Highly Glycosylated Flavonols at the Genistoid Boundary and The

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South African Journal of Botany 89 (2013) 181–187

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South African Journal of Botany

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Highly glycosylated ﬂavonols at the genistoid boundary and the systematic position of Dermatophyllum

G.C. Kite a,⁎, N.C. Veitch a, M. Soto-Hernández b, G.P. Lewis a a Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK b Colegio de Postgraduados, Campus Montecillo, Texcoco, Estado de México 56230, Mexico article info abstract

Available online 1 July 2013 Among papilionoid legumes known to express the phenotype of quinolizidine alkaloid production, only Dermatophyllum occurs outside of the genistoid clade in phylogenetic analyses of DNA sequence data. Analysis Edited by B-E Van Wyk of the foliar flavonoid glycosides of Dermatophyllum and possibly related clades, by liquid chromatography-UV spectrophotometry-mass spectrometry, revealed that taxa sampled from Dermatophyllum, Amphimas and Keywords: the Cladrastis, lecointeoid and vataireoid clades contained mostly flavonol O-glycosides whereas taxa sampled Leguminosae from early-branching genistoid clades, the Andira clade and Aldina contained mostly flavone C-glycosides. Dermatophyllum Furthermore, leaves of Dermatophyllum secundiflorum and Dermatophyllum arizonicum contained, as their Calia fl fl α → α Genistoids main avonoids, two highly glycosylated avonols: kaempferol 3-O- -rhamnopyranosyl(1 2)[ - Flavonol glycosides rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside and its quercetin analogue. These compounds also occurred in Cladrastis kentukea, Styphnolobium japonicum and Pickeringia montana in the Cladrastis clade, Uribea tamarindoides and some samples of Zollernia in the lecointeoid clade, and in Amphimas pterocarpoides (another genus of uncertain relationships). The alkaloid and flavonoid pheno- types of Dermatophyllum each suggest affinities to different groups — aconflict which is accommodated by the current phylogenetic hypothesis, based on molecular data, that the genus is a possible sister to the genistoid clade but not a member of it. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction et al., 2011). Quinolizidine alkaloids have been reported in leaves, stems, roots and seeds of Dermatophyllum secundiﬂorum (Ortega) Within Leguminosae subfamily Papilionoideae, phylogenetic anal- Gandhi & Reveal (Chavez and Sullivan, 1984; García-Mateos et al., yses of DNA sequence data (‘molecular analyses’) have generally re- 2007; Izaddoost et al., 1976; Ketter, 1975) and recently in leaves of solved a well-supported genistoid clade (Cardoso et al., 2012; Doyle Dermatophyllum arizonicum (S. Watson) Vincent and Dermatophyllum et al., 1997; Pennington et al., 2001; Wojciechowski et al., 2004). Stud- gypsophilum (B.L. Turner & A.M. Powell) Vincent (Lee et al., 2013). ies on the chemistry of members of this clade suggest that they share In molecular analyses to date, Dermatophyllum has been placed close the common phenotype of quinolizidine alkaloid production, except to the genistoid clade of other quinolizidine alkaloid-containing taxa for some genera in tribes Crotalarieae and Genisteae which instead but never within it. Initial trnL sequence analyses placed the genus produce pyrrolizidine alkaloids (Van Wyk, 2003). Although legume among several unresolved sister groups to the genistoids (Pennington taxa outside of the genistoid clade may possess the genes for et al., 2001). A subsequent matK analysis provided resolution of some quinolizidine alkaloid synthesis (Wink and Mohamed, 2003), only of these potential sister groups and placed members of the lecointeoid one genus, Dermatophyllum, is known to produce these metabolites clade between the genistoid clade (and other unresolved groups) and (García-Mateos et al., 2007; Ketter, 1975; Lee et al., 2013). Reports Dermatophyllum, although with only moderate support (Wojciechowski of other legume taxa outside of the genistoid clade accumulating et al., 2004). In the most recent family-wide analysis of matK sequence quinolizidine alkaloids have proved to be erroneous (Kite and data, which included increased sampling among taxa in the boundary Pennington, 2003). between the genistoid clade and related groups, phylogenetic resolution Dermatophyllum Scheele, formerly segregated as Calia Terán & again collapsed and Dermatophyllum was a monogeneric group among Berland from Sophora sensu lato, is a genus of four species of trees several well-supported but unresolved clades that comprised the large and shrubs occurring from south-western USA to Mexico (Gandhi unresolved 50 kb-inversion clade (containing most papilionoid legumes); this clade also included Amphimas Pierre ex Harms and Aldina Endl. as the two further unresolved genera (Cardoso et al., 2012). The ⁎ Corresponding author. Tel.: +44 208 332 5368; fax: +44 208 332 5310. similarity in alkaloid chemistry between Dermatophyllum and the E-mail address: [email protected] (G.C. Kite). genistoid clade, and the poor support in molecular data separating

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.06.003 182 G.C. Kite et al. / South African Journal of Botany 89 (2013) 181–187 them, creates uncertainty in the placement of Dermatophyllum within analogue, quercetin 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopy- the papilionoid phylogeny. ranosyl(1 → 6)]-β-glucopyranoside-7-O-α-rhamnopyranoside (4), A new line of enquiry, using chemical characters, on the relation- was again isolated from leaves of S. japonicum (Kite et al., 2007). ships of Dermatophyllum was recently suggested following the de- Analysis by liquid chromatography-UV spectrophotometry-mass tection of highly glycosylated flavonols in leaves of D. secundiflorum spectrometry (LC-UV-MS) has shown that leaves of C. kentukea con- (Barrón-Yánez et al., 2011). These compounds were examples of tain all of 1–4 (Kite et al., 2011), so for convenience we will refer to tetraglycosides of kaempferol and quercetin bearing a branched tri- flavonol tetraglycosides with this glycosylation pattern as Cladrastis- saccharide at C-3 (glucose or galactose with both a (1 → 2) and a type highly glycosylated flavonols (CHGFs). (1 → 6) terminally linked rhamnose) and a rhamnose at C-7 (Fig. 1). To understand further the significance of the occurrence of CHGFs Although not particularly unusual in structural terms, flavonols with in Dermatophyllum, we have carried out a survey of the flavonoid these glycosylation patterns have not been widely reported in glycosides of 28 taxa selected from across the genistoid boundary; plants, indeed the first records were from legumes placed close to i.e. early-branching taxa of the genistoid clade and taxa from po- Dermatophyllum in molecular analyses. Kaempferol 3-O-α-rhamnopy- tential sister groups of the genistoid clade. Leaf extracts were ana- ranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7- lysed by LC-UV-MS specifically for CHGFs 1–4, although the data O-α-rhamnopyranoside (1)wasfirst described from leaves of acquired could also be used to determine the general types of Zollernia ilicifolia (Brongn.) Vogel (Coelho et al., 2003), a member flavonoid glycoside present. While the flavonoid chemistry of of the lecointeoid clade, and the glucose analogue of this com- temperate members of the core genistoids is reasonably well pound, kaempferol 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopy- known (e.g. Harborne, 1969), there have been fewer studies of the ranosyl(1 → 6)]-β-glucopyranoside-7-O-α-rhamnopyranoside (2), flavonoids of early-branching genistoid clades, as many are less was described from leaves of Styphnolobium japonicum (L.) Schott readily accessible tropical tree species (Van Wyk, 2003). Tropical (Kite et al., 2007), a member of the Cladrastis clade; these clades are woody taxa are also prevalent among potential sister groups to the the sister groups to Dermatophyllum in the analysis of Wojciechowski genistoid clade (Lewis et al., 2005), and their flavonoid chemistry is et al. (2004). Quercetin 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopy- likewise poorly known. Thus the present study also aimed to com- ranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside (3) pare the general profile of flavonoid glycosides of early-branching was detected in leaves of Cladrastis kentukea (Dum. Cours.) Rudd genistoids and potential sister groups, including Dermatophyllum. (Kite et al., 2011), although not formally described, while its glucose 2. Materials and methods

2.1. Plant material and sample preparation R 3' CH3 OH Details of the taxa analysed and the source of material are listed in HO 4' the Appendix 1. Dry leaf material (20–100 mg, weighed accurately) O O O HO 7 OH was powdered in a pestle and mortar with sand and transferred to O CH3 an Eppendorf tube. Aqueous 80% methanol was then added (1 μl per HO 3 O OH 5 OH mg of plant material) and the sample was left overnight (ca 18 h) at 7-O-Rha OH O O 1'' 2'' 2''-O-Rha room temperature (ca 22 °C). Following centrifugation, the superna- O OH tant was poured into a second Eppendorf tube and diluted with an Gal OH equal volume of water. After an hour standing at room temperature, CH3 any precipitate that had formed was removed by centrifugation and HO 6'' the supernatant was poured into an autosampler vial for LC–MS 6''-O-Rha O HO O analysis. HO 2.2. Analysis by LC-UV-MS 1 R = H 3 R = OH Samples were analysed using a Thermo Scientific LC-UV-MS system comprising an ‘Accela’ 1290 pump, autosampler and PDA de- R tector interfaced to an ‘LTQ-Orbitrap XL’ hybrid mass spectrometer 3' ‘ ’ CH3 OH via an Ion-Max electrospray source. Separation of the glucose and HO 4' galactose analogues of CHGFs 1 & 3 and 2 & 4 was achieved by O O O performing high resolution chromatography of 2 μl injections on a HO 7 OH O CH3 150 mm × 2.1 mm, 1.9 μm Hypersil GOLD C18 column (Thermo Sci- HO 3 OH fi μ 5 O OH enti c) using a 400 l/min mobile phase gradient of 95:0:5 to 0:95:5 7-O-Rha OH O O water/methanol/acetonitrile + 1% formic acid over 100 min, follow- 1'' 2'' 2''-O-Rha ing 3 min pre-injection equilibration in start conditions. Tuning of O OH Glc the electrospray source and calibration of the mass spectrometer CH followed the manufacturer's procedures and recommended settings. 3 6'' 1 HO OH High resolution (30,000) first order mass spectra (MS ) were acquired 6''-O-Rha O in negative mode over the range m/z 250 to 2000 by the orbitrap HO O analyser while, simultaneously, the ion trap acquired low resolution HO MS1 (m/z 125–2000) and serial mass spectra (MS2 and MS3) in both negative and positive modes. For serial mass spectrometry, the most 2 R = H abundant three or four ions in the preceding lower order scan were 4 R = OH selected successively and fragmented using an ion isolation window of ±2 m/z units and a relative collision energy of 35%. These data Fig. 1. Structures of flavonol tetraglycosides 1–4. The homologous triglycosides 1a–4a lack the 7-O-Rha, the triglycosides 1b–4b lack the 2″-O-Rha, and the diglycosides 1c–4c dependent acquisitions were directed by the instrument software lack both Rha residues (7-O-Rha and 2″-O-Rha). (Xcalibur 2.0.7), which was also used to analyse the data. G.C. Kite et al. / South African Journal of Botany 89 (2013) 181–187 183

Screening samples for CHGFs 1–4 was achieved by searching 3 1 12.8 the high resolution MS data for ions within 2 ppm of the theoretical 100 D.sec A − m/z value of the deprotonated molecules [M–H] (i.e. m/z 885.2670 80 for 1 & 3 and m/z 901.2619 for 2 & 4) at the expected retention 60 1 times. The latter were obtained from the analysis of a leaf extract 40 15.1 of C. kentukea (Kite et al., 2011). The identification of 1–4 was 20 3a confirmed through examination of UV absorbance and serial 0 mass spectra (Kite and Veitch, 2009, 2011). General detection of 1 certain flavonol O-glycosides, flavone O-glycosides and flavone 15.1 C.ken B 100 4c C-glycosides was achieved by filtering the serial mass spectrometric 80 20.1 data. O-Glycosides of quercetin, kaempferol, luteolin and isobaric 60 1a flavonoids were detected by filtering the positive mode MS2 data for 40 3 4a spectra containing aglycone product ions at m/z 303 or 287, resulting 12.9 2 17.2 from loss of all the O-linked sugars. Flavone C-glycosides were 20 4 22.8 fi 2 0 detected by ltering the negative ion MS spectra for a neutral loss 1b of 120 Da, resulting from internal ring cleavage of a C-linked hexose 20.0 100 P.mon C sugar. For the most abundant signals detected by these filtering 80 methods, the chromatographic peaks responsible were located and their mass spectrometric and UV data were examined manually both 60 3b 1 to confirm the general identification and distinguish between O- 40 17.1 15.1 glycosides of flavonols and flavones. Data for any major UV absorbing 20 3 peaks not accounted for by the above filtering methods were also 0 1 examined manually to record the occurrence of other flavonoid 15.0 U.tam Relative Absorbance, 350 nm (%) 100 D glycosides. 80 3. Results and discussion 60 40 1a 3.1. CHGFs in Dermatophyllum and the Cladrastis clade 20 19.6 0 Comparing the flavonoids present in leaf extracts of D. secundiflorum 1 15.1 100 A.pte E with those of C. kentukea by high resolution LC-UV-MS revealed that 1a the major CHGFs in D. secundiflorum were 1 and 3 (Fig. 2A & B); 80 3a 19.6 21.4 previously in the low resolution analyses of Barrón-Yánez et al. 60 16.5 (2011) the hexose sugars of these CHGFs had been assigned 40 arbitrarily to glucose. Support for the identificationof1and3came 20 3 22.7 from serial mass spectrometric data using the methods of Kite and 0 Veitch (2009).CHGFs1 and 3 were estimated from UV absorbance to 10 15 20 25 be the major flavonoid glycosides in all three samples analysed of Time (min) D. secundiflorum (Table 1). Quercetin 3-O-α-rhamnopyranosyl(1 → 2) [α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside (3a), i.e. 3 lacking Fig. 2. LC-UV (350 nm) chromatograms of leaf extracts of Dermatophyllum secundiflorum (A), Cladrastis kentukea (B), Pickeringia montana (C), Uribea tamarindoides the 7-O-Rha, was also present in these samples together with, as (D) and Amphimas pterocarpoides (E). See Fig. 1 for compound numbering. minor components, 1a (the homologous triglycoside of 1) and the 3-O-α-rhamnopyranosyl(1 → 6)]-β-galactopyranosides of kaempferol and quercetin (1cand3c). These tri- and diglycosides were observed in trace levels in the samples studied. Pickeringia therefore contained C. kentukea, together with minor triglycosides lacking the 2″-O-Rha (1b the same triglycosides as C. kentukea but in different proportions. and 3b) and the glucose analogues of all these (2a–c, 4a–c) (Kite et al., Notably, triglycosides lacking the 6″-O-Rha (e.g. kaempferol 3-O-α- 2011). Like C. kentukea, leaves of D. secundiflorum contained acylated rhamnopyranosyl(1 → 2)-β-galactopyranoside-7-O-α-rhamnopyran- derivatives of flavonol glycosides, although these had different re- oside) were missing in all the above mentioned CHGF-producing taxa. tention times to those described from C. kentukea; they are not charac- Molecular phylogenies indicate that Cladrastis is not monophyletic, terized further here. The one sample of D. arizonicum examined with Cladrastis platycarpa (Maxim.) Makino separated from C. kentukea contained 1 and 3 as the major flavonoid glycosides together with the by Styphnolobium and Pickeringia (Wojciechowski et al., 2004). Anal- same flavonol triglycosides observed in D. secundiflorum. This sample ysis of C. platycarpa indicated that it also contained a kaempferol also contained 2 (the glucose analogue of 1)and2a as minor compo- tetraglycoside but the retention time of this compound was different nents, but not 4 (the glucose analogue of 3). Acylated flavonol glyco- from that of 1 or 2, and serial mass spectrometry indicated that it sides were minor components in this sample. bores a linear trisaccharide at C-3. The major flavonoid glycoside was The presence of CHGFs in leaves of Styphnolobium, a second genus the triglycoside homologue that lacked the 7-O-Rha. The triglycosides in the Cladrastis clade, has been reported on previously (Kite et al., noted among other members of the clade were absent. Further sam- 2007). Levels of CHFGs in S. japonicum leaves are generally low rela- pling of this taxon is required to confirm these initial observations. tive to the high accumulation of rutin (4c) (Table 1). A third genus in the clade is the monospecific Pickeringia Torr. & Gray. The leaf 3.2. CHGFs in Zollernia, Uribea and Amphimas extracts of Pickeringia montana Torr. & Gray examined here contained 1 and a lower level of 3; the major flavonoid glycoside was 1b CHGF 1 was first described from leaves of Z. ilicifolia (Brongn.) Vogel, (kaempferol 3-O-α-rhamnopyranosyl(1 → 6)-β-galactopyranoside- a member of the lecointeoid clade (Coelho et al., 2003). Leaf samples 7-O-α-rhamnopyranoside) with lower levels of 3b(Fig. 2C). The from two specimens of Z. ilicifolia together with single samples from triglycosides 1a and 3a were relatively minor components of the three other species (Zollernia glabra (Spreng.) Yakovlev, Zollernia flavonoid glycoside profile, and acylated flavonol glycosides were at grandifolia Schery and Zollernia latifolia Benth.) were examined here 184 G.C. Kite et al. / South African Journal of Botany 89 (2013) 181–187

Table 1 Flavonoid glycosides in early branching genistoids and related clades.

Ref. Species Relative abundance (%) Flavonoid glycosides

1234Flavonol O-glyc. Flavone O-glyc. Isoﬂavone O-glyc. Flavone C-glyc.

Dermatophyllum 22377 Dermatophyllum arizonicum 59 9 100 ++ 15586 Dermatophyllum secundiﬂorum 36 100 ++ 15587 ″ 50 100 ++ 22378 ″ 45 100 ++

Cladrastis clade 15514 Cladrastis kentukea 100 18 20 9 ++ 18552 Cladrastis platycarpa ++ 18565 Pickeringia montana 19 3 ++ 14415 Styphnolobuim japonicum 34b1 b1++

Lecointeoid clade 22467 Exostyles amazonica ++ 22466 Harleyodendron unifoliolatum + 22375 Holocalyx balansae ++ 22376 Lecointea amazonica ++ 22379 Uribea tamarindoides 100 1 ++ 22393 ″ 100 95 ++ 22391 Zollernia glabra ++ 22374 Zollernia grandifolia ++ 22391 Zollernia ilicifolia ++ 22392 ″ 7++ 22390 Zollernia latifolia 38 6 ++

Amphimas 22468 Amphimas pterocarpoides 100 – 5 – ++ ++ 22698 ″ 100 – 3 – ++ ++

Vataireoid clade 22381 Luetzelburgia auriculata ++ 22380 Sweetia fruticosa ++ ++

Ormosia clade 22382 Ormosia nobilis ++

Brongniartieae 22388 Brongniartia inconstans + 22387 Brongniartia ulbrichiana + 22386 Harpalyce rupicola +++ 14084 Poecilanthe parviﬂora ++

Bowdichia clade 22469 Leptolobium dasycarpum ++ 22384 Leptolobium panamense +++ 22385 Guianodendron praeclarum ++ +

Aldina 22470 Aldina insignis +++

Andira clade 4705 Andira legalis +++

Relative abundance of 1–4 is the UV 350 nm chromatographic peak height expressed as a percentage of the peak height of the most abundant flavonoid glycoside present. For flavonoid glycosides, ++ indicates numerous and/or high abundance, + indicates few and/or low abundance, based on LC-UV–MS data. to investigate this occurrence further. Kaempferol O-glycosides were individuals especially in the degree to which the leaf margin is serrated; found to be the major flavonoid glycosides in these samples of Zollernia sample BI 22391 bore leaves with a rounded apex and strongly serrate with quercetin O-glycosides and acylated flavonol glycosides being margin with prickles, and sample BI-22392 bore leaves with rounded only minor components or undetectable. CHGF 1 was only detected to acute apices and a weakly serrate leaf margin with prickles. Further in Z. latifolia and one of the two samples of Z. ilicifolia (BI-22392). In sampling is required to determine whether the variation in flavonoid both these samples containing 1,andthesamplesofZ. glabra and chemistry in Z. ilicifolia correlates with this morphological variation, Z. grandifolia, the major flavonoid glycoside was 1a; a minor glucose an- although it is clear from the current sampling of taxa that presence of alogue (2a) of this kaempferol triglycoside was also present. The other CHGFs is not a consistent feature of the genus Zollernia as a whole. sample of Z. ilicifolia (BI-22391), however, contained an abundant Among the other taxa sampled in this study, CHGFs were only kaempferol triglycoside with a different glycosylation pattern, namely detected in leaves of two species: Uribea tamarindoides Dugand & kaempferol 3-O-α-rhamnopyranosyl(1 → 2)-β-galactopyranoside-7- Romero and Amphimas pterocarpoides Harms (Table 1; Fig. 2D&E). O-α-rhamnopyranoside. As noted above, flavonol triglycosides with In both specimens sampled of the monospecific Uribea, 1 was the this glycosylation pattern were not detected among members of the major flavonoid glycoside component, together with the triglycoside Cladrastis clade producing CHFGs. The major flavonol glycoside in 1a. CHGF 3 was also a major flavonoid glycoside in specimen Z. ilicifolia (BI 22391) was kaempferol 3-O-β-galactopyranoside-7-O- BI-22393 together with 3a, but specimen BI-22379 contained only α-rhamnopyranoside. Leaf morphology in Z. ilicifolia,ascurrently low relative amounts of these compounds. Glucose analogues of circumscribed (Mansano et al., 2004), varies greatly between these flavonoid glycosides could not be detected in either specimen. G.C. Kite et al. / South African Journal of Botany 89 (2013) 181–187 185

The flavonoid glycoside chemistry of the two specimens of A. leaves of Luetzelburgia auriculata (Allemão) Ducke also contained fla- pterocarpoides examined had 1, 1aand3aasmajorflavonoids, with vonol O-glycosides (mainly O-glycosides of kaempferol), while leaves 3 as a minor component. Leaves of both species contained abundant of Sweetia fruticosa Spreng. contained flavonol O-glycosides and acylated flavonoid glycosides. prenylated isoflavone O-glycosides. Uribea is placed in the lecointeoid clade on the basis of molecular In contrast, the taxa sampled from early-branching genistoid clades evidence (Cardoso et al., 2012; Pennington et al., 2001; Wojciechowski (the Ormosia, Brongniartieae and Bowdichia clades) contained flavone et al., 2004), but within this clade CHGFs were only otherwise detected C-glycosides, although in some specimens (e.g. the two specimens in the specimens of Zollernia mentioned above. The affinities of of Brongniartia) these were not always abundant or numerous, based Amphimas are uncertain (Lewis et al., 2005); recently DNA sequence on UV absorbance. In some members of the Brongniartieae and the data for Amphimas (represented by A. pterocarpoides) has been obtained Bowdichia clade, the flavone C-glycosides co-occurred with flavone for the first time and this places it as an unresolved genus in the large O-glycosides, the latter as minor or single components; Guianodendron 50 kb-inversion clade (Cardoso et al., 2012). praeclarum (Sandwith) R. Schütz & A.M.G. Azevedo additionally contained isoflavone O-glycosides. Flavone C-glycosides were also pro- 3.3. Flavonoid glycoside types in early branching genistoids and duced by the non-genistoids Aldina insignis (Benth.) Endl. and Andira related clades legalis (Vell.) Toledo, representing the isolated genus Aldina and the Andira clade — two other unresolved members of the 50 kb inversion All members of the lecointeoid clade examined produced flavonol clade in the phylogenetic analysis of Cardoso et al. (2012). In both O-glycosides as the major class of flavonoid glycosides. Except for taxa the flavone C-glycosides co-occurred with minor flavonol Uribea and some samples of Zollernia, none contained flavonol O-glycosides. tetraglycosides, and the glycosylation patterns of the major flavonol tri- and diglycosides present were generally different to those of 3.4. Flavonoid glycoside affinities of Dermatophyllum CHGF-producing taxa. For example the main flavonol glycosides in Lecointea amazonica Ducke and Exostyles amazonica Yakovlev had Based on the limited sampling performed in this study, early a C-3 disaccharide with a (1 → 2) linkage (namely kaempferol branching genistoids and their possible sister groups can be divided 3-O-β-glucopyranosyl(1 → 2)-β-galactopyranoside and its 7-O-α- into those that produce flavone C-glycosides as the main type of flavo- rhamnopyranoside, respectively) whereas in CHGF-producing taxa noid glycosides (the early branching genistoids comprising the Ormosia, the glycosides having a disaccharide at C-3 had a (1 → 6) linkage Brongniartieae and Bowdichia clades, and the non-genistoids Aldina and and were terminated by rhamnose rather than a hexose sugar. A flavo- the Andira clade) and those that do not (Dermatophyllum, Amphimas nol glycoside with an unusual disaccharide at C-3 has been reported and the Cladrastis, lecointeoid and vataireoid clades). This distinction from Holocalyx balanse Micheli (as Holocalyx glaziovii Micheli), namely can be visualised when the data acquired by LC–MS are filtered for kaempferol 3-O-β-glucopyranosyl(1 → 4)-α-rhamnopyranoside-7- mass spectrometric signals indicative of flavonoid C-glycosides and flavo- O-α-rhamnopyranoside (Haraguchi and Gimaraes, 1992), and this noid O-glycosides (Fig. 3). compound was detected as a major flavonoid in the LC–MS analyses The flavonoid phenotype of Dermatophyllum, therefore, is differ- performed here. Of the two taxa sampled from the vataireoid clade, ent from early-branching genistoids even though these taxa share

Flavonoid O-glycosides Flavonoid C-glycosides

100 100 D.sec A

H.bal B

L.aur C

O.nob D

flavone H.rup E

flavone L.pan F Relative abundance (%)

A.ins G

A.leg H

0 0 010203040010203040 Time (min) Time (min)

Fig. 3. LC–MS2 analyses showing mass spectrometric signals for O- and C-glycosides of flavonoids in leaf extracts of Dermatophyllum secundiflorum (A), Holocalyx balanse (B), Luetzelburgia auriculata (C), Ormosia nobilis (D), Harpalyce rupicola (E), Leptolobium panamense (F), Aldina insignis (G) and Andira legalis (H). Signals for O-glycosides were flavonol O-glycosides except where indicated in E and F. 186 G.C. Kite et al. / South African Journal of Botany 89 (2013) 181–187 the alkaloid phenotype of quinolizidine alkaloid expression, a phe- BI-22378. Exostyles amazonica Yakovlev: Brazil, 24/03/1988, M.J. notype that otherwise strongly supports the genistoid clade. This Pites & N.T. Silva 2054 (K), BI-22467. Guianodendron praeclarum conflict in the chemical affinities of Dermatophyllum tends to sup- (Sandwith) R. Sch. & A.M.G. Azevedo: Guyana, 17/05/1943, D.C.O. port the current phylogenetic position of the genus, i.e. as a possible 65,759 (K), BI-22385. Harleyodendron unifoliolatum R.S. Cowan: sister to the genistoid clade but not within it. The flavonoid data on Brazil, 03/02/1999, V.F. Mansano, J.G. Jardim & S.C. de Sant'Ana 45 Dermatophyllum suggests that, of the possible related non-genistoid (K), BI-22466. Harpalyce rupicola Donn. Sm.: Guatemala, 09/04/ groups, the genus shows greater affinity with the Cladrastis, lecointeoid 1991, C.E. Hughes 1470 (K), BI-22386. Holocalyx balansae Micheli: and vataireoid clades and Amphimas, rather than Aldina and the Andira Bolivia, 17/03/2005, J.R.I. Wood & A. Haigh 21887 (K), BI-22375. clade. The prominence of CHGFs in the flavonol O-glycoside profile of Lecointea amazonica Ducke: Costa Rica, 11/11/1992, K. Thomsen 531 Dermatophyllum might further suggest affinities with Amphimas and (K), BI-22376. Leptolobium dasycarpum Vogel: Brazil, 16/11/2009, B.B. some members of the Cladrastis and lecointeoid clades, although incon- Klitgaard 1185, BI-22469. Leptolobium panamense (Benth.) Sch. Rodr. & sistencies in the presence of CHGFs among closely related taxa A.M.G. Azevedo: El Salvador, 20/02/1989, G.P. Lewis & C.E. Hughes (e.g. Cladrastis platycarpa in the Cladrastis clade and among mem- 1739 (K), BI-22384. Luetzelburgia auriculata (Allemão) Ducke: Brazil, bers of the lecointeoid clade) casts doubt on the reliability of this 19/07/1997, J.A. Ratter, J.F. Ribeiro, S. Bridgewater & J. Fonsêca Filho character as a phylogenetic indicator of wider relationships. R7719 (K), BI-22381. Ormosia nobilis Tul.: Venezuela, 28/01/2005, K.M. There is also evidence that CHGFs occur among members of the Redden 3370 (K), BI-22382. Pickeringia montana Torr. & A. Gray: USA, earliest-branching papilionoid lineages. CHGFs have been reported from 09/08/1932, A. Eastwood 18817 (K), BI-18565. P. montana:USA,28/4/ leaves of Cordyla madagascariensis R. Vig. and Cordyla haraka Capuron 2013, M.F. Wojciechowski 1953 & J.L. Wojciechowski (ASU). P. montana: (Veitch et al., 2008). In the latter species they occurred with two flavo- USA, 28/4/2013, M.F. Wojciechowski 1954 & J.L. Wojciechowski (ASU). nol O-pentaglycosides: kaempferol 3-O-α-rhamnopyranosyl(1 → 3)-α- Poecilanthe parviflora Benth.: Cult. (RBG Kew), 6/4/2005, 1994–1230, rhamnopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyra- BI-14084. Styphnolobium japonicum (L.) Schott: Cult. (RBG Kew), 11/ noside-7-O-α-rhamnopyranoside and its quercetin analogue; i.e. CHGFs 08/2005, 1905–51006, BI-14415. Sweetia fruticosa Spreng.: Paraguay, bearing an additional rhamnosyl group. The Cordyla pentaglycosides undated, Hassler 6211 (K), BI-22380. Uribea tamarindoides Dugand & and their glucose analogues were subsequently reported from pods of Romero: Costa Rica, 02/02/1995, R.T. Pennington, N. Zamora & R. Aguilar Bobgunnia madagascariensis (Desv.) J.H. Kirkbr. & Wiersema (Stevenson 579 (K), BI-22379, U. tamarindoides: Costa Rica, 31/01/1991, T.D. et al., 2010), although the presence of CHFGs was not mentioned. Flavo- Pennington & N. Zamora 13384 (K), BI-22393. Zollernia glabra (Spreng.) nol tetraglycosides having a different glycosylation pattern to CHFGs Yakovlev: Cult. (RBG Kew), 15/08/2003, 1993–3632,BI-12670.Zollernia have been reported from leaves of Mildbraediodendron excelsum Harms. grandifolia Schery: Brazil, June 1995, W. Milliken 2367 (K), BI-22374. (Veitch et al., 2005a)andAteleia chicoasensis J. Linares (Veitch et al., Zollernia ilicifolia (Brongn.) Vogel: Brazil, 19/11/1978, S.A. Mori, T.S. dos 2005b). Santos & C.B. Thompson 11197 (K), BI-22391. Z. ilicifolia: Brazil, 21/09/ It is interesting that highly glycosylated flavonoids are being 2005, G. Hatschbach & E. Barbosa 79402 (K), BI-22392. Zollernia latifolia reported among the early-branching papilionoid legumes but are oth- Benth.: Brazil, 27/09/1984, P.M. Andrade & M.A. Lopes 431 (K), BI-22390. erwise not widely reported in most members of the subfamily. 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