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Flavonoid variation in Lasthenia california (Asteraceae)
Biochemical SystemaEcs and Ecology, Vol. 21, No. 4, pp. 449-453, 1993. Printed in Great Britain.
0305-1978/93 $6.00+0.00 © 1993PergamonPressLtd.
Flavonoid Variation in Lasthenia californica (Asteraceae) ANDRI~E M. DESROCHERS and BRUCE A. BOHM Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
Key Word Index--Lasthenia californica; Asteraceae; flavonoids; populational variation. Abstract--Five-hundred and sixty-three individuals of Lasthenia californica representing the entire range of the species in California, Arizona, Oregon and Baja California were examined for their flavonoid chemistry. With only a few minor exceptions our findings agree with the view that four pigment profiles exist in the species (Bohm et al., 1989). An additional 33 populations of L. californica were also sampled. The known flavonoid profiles were found to predominate, but there was a distinct polarity to the occurrence of the flavonoid types: simpler profiles tend to occur in the northern part of California and in Oregon, while the more complex profiles appear in southern California, Arizona and Baja California. A new flavonoid profile was detected in two populations along the coast north of San Francisco. Minor variants of the basic flavonoid types have also been noted; further structural work is indicated. Our observations support the view that extensive differentiation within the species is now reflected in the existence of numerous morphological, ecological, cytological and chemical races.
Introduction Lasthenia californica DC. ex Lindley is the most widespread of the 17 species in this mostly Californian genus. The distribution of L. californica ranges from southern Oregon throughout California, from the foothills of the Sierra Nevada to the coast, east into Arizona and in the northern part of Baja California (Ornduff, 1966). This species varies in several features: it occupies the widest range of habitats within the genus; it displays a considerable amount of morphological variation; and it includes both diploid (n = 8) and tetraploid (n = 16) populations (Ornduff, 1966). Previous studies on the flavonoid chemistry of L. califomica showed a variable profile of compounds at both the inter- and intrapopulation levels (Ornduff etal., 1974; Bohm et aL, 1989). In the earlier study, however, only a few populations were examined, and pooled samples were used. In the more recent study we examined a population of L. califomica located in the Jasper Ridge Biological Preserve of Stanford University over a period of 6 years. Since the populations examined in these two studies represented only a small part of the range of L. californica, we undertook the current study in an effort to assess the overall flavonoid variability of this species. Materials and Methods Ray florets of L. ca/ifornica were removed from individual capitulescences from herbarium sheets, placed in vials, covered with a few drops of MeOH and allowed to stand at room temperature for at least a day. Extracts from ca 25 florets were spotted on 20;<20 cm Polyamid 6.6 plates (home-made) and developed onedimensionally in a mixture of water-n-butsnol-acetone-dioxane (70:15:10:5). After drying, the plates were sprayed with aminoethyl-diphenylborate, again allowed to dry in the air and scored under 366 nm UV. Populations were sampled by collecting, at random, at least 30 individuals. The locations of the populations studied are presented in Table 1. In a few cases, individuals were lost, so Table 1 shows lower numbers for three populations. Larger samples were taken from proportionately larger populations, i.e. Jasper Ridge, Mt Tamalpais and Tehachapi. Population samples were extracted and chromatographed as described above.
Results and Discussion Examination of the Jasper Ridge population of L. calfforn/ca (Bohm et aL, 1989), using individual plants, showed the presence of four flavonoid profiles, which we have designated A-D. Profile C consists of aurone and chatcone monoglycosides and a set (Received3 November 1992)
449
450
A.M. DESROCHERS AND B. A. BOHM
TABLE 1. LOCATION OF THE POPULATIONS USED FOR FLAVONOID ANALYSIS OF LASTHENIA CALIFORNICA
Arizona
Counties
Location
Population
Yavapai Yavapai Pima
Bagdad Hillside Tanque Verde
433 434 435
Mission (near) Cantamer
436 437
Baja California
California
San Benito Monterey Kern San Diego Ventura Marin Sonoma Tehama Riverside Santa Barbara San Luis Obispo San Benito Santa Clara Tuolumne Amador Sacramento San Mateo Marin Marin Sonoma Shasta Fresno San Diego Riverside Los Angeles Fresno
Panoche (near) Priest Valley (near) Tehachapi Cuyamaca Lake Ventucopa (South of) Mt Tamalpais Salt Point Station Pk Red Bluff Lake Matthew Los Olivos Santa Margarita Hollister Hwy 152, East of Santa Nella Jamestown Jackson Clay Jasper Ridge Point Reyes Dillon Beach Fort Ross Millville Hwy 198 Anza Borrego Murrieta Palmdale Dunlap
323 325 328 330 331 333 335 425 438 439 440 441 442 443 444 445 447-462 449 450 451 460 464 468 471 473 475
Oregon
Jackson Jackson
Gold Hill Table Rock
453 454
of flavone and flavonol glucuronides. Profile B adds luteolin 7-glucoside. Profile A has the fundamental C profile plus sulphated kaempferol and quercetin diglycosides plus prominant eriodictyol glycosides. Profile D, the rarest of the four, is similar to the A profile except that the flavonol diglycosides lack the sulphate group. The present study addressed two questions. (1) Does the existence of the four flavonoid profiles, as reported from Jasper Ridge, represent the range of structural variants in the species, or do other profiles exist? (2) What is the populational structure, with regard to flavonoids, of L. californica in other parts of its range? The first question was addressed by examining individual flower heads of L. califomica taken from the entire range of the species. This was done by removal of one ray floret per plant from herbarium specimens. This sampling provided information for plants from seven counties in Arizona, 44 counties in California and three counties in Oregon, plus collections from Baja California. In all, 563 individuals were tested (398 herbarium sheets). The Arizona specimens (76 individuals) exhibited only type A profiles; the California specimens exhibited profiles based on types A (321 individuals) and C (124 individuals); and specimens from Oregon (15 individuals) exhibited consistently type C profiles. Specimens from Baja California (24) exhibited 22 type A profiles and two type B profiles. Profile types E} and D were seen only rarely in Californian specimens (3) and not at all in those from Arizona and Oregon. This study
FLAVONOID VARIATION IN LASTHENIACALIFORNICA(ASTERACEAE)
451
satisfied us that the four pigment profiles observed to date represent the range of variation present in the taxon. Subsequent observations caused us to modify this conclusion, however (vide infra). The second question concerned the structure of populations of L. californica. Detailed examination of flavonoid variation of L. californica sampled along fixed transects in the Jasper Ridge Biological Preserve of Stanford University showed the persistence of four pigment profiles over a period of 6 years. Two of these profiles, A and C, were the predominant profiles seen. Profiles B and D were seen much less frequently. The most striking outcome of our work in Jasper Ridge was the observation that the spatial distribution of the two major types, A and C, remained remarkably constant over a period of six seasons, 1982-1987 (Bohm et al., 1989). During the 1989 and 1990 seasons, plants were again collected along the original Jasper Ridge transects. Flavonoid analyses of these plants showed that the patterns of occurrence of the A and C profiles had not changed (profiles B and D were again only minor components). If we are allowed to assume that nothing unusual happened in the 2 years during which collections were not made (1987 and 1988) the 1989-1990 results suggest that the spatial distribution of pigment types has remained essentially constant for a period of 10 years. Recent morphometric studies of the fruits, plus analysis of electrophoretic banding patterns of leaf isozymes of plants collected in the Jasper Ridge Preserve yielded patterns of occurrence that correspond very closely with the occurrence of the flavonoid profiles (Desrochers, unpublished results). These observations will be presented and discussed in detail elsewhere. Thirty-two additional populations of L. californica have now been examined for their flavonoid profiles. Three of these populations were from Arizona, two from Baja California, two from Oregon and the rest were from throughout the species' range in California. With the exception of two populations (nos 449, 451) the profiles were clearly discernible as A, B, C or D. The two unusual profiles were modified B profiles that require additional detailed chemical study and represent the only significant deviation from the original four profiles that we have seen. We have also noticed minor variants within the two major profile types, A and C. These variants involve compounds present at lesser to barely detectable levels. Structural work on these compounds has not been undertaken owing to their low concentrations and the limited amount of plant material available. The distribution of profile types in the populations is presented in Table 2. The geographical distribution of the pigment types is presented in Fig. 1. Reference to the figures shows that, although there are a few mixed populations, with both A- and B/C types present, the majority of populations exhibit either the A- or B/C-type profile. The distribution of profile types is not random within the range of the species. The two Oregon populations and the two most northerly populations from California exhibit the B/C profile. The populations from central California are either A- or B/C-types while the populations in southern California, here defined as south of San Francisco Bay, are predominantly A-type; 86-100% of the individuals exhibit type A profiles in these populations. Populations 440 and 475 are the only exceptions for this region. One Arizona and one Baja California population are also exclusively A types. The other three populations from these areas are mixed types having mostly A profile plants. We do not have an explanation for the variability of the flavonoid,profiles exhibited by L. californica. At this time we can only point out that our observations of flavonoid, electrophoretic and morphological variability of the species are in agreement with Ornduff's (1966) comments concerning its heterogeneity. The distribution of the three variable features is not consistent throughout the species' range in that different combinations of flavonoid, electrophoretic and morphological features occur in different populations. Furthermore preliminary findings of breeding experiments
452
A. M. DESROCHERS AND 8. A. 8OHM TABLE 2. FLAVONOID PROFILESOCCURRING IN EACH POPULATION OF LASTHENIA CALIFORNICA
Populations 323 325 328 33O 331 333 335 425 433 434 435 436 437 438 439 440 441 442 443 444 445 447* 449 450 451 453 454 460 462* 464 468 471 473 475
A
B
40 36 65 33 35 54 25
D
1
1 10 6
2
4 1 30 2
1 5 30t
8 4 123
30 30t
69 30 30 30 32 11
1
6 5 33
32 25 20 19 30 26 29 27 30 30 22 22 54
C
18
30 30 33 98
24
1
Total no. of individuals 40 36 65 35 35 60 30 33 32 28 30 25 30 30 30 30 30 30 30 30 27 182 30 30 30 30 30 33 185 30 30 30 32 35
*Jasper Ridge population, tModified profile.
suggest that plants with the A-type flavonoid profile and plants with the C-type profile do not appear to cross (Desrorchers, unpublished results). This also agrees with Ornduff's (1966) observation that plants from different populations exhibit a low level of crossability. Ornduff (1966) attributed the diversity of this species to evolutionary responses to climatic and geological changes that have occurred in California and to the relative age of the species. Lasthenia californica occupies several areas that became available recently, in terms of geological time. These areas were submerged during the Pliocene, or as late as the Pleistoc( me in some cases, and include the San Joaquin Valley, the southern part of the Sacramento Valley, large areas of southern California and most valleys of western Oregon. It is likely that L. califomica has undergone a considerable degree of differentiation as it migrated to newly available sites. Given the relatively old age of the species this differentiation is now reflected in the existence of numerous morphological, ecological, cytological and chemical races. Only time will tell which or how many of these populations will differentiate sufficently to be recognized as species in their own right.
FLAVONOID
VARIATION
IN
453
LASTHfN/A CALIf0ffNK.X (ASTERACEAE)
l
9
0
FIG. 1. GEOGRAPHICAL
DlSTFtl6UTlON
location of the sampled
population.
OF FLAVONOID
The percentage
ing, with solid circles = 100%. 0 = Modified
PROFILE TYPES FOR
of flavonoid
433 434
LASTHENM C4LFORNC4. Each circle indicates the
profile type-A in a population
is displayed
by increased
darken-
profile type.
Acknowledgements-This work was supported by operating and equipment grants from the Natural Sciences and Engineering Research Council of Canada. We wish to acknowledge the curators of the herbaria from which specimen sheets of L. califomica were borrowed and for permission to sample these sheets for ray florets. The Administration and staff of Jasper Ridge Biological Preserve deserve special thanks for making the facilities at the Preserve available to us; we are much in their debt. Professor Ornduff’s interest in our work over the years is also much appreciated.
References Bohm, B. A., Herring, A., Nicholls, K. W., Bohm, L. Ft. and Ornduff, R. (1989) A six-year study of flavonoid variation in a population of Lasthenia caliknica. Am. J. Bat. 76, 157-163. Ornduff, R. (1996) A biosystematic study of the goldfield genus Lasthenia. Univ &/if. Pub. Bot. 46, I-92. Ornduff, R., Saleh, N. A. M. and Bohm, B. A. (1974) Intraspecific variation of flavonoids in Lasthenia. Brittonia26, 411-420.
0305-1978/93 $6.00+0.00 © 1993PergamonPressLtd.
Flavonoid Variation in Lasthenia californica (Asteraceae) ANDRI~E M. DESROCHERS and BRUCE A. BOHM Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
Key Word Index--Lasthenia californica; Asteraceae; flavonoids; populational variation. Abstract--Five-hundred and sixty-three individuals of Lasthenia californica representing the entire range of the species in California, Arizona, Oregon and Baja California were examined for their flavonoid chemistry. With only a few minor exceptions our findings agree with the view that four pigment profiles exist in the species (Bohm et al., 1989). An additional 33 populations of L. californica were also sampled. The known flavonoid profiles were found to predominate, but there was a distinct polarity to the occurrence of the flavonoid types: simpler profiles tend to occur in the northern part of California and in Oregon, while the more complex profiles appear in southern California, Arizona and Baja California. A new flavonoid profile was detected in two populations along the coast north of San Francisco. Minor variants of the basic flavonoid types have also been noted; further structural work is indicated. Our observations support the view that extensive differentiation within the species is now reflected in the existence of numerous morphological, ecological, cytological and chemical races.
Introduction Lasthenia californica DC. ex Lindley is the most widespread of the 17 species in this mostly Californian genus. The distribution of L. californica ranges from southern Oregon throughout California, from the foothills of the Sierra Nevada to the coast, east into Arizona and in the northern part of Baja California (Ornduff, 1966). This species varies in several features: it occupies the widest range of habitats within the genus; it displays a considerable amount of morphological variation; and it includes both diploid (n = 8) and tetraploid (n = 16) populations (Ornduff, 1966). Previous studies on the flavonoid chemistry of L. califomica showed a variable profile of compounds at both the inter- and intrapopulation levels (Ornduff etal., 1974; Bohm et aL, 1989). In the earlier study, however, only a few populations were examined, and pooled samples were used. In the more recent study we examined a population of L. califomica located in the Jasper Ridge Biological Preserve of Stanford University over a period of 6 years. Since the populations examined in these two studies represented only a small part of the range of L. californica, we undertook the current study in an effort to assess the overall flavonoid variability of this species. Materials and Methods Ray florets of L. ca/ifornica were removed from individual capitulescences from herbarium sheets, placed in vials, covered with a few drops of MeOH and allowed to stand at room temperature for at least a day. Extracts from ca 25 florets were spotted on 20;<20 cm Polyamid 6.6 plates (home-made) and developed onedimensionally in a mixture of water-n-butsnol-acetone-dioxane (70:15:10:5). After drying, the plates were sprayed with aminoethyl-diphenylborate, again allowed to dry in the air and scored under 366 nm UV. Populations were sampled by collecting, at random, at least 30 individuals. The locations of the populations studied are presented in Table 1. In a few cases, individuals were lost, so Table 1 shows lower numbers for three populations. Larger samples were taken from proportionately larger populations, i.e. Jasper Ridge, Mt Tamalpais and Tehachapi. Population samples were extracted and chromatographed as described above.
Results and Discussion Examination of the Jasper Ridge population of L. calfforn/ca (Bohm et aL, 1989), using individual plants, showed the presence of four flavonoid profiles, which we have designated A-D. Profile C consists of aurone and chatcone monoglycosides and a set (Received3 November 1992)
449
450
A.M. DESROCHERS AND B. A. BOHM
TABLE 1. LOCATION OF THE POPULATIONS USED FOR FLAVONOID ANALYSIS OF LASTHENIA CALIFORNICA
Arizona
Counties
Location
Population
Yavapai Yavapai Pima
Bagdad Hillside Tanque Verde
433 434 435
Mission (near) Cantamer
436 437
Baja California
California
San Benito Monterey Kern San Diego Ventura Marin Sonoma Tehama Riverside Santa Barbara San Luis Obispo San Benito Santa Clara Tuolumne Amador Sacramento San Mateo Marin Marin Sonoma Shasta Fresno San Diego Riverside Los Angeles Fresno
Panoche (near) Priest Valley (near) Tehachapi Cuyamaca Lake Ventucopa (South of) Mt Tamalpais Salt Point Station Pk Red Bluff Lake Matthew Los Olivos Santa Margarita Hollister Hwy 152, East of Santa Nella Jamestown Jackson Clay Jasper Ridge Point Reyes Dillon Beach Fort Ross Millville Hwy 198 Anza Borrego Murrieta Palmdale Dunlap
323 325 328 330 331 333 335 425 438 439 440 441 442 443 444 445 447-462 449 450 451 460 464 468 471 473 475
Oregon
Jackson Jackson
Gold Hill Table Rock
453 454
of flavone and flavonol glucuronides. Profile B adds luteolin 7-glucoside. Profile A has the fundamental C profile plus sulphated kaempferol and quercetin diglycosides plus prominant eriodictyol glycosides. Profile D, the rarest of the four, is similar to the A profile except that the flavonol diglycosides lack the sulphate group. The present study addressed two questions. (1) Does the existence of the four flavonoid profiles, as reported from Jasper Ridge, represent the range of structural variants in the species, or do other profiles exist? (2) What is the populational structure, with regard to flavonoids, of L. californica in other parts of its range? The first question was addressed by examining individual flower heads of L. califomica taken from the entire range of the species. This was done by removal of one ray floret per plant from herbarium specimens. This sampling provided information for plants from seven counties in Arizona, 44 counties in California and three counties in Oregon, plus collections from Baja California. In all, 563 individuals were tested (398 herbarium sheets). The Arizona specimens (76 individuals) exhibited only type A profiles; the California specimens exhibited profiles based on types A (321 individuals) and C (124 individuals); and specimens from Oregon (15 individuals) exhibited consistently type C profiles. Specimens from Baja California (24) exhibited 22 type A profiles and two type B profiles. Profile types E} and D were seen only rarely in Californian specimens (3) and not at all in those from Arizona and Oregon. This study
FLAVONOID VARIATION IN LASTHENIACALIFORNICA(ASTERACEAE)
451
satisfied us that the four pigment profiles observed to date represent the range of variation present in the taxon. Subsequent observations caused us to modify this conclusion, however (vide infra). The second question concerned the structure of populations of L. californica. Detailed examination of flavonoid variation of L. californica sampled along fixed transects in the Jasper Ridge Biological Preserve of Stanford University showed the persistence of four pigment profiles over a period of 6 years. Two of these profiles, A and C, were the predominant profiles seen. Profiles B and D were seen much less frequently. The most striking outcome of our work in Jasper Ridge was the observation that the spatial distribution of the two major types, A and C, remained remarkably constant over a period of six seasons, 1982-1987 (Bohm et al., 1989). During the 1989 and 1990 seasons, plants were again collected along the original Jasper Ridge transects. Flavonoid analyses of these plants showed that the patterns of occurrence of the A and C profiles had not changed (profiles B and D were again only minor components). If we are allowed to assume that nothing unusual happened in the 2 years during which collections were not made (1987 and 1988) the 1989-1990 results suggest that the spatial distribution of pigment types has remained essentially constant for a period of 10 years. Recent morphometric studies of the fruits, plus analysis of electrophoretic banding patterns of leaf isozymes of plants collected in the Jasper Ridge Preserve yielded patterns of occurrence that correspond very closely with the occurrence of the flavonoid profiles (Desrochers, unpublished results). These observations will be presented and discussed in detail elsewhere. Thirty-two additional populations of L. californica have now been examined for their flavonoid profiles. Three of these populations were from Arizona, two from Baja California, two from Oregon and the rest were from throughout the species' range in California. With the exception of two populations (nos 449, 451) the profiles were clearly discernible as A, B, C or D. The two unusual profiles were modified B profiles that require additional detailed chemical study and represent the only significant deviation from the original four profiles that we have seen. We have also noticed minor variants within the two major profile types, A and C. These variants involve compounds present at lesser to barely detectable levels. Structural work on these compounds has not been undertaken owing to their low concentrations and the limited amount of plant material available. The distribution of profile types in the populations is presented in Table 2. The geographical distribution of the pigment types is presented in Fig. 1. Reference to the figures shows that, although there are a few mixed populations, with both A- and B/C types present, the majority of populations exhibit either the A- or B/C-type profile. The distribution of profile types is not random within the range of the species. The two Oregon populations and the two most northerly populations from California exhibit the B/C profile. The populations from central California are either A- or B/C-types while the populations in southern California, here defined as south of San Francisco Bay, are predominantly A-type; 86-100% of the individuals exhibit type A profiles in these populations. Populations 440 and 475 are the only exceptions for this region. One Arizona and one Baja California population are also exclusively A types. The other three populations from these areas are mixed types having mostly A profile plants. We do not have an explanation for the variability of the flavonoid,profiles exhibited by L. californica. At this time we can only point out that our observations of flavonoid, electrophoretic and morphological variability of the species are in agreement with Ornduff's (1966) comments concerning its heterogeneity. The distribution of the three variable features is not consistent throughout the species' range in that different combinations of flavonoid, electrophoretic and morphological features occur in different populations. Furthermore preliminary findings of breeding experiments
452
A. M. DESROCHERS AND 8. A. 8OHM TABLE 2. FLAVONOID PROFILESOCCURRING IN EACH POPULATION OF LASTHENIA CALIFORNICA
Populations 323 325 328 33O 331 333 335 425 433 434 435 436 437 438 439 440 441 442 443 444 445 447* 449 450 451 453 454 460 462* 464 468 471 473 475
A
B
40 36 65 33 35 54 25
D
1
1 10 6
2
4 1 30 2
1 5 30t
8 4 123
30 30t
69 30 30 30 32 11
1
6 5 33
32 25 20 19 30 26 29 27 30 30 22 22 54
C
18
30 30 33 98
24
1
Total no. of individuals 40 36 65 35 35 60 30 33 32 28 30 25 30 30 30 30 30 30 30 30 27 182 30 30 30 30 30 33 185 30 30 30 32 35
*Jasper Ridge population, tModified profile.
suggest that plants with the A-type flavonoid profile and plants with the C-type profile do not appear to cross (Desrorchers, unpublished results). This also agrees with Ornduff's (1966) observation that plants from different populations exhibit a low level of crossability. Ornduff (1966) attributed the diversity of this species to evolutionary responses to climatic and geological changes that have occurred in California and to the relative age of the species. Lasthenia californica occupies several areas that became available recently, in terms of geological time. These areas were submerged during the Pliocene, or as late as the Pleistoc( me in some cases, and include the San Joaquin Valley, the southern part of the Sacramento Valley, large areas of southern California and most valleys of western Oregon. It is likely that L. califomica has undergone a considerable degree of differentiation as it migrated to newly available sites. Given the relatively old age of the species this differentiation is now reflected in the existence of numerous morphological, ecological, cytological and chemical races. Only time will tell which or how many of these populations will differentiate sufficently to be recognized as species in their own right.
FLAVONOID
VARIATION
IN
453
LASTHfN/A CALIf0ffNK.X (ASTERACEAE)
l
9
0
FIG. 1. GEOGRAPHICAL
DlSTFtl6UTlON
location of the sampled
population.
OF FLAVONOID
The percentage
ing, with solid circles = 100%. 0 = Modified
PROFILE TYPES FOR
of flavonoid
433 434
LASTHENM C4LFORNC4. Each circle indicates the
profile type-A in a population
is displayed
by increased
darken-
profile type.
Acknowledgements-This work was supported by operating and equipment grants from the Natural Sciences and Engineering Research Council of Canada. We wish to acknowledge the curators of the herbaria from which specimen sheets of L. califomica were borrowed and for permission to sample these sheets for ray florets. The Administration and staff of Jasper Ridge Biological Preserve deserve special thanks for making the facilities at the Preserve available to us; we are much in their debt. Professor Ornduff’s interest in our work over the years is also much appreciated.
References Bohm, B. A., Herring, A., Nicholls, K. W., Bohm, L. Ft. and Ornduff, R. (1989) A six-year study of flavonoid variation in a population of Lasthenia caliknica. Am. J. Bat. 76, 157-163. Ornduff, R. (1996) A biosystematic study of the goldfield genus Lasthenia. Univ &/if. Pub. Bot. 46, I-92. Ornduff, R., Saleh, N. A. M. and Bohm, B. A. (1974) Intraspecific variation of flavonoids in Lasthenia. Brittonia26, 411-420.