Chemosyndrome Variation in Mycobiont Culture and Thallus of Parmelina carporrhizans and Parmelina quercina

We cultured Parmelina carporrhizans and P. quercina in Corn Meal Agar and 0.2% glucose Malt Yeast Agar for 160 days. Chemosyndrome of natural thalli and mycobiont cultures were analyzed by HPLC. Lecanoric acid, atranorin, chloratranorin and ergosterol were detected in P. carporrhizans thalli, while lecanoric acid, chloratranorin and aliphates were found in P. quercina thalli. The secondary methabolites pattern between thalli and mycobiont culture was completely different in both species. Both species secreted the phenalenone myeloconone C in culture media and was also detected in P. quercina mycobiont aggregates. Interestingly, the phenolic compounds produced by the mycobiont culture of P. carporrhizans are related to those produced by natural thallus by the same biosynthetic pathway, while the chemosyndrome of P. quercina mycobiont implies switch of biosynthetic pathway from acetate-polymalonate pathway to shikimic acid pathway, with pulvinic acid as major compound of mycobiont culture. The role of Myelochonone C, confluentic acid and pulvinic acid produced by mycobiont culture is discussed as possible adaptive vantage in field as photoprotective agent or as byproduct result of stressing artificial culture conditions.


Introduction
Lichen forming fungus produce a wide range of extrolites (secondary metabolites) and whole lichens have had several folk applications as dyes, food, and health-promoting teas by herbalism, Indian Ayurveda medicine and Tradicional Chinese Medicine [1][2][3][4].Many of the interesting lichen substances are aromatic polyketides [5] with marked antioxidative properties [6] conferring: allelopathy properties against bacteria [7][8][9] and fungi [10,11] and enabling specific pharmacological applications as photoprotection [12,13] cardioprotection [14] neuroprotection [15] antiviral [16] and antitumoral applications [9,17] in mammals by modulating intracellular concentration of hydrogen peroxide [18].While lichen substances have interest by the properties exhibited, these substances have had importance in lichen determination by identifying lichen substances as a character in chemotaxonomy.In the second half of the 20th century thin layer chromatography (TLC) has been widely used to identify lichen substances, in the last decades the number of identified lichen substances increased by using more powerful methods [5] as high performance liquid chromatography (HPLC), which have higher sensibility to detect phenolic secondary metabolites [19].
Many of these lichen extrolites are biosynthetically related since they are produced in different steps of the same biosynthetical pathway, consisting in a set of substances called chemosyndrome [20].Lichen substances are classified in three major classes: i) acetate-polymalonate pathway, ii) mevalonic acid pathway and iii) shikimic acid pathway.[21,22].The chemosyndrome varies between and within species, depending on environmental conditions as responses of the lichens to their variable environments [5].Indeed, the phenolic pattern can vary substantially between the mycobiont growing in aposymbiotic conditions and the mycobiont associated with its photosynthetic partner (lichen symbionts).These variations have been explained as differences in abiotic factors (nutrients, temperature, pH) [19,23,24] and the modulating effect of the photobiont [25].
For this study, we choose two sexually obligated species of a small lichen genus Parmelina Hale, characterized by containing lecanoric acid, atranorin and chloratranorin -at a trace level - [26].Parmelina carporrhizans (Taylor) Hale and Parmelina quercina (Willd.)Hale are morphochemically similar and have Mediterranean distribution but with different ecological preferences.P. carporrhizans grows in more oceanic sites with higher incidence in deciduous phorophytes as Castanea sativa while P. quercina grows in more continental habitats typically growing on perennial oaks as phorophyte.Although it is reported the coincidence of the two species in the same branch tree, the two species rarely coexist.
Our aims are i) characterize the chemosyndrome composition of these two related species by HPLC and ii) comparing the chemosyndrome variation between species and between natural thallus and mycobiont aposymbiotic culture.

Results
After 80 days on culture, Parmelina mycobiont cultures produced striking brownie drops (figure 1A and 1C) and after 160 days in most of the cases mycobiont aggregates stained the culture medium around the mycobiont culture (figure 1B), in other cases dark brown substances crystalized in the contact zone of agar with mycobiont (figure 1D).The high-performance liquid chromatography (HPLC) showed that the P. carporrhizans aggregated on culture (both in CMA and 0.2G-MY) produced as a major compound confluentic acid and others from the acetate-polymalonate pathway (Table 1).However, thallus of Parmelina carporrhizans collected from the field contained lecanoric acid, atranorin, chloratranorin, ergosterol and depsides.
The P. quercina aggregated on culture (0.2G-MY) produced as major compound pulvinic acid (Mevalonic acid pathway) and others substances as 2'-O-Methylphysodic acid and Myelochonone C (Table 1 and Fig. 2).P. quercina thallus contained lecanoric acid, chloratranorin and aliphates (Table 1).Both P. carporrhizans and P. quercina secreted Myelochonone C in culture medium.Unfortunately, we disposed of limited P. quercina mycobiont aggregates, and lyophilized samples of P. quercina growing on CMA were broken in the travel from Madrid to Frankfurt, and thus we were unable to analyze, for this reason the data from P. quercina cultured in CMA is missing in Table 1.

Discussion
We found similar, almost the same chemosyndrome in Parmelina carporrhizans and Parmelina quercina in natural thalli as previously described.In addition to lecanoric acid, atranorin and chloratranorin, we found ergosterol in natural thallus of P. carporrhizans, which is part of fungal membrane and have antitumoral activity [27].However, we did not found gyrophoric acid that was reported at trace level by Clerc and Truong [28].In P. quercina thallus, in addition to the lecanoric acid and chloratranorin we found aliphates.While chemosyndrome was completely different between species in mycobiont cultures, both mycobiont cultures secreted myeloconone C to the media (see Table 1).Myeloconone C was described for first time in thallus of Myeloconis parva with the deduced formula (C14H10O7) [29].The myeloconones are phenalenones [30,31] found also in other lichen species as medullary pigments (Myeloconis [29]; Peltula [32] and Buellia [33]) but we reported here for first time the production and secretion of a myeloconone in mycobiont aposymbiotic culture.Cultures of both species produced and secreted the phenalenone myeloconone C to the culture media, which probably have allelopathic properties similar to those exhibited by plant phenalenones [34].Moreover, by the photosensitizer effect against oxygen singlet which has been produced several patents (US4803161A [35] US7253206B2, [36] ES 2357926B2 [37]).The secretion of myeloconone C by Parmelina mycobiont culture make more easy obtaining the substance.Further research can establish Parmelina mycobiont cultures as a myeloconone obtaining system.Further, the chemosyndrome within species and between the aposymbiotic culture and thallus were different, being different and more numerous the secondary metabolites of mycobiont culture than those produced in natural thalli (Table 1, Fig 2).Similar results of variation of chemosyndrome between thallus and culture were reported [19,25,38] arguing that the switch of biosynthetic pathway changed the chemosyndrome [38] by expression of different polyketide synthases [19].The chemosyndrome variation between thallus and mycobiont culture can be attributed to the absence of carbon sources provided by the photobiont or suboptimal environmental conditions producing stress.In resynthesis studies, the decreasing amount of substances in culture after photobiont contact was registered [25].Moreover, the importance of environmental conditions in chemosyndrome variation in culture was revealed by carbon source in culture media and simulated environmental stress [39] changing the expression of the polyketide synthase genes and chemosyndrome [19].However, the natural thallus and mycobiont culture can produce the same chemosyndrome [40] at least under certain culture conditions such as at variable temperatures [23,24,39].It has been seen that the age of the culture can change the chemosyndrome and can allow mycobiont to produce common substances present in lichen thallus for example atranorin, which was produced in culture only after five months and desiccation treatment in Parmotrema reticulatum [41].
Attending to major substances, Parmelina carporrhizans thalli produced lecanoric acid in natural condition while in aposymbiotic stage it produced confluentic acid.Both substances are related by the acetate-mevalonate pathway being a minor chemosyndrome variation.These are produced in the two culture media accompanied by almost the same chemosyndrome according to Stocker-Wörgötter et al. [40] but contrasting with other previous reports [41].Conversely, in Parmelina quercina the biosynthetic pathway of the major substance produced changes from the acetate-mevalonate pathway in natural thallus (Lecanoric acid) to shikimic acid pathway in aposymbiotic stage (pulvinic acid).
We do don't have a clear explanation of why two close species which produce the same chemosyndrome in natural thallus produced different chemosyndrome in culture.We can speculate that the mycobiont culture chemosyndrome could be very similar to the chemosyndrome of aposymbiotic pre-thallus stage in nature being related to adaptation to different habitats and different ecological valence.In this case make sense that Parmelina carporrhizans inhabiting more oceanic habitats is producing confluentic acid with biocide properties [42] very useful in humid places while P. quercina inhabiting continental drier habitat is producing pulvininic acid as major compound, which confers vantage by its photoprotective properties and DNA protection against oxidative stress [43] as an adaptation to drought and insolation.
Frequently the environmental conditions as carbon source, PH levels, temperature variation and stress in general modifies the chemosyndrome in mycobiont culture [19,23,24] however P. carporrhizans produced confluentic acid as major compound and practically the same chemosyndrome in both media assayed.How much is the relative weight of genetics and environment in determining the chemosyndrome substances of aposymbiotic mycobiont in laboratory culture and natural pre-thallus stage?Further studies are needed to unravel if we can use chemosyndrome variation in mycobiont culture of Parmelina to deduce adaptive strategies of aposymbiotic phase in nature or if we can produce in culture a set of different chemosyndromes by changing culture conditions and activating different polyketide synthases.

Isolation and culture
We isolated P. carporrhizans mycobiont from ascospores following the inverted Petri dish methods of Ahmadjian [44] Apothecia were mechanically cleaned and washed following the protocols established by Molina and Crespo [45] and attached to the inner side of inverted Petri dish lids with petroleum jelly.The plates contained Bold's basal medium (BBM, [46]) for spore germination [47].Twenty-five to thirty days after spore germination plurisporic mycelia were transferred to new plates on two different culture media: 0.2% glucose malt-yeast extract (0.2G-MY) according to Molina et al. [48] and cornmeal agar (CMA) following the manufacturer's instructions (Difco Laboratories, Detroit, MI, USA).Cultures were incubated at constant 20ºC in the dark [44] for 160 days.

DNA extraction and molecular identification
Prior to DNA extraction, we extracted secondary metabolites with acetone, and then crushed the samples with pestles in liquid nitrogen and extracted genomic DNA with the DNeasy Plant Kit (QIAGEN, Redwood City, California, USA) according to the manufacturer's instructions.To confirm the identity of the mycobiont cultures, we amplified the internal transcribed spacer (ITS) region of the nuclear rDNA from the axenic cultured tissues.The identity of the sequences and specimens were confirmed using the MegaBLAST search function in GenBank.ITS sequences were deposited in GenBank (accession numbers KM357892 and KM357893), and Parmelina carporrhizans genome was sequenced using Illumina from these samples [49].

High-performance liquid chromatography
Approximately 35 mg of pure mycobiont culture and agar pieces of 5 mm 2 were extracted in 0.5 ml of acetone for 60 min and the extract was freeze-dried and sent from Madrid to Frankfurt to be analyzed.Extracts were resuspended with 200 µl methanol at room temperature for 1 hour prior analysis.For HPLC, an Agilent Technologies 1260 quaternary system with incorporated degasser and a DAD was used.The detection of compounds was done using OpenLAB CDS ChemStation software (Agilent Technologies) by comparing spectra and retention time against a library of spectra from authentic metabolites.For more details see [50]

Figure 1 .
Figure 1.Plurisoporic mycobiont culture in axenic conditions.A) P. carporrhizans at 80 days, showing drops of phenolic nature, B) P. carporrhizans at 160 days showing agar media stained by myeloconone C, C) Parmelina quercina at 80 days showing drops of phenolic nature and D) Parmelina quercina at 160 days, * marks crystalized brown substance.Scale bar: 1mm

Table 1 .
Lichen substances and retention time of Parmelina carporrhizans and Parmelina quercina found in natural thalli and mycobiont culture.