Phylogenetic Studies and Metabolite Analysis of Sticta Species from Colombia and Chile by Ultra-High Performance Liquid Chromatography-High Resolution-Q-Orbitrap-Mass Spectrometry

Eleven species of lichens of the genus Sticta, ten of which were collected in Colombia (S. pseudosylvatica S. luteocyphellata S. cf. andina S. cf. hypoglabra, S. cordillerana, S. cf. gyalocarpa S. leucoblepharis, S. parahumboldtii S. impressula, S. ocaniensis) and one collected in Chile (S. lineariloba), were analyzed for the first time using hyphenated liquid chromatography with high-resolution mass spectrometry. In the metabolomic analysis, a total of 189 peaks were tentatively detected; the analyses were divided in five (5) groups of compounds comprising lipids, small phenolic compounds, saturated acids, terpenes, and typical phenolic lichen compounds such as depsides, depsidones and anthraquinones. The metabolome profiles of these eleven species are important since some compounds were identified as chemical markers for the fast identification of Sticta lichens for the first time. Finally, the usefulness of chemical compounds in comparison to traditional morphological traits to the study of ancestor–descendant relationships in the genus was assessed. Chemical and morphological consensus trees were not consistent with each other and recovered different relationships between taxa.


Introduction
Lichens constitute a mutualistic symbiosis with green algae and/or cyanobacteria [1], and in some cases present a tripartite symbiosis between different mycobionts and photobionts known as photosymbiodemas [2][3][4]. The genus Sticta (Schreb.) Ach. is the most species-diverse group of macrolichens in the family Lobariaceae, with about 120 species, and is characterized by a heteromeric thallus, with wide and rounded or elongated and

Results and Discussion
In this study, the metabolome profile of eleven species of Sticta lichens are reported for the first time. Lichen substances have gained considerable attention due to their potential health benefits and possible food nutraceutical or biotechnological applications [31]. Such compounds consist mostly of aliphatic and aromatic substances which proved to have biological and pharmacological activities compared to higher plants, in particular

Results and Discussion
In this study, the metabolome profile of eleven species of Sticta lichens are reported for the first time. Lichen substances have gained considerable attention due to their potential health benefits and possible food nutraceutical or biotechnological applications [31]. Such compounds consist mostly of aliphatic and aromatic substances which proved to have biological and pharmacological activities compared to higher plants, in particular several depsidones and depsides which proved to be antioxidant, cytotoxic and anti-inflammatory agents, among several other bioactivities reported from the genus [31][32][33][34][35]. Their identification by hyphenated mass spectrometry liquid chromatography techniques is highly important for the fast metabolite profiling of these important biodiverse organisms, and the possibility of finding biomarkers that could be of help for their identification.

Identification of Metabolites in 11 Lichen Species
In this study, eleven species of the genus Sticta were analyzed and Figure 2 shows the chromatograms for the species S. pseudosylvatica and S. lineariloba. In the metabolomic analysis, a total of 189 peaks were detected, 41 of which were unknown compounds, and the analyses were divided in five groups of compounds as explained below (Table 1).

Results and Discussion
In this study, the metabolome profile of eleven species of Sticta lichens are reported for the first time. Lichen substances have gained considerable attention due to their potential health benefits and possible food nutraceutical or biotechnological applications [31]. Such compounds consist mostly of aliphatic and aromatic substances which proved to have biological and pharmacological activities compared to higher plants, in particular several depsidones and depsides which proved to be antioxidant, cytotoxic and anti-inflammatory agents, among several other bioactivities reported from the genus [31][32][33][34][35]. Their identification by hyphenated mass spectrometry liquid chromatography techniques is highly important for the fast metabolite profiling of these important biodiverse organisms, and the possibility of finding biomarkers that could be of help for their identification.

Identification of Metabolites in 11 Lichen Species
In this study, eleven species of the genus Sticta were analyzed and Figure 2 shows the chromatograms for the species S. pseudosylvatica and S. lineariloba. In the metabolomic analysis, a total of 189 peaks were detected, 41 of which were unknown compounds, and the analyses were divided in five groups of compounds as explained below (Table 1).     Peak 1 was tentatively identified as gluconic acid (C 6 H 11 O 7 ), peak 3 as manitol, peak 4 as arabic acid, peak 5 as citric acid and peak 10 as its isomer isocitric acid (C 6 H 7 O 7 ), peak 7 as 4-ethyl-2-ethylisophthalic acid (C 10 H 9 O 4 ) with peak 2 as its isomer 2-ethylisophthalic acid, peak 15 as the derivative 2-hydroxyisophthalic acid, peak 17 was identified as 4-Odemethylglomellic acid (C 24 H 25 O 9 ), peak 30 as 1,5-pentanedicarboxylic acid (C 7 H 11 O 4 ) and peak 44 as 2,4-dicarboxy-3-hydroxy-5-methoxytoluene (C 10 [44], and finally, peak 187 was identified as the cytotoxic compound perlatolic acid [45], peak 188 as the antibacterial compound caperatic acid [46] and peak 189 as atranorin.

Terpenes
Peak 112 was identified as retigeric acid B (C 30 H 45 O 6 ), while peak 111 with an ion at m/z: 515.3025 was tentatively identified as a retigeric acid derivative (C 30 H 43 O 7 ).

Lipids
Oxylipins polyunsaturated fatty acids are an important dietary compounds, and can be found in edible fruits by HPLC orbitrap mass spectrometry [47] and also can be found in useful plants [48] and lichens [49]. In this study, several fatty acids including saturated fats and oxylipins were found using this technique in Sticta lichens. Peak 33, with a parent ion at m/z: 555.3047, was identified as decahydroxyoxopentacosanoic acid (C 25 H 47 O 13 ); peak 43, with a parent ion at m/z: 187.0977, was determined to be 4,5-dihydroxy-2-nonenoic acid (C 9 H 15 O 4 ); peak 72 as 12,13,15-trihydroxy-9-octadecenoic acid (C 18 ). In the same manner, peak 161 and 162 were attributed to pentadecatetraenoic acid and 9-hydroxyoctadecatrienoic acid, respectively. Finally, peak 171 was identified as trihydroxyheptacosa pentaenoic acid; peak 173 as hydroxytrioxodocosanoic acid; and peak 183 as dihydroxyicosahexaenoic acid (C 20 H 27 O 4 ).
In this study, we worked on 11 species of the genus Sticta from Colombia and Chile. It should be noted that the species were collected in different ecosystems and environmental conditions in South America. The analyses includes 189 compounds, 41 of which had not yet been identified, of which the most representative are gluconic acid (1), citric acid (5), 2-Ethylisophthalic acid (13), orsellinic acid (34), lecanoric acid (89), stictic acid (105), parietin (106), gyrophoric acid (123) and usnic acid (184) (Figure 3). It should be noted that none of the identified and unidentified compounds are present simultaneously in all 11 species.
Metabolites 2022, 12, x FOR PEER REVIEW 11 of 19 In this study, we worked on 11 species of the genus Sticta from Colombia and Chile. It should be noted that the species were collected in different ecosystems and environmental conditions in South America. The analyses includes 189 compounds, 41 of which had not yet been identified, of which the most representative are gluconic acid (1), citric acid (5), 2-Ethylisophthalic acid (13), orsellinic acid (34), lecanoric acid (89), stictic acid (105), parietin (106), gyrophoric acid (123) and usnic acid (184) (Figure 3). It should be noted that none of the identified and unidentified compounds are present simultaneously in all 11 species.
In general, maximum-parsimony strict consensus trees from morphological and chemical characters were not consistent with each other and did not recover the same relationship between taxa (Figure 4), whereas the morphological tree recovers most of the evolutionary relationships (positions in phylogeny) documented in the published molecular phylogeny of Colombian Sticta [50] and the chemical-compound tree mirrors the geographic clusters of collected samples exactly.
In general, maximum-parsimony strict consensus trees from morphological and chemical characters were not consistent with each other and did not recover the same relationship between taxa (Figure 4), whereas the morphological tree recovers most of the evolutionary relationships (positions in phylogeny) documented in the published molecular phylogeny of Colombian Sticta [50] and the chemical-compound tree mirrors the geographic clusters of collected samples exactly.   In the last two decades, the study of secondary metabolites in lichens has represented an input for the determination of specimens in different complex groups, through their intervention in taxonomic keys. These compounds are mostly aromatic derivatives such as depsides, depsidones, dibenzofurans, dibenzoquinones and usnic acid among others that derive from the biochemical pathways generated by malonic, mevalonic and shikimic acids [51]. The morphological data used in the present analysis of phylogenetic relationships within species of Sticta demonstrate the relevance of morphological traits in lichen taxonomy. Nevertheless, the chemical characters offer the possibility of an alternative comparison, independent from the morphology-based classification system [52]. In this study, the analysis of chemical traits recovered more geographic than ancestor-descendant relationships among taxa. These results enrich the discussion of the role of the local environment on lichen adaptation through the actions of natural selection on biochemical pathways.
In other studies, groupings based on chemical compounds such as the case of S. cf ocaniensis, S. cf. pseudolobaria, and S. canariensis, were consistent with the known molecular phylogeny of Sticta, as was the case with S. pulmonarioides and S. cf. weigelia [53]. For the genus Cetrelia, an assessment of the composition of secondary metabolites allowed for a confirmation of the presence of species only reported in America in Europe, as in the case of C. chicitae. Metabolite composition has also facilitated the confirmation of new species for the genus by chemical fingerprinting, which contrasts the phenotypic plasticity of some morphological characters used for identification [54]. In the genus Psoroma, an analysis of the distribution of secondary metabolites has validated the presence of chemical markers unique to the group, and the presumed description of subgenera by taxa heterogeneity, causing spatial segregation [55].
On the other hand, in the genus Cladonia, there is evidence of the use of chemotaxonomic methods to determine and differentiate phylogenetically related species (C. arbuscula, C. borealis, C. chlorophaea, C. coccifera, C. coniocraea, C. cornuta, C. fimbriata, C. mitis, C. monomorpha, C. pyxidate, C. rangiferina, C. stellaris, and C. stygia), which contain chemical markers exclusive to the group [56]. In this way, the morphological-anatomical data are complemented, and discriminatory characters are provided to distinguish the species. In the genus Blastenia, reduced chemotypes are also reported in some lineages with particular genetic characteristics and distribution [57].
Currently, the process of chemotaxonomic discrimination analysis in lichen groups requires reinforcement with complementary techniques, such as the use of pigments derived from anthraquinone-type compounds in specimens of the family Teloschistaceae (Pyrenodesmia sensu lato) [58] and optical-sensor profiles for metabolic profiling in species of the genera Cladonia, Stereocaulon, Lichina, Collema and Peltigera [59]. In addition, advances in analytical chemistry and mass spectrometry have allowed for a greater specificity in the elaboration of bioactive compounds profiles, which, together with modern DNAsequencing techniques and the extension of morphological descriptions as a "polyphasic approach", provide objectivity in the delimitation of lichen species [60,61]. However, due to the wide variation in lichen chemotypes, the use of new compounds such as fatty acids is proposed in chemotaxonomy and phylogeny analyses, and in building an understanding of molecular-complex communication and compound biosynthetic pathways [62].

Chemicals
Ultrapure water was obtained from a water purification system brand Millipore (Milli-Q Merck Millipore, Santiago, Chile). Analytical reagents were all purchased from Sigma Aldrich Co. (Santiago, Chile). Ethanol, Methanol, formic acid, acetone, and acetonitrile were of chromatographic grade for HPLC analysis. Analytical lichen standards (purity: 98% by HPLC) were purchased from Sigma-Aldrich Chemical Company (Santiago, Chile).

Preparation of the Sample for Analyses
Fresh samples were weighed and frozen for two days at −80 • C. Then, the samples were taken to a freeze-evaporation system (Model 7670541 FreeZone 2.5 Liter Labconco Freeze Dry Systems) and all the water contained in the original product was removed by freeze-evaporation cycles. A total of 3 g of each dried lichen was macerated with methanol (3 times, 30 mL each time, 3 days/extraction). The solutions were concentrated to obtain 11 mg of extract from S. pseudosylvatica; 9 mg S. luteocyphellata; 14 mg S. cf. andina; 12 mg S. cf. hypoglabra; 10 mg S. cordillerana; 9 mg S. cf. gyalocarpa; 13 mg S. leucoblepharis; 13 mg S. parahumboldtii; 8 mg S. impressula and 9 mg S. ocaniensis, respectively. Then, the lichen extracts were processed individually for HPLC-MS analyses (redissolved in methanol at a concentration of 1 mg/mL for the analyses).

Instrument
A Thermo Scientific Ultimate 3000 UHPLC with a PDA (photodiode array detector) detector controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA) in conjunction with a Thermo high resolution Q-Exactive focus mass spectrometer (Thermo, Bremen, Germany) were used for analysis. The chromatographic system was coupled to the MS using a type II heated electrospray ionization source. Nitrogen obtained (purity >99.999%) from a nitrogen generator (Genius NM32LA, Peak Scientific, Billerica, MA, USA) was employed as both the collision and damping gas. Mass calibration for Orbitrap was performed once a day, in both negative and positive modes, to ensure working mass 5 ppm of accuracy. Sodium dodecyl sulfate, caffeine, N-butylamine, buspirone hydrochloride, and taurocholic acid sodium salt (Sigma Aldrich, Saint Louis, MO, USA) plus Ultramark 1621 (Alpha Aezar, Stevensville, MI, USA), a phosphazine fluorinated solution, was the standard mixture used to calibrate the mass spectrometer. These compounds were dissolved in a mixture of acetic acid, acetonitrile, water, and methanol (Merck, Darmstadt, Germany) and were infused using a Chemyx Fusion 100 syringe pump, XCalibur 2.3 software and Trace Finder 3.2 (Thermo Fisher Scientific, San José, CA, USA), which were used for control and data processing. Q Exactive 2.0 SP 2 from Thermo Fisher Scientific was used to control the mass spectrometer. The lichens extracts were individually redissolved in methanol (at a concentration of 1 mg/mL), each solution was filtered (PTFE filter, Merck) and then 10 microliters were injected in the UHPLC instrument for UHPLC-MS analysis. XCalibur 2.3 software (Thermo Fisher Scientific, Bremen, Germany) and Trace Finder 3.2 (Thermo Fisher Scientific, San José, CA, USA) were used for UHPLC control and data processing, respectively. Q Exactive 2.0 SP 2 from Thermo Fisher Scientific was used to control the mass spectrometer.

MS Parameters
The HESI parameters were as follows: sheath gas-flow rate of 75 units; aux. gas unit flow rate of 20; capillary temperature of 400 • C; aux gas heater temperature of 500 • C; spray voltage of 2500 V (for ESI−); and S lens RF level of 30. Full scan data in both the positive and negative modes were acquired at a resolving power of 70,000 FWHM (full width half maximum) at m/z 200. For the compounds of interest, a scan range of m/z 100-1000 was chosen; the automatic gain control (AGC) was set at 3 × 10 6 and the injection time set to 200 ms. Scan-rate was set at 2 scans s −1 . External calibration was performed using a calibration solution in the positive and negative modes. For confirmation purposes, a targeted MS/MS analysis was performed using the mass inclusion list, with a 30 s time window, with the Orbitrap spectrometer operating both in the positive and negative mode at 17,500 FWHM (m/z 200). The AGC target was set to 2 × 10 5 , with the max. injection time of 20 ms. The precursor ions were filtered by the quadrupole, which operates at an isolation window of m/z 2. The fore vacuum, high vacuum and ultrahigh vacuum were maintained at approximately 2 mbar, from 10 5 and below 10 10 mbar, respectively. Collision energy (HCD cell) was operated at 30 kv. Detection was based on calculated exact mass and on retention time of target compounds, as shown in Table 1. The mass tolerance window was set to 5 ppm for the two modes for most compounds.

Similarity and Phylogenetic Analyses
We carried out a phylogenetic study to analyze if the chemical compounds found in Sticta specimens recovered a phylogenetic signal consistent with the current taxonomic relationships in the genera. First, we built two character-state matrices. One matrix included the 189 chemical compounds reported in this paper, and the other encompassed 16 morphological traits (Suppl. Tables S1 and S2). Then, the compounds were coded as binary characters (presence/absence), whereas morphological traits were coded as multistate characters. All character states receive the same weight and were set as unordered. Next, exploratory Neighbor-Joining distance trees were built. After that, we used a maximum parsimony phylogenetic approach to perform an exhaustive search of optimal trees. A maximum of 100 trees were retained after evaluating ca. 34 million trees per matrix. Trees were unrooted given the absence of descriptions of chemical compounds and morphological traits for potential outgroups. Then, the consistency and retention indexes were calculated. Finally, we obtained the strict consensus of optimal trees. All the procedures were performed in PAUP 4a168 for mac.

Conclusions
Eleven lichens of the Sticta genera from two different country zones were phytochemically investigated. More scientific data on chemistry is presented for these interesting lichens that can significantly increase the knowledge and potential for sustainable applications and industrial interest. This valuable natural-product biomass has potential applications in food, medicine, biotechnology, pharmaceuticals, and cosmetics, with many possible applications from food-conserving agents to anticancer biomaterials. The morphological data used in the present analysis of phylogenetic relationships within genus of Sticta demonstrate the relevance of morphological traits in lichen taxonomy. Nevertheless, chemical characters for chemotaxonomic studies offer the possibility of an alternative comparison, independent from the morphology-based classification system. In this study, chemical traits' analysis recovered more geographic than ancestor-descendant relationships among taxa, and these results enriched the discussion of the role of the local environment on lichen adaptation through natural selection acting on biochemical pathways.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/metabo12020156/s1, Table S1: Description of character and character states used in the present study of Sticta, Table S2: Character state matrix of morphological traits used to recover phylogenetic relationships in species of Sticta.