualitatively and quantitatively in axenic that our methodology may be a eco-friendlier option of culture. obtaining this active biomolecule, although the process must Chemosyndrome variations between mycobiont culture be improved to implement the efficacy. P. saxatilis axenic and natural thalli where observed in all species. This can be culture produced nordivaricatic acid, which could be an inhib- attributed to change in abiotic factors such as nutrients, tem- itor of human leukocyte elastase (HLE) implicated in several perature, pH substrate, day–night cycles, etc., between sym- inflammatory diseases (Zheng et al. 2012); lobaric acid which biotic and aposymbiotic conditions (Behera and Makhija exert various biological activities, including antitumor, anti- 2001;Molinaetal.2003; Stocker-Wörgötter 2008) and the proliferative, anti-inflammatory, and antioxidant activities absence of carbon sources provided by the photobiont (Hong et al. 2018) and physodic acid, which has anticancer (Brunauer et al. 2007). The production of phenols in natural activity (e.g. Studzińska-Sroka et al. 2016). conditions is also highly variable within species and depends on environmental factors (Paukov et al. 2019; Latkowska et al. 2019). Shanmugam et al. (2016) report that the best 4 Conclusions condition to obtain the own compounds of natural thallus in axenic cultures is to grow the mycobiont with the photobiont The results confirm that the compounds synthesized by the and in the absence of an additional carbon source, however, mycobiont under symbiotic and aposymbiotic conditions vary the co-culture (mycobiont-photobiont) is an added difficulty. significantly, possibly due to complex multifactorial variables The similarity in the chemical pattern between P. tiliacea and that should continue to be investigated. Successful culture X. tinctina is surprising (aspicilin, usnic acid, α-alectoronic conditions for the growth in axenic conditions of four and β-alectoronic acid) because these species that do not share lichen-forming fungi (A. centrifuga, P. saxatilis, P. tiliacea phenolic compounds when they form a natural thallus. This and X. tinctina), which produced phenolic compounds of pattern is not related to the culture media (Figs. 7 and 8)sothe pharmacological and industrial interest (usnic acid, aspicilin, Axenic culture and biosynthesis of secondary compounds in lichen symbiotic fungi, the Parmeliaceae 91

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