molecules Article Chemotypes and Biomarkers of Seven Species of New Caledonian Liverworts from the Bazzanioideae Subfamily Benjamin Métoyer 1, Nicolas Lebouvier 1, Edouard Hnawia 1, Gaëtan Herbette 2 ID , Louis Thouvenot 3, Yoshinori Asakawa 4, Mohammed Nour 1 and Phila Raharivelomanana 5,* 1 Institut des Sciences Exactes et Appliquées (ISEA) EA 7484, Université de la Nouvelle-Calédonie, 98851 Nouméa, New Caledonia; [email protected] (B.M.); [email protected] (N.L.); [email protected] (E.H.); [email protected] (M.N.) 2 Aix Marseille Univ, CNRS, Centrale Marseille, FSCM, Spectropole, Service 511, Campus Saint-Jérome, 13397 Marseille CEDEX 20, France; [email protected] 3 Independent Researcher, 11, Rue Saint-Léon, 66000 Perpignan, France; [email protected] 4 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 7708514, Japan; [email protected] 5 UMR 241 EIO, Université de la Polynésie Française, 98702 Faaa, Tahiti, French Polynesia * Correspondence: [email protected] Received: 21 May 2018; Accepted: 31 May 2018; Published: 5 June 2018 Abstract: Volatile components of seven species of the Bazzanioideae sub-family (Lepidoziaceae) native to New Caledonia, including three endemic species (Bazzania marginata, Acromastigum caledonicum and A. tenax), were analyzed by GC-FID-MS in order to index these plants to known or new chemotypes. Detected volatile constituents in studied species were constituted mainly by sesquiterpene, as well as diterpene compounds. All so-established compositions cannot successfully index some of them to known chemotypes but afforded the discovery of new chemotypes such as cuparane/fusicoccane. The major component of B. francana was isolated and characterized as a new zierane-type sesquiterpene called ziera-12(13),10(14)-dien-5-ol (23). In addition, qualitative intraspecies variations of chemical composition were very important particularly for B. francana which possessed three clearly defined different compositions. We report here also the first phytochemical investigation of Acromastigum species. Moreover, crude diethyl ether extract of B. vitatta afforded a new bis(bibenzyl) called vittatin (51), for which a putative biosynthesis was suggested. Keywords: liverwort; Bazzania; Acromastigum; sesquiterpene; diterpene; bis(bibenzyl); biosynthesis; zierane; vittatin 1. Introduction Liverworts are part of Bryoflora (mosses: 14,000 species, liverworts: 6000 species and hornworts: 300 species), considered as the first terrestrial plants and taxonomically indexed between algae and pteridophytes. Bryophytes possess archaic characteristics such as the absence of seeds and vascularized leaves [1]. Morphological traits such as small size of organs or relatively simple structure or high intraspecies variability and fugacity of some microscopic details (which may disappear within the plant dryness such as oil bodies), add difficulties for liverwort’s taxonomic identification. Nevertheless, many liverworts have unique organelles called oil bodies in their cells which are linked to the biosynthesis of original secondary metabolites such as mono-, sesqui- and diterpenes or phenolic compounds that could be cladistic biomarker. Most of liverwort’s sesquiterpenes are enantiomers of those found in higher plants [2]. Molecules 2018, 23, 1353; doi:10.3390/molecules23061353 www.mdpi.com/journal/molecules MoleculesMolecules 20182018,, 2323,, 1353x 2 of 27 2 of 27 New Caledonia is an archipelago of 18,600 km2 located in South Pacific region and considered as a Newhotspot Caledonia of biodiversity is an archipelago [3]. In New of Caledonia, 18,600 km2 482located species in South and infraspecific Pacific region taxa and of considered liverworts ashave a hotspot been described of biodiversity in a recent [3]. Inchecklist New Caledonia, [4]. The rate 482 of species endemism and is infraspecific comprised taxabetween of liverworts 13% and have39% that been makes described New in Caledonia a recent checklist as one of [4 ].the The richest rate of liverwort endemism areas is comprised in the world, between together 13% with and 39%Japan, that New makes Zealand, New Caledoniaand Costa asRica one [5]. of the richest liverwort areas in the world, together with Japan, NewBazzanioideae Zealand, and Costa is a Ricasubfamily [5]. of the Lepidoziaceae family including three genera Bazzania, AcromaBazzanioideaestigum and Mastigopelma is a subfamily, this of latter the Lepidoziaceaeone had never familybeen inventoried including threein New genera CaledoniaBazzania [4],. AcromastigumThrough the world,and Mastigopelma nearly 280 ,Bazzania this latter species one had and never 40 Acromastigum been inventoried species in are New described Caledonia [6]. [ 4In]. ThroughNew Caledonia, the world, 20 nearlyBazzania 280 speciesBazzania or speciesvarieties and including 40 Acromastigum 12 endemicsspecies and are 16 describedAcromastigum [6]. Inspecies New Caledonia,or varieties 20includingBazzania 10species endemics or varieties have been including described 12 endemics[4]. Usually, and Bazzania 16 Acromastigum species arespecies divided or varietiesinto two includingchemotypes 10: endemics albicanyl(drimenyl) have been described-caffeate-cuparane [4]. Usually, (I) Bazzaniaand calamenanespecies are(II) divided [7]. into two chemotypes:Volatile albicanyl(drimenyl)-caffeate-cuparanecompounds of 27 specimens belonging (I)and to seven calamenane different (II) [species,7]. including three endemicsVolatile (marked compounds with an of 27asterisk) specimens were belonging investigated to seven in order different to check species, intra including- and inter three-variability endemics of (markedmolecular with contents. an asterisk) Studied were Bazzanioideae investigated inspecies order belong to check to intra- the following and inter-variability genera: Acromastigum of molecular contents.(A. tenax*, Studied A. caledonicum Bazzanioideae*) and Bazzania species ( belongB. parisii to, B. the marginata following*, B. genera: vittata, AcromastigumB. francana, B. bernieri (A. tenax and*, A.B. caledonicumserrifolia). B.*) serrifolia and Bazzania is a synonym (B. parisii of, B. B. marginata bernieri according*, B. vittata ,toB. Kitagawa francana, B.(1973) bernieri [8] andbut B.we serrifolia studied). B.here serrifolia its putativeis a synonym specific status of B. by bernieri separatingaccording the samples to Kitagawa in two (1973) lots of [8 ]specimens but we studied on the herebasis itsof putativetheir morphological specific status traits. by separating the samples in two lots of specimens on the basis of their morphologicalVolatile components traits. of diethyl ether extracts were analyzed by GC-MS-FID in order to index theseVolatile plants componentsinto knownof Bazzania diethyl etherchemotypes. extracts wereIn the analyzed present bywork, GC-MS-FID two new in ordermolecules: to index an theseoxygenated plants intodimer known of Bazzanialunularicchemotypes. acid (51) and In the an present alcohol work, zierane two new-type molecules: sesquiterpene an oxygenated (23) are dimercharacterized of lunularic for the acid first (51 time) and (Tables an alcohol 1 and zierane-type 2). They were sesquiterpene respectively (23 isolated) are characterized from diethyl for ether the firstextract time of (Tables B. vittata1 and and2). TheyB. francana were respectively. To our best isolated knowledge from diethyl, this etheris the extract first ofphytochemicalB. vittata and B.investigation francana. To ourof all best these knowledge, species this and is thethe first first phytochemical chemical analysis investigation of those of all belonging these species to andthe theAcromastigum first chemical genus. analysis of those belonging to the Acromastigum genus. InIn orderorder toto sortsort thethe detected volatile compounds in this study, diterpene-typediterpene-type contentscontents areare listedlisted (Table(Table 33),), listedlisted sesquiterpenesesquiterpene moleculesmolecules werewere classifiedclassified followingfollowing theirtheir firstfirst cyclizationcyclization precursorsprecursors (Figure(Figure1 1),), and and their their sesquiterpene sesquiterpene types types (Tables (Table4–6)s related4–6) related to the correspondingto the corresponding biosynthesis biosynthesis pathway schemepathway from scheme the literature from the data literature [9–19]. data So, compilation [9–19]. So, ofcompilation chemical composition of chemical of composition all studied species of all arestudied gathered species in Tableare gathered7. List of in identified Table 7. andList unknownof identified compounds and unknown is shown compounds in Tables is8 –shown10. Most in importantTables 8–10. detected Most important compounds detected for chemotaxonomy compounds for are chemotaxonomy shown in Figure are2. shown in Figure 2. 14 5 8 7 4 ionization isomerization 6 + 9 3 H C 10 2 2 15 11 + 12 1 CH2 13 OPP Farnesyl diphosphate Farnesyl Nerolidyl RC1 10,1 RC1 11,1 RC1 6,1 RC1 10,1 RC1 11,1 + C + + + C CH CH + C (E-E)-Germacradienyl (E-E)-Humulyl Bisabolyl (Z-E)-Germacradienyl (Z-E)-Humulyl FigureFigure 1. SchemeScheme of ofcationic cationic first first cyclizat cyclizationion precursor precursor for the for detected the detected sesquiterpenes sesquiterpenes (1 ring closure) (1 ring closure)[19]. [19]. Molecules 2018, 23, 1353 3 of 27 Molecules 2018, 23, x 3 of 27 OH HO 1 2 3 4 5 6 7 OH 8 9 10 11 12 13 H H 14 15 16 17 18 H H HO H HO OH H H HO 19 20 21 22 23 H H O 24 25 26 27 28 29 H H HO OH H H H H 30 31 32 33 34 35 36 OH OH OH