Fig 1.
Chemical structures of major compounds in Icelandic Melanelia lichens.
Compounds include cryptostictic acid 1, stictic acid 2, norstictic acid 3, usnic acid 4, stenosporic acid 5, rangiformic acid 6 and perlatolic acid 7.
Fig 2.
Base peak MS chromatograms of lichen acetone extracts in negative ion mode.
(A) MS chromatogram of Melanelia hepatizon containing cryptostictic acid 1, stictic acid 2 and norstictic acid 3. (B) MS chromatogram of one Montanelia disjuncta chemotype that contains usnic acid 4, stenosporic acid 5, rangiformic acid 6 and perlatolic acid 7. (C) MS chromatogram of the other Montanelia disjuncta chemotype without usnic acid 4 and rangiformic acid 6. Chromatograms of Melanelia stygia and M. agnata are not shown since no major lichen acids were detected.
Table 1.
Major lichen metabolites (1–7) and unknown minor compounds (a-c) in Icelandic Melanelia taxa.
Fig 3.
MS fragmentation patterns of depsidones in the lichen Melanelia hepatizon.
Compounds include cryptostictic acid 1, stictic acid 2 and norstictic acid 3.
Fig 4.
MS fragmentation patterns of depsides in the lichen Montanelia disjuncta.
Compounds are stenosporic acid 5 and perlatolic acid 7. Both shared the same fragment ions.
Fig 5.
MS fragmentation pattern of rangioformic acid.
Rangiformic acid 6 was found in one chemotype of Icelandic Montanelia disjuncta taxon, as shown in Fig 2B.
Fig 6.
MS fragmentation pathway of usnic acid.
Usnic acid was detected in one chemotype of Icelandic Montanelia disjuncta taxon. Structure a, b, d, e and f are characteristic fragment ions in MS2 spectrum (S3 Fig). Structure b is a resonance contributor of structure a but not a true structure of usnic acid. Structure c is only reported in LDI or FAB-MS. (Abbreviation: RDA: retro-Diels—Alder reaction; LDI/FAB: laser desorption ionization/fast atom bombardment).
Fig 7.
Multivariate analysis of LC-MS metabolite data.
(A) PCA plot of chemical profiles of Icelandic Melanelia lichens, where M. agnata and M. stygia are clustered. (B) OPLS-DA plot shows the separation of the two Melanelia taxa. The metabolome of M. agnata and M. stygia can be differentiated with a high level of prediction value: R2Y(cum) = 1, Q2(com) = 0.99. (C) Loading S-plot from LC-MS data of M. agnata and M. stygia. Cut-off values of p(corr) < |0.8| were selected to designate the metabolites contribuiting significantly to the overall difference (area in color) between M. agnata and M. stygia. Two metabolites were thus identified from each species.
Table 2.
Genetic distances, alignment length and number of specimens (haplotypes) for the genera Melanelia and Montanelia.
Fig 8.
Genetic distance histograms and barcoding gap analysis for reported Melanelia species in Iceland.
(A and B) p-Distance histogram for the genus Melanelia and Montanelia, respectively; (C and D) Barcoding gap analysis for Melanelia species and Montanelia disjuncta.
Fig 9.
Fungal nrITS gene tree obtained from 116 Melanelia and Montanelia specimens.
(A) Neighbor-joining tree, bootstrap values over 80% are labelled on the branches; (B) Maximum-likelihood tree, posterior probability/bootstrap values are labelled on branches.