Paper chromatography for determining palatability differences in various strains of big sagebrush

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USDA Forest Service
Research Paper INT-101
1971

PAPER CHROMATOGRAPHY FOR
DETERMINING PALATABILITY DIFFERENCES IN

VARIOUS STRAINS OF BIG SAGEBRUSH

_ David L. Hanks, James R. Brunner,
Donald R. Christensen, and A. Perry PI

AND RANGE EXPERIMENT STATION

THE AUTHORS

DAVID L. HANKS was a Plant Physiologist in 1969 for the Intermountain
Forest and Range Experiment Station in Ephraim, Utah. He received
his B.S. degree in Botany (1962) and M.S. degree in Chemistry (1963)
from Brigham Young University. He obtained his Ph.D. in Plant
Physiology (1966) from the University of Michigan. Between 1966 and
1969 he taught mycology and plant physiology at Brigham Young Uni-
versity. He is presently teaching plant physiology at the University
of Missouri, Kirksville.

JAMES R. BRUNNER is a Range Conservationist for the Bureau of Land
Management, which position he has held since 1957. Prior to that
time, he served in a similar capacity for the Soil Conservation Serv-
ice in Texas. He received his B.S. degree in 1941 from Colorado
State University at Fort Collins.

DONALD R. CHRISTENSEN is a Game Biologist for the Utah State Divi-
sion of Fish and Game. He received his B.S. degree from the Uni-
versity of Utah in Zoology (1955). From 1955 to 1957, he worked as
a chemist for Kennecott Copper Corporation and since 1958, he has
worked for the Utah State Division of Fish and Game as a Research
Game Biologist.

A. PERRY PLUMMER is a Research Scientist for Intermountain Station
at Ephraim, Utah. He has worked in range research for the Station
since 1936. He received his B.S. degree (1935) and his M.S. degree
(1939) in Botany from the University of Utah. His research has been
principally concerned with the restoration of western ranges.

USDA Forest Service
Research Paper INT-101
July 1971

PAPER CHROMATOGRAPHY FOR DETERMINING PALATABILITY DIFFERENCES
IN VARIOUS STRAINS OF BIG SAGEBRUSH

David L. Hanks, James R. Brunner, Donald R. Christensen, and A. Perry Plummer

Federal aid in wildlife restoration funds
was provided through Project W-82-R

INTERMOUNTAIN FOREST AND RANGE EXPERIMENT STATION
Forest Service
U.S. Department of Agriculture
Ogden, Utah 84401
Robert W. Harris, Director

CONTENTS

Page

INTRODUCLION cise a) ser cere s ie iol onae momen:
EXPERIMENTAL PROCEDURE ..... 2
RESULTS ey eset cc: aitts tlone nl soles) Memon ere 3
Chromatograms) 525)". 0... savenve mck ne eek ao
(Cre VAD e eee EOMEC TE Ol to Go) Heo. oo SB oO.
DISCUSSION: (2 --s20e pee bea tient 8 Lomi cpt eee eet eee
LITERATURE; CLL ED year cueeintel tones g

ABSTRACT

Two-dimensional phenolic extraction on 9-inch squares of chromatographic
paper was discovered to be a simple laboratory technique for quickly classifying
more than 100 foliage collections of big sagebrush from over the Intermountain
area into two major palatability classes. These agreed readily with field obser-
vations of preferences by deer and livestock on winter ranges. The technique
was also in close agreement with the observed preferences by deer of 10 geo-
graphic sources transplanted to a common area having a uniform soil within their
winter range. It was also useful for quickly recognizing four subgroups in sub-
species of Artemisia tridentata vaseyana and two subgroups in Artemisia
tridentata tridentata.

The solvent system for the first dimension was n-butanol, acetone, water
(4:1:3) and for the second dimension acetic acid and water (15:85). Chromato-
grams were viewed under longwave ultraviolet light before and after exposure to
ammonia fumes and in daylight following the application of ammonia. The strong
association that was found between chromatographic variation and palatability
suggests that the procedure may be used to predetermine the grazing potential of
any collection of big sagebrush.

The most obvious chromatographic characteristic was the size and intensity
of the blue in spot 9. It ranged from large to small and iridescence varied from
brilliant to dull. This spot was successfully used to classify all big sagebrush
samples into the two basic groups with the other spots being used to make
subgroups.

Introduction

Common big sagebrush (Artemista tridentata) is one of the most widely distributed
and abundant shrubs of the Western United States (Hall and Clements 1923; Beetle 1960;
Dayton 1931; and Plummer et al. (1968). It is particularly abundant over much
of the Great Basin and in many areas may comprise up to 90 percent of the shrubby
vegetation. It is an important browse for big game on their winter ranges. (The
nomenclature in this publication has followed Holmgren and Reveal 1966.)

Past observations have revealed that there is considerable variation in the pala-
tability of big sagebrush for big game and livestock. This variation in palatability
is often associated with the geographical source of the sagebrush, but in some cases
palatable and unpalatable plants may be found growing on a common site. These obser-
vations indicate that there may be a real opportunity for development of improved
strains through selection and breeding.

Apparently, considerable genetic diversification has occurred in the big sagebrush
complex as a result of hybridization and back crossing. Genetic makeup is probably
associated with dissimilarities in the chemistry of the plants involved; thus, if
chemical differences are detected in plants collected from various geographical sources,
this indicates that some degree of genetic divergence exists. Furthermore, the amount
of chemical difference may be an index of the extent to which strains have become
separated. Such differences in the chemistry of plants have been utilized extensively
in recent years to supplement morphological differences in the grouping of closely
related plants in some of the major taxa. For example, Hollis (1966, 1967a, 1967b, and
1967c) made comparative analyses of the polyphenols from leaf extracts of Eucalyptus
using paper chromatography. As a result of these analyses, he resolved many of the
problems relating to the taxonomy of this genus. The solution of these problems was
not possible on the basis of morphological characters alone.

Similar studies on the genus Baptista were conducted by Alston and Turner (1962,
1963) and Brehm and Alston (1964). In addition, Holbo (1965) completed a comparable
study of Artemtsta section Tridentatae in which he readily separated the individual
species by chromatography. Using thin-layer chromatography, Brunner! showed that strain
variation in big sagebrush could be readily detected. Consequently, it appeared likely
that similar techniques could be used to separate strains of big sagebrush into pala-
tability classes.

1J. Brunner. Some observations on Artemisia in Nevada. Bureau of Land Management ,
Las Vegas, Nevada. (Manuscript in preparation. )

Experimental Procedure

From February through September 1969, more than 100 foliage collections of big
sagebrush were taken from widely scattered areas in Utah, Nevada, Idaho, Wyoming, and
Colorado. We obtained these from a wide variety of sites so that the collections would
be fairly representative of the species distribution. In addition to using them in the
chemical analyses described below, the degree of grazing by big game was determined to
discover if palatability, or preference, was associated with geographical source or
ecotype. Observations were made at locations where grazing pressure had not been severe
enough to force utilization of unpalatable plants. Generally, the degree of grazing
was determined in January, but some observations were made in February and March, and
a few were made in April. Palatability was given a low rating where less than 15
percent of the current growth was removed, medium where utilization was 15 to 40 per-
cent, and high where utilization was more than 40 percent. The degree of herbage remov-
al was determined by the technique described by Pechanec and Pickford (1937).

Since we were aware that palatability may be affected by soil or other environ-
mental factors, about 100 plants under 2 years old were collected from each of ten
geographical sources of big sagebrush. These plants (totaling about 1,000) were trans-
planted from their natural sites to comparable randomized rows spaced 3 feet apart on
State Fish and Game land located on deer winter range near Price, Utah. Transplanting
was done in April and early May of 1968. Plants from three of these sources had been
observed to be especially palatable to deer; plants from seven sources had been cate-
gorized as unpalatable while growing on their natural sites. The degree of grazing on
these transplanted rows was determined in January 1969 and 1970 by the same technique
described above.

Foliage collections from plants of the more than 100 sources were placed in brown
paper bags and dried in the absence of light. Then, by use of a mortar and pestle,
0.5 grams of the dried leaves were pulverized and placed in brown 30 ml. bottles, and
7.0 ml. of absolute methanol was added to extract phenolic constituents. After 24 hours
at room temperature, the extract was decanted and concentrated by evaporation to 2.0 ml.
Twenty-five ul of this extract was applied to duplicate 9-inch squares of Whatman No. 3
MM chromatographic grade filter paper in two dimensions. The solvent system for the
first dimension was n-butanol, acetone, water (4:1:3) and for the second dimension,
acetic acid, water (15:85). Chromatograms were viewed under longwave ultraviolet light
before and after exposure to ammonia fumes and in daylight following the application of
ammonia in order to note the appearance and color changes of the resulting spots. Each
spot was given an arbitrary number for identification purposes and the Re value of each
was computed for both directions of the finished chromatogram.

Rees Distance of spot from starting point
f Distance of solvent front from starting point

The R, value of a given spot is then expressed as:

f
Re = Re (first dimension) /R, (second dimension)

Results
CHROMATOGRAMS

As might be expected when dealing with a large and complex species such as big
sagebrush, considerable chromatographic variation was found. Presently, the plant
collections have been divided into two major groups, I and II. Group I has 4 subgroups
(Ila, Ib, Ic, and Id). Group II has 2 subgroups (IIa and IIb). These groups are based
on differences in the chromatographic spots. Chromatograms of each collection always
display a. basic compliment of ‘ten spots (1, 2, 3, 4, 7, 8, 9, 11, 13, 27) plus varying
combinations from: an additional eleven spots (5, 6, 10, 12, 14, 16, 22, 25, 26, 33, 36)
(table 1 and figs. 1-6). Some of these spots exhibit marked differences in size and
intensity of color; therefore, the chromatograms were organized into groups taking into

consideration both qualitative and quantitative variations.

Table 1.--Rf

f values and color of the chromatographte spots in Artemisia tridentata

: Color
SPOG INO. Me Ultraviolet NH. + Ultraviolet NH. + Daylight
1 355/346 Blue Yellow-green Gray
2 .90/.78 Violet Violet --
3 88/.71 Blue-green Blue-green --
4 ce Asal Violet Violet-brown Yellow
5 pal ce Yellow or Yellow or --
Yellowish-brown Yellowish-brown
6 528) O13 Yellow or Yellow or --
Yellowish-brown Yellowish-brown
UD, 52/ 308 Violet Violet Yellow-brown
8 51/287 Dark blue Dark blue --
9 x4 [78 Light iridescent Light iridescent --
blue-green blue-green
10 .18/.84 Blue Blue --
ae a4). 15 Blue Yellow-green Yellow
uy 226/71 -- Blue --
13 .29/ .64 Pink Yellow-pink ae
14 .35/.59 Blue or violet Gray-blue or violet --
16 .32/.90 Dark blue Dark blue --
22 252/\.84 Blue Blue-green --
25 WOo/s0/ Blue-green Blue-green --
26 138/515 Blue Blue --
27 sol S57 Blue Yellow-green Gray
33 hs 00 Pink Pink --
36 .26/.84 Bright blue-green Bright blue-green --

Figures 1-6.--Representative two-dimenstonal chromatograms of methanol-soluble extracts
from the leaves of the six subgroups (under groups I and II) of big sagebrush. For
spot coloration and Re values, see table 1.

Figure 1 Figure 2

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Figure 5 Figure 6

Chromatograms of all collections in Group I contain a large, iridescent blue spot
9 (R¢ = .54/.78) which is the most prominent in the entire chromatogram and most char-
acteristic of this major group. In addition, displayed in all specimens of Group I
were spots 25 and either 16 or 22 (.83/.67, .32/.90, and .32/.84, respectively). On the
other hand, spot 9 of Group II is usually smaller and always less brilliant than in
Group I. Furthermore, spot 16 was always absent from the chromatograms of Group II and
22 was found only occasionally. However, spots 5, 6, and 26 (.47/.11, .28/.13, and
.38/.73, respectively) were always present in Group II as shown in figures 5 and 6.

Group I.--This major group was subdivided into its four distinct subgroups of la,
Ib, Ic, and Id (figs. 1-4), on the basis of the following characteristics. In addition
to the brilliancy of spot 9, and the presence of spots 16, 22, and 25 mentioned pre-
viously, chromatograms of Ia contain spots 5, 6, 10, 12, and 14 (fig. 1). Spots 5 and
6 of this group are relatively small and dull and yellowish-brown in color.

Subgroup Ib differs from Ia only in the size and color of spots 5 and 6, which in
Ib are large and bright yellow (fig. 2).

Chromatograms of Ic lack spots 5, 6, and 12 but always contain the prominent pink
spot, 33. Spot 9, however, is larger and more brilliant in Ic than in any of the other
3 subgroups of group I (fig. 3).

Subgroup Ic appears to be made up of collections of subspecies A. tridentata
vaseyana or Closely related intermediates within group I. Since this subspecies
is such a polymorphic complex, it is possible that Ia and Ib, as well as Ic, may be
considered within it.

Group Id lacks spots 10, 12, 14, and 16. Here, spot 9 is intermediate in size but
remains highly iridescent. In Id, spots 5 and 6 are large and bright yellow as in Ib.
In addition, a bright blue-green spot, 36, is prominent in subgroup Id (fig. 4).

Group II.--This major group was divided into the two subgroups Ila and IIb (figs.
5 and 6), both of which lack spots 16 and 36 but contain spots 5, 6, and 26.

In subgroup IIa, spot 9 is smaller than in any of the previously described sub-
groups except Id where it is of similar size. However, the brilliancy of this spot in
IIa is decidedly less intense than that found in any of the collections placed in
group I; nevertheless, spot 9 in IIa remains somewhat iridescent. Spots 12, 14, and
25 occur in this subgroup (fig. 5). In both IIa and IIb, spots 5 and 6 are small and
dull yellowish-brown. They are similar in size and color to those found in Ia.

In subgroup IIb, spot 9 is very small, often not exceeding one-half inch in diam-
eter, and it exhibits little or no iridescence. In most chromatograms of subgroup IIb,
spots 12 and 25 are missing; and when present they are small and only faintly colored.
Spot 14 is always present in IIb, although occasionally the color of this spot is dark
violet rather than the usual blue-gray found in all other groups (fig. 6). On the basis
of morphological examinations, it seems apparent that collections representing sources
in subgroups IIa and IIb would be in the subgenus 4. tridentata tridentata. Further
observations may show IIa to be considerably integrated with Ia.

GRAZING

The groupings already described are related to the degree of grazing by deer and
livestock. However, observations indicate that plants in group I are much more pala-
table than those in group II (fig. 7). This was true on the row plantings on State
Fish and Game lands northwest of Price, Utah, in January 1969 and 1970, and also where
natural representatives of these two major groups were found-on the local winter range.

Figure 7.--Comparison of subgroup Ie from Hobble Creek (left) with subgroup IIb
from Indianola, in January 1970, when grazing averaged 60 and 5 percent,
respectively. Plants of these sources had attained a similar height of
about 24 inches when deer began to graze them in mtd-November.

We are confident that differences in palatability exist within groups I and II, but our
observations have not yet been intensive or refined enough to detect this and correlate
it definitely with chromatographic analyses.

Three of the ten collections planted near Price chromatographed as group I. By
mid-January in both 1969 and 1970, all of these were grazed by deer to an average of
about 60 percent. None of the big sagebrush plants were grazed less than 40 percent;
and on several of the plants, grazing was in excess of 80 percent. In contrast, of the
seven collections chromatographed as group II, none were grazed more than 40 percent,
and most were grazed less than 5 percent.

All collections from individual A. tridentata plants or populations on winter
ranges which had been grazed in excess of 50 percent invariably chromatographed as
group I except for the single exception represented by a collection from Utah County
(Hobble Creek trial site) and involving some big sagebrush transplants. Moreover, all
collections from plants or populations observed to be relatively unpalatable on the
open range, chromatographed as group II. This was especially noticeable in the area
from which collections of subgroups Id and Ila were taken in northwestern Nevada where
these two subgroups grow in an intermixed population on a foothill winter range (fig. 8).
Subgroup Id on this same range was palatable to the cattle that graze on the area, but
IIa was unpalatable and grazed very little. By March 15, 1968, subgroup Id on this
range had been grazed slightly in excess of 60 percent, and grazing on IIa was less
than 15 percent. The partiality for Id was also exhibited by sheep and deer in that
area. The difference between subgroups Id and Ila was also evident on the transplanted
areas near Price, Utah. A similar selectivity was also noted where subgroups Ia and Ib
intermix with IIb in Ephraim Canyon. Under these intermix conditions, Ia and Ib were
grazed in excess of 70 percent by late January of 1969 and 1970, but none of the plants
in subgroup IIb were grazed more than 15 percent.

Id IIa

Figure 8.--Marked differences were noted tn the size of Artemisia tridentata
subgroups Id (left) and IIa (right) as a result of cattle grazing in the
Jackson mountains tn northwestern Nevada.

The most obvious chromatographic characteristic of collections that were selected
by deer and livestock was the presence of a large and highly iridescent spot 9. This
marker is particularly obvious in subgroups Ia, Ib, and Ic where its brilliance is
considerably greater than in the unpalatable subgroups; however, this distinction is
not so clear in regard to Id and IIa. Since subgroups Id and IIa are found in close
physical association, the use of additional characters is frequently necessary. Other
indicators of palatability for deer and livestock are the presence of spots 16 and 22
in subgroups Ia, Ib, and Ic; the presence of 22 in Id; and the absence of 26 in all
groups.

Discussion

At this point in our study of the big sagebrush complex, we believe it is signifi-
cant that chromatograms of the various collections allow rather well-defined groupings.
The existence of these groups not only supports our initial hypothesis regarding the
occurrence of genetic divergence within subspecies of big sagebrush, but provides
evidence that this genetic change is following quite discrete lines. However, we must
point out that some of the collections do not fall into any of the designated groupings
but appear to occupy positions intermediate between them. Chromatograms of these inter-
mediates most commonly contain both spot 22, found primarily in group I, and 26 occur-
ring in group II; but such chromatograms lack spot 16 found in group I, and spot 9 is
usually intermediate in brilliancy. This combination suggests hybridization between
groups I and II and the retention by the progeny of some characteristics from each
parent. Nevertheless, the majority of collections can be readily separated into the
previously described groupings by the methods given.

In regard to the ecological distribution of the major groups and subgroups, the
following observations have been made. Collections of group I were obtained primarily
in mountain habitats. Those of subgroup Ic were obtained in the lower mountains and
higher foothill areas. Individuals from subgroups Ia and Ib of the lower foothills
area have come from areas extending from the lower limits of Ic to the base of the
foothills where they overlap with subgroup IIb, the prevalent big sagebrush (A.
tridentata tridentata) of the more extensive lowlands.

The two subgroups Ia and Ib occupy the lower foothill areas, and it appears that
Ia predominates in the upper portion while most collections of Ib have come from the
lower part. However, specimens of each have been collected throughout this entire
range.

Subgroups Id and IIa have only been collected from areas on the Jackson and Pine
Forest mountains in northwest Nevada where they occupy similar habitats.

Chromatograms of the tall bushes commonly observed growing along such places as
fencerows and arroyos differ slightly from those of IIb, among which they are frequently
found. However, too few collections have been analyzed to warrant the formation of a
separate subgroup at this time, although it appears likely that this may be advisable
following a more thorough study. In the present paper, this tall fencerow type is
included in IIb. Thus, on the basis of source material, it seems significant that the
distribution pattern fits fairly close to the chromatographic divisions, especially
between highland and lowland sources.

There is little likelihood that phenols observed in the big sagebrush of this
study are responsible for the relative palatability of these plants. However, the
strong association that has been found between chromatographic variation and palatability
suggests that this laboratory procedure may be utilized to quickly evaluate the grazing
potential of any collection of big sagebrush. Consequently, chromatography can be a
useful tool in selecting strains of big sagebrush for specific purposes.

Literature Cited

Alston Re Ese and Be Ls Turner
1962. New techniques in analysis of complex natural hybridization. Nat. Acad.
Sei. Proc. 482 150-137. illus.

1963. Natural hybridization among four species of Baptista. Amer. J. Bot. 50:
159-173, illus.

Beetle, A. A.
1960. A study of sagebrush, the section Tridentatae of Artemista. Univ. Wyo.
Agr. Exp. Sta. Bull. 368, 88 p., illus.

Brehm, B. G., and R. E. Alston
1964. A chemotaxonomic study of Bapttsta leucophaea var. laevicaulis (Leguminosae).
Amer. J. Bot. 51: 644-650, illus.

Dayton, W. A.
1931. Important western browse plants. USDA Misc. Pub. 101, 214 p., illus.

Hall, H. M., and F. E. Clements
1923. The phylogenetic method in taxonomy. The North American species of
Artemista, Chrysothamnus, and Atriplex. Carnegie Inst. Wash., illus.

Holbo, H. R., and H. N. Mozingo
1965. The chromatographic characterization of Artemista, Section Trtdentatae.
Amer. J. Bot. 52: 970-978, illus.

Holiaiss Wee B*
1966. Polyphenols in the leaves of Eucalyptus L'Herit (Myrtaceae). A chemotaxon-
omic survey I: Introduction and study of the series Golbulares. Phyto-
chemistry 5: 1075-1090, illus.

1967a. Polyphenols in the leaves of Eucalyptus (Myrtaceae): A chemotaxonomic survey
II: The sections of Renantheroideae and Renantherae. Phytochemistry 6: 259-
274, illus.

1967b. Polyphenols in the leaves of Eucalyptus: A chemotaxonomic survey III.
The series Transversae, Argurophyllae, and Paneculatae of the section
Macrantherae. Phytochemistry 6: 275-286, illus.

1967c. Polyphenols in the leaves of Eucalyptus: A chemotaxonomic survey IV. The
sections Porantheroides and Terminales. Phytochemistry 6: 373-382, illus.

Holmgren, Arthur H., and James L. Reveal
1966. Checklist of the vascular plants of the Intermountain region. U.S. Forest
serv. Res. Bap. INT-32.,:160 p.

Pechanec, J. F., and G. D. Pickford
1937. A comparison of some methods used in determining percentage utilization of
range grasses. J. Agr. Res. 54(10): 753-765.

Plummer, A. P., D. R. Christensen, and S. B. Monsen
1968. Restoring big game range in Utah. Utah Div. of Fish and Game Pub. 68-3,
183: 'p.

9

AFLC/HAFB, Ogden

Headquarters for the Intermountain Forest and
Range Experiment Station are in Ogden, Utah.
Field Research Work Units are maintained in:

Boise, Idaho

Bozeman, Montana (in cooperation with
Montana State University)

Logan, Utah (in cooperation with Utah
State University)

Missoula, Montana (in cooperation with
University of Montana)

Moscow, Idaho (in cooperation with the
University of Idaho)

Provo, Utah (in cooperation with
Brigham Young University)