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.

1. 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 truncated lobes, sometimes overlapping and rarely polyphilic, with smooth upper surface or with light ribs that may carry isidia or soralia, and the presence of cyphelas on the ventral surface of variable sizes dispersed in the tomentum [5,6,7,8,9,10,11]. In South America, the genus is found in Andean, sub-Andean, and moorland ecosystems, and develops in substrates of bark, soil, wood and rocks [5,6,7,8,9,10,11,12].

Chemical studies of the genus Sticta are scarce. In Sticta and other genera, most compounds are of fungal origin and their chemical variety is related to the environmental conditions in which the species develops [13,14], which is observed in this work. From the earliest reports found for this genus, triterpenes were identified in the species S. billardierii, S. coronata, S. colensoi, and S. favicans [15,16]. The latest reports on species such as S. fuliginosa, S. weigelia, S. caulescens and S. santessonii show the presence of substances such as 7β-acetoxy-22-hydroxy hopane usnic acid, ursolic acid, ergosterol peroxide and β-sitosterol [17]. The S. nylanderiana ethyl-3-formyl-2,4-dihydroxy-5,6-dimethylbenzoate, methyl-2,4-dihydroxy-3,5,6-trimethylbenzoate, orsellinic acid, methyl orsellinate, ethyl orsellinate, lecanoric acid, 4-O-methyl gyrophoric acid and retigeric acid B compounds were isolated and identified [18]. In an unidentified species of the genus Sticta, the compounds stictamides A-C were isolated and identified. In addition, stictamide A acts as an inhibitor of the MMP12 protease (metallopeptidase 12) [19].

Currently, for the identification and elucidation of metabolites in complex extracts, the technique of ultra-high performance liquid chromatography-diode array detection (UHPLC-PDA) coupled to an electrospray ionization tandem mass spectrometer (ESI–MS–MS) [20,21,22,23,24,25,26] is used. The Q-Exactive Focus is a hybrid high-resolution mass spectrometer used to detect and quantify small organic compounds via high-resolution accurate mass spectrometry. This machine combines UHPLC-PDA (ultrahigh pressure liquid chromatography with photodiode array) with an orbitrap, a quadrupole (Q) and a high-resolution collision cell (HRCD), which allows for high resolution diagnostic untargeted metabolomics and accurate determination of fragments [15,16,17,18,19,20,21,22,23,24,25,26,27,28]. This technique has been useful for the chemical study of several lichens of the genera Ramalina, Parmotrema and Usnea [23,24,25,26,27,28,29,30] conducted by our research group and others.

In this research, we analyzed the phytochemical profile of several species of the Sticta genus collected in different geographical regions of Colombia and Chile, based on UHPLC-DAD coupled with high-resolution electrospray ionization tandem mass spectrometry (ESI-MS-MS) for the first time. The eleven (11) lichens of the genus Sticta analyzed were S. pseudosylvatica S. luteocyphellata S. cf. andina S. cf. hypoglabra, S. cordillerana, S. cf. gyalocarpa S. leucoblepharis, S. parahumboldtii S. impressula, S. ocaniensis and S. Lineariloba (Figure 1). Based on the quantity and variety of chemical compounds found in the studied specimens, we complemented the chemical characterization with a maximum parsimony analysis based on the hypothesis that phytochemical compounds in lichens may show a phylogenetic signal, thus proving utility for chemotaxonomy.

Figure 1

(A) S. pseudosylvatica (Colombia); (B) S. lineariloba (Chile).

2. 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.

2.1. 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).

Figure 2

Chromatogram of the species S. pseudosylvatica and S. lineariloba.

Table 1

Identification of metabolites in 11 species of the genus Sticta from Colombia and Chile.

Peak	Tentative Identification	[M-H]-	Retention Time (min)	Theoretical Mass (m/z)	Measured Mass (m/z)	Accuracy (ppm)	Metabolite Type **	MS Ions (m/z)	Lichen Species *
1	Gluconic Acid	C6H11O7	1.28	195.0509	195.0505	2.0	Acid	165.0401;	2; 3; 7; 9; 10; 11
2	Unknown	C11H5O5N	1.28	231.0184	231.0195	−4.8	-	---	6
3	Manitol	C6H13O6	1.31	181.0712	181.0714	−1.1	Carbohidrate	181.0717; 163.0606	7; 9; 10; 11
4	Arabic acid	C5H9O6	1.29	165.0399	165.0401	−1.2	Acid	147.0293; 113.0237; 129.0196	6
5	Citric Acid	C6H7O7	1.38	191.0196	191.0192	2.0	Acid	111.0080	1; 2; 3; 4; 5; 6; 7; 8; 9; 11
6	Unknown	C15H5O3N2	1.38	261.0289	261.0300	−4.2	--	---	1; 7; 8; 10
7	4-ethyl-2-Ethylisophthalic acid	C10H9O4	1.44	193.0504	193.0501	1.6	A	133.0288	1; 2; 3; 4; 6
8	Unknown	C8H10O5N	1.56	200.0563	200.0559	2.0	-	--	1; 3; 4; 6
9	Unknown	C15H5O3N2	1.64	261.0247	261.0278	−11.8	-	---	7; 8; 10
10	Isocitric Acid	C6H7O7	1.77	191.0195	191.0192	1.6	Acid	111.0079;	1; 2; 3; 4; 6; 7
11	Unknown	C7H9O7	1.91	205.0352	205.0348	2.0	-	187.0245; 173.0087; 121.1131	2
12	Unknown	C7H7O6	2.24	187.0246	187.0243	1.6	-	125.0237; 137.2503;	2
13	2-Ethylisophthalic acid	C10H9O4	2.85	193.0505	193.0501	2.0	A	161.0240; 133.0290	1; 2; 3; 4; 6; 7; 8; 9; 10
14	Trihydroxy benzaledehyde	C7H5O4	4.81	153.0188	153.0189	−0.7	A	137.0238	1; 2; 3; 4
15	2-Hydroxyisophthalic acid	C8H5O5	7.63	181.0137	181.0141	−2.2	A	137.0238	4
16	2,4-dihydroxy benzaldehyde	C7H5O3	8.02	137.0238	137.0239	−0.7	A	121.0289	1; 2; 3; 4
17	4-O-Demethylglomellic acid	C24H25O9	8.65	457.1476	457.1499	−5.0	d	---	5; 8
18	Unknow	C22H23O7	8.77	399.1444	399.1413	7.8	-	---	8
19	Unknow	C7H11O5	8.86	175.0606	175.0611	−2.9	-	---	5
20	Grayanic acid	C23H25O7	8.91	413.1600	413.1569	7.5	d	181.06503	8
21	Unknown	C13H16O7N	8.98	298.0940	298.0927	4.4	-	175.0609; 283.0210; 202.0696;	5
22	Unknown	C11H9O7	9.33	253.0361	253.0348	5.1	-	137.0603; 181,0505; 249.3808; 173.4203;	5
23	Atranol	C8H7O3	9.44	151.0395	151.0396	−0.7	A	123.0445; 135.0445	3
24	Unknown	C24H23 O8	9.48	439.1371	439.1393	−5.0	-	151.0397; 105.0948; 121.4871;	5
25	5,7-Dihydroxy-6-methylphthalide	C9H7O4	9.64	179.0344	179.0347	−1.6	A	135.0444; 107.0494	4; 5; 7; 8; 10
26	Unknown	C16H15O10	9.73	367.0665	367.0639	7.1	-	---	1; 4
27	Unknown	C30H27O13N	10.03	609.1475	609.1482	−1.1	-	---	1; 4; 6
28	Unknow	C18H15O4	10.31	295.0970	295.0935	11.8	-	---	9
29	Unknow	C17H13O6	11.31	313.0712	313.0724	−3.8	--	---	1; 2; 3; 4; 5
30	1,5-Pentanedicarboxylic acid	C7H11O4	10.56	159.0657	159.0660	−1.9	L	115.0758	5
31	Unknown	C29H25O13N	10.54	595.1316	595.1326	−1.7	-	---	3
32	Didechlorolecideoidin	C17 H13O7	10.64	329.0661	329.0676	−4.6	D	209.0456; 285.0776; 151.0396; 179.0347; 123.0443	3
33	Decahydroxyoxopentacosanoic acid	C25H47O13	10.71	555.3017	555.3047	−5.4	L	---	8
34	Orsellinic acid	C8H7O4	11.04	167.0347	167.0344	1.8	A	123.0442	2; 3; 4; 5; 6
35	Unknow	C21H19O12	11.04	463.0877	463.0893	−3.4	-	---	3
36	Unknow	C10H9O5	11.07	209.0450	209.0458	−3.8	-	---	5; 6
37	Nor 8′-methylconstictic acid	C21H19O11	11.11	447.0927	447.0942	−3.4	d	209.0455	2
38	Unknow	C17H13O6	11.20	313.0712	313.0720	−2.5	-	---	7
39	Metil-2,6-dihidroxibenzoate	C8H7O4	11.21	167.0344	167.0346	−1.1	A	109.0287; 137.0238	9
40	Hypostictic acid isomer	C19H15O8	11.36	371.0778	371.0782	−1.1	D	195.0665; 327.0885; 341.0679; 179.0347	1; 3; 4; 7
41	Unknow	C19H16O9 N	11.69	402.0825	402.0841	−3.98	-	---	11
42	Fumarprotocetraric acid derivative	C17H11O6	11,70	311.0556	311.0564	−2.5	d	---	7; 10
43	4,5-Dihydroxy-2-nonenoic acid	C9H15O4	12.11	187.0974	187.0977	−1.6	L	171.1025; 143.1072	1; 2; 3; 4; 9; 10
44	2,4-Dicarboxy-3-hydroxy-5-methoxytoluene	C10H9O6	12.14	225.0407	225.0399	3.5	A	181.0503; 167.0346; 149.0240	4
45	Unknown	C17H13O6	12.16	313.0724	313.0727	−1.0	-	---	4
46	Unknown	C18H15O7	12.37	343.0818	343.0826	−2.3	-	---	7; 9
47	Unknown	C21H17O12	12.40	461.0737	461.0720	3.7	-	---	6
48	Unknown	C20H17O8	12.52	385.0939	385.0923	4.1	-	---	1; 2; 3
49	Unknow	C10H9O4	12.81	193.0501	193.0502	−0.5	-	---	1; 2; 3; 4; 5; 6; 7; 8; 9; 10
50	Unknow	C27H45O6	13.04	465.3216	465.3231	−3.2	-	---	3
51	2,4-dihydroxy benzaldehyde	C7H5O3	13.07	137.0237	137.0239	−1.5	A	121.0288	5
52	Consalizinic acid derivative I	C19H13O11	13.36	417.0458	417.0474	−3.8	D	373.0573; 387.0373; 225.0406, 177.0193	11
53	4-Ethoxy-3-formyl-2-hydroxy-6-methylbenzoic acid	C11H11O5	13.41	223.0614	223.0606	3.6	A	177.0190; 133.0296;	1; 2; 3; 4; 7; 9; 10
54	Unknow	C20H15O8	13.47	383.0767	383.0781	−3.6	-	---	1
55	Consalizinic acid derivative II	C20H17O11	13,47	433.0771	433.0787	−3.7	D	401.0524; 417.0474; 373.0574	11
56	Cynodontin or Citreorosein isomer,	C15H9O6	13.71	285.0399	285.0410	−3.9	Anthraquinone	151.0396; 137.0237	2
57	Consalizinic acid derivative I isomer	C19H13O11	13.78	417.0458	417.0474	−3.84	D	373.0573; 343.0467, 77.0190; 401.0523	10
58	Unknow	C30H47O7	13.79	519.3322	519.3337	−2.9	-	---	3
59	1,4,5,6,8-Pentahydroxy-3-ethylanthraquinone	C15H9O7	13.87	301.0348	301.0361	−4.3	Anthraquinone	---	4
60	Unknow	C19H15O4	13.87	307.0970	307.0939	10.0	-	---	8
61	Unknow	C14H13O7	13.99	293.0661	293.0674	−4.4	-	---	3
62	Haemathamnolic acid isomer	C19H15O10	14.18	403.0665	403.0681	−3.97	D	359.0781; 371.0414; 209.0455	11
63	Fumarprotocetraric acid derivative	C17H11O6	14.78	311.0567	311.0569	−0.6	d	---	1; 2; 3; 7; 10
64	Constictic acid	C19H13O10	14.61	401.0509	401.0528	−4.74	D	357.0625; 313.0726; 283.0619; 255.0670; 121.0289	11
65	Hypostictic acid isomer	C19H15O8	14.98	371.0781	371.0767	3.8	D	327.0883; 195.0664; 179.0347	5
66	Thelephoric acid	C18H7O8	14.81	351.0141	351.0154	−3.7	Terphenylquinones	---	3
67	Methylstictic acid	C20H15O9	15.26	399.0716	399.0728	−3.0	D	371.0779; 193.0504	2; 7
68	Nor 8′-metilconstictic acid	C21H19O11	15.28	447.0927	447.0944	−3.80	D	401.0524; 209.0455	11
69	Protocetraric acid	C18H13O9	17.17	373.0560	373.0574	−3.75	D	355.0468; 329.0674; 311.0568; 227.0352; 267.0669; 285.0777	11
70	Hypoconstictic acid	C19H15O9	17.27	387.0716	387.0729	−3.4	D	267.0673; 311.0552; 149.0238; 343.0827;167.0345	3
71	Unknow	C14H13O6	17.84	277.0712	277.0724	−4.3	-	--	3; 6
72	12,13,15-Trihydroxy-9-octadecenoic acid	C18H15O5	18.11	329.2328	329.2340	−3.6	L	285.1716;	2; 6
73	Unknow	C20H13O8	18.19	381.0610	381.0626	−4.2	-	---	3; 4
74	Salazinic acid	C18H11O10	18.21	387.0352	387.0368	−4.13	D	343.0468; 269.0458; 241.0507; 325.0365; 299.0569	11
75	Unknown	C10H9O4	18.45	193.0505	193.0501	2.1	-	---	3; 4
76	Unknown	C30H47O7	18.61	519.3322	519.3319	0.6	-	--	3
77	Menegazziaic acid	C18H13O9	18.79	373.0560	373.0575	−4.0	D	311,0570; 255.0666; 329.0679	3
78	Norstictic acid	C18H11O9	18.86	371.0403	371.0417	−3.8	D	327.0526; 151.0396; 123.0444;	3
79	Unknow	C22H19O10	18.88	443.0978	443.0996	−4.1	-	---	3
80	Physodalic acid	C20H15O10	18.99	415.0665	415.0681	−3.85	D	359.0417; 315.0520; 343.0832; 387,0367; 373.0573; 401.0525	11
81	Unknow	C26H19O10	19.00	491.0978	491.0997	−3.9	-	---	3
82	Derivative methyl 8-hydroxy-4-0-demethylbarbatate	C19H19O9	19.05	391.1045	391.1029	4.1	d	359.0788	3
83	12,13,15-Trihydroxy-9-octadecenoic acid	C18H33O5	19.05	329.2328	329.2336	−2.4299	L	---	7
84	Haemoventosin	C15H11O7	19.16	303,0519	303.0505	4.7	Naphthaquinone	259.0619; 231.0667; 189.0560;	3
85	α-acetilconstictic acid derivative I	C21H17O11	19.22	445.0771	445.0786	−3.3	D	415.0680; 371.0780; 427.0676; 343.0830;193.0504, 401.0522	11
86	Conhypoprotocetraric acid or Convirensic acid	C18H15O8	19.25	359.0781	359.0767	3.9	D	344.0545; 302.0442	3
87	4-O-dimethylbaemycesic acid	C18H15O8	19.27	359.0781	359.0767	3.9	d	181.0714; 163.0397; 137.0236	1; 2; 3;5; 6
88	Orsellinic acid Isomer	C8H7O4	19.45	167.0344	167.0346	−1.1974	A	123.0440; 149.0235	9; 10
89	Lecanoric acid	C16H13O7	19.51	317.0661	317.0671	0.6	d	167.0345; 123.0443; 149.0238;	1; 2; 3; 4; 5; 6
90	Constictic acid isomer	C19H13O10	19.56	401.0509	401.0524	−3.74	D	357,0626; 313.0726; 343.0831; 255.0622	11
91	Pentahydroxytetracosanoic acid	C24H47O7	19.67	447.3322	447.3336	−3.1	L	---	1; 3; 7; 9; 10
92	2-Methyl-5-hydroxy-6-hydroxymethyl-7-Methoxychromone	C12H11O5	19.73	235.0606	235.0615	−3.8	C	181.0504	3
93	Unknown	C20H17O8	19.79	385.0939	385.0923	4.1	-	---	1; 4; 5; 10
94	Heptahydroxytrioxooctadecanoic acid	C18H29O12	19.86	437.1664	437.1645	4.3	L	---	1; 4; 5; 6; 7; 9; 10
95	5,7-Dihydroxy-6-methylphthalide derivative	C9H7O3	19.88	163.0395	163.0392	1.8	A	119.0492	10
96	Criptostictic acid derivative	C18H11O8	20.04	355.0454	355.0462	−2.2	D	133.0288; 239.0715; 311.0572; 179.0345;	7
97	Unknow	C18H17O6	20.08	329.1025	329.1032	−2.1	-	---	9
98	Unknow	C20H15O8	20.16	383.0767	383.0775	−2.0	-	---	9; 10
99	Unknow	C21H19O9	20.18	415.1045	415.1029	3.9	-	---	3; 5; 9; 10
100	Unknow	C28H23O11	20.12	535.1240	535.1257	−3.1	-	---	3
101	Unknow	C15H13O3	20.13	241.0872	241.0874	−0.8	-	----	1; 2
102	Heptahydroxytetraoxoicosanoic acid	C20H31O13	21.19	479.1765	479.1746	3.9	L	---	7
103	Tetrahydroxytricosanoic acid	C23H45O6	20.26	417.3232	417.3216	3.9	L	403.3073	1; 3; 4; 7
104	Tetrahydroxytrioxoundecanoic acid	C11H15O9	20.30	291.0716	291.0699	5.8	L	---	8
105	Stictic acid	C19H13O9	20.34	385.0560	385.0576	−4.1	D	341.0674; 357.0622; 297.0774; 313.0721; 193.0504; 269.0826	11
106	Parietin	C16H11O5	20.39	283.0606	283.0617	−3.9	Antraquinone	179.0345	1; 2; 6
107	Unknow	C24H47O11N2	20.39	539.3157	539.3180	−4.3	-	---	3
108	Evernic acid isomer	C17H15O7	20.46	331.0818	331.0830	−3.6	d	167.0347; 123.0447; 149.0240	1
109	Hypoconstictic acid	C19H15O9	20.50	387.0716	387.0732	−4.1	D	149.0238; 343.0836; 167.0345	4
110	Cryptostictic acid	C19H15O9	20.50	387.0716	387.0725	−2.3	D	267,0661; 343,0825; 311.05067; 239.0710	7; 8
111	Retigeric acid derivative	C30H43O7	20.51	515.3009	515.3025	−3.1	Triterpene	---	3
112	Retigeric acid B	C30H45O6	20.56	501.3216	501.3236	−4.0	Triterpene	---	3
113	Salazinic acid isomer	C18H11O10	20.58	387.0352	387.0368	−4.13	D	343.0468; 299.0565	11
114	Unknown	C23H22O10N	20.68	472.1244	472.1259	−3.2	-	-----	6
115	9,10-dihydroxyoctadecatrienoic acid	C18H29O4	20.69	309.2081	309.2066	4.9	L	291.1975	1; 2; 4
116	Unknow	C17H13O6	20.84	313.0712	313.0727	−4.8	-	----	1; 2; 6
117	Pulvinic acid derivative I	C18H11O6	20.97	323.0556	323.0556	0.0	Pulvinic acid y derivates	133.0286; 117.0335	10
118	9,10,12 Trihydroxytriacontaheptaenoic acid	C30H45O5	20.99	485.3284	485.3267	3.5	L	---	3
119	4-0-Demethylbarbatic acid	C18H17O7	20.99	345.0974	345.0989	−4.3	d	181.0505; 163.0396; 137.0603	4
120	Unknow	C24H23O10N	21.02	485.1322	485.1319	0.6	-	---	1; 5
121	Methyl orsellinate	C9H9O4	21.05	181.0502	181.0501	0.5	A	163.0389	1
122	Heptahydroxyetraoxoicosanoic acid	C20H31O13	21.17	479.1752	479.1765	−2.7	L	---	1; 2; 3; 4; 5; 6; 9
123	Gyrophoric Acid	C24H19O10	21.25	467.0991	467.0978	2.8	d	167.0346; 317.0673; 123.0445; 149.0238;	1; 3; 4; 6; 11
124	Galbinic acid	C20H13O11	21.27	429.0458	429.0474	−3.73	D	403.0681; 371.0417; 401.0524; 327.0518; 149.0239	11
125	Hyposalazinic acid	C18H13O8	21.28	357.0610	357.0623	−3.6	D	313.0723; 135.0444; 179.0348	1
126	Hydroxytetracosapentaenoic acid	C24H37O3	21.42	373.2743	373.2743	0.0	L	---	10
127	Orsellinic acid isomer	C8H7O4	21.47	167.0347	167.0344	1.8	A	149.0239; 123.0443	1; 4; 6
128	Dihydroxyoctadecenoic acid	C18H33O4	21.48	313.2390	313.2395	−1.6	L	----	2; 5; 6
129	Norstictic acid	C18H11O9	21.64	371.0403	371.0417	−3.77	D	27.0517; 227.0716; 151.0390; 243.0297	11
130	Dihydroxyoctadec-6-enoic acid	C18H33O4	21.58	313.2379	313.2379	0.0	L	---	10
131	Loxodinol isomer	C25H29O9	21.64	473.1812	473.1818	−1.2	DE	429.1919	9
132	EthyI 2,4-dihydroxy-6-n-nonylbenzoate	C18H27O4	21.65	307.1909	307.1922	−4.2	A	263.1659	1; 2; 3; 4
133	Evernic Acid	C17H15O7	21.81	331.0828	331.0818	3.0	d	167.0345; 123.0444; 149,0238;	1; 2; 3; 4; 5
134	Protocetraric acid Isomer	C18H13O9	21.85	373.0560	373.0573	−3.48	D	355.0460; 329.0674; 285.0780, 311.0567; 255.,0672	11
135	Unknown	C22H22O8N	21.83	428.1360	428.1345	3.5	-	----	6
136	Strepsilin	C15H9O5	21.89	269.0450	269.0462	−4.5	DBF	225.0554	2; 5
137	Unknown	C30H29O4	21.97	453.2066	453.2061	1.1	-	---	3
138	Unknown	C18H11O6	22.01	323.0556	323.0570	−4.33	-	---	11
139	Hexahydroxytrioxooctacosatrienoic acid	C28H43O11	22.12	555.2805	555.2841	−6.4832	L	---	9
140	Nonahydroxyoctacosatetraenoic acid	C28 H47O11	22.26	559.3124	559.3132	−1.4	L	---	2
141	Unknow	C28H41O9N2	22.26	549.2849	549.2812	6.8	-	---	3
142	Norsolorinic acid	C20H17O7	22.44	369.0974	369.0989	−4.06	-	---	11
143	Unknow	C25H33O13	22.46	541.1921	541.1909	2.2	-	---	1; 2; 3; 6; 10
144	Hydroxytetracosapentaenoic acid derivative	C24H37O3	22.61	373.2743	373.2741	0.5	-	---	10
145	Hydroxytrioxotricosanoic acid	C23H39O6	22.53	411.2747	411.2757	−2.4	L	---	8
146	Squamatic acid	C19H17O9	22.89	389.0873	389.0886	−3.3	d	343.0836; 163.0396; 193.0139; 149.0238; 121.0286	1; 3; 4
147	Picrolichenic acid	C25H29O7	22.72	441.1913	441.1926	−3.0	Depsones	---	1
148	Heptahydroxydioxohexacosanoic acid	C26H47O11	22.76	535.3118	535.3134	−3.0	L	---	6
149	Unknow	C30H45O4	22.83	469.3319	469.3335	−3.4	-	---	3
150	2,2′-Di-O-methylanziaic acid	C26H33O7	22.85	457.2226	457.2244	−3.9	d	413.2345;	4
151	Dihydroxytetracosahexaenoic acid	C24H35O4	22.85	387.2535	387.2552	−4.4	L	---	5
152	Hydroxyoctadecadienoic acid	C18H31O3	22.90	295.2273	295.2273	0.0	L	---	10
153	Orsellinic acid Isomer	C8H7O4	22.92	167.0344	167.0348	−2.3	A	149.0240; 123.0445	11
154	Pulvinic acid	C18H11O5	22.98	307.0606	307.0613	−2.2	Pulvinic acid y derivates	117.0338; 263.0713	9
155	4-0-Demethylbarbatic acid	C18H17O7	23.02	345.0974	345.0986	−3.5	d	123.0443; 137.0237; 181.0502	1
156	Psoromic acid	C18H13O8	23.06	357.0610	357.0626	−4.4	D	313.0726; 181.0502; 179.0347; 327.0520; 269.0826; 285.0776	11
157	Methylgyrophoric acid	C25H21O10	23.15	481.1135	481.1147	−2.5	d	149.0238; 123.0442; 167.0346; 317.0671	1; 4
158	Evernic acid isomer	C17H15O7	23.22	331.0818	331.0832	−4.2	d	149.0239; 123.0443; 167.0346; 105.0337	11
159	Skyrin	C30H17O10	23.28	537.0822	537.0840	−3.4	Anthraquinones	----	3
160	Angardianic acid	C19H35O4	23.36	327.2543	327.2547	−1.2	Acids	283.2649; 309.2081	2; 4
161	Pentadecatetraenoic acid	C15H21O2	23.38	233.1542	233.1545	−1.2	L	---	9; 10
162	9-hydroxyoctadecatrienoic acid	C18 H29O3	23.45	293.2117	293.2130	−4.4	L	277.2180	6
163	Unknow	C18H15O7	23.53	343.0818	343.0824	−1.7	-	---	9
164	Pulvinic acid derivative II	C19H13O5	23.68	321.0763	321.0770	−2.1	Pulvinic acid y derivates	117.0337	9; 10; 11
165	Pulvinic acid	C18H11O5	23.77	307.0606	307.0620	−4.5	Pulvinic acid y derivates	263.0720; 117.0339	11
166	Furfuric acid isomer	C28H23O12	23.82	551.1190	551.1197	−1.2	D	371.0784; 193.0504; 179.0347; 207.0297; 193.0504	8
167	Unknow	C26H47O5N2	23.82	467.3485	467.3492	−3,9	-	---	3
168	Unknow	C30H27O6	23.89	483.1808	483.1820	−2,5	-	---	1
169	Unknow	C30H25O6	24.01	481.1651	481.1663	−2,5	-	---	1
170	Unknow	C15H13O3	24.02	241.0872	241.0872	0,0	-	---	1
171	Trihydroxyheptacosa pentaenoic acid	C27H43O5	24.05	447.3110	447.3127	−3.8	L	---	8
172	Barbatic Acid	C19H19O7	24.26	359.1141	359.1131	2.8	d	137.0603; 163.0396; 181.0509	1; 4
173	Hydroxytrioxodocosanoic acid	C22H37O6	24.29	397.2590	397.2601	−2.8	L	---	8
174	Thamnolic acid isomer	C19H15O11	24.41	419.0614	419.0630	−3.8	d	375.0730; 167.0344; 209.0455; 181.0503	11
175	Orsenillic acid derivated II	C8H7O4	24.73	167.0344	167.0347	−1.80	-	149.0239; 1230444;	11
176	Unknow	C26H33O8	24.73	473.2190	473.2175	3.2	-	---	2
177	Lobaric acid	C25H27O8	24.81	455.1706	455.1718	−2.6	D	411.1824; 367.1811	1
178	Unknow	C22H27O7	24.97	403.1770	403.1757	3.2	-		2
179	Unknow	C30H41O8	25.27	529.2819	529.2801	3.4	-	---	4
180	Hypothamnolic acid	C19H17O10	25.49	405.0822	405.0832	−2.5	d	209.0456; 181.0499	1
181	Unknow	C25H11O7	25.53	423.0505	423.0497	1.9	-	---	4
182	Pulvinic acid derivative III	C19H13O5	25.67	321.0763	321.0777	−4.3	Pulvinic acid y derivates	117.0338	11
183	Dihydroxyicosahexaenoic acid	C20H27O4	26.02	331.1909	331.1925	−4.8	L	-----	11
184	Usnic acid	C18H15O7	26.05	343.0818	343.0831	−3.8	DBF	231.0658; 328.0585; 259.0604	3; 4; 6; 7; 8
185	Nephromopsic acid orRoccellaric acid	C19H33O4	26.32	325.2392	325.2379	4.0	Acids	281.2494	3; 4
186	Unknow	C28H25O5N	26.87	455.1733	455.1723	2.20	-	---	11
187	Perlatolic acid	C25H31O7	26.98	443.2070	443.2078	−1.8	d	205.0867; 179.1073; 223.0973	7; 8
188	Caperatic acid	C21H37O7	28.14	401.2539	401.2549	−2.4	Acids	255.2327	8
189	Atranorin	C19H17O8	29.64	373.0923	373.0937	−3.75	d	177.0192; 163.0397	9; 11

* Identified by addition experiments with a genuine compound. A = Aromatic compound; L = Lipid; D = depsidone; d = depside; DE = diphenilether; DBF = dibenzofurane. C = Chromone. ** 1 = S. cf. andinaa; 2 = S. cf. hypoglabra; 3 = S. cordillerana; 4 = S. cf. gyalocarpa; 5 = S. leucoblepharis; 6 = S. parahumboldtii; 7 = S. impressula; 8 = S. ocaniesnsis; 9 = S. speudosylvatica; 10 = S. luteocyphelata; 11 = S. lineariloba.

2.1.1. Saturated Organic Acids

Peak 1 was tentatively identified as gluconic acid (C6H11O7), peak 3 as manitol, peak 4 as arabic acid, peak 5 as citric acid and peak 10 as its isomer isocitric acid (C6H7O7), peak 7 as 4-ethyl-2-ethylisophthalic acid (C10H9O4) with peak 2 as its isomer 2-ethylisophthalic acid, peak 15 as the derivative 2-hydroxyisophthalic acid, peak 17 was identified as 4-O-demethylglomellic acid (C24H25O9), peak 30 as 1,5-pentanedicarboxylic acid (C7H11O4) and peak 44 as 2,4-dicarboxy-3-hydroxy-5-methoxytoluene (C10H9O6)

2.1.2. Small Phenolic Compounds

Peaks 14 and 16 were identified as trihydroxy benzalaldehyde (C7H5O4) and 2,4-dihydroxy benzaldehyde (C7H5O3), peak 25 as 5,7-dihydroxy-6-methylphthalide (C9H7O4), peak 39 as metil-2,6-dihidroxibenzoate (C8H7O4), peak 51 as 2,4-dihydroxy benzaldehyde (C7H5O3), peak 53 as 4-ethoxy-3-formyl-2-hydroxy-6-methylbenzoic acid (C11H11O5), peak 95 5,7-dihydroxy-6-methylphthalide derivative (C9H7O3), peak 132 as ethyl 2,4-dihydroxy-6-n-nonylbenzoate (C18H27O4).

2.1.3. Typical Lichenic Phenolic Compounds (Depsides, Depsidones and Anthraquinones)

Several compounds were identified as the typical types of compounds occurring in lichens in our Sticta species. Peak 20, with a deprotonated molecule at m/z: 413.1569, was identified as grayanic acid (C23H25O7), peak 23 was identified as atranol (C8H7O3) [25], peak 32 as didechlorolecideoidin (C17 H13O7) [36] showing diagnostic fragments at m/z: 209.0456; 285.0776; 151.0396; 179.0347 and 123.0443, peak 34 was identified as orsellinic acid (C8H7O4), peak 37 as nor 8′-methylconstictic acid (C21H19O11), showing a diagnostic daughter fragment at m/z: 209.0455, peak 40 as hypostictic acid isomer (C19H15O8), peak 42 as fumarprotocetraric acid derivative (C17H11O6), peak 52 as consalizinic acid derivative I (C19H13O11, with diagnostic daughter ions at m/z: 373.0573; 387.0373; 225.0406 and 177.0193, and peak 55 as consalizinic acid derivative II, with ions at m/z: 401.0524; 417.0474 and 373.0574), peak 56 and 57 as an cynodontin, citreorosein isomer or consalizinic acid derivative I isomer, respectively, peak 59 as 1,4,5,6,8-pentahydroxy-3-ethylanthraquinone (C15H9O7), peak 62, with an ion at m/z: 403.0681 was identified as a haemathamnolic acid isomer (C19H15O10) and peak 64 as constictic acid (C19H13O10), peak 63 as a fumarprotocetraric acid derivative (C17H11O6), peak 65 as hypostictic acid isomer (C19H15O8), producing daughter ions at m/z: 327.0883, 195.0664 and 179.0347 peak 66 as terphenylquinones thelephoric acid (C18H7O8) and peak 67 as methylstictic acid (C20H15O9, ions at 371.0779 and 193.0504). Peak 68 was tentatively identified as 8′-metilconstictic acid isomer (C21H19O11), and peak 69 as protocetraric acid (C18H13O9) [29], peak 70 as hypoconstictic acid (C19H15O9), peak 77 as menegazziaic acid (C18H13O9) [37] with ionic fragments at m/z: 311.0570; 255.0666; 329.0679, peak 78 as norstictic acid (C18H11O9 ions at m/z: 327.0526; 151.0396 and 123.0444), peak 80 as the antioxidant agent physodalic acid [35] (C20H15O10, MS2 peaks at m/z: 359.0417; 315.0520; 343.0832; 387.0367; 373.0573 and 401.0525), peak 82 as derivative methyl 8-hydroxy-4-O-demethylbarbatate (C19H19O9), peak 83 as 12,13,15-trihydroxy-9-octadecenoic acid (C18H33O5), peak 84 as the cytotoxic compound haemoventosin (C15H11O7) [38], with ions at 259.0619, 231.0667, 189.0560, peak 86 as conhypoprotocetraric acid or convirensic acid (C18H15O8) [29], peak 87 as 4-O-dimethylbaeomycesic acid (C18H15O8, 181.0714; 163.0397 and 137.0236), a methyl derivative of baeomycesic acid [39], peak 88 as orsellinic acid isomer (C8H7O4, 123.0440; 149.0235), peak 89 as lecanoric acid (C16H13O7, 167.0345; 123.0443; 149.0238) [23], peak 90 as constictic acid isomer (C19H13O10), peak 92 with a deprotonated molecule at m/z: 235.0615 and daughter ion at m/z: 181.0504 was identified as 2-methyl-5-hydroxy-6-hydroxymethyl-7-methoxychromone (C12H11O5), peak 96 was identified as criptostictic acid derivative (C18H11O8), peak 105 as stictic acid (C19H13O9) [25], peak 106 as the typical lichen anthraquinone parietin (C16H11O5), while peak 108 was identified as evernic acid isomer (C17H15O7), peak 109 as hypoconstictic acid and peak 110 as cryptostictic acid [40] (C19H15O9, diagnostic daughter ions at m/z: 267.0661; 343.0825, 311.05067 and 239.0710). peak 113 as salazinic acid isomer (C18H11O1), peak 117 with a deprotonated molecule at m/z: 323.0556 was identified as pulvinic acid derivative I (C18H11O6), producing ions at m/z: 133.0286; 117.0335. Peak 119, with a parent ion at m/z: 345.0989, was identified as 4-O-demethylbarbatic acid (C18H17O7) [29], peak 121 as methyl orsellinate [37] and peak 123 as gyrophoric acid. Peak 125, producing fragments at m/z: 313.0723; 135.0444 and 179.0348, was identified as hyposalazinic acid (C18H13O8) [37], peak 127 as an isomer of orsellinic acid [23], peak 124 as galbinic acid (C20H13O11, 403.0681; 371.0417; 401.0524; 327.0518 and 149.0239) [37], peak 129 as norstictic acid (C18H11O9, 27.0517; 227.0716; 151.0390; 243.0297) [1,2], peak 133 as evernic acid (C17H15O7) [25], peak 131 as loxodinol isomer (C25H29O9), peak 136 as the dibenzophenone strepsilin (C15H9O5), peak 146 as squamatic acid and peak 147 as the depsone picrolichenic acid (C25H29O7), peak 144 and 145 as hydroxytetracosapentaenoic acid derivative (C24H37O3) and hydroxytrioxotricosanoic acid (C23H39O6), respectively. Peak 150, with an ion at m/z: 457.2244, was identified as 2,2′-di-O-methylanziaic acid (C26H33O7) [41], peak 154 was determined to be pulvinic acid, (C18H11O5, ions at m/z: 117.0338; 263.0713), peak 164 as a pulvinic acid derivative (C19H13O5), peak 164 as a pulvinic acid derivative of 321.0770 (C19H13O5), and peak 165 as another isomer of pulvinic acid (C18H11O5), while peak 155 was identified as 4-O-demethylbarbatic acid (C18H17O7, diagnostic ions at m/z: 123.0443; 137.0237; 181.0502). Peak 156 was determined as soromic acid (ions at m/z: 313.0726; 181.0502; 179.0347; 327.0520; 269.0826 and 285.0776), peak 157 as methylgyrophoric acid (C25H21O10, diagnostic ions at m/z: 149.0238; 123.0442; 167.0346 and 317.0671), and peak 158 as evernic acid isomer. Similarly, peak 159 was identified as anthraquinones skyrin (C30H17O10), peak 160 as angardianic acid (C19H35O4) [42], peak 166, with an ion at m/z: 551.1197, was identified as furfuric acid isomer (C28H23O12, producing fragments at m/z: 371.0784; 193.0504; 179.0347; 207.0297 and 193.0504), peak 172 as barbatic acid (C19H19O7) [25], peak 174 as thamnolic acid, peak 175 as orsenillic acid derivative II (C8H7O4), peak 177 as lobaric acid (C25H27O8 with ions at m/z: 411.1824; 367.1811), peak 180 as hypothamnolic acid (C19H17O10 with ions at m/z: 209.0456; 181.0499) [43], peak 184 as usnic acid (C18H15O7) [25], peak 185 as either nephromopsic acid or roccellaric acid (C19H33O4) [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.

2.1.4. Terpenes

Peak 112 was identified as retigeric acid B (C30H45O6), while peak 111 with an ion at m/z: 515.3025 was tentatively identified as a retigeric acid derivative (C30H43O7).

2.1.5. 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 (C25H47O13); peak 43, with a parent ion at m/z: 187.0977, was determined to be 4,5-dihydroxy-2-nonenoic acid (C9H15O4); peak 72 as 12,13,15-trihydroxy-9-octadecenoic acid (C18H15O5), while peaks 91 and 94 were determined as pentahydroxytetracosanoic acid (C24H47O7) and heptahydroxytrioxooctadecanoic acid (C18H29O12), respectively. In the same manner, peaks 102–104 were identified as heptahydroxytetraoxoicosanoic acid (C20H31O13), tetrahydroxytricosanoic acid (C23H45O6), and tetrahydroxytrioxoundecanoic acid (C11H15O9), respectively. Peak 115 was assigned as 9,10-dihydroxyoctadecatrienoic acid (C18H29O4) and peak 118 as 9,10,12 trihydroxytriacontaheptaenoic acid; peak 122 as eptahydroxyetraoxoicosanoic acid (C20H31O13); peak 126 as hydroxytetracosapentaenoic acid (C24H37O3); and peak 128 as dihydroxyoctadecenoic acid (C18H33O4). Peak 130 was tentatively identified as dihydroxyoctadec-6-enoic acid (C18H33O4); peak 134 as a protocetraric acid isomer (C18H13O9) [29]; peak 139 as hexahydroxytrioxooctacosatrienoic acid (C28H43O11); peak 140 as nonahydroxyoctacosatetraenoic acid (C28H47O11); peak 142 as norsolorinic acid (C20H17O7); peak 148 as heptahydroxydioxohexacosanoic acid (C26H47O11); peak 151 as dihydroxytetracosahexaenoic acid (C24H35O4); and peak 152 as hydroxyoctadecadienoic acid (C18H31O3). 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 (C20H27O4).

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.

Figure 3

Structures of some representative compounds found in Sticta lichens.

2.1.6. Distance and Phylogenetic Analysis

We found that 69/189 (37%) chemical characters and 7/16 (44%) morphological characters were parsimony informative. Optimally retained trees had a minimum parsimony score of 256 (chemical compounds) and 87 (morphology). The chemical compounds tree showed higher consistency and retention indexes and lower homoplasy (CI = 0.966, RI = 0.786) than the morphology tree (CI = 0.738., RI = 0.531).

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.

Figure 4

Neighbor joining trees (A,B) and unrooted strict consensus trees from maximum parsimony analysis (C,D) with morphological traits (A,C) and chemical compounds (B,D) in some Colombian species of Sticta. Colored lines represent localities from collected specimens. Colored circles represent their position in the molecular phylogeny of Moncada et al. (2014); grayish colors in the color scale refer to more basal taxa, whereas bluish colors are more recent taxa. Although S. lineariloba occurs in Colombia, the specimen in this study was collected in Valdivia (Chile). Independent of the grouping method, trees built based on chemical compounds recovered the geographic relationships, while trees built with morphological traits resemble the molecular phylogeny of Colombian Sticta.

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 DNA-sequencing 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].

3. Materials and Methods

3.1. 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).

3.2. Lichen Material

The lichen specimens S. pseudosylvatica Moncada & Suárez (35 g) and S. luteocyphellata Moncada & Lücking (28 g) were collected by Olimpo García Beltrán and Alfredo Torres Benítez in 2018 in Villahermosa, in the department of Tolima-Colombia, at the farm La Estrella (5°02′48.63″ N–75°07′37.98″ W). The species S. cf. andina Moncada & Lücking (31 g), S. cf. hypoglabra Moncada & Lücking (42 g), S. cordillerana Gyeln (37 g), S. cf. gyalocarpa (Nyl.) (29 g), S. leucoblepharis (Nyl.) Tuck. & Mont (34 g). y S. parahumboldtii Moncada & Lücking (40 g) were collected by Alfredo Torres Benítez and Emmanuel Campos in 2018 in the Semillas de Agua Civil Society Nature Reserve in the Anaime páramo (4°15′18.09″ N–73°33′23.27″ W) and the species S. impressula (Nyl.) Zahlbr (29 g) and S. ocaniensis (33 g) Moncada & Simijaca were collected by Alfredo Torres Benítez and María Rivera Montalvo in 2017 in the “Combéima river basin”, Ibagué-Tolima, Colombia (4°36′02.35″ N–75°19′50.45″ W). All voucher specimens were deposited in the herbarium of Universidad Distrital Francisco José de Caldas (Colombia) and Prof. Alejandra Suárez Corredor confirmed their identity.

3.3. 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).

3.4. 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.

3.5. LC Parameters

Liquid chromatography on a UHPLC C-18 column (Acclaim, 150 mm × 4.6 mm ID, 2.5 μm, Thermo Fisher Scientific, Bremen, Germany) was performed as reported previously. The mobile phases were 1% formic aqueous solution, (A) methanol 1% formic acid (B) and acetonitrile 1% formic acid (C). The gradient program time were as follows: 0.00 min, 18 B, 75 C; 5.00 min, 18 B, 75 C; 15.00 min, 40 B, 60 C; 20.00 min. B, 100 C; and 12 min for column equilibration at starting conditions.

3.6. 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 × 106 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 × 105, 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 105 and below 1010 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.

3.7. 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.

4. 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.