Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Discussion: Full Siblings of Tree AS-A95 

The number of actual modes in the frequency distribution of individual 

monoterpenes—as derived from modal analysis—equalled or exceeded the 

expected number of modes—as derived from the assignment of additive alleles; 

this supported the additive allele hypothesis and generally supported the 

assignment of alleles to Tree AS-A95 and the pollen parents. Mode proportions 

were generally fairly close to the expected; this was additional support to the 

hypothesis. 

In most families there was a marked difference between the composition of 

nursery trees and those in containers or a container/plantation. This difference 

was evidenced by greater limonene in the nursery trees and by greater αpinene and ß-pinene in the container or container/plantation trees. The 

number of samples in the containers and container/plantation was usually 

too small for confident assessment. Nevertheless, only nursery data were 

used in further analysis. 

The average composition of the five sets of progeny of Tree AS-A95 was 

about as expected from the assignment of alleles to the parents and the action of 

several modifying factors. 

There was an unusually large number of ultra-high limonene progeny in 

crosses with high limonene trees. The assignment of a (1-1) set of alleles to 

limonene in Tree AS-A95 seemed correct, and it did perform as a high limonene 

tree. It was a high limonene tree with one allele (0-1) each for α-pinene and ßpinene. 

It is interesting that no ultra-high limonene trees were found in a 1600-tree 

sample at Adin Summit, yet many of them were produced by crosses of trees in 

the area, such as Trees AS-3139, AS-A78, and AS-A95. Have trees of this type 

failed to survive, or is this just a juvenile characteristic? 

Crosses of P. ponderosa AS-5789 with Five Sources of Pollen 

Tree AS-5789, growing near Adin Summit, was 12 inches in diameter and had 

good growth and form. It was classed as near-high limonene. Composition was 

quite constant over time and location of the sample on the tree (table 6-2). It had 

moderately abundant cone crop during the years of breeding, and

comparatively larger families were obtained. It was also an easy and 

comfortable tree for climbing and breeding. It was crossed with five male trees, 

and progeny were grown in the nursery, in containers, and in a container/ 

plantation (table 6-7). The progeny of this female were of particular interest 

because of the assignment of a (0-1) set of alleles for myrcene.

Progeny of P. ponderosa AS-5789 x AS-3139

This was a cross of a near-high with a high limonene tree that were growing

near each other. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-5789 female 1 23 10 0 29 37 

AS-3139 male 1 18 1 0 22 59 

F-1 progeny 145 32 18 * 10 39 

There was a sharp decrease in limonene in both container-grown trees 

and in container/plantation trees. Therefore only nursery data were used in 

further analysis. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 345 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-27—Frequency distribution of 

the amount of the noted monoterpenes 

in the progeny of P. ponderosa AS-A95 x 

BL-6074; each dot is measurement of one 

tree; [▲] is average of set. 

346 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parent trees (fig. 6-28). A better 

assessment of the modal frequency distribution was possible because of the 

large number of samples (fig. 6-28). These analyses showed three to four readily 

discernible modes for four components. As expected, 3-carene was essentially 

zero. Mode proportions were close to the expected, and could be improved by 

reducing the modes to the expected number. If the two higher modes of 

myrcene were combined, to get the expected two modes in the distribution, 

mode proportions were 53:47. The two distinct modes for myrcene supported 

the assignment of the (0-1) set of alleles to Tree AS-5789. The upper mode was 

split into two by the modal analysis, probably because of the action of the 

modifying factors and the interactions of the basic monoterpene synthetic 

system. This two-mode distribution was achieved for all families of Tree AS5789 to further support the assignment of (0-1) alleles to myrcene. 

Ultra-high limonene progeny and high limonene progeny were produced 

by this cross of a near-high limonene with a high limonene tree (fig. 6-3). There 

were two ultra-high limonene and several high limonene trees. The large 

sample enabled a better assessment of types of composition. The incidence of 

ultra-high and high limonene trees was about the same for the reciprocal of this 

pair of trees, AS-3139 x AS-5789, as was the average of components. The two 

sets of reciprocals—even with small number of samples—were quite similar in 

response to the stress of container growth and growth in a container/plantation 

(table 6-8). Thus, monoterpene genes are not sex linked. 

Progeny of P. ponderosa AS-5789 x WP-3347 

This was a cross of two near-high limonene trees that were about 400 miles 

apart and that differed in other components. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-5789 female 1 23 10 0 29 37 

WP-3347 male 1 4 17 25 3 48 

F-1 progeny 71 7 39 29 6 17 

Seventy-one progeny were examined (table 6-7). Container growth caused a 

decrease in limonene and an increase in α-pinene; container/plantation growth 

caused a slight increase in limonene and a large decrease in 3-carene. Again, the 

small number of trees for container and container/plantation trees may not 

warrant any strong conclusions. The container and container/plantation trees 

were not included in the modal analysis. The frequency distributions were 

large for α-pinene, ß-pinene, 3-carene, myrcene, but unusually small for 

limonene (fig. 6-29). The three modes of limonene did not have the usual spread 

of more than 50 to 60 percent; instead it was only about 30 percent. 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parents (fig. 6-29). Mode 

proportions were reasonably close to the expected for myrcene, 45:55, and for 

ß-pinene, 63:37, if two modes were used. These results support the additive 

allele hypothesis and the assignment of alleles to the parent trees. 

There were no high limonene trees nor potentially high ones, though such 

would be expected (fig. 6-3). All trees had more than 20 percent ß-pinene and 

20 percent 3-carene. There should have been some trees lacking in ß-pinene or 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 347 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Table 6-7—Average xylem monoterpene composition of the progeny of tree P. ponderosa AS-5789 when crossed with five different 

males and when grown under three different conditions. N = number of trees. 

Monoterpene composition of progeny2 

Male 

parent 

Growth 

place1 N α-p cam ß-p car myr lim ß-ph ter 

percent3 

AS-3139 nur 145 32.2 0.1 18.2 0.1 10.0 39.3 0.1 0 

AS-3139 con 16 25.2 0.1 34.7 9.5 5.6 23.9 0.3 0.5 

AS-3139 copl 17 43.9 0.1 29.2 0 7.6 18.7 0.4 0 

WP-3347 nur 71 7.1 0.2 38.6 29.4 5.6 17.2 0.4 1.5 

WP-3347 con 7 8.0 0.1 45.5 28.3 3.0 12.1 0.9 1.8 

WP-3347 copl 8 9.7 * 44.8 19.4 4.9 19.9 0 1.3 

AS-3194 nur 75 16.5 0.1 27.0 25.2 7.7 21.7 0 1.0 

AS-3194 con 7 8.2 0 36.3 39.8 6.6 5.3 1.3 2.6 

AS-3194 copl 5 41.1 0.1 32.0 12.7 5.7 6.8 0.6 1.0 

WP-3318 nur 68 21.8 0.2 26.6 23.6 4.0 15.6 0.2 1.3 

WP-3318 con 9 29.3 0.2 32.0 23.7 3.0 10.3 0.4 1.2 

WP-3318 copl 8 30.6 0.2 35.1 14.7 3.5 14.6 0.4 0.9 

WP-3352 nur 9 32.1 * 28.0 0 11.3 28.1 * 0 

1

nur = nursery 6 to 8 years; con = 9-year container; copl = 9-year container/plantation. 2

α-pinene, camphene, ß-pinene, 3-carene, myrcene, limonene, ß-phellandrene, terpinolene. 3

Normalized; * = trace. 

Table 6-8—Average monoterpene composition of sets of reciprocal crosses: (A) AS-3139 x AS-5789, and (B) AS-5789 x AS-3139 grown in 3 locations. 

N=number of trees. 

Monoterpene1 

Cross Place of 

growth 

N α-p cam ß-p car myr lim ß-ph ter 

percent2 

A Nursery 35 27.0 * 15.5 0.1 10.9 46.5 0 0 

B Nursery 145 32.2 0.1 18.2 0.1 10.1 39.3 0.1 0 

A Container 9 32.6 0.2 36.8 0.1 8.2 21.3 0.8 0 

B Container 16 25.2 0.1 34.7 9.5 5.6 23.9 0.3 0.5 

A Copl3 7 41.4 0.1 28.9 5.1 8.8 19.0 0.2 0.6 

B Copl 17 43.9 0.1 29.2 0 7.6 18.7 0.4 0 

1

α-pinene, camphene, ß-pinene, 3-carene, myrcene, limonene, ß-phellandrene, terpinolene. 2

Normalized; * = trace. 3

Container Plantation.

348 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-28—Distribution and analyses of the monoterpenes in the progeny of P. ponderosa AS-5789 x AS-3139 ; see 

caption of figure 6-2 for explanation of the format below. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 349
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

3-carene, or both; in such trees, limonene would have been high, but there were 

no such trees. I attributed this to the great distance between parents; this 

caused a type of stress that was reflected in greater ß-pinene and 3-carene. The 

absence of a mode for 3-carene at or near zero supports the assignment of a (1-

1) set of alleles for 3-carene to WP-3347. 

Progeny of P. ponderosa AS-5789 x AS-3194

Both trees, growing near Adin Summit, were classed as near-high limonene, 

with 37 percent and 39 percent, respectively (table 6-1). 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-5789 female 1 23 10 0 29 37 

AS-3194 male 1 7 6 33 13 39 

F-1 progeny 75 17 27 25 8 22 

Seven trees, grown in containers for 7 years, had a sharp reduction in 

limonene and increases in ß-pinene and 3-carene, but there was a surprising 

decrease in α-pinene (table 6-7). The magnitude of the reduction in limonene 

was so great that one might speculate that the alleles for limonene did not 

function at all in either containers or in a container/plantation because of the 

stress of root containment and/or the over application of a phytocide. There was 

a sharp decrease in α-pinene in containers and a large increase in container/ 

plantation. The effects of stress, therefore, seemed to be both large and 

unpredictable. Thus, only data for nursery plants were used in further analysis. 

The analysis of the frequency distributions showed that the number of 

modes actually found in the analysis equalled or exceeded the number expected 

from the assignment of alleles to the parents (fig. 6-30). Mode proportions were 

fairly close to the expected. If the upper two modes of α-pinene were combined, 

proportions of 47:53 resulted; the two existing proportions for myrcene were 

39:61. The proximity of the parents was reflected in a relatively high average 

for limonene and for the incidence of high limonene trees. These results support 

the additive allele hypothesis and the assignment of alleles to the parent trees. 

There were no ultra-high limonene trees, though such was possible, as shown 

by other crosses of near-high limonene trees (fig. 6-3). When both ß-pinene and 

3-carene were low or lacking, there was an increase in α-pinene rather than 

limonene. It seemed that in a few instances, at least, the increase would have 

been in limonene. This could be an effect of the juvenile factor, and, in time, 

trees like types #9 and #20 (fig. 6-3) might become ultra-high limonene trees. 

This cross also illustrates the effect on composition by the interaction of the 

monoterpene synthesis system. Though parents were relatively high in 

limonene, the limonene composition was relatively low in the progeny because 

each parent brought another allele into the family to compete with limonene for 

the limited amount of the precursor. 

350 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-29—Distribution and analyses of the monoterpenes in the

progeny of P. ponderosa AS-5789 x W P-3347; see caption of figure 6-2 for

explanation of the format below. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 351
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-30—Distribution and analyses of the monoterpenes in the 

progeny of P. ponderosa AS-5789 x AS-3 194; see caption of figure 6-2 for

explanation of the format below. 

352 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Progeny of P. ponderosa AS-5798 x WP-3318

Tree WP-3318, like AS-5789, was classed as near-high limonene, but the two 

trees differed in the other components.

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-5789 female 1 23 10 0 29 37 

WP-3318 male 1 27 10 18 3 40 

F-1 progeny 68 22 27 24 4 16 

Thus, Tree WP-3318 had 18 percent 3-carene and 3 percent myrcene while 

Tree AS-5789 had 0 percent 3-carene and 29 percent myrcene (table 6-1). Trees 

grown in containers or in the container-plantation had lower limonene, higher 

α-pinene and ß-pinene, but about the same amount of 3-carene (table 6-7). 

Therefore, only data on nursery trees were used in further analysis. 

The analysis of the frequency distributions showed that the number of 

modes found by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-31). Mode proportions were fairly 

close to the expected: 47:53 for both 3-carene and myrcene. Mode proportions 

for α-pinene and limonene were not very close to the expected. The low 

amount of limonene in the progeny and the lack of the expected high limonene 

trees were attributed to the great distance between the parent trees. 

The lack of a mode near zero for 3-carene would appear to be an anomaly. 

That is, with a (0-0) set of alleles for Tree AS-5789 and a (0-1) set for Tree WP3318, one would have expected a moderate sized mode at or near 0 percent from 

a (0-0) set in the progeny. The lack of such results could be caused by some 

activity by the (0-0) set in the progeny due to the juvenile effect, or the incorrect 

assignment of a (0-1) in the male parent. However, other results in the study, 

which supported the (0-1) assignment, strongly suggested the juvenile effect as 

the cause and not an anomaly. This anomaly with WP-3183 as a male occurred 

in two other families. 

Progeny of P. ponderosa AS-5789 x WP-3352 

This was another cross of near-high limonene with high limonene, as in the 

previous cross; however the parents were about 400 miles apart. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-5789 female 1 23 10 0 29 37 

WP-3352 male 1 24 * * 18 57 

F-1 progeny 19 32 28 * 11 28 

Nineteen progeny were sampled and the average composition was 

somewhat different from the previous set, Trees AS-5789 x AS-3139 (table 6-7). 

This was attributed to the factor of the distance between parents—Tree AS-3139 

was a couple of miles from Tree AS-5789, while Tree WP-3352 was about 400 

miles away. The number of trees examined was insufficient for a good 

interpretation of frequency distributions (fig. 6-32). However, much like the 

progeny of Trees AS-5789 x AS-3139, the range of frequencies was large for αpinene, ß-pinene, myrcene, and limonene. Even with the small numbers, three 

modes for α-pinene and two for myrcene were easily discernable in this very 

small family (fig. 6-32). There was considerable similarity between this family 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 353 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-31—Distribution and analyses of the monoterpenes 

in the progeny of P. ponderosa AS-5789 x WP-3318; see caption

for figure 6-2 for explanation of the format below. 

354 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smi

th Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

and the family of Trees AS-5789 x AS-3139 in both average composition and in 

the characteristics of the frequency distributions, when the factor of distance 

between parents is considered. Both male parents—WP3352 and AS-3139— 

were high limonene. 

There were no ultra high limonene progeny (not tabled), again attributed to 

stress caused by the great distance between parent. Though high limonene 

trees were relatively common, they were not nearly as common as in the 

progeny of the previous cross of Trees AS-5789 x AS-3139. 

Discussion: Full Siblings of Tree AS-5789 

There were large differences in composition between nursery trees and trees 

grown in containers or in a container/plantation. This was a reflection of stress 

in the containers and container/plantation, and by the additional stress of the 

over application of a phytocide. This effect was evidenced by nursery trees 

having greater limonene and lesser α-pinene and ß-pinene. Therefore only data 

on nursery trees were analyzed. 

The actual number of modes in the frequency distribution of individual 

monoterpenes—as derived from modal analysis—equalled or exceeded the 

expected number—as derived from the assignment of alleles; this supported 

the additive allele hypothesis. Mode proportions were usually close to the 

expected to provide additional support to the hypothesis. 

The data for all crosses supported the assignment to Tree AS-5789 of (0-1), 

(0-1), (0-0), (0-1), and (1-1) sets of additive alleles, respectively, for α-pinene, ß– 

pinene, 3-carene, myrcene, limonene. Thus, though Tree AS-5789 was assigned 

a (1-1) set of alleles for limonene, the presence of three other alleles prevented it 

from functioning as a typical high limonene tree. 

The average for limonene and the number of high and ultra-high limonene 

trees was always higher in comparable crosses when the two parents were in 

proximity than when they were far apart (fig. 6-3). This difference was expressed 

much like that attributable to stress. It is postulated that there was a level of 

stress in progeny of the parents that were far apart. However, progeny of both 

near and distant parents responded similarly to the additional stress of 

container growth and over application of phytocide, which was further 

evidence that the difference between near and distant parentage was stress or 

stress-like. 

When the frequency distributions of the individual monoterpenes were 

compared for the families of Tree AS5789, it was clear that the averages and 

position of the modes of multimodal distributions varied according to the other 

alleles in the cross, though the number of modes did not change (fig. 6-33). Also, 

that portion of figure 6-33 for myrcene illustrated its bimodal characteristic for 

all the families of Tree AS-5789. Such crosses always resulted in two modes of 

equal proportions, thus supporting the assignment of a set of (0-1) for myrcene 

in AS5789. However, it also showed that myrcene was a rare allele. 

Tree AS-5789 had ultra-high limonene progeny when crossed with a high 

limonene male. This again showed that it had a (1-1) set of alleles for limonene. 

Reciprocal crosses were generally similar in average composition, in the 

occurrence of types of composition, and in the general characteristics of the 

frequency distributions. Likewise, both sets of reciprocals responded very 

similarly to the same location of growth. 

Tree AS-5789 was pollinated with two males. One of them was within 2 

miles of Tree AS-5789, and one was about 400 miles from Tree AS-5789. The 

ratios of limonene in the progeny vs. that in the male were calculated (table 6-9). 

These ratios again show that limonene in progeny of nearby parents was much 

greater than that in progeny of parents that were far apart. In general, the 

amount in nearby progeny was about twice as much. This was again an example 

of the effect of the distance between parents. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 355 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-32—Frequency distribution of 

the amount of the noted monoterpenes in 

the progeny of P. ponderosa AS-5789 x WP3352; each dot is measurement of one 

tree; [▲] is average of set. 

356 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-33—Comparative frequency distribution of the amount of 

the noted monoterpenes in the progeny of P. ponderosa AS-5789 when 

crossed with the five males listed on 

the right;

each is n trees; [▲] is average of set. 

each dot is measurement of 

one tree except where indicates 

(fig 6-33 continues on next 3 pages) 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 357 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

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Siblings of P. ponderosa 

Table 6-9—Ratio of limonene in male parent tree to average limonene in four sets of progeny of AS-5789: two sets with nearby male 

(AS-3139, AS-3194), two sets with distant males (WP-3347, WP-3318) 

Male tree Distance: female-male Limonene in: Ratio for limonene: 

male progeny progeny to male 

miles pct1 

AS-3139 3 59 39 1:1.5 

AS-3194 1 39 22 1:1.7 

WP-3347 400 48 17 1:2.8 

WP-3318 400 40 16 1:2.5 

1

Normalized. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 359 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

With the larger number of trees in the families of Tree AS-5789, mode 

assessment, analysis, and interpretation were made with greater confidence. But 

the conclusions reached from the results with the families of Tree AS-5789 were 

essentially the same as those made with much smaller families of other females. 

Thus, the results with Tree AS-5789 support the conclusions made with much 

smaller families. Though the larger families provide more confidence, they may 

not be all that much better in making correct conclusions. 

Crosses of P. ponderosa AS-A295 X Three Pollen Sources 

Tree AS-A295 was growing at Adin Summit (fig. 6-1). But the breeding was 

done on grafts in the Arboretum at IFG. Tree AS-A295 was selected for breeding 

because it had reasonable amounts of all the major components except ß-pinene. 

Progeny of P. ponderosa AS-A295 x AS-5789 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-A295 female 1 13 3 32 19 32 

AS-5789 male 1 23 10 0 29 37 

F-1 progeny 34 35 25 15 7 16 

The average composition of 34 nursery-grown progeny was much higher 

than expected in α-pinene and ß-pinene, and much lower than expected in 

limonene and myrcene (table 6-10). Container and container/plantation trees were 

even much higher in α-pinene or ß-pinene and much lower in limonene: this was 

attributable to stress. Thus, only nursery data were used for further analysis. 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parents (fig. 6-34). Mode 

proportions were very close to the expected. The three expected modes for αpinene were 21:51:28 which is very close to the expected 25:50:25. Mode 

proportions of 30:70 were achieved for 3-carene by combining modes to get the 

expected two. The two modes for myrcene were 57:43, which was quite close to 

the expected, and proportions of 35:65 were obtained for limonene by 

combining modes. 

Four trees had more than 40 percent limonene—as would be expected 

from a male lacking 3-carene and a female lacking ß-pinene—and could be 

classed as high limonene (fig. 6-3), but there was a moderate amount of ßpinene in all of them. Trees without 3-carene and with small amounts of 

3-pinene were high in α-pinene. These departures from the expected were 

probably due to the juvenile effect. 

Progeny of P. ponderosa AS-A295 x AS-A95

Tree AS-A95 was much like Tree AS-5789, the previous tree, in having nearhigh limonene. 

Percent normalized compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-A295 female 1 13 3 32 19 32 

AS-A95 male 1 19 21 0 15 45 

F-1 progeny 8 26 44 9 3 17 

The average composition of eight 4-year-old container trees was somewhat 

similar to progeny of Trees AS-A295 x AS-5789 (table 6-10). Some high limonene 

360 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Table 6-10—Average monoterpene composition of the progeny of AS-A295 with three males, grown in different locations, for 

varying periods of time. N - number of trees. 

Monoterpene composition of progeny2 

Male 

parent 

N Place1 of 

growth α-p cam ß-p car myr lim ß-ph ter 

percent3 

AS-5789 34 nur 34.8 0.1 25.4 15.3 7.2 15.9 0.4 0.9 

AS-5789 6 con 24.4 0.1 39.6 24.4 4.8 3.9 0.8 1.9 

AS-5789 4 copl 51.4 * 26.3 9.6 7.1 4.2 0.6 0.8 

AS-A95 8 con-4 25.9 0.1 43.9 9.3 2.6 16.6 0.7 0.9 

WP-3298 10 con-4 42.6 0 20.7 21.2 2.4 11.7 0.2 1.3 

1

nur = nursery; con = container; copl = container plantation. 2

α-pinene, camphene, ß-pinene, 3-carene, myrcene, limonene, ß-phellandrene, terpinolene. 

3Normalized; * = trace. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 361 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-34—Distribution and analyses of the monoterpenes 

in the progeny of P. ponderosa AS-A295 x AS-5789; see caption 

of figure 6-2 for explanation of the format below. 

362 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

trees were expected, but none was found (fig. 6-3); the 8-tree sample was too 

small to express the full range of composition types. The small sample also

precluded an analysis of the frequency distributions (fig. 6-35). Even with so 

few trees, there was a large range in amounts of the major monoterpenes. 

Progeny of P. ponderosa AS-A295 x WP-3298

Tree WP-3298 was high in limonene. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-A295 female 1 13 3 32 19 32 

WP-3298 male 1 26 * 0 16 58 

F-1 progeny 10 43 21 21 2 12 

Only 10 4-year progeny, which were grown in containers, were examined. 

The average composition was much higher in α-pinene, 43 percent, and much 

lower in limonene, 12 percent, than would be expected from additive alleles 

(table 6-10). Limonene was so low that one might suspect that one allele for it 

had not yet become functional. There were no high limonene progeny, though 

some were expected (fig. 6-3). The sample size was too small for an analysis of 

the frequency distributions (fig. 6-36); however, the distributional spread of 

four of the components was large, even with a small sample size. The expected 

two modes of α-pinene were discernible with proportions of 60:40. 

Here, as with the progeny of Trees AS-A295 x AS-A95, the lack of high 

limonene progeny might be attributed to the small number of samples, or it 

might be an expression of the great distance between the two parents. 

Discussion: Full Siblings of Tree AS-A295 

For the one set of progeny large enough for analysis, the actual number of 

modes in the frequency distribution of individual monoterpenes—as derived 

from modal analysis—equalled or exceeded the expected number of modes— 

as derived from the assignment of additive alleles; this supported the additive 

allele hypothesis and the assignment of alleles to Tree AS-A295 and to the 

male parent. Though classed only as moderate high limonene, there were 

progeny, when crossed with another near-high male, Tree AS-5789, that were 

high in limonene. Breeding with Tree AS-A295 was too limited to permit 

additional discussion. 

Crosses of P. ponderosa AS-3187 With 10 Pollen Sources 

Tree AS-3187 had moderate amounts of ß-pinene, 23 percent; 3-carene, 23 

percent; myrcene, 14 percent; limonene, 29 percent (table 6-1). It was growing 

near Adin Summit, had good growth, but was rather limby. Its compostion was 

quite constant over time and with location of growth (table 6-2). It had good 

cone crops during the period of breeding. As noted previously, it was a 

"comfortable" tree for breeding studies. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 363 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-35—Frequency distribution of 

the amount of the noted monoterpenes in 

the progeny of P. ponderosa AS-A295 x ASA95; each dot is measurement of one tree; 

[▲] is average of set. 

364 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-36—Frequency distribution 

of the amount of the noted monoterpenes in the progeny of P. ponderosa 

AS-A295 x WP-3298; each dot is measurement of one tree; [▲] is average 

of set. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 365
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Ten sources of pollen were used in controlled breeding with AS-3187, and 

progeny were grown in the nursery, containers, and in a container/plantation 

for 6 to 9 years (table 6-11). 

Progeny of AS-3187 x AS-3139 

Tree AS-3139 was a high limonene tree growing near Tree AS-3187 at Adin 

Summit. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

AS-3139 male 1 18 1 0 22 59 

F-1 progeny 88 17 29 17 4 32 

There were 88 nursery-grown trees and only five container and container/ 

plantation trees (table 6-11); therefore only the 88 nursery trees were used for 

further analysis. 

There were no large shifts in average composition between 6 years and 11 

years (table 6-12), or in the configuration of frequency distributions (fig. 6-37). 

Therefore, all nursery trees were aggregated for averages, frequency 

distributions, and for modal analysis. 

The analysis of the frequency distributions showed that the number of 

modes equalled or exceeded the number expected from the assignment of alleles 

to the parents (fig. 6-38). Results with mode proportions were mixed. The two 

proportions for α-pinene, 76:24,. probably were skewed because of the one 

measurement above 75 percent which drew the second mode too far up scale and 

shifted several measurements to the lower mode. By combining modes for ßpinene, proportions were 33:67; the proportions for the two modes for 3-carene 

were 42:58. By combining modes for limonene, proportions were 44:56. 

High limonene trees were common, but there were no ultra-high ones 

because a low percent 3-carene was never linked with low ß-pinene, a 

requirement of ultra-high limonene (fig. 6-3). There were 48 trees with 0 percent 

3-carene and eight with less than 15 percent ß-pinene, but there were no trees 

with both requirements. If this was a product of the juvenile factor, a few ultrahigh limonene trees may develop as the trees mature. 

Progeny of P. ponderosa AS-3187 x WP-3352 

Tree WP-3352 was a high limonene tree at Wheeler Peak with a composition 

essentially the same as that of the previous male, Tree AS-3139. 

Normalized percent compositions for parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

WP-3352 male 1 24 18 57 

F-1 progeny 29 16 39 20 4 20 

The average composition of the progeny differed from the progeny of Trees 

AS-3187 x AS-3139, the previous male (table 6-11); ß-pinene was much higher, 

39 percent, and limonene was much lower, 20 percent, than the corresponding 

averages for the progeny of Trees AS-3187 x AS-3139 that were 29 and 32 

percent, respectively. The data for all nursery trees were aggregated for 

analysis, though there was an appreciable difference in 3-carene for the two 

periods of nursery growth (table 6-12). There was no difference in the location 

366 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
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Siblings of P. ponderosa 

Table 6-11—Average monoterpene composition of progeny of P. ponderosa AS-3187 when crossed with 10 different sources of 

pollen and grown in three locations: nursery (nur), container (con), container/plantation (copl). N = number of trees. 

Monoterpene composition of progeny1 

Male Location of N α-p cam ß-p car myr lim ß-ph ter 

growth 

percent2 

WP-3352 nur 29 15.9 * 38.8 20.3 3.9 20.0 * 1.1 

WP-3347 nur 77 7.7 0.1 34.1 31.1 2.8 21.5 * 1.8 

WP-3347 con 16 8.2 * 38.5 32.3 2.4 16.6 0.3 1.6 

WP-3347 copl 10 12.2 0.1 38.0 23.0 4.3 20.1 0 1.6 

WP-3298 nur 52 22.4 * 37.3 19.4 3.2 16.1 0.1 1.0 

WP-3298 con 8 32.7 0.2 33.7 20.6 2.7 10.0 0.5 1.0 

WP-3298 copl 9 33.2 0.1 30.0 20.4 4.8 9.8 0.2 1.4 

AS-3194 nur 89 13.2 * 31.1 29.5 3.1 21.2 0.1 1.7 

AS-3194 con 8 8.4 * 34.3 39.0 1.9 14.4 0.4 1.6 

AS-3194 copl 1 26.8 0.1 31.6 23.1 3.9 12.8 * 1.7 

AS-3139 nur 88 16.6 0.1 28.7 17.0 4.3 32.2 * 1.1 

AS-3139 con 1 21.4 0.2 36.9 0 4.4 36.9 * * 

AS-3139 copl 4 29.9 0.1 36.5 6.4 5.3 21.3 0 0.6 

AS-5789 nur 34 17.5 * 42.9 15.4 6.5 16.6 0.1 0.8 

AS-5789 con 8 16.1 0.1 54.5 8.7 6.1 12.5 1.4 0.6 

AS-5789 copl 6 31.6 0.1 33.5 21.1 5.2 7.0 0.2 1.2 

WP-3299 nur 33 13.5 * 28.0 25.9 4.9 26.2 0 1.6 

WP-3299 con 5 14.7 0.4 30.5 34.9 3.6 12.8 0.7 2.2 

WP-3312 nur 34 12.3 * 20.6 35.0 3.7 26.4 0 1.9 

WP-3312 con 7 12.8 * 19.6 40.2 2.3 22.7 0 2.1 

WP-3312 copl 8 17.3 * 26.9 23.1 3.9 27.0 0.2 1.6 

WP-3318 nur 34 16.3 * 29.3 28.7 3.4 20.7 0 1.7 

WP-3318 con 8 8.5 * 42.0 28.7 2.4 16.8 0.3 1.2 

WP-3318 copl 9 23.7 0.1 33.5 17.0 3.4 20.8 0.2 1.2 

AS-3178 nur 18 9.7 0 18.8 46.1 8.0 14.6 0 2.7 

AS-3178 con 8 9.0 0.1 22.5 41.4 6.7 17.2 0.4 2.5 

AS-3178 copl 9 19.0 * 21.2 36.1 6.1 14.6 0.1 2.7 

1

α-pinene, camphene, ß-pinene, 3-carene, myrcene, limonene, ß-phellandrene, terpinolene. 2

 Normalized; * = trace. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 367 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Table 6-12—Average monoterpene composition of progeny of P. ponderosa AS-3187 when crossed with five different males with 

two ages (years) of nursery growth and. for aggregates (x). N = number of trees. 

Monoterpene composition of progeny1 

Male Age N α-p cam ß-p car myr lim ß-ph ter 

parent 

percent2 

WP-3352 6 19 15.6 * 42.2 14.6 4.0 22.7 * 0.8 

11 10 16.4 0 32.2 31.1 3.6 14.8 0 1.9 

x (=29) 15.9 * 38.8 20.3 3.9 20.0 * 1.1 

WP-3347 6 34 7.1 0.2 33.7 33.2 2.3 21.6 0.1 1.8 

11 43 8.0 0.1 36.2 28.9 3.1 22.1 0 1.7 

x ( =77) 7.7 0.1 34.9 31.1 2.8 21.5 * 1.8 

WP-3298 6 43 22.1 * 37.8 19.4 3.4 16.3 0.1 1.0 

11 9 23.9 * 34.8 19.8 5.0 15.4 0 1.1 

x ( =52) 22.4 * 37.3 19.4 3.7 16.1 0.1 1.0 

AS-3194 6 43 12.4 0.1 32.9 27.4 2.9 22.6 0.2 1.5 

11 46 14.0 * 29.3 31.5 3.4 19.9 0 1.8 

x ( =89) 13.2 * 31.1 29.5 3.1 21.2 0.1 1.7 

AS-3139 6 42 16.1 0.1 31.4 15.4 3.3 32.6 0.1 0.9 

11 46 17.1 0.1 26.2 18.4 5.2 31.9 0 1.2 

x ( =88) 16.6 0.1 28.7 17.0 4.3 32.2 * 1.8 

1

α-pinene, camphene, ß-pinene, 3-carene, myrcene, limonene, ß-phellandrene, terpinolene. 2

Normalized; * = trace. 

368 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-37—Comparative frequency distribution of the noted monoterpenes in the progeny of P. ponderosa 

AS-3187 x AS-3139 for two periods 

(years) of nursery growth listed on the 

right; each dot is measurement of one 

tree; [▲] is average of set. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 369 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-38—Distribution and analyses of the

monoterpenes in the progeny of P. ponderosa AS-3187 x 

AS-3139; see caption of figure 6-2 for explanation of the 

format below. 

370 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

of the modes because of the large separation of the two modes of 3-carene (fig. 

6-39). The characteristics of the frequency distributions of the progeny of Trees 

AS-3187 x WP-3352 were quite similar to those of the progeny of Trees AS-3187 

x AS-3139 except that the percent scale shifted. 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parents (fig. 6-39). Mode 

proportions were reasonably close to the expected for ß-pinene and limonene, 

but poor for α-pinene. The distribution of α-pinene was skewed far to the right 

because of two values greater than 26 percent; this caused part of the mode 

between 18 and 20 percent to be included in the low mode, therefore causing 

poor mode proportions. Without the two extreme values above 26 percent, the 

two modes would be separated at about 16 percent, and mode proportions 

would be nearly equal. By combining modes for ß-pinene to get the expected 

two modes, mode proportions were 63:37. Combining modes for limonene 

resulted in proportions of 46:54. 

Only one tree had more than 40 percent limonene and could be classed as 

high limonene; it had 36 percent ß-pinene, making it different from other high 

limonene trees (fig. 6-3). The whole set of progeny was low in limonene; this 

could be attributed to the juvenile factor, which caused greater amounts of αpinene, ß-pinene, and 3-carene, and to the great distance between parents 

which appeared to cause the same kinds of shifts—increase in ß-pinene and αpinene, and decrease in myrcene and limonene. These kinds of shifts were 

usually attributed to stress. 

Progeny of P. ponderosa AS-3187 x WP-3298 

The composition of the progeny when crossing Tree AS-3187 with Tree WP3298, another high limonene tree at Wheeler Peak (table 6-1), was nearly

identical to the composition of the previous cross of Trees AS-3187 x WP-3352. 

Normalized compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

WP-3298 male 1 26 0 16 58 

F-1 progeny 52 22 37 19 3 16 

Though the average compositions of the two sets of progeny, Trees AS-3187 

x WP-3298 and AS-3187 x WP-3352, were very similar, as expected (table 6-11), 

there were some small differences in the characteristic of the frequency 

distributions. The frequency distributions of the progeny of Trees AS-3187 x 

WP-3298 were very similar for two periods of nursery growth, though the 

number of trees for the 7- to 11-year period was probably too small for reliable 

interpretation (fig. 6-40, table 6-12); only data on the aggregated nursery-grown 

trees were analyzed further. 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parents (fig. 6-41). Mode 

proportions were fairly close to the expected. When combining modes for αpinene, ß-pinene, and 3-carene, and limonene, proportions were 47:53 for 

α-pinene, 36:64 for ß-pinene, 52:48 for 3-carene, and 53:47 for limonene. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 371 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-39—Distribution and analyses of the

monoterpenes in the progeny of P. ponderosa AS-3187 x 

WP-3352; see caption of figure 6-2 for explanation of the

format below. 

372 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-40—Comparative frequency 

distribution of the noted monoterpenes 

in the progeny of P. ponderosa AS-3187 x 

WP-3298 for two periods (years) of 

nursery growth listed on the right each 

dot is measurement of one tree; [▲] is 

average of set. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 373 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-41—Distribution and analyses of the monoterpenes in the progeny of P. ponderosa AS-3187 x WP-3298; see 

caption of figure 6-2 for explanation of the format below; 

** = mode at zero was not used in the modal analysis, but 

was included in the mode count. 

374 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

There were no high limonene trees, though a few were expected, and again 

the parental distance factor could have been the cause, as noted earlier for the 

progeny of Trees AS-3187 x WP-3352 (fig. 6-3). 

Progeny of P. ponderosa AS-3187 x WP-3299 

Tree WP-3299 was another near-high limonene tree. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

WP-3299 male 1 6 * 43 8 40 

F-1 progeny 33 14 28 26 5 26 

The average composition of 33 nursery trees generally failed to show 

intermediacy, with limonene and myrcene lower, and α-pinene and ß-pinene 

higher; 3-carene was about at the intermediate level (table 6-11). Only nurserygrown trees were used for further analysis. 

The analysis of the frequency distributions showed that the number of 

modes found by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-42). Mode proportions were 

reasonably close to the expected. By combining modes to the expected number, 

mode proportions for ß-pinene were 48:52, and for limonene the proportions 

were 55:45. The proportions of 33:30:27 for 3-carene were not too strong, but 

probably acceptable under the conditions of the various modifying factors. 

Though there were high limonene trees, they were not as common as 

expected of a cross of two such trees (fig. 6-3). Again, it was postulated that the 

parental distance factor caused a level of stress in the progeny, expressed by 

lower limonene and higher α-pinene and ß-pinene. 

Progeny of P. ponderosa AS-3187 x AS-3194

These two trees were growing within 50 yards of each other near Adin Summit. 

Tree AS-3194 was classed as a near-high limonene. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

AS-3194 male 1 7 6 33 13 39 

F-1 progeny 89 13 31 30 3 21 

The average for α-pinene and ß-pinene was much higher than expected, 

and myrcene and limonene were much lower than expected in the progeny 

(table 6-11). The composition of the nursery trees differed from container and 

container/plantation trees; therefore, only nursery data were analyzed further. 

The characteristics of the frequency distributions of the two ages of nursery 

growth were quite similar (table 6-12, fig. 6-43), and the two sets were aggregated 

for further analysis. The larger population seemed to improve the resolution of 

modes and mode proportions. 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parents (fig. 6-44). Mode 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 375 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-42—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa AS-3187 x 

WP-3299 ; see caption of figure 6-2 for explanation of 

the format below;**= mode at zero was not used in the 

modal analysis, but was included in the mode count. 

376 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-43—Comparative frequency 

distribution of the noted monoterpenes 

in the progeny of P. ponderosa AS-3187 x 

AS-3194 for two periods (years) of nursery growth, listed on right; each dot is 

measurement of one tree; [▲] is average of set. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 377 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-44—Distribution and analyses of the monoterpenes in the progeny of P. ponderosa AS-3187 x AS-3194; see 

caption of figure 6-2 for explanation of the format below; ** 

= mode at zero was not used in the modal analysis, but was 

included in the mode count. 

378 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

proportions were reasonably close to the expected when the possible effects of 

the modifying factors were considered. The proportions for the three modes of 

3-carene were very good at 23:54:23, but the proportions for limonene were not 

too good at 13:63:24. The proportions probably were still acceptable under the 

conditions of the several modifying factors. 

Neither parent was high limonene; yet there were a few high limonene 

progeny (fig. 6-3). One progeny had limonene near 60 percent, and if the 

juvenile effect lessens with age, this tree could become ultra-high limonene, 

because it lacked ß-pinene. 

Progeny of P. ponderosa AS-3187 x AS-S789 

Both parents were growing near Adin Summit (fig. 5-1). Tree AS-5789 was

classed as near-high limonene.

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

AS-5789 male 1 23 10 0 29 37 

F-1 progeny 34 18 43 15 7 17 

Tree AS-5789 had the highest percentage of myrcene of any tree used in the 

controlled breeding program and was assigned a (0-1) set of alleles. The average 

composition of 34 nursery-grown trees (table 6-11) was much higher than the 

intermediate level for ß-pinene and 3-carene, and much lower than the 

intermediate level for limonene and myrcene; the difference was attributed to 

the juvenile factor. The average for α-pinene was about as expected for 

intermediacy. The effects of being grown in containers and in a container/ 

plantation on trees were mixed; limonene was greatly depressed, especially in 

container/plantation trees; α-pinene was depressed slightly in container trees 

but was greatly increased in container/plantation trees; both ß-pinene and 3-

carene increased and decreased differently in container trees and in the 

container/plantation trees. This inconsistency of expression in container trees 

and in the container/plantation trees was probably caused by variation in the 

dosage of phytocide and the effects of prolonged root containment in the 

container-grown trees. Thus, only the 34 nursery-grown trees were used in 

further analysis. 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parents (fig. 6-45). Mode 

proportions were fairly close to the expected. The proportions for limonene 

were 62:38 by combining the frequency distribution to the expected two modes. 

When combining modes of the frequency distribution to the expected two 

modes, proportions for α-pinene and ß-pinene were 45:55 and 60:40, 

respectively. Proportions for myrcene were 60-40. There were two clearly 

separated modes for myrcene (fig. 6-45), adding support to the assignment of a 

set of (0-1) alleles to myrcene in Tree AS-5789. This characteristic of Tree AS5789 prevailed when the tree was male or female—as noted earlier when Tree 

AS-5789 was the female parent. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 379 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-45—Distribution and analyses of the monoterpenes in the progeny of P. ponderosa AS-3187 x AS-5789; 

see caption of figure 6-2 for explanation of the format

below. 

380 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Most trees were high in ß-pinene, and this could have been caused by the 

juvenile effect and by the presence of ß-pinene in both parents (fig. 6-3). I 

would expect ß-pinene to decrease with age and limonene to increase, though 

it is not likely that any will become high limonene. 

Progeny of P. ponderosa AS-3187 x WP-3318 

Tree WP-3318 was another near-high limonene tree (40 percent) growing at 

Wheeler Peak, with moderate α-pinene.

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

WP-3318 male 1 27 10 18 3 40 

F-1 progeny 34 16 29 29 3 21 

The average composition of 34 nursery progeny was not intermediate to the 

parents for any component (table 6-11). There were some differences in 9-

year container and container/plantation trees; therefore, only the data on 

nursery trees were analyzed further. 

The analysis of the frequency distributions showed that the number of 

modes found by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents, except for 3-carene (fig. 6-46). The 

modal characteristics of 3-carene did not fit the expected and would have to be 

considered an anomaly. This possible anomaly was discussed in the section 

"Progeny of P. ponderosa AS-5789 x WP-3318." Thus the two anomalies occurred 

when Tree WP-3318 was a male. The third case of a possible anomaly, which 

will be reported later, also involved Tree WP-3318 as the male parent. Mode 

proportions were not as close to the expected ones as was found in most other 

families, probably because of the small sample number and the effects of the 

modifying factors. By combining modes of the frequency distribution to get the 

expected number of modes, proportions of 39:61 and 51:49 were achieved for 

α-pinene and ß-pinene, respectively. The 3-mode proportions for 3-carene and 

limonene were only moderately close to the expected. 

There were no high limonene progeny, though a few were expected (fig. 6-

3). However, the parental distance factor probably accounted for this lack of 

high limonene. 

Progeny of P. ponderosa AS-3187 x WP-3312 

Tree WP-3312 was also classed as near-high limonene. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

WP-3312 male 1 5 7 34 4 48 

F-1 progeny 34 12 21 35 4 26 

The average composition of 34 nursery-grown trees was not intermediate 

to the two parent trees (table 6-11); α-pinene and ß-pinene were much higher 

than expected, limonene and myrcene were much lower, and 3-carene was 

about as expected. Again, these differences were attributed to the juvenile and 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 381 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-46—Distribution and analyses of the

monoterpenes in the progeny of P. ponderosa AS-3187

x WP-3318; see caption of figure 6-2 for explanation of

the format below.

382 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
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Siblings of P. ponderosa 

the parental distance factors. Results with container and container/plantation 

trees were variable. Container trees were higher in 3-carene and lower in 

limonene (table 6-11); α-pinene did not show the usual increase in containers. 

Only data on nursery-grown trees were included in further analyses. 

The analysis of the frequency distributions showed that the number of 

modes found by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-47). Mode proportions were very 

close to the expected when mode numbers were reduced to the expected. By 

combining modes of the analysis to get the expected number, proportions were 

48:52 for ß-pinene, 52:48 for 3-carene, and 50:50 for limonene. 

There were two high limonene trees in contrast to none in other sets of 

progeny between Tree AS-3187 and other distant males (fig. 6-3). Therefore, the 

speculation that there could be such trees in the other sets of progeny between 

Tree AS-3187 and distant near-high limonene males seems valid. 

Progeny of P. ponderosa AS-3187 x AS-3178

Tree AS-3178 had only moderate limonene, and was about 50 yards from Tree 

AS-3187. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

AS-3178 male 1 3 1 55 18 19 

F-1 progeny 18 10 19 46 8 15 

Though intermediacy was not found in the average composition of 18 

nursery progeny, it was not all that far from it (table 6-11). There were some 

differences in the composition of container and of container/plantation trees. 

Only the nursery-grown trees were used further. But the small number of 

samples precludes an analysis of frequency distribution (fig. 6-48), and types of 

composition were not listed in a table. 

There were no high limonene progeny. Though some were expected, 

they would have shown that such high limonene was possible in the progeny of 

two moderate-limonene trees growing in the same locality. There may not have 

been enough progeny for a high limonene progeny to occur. 

Progeny of P. ponderosa AS-3187 x WP-3347 

Tree WP-3347, growing at Wheeler Peak, was classed as near-high limonene. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

AS-3187 female 1 9 23 23 14 29 

WP-3347 male 1 4 17 25 3 48 

F-1 progeny 77 8 34 31 3 22 

The average composition of the progeny, as with other sets of progeny 

between Tree AS-3187 and males located at Wheeler Peak about 400 miles 

away, was higher than expected in ß-pinene and 3-carene and lower in myrcene 

and limonene (table 6-11). Neither the average composition nor the 

characteristics of the frequency distributions seemed to be affected by age of 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 383 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-47—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa AS-3187

x WP-3312; see caption of figure 6-2 for explanation of

the format below.

384 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-48—Frequency distribution 

of the amount of the noted monoterpenes in the progeny of P. ponderosa AS3187 x AS-3178; each dot is measurement of one tree; [▲] is average of 

set. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 385 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-49—Comparative frequency 

distribution of the amount of the noted 

monoterpenes in the progeny of P. ponderosa AS-3187 x WP-3347 for two 

periods of nursery growth (years), listed on the right; each dot is measurement of one tree; [▲] is average of 

set. 

386 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

nursery growth (table 6-12, fig. 6-49), though an age span of only 4 to 5 years 

may not be too meaningful. There were some rather small differences between 

nursery trees and container trees, particularly in 3-carene (table 6-12). However, 

the two sources were not aggregated for further analysis. 

The analysis of the frequency distributions showed that the number of 

modes found by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-50). By combining modes to get 

the expected number, proportions were 33:67 for ß-pinene and 45:55 for 3-

carene. Proportions for limonene were poor even by combining modes. 

None of the 98 progeny had more than 35 percent limonene (figs. 6-3, 6-49). 

One might speculate that because there were alleles for both ß-pinene and 3-

carene in both parents along with alleles for limonene, the progeny would tend 

to have alleles for all three components in most or all trees. Thus, through 

competition for a limited amount of precursor, limonene would tend to be less 

than 40 percent. That limonene was generally less than ß-pinene and 3-carene 

may be caused by the juvenile effect or by limonene being unable to compete 

equally with ß-pinene and 3-carene when they were both present during the 

juvenile period. 

Discussion: Full Siblings of Tree AS-3187 

The composition of nursery trees generally differed from that of container and 

container/plantation trees; therefore, only data on nursery trees were analyzed 

further. There was generally little or no difference due to age of growth in the 

nursery; therefore all nursery data were aggregated for further analysis. 

However, the differences due to place of growth were greatest for female trees 

with higher limonene. That is, limonene seemed to be more adversely affected 

by the juvenile factor than were other monoterpenes. Though the juvenile and 

parental distance factors had an appreciable effect on composition, the effect 

was relative and simply moved the modes to other positions on the percent 

scale; the number of modes was not changed, but their positions were. 

The actual number of modes, as derived from modal analysis, with one 

exception, always equalled or exceeded the expected number, as derived from 

the assignment of additive alleles to the parents; this supported the additive 

allele hypothesis. The aggregate data also reaffirm the assignment of alleles to 

tree AS-3187 (table 6-1). Most mode proportions were reasonably close to the 

expected when using the expected number, providing additional support to 

the hypothesis. 

No ultra-high limonene trees were found in the progeny of Tree AS-3187 

(fig. 6-3), and high limonene progeny were not at all common, though high 

limonene males were used. 

The average for limonene and the number of high limonene trees were 

much higher in sets of progeny with nearby males than with distant males. 

Again, a stress factor and the distance between parents were postulated as the 

causes. 

Tree 3187 was an excellent tree for the breeding studies, and the results with 

its progeny added much to the conclusions. It was also a desirable tree because of 

its good cone crops, easy breeding, and ease with which it could be climbed. 

Crosses of P. ponderosa IFG-4B63 with 12 Pollen Sources 

Tree IFG-4B63 was growing at the Institute of Forest Genetics (fig. 5-1). It had 

been used for much breeding and other studies of P. ponderosa at IFG. Its 

monoterpene composition (table 6-1) was characterized as having 0 percent 3-

carene; having a large amount of ß-pinene, 55 percent; and having smaller 

amounts of α-pinene, 9 percent; myrcene, 12 percent; and limonene, 22 percent. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 387 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-50—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa AS-3187 x

WP-3347; see caption of figure 6-2 for explanation of

the format below.

388 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Twelve different sources of pollen were used with it (table 6-13). Though there 

was considerable range in the percent myrcene in the male parents, there was 

little range in the various sets of progeny. It was assumed that the juvenile 

effect caused a reduction of the full effect of myrcene and possibly limonene. 

The average composition of 5- to 7-year-old nursery trees was nearly the same 

as 4-year-old container trees; therefore, nursery and 4-year container trees were 

aggregated, but 9-year container and container/plantation trees were not 

aggregated because they differed somewhat and there were so few of them. 

Progeny with moderate to high limonene had been expected in crosses with 

Tree IFG-4B63, particularly males with high or near-high limonene. But these 

progeny did not occur, though there were a few individual trees with 45 to 50 

percent limonene. This could again point to the possibility of 13-pinene, which 

was very high in Tree IFG-4B63, being a strong factor in the juvenile effect. But 

just how this factor works was not clear. That is, does it operate by "turning 

down" limonene and myrcene, or by "turning up" α-pinene and ß-pinene. 

However, the results with IFG-4B63 were satisfactory. Though the frequency 

distribution was moved up or down the percent scale, the modal structure 

generally remained unchanged. 

Progeny of P. ponderosa IFG-4B63 x AS-3178 

Tree AS-3178 was the reverse of Tree IFG-4B63 with respect to ß-pinene and 3-

carene. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

AS-3178 male 1 3 1 55 18 19 

F-1 progeny 70 11 45 22 5 17 

There were only slight differences between data on nursery and 4-year-old 

trees; therefore they were aggregated. The average composition of the 70 trees 

was expected to have about equal amounts of ß-pinene and 3-carene, because 

IFG-4B63 had two alleles for ß-pinene and AS-3178 had two alleles for 3-

carene. Instead, the average amounts in the progeny were 45 percent ß-pinene 

and 21 percent 3-carene. This suggests, again, that ß-pinene was the strong 

element in the juvenile factor. This was evident in all the progeny of IFG-4B63. 

The analysis of the frequency distributions showed that the number of 

modes actually found by the analysis equalled or exceeded the number 

expected from the assignment of alleles to the parent trees (fig. 6-51). This 

supports the additive allele hypothesis. By combining modes to get the expected 

number, mode proportions were 48:52 for ß-pinene, and 39:61 for 3-carene. 

The frequency distribution of 3-carene had two modes, one at 0 and one at 

about 30 percent, where one mode was expected; i.e., with a (1-1) set of alleles 

for 3-carene in AS-3178, all progeny should have at least one allele for 3-carene. 

The one mode at 30 percent was expected, but not the mode at 0. I attribute this 

to the juvenile factor which seemed unusually strong when ß-pinene was a 

large percent, and the percent of ß-pinene was high in all progeny (fig. 6-51, 

table 6-13). This condition was also found in the progeny of IFG-4B63 x AS-3312 

(discussed later in this section). And here too there was a large percent of ßpinene. However, when there were two alleles for 3-carene, and 3-pinene was 

absent or very small, there was no mode at 0 for 3-carene (See AS-3139 x 3178, 

AS-3187 x AS- 3178, AS-3139 x WP-3347, AS-3139 x WP-3312). AS-3178 was the 

male parent in two of the examples, and in each of these two, the absence of a 0 

mode for 3-carene supports the assignment of a (1-1) set of alleles for 3-carene 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 389 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Table 6-13—Average xylem monoterpene of progeny of P. ponderosa 1FG-4B63 when used with 12 sources of pollen and 

grown under different conditions. N = number of trees. 

Monoterpene composition of progeny1 

Male 

parent 

N Place of 

growth2 

α-p cam ß-p car myr lim ß-ph ter 

percent

3 

AS-3178 51 nur 10.6 0.1 42.1 22.4 5.6 17.8 0.2 0.9 

AS-3178 19 con-4 11.7 0 53.3 17.0 1.6 15.1 0.3 1.0 

AS-3194 12 nur 13.0 * 48.6 22.5 1.4 13.0 0.4 1.1 

AS-3194 11 con-4 10.4 0 52.5 14.6 1.7 19.9 0 1.0 

AS-3187 23 nur 7.1 * 31.9 36.3 7.1 15.7 0 1.9 

AS-3187 27 con-4 7.3 0 34.2 39.5 5.2 12.3 0.1 2.4 

WP-3318 24 nur 21.0 0.1 39.2 18.1 2.4 18.3 0 0.9 

WP-3318 8 con-9 21.8 0.2 43.0 19.8 1.7 11.7 1.0 1.0 

WP-3318 7 con-4 21.2 0 43.5 20.2 1.3 12.8 0 1.0 

WP-3318 3 copl 23.4 0.1 48.7 10.6 1.8 14.9 0 0.6 

WP-3312 74 nur 13.3 * 40.0 18.8 3.3 23.4 0.2 0.8 

WP-3312 9 con-4 14.0 0.1 34.7 30.0 1.7 18.2 0 1.2 

WP-3299 8 nur 12.7 0 41.6 19.4 3.3 22.1 0 0.9 

WP-3299 l con-4 9.7 0 36.5 33.2 6.2 10.4 1.6 1.8 

AS-5759 14 nur 18.0 0 66.5 0 4.0 11.3 0.1 0 

AS-5789 16 con-4 16.8 0.1 67.3 0 4.6 9.5 1.0 0.8 

AS-A95 26 nur 18.4 0.1 52.6 0 1.9 27.0 0 0 

AS-A95 27 con-4 16.1 0 55.8 2.9 1.5 23.3 0 0.1 

AS-3139 26 nur 19.6 0 49.7 2.6 2.6 28.0 0 0 

AS-3139 21 con-4 16.3 0 49.0 5.5 2.0 27.0 0 0.3 

WP-3298 23 nur 30.4 0 56.0 0 2.5 11.0 0 0 

WP-3298 6 con-9 31.8 0.2 51.9 0.8 2.7 11.5 0 1.2 

WP-3298 5 copl 45.4 0.1 44.2 0 3.2 7.1 0 0 

WP-3298 25 con-4 28.0 0.1 55.0 3.1 2.1 10.0 1.4 0.2 

BL-6074 2 nur 18.6 0.1 49.5 0 3.1 28.6 0 0 

WP-3352 60 nur 18.1 0.1 60.8 0 2.3 18.1 0.4 0 

WP-3352 8 con-9 19.4 0.1 63.2 1.4 1.9 12.7 1.3 0.1 

1

α-pinene, camphene, ß-pinene, 3-carene, myrcene, limonene, ß-phellandrene, terpinolene. 2

nur = nursery; con = container; copl = container-plantation. 3

Normalized; * = trace. 

390 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Fig. 6-51—Distribution and analyses of the

monoterpenes in the progeny of P. ponderosa IFG4B63 x AS-3178; see caption of figure 6-2 for explanation of the format below. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 391
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

in AS-3178. It might be that, in the juvenile period, an allele for ß-pinene 

prevents the action of an allele for 3-carene. Very little is known about the 

effects of the juvenile factor and just how it operates. 

Progeny of P. ponderosa IFG-4B63 x AS-3194 

Tree AS-3194 was classed as a near-high limonene tree. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

AS-3194 male 1 7 6 33 13 39 

F-1 progeny 23 12 50 19 2 17 

Though the data for nursery and 4-year container trees differed a bit (table 

6-13), they were aggregated for averages and for frequency distribution display. 

The small sample number ruled out modal analysis. The averages for the 

progeny of Trees IFG-4B63 x AS-3178 and IFG-4B63 x AS-3194 were quite 

similar. Even with a small sample, a bimodal distribution was discernible for 

ß-pinene and 3-carene (fig. 6-52). 

Progeny of P. ponderosa IFG-4B63 xAS-3187

Tree AS-3187 had moderate amounts of ß-pinene, 3-carene, myrcene, and

limonene, and a small amount of α-pinene (table 6-1). 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

AS-3187 male 1 9 23 23 14 29 

F-1 progeny 50 7 33 38 6 14 

There was little difference between nursery and 4-year container data; 

therefore, they were aggregated. The average for 50 progeny (table 6-13) showed 

the usual juvenile effects in the reduced amounts of myrcene and limonene and 

the increased amount of ß-pinene. However, the amount of 3-carene, 38 

percent, was surprising because 3-carene was absent in Tree IFG-4B63 and 

present in only a moderate amount in Tree AS-3187. Most notable about the 

frequency distributions was their unusually small range (fig. 6-53). The 

combination of these two parents seemed to be different from most other 

combinations of parents, in the narrow range of the frequency distribution of 

individual components. There could have been a cancelling out of the factors 

that can cause variation. Though the ranges in the distributions were usually 

small, compared to other sets of progeny, they were still suitable for modal 

analysis. This narrow range in frequency distributions seemed to be more 

common in the progeny of Tree IFG-4B63 than in the progeny of other females. 

The analysis of the frequency distributions showed that the number of 

modes found by the analysis equalled or exceeded the number expected based 

on the assignment of alleles to the parents (fig. 6-53). This supports the additive 

allele hypothesis. Mode proportions were very close to the expected: for αpinene, 55:45; ß-pinene, 44:56; and 3-carene, 64:36. The 3-mode proportions for 

limonene were not very close to the expected, probably because of the aggregate 

effect of the modifying factors. 

392 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-52—Frequency distribution 

of the amount of the noted monoterpenes in the progeny of P. ponderosa 

IFG-4B63 x AS-3194; each dot is measurement of one tree; [▲] is average 

of set. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 393 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-53—Distribution and analyses of the monoterpenes in

the progeny of P. ponderosa IFG-4863 x WP-3187; see caption of figure 6-2 for explanation of the format below. 

394 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

The lack of potentially high limonene trees was not surprising with the 

composition of the two parent trees (fig. 6-3). 

Progeny of P. ponderosa IFG-4B63 x WP-3318 

Tree WP-3318 was near-high limonene. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene

IFG-4B63 female 1 10 54 0 11 23 

WP-3318 male 1 27 10 18 3 40 

F-1 progeny 31 21 40 18 2 18 

There was little difference between nursery and 4-year container data (table 

6-13); therefore they were aggregated for further analysis. The 9-year container 

and container/plantation data did differ, somewhat, and were not included in 

further analysis. 

The analysis of the frequency distributions showed that the number of 

modes found by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-54). Mode proportions were 

somewhat close to the expected, when all conditions were considered. The 

proportions for α-pinene at 44:56 and for 3-carene at 33:67 were fairly close to 

the expected. By combining to get the expected two modes for ß-pinene, 

proportions were 41:59. The three modes for limonene were 24:60:16 and quite 

close to the expected. 

None of the progeny had a mode at or near 0 percent for 3-carene where 

one would be expected. This was the third case of an anomaly with 3-carene as 

discussed previously for the progeny of Trees AS-5789 x WP-3318 and AS-3187 

x WP-3318. All three cases involved Tree WP-3318 as a male which was assigned 

a (0-1) set of alleles for 3-carene based on the 18 percent 3-carene in its 

composition. Could there have been a mistake in the processing of Tree WP3318? Or was there something unusual about this tree? High 3-carene and high 

ß-pinene were the most common types of composition (fig. 6-3.) 

Progeny of P. ponderosa IFG-4B63 x WP-3312 

The composition of Tree WP 3312 was similar to that of Tree AS-3194 except

Tree WP-3312 had somewhat higher limonene. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene

IFG-4B63 female 1 10 54 0 11 23 

WP-3312 male 1 5 7 34 4 48 

F-1 progeny 83 13 40 20 3 23 

There were small differences between nursery and 4-year container data; 

however, it was not considered too large, and the two sets of data were 

aggregated for further analysis (table 6-13). The average composition of 83 

nursery and container progeny was higher in limonene than the progeny of 

Trees IFG-B63 x AS-3194; the former was 23 percent vs. 13 percent for the latter. 

The analysis of the frequency distributions showed that the number of 

modes derived by the analysis equalled or exceeded the number expected from 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 395 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-54—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa IFG4B63 x WP-3318; see caption of figure 6-2 for explanation of the format below. 

396 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

the assignment of alleles to the parents (fig. 6-55). Mode proportions were only 

moderately close to the expected. Combining for the expected two modes for 3-

carene, proportions were 30:70, and combining for the expected two modes for 

limonene, proportions were 55:45. 

The absence of a mode at or near 0 percent for 3-carene supported the 

assignment of a (1-1) set of alleles to Tree WP-3312. 

Again, as in other sets of progeny of Tree IFG-4B63, the ranges in the 

frequency distributions were relatively narrow. However, the modal 

configuration was suitable for good analysis. Potentially high limonene trees 

were expected and were present, but were not common (fig. 6-3). 

Progeny of P. ponderosa IFG-4B63 x WP-3299 

Tree WP-3299 was near-high limonene with a large amount of 3-carene.

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

WP-3299 male 1 6 43 8 40 

F-1 progeny 8 13 42 19 3 22 

There was only one 4-year container tree, and its composition was quite 

different from that of nursery trees; therefore, the container tree was not added 

to the nursery trees. Only eight progeny were sampled, and the average 

composition was higher in ß-pinene and lower in 3-carene than expected (table 

6-13), i.e., near-equal amounts would be expected. This again pointed to a 

juvenile increase in ß-pinene. This same result was found in the progeny of 

Trees IFG-4B63 x WP-3178. With only eight samples, little can be said about the 

frequency distributions except that the range was quite large even with so few 

samples, and that the multimodal characteristics were beginning to be evident 

for 3-pinene, 3-carene, and limonene (fig. 6-56). 

Types of composition were not put in a table, but trees with and without 3-

carene were expected and found. 

Progeny of P. ponderosa IFG-4863 x AS-5789

Tree AS-5789, like Tree IFG-4B63, had no 3-carene, but had near-high limonene.

Normalized percent compositions for parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

AS-5789 male 1 23 10 0 29 37 

F-1 progeny 30 18 67 0 4 10 

Data on nursery and 4-year containers data were aggregated. The average 

of the aggregated 30 nursery and container trees was not as expected (table 6-

13) when compared with the previous sets of progeny. Limonene was only 

about half as much as expected, and myrcene was much lower than expected. 

The average for limonene was 10 percent, while 20 percent was expected. It 

could be that one of the alleles for limonene had not yet become functional in 

the progeny of this particular combination of parents. However, if this were so, 

one would expect a reduction in the number of modes. Because this was not so, 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 397 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-55—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa IFG-4863 

x WP-3312; see caption of figure 6-2 for explanation of 

the format below; **= mode at zero not used in modal 

analysis, but was included in mode count. 

398 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-56—Frequency distribution 

of the amount of the noted monoterpenes in the progeny of P. ponderosa 

IFG-4863 x WP-3299; each dot is measurement of one tree; [▲] is average of 

set. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 399 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

it appeared that any allele for limonene was affected. The composition of the 

progeny does reflect the high ß-pinene and the absence of 3-carene, which was 

expected. The two modes for ß-pinene support the assignment of a (1-1) set of 

alleles to Tree IFG-4B63 and a set of (0-1) alleles to Tree AS-5789. Myrcene had 

two modes of equal proportions which again supports the assignment of a (0-1) 

set of alleles to Tree AS-5789, as shown in other families where this tree was 

male or female. 

The analysis of the frequency distributions showed that the number of 

modes derived by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-57). Mode proportions were 

reasonably close to the expected. By combining modes for α-pinene and 

limonene, proportions of 39:61 and 40:60, respectively, were found. 

It appeared that nearly all trees went to ß-pinene rather than to limonene 

and myrcene (fig. 6-3). This could have been caused by the juvenile effect. As 

expected, all trees lacked 3-carene. There were not even small amounts; this 

suggested that 3-carene was not produced by isomerization in this parental 

combination. It will be shown shortly that the progeny of Trees IFG-4B63 x WP3298 (both trees had no 3-carene) did have small amounts of 3-carene. This was 

suggestive of isomerization, but this is an uncommon occurrence. 

Progeny of P. ponderosa IFG-4863 x AS-A95 

Tree AS-A95 was near-high limonene. It was discussed earlier in this chapter as

a male and as a female. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

AS-A95 male 1 19 21 0 15 45 

F-1 progeny 53 17 54 2 2 25 

The data for nursery and 4-year container plants were similar and were 

aggregated for further analysis (table 6-13). The average composition of 53 

nursery and container progeny was much higher in limonene, 25 percent, than 

the progeny of crosses with Trees WP-3312 and WP-3298, all having higher 

limonene than Tree AS-A95. This could have been an expression of parents in 

different ecological (resin) zones. Trees IFG-4B63 and AS-A95 are about 150 

miles apart, but both are in the Sierra Pacific Region (see Chapter 3 of this 

report). The other two trees were more than 400 miles from Tree IFG-4B63 and 

are in the Escalante North Transition Zone. 

The progeny of Trees IFG-4B63 x AS-A95 had little or no 3-carene, as 

expected (fig. 6-58). There were, however, nine trees with about 8 percent 3-

carene, and all were in the 4-year container group. This was much like the 

transient time-of-growth anomaly found for camphene in Chapter 5. 

The analysis of the frequency distributions showed that the number of 

modes derived by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-58). Mode proportions were 

reasonably close to the expected if modes were combined to get the expected 

number. This resulted in proportions of 35:65 for α-pinene, 45:55 for ß-pinene, 

and 39:61 for limonene. 

400 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-57—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa IFG4B63 x AS-5789; see caption of figure 6-2 for explanation of the format below. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 401
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-58—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa IFG4B63 x AS-A95; see caption of figure 6-2 for explanation of the format below. 

402 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Though there were only two high limonene trees, this was more than in the 

sets of progeny with a distant male tree with much more limonene than Tree 

AS-A95 (fig. 6-3). Again, the distance between parents was a possible reason. 

Progeny of P. ponderosa IFG-4B63 x AS-3139 

Tree AS-3139 was high limonene with essentially neither ß-pinene or 3-carene. 

Normalized percent compositions for parents and progeny were as follows: 

Tree and type Number a pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

AS-3139 male 1 18 1 0 22 59 

F-1 progeny 47 18 49 3 2 28 

Data for nursery and 4-year container trees were quite similar (table 6-13) 

and were aggregated for further analysis; 9-year container and container/ 

plantation progeny were quite different and were not used for further analysis. 

The analysis of the frequency distributions showed that the number of 

modes derived by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-59). Mode proportions were not 

as close to the expected as in other families. The proportions for α-pinene were 

strongly affected by one measurement at 42 percent. The combined modes for 

limonene had proportions of 78:22, which was marginally good. Again, as with 

the progeny of Tree AS-A95 above, limonene was much greater when parents 

were in the same ecological (resin) zone than when they were in different zones. 

High limonene progeny were expected but none were found, although the 

level of limonene was better than most other crosses (fig. 6-3). Most of the 

composition went to ß-pinene, possibly because of the juvenile factor. More 

trees may change to higher limonene as the trees age. 

Progeny of P. ponderosa IFG-4863 x WP-3298 

Tree WP-3298 was another high limonene tree with a composition almost

identical to that of Tree AS-3139. However, it was growing at Wheeler Peak, 

more than 400 miles to the east. It was reported earlier in this chapter as a 

pollen source with other females. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

WP-3298 male 1 26 0 16 58 

F-1 progeny 48 29 56 2 2 11 

Data on nursery and 4-year container trees were nearly identical and were 

aggregated for further analysis (table 6-13); the data for 9-year-old container 

and container/plantation trees were not aggregated with the nursery data. The 

average composition of the progeny of Trees IFG-4B63 x WP-3298 was quite 

different from the progeny of Trees IFG-4B63 x AS-3139; both males were high 

in limonene and had about the same composition for all other components. The 

progeny with WP-3298 were quite low in limonene, 11 percent, and were quite 

high in α-pinene, 29 percent, whereas the progeny with Tree AS-3139 averaged 

about 28 percent in limonene (table 6-13). The factor of distance between parents, 

and/or the juvenile effect, or the location of the parents in different ecological 

(resin) regions could explain this difference. 

The analysis of the frequency distributions showed that the number of 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 403 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-59—Distribution and analyses of the

monoterpenes in the progeny of P. ponderosa

IFG-4B63 x AS-3139; see caption of figure 6-2

for explanation of the format below. 

404 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

modes found by the analysis equalled or exceeded the number expected from 

the assignment of alleles to the parents (fig. 6-60). Mode proportions were 

comparatively good: 43:57 for α-pinene, and 48:52 for limonene by combining 

modes to get the expected number. 

There were no high limonene trees, and only a rare potential one (fig. 6-3). 

Here again the composition went to higher α-pinene or ß-pinene when 3-

carene was lacking or very low. Though most trees had no 3-carene, as expected, 

there were several trees with about 5 to 10 percent; this could have been caused 

by isomerization, or by some minor genetic structure. 

Progeny of P. ponderosa IFG-4B63 x WP-3352 

Tree WP-3352 was another high limonene tree with nearly the exact 

composition of Trees WP-3298 and AS-3139, the two previous males used on

Tree IFG-4B63 (table 6-1). WP-3352 was reported in this chapter as a pollen

source with other females. 

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

WP-3352 male 1 24 * * 18 57 

F-1 progeny 60 18 61 0 2 18 

The average composition of sixty nursery and container progeny was (table 

6-13) very similar to the previous family where WP-3298 was the male parent. 

The progeny with Tree WP-3298 had the highest average ß-pinene of the two, 

66 percent, despite having only one allele for ß-pinene, which it got from the 

female. Average limonene in this family, with WP-3352 as the male, was 18 

percent; limonene was only 11 percent in the family with WP-3298 as the male. 

The frequency distributions of the progeny of Tree IFG-4B63 x WP-3352 differed 

only slightly from the previous set of progeny in mode configuration (fig. 6-61), 

though the percent scales were somewhat different. 

The analysis of the frequency distributions showed that the number of 

modes derived by the analysis equalled or exceeded the number expected, based 

on the assignment of alleles to the parents (fig. 6-61). Mode proportions were 

fairly good. By combining to get the expected two modes for α-pinene and 

limonene, proportions were 46:53 and 37:63, respectively. 

There were no high limonene trees, nor any potential ones (fig-6-3). If the 

juvenile factor was the cause of the lack of such trees, some may develop as the 

trees mature. 

Progeny of P. ponderosa IFG-4B63 x BL-6074 

Tree BL-6074 was high limonene, 57 percent, with a small amount of α-pinene

and ß-pinene (table 6-1). It was reported earlier in this chapter as a pollen

source with other females.

Normalized percent compositions of parents and progeny were as follows: 

Tree and type Number α-pinene ß-pinene 3-carene myrcene limonene 

IFG-4B63 female 1 10 54 0 11 23 

BL-6074 male 1 7 11 0 27 56 

F-1 progeny 2 18 50 0 3 29 

Though there were only two trees (table 6-13), the results still strongly show 

that progeny of parents with about the same composition, from different 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 405 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

Figure 6-60—Distribution and analyses of the 

monoterpenes in the progeny of P. ponderosa IFG 

4B63 x WP-3298; see caption of figure 6-2 for expla 

nation of the format below. 

406 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

Siblings of P. ponderosa 

Figure 6-61—Distribution and analyses of 

the monoterpenes in the progeny of P. ponderosa IFG-4B63 x WP-3352; see caption of 

figure 6-2 for explanation of the format 

below. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 407 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

ecological regions, may have progeny with somewhat different composition 

than progeny of parents in the same ecological region when both sets of parents 

have the same primary structure of alleles. This difference in composition 

could be due to a stress factor of early growth and to parents who are from 

different ecological regions. The average for limonene was 28 percent in the 

progeny of IFG-4B63 with three different high limonene males in the Sierra 

Pacific Region: Trees AS-3139, AS-A95, and BL-6074 (table 6-13). On the other 

hand, limonene averaged only 11 to 18 percent in the progeny with high 

limonene males from the Escalante North transition zone, Trees WP-3298 and 

WP-3352 (table 6-13). Again, this points to the effect of distance between parents. 

Discussion: Full Siblings of Tree IFG-4B63 

The compositions of nursery and 4-year container trees of the families of IFG4B63 were usually about the same and were usually aggregated for analysis. 

The compositions of 9-year container and 9-year container/plantation trees 

nearly always differed from that of nursery trees and were not aggregated with 

the nursery and 4-year container trees for further analysis. 

The actual number of modes, as derived from modal analysis, always 

equalled or exceeded the expected number, as derived from the assignment of 

additive alleles to the parent trees; this supported the additive allele hypothesis. 

In general, the data also confirm the assignment of alleles to Tree IFG-4B63 and 

to the males used with it. Mode proportions were generally close to the expected 

when the various modifying factors were considered; this provided additional 

support to the hypothesis. Mode proportions were usually improved by using 

the expected number of modes rather than the number actually derived from the 

modal analysis. 

There were no ultra-high limonene trees among the progeny and very few 

high limonene ones. What few there were showed a tendency to be more 

common in sets of progeny with nearby males, again supporting the postulate 

that there is greater stress in progeny of more distant parents. 

A higher average limonene had been expected in the progeny with high 

limonene males because Tree IFG-4B63 had no 3-carene and a moderate amount 

of limonene. Instead, the composition of most progeny was much greater than 

expected in α-pinene and ß-pinene. This might be caused by the juvenile 

effect, particularly with large amounts of ß-pinene present in all progeny; the 

average for limonene could increase with age. This was the only parent with 

two alleles for ß-pinene, and the large amount of ß-pinene in all progeny 

supports the assumption that ß-pinene is a very strong element in the juvenile 

factor. Though this had a strong effect on composition, it apparently did not 

change the basic modal characteristics; instead, the modes were shifted strongly 

to the lower end of the percent scale. 

The effect of parental distance on the level of limonene was clearly shown 

in sets of progeny with Tree IFG-4B63. The progeny with high limonene males 

in the same ecological region—Trees AS-3139, AS-A95, BL-6074—averaged 25 

to 30 percent, with individual trees as high as 50 percent. The progeny with high 

limonene males from another ecological region—Trees WP-3298 and WP3352—averaged about 14 percent, with individual trees less than 36 percent. 

The aggregate data support the assignment of sets of alleles of (0-0), (1-1), 

(0-0), (0-0), (0-1), respectively, for α-pinene, ß-pinene, 3-carene, myrcene, 

limonene to Tree IFG-4B63. 

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th Monoterpene Composition of Populations of 4- to 11-Year-Old Full Chapter 6 

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Discussion: Full Siblings of P. ponderosa 

The breeding program of seven females with 4 to 14 sources of pollen produced 

56 full sibling families; 42 of these families were large enough for modal 

analysis. Both male and female were selected primarily on xylem monoterpene

composition and particularly on varying percentages of limonene. The

monoterpene composition of these young progeny was close to the predicted

one based on the assignment of additive alleles to the parent trees for the five 

major monoterpenes—α-pinene, ß-pinene, 3-carene, myrcene, limonene—, but 

within the constraints of several modifying factors or conditions as follows:

• forms of stress • a juvenile factor which was particular

ly strong in progeny with large 

amounts of α-pinene and ß-pinene; 

• a parental distance effect which really seemed to be somewhat related 

to parents being in different ecological (resin) zones as well as on the 

linear distance; 

• seed growing outside the provenance of its parents; 

• some degree of isomerization and biosynthetic by-products; 

• competition within the genetic structure for a fixed amount of C5 

precursor, resulting in a type of de facto complex gene; and 

• the possibility of a secondary, and minor, genetic structure, as yet 

unidentified. 

Both the parental distance effect and the juvenile effect were manifested in 

the same way: increase in α-pinene and ß-pinene and decrease in myrcene and 

limonene. These same shifts were also associated with various forms of stress, 

such as prolonged root containment and over-application of a phytocide. Could 

it be that juvenile growth was "stressful" and that there was a degree of stress 

in the progeny of parents that were located at a great distance? The question of 

stress and its effect would be interesting and valuable research. 

These modifying factors changed to some degree the quantitative 

composition, but the basic modal characteristics of the frequency distributions 

of individual components were not changed. Thus, the interpretive value of the 

data was considered good and valid. 

The juvenile factor was a large one in the breeding study, and there was 

little evidence that its effect was weakening over the first eleven years of 

growth. The one possible exception was some evidence that the quantity of 

myrcene was increasing slightly over the 11-year period. The effect of the 

juvenile factor seemed to be dependent of the composition itself, being stronger 

in progeny with sizeable amounts of α-pinene and/or 3-pinene. 

Numbers of progeny in 42 families were considered adequate for modal 

analysis of the frequency distribution of the individual monoterpenes. In every 

family so analyzed, the number of modes derived by the analysis equalled or 

exceeded the number expected based on the assignment of additive alleles to 

the parents. This was strong support for the additive allele hypothesis and for 

the preliminary assignment of alleles to parent trees. The number of modes in 

excess of the expected number was attributed to the interaction within the 

biosynthetic structure of the monoterpenes and the several modifying factors 

noted above. Some 200 frequency distributions were analyzed in the 42 families; 

in about 100 of them the actual number of modes was the same as the expected 

number; in the remaining 100, the actual number was greater than the expected 

number. Mode proportions were generally close to the expected, considering 

the effects of conditions and factors just noted. 

There were 32 cases where each parent was assigned a (0-1) set of alleles for 

the same component — either α-pinene, ß-pinene, 3-carene, or limonene. In 

nearly all 32 cases, the modal separation was judged good to very good and 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 409 
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Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

suitable for calculation of mode proportions. In these cases — of an (0-1) set of 

additive alleles for each parent — the modal proportions should be 25-50-25 for 

the low, middle and high mode. In many cases there was a marked 

improvement in mode proportions by using the expected number of modes 

instead of the ones derived from the modal analysis. 

The following is a summary listing of the average mode proportions for 

each monoterpene when both parents were assigned a (0-1) set of additive 

alleles for that component—and when proportion of 25-50-25 are expected: 

Number of crosses Average mode proportions 

with alleles of 

Component (0-1) x (0-1) low middle high 

α-pinene 12 34 39 27 

ß-pinene 10 23 44 33 

3-carene 5 36 36 28 

limonene 5 32 48 20 

These proportions were considered quite good in these young trees 

considering the effects of all the above factors and conditions. 

An examination was made of 73 crosses of parents with a combination of 

(0-0) x (0-1) alleles for a given component, where there should be two modes of 

equal proportions. Where there were more than two modes, adjacent modes 

were combined to get the expected two modes. The following is a listing of that 

examination, showing the component, the number of cases of such a cross, the 

percent of such cases where the proportions were better than 35-65, and those 

better than 40-60. 

Number Proportions Proportions 

of better than better than 

Component cases 35-65 or 65-35 40-60 or 60-40 

-------------- percent 

α-pinene 21 71 48 

ß-pinene 18 83 72 

3-carene 16 94 56 

myrcene 8 100 75 

limonene 10 74 37 

Thus both the number of modes and their proportions supported the 

additive allele hypothesis. The data do not support two alternative hypotheses: 

dominant-recessive alleles or multiple genes. Myrcene was not included in the 

three-mode examination because only one tree, AS-5789, had an assignment of 

(0-1) set of alleles; all other parents had a (0-0) set. This tree was used as female 

and male, and mode proportions averaged 53-47 and 46-54 respectively. Thus 

myrcene, though a rare allele, fitted the additive allele hypothesis along with 

the other four major components noted above. 

One might hypothesize a multiple gene structure for the individual 

monoterpenes because of the large range in their frequency distribution in a 

stand. This study showed that several factors (noted above) can cause 

quantitative changes. All these factors in aggregate could cause a substantial 

increase in variation. However, there really was a de facto multiple gene effect 

because of the competition among the sets of additive alleles of the five major 

genes for a fixed amount of precursor; the whole gene structure was itself a 

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multiple gene. And there may be additional variation because of unique 

interactions of certain components or combinations of components. However 

data in this report strongly supported the additive allele hypothesis for each monoterpene, and explained the large variation. 

One apparent anomaly was found with 3-carene: the absence of a distinct 

mode at zero percent when one would be expected with additive alleles; i.e. 

combinations of a (0-1) and (0-0) set of alleles; with such a combination there 

should be a (0-0) set in the progeny, resulting in a mode at or near 0 percent and 

an (0-1) set resulting in a mode well along the percent scale. In 28 families 

identified with this condition, (0-1)x(0-0), the zero mode for 3-carene was 

present in 25 families, as expected, but was not present in three families all 

of the same male. Thus it was a rare condition, and it might be explained by 

the juvenile factor, or by some degree of activity by the (0-0) set of alleles 

under certain circumstances that have not been identified for this particular 

male parent. In 21 families, both parents were assigned an (0-0) set of alleles 

for a given component; and in all families, there was only a single mode at 0 

percent, as expected. 

There were at least 58 types of individual tree composition among the more 

than 2,000 progeny of full sibling families; this was expected, given the range of 

parental variation, the action of additive alleles, and the presence of several 

modifying factors. Likewise, these types of composition were easily predictable 

on the basis of the assignment of additive alleles to the parents. This large array 

of types accomplished the objective of the study: to produce a large supply of 

trees with a diverse array of compositions at IFG for further study and 

experimentation on host relationship to bark beetles and on the genetics of 

monoterpenes. The types of composition ranged from 7-2-0-2-88 percent to 17-

80-0-1-1 percent respectively for α-pinene, ßpinene, 3-carene, myrcene, 

limonene. The range of compositions was very large (fig. 6-3). Nearly all the 58 

types were found in the range-wide study of P. ponderosa as reported in 

Chapter 3. However, one type classed as ultra-high limonene— 7-2-0-2-88 

percent noted above— was found in the breeding study but was not found in the range-wide study; and such a composition was expected. Why such trees 

have not been found in natural stands is an interesting question. Is such a 

composition only a juvenile characteristic that changes as the tree matures? Do 

such trees fail to survive? Has the search of natural stands been inadequate? 

The breeding study found this type of composition to be common among the 

progeny of high limonene x high limonene trees; but it was present but not 

common with other combinations of parents. Thus, the rarity of high limonene 

trees could explain the lack of ultra-high limonene in natural stands. For 

example only one tree in about 500 was high limonene at Adin Summit; and 

there were two in about 80 at Wheeler Peak. 

Several parent trees were found which could be of considerable value in the 

study of the variation in tree characteristics, further studies of monoterpenes, 

other genetic studies, such as host resistance, and plant adaptation to name a 

few. These trees were AS-3139, AS-A95, AS-3194, and AS-5789 at Adin Summit, 

Tree BL-6074 near Blue Lake, and several trees at Wheeler Peak. Additionally 

there are two plantations of about 300 young progeny at IFG as well as the 14 

grafts of the Adin Summit trees which are now growing at IFG. The plantations 

contain most of the types of composition found in the study. More work with 

tree BL6074 would have been of considerable value; it was the only high 

limonene tree with low α-pinene. This tree was found in a 40-tree stand that 

had the average limonene of Sierra Pacific Region. The stand also had a tree 

with about 40 percent limonene. 

The breeding study found that there was little or no incompatibility 

between Wheeler Peak trees in eastern Nevada and Adin Summit trees in 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 411 
Chapter 6 Monoterpene Composition of Populations of 4- to 11-Year-Old Full Smith 

Siblings of P. ponderosa 

northeastern California, though the progeny of trees between the two areas 

seemed to be under some degree of stress. The study also found that the 

progeny of grafts had about the same average composition and about the same 

types of composition as the progeny of the parent tree when crossed with the 

same male or female tree. 

Analysis of the composition of the parent trees at Adin Summit strongly 

showed that the compositions of these selected trees were, essentially, constant 

over time and for different locations on the tree. Some scientists have called 

these compositions spurious and invalid. The reanalysis and the large body of 

results on half and full sibling populations in this report clearly proves this 

view to be incorrect. 

412 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Chapter 7 

Grafted Pines 

Summary: Thirty years after grafting, the monoterpene composition of the 

scion of interspecific grafts-P. ponderosa-on-P. jeffreyi, P. jeffreyi-on-P. ponderosa, 

P. sabiniana-on-P. ponderosa, P. torreyana-on-P. ponderosa-was about the same as 

that of the parent species. However, the composition of the root stock was 

being affected by the scion, and the effect was proportional to the distance from 

the graft union. On the other hand, the qualitative and quantitative composition 

of the scions of 6- to 15-year-old intraspecific grafts of P. ponderosa were radically 

different from those of the parent tree for about 10 years after grafting. The 

greatest differences were in grafts in which the compositions of the root stock 

and the scion were most widely divergent. Three possible causes are postulated: 

(1) the severe stress of grafting, (2) a strong effect of the root stock, and (3) an 

expression of a minor genetic structure through the (0-0) set of alleles. Between 

10 and 15 years after grafting, the composition of the scion suddenly and 

rapidly became much like that of the parent tree. After this sudden change, the 

scion began to affect the composition of the root stock much as in older 

interspecific grafts noted above. 

The composition of xylem monoterpenes of grafted pines was discussed 

separately because published information was limited, and rather unexpected 

results were found. In general, success in grafting pines in the past has been 

variable and often poor. 

Measurements of two sets of grafts were given. One set consisted of four 

interspecific grafts; the other set consisted of 14 intraspecific grafts of P. 

ponderosa. The interspecific grafts were made by Mirov in about 1940 using 

approach grafting of rooted trees. When grafting was judged successful, the 

root stock was cut off above the union, and the scion below the union. 

The intraspecific grafts were made in 1971 with four of the P. ponderosa 

pines that were used in the P. ponderosa breeding program (see Chapter 6); 14 

were successful. Bottle grafts were made for 12 of these trees, and approach 

grafts were made for the remaining two. All scions were cuttings from parent 

trees at Adin Summit. All root stocks were 2-year-old potted trees of unknown 

parents from the central Sierra Nevada. Graft unions were made in the first 

internode of the root stock. Root stock branches below the union were removed 

after the first year of grafting. Both root and scion were allowed to grow in the 

two remaining grafts, thereby producing two forked trees. All trees were 

maintained in pots in a greenhouse for two seasons and then outplanted in an 

arboretum. 

The interspecific grafts were sampled by macro taps at about 30 years after 

grafting when the trees were 6-8 inches in diameter; the intraspecifics were 

sampled by mini taps in the basal internode of the scion at 10, 15 or 16, and 17 

years (see Chapter 2 for procedures). At that time the scions were 2 inches to 3 

inches in diameter at the basal internode. By waiting until 10 years after 

intraspecific grafting, it was assumed that any period of juvenile instability in 

xylem monoterpene composition would have passed and generally stable 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 413 
Chapter 7 Gr

afted Pines Smith 

monoterpene conditions would exist. The xylem monoterpene composition of 

the scions was not determined before the 1 0-year sampling, but the composition 

of the parent trees was known (table 7-1). In studies of side branch resin of 

young P. ponderosa, the resin composition was found to be the same as or nearly 

the same as the xylem resin of the main stem (see Chapter 8). Thus the 

assumption was made that the monoterpene composition of the scion was the 

same as that of the parent tree. 

These same intraspecific grafts were used in a breeding program in 1978 

and 1979. The evidence from their progeny shows that the scions' reproductive 

tissue was the same as the parent tissue. The scions produced sexually 

reproductive tissue in less than 6 years after grafting. 

I believe that there were at least five conditions in grafts that were different 

from normal growth conditions, and that might alter quantitative monoterpene 

composition of xylem resin to some degree. I would call these the grafting effects. 

1. Wounding of root stock and scion could alter some of the processes that 

result in monoterpene production and deposition in both tissues; this might be 

particularly true over long periods of months and years as the wounds heal. 

2. Juvenile instability had been shown for very young tissue of P. ponderosa 

(see Chapter 8); the scion tissue was 1-year-old shoots. 

3. There was severe stress in both scion and root stock for at least 2 years 

and possibly for several more until the union is fully functioning and the scion 

vegetative growth was fully expressed. 

4. There could have been antagonism, or a type of rejection response, when 

joining tissues from two different trees. These four growth conditions, singly or 

combined, could produce the large changes that were found in the production and deposition of monoterpenes in the scions. 

5. Inter-tissue exchange could result in the movement of biosynthetic 

materials or signals. It will be shown later in this chapter that the scion has an 

effect on the composition of the rootstock; the opposite might take place, i.e., 

root to scion, under the unusual conditions of grafting. 

Interspecific Grafts 

The results of the analysis of the xylem resin of interspecific grafts were taken 

from Smith (1982a) (table 7-1). 

The composition of the scion portion of the P. jeffreyi-on-P. ponderosa graft 

was clearly only P. jeffreyi at all heights sampled above the graft union. There 

were no characteristics of P. ponderosa xylem monoterpenes in the scion. The 

composition of the P. ponderosa root stock xylem one foot below the graft union 

was still largely that of P. jeffreyi. However, there were small and measurable 

amounts of ß-pinenes, 3-carene, and myrcene which were characteristic of P. 

ponderosa. The amounts of ß-pinene, 3-carene, and myrcene increased at 5 feet 

below the union, and there were measurable amounts of α-pinene and 

limonene. Thus, the effect of the scion was decreasing with increasing distance 

below the graft union. Measurements of the P. sabiniana-on-P. ponderosa graft 

were very similar to the P. jeffreyi-on-P. ponderosa graft: above the graft union, 

the P. sabiniana-on-P. ponderosa scion was typical of P. sabiniana; below the 

union the characteristics of P. ponderosa increased with distance below the 

union. 

The composition of the P. ponderosa-on-P. jeffreyi graft was quite different 

than those of the previous two grafts. The composition was only P. ponderosa at 

all points above and below the graft union. The composition of the P. torreyana-on-

P. ponderosa graft was all P. torreyana above the graft union. No measurement 

could be made below the graft union because it was essentially at the ground line. 

The presence of scion characteristics in the root stock pointed to the 

414 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Grafted Pines Chapter 7 

Table 7-1—Monoterpene composition of scion and root stock of 30-year-old interspecific pine grafts: P. jeffreyi-on-P. ponderosa (J/P), P. 

ponderosa-on-P. jeffreyi (P/J), P. sabiniana-on-P. ponderosa (S/P), P. torreyana-on-P. ponderosa (TIP); --- = graft union (GU) at about 7 feet 

(7GU), except for TIP which was near the ground line. 

Monoterpene1 

Graft Scion Feet 

or root above hep α-p cam ß-p car myr lim ß-ph γ-ter terp 

ground 

percent2 

J/P Scion 16 98 * * * * 2 * - - -

Scion 10 98 * * * * 2 * * - -

G.U.3 7 ---

Root 6 94 * * 2 2 2 * * * * 

Root 2 70 2 * 10 12 4 2 * * * 

P/J Scion 20 - 8 - 29 50 7 3 1 * 2 

Scion 16 - 8 - 29 49 7 3 * * 3 

Scion 10 - 8 - 29 49 7 3 1 * 3 

G.U. 7 ---

Root 6 - 8 - 29 50 7 3 1 * 3 

Root 2 8 29 49 7 4 * * 3 

S/P Scion 15 99 * 1 - - - - - - -

Scion 8 99 * 1 - - - - - - -

G.U. 7 ---

Root 6 97 * 1 1 1 * * - - -

Root 2 81 1 1 4 8 2 3 - - -

T/P Scion 6 - 3 - 10 2 4 81 - - -

Scion 2 3 - 9 - 4 85 - - -

G.U. 0 ---

1

heptane, α-pinene, camphene, ß-pinene, 3-carene, myrcene, limonene, ß-phellandrene, γ-terpenene, terpinolene. 2

Normalized; * = trace, - = zero. 3

Graft Union. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 415 
Chapter 7 Grafted Pines Smith 

downward movement across the graft union of: (a) terpene-specific precursors, 

(b) biosynthetic signals, and/or (c) monoterpenes. The general assumption is 

that resin itself does not move except at wounds, though one cannot rule out 

slow diffusion over long periods of time. We do not know enough about 

monoterpene biosynthesis to evaluate the other two possibilities. 

Intraspecific grafts of P. ponderosa

More than 50 grafts were made of trees at Adin Summit; but only 14 survived 

(table 7-2). Attempts to graft scions from Wheeler Peak trees were not successful. 

Most of the Adin grafts that died did so during the first season, but several died

after outplanting. The xylem monoterpene composition of the root stock was 

not known. However, they were seedlings from central Sierra Nevada parents and,

I assumed, had a generalized composition of 10:35:35:10:10, respectively, for αpinene, ß-pinene, 3-carene, myrcene, limonene. The composition of the root stock

forks of the two forked grafts supports this assumption (fig. 7-1). (One had a 

composition of 9:19:49:7:12 for the five major components; the other was 8:36:40:6:7.)

The analysis of the composition by years and internodes will be covered for 

the 14 grafts derived from each of the four parent trees as noted (table 7-2) and 

for the two root stocks of the scions of Tree AS-A78 that were allowed to grow 

as forked tree. Two of the four parent trees of the scions—Trees AS-A78 and 

AS-3139— were without ß-pinene and 3-carene. Therefore, they were high in 

limonene, myrcene, and α-pinene. The other two were classed as moderately 

high limonene: AS-A95 had moderate ß-pinene but lacked 3-carene, Tree ASA295 had moderate 3-carene but lacked ß-pinene. Thus, each of the four parent 

trees lacked either or both ß-pinene and 3-carene. 

Results 

Ten years after grafting, the composition of nearly all scions was markedly 

different from that of the parent stock (fig. 7-1). All four sets of scions had 15 to 

20 or 20 to 30 percent ß-pinene and 25 to 40 percent 3-carene despite the fact 

that parent Trees AS-3139 and AS-A78 essentially lacked both components, and 

that parent Trees AS-A95 had no 3-carene, and AS-A295 had no ß-pinene. 

Between 10 and 15 years after grafting, a pattern of change appeared: ß-pinene 

and 3-carene decreased, and α-pinene, myrcene, and limonene increased. The 

magnitude of the change was not large, but it was consistent for nearly all 14 

scions and for all components. In general, this change seemed to start with a 

decrease in ß-pinene and 3-carene. Then, between 15 and 17 years, this change 

was large and clearly obvious, and the composition of all scions moved rapidly 

toward that of the parent stock (fig. 7-1). 

Many studies, some reported in Chapter 8, were made to determine the 

validity of basal sampling of P. ponderosa for xylem monoterpene composition. 

Three studies are of note: (a) 75 trees, more than 40 years old, and with a great 

range in variation of composition, were re-sampled after 9 years; (b) 72 trees 6 

years old were re-sampled after 2 years; (c ) 340 trees 8 - 11 years old were resampled after about 4 years. All studies showed no change or very little 

changes in composition between the two sampling periods. Thus one can 

assume from these studies that there was little or no change in the composition 

of the basal sample of P. ponderosa over periods of 5-9 years. 

The following discussion is based on the sets of scions of the four parent 

trees. Generally the changes took place throughout the tree (fig. 7-2) and not just 

in the basal sample. Also, of the 14 scions, two made the large change prior to the 10th 

year, three between 10 and 15 years, and nine between 15 and 17 years. 

P. ponderosa AS-A 78 

There were eight grafts of this parent. The root stock had not been cut off from 

416 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 415 
Chapter 7 Grafted Pines Smith 

two of them. Thus these two were a type of forked tree. 

At 10 years, the composition of seven of the scions was quite similar to each 

other but sharply different from that of the parent tree (table 7-2, fig. 7-1). The 

parent had essentially no ß-pinene or 3-carene; the scions had an average of 18 

percent and 24 percent, respectively. The amount of myrcene and limonene 

was much lower in the scions than in the parent, and there was not much 

variation in composition among the scions. The amount of α-pinene was about 

the same in both scion and parent. The composition of the 8th scion was 

somewhat similar to that of the parent; apparently it had changed before the 

10th year. 

By 15 years, a trend seemed to be developing in the seven scions noted above: 

α-pinene, myrcene, and limonene were increasing slightly while ß-pinene and 3-

carene were clearly decreasing. 

At 17 years, all trends in all scions were pronounced, as ß-pinene and 3-

carene decreased to only 2 percent and 5 percent from the previous 18 and 24 

percent, respectively. Limonene and α-pinene increased to nearly 40 percent 

and myrcene to about 15 percent. With these changes the compositions were 

starting to resemble that of the parent stock. Two scions still attached to the 

root stock responded in a similar manner and were averaged with the others. 

The composition above the union of the two attached root stocks did not show 

these sharp changes for the resampling between 15 and 17 years. In fact, the 

small changes, as recorded, were in the opposite direction of the changes in the 

first internode of the scions just a few inches away. The compositions of the 

internodes above the basal scions were essentially the same as the basal ones 

(fig. 7-2). Thus the changes appeared to be taking place throughout the whole 

scion for all seven scions (fig. 7-2). 

The composition of the root stock at 17 years just below the graft union was 

nearly identical to that of the basal internode of the scion just above the union. 

This suggests that, like the 30-year-old interspecific grafts discussed above, the 

scion apparently was now starting to affect the root stock. No measurements 

were made of the root stock prior to 17 years, but on the basis of prior studies, I 

was quite certain that it was not like that of the scion. Of course, the 

measurements of the basal internode of the scion and root stock were only a 

few inches apart. 

P. ponderosa AS-3139 

There were three grafts of this parent tree, and all had the root stock cut off 

above the union. Like Tree AS-A78, Tree AS-3139 had very little or no ß-pinene 

or 3-carene. And again like Tree AS-A78, all scions averaged about 20 percent 

ß-pinene and 28 percent 3-carene at the 10-year sampling. At 15 years, all 

scions showed the same trends as Tree AS-A78: less ß-pinene and 3-carene,

and greater α-pinene, myrcene, and limonene. And again, the changes between

10 and 15 years were not large, though they were consistent for all scions. 

Between 15 and 17 years, the direction of all trends remained the same, but the 

magnitude was much greater (fig. 7-1). Thus at 17 years, the composition of the

scions was shifting rapidly toward that of the parent stock. The composition of 

the scions farther up the tree was essentially the same as that of the basal

internode of the scion. This strongly suggests that the changes were taking 

place throughout the whole scion (fig. 7-2). Again, like Tree AS-A78, the changes

took place in all scions at about the same time. 

P. ponderosa AS-A295 

There were two scions of this parent tree, and both had the rootstock removed 

above the union to produce single stems. Tree AS-A295 had moderately high 

limonene, no ß-pinene, but moderate 3-carene. At 10 years, the composition of 

418 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Grafted Pines Chapter 7 

Table 7-2—Monoterpene composition of the xylem resin of the four parent trees that were the source of scions for the intraspecific 

grafts of P. ponderosa1 

Tree Number Tree Class of 

designation of grafts Approximate composition of parents2 diameter tree 

α-p ß-p car myr lira ter 

percent3 in. 

AS-A78 8 20 - - 20 60 8 suppressed 

AS-3139 3 18 - - 22 60 - 20 dominant 

AS-A295 2 15 1 32 19 32 2 12 co-dominant 

AS-A95 1 22 23 - 10 35 - 10 co-dominant 

1 All grafts are now growing at the Camino Arboretum of IFG. Trees AS-3139 and AS-A95 are still at Adin Summit; Trees 

AS-A78 and AS-A295 have been removed. 

2 α-pinene, ß-pinene, 3-carene, myrcene, limonene, terpinoline. 

3 Normalized. 

Figure 7-2—Average percentage of 5 monoterpenes, as 

noted in the key, at 17 years after grafting, in the internodes 

about the basal one of the 4 listed P. ponderosa reported in 

fig. 7-I and for the 2 forked-trees root stocks. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 419 
Chapter 7 Grafted Pines Smith 

one scion was somewhat like that of the parent tree (table 7-2, fig. 7-1). The 

second scion had about 18 percent ß-pinene, while the parent tree had only 1 

percent. Between 10 and 15 years, the same general pattern of increases and 

decreases prevailed for this second scion as in the previous sets of scions (fig. 7-

1). Between 15 and 17 years the trends continued in the same direction in the 

second scion but at a much greater magnitude for α-pinene, ß-pinene, myrcene, 

and limonene. However, this trend did not prevail for 3-carene which changed 

only slightly; 3-carene was present in the parent stock. Here again, the 

composition of the scion rapidly shifted toward that of the parent tree. As with 

the previous sets, the composition of the monoterpenes was fairly constant 

throughout the tree at 17 years (fig. 7-2). Also, at 17 years, the composition of 

the root stock was nearly identical to that of the basal internode of the scion. 

Thus, the scion appeared to be affecting the root stock. 

P. ponderosa AS-A95 

There was only one successful graft of this tree. With the parent lacking 3-

carene but having 23 percent ß-pinene, it was, therefore, the reverse of Tree 

AS-A295 that lacks ß-pinene but had 32 percent 3-carene. The composition of 

the one scion at 10 years did not look like the parent because it contained 35 

percent 3-carene (fig. 7-1). By 15 years, a decrease in 3-carene was perceptible,

while at 17 years it had dropped to 8 percent from the 34 percent at 15 years. 

There were slight increases in α-pinene, myrcene, and limonene at 15 years

with large increases by 17 years. There was only a slight decrease in ß-pinene,

but the amount was still somewhat near that in the parent tree. 

Root Stock 

Two sets of root stock were sampled for comparison with the composition of 

the scions: one set consisting of the 12 root stocks of the single-stemmed cleft 

grafts just discussed, and the other set consisting of the two root stocks that had 

been allowed to grow to form a type of forked tree with the attached scion. The 

12 single-stemmed root stocks were sampled only at 17 years. The composition 

of all 12 closely resembled that of the respective basal internode of the scion just 

above the union (fig. 7-3), strongly suggesting that the scion was affecting the 

root stock. The composition of the forked root stock was determined above and 

below the graft union. Above the union the composition was much like that of 

a Central Sierra Nevada tree: high in ß-pinene and 3-carene and moderate in 

α-pinene, myrcene, and limonene; below the union the composition was much 

like that of Tree AS-A78: little or no ß-pinene and large amounts of α-pinene, 

myrcene, and limonene. Thus composition of the root stock tissue was different 

above and below the graft union. 

Discussion: Intraspecific Grafts 

The 14 intraspecific grafts of ponderosa pine could be considered interracial 

because of the great difference in qualitative and quantitative monoterpene 

composition between the scion and root stock. The differences were in the 

order of magnitude noted for the interspecific grafts. 

At 10 years after grafting, the qualitative and quantitative composition of 

12 of the scions was very different from the parent tree. Then between 10 and 15 

years, the composition in all scions began to shift toward that of the parent tree. 

These changes greatly accelerated between the 15th and 17th year so that the 

composition of the scion nearly resembled that of the parent tree. Thus, these 

changes held for all 12 scions regardless of the composition. Within one set of 

clones of one scion source there were some small differences in the year of these 

changes. I have not found such changes in unaltered trees, and I know of no 

other such reports. 

420 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Grafted Pines Chapter 7 

Figure 7-3—Regression of the 

amount of monoterpene, at 17 

years after grafting, in the basal 

internode of the scion with the 

amount in the root stocks; 

limonene and 3-carene not plotted 

but fall within the ranges of the 

plotted components. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 421 
Chapter 7 Grafted Pines Smith 

Following are some of the factors or conditions that might cause or 

contribute to the changes. 

(a) After grafting, the scion does not have the expected monoterpene 

composition; the composition it does have suggests that a combination of 

postulated grafting effects places a very severe stress on the scion, or that the 

root stock has a very strong effect on the scion. The shifts in composition 

resembled that found for other forms of stress. 

(b) The scion also is juvenile tissue under severe stress, and there could be 

a juvenile effect as well as a root stock effect. 

(c) Both root and scion might also respond to severe wounding. 

(d) An interaction of root stock effect and juvenile scion effect varied with 

time and possibly with the degree of divergence of the composition of the two 

different sources of tissues. 

(e) Usually between 15 and 17 years after grafting, infrequently at about 10 

years, there was a dramatic change as the scion appeared to start to function 

normally, resulting in a monoterpene composition that began to resemble that 

of the parent stock. 

(f) Seventeen years after grafting, the scion stock appeared to be strongly 

affecting the composition of the root stock within 6 inches of the graft union; 

this is similar to the effect noted in the 30-year-old interspecific grafts noted 

earlier. 

(g) Because the effect of scion on root in interspecific grafts was progressive 

and proportional to distance from the graft union, it might be assumed that the 

effect of root on scion was similar. But, in general, the effect on the composition 

of intraspecific grafts was not proportional to distance. Therefore, the effect 

observed in the scions of intraspecific grafts of P. ponderosa was an interaction 

of root effect and juvenile instability of the scion; an alternative is that there 

were unknown effects. There were limited indications in a few trees of distinct 

and separate root and scion effects; thus, an interaction seems likely. 

(h) In intraspecific grafts, ß-pinene and 3-carene decreased, and α-pinene 

increased sharply, whereas myrcene and limonene increased in large or 

moderate amounts. 

(i) Though the vegetative tissue of the scions went through these changes, 

the reproductive structure was unaffected and, breeding was predictable, as 

noted in Chapter 6 on full siblings. 

(j) A grafting study could be devised to elucidate some of the uncertainty 

in this discussion of intraspecific grafts of ponderosa pine. The study would 

use root and scion stock of different and similar monoterpene composition, 

would use different types of grafting and post grafting treatments (particularly 

to provide greater length of root stock), and would require annual monitoring 

of monoterpene composition and of the phloem tissue by electrophoresis. 

Approach grafts of already-established trees in the 1979 nursery could be made 

between trees of very different resin composition. 

The grafting effect did not appear to be fully the same as the juvenile effect 

noted for young ponderosa pine in Chapters 5 and 6, but the juvenile effect may 

be part of the grafting phenomenon. In the juvenile effect, the expected 

components were present but not in the expected amounts, and the changes are 

slow. In the grafting effect, unexpected components were present in large 

quantities, and the changes were very sudden, very rapid, and very large. 

However, the juvenile effect may be involved as part of the grafting effect as 

suggested by some difference between parent and scion. 

There is another remotely plausible explanation for these great changes in 

grafts for which I have no evidence. Could there be activity by the (0-0) set of 

alleles? Such monoterpenes as 3-carene and/or ß-pinene were lacking in all 

422 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Grafted Pines Chapter 7 

scions, and the breeding work reported in Chapter 6 validates an assignment of 

the (0-0) set of alleles to these two components in these trees. In all scions, 

where there was an (0-0) set of alleles for ß-pinene and/or 3-carene, there 

initially were substantial amounts of these two components. Could the (0-0) set 

of alleles have a small amount of activity under normal conditions, as has been 

proposed in the report of young half- and full-siblings of P. ponderosa? Could 

the (0-0) set have much greater activity under the severe conditions of the stress 

of grafting? One of the responses to stress reported earlier was an increase in αpinene, ß-pinene, and sometimes 3-carene. If such a mechanism operates, it 

might also be the minor genetic structure proposed earlier in this report. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 423 
Chapter 8 

Measuring the Effects of Sampling and 

Operational Parameters: Regression, 

Trends Lines, Tabulation 

Summary: Questions have been raised about the validity of a sample of xylem 

resin. When and where was the sample taken from the tree, how was it prepared 

for analysis, and how was it analyzed and stored? These questions were 

particularly cogent in the early years of gas chromatography when very small 

samples were used and much attention was given to individual tree 

composition. Much of Mirov's work (1961) was with large samples which were 

often secured by aggregating resin from several trees. Thus, the early work was 

not too helpful in addressing these concerns. 

Much of the early work reported in this publication was with trees greater 

than 40 years old. Much later, as research included studies of siblings and half 

siblings, attention shifted to trees less than 15 years old. Results of the study 

with grafted pines were covered separately in Chapter 7 because of the 

unusual results. 

With questions on validity not adequately answered, studies were made 

primarily in the early years of the research to determine the effects of (1) 

variation in temporal and spatial sampling and of (2) operational parameters 

and sample preparation and analysis. The results of all studies clearly show 

that there was little or no variation in monoterpene composition due to any of 

the variables examined in trees greater than 40 years old except for a very small 

increase from top to bottom of P. Ponderosa in myrcene and limonene and a 

decrease in ß-pinene and α-pinene. The results of 33 studies were given in 

seven reports (Smith R.H., 1964b, 1966 a and b,1967h,1968,1977,1982a). 

The basic objective of the 17 studies reported below, generally, was to see 

how well the plotted data were distributed with reference to the theoretical 

regression of 1.0 or how well they fitted a trend line. 

Three procedures were used to analyze and display the data of these 

studies: simple regression, trend lines, and tabulation. 

Simple regression was applied to all but a few studies when the objectives 

were: 

(1) to compare the composition of a given tree at different locations on the 

tree at a given sampling time; 

(2) to sample the same locations on the tree at different times; 

(3) to compare two different operational procedures using the same resin 

sample. Therefore, each plotted point was the intersection of the two 

measurements being compared. In all cases the reference line was the 1.0 

regression and not the regression line of the data. 

Trend Lines ware applied to the vertical sampling of a tree at several points 

along the trunk. Straight lines were drawn that represented the trend lines. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 425 
Chapter 8 Measuring the Effects of Sampling and Operational Parameters: Smith 

Regression, Trends, Lines, Tabulation 

Tabulation was used to record data not applicable to either of the above 

procedures, or to summarize large amounts of data. 

Interpretive text was included only in the headings or captions. Thus, there 

was no separate text per se. Each study illustration in the chapter, or the single 

table, given below has a caption that will be a brief discussion of the illustration. 

The report was divided into two sections: (a) sampling parameters and (b) 

operational parameters. 

The basic GLC procedures are given in Chapter 2. 

426 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Measuring the Effects of Sampling and Operational Parameters: Chapter 8 

Regression, Trends, Lines, Tabulation 

Table 8-1 and Study. 8-1. Height of sample: The composition of one tree each for 11 pine species at the 

Institute of Forest Genetics was compared at two sampling heights: (a) 3 feet (b) 20 feet. All trees were more 

than 40 years old. The data as listed below show little or no difference in normalized composition between the 

two heights even though there was wide range in the composition among the 11 species. 

Pinus Monoterpene1 

α-p cam ß-p car sab myr lim ß-ph oci ter 

pct 2 

attenuata a3 94.5 0.3 2.7 0 0 0.9 0.6 0.9 0 0.1 

a ́ 94.4 0.4 2.8 0 0 0.9 0.6 0.9 0 0.1 

b 94.3 0.4 2.8 0 0 0.9 0.6 0.9 0 0.1 

b ́ 94.2 0.3 2.8 0 0 1.0 0.6 1.1 0 0 

contorta a 4.6 0.3 11.5 25.9 0 3.9 0.7 51.2 0 1.8 

b 5.6 0.2 12.5 25.7 0 3.6 0.6 53.7 0 1.0 

coulteri a 25.1 0 3.8 0.5 0 23.5 14.4 32.7 0 0 

b 23.7 0 3.9 0.6 0 23.4 14.7 33.7 0 0 

edulis a 79.1 ** 0.4 15.5 0 1.5 0 0 2.0 1.5 

b 76.2 ** 0.4 18.6 0 0.9 0.1 0 2.0 1.6 

flexilis a 46.5 ** 27.9 16.7 0 2.5 0.7 1.2 0 4 

b 49.5 1.0 30.8 11.2 0 2.1 0.7 1.2 0 3.5 

lambertiana a 64.9 0.1 24.6 0 0 9.9 0 0.4 0 ** 

b 66.3 0.2 23.3 0 0 9.8 0 0.5 0 ** 

monophylla a 71.1 ** 0.6 0 10.4 3.0 4.2 0 4.7 6.0 

b 70.8 ** 0.7 0 10.0 2.9 3.7 0 4.7 7.2 

muricata a 3.3 0 0.3 86.0 0 1.2 ** 0.3 ** 8.4 

b 4.5 0 0.3 84.8 0 1.3 ** 0.5 ** 8.1 

quadrifolia a 82.2 0.1 0.1 7.9 0 3.0 0 0 5.4 0.8 

b 82.9 0.1 0.5 8.2 0 3.0 0 0 4.8 0.9 

radiata a 24.4 0.2 73.8 ** 0 0.6 0.4 0.7 0 0 

b 23.3 0.1 75.5 ** 0 0.5 0.4 0.4 0 0 

washoensis a 10.2 0 44.7 25.8 0 11.1 4.0 1.4 0 1.4 

b 10.1 0 40.8 28.3 0 13.6 4.0 1.9 0 1.3 

1 α-pinene, camphene, ß-pinene,3-carene, sabinene, myrcene, limonene, ß-phellendrene ocimene ,terpinolene 2 Normalized. **= trace 3 a ́ and b ́ are forks of a and b. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 427 
Chapter 8 Measuring the Effects of Sampling and Operational Parameters: Smith 

Regression, Trends, Lines, Tabulation 

Study 8-2. Height of sample: Trend lines were drawn for the amount of the individual monoterpenes in 

the internodes of three P. ponderosa (a,b,c) 60 to 80 years old; internodes were counted from the top 

of the tree to the base, thus were essentially tree age at a given internode; each trend line has a separate percent base; trees were standing when sampled. All three trees were very similar in the slope 

and relative magnitude of the increase or decrease from 2-5% in the trend of the amount of four major 

monoterpenes. It should be pointed out that, though the sampling covered 20 to 50 years of internodes, the actual xylem layer sampled was essentially the same age, about 1 to 3 years for all samples. It

is obvious that α-pinene and ß-pinene decreased from top to base while myrcene and limonene 

increased. 

428 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Measuring the Effects of Sampling and Operational Parameters: Chapter 8 

Regression, Trends, Lines, Tabulation 

Study 8-3. Height of sample: Comparisons were made of the monoterpenes (in percent) between pairs of internodes in a population 

of 7-year old P. ponderosa. All samples were obtained by severing the tree at the internodes; internodes were counted from top of tree to 

base which was equivalent to tree age at a given internode. Diagonal line in regression of 1.0. Regression improved with age of internode. 

Study 8-4. Height of sample: Internode comparisons were made of the amount of the individual monoterpenes with different internode source of resin of 4-year old P. ponderosa; internodes were counted from the top to base of the tree, which is equivalent to tree 

age at an internode; diagonal line is regression of 1.0. Again, regression improved with age of internode. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 429 
Chapter 8 Measuring the Effects of Sampling and Operational Parameters: Smith 

Regression, Trends, Lines, Tabulation 

Study 8-5. Height of sample: Internodal comparison of the monoterpenes of pairs of internodes of a 9-year old full sibling population of P. ponderosa. Samples obtained by severing trees at internodes; internodes counted from top of tree to base. Again, regression improved with age of internode. Regression of 1st and 9th internodes were particularly poor. The results with these studies of 

internodes suggest that measurements of the composition of the first three internodes may not be valid, except for comparisons. 

Study 8-6. Annual ring: Comparisons were made of the monoterpene composition of the 1981 and 1987 annual ring of the nursery-grown full siblings of 6-year old P. ponderosa IFG-4B63 x AS-5789; diagonal line is slope of 1.0. Clearly, all points were on the 1.0 

diagonal or within 2 percent of it. 

430 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Measuring the Effects of Sampling and Operational Parameters: Chapter 8 

Regression, Trends, Lines, Tabulation 

Study 8-7. Annual ring: Comparison were made of the monoterpene composition of the 1977 and 1983 annual ring at the basal internode of 72 6-year old P. ponderosa. Diagonal line is regression of 1.0. Over all there was very little difference, if any at all, between the composition of the 1977 and 1983 annual ring of the basal internode of these young trees; the results with myrcene and limonene were very 

similar. 

Study 8-8. Time of resin flow and sample storage: Comparisons were made of the monoterpene composition with differing time of resin 

flow and time of storage of prepared sample. Resin of seven P. rigida more than 40 years old was obtained at 4 hours, 2 days, and 7 days; and 

for 5 years of storage of prepared samples of P. lambertiana. Diagonal line is regression of 1.0. Data were regressed for pairings of 

time of flow or storage. There was, essentially, no difference in composition of three noted monoterpenes with time of flow of resin or for time of 

storage for five monoterpenes in prepared samples. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 431 
Chapter 8 Measuring the Effects of Sampling and Operational Parameters: Smith 

Regression, Trends, Lines, Tabulation 

Study 8-9. Time of storage--Effect of storage time of pentane-prepared samples of 226 P. ponderosa was determined. Diagonal is regression of 

1.0. A regression was made of the first analysis and the analysis after 7 years storage at 0 c. There was little, if any, change during storage. The data 

for myrcene and limonene were nearly identical to ß-pinene and 3-carene. 

432 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smith Measuring the Effects of Sampling and Operational Parameters: Chapter 8 

Regression, Trends, Lines, Tabulation 

Study 8-10. Source of resin: Correlations were made of the amount of monoterpenes with source of resin of P. ponderosa: cortex vs. xylem using (A nursery-grown full siblings of Adin x Wheeler, and of (B) nursery-grown half siblings from Ranger Peak, diagonal line is slope of 1.0. Clearly, there was very poor relation between the components in xylem and cortex. The set from Ranger 

Peak was not as poorly related as the set from Adin/Wheeler full siblings. This suggested that composition itself can be a factor in 

the differences. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 433 
Chapter 8 Measuring the Effects of Sampling and Operational Parameters: Smith 

Regression, Trends, Lines, Tabulation 

Study 8-11. GLC column, time of resin flow, analytical preparation: Three studies were made, all using correlation, of the following operational procedures: (A) the packing of the chromotographic column (LAC vs. ß ß ́ oxydipropionitrile), (B) the type of preparation of the sample (pentane vs. short path distillation), (C) and time of flow of resin (one hour vs. 12 hours). All studies used different samples of P. ponderosa 

greater than 40 years old. Diagonal is slope of 1.0. It was clear that none of the variables had a noticeable effect on the measurements. 

434 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Chapter 9 

The Function of Xylem Monoterpenes of 

Young P. ponderosa during Western Pine 

Beetle Attack (Coleoptera: Scolytidae): A 

Bioassay 

Summary: A series of laboratory and field experiments were carried out to 

develop a working model for the resistance of P. ponderosa to western pine 

beetle (Dendroctonus brevicomis). The procedure was to force beetle attacks on 

irreversibly stressed 8- to 12-year-old trees, derived from the half- and fullsibling studies, as reported in Chapters 5 and 6. Assessment was by adult 

gallery length at 2 weeks. An inverse association was found between the 

amount of beetle boring and both the amount of resin flow and the percentage 

of limonene in trees that were 7.5-10 cm in diameter. Trees less than 5.0 cm in 

diameter did show the beetle-success/percent-limonene association but did 

not show the beetle-success/resin-flow association, probably because the trees 

were too small for testing. 

A working model of the resistance of P. ponderosa to western pine beetle (WPB) 

(Dendroctonus brevicomis LeConte) was developed after a series of laboratory 

and field experiments (Smith 1975). The factors in the model were the quantity 

and quality of the xylem resin of P. ponderosa, and the quantity and quality of 

western pine beetle (WPB). The experiments measured three of the parameters 

of the model; beetle quality was not measured but was added to the equation 

simply on the presumption that both beetle and resin should have a qualitative 

property. A candidate resistant type of tree was found in a range-wide study of 

the variation and distribution of the xylem monoterpenes of P. ponderosa (Smith 

1977). This chapter reports on a bioassay of young progeny that were derived 

from the studies of wind and controlled pollination of those selected trees (see 

Chapters 5 and 6). 

The concept of host resistance did not appear in the early years of research 

and control of WPB on P. ponderosa. The first two decades, from about 1905 to 

1925, were chiefly devoted to large-scale direct control programs to reduce 

beetle populations. It was assumed that there would be a proportionate 

reduction in the subsequent level of tree mortality. The results of the early 

programs were good, except they were quite variable, and usually temporary. 

In general, the results were not as effective as anticipated or hoped for 

(Craighead 1925, Craighead and others 1931; Miller 1926). About 1930, a concept 

started to emerge that could account for the variable and temporary results of 

direct control. This concept might be called host susceptibility and suitability. 

J.M. Miller, in a letter written in 1924, formalized the concept as follows, "The 

only answer [to variable and temporary results] seems to be through methods 

of stand management which will reduce or eliminate in the stand those trees 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 435 
Chapter 9 

The Function of Xylem Monoterpenes of Young P. ponderosa during Smith 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

which are especially susceptible to western pine beetle attack." Many years 

later, Keen (1936) added the factor of population dynamics to the host 

susceptibility concept as follows, "Probably the most important factor in 

building up (bark) beetle populations is an abundance of suitable breeding 

material.... The beetles prefer certain trees that are in a susceptible condition for 

attack." 

The concept of susceptibility launched a research effort to identify those 

tree characteristics which could be most strongly and consistently associated 

with western pine beetle attack and success. Much of that research was covered 

by Miller and Keen (1960). Shortly after the concept of host susceptibility was 

stated, and even before preliminary basic research results were available, initial 

attempts were made to apply forms of stand management to reduce subsequent 

losses due to WPB. Those initial efforts with considerable modification and 

adjustment were enormously successful and led to at least three landmark 

concepts: (a) the California Pine Rating System and its several derivations 

(Salman and Bongberg 1942, Smith and others 1981, Wickman and Eaton 1962; 

(b) the Keen Tree Vigor Classification (Keen 1936); and (c) stand hazard 

classification (Johnson 1949). These concepts applied primarily to P. ponderosa 

and WPB, though P. jeffreyi and the Jeffrey pine beetle (JPB)were added later. 

Thus, the concept of host susceptibility—that certain trees in a stand were more 

susceptible than others to western pine beetle attack—was shown to be valid. 

The removal of these high-risk trees greatly reduced subsequent losses for 

many years. The concept of susceptibility paid excellent dividends in both 

research and control. The success of this program seems to have been the 

impetus and justification for many of the subsequent studies of the effect of 

the host on the dynamics of both bark beetles and defoliators. 

Though much of the early work emphasized susceptibility, there was an 

underlying assumption that host resistance might also be important. The 

abundance of susceptible and suitable trees fostered epidemics or 

maintained endemics, whereas resistant trees deterred epidemics. Hopkins 

(1902) noted that until the flow of resin was overcome, the mountain pine 

beetle (MPB) (Dendroctonus ponderosae Hopkins) was unable to extend 

galleries rapidly or to oviposit. Thus from early research onward, xylem 

resin was a suspected resistance factor, but little was done because of the 

preoccupation with susceptibility.

Miller (1950) was the first to consider the causes of resistance of P. ponderosa 

to WPB. Miller first turned to the host relationship of pine hybrids being 

developed at that time by the Institute of Forest Genetics. He was particularly 

intrigued by host x non-host hybrids. That is, what was the relationship of WPB 

to a hybrid of P. ponderosa x P. jeffreyi, a host x non-host hybrid? Miller (1950) 

conceded that it might be 20 years before these trees could be properly and 

reliably tested, because WPB typically did not attack trees under 6 inches in 

diameter. 

Then in the early 1950's, Callaham (1955) studied quantitative 

characteristics of the xylem resin of P. ponderosa of different risk categories as 

defined by the California Pine Risk Rating System (Salman and Bongberg 

1942). Callaham (1955) found that the duration of resin flow was greater in 

trees classed as lower risk, i.e., those trees least likely to be attacked and killed. 

Vite' and Wood (1961) subsequently found that P. ponderosa with high oleoresin 

exudation pressures (o.e.p.) were less frequently killed by mountain pine beetle 

than trees with low o.e.p. Results of both studies—resin flow and o.e.p.—could 

best be expressed as probabilities. The probabilities were greater that trees with 

a longer period of resin flow and higher o.e.p. were less likely to be attacked 

and killed than trees with shorter flow and lower o.e.p. In both studies some 

trees with the assumed resistant feature(s) were killed, and some with the 

assumed susceptibility features survived. One might logically conclude that 

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Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

other unknown factor or factors had an appreciable effect on resistance. 

The work started by Miller was continued with the emphasis on properties 

of xylem resin. Smith (1965) undertook laboratory studies with western mountain, and Jeffrey pine beetles. He concluded, in general, that: (a) beetles 

were able to tolerate the resin of host species of pine better than the resin of 

non-host species; (b) resin of hybrids of host and non-host was intermediate in 

its effect on beetle tolerances; and (c) there was a difference in the tolerance 

that WPBs have to the individual monoterpene components of their host 

resin, P. ponderosa. Extensive analysis of native P. ponderosa pine revealed 

high levels of local variation among trees and significant regional differences 

among stands in the monoterpene composition of P. ponderosa (Smith 1977) 

(see Chapter 3, #14). 

In forced-attack studies on P. ponderosa—no-choice testing on irreversibly 

stressed trees of suitable size for beetle attack—WPB was found to be far less 

successful in constructing galleries and ovipositing in trees with high-limonene 

content than in trees with low limonene content; the trees used in these studies 

were 15-20 cm in diameter. Quantitative flow of resin was also important, with 

success of attack inversely related to flow (Smith 1966b). These studies were the 

basis of this model: resistance is a function of [beetle quantity x beetle quality] -

[resin quantity x resin quality] (Smith 1975). 

A tree selection and breeding program of P. ponderosa, as reported in 

Chapters 5 and 6, was carried out from 1972 to 1978. The program used trees 

selected for monoterpene composition to produce a readily available supply of 

trees having a large array of monoterpene compositions suitable for resistance 

bioassay and for chemical and genetic studies. By 1987, the progeny derived 

from the controlled breeding, along with trees from wind-pollinated seed of 

some of the parents used in the controlled breeding, were 10 to 12 years old and 

thought to be suitable for resistance testing, but the diameter of these trees at 

7.5 to 10 cm was still far below the presumed size for western pine beetle attack. 

This section briefly reports on early results in the evaluation of the 

resistance of these trees and on the degree of agreement of the results with the 

resistance formula that was developed from experiments with much larger and 

older trees. Favorable results could substantiate the early results on resistance, 

decrease the waiting time for evaluation of trees, more strongly link resistance 

with bioassay and with chemical assay, and, by using small trees, increase the 

replication and lower the cost for this type of testing. 

Material and Methods 

Selection of Parent Trees 

Trees for the experiments were the products of the long-term selection and 

breeding program. Selection of the parent trees was difficult and uncertain. P. 

ponderosa and most other forest trees have not been fully cultured, and the 

effects of stand and site conditions on the desired characteristic are not at all 

well understood. Nevertheless, test trees were selected on the basis of the 

monoterpene composition of the xylem resin (Smith 1982b). Wind-pollinated 

seeds were collected from parent trees in 1970 and 1971 (see Chapter 5). 

Controlled breeding was carried out from 1973 to 1979 (see Chapter 6) using 

standard pine breeding procedures (Anonymous 1948, Cumming and Righter 

1948). Seeds were planted and trees were cultured by standard nursery 

practices at the Institute of Forest Genetics (IFG) near Placerville, California 

(see Chapter 6 for breeding and culturing procedures). 

Four sets of 9- to 12-year-old trees were selected, one set each from four 

different nurseries: 1974, 1975, 1976, 1979 (table 9-1). Each set had 10 to 16 trees 

that were selected on the basis of either high- or low-limonene content of the 

xylem resin. An effort was made to have comparable tree diameters for the two 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 437 
Chapter 9 The Function of Xylem Monoterpenes of Young P. ponderosa during Smith 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

Table 9-1—Average xylem monoterpene composition of four sets of P. ponderosa classed as high and low limonene used in forced-attack 

bioassay of resistance to western pine beetle 

Average Monoterpene Composition 

Year of Age of

nursery1 trees2

 Trees Replicate α-pinene ß-pinene 3-carene myrcene limonene 

yr number percent

3 

1974 14 5 14 12 11 20 8 48

5 13 

13 7 21 

6 19 

12 6 23 

5 18 

9 10 10 

6 6 

1975 

1976 

1979 

12 44 30 8 1 

29 1 0 4 52 

6 33 42 13 1 

31 2 0 7 59 

5 28 47 13 2 

24 5 0 9 64 

32 33 7 6 14 

1

1974, 1975, 1976 had a regular sequence of 3 to 4 replicates per tree; 1979 had only the base section. 2

(x) = age of trees in years. 3

Normalized; small amounts of camphene ß-phellandrene and terpinolene make up the remainder of 100 percent.

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Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

subsets, i.e., high and low limonene. However, despite a good number of trees 

to select from, there still was a small range in diameters of 7.5-10.0 cm in the 

1975 and 1976 sets, and 5.0-10.0 cm in the 1974 set. The trees in the 1979 set were 

about 5.0 cm in diameter. Thus all trees in the four sets were well below the size 

of 15 cm considered a minimum size for western pine beetle. 

Very little is known about the effects of phloem thickness on gallery 

construction. All research on western pine beetle has been on large trees where phloem thickness was not considered an important factor in attack and 

development. Phloem thickness was about 2 mm in the test trees, was probably marginal for gallery construction in the experiment, and may have been a 

confounding factor in the 1979 set of trees. Bioassay: The procedure for preparing the young trees for tests was q

uite 

similar to that used on larger trees (Smith 1966b), except that trees in this report 

were less than 10 cm in diameter and were growing in a nursery. This procedure 

forced a group of beetles to attack a small, caged, circumferential section of a 

tree whose crown had just been cut off. The procedure, in brief, was as follows: 

The basal 1 to 2 m of the tree were caged with four contiguous but separated 

sleeve cages. The length of each caged section was selected to give about 300 to 

450 cm2 of bark area inside the cage. Caging material was 20 mesh plastic 

screening. Bands of polyurethane foam about 2.5 cm thick and about 10 cm 

wide were used to hold the screening away from the bark and to separate the 

sections of the tree (fig. 9-1). The bark under these bands was shaved smooth to 

presumably make an escape-proof fit; bark within the caged portion remained 

untouched. The beetle needs roughened bark to attack. Roughening of P. 

ponderosa bark starts at about 4-7 years of age. 

Beetles used in the tests were reared from infested bark taken from trees on 

the Sierra and Eldorado National Forests in California. The bark was gathered 

in early spring when the over-wintering brood was in the late larval stage. The 

brood bark was held in a cold room at 0 °C. As beetles were needed, bark was 

placed in a roofed, screened outdoor insectary at IFG. Beetles were collected in 

the evening in cooled petri dishes, 20 beetles per dish, held at about 5 °C during 

collection, and held at 0 °C overnight. These beetles had emerged over the 

previous 24-hour period. Beetles were sexed under cooled conditions the day 

after collection. Only females were used and were randomly apportioned to the 

replicates in a test; the number of beetles was proportional to bark area of the 

section being tested. 

When a set of trees was ready to be tested, the trunk of each tree was cut off 

just above the caged portion early in the morning. The term "irreversibly 

stressed trees" was applied to trees treated in this way. Six hours later, the resin 

that had exuded to the cut surface along the outer half inch was weighed as a 

measure of resin flow. Twelve hours after cutting, i.e., early evening, female 

beetles were introduced into the top caged section of each tree at the rate of 

about one female per 30 cm

2

. Thus each sleeve cage was charged with 7 to 10 

females. An umbrella-like structure was placed on the top of the cut section to 

provide shade for the top section. 

Two weeks later the sequence was repeated. The caging was removed from 

the top section, which was the section that had been under attack and was cut 

off for subsequent examination of beetle performance. Resin flow on the new 

cut surface was measured at 6 hours, and the new top caged section was 

charged with beetles early in the evening. Thus, three to four replicates were 

obtained from each tree. Each replicate of a tree had the same resin monoterpene 

composition (quality), but a different flow of resin (quantity). That is, the rate of 

flow of resin usually changed from section to section, but the monoterpene 

composition of all the sections of a given tree remained about the same. 

Each section was examined within 4 hours after it was cut from the stem of 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 439 
Chapter 9 The Function of Xylem Monoterpenes of Young P. ponderosa during Smith 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

Figure 9-1—Schematic diagram of 

a longitudinal section of a sleevecaged portion of P. ponderosa (a) and 

the portion of the section above (b) 

and below (c) the section. 

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th The Function of Xylem Monoterpenes of Young P. ponderosa during Chapter 9 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

the tree. The bark was carefully shaved off down to the xylem, and all attacks 

that reached the phloem were recorded and measured for gallery length, the 

measure of beetle success. If there was no evidence of any attacks on a section, 

it was discarded from the test. The lack of any evidence of attack in a section 

was rare; such cases could have been caused by the escape of all beetles. The 

necrosis associated with the beetle gallery was also measured. Though there 

was some difference in the extent of this necrotic area, there seemed to be a 

fairly strong positive relationship between gallery size and the size of the 

associated necrotic area. 

Data Analysis: The average length of gallery for each caged section of each 

tree was calculated and paired with the value for the quantitative flow of resin 

in the outer 0.6 cm of the top cut of that section. The average gallery length for 

a caged section was based on 3 to 12 galleries, and this was considered a 

replicate. The resin flow value was based on only a single measurement. 

The data for each of the four sets of trees were analyzed separately. A 

regression was made between average gallery length and resin flow for each 

class of limonene. A one-way analysis of variance was made for the gallery 

lengths of high- and low-limonene trees and for resin flow from high- and 

low-limonene trees for each set of trees. A modified t-test was the basis for 

judging two populations of replicates to be different. 

Results and Discussion 

The results, though positive, were not as indicative as expected. This seems to 

be attributable to the small size of the trees; i.e., all trees were relatively 

resistant to beetle attack because of their small size. However, within this 

condition of "relative resistance" it was possible to compare two parameters, 

resin flow and resin quality, while holding beetle quantity constant. The 

primary reason for the experiments at this time was to see if bioassay could be 

made of small trees, thus reducing the waiting time for trees to reach suitable 

size for conventional testing. This hypothesis should be kept in mind as the 

results are discussed. 

The regression data for the trees in the 1975 and 1976 nurseries show that 

adult gallery length was inversely related to resin flow (fig. 9-2), and that the 

average adult gallery length was significantly shorter in the high-limonene set 

of trees for all four experiments (table 9-2). This supported the resistance 

formula (Smith 1975). However, there were individual values for high and low 

limonene within the one standard deviation band for the opposite set. For 

example, in the 1975 nursery data, the value for two high-limonene replicates 

fell within the limits of one standard deviation of the low-limonene set of trees, 

and two low-limonene replicates fell within the limit of one standard deviation 

of the high-limonene set of tests. Obviously, variation was large and the number 

of replicates that were used was necessary. 

The regression data for the 1974 nursery were inconsistent in that they 

showed a direct relation between resin flow and gallery length for only the lowlimonene trees, but an indirect relationship—greater flow with shorter 

galleries—for the high-limonene trees, suggesting that success of attack (gallery 

length) was still inversely related to the percentage of limonene (fig. 9-2). There 

is a significant grouping of the high-limonene replicates toward the lower 

range of gallery length and a grouping of the low-limonene replicates toward 

the higher range of gallery length. In this experiment, the low-limonene trees 

were generally larger in diameter than the high-limonene trees. 

The results of the 1974 nursery must be viewed with some question because 

of the temperature problems and because the trees within a set were quite 

variable in size. The umbrella-like structure did not seem fully adequate. It did 

screen out direct sunlight from the caged section, but it did not shade the 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 441 
Chapter 9 The Function of Xylem Monoterpenes of Young P. ponderosa during Smith 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

Table 9-2—Average adult gallery length (x) and resin flow (y) for four sets of replicates of high- and low-limonene P. ponderosa less than 3 inches 

in diameter 

Resin flow 

Gallery length cc/10 cm circumference 

Nursery Limonene class Replicate X S.D.2 y2 S.D.2 

number mm cc 

1974 high

low 

14 

13 

4.1*1 

12.2 

3.0 

8.3 

4.0 

5.3 

1975 high

low 

21 

19 

6.7* 

10.8 

3.6 

4.3 

1.9* 

4.2 

1976 high

low 

23 

16 

5.7* 

12.0 

3.6 

8.9 

3.2 

5.2 

1979 high

low 

10 

6 

2.7* 

11.6 

2.3 

7.9 

1.8 

3.1 

2.1 

4.3 

1.4 

2.9 

2.5 

4.8 

1.8 

2.9 

1

*Significantly lower at 5 pct level under the modified t-test. 2

S.D. = standard deviation. 

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Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

section from the heat reflected from the open ground of the nursery. The 1974 

nursery bed had been opened up to light too much by the previous season's 

work in attempting to use attacks by individual females. The residual trees in 

the 1975 and 1976 beds (as will be described below) provided more shade than 

did the residual trees in the 1974 bed. Additionally, as a result of the experience 

with the 1974 bed, cloth shading was erected along the east, south, and west 

sides of the 1975 and 1976 nurseries. Thus, in a way, the 1974 nursery was a 

learning process. Local temperature control was not a problem in the prior 

work (Smith 1966b, 1969) with larger trees in natural stands because the sections 

were not close to the ground, and the natural stand provided general shading. 

A series of temperature readings showed that the temperature under the 

umbrella and the caged section was about 5 °C higher than at an official 

weather station about 300 ft away. On the other hand, the temperature at the 

weather station was about 5 °C higher than in the shade of a well-stocked 

ponderosa pine stand about 150 ft away. 

The results with the 1975 and 1976 nurseries were strong support for the 

working model—the differences between low and high limonene were 

significant (table 9-2), and the correlation of decreased gallery length with resin 

quantity was evident (fig. 9-2). 

The regression data of the 1979 set of replicates were the reverse of the 1975 

and 1976 data; the correlation of gallery length with resin flow was direct rather 

than inverse. Yet gallery length for the high-limonene replicates was 

significantly shorter than the low-limonene replicates. This experiment suggests 

quite strongly that this tree size, less than 5.0 cm in diameter, was not suitable 

for this type of testing. Also, phloem thickness may have been a confounding 

factor in the 1979 set. 

For all experiments, average gallery length was 4.8 mm for high-limonene 

trees and 11.7 mm for low-limonene trees (table 9-2). Though resin flow was 

always greater in the low-limonene set, it was at the significant level only in the 

1975 nursery. Thus, it appeared that limonene content may be more effective 

than resin flow in reducing success of beetle attack in small trees. 

Conclusions 

From the results and my observations of all previous work with trees 

greater than 15 cm in diameter and of the trees used for this report, it 

appeared that trees with a diameter less than 7.5 cm were not suitable, that 

trees with diameters greater than 7.5 cm but less than 12.5 cm are adequate 

for comparative testing, and that trees with diameters greater than 15 cm 

will probably give both comparative and absolute classification of the 

resistance of trees. 

It was quite clear from all the data on gallery length that the type of trees 

used in the experiments was generally resistant to western pine beetle attack, 

and experiments on such trees can be only comparative and not absolute. From 

extensive previous work with forced attacks on larger trees, I would have 

expected gallery length to be well above 100 mm in sections of low limonene 

with low flow of resin for the period of the test. Yet in the experiments 

reported here, gallery length for low-limonene and low-resin flow ranged 

from 10 to 20 mm. One can speculate that only one or more of the following 

factors could have caused this phenomenon: small diameter, thin phloem, 

thin bark, the high temperature of the micro-environment of the nursery. The 

large variation in gallery length, even within a single replicate, might also be 

explained by these same factors. 

In the earlier work with larger trees using the irreversible stress procedure, 

the change in resin flow was fairly predictable; flow decreased as each 

successive section was cut from the tree. This did not prove to be so with the 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 443 
Chapter 9 The Function of Xylem Monoterpenes of Young P. ponderosa during Smith 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

Figure 9-2—Regression of gallery length of western pine beetle on resin flow of P. ponderosa with

sets of high- and low-limonene trees in the 1974, 1975, 1976, 1979 nurseries; each plot point is the

average gallery value for one replicate (i.e., one log); ▲ = high limonene, ○ = low limonene. 

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Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

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Chapter 9 

The Function of Xylem Monoterpenes of Young P. ponderosa during Smith 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

small, nursery-grown trees. Though flow did change from section to section of 

the same tree, it was not at all as regular and predictable as in larger trees. The 

nearness of the trees to each other and the possible intertwining of the root 

systems are the most logical explanations of this phenomenon. 

It was observed in these series of experiments that nearly all galleries, short 

or long, were oriented in a vertical direction, rather than the sinuous, typically 

inclined, direction for WPB. Short vertical galleries were typical of forced 

attacks of WPB on non-hosts, hybrids of host x non-host, and on ponderosa 

pine with a large flow of resin. The large variation in gallery length also could 

have been caused by the irregular response of the resin system to the sequence 

of cutting. Variation in gallery length was not as great when using larger trees 

and larger sections (Smith 1975). The factor of phloem thickness may be 

important for typical gallery construction. Phloem thickness was 2 to 3 mm in 

larger trees and about 2 mm in smaller trees. Beetles will construct galleries in 

phloem less than 2 mm thick, but their boring may not be typical. 

The range in gallery length was usually greater for low-limonene replicates 

than for high-limonene replicates. This might have been caused by greater 

variation in qualitative monoterpene composition of the low-limonene 

replicates. That is, low-limonene trees were selected only on that basis, with the 

remainder of the composition varying widely among the other four major 

components: α-pinene, ß-pinene, 3-carene, and myrcene. Earlier work (Smith 

1965) showed a difference in toxicity between the four monoterpenes and the 

adult WPB. On the other hand, in selecting for high limonene, the remaining 

composition was almost always α-pinene and myrcene, and very little ßpinene and 3-carene. Of course, the factor of beetle quality could be important in causing such great variation in gallery length. The factor of beetle quality has 

not yet been addressed in this work. 

In early exploratory work on testing trees for resistance to WPB attack, 

attempts were made to cage individual females or pairs instead of groups of 

beetles so that the performance of individual beetles could be measured; i.e., 

each beetle was a replicate. The size of the cage area ranged from about 0.4 cm2 

to about 6.0 cm

2

 and varied from a circular to a rectangular shape. No effort was 

made to encourage boring in the small cages by starting an entry into the bark, 

but pheromones were used in some tests to encourage boring. However, the 

results were not as satisfactory as group testing, and the use of small cages and 

individual females was not pursued. 

One might also question the absence of male beetles in the tests. All reports 

agreed that the female initiated the attack and did all the boring and that the 

male may help a bit in moving frass and boring particles about the galleries. My 

observations of males with females in 1985 and 1986 suggested that the males 

were not important in attack initiation and gallery excavation for the 2-week 

testing period and that the addition of males could add another variable. 

The results of the 1987 experiments, when compared to prior studies, were 

not as indicative as expected in proving that reliable bioassay could be made 

with small trees and projected to large trees. There may be limitations on this 

type of testing on small trees. However, the results should be compared with 

what has been obtained from tests on large trees and with what will be obtained 

for this same genetic stock when they are tested as larger trees. In time, both 

chemical assay and bioassay of young trees could be highly indicative of the 

results which might be expected from tests with large trees. When this is 

accomplished, then testing at a young age will be adequate. 

Procedures other than no-choice testing on irreversibly stressed trees were 

tried early in the program, but none proved to be suitable. Four of these are 

reviewed very briefly as follows: 

(a) Pairs of beetles were caged on a small portion of the bark of a large P. 

446 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
Smi

th The Function of Xylem Monoterpenes of Young P. ponderosa during Chapter 9 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

ponderosa, quantity and quality of resin were determined, and boring success 

was determined. The procedure failed because bark beetle attack functioned as 

a mass attack phenomenon, and individual attacks are impossible to evaluate 

under these conditions. 

(b) Natural populations of beetles were attracted to groups of trees selected 

on the basis of resin properties. Progress of attack was monitored and associated 

with resin properties. Evaluation was poor because beetle quantity was not 

controlled, and the mass attack phenomenon masked the effects of the resin 

characteristics. Attempts were not successful in "turning off the beetle attack" 

when it seemed that some trees would live and others would die. 

(c) Regulated numbers of beetles were caged on unaltered trees of the 

proper size. The beetles were added to successive caged units working from the 

top of the caged portion, i.e., much like the course of a natural attack. The plan 

was to halt the addition of beetles when some trees were judged as near to 

being successfully attacked based on the occurrence of beetle oviposition. This procedure also failed because the time to stop the attack could not be 

determined accurately or reliably. Oviposition was not a reliable sign that 

the attack was successful, and all trees survived when the attack sequence 

was stopped. 

(d) Small trees about 6 inches in diameter were selected for resin properties 

and were cut off at about 5 feet above the ground. A rigid, portable cage was 

placed over the "high" stump and beetles introduced into the cage on the basis 

of bark area of the stump. The stump was cut off at the ground after 3 weeks, 

and all beetle activity was evaluated by shaving off the bark. Again evaluation 

was difficult. There were insufficient replicates, and there was usually a very 

strong and irregular flow of resin the first few weeks after the tree was cut. 

With this unpredictable flow of resin and the lack of replicates, it was difficult 

to evaluate beetle activity. However, these unsuccessful results led to the nochoice, irreversibly stressed, multiple-section procedure because the active tree physiology was largely nullified, beetle quantity was regulated, there were 

several replicates for a given tree, and resin quality was constant within a given 

tree but differed from tree to tree by choice. Resin quantity usually varied with 

each successive section so that it could be treated as a variable. 

The nursery bed may not be the most appropriate place for this type of 

testing. However, it is very convenient and most economical with regard to 

time and money expended. Future tests in a nursery might consider a sunscreening structure over the bed or other means for controlling the micro 

environment. A plantation-type area should be tried. Though this would cost 

more, it could be more reliable. Pheromones might be used to attract beetles to 

uncaged and unmodified trees in plantations. Other procedures that should be 

pursued are: (a) further attempts to use individual females or pairs despite the 

poor results with preliminary testing; (b) other measurements of resin flow; (c) 

as mentioned earlier, use of larger trees; and (d) testing periods other than 2 

weeks. With procedural improvements, it might be possible to use a larger 

array of compositions to associate boring success with various resin qualities. 

Miller (1950) may have been correct that good and confident testing may 

require trees greater than 15 cm in diameter. However, one purpose of the 

experiments was to shorten the period between seed germination and testing 

from 15-20 years to between 8 and 10 years. The results show that the time 

interval for testing can be shortened but that improved culturing of trees 

might help. 

These experiments culminated a long program of (a) identifying xylem 

resin limonene as a component of ponderosa pine that increases its resistance to 

western pine beetle attack, (b) similarly testing native trees, (c) searching for 

parent trees for breeding, and (d) culturing and testing the progeny of the 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-177. 2000 447 
Chapter 9 The Function of Xylem Monoterpenes of Young P. ponderosa during Smith 

Western Pine Beetle Attack (Coleoptera: Scolytidea): A Bioassay 

breeding program. The results of the testing, in general, supported the 

resistance model, demonstrating that other factors being equal, the success of 

attack of WPB on P. ponderosa is significantly less in trees with greater 

limonene content of xylem resin and in trees with greater flow of resin. It was 

also obvious that there is much room for improving the procedure. Resin flow 

seemed to be less of a factor in trees with a diameter well below that associated 

with normal beetle activity. The resistance model might be used in pest 

management as follows: (a) keep resin flow high by regulating stand density 

and composition; (b) keep beetle numbers low whenever economically and 

ecologically possible; (c) consider high-limonene sources of ponderosa pine 

for planting stressful sites. And it is quite likely that there are a number of 

other factors that could bear upon each of the four primary elements of the 

resistance model. 

448 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
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454 USDA Forest Service Gen. Tech. Rep. PSW-GTR-177.2000 
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