A chemosystematic survey of the fern genus Bommeria

Biochemical SystematicsandEcology, Vol. 10, No. 2, pp. 107-110, 1982.

0305-1978182/020107-04 $(]8.00/0 ¢) 1982 Pergamon Press Ltd.

Printed in Great Britain.

A Chemosystematic Survey of the Fern Genus Bommeria CHRISTOPHER H. HAUFLER" and DAVID E. GIANNASIt • Department of Botany, University of Kansas, Lawrence, KS 66045, USA; t Botany Department, University of Georgia, Athens, GA 30602, USA

Key Word Index - B o m m e d a ; Pteridaceae; ferns; flavonoids; chemosystematics. /Idael~¢t - A phytochemical survey of the species of B o m m e # a showed that foliage of the members of this genus contains a variety of flavonol and flavone glycosides. The distributions of chemical and morphological characters in B o m m e r i a correlate to circumscribe infrageneric affinities: B. hispida and B. subpaleacea comprise one evolutionary line, while B. p e d a t a and B. e h r e n b e r g i a n a form a second. Flavonoid data may also be applied in concert with spore morphology in defining intergeneric species groups within B o m m e r i a and Hemionitis.

IWo'oducllJon The fern genus Bommefia consists of four small terrestrial species that are restricted to xeric or seasonally dry habitats in the south-western United States, Mexico and central America. Recent systematic treatment of Bommeria [1] shows it to be closely related to both the gymnogrammoid and cheilanthoid ferns. The interpretation of affinities both between and within the genera that comprise these groups has proved to be quite challenging. Perhaps more than in any other group of ferns, morphological characters fail to correlate in delimiting genera and in erecting logical hierarchies of relationship. This apparent evolutionary complexity has led investigators to consider evidence from a broad spectrum of sources. Recent investigations have demonstrated the value of chemosystematics in work with both gymnogrammoid [2-4] and cheilanthoid [5] genera and have shown that chemical data often correlate with morphological and cytological data [3-6]. In the present paper, we will discuss the identification and systematic significance of the flavonoid components of Bommeria foliage.

Results Composite chromatographic flavonoid patterns for the four species of Bommeria recognized by Haufler [1] are shown in Fig. 1. No qualitative differences in flavonoid profiles were detected between mature and immature (without ripe


®

®

o

® TBA FIG. 1. TWO-DIMENSIONAL COMPOSITE CHROMATOGRAM OF SPOT DISTRIBUTIONS IN BOMMERIA SPECIES: See Table 2 for identification of compounds.

TABLE 1. DISTRIBUTION OF FLAVONOIDS IN BOMMERIA SPECIES. Flavonols O° B B B. B

h/spida subpaleacea peclata ehrenberglana

K"

+ + +

+

1

2

+ + + +

+ + + +

3

4

+ +

Flavones

5

6

7

8

+

+

+ + + +

+ + + +

9

• Found as tree aglycone on chromatograms.

(Received 9 N o v e m b e r 1981 ) 107

Glycoflavones

Unknown

L°

A°

10

11

12

13

14

15

16

17

+

+ +

+

+

+ + + -t-

+ -t-

+ +

+

+

+

108

CHRISTOPHER H HAUFLER AND DAVID E GIANNASI

TABLE 2. IDENTITY AND PERTINENT CHROMATOGRAPHY AND SPECTRAL DATA OF FLAVONOIDS IN BOMMERIA

Solvents 1

Colors ~:

Absorption maxima in nm§

UV

UV NH3

0.63

O

GY

0.46

0.46

D

GY

0.72

0.73

D

G

4, O-3-O-

0.36

0.73

D

D

5. Q-3-O-

0.41

0.73

D

log

6. Q-3-O-

0.53

0.62

D

YG

7. K-3-O-

0.53

0.69

D

G

0.56

0.52

D

G

0.65

0.58

P

G

0.50

0.34

D

D

0.45

0.15

P

D

0.25

0.65

P

G

0.39

0.31

P

pG

0.31

0.18

P

pY

0.15

0.43

P

YG

0,65 0.06

0,52 0.01

P Y

G Y

No. Identity* 1. Q-3-O-

I

II

0.40

rutinoside 2. Q-3-O-

glucoside 3. Q-3-O-

glycoside glycoside

glycoside II glycosiclell rutinoside

8. K-3-O-

glucoside g. K-3-O-

glycoside 10. A-4'-O-

arabinoside 11. L-3'-O-

glucoside 12. A-6-C-

glycoside 13. A-8-C-

glycoside 14. L-8-C-

glycoside 15. L-6,8-di -

C-glycoside 16, Unknown 17. Unknown

MeOH

AICI3

AICI3 HCI

NaOMe

NaOAc NaOMe

H3BO3

356 254 356 254 355 256 353 253

432

399

407

265

377

436

401

413

268

379

435

401

409

269

374

429

397

407

265

373

435

401

410

250

371

432

399

407

270

375

392

392

401

271

348

400

397

403

272

350

402

395

403

274

356

385

380

365

278

320

385

383

395

270

340

384

386

402

282

331

387

380

398

278

330

432

400

401

261

260

428

395

418

279

368

352 251 354 253 346 264 351 265 350 268 322 269 337 240 333 274 330 259 350 254 350 256

"A = aptgenin, L = luteolin, K = kaempferol, O = quercetin. t Solvents: I - tert-butanol-acetic a¢id-water (3:1:1, v/v); II - acetic acid-water (15:85, v/v). ¢ P = purple, Y =yellow, G =green, D =dark, p= pale. §All maxima are band I (visible) except for MeOH which includes band II for calculation ot shift against band II in NaOAc for the 7-hydroxyl position.

IISugars at position 3: compound 5, galactose and rhamnose; compound 6, glucose and arabinose. Sequence of sugars not determined.

sporangia) leaves of the plants investigated. Each species possessed a distinct flavonoid pattern (Table 1) comprised of flavonols and flavones, the former being more ubiquitous and diverse than the latter (Table 2). All species contained quercetinand kaempferoI-O-glycosides (compounds 1-9) and an apigenin-6-C-glycoside (compound 12). In addition Bommeria hispida and B. subpaleacea contained luteolin O- and C-glycosides (compounds 11,14, 15). Intraspecific variation was found in the glycoside patterns but this variability was not systematically significant. It is interesting to note, however, that B. hispida showed more intraspecific glycosidic variability than any of the other species and was the only species to have the rare B-ring 4'- or 3'-O-glycosylations (compounds 10 and 11, respectively). In B. subpaleacea and B. ehrenbergiana, this lack

of variability may be attributed to the limited geographic range and consequent small sample size of these species. In B. pedata, however, low intraspecific variation may be a result of its apomictic life cycle [7]. Overall, the chemical data circumscribe two species groups within Bommeria: B. pedata and B. ehrenbergiana confain kaempferol, quercetin and apigenin glycosides while B. hispida and B. subpaleacea confain these constituents together with luteolin glycosides. Dilcu=don Chemical investigations of ferns have not been extensive (see [8, 9] for reviews) and many have been concerned with individual species (e.g. [2, 1012]) or with broad overviews (e.g. [13-15]). Although these studies serve to introduce the

A CHEMOSYSTEMATICSURVEYOF BOMMERIA

diversity of chemical compounds in ferns, they do not demonstrate the value of phytochemistry in fern systematics at the generic level. The few available chemosystematic studies of whole genera [3, 16] or groups of related species [5, 17-20] do indicate, however, that chemical analyses can be applied in assessing relationships among such fern taxa. The present study of Bommefia shows the value of chemosystematic data both in determining intrageneric relationships and in suggesting supragenic alliances. Previous studies of Bommeria [1, 21] have shown that, as with other members of the gymnogrammoid ferns [22], the majority of morphological characters do not correlate to define species alliances. The present chemical survey, in conjunction with an earlier study of spore micromorphological features [21], indicates that the distribution of flavonoid compounds corresponds mainly to differences in spore morphology. The species containing luteolin glycosides (B. hispida and B. subpaleacea) have cristate spores while those lacking these compounds (B. pedata and B. ehrenbergiana)have reticulate spores. The flavonoid and spore data along with scale morphology [1] define two evolutionary lines in Bommetia. The flavonoid chemistry of Bommeria has implications in interpreting other infrageneric relationships. The disjunct distribution of B. subpaleacea, for example, was originally defined as representing two distinct species [23]. Recent morphological comparisons [ 1], however, showed a breakdown in the distinctness of these putative species. Chemical analyses corroborated these findings since collections representing both "species" had a similar set of flavonoid profiles. Chemical evidence (i.e. lack of luteolin and possession of a similar set of compounds) also indicates the diploid B. ehrenbergianato be the most likely species to serve as the parent of the hybrid-derived, triploid apomict B. pedata. Aspects of spore morphology and lamina indument indicate that Bommeria hispida and B. subpaleacea are the more advanced members of the genus. Both of these species have cristate spores which may be ontogenetically derived from the reticulate ones found in the other two Bommeria species [21]. Bommefiahispida and B. subpaleaceaalso have more robust scales than the other species [1], a situation that indicates evolutionary advancement [24]. Evidence from flavonoid chemistry corroborates this morphological evidence. The overall greater diversity of chemical compounds and especially the elaboration of flavone O- and C-glycosides [25,

109

26] indicate that B. hispidaand B. subpaleaceaare the more advanced species. Intergenerically, Copeland [27] considered Bornrnefia to be most closely allied to Hemionitis but this close relationship was not precisely defined. As discussed by Giannasi [4], the flavonoid composition and spore morphology of Bommefia and Hemionitis agree in suggesting affinities between the two genera. All Bommetia species and a majority of Hemionitisspecies have either cristate spores or a closely related reticulate type [21,22] and are characterized by the presence of flavonol 3-O-glycosides. The remaining Hemionitis species have tuberculate spores [22], ar,d only these species uniquely contain flavonol3,4'-O-diglycosides. Similar B-ring substitutions in Bommetia occur only in B. hispida and only in flavones. In both Bommefiaand Hemionitis, however, morphological features fail to correlate in forming species clusters like those delimited by spore morphology and chemical evidence. We have shown that flavonoid chemical evidence can be valuable in sorting out specific and generic relationships in a systematically complex group of ferns. Chemical surveys of other.qenera in the gymnogrammoid-cheilanthoid alliance coordinated with a re-evaluation of morphological features may provide new insights into the natural classification of these taxa.

Experimental Leavesfor chemical analyseswere collected from greenhouse cultures of adult sporophytes. Vouchers of the collections that originated these cultures (Bommeria hispida: Haufler 6` Haufler 1-3, Texas, USA; 4-6, Arizona, USA; 7, New Mexico, USA; Haufler 6" Brown 15, Chihuahua, Mexico; Gestony 6` Haufler 1073, Jalisco, Mexico. Bornmeda pedata: Haufler 6` Brown 8, Jalisco, Mexico; 9-10, Michoacan, Mexico; 11, Guerrero, Mexico; Gestony 6` Haufler 1072, Jalisco, Mexico. Bommeria subpaleacea: Haufler 6` Brown 14, Hidalgo, Mexico; Haufler25, 28, Chihuahua, Mexico. Bommeriaehrenberg~ana." Gastony 6" Haufler 1086, Hidalgo, Mexico) are deposited at IND and GH. Vouchers of the Mexican collections are also deposited at M EXU. These cultured sporophytea were grown from field collections of rhizornes. Approximately3 g of dried, powdered leaf material from each of these cultured sporophytes was extracted in absolute methanol. The extracts were concentrated in vacuo and comparative 2-D PC flavonoid profiles were obtained using standard procedures and solvents [28]. Flavonoid analysis employed further preparative 2-D PC (samples of 5 g or less) or CC (samples of 5 g or more). For preparative CC, glass columns of various sizes were used depending on the sample size. Dried leaf material was first extracted in 80% MeOH (aq.). The extract was reduced In vacuo until only an aq. slurry remained in the flask. This was diphased several times against chloroform in a separatory funnel to remove chlorophyll and other non-flavonoid constituents [29]. The chloroform portion was monitored by

110 PC or cellulose TLC to ensure that no flavonoids escaped our attention. The flavonoid-containing aq. portion was taken to dryness and redissolved in 10% aq. methanol and an aliquot applied to a glass column packed with Sephedex LH-20 in the same solvent. Development of the column used increasing increments (10%) of MeOH in water until all flavonoids and other phenolics (observed under UV light) were collected. The column was finally regenerated with acetone. Purification of individual flavonoids in each column fraction was carried out with PC and cellulose and/or polyamide TLC [30]. UV spectra of flavonoids followed standard procedures [28]. Hydrolysis of glycosides employed trifluoroacetic acid followed by 1-D PC or TLC of the sugars against known standards [30]. Sugars were visualized by spraying the chromatograms with p-anisidine hydrochloride followed by heating in an oven at 100 ° for 30 min [31]. In a few cases, glycosides were treated with f3-glucosidase for sugar identification or confirmation [28, 32].

Acknowl~lgqmNmm-Our thanks to Becky Wilcox and James Manhart for their technical assistance on aspects of the chromatographic and spectral analyses of the flavonoids. Chemical analyses were supported in part by NSF Grant DEB7904551 awarded to D. E. Giannasi and the Systematics Training Grant, Indiana University. Field work was partially funded by grants-in-aid of research to C. H. Haufter from the Indiana Academy of Science, the Office of Research and Advanced Study at Indiana University, and Sigma Xi, the Scientific Research Society of America.

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