_ GENETIC VARIATION AND INTERRELATIONSHIPS OF THE CORTICAL MONOTERPENES FOLIAR MINERAL ELEMENTS AND GROWTH ; <.;]_,_._ a CHARACTERISTICS OF EASTERN ....... ’ “ -' WHITE PINE " ‘ ' " IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII”III"III ‘; 31293 10681 6014 This is to certify that the thesis entitled Genetic Variation and Interrelationships of the Cortical Monoterpenes, Foliar Mineral Elements, and Growth Characteristics of Eastern White Pine presented by Robert Louis Hilton has been accepted towards fulfillment of the requirements for . _.;;_l.__._/_L_J_degree inM ”7"“77/414/ 5411;,3l/ [/4 //// /;:f /-’/ Major professor ’J (“\I I I 25/], "7 (’4 / o / (é / Damn/gr: ’1 v2: /.. of} 0-169 ABSTRACT GENETIC VARIATION ANDIINTEBRELATIONSHIPS OF THE CORTICAL MONOTERPENES, FOLIAR MINERAL ELEMENTS, AND GROWTH CHARACTERISTICS OF EASTERN WHITE PINE By Robert Louis Hilton Analysis of the cortical monoterpenes in 23 geographic sources - part of a range-wide provenance study - at four test sites in Lower Michigan revealed that sources differed significantly in their concentrations of alpha-pinene, limo- nene. nyrcene, 3-carene and beta-phellandrene. Absence of distinct geographic patterns suggests that genetic drift is responsible for these differences. The large percentage of variation in terpene levels due to site and source 1 site interaction demonstrates that mono- terpene composition is highly dependent upon environmental influences. Only limonene and myrcene appear to be under strong genetic control. However, it is hypothesized that the large influence of environment on terpene composition was due to sampling widely diverse genotypes growing under drastically different conditions. Simple correlations were calculated between each of several morphological traits and the concentrations of the individual nonoterpenes for one southern plantation. There was a positive relationship between 3-carene and height growth. However, this relationship may be of no biological significance. Robert Louis Hilton The cortical oleoresin was also analyzed in 3? half- sib families representing six geographic areas in Michigan. There were significant differences among families in the concentrations of eight out of nine monoterpenes. Seven of these terpenes varied significantly among families within stands. Among the six geographic areas there were significant differences in the concentrations of alpha-pinene, 3-carene, myrcene and limonene. It is apparent that separation of Hichigan's Lower and Upper Peninsulas by five miles of water has allowed differentiation with regard to the levels of three of these terpenes. There was very little variation among stands within the Lower and Upper Peninsulas. This probably reflects the continuity of eastern white pine with- in these areas. Frequency distributions for the major terpenes in 131 trees strongly indicate multigenic inheritance patterns for alpha- and beta-pinenes. On the other hand, the concentra- tions of myrcene, 3-carene, limonene. and beta-phellandrene appear to be controlled by relatively few genes. These data for 131 trees were also used to compute simple correlations between monoterpenes. There was a posi- tive correlation (r - .7“; significant at the 1% level) be- tween 3-carene and terpinolene. Pour negative correlations were significant at the 5 or 1% levels, i.e. alpha-pinene and 3-carene, beta-pinene and 3-carene, beta-pinene and Robert Louis Hilton terpinolene, alpha-pinene and myrcene. It is pointed out that the exact relationships between these terpenes in biosynthetic pathways is difficult to de- termine from correlation analyses alone. Biochemical studies are needed to further define the exact modes of biosynthesis. At two test sites of the range-wide provenance study referred to above cortical monoterpene concentrations were investigated at two different times of the year. It was de- termined that, with the exceptions of gamma-terpinene and terpinolene, reasonably accurate estimates of cortical mono- terpene concentrations can be obtained by sampling only once during the dormant period. Likewise, monoterpene levels in 1- and 2-year-old cortex were studied at these test sites. From the results it is hypothesized that there are differences in enzymatic activity between 1- and 2-year- old cortical tissues. Here, also, 12 mineral elements were analyzed in the foliage of 15 geographic sources. There were significant differences among 14 seedlots growing at both locations in the concentrations of N, Cu and Zn. However, there were no geographic patterns. Likewise, there were no relationships between the concentrations of these three elements and any growth or morphological traits. In view of the author's main objectives two general conclusions can be stated. First, there is genetic varia- tion in both the cortical monoterpene and foliar mineral Robert Louis Hilton element compositions of eastern white pine. Second, in spite of these genetic differences there appear to be no correlated responses of biological significance with regard to the growth characteristics studied. GENETIC VARIATION AND INTERRELATIONSHIPS OF THE CORTICAL MONOTERPENES, FOLIAR MINERAL ELEMENTS, AND GROWTH CHARACTERISTICS OF EASTERN WHITE PINE By Robert Louis Hilton A THESIS Submitted to Michigan State University in partial fulfillemnt of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Forestry 1968 ACKNOWLEDGMENTS The author sincerely thanks the members of the Gui- dance Committee -- Drs. A.A. DeHertogh, J.E. Grafius, J.W. Hanover, C.J. Pollard, and J.W. Wright (Chairman) -- for their assistance. Thanks are also due to W. Lemmien, J. Tobolski and J. Bright for their assistance in collecting oleoresin samples at the W.K. Kellogg and Fred Russ Forests. In addition, I am grateful to the Glidden Company of Jacksonville, Florida and the Hercules Powder Company of Wilmington, Delaware for supplying pure samples of monoterpenes. My wife, Carol, provided much inspirational encourage- ment during the course of this study and actively partici- pated in the preparation of the manuscript. To her I owe a special debt of gratitude. This study was financed by the Cooperative Stats Re- search Service of the 0.8. Department of Agriculture as part of the regional project NC-51 entitled, “Tree Improvement Through Selection and Breeding”, and by the McIntire-Stennis Cooperative Forestry Research Program. 11 TABLE OF CONTENTS ACKNOWLEDGEMENTS...................................... LIST OF TABLES........................................ LIST OF FIGURES....................................... CHAPTER" I. INTRODUCTION.................................... Geographic variation in Eastern White Pine... Physiological traits...................... Monoterpenol................................. Mineral Elementseeeeeeeeeeeoeeeeeeeeoeeoeeeoe 0bJ9°t1ve5 Of Study.......................... II. GEOGRAPHIC VARIATION IN THE MONOTERPENE COMPOSI- TION OF EASTERN WHITE PINEOOOCCOOOCOO00.0.0.0... PiethOdsOIOOOOOOOOOOOO..OOOOIOOOOOOOOOOOOOO... Variation Among Geographic Sources........... RCIUItBeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Plantation Differences....................... Source x Plantation Interaction.............. Relationships Between Terpene Concentrations and Othfir Traits at KOIIOgg................. Comparison of Cortical and Xylem Oleoresin... III. VARIATION, INHERITANCE AND RELATIONSHIPS OF THE CORTICAL MONOTERPENES IN HALF-SIB FAMILIES OF EASTERN WHITE PINE FROM SIX AREAS IN MICHIGAN... MethOdBOOOOOOCOOOOOOOOOOOOOOOUOOOOOOOOOOOOOOO Sources of Variation in Terpene Concentrations Genetic Control of Tsrpene Levels............ Growth Rate and Terpene Concentrations....... Half-31b Families at Allegan................. Inheritanc. PattCPnBeeeeeeeeeeeeeeeeeeeeeeeee 111 PAGE 11 viii mmamw H 49 54 55 6O CHAPTER III. (cont'd.) Simple Correlations Between Monoterpenes..... nothOdaoOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOO... Results...OOOOOOOOOOOOOOCOOOOOOOOO00...... DlacuasionOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOO IV. EFFECTS OF SAMPLING TIME AND AGE OF TISSUE ON CORTICAL MONOTERPENE LEVELS IN EASTERN WHITE PINEOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOO...O. Time of Suplm............................. nethOdseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee n.8u1t8 md diaou881on0000OOOOOOOOOOOOOOOO A8. or TisauaOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Methad'ODOOOOOOOOOOO0.0...00....0.0.0.0... n.8u1t8 and discussionOOOOOOOOOOOO0..CO... conclusions...’000......OOOOOOOOOOOOOOOOOO... V. GEOGRAPHIC VARIATION IN THE FOLIAR MINERAL COM- mSITon OF EASTERN WHITE PINEOOOOOOOOOOOOOOOOOO HuthOdBOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOO. Results and Discussion....................... Plantation differences.................... Relative importance of geographic origin, planting location and interaction of theao r‘OtorIOOOOO.OOOOOOOOOOOOOOOOOOOOOO anm CITED.0.0.0....0.00.0.0...OOOOOOOOOOOOOOOOO VITAOOCOODOOOOOOOOCOOOOOOOOOOOO...OOOOOOOOOOOOOOO0..O. APPENDIXeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee iv PAGE 87 89 9o 93 95 99 10h 106 LIST OF TABLES TABLE PAGE 1.--Concentrations of five monoterpenes in 23 seedlots growing at four locations in southern Michigan. 22 2.--Ccmparison of F values derived from analyses of variance of the terpene concentrations among seedlots growing in either two or four plantations. 23 3.--Compariscn of monoterpene compositions at four plantations in southern Michigan. 25 4.--Percent of variation in the terpene composition of geographic origins due to seedlot, planta- tion, seedlot x plantation interaction, and er- ror (calculated from the data for eight seed- lots in all four plantations). 32 5.--Percent of variation in the terpene composi- tion of geographic origins due to seedlot, plan- tation, seedlot x plantation interaction and error (calculated from the data for 12 seedlots at the Kellogg and Russ Forests). 34 6.--Geographic variation in growth, female flower production and foliar characteristics among 1” seedlots at the W.K. Kellogg Forest (from both personal measurement and wright, 1968). “0 7.--Monoterpene composition of half-sib families growing at the w.x. Kellogg Forest in south- western Michigan. 50 8.--Monoterpene composition of progeny originating from six areas in Michigan and growing at the N.K. Kellogg Forest. 52 9.--Sources of variation in the terpene composition of half-Bib families growing at the W.K. Kel- logg Forest. . 53 V TABLE iO.--Leve1s of three monoterpenes in half-31b fami- lies growing at the Allegan State Recreation Area in west-central Michigan. 11.--Monoterpene composition of progeny originating from five areas in Michigan and growing at the Allegan State Recreation Area. 12.--Sources of variation in the terpene composi- tion of half-eib families growing at the Alle- gan State Recreation Area. 13.--Simple correlations between monoterpenes ana- lyzed in 84 trees from half-Bib families at the W.K. Kellogg Forest. 1#.--Percent of variation in terpene composition due to seedlot, time of sampling, and error, and the reliability of sampling at only one time of the year. 15.--Average monoterpene levels in 1- and 2-year- cld cortex of geographic origins at two loca- tions in southwestern Michigan. 16.--Percent of variation in the terpene composi- tion of geographic origins due to seedlot, age of tissue, seedlot x age of tissue interaction, and error. ‘ 17.--Foliar nitrogen, capper, and zinc concentra- tions of 15 seedlots growing at two places in ,southwestern Michigan. 18.--Comparison of foliar mineral element concen- trations at two plantations in southwestern Michigan. 19.--Percent of variation in mineral element con- centration due to seedlot, plantation, seed- lot x plantation interaction, and error, and the reliability of analyzing foliage from #0 trees. 20.--Comparison of foliar mineral element concen- trations in eastern white pine and in four other pine species (From Wright, personal com- munication). v1 PAGE 57 58 59 66 76 81 91 9“ 96 97 TABLE PAGE 21.--Monoterpene composition of #7 trees from five half-sib families at Allegan State Recreation Area. 106 22.--Monoterpene composition of SA trees from five half-sib families at the w.K. Kellogg Forest. 109 _vii LIST OF FIGURES IPIKGURE i.-—Natural distribution of eastern white pins in the United States and Canada (shaded) and ori- gin of 23 geographic sources sampled in this study. 2.--Locations of provenance test plantations in Michigan - Fred Russ Forest (R), N.K. Kellogg Forest (K), Newaygc Research Forest (N), Pine River Research Forest (P). 3.-4The Kellogg plantation pictured here is eight years old from seed. Ih$--The Newaygo plantation pictured here is nine years old from seed. Note the poor growth in comparison with that at the Kellogg plantation in Figure 3. 5.--Locations of 48 parents in Michigan. Grand Traverse County (G), Iron County (I), Lake County-(L), Newaygo County (N), and School- craft County (8). 6.--Frequency distributions of the six major corti- cal monoterpenes in eastern white pine. Basis, 131 trees. 7.--Hypothesized pathways for the biosynthesis of eight monoterpenes (after Juvonen, 1966). viii PAGE 12 15 29 31 45 62 83 CHAPTER I INTRODUCTION Provenance studies have demonstrated that eastern white pine (Pings strobus) is genetically variable with regard to growth rate and a number of other morphological as well as physiological traits. The best evidence comes from a U.S. Forest Service study initiated in 1955. Interim results have been published for plantations in the southern Appala- chians by Sluder (1963), in the Northeast by Santamour (1960), and in the Midwest by Funk (1964). The 10-year results of this investigation as well as other studies are thoroughly reviewed by Wright (1968). A synopsis of this and other pa- pers is given below. GEOGRAPHIC VARIATION’;N EASTERN WHITE PINE The earliest geographic origin test, established in Massachusetts in 1937, was described by Pauley 22.21, (1955). Eighty of the 159 seed sources were local collections from Massachusetts. In addition there were 27 New Hampshire, 17 New York, 21 Ontario, 3 Michigan, 9 Minnesota and 2 Virginia sources. The Massachusetts origins displayed the best growth rate. In 16 plantations of the U.S. Forest Service study lo- cated from Georgia to Pennsylvania and in central Michigan 1 .2 and southern Ontario, seedlots 1-CA, 2-NC and 3-TENN grew 25 percent faster than the average for all seedlots. Sources 6-PENN, l6-OHIO and 24-ONT grew at an intermediate rate. Eight other seedlots grew at a rate below the plan- tation averages. Thus, with few exceptions, seedlots from the southern Appalachians grew the fastest. In contrast to the above, seedlots 31-WISC.and 32-MICH grow well while seedlots i-CA, 3-TENN, lu-ME and 23-QUE grew poorly in four plantations located in the northern Lake States and in northern Ontario. In general, the fastest growing sources originated south of the test sites. Genys (1968) reported on another provenance test in Maryland in which 99 geographic sources were studied. Two- year nursery data revealed the same general results reported above for 16 south-central test plantations in the U.S. Forest Service study. Growth was fastest in seedlots from the southern Appalachians, Pennsylvania, Ohio, and southern Ontario and slowest in seedlots from the northen Lake States and portions of Canada. In all three studies Virginia sources grew the poorest. Hence, one should not generalize concerning the performance of southern Appalachian white pine. Similarly, the superior growth rate of southern Ontario sources does not allow one to generalize concerning growth rates among northern sources. Variation among individual trees and stands was investi- gated in a half-sib progeny test started by Michigan State University in 1962 (Wright, 1968). Seed was collected from 123 parents over lb counties in both the Lower and Upper Peninsulas of Michigan. Height measurements in four south- ern Michigan plantations at age 6 revealed that there were no significant differences in growth rate among progenies from the same stand. However, there were significant dif- ferences among progenies due to region of origin. Seedlots from the west-central Lower Peninsula grew fastest. Growth rate was significantly less among progenies from the north- eastern Lower Peninsula and particularly among those from the Upper Peninsula. Morphological characteristics other than growth rate have also been studied. Wright 35 5;. (1963) reported significant differences in lemmas growth (1 percent level) among 15 origins of the U.S. Forest Service study in a test plantation in southern Michigan. However, there was no pat- tern to these differences. Likewise, Santamour (1960) dis- covered no correlation between latitude of seed source and percent of seedlings with lemmas growth at a plantation site in New Jersey. Wright (1968) summarized the data for female flower production at five plantation sites of the U.S. Forest Ser- vice study. Slow growing seedlots 20-NS, 21-NB and 28-MINN produced the greatest quantities of female flowers. Other slow growing and all five southern Appalachian origins dis- played very little flowering. Numerous foliar traits were investigated among 15 geo- graphic origins of the U.S. Forest Service study in southern a Michigan. Seedlots differed significantly but to a very small degree in most traits (Wright 22,51,, 1963; Wright, 1968) . Although there were significant differences among seed- lots in the width and length of the needles, number of endo- dermal cells, number of resin canals, number of stomata, and period of needle retention, no patterns were observed. How- ever, southern origins generally possessed the greatest num- ber of serrations per millimeter of needle length. Likewise, eastern origins could be classified as having blue foliage and northwest origins plus seedlot lh-ME as having yellow- green foliage. In contrast to the above, a study, conducted in Connec- ticut, of eight seed sources at age three by Morgen (1963) revealed that northern trees had the greatest number of ser- rations. Also, there was a strong relationship between lati- tude of seed source and number of stomata, number of resin canals and average needle length. Southern origins posessed the largest number of stomata and the longest needles, but the fewest resin ducts. In addition, Genys (1965) studied cotyledon numbers among 16 geographic sources of a 99-origin Maryland pro- venance test. Although none of the progenies had uniform numbers of cotyledons, seedlings from the most northern and southern locations possessed the greatest numbers and differ- ed significantly (1 percent level) from sources representing the more central parts of the species' range. 5 PHYSIOLOGICAL TRAITS Mergen (1963) reported the responses of several geo- graphic sources, grown in Connecticut, to varying photo- periods and temperatures. In one investigation he brought three-year-old seedlings of eight seed sources into the greenhouse each month from September to January and exposed them to light periods of 8- and l6-hours. For the 8-hour light treatment there were significant differences among seedlots in the initiation of growth. Northern sources were the first to break dormancy but grew less than southern sources. For the 16-hour light treatment there were no sig- nificant differences among seed sources in time of bud break. However, southern Appalachian seedlots grew, on the average, 50 percent more than northern seedlots. Using the same materials specified above Mergen placed 3-year-old seedlings in growth chambers during January and grew them at a day temperature of 80° F. and night tempera- tures of 39°, 60° and 80° F. All origins grew best at a night temperature of 60° F. At this temperature southern origins grew the fastest. Growth was noticeably poor in northern origins grown at a night temperature of 80° F. and in southern origins grown at 39° F. These data may explain why southern Appalachian seedlots have not performed well in provenance test plantations established in the northern Lake States. Mergen also reported that the foliage of southern Appa- lachian seedlots was severely discolored when subjected to a 6 temperature of -75° F. On the other hand, northern seedlots suffered no damage. Furthermore, Mergen (1963) studied the stratification requirements of seedlots from North Carolina, New Brunswick and New Hampshire. Seed of the latter origin needed very little stratification while that of the New Brunswick and North Carolina origins germinated only when stratified 28 or more days. A.more intensive study of 11 seedlots by Fowler and Dwight (196%) demonstrated that the percent germination of all seedlots was positively correlated with length of the stratification period. However, seed of four northern origins germinated fairly well even with no treatment while the seed of most southern origins germinated poorly unless stratified at least 60 days. Apparently geographic origins of eastern white pine are adapted to local climatic conditions. The seed of southern origins possesses a greater degree of dormancy to prevent germination and subsequent mortality during the relatively warm but often harsh winter weather characteris- tic of southern areas. Thus, provenance testing has established the presence, and in some instances, the patterns of genetic variation in numerous morphological and physiological traits of eastern white pine. However, there is a noticeable absence of data pertaining to chemical constituents of this species. MONOTERPENES Monoterpenes are 10-carbon compounds classified among the isoprenoids because they may be considered derivatives of isoprene, C5H8. The simplest monoterpenes, the aliphatic compounds myrcene and ocimene, are thought to be formed from geraniol pyrophosphate by phosphatase activity and dehydra- tion. Most of the monoterpenes are cyclic and are presumed to be derived by cyclization of geraniol or geraniol pyro- phosphate (Bonner and Varner, 1965). The importance of monoterpenes in taxonomic investiga- tions has been stressed by Mirov (1961; 1963). Studies which have shown that the terpenes are under strong genetic control have justified their use in a phylogenetic approach to plant classification. Already there have been several successful attempts to use the qualitative and quantitative monoterpene composition of xylem oleoresin as an aid to taxonomic study in pine species (Fords and Blight, 196“; Iconomou g§_g;.. 196“; Mirov, 1961; Mirov gt_gl.. 1965; 1966a: 1966b; Pelo- quin, l96h; Squillace and Fisher, 1966; Tobolski, 1968; Williams and Bannister, 1962; and Zavarin g§,gl., 1966). More recently the monoterpene composition of cortical oleoresin has been studied (Hanover, 1966a; 1966b; 1966c; Squillace and Fisher, 1966; and Tobolski, 1968). As stated by Hanover (1966b) the close proximity of the cortical ter- penes to the site of their synthesis, the epithelial cells, and to the photosynthetic tissue renders them most relaible for genetic studies. The function(s) of terpenes in the plant has not been resolved. Recently they have been implicated as factors in- fluencing insect resistance. Although.some investigators regard terpenes only as waste products of normal metabolism, others reject this hypothesis because of the large amounts of energy expended by the plant in producing considerable quantities of terpenes. MINERAL ELEMENTS Mineral elements analyses of whole plants or parts thereof have been previously used to investigate the role of nutrients in plant metabolism. More recently foliar analyses in replicated provenance tests have been used to detect genetic differences among geographic sources (Ger- hold, 1959; Lee, 1966; Mergen and Worrall, 1965; and Stein- beck, 1966). OBJECTIVES Q: STUD; Following, in Chapters 2 to 5, are the results from investigations of the cortical monoterpenes and foliar mineral elements found in eastern white pine. These studies were conducted in lower Michigan in four provenance test plantations which are part of the 1955 U.S. Forest Service study, and in two other half-sib progeny test plantations established by Michigan State University in 1962. The purpose of these investigations was fourfold: (1) To provide further knowledge concerning the patterns of genetic differentiation among geographic origins. (2) To 9 uncover some correlated responses which would aid in the genetic improvement of eastern white pine by selection and breeding. (3) To determine possible modes of inheritance and biosynthesis of the cortical monoterpenes through analyses of their concentrations in individual trees. This information is important to understanding the progress and patterns of differentiation among geographic sources in re- gard to terpene levels. (A) To determine the direction and magnitude of the effects of sampling time and age of tissue sampled on terpene concentrations. These factors must be investigated if one is to make valid comparisons among dif- ferent studies of the cortical monoterpenes. CHAPTER II GEOGRAPHIC VARIATION IN THE MONOTERPENE COMPOSITION OF EASTERN WHITE PINE A total of 23 geographic sources of eastern white pine (giggg strobus) were planted in four experimental plantations in Lower Michigan in 1960 and 1962. These sources are a por- tion of 33 original seedlots assembled for a range-wide pro- venance test by the U.S. Forest Service in 1955. The origins of these seedlots are shown in Figure 1. Fifteen seedlots are represented at the W.K. Kellogg Forest in Kalamazoo County, 1“ at the Fred Russ Forest in Cass County, 16 at the Newaygo Research Forest in Newaygo County, and 18 at the Pine River Research Forest in Wexford County. Each of the four plantations follows a randomized com- plete block design. The Kellogg and Russ plantations contain ten replications of h-tree plots. Both were established with 2-1 stock in the spring of 1960 and were ten years old from seed when this study was conducted. The Newaygo Plantation consists of four replications with 81-tree plots; that at Pine River contains 25 replications with 1-tree plots. These latter two plantations were established with 2-2 stock in the spring of 1962 and were nine years old from seed when sampled. The location of these four plantations is shown in 10 11 Figure 1.--Natura1 distribution of eastern white pine in the United States and Canada (shaded) and origin of 23 geographic sources sampled in this study. _ -—- —‘“H_ __—_._a—- a-» “In — —_— —-—~‘ ,- ——_‘—‘R 11 Figure 1.--Natural distribution of eastern white pine in the United States and Canada (shaded) and origin of 23 geographic sources sampled in this study. 13 Figure 2. Methods Cortical oleoresin was collected at the four plantations during the spring of 1967. For each seedlot oleoresin sam- ples from individual trees were bulked over each half of a plantation. This provided two replicate determinations for each seedlot. The values obtained therefrom were used in subsequent analyses of variance to compute error mean squares for testing seedlot x plantation interaction. At the Kellogg and Russ plantations the tallest tree of each plot was sampled at the 3-year-old node. The cortex of the previous year's (1965) lateral growth was cut with a razor blade and 20 microliters of the exuding resin was drawn into capillary tubes one mm. in diameter. The oleo- resin from five tress comprised each bulk sample. The Kel- log samples were collected March 2“, 1967, stored at h0° F. and analyzed March 29, 1967. The Russ samples were collect- ed March 31, 1967, similarly stored, and analyzed April 26, 1967. At the Newaygo plantation the tallest three trees per plot were similarly sampled at the 2-, 3- and h-year-old branch whorls. A bulk sample consisted of 120 microliters of oleoresin from a total of six trees. Bulk collections at the Pine River plantation contained a total of 100 micro- liters of oleoresin from the 2-, 3-, and h-year-old branch whorls of five trees per source selected over 11 replicates 1n Figure 2.--Locations of provenance test plantations in Michi- gan - Fred Russ Forest (R), W.K. Kellogg Forest (K), Newaygo Research Forest (N), Pine River Re- search Forest (P). 16 for good thrift. The Newaygo samples were collected April 19, 1967, stored at UO° F., and analyzed April 26, 1967. The Pine River samples were collected April 23, 1967, simi- larly stored and analyzed April 27, 1967. Each sample was diluted 1:1 with pentane and analyzed by gas-liquid chromatography. Three microliters of the di- luted oleoresin were injected into an F e M gas chromato- graph, Model 700, having a thermal conductivity detector. A stainless steel column measuring 6 feet x 1 inch and con- taining 15%fl ,fl -oxydipropionitrile on chromosorb C support was used. The analytical conditions were as follows: column temperature, 61° to 65° 0.: injection port temperature, 150° to 151° 0.; detector temperature, 15h° to 160° 0.: and helium flow rate, 100 to 110 ml. per minute. All of the individual monoterpenes, except beta-phellan- drone, were identified by comparing relative retention times with those of known compounds at the same operating condi- tions and by comparison with values published by Klouwen and ter Heide (1962). Beta-phellandrene was identified only by the latter procedure. magnet! mg 6306 sousgss Several investigators have demonstrated intraspecific variation in the monoterpene composition of pine species. Samples of xylem oleoresin were collected from Monterey pine (Zigpg,;ggiata) trees growing in three native California stands. The stand average varied from 23 to 3“ percent for 17 alpha-pinene: from 6h to 76 percent for beta-pinene. Other terpenes were present in very minute quantities (Bannister 31:, 51.. 1962). Mirov 23,31. (1965) analyzed a composite sample of xylem oleoresin from #00 slash pine (21235 elliottii var. elliottii) trees from northern Florida. Beta-phellandrene accounted for only 3 percent of the total monoterpene con- tent in contrast to 19 percent found earlier by Mirov in a composite sample of oleoresin from 21325 elliottii var. 2223;,from southern Florida. Squillace and Fisher (1966) studied the xylem oleoresin from five geographic sources of slash pine. There were significant differences among sources in the concentrations of beta-pinene and myrcene. No geogra- phical patterns were observed. Squillace and Fisher (1966) also investigated the cor- tical oleoresin of slash pine. They sampled 12 geographic sources distributed from central Florida to central Georgia. There were significant differences in myrcene content. How- ever, no geographic pattern was evident. There were signifi- cant differences in the levels of beta-pinene and beta-phal- landrene as well. The latter two terpenes followed north- south patterns that can be compared to similar variation patterns for several traits as shown below. These authors did not imply any cause and effect relationships. 18 Height Needle Stomata Beta- Beta-phel- Region (Age 1) Length per mm. pinene landrene 931,. 23. number percent percent S. Florida 9-19 16-20 8.6-8.8 -- -- Central Florida 10-28 16-19 8.2-8.8 10-35 22-h0 N. Florida and 8.0. to La. 24-30 1u-15 8.8-9.2 36-55 9-18 Fords and Blight (196k) analyzed 58 samples of xylem oleoresin from Bishop pine (gigg§,murigata) collected in eight native stands. Variation in terpene concentrations was strongly related to morphological differences. These authors recognized three chemical races, i.e. a northern (96 to 99 percent alpha-pinene), central (73 to 89 percent 3-carene), and a southern race (50 to 75 percent sabinene and 18 to #3 percent terpinolene). Mirov g§_gl. (1966b) further defined the geographic limits of these three chemical races after sampling 127 trees at 16 additional locations. These inves- tigators regard alpha-pinene as the "ancestral” terpene of pines belonging to the Subsection Oocarpg . On this basis they deduced that Bishop pine originated in the northern portion of its range and differentiated into chemical races as it spread southward. Smith (1967b) studied the monoterpene composition of the xylem oleoresin from 20 Coulter (Ping; poulteri), #6 19 Washoe (Flags washoensis), and 128 Jeffrey (Pings jeffrezi) trees which represented a large portion of the range of each species. The terpene fraction of Jeffrey pine oleoresin varied little over the locations sampled. It consisted of 88 to 99 Percent heptane. On the other hand, there was con- siderable variation in Coulter pine with regard to the levels of alpha-pinene (25 to 51 percent), beta-phellandrene (21 to 37 percent), and myrcene (6 to #2 percent). Samples of Washoe pine displayed considerable variation in the amounts of beta-pinene (0 to 33 percent), 3-carene (45 to 78 percent), and myrcene (5 to 20 percent). However, for both Coulter and Washoe pines there were no distinct geographical patterns in terpene composition. Xylem oleoresin was collected from 9# Douglas-fir (Pseudotsggg menziesii var. glauca) trees growing at three locations in Idaho and Montana. One Idaho population con- tained the highest levels of alpha-pinene and the lowest levels of beta-pinene, 3-carene, and limonene (Hanover and Furniss, 1966). Zavarin g§_§;. (1966) examined the monoterpene fraction of xylem oleoresin from giggg khasza in Southeast Asia. Four samples were obtained from Assam and Burma and one from Viet Nam. Each sample contained the oleoresin from several trees. As shown in the following tabulation some of the stands from Assam and Burma differed considerably from the Viet Nam stand. 20 Terpene Assam and Burma Viet Nam percent 2; monoterpenes Beta-pinene 2-52 2 Longifolene 1-5 trace Beta-phellandrene 1-3 17 Smith (1964b) studied the xylem oleoresin in 6“ pon- derosa pine (Ppppp ponderosa) trees growing at eight places in the mountains of California. There was a wide range in monoterpene composition among the individual trees, and no geographic patterns were observed. Terpene analysis of ponderosa pine trees from no loca- tions was reported by Peloquin (1964). Trees from southern California and Arizona were different from trees over the rest of the range. Tobolski (1968) reported the cortical monoterpene com- position among 108 seed sources of Scotch pine (Ppppp s 1- vestris) from Europe and Asia. There were significant dif- ferences among sources in all 11 monoterpenes. Alpha-pinene and 3-carene varied the most. The latter compound was ab- sent in most southern populations and increased northward, reaching a high of 63 percent. Alpha-pinene showed a dif- ferent trend. It varied from 5 percent in Scandinavia to 69 percent in Spain. These two terpenes were the most useful in distinguishing previously named varieties of Scotch pine. 21 RESULTS In this investigation the concentrations of five mono- terpenes differed among seedlots (Table 1). Eight seedlots were represented in all four plantations. Most of the other 15 seedlots were represented only at the Kellogg and Russ or only at the Newaygo and Pine River plantations. values for sources not present at all four locations were adjuested to account for plantation differences. Analyses of variance were calculated using only data obtained from the eight seedlots growing at all four test sites. There were significant differences among seedlots in levels of alpha-pinene, limonene, and myrcene. However, further analyses for 12 seedlots growing in the Kellogg and Russ plantations revealed significant differences in concen- trations of two additional terpenes, i.e. 3-carene and beta- phellandrene (Table 2). The significant variation among sources in limonene con- tent can largely be attributed to the high concentrations of this chemical in seedlots 18-WISC and 20-NS. Fast-growing southern seed sources contained relatively high amounts of 3-carene. Seedlots 2h-ONT and 32-MICH, both fast-growing, contained the highest levels of 3-carene among northern seed sources. However, seedlots i3-MASS, 15-IOWA and 21-NB, characterized by slow to moderate growth rates, also had relatively high concentrations of 3-oarene. There were no other terpenes in which it was possible to recognize geographic trends. Seedlots differed, but 22 Table 1.--Ccncentrations of five monoterpenes in 23 seedlots growing at four locations in southern Michigan. Monoterpene P1 tgd Alpha- 3- Myr- Limo- Beta-phel- At a Seedlct pinene carene cene nene landrene ----- perpent 2; total monoterpenes----- 1 GEO K a N P 22 1b 11 6 7 2 NC P 17 25 1a 5 4 3 TENN K a N P 29 16 9 7 8 6 PENN K a N P 33 15 u 3 8 9 PENN K a 21 25 22 3 3 10 N! K a 36 23 5 4 3 12 N! K s N P 29 5 1g 4 8 1 MASS K s 41 10 3 3 1 MAINE K P 35 6 10 h 5 15 IOWA N P 26 11 9 6 8 16 OHIO N P 27 2 12 2 6 18 use N P 27 u 12 12 8 19 MINN K N P 29 2 1h 3 6 20 NS K a N P 2h 6 7 16 8 21 NB K a 28 11 9 9 u 2 QUE N P 26 6 36 3 5 2 our K s 28 16 u 5 II 25 ONT K s N P 26 5 15 3 6 28 MINN K R N P 31 7 12 a 6 29 MICH K a N P 26 a 19 7 31 WISC N P 35 5 1“ 3 9 32 MICH N P 21 11+ in 6 5 35 MINN N P 30 2 12 LI 10 LSD (.05)(b) 10.7 17.3 12.u 5.3 5.3 LSD (.01) 15.8 25.6 1u.3 7.9 7.9 (a) Letters denote Kellogg, Russ, Newaygo and Pine River pl?n§ations. b ‘These LSD values are conservative insofar as it was assumed that each seedlot was represented at only two lo- cations. To compute the smaller values applicable to seed- lots at four locations it is necessary only to multiply by .7. 23 Table 2.--Comparison of F values derived from analyses of variance of the terpene concentrations among seid- lots growing in either two or four plantations. 3’ F value Kellogg A11 Monoterpene and Russ plantations Alpha-pinene 2. 58 2.81* Beta-pinene 2.01 .66 Camphene 1.86 1.7“ 3-carene 6.93** 1.76 macho 13. 9i" 5. 56*“ Limonene 17.72*9 16.22** Beta-phellandrene 3.99. .57 Gamma-terpinene .9h .63 Terpinolene 1.99 .77 v '— (9) F values for Kellogg and Russ are based on 12 seedlots represented at both test sites: those for all four plantations are based on eight seedlots represented at each test site. 9, 9* - Significant at the 5 and 1 percent levels, respectively. 24 distant sources were often as similar to each other as were neighboring sources. In other words, differentiation into distinct varieties has not occurred. The extensive range of eastern white pine from Newfound- land to Georgia has provided enough geographic isolation so that lack of gene exchange could permit seed sources to dif- ferentiate with regard to cortical monoterpene composition. The absence of geographical patterns suggests that genetic drift has been responsible for the differentiation observed in this study. PLANTATIQN QIEZERENQES There were significant differences among the four plan- tations in the concentrations of alpha-pinene, beta-phellan- drone, gamma-terpinene, and terpinolene (Table 3). 'Apparent- 1y environmental factors influenced either the formation or the stability of these four terpenes. Major differences occurred between the two southern and two central Michigan plantations. Alpha-pinene was low- er and beta-phellandrene, gamma-terpinene and terpinolene were higher at Kellogg and Russ than at the Newaygo and Pine River plantations. Thus, it is inviting to conclude that environmental factors which vary with latitude, especially light and temperature, influenced monoterpene levels. Reported variability in leaf oil composition of pepper- mint clones (Montha piperita var. Black Mitcham) due to lo- cation, year, and cultural treatment prompted Burbott and Loomis (1967) to investigate the effects of light and 25 Table 3.--Comparison of monoterpene compositions at four plantations in southern Michigan. Monoterpene composition at W.K. Kellogg Fred Russ Newaygo Pine River Forest Forest Research Research Terpene Forest Forest --------pprcent p; pppgl_monoterpenes- ------- Alpha-pinene** 24.0 24.5 27.3 31.9 Beta-pinene 32.9 36.3 37.0 32.6 Camphene 3.2 3.4 3.3 3.6 3-cerene 9.7 7.2 9.? 9.0 Myrcene 12.2 11.9 10.9 11.4 Limonene 5.6 5.5 6.6 5.5 Beta-phellendrene** 9.4 9.3 4.5 4.8 Gamma-terpinene** 1.2 .7 .1 .4 Terpinolene* 1.8 1.2 .6 .8 Total 100.0 100.0 100.0 100.0 *, ** - Indicate significant differences among plan- tations at the 5 and 1 percent levels, respectively. 26 temperature on monoterpene concentrations. Using plants of a single clone of the Black Mitcham variety these investiga- tors employed several combinations of temperature and photo- period in a growth chamber. A minimum of six analyses were made for each set of conditions. It was discovered that short or cool nights increased the levels of methcne and de- creased the levels of menthofuran and pulegone. These effects were thought to be indirect and mediated through the concen- trations of respiratory substrates in the cells producing terpenes. Based on analyses of four trees Juvonen (1966) reported that levels of alpha-pinene in Scotch pine foliage are posi- tively correlated with temperature. The data for peppermint and Scotch pine support the hy- pothesis that light and/or temperature affect the cortical monoterpene composition of eastern white pine. However, as shown in the following tabulation there are only slight dif- ferences in temperature and length of growdng season between southern and central Michigan. Although I have no data, Av. Temperature Growing Plantations Jan. July Season :1. 31. days Kellogg and Russ 24.2 73.3 138 Newaygo and Pine River 20.6 70.6 1 2 2? differences in photoperiod are probably slight also. Other environmental factors may influence monoterpene levels. The Kellogg and Russ plantations have grown four to five times faster than the Newaygo and Pine River plantations. Height growth differences between the Kellogg and Newaygo plantations are shown in Figures 3 and 4. Possibly dif- ferences in vigor are somehow responsible for the quantita- tive differences in terpene composition. Evidence for this viewpoint can be obtained from a study of Scotch pine by Tobolski (1968). He demonstrated that defoliation by the European sawfly (Neodiprion sortifer) significantly affected the levels of alpha-pinene, beta-pinene, 3-careno and total monoterpenes in the cortical tissues. Within trees terpene concentrations were higher in moderately attacked branches (35 to 65 percent) than in those only lightly attacked (5 to 25 percent). Positive identification of the environmental factors influencing terpene levels in this study and the mechanism of such action must await further experimentation. SOURCE 1‘. PLANTATION W The importance of geographic origin (genotype), planta- tion (environment), and the interaction of these two factors (genotype x environment interaction) upon monoterpene compo- sition is shown in Table 4. Most striking is the amount of interaction for six of the nine monoterpenes. This, combined with a significant effect of location on the levels of four 28 Figure 3.--The Kellogg plantation pictured here is eight years old from seed. 30 Figure 4.--The Newaygo plantation pictured here is nine years old from seed. Note the poor growth in comparison with that at the Kellogg plantation in Figure 3. 31 32 Table 4.--Percent of variation in the terpene composition of geographic origins due to seedlot, plantation, seedlot x plantation interaction, and error (calcu- lated from the data for eight seedlots in all four plantations). Percent of Variance Due to: Seedlot Plantation Seedlot x Error Monoterpene Plantation Alpha-pinene 20* 22** u3ss 15 Beta-pinene 0 O 87*& 13 Camphene 13 O 68** 19 3-carene 14 o 73ee 13 Myrcene 46** O 40** 14 Limonene 71a 0 19* 10 Beta-phellandrene O 67** 26es 7 Gamma-terpinene O 68*! 25s» 7 Terpinolene O 44* 46*. 10 *, 9* - Significant at the 5 and 1 percent levels, respectively. . 33 terpenes illustrates that monoterpene composition is highly dependent upon environmental influences. Only the variabi- lity in concentrations of limonene and myrcene appear to be largely due to genetic differences. In comparison with the combined analyses for all four plantations, data for the Kellogg and Russ Forest plantations only revealed that environmental factors had comparatively small effect on terpene levels (Table 5). Hence, much of the environmental influence on terpene composition reported above is the result of differences in terpene values between the southern and central plantations. The striking differen- ces in growth between the Kellogg and Newaygo plantations exemplifies these widely different environments. Several authors have reported that the monoterpene com- position cf Pippa wood oleoresin is determined predominantly by the genotype of the organism. In his investigation of the turpentine composition in Ppppp attenuate x radiate hy- brids Fords (1964) found that when two individuals of a clone were sampled, the monoterpene composition agreed quite close- 1?. Fords and Blight (1964) collected 58 oleoresin samples from BishOp pine growing in eight locations. They discover- ed that variation among samples from the same area was in- significant in comparison with.differences among areas. For example, differences of approximately 90 percent in alpha- pinene content were evident among some areas while differences within areas were generally on the order of 5 percent or less. Table 5.--Percont of variation in the terpene composition of geographic origins due to seedlot, plantation, seedlot x plantation interaction and error (calcu- lated from the data for 12 seedlots at the Kellogg and Russ Forests). Percent of Variance Due to: Monoterpene Seedlot Plantation Seedlot x Error Plantation Alpha-pinene 34 10 43** 13 Beta-pinene 29 O 59** 12 Camphene 16 33** 37* 14 3-carone 64** 0 21 15 Myrcene 75** O 12 13 Limonene 77*! o 9 14 Beta-phellandrene 49* O 32 19 Gamma-terpinene O 32* 53** 15 Terpinolene 21 15* 46* 18 *, ** - Significant at the 5 and 1 percent levels, respectively. 35 Also, the turpentine from both wild and planted trees of the same geographic origin agreed quite closely. Based upon data from previous investigations involving several P;ppp_3pecies which illustrated the gross similarity of turpentine composition of trees growing within and outside their native ranges, Mirov (1961) concluded that the terpene composition of xylem oleoresin is a genetically fixed charac- ter. Likewise, based upon samples obtained from several lo- cations in Canada, Michigan and Minnesota von Rudloff (1967) reported that ecological differences did not affect leaf oil composition of white (gpppg glauca) and black spruce (£123; mar ana). Von Rudloff and Hefendehl (1966) found almost no variation in the terpene composition of the oil of Mentha ppvensip var. glpbrata collected from different localities. Baker and Smith (in Mirov, 1948) also have reported the con- stancy of the oil of Eucelzptus globulus from several locali- ties. Squillace and Fisher (1966) obtained intraclass correla- tions for both progeny and clonal data from which they con- cluded that most of the cortical terpenes in slash pine are strongly inherited. Particularly convincing are the data for two representative clones; as an example, differences among these clones in alpha-pinene content were five times greater than differences among ramets withbn clones. In the case of each clone some of the remote were rooted cuttings from one location while others were grafted trees from another loca- tion. 36 Hanover (1966a; 1966b) has given similar evidence for 5 of 6 cortical monoterpenes studied in western white pine (£figgg_montico1a). Hanover's conclusions are primarily based on analyses of two grafted trees from each of four clones growing in three widely different environments in northern Idaho in addition to heritability estimates derived from breeding data. For each clone percentages of any given ter- pene component generally varied by only 2 or 3 percent among the three sites. On the other hand, differences among clones were considerably greater except in the case of alpha-pinene. Using data from the progeny of 17 crosses among nine parents at one location in Idaho heritability estimates were derived from regression analyses of offspring on.mid-parent values. For all terpenes, except camphene, narrow sense heritability estimates approximating unity were obtained. However, large standard errors averaging 0.5 were also reported. Tobolski (1968) investigated ten seed sources of Scotch pine growing at two locations in Michigan. He determined that location and seed source x location interaction accounted for an insignificant amount of the variation in 10 cf 11 cortical terpene components in comparison with variation attributable to differences among geographic sources. In view of this evidence for strong regulation of both wood and cortical terpenes by gene action it might be assumed that collection time, collection technique, storage, nondran- dos distribution of genotypes within seedlots over locations, sampling error or all five factors have influenced the results 37 presented in Tables 3 and 5. But all available evidence implicates these factors only to a small degree. Seedlings of each seedlot were randomly assigned to a plantation at time of outplanting. Thus, there is no reason to suspect that genotypes within seedlots were stratified by location. Sampling error is expected due to the presence of dif- ferent genotypes from one plantation to the next. This is particularly expected for terpenes controlled by relatively few genes having low frequencies such as 3-carene, myrcene, limonene and beta-phellandrene (Chapter 3). However, source x replication interaction variance within plantations should also increase as a result of sampling error. Since this variance tests source x plantation interaction and contri- butes to the total variance, there is little reason to sus— pect that sampling error is the cause of the large and sta- tistically significant interaction variances found in this study. The fact that oleoresin was collected at the 2-, 3-, and h-year-old branch whorls at the Newaygo and Pine River plantations and only at the 3-year-old branch whorl at the Russ and Kellogg Forest plantations must be considered. However, preliminary investigations on two trees of each of five different geographic origins at the Kellogg Forest re- vealed no significant variation in the monoterpene composi- tion of 2-year-old cortex of lateral branches located at the 3- and 5-year-old nodes. 38 Reasonable care was exercised to collect equal amounts of oleoresin from each tree, and all samples, especially those from the same plantation, were stored for approximate- ly equal periods under the same conditions. Finally the influence of sampling time was probably negligible since samples were collected during a 30-day period in March and April. A study of the effects of sam- pling time (Chapter h) demonstrated that monoterpene con- centrations did not differ appreciably when samples were collected in July and March or in October and March. It should be noted that some of the environmental in- fluences on terpene concentration may be more apparent than real. Inasmuch as the terpene concentrations were measured as percentage of total monoterpene content, it is highly probable that significant changes in the levels of one or more terpenes caused others to change also. However, it is most likely that there were some real environmental influ- ences on terpene composition. The differences in vigor and growth between the southern and central Michigan plantations further reflect these environmental influences. In reconciling the results of this investigation with those reported by other authors, it should be noted that clonal data provide the most convincing evidence for lack of environmental influences on terpene composition. However, clonal and seedling data may not be comparable. Ideally clones are better suited to investigations of environmental effects. But unless one employs several clones genetically 39 very dissimilar, seedlings with their wide genetic base may better depict the influence of environment under natural conditions. Hence, it is possible that environmental in- fluences on terpene composition occurred in this study largely as a result of sampling widely diverse genotypes growing under drastically different conditions. RELATIONSHIPS BETWEEN TEHPENE CONCENTRATIONS LN}; m BAITS A: KELLOGG Growth and morphological data for the Kellogg Forest plantation are presented in Table 6. Using seedlot means as items, simple correlations were calculated between those data and the concentrations of the individual monoterpenes. Seed- lots which were high in terpinolene contained the fewest resin canals. This negative correlation (r s -.62) is sig- nificant at the 55 level. However, it may not be biologi- cally meaningful because one expects some significant cor- relations on the basis of chance alone. Other correlations were non-significant. These correlations based on Kellogg data alone may not have revealed all the growth-terpene relationships. Wright (1968) has summarized growth data for all origins tested in numerous southern plantations. Seedlots i-GA, Z-NC, 3dTENN, 6-PENN and 9-PENN grew rapidly. All ahd high quantities of 3-carene (Table 1). Similarly, fast-growing northern seed- lots 10-NI, 2#-ONT, and 32-HICH were high in 3-carene. Tobolski (1968) found that high.amounts of 3-carene doped essence H on» as pssoauaswum I as macaw reduce: use acauaepea assauss a ma ”seduces c.9sch msoabeha no mood opsaaaoo s 0 An. #0 acenmnosdn I OH unconm I « Adv sea.” eeo.n sew.n eem.m es#.n ssm.~ sso.Na ssw.m can.m ssN.m ozam> m «a m.~ em mm :a. me o“ a mg « e.oH sees no ~.~ on on ma. co m m an an :.m mon mm ma n.~ me on «a. :0 a m aw om e.m zzH: mm on m.~ «a mm «a. we on m «a co o.«« 920 mm m” H.~ nu om «a. on m on «N o: m.aa 92o aw ma m.~ «N we we. we on a mu om a.m mz «m ma n.~ mm «o ea. me n" m m: an e.m ex on ma n.~ ma mm me. me on n n on m.m azuaz H ma ~.~ e« an “a. a» «a m an o: m.oa mm scH asHsonhcoTHH soH sstonasolvoaosos—HH if / \ HHH> noH asHsonaso osoanIsnnfld 84 hypothesis that the primary precursor of this terpene is carbonium ion II rather than carbonium ion V. On the other hand, one might expect more 3-carene in the 2-year-old cor- tex due to less conversion of carbonium ion II to carbonium ion V. This was not the case. Several factors could be Operating to maintain relatively constant levels of 3-carene in different age cortical tissues. These include the af- finity of an enzyme for its substrate, rate of metabolism, feedback inhibition, etc. The contribution of tissue age to the variability of a particular monoterpene appears to vary with planting loca- tion as the data for beta-phellandrene and terpinolene in both plantations, of limonene and gamma-terpinene in one or the other of the plantations indicate (Tables 15 and 16). W Although the monoterpene composition of any given seed- lot is expected to vary with age of the tissue sampled, the lack of seedlot x tissue age interaction clearly illustrates that the relative seedlot values for all monoterpenes will be the same as long as oleoresin is collected from cortex of identical age. Thus, consistent sampling of either 1- or 2-year-old cortex should be used to determine cortical mono- terpene composition. As previously determined, sampling need be carried out only once during the dormant period for determination of the levels of the major monoterpenes. How- ever, the choice of age of tissue to sample and time of sam- 1ing must necessarily depend on the objective of the 35 Table 16.--Percent of variation in the terpene composition of geographic origins due to seedlot, age of tis- sue, seedlot x age of tissue interaction, and er- I‘OI‘. Percent of Variance Due to: Monoterpene Seedlot Age of Seedlot x Error Tissue Tissue Age W.K. Kellogg Forest Alpha-pinene 49** l O 50 Beta-pinene 28* 16** O 56 Camphene 18* 53** O 29 3-carene 61** O O 39 Myrcene 72** O O 28 Limonene 46** O O 54 Beta-phellandrene 31** 33** O 36 Gamma-terpinene 8 14* O 78 Terpinolene 39* 12* 3 46 Fred Russ Forest Alpha-pinene 56* O 6 38 Beta-pinene 42** 21** O 37 Camphene 17* 61** 1 21 3-carene 62** l O 37 Myrcene 64** 2 O 34 Limonene 47** 15** O 38 Beta-phellandrene ll 54** O 35 Gamma-terpinene 6 5 O 89 Terpinolene 49** 3* O 48 *, ** - Indicates significance at the 5 and 1 percent levels, respectively. 86 investigator. It is only logical that many studies of phys- iological significance of the monoterpenes should be carried out during the growing season on actively growing tissue. CHAPTER V GEOGRAPHIC VARIATION IN THE FOLIAR MINERAL COMPOSITION OF EASTERN WHITE PINE Provenance studies have established that eastern white pine (gIggg strobus) is genetically variable with regard to numerous morphological and physiological traits (Bourdeau, 1963; Fowler and Dwight, 1964; Funk, 1964; Genys, 1965; 1968; Mergen, 1963; Pauley g; g;., 1955; Santamour, 1960; Sluder, 1963; Wright g§_g;., 1963; and Wright, 1968). A detailed review of specific traits was given in Chapter 1. To further define the variation patterns within this species and to dis- cover why certain geographic sources exhibit superior growth rates, this study of the foliar mineral elements was per- formed on 15 seedlots of the 1955 U.S. Forest Service prov- enance test at two southern Michigan locations. Investigations of a similar nature have been carried out on several other tree species. Gerhold (1959) reported that six geographic origins of Scotch pine (gIngg sylvestris) sampled at 19 years of age differed significantly in the foliar contents of N, Ca, Mg and Fe. Steinbeck (1966) determined the foliar mineral element levels in 45 origins of 4-year-old Scotch pine growing at three locations in southern Michigan. All 12 elements measured varied significantly among the three test sites. 87 88 However, there were significant differences among seed sources only in the concentrations of N, P, Na, Mg and B. Of these elements nitrogen and magnesium were related to the current year's internode growth. In the case of ni- trogen a positive relationship was found at one site and a negative relationship at another. On the other hand, fast- growing origins contained high levels of magnesium at all sites. A cause and effect relationship was suggested. Corsican seedlots of Austrian pine (g;gg§.glggg) were found to differ considerably from other seedlots in several foliar mineral elements. However, there was no significant correlation between height growth and the levels of these elements (Lee, 1966). Wright (personal communication) found differences be- tween two varieties of ponderosa pine (2I22§_ponderosa) in the levels of N, K, P, Ca, B, Zn, Na and Mg. In comparison with Rocky Mountain seedlots, those from the Pacific Coast contained higher concentrations of all these elements. There were significant intra-varietal differences for N, x. P, Mg and B as well, especially with regard to the Pacific Coast variety. Wright also discovered that various geographic sources of Virginia pine (gIngs'virginiana) differed sig- nificantly in the levels of N, P, Mg, B, Zn, Al and Cu. Mergen and Worrall (1965) studied the foliar mineral element contents of jack pine (Pgngs banksIana) seedlots from the eastern, central and western portions of the species' range. Seedlings were grown under three photoperiods and 89 three combinations of day/night temperatures and harvested after 90 days. There were significant differences among seed sources in the N, P, K and ash contents. However, these genetic differences were both enhanced and obscured by a particular set of environmental conditions. memes During the last week of March 1966 foliage samples were collected from 14 geographic sources of the range-wide prov- enance study located at the Fred Russ Experimental Forest in Kalamazoo County, Michigan. The study consists of ten rep- lications of 4-tree plots. Needle fascicles were removed from an area extending two inches below the terminal bud of a branch located at the 3-year-old node. The foliage from replications 1-5 and 6-10 were bulked for seven origins. The foliage of the remaining seven origins was bulked over all ten replications. A minimum of 100 fascicles was obtain- ed for each bulk sample and each tree and 4-tree plot was equally represented. The foliage samples were stored at 40° F. for four days and then dried at 70° 0. for 48 hours. Later the samples were ground in an intermediate Wiley mill fitted with a 20- mesh sieve. Mineral element analysis was performed by Michi- gan State University's Plant Analysis Laboratory. Nitrogen concentrations were determined with a micro-Kjeldahl appa- ratus while the concentrations of 11 other elements were made using a direct-reading spectrograph. 90 Similar procedures were employed at the W.K. Kellogg Forest in Kalamazoo County, Michigan where 15 geographic origins were sampled on December 8, 1965. The season, position on the tree, and drying procedures specified above are in accordance with the recommendations set forth by White (1954) for foliar analyses of pine species. The locations of the Russ and Kellogg Forests, and the origins of the geographic sources sampled in this study are shown in Figures 1 and 2 (Chapter 1). Results and Discussion The 14 seedlots growing at both locations differed sig- nificantly only in the concentrations of N, Cu and Zn (Table 17). There are no consistent north-south or east-west trends in the levels of these elements. However, seedlots 6-PENN, and 19-MINN contained relatively low concentrations of all three elements. Any one or a combination of three factors could be responsible for the differences among seedlots. As pointed out by Steinbeck (1966), variation in foliar mineral element concentrations may reflect differences among trees in their ability to extract, translocate or metabolize nu- trients. . Although N, Cu and Zn varied among seedlots there are no clear-cut relationships between the concentrations of these elements and any growth or morphological traits. In contrast, Mitchell (1939) found strong positive relation- ships between the internal concentrations of N, P, K, and 91 Table l7.--Foliar nitrogen, copper, and zinc concentrations of 15 seedlots growing at two places in south- western Michigan. Element Seedlot Nitrogen Copper Zinc ----- peggeg; g; geaQ------ l GEO 91 123 103 3 TENN 91 150 105 6 PENN . 98 96 92 9 PENN 102 118 101 10 NY 102 107 101 12 NY 102 96 101 13 MASS 102 86 110 14 MAINE 105 54 79 19 MINN 98 86 94 20 NS 101 96 97 21 NB 102 80 88 24 ONT 102 93 101 25 ONT 103 96 108 28 MINN 102 94 110 29 MICH 99 123 110 Mean 1e87% 6.2ppm 46pm 92 Ca and the dry weight yield of 4-month-old eastern white pine seedlings grown in sand cultures. More than 200 seed- lings were analyzed for each element. Corrections were made for differences in food reserves of the seed. Dry weight yield reached a maximum but varied little over the ranges in concentration shown below. Element Range in percent of dry weight Nitrogen 2.50 to 3.26 Phosphorus .56 to .67 Potassium 1.50 to 1.72 Calcium .28 to .33 As pointed out by Mitchell, these values for i-year-old seedlings are probably too low for Optimum yields in older trees. Yet the average foliar levels of these four elements in 10-year-old trees at the Kellogg and Russ Forests were considerably below those given above as shown in the follow- ing tabulation: Plantation Element Kellogg Russ ---percent---- Nitrogen 1.90 1.85 Phosphorus .24 .18 Potassium .56 .47 Calcium .40 .47 93 Thus, one cannot explain a lack of correlation between nu- trient concentrations and growth in this study on the basis that nutrient levels were optimum. Apparently these rela- tionships became obscured after the first year and particu- larly after outplanting as competition for light, moisture and growing space became more acute. There is an inverse relationship between the concentrations of N and Cu (r = -.80; significant at the 1% level). However, this may be of no physiological importance. Plantation Qifferences The concentrations of all the mineral elements, except aluminum, differed significantly at the 1% level between the two plantations (Table 18). Geographic origins growing at the Fred Buss Forest contained lower concentrations of all the macro elements measured except calcium. 0n the other hand, with the exception of capper, the concentrations of the micro elements were higher at the Russ Forest. Aluminum, a non-essential element, was found in equal concentrations in both plantations. Growth at the Fred Buss plantation lags approximately two years behind that at the W.K. Kellogg plantation. One possible explanation lies in the higher concentrations of the macro elements at Kellogg. However, it is likely that both growth rate and uptake of the macro elements were en- hanced in the Kellogg plantation as a result of superior weed control. While chemicals were employed at the W.K. Kellogg Forest plantation to eliminate weeds completely, 94 Table 18.--Comparison of foliar mineral element concentra- tions at two plantations in southwestern Michi- gan. Foliar concentration at: 8183:3213) Unit W.Kéogzilogg Fgggeggss Nitrogen % 1.90 1.85 Potassium % .56 .46 Phosphorus % .24 .18 Magnesium % .16 .13 Calcium % .40 .47 Sodium ppm 339 518 Manganese ppm 301 405 Iron ppm 141 196 Copper ppm 7.0 5.7 Boron ppm 24 31 Zinc ppm 44 48 Aluminum ppm 252 252 (a) Between plantation differences were significant at the one percent level for all elements except aluminum which was nonsignificant. 95 only mowing was used to control competition at the Fred Russ plantation. Relative lmportance 9_f_ Geographic on in, Planting Location and Interaction g£_zgg§g Factors With the exceptions of N, Cu and Zn, which vary sig- nificantly among geographic origins, the variability in con- centrations of the mineral elements in the foliage of eastern white pine is largely due to plantation differences (Table 19). Seedlot x plantation interaction, although significant for 8 of the 12 elements analyzed, accounted for very little of the variability encountered with the exception of manganese and particularly aluminum. Thus, the geographic origins utilized in this study accumulated vastly different but relatively the same amounts of the majority of the mineral elements at the two locations. The coefficients of variation in Table 19 illustrate that reliable estimates of the foliar mineral element levels can be obtained using 40-tree samples. These values were computed by dividing the standard deviation of a 40-tree sample by the mean of the experiment. This result was ex- pressed as a percentage. With the exception of two elements these values ranged from one to ten percent. The coefficients of variation for copper and manganese were 16 and 18 per- cents, respectively. Hence, if these elements are of special interest, sampling of more than 40 trees is warranted. Coefficients of variation have been calculated for 12 96 Table l9.--Percent of variation in mineral element concen- tration due to seedlot, plantation, seedlot x plantation interaction, and error, and the reli- ability of analyzing foliage from 40 trees. Percent of Variance Due to: Coefficient Mineral Seedlot Plantation Seedlot x Error Varigiion(a) Element Plantation (%) Nitrogen 74** 16*“ 1 9 1.4 Potassium 3 90** 5* 2 3.5 Phosphorus 0 92** 7** 1 5.7 Magnesium 7 78** 8 7 7.2 Calcium 7 65*“ 22** 6 7.6 Sodium 3 85** 10** 2 11.1 Manganese 0 57** 37** 6 17.? Iron 9 76** 6 9 10.2 Copper 46** 23** 16 15 16.1 Boron 12 71** 13* 4 8.8 Zinc 37* 29** 23* 11 6.8 Aluminum 2 O 74* 24 8.7 (a) Coefficient of variation is the standard devia- tion of a 40-tree sample divided by the mean of the experi- ment and expressed as a percentage. *, ** - Indicates significance at the 5 and 1 per- cent levels, respectively. 97 Table 20.--Comparison of foliar mineral element concentra- tions in eastern white pine and in four other pine species (from Wright3 personal communica- tion . Foliar Concentration in: Mineral Eastern White Four Other Element Unit Pine gigs Species (a) Nitrogen % 1.87 1.38 to 1.88 Potassium f .51 .46 to .64 Phosphorus % .21 .17 to .22 Magnesium % .15 .08 to .14 Calcium % .43 .22 to .41 Sodium ppm 433 67 to 514 Manganese ppm 353 121 to 728 Iron ppm 170 53 to 176 Ccpper ppm 6.1 6.9 to 15.2 Boron ppm 28 18 to 32 Zinc ppm 46 29 to 63 Aluminum ppm 252 158 to 1164 (‘) ginus virginiana, g, onderosa, 2. szlvestris, .2- hi ‘9 and 29 2222222- 98 foliar elements analyzed in geographic origin studies of Virginia, ponderosa, Scotch, Austrian and eastern white pines growing at the W.K. Kellogg Forest (unpublished data at Mich. State Univ.). The collection and analytical methods were the same as described above. Although the coefficients of variation for a particular element varied among the five species, there were no significant differences. These results indicate that one can use an average experimental error for all species to conserve time and expense in future foliar analysis studies. The results obtained in this study generally indicate that eastern white pine is less variable genetically with regard to foliar mineral element levels than are Scotch, Eurcpean black, ponderosa, and Virginia pines. Compared to these other four species eastern white pine is relatively high in N, Ca, Mg and P and low in On. It ranks with all the others in the concentrations of seven additional elements studied (Table 20). If foliar concen- trations of the mineral elements reflect to a high degree the ability of each species to extract nutrients from the soil, one may conclude that eastern white pine is at least slight- ly more site demanding with regard to soil nutrients. As is the case with Scotch pine site has a pronounced effect on the foliar mineral element composition of eastern white pine. LITERATURE CITED LI TERATURE CI TED Bannister, M.H., H.V. Brewerton, I.R.C. McDonald. 1959. Ve- pour-phese chromatography in a study of hybridism in Pinus. Svensk Papp Tidn. 62: 567-573. , A.L. Williams, I.R.C. McDonald, and M.B. Forde. 1962. variation of turpentine composition in five population samples of Pinus radiate. New Zealend J. 501 e 5: 486-495e Blight, M.M. and I.R.C. McDonald. 1964. Sample reproducibil- ity in Pinus essential oil studies. New Zealend J. Sci. 7: 212—220. Bonner, J. and J. Verner. 1965. Plant biochemistry. Academic Press, N.Y. 1054 pp. Bourdeau, P.F. 1963. Photosynthesis and respiration of Pinus strobus L. seedlings in relation to provenance and treatment. Ecology 44: 710-716. Burbott, A.J. and W.D. Loomis. 1967. Effects of light and temperature on the monoterpenes of peppermint. Plant Physiol. 42: 20-28. Emboden, W.A., Jr. and H. Lewis. 1967. Terpenes as taxonomic characters in Salvia section Audibertia. Brittonia 19: 152-160. Forde, M.B. 1964. Inheritance of turpentine composition in Pinus attenuate x radiate hybrids. New Zealend J. Bate 2: 53-590 and M.M. Blight. 1964. Geographical variation in theAZErpentine of bishop pine. New Zealend J. Bot. 2: 52. Fowler, D.P. and T.W. Dwight. 1964. Provenance differences in the stratification requirements of white pine. canade Je Bate 42: 669-675. Funk, D.T. 1964. Southern Appalachian white pine off to a good start in the mid-west. Fourth Central States Forest Tree Improv. Conf. Proc.: 26-28. 99 100 Genys, J.B. 1965. Variation of cotyledon numbers in eastern white pine. Natural resources Instit., Univ. of Mary- land Ref. 65'19e 4 pp. . 1968. Geographic variation in eastern white pine: Two-year results of testing r e-wide collections in.Maryland. Silvae Genetics 17 1): 6-12. Gerhold, H.D. 1959. Seasonal variation of chloroplast pig- ments and nutrient elements in the needles of eo- grephic races of Scotch pine. Silvae Genetics 3 113'121e Hanover, J.W. 1966a. Genetics of terpenes 1. Gene control of monotergzne levels in Pinus monticola Dougl. Heredity 21: 73' e . 1966b. Environmental variation in the monoterpenes of Pinus monticola Dougl. Phytochem. 5: 713-717. . 1966c. Inheritance of 3-cerene concentration in Pinus monticola. Forest Sci. 12: 447-450. and M.M. Furniss. 1966. Monoterpene concentration in Douglas-fir in relation to geographic location and resistance to attack by the Douglas-fir beetle. Joint Proc. Second Genetics Workshop of S.A.F. and Seventh Lake States For. Tree Improv. Conf. U.S. Forest Service Res. Paper NC-6: 23-28. Iconomou, N., G. Valkanes and J. Buchi. 1964. Composition of gum turpentines of Pinus halepensis and Pinus brutia grown in Greece. J. Chromatog. 16: 29-33. Ikeda, 3.14.. w.L. Stanley, 3.11. Vannier, and L.A. Rolle. 1961. Deterioration of lemon oil. Formation of p- cgmene from gemma-terpinene. Food Tech. 15: 379- 3 O. Juvonen, S. 1966. Uber die die Terpenbiosynthese beeinflus- senden Faktoren in Pinus silvestris L. Acta Botanice Fennica 71. 92 pp. Klouwen, M.H. and R. ter Heide. 1962. Studies on terpenes. I. A systematic analysis of monoterpene hydrocarbons by gas-liquid chromatography. J. Chrometog. 7: 297-310. Lee, 0.3. 1966. Anatomical characters and chemical composi- tion of EurOpeen black pine needles as influenced by geographic origins and nitrogen fertilization. Ph.D. Thesis, Mich. State Univ., 158 pp. 101 Mergen, F. 1963. Ecotypic variation in Pinus strobus L. Ecology 44: 716-727. and J. Worrall. 1965. Effect of environment and seed source on mineral content of Jack pine seed- lings. Forest Sci. 11: 393-400. Mirov, N.T. 1948. The terpenes (in relation to the biology of genus Pinus). Ann. Rev. Biochem. 17: 521-540. . 1961. Composition of gum turpentines of pines. U.S. D.A. Tech. Bull. 1239. 158 pp. . 1963. Chemistry and plant taxonomy. Lloydia 26: 117-124e , E. Frank and E. Zavarin. 1965. Chemical composi- tion of P. elliottii var. elliottii turpentine and its possible reIation to taxonomy of several pine species. Phytochem. 4: 563-568. , E. Zavarin and K. Snejberk. 1966a. Chemical compo- sition of the turpentines of some eastern Mediter- ranean pines in relation to their classification. Phytochem. 5: 97-102. , , and K. Costello. 1966b. Further studies of turpentine composition of Pinus muricate in relation to its taxonomy. Phytochem. 5: 343-355. Mitchell, H.L. 1939. The growth and nutrition of white pine (Pinus strobus L.) seedlings in cultures with vary- ing nitrogen, phosphorus, potassium, and calcium. Black Rock Forest Bull. No. 9. 135 PP. Pauley, 8.8., S.H. Spurr and F.W. Whitmore. 1955. Seed source trials of eastern white pine. Forest Sci. 1: 244-256. Peloquin, R.L., Jr. 1964. Geographic variation of the mono- terpenes of Pinus ponderosa. M.A. Thesis, Stanford Univ. von Rudloff, E. 1967. Chemosystematic studies in the genus Picea (Pinaceae). II. The leaf oil of Picea glauca 8.113 _P_e marianae Can. Je Bette 45: 1703-171Ee and F.W. Hefendehl. 1966. Gas-liquid chromatography of terpenes. xv. The volatile oil of Mentha arvensis var. glebrete Hey. Can. J. Chem. 44: 2015-2022. 102 Santamour, F.S., Jr. 1960. Seasonal growth in white pine seedlings from different provenances. U.S. Forest Service. Northeast. Forest Expt. Ste. Res. Note 105. 4 pp- Sluder, E.R. 1963. A white pine provenance study in the southern Appalachians. U.S. Forest Service Res. Pe- per SE-2. 16 pp. Smith, R.H. 1964s. Perennial constancy of the monoterpene synthesis in the wood oleoresin of Pinus ponderosa. Nature 202: 107-108. . 1964b. Variations in the monoterpene composition of ponderosa pine wood oleoresin. U.S. Forest Service Res. Paper PSW-lS. 17 pp. . 1967a. Monoterpene composition of pine species and hybrids ... some preliminary findings. U.S. Forest Service Res. Note PSW—l35. 14 pp. . 1967b. Variations in the monoterpene composition of the wood resin of Jeffrey, Washoe, Coulter and lodgepole pines. Forest Sci. 13: 246-252. Squillace, A.E. and G.S. Fisher. 1966. Evidences of the in- heritance of turpentine composition in slash pine. Joint Proc. Second Genetics Workshop of S.A.F. and Seventh Lake States For. Tree Improv. Conf. U.S. Forest Service Res. Paper NC-6: 53-59. Steinbeck, K. 1966. Site, height, and mineral nutrient con- tent relations of Scotch pine provenance. Silvae Genetics 15: 42-50. Tobolski, J.J. 1968. Variations in monoterpenes in Scotch pine (Pinus s lvestris L.). Ph.D. Thesis, Mich. State Univ. 125 pp. White, D.P. 1954. Variation in the nitrogen, phosphorus, and potassium contents of pine needles with season, crown portion, and sample treatment. Soil Sci. Amer. Proc. 18: 326-330. Williams, A.L. and M.H. Bannister. 1962. Composition of gum turpentines from twenty-two species of pines grown in New Zealend. J. Pharmaceutical Sci. 51: 970-975. wright, J.W. 1968. Genetics of eastern white pine (Pinus strobus L.). In press. 103 Wright, J.W., W.L. Lemmien, and J. Bright. 1963. Geographic variation in eastern white pine - 6-yesr results. Mich. Agr. Expt. Sta. Quart. Bull. 45: 691-697. Zavarin, E.. N.T. Mirov and K. Snanerk. 1966. Turpentine chemistry and taxonomy of three pines of southeast- ern Asis. Phytochem. 5: 91-96. VITA ROBERT LOUIS HILTON Candidate for the degree of Doctor of PhilosOphy Guidance Committee: AeA. DeHertogh, J.B. Grafius, J.W. Hanover, C.J. Pollard, J.W. Wright (Major Professor). Dissertation: Genetic variation and interrelationships of the cortical monoterpenes, foliar mineral elements, and growth characteristics of eastern white pine. Outline of studies: Major subject: Forestry, Minor subjects: Forest genetics, Plant physiology. University of Massachusetts, B.S., 1963 University of Massachusetts, M.S., 1965 Michigan State University, Ph.D., 1968 Biographical items: Born.May 22, 1941, in Boston, Massachusetts. Married Carol A. Keirstesd on August 25, 1962. Two children: Heather Joan, born Nov. 16, 1963 and Scott Andrew, born July 14, 1966. Experience: Research assistant at the University of Massachu- setts from September 1963 to June 1965, and 104 105 research associate at Michigan State University from June 1966 to September 1967. Timber stand improvement work for the U.S. Forest Service on the Stanislaus National Forest, California from June to August 1961. Cortical oleoresin analyses at Michigan State University from June to August 1966. Assistent Professor of Biology, State Col- lege at Boston, September 1967 to present. Memberships: Alpha Zeta Xi Sigma Pi Phi Kappa Phi Sigma Xi Society of American Foresters APPENDIX Table 21.-Monoterpene composition of 47 trees from five half-sib families at Allegan State Recreation Area. Monoterpenes Terpin- olene terpinene Beta-phel- landrene Limo- nene Myr- cene rene Cam- phene Beta- pinene Alpha- pinene Tree No. Grand Traverse County MSFG 5038 106 conooaone-iinout. HNHH N (DONMOONNON eeeeeeeeee H NQQO‘HCDWNWQ H HHHd itcooacaaotnbao N JHHJNHF‘F‘ 0303:}.QN03H MNNNJHNde-‘e: NN N NN “Hummoahmm H O\O Osw NH (““5 O I O O O O O O O O fiMMMQO-Q “B0 03“ (\ino HHH O V" H N V‘ N 10.0 17.6 10.0 11.6 14.8 \ONHBOQOMQ'MO‘ 000000.000 NNo odwmmd NN“ mnmmum HNMiVVOBQQS Grand Traverse County MSFG 5042 4700000 OOOOMO SBOIN-NB ee ee ee Pie-O 1'4 ON O‘BMR O “Nam: \O‘OOKOINO O O O O 0 30'4de :1 NM concomsoo (n H NMNCOMB e e e e e e MCI-Ink.“ Nm 00303 O O O #mNNnH "Ne-MOM \0 MO MN: m m NON-3!": 12 {2 15 16 107 m. H. a.” ~.~ m.“ m. e.n m.s~ ~.oo an m. o. «.3 0.“ N.u m. w.N N.n: m.:: on N. o. m.N w. o.« a. H.N «.m: m.:: 0N season countess cacao meow 0mm: o. o. a.~ H.~ a.” a. «.e m.on «.mn mm o. o. o. n.« n.« m. w.: m.«m o.o: NN N. on m. N.« a." m. N." N.oa n.3n 0N N. o. N.N m.” w.a m. N.“ «.mN n.Nw mN o. o. N. m. o.« a.” «.N m.on ¢.nm N N. o. a. «.a o.« N. 5.: m.nm a.“ N N. o. N.m o.« m.« w. w.m N.mm m.on NN N. o. n.“ n.” n.a o.« o.n «.nn .om «m o. o. m. n. e. N. m.~ «.mn a.an on o. o. n. m. a. N. a.« a.om e.me as sassoo onsetsaa cease neon can: o. m. e.n n.~ o.~ m. m.~ n.oo o.m~ on N. o. m. w.N v.” N. :.w m.mu N.mw Nu haasoo emaebeaa undue Neon 0mm: 3-..-........---..-----------------§ mm g---:::--3-------u...i.....- cacao eaoaaaaea oooaoaea one: once each enema enenaa encode .oz nodanea nausea uHenAIepem ioaaq nah: twain also venom nosed< eeha neneaaeposcz A.e.esco. am canes Table 21 (ccnt'd.) Monoterpenes Terpin- olene terpinene Beta-phel- landrene Limo- nene Myr- cene Beta- pinene Alpha- pinene Tree No. Grand Traverse County' MSFG 5048 108 (“N MN MN 0 0 000000 MMNNN: eeeeee #e-IOt I" :mowon eeeeee v-lv-Ifiv-OH (““50 MW He'le-‘le-ie-IH cove O\\O\O F: \03 e-IV‘O‘Q 330-3 “‘1‘ Grand Traverse County MSFG 5056 OONNMOOMON OOOOOOONOO O\O\O WNB‘O N O 05 FINN file-I O‘NOON‘OMOF‘Q NNNNNNHHH Us: CNN (“v-HO: M e-INe-ONHHQ N “m (fie-l: [\MO (\‘0 Hal-OH H H ounvaouecaeasoavx 0.0.0.0... mnemeNa: OHAC‘NQ-fl’ “0‘0““ 0 O O O 0 O O O C \OOV‘WO“ cc’ou-nn “33-3 mmnm QNNHO~T “NCO com NON #01 “do: man an mace-ON“: 100$- comes: 3.3:! 109 I I £0 I «at: I nascooeaaxodommonmoaNr-c A. I eeeeeeeeeeeeeeeeee ° HF'I I PIéPIdPIPIv-IWMN Nv‘NPIPIPIN M 3° : 3 I o e I E I NPINNPIv-In c-n-c v-u-o HO] .9 as. u A C O I D I h 0 I I 43 PI H0 I 5 PI On I v4 :0 I O QMHMMNaNQV-IQQNMWN‘O a as IO eeeeeeeeeeeeeeeeee 5. & : 0 annmmmmmnNMv-INMMNMQ p .3 . .. ... :3 I .3 Q I I I H I U a: o nNnV‘NNNNNNNflHMNN3H e i A P0 0 «up a 4 a H" O a: o OQOOOOOOOOOOOOOOOO 0 fl 0 g eeeeeeeeeeeeeeeeee 8‘0 0 a 0 O Q 00 8 HE R C u o d H m a 2 0 UN 0 H O‘DQWN0500300HMCDQI‘N 0° :1 .9: 1.3 eeeeeeeeeeeeeeeeee 0H o E-4 s: PIPIe-IPIPI .4 RH 2 N 0 "a3 3 g I: Q‘OQ:QNN‘OMO°Q:QQNM eeeeeeeeeeeeeeeeee ° : Q I I 0 O “a I M NQQMNNHHON‘ONNNOQN‘O PI 0 Io eeeeeeeeeeeeeeeeee ‘3 I. I “ PImPIPIe-IPIPIPIPI H M PI PI 6 n“ I N m U I 0 8. e S "‘ 8 In I g “domocfidmoééaNGmC‘m o I eeeeeeeeeeeeeeeeee 3 3 0 d I “\OCNNBQPIONQQW‘V‘V‘mNQ a n : sas"ss's's assess 3 g I 3H3§§303333 H #MNN 3 8 I g a : mmmoo¢mmoonwddéamm I Nv-I'ABOsNNfiv-Imv-INO‘: N N C H O- 9 ea HNMdmwNQQONNmam M0 Sm Table 22 (oont'd.) Monoterpenes Plra- Terpin- oymene olene Beta-phal- landrene Limo- Gamme nene Myr- Ter nenes oene. A p 3-03- Can- phn Be a phene rene Tree No. Lake County MSFG 5036 8.1 13 2.8 12 Grand Traverse County m1 2.0 46.2 28.1 19 110 MSFG 5051 OQQQnfiQéNOQWQONN O O O O O O O O O . O O O O O 0 PIN NMHMNNPIPIMM OQMNQNNO‘NNO-flNQV‘O PI PI PIPIPI PI ONHV‘QOQWQNONMV‘QN O I Gammmmnmmmsdkagd QMQMNNHNQNQVEMQOB PIMPIMMNé‘Oan-INNMNPI OOOOQOOOOOOOONOO O O O C I O O O O O O O O O 0 PI OOV‘QNQOQOQ'NNNNOM PIPIPI PIPI NM‘OMOHFQOQQnoauk eeeeeeeeooeeeeee mmmnmnmgammmmmmm “GEQMQOMS‘NOWPINNOON PI Hfigflfif‘ PI PIPI \OBMQONWQQQNHQNOB O O O O O O C O O O O O O O O O ‘n HWNN “NQ MN N mans nmmommmdmdumummn wdfifli駧ddé£ééd° :nwmmnmn3303333 “NW-fiflQOOmQ “314-33": 0 O O . «mmaoaoxfiaoéaévvozhom (“NH-##PIQHMQNQMNNM OPINNV‘OBQQOPINni‘n NNNNNNNNNNMMMM 0" Table 22 (eont'd.) Monoterpenes Para- Terpin- oymene olene Lino- Beta-phel- landrene Ter 1nenee oene Alpha Gamma nene Myr- Can- 3-ca- P1nenea Alpha Beta phene rene Tree Nm Grand Traverse County MSFG 5059 111 HMQHMQ’QHQWQ: (\“OQPMO no 0 O O O . NMNNMMHNNmB-MNNNHHv-cfim WNWNHQOBOGéW‘ONO‘QNOOB PIPIPIPINNPI NPIPIPI PIPI P'I amommonvx30N3Hooo~ouN 0.000.000.0000000000 NNMMthNQNééM-fl'mwdngm Fun:rvueoL3eabuovdawovabmbou“vmo 000000000000 Oeoeeee maaéwMMMNNnafiMMNNmH: OOWQOOOOOOOOOOOOMO‘OO O O O C QONQBOON-‘IOOVNS'O‘OSBWOO O 0 PI PI PIPIPI V‘NOWQ03QBWQNQHQNO‘HNO MgNMN-QMMNMNMNMNMMM NOQOQQQO‘OQN‘A-fl'd‘ONQQv-CN H u-n-I H (“\OPIPIPI H «H n: omNn:MNmnmannomommom OeeOee.eeOeee eeeeee d'MHQHV‘NNNv-IHNQMHMOO N-Q‘QW “\O \nounrnmxoaxooonN Os“ md°éo6omNam NgNénmdeo -$¢Wfl4“"\33 #V\#Cfi CVJWWVX3:IVW“ mnwaHmooommwmanmon O O O O O O O 0 C O O O O mmmmNNNm:QNMH:m¢mNaw nNmaamaNdua:nm¢Nmmma Table 22 (oont'd.) Monoterpenes Terpin- olene Para- oymene Beta-phal- landrene Limo- Ter 1nenes oene Alpha Gamma nene 3-ca- Myr- rene Cam- phene Pinenea A pha Beta I Tree No. Grand Traverse County MSFG 5063 112 \ON\O\O N‘OQPI (\‘O 0P0: MN: e e e P'IMNI'I PINNN mNNHv-I 3‘0““0 5500-30 PIQO Ofio PIPIPIPI PI PIv'IN PI “NOOHWOQQBMNOHV‘ eeeeeeeeeeeeeeee 300MGQOMHQ350©PIN C O eeeeeeeeeeeeee OOOOOOOOWOOOOQOO O 0 PI PII‘NOOB‘AQWNPIQ'OOQQN O PIPI PIPIPIPIPI “fidéNQMO (\3 (\m‘APIN e e e e e e e e e e e e e e e 0 mm NPINM‘ON N v-I HWBOOBQONQOONQQWQ O O I PIPI PI: PI N N-a'OPIONv-Inmmnmoaafi eeeeeeeeeeeeeeee nmnNfiéaaNnm-éNBnW bthhQOflfi‘fiOéQOOQN eeeeeeeeeeeeeeee momw‘nmomv‘HmNmmmm Table 22 (oont'd.) Monoterpenes Para- Terpin- oymene olene Beta-phal- landrene Limo- Gamma nene Myr- Ter 1nenee oene A p 3-oa- Cam- Be a phene rene Tree No. Schoolcraft County MSFG 5100 113 “3.33““QOFMOO:V\N '45wa 0" PIMMQ O\NO“\F\Q3QQ30\OQ e e N PIPIN N PIPI‘O PIQNNONPI3\ONN\O3 O O 0 O O O O O O O O O O NMN MnngNBv-INBM eeeeeeeeeeeee 8MNNNMPI‘nPImmPIm oooooqooooooo O‘DOHNQ‘OO‘ONNHB H HHH HH zbabanw03Nmam C O O O O O O O O O O O O MNMHNWMQMNHNN H N H MNOOS3NNOQ3hb-O e e e e e e e e e e e e e PINPI\OO\OPIN PINQ 0" NMM “MN NOfiO-fl‘ml‘NV‘NNNON eeeeeeeeeeeee QONQONN‘O30‘Q “\O O O O O O O O O O O 0 O O nnmmwonoomanoH 3d3Mv-IPIWN3PIPIPIN ".7.":- Iii 1.": . "IlE’EflflflMl’fllW/WfififiITS