Flavonoid chemistry of Calyceraceae

Bruce A. Bohm, Alan Reid, Melanie DeVore, and Tod F. Stuessy

Abstract: Flavonoid profiles were determined for I I species representing five genera of Calyceraceae: Acicatpha, Boopis, Calycera, Garnocarpha, and Nastanthus. Kaempferol, quercetin, 6-methoxykaempferol, and 6-methoxyquercetin (patuletin) were unequivocally identified. Kaempferol and quercetin occurred as 3-0-mono- and 3-0-diglycosides, whereas the latter two flavonols were observed only as aglycones. Several unidentified phenolic compounds were also noted. The simplest profile in the family consisted solely of the kaempferol and quercetin glycosides in Acicatpha, Gamocatpha, and one specimen of Calycera leucanthema. Morphological evidence suggests that Acicatpha represents prototypical Calyceraceae and that a close relationship exists between primitive Calyceraceae and some basal members of Asteraceae (subfamily Barnadesioideae). This last possibility is reinforced by the similarity of flavonoid profiles of Acicatpha and members of Barnadesioideae.

Key words: Calyceraceae, Asteraceae, Compositae, Barnadesioideae, flavonoids.

RCsumC : Les auteurs ont ditermint les patrons des flavono'ides chez 11 espbces reprksentant 5 genres de Calyceraceae : Acicatpha, Boopis, Calycera, Gamocatpha et Nastanthus. Le kaempferol, la quercCline, le 6-mCthoxykaempf6rol et la 6-mCthoxyquercttine (patulktine) ont CtC clairement identifits. Le kaempfkrol et la quercCtine se retrouvent sous forme de 3-0-mono- et de 3-0-diglycosides alors que les deux autres flavonols se presentent sous forme d'aglycones. Plusieurs autres composts phCnoliques non-identifies ont t t t Cgalement observCs. Le profile le plus simple observe dans la famille ne comporte que les glycosides de kaempferol et de quercttine chez l'Acicatpha, le Gamocatpha et un specimen du Calycera leucanthema. Les donnCs morphologiques suggbrent que le genre Acicarpha reprCsente les Calyceraceae prototypiques, et qu'il existe une Ctroite relation entre les Calyceraceae primitives et certains membres de base des Asteraceae (sous-famille Barnadesioideae). Cette dernibre possibilitC est supportee par la similarit6 des patrons des flavono'ides de I'Acicatpha et des membres des Barnadesioideae.

Mots elks : Calyceraceae, asteraceae, compositae, Barnadesioideae, flavono'ides. [Traduit par la rCdaction]

Introduction

Calyceraceae L. C. Richard comprises six genera with 50 (Hansen 1992) to 60 species (Cronquist 1981). Members of the family occur widely in South America with one species known on the Falkland Islands. The constituent genera are Acicarpha Juss., Boopis Juss., Calycera Cavanilles, Gamo- carpha DC., Moschopsis Phil., and Nastanthus Miers. Boopis and Calycera are the largest genera with over a dozen species each. The family has attracted a good deal of atten- tion in recent years owing to its candidacy as a sister group to the Asteraceae (Hansen 1992; Gustafsson and Bremer 1995; DeVore and Stuessy 1995).

Received April 12, 1995.

B.A. Bohml and A. Reid. Department of Botany, The University of British Columbia, Vancouver, BC V6T 124, Canada. M. DeVore. Department of Biology, Sam Houston State University, Huntsville, TX 77341, U.S.A. T.F. Stuessy2. Department of Plant Biology, Ohio State University, Columbus, OH 43210, U.S.A.

' Author to whom all corresondence should be addressed. Natural History Museum of Los Angeles Co., 900 Exposition Blvd., Los Angeles, CA 90007, U.S.A.

A review of the taxonomic history of Calyceraceae shows that in most cases, the family has been accorded a position near Asteraceae, if not in the same order, then in a closely related one. For instance, Wagenitz (1964) placed Caly- ceraceae and Asteraceae in his Campanulales. Cronquist (1981), after having placed Calyceraceae in Dipsacales in an earlier publication, reconsidered the situation and accorded it its own order, Calycerales, which agrees with Takhtajan's (1986, 1987) views. Dahlgren (1983) spread these various families around with the Asteraceae in Asterales, Calycera- ceae in Dipsacales, and Goodeniaceae in Goodeniales. These three orders were assigned to Asteriflorae (along with Cam- panulales), Corniflorae, and Gentianiflorae, respectively (Dahlgren 1983). Thorne (1992) sees Asterales as com- prising Asteraceae and Calyceraceae. A concise summary of the features characterizing the family and its taxonomic history appeared in Larnrners' (1992) circumscription of Campanulales.

We undertook an examination of the flavonoids of Caly- ceraceae with the hope that these additional data might provide a better understanding of the relationships with Asteraceae. This seemed timely in view of recent efforts to arrive at phylogenetic relationships among Asteraceae and related families (Gustafsson and Bremer 1995 and citations therein). Also, a recent examination of the flavonoids of Bar-

Can. I. Bot. 73: 1962-1965 (1995). Printed in Canada / IrnprirnC au Canada

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Table 1. Phenolic compounds in Calyceraceae.

Flavonoidst Unknown phenols

Taxon Coll.No.* A B C D E F G 1 2 3 4 5 6 7

Acicatpha tribuloides 1845 + + + + Boopis graminea 1442 + + + + + + + + + Calycera crassifolia 1833 + + + + +

1838 + + + + + Calycera eryngioides 1513 + + + + + Calycera herbacea 1722 + + + + + + + + Calycera leucanthema 1486 + + + + + + + +

1431 + + + + Calycera spinulosa 1728 + + + + +

1729 + + + + Calycera sympanganthera 161 1 + + + + + Gamocatpha alpina 1447 + + + + Nastanthus spathulatus 1723 + + + + + + + t Barnadesioideae + + + +

*Collection numbers are from DeVore (1994). See Materials and methods for details. t ~ , kaempferol 3-0-glucoside; B, quercetin 3-0-glucoside; C, quercetin 3-0-galactoside; D, kaempferol 3-0-rutinoside;

E, quercetin 3-0-rutinoside; F, 6-methoxykaempferol; G, 6-methoxyquercetin.

nadesioideae (Bentham & Hooker) Bremer & Jansen provided a view of what the primitive flavonoid profile of the Astera- ceae might have been (Bohm and Stuessy 1995). It is the pur- pose of the present paper to describe flavonoids encountered in Calyceraceae and to speculate on possible relationships between Barnadesioideae and prototypical Calyceraceae.

Materials and methods

Sources of plant material Acicarpha tribuloides Juss., DeVore 1845: Argentina, Buenos Aires, Punta Lara: Boopis graminea Phil., DeVore 1442: Argentina, Buenos Aires, Punta Blanko; Calycera crassifolia (Miers) Hicken, DeVore 1833: Argentina, Buenos Aires, Cabo San Antonio; DeVore 1838: Argentina, Buenos Aires, Mar del Plata; Calycera eryngioides Remy, DeVoire 1513: Chile, Santiago, Lagunilles; Calycera her- bacea Cav., DeVore 1722: Argentina, Mendoza, E of Puenta del Inca; Calycera leucanthema (Poepp. ex Miers) Kuntze, DeVore 1431: Chile, Bio-Bio, Village of Recinto; DeVore 1486: Chile, Talca, S of Vilches Alto; C. spinulosa C. Gillies ex Miers, DeVore 1728: Argentina, Mendoza, NE of Los Arboles; DeVore 1729: Argentina, Mendoza, E of Los Arboles; Calycera sympanganthera (Ruiz & Pav.) Kuntze, DeVore 1611: Chile, Piedra del Aquila; Gamo- carpha alpina (Poepp. ex Less.) H. V. Hansen, DeVore 1447: Chile, Bio-Bio, Termas de Chillan; Nastanthus spathulatus (Phil.) Hicken, DeVore 1723: Argentina, Men- doza, Las Cuevas. Duplicate voucher specimens are deposited at the Ohio State University (0s ) and the University of Wisconsin -0shkosh (OSH). Aerial parts of the plants to be used for chemical analysis were dried over silica gel in the field.

ldentification of flavonoids Plant material was extracted with 80% methanol in water and the extract was evaporated to dryness in vacuo. The flavo- noid fraction was isolated from the resulting gummy residue

by extraction with boiling water followed by extraction of the aqueous solution with water-saturated n-butanol. Evapora- tion of the butanol extract afforded a residue that was exam- ined by two-dimensional thin layer chromatography and then subjected to column chromatography using Sephadex LH-20 according to procedures described in detail by Gornall and Bohm (1980). Structures of purified flavonoids were deter- mined using standardized ultraviolet (Mabry et al. 1970) and electron impact mass spectroscopic (Markham 1982) methods.

Results and discussion

Only limited information on chemical constituents of Caly- ceraceae has appeared in the literature. Members of the family accumulate inulin as storage polysaccharide (Pollard and Amuti 1981), and seco-loganin, an iridoid derivative, was reported from A. tribuloides (Jensen et al. 1975). Mabry and Bohlmann (1977) stated that Calyceraceae do not pro- duce acetylenes. Additional absences recorded for Acicarpha pinnatifida (syn. A. tribuloides) include sedoheptulose, alu- minum accumulation, raphides, tannins and leucoantho- cyanins, cyanogenic glycosides, saponins, and I-inositol (Gibbs 1974). The present work appears to be the first description of flavonoids of the family. Brief reference to these data was made in our recent discussion of Bar- nadesioideae (Bohm and Stuessy 1995).

Seven flavonoids were conclusively identified in the present study: kaempferol and quercetin 3-0-glucosides, quercetin 3-0-galactoside, kaempferol and quercetin 3-0-rutinosides, and the 6-methoxy derivatives of kaempferol and quercetin (patuletin). The latter two compounds were found to occur as free phenols. An additional eight phenolic compounds, as judged by fluorescent behaviour and response to spray rea- gents, were observed on chromatograms but were not identi- fied owing to insufficient material. The flavonoid profiles of the taxa studied were based upon glycosides of kaempferol, quercetin, 6-methoxykaempferol, and 6-methoxyquercetin (patuletin). Details of the occurrence of these compounds are

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given in Table 1. For comparison purposes typical flavonoid data for Barnadesioideae are included in the table.

Recent studies relied on macromolecular data (Chase et al. 1993; Michaels et al. 1993; Olmstead et al. 1993) to examine relationships among various groups of angiosperms including Asteraceae, Calyceraceae, and related families. Two additional studies utilized morphological and selected micromolecular characters of Calyceraceae (Hansen 1992; Gustafsson and Bremer 1995). Hansen's (1992) study of Calyceraceae focused on pollen and petal epidermis features and although directed primarily at gaining a better under- standing among genera within the family, also discussed relationships among Calyceraceae and other families, most prominent of which were Asteraceae and Lobeliaceae.

Flavonoid data collected in the present study provide addi- tional information for examining relationships among the various families in question, particularly Asteraceae. Owing to the vast amount of information available on flavonoids of the Asteraceae (B.A. Bohm and T.F. Stuessy, unpublished data), almost any combination of flavonoid structural types and modifications may be found in some member or group within the family. It is not surprising, then, that the flavonoid chemistry of Calyceraceae falls well within the bounds of structural variation known of Asteraceae. Flavonols, which constitute the major class of flavonoids present in Caly- ceraceae, are common in all tribes of Asteraceae, and 6-substitution is also a common structural feature of astera- ceous flavonoid profiles. A discussion of evolutionary rela- tionships between Asteraceae and Calyceraceae as entire units in terms of these flavonoid observations would be only marginally informative. However, a closer look at flavonoid profiles of relevant groups within Asteraceae proved of greater value.

The observation by Jansen and Palmer (1987) that mem- bers of the Barnadesia group of genera (Barnadesia Mutis ex L. f., Chuquiraga Juss., and Dasyphyllum Kunth.) lack a chloroplast DNA inversion present in all other members of Asteraceae led to a profound change in thinking about what constituted the basal group in the family. These recon- siderations led to establishment of a new subfamily, Barna- desioideae (Bentham & Hooker) Bremer and ans sen, to accommodate genera formerly placed in Mutisieae subtribe Barnadesiineae (Bremer and Jansen 1992). With the avail- ability of material of 31 species (of ca. 90) representing seven of the nine recognized genera of ~arnadesioideae, we previously undertook a study of the flavonoid chemistry of the subfamily (Bohm and Stuessy 1995). The flavonoid pro- file of Barnadesioideae is simple, consisting of kaempferol and quercetin 3-0-mono- and-3-0-diglycos~des with infre- quent occurrence of eriodictyol and a single instance of an isorhamnetin derivative. We concluded in that paper (Bohm and Stuessy 1995, p. 23) that ". . . no other major taxon within the Asteraceae (i.e., other subfamily, tribe, or sub- tribe) has such a consistently simple pigment profile . . ."

Turning our attention back to Calyceraceae, we see that three specimens exhibit a flavonoid profile consisting solely of kaempferol and quercetin 3-0-mono- and 3-0-diglyco- sides, viz. A. tribuloides, G. alpina, and one specimen of C. leucanthema. These observations are of some signifi- cance insofar as Acicalpha has been advanced as representa- tive of prototypical Calyceraceae (DeVore 1994). This

position is based on its pattern of floral development (centri- petal), its diploid chromosome number (n = 8), which repre- sents the only diploid known so far from the family (Sugiura 1936, 1937; Raven et al. 1965; Rodrigues-Correia et al. 1977), and its range of occurrence, which is similar to that of Schlectendalia Less. (Barnadesioideae) in the grasslands of Uruguay and adjacent regions. The limited number of samples available to us notwithstanding, addition of very similar flavonoid profiles to this list of features certainly strengthens the idea of close relationship between primitive elements in Calyceraceae and similarly ancestral members of Asteraceae.

It has not escaped the authors' notice that despite macro- molecular efforts, which certainly speak for a close relation- ship between Calyceraceae and Asteraceae, it is a set of secondary metabolite data that gives us an even closer look at potential evolutionary relationships within this interesting group of organisms. A detailed macromolecular study of representatives of these groups might, nonetheless, be infor- mative.

Acknowledgements

Laboratory studies were supported by operating and equip- ment grants from the Natural Sciences and Engineering Research Council of Canada (to B.A.B.). Field studies were supported by the Beatley Herbarium Fund and Latin Ameri- can Studies Program of Ohio State University. Additional support was provided by National Geugraphic Society grant NO. 4459-91 (to T.F.S.).

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