Biochemical Systematics and Ecology 42 (2012) 49–58 Contents lists available at SciVerse ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Flavonoids as chemosystematic markers for the genus Adenocarpus Rafaa S. Essokne a, Renée J. Grayer b, *, Elaine Porter b, Geoffrey C. Kite b, Monique S.J. Simmonds b, Stephen L. Jury a a b Centre for Plant Diversity and Systematics, School of Biological Sciences, The Harborne Building, University of Reading, Whiteknights, Reading RG6 6AS, UK Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK a r t i c l e i n f o a b s t r a c t Article history: Received 12 July 2011 Accepted 26 December 2011 Available online 14 March 2012 Twenty-four species of the genus Adenocarpus (Leguminosae), sampled in Mediterranean and tropical African regions, were surveyed for their flavonoids by means of high performance liquid chromatography with diode array detection and atmospheric pressure chemical ionization-mass spectrometry. For ten of these species, 2–8 samples were examined to investigate a possible infraspecific variation in flavonoids. Nineteen flavonoids belonging to various different classes (flavone C- and O-glycosides, flavonol glycosides, isoflavone glycosides and flavanones) were detected. Previous DNA sequence analyses have resulted in the grouping of Adenocarpus species into four different clades, so that the results of the flavonoid survey could be compared with that of molecular studies. A good correlation was found in that each of the clades could also be characterised by a combination of flavonoids. For example, the species in clade 4, which are distributed in South and South-East Europe and tropical Africa, are characterised by the presence of flavonol O-glycosides and 5-hydroxyisoflavone O-glycosides. In contrast, flavonol O-glycosides were absent from all but one species of the other three clades, which have a mainly North Africa distribution, and these species produce flavone mono-C-glycosides and/or flavone 7-O-glycosides instead. In addition flavone 40 -O-glycosides were only found in species of clade 1 and flavanones were mainly restricted to clade 3. The relationships among species of Adenocarpus suggested by flavonoid profiles and DNA sequence analyses provide a framework that can be used as a basis for a new classification of the genus. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: Adenocarpus Leguminosae Papilionoideae Flavonoids Isoflavonoids Chemosystematics 1. Introduction Adenocarpus belongs to subfamily Papilionoideae of the family Leguminosae, tribe Genisteae. The genus is centred in the western Mediterranean region and presently consists of ca. 25 species. The genus was last fully revised by Gibbs (1967), who recognized 14 species; two in the Canary Islands, seven in North Africa, four in the Mediterranean region of Europe, extending into the Atlantic region (Spain, Portugal, France), and one species in Tropical Africa, Adenocarpus mannii Hook.f. The major floristic inventory of the Mediterranean, Med-checklist (Greuter et al., 1989), enumerated a total of 11 species and six subspecies. Since then Castroviejo (1999a) has undertaken a revision of the genus for the Iberian Peninsula for his account in Flora Iberica (Castroviejo, 1999b) and added two further species. In addition, he raised some subspecies to species status. Furthermore, Brullo and De Marco (2001) have described two new species of Adenocarpus from Italy. Many of the new species used to be subspecies or forms of the polymorphic circum-Mediterranean taxon Adenocarpus complicatus (L.) Gay, e.g. Adenocarpus anisochilus Boiss., Adenocarpus aureus (Cav.) Pau, Adenocarpus bivonii (Presl.)Presl., Adenocarpus brutius Brullo, Adenocarpus commutatus Guss., Adenocarpus desertorum Castrov., Adenocarpus gibbsianus Castrov. & Talavera, Adenocarpus * Corresponding author. Tel.: þ44 (0) 20 8332 5312; fax: þ44 (0) 20 8332 5310. E-mail addresses: r.grayer@kew.org, r.grayer@rbgkew.org.uk (R.J. Grayer). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.12.023 50 R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 lainzii (Castrov.) Castrov. and Adenocarpus nainii Maire. Furthermore, Adenocarpus argyrophyllus (Rivas Goday) Caball. used to be a subspecies of Adenocarpus hispanicus (Lam.) DC. Harborne (1969) carried out a comprehensive survey of the flavonoids and isoflavonoids in the tribe Genisteae. He studied the acid-hydrolysed leaf extracts of 128 species belonging to 22 genera and found in the majority of Genisteae species the isoflavones, daidzein, genistein and 5-methylgenistein (¼isoprunetin). Flavonols were also common, such as kaempferol, quercetin and fisetin, but the flavones luteolin and apigenin occurred less frequently. Glycoflavones were common in some genera, e.g. Ulex and Chamaecystus, but absent or rare in others. In this study of the Genisteae, Harborne (1969) also investigated one species of Adenocarpus, Adenocarpus foliolosus, and reported the isoflavones, daidzein, genistein and 5methylgenistein from this species, but no flavonols or flavones. Most other chemical studies of the genus Adenocarpus have involved alkaloids rather than flavonoids, and many of these investigations were carried out more than five decades ago (summarized in Mears and Mabry, 1971). Sparteine-type quinolizidine alkaloids were found in A. argyrophyllus, Adenocarpus decorticans and A. hispanicus; the pyrrolizidine alkaloid, decorticasine, in the same three species and in Adenocarpus grandiflorus and the ammodendrine-hystrine alkaloids, adenocarpine, isoorensine and santiaguine in A. grandiflorus, Adenocarpus intermedius and A. mannii. In addition, adenocarpine and santiaguine were also detected in Adenocarpus anagyrus (¼Adenocarpus viscosus) and A. foliolosus. In the present work, flavonoids and isoflavonoids were surveyed in 24 species of Adenocarpus, collected in nine countries or geographic areas (Morocco, Algeria, Canary Islands, Spain, Portugal, Italy, European Turkey, Malawi and Tanzania). For ten of the species two or more accessions were investigated, sometimes from different geographic regions, to study possible infraspecific chemical variation. These included eight accessions of Adenocarpus telonensis (six from Morocco, one from Portugal and one from Spain), four accessions of A. decorticans (two from Morocco, one from Algeria and one from Spain), six accessions of A. complicatus (one from Morocco, two from Spain, one from Portugal and two from Turkey) and four accessions of A. mannii (two from Malawi and two from Tanzania). The results of the flavonoid and isoflavonoid survey were projected onto dendrograms based on a DNA sequence analysis of several genes by Cubas et al. (2010) and of the ITS region by one of us (Essokne, 2011). This paper forms part of a study to provide a new monographic treatment of Adenocarpus that reflects the morphology, geographic distribution, phytochemistry and DNA sequences of the genus. 2. Material and methods 2.1. Plant material In order to revise and understand the variation in Adenocarpus species, field collections were made from the end of May until mid June 2006 in Morocco, GPS readings for all the sites were recorded and the specimens transported to the Herbarium of the University of Reading (RNG), where they were dried ready for morphological and phytochemical analysis. Permission to collect and export the material was obtained from Professor Sherif Harouni (IAV-Agadir) and Professor Mohammed Fennane (RAB), respectively. A total of 23 specimens were collected at each site by two of us (S.L.J. & R.S.E.) from natural habitats in Morocco. Voucher specimens have been deposited in RNG. In addition, for the other European and Tropical African Adenocarpus species, herbarium specimens were used from the Herbaria of RNG and E. Details of the specimens studied are presented in Table 1. 2.2. Extraction Dried leaves of each Adenocarpus species (ca. 0.5–1.0 g) were crushed and extracted with ca. 10 ml of 80% MeOH in a test tube. The plant material was boiled for 10 min in a heating block at 85  C. The samples were left to stand overnight at room temperature. After filtration, 3 ml of the solvent from each sample was removed to a small beaker to dry. The dried extract was redissolved in 1 ml of 80% MeOH in preparation for analysis by HPLC. 2.3. HPLC The HPLC system consisted of a Waters LC 600 pump and 996 photodiode array detector. A Merck LiChrospher 100RP-18 (5 mm) column was used; the dimensions were 4.0 mm (i.d)  250 mm. Two solvent solutions, denoted A and B, were used for elution. Solvent A consisted of 2% HOAc and solvent B of MeOH:HOAc:H2O, 18:1:1. The gradient pogramme started at 75% A and 25% B, and progressed with a linear gradient reaching B ¼ 100% at t ¼ 20 min. This was followed by isocratic elution (B ¼ 100%) to t ¼ 24 min, after which the programme returned to the initial solvent composition. Column temperature was maintained at 30  C and a flow rate of 1.0 ml/min was used. Injections of 20 ml were made by autosampler. Spectral scanning took place over the range 200–400 nm and chromatograms were printed at 340 nm. Retention times and UV spectra of flavonoids were compared with those of standards and published data. 2.4. LC–MS Positive ion Atmospheric Pressure Chemical Ionization (APCI)-mass spectra were obtained with a quadrupole ion-trap instrument (Thermo Scientific LCQ ‘Classic’) using a vaporiser temperature of 550  C, sheath and auxiliary nitrogen 51 R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 Table 1 Adenocarpus species used for flavonoid studies, accession numbers and country of origin. Adenocarpus species Accession number; Reading (RNG) & Edinburgh (E) BI number RBG Kew Country of origin A. anagyrifolius Coss. & Ball A. anagyrifolius Coss. & Ball A. anagyrifolius A. anagyrifolius var. leiocarpa Maire A. anisochilus Boiss. A. argyrophyllus (Rivas Goday) Caball. A. artemisifolius Jahand., Maire & Weiller A. artemisifolius Jahand., Maire & Weiller A. aureus (Cav.) Pau A. bacquei Batt. & Pit. A. bivonii Brullo A. boudyi Batt. & Maire A. boudyi Batt. & Maire A. bracteatus Font Quer & Pau A. bracteatus Font Quer & Pau A. brutius Brullo A. cincinnatus Maire A. cincinnatus Maire A. commutatus Guss. A. complicatus J. Gay ex Gren. & Godr. A. complicatus J. Gay ex Gren. & Godr. A. complicatus J. Gay ex Gren. & Godr. A. complicatus J. Gay ex Gren. & Godr. A. complicatus J. Gay ex Gren. & Godr. A. complicatus J. Gay ex Gren. & Godr. A decorticans Boiss. A decorticans Boiss. A decorticans Boiss. A decorticans Boiss. A. desertorum Castrov. A. faurei Maire A. foliolosus DC. A. gibbsianus Castrov. & Talavera A. hispanicus (Lam.) DC. A. lainzei (Castrov.) Castrov. A. lainzei (Castrov.) Castrov. A. mannii Hook.f. A. mannii Hook.f. A. mannii Hook.f. A. mannii Hook.f. A. nainii Maire A. telonensis (Loisel.) DC A. telonensis (Loisel.) DC A. telonensis (Loisel.) DC A. telonensis (Loisel.) DC A. telonensis (Loisel.) DC A. telonensis (Loisel.) DC A. telonensis (Loisel.) DC A. telonensis (Loisel.) DC A. viscosus Webb & Berthel. S.L. Jury & R. Shkwa-20840 S.L. Jury & R. Shkwa-20847 S.L. Jury & R. Shkwa-20841 E00245708 P0033927 P0001495 S.L. Jury & R. Shkwa-20852 P0030758 P0030718 S.L. Jury & R. Shkwa-20894 P003910 S.L. Jury & R. Shkwa-20918 S.L. Jury & R. Shkwa-20931 S.L. Jury & R. Shkwa-20995 S.L. Jury & R. Shkwa-21003 P0030913 S.L. Jury & R. Shkwa-20851 Davis 53691 P0001544 S.L. Jury & R. Shkwa-20890 E00245630 P0030684 P0030800 E00245668 P0030792 S.L. Jury & R. Shkwa 20980 S.L. Jury & R. Shkwa 21013 E00245721 S.L. Jury-18571 P0030802 E00245670 P0030758 P0005834 S.L.Jury-11176 P0030729 P0030797 E00245695 E00245696 E00245692 E00245693 S.L. Jury & R. Shkwa-21004 S.L. Jury & R. Shkwa-20847 S.L. Jury & R. Shkwa-20862 S.L. Jury & R. Shkwa-20870 S.L. Jury & R. Shkwa-20873 S.L. Jury & R. Shkwa-20878 S.L. Jury 20947 P0030795 P0022614 P0030775 18053 17823 17824 17825 17811 17814 17827 18059 17807 18060 17817 18048 18050 18056 18057 17819 18062 17822 17818 18049 17830 17832 17833 17831 17835 18061 18063 17836 17815 17809 17816 17828 17810 17813 17806 17808 17820 17821 17837 17838 18058 18047 18051 18052 18054 18055 17826 17834 17812 17829 Morocco Morocco Morocco Morocco Portugal Spain Morocco Morocco Spain Morocco Italy Morocco Morocco Morocco Morocco Italy Morocco Morocco Italy Morocco Spain Spain Portugal Turkey Turkey Morocco Morocco Algeria Spain Spain Algeria Canary Islands Spain Spain Spain Spain Malawi Malawi Tanzania Tanzania Morocco Morocco Morocco Morocco Morocco Morocco Morocco Spain Portugal Canary Islands RNG RNG RNG E RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG E RNG RNG E RNG RNG RNG E RNG RNG E RNG RNG RNG RNG RNG E E E E RNG RNG RNG RNG RNG RNG RNG RNG RNG RNG pressures of 80 and 20 psi, respectively, a needle current of 5 mA and a capillary temperature of 150  C. Samples were introduced via an HPLC and chromatography was performed in a similar manner to analytical HPLC, except that the concentration of acetic acid in the mobile phases was 1%. The mass spectrometer was controlled by Xcalibur 1.0 software (Thermo Scientific) which was programmed to record the first order mass spectra (m/z 125–1200) and then the MS/MS spectra of three or four of the most abundant ions in each first order spectrum by dat dependent acquisition. CID spectra were obtained by prior isolation of the precursor ion in the trap (isolation width 5 amu) and then applying a collision energy of 45% (without wide band activation), to obtain a fingerprint of the aglycone which was compared with those in an in-house database for the identification of the aglycone. 2.5. Standards Commercial standards for chromatographic and spectral comparison were available for orientin, vitexin, luteolin 7-Oglucoside, apigenin 7-O-glucoside, quercetin 3-O-glucoside and isorhamnetin 3-O-rutinoside (all from Apin Chemicals, Abingdon, UK), genistein 7-O-glucoside and daidzein 7-O-glucoside (both from Plantech, Reading, UK). 52 R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 3. Results and discussion 3.1. Identification of the compounds A total of 19 flavonoid and isoflavonoid glycosides were identified or characterised from the species Adenocarpus investigated by HPLC and LC-MS by a combination of UV spectra generated by the diode array detector and APCI-MS data (Grayer et al., 2000). Identifications could be confirmed by comparison with standards when available (see Section 2.5). Table 2 shows the HPLC retention times of the characterised compounds, their UV lmax (nm), APCI-MS data and the identification or tentative identification of the glycosides. First order APCI-mass spectra in positive mode provided the protonated molecule [M þ H]þ and, for flavonoid O-glycosides, fragment ions corresponding to the aglycone [A þ H]þ and sugar-loss intermediates [I þ H]þ if more than one sugar was attached to the flavonoid. Aglycones with the same molecular mass such as luteolin and kaempferol (Mr ¼ 286) were distinguished by the characteristic product ion spectra of [A þ H]þ. The compounds were characterised as follows. The UV spectra and lmax of compounds 1 and 3 (271, 334 nm and 268, 337 nm, respectively, see Table 2) suggested that they were apigenin derivatives. The protonated molecular masses of 1 and 3 after APCI-MS were 595 and 433, respectively, suggesting that 1 was an apigenin dihexoside and 3 an apigenin monohexoside. However, MS/MS of 1 and 3 did not yield the aglycone ion [A þ H]þ at m/z 271 expected for apigenin glycosides, but instead losses of 90 and 120 amu, which are characteristic of flavone C-glycosides with glucose as a sugar (Grayer et al., 2000). Therefore, 1 is an apigenin di-C-hexoside, tentatively identified as apigenin 6,8-di-C-glucoside (¼vicenin-2). From the APCI-MS data, compound 3 could be either apigenin 6-C-glucoside (isovitexin) or apigenin 8-C-glucoside (vitexin). Comparison with isovitexin and vitexin standards showed that 3 was vitexin. In a similar way, flavonoid 2, which had a very similar UV spectrum to that of luteolin (lmax 256, 267, 348), was shown to be luteolin 8-C-glucoside (orientin) by comparison with a standard. Flavonoids 4 and 5 produced aglycone ions [A þ H]þ at m/z 287 and the fragmentation of [A þ H]þ matched that of protonated luteolin under identical collision energies and isolation widths (Grayer et al., 2000). This information in combination with the UV spectra suggested that 4 and 5 were luteolin 7-O-glycosides as 5-O-, 30 -O- and 40 -O-glycosides of luteolin have different UV spectra (Grayer and de Kok, 1998; Grayer et al., 2002). A loss of 162 amu from the protonated molecule of 5 indicated that the sugar was a hexose, which is usually glucose for flavone glycosides in the Genisteae, so that flavonoid 5 was tentatively identified as luteolin 7-O-glucoside. This identification was confirmed by comparison with a standard. Luteolin glycoside 4 yielded one intermediate ion [I þ H]þ at m/z 449 (see Table 2) and therefore contains two sugars, a deoxyhexose (loss of 146 amu, probably rhamnose), and a hexose (loss of 162 amu, probably glucose), so that 4 was tentatively identified as a luteolin 7-O-rhamnosylglucoside. The sugar was likely to be rhamnosyl(1 / 6)glucose (¼rutinose) as the retention time of 4 Table 2 Flavonoids found in Adenocarpus species, their HPLC retention times, UV and mass spectra. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a b c Compound name i) Flavone di-C-glycosides Vicenin-2 (Apigenin 6,8-di-C-glucoside) ii) Flavone mono-C-glycosides Orientin (Luteolin 8-C-glucoside) Vitexin (Apigenin 8-C-glucoside) iii) Flavone 7-O-glycosides Luteolin 7-O-rutinoside Luteolin 7-O-glucoside Apigenin 7-O-rutinoside Apigenin 7-O-glucoside Chrysoeriol 7-O-glucoside iv) Flavone 40 -O-glycosides Luteolin 40 -O-glucoside Chrysoeriol 40 -O-glucoside v) Flavonol 3-O-glycosides Quercetin 3-O-glucoside Kaempferol 3-O-rutinoside Isorhamnetin 3-O-rutinoside vi) 5-Hydroxy-isoflavonoid glycosides Genistein 7-O-glucoside Genistein 7-O-malonylglucoside vii) 5-Deoxy- or 5-Methoxy-isoflavonoid glycosides 5-Methylgenistein 7-O-glucoside Daidzein 7-O-glucoside Daidzein 7-O-malonylglucoside viii) Flavanone glycosides Naringenin O-glycoside? UV lmax (nm) [M þ H]þ (m/z)a [I þ H]þ (m/z)b [A þ H]þ (m/z)c 271, 334 595 505, 475, 385, 355 – 10.7 11.7 256, 267, 348 268, 337 449 433 359, 329 343, 313 – – 12.4 12.6 13.7 13.9 14.0 256, 256, 267, 267, 254, 267, 346 267, 348 337 338 267, 343 595 449 579 433 463 449 287 287 271 271 301 14.3 14.6 249, 268, 337 248, 268, 333 449 463 13.0 14.3 14.5 256, 354 265, 344 255, 352 465 595 625 11.8 14.1 260, 325sh 260, 325sh 433 519 271 271 9.6 9.7 11.9 255, 320sh 255, 320sh 255, 320sh 447 417 503 285 255 255 9.5 290, 330sh n.d. Retention time (min) 8.8 [M þ H]þ protonated molecule obtained by APCI-LC-MS. [I þ H]þ intermediate ions. [A þ H]þ aglycone ion. 433 287 301 449 479 303 287 317 R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 53 was only 0.2 min shorter than that of 5, whereas the retention time of the corresponding rhamnosyl(1 / 2)glucoside (¼neohesperidoside) would have been ca. 1 min shorter (Heneidak et al., 2006). In a similar way, compound 7 was identified as apigenin 7-O-glucoside, which was confirmed by comparison with a standard, and compounds 6 and 8 were tentatively identified as apigenin 7-O-rutinoside and chrysoeriol 7-O-glucoside, respectively. Glycosides 9 and 10 produced the same APCI-MS spectra in positive mode as 4 and 8, respectively, but the retention times were longer and the UV spectra different. This combined information suggested that 9 and 10 were the 40 -O-glucosides of luteolin and chrysoeriol, respectively. The UV spectrum of compound 11 suggested that it was a quercetin 3-O-glycoside (Mabry et al., 1970). The aglycone ion [A þ H]þ at m/z 303 and its product ion spectrum confirmed this assumption. A loss of 162 amu of the protonated molecule of 5 indicated that the sugar was a hexose, and after comparison with a standard, glycoside 11 was identified as quercetin 3-O-glucoside. Compounds 12 and 13 appeared to be a kaempferol and an isorhamnetin 3-O-glycoside, respectively, on the basis of their UV spectra, aglycone ions [A þ H]þ at m/z 287 and 317 and their product ion spectra. APCI-MS data (production of an intermediate ion, see above for compound 4) indicated that two sugars were present, rhamnose and glucose, so that 12 was tentatively identified as kaempferol 3-O-rutinoside and 13 as isorhamnetin 3-O-rutinoside. The latter identification was confirmed by comparison with a standard. The UV spectra of compounds 14 and 15 (lmax 260 nm, shoulder at 325 nm) suggested that these were derivatives of the isoflavone genistein. The aglycone ion [A þ H]þ at m/z 271 (Table 2) and its product ion spectrum confirmed this. A loss of 162 amu (hexose) from the protonated molecule to yield the aglycone ion, suggested that 14 was a genistein glucoside and comparison with a standard resulted in the identification of 14 as genistein 7-O-glucoside. The protonated molecule ion [M þ H]þ of 15 was 519, which was 86 amu more than that of glycoside 14, suggesting that 15 was acylated with malonic acid. A loss of 248 amu from the protonated molecule to yield the aglycone ion also suggested the presence of malonylglucose in the glycoside. Therefore, 15 was tentatively identified as genistein 7-O-malonylglucoside. The APCI-MS data of compounds 17 and 18 (aglycone ion [A þ H]þ at m/z 255, MS3 product analysis) suggested that these compounds were glycosides of the 5deoxyisoflavone, daidzein (¼5-deoxygenistein). The UV spectra were as expected for daidzein (lmax 255 nm, shoulder at 320 nm) (Mabry et al., 1970). After comparison with the standard, isoflavone 17 was identified as daidzein 7-O-glucoside. A loss of 248 amu from the protonated molecule to yield the aglycone ion, as in the case of compound 15, suggested that compound 18 would probably be daidzein 7-O-malonylglucoside. The UV spectrum of compound 16 was very similar to that of glycoside 17, but the protonated molecule and aglycone ions were both 30 amu more (see Table 2). This suggested that 16 was a methoxylated derivative of 17. As 5-methoxyflavonoids generally have very similar UV spectra to 5-deoxyflavonoids, and 5-O-methylgenistein (¼isoprunetin) has been reported from the acid-hydrolysed extracts of species of Adenocarpus and other species of the Genisteae (Harborne, 1969), 16 was tentatively identified as 5-methylgenistein 7-O-glucoside. Compound 19 showed the typical UV spectrum of a flavanone (lmax 290 nm, shoulder at 330 nm), but no APCI-MS data were obtained for this compound, so that it could not be identified further. The short retention time suggested that it may be a naringenin glycoside. 3.2. Distribution of the compounds in the Adenocarpus species investigated Nineteen flavonoids and isoflavonoids were identified or tentatively identified in species of Adenocarpus (see Table 2) and representative structures are presented in Fig. 1. When the distribution of these compounds in the specimens of Adenocarpus was analysed, it appeared that compounds belonging to the same group of flavonoids generally occurred in the same plants. For example, plants that contained kaempferol glycosides generally also produced other flavonols such as quercetin and isorhamnetin glycosides, and plants that contained the flavone mono-C-glycoside orientin, generally also contained the flavone mono-C-glycoside vitexin. For that reason we combined the compounds into eight biosynthetic groups to make the distribution table more concise and clearer. Table 2 shows these groups, i) flavone di-C-glycosides (representing compound 1); ii) flavone mono-C-glycosides (2 and 3), iii) flavone 7-O-glycosides (4–7), iv) flavone 40 -O-glycosides (9 and 10), v) flavonol 3-O-glycosides (11–13), vi) 5-hydroxyisoflavone glycosides (14 and 15), vii) 5-deoxy- and 5-methoxyisoflavone glycosides (16–18) and viii) flavanone glycosides (19). Table 3 shows the occurrence of these eight compound groups in the plant specimens of Adenocarpus investigated. The species are arranged in this table according to the four clades shown by molecular analyses of the genus by a recent publication by Cubas et al. (2010). The country of origin of every studied plant accession is also given in this table and in the following discussion the following abbreviations will be used: MO ¼ Morocco, AG ¼ Algeria, SP ¼ Spain, CI ¼ Canary Islands, PO ¼ Portugal, TU ¼ Turkey, IT ¼ Italy, TA ¼ Tanzania, M ¼ Malawi. 3.3. Comparison of flavonoid results of Adenocarpus with those of molecular studies We did not do a cladistic analysis using the flavonoid profiles obtained for the species, as chemical features in general are not very suitable for this type of analysis because of the low number of characters available in comparison to those provided by morphological or molecular investigations. Therefore it is preferable to compare chemical profiles with arrangements provided by morphological studies or DNA sequence analyses and see whether there are any correlations. Thus we compared the results of the flavonoid research with a maximum likelihood tree produced from combined 30 end ETS and ITS data by Cubas et al. (2010), who distinguished four clades in Adenocarpus. Our own preliminary ITS analysis gave very similar results (Essokne, 2011). However, Cubas et al. (2010) were not certain of the placement of A. mannii, as this species was sister to clade 54 R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 R2 OH Glucose O HO R1 OH O Flavone C -glycosides 1 R1 = Glucose; R2 = H 2 R1 = H; R2 = OH 3 R1 = H; R2 = H R2 OR3 O R1O OH O Flavone O -glycosides 4 R1 = Rutinose; R2 = OH; R3 = H 5 R1 = Glucose; R2 = OH; R3 = H 6 R1 = Rutinose; R2 = H; R3 = H 7 R1 = Glucose; R2 = H; R3 = H 8 R1 = Glucose; R2 = OCH ; R3 = H 9 R1 = H; R2 = OH; R3 = Glucose 10 R1 = H; R2 = OCH ; R3 = Glucose R1 OH O HO OR2 OH O Flavonol O-glycosides 11 R1 = OH; R2 = Glucose 12 R1 = H; R2 = Rutinose 13 R1 = OCH3; R2 = Rutinose O R2O R1 O OH Isoflavone O-glycosides 14 R1 = OH; R2 = Glucose 15 R1 = OH; R2 = Acetylglucose 16 R1 = OCH ; R2 = Glucose 17 R1 = H; R2 = Glucose 18 R1 = H; R2 = Acetylglucose Fig. 1. Structures of the flavonoids identified from species of Adenocarpus. 3 in the ETS tree, but sister to clade 4 in the ITS tree. According to our ITS analysis this species was sister to the species in clade 4 and we have positioned it after clade 4 in Table 3 (Essokne, 2011). The species of Cubas’ clade 1, Adenocarpus anagyrifolius (MO, 4 samples), Adenocarpus cincinnatus (MO, 2 samples), Adenocarpus bacquei (MO), Adenocarpus faurei (AG) and Adenocarpus artemisiifolius (MO, 2 samples), all occur in Northern Africa and are characterised by the presence of 2–4 different types of flavone glycosides and the absence of flavonol and flavanone glycosides (see Table 3). In the majority of species isoflavanone glycosides were also absent (except for A. artemisiifolius). In all species of this clade flavone di-C-glycosides were present, but there was some infra- and interspecific variation in the presence of the other types of flavone glycosides. Flavone mono-C-glycosides were detected in all the species apart from A. faurei from Algeria and flavone 7-O-glycosides were generally present except in A. bacquei and both samples of A. artemisiifolius. All four samples of A. anagyrifolius, one sample of A. cincinnatus and two samples of A. artemisiifolius contained flavone 40 -O-glycosides, compounds which were absent from all the other clades, so that they can be considered characteristic for clade 1. However, they were absent from A. bacquei, A. faurei and one of the two samples studied of Table 3 Distribution of flavonoid glycosides in species of Adenocarpus from Europe and Africa. Adenocarpus Species Country of origin Flavone diC-glycosides Flavone monoC-glycosides Flavone 7O-glycosides Flavone 40 O-glycosides Flavonol O-glycosides 5-OH-isoflavone O-glycosides 5-Deoxy- and 5OMe-isoflavone O-glycosides Flavanone O-glycosides 18053 17823 17824 17825 Morocco Morocco Morocco Morocco 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18062 17822 18060 17816 17827 18059 Morocco Morocco Morocco Algeria Morocco Morocco 1 1 1 1 1 1 1 1 1 0 1 1 1 1 0 1 0 0 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 0 0 0 0 0 0 17814 17813 18061 18063 17836 17815 18056 18057 Spain Spain Morocco Morocco Algeria Spain Morocco Morocco 0 0 1 1 1 0 1 1 0 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 1 0 0 1 1 1 1 0 1 0 0 0 0 0 0 0 0 1 1 18048 18050 18047 18051 20870 18054 18055 17826 17812 17834 18058 18049 Morocco Morocco Morocco Morocco Morocco Morocco Morocco Morocco Portugal Spain Morocco Morocco 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 0 0 0 1 1 1 0 0 0 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 1 1 17830 17832 17833 17831 17835 17807 17808 17806 17809 17810 17828 Spain Spain Portugal Turkey Turkey Spain Spain Spain Spain Spain Canary Islands 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 Clade 1 A. anagyrifolius A. anagyrifolius A. anagyrifolius A. anagyrifolius var. leiocarpa A. cincinnatus A. cincinnatus A. bacquei A. faureia A. artemisiifolius A. artemisiifolius Clade 2 A. argyrophyllus A. hispanicus A. decorticans A. decorticans A. decorticans A. decorticans A. bracteatusb A. bracteatusb Clade 3 A. boudyi A. boudyi A. telonensis A. telonensis A. telonensis A. telonensis A. telonensis A. telonensis 24. A. telonensis 46. A. telonensis A. nainii A. complicatusc Clade 4 A. complicatus A. complicatus A. complicatus A. complicatus A. complicatus A. aureus A. lainzii A. lainzii A. desertorum A. gibbsianus A. foliolosus BI number (Kew ref.) (continued on next page) 55 56 Table 3 (continued ) BI number (Kew ref.) Country of origin Flavone diC-glycosides Flavone monoC-glycosides Flavone 7O-glycosides Flavone 40 O-glycosides Flavonol O-glycosides 5-OH-isoflavone O-glycosides 5-Deoxy- and 5OMe-isoflavone O-glycosides Flavanone O-glycosides A. viscosus 17829 Canary Islands Portugal Italy Italy Italy 1 0 0 0 1 1 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 Malawi Malawi Tanzania Tanzania 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 A. anisochilus 17811 A. bivonii 17817 A. commutatus 17818 A. brutius 17819 Sister to clade 3 or 4 A. mannii 17820 A. mannii 17821 A. mannii 17837 A. mannii 17838 a b c According to the molecular analysis by Cubas et al. (2010) A. faurei belongs to clade 4, but according to ours it belongs to clade 1 (Essokne, 2011). A. bracteatus was not analysed by Cubas et al. (2010). According to our ITS analysis (Essokne, 2011), the Moroccan sample of A. complicatus belonged to clade 3 and the European samples to clade 4. R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 Adenocarpus Species R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 57 A. cincinnatus. According to our ITS analysis (Essokne, 2011), A. faurei belongs to clade 1, but according to Cubas et al. (2010), it is a member of clade 4. The flavonoid profile of this species did not fit in completely with the species of clade 1, but much less so with the species of clade 4. The placement of A. faurei is thus unresolved. The species of clade 2 investigated for flavonoids were A. argyrophyllus (SP), A. hispanicus (SP), A. decorticans (AG, SP, MO, 2 samples) and Adenocarpus bracteatus (MO, 2 samples). A. bracteatus (¼A. complicatus var. bracteatus) was not investigated by Cubas et al. (2010), but in our ITS analysis it came out together with A. decorticans (Essokne, 2011), so that we have included this species in clade 2. The flavonoid profiles of A. argyrophyllus and A. hispanicus were identical, which is not surprising as A. hispanicus used to be a subspecies of A. argyrophyllus. All species in clade 2 produced flavone 7-O-glycosides and did not contain flavone 40 -O-glycosides nor flavonol glycosides. Flavanone glycosides were absent from all species except A. bracteatus. Clade 2 was the only group in which some of the species did not contain flavone di-C-glycosides (absent from A. argyrophyllus, A. hispanicus and the Spanish sample of A. decorticans, so absent from all the Spanish samples of clade 2). Flavone mono-C-glycosides were present in all four samples of A. decorticans (MO, AG, SP), but absent from A. argyrophyllus, A. hispanicus and A. bracteatus. On the other hand, isoflavone glycosides were present in A. argyrophyllus, A. hispanicus and three samples of A. decorticans. The species of clade 3 investigated comprise Adenocarpus boudyi (MO, 2 samples), A. telonensis (seven samples from MO; one sample from SP) and A. nainii (MO, one sample), but we also included the Moroccan sample of A. complicatus on the basis of our ITS analysis results (Essokne, 2011). The Spanish sample of A. telonensis was the only sample investigated for this clade to contain flavonol glycosides, as all the Moroccan samples lacked flavonols and contained flavone mono-C-glycosides and/or flavone 7-O-glycosides instead. All the species in this clade were chemically characterised by the presence of flavone di-Cglycosides and the absence of flavone 40 -O-glycosides and isoflavone glycosides. Flavanone glycosides were found in samples of all four species, but in three of the six samples of A. telonensis from Morocco and in the Portuguese and Spanish samples of this species flavanones could not be detected. The flavonoid profiles of the two samples of A. boudyi, A. nainii and the Moroccan specimen of A. complicatus were identical, indicating that the placement of the Moroccan sample of A. complicatus in clade 3 is justified, although all the other samples of A. complicatus studied (all from Southern Europe) belong to clade 4. In this respect it is interesting that the two Moroccan samples of A. bracteatus (¼A. complicatus var. bracteatus), which we included in clade 2, also have the same flavonoid profiles as A. complicatus from Morocco. Perhaps these samples of A. bracteatus should also be placed in clade 3, especially as they contained flavanone glycosides, which can be considered as a characteristic chemical feature of clade 3. There was much infraspecific variation in flavonoids in A. telonensis. There was not only variation in the presence and absence of flavanone glycosides among the samples, but also in the presence or absence of flavone mono-C-glycosides and flavone 7-O-glycosides. The Spanish sample of A. telonensis was the only sample in this group to produce flavonol glycosides, which compounds are characteristic for clade 4. The most homogeneous species group regarding flavonoid profiles was clade 4 of Cubas et al. (2010). The species and samples in this clade investigated for flavonoids were A. complicatus (two samples from SP, one from PO and two from TU), A. aureus (SP), A. lainzii (SP, two samples), A. desertorum (SP), A. gibbsianus (SP), A. foliolosus (CI), A. viscosus (CI), A. anisochilus (SP), A. bivonii (IT), Adenocarpus communtatis (IT) and A. brutius (IT). None of these samples were obtained from Northern Africa; they all came from Southern Europe. All these species and samples showed identical flavonoid profiles: the presence of flavone di-C-glycosides, flavonol O-glycosides and 5-hydroxyisoflavone O-glycosides and absence of flavone mono-C-glycosides, flavone 7-O- and 40 -O-glycosides, 5-deoxy- and 5-methoxyisoflavone O-glycosides and flavanone O-glycosides. Not only is there no chemical divergence in this group of species, but according to Cubas et al. (2010), the molecular divergences of the ITS and ETS sequences in this group are also almost zero, although many of the species are morphologically distinct, especially the Canary Island taxa. Many of the present species used to be subspecies of the polymorphic A. complicatus and whether they all warrant to be considered as separate species is a matter of discussion. Cubas et al. (2010) were not certain whether A. mannii was related to the species in clade 4 as it came out as sister to clade 3 in the ETS tree, but to clade 4 in the ITS tree. The four samples studied for flavonoids for this species, two from Malawi and two from Tanzania, all showed identical flavonoid profiles and these were very similar to those of the species in clade 4; presence of flavone di-C-glycosides, flavonol O-glycosides, and 5-hydroxyisoflavone O-glycosides and absence of flavone mono-C-glycosides, flavone 40 -O-glycosides and flavanone O-glycosides. However, flavone 7-O-glycosides and 5-deoxy- and 5-methoxyisoflavone O-glycosides were present in these samples, whereas they were absent from the species of clade 4. The chemical similarity between A. mannii and the species of clade 4, especially the presence of flavonol and 5-hydroxyisoflavone glycosides, suggest that A. mannii is closer to clade 4 than to clade 3, where these compound groups were virtually absent. Summarising, the flavonoid profiles in species of Adenocarpus are on the whole closely correlated with the arrangement of the species in clades on the basis of DNA sequence analyses, although the flavonoids of a few species, or samples of species, do not fit in. For instance, the Spanish sample of A. telonensis showed a flavonoid profile similar to that of many other Spanish Adenocarpus species (presence of flavonol glycosides) rather than to the Moroccan samples of A. telonensis, which only produce flavone glycosides. Furthermore, the Moroccan sample of A. complicatus lacked flavonol glycosides, whereas all South European samples of this species produced these compounds. The phenomenon that flavonoid profiles sometimes show stronger correlations with geography than lineages is well known (Marin et al., 2003) and this is thought to be caused either by introgressive hybridisation or by influences of microclimate which have become genetically fixed. Another explanation of the fact that A. complicatus from Morocco belonged to clade 3 on the basis of both our molecular and chemical evidence, whereas the European samples belonged to clade 4 could be that an ancestor of A. complicatus from Morocco (ITS sequences similar to those of clade 3 species and lacking flavonols) also was the ancestor of offspring that moved to Southern Europe, 58 R.S. Essokne et al. / Biochemical Systematics and Ecology 42 (2012) 49–58 switching to flavonol production and gave rise to the European A. complicatus aggregate with clade 4 ITS sequences. The other species of clade 4 could have evolved from this aggregate, but their chemical profiles stayed more or less the same. This hypothesis is consistent with the molecular evidence of Cubas et al. (2010). On the basis of their ETS and ITS ML tree, Cubas et al. (2010) estimated that a time-extended lineage of Adenocarpus split abruptly into two branches at the middle Miocene. The first branch of Adenocarpus separated rapidly into clades 1 and 2, whereas divergence in the second branch might have occurred during the last stages of the Middle Miocene and led to the separation of clade 3, A. mannii and clade 4. Diversification of clades 1–3 probably also occurred in the Miocene, whereas clade 4 diversified much later, during the Pliocene. This may explain why the flavonoid profiles of clades 1–3 are much less homogeneous than those of clade 4, which are all identical. The fact that the profile of A. mannii is so similar to that of the species of clade 4 suggests that A. mannii and the species of clade 4 share an ancestor in which the production of flavone glycosides was switched to flavonol glycosides, and that A. mannii is thus likely to be sister to clade 4 rather than to clade 3. According to Gibbs (1967), the genus originated either in North Africa with the ancestors of A. mannii moving to Tropical Africa and A. complicatus to Southern Europe, or the genus originated in Tropical Africa and extended to Northern Africa and Southern Europe. According to the molecular and chemical data the first explanation is much more likely. References Brullo, S., De Marco, G., 2001. Taxonomical notes on the genus Adenocarpus DC. (Leguminosae) in Italy. Bocconea 13, 425–436. Castroviejo, S., 1999a. Apuntes sobre algunos Adenocarpus (Leguminosae) Ibericos. Anales Jardín Botánico de Madrid 57, 37–46. Castroviejo, S., 1999b. Adenocarpus DC. In: Castroviejo, et al. (Eds.), Flora Iberica, 7. CSIC, Madrid, pp. 189–205. Cubas, P., Pardo, C., Tahiri, H., Castroviejo, S., 2010. Phylogeny and evolutionary diversification of Adenocarpus DC. (Leguminosae). Taxon 59, 720–732. Essokne, R.S., 2011. A Taxonomic Treatment of the Genus Adenocarpus (Leguminosae). 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