Biochemical Bystematics and Ecology, Vol. 14, No. 3, pp. 291-298, 1986. 0305-1978/86 $3.00+0.00 Printed in Great Britain. Pergamon Journals Ltd.

Environmental and Genetic Components of Flavonoid Variation in Red Oak, Quercus rubra*

KAREN M. McDOUGAL and CLIFFORD R. PARKS1" Biology Department, Lenoir-Rhyne College, Hickory, NC 28603, U.S.A.;

tDepartment of Biology, Coker Hall 010-A, University of North Carolina, Chapel Hill, NC 27514, U.S.A.

Key Word Index--Quercus rubra; Fagaceae; flavonoids; elevational variation; chemotypes; environmental variation; chemo- taxonomy.

Abstract--Two elevation-related chemotypes of red oak (Ouercus rubra L.) occur in the southern Appalachians. Studies were car- ried out to evaluate the effects of genetic and environmental factors on these flavonoid profiles. Low elevation trees which con- tain only kaempferol and quercetin glycosides were used for assessment of within-tree variation. Within a single tree, sun, shade and branch position have only minor quantitative effects on the flavonoid array. To assess the effect of growing locality on flavon- old profiles we collected red oak acorns from eight locations spanning 1455 m elevation in NC and VA, and divided them equally between nursery plots at 75 and 1140 m in elevation. After five years, leaves of each tree were collected and analysed for flavon- old content and quantity. Myricetin-rich high elevation and quercetin-rich low elevation individuals retain their distinctive profiles regardless of planting size. Some quantitative intrapopulational variation in flavonoid constituents exists between high and low elevation grown trees, but is slight and primarily restricted to minor components. Populations from middle elevations are more heterogeneous than those from either high or low elevations. Some of these trees have lower quantities of myricetin glycosides than high elevation trees while others lack them entirely.

Introduction Red oak, Quercus rubra L., is a major forest tree of eastern North America, occurring from Ontario southward to the Gulf Coast states [1-4]. The species is quite variable in leaf and acorn shape over its range, and two varieties have been separated based on these characters [1, 4]. However, they intergrade in the field [5] and are reported not to breed true [6]. Quercus rubra var. borealis (Michaux f.) Farwell occurs farther north than the more widespread typical variety, and at higher elevations in the southern Appalachians where the two varieties are reported to inter- grade between 1200 and 1350 m [4, 7]. Our earlier studies revealed two chemotypes of red oak in this area which intergrade between 600 and 1050 m [8]. The low elevation form produces only quercetin- and kaempferol-3-O-glycosides, whereas the high elevation form can also syn- thesize myricetin-3-O-glycosides.

*This paper constitutes part of a dissertation completed under the direction of C.R.P. and submitted by K.M.M as par- tial fulfillment for the degree of Doctor of Philosophy at the University of North Carolina at Chapel Hill.

(Received24 March 1985)

In 1974, we initiated a programme to deter- mine if the chemical differences in red oaks from high and low elevations are genetically deter- mined or environmentally induced. Concurrent with it, we conducted an additional study to determine the nature and extent of variation within single trees. This provided us with an estimate of within-tree variation due to shading and leaf position prior to sample collection for the larger study. We present here results of the effects of environmental conditions on the foliar flavonoids of red oak.

Results and Discussion

Within-Tree Variation Although there are stunted stands of red oak in the high elevation forests of the southern Appa- lachians [9, 10], red oak is usually found in meso- phytic communities where it often exceeds 21 m in height and may reach 45 m [4, 11, 12]. Conse- quently leaf material for vouchers or chemical analysis is often difficult if not impossible to obtain from canopy (full sun) branches. The fol- mar flavonoids of Quercus rubra are qualitatively identical throughout a tree, regardless of the

291

292 KAREN M. McDOUGAL AND CLIFFORD R. PARKS

km km

L GA

480 I

FIG. 1. SEED COLLECTION AND PLANTING LOCALITIES FOR RED OAK ACORNS. O~collection site; O~collection site and planting site. Figures give elevation in m.

position of a leaf on a branch or the amount of sunlight it receives. Concentrations of individual flavonoids from mature trees at Chapel Hill (75 in Fig. 1) appear to be the highest in apical leaves of sun branches, and decrease slightly with increasing distance from the apex as well as in shade leaves (Tables 1 and 2). Some minor com- pounds are so faint that they are not seen at all on plates from basal or shade leaves (e.g. Q-7 in tree 1 sun; K-7 in tree 1 shade), but this is a func- tion of visualization since higher spotting con- centrations reveal their presence. With a single exception (Q-10) all 16 flavonol glycosides assayed are present in at least some leaves of the two study trees.

Total flavonoids per leaf show the effect of shading (both due to sun-shade differences and to position) more clearly than individual com- pounds (Table 1). Within a branch, flavonoid values are generally highest in the apical leaf of the terminal branch and somewhat lower in the sub-apical leaves. The maximum, minimum and mean values are higher for the two sun collec- tions than for the shade collection, and the most shaded leaf of tree 1-shade (#13) has the lowest total. Quantities of most compounds differ slightly in trees 1 and 2, although the flavonoid

profiles are so similar that TLC plates from them can only be distinguished visually with difficulty.

Total flavonoids show no relationship to leaf weight in tree 1 but are inversely related to leaf weight in tree 2 (Y=7.22+3.83/X; R2=0.77, where X=leaf wt and Y=total flavonoids). Leaf weights and leaf position are not correlated.

Total flavonoid quantities decrease in sub- terminal and shade leaves, but this seems more likely to be a function of the distance from the branchlet apex and therefore of shading by nearby leaves, than of actual branch position, since apical leaves on side branches have fla- vonoid scores nearly equal to apical leaves of main branches. In fact, the observed quantitative differences are very likely greater than actual dif- ferences in flavonoid content per leaf, due to the use of wet weights rather than dry weights. Higher water content in shade leaves [13, 14] may dilute the flavonoids and cause them to appear less concentrated.

Reciprocal Transplant Study In Quercus rubra differences in foliar flavonoids associated with elevation are both qualitative and quantitative. High elevation trees have the ability to synthesize myricetin glycosides, while low elevation trees can only produce kaempferol and quercetin glycosides [8]. Quantities of myri- cetin glycosides decrease with decreasing ele- vations, so that high and low elevation forms appear to intergrade between 600 and 1050 m in the North Carolina mountains.

Sixteen major flavonol glycosides are consist- ently visible in red oak foliar extracts although nearly 50 more can be seen less frequently and in trace amounts. Many of these latter com- pounds appear from R~s to be triglycosides (Fig. 2). Regardless of the planting site (Table 3, Fig. 1) trees grown from seed collected from 75 and 390 m produce only quercetin and kaempferol glycosides, while those from 1230 to 1530 also contain myricetin glycosides (Table 4).

Trees from 1110 m display the typical myri- cetin-rich high elevation pattern, except that quantities of several compounds, notably M3 (myricetin-3-O-galactosylrhamnoside), are dimi- nished in some individuals and absent in others. In addition, population means for individual compounds are often intermediate between

FLAVONOID VARIATION IN RED OAK

TABLE 1. FLAVONOID QUANTITIES OF INDIVIDUAL RED OAK LEAVES GROWN IN SUN AND SHADE

293

Concentration--(0-3 scale)* Lf. wt.

Leaf Q1 0.2 Q3 Q4 Q5 Q6 Q7 08 Q9 Q10 K1 K2 K3 K4 K5 K6 K7 Total:l: (g)

Tree 1 (Sun)

1 2.67 1.67 1.67 0.83 - - 0.50 0.50 0.16 - - - - 1.00 1.00 1.00 0.67 0.50 0.16 - - 12.33 1.40 6 2.00 1.00 1.30 0.67 0.67 0.17 0.50 - - - - - - 1.00 1.00 1.00 0.67 - - 0.17 0.17 10.32 0.90

7 2.00 1.00 1.00 0.50 0.50 0.17 0.17 0.17 0.17 0.17 1.00 1.00 1.00 0.83 0.33 0.33 - - 10.34 0.70

11 2.00 1.00 1.00 1.00 0.17 0.50 0.50 0.17 - - - - 1.00 1.00 1.00 0.50 0.50 0.50 - - 10.67 1.15

14 2.00 1.00 1.00 0.50 0.83 0.67 0.50 0.33 - - - - 1.00 1.00 1.00 0.67 0.16 0.33 - - 10.99 1.70

18 2.00 1.33 1.00 0.50 0.67 0.33 0.33 0.33 0.17 - - 1.00 1.00 1.00 0.67 0.33 0.17 - - 10.83 0.85

22 2.00 1.33 1.00 0.67 0.67 0.33 - - 0.50 0.17 - - 1.00 1.00 1.00 1.00 0.50 - - - - 11.17 1.25

)~ 2.10 1.19 1.00 0.64 0.53 0.38 0.36 0.24 0.07 0.02 1.00 1.00 1.00 0.72 0.33 0.24 0.02 10.95 1.14

s.d. 0.25 0.26 0.00 0.19 0.30 0.19 0.20 0.16 0.09 0.06 0.00 0.00 0.00 0.16 0.19 0.16 0.t6

Tree 1 (Shade) 1 2.00 1.00 1.00 0.67 0.50 0.67 0.67 - - 1.00 1.00 1.00 0.50 0.50 0.50 0.50 11.51 1.05

4 2.00 1.00 1.00 0.50 0.50 0.50 0.50 - - 1.00 1.00 1.00 0.50 0.50 0.50 0.50 11.00 1.70

6 2.00 1.00 1.00 0.50 0.60 0.50 0.50 0.17 - - - - 1.00 1.00 0.83 0.50 0.50 0.33 0.17 10.50 0.60

9 2.00 1.00 1.00 0.50 0.50 0.50 0.50 - - 0.17 - - 1.00 1.00 0.50 0.50 0.67 0.50 - - 10.34 1.10

13 2.00 1.00 1.00 0.33 0.67 0.50 0.50 0.17 0.83 0.83 1.00 0.33 0.33 0.33 - - 9.82 1.15

)~ 2.00 1.00 1.00 0.50 0.53 0.53 0.53 0.03 0.03 0.03 0.97 0.97 0.87 0.47 0.50 0.43 0.23 10.63 1.12 s.d. 0.00 0.00 0.00 0.12 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.12 0.09 0.08

Tree 2 (Sun) 1 2.00 2.00 1.33 0.67 - - 1.00 1.00 0.30 - - - -

3 2.00 2.00 1.00 0.67 0.83 1.00 0.83 0.33 0.50 - -

6 2.00 0.83 0.50 0.50 0.50 0.33 0.67 0.17 0.50 - - 12 2.00 1.00 1.00 1.00 0.67 0.33 1.00 - - 0.17 - -

16 2.00 1.00 1.00 1.00 0.50 0.50 0.50 - - 0.33 - -

1.00 0.83 0.50 0.83 0.33 0.17 0.50 12.46 0.80

1.00 0.83 0.67 1.00 0.33 0.33 0.33 13.65 0.90

1.00 0.50 1.00 0.67 0.33 0.50 0.17 10.17 1.55

1.00 0.50 1.00 0.50 - - 0.17 0.17 10 .51 0.90

1.00 0.50 1.00 0.50 0.17 0.50 - - 10.50 1.00

2.00 1.37 0.97 0.77 0.50 0.63 0.80 0.16 0.30 - - 1.00 0.67 0.80 0.70 0.23 0.33 0.23 11.46 1.03

s.d. 0.00 0.58 0.30 0.22 0.31 0.34 0.22 0.16 0.22 - - 0.00 0.17 0.22 0.22 0.15 0.17 0.19

*Q1 through Q10 are quercetin glycosides; K1 through K7 are kaempferol glycosides: Concentration scale:---not present on any TLC plate for that sample; 0.05--trace, visible only after spraying with diphenylborate; 1 -minor compound, visible as a small pale spot in UV, 2--moderately con- centreted, visible as a large dark spot in UV; 3-highly visible as a very large, very dark spot in UV, a major compound. Values are averages from 3 two-dimensional plates per sample.

tLeaf positions are shown in Table 2: weights are wet weights. :l:Total-total flavonoids per leaf, including some trace components not shown.

those for low and high elevation populations. Due to this heterogeneity, this population exhib- its the highest degree of variability of any studied (Table 4). Estimates of infrapopulation variability (based on average coefficients of vari- ation for each population) indicate that popu- lations from 75, 1230, 1260 and 1350 m are the least variable, those from 390 and 1530 m are

Although myricetin-rich high elevation and quercetin-rich low elevation individuals retain their profiles regardless of planting site, there is measurable quantitative variation. MANOVA results indicate that flavonoid concentrations are strongly affected by the elevation of origin and by the planting elevation as well. Using Wilk's criterion [35] main effects due to original eleva-

slightly more so, and the 1110 m population is tion are significant at the 0.0001 level (F--8.04), the most variable. There are no apparent differ- and those due to planting site are significant at ences in heterogeneity in samples collected the 0.01 level (F==2.76). An unexpected result is a from individual trees (one female parent) or highly significant interaction between original those from the ground (multiple male and elevation and planting site (F==2.14; p==0.0001). female parents). The analysis of variance of source-planting

294

TABLE 2. POSITIONS OF LEAVES USED FOR WITHIN-TREE VARIATION

Leaf No. Branchlet Leaf position

Tree 1 (Sun)

1 terminal apical 6 terminal 6th

7 1 st lateral apical

11 1 st lateral 5th

14 2nd lateral 2nd 18 2nd lateral 6th

22 tertiary off 2nd lateral basal

Tree 1 (Shade)

1 terminal apical

4 terminal 4th 6 1 st lateral apical

9 tertiary off 1st lateral 2nd

13 2nd lateral 2nd

Tree 2 (Sun l

1 terminal apical

3 terminal 3rd

6 terminal 6th 12 terminal 12th

16 5th lateral basal

effects on individual flavonoids indicates that most myricetin and quercetin glycosides are highly correlated with original elevation (Table 5). Only 4 of the 16 major flavonoids show sig- nificant responses to planting site (Q3, Q4, Q5 and M3, see "Fable 5). Q3 and Q4 can only be

TABLE 3. LOCALITIES AND ELEVATIONS OF RED OAK ACORNS USED

IN THE RECIPROCAL TRANSPLANT STUDY

Collection site No.

Elevation parental No. surviving in 1980 (m) Co., State trees* Low High Total

75 Orange, NC 1 6 7 13

390 Rockbridge, VA ~> 1 4 4 8 1110 Macon, NC > 1 5 5 10

1230 Macon, NC > 1 6 8 14

1260 Avery, NC ~> 1 3 1 4

1290 Macon, NC 1 1 1 2 1350 Haywood, NC > 1 1 3 4 1530 Haywood, NC 1 3 4 7

Total 29 33 62

Plots were established in 1975 by CRP at 75 m (low) and 1140 m (high) in elevation. Leaves were sampled for flavonoid analysis in August,

1980. Collections are those of CRP except for the 390 m site which was collected by D. Boufford and E. Wood.

*Where possible, acorns were removed from single trees or below the only reproductive tree in an area. In some cases it was possible only to make mass collections from beneath several trees, in which cases the parentage was uncertain.

KAREN M. McDOUGAL AND CLIFFORD R. PARKS

detected in trees from 75 and 390 m. In both cases, production levels are enhanced in trees grown at low elevations. Q5 (quercetin-3-O- arabinosylxyloside and quercetin-3-O-diarabino- side), present in all populations, shows small but consistent increases in quantity when trees are low elevation grown. M3 is only present in trees from 1110 m and above, and appears to be enhanced when they are grown at high eleva- tions. M1 shows an interactive effect between original and planting elevations, but its responses are not consistent with regard to planting site. In addition, it is a pale minor com- ponent which is difficult to distinguish reliably from a quercetin glycoside with which it co- chromatographs. When overall population-plant- ing site means for these compounds are examined (Table 4, column 7), it becomes clear that total planting site differences exceed 0.5 for only one (difference in low and high elevation means for M3=0.57).

In general, after five years' growth at either high or low elevation sites, red oak foliar flavon- old profiles primarily reflect the elevation of the seed source, not that of the planting site. Myri- cetin dimorphisms in red oak are therefore genetically determined and not environmentally caused. The trees sampled here are from a much wider geographic area than in our earlier study [8], and demonstrate that the variation in myricetin glycosides associated with elevation is not restricted to one small area in the North Carolina mountains.

Several minor quantitative differences in fla- vonoids produce a cumulative effect due to planting site. However, these differences are small (<0.5 units) and TLC plates from high and low elevation-grown trees cannot be distin- guished visually. Consequently it is our opinion that the planting effects on flavonoid profiles, while statistically significant, are due primarily to the spurious effects of small sample sizes and are probably not biologically significant. We do not discount, however, the possibility that more sensitive detection devices such as are available with HPLC may find small but real differences, especially at intermediate elevations.

In our earlier studies of red oak in the south- ern Appalachians [8], we found chemically inter- mediate populations between 600 and 1050 m at a locality very close to that of the present 1110 m

FLAVONOID VARIATION IN RED OAK 295

0 0 0

A

M6 Q7

0 ~

I m

° I O 0 ,,

Q2 Q3 Q4

o~0 oO K5

~ ° O~ O

O

& M3 &M4 &MS

o ~ o~° oC~ o o Mt Q ! K2

O ~ O o o

O

@ O

O O O O

oo O o O

E) O O

K1 <>

Myr ice t in glycosides (and quercetin replacements)

O Quercet in glycosides

O Kaempferol glycosides

FIG. 2. COMPOSITE CHROMATOGRAPH OF FLAVONOIDS FOUND IN RED OAK LEAVES FROM ALL ELEVATIONS. Myricetin glycosides are only present in trees from high elevations and are replaced, in mo~t cases, by quercetin glycosides with nearly identical R~ (e.g. M4 by Q3). Numbered spots except K6, Q7 and M6 were used in statistical analyses; most were identified; K1 and K2--K-3-gly, K3=K-3-glu, K4--K-3-arab, K5=K-3-rha-glu, K6=l-3-rha-glu; Ql--Q-3-rha, 02 and Q3=Q-3-gly, Q-4-Q-3-gal-glu, Q5=Q-3-arab-xyl, Q6=Q-3-diarab, Q7=Q-3-rha-glu; Ml=M-3-arab, M2=M-3- rha, M3=M-3-gal-rha, M4-M-3-gal-glu, M5--M-3-diarab and M-3-arab-xTI, M6=M-3-rha-glu. Key: I=isorhamnetin, K=kaempferol, M--myricetin, Q=quercetin. Solvent I: CHCI3-iso-PrOH-MEK-HOAc (10:3:3:4); Solvent I1: H20-n-BuOH-dioxane-Me2CO (42:9:4:7).

site. We suggested then that their intermediacy could be due to heterogeneity of the major agly- cones present, or to actual intermediate or 'hybrid type' individuals. No further conclusions could be drawn at that time because populations rather than individuals were analysed. Now we see, however, that chemically intermediate populations may result from altered quantities of

the flavonol glycosides as well as from qualita- tive differences in aglycones within the popula- tion. Indeed, both of these aspects of variation occur in the 1110 m population. In addition, indi- vidual components display somewhat greater quantitative responses to environmental factors in this heterogeneous population. Clearly, popu- lations at elevational extremes are more homo-

296 KAREN M. McDOUGAL AND CLIFFORD R. PARKS

TABLE 4. MEAN FLAVONOID CONCENTRATIONS IN RED OAK LEAVES TABLE 4. CONTINUED BY POPULATION AND PLANTING SITE (LOW OR HIGH)

Original elevation (m) No. of Original elevation (m)

No. of trees 75 390 1110 1230 1350§ 1530 Total trees 75 390 1110 1230 1350§ 1530 Total low/high 6/7 3/4 5/5 5/8 5/5 3/4 27/33 low/high 6/7 3/4 515 518 515 314 2 7 • 3 3 Compdt Means$

Compdl- Means:[: Concentration (0-4 scale)ll

K1

K2

K3

K4

K5

Q1

Q2

Concentration (0-4 scale)ll

M4 )(L - - - - 3.60 3.20 2.94 3.00 2.14 )(L 2.28 2.78 2.40 1.87 1.80 1.89 2.15

)(H - - - 2.80 2.88 3.13 3.25 1.99 XH 2.38 2.33 2.53 2.37 2.13 2.08 2.32

)(T 0.00a 0.00a 3.20ab 3.00b 3.03b 3.14b )(T 2.38a 2.52a 2.45a 2.18a 1,97a 2.00a

M5 )(L . . . . 3.93 4.00 3.67 4.00 2.59 )(L 2.17 1,78 0.67 1.80 0.80 0.89 1.38

XH -- 3.67 4.00 3.87 4~00 2.60 XH 1.76 0.91 1.60 1.87 1.33 1.50 t.57

XT 0.00a 0.00a 3.80b 4.00b 3.77b 4.00b XT 1.95a 1.26a 1.13a 1.85a 1.07a 1.24a

)(L

;~H

)(L ,~H

)(T

)(L

)(H

XT

XL

)(T

2.78 2.22 3.00 2.93 2.34 2.56 2.68

2.78 2.83 2.80 2.96 2.74 2.42 2.78

2.78a 2.57a 2.90a 2.95a 2.53a 2,48a

3.00 2.67 2.33 2.93 2.80 2,22 2.70

3.00 2.75 2.40 2.58 2.20 2.08 2.55

3.00a 2.71ab 2,37ab 2.72ab 2.50ab 2.14b

1.50 1.56 1.27 1.27 1.06 1.00 1.28

1.43 1.50 1.20 1.21 1.67 1.08 1.34

1.46a 1,52a 1.23a 1.23a 1.37a 1.05a

2.11 2.45 1.93 1.87 1.26 1.44 1.84

2.09 1.75 1.93 2.00 1.67 1.75 1.90

2.10a 2.05a 1.93a 1.95a 1.47a 1.62a

2.22 2.44 0.40 -- - - - - 0.84

2.38 1.92 -- - - - - 0.74

2.31a 2.14a 0.20b O.OOb O.OOb O.OOb XH

kT

tCompound designations are as in Fig. 2.

tXL and )(H are means for individuals planted at low and high ele-

vations, respectively. ~ is the total mean for a compound regardless of planting site.

§Populations from 1260, 1290 and 1350 m were pooled for analysis due to small sample sizes (See Table 3).

II Concentration scale: - -=not present on any TLC plate for that sam-

ple: 1 =trace, visible only after spraying with diphenylborate; 2=minor compound, visible as a small pale spot in UV; 3~moderately concen-

trated, visible as a large dark spot in UV; 4~highly concentrated, visible

as a very large, very dark spot in UV, a major compound. For each com-

pound elevational means with different italicized, lower case letters (a, ab, b) are significantly different (p<0,05) by Scheffe's multiple compar- ison test. Low and high elevation means for each compound (all individ-

uals planted at low vs all planted at high elevations) with an asterisk(*) differ significantly (p<0.05) by the same test.

Q3

Q4

Q5

Q6

M1

M2

M3

)(L 2.50 2.67 - - -- - - 0.85*

)(H 1.95 1.67 . . . . 0.62*

)~T 2.20a 2.09a 0.00b 0.00b 0.00b 0.00b

,~L 3.17 3.11 . . . . . 1.05

)(H 2.29 2.50 . . . . 0.79 )(T 2.69a 2.76a 0.00b 0.00b 0.00b 0.00b Flavonoid

K1 )(L 3.89 4.00 3.53 3.13 2.87 3.22 3.43* K2 )(H 3.14 3.50 2,87 3.00 3.06 3.00 3.08* K3

)(T 3,49ab 3.71a 3.20ab 3.05b 2.97b 3.09b K4 K5

)(L 3.94 4.00 3.87 4.00 3.40 3.89 3.84 Q1 XH 4.00 4.00 3.67 4,00 3.80 4.00 3.92 Q2 )(T 3.97a 4.00a 3.77a 4.00a &60a 3.95a Q3

Q4 XL 080 1.40 0.80 1.11 0.73* Q5 ,XH - - - - 0.67 0.00 1.40 1.08 0.44* Q6 ,~T 0.00a 0.00a 0.73ab O.64ab 1.10b 1.09b M1

M2

)(L - - - - 1.47 0.87 0.27 0.44 0.53 M3 )(H - - - - 0.93 0.96 0.93 0.75 0.61 M4 ;~T 0.00a 0.O0a 1.20b 0.92b 0.60ab 0.62ab M5

;~L )(H

)(T

-- - - 0.80 2.67 2.27 1.78 1.18" - - - - 2.47 2.75 2.40 2.83 1.75" 0.00a 0.00a 1.63b 2.56b 2.33b 2.38b

TABLE 5. SIGNIFICANCE OF EFFECTS ON RED OAK FLAVONOIDS DUE

TO ORIGINAL ELEVATION, PLANTING ELEVATION AND INTERACTIONS

Effect of elevation1- Orig./planting

Original Planting interactions

* NS NS NS NS NS

NS NS NS

* NS NS NS NS NS NS NS NS

* * * NS NS

** * NS ** ** NS * NS NS *** NS **

* * * NS NS * * * * NS *** NS NS *** NS NS

Effects were modelled using ANOVA.

tSignificaoce levels (p=): *=0.01, **=0.001, ***=0.0001, NS=not significant at the 0.01 level.

FLAVONOID VARIATION IN RED OAK 297

geneous than the one mid elevation population represented here. Further studies must be done to determine if this pattem holds over the range of the species. High and low elevation habitats for red oak are clearly different [7, 8], and the intermediacy in chemistry and morphology may be another case of intergrading forms separated by different habitats [15].

Conclusions These studies confirm our earlier reports of ele- vational differences in the flavonoids of Quercus rubra. Reciprocal plantings show that the chemo- types of red oak are under strong genetic con- trol, Flavonoids are generally considered to be under strong genetic control and therefore resis- tant to environmental influences [16-20], There are, however, reports of quantitative variation in fiavonoids due to aging [17, 21, 22], nutrient or light regimes [23-25] and time of season [23, 26-32]. In some cases glycosidic patterns or compound ratios seen in field grown plants dis- appear when plants are grown in the green- house [23, 30, 33, 34]. One of the best examples of this is Lupinus sericeus, whose geographically distinct chemotypes disappear under controlled conditions [34].

Progeny from myricetin-producing high eleva- tion red oak trees produce myricetin glycosides after 5 years at low elevations, and myricetin- lacking low elevation progeny still produce only kaempferol and quercetin derivatives after five years at high elevations.

While high and low elevation chemotypes appear to correspond with varieties borealis and rubra, respectively, of Quercus rubra, in the southern Appalachians, the zone of chemical intergradation occurs at a lower elevation than the described region of morphological overlap (1200-1350 m). Consequently it is premature to extrapolate beyond this geographic area.

Experimental Within-tree variation. Sun and shade branches were collected from two mature trees in Chapel Hill, NC on 6/18/78, and leaf positions and wet weights recorded (Tables 1 and 2). Individ- ual leaves were extracted in 80% MeOH on a rotary shaker at 4 ° for 4 days. Samples were then filtered, washed with MeOH, and the elulate taken to dryness and resuspended in MeOH, (2:1 v/w). Two drops H20 were added to improve solubility. Solns were stored covered at 4 ° until applied to triplicate poly- amide thin layer plates and developed 2D (Fig. 3). Plates were

marked under UV, sprayed with diphenylborate ethanolamine and marked again [36, 37]. For each spot, colour in UV and diphenylborate, position on a grid, and a concentration (visual estimation of spot density on a 0.5 to 3 scale) were averaged for 3 plates and recorded. Compounds were identified by comparison with fiavonol glycosides previously identified in Quercus rubra [8].

Total flavonoid quantities per leaf were estimated by sum- ming the average values for all 16 compounds, then regressed against leaf position and weight,

Reciprocal transplant study. Acorns were collected from eight localities in NC and VA (Fig. 1, Table 3) during autumn, 1974 and stratified under cool moist conditions until planting in spring of 1975. Viable acorns from a single provenance were divided equally between plots at 75 and 1140 m in elevation (Orange and Macon counties, N.C,, respectively, Table 3). Plots were given minimum maintenance. After 5 years leaves were collected from each tree and dried for flavonoid analysis. Vouchers of parental trees have been deposited at NCU.

Dried leaves (1.0 g dry wt each) were extracted in MeOH at 4 ° for 2 weeks, Further sample preparation and chromato- graphy were carried out as described above except that no H20 was added and a 1-4 concentration scale was used.

The effects of original elevation, planting elevation, and interactions between these two on flavonoid composition were tested using multivariate analysis of variance [38] which provides an analysis of variance of each compound in addition to tests of main effects. Dependent variables of 16 flavonol gly- cosides were modelled against main effects of original eleva- tion, planting elevation and interactions between these two.

Acknowledgements--Support for this project was provided by the University of North Carolina Research Council and the Highlands Biological Foundation. The authors would like to thank Chicita F. Culberson, James D. Doyle, James W. Hardin, Robert K. Peet, Karl A. Suiter and Susan S. Whitfield for assis- tance at various stages of this project.

References 1. Fernald, M. L. (1950) Gray's Manual of Botany. American

Book Company, New York. 2. Fowells, H. A. (1965) Silvics of Forest Trees of the United

States. U.S. Division of Timber Management Research, U.S. Forest Service. Agriculture Handbook No. 271.

3. Little, E. L. (1971) Atlas of US. Trees. VoL 1. Conifers and Important Hardwoods. USDA Misc. Publ. No. 1146.

4. Sargent, C. S. (1965) Manual of the Trees of North America. 2nd edn., Dover Publications, New York.

5. Jensen, R. J. (1977) Taxon 26, 399. 6. Palmer, E. J. (1942) Am. Mid/. Nat. 27, 732. 7. Whittaker, R. H. (1956) Ecol. Mon. 26, 1. 8. McDougal, K. M. and Parks, C. R. (1984) Am. J. Botany71,

331. 9. Braun, E. L. (1950) Deciduous Forests of Eastern North

America. Hafner Press, New York. 10. Delapp, J. (1978) Gradient Analysis and Classification of the

High Elevation Red Oak Community of the Southern Appala- chians. M.S, Thesis, Department of Botany, North Carolina State University, Raleigh, NC.

11. Gleason, H. A. and Cronquist, A. (1963) Manual of Vascular

298 KAREN M. McDOUGAL AND CLIFFORD R. PARKS

Plants of Northeastern United States and Adjacent Canada. D. Van Nostrand, Princeton, NJ.

12. Harlow, W. M. and Harrar, E. S. (1968) Textbook of Dendrol- ogy. McGraw-Hill, New York.

13. Feeny, P. (1970) Ecology51, 565. 14. Kramer, P. J. and Kozlowski, T. T. (1979) Physiology of

Woody Plants. Academic Press, New York. 15. Coker, W. C. and Totten, H. R. (1934) Trees of the Southeast-

ern States, 3rd edn. University of North Carolina Press, Chapel Hill.

16. Harborne, J. B. (1967) Comparative Biochemistry of the F/av- onoids. Academic Press, New York.

17. McClure, J. W. (1975) Aquatic Botany 1, 395. 18. McClure, J. W. (1975) The Flavonoids (Harborne, J. B.,

Mabry, T. J. and Mabry, H., eds). Vol 2, p. 970. Chapman & Hall, London.

19. McClure, J. W. and Alston, R, E, (1966) Am. J. Botany 53, 849.

20. Parks, C. R., Sandhu, S. S. and Montgomery, K. R. (1972) Am. J. Botany59, 158.

21. Asen, S. (1979) J. Am. Soc. Hort. Sci. 104, 223. 22. Crawford, D. J. (1970) Britonnia 22, 93. 23. Aaen, S. (1982) J. Am. Soc. Herr. Sci. 107, 744. 24. Ball, G. A., Beal, E. O. and Flecker, E. A. (1967) Brittonia 19,

273.

25. Tso, 1-. C., Sorokins, T. P., Engelhaupt, M. E., Anderson, R. A., Bortner, C. E., Chaplin, J. F., Miles, J. D., Nichols, B. C., Shaw, L. and Street, O. E. (1967) Tobacco ScL 11, 133.

26. Cooper-Driver, G., Finch, S. and Swain, T. (1977) Biochem. Syst. Ecol. 5, 177.

27. McDougal, K. M. Ph.D. dissertation, University of North Carolina, Chapel Hill, NC.

28. Parker, J. (1977) J. Chem. Ecol. 3, 489. 29. Schultz, J. C. and Baldwin, I. T. (1982) Science217, 149. 30. Swain, T. (1960) Phenolics in Plants in Health and Disease

(Pridham, J. B., ed.). Pergamon Press, New York. 31. Tissut, M. (1967) Phytochemistry6, 1291. 32. Tissut, M. and Egger, K. (1972) Phytochemistry11, 631. 33. Bohm, B. A. and Stuessy, 1-, F. (1981) Phytochemistry20,

1573. 34. Nicholls, K. W. and Bohm, B. A. (1982) Biochem. Syst. Ecol.

10, 225. 35. Rao, C. R. (1973) Linear Statistical Inference. John Wiley,

New York. 36. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) The

Systematic Identification of Flavonoids, Springer, New York. 37. Wilkins, C. K. and Bohm, B. A. (1976) Can. J. Botany 54,

2133. 38. SAS Institute, Inc. (1982) SAS User's Guide: Statistics. Cary,

NC.