VARIATIONS iN MONOTERFENES EN SCOTCH PINE _ (PINUS SYLVESTRES L) Thesis for the Degree of Ph, D. MICHIGAN STATE UNIVERSITY JAMES J.. TOBOLSKI 1968 LIBRARY meets Michigan S ta ta University This is to certify that the thesis entitled VARIATIONS IN MONOTERPENES IN SCOTCH PINE (PINUS SYLVESTRIS L.) presented by James J. Tobolski has been accepted towards fulfillment of the requirements for Ph.D. Forestry degree in \ gin/who KO “5in / / Major professor 0-169 ABSTRACT VARIATIONS IN MONOTERPENES IN SCOTCH PINE (PINUS SYLVESTRIS L.) by James J. Tobolski The objectives of this study were: (1) to determine the qualitative and quantitative variations in monoterpenes of Scotch pine, (2) to determine the following physiologi- cal and environmental sources of variation in monoterpenes: (a) the variation in monoterpene composition among needle, xylem and cortex tissue; (b) the effects of cortex age; (c) effects due to site; (d) the changes in monoterpene compo- sition due to defoliation by the EurOpean pine sawfly, and (e) the effects of season on monoterpene composition, (3) to determine the variation between and within half-sib families and seed lots, and (4) to determine the degree of correlation between monoterpenes, growth rate and existent varietal classes. Variation in Scotch pine monoterpenes was investi- gated in 7 to 9 year-old trees growing in replicated pro- venance tests in southern Michigan. Quantitative and qualitative analyses were performed using gas-liquid chromotography. Scotch pine normally contained the James J. Tobolski following 11 monoterpenes: a-pinene, camphene, B—pinene, myrcene, 3-carene, a-terpinene, limonene, 8-phellandrene, y-terpinene, cymene and terpinolene. Cortical oleoresin was found to contain larger concentrations of limonene, B-phellandrene, B-pinene, 3— carene, and terpinolene than xylem or needle oleoresin. Needle tissue was high in a-pinene (averaging 61 percent) and was characterized by having the highest concentrations of cymene and camphene. Xylem oleoresin contained high concentrations of a-pinene with some trees having a con- centration of 94 percent. Differences between tissues were statistically significant (at the 1 percent level) for all monoterpenes except y-terpinene. Cortical monoterpenes were found to vary with age of the cortex tissue. Proceeding from current-year tis- sues formed in 1967 to tissue formed in 1965 the concentra- tion of a-pinene and B-pinene increased while myrcene, 3-carene and limonene decreased. The effects of site and sawfly defoliation on cor- tical terpenes were small. Only for B-phellandrene was there a significant interaction between seed source and site. Defoliation by the European pine sawfly significantly increased the a-pinene concentration and the total terpene concentration in defoliated branches as compared to foliated branches within a tree. The effect was most pronounced on moderately defoliated (35-65 percent) trees. James J. Tobolski Seven terpenes were found to vary significantly from season to season as determined from eight trees sampled at nine intervals during a thirteen-month period. This variation, however, was small and never exceeded 5 percent for any terpene. The between—tree variation was considerably larger than the between-season variation for the principal monoterpenes. From studies of individual trees and half-sib- families the monoterpenes 3-carene, myrcene, limonene, 8-phellandrene and terpinolene appeared to be under simple genetic control. Multiple inheritance patterns were indi- cated for the remaining terpenes. The variation among half-sib families was significant for the principal terpenes. Tree-to-tree variability within seedlots was large for simply inherited monoterpenes having low gene frequen— cies. Thus, large sample sizes (100-200 trees) may be necessary to detect small differences between pOpulations. Simple correlations between monoterpenes support the hypothesis that all terpenes are derived from the stable precursor geranylperphosphate. A consistent, positive cor- relation was found only between 3-carene and terpinolene. The cause of this association is unknown. Geographical variation of cortex monoterpenes was determined from 108 Scotch pine provenances from Europe and Asia. Monoterpene composition was found to parallel James J. Tobolski geographic variation. Thus, terpenes are a valuable chemo- taxonomic aid in Scotch pine. Of the 11 terpenes the most variable were a-pinene and 3-carene which were inversely correlated in a general north-south direction. For example, 3-carene varied from 0-60 percent south to north, respec- tively. The absence of 3-carene in most isolated southern populations indicates that they are probably Tertiary relics. Apparently little gene exchange has taken place between them and the more continuous northern pOpulations. Similarity in terpene composition between Middle Europe, southern Scandinavia and Scotland, lends credence to the hypothesis that Scotch pine migrated from middle EurOpe across land bridges which had once connected these land masses . VARIATIONS IN MONOTERPENES IN SCOTCH PINE (PINUS SYLVESTRIS L.) BY tv 669 James JEPTobolski A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Forestry 1968 ACKNOWLEDGMENTS The author is indebted to the members of the Guidance Committee--Drs. M. W. Adams, A. DeHertogh, C. J. Pollard, J. W. Wright and J. W. Hanover (Chairman)-- for their encouragement and assistance during the course of this study. Thanks are also due to W. Lemmien and R. M. Hilton for their assistance in collecting oleoresin samples at the W. K. Kellogg Forest. Further, the author extends his appreciation to the Glidden Company, Jacksonville, Florida and the Hercules Powder Company, Wilmington, Delaware for supplying pure samples of monoterpenes used as standards in this study. Finally I wish to thank my wife, Marilyn, for her sacrifices and typing assistance. The financial support for this study was provided through project number 936 of the McIntire-Stennis CooPera- tive Forestry Research Program. ii TABLE OF CONTENTS AC KNOWLE DGMEN T S O O O O O O O O O O O O O O I 0 LIST OF LIST OF CHAPTER I. II. TABLES O O O O O O O O O O I O O C I O FIGURES O O O O O O O C O O O O O O O 0 INTRODUCTION 0 O O O O O C O C O O O . Provenance Testing in Scotch Pine Monoterpenes in Pines . . . . . . Objectives of Study . . ._. . . . NON-GENETIC VARIATION o o o o o o o o 0 Materials and Methods . . . . . . VARIATION IN NEEDLE, CORTEX AND XYLEM TISSUES O O O O O O O O O O C O 0 Results and Discussion . . . . . VARIATION ASSOCIATED WITH TISSUES OF DIFFERENT AGE 0 I O O O O O O O 0 EFFECTS OF DEFOLIATION ON CORTICAL MONOTERPENES . . . . . . . . . . Results and Discussion . . . . . EFFECTS OF SITE ON CORTICAL MONOTERPENES O O O O O O O O O 0 Results and Discussion . . SEASONAL VARIATION OF CORTICAL MONOTERPENES . . . . . . . . . . Results and Discussion . . . . . Sampling Errors Methods of Expressing Data Defoliation Effects of Tissue Age iii Page ii vii \DmH l—' 11 11 13 14 17 19 20 23 24 27 28 CHAPTER Page III. VARIATION WITHIN SEEDLOTS . . . . . . . . . 36 Materials and Methods . . . . . . . . 36 Results and Discussion . . . . . . . 38 Inheritance of Monoterpenes Minimum Sample Size IV. VARIATION IN HALF-SIB FAMILIES . . . . . . 47 Materials and Methods . . . . . . . . 47 Results and Discussion . . . . . . . 48 V. INTERRELATIONSHIPS AMONG MONOTERPENES . . . 53 Results and Discussion . . . . . . . 55 VI. GEOGRAPHIC VARIATION IN SCOTCH PINE MONOTERPENES . . . . . . . . . . . . . . 58 Introduction . . . . . . . . . . . . 58 Monoterpene Variation Within Species Taxonomy of Scotch Pine Sampling Considerations Materials and Methods . . . . . . . . 62 Results and Discussion . . . . . . . 63 Geographic Variation Evolution of Scotch Pine VII. CONCLUSIONS AND RECOMMENDATIONS . . . . . . 76 LITERATURE CITED 0 O O O O I O O O O O O O O O O O 81 VITA O O O O O O O O O O O O O O O O O O O O O O O 8 4 APPENDIX 0 O O O O O O O O O O O p O O O O O O O O C 85 iv LIST OF TABLES TABLE Page 1. Monoterpene composition in different tissues in 10 trees from 9 seed sources . . . . . . . . . . ._. . . . . . 15 2. The effect of individual trees and de- foliation by the European pine sawfly on the monoterpene composition of Scotch pine . . . . . . . . . . . . . . . 21 3. Changes in monoterpene concentration associated with different degrees Of defOlj-ation O O O '0 O O C O O O O O O 22 4. Monoterpene composition of 10 seed sources growing at two locations . . . . . . . . 25 5. Analysis of seasonal variation of monoter- penes from eight Scotch pine trees sampled on nine dates . . . . . . . . . . 29 6. Concentration of five cortical monoter- penes of western white pine sampled at different seasons . . . . . . . . . . 34 7. Variation in cortex monoterpenes of individual trees from three seedlots grown at the Rose Lake Wildlife Ex- periment Station, Shiawassee County, Michigan . . . . . . . . . . . . . . . . 39 8. Size sample needed to detect significance (5 percent level) of a given differ- ence in monoterpene c0ncentration between seedlots . . . . . . . . . . . . 45 9. Concentrations of the principal monoter- penes in half-sib families growing at the Fred Russ Forest . . . . . . . . . 49 TABLE 10. 11. 12. 13. 14. Page Analysis of monoterpene variation in half-sib families growing at the Fred Russ Forest . . . . . . . . . . . . 51 Correlations between monoterpenes for 54 trees from southern Sweden, western Germany and Yugoslavia . . . . . 55 Classification of Scotch pine into varieties according to Wright, et. a1. (1966) O I O O O O O O O O O O O O 70 Variation in monoterpene concentrations in Scotch pine varieties grown at the W. K. Kellogg ForeSt O O O O O O O O O O 71 Percentage of variation in monoterpene concentration accounted for by dif- ferences between and within varieties . . . . . . . . . . . . . . . . 72 vi FIGURE 1. LIST OF FIGURES Natural distribution of Scotch pine in EurOpe (shaded) and provenances in- cluded in this study (numbered dots) Natural distributions of Scotch pine in Asia (shaded) and provenances in- cluded in this study (numbered dots) The mean seasonal variation in the principal monoterpenes in 8 Scotch pines . . . . . . . . . . . . . . . Frequency distributions of the major cortical monoterpenes. Basis, 54 trees 0 O O ‘ O I O O O O O I O O O 0 Geographic variation in the principal monoterpenes in Scotch pine from Europe 0 O O O O O O O O I O O O O 0 Geographic variation in the principal monoterpenes in Scotch pine from ASia O O O O I O I O O O O O O O O 0 vii Page 31 42 64 66 CHAPTER I INTRODUCTION Scotch pine (Pinus sylvestris L.) is one of the most variable tree species in the world, and during the past 154 years numerous varieties have been described. Ruby (1964) recognized 21 geographic variables as valid. The native range of Scotch pine includes most of Europe and northern and west-central Asia (Figures 1 and 2). In the north, it is a valuable timber and pulp species where its range is continuous over large areas of low and medium elevation. It is less important in southern EurOpe where it is confined to scattered mountainous areas. Scotch pine has been widely planted outside its natural range both as an ornamental and forest tree. It is an important exotic in the United States, especially in the northeastern and northcentral states, where millions of seedlings have been planted. Recently, it has become one of America's most important Christmas trees. Provenance Testing of Scotch Pine A provenance in forestry, as I use the term, refers to a population of trees growing at a specific place of origin. Provenance research provides information about the 1 Figure l.——Natural distribution of Scotch pine in Europe (shaded) and provenances included in this study (numbered dots). '0’ IO‘ 15' 10’ 28' 0. 1:... . t .1 1 u ._. .c w.......em,w 7%."... z? .. . 2... . .. 5. '1‘... /,,H a .w/ . :2 z , . r/mZ/z/ ”7/2 a ,7/ .40 ’« ion Figure 2.--Natural distribution of Scotch pine in Asia (shaded) and provenances included in this study (numbered dots). W\s\ \ I..."- 3“ \ \ at fa§\\/ \ g . \\\\\\\‘ -. \‘\\\ \\\\\\" ‘3: \\\\\ wt}: \\§:K‘ \\\‘ § \\\ \\\\ g “\M \\ §\‘ \\ \\ \\~“ \\‘\\\ .. \ fl. '0' environmental and genetic components of variation associated with geographic source and thus can be useful in directing breeding programs. Furthermore, these tests are an adjunct in defining the evolutionary patterns of species or higher taxon. Some of the earliest provenance testing of forest trees was done on Scotch pine by the French seedsman, DeVilmorin between 1820 and 1850. He demonstrated that large growth differences existed between seedlings from different regions. Since that time, many other tests have been conducted, but most were not replicated and either have limited genetic value or are of historical interest only. I In the 1930's Langlet (cited from Wright and Bull, 1963) conducted a study of dry matter content in Scotch pine needles of 2-year old seedlings derived from 582 stands in Sweden. In less extensive studies, he also mea- sured height growth, needle length, and needle color of Scotch pine provenances of Swedish origins. He showed that trees from northern Sweden grew very slowly, had short needles with a high dry matter content and yellow winter coloration. Widespread replicated tests were initiated by the International Union of Forest Research Organizations (IUFRO) in 1907 and 1938. The first of these tests included 13 origins and 16 outplantings in Sweden, Germany, Belgium, Hungary, and the Netherlands. Wiedemann in 1930 (cited from Wright and Bull, 1963) summarized the separate pub- lished reports for the entire experiment. The 1938 IUFRO test included 55 provenances from Scotland to Latvia and from Germany to northern Scandinavia: Each c00perator es- tablished one or more plots of as many origins as he chose to include. All 55 origins were planted in a replicated test at Hillsboro, New Hampshire and some origins were also planted in New York and Michigan. Results of various as- pects of these test plantations are summarized in a series of reports by Baldwin (1956), Wright and Baldwin (1957), Echols (1958), Gerhold (1959). The IUFRO investigations showed that the Belgian origins consistently grew faster but were sometimes inferior in stem form. Latvian origins had good stem form but grew more slowly. Differences were also observed in carotenoid and mineral content of foliage. Slow growing Scandinavian trees produced wood of high density, but cellulose produc- tion was much greater for faster growing origins from Bel- gium and Germany. The potential of Scotch pine on many infertile sites of the north-central region of the United States as well as inquiries by Christmas tree growers prompted the NC-Sl Regional Tree Improvement Committee to initiate ex- tensive provenance tests of the species. Three of these test plantations were used in this study. They were estab— lished from seed collected from 108 native Scotch pine stands throughout Europe and Asia. Monoterpenes in Pines The monoterpenes and their oxygenated derivatives have been found in over 350 plant species. Over 150 mono— terpenes are known to exist. They are among the oldest studied natural products because of their fragrance and use in medicinal preparations. In Pinus, Picea, Larix and Pseudotsuga the monoter- penes comprise a fraction of the oleoresin which is synthe— sized in the thin-walled epithelial cells surrounding the resin canals. These ducts are found in the needles, cortex of the bark, and in the heartwood and sapwood. The oleo- resin within the canals is normally under pressure and when they are severed dr0plets of oleoresin are exuded. Oleoresin can be divided into two components: turpentine and rosin. Turpentine is a mixture of monoter- penes (C10H16); rosin is a mixture of resin acids which are diterpene derivatives (C20H3002), and are classified into two types, abietic and pimaric. The role of monoterpenes in survival and growth of pines has been a topic of lively conjecture for many years. Some workers feel that the monoterpenes are merely metabolic wastes while others believe they may be important in disease resistance, insect resistance or as useful metabolic constitutents in the growth processes. Since it is postu- lated that the pines originated in the Mesozoic era some 150,000,000 years ago, the resin duct system may be nothing more than a "human appendix," once important, but now simply a relic of the past. Objectives of Study This study is part of a long range research program directed towards defining the patterns of genetic variation and inheritance of physiological traits of forest trees. It focuses on the monoterpene compounds in Scotch pine and seeks to provide genetic and physiological information about these compounds in the species. HOpefully, this information will contribute to our general knowledge about genetic dif- ferentiation in trees and eventually to the improvement of Scotch pine planted in the United States. The specific objectives of this study were: 1. To determine the qualitative and quantitative variations in monoterpenes of Scotch pine. 2. To determine the following physiological and environ- mental sources of variation in monoterpenes: a) the effects of season on monoterpene composition; b) the changes in monoterpene composition due to de- foliation by the European pine sawfly; c) the variation in monoterpene composition among needle, xylem and cortex tissue; 10 d) the effects of tissue age on cortical monoterpenes, and e) the effects of site on cortical monoterpenes. To determine the variation between and within half-sib families and seedlots. To determine the degree of correlation between monoter- penes, growth rate and existent varietal classes. CHAPTER II NON-GENETIC VARIATION Investigations in pine monoterpenes have been di- verse and unrelated. Some studies have implicated the monoterpenes as having a role in disease and insect resis- tance. Others were directed at terpene physiology, inher- itance mechanisms or chemosystematics. It is essential therefore, that factors affecting monoeterpene levels be clarified in order to plan and interpret experiments meaningfully. The environmental sources of variation which I studied included tissue dif- ferences (needles, cortex and xylem), effects associated with the age of cortex tissue, time of sampling, and ef- fects due to site and defoliation. These factors are considered in more detail under the respective investigations. Materials and Methods Oleoresin samples were obtained from a provenance- test plantation established at the Rose Lake Wildlife Experiment Station, located 10 miles northeast of East Lansing, Michigan. This plantation was established in 1961 with 2-0 stock and was 7-9 years old at the time of samp- ling. The planatation contains 75 origins of trees planted 11 12 in an 8-by-8 foot spacing in four-tree plots and eight repli- cations (Wright and Bull, 1963). The 75 origins represent 75 stand collections from throughout the natural range of the species. A single row of trees composed of Austrian and Swedish sources borders the planting. A similar plantation at the Fred Russ Memorial Forest was also sampled. The sam- ples were placed in small vials or centrifuge tubes stored at 35°C and analyzed by gas-liquid chromatography within 10 days. Details of oleoresin collection varied and are des— cribed separately under each study. At the time of analysis each sample was diluted with acetone or pentane and a three microliter aliquot was immediately injected into an F and M model 700 gas chroma- tograph. It was equipped with a thermal conductivity de- tector, a Hewlett Packard automatic attenuator model 50B, and a Honeywell disc—chart integrator Model 227. The chromatograph column was stainless steel, 1/4-inch in diam- eter and 8 feet long, and packed with 10% polypropylene glycol on 60/80 mesh chromasorb G-AW. Column temperatures were 95°C-105°C, injection port and detector temperatures were 190°C-200°C, and the helium flow rate was 100-110 ml/ min. The monoterpenes were tentatively identified by comparing relative retention times of the unknowns with those of known compounds. To check identification, several 13 samples were rerun at 60°C on a column containing the polar substrate 8, B'-oxydipr0pionitri1e. When the data is expressed as a percent of the total monoterpenes, concentration was determined by integration and summation of areas under the peaks. In one study (Ef— fect of Defoliation) monoterpene concentrations are expressed as a percentage of the oleoresin. Here, concentrations were derived from area integrator values on a column in which standard curves were prepared from a series of known mono- terpene concentrations. The same conditions were used when analyzing the unknown oleoresin samples. VARIATION IN NEEDLE, CORTEX AND XYLEM TISSUES Several investigations have shown that monoterpene composition varies between tissues of the same tree (Squil- lace and Fisher, 1966; Juvonen, 1966). To date, however, differences among all three major tissues (needles, cortex and xylem) of individual trees in unknown. To obtain such information the following investigation was undertaken. Ten trees, representing nine diverse seed sources were sampled at the Rose Lake plantation in June and August 1967. From each tree, 20 ul. of cortex oleoresin was ob— tained from incisions on year-old branches of the 1965 or 1966 whorl. The same branches were cut into 6 or 8 cm. sections and from 3 to 12 pl. of xylem oleoresin was obtained from the cut surfaces. Finally, the foliage was removed from 14 these sections, placed in polyethylene bags and extracted the same day in the laboratory. The monoterpenes were extracted from 10 grams of needles by homogenizing them in a Waring blendor for two minutes in a sufficient volume of pentane to cover the foliage. The extract was filtered into a beaker and the remaining homogenate was washed three times with 15 m1. of pentane, filtered and added to the initial extract. To re- move water, 4 to 5 grams of anhydrous sodium sulfate were stirred into the extract which was then evaporated with an air stream down to 15 or 20 ml. This remaining solution was filtered into a test tube and further evaporated to a final volume of .5 ml. Results and Discussion Differences between the June and August collections were less than 2 percent for all monoterpenes with the ex- ception of a-pinene and 3—carene in the xylem. In this tissue a-pinene was 8.4 percent lower and 3-carene was 6.6 percent higher in the June collection. Since differences in sampling time were generally small,the data was combined and the mean variation among tissues is presented in Table l. The differences between tissues were significant for all monoterpenes except y-terpinene. Needle tissues was consistently higher in camphene and cymene than xylem or cortex. The mean concentrations 15 Table l.--Monoterpene composition in different tissues in 10 trees from 9 seed sources. Monoterpenes Tissue Needle Cortex Xylem a-Pinene** Camphene** B-Pinene** Myrcene** 3-Carene** a-Terpinene** Limonene** B-Phellandrene** Cymene** y-Terpinene Terpinolene** Mean Percent of Total Monoterpenes 61.0 11.4 8.5 i i H- H- H- H- H- H- H- H- H- 5 % 11 21 10 22 25 8 11 17 25 23 1 14.2 .5 12.2 15.0 32.6 1.0 + i H- H- H- H- H- H- H- H- H- 29% 40 25 35 24 20 36 43 13 33 24 59.5 + H- H- H- H- H- H- H- H- H- H- 12% 23 29 29 24 33 20 33 25 26 **Between-tissue differences significant at the 1 percent level. 1 Standard error of the mean expressed as a percent. 16 of a-pinene were equally high in xylem and cortex tissue (60 percent); however, the variation between trees for each tis- sue was considerably different. In the xylem a-pinene ranged from 28 to 94 percent with a standard error of 12 percent. The a-pinene concentration in the foliage was more consistent. It varied from 46-66 percent with a standard error of only 5 percent. The concentration of a-pinene was consistently lower in the cortex ranging from 6 to 46 per- cent but the tree-to-tree variability was highest with a standard error of 29 percent. The high a-pinene concentra— tion in xylem oleoresin was also found in Pinus elliottii Englem. (Squillace and Fisher, 1966). 'Juvonen (1966) also found that a-pinene was high in needle tissue from several sources of Scotch pine. In the cortex limonene ranged from 0-40 percent and B-phellandrene varied from 1-31 percent. Apparently, the large tree-to-tree variability for these terpenes is charact- eristic of this tissue. Limonene and B-phellandrene in the other tissues varied from only 0—3 percent. At this time, I can only speculate on the physio- logical basis accounting for these differences. Sucrose, the most abundant and highly transported product of photo- synthesis, is thought to be a precursor for monoterpene synthesis. Thus, the monoterpene concentration in a parti- cular tissue may reflect the utilization of sucrose which could be influenced by such factors as enzyme levels, enzyeme 17 activities and conditions causing spontaneous conversions. For example, cymene and camphene are spontaneously formed from a-pinene under acid conditions (Mutton, 1962). The higher cymene and camphene concentrations found in the fol- iage is associated with higher pH levels which may subse- _ quently influence enzyme activities or terpene conversion involving these terpenes. In chemosystematic studies, the choice of tissue to be sampled is an important consideration. To distinguish trees or populations from one another, variation in monoter— pene composition must be present. Thus, the monoterpenes from needle tissue would be of little value since there was only slight variation among these 10 trees. The varia- tion between trees in the xylem and cortex monoterpenes was much larger. In these tissues, a-pinene, B-pinene, myrcene, and 3—carene would be equally effective in the separation of these trees. However, the cortical monoterpenes were the most suitable because of their additional variation in the concentration of limonene and B-phellandrene. Cortical oleoresin is also more abundant and easier to collect than xylem or needle oleoresin in young branches of Scotch pine. VARIATION ASSOCIATED_WITH TISSUES OF DIFFERENT AGE Cortical monoterpene concentration has been shown to vary among different age tissues. Hanover (1966a) re- ported that in three trees of western white pine (Pinus 18 monticola Dougl.), current year cortex tissue had less a- pinene, B-pinene and limonene and more myrcene and 3-carene than that of older tissue. In contrast, R. M. Hilton (per- sonal communication) found that 1966 cortex tissue of 23 Pings strobus L. provenances had significantly higher con- centrations of B-pinene and y-terpinene and lower amounts of camphene, B-phellandrene and terpinolene than 1965 tissue. To determine the effects of tissue age in Scotch pine, four trees from different seed sources were sampled. Approximately 20 microliters of oleoresin were collected from cortical incisions made on a lateral branch of the 1964 whorl. Collections were made only from the 1965, 1966 and 1967 internodes. The concentration of the major monoterpenes was as follows: Year Monoterpenes Tissue Formed a-Pinene B-Pinene Myrcene 3-Carene Limonene Percent of Monoterpenes 1965 23.8 30.1 15.1 23.4 2.0 1966 18.5 29.9 18.7 24.8 2.5 1967 15.9 21.2 21.0 29.7 4.2 Proceeding from current-year tissue to older tis— sue, the concentrations of a-pinene and B-pinene increased while those of myrcene, 3-carene and limonene decreased. 19 For most monoterpenes, the largest change occurred between the current-year tissue formed in 1967 and year-old tissue formed in 1966. These results are in general agreement with Hanover's (1966a) data on western white pine. EFFECTS OF DEFOLIATION ON CORTICAL MONOTERPENES The use of monoterpenes as a taxonomic tool is based in part on the evidence that terpenes.are only slightly in- fluenced by the environment. Thus, factors affecting cor- tical monoterpene composition, which have not been previously studied, such as defoliation, are important if valid inter- pretations are to be made. As will be shown from the study on site effects, the average level of a-pinene concentration at the Rose Lake plantation was 4.9 percent higher than at the Russ Forest plantation. It was suspected that this difference as well as other changes may have been due to defoliation at Rose Lake by the European pine sawfly (Neodiprion sertifer (Geoff.)). In addition, two of the three replicates sampled at Rose Lake, which were heavily defoliated, were also higher in a-pinene concentration. To determine the effects of defoliation, samples were obtained from defoliated and non-defoliated branches of each of 31 trees. Of the trees sampled, 9 were classi- fied as lightly defoliated (5 to 25 percent of the branches defoliated) while 12 and 10 trees were classified in the 20 medium to heavy defoliation classes, 35-65 and 75-95 percent of branches defoliated, respectively. Normally only year-old old needles are eaten by the sawfly and current-year needles are not damaged. Because the plantation has been infested by sawflies for the past four years, repeated defoliation had removed all but the current year needles on some trees. The samples were collected in mid-July 1967 and from 1966 cortex tissue in the upper two-thirds of the crown. Twenty microliters of oleoresin were obtained from cortical inci- sions on a defoliated and non-defoliated branch of each tree. Results and Discussion Differences in monoterpene concentrations were small and not significant between defoliated and normal (non-defoliated) branches of lightly defoliated trees. How- ever, differences did occur within the medium and heavy de- foliated trees. Since the effects were similar in both classes of defoliation, their data was combined and ana- lyzed together (Table 2). There was a significant increase in the concentra- tion of a-pinene (1.26 percent) and in the total monoterpene concentration (2.17 percent). To explain these changes, it is helpful to compare differences between defoliated and normal branches in each of the three defoliation classes. The monoterpene concentration in defoliated branches increased (+) or decreased (-) as indicated in Table 3. 21 oOGQCOEflH HON 0>H50 COMfiMHQHHMO GS». EOHH Mumfifl—FHQUGU SOflHMHHQGOGOUN .mflmhamcm on» cw boondocw mum Ammapooc pHOIummm mo ucmouwm mmImmv mommmHo nowadaaommc abomn ou endows on» waned .Hm>ma ucoouom H ecu um ucm0flwwcmflmss mm .sm «sow sa.~+ o.ov I m.m~ mmamaumuoaozamupa HM II II Mddlfl h. I o. mcmcmemBIe II II II mo. + H.H I m. mcmnmsmo II II II «C. + N.m I o. . mamaocfimuoa II II II Ho. + m. I o. msmfiho II II II oo. v. I o. mcwcflmumBI» II II II mo. I e.v I v. monoupcmaamchm m o «smm ma. + H.HH I m. mamGOEflq w o ssmm mv. + m.HN I o. OGOHMUIM N o Isms mo. + m.mH I ~.H masons: v o ssmm mm. + o.om I o.H mcmchIm v ««m ssmm 0N.H+ m.vN I H.N mcwcwmld unmoumm :wmmuomao mo unmoumm Houum coaumflHOMmo some cowuwwHOMmo momma mmcmmumuocoz 0» 050 done no ”on one mocmwnm> AIL wmmmnoma Ho mmsmm Hmuoa mo cofluuom A+v mmmmuocH com: cowpmuucmocoo mcmmumuocoz .mswm couoom mo cowuflmomEoo mcmmuouocoe on» so Manson Hon» an coaumflaomme one mmmuu Hmsefl>aeqa «0 powwow mnauu.m magma scam cmmmousm 22 Table 3.--Changes in monoterpene concentration associated with different degrees of defoliation. Monoterpene. Degree of Defoliation a-Pinene B-Pinene 3—Carene a-Terpinene Total Percent of Oleoresin Light + .18 + .12 + .51 +.02 + .49 Medium +1.36*** +1.06* +1.23* , -.06 +3.86* Heavy +1.16** - .59 — .40 . + .02 + .45 *, **, ***Significant at the 10, 5, and 1 percent 1eve1s,respectively. Note that trees which were moderately defoliated ex- hibited the largest changes in monoterpene composition. In lightly defoliated trees no significant change occurred, and the small differences shown are primarily due to experimental error. With heavy defoliation, differences between defoliated and non-defoliated branches decreased and only the concentra- tion of a-pinene was significantly different. Apparently the almost total lack of foliage also influenced the monoterpenes in the few remaining foliated branches decreasing the differ- ence between the normal and defoliated branches. This effect seems to be reversed in lightly defoliated trees where the presence of foliage overrides the effect of defoliation. Thus, changes in monoterpene composition reflect the degree of defoliation. As defoliation increases the mononter- pene composition is subsequently altered, but the exact degree of this change in unknown in heavy defoliated trees since the 23 monoterpene composition in the remaining foliated branched is also affected. Thielges (1968) reported that in the same pro- venances of Scotch pine defoliation caused a similar general response in phenol metabolism. An unknown compound al- most doubled in the foliage of partially defoliated trees. The mechanisms involved in these monoterpene changes are unknown. Foliage or its absence may be altering sub- strates, enzyme activities and/or enzyme levels involved in monoterpene metabolism. EFFECTS OF SITE ON CORTICAL MONOTERPENES A number of studies have indicated that oleoresin derived from wood has a stable monoterpene composition whether the tree is growing in its native habitat or planted in a foreign environment (Mirov, 1961; Williams and Bannister, 1962). Recent studies of the monoterpenes derived from cor- tex oleoresin also indicate that site has little influence (Squillace and Fisher, 1956; Hanover, 1966a). However, site affects were apparent in provenances of Pinus strobus (R. M. Hilton, personal communication) which were growing in several Michigan planatations. The influence of site on monoterpene composition is an important consideration for their use in systematic studies. This study was undertaken to determine if different sites affect the monoterpene composition in provenances of Scotch pine. 24 Ten selected seed sources encompassing the entire. range of Scotch pine were sampled in early July, 1967 at both the Fred Russ Forest and at Rose Lake. The Fred Russ plantation is near Dowagiac, Michigan, about 120 miles southwest of the Rose Lake plantation. The Russ plantation is level and the soil is a fertile sandy loam. Chemical weed control had been used to eliminate competing vegetation since plantation establishment. The Rose Lake plantation had rolling hills with slopes up to 10 percent. The soil is an infertile loamy sand. No weed control was used. The growth rate in both plantings was similar. Twenty microliters of oleoresin were collected from cortical incisions made on year-old branches of the 1964 whorl. Three 8-tree bulked samples (totaling 24 trees) were collected from each seed source in each plantation. Results and Discussion The mean difference between plantations ranged from zero (camphene and cymene) to 4.9 percent (a-pinene). The variation between sources is highly significant and it ac- counts for the major portion of the total variance (Table 4). There were significant differences between locations in the concentration of d-pinene, myrcene and B-phellandrene. The changes in d-pinene and myrcene concentrations were con- sistent between plantations for most sources and thus no interaction occurred. For B-phellandrene, however, irregular source differences between plantations resulted in a small '25 Table 4.--Monoterpene composition of 10 seed sources growing at two locations. Location Percent of Variance Due to Rose . Source x Monoterpenes Russ Lake Source Location Location Error Percent Of Monoterpenes . d-Pinene 14.9 19.8 73.7** 10.8** 0 '15.5 Camphene .7 .7 52.4** 0 0 47.6 B-Pinene 28.0 26.4 79.4** 0 0 20.6 Myrcene 17.7 14.6 57.8** 5.0** 3.8 33.4 3-Carene 18.6 20.7 87.2** 0 0 12.8 a-Terpinene .9 .8 83.2** 0 0 16.8 Limonene 9.3 8.5 58.4** .4 1.2 40.0 B-Phellandrene 6.3 5.1 77.5** l.0** 9.4** 12.1 Cymene .9 .9 21.5** 0 ‘37.8 40.7 y-Terpinene .5 .4. 81.0** .7 .9 17.4 Terpinolene 2.1 2.0 90.3** 0 .6 9.1 1A mean value of 24 trees; 8 in each of 3 replicates. **Significant at the 1 percent level. 26 but significant source X location interaction. Because of the small sample size, 24 trees, this apparent interaction may have been due to chance. B-phellandrene is a simply in- herited monoterpene (Chapter III) whose genes were in low frequency in 9 of the 10 sources sampled. In such a case, a much larger sample is necessary to obtain a precise esti- mate of its true mean concentration. A portion of the location differences was thought to be due to the heavy defoliation of the Rose Lake planta- tion by the European pine sawfly. The Russ Forest planta- tion was lightly attacked in 1963 and 1964. The attack at Rose Lake began in 1963 and was heavy in 1965, 1966, and 1967 (Wright et al., 1967). A portion of the data from a study on defoliation affects (the preceding investigation in this chapter) indicated that differences between locations were due partly to defoliation. The mean increase (+) or decrease (-) in monoterpene concentration at Rose Lake was comparable to changes due to defoliation. These differences are illustrated in the following array of data: Monoterpenes Average Difference in a-Pinene Myrcene B—Phellandrene Percent of Total Monoterpenes Sources at Rose Lake +4.9 -3.1 —1.2 Defoliated 27 This study indicates that the monoterpenes in Scotch pine are under strong genetic control. Site had little in- fluence on monoterpene composition and defoliation may have been responsible for the significant location differences in the concentrations of a-pinene, myrcene and B-phelland- rene. The small size of the sample may explain the source X site interaction in B-phellandrene. SEASONAL VARIATION OF CORTICAL MONOTERPENES A knowledge of seasonal variation in monoterpenes is a fundamental prerequisite to planning and interpreting other investigations on these compounds. This is especially true for studies related to insect and disease resistance, chemo- systematics, inheritance mechanisms and physiology. Two previous studies (Bannister et a1., 1962, Blight and McDonald, 1964) on wood oleoresin of Pinus radiata D. Don have indicated that monoterpene composition is highly stable, varying only 1-3 percent between any sampling date. Monthly samples obtained over a one-year period from a Pinus ponderosa Dougl. var. ponderosa tree also showed little variation in wood monoterpenes (Smith, 1964). In contrast to this evidence for seasonal stability, a study (Hanover and Furniss, 1966) of monoterpenes obtained from wood oleoresin of 15 Douglas-fir (Psueudotsuga menziesii (Mirb.) Franco var. menziesii) showed that a-pinene and B-pinene increased significantly between June and October. 28 Some seasonal variation of cortical monoterpenes was also detected by R. M. Hilton (personal communication) in a study involving 23 seed sources of eastern white pine. Camphene, y-terpinene, and terpinolene varied significantly between the March and October collections. To determine the degree and pattern, if any, of Seasonal variation in Scotch pine, eight border row trees, four from each of two seed sources, were selected for sampling because of their wide variation in monoterpene composition. Oleoresin samples were collected at nine intervals from December 1966 through December 1967. Five to twenty microliters of oleoresin were obtained from cortex tissue of lateral branches by either scraping off a drOplet of oleoresin from a severed branch or by drawing up a droplet in a capillary tube from a small cortical incision made with a razor blade. The means of 14 paired collections are included in this study. These samples were obtained from seven of the trees on three of the sampling dates to deter- mine the repeatability of multiple samples taken within a tree. Results and Discussion Seven of the eleven monoterpenes varied significantly between sampling times (Table 5). For the major monoterpenes, the amount of seasonal variation was slight compared to the between-tree variation. Camphene, cymene and y-terpinene 29 mom msmmflu .>Hm>wuommmmu .Hm>ma unmonmm H can m on“ an unmoHMHcmHmss.« .mHm3mm man an scandaaommp com coauwmom use 0» map muommmo oEOm mopsaocfl Oman mcHHmEMm mo saw» you msHm>Im a o.o «.mm mm.H ..m~.Hmw smH.o ~.m Ia. mamaocflauma m.~ H.mm .mm.m .ssm.¢m mmo.o H.H Io. mcmcfldumau> H.4H H.om ..om.e ..os.a~ mHH.o m.a um. mcmssu o.o m.mm Hm.a «.Hs.mom mmH.H H.5muo. mcmueemaamzmum m.o m.mm .Nm.m «.mo.mma ems.m m.sHIH.H mammossq m.~ m.am mw.H «.mo.o~ mma.o m.H no. mcmcflaumaua 0.0 m.mm mo.H «smv.~4~m mme.m v.moI~. mamnmoum m.o m.mm .mm.~ s.am.sma mmm.m m.H~Im.N memos»: m.o 5.5m .me.m Isom.~mm Hms.m m.HsIH.m mcmaflmtm m.~m m.H~ s4m~.e 4.4m.m «mo.o o. IN. mcmndsmo m.m s.am .«mm.m ..m~.ow amm.m o.mHIm.o mamcflmua mcwmnwuocoz ucmoumm mcflamfimm moms madamEMm owns mumswm made: some mmcmmnmuosoz mo mafia Hmo.mfifla and: mo mmcmm Hounm on one cocoaum> mo usmouom on man msam> m .mmump mcflc so conEMm moon» ocflm couoom unmflm Eoum mocmmumuosoe mo :oflumwum> HMGOmmmm mo mammamchI.m manna 30 varied significantly although they were present in very small quantities which made their precise measurement difficult. The pattern of seasonal variation for the major monoterpenes is shown in Figure 3. Alpha-pinene and myrcene decreased from December to March, increased from April to August and then decreased again. However, the maximum varia- tion throughout the year was only 2.9 and 3.4 percent for a-pinene and myrcene, respectively. From December, 1966 to March, 1967 a-pinene averaged 2.3 percent higher than during the May to December, 1967 period. Variation in the other monoterpenes appears to be random with respect to season. In addition to sampling time other factors may have influenced the results. These are: sampling errors, the methods of expressing monoterpene concentration, defoliation of a sampled branch and tissue age. SamplingiErrors During the course of the seasonal variation study, four to six branches were sampled on each tree. For the majority of these collections, only one sample was obtained for each tree on a specific date. However analyses of 14 paired samples obtained to estimate branch-to—branch dif- ferences indicated that some within-tree variation was present. The detailed results are given below. Figure 3.--The mean seasonal variation in the principal monoterpenes in eight Scotch pines. The maxi- mum standard error of the means is 1.8 percent. 31 PERCENT MONOTERPENE 32 45 BETA-PINENE 40 35 L / / 2° 3-CARENE l5 ALPHA- PINENE W LIMONENE / \ . 1.” ‘,-.---\\v’ .0 ‘3:\~ ’ ’- ...... I' ~_ _____ .— ‘~~- -_Mmceus ,4” ~‘s ....... J 5 W #- TERPINOLENE 0 as MS 3-IO 4-9 s- 24 6-25 9- I0 lO-3 l2-4 l966 Iss7 DATE SAMPLE!) 33 Monoterpenes a- B- 3- B- Pinene Pinene Myrcene Carene Limonene Phellandrene Percent of Monoterpenes Mean 12.9 36.3 10.5 13.6 13.6 9.1 Standard Error 1.9 5.3 2.9 4.0 4.8 2.6 Camphene, a-terpinene, cymene, y-terpinene and ter- pinolene occurred in very low concentrations and had standard errors of less than 0.5 percent. In addition to the varia- tion between branches, a portion of these standard errors is due to errors in the chromatographic analysis. Methods of Expressing Data Expressing oleoresin monoterpene composition either as a percent of the total monoterpene fraction or as a per- cent of the total oleoresin can affect the results. Table 6 illustrates how this may occur using data from western white pine. Expressed as a percent of oleoresin, all monoterpenes decreased from February to May, while little change is evi- dent from the data when expressed as a percentage of the monoterpene fraction. The latter conclusion could only result if all monoterpenes decreased, in a like manner, either from evaporation, translocation or metabolism, or if the resin acid fraction increased while the monoterpene 34 Table 6.--Concentration of five cortical monoterpenes of w western white pine sampled at different seasons (Hanover, personal communication). Monoterpenes Month Sampled a-Pinene B-Pinene Myrcene 3-Carene Limonene Total Percent of Oleoresin February 7.6 21.3 2.5 13.3 3.9 48.6 May 5.7 17.0 1.4 11.2 3.4 38.7 August 4.3 12.2 0.8 11.6 3.0 31.9 Percent of Monoterpenes February 15.6 43.9 5.1 27.4 8.0 100.0 May 14.6 44.0 3.7 29.0 8.7 100.0 August 13.5 38.3 2.6 36.3 9.3 100.0 35 fraction remained unchanged. All monoterpenes except 3-carene and limonene decreased from May to August regardless of the method of expression.‘ Based on percent of oleoresin, 3-carene increased slightly, while limonene decreased. However, both 3-carene and limonene increased considerably when the results are expressed as a percentage of the monoterpene fraction. The seasonal variation data of Scotch pine is ex- pressed as a percentage of the total monoterpenes. Thus changes in the absolute amount of the monoterpenes or resin acids could have occurred but would not be detected. How— ever, as indicated from the western white pine data, any dissimilar change in one or more monoterpenes would be evi- dent although the results are considerably different depend- ing on the manner of expressing the monoterpene concentrations. Defoliation All sample trees were heavily defoliated by the EurOpean pine sawfly in June and July, 1967, and thus the August and October samples were collected from defoliated branches. Defoliation, as indicated by the previous study, significantly increases the a-pinene concentration and also affects other terpenes in an unpredictable manner. Effects of Tissue Age Small changes in monoterpene composition are known to occur between cortex tissue of different ages as indi- cated by the previous study. Changes associated with tissue 36 age may have influenced the seasonal variation data for Scotch pine in two ways. First, the sampling began on the current, 1966 tissue and as the sampling progressed the tissue aged one year. Second, in order to terminate the sampling cycle with the current year's growth, the December,1967 samples were collected from 1967 cortex tissue. Thus, part of the variation observed between the October and December 1967 sam- ples could have been due to a change in tissue age. Thus, this study indicates that although 7 of the 11 monoterpenes varied significantly between sampling times, changes in their concentrations were small and may in part be due to tissue aging, sampling errors and defoliation. With a well designed, replicated experiment, these sources of variation can be eliminated in a seasonal variation study. Further, additional information may be obtained by collecting known volumes of oleoresin and expressing the monoterpene concentration as a percent of the oleoresin. CHAPTER III MONOTERPENE VARIATION WITHIN SEEDLOTS The variability of the monoterpenes between and within pOpulations may provide useful information for such studies as the modes of monoterpene inheritance, the bio- synthesis of monoterpenes and the taxonomy and evolution of natural pOpulations. My reasons for sampling individual trees within seed- lots were twofold. First, information on the magnitude of ' monoterpene variation is indispensable in predicting the sample size necessary to achieve given levels of signifi- cance. Second, for traits under gene control, individual tree data can be used to obtain information on inheritance patterns. For a monoterpene that is simply inherited, concentration may indicate directly the genotype of an in- dividual. Thus, by sampling a number of trees in a popula- tion it may be possible to determine gene frequency. Materials and Methods Oleoresin samples for this study were obtained from trees growing in the Rose Lake provenance plantation. De- tails of the planting site are discussed in Chapter II. 37 38 In early December, 1967, samples were collected from the following seedlots: Southern Sweden, No. 541 (14 trees); western Germany, No. 252 (19 trees) and Yugoslavia, No. 242 (21 trees). Several small incisions were made on a tree in the cortical tissue of the current years growth of the 1966 (next- to-t0p) whorl. A 20-microliter sample of oleoresin was col- lected from these incisions on each tree. The collection and analytical procedures were the same as those described previously (Chapter II under the section on defoliation). Results and Discussion Inheritance of Monoterpenes The concentrations of monoterpenes in all 54 trees are shown in Table 7. Camphene, a-terpinene, cymene and y-terpinene occurred in relatively small concentrations and varied little. Alpha-pinene is more variable than the above terpenes and its normal pattern of distribution is suggestive of multiple gene inheritance. Similar patterns in concentra— tions of a—pinene have been shown for individual trees of Pinus elliottii (Squillace and Fisher, 1966) and for indi- viduals of half-sib families of Riggs strobus (Hilton, per- sonal communication). However, the normal pattern of distribution in the concentration of a-pinene could be due to environmental modifications such as defoliation. Another possibility is that a-pinene may be simply inherited, but it Table 7.--Variation in cortex monoterpenes of individual trees from three seedlots grown at the Rose Lake Wildlife Experiment Station, Shiawassee County, Michigan. 39 4(3 Monoterpene e~ C.- 8- Myr- 3- e-‘l‘er- Lino- B-Phel- Cyn- y-Ter- Ter- __Pin C n lendrene ene Total ...................... ”cutofolmgu-------------—--—-- Seedlot. 581 Southern Sweden .16 .21 .89 .98 19.86 .33 .38 .51 .18 .10 1.16 25.93 1.39 .88 1.28 1.86 20.61 .26 .38 3.82 .18 .31 2.30 32.05 1.51 .33 2.55 1.21 19.01 .81 .38 2.31 .11 .19 1.98 30.01 3.21 .33 9.65 6.11 10.36 .26 .51 .11 .11 .01 .11 32.13 1.88 .33 2.98 1.86 19.91 .81 .51 2.18 .18 .28 1.16 32.80 2.28 .33 3.91 6.59 8.65 .81 1.88 8.02 .26 .86 1.13 29.91 1.03 .21 1.88 1.36 28.86 .81 .38 .60 .03 .19 2.15 32.88 1.93 .21 1.61 1.86 21.13 .81 .38 .60 .03 .31 2.93 31.06 2.28 .33 1.12 1.21 11.83 .33 .38 2.56 .03 .01 .95 26.65 1.15 .21 .99 13.90 .21 .03 .85 6.50 .11 .00 .23 28.18 1.30 .33 3.82 .89 18.18 .33 .38 .51 .16 .10 1.80 23.53 1.51 .88 1.91 1.86 18.10 .81 .51 3.82 .89 .28 2.12 26.15 1.21 .33 .99 .98 18.90 .81 .82 .51 .11 .19 1.98 25.99 1.8“ e33 be“ 1.21 11.32 e26 .52 5.05 e26 e10 1e“ “e76 x 1.11 .29 3.13 2.91 15.16 .33 .51 2.39 .16 .20 1.68 29.06 Seedlet 252 Heetern Ger-w 2.01 .33 8.01 1.21 .00 .03 18.29 .51 .81 .01 .05 22.92 1.88 .33 5.18 1.21 11.96 .26 8.88 .85 .26 .19 1.31 28.31 1.88 .33 1.02 1.11 11.09 .81 .51 .60 .11 .10 1.61 30.81 1e” 033 Sela .89 l~.~2 e26 e~2 e51 e18 e10 1e“ 25.“ 1.15 .21 1.61 1.18 22.15 .56 .60 3.85 .81 .31 2.51 36.81 2.19 .33 2.55 8.59 13.28 .81 .51 .60 .26 .10 1.80 26.11 2.91 .33 10.08 6.30 8.88 .26 .38 .11 .26 .10 1.22 30.95 1.93 .33 .89 1.11 11.13 .56 8.11 .11 .11 .19 1.85 30.23 1.39 .33 2.18 1.21 26.89 .68 .51 .60 .18 .19 2.39 36.11 2.28 .33 1.38 8.89 10.51 .89 .51 1.02 .18 .10 1.22 26.51 3.85 .33 11.60 10.11 .32 .18 3.68 .60 .11 .00 .18 31.16 3.85 .33 12.38 1.25 .32 .18 2.82 .11 .33 .00 .18 21.91 3.00 .88 8.88 1.08 8.33 .18 5.05 .68 .81 .10 .86 28.61 .85 .33 1.19 1.86 25.31 .56 .82 .51 .18 .31 2.5: 33.12 3.58 .33 5.16 9.63 .32 .18 5.65 .51 .81 .01 .1 26. 2.31 .33 1.32 1.18 9.18 .18 3.51 3.16 .18 .19 1.22 29.98 1.88 .33 3.13 3.68 6.83 .18 .38 1.91 .68 .01 .59 19.13 2.31 .33 8.88 1.08 11.96 .26 2.39 .51 .11 .01 1.13 25.02 1.03 e33 2e35 1e11 19e12 056 e~2 e“ e“ e28 2e” 28050 x 2.18 .30 5.15 3.88 11.80 .35 2.61 1.06 .30 .13 1.21 28.65 8.6816: 282 Central rugooiuvis 2.91 .33 3.81 9.06 .53 .26 6.01 3.85 .81 .10 .18 21.81 1.93 .21 .80 10.96 .32 .26 1.31 3.16 .18 .10 .18 20.02 3.58 .33 12.38 1.65 .11 .03 15.06 .85 .81 .01 .05 38.80 1.8“ 021 2e35 6e18 0” en he“ he“, en e01 elk 21.18 2.31 .33 8.59 8.11 .21 .11 11.30 .68 .33 .01 .18 28.88 1.51 .33 .10 6.30 .32 .18 10.88 8.62 .33 .10 .18 25.03 2.86 .33 1.58 9.15 .83 .18 9.16 6.59 .89 .19 .23 30.11 1.66 .33 .12 1.36 .11 .03 11.12 1.11 .81 .28 .18 23.85 3.18 .33 8.29 1.65 .21 .11 9.81 3.59 .81 .01 .18 27.32 3.89 .33 13.15 6.68 .68 .18 5.81 2.22 .26 .10 .18 33.07 3.30 .88 .99 8.68 .53 .18 3.68 8.19 .33 .10 .32 23.25 3.63 .33 1.61 18.38 .32 . .25 8.88 .33 .00 .18 26.18 3.89 .33 1.09 6.21 .21 .03 5.56 8.02 .11 .00 .18 21.59 2.68 .33 1.19 6.18 .21 .11 2.18 6.16 .18 .00 .18 20.81 1.51 .21 .51 1.36 .11 .11 15.15 2.18 .18 .01 .18 22.01 3.12 .88 11.10 8.30 1.01 .81 2.91 1.19 .33 .19 .23 30.88 5.68 .33 6.68 18.16 .00 .18 2.82 .98 .18 .01 .05 31.58 8.88 .88 3.81 18.38 1.01 .81 .85 5.30 .26 .01 .18 31.55 2.13 .21 5.86 6.59 83 .18 5 39 8.85 .26 .01 .18 26.23 1.88 .21 1.61 1.93 .21 .11 13 88 6.33 .26 .01 .18 26.18 3.63 .33 13.06 1.55 .83 .18 11.08 .82 .33 .01 .05 31.02 2' 3.01 .29 8.58 1.03 36 .18 1 31 3.87 .29 .06 .12 26.19 I 41 is metabolized at different rates in individual trees; conse- quently, a normal distribution is manifested. Obviously, to confirm the exact nature of inheritance for any of the ter- penes, parent-progeny relationships obtained from selected crosses are necessary. Indications of the inheritance patterns were more easily discerned by plotting histograms of the data (Figure 4). Such frequency distributions were particularly interest- ing for B-pinene, 3-carene, myrcene, limonene, B-phellandrene and terpinolene. The concentrations of these monoterpenes formed bimodal or trimodal patterns suggestive of control by one or two pairs of genes. However, distinct classes were not always evident because of the small size of the sample. Simple inheritance has also been suggested for 3- carene in Pinus monticola (Hanover, 1966b); for B-pinene and B-phellandrene in Pinus elliottii (Squillace and Fisher, 1966) and for myrcene and limonene in REESE strobus (Hilton, personal communication). When the exact modes of simply inherited monoterpenes become known, it may then be possible to measure gene fre- quencies for these terpenes in a pOpulation. The frequencies of such genes could be a valuable aid in determining the genetic structure and evolution of natural populations. Minimum sample size A knowledge of individual tree variation within a population is fundamental to studies of geographic variation. Figure 4.-'Frequency distributions of the major cortical monoterpenes. Basis, 54 trees. 42 43 §§§§ o \ I 24 27 w m §m m m V\\\\\m w .r V\... m E m .. \\\ ... m N ‘\ H . M m V\ .. e u p V\\ 7 m a V\\\\\ . \\\\\\\\\ .. V\\\\\\\\\\\\\\\\ - V\\m e m. \\\\\ u m m. \\\\ w c m §\\\\ 1 m a .\\\\\\\\\\ m u \\\\\\\\ ‘\\\\ 5 § 8 .7. \\ V\\\\\\\\\\\\\\\\\\\\\ - §§§§ \w §\9 V\\\\\\\.. 0 § m §\\\\\\§\\§ 30- 25' - b 5 9 5 I m s o 2 2 20' mwmmh no mmmZDz O 5 0 2 I I 5 MONO TERPENE CONCENTRATION (PERCENT) 44 Obviously, differences in terpene composition between pOpula— tions can be discerned only if an adequate number of samples is analyzed. Therefore, the number of trees needed to show significance for a certain difference between seedlot means was calculated using the following formula: (SE-SE where R = number of trees, t = Students' t, V = within-seedlot variance and (fl - 22) = minimum detectable difference between seedlot means. It is evident from Table 7 that simply inherited monoterpenes are highly variable within a seedlot and to obtain a precise estimate of their mean concentrations a large sample size is necessary (Table 8). To detect a dif- ference of 1/10 of the range of concentration between seed- lots from 8 to 238 individuals would have to be sampled. The 8-tree sample for camphene is a reflection of its low variability within a seedlot while a highly variable terpene like myrcene requires a sample size of 238 trees. The sample sizes indicated are probably underestimated because the vari- ances of simply inherited terpenes are unequal. The sample sizes presented in Table 8 illustrate one of the considera- tions involved in monoterpene studies and the detectable pOpulation differences (l/lO of the total range in concentra- tion) were arbitrarily chosen. Smaller or larger sample 45 Table 8.--Size of sample needed to detect significance (5 percent level) of a given difference in monoter- pene concentration between seedlots. Error Trees Needed to Mean Range in Detect a Difference Monoterpenes Square Concentration of 1/10 of Range Percent of 2 Oleore51n Number d-Pinene .82 17.86 10 Camphene .003 .59 8 B-Pinene 14.16 12.79 68 Myrcene 14.77 6.92 238 3-Carene 36.97 17.39 94 a-Terpinene .02 .64 4O Limonene 14.82 9.44 132 B-Phellandrene 2.89 6.22 60 Cymene .018 .53 52 y-Terpinene .015 .34 103 Terpinolene .39 1.60 14 lDetermined from the 108 seedlots of the geographic variation study. 2These data and the formula on which they are based probably underestimates the number of samples needed. For a more conservative estimate, the following formula would be more appropriate: 2 2v(tO + t1) 2 r = _ _ (X1 - X2) 46 sizes may be necessary depending upon the objectives of a par- ticular study. In conclusion, this investigation indicates that several monoterpenes appear to be under simple genetic con- trol, and therefore, are highly variable within a pOpulation. Thus, to detect small differences between populations for these monoterpenes, large sample sizes are necessary. Monoterpene analyses may be conducted on samples obtained from individual trees or replicated bulked samples. The choice will depend upon such factors as the specific ob- jectives of the study, the number of trees available, and the costs involved. CHAPTER IV MONOTERPENE VARIATION IN HALF-SIB FAMILIES In the past few years several studies have been con- ducted on the inheritance of monoterpenes in pines. Such data have been obtained from parent-progeny and other rela- tionships studied in full-sib families (Squillace and Fisher, 1966; Hanover, 1966c) or from the progeny of half-sib fami- lies (Hilton, personal communication). In addition, analyses of half-sib families derived from natural stands may provide information about the genetic structure and evolution of a species. The purpose of this study was to examine the varia- tion in monoterpene composition among 30 half-sib families derived from three European stands. Materials and Methods The trees sampled are a portion of a larger study involving 140 open-pollinated families derived from nine European stands (Wright, 1963). The seed was collected in 1958 by European cooperators and was kept separate by parent tree and by stand. The identity of the seedling progeny derived from these parent trees was maintained in the nursery and in the plantation. 47 48 The trees used in this study are growing at the Fred Russ Memorial Forest. The plantation was established in 1961 with 2-0 stock, and the 10 families derived from each stand were planted in a replicated design. The families sampled came from a native stand in southern Norway (Nos. 275 to 284); and from two native stands in East Germany (Nos. 341 to 350 and Nos. 501 to 510). Cortical oleoresin was collected in late July, 1966 from one-year-old lateral branches of the 1964 whorl. DrOp- lets of oleoresin were collected from the cut surfaces of severed branches and bulked for each four-tree replicate. Four replicates were obtained for 10 families and five rep- licates were obtained for the remaining 20 families to give a total of 140 samples. The samples were placed in 2 ml. stoppered vials and stored at 2°C until analysis. Details of the analytical procedures are described in Chapter II. Results and Discussion The mean monoterpene concentrations for each family are shown in Table 9. Two of the most variable monoterpenes were limonene (1.2 to 25.8 percent) and B-phellandrene (1.6 to 17.9 percent). Considering all of the monoterpenes, the 10 families in the Norwegian stand are less variable than the 20 families of the combined East German stands; however, the variability of the Norwegian families is similar to that in families of either East German stand. 49 Table 9.--Concentrations of the principal monoterpenesl in half- sib families growing at the Fred Russ Forest. Family (MSFG a- 8— Myr— 3- Limo- B—Phel- Ter- Number) Pinene Pinene cene Carene nene landrene pinolene Mean Percent of Total Monoterpenes Norwegian Stand 275 9.5 26.6 4.4 39.9 11.9 3.2 1.4 276 7.4 14.1 6.6 56.2 4.6 6.4 2.5 277 11.5 12.2 11.6 44.6 5.6 10.6 1.8 278 10.3 17.7 7.0 48.8 4.8 7.2 1.5 222------12.2___2§.1___§.§___2§.2---1§.2_--_§.2-____-1.1_-_ 280 9.3 24.9 4.9 35.2 17.9 3.3 2.5 281 15.6 34.7 6.2 21.8 17.5 1.6 .9 282 8.3 16.1 8.8 42.6 10.3 8.5 2.5 283 6.2 12.0 3.2 66.1 1.9 5.2 2.8 284 12.8 13.8 5.1 51.7 5.6 4.4 2.6 East German Stand 341 6.9 16.0 5.1 61.0 1.2 3.5 3.9 342 7.7 11.1 3.4 45.7 9.8 15.5 4.4 343 10.6 11.5 3.4 40.5 12.8 16.2 2.9 344 6.1 13.4 3.6 66.1 3.2 2.9 2.8 323----_--2.9__-12.2-__2.z___§é.9---_2.§_-__z.2__-___§.9_-- 346 10.2 18.6 4.0 37.8 14.4 9.5 2.5 347 8.7 27.5 7.9 41.7 3.5 4.3 4.0 348 8.5 21.4 2.7 40.7 16.9 4.4 2.7 349 7.2 19.0 3.3 50.8 4.1 9.5 3.9 350 8.3 8.6 3.6 61.4 1.7 8.3 5.3 East German Stand 501 13.7 28.1 6.7 38.2 2.7 6.7 1.7 502 4.9 11.5 3.7 70.7 1.3 2.5 3.1 503 8.6 14.9 3.1 56.4 6.4 5.8 2.4 504 9.1 20.6 4.0 57.5 2.1 1.8 2.7 §9§-___--_Z;1____§;Q--_§;§_-_§£12_---§;Z___-§12_-_--_§;Z___ 506 12.1 17.8 3.3 56.0 3.6 2.4 3.0 507 8.2 15.2 5.1 50.9 13.3 3.3 2.1 508 6.1 16.0 2.9 67.0 1.8 1.6 3.4 509 9.3 16.3 4.5 54.6 6.4 3.5 2.5 510 11.0 19.5 6.7 27.4 25.8 6.3 1.7 1Does not include camphene, cymene and y-terpinene which were present in small concentrations (0.2-2.4 percent). 50 In order to determine the significance of the varia— tion between families, the data were subjected to an analysis of variance. The percentage of the total variation within and between families was calculated for each monoterpene. The results show a consistent significant, difference be- tween families of each stand in the concentrations of d- pinene, B-pinene, 3-carene, limonene, and B-phellandrene. The remaining terpenes were significantly different between families in only one or two stands (Table 10). Some indication of the modes of inheritance of the monoterpenes is given by the amount of within-family varia- tion. Large differences within families were observed for myrcene, 3-carene, limonene, B-phellandrene, and terpin- olene. These differences may be due to the segregation of a few genes. Consistently large differences were not ob- served within families for a-pinene, camphene, cymene and y-terpinene which suggests a more complex type of gene control. The mode of inheritance of B-pinene is not clari- fied by this data. The within-family differences were not as large as they would have been if one rather than four- tree bulked samples had been used. The gene frequency of simply inherited monoterpenes like myrcene and 3-carene have a major influence on the within and between variance components. For example, in the German progenies only one sample each in family 501, 507, and 510 had high concentrations of myrcene (averaging 51 .smzuoz .6mmumhm Imameumo .ommnflem 15262660 .oamuflom .>H0>Huommmmu 1H0>ma ucmuumm H can m on» um uGMOflMflcmwm188 .8 snaps; smw3umm moccaum> mo unmouwm 62:06: cmm3uwm mocmaum> mo usmonwm canvas cmmaumm mommaum> mo ucmoumm #mNIth Uhmz omMIva 0mm: HOHmIHom whmz H as 88mm o.~ ma 86~ >.m mm o m.m mamaocnduma 6m m 8. m6 «.aa m. mm o G. mcmcnmumau> hm «1mm m.H mm Ga v.H Ha m H.H mamsmo as 82mm e.m om 11mm 6.6 an «Hm o.6 mamuoamaamnmum mm «85m m.m mm .856 H.a mm 18mm a.m mcmcosfiq mm .106 H.6v H6 11mm o.Hm mm «.mv n.6m mcmumoum mm «8am e.m mm ma o.¢ ooa o m.v 0:800»: cm 11cm m.mH am 12mm H.GH an 2mm m.ma mqmcflmum «m m 6. mm 5H m. 04 «.om v. mamamEmo vw «8mm v.oa vm «smv m.h an 88mm N.m mcmcmmld mGHHHEmm meHHEmm GMT: meHHEmm meHHEmrm Gmwz meHHEmm mmfldflfimm Gmwz mmdmmhmfiocoz omnm on» um mcfi3oum mmHHwEMM nfimlmams Ca coflDMHHm> .ummuom mmsm mammnmuocofi mo mflmhamc¢ll.oa DHQMB 52 15.9 percent). Thus,the gene frequency for myrcene is very low in this stand and 100 percent of the total variance is within families. If myrcene is simply inherited, the high concentration in a sample is probably a result of pollina- tion by a heterozygous male. A decrease in within-family variance and significant between-family differences would result if by chance progency from this heterozygote parent were included in the sample. The gene frequency for myrcene is higher in the families of the Norwegian stand. Possibly the parent trees of families 277 and 282 were heterozygous because 3 of the 5 samples in each of these families had high myrcene concentrations. In this stand 65 percent of the variance was within families. The remaining between- family differences (35 percent) were significant. The gene(s) for 3-carene are intermediate in these three stands resulting in significant between-family differences. In addition, the proportion of the total variance is more equally divided between the two variance components. For a terpene like a-pinene which appears to be under multiple gene control, the between-family differences were significant in all stands. The within-family variances are intermediate to high. Thus, this study indicates that the three stands sampled are highly heterozygous for genes controlling the synthesis of myrcene, 3-carene, limonene, B-phellandrene and terpinolene. This is evident from the large within-family variances for these simply inherited monoterpenes. CHAPTER V INTERRELATIONSHIPS AMONG MONOTERPENES The occurence and quantity of cortical monoterpenes are known to be under strong genetic control in Scotch pine as well as several other pine species (Squillace and Fisher, 1966; Hanover, 1966c). The enzymes, which are the functional products of these genes, apparently regulate the synthesis and ultimate concentration of the various terpenes. These facts suggest that the biosynthetic pathways leading to the formation of the monoterpenes may be indirectly determined by examining their relationship to one another. The work of several investigators has provided some information on monoterpene biosynthesis. Stanley (1958) showed that labeled mevalonic acid gave rise to radioactive a-pinene in shoots of Pinus attenuata Lemm.. Other investi— gations indicated that the active isoprene molecule (derived from mevalonic acid) was iSOpentenylperphosphate and that it condensed with dimethylallylpyrophosphate to form the C10 compound geranylpyrophosphate (Lynen et a1., 1959; Chaykin et a1., 1958). This compound can be converted to myrcene by the loss of the perphospha e unit or through intramolecular electrOphilic rearrangements give rise to monocyclic (limonene) 53 54 'and bicyclic (a-pinene) terpenes (Sanderman and Schweers, 1962). Juvonen (1966) hypothesized a very complete and de- tailed scheme of monoterpene biosynthesis. The pathways were based in part on the above work and the observations by many investigators of terpene fluctuations.~ The latter correla- tions suggested the idea that several common precursors were involved in the synthesis of the monoterpenes. The precursors, believed to be carbonium ions, gave rise to two or more structurally similar terpenes. The involvement of enzymes in this scheme was completely ignored. To determine interrelationships between the mono- terpenes, simple correlation analyses were performed on the cortical terpene composition of 54 trees. The origins and methods of oleoresin collection and analysis of these trees were described in Chapter III. Three simple correlation analyses were performed. One on all 54 trees, one on 28 of these trees with high concentrations of 3-carene and ter- pinolene and the third on the 26 remaining trees with only trace amounts of 3-carene and terpinolene. All correlations were performed on transformed data (arc sine) which had been expressed as a percent of the total monoterpenes. Results and Discussion The correlation coefficients (Table 11) based on all trees or only on those containing large concentrations of 55 Table ll.--Correlations between monoterpenes for 54 trees from southern Sweden, western Germany and Yugoslavia. Monoterpenes a— 8. Myr- 3- Pinene Pinene cene Carene a-Ter- pinene nene Limo- B-Phel- landrene pinene y—Ter- B-Pinene Myrcene 3-Carene a-Terpinene Limonene B-Phel- landrene Y-Terpinene Terpinolene .44 NS .69 .64 NS .53 -.71 NS -087 -.39 NS -.54 NS -.59 * NS NS NS _o6l NS -.56 -.71 NS -084 -.74 NS NS -069 NS NS NS -034 -066 NS NS NS -087 NS .45 NS NS .50 NS NS -058 * -050 .73 .67 .61 -.71 -.51 -.42 NS * .60 NS * .98 .61 .89 I -054 * NS NS .55 NS * .72 NS .63 NS NS -.71 NS * NS NS NS -.33 .61 NS .62 NS .63 1Values listed are significant at the 1 percent level. NS and * indicate non-significance and significance at the 5° level, respectively. 2Upper value based on 54 trees. Middle value based on 26 of the above trees which have only a trace amount of 3-carene and terpinolene. Lower value based on the 28 remaining trees which contain large amount of 3-carene and terpinolene. S6 3-carene and terpinolene were similar except in the case of B-pinene. Correlation of that chemical with 3-carene was r = 7.74, with a-terpinene r =-=69, with y-terpinene r = —.65 and with terpinolene r =-=79 when based only on trees with high 3-carene and terpinolene concentrations. But B-pinene was not correlated with those same terpenes if all trees were considered. On the other hand, monoterpene correlations with a-pinene varied drastically among the three sets of analyses. When all trees were included or those containing 3-carene, most correlations were significant. However, these corre- lations were not significant when based on trees lacking 3- carene. The two notable exceptions to this trend are evident in the correlations between limonene and d-pinene (r = -.57) and limonene and myrcene (r = -.87). These correlations were either not significant or significant at the 5 percent level when performed on trees containing 3-carene. A negative correlation between any two terpenes sup- ports the hypothesis that both are derived from a common precursor. Thus, as one or two terpenes greatly increases others are driven down since the total monoterpenes synthe- sized is fairly constant. Negative correlations by them- selves do not indicate whether the precursors are separate and distinct carbonium ions or if all terpenes are derived from a single substrate such as a pool of geranylpyrophosphate. Positive correlations may be due to one of several factors. For example the presence of 3-carene is apparently 57 responsible for the positive correlation of a-pinene with B-pinene and with myrcene. Both of these correlations are not significant when 3-carene is absent. Only the positive correlation between 3-carene and terpinolene was consistent regardless of the variation in all other terpenes. This association may be due to a specific common precursor (other than geranylpyrophosphate) or one of these terpenes may serve as a precursor in the synthesis of the other. Finally, this and other positive correlations could be a result of genetic linkage. Correlations were performed on trees with or without 3-carene and terpinolene as a technique to clarify relation- ships which may have been obscured when all trees were analyzed together. However, these groupings unexpectedly showed that many correlations could be completely altered by including or excluding one of the terpenes. Thus, many correlations given here and published elsewhere have no real significance in relation to biosynthetic pathways. I feel that the data of this study is consistent with the hypothesis that all monoterpenes are derived from one precursor, geranylpyrophosphate. Hypothesizing that several other intermediates are involved, based on terpene correlations, is not supported by the fact that these cor- relations can be easily altered. The only consistent association observed was between 3—carene and terpinolene. CHAPTER VI GEOGRAPHIC VARIATION IN SCOTCH PINE MONOTERPENES Introduction Since the advent of gas chromatography, the monoter- pene composition of very small samples can be measured pre- cisely and rapidly. This fact along with the evidence that monoterpenes are under strong genetic control makes it pos- sible to undertake intensive studies of variation within species or local populations. Historically, taxonomy and classification have been based on morphologiCal characteristics. The more character- istics used in a system the more probable it is that the system will express the natural relations among populations. In the last decade plant chemicals have been used increasingly as another dimension in systematic classification. This has come to be known as chemotaxonomy or chemical systematics. Chemical studies in several genera and species have been helpful in elucidating their classification and evolution. In fact, chemicals may be particularly useful in delineating subtle differences between races, varieties and hybrid pOpu- lations where morphological features, influenced by the environment, may be of little value. 58 59 Monoterpene Variation Within Species Geographic variation in monoterpenes has been reported in several species of Pinus. Mirov (1961) reported consider- able variation in monoterpenes of Pinus ponderosa oleoresin samples collected from 12 localities. Peloquin (1964) con- ducted a more intensive study in Pinus ponderosa and showed that there was a general correspondence between morphologi- cal and monoterpene variation patterns. Squillace and Fisher (1966) determined that the cortical oleoresin in Pinus elli- ottii var. elliottii in northern Florida was low in B-phel- landrene and high in B-pinene. These trends were reversed in Pinus elliottii var. densa in southern Florida. Varieties of Pinus muricata exhibit especially interesting monoterpene patterns (Forde and Blight, 1964; Mirov et a1., 1965). With- in this California species, the northern variety consists largely of a-pinene, the central variety is composed of 3- carene and terpinolene, and the southern varieties are com- posed largely of sabinene and terpinolene. The insular populations are high in a-pinene, sabinene and terpinolene. Geographic variation has also been shown in three populations of Pseudotsuga menziesii in Montana and Idaho (Hanover and Fur- niss,l966); and in five populations of Pinus radiata in Cali- fornia (Bannister et a1., 1962). R. M. Hilton found differ- ences between Pinus strobus from Michigan's Upper and Lower Peninsulas. 60 Variation in Scotch pine monoterpene composition throughout Europe and Asia was summarized by Mirov (1961) and more recently by Juvonen (1966). These reviews indi- cate that Scotch pine monoterpenes vary extensively from region to region. However, these investigations usually consisted of analyzing the oleoresin in only a few native trees. In addition, some of the analyses were conducted on oleoresin obtained from xylem tissue, other investiga- tors analyzed the oleoresin from needle or cortex tissue, and still others determined the monoterpene composition from two or more tissues combined. There have been no ex- tensive systematic studies of the monoterpenes in Scotch pine. Taxonomy of Scotch Pine Numerous varieties of Scotch pine have been desig- nated by different taxonomists and many variants have been named. The NC-Sl Scotch pine provenance test is the most extensive one to date. As such, it has been a valuable aid in clarifying the taxonomy of the species. Recently, the assigning of valbd varietal names was based on multivariate analysis of several traits measured in two-year old seedlings and from cone and needle characteristics of wild tress (Ruby, 1964). Ruby recognized 21 geographic varieties of Scotch pine as valid. However, the type variety, Pinus sylvestris var. sylvestris is still in doubt because it is unknown 61 whether Linneaus' type specimen was collected in Germany or southern Sweden. Sampling Considerations Before embarking upon a study of geographic varia- tion, several sampling procedures should be considered. Some of the more important of these are choice of tissue to be sampled, size of sample needed to characterize a population and whether analyses should be based on bulked or individual tree samples. On the basis of preliminary investigations, oleoresin from cortex tissue was selected for sampling. Cortical oleoresin was more abundant than xylem oleoresin, and also cortex monoterpenes were more variable--between trees--an important chemotaxonomic consideration. Needle tissue requires a time consuming extraction procedure, and further, the monoterpenes appear to be less variable than those in cortex tissue. This portion of the study was primarily concerned with characterizing the monoterpene composition of a popula- tion or seedlot. Ideally, sampling every tree of a seedlot would provide the maximum information. Since a sample size of this magnitude was impossible, bulking the oleoresin for a number of trees of each seedlot appeared to be a logical compromise. In a preliminary study the variation between replicates of five-tree bulked samples indicated that a 20- tree bulked sample would provide a good estimate of the monoterpene composition of a seedlot. 62 In the near future, individual tree analyses may pro- vide information that is lost when samples are bulked (Chapter III). This is especially true for monoterpenes, such as 3- carene, which are simply inherited (Hanover, 1966b). The con- centration of 3-carene could indicate whether an individual is homozygous or heterozygous for the genes controlling 3- carene synthesis. Thus, the gene frequency for 3-carene could easily be determined in a population. This knowledge would furnish the means for more directly measuring the degree of relatedness between populations. Materials and Methods Samples were collected from 108 seedlots of an NC-51 plantation growing at the W. K. Kellogg Forest, located in Kalamazoo county, two miles west of Augusta, Michigan. The plantation was established in 1961 with 2-0 stock and each seedlot is represented in lO-replicated, four-tree plots. Details of the nursery procedures and other establishment information are given by Wright and Bull (1963). A 20-tree bulked sample was obtained by collecting oleoresin from two trees in each of the 10 replicates. Sam- ples were obtained by scraping off a droplet of oleoresin from the cortex tissue of a one-year old lateral branch severed at the 1964 whorl. The oleoresin was placed in one- half dram vials, tightly sealed and stored at 35°C. Samples were collected during a four-day period in June, 1966, and 63 analyzed in December, 1966, and January, 1967. In order to determine the amount of variation within a source, two repli- cated, bulked samples were collected from 21 seedlots. Each replicate consisted of oleoresin bulked from 10 trees collected from plantation replicates 1-5 and 6-10. Samples were analyzed by gas-liquid chromatography as described in Chapter II under the section on seasonal variation. The column used was 6' x 1/4" stainless steel, packed with 10 percent polyproplyene glycol on 60/80 mesh chromasorb G-AW. Column temperatures were 95°C to 97°C, in- jection port and detector temperatures were 200°C and the helium flow rate was 110 ml/min. Twenty-four samples were rerun on a 6' x 1/4" column containing 8, B'-oxydipr0pioni- trile at 60°C. The re-chromatographed samples further sub- stantiated identification of the monoterpenes and also aided in the quantitative determination of limonene and B-phellan- drene. These monoterpenes are not completely separated by polypropylene glycol. Results and Discussion Geographic Variation The variation in monoterpenes among the 108 seed sources sampled was amazingly large. One of the most vari- able monoterpenes was 3-carene as indicated from Figures 5 and (1 Most of the isolated southern populations either lacked 3-carene or possessed it in small amounts, while 64 Figure 5.--Geographic variation in the principal mono- terpenes in Scotch pine from EurOpe. The ‘ key to the symbol is shown below. Each monoterpene is divided into nine equal classes and the amount of each is represented by the length of bar or degree of shading of the circle. d-Pinene (5-70 percent) B-Phellandrene (0—27 percent) B-Pinene (8-58 percent) ___——w—-3-Carene (0-65 percent) Limonene Myrcene (0-26 percent) (2-28 percent) 65 IO' I8' 20' 30° 38° ”I /) 40‘ .18.!- . 8.. 3- IO‘ on .....\.... 10’ 00' If 48‘ 66 Figure 6.--Geographic variation in the principal mono- terpenes in Scotch pine from Asia. The key to the symbol is shown below. Each monoter- pene is divided into nine equal classes and the amount of each is represented by the length of bar or degree of shading of the circle. a-Pinene (5-70 percent) ”._.. B-Phellandrene (0-27 percent) B-Pinene (8-58 percent) ,fl. 3-Carene (0-65 percent) Myrcene Limonene (2-28 percent) (0-26 percent) °8 ‘ 6 8 2 1"th \ N ((8. '5 f! 1 fi\\ \\\\§ . ‘ \\\V\\\\§ a ‘1' NW ’a V \ .. (8“ V. 3 x“ Q“ ,‘ \§\\\\\‘ ...::\\\'I..o.. 1:..- “. {‘9 ~\ \\\\ o... ‘ ” 68 northern populations generally contained large concentrations of 3-carene. Within each of these two major groups, the varia- tion in other monoterpenes exhibit distinct geographic pat- terns. This is especially evident in the southern sources. Spanish sources (var. iberica) are high in a-pinene; sources from southern France (var. aquitana) are high in B-pinene; 'N. Italy' sources are high in B—pinene and myrcene; Greek sources (var. rhodopaea) are high in B-pinene and limonene and Turkish and Georgian SSR sources (var. armena) are high in B-pinene and myrcene. The monoterpene composition of the latter two varieties are not clearly distinguishable; however, since they are not adjacent to each other, their separate identities are maintained. In the north where Scotch pine is more continuous, geographic patterns exist but no sharp divisions are evident. The degree of correlation between monoterpene com- position and existent varietal classes was determined by grouping the sources according to Ruby's (1964) varietal classification and subjecting the data to an analysis of variance. Three sources adjacent to varietal boundaries were regrouped to improve the fit of the data. Seedlots 254 and 255 were removed from their respective varieties, combined and analyzed as a separate group. Seedlot 542 was regrouped from variety rigensis into variety septentri— onalis and variety rigensis was divided into Latvian and 69 Swedish sources-~a division that had been made in the 1938 IUFRO tests. These minor changes did not conflict with Ruby's data and the naturalness of the geographic varieties was maintained. Planted stands and varieties represented by a single seedlot were omitted from the analysis. Seedlots comprising each variety are given in Table 12 and the varie- tal means are given in Table 13. An analysis of variance of the replicated lO-tree bulked samples of 21 seedlots showed that the within-seedlot variance was small. The variation between varieties (Table 14) was significant at the 1 percent level for all monoter- penes except cymene. The most suitable monoterpenes for distinguishing varieties was 3-carene and a-pinene. The between-variety variances for these compounds were 87 and 86 percent of the total variances, respectively. The middle European variety hercynica contributed substantially to the within variety variation; 3~carene varied between 13 and 54 percent and a-pinene between 6 and 15 percent. Also, the central Scandinavian variety septen- trionalis varied from 32 to 63 percent in 3-carene. Both of these varieties encompass regions of considerable geographic diversity and complex patterns of plant distribution. Ruby separated the East German and Czechoslovakian sources of hercynica. However, a simple natural division was not ob- vious on the basis of monoterpene variation. Within Czecho- slovakia, 3-carene varied between 16 and 54 percent. On the 70 Table 12.--Classification of Scotch pine into varieties according to Wright et al. (1966). Seedlot Variety Countries MSFG Nos. lapponica Northern Finland 229 Northern Sweden 546,547,548,549 mongolica Eastern Siberia 254 altaica Southern Siberia 227, 234, 255, 256 septentrionalis Central Sweden 222, 521, 522, 523, 524, 543, 544, 545 Central Norway 201, 273, 274 Southern Finland 228, 230, 232, 233 rigensis Latvian SSR 223, 224 Southern Sweden 541,.542, 543 uralensis Ural Mtns., Russia 257, 258, 259, 260 olonica Poland 211, 317 boru351ca Northeastern Germany 202, 210 hercynica Germany 203, 204, 207, 208, 209, 525, 526, 527, 528, 529 Czechoslovakia 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 haguenensis Western Germany 206, 250, 251, 252, 253 Vosges Mtns., France 236, 237, 241 Belgium 318, 530 'East Anglia1 England 269, 270 pannonica Hungary 552, 553 'North Italy' Italy 554, 555, 556, 557 illyrica Yugoslavia 242 scotica Scotland 265, 266, 267, 268 iBerica Spain 218, 219, 246, 247 aquitana Central Massif, 212, 238, 239, 240, France 316, 320 rhodopaea Greece 243, 244, 271, 272, 551 armena Caucasus Mtns., 213, 214, 220, 221 Turkey Georgian SSR 261, 262, 263, 264 71 Table l3.--Variation in monoterpene concentrations in Scotch pine varieties grown at the W. K. Kellogg Forest. Concentration of a- 8— Myr- 3- Limo- B-Phel- Terpin- Variety Pinene Pinene cene Carene nene landrene olene Percent of Total Monoterpenes Scandinavian and Siberian Varieties lapponica 9.1 17.0 5.5 41.1 9.3 10.9 3.2 mongolica 15.4 28.7 11.5 8.7 16.2 16.4 1.3 altaica 10.6 17.3 7.4 26.3 14.1 17.4 3.7 —E——se Fent'. 7.6 17.3 8.2 44.1 6.4 9.5 4.0 rionalis rigensis Sweden 5.6 13.6 4.3 58.8 4.4 5.5 4.6 Latvia 8.2 14.4 15.3 29.7 6.9 19.6 2.7 uralensis 9.1 19.9 11.3 25.0 11.3 17.2 3.1 Central EurOpean Varieties olonica 9.5 22.1 8.9 38.5 8.1 6.4 3.0 Eorussica 10.1 22.8 8.7 41.0 3.7 6.1 4.8 Hercynica 10.4 22.9 11.9 30.7 8.6 8.9 2.9 Haguenensis 9.5 19.5 9.7 41.6 4.6 4.9 3.2 rEast AnglIa' 17.2 31.6 11.4 26.4 4.4 3.3 2.4 pannonica 8.3 12.9 5.5 49.9 6.0 8.9 4.7 'N. Italy‘ 17.1 41.6 23.7 4.4 6.4 3.7 .7 illyrica 11.8 24.3 17.9 20.2 12.9 7.9 1.8 West and South European Varieties scotica 10.6 21.9 11.8 39.7 5.0 4.5 3.1 iberica 46.1 15.3 12.7 .8 7.1 14.7 .3 a uitana 18.5 47.1 10.4 4.9 11.7 3.4 .3 rhodopaea 12.4 34.9 11.5 5.8 23.1 7.9 .9 armena 19.1 42.7 17.7 4.4 9.8 3.4 .5 l Camphene, cymene, a-terpinene, and y-terpinene are omitted because they were present in very small amounts (0-2 percent). 72 Table l4.--Percentage of variation in monoterpene concen- tration accounted for by differences between and within varieties. Percent of Variance Due to Differences Between Within Monoterpenes Varieties Varieties a-Pinene 86**‘ 14 Camphene 61** 39 B-Pinene 73** 27 Myrcene 41** 59 3-Carene 87** 13 a-Terpinene 50** 50 Limonene 53** 47 B-Phellandrene 72** 28 Cymene 22 78 y-Terpinene 50** 50 Terpinolene 73** 27 **Significant at the 1 percent level. 73 basis of terpenes alone, hercynica could be divided into 3 or 4 subunits, but this additional splitting would not be supported by morphological differences. The high correspondence between existent varietal classes and monoterpene composition indicates that the monoterpenes are indeed a useful taxonomic tool in Scotch pine. Evolution of Scotch Pine The evolution of monoterpenes and the resin duct system is strictly a matter of conjecture. However, the patterns of geographic variability in monoterpenes do re- flect the evolution and migration of Scotch pine. In the south, tree growth is confined to isolated stands located at high elevations. These forests were south of the maximum extent of glaciation during the Pleistocene. Evolution has probably proceeded uninterrupted for a much longer time than in northern Europe where the species was obliterated'during glaciation. Two opposing theories of plant distribution in re- sponse to glaciation are evident from the literature. Bertsch (1953) in his description of the German forests con- tends that middle Europe, directly south of the glacier of the last ice age, was covered with tundra and below this there was a scrub zone. The forest survived only in the 74 lower elevations of the Mediterranean region. All of middle Europe was devoid of forest. A more recent hypothesis contends that the glaciers had little influence on plant distribution in unglaciated areas. The zone of tundra and scrub was very narrow and the retreating Scotch pine never came in contact with the isolated southern pOpulations. The distribution of 3-carene appears to support the later theory, assuming that 3-carene was pre- sent in the pre-Pleistocene populations in northern Europe. The genes for 3-carene synthesis are entirely absent in most southern populations. This would not be the case if the northern pOpulations had come into contact with the southern stands or if the northern forest was derived from the southern stands. The exchange of genetic material has also been limited between Spain, the Central Massif of France, Italy, Yugoslavia, Greece, Turkey and Georgian SSR. Apparently these populations are Tertiary relics and their extended isolation is reflected in their distinct morphological and chemical differentiation. During the Pleistocene glaciation, probably a number of refugia existed just south of the ice and perhaps some within the glaciated area (Wulff, 1943). Some of the present patterns of variation in northern Europe and Asia reflect the migration of several well-differentiated populations. Paleobotanical evidence indicates that refugia probably existed in the Carpathean and Ural mountains, in the south 75 of western Siberia and even possibly in the Scandinavian highlands and the Gulf of Finland. Reinvasion eventually formed a more or less continuous forest in a complex arrange- ment. The free exchange of genetic material created addi- tional diversity. Selection pressures acting upon this complex gene pool have allowed more recent morphological and chemical differentiation. The heterogeneous monoterpene patterns found especially in Middle Europe manifest these recent geological events. The retreat of the ice coincided with a rise of the land, connecting the Scandinavian peninsula with western Eur0pe via Denmark. Also, the shallow southern part of the North Sea formed a land bridge between England and the con- tinent. The high concentrations of 3—carene in southern Scandinavia and in some populations in Germany, lend further support to the concept of the formation of a land bridge. The similarity of Scottish populations and those of middle Europe also indicate recent gene exchange between these pOpulations. The present distribution of Scotch pine and perhaps some of its complexity may be accounted for by early man. The complete extermination of Scotch pine in England and Denmark happened recently after man arrived there (Godwin, 1956). A change from a continental to an oceanic climate contributed to this obliteration. Further, man has artifi- cially established populations of trees by either a directed program of reforestation or as a matter of chance, and it is often difficult to distinguish them from natural populations. CHAPTER VII CONCLUSIONS AND RECOMMENDATIONS The environmental effects on monoterpene composition in Scotch pine were small but usually predictable. Although 7 of the 11 monoterpenes varied significantly between sampling times, these changes were confounded by the effects of defolia- tion, errors in sampling and tissue age effects. The effects of sawfly defoliation and errors in sampling can be easily eliminated from seasonal variation studies by obtaining repli- cated samples on protected trees. Tissue age effects are difficult to separate from seasonal effects on plantation grown trees. However, the major environmental influences (soil moisture, temperature and photoperiod) could be elimi— nated from the effects of tissue age by using clonal material in growth chamber investigations. In such studies, material of the same age could be tested simultaneously under various conditions. Cortical monoterpene changes associated with different aged tissue and defoliation were small. Oleoresin from older tissue formed in 1965 was higher in d-pinene and B-pinene and lower in myrcene, 3-carene and limonene than younger tissue formed in 1967. Defoliation significantly increased (at the 5 76 77 or 10 percent level) the concentration of a-pinene, B-pinene, 3-carene and the total monoterpenes. This effect was most pronounced on moderately defoliated trees. In heavy defo- liated trees, branches with and without foliage differed only in d-pinene concentration. I believe the almost total lack of foliage altered the monoterpene concentrations throughout the tree, including the few remaining foliated branches. Such changes would not be detected by the within-tree sampling scheme that was utilized. Therefore, to investigate this hypothesis and to determine the effects of defoliation more precisely, clonal material should be used. In such a study, monoterpene concentrations would be determined before and after artificial defoliation. The mechanism(s) of these changes would be difficult to determine since they probably involve enzyme activities, enzyme concentrations or changes in substrates. In order to plan and interpret other related investi- gations on terpenes, a knowledge of changes associated with tissue age and defoliation may be important. For example, to obtain valid comparisons in chemosystematic studies, I would recommend that all oleoresin samples be collected from the same aged tissue of uninjured trees. Site effects, another source of environmental influ- ence, were minor. Although a significant seed source X site interaction was determined for B-phellandrene, the small size for this simply inherited terpene may have been responsible 78 for this interaction. It is apparent that in future investi- gations site effects can be determined more precisely and with fewer samples from clones established in diverse location. Different tissues (cortex, xylem and needle) had dis- tinct monoterpene compositions. Each tissue appears to act as a separate compartment having a distinct terpene metabolism. Probably little or no monoterpene transport occurs between tissues. Tree-to-tree variability of terpene composition was smallest in needle tissue and largest in cortex tissue. Thus, in chemosystematic studies the analysis of cortical monoter- penes is recommended since the presence of variability is essential in distinguishing trees or populations from one another. From studies on individual trees and half-sib fami- lies, the monoterpenes 3-carene, myrcene, limonene, B-phel- landrene and terpinolene appeared to be under simple genetic control. More complex gene action was indicated for a-pinene and B-pinene, camphene, a-terpinene, cymene and y-terpinene. To clarify the nature of these inheritance patterns, parent- progeny relationships obtained from selected crosses are needed. Large tree-to-tree variation occurred in the simply inherited terpenes, especially when their gene frequency in a population was low. In such cases large sample sizes are necessary to detect small difrerences between populations. This is most easily accomplished by replicated bulked samples. 79 However, in the near future additional knowledge of terpene inheritance may make it possible to determine the genotype of an individual directly from terpene concentration. Thus, gene frequencies of populations could be determined which would aid in chemosystematic studies and especially in in- vestigations related to the evolution of natural populations. Simple monoterpene correlations support the hypo- thesis that all the terpenes are enzymatically derived from the stable precursor geranlypyrophosphate. The enzyme and subsequent terpene concentrations are under strong genetic control, and the presence or absence of a terpene reflects the gene status. The only positive correlation that per- sisted, despite changes in any other terpene, was between 3-carene and terpinolene. At present the cause of this common association is entirely speculative. From time course studies using 14C02, it may be possible to directly determine terpene biosynthetic pathways and turnover. Se- lected genotypes which are lacking in one or more terpenes could aid such studies. The monoterpenes in Scotch pine exhibited wide geo- graphical variation. They proved to be an excellent chemo- taxonomic tool based on the fact that there was a high correspondence between terpenes and existent varietal classes. The highly variable middle European area, in morphology and in terpene composition, indicates that additional sampling is necessary to further delineate natural groupings. 80 In addition, geographic variability of the monoter- penes is useful in explaining evolution and migration patterns in Scotch pine. For example, the absence of 3— carene in most isolated southern populations indicates that little gene exchange has taken place and that these popula- tions are probably Tertiary relics. The similarity in terw pene composition between Germany and lower Scandinavia lend further support to the hypothesis that Scotch pine migrated across a land bridge which had once connected these land masses. Also there probably has been recent gene exchange between the Scottish populations and those of Middle EurOpe since they have similar terpene compositions. LITERATURE CITED Baldwin, H. I. 1956. Winter foliage color of Scotch pine. Northeast For. Tree Improv. Conf. Proc. 3: 23-28. Bannister, M. H., A. L. Williams, I. R. C. McDonald, and Margot B. Forde. 1962. Variation of turpentine composition in five population samples of Pinus radiata. New Zealand Jour. of Sci. 7(2): 2I2-220. Bertsch, Karl. 1953. Geschichte des deutschen Waldes (3rd ed.) Gustav Fischer, Jena, 124 pp. Blight, Margaret M. and I. R. C. McDonald. 1964. Sample reproductibility in Pinus essential oil studies. New Zealand Jour. of Sci. 7(2): 212-220. Chaykin, S., J. Law, A. H. Phillips, T. T. Tchen and K. Bloch. 1958. Phosphorylated intermediates in the synthesis of squalene. Proc. Natl. Acad. Sci. 44: 998-1004. Echols, R. M. 1958. Variation in tracheid length and wood density in geographic races of Scotch pine. Yale Univ. School of Frestry Bull. 64, 52 pp. Forde, Margot B., and Margaret M. Blight. 1964. Geographi- cal variation in turpentine of bishop pine. New Zeal. Jour. Bot., 2(1): 44-52. Gerhold, H. D. 1959. Seasonal variation of chloroplast pigments and nutrient elements in the needles of geographic races of Scotch pine. Silvae Genetica 8: 112-123. Godwin, H. 1956. The history of the British flora. Cam- bridge Univ. Press, London. 383 pp. Hanover, J. W. 1966a. Environmental variation in the mon- terpenes of Pinus monticola Dougl. Phytochemistry 5: 713-717. . 1966b. Inheritance of 3-carene concentration in Pinus monticola. For. Sci. 12: 447—450. 81 82 Hanover, J. W. 1966c. Genetics of terpenes. I. Gene con- trol of monoterpene levels in Pinus monticola Dougl. Heredity 21: 73-84. , and M. M. Furniss. 1966. Monoterpene concentra- tion in Douglas-fir in relation to geographic location and resistance to attack by the Douglas-fir beetle. In: Joint Proceedings, Second Genetics WorkshOp of the Soc; of Amer. For. and the Seventh Lake States For. Tree Impr. Conf. U. S. For. Serv. Res..Pap. NC-6. pp. 23—28. 1 Hilton, R. M. 1968. Michigan State University Ph.D. thesis in preparation. Juvonen, S. 1966. fiber die die terpenbiosynthese Beeinflus- senden faktoren in Pinus sylvestris L. Acta Bot. Fennica, Helsinki. 92 pp. Lynen, F., B. W. Agranoff, H. Eggerer, U. Henning and E. M. Moslein. 1959. Dimethyl-ally1-pyrophosphate and Geranyl-pyrophosphate, biologische Vorstufen des Squalens. Angew. Chem. 70: 738—742. Mirov, N. T. 1961. Composition of gum turpentines of pines. U.S.D.A. Tech. Bull. No. 1239. 158 pp. . 1967. The genus Pinus. The Ronald Press, New York. 602 pp. , E. Zavarin, K. Snajberk and K. Costello. 1966. Further studies of turpentine composition of Pinus Muricata in relation to its taxonomy. Phytochemistry 5: 343-355. Mutton, D. B. 1962. Wood resins. pp. 331-363. Hillis, W. E. (ed.). In Wood extractives and their signifi- cance to the pulp and paper industries. Academic Press, N.Y. 513 pp. Peloquin, R. L., Jr. 1964. Geographic variation of the monoterpenes of Pinus ponderosa. Stanford Univ. M.A. thesis. Ruby, J. L. 1964. The correspondence between genetic, mor- phological, and climatic variation patterns in Scotch pine. Mich. State Univ. Ph.D. thesis. Sandermann, W. and W. Schweers. 1962. fiber die biogenesis von a-pinen in Pinus nigra Austriaca. Tetrahedron Letters. 257—259. 83 Smith, R. H. 1964. Variations in the monoterpene composition of ponderosa pine wood oleoresin. U.S. For. Serv. Res. Pap. PSW-lS. 17 pp. Squillace, A. E., and G. S. Fisher. 1966. Evidences of the inheritance of turpentine composition in slash pine. In: Joint Proceedings Second Genetics Workshop of the Soc. of Amer. For. and the Seventh Lake States Forest Tree Improv. Conf. U.S. For. Serv. Res. Pap. NC-6. pp. 53-62. Stanley, R. G. 1958. Terpene formationjxnpine from mevalo- nic acid. Nature 182: 738-739. Thielges, B. A. 1968. Altered polyphenol metabolism in the foliage of Pinus sylvestris associated with European pine sawfly attack. Can. J. Botany 46: 724. Williams, A. L., and M. H. Bannister. 1962. Composition of gum turpentines from twenty-two species of pines grown in New Zealand. J. Pharmaceutical Sciences 51: 970-975. Wright, J. W. 1963. Genetic variation among 140 half-sib Scotch pine families derived from nine stands. Silvae Genetica 12: 73-104. , and H. 1. Baldwin. 1957. The 1938 International Union Scotch pine provenance test in New Hampshire. Silvae Genetica 6: 2-14. , and W. I. Bull. 1963. Geographic variation in Scotch pine, results of a 3-year Michigan study. Silvae Genetica 12: 1-25. , S. S. Pauley, R. B. Polk, J. J. Jokela, and R. A. Read. 1966. Performance of Scotch pine varieties in the North Central region. Silvae Genetica 15: 101-140. , L. F. Wilson and W. K. Randall. 1967. Differences among Scotch pine varieties in susceptibility to European pine sawfly. For. Sci. 13: 175-181. Wulff, E. V. 1943. An introduction to historical plant geography. Chronica Botanica Co., Waltham, Mass. 223 pp. VITA James J. Tobolski Candidate for the Degree of Doctor of PhiloSOphy Final Examination: July 26, 1968 Guidance Committee: Drs. M. W. Adams, A. DeHertogh, C. J. Pollard, J. W. Wright, and J. W. Hanover (Chairman) Dissertation: Variations in Monoterpenes in Scotch pine (Pinus sylvestris L.) Biographical Items: Born July 4, 1935, South Bend, Indiana Married Marilyn Bock, September 1, 1962 Children: Erica, born August 3, 1963 Jessica, born August 17, 1967 Education: Michigan State University, BSF, 1958 Yale University, MF, 1961 Indiana University, Secondary Teacher Certificate, 1963 Michigan State University, Ph.D., 1968 Experience: . U. S. Forest Service: General Forestry Aide, Ely, Minnesota, summer 1955; Timber Management Aide, North Bend, Washington, summer 1956; Research Forester, Macon, Georgia, spring 1958; Research Forester, Rhinelander, Wisconsin, summer 1965. U. 8. Army: Meteorological Observer, Fort Huachuca, Arizona and Houghton, Michigan, 1958-1960. Research Technician, Cold Regions Research Engineer- ing Laboratory, Greenland, summers 1960, 1961, and 1963. Teacher, Secondary Science, South Bend Community School Corporation, South Bend, Indiana, 1963- 1965. Graduate Research Assistant, Michigan State Univer- sity, East Lansing, Michigan, 1965 to date. 84 APPENDICES 85 1 cam N.o w.o n.m -o.o m.o ¢.o m.o ,wma fi.n n.o c.c n.m-. o.o: a.m m.m;- one Io.” c.u m.u v.0 fi.m o.o m.o fi.m v.¢ fi.« m.o m.e c.o :Ilim.o 0.0. m.m one 0.0 5.x 1 1m o .m.a m o c.o 0.5 m. o 1,:m.o Ito.a 12m. m wzwn (mzmz~ mam 1c?” 31mm; Ix»u 301mmh M4z2x wrmmmmpozoz Hzmumud . .oooflcmamm~» aro m4m»-;ll. m14124m mwak m3oz_m noofi\~m\o autuuolou m.cm 7.1 v.mm w.oo o.mm 6.60 v.fio ~.ov F.0m mrm: n—Q .wqx644 I.IIiII::l-mu24m .xmlmou z. zo~t mamasm ¢:zox V r-d V o, cmm .zm4yx n cmm xmhmou o t omm mDommz; n emu rm4>x m cmm xmpaou- n cmm chmw: m 0mm rm4yx a 0mm xmkmoc m . onm wJmez a opm rm4>x H cam xmkqou H 1.01w whammz o . mam zm4>x 0 arm xmtaoe o.!-mfim mammmz .02 amm uumzlnwnmmmh-ill -- .ak 0H m4m

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