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Regional, provenance and family variation in cold hardiness of western white pine (Pinus monticola Dougl.… Thomas, Barbara R. 1990

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REGIONAL, PROVENANCE AND FAMILY VARIATION IN COLD HARDINESS OF WESTERN WHITE PINE (Pinus monticola D o u g l . e x . D . Don) by Barbara R. Thomas B.Sc.(Agr.), The U n i v e r s i t y of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (For e s t Science, F o r e s t Tree Improvement) F a c u l t y of F o r e s t r y We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1990 © B a r b a r a Ruth Thomas, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date Qd /2 /*ft£> DE-6 (2/88) ABSTRACT Thirty-seven seedlots of western white pine (Pi nus mont i col a Doug. ex. D. Don) were tested for frost hardiness to determine how transferable seed would be from d i f f e r e n t seed sources within white pine's coast and i n t e r i o r ranges in B r i t i s h Columbia. Twenty-nine seedlots represented the coast and i n t e r i o r of B r i t i s h Columbia (BC), two were from coastal United States (US), three were from i n t e r i o r US and three were hybrids between i n t e r i o r US and i n t e r i o r BC parents. Detached needles were exposed to a series of freezing temperatures in a programable freezer and r e l a t i v e hardiness was calculated as the length of injured needle expressed as a percentage of t o t a l needle length 10 days after freezing. Seasonal progress in hardening was tested using fiv e dates in the autumn of 1989. Seedlings were maintained at the University of B r i t i s h Columbia nursery. Testing also was carried out from samples c o l l e c t e d on separate dates from Nakusp in the BC i n t e r i o r and from Ladysmith, a coastal BC s i t e . There was a s t a t i s t i c a l l y s i g n i f i c a n t (p<0.0l) regional difference between the BC coast and BC i n t e r i o r sources in a l l test runs, excluding the f i r s t UBC run and the Ladysmith run. In the runs where regions d i f f e r e d s i g n i f i c a n t l y , the difference in percent damage response of needles to freezing was approximately 20%. /" / /' Measurements of shoot growth phenology were planned as an additional component of growth rhythm. Injury from uncontrolled freezing forced a change of objective to evaluation of genetic differences in recovery from freezing. Those evaluations did not reveal genetic differences in recovery. / V TABLE OF CONTENTS ABSTRACT // ACKNOWLEDGEMENTS xvi PREFACE xvii TABLE OF CONTENTS / v LIST OF TABLES vii LIST OF FIGURES x i i i 1.0 INTRODUCTION 1 2.0 LITERATURE REVIEW 5 2.1 PHENOTYPIC PLASTICITY 5 2.2 PLASTICITY AND COLD HARDINESS 10 2.3 COLD HARDINESS AND DORMANCY 12 2.4 COLD HARDINESS AND THE ASSOCIATED PHYSIOLOGICAL CHANGES 16 2.5 GENETICS OF COLD HARDINESS 20 2.6 TECHNIQUES TO MEASURE HARDINESS 22 2.6.1 Methods of Evaluation 22 2.7 COLD HARDINESS, PHENOTYPIC PLASTICITY AND Pw 27 3.0 MATERIAL 32 4.0 METHODS AND EXPERIMENTAL DESIGN 37 4.1 COLD HARDINESS 37 4.2 PHENOLOGY AND DAMAGED MATERIAL 40 5.0 DATA ANALYSIS 43 5.1 CONTROLLED FREEZING 43 5.2 HYBRIDS 45 5.3 PHENOLOGY 45 5.4 UNCONTROLLED FREEZING 46 V 6.0 RESULTS 48 Section I. 48 6.1 COLD HARDINESS OVER TIME AT ONE LOCATION 48 6.1.1 A l l dates combined, a l l regions, Hypothesis 1 (Appendix C) 49 6.1.2 Individual dates, a l l regions 51 6.1.3 A l l dates, BC regions, Hypothesis 2 (Appendix C) 52 6.1.4 Individual dates, BC regions 53 Section I I . 54 6.2 COLD HARDINESS AT THREE LOCATIONS 54 6.2.1 A l l locations, a l l regions, Hypothesis 3, (Appendix C) 54 6.2.2 Individual locations, a l l regions 55 6.2.3 A l l locations, BC regions,-Hypothesis 4, (Appendix C) 56 6.2.4 Individual locations, BC regions 57 Section I I I . 58 6.3 HYBRID COLD HARDINESS, 5 DATES 58 Section IV. 60 6.4 PHENOLOGY 60 6.5 DAMAGE ASSESSMENT 61 7.0 DISCUSSION 63 7.1 RECOMMENDATIONS BASED ON FREEZE TESTING 63 7.2 PHENOLOGY 66 7.3 Pw, COLD HARDINESS AND PHENOTYPIC PLASTICITY 69 7.4 FUTURE RESEARCH 75 V /" 8.0 LITERATURE CITED 118 APPENDIX A 124 APPENDIX B 125 APPENDIX C 126 V / / LIST OF TABLES Table 1 Descriptive information for Pi nus monticola seed sources ..76 Table 2 Location of sampled trees, test date, day (from Sept. 7, 1989) and test temperatures 78 Table 3 Number of seedlings available, by provenance, which were included in the damage assessment analysis 79 Table 4 a) Results from analysis of variance for combined data from controlled freezing on 5 test dates using 2-year-old seedlings grown at UBC (See Table 2 for test temperature and dates) 80 b) Multiple-comparison testing of regional means..80 Table 5 a & b) Results from analysis of regression for f o l i a r injury and lat i t u d e or elevation of seedling o r i g i n . Data on f o l i a r injury were from combined dates of cont r o l l e d freezing using 2-year-old seedlings grown at UBC 81 Table 5 c) Results from analysis of regression for f o l i a r injury with latitude and elevation of seedling o r i g i n . Data on f o l i a r injury were V / / / from combined dates of controlled freezing using 2-year-old seedlings grown at UBC... 82 Table 6 a) Results from analysis of variance for single test dates from controlled freezing using 2-year-old seedlings grown at UBC (See Table 2 for test temperatures) 83 b) P r o b a b i l i t y of a larger value for F in orthogonal contrasts among diff e r e n t combinations of regions at five d i f f e r e n t dates of te s t i n g 84 Table 7 a) Analysis including highest order interaction and excluding P(R), R x D, R x T(D), P(R) x D, P(R) x T(D). The exclusions were imposed by lim i t a t i o n s on computing capacity...85 b) Results from analysis of variance for combined data from controlled freezing on 5 test dates using 2-year-old seedlings grown at UBC (BC material only, H Q2) (See Table 2 for test temperatures and dates) (analysis excludes highest order in t e r a c t i o n T x F(D P R)) . 86 / X Table 8 Results from analysis of variance for single test dates from c o n t r o l l e d freezing using 2-year-old seedlings grown at UBC (See Table 2 for test temperatures) (BC material only, H 2) 87 Table 9 a) Results from analysis of variance for combined data from con t r o l l e d freezing using 2-year-old seedlings from UBC (day 66) and 3-year-old seedlings from Ladysmith and Nakusp (See Table 2 for test temperature and dates) 89 b) Results from analysis of variance for combined data from controlled freezing using 2-year-old seedlings from UBC (day 45) and 3-year-old seedlings from Ladysmith and Nakusp (See Table 2 for test temperature and dates) 89 Table 10 a) Results of analysis of variance Tables for single test dates and locations from controlled freezing using 2-year-old seedlings from UBC (days 45 and 66) and 3-year-old seedlings from Ladysmith and Nakusp (See Table 2 for test temperatures and dates) 90 X b) P r o b a b i l i t y of a larger value for F in orthogonal contrasts among di f f e r e n t combinations of regions at di f f e r e n t test dates and locations.... 91 Table 11 a) Results for analysis of variance for combined data from controlled freezing using 2-year-old seedlings from UBC (day 66) and 3-year-old seedlings from Ladysmith and Nakusp (BC material b) Results from analysis of variance for combined data from controlled freezing using 2-year-old seedlings from UBC (day 45) and 3-year-old seedlings from Ladysmith and Nakusp (BC material only, H Q4) (See Table 2 for test temperatures and dates) 92 Table 12 a) Results from analysis of variance for single test dates and locations from controlled freezing using 2-year-old seedlings from UBC (days 45 and 66) and 3-year-old seedlings from Ladysmith and Nakusp (BC material only) (See Table 2 for test Table 12 b) Summary of probability of differences by source only, H Q4) (See Table 2 for test temperatures and dates) 92 temperature and dates) 94 of v a r i a t i o n for a l l four hypotheses 96 xi Table 13 a) Results from analysis of variance for combined data of the Galena-Moscow hybrid, Galena-Arrow provenance and the US i n t e r i o r seedlots from controlled freezing on 5 test dates using 2-year-old seedlings grown at UBC (See Table 1 for location descriptions, F i g . 7 for pollen source and Table 2 for test temperatures and dates) 97 b) Multiple-comparison testing of provenance means 97 Table 14 Results from analysis of variance for individual measurement dates for needle elongation phenology (cm) on fi v e 2-year-old seedlings per seedlot per block grown at UBC (Day 0 i s March 3/89) 98 Table 15 a) Results from analysis of variance for individual measurement dates for shoot length (cm) 2-year-old seedlings per seedlot per block grown at UBC (Day 0 i s March 3/89) 99 b) Probabil i t y of a larger value for F in orthogonal contrasts amoung d i f f e r e n t xi i combinations of regions at two measurement dates for shoot length (cm) on five 2-year-old seedlings per seedlot per block grown at UBC. (Day 0 is March 3/89) 101 Table 16 a) Results from analysis of variance for injury assessment by a quantitative measure of % injury of t o t a l needles scored on a l l seedlings in both blocks exposed to the uncontrolled freezing in Jan./Feb. 1989 102 b) Results from analysis of variance for injury assessment by shoot length measurements on a l l seedlings in both blocks exposed to the uncontrolled freezing in Jan./Feb. 1989 102 Table 17 Results from analysis of variance for recovery assessment by shoot length difference between May 17, 1989 and July 19, 1989 103 Table 18 Shoot length (mm) measurement of undamaged and damaged seedlings in blocks 1 and 2 for day 65 and 128 104 xi i i LIST OF FIGURES Map of seedlot o r i g i n s (•)>, natural range of western white pine ( s t i p p l e d area) and region delineation (numbers), (modified from Fowells 1965). 105 F i g . 2 Mean needle injury (%) and standard error by region for combined data from c o n t r o l l e d freezing on 5 test dates using 2-year-old seedlings grown at UBC 106 F i g . 3 a) Mean needle injury (%) and standard error of 7 coastal provenances by l a t i t u d e using combined data from 5 dates of c o n t r o l l e d freezing using 2-year-old seedlings grown at UBC... 107 b) Mean needle injury (%) and standard error of 8 i n t e r i o r provenances by l a t i t u d e using combined data from 5 dates of c o n t r o l l e d freezing using 2-year-old seedlings grown at UBC. 107 F i g . 4 a) Mean needle injury (%) and standard error by region for 5 dates of c o n t r o l l e d freezing using 2-year-old seedlings grown at UBC 108 F i g . 4 b) Mean needle injury (%) and standard error showing xi v s i g n i f i c a n t region x temperature interaction for the ind i v i d u a l test day 20 for regions 1 and 2 only 109 F i g . 5 Mean needle injury (%) and standard error by region from c o n t r o l l e d freezing using 2-year old seedlings from UBC (day 66) and 3-year-old seedlings from Ladysmith and Nakusp 110 F i g . 6 a) Mean needle injury (%) and standard error by region from controlled freezing using 2-year old seedlings from UBC (day 45) and 3-year-old seedlings from Ladysmith and Nakusp 111 F i g . 6 b) Mean needle injury (%) and standard error showing s i g n i f i c a n t region x temperature interaction for the Ladysmith regions 1 and 2 only 112 F i g . 7 Location of pollen source from Moscow, Idaho 113 F i g . 8 a) Mean needle injury (%) and standard error by provenance for combined data from controlled freezing on 5 test dates using 2-year-old seedlings grown at UBC. BC-I = Galena-Arrow provenance, Hybrid = Galena-Moscow cross, US-I = Flower, Cr y s t a l and Beaver Creek provenances combined 114 F i g . 8 b) Mean needle injury (%) and standard error by provenance for 5 dates of cont r o l l e d freezing using 2-year-old seedlings grown at UBC. BC-I = Galena-Arrow provenance, Hybrid = Galena-Moscow cross, US-I = Flower, Crystal and Beaver Creek provenances combined 115 Fig. 9 a) Mean size and standard error of needle elongation (mm) by region over 5 measurement dates in 1989 on 5 seedlings per seedlot in block 1 116 Fig. 9 b) Mean size and standard error of needle elongation (mm) by region over 5 measurement dates in 1989 on 5 seedlings per seedlot in block 2 117 xv i ACKNOWLEDGEMENTS I would l i k e to thank Dr. Don Lester for his thoughtful advising and careful revising of thi s thesis, Dr. Mike Meagher for his time and information provided and Dr. John Worrall for his helpful discussions. Many thanks to Dr. Judy Loo-Dinkins for her assistance with the computing, data analysis and encouragement. In addition, thank you to my fellow graduate students, John Russell, David Kolotelo, Greg O'Neill, Marilyn Cherry, George Harper and Donna Robertson for their help in data c o l l e c t i o n . I would also l i k e to thank Dr. Stan Boutin for his support, encouragement and s k i l f u l editing of sections of thi s thesis. PREFACE This study was funded by the Canadian Forestry Service under the Canada-British Columbia Forest Resource Development Agreement (1985-1990) and by the NSERC/Industrial Chair in Forest Genetics and Tree Improvement. 1 1.0 INTRODUCTION Western white pine (Pinus monticola Dougl. ex. D. Don) (Pw) has a wide ecological d i s t r i b u t i o n (Powells 1965), with both a coastal (drier coastal western hemlock zone) and an i n t e r i o r range ( i n t e r i o r cedar hemlock zone) in B r i t i s h Columbia (BC) (Krajina et al. 1982) and western United States (US)(Fig. 1). Although Pw has been devastated by the white pine b l i s t e r rust fungus (Cronartium r i b i c o l a J.C. Fisch. ex. Rabenh.), introduced to Vancouver about 1910, i t has recently been reinstated for commercial use in BC (Hunt 1988, Muir 1988) as i t has many desirable c h a r a c t e r i s t i c s for use as a commercial species. Favourable response to genetic selection for rust resistance has reduced the risk of loss from b l i s t e r rust (Bingham et al. 1972, Bingham 1983) as have s i l v i c u l t u r a l techniques including pruning and hazard rating of potential planting s i t e s (Hunt 1982, Hunt 1983, Hungerford et al. 1982). At present, a consideration to the reintroduction i s how transferable, regarding cold hardiness, seed from d i f f e r e n t seed sources w i l l be within Pw's coast and i n t e r i o r ranges in BC. In general, Pw, r e l a t i v e to many other conifers, has shown r e l a t i v e l y l i t t l e genetic variation in phenology and cold hardiness associated with provenance (Townsend & Hanover 1972, Steinhoff 1979, Rehfeldt 1979, Rehfeldt et al. 1984, Campbell & Sugano 1989). Both Douglas-fir 2 {Pseudotsuga menziesii (Mirb.) Franco) (Df) and grand f i r (Abies grandis [Dougl.] L i n d l ) , which also have coastal and i n t e r i o r d i s t r i b u t i o n s , have shown marked differences in survival and height growth when transferred between these two zones. Rehfeldt (1977), found that coastal Df grew faster than i n t e r i o r Idaho sources in Idaho, but winter survival was very poor. S i m i l a r l y , Steinhoff (1980), found coastal grand f i r to grow twice as fast as i n t e r i o r grand f i r in Idaho. The degree of winter survival was higher, however, than that of the Df. Pw transferred from the coast to the i n t e r i o r (Steinhoff 1981), (12-year-old Olympic Peninsula stock), and from the i n t e r i o r to the coast of BC (Bower 1987) (11-year-old Idaho stock), showed no s i g n i f i c a n t differences in either height growth or winter s u r v i v a l . Isozyme analysis, by Steinhoff et al . (1983) showed that the major genetic variation in Pw i s associated with two l a t i t u d i n a l l y d i s t i n c t subgroups. The Rocky Mountain and northern Cascade ranges form the major subgroup of Pw, with a smaller subgroup in the Sierra Nevada Mountain range. The population of Pw in the southern range of the Cascade Mountains appears to be intermediate between the two subgroups. The small amount of v a r i a t i o n which does occur within the populations north of 44° lat i t u d e appears to be unrelated to geographic variables (Rehfeldt 1979, Steinhoff et al . 1983, Rehfeldt et al. 1984). Further, variation predominantly has been found to be among 3 individuals (Townsend & Hanover 1972, Steinhoff 1979, Campbell & Sugano 1989). Squillace and Bingham (1958), reported ecotypic variation in Pw for both growth rate and seedling establishment. Results discussed above however, provide evidence contrary to Squillace and Bingham's (1958) findings (Rehfeldt 1979). These isozyme and survival studies discussed above support the view that Pw repopulated i t s northern range from a single refugium, possibly in southwestern Oregon, during the l a s t (Wisconsin) g l a c i a t i o n ( C r i t c h f i e l d 1984). Further, the north-south s p l i t in the degree of variation found in t h i s species i s also present in Df which occupies a similar geographic range and which was also subjected to an ice-age refugium ( C r i t c h f i e l d 1984). Rehfeldt and Steinhoff (1970, Rehfeldt 1979, Rehfeldt et al . 1984), suggest that the a b i l i t y of Pw to adapt to these very d i f f e r e n t environments (coast and i n t e r i o r ) i s governed predominately by phenotypic p l a s t i c i t y rather than genetic v a r i a t i o n . The di r e c t r e l a t i o n s h i p between phenotypic p l a s t i c i t y and cold hardiness however, i s only speculative. This study, provides a preliminary examination of t h i s r e lationship. The objective of t h i s study was to address the question of Pw seed t r a n s f e r a b i l i t y , in terms of cold hardiness, between the coast and i n t e r i o r of BC. Controlled 4 c o l d h a r d i n e s s t e s t i n g w a s u s e d t o e s t i m a t e t h e r e l a t i v e d i f f e r e n c e s i n c o l d h a r d i n e s s u s i n g s e e d l i n g s g r o w n f r o m s e e d c o l l e c t e d i n s e v e r a l d i f f e r e n t l o c a t i o n s n o r t h o f 4 4 ° ( F i g . 1 ) l a t i t u d e a n d g r o w n u n d e r n u r s e r y a n d f i e l d c o n d i t i o n s . G e n e t i c d i f f e r e n c e s w e r e e s t i m a t e d a t t h e r e g i o n a l , p r o v e n a n c e a n d f a m i l y l e v e l s . A l t h o u g h f r o s t h a r d i n e s s i s a c o m p l e x p h y s i o l o g i c a l c h a r a c t e r i s t i c , i t h a s a n u n d e r l y i n g g e n e t i c b a s i s ( G l e r u m 1 9 7 5 ) w h i c h m a k e s f r o s t h a r d i n e s s t e s t i n g a u s e f u l t o o l w h e n a d d r e s s i n g q u e s t i o n s o f s e e d t r a n s f e r a b i l i t y . F r o s t h a r d i n e s s i s a l s o a n i m p o r t a n t f a c t o r i n u n d e r s t a n d i n g t h e d i s t r i b u t i o n o f a s p e c i e s . T h e r e s u l t s w i l l h a v e s i g n i f i c a n t m a n a g e m e n t i m p l i c a t i o n s f o r b o t h t r e e b r e e d e r s a n d r e f o r e s t a t i o n p l a n n e r s ( B o n g a r t e n 1 9 8 6 ) . P r e v i o u s l y , f r o s t h a r d i n e s s h a d b e e n t e s t e d u n d e r c o n t r o l l e d c o n d i t i o n s a t o n e t i m e o n l y i n t h e e a r l y p h a s e o f h a r d i n e s s d e v e l o p m e n t . T h e r e s u l t s i d e n t i f i e d a t e n t a t i v e l i n e o f d i f f e r e n c e b e t w e e n t h e c o a s t a n d i n t e r i o r ( R e h f e l d t et al . 1 9 8 4 ) . F u r t h e r , M e a g h e r ( 1988 ) , c i t e d a n g r e a t e r i n d a m a g e t o I d a h o F 2 s e e d l i n g s r e l a t i v e t o l o c a l s e e d l i n g s , a t a p l a n t a t i o n i n B a r r i e r e , B C , w h i c h w a s s u b j e c t e d t o a s e v e r e s p r i n g f r o s t i n 1 9 8 5 . 5 2.0 LITERATURE REVIEW 2.1 PHENOTYPIC PLASTICITY The d i s t r i b u t i o n of a s p e c i e s i s determined by b i o t i c i n t e r a c t i o n s , ( e . g . : c o m p e t i t i o n , p r e d a t i o n , p a r a s i t i s m , d i s e a s e ) , and p h y s i o l o g i c a l t o l e r a n c e to a b i o t i c c o n d i t i o n s (eg:drought, c o l d , h e a t ) . P h y s i o l o g i c a l t o l e r a n c e i s determined by a combination of g e n e t i c v a r i a t i o n i n a s p e c i e s and/or phenotypic p l a s t i c i t y . The r e l a t i v e c o n t r i b u t i o n s of these two components i s o f t e n very d i f f i c u l t to a s s e s s . Phenotypic p l a s t i c i t y , as d e s c r i b e d by Bradshaw (1965), i s a c h a r a c t e r i s t i c of the i n d i v i d u a l . I t i s the a b i l i t y of an i n d i v i d u a l to produce more than one morp h o l o g i c a l a l t e r n a t i v e , or p h y s i o l o g i c a l s t a t e and to measure i t s importance, the same genotype should be grown under d i f f e r e n t environments (normal and s t r e s s e d ) . T h i s process i s s t r a i g h t - f o r w a r d f o r s p e c i e s which can be cloned but i s more d i f f i c u l t with other s p e c i e s where d i f f e r e n t i n d i v i d u a l s from a p o p u l a t i o n must be used. Large numbers of f u l l or h a l f - s i b i n d i v i d u a l s are needed i n these i n s t a n c e s , to compare responses (means) acr o s s environments ( t r e a t m e n t s ) . The c l a s s i c phenotypic p l a s t i c i t y experiment i n 6 p l a n t s i n v o l v e s g r o w i n g t w o o r m o r e t a x a ( c l o n e s , g e n o t y p e s , f a m i l i e s , p o p u l a t i o n s o r s p e c i e s ) i n a s e r i e s o f d i f f e r e n t e n v i r o n m e n t s ( S c h l i c h t i n g 1 9 8 6 ) . M e a s u r e m e n t s o f p h e n o t y p i c c h a r a c t e r s ( l e a f s i z e , n u m b e r o f f l o w e r s , s i z e o f f l o w e r s e t c . , p r o d u c e d i n a g i v e n e n v i r o n m e n t ) a r e t h e n t y p i c a l l y a n a l y s e d b y a n a l y s i s o f v a r i a n c e ( A N O V A ) a n d t h e l e v e l o f p h e n o t y p i c p l a s t i c i t y i s d e t e r m i n e d . A N O V A p r o v i d e s a m e a n s t o s e p a r a t e g e n e t i c a n d e n v i r o n m e n t a l c o m p o n e n t s a n d t h e g e n o t y p e X e n v i r o n m e n t i n t e r a c t i o n c o m p o n e n t s . I n a o n e - w a y A N O V A , a s i g n i f i c a n t t r e a t m e n t e f f e c t i n d i c a t e s t h a t d i f f e r e n t g e n o t y p e s r e s p o n d d i f f e r e n t l y i n o n e e n v i r o n m e n t a n d t h e c h a r a c t e r b e i n g m e a s u r e d i s d e t e r m i n e d t o b e p l a s t i c . I n a t w o - w a y A N O V A a s i g n i f i c a n t e n v i r o n m e n t e f f e c t i n d i c a t e s a p l a s t i c r e s p o n s e a n d a s i g n i f i c a n t g e n o t y p e X e n v i r o n m e n t i n t e r a c t i o n i n d i c a t e s t h a t d i f f e r e n t g e n o t y p e s r e s p o n d d i f f e r e n t l y u n d e r d i f f e r e n t e n v i r o n m e n t s ( t h e p l a s t i c r e s p o n s e s a r e d i f f e r e n t ) . A t p r e s e n t h o w e v e r , t h e r e i s n o t a s a t i s f a c t o r y m e t h o d o f a n a l y s i s t o d i f f e r e n t i a t e b e t w e e n p a t t e r n s o f p l a s t i c i t y v e r s u s a f i x e d g e n e t i c r e s p o n s e w h e n a g e n o t y p e r e s p o n d s t h e s a m e w a y i n d i f f e r e n t e n v i r o n m e n t s . A m o n g t h e m a n y t e c h n i q u e s a v a i l a b l e f o r f u r t h e r a n a l y z i n g s i g n i f i c a n t i n t e r a c t i o n s , (e.g.: e c o v a l e n c e = t h e d e v i a t i o n o f a n i n d i v i d u a l g e n o t y p e f r o m t h e e x p e c t e d m e a n s f o r e a c h g e n o t y p e a n d e n v i r o n m e n t c o m b i n a t i o n , j o i n t r e g r e s s i o n , p e r f o r m i n g a l l p a i r w i s e A N O V A ' s a n d e s t i m a t i n g t h e a v e r a g e c o n t r i b u t i o n o f a g e n o t y p e t o t h e o v e r a l l 7 i n t e r a c t i o n ) , analyses can be performed by p r o f i l e a n a l y s i s (compares p a t t e r n s of the i n d i v i d u a l l i n e segments connecting genotype means i n d i f f e r e n t environments), or Spearmans c o r r e l a t i o n ( r e c o r d i n g changes i n rank of treatments between two genotypes) of treatment means f o r a s i n g l e c h a r a c t e r between two genotypes ( s p e c i e s , p o p u l a t i o n s ) or by p r i n c i p a l component a n a l y s i s ( d e v i a t i o n s from r e g r e s s i o n ) , to determine the amount and d i r e c t i o n of the p l a s t i c response. For c l a s s i c q u a n t i t a t i v e g e n e t i c s t u d i e s with f o r e s t t r e e s , estimates of environmental v a r i a t i o n and f a m i l y X environment i n t e r a c t i o n s can be compared to determine p l a s t i c i t y l e v e l s among taxa ( S c h l i c h t i n g 1986). The use of c l o n a l samples i n c r e a s e s the p r e c i s i o n of the c a l c u l a t e d measures of v a r i a b i l i t y (standard d e v i a t i o n , v a r i a n c e , c o e f f i c i e n t of v a r i a t i o n ) , by c o n t r o l l i n g the amount of within-treatment g e n e t i c v a r i a t i o n (Bradshaw 1965, S c h l i c h t i n g 1986). The b a s i s of phenotypic p l a s t i c i t y i s p h y s i o l o g i c a l changes i n response of the i n d i v i d u a l to f l u c t a t i o n s i n i t s immediate environment ( s t r e s s ) (Bradshaw 1965, Bradshaw & Hardwich 1989). The g e n e t i c system r e s p o n s i b l e f o r the observed p h y s i o l o g i c a l changes i s that which c o n t r o l s development ( S c h l i c h t i n g 1986). The a l t e r e d p h y s i o l o g i c a l response or phenotype i s thought to be e n v i r o n m e n t a l l y s p e c i f i c and not a random s h i f t i n the c h a r a c t e r (Bradshaw 1965). Although the change i s s p e c i f i c i n p a t t e r n and d i r e c t i o n , (Bradshaw & Hardwich 1989) t h i s does not imply 8 that i t i s n e c e s s a r i l y a d a p t i v e and that i t i n c r e a s e s f i t n e s s ( S m i t h - G i l l 1983). However, p l a s t i c i t y i s u s u a l l y thought to a f f o r d p l a n t s an adaptive b a s i s to deal with the changes i n environment experienced by an i n d i v i d u a l genotype (Bradshaw 1965). E x p r e s s i o n of p l a s t i c i t y or changes i n the phenotype, do not have an accompanying g e n e t i c change ( s i n c e the ge n e t i c system i n somatic c e l l s i s g e n e r a l l y f i x e d ) a s s o c i a t e d with that e x p r e s s i o n . F i n a l l y , phenotypic p l a s t i c i t y i s under a d d i t i v e g e n e t i c c o n t r o l and i s , t h e r e f o r e , h e r i t a b l e ( J a i n 1978) and a f f e c t e d by s e l e c t i o n (Bradshaw 1965, Stearns 1983, Bradshaw & Hardwick 1989) . In much of the l i t e r a t u r e , there appears to be a d e s i r e to l i n k phenotypic p l a s t i c i t y to l e v e l s of h e t e r o z y g o s i t y . T h i s i s based on the hypothesis that i f a p o p u l a t i o n or s p e c i e s has low genetic v a r i a t i o n and s e l e c t i o n p r e s s u r e s f l u c t u a t e , only those i n d i v i d u a l s which are p l a s t i c , w i l l s u r v i v e . H i g h l y f i t or s p e c i f i c a l l y adapted i n d i v i d u a l s (e.g.: with s p e c i f i c adaptive gene complexes) may not be present f o r n a t u r a l s e l e c t i o n to act upon. Some s t u d i e s have found g e n e t i c v a r i a t i o n and phenotypic p l a s t i c i t y to be i n v e r s e l y r e l a t e d ( J a i n 1979, S i l a n d e r 1985), but others have found no c o r r e l a t i o n (Scheiner and Goodnight 1984, S c h l i c h t i n g and Lev i n 1984, S c h l i c h t i n g 1986, Macdonald & Chinnappa 1989), or a p o s i t i v e c o r r e l a t i o n (Wilken 1977, S c h l i c h t i n g 1986). 9 T h e r e l a t i o n s h i p b e t w e e n p h e n o t y p i c p l a s t i c i t y a n d g e n e t i c b u f f e r i n g i s a l s o n o t a c l e a r o n e . W r i g h t ( 1 9 3 1 ) , s u g g e s t e d t h a t h i g h a m o u n t s o f p l a s t i c i t y i n a t r a i t m a y d a m p e n t h e e f f e c t s o f s e l e c t i o n . T h e m e a n f o r a g i v e n t r a i t w o u l d b e b u f f e r e d f r o m d i r e c t i o n a l s e l e c t i o n t h e r e b y s l o w i n g t h e p r o c e s s o f p o p u l a t i o n d i f f e r e n t i a t i o n ( B r a d s h a w 1 9 6 5 ) . S t e a r n s ( 1 9 8 3 ) h o w e v e r , h a s s h o w n t h a t i n m o s q u i t o f i s h , a g e a t m a t u r i t y w a s b o t h t h e m o s t r a p i d l y e v o l v i n g t r a i t s t u d i e d a n d t h e m o s t p l a s t i c t r a i t s t u d i e d . H o w e v e r , S t e a r n s ( 1 9 8 3 ) s u g g e s t s t h a t p l a s t i c i t y i n a g i v e n t r a i t m a y c o n f e r s t a b i l i t y o r s t a s i s i n a n o t h e r , t h u s a p p e a r i n g t o b e r e s p o n s i b l e f o r t h e l a c k o f m o r p h o l o g i c a l c h a n g e s o v e r g e o l o g i c a l t i m e i n a g i v e n s p e c i e s . 10 2.2 PLASTICITY AND COLD HARDINESS Phenotypic p l a s t i c i t y i s s p e c i f i c f o r i n d i v i d u a l c h a r a c t e r s and may apply to only a few or many c h a r a c t e r s w i t h i n a given i n d i v i d u a l . As w e l l , the degree of p l a s t i c i t y may vary between c h a r a c t e r i s t i c s and the p l a s t i c i t y of one c h a r a c t e r i s t i c may r e s u l t i n the s t a b i l i t y of another ( S c h l i c h t i n g 1986). Most p h y s i o l o g i c a l r e s e a r c h on phenotypic p l a s t i c i t y has focused on r e l a t i o n s h i p s between d i r e c t environmental cues and the a s s o c i a t e d response. An environmental s t r e s s such as drought, can cause a d i r e c t response i n stomatal c l o s u r e (Bradshaw & Hardwick 1989). How t h i s response v a r i e s f o r a given genotype i n d i f f e r e n t drought environments would be examined to determine the l e v e l of p l a s t i c i t y . C o l d h a r d i n e s s however, i s a response to an i n d i r e c t cue (Bradshaw & Hardwick 1989). P h y s i o l o g i c a l changes a s s o c i a t e d with c o l d h a r d i n e s s (a s t r e s s r e s i s t a n c e mechanism to the s t r e s s of c o l d ) , are a response to the environmental cues of photoperiod and temperature r e s p e c t i v e l y , i n d i r e c t cues of impending s t r e s s (cold) (Bradshaw & Hardwick 1989). The cues are not the s t r e s s i t s e l f i n t h i s case and the r o l e of phenotypic p l a s t i c i t y i n a c o l d h a r d i n e s s response i s unknown. . In a d d i t i o n , how photoperiod a f f e c t s t r a i t s such as 11 dormancy, a p o s s i b l e key component or p a r a l l e l process i n the development of c o l d h a r d i n e s s , i s unknown. Since most re s e a r c h on phenotypic p l a s t i c i t y has focused on the d i r e c t m o r p h o l o g i c a l l y and p h y s i o l o g i c a l l y a l t e r e d c h a r a c t e r i s t i c s (Caswell 1983), i n d i r e c t p h y s i o l o g i c a l responses remain p o o r l y understood. Caswell (1983), d i s c u s s e s a model of dormancy i n r e l a t i o n to p l a s t i c i t y whereby f i t n e s s i s i n c r e a s e d . However, t h i s model has yet to be t e s t e d or s t u d i e d i n terms of the l i f e h i s t o r y of a s p e c i f i c s p e c i e s . 1 2 2.3 COLD HARDINESS AND DORMANCY Timing of a c c l i m a t i o n , the i n c r e a s i n g a b i l i t y to t o l e r a t e c o l d , i s c r i t i c a l to the s u r v i v a l of woody p l a n t s i n the temperate r e g i o n s . T h i s a c c l i m a t i o n process has been w e l l documented as being a secondary r e a c t i o n to changes i n temperature and/or photoperiod (Weiser 1970, L e v i t t 1980, Blum 1988 and o t h e r s ) . However, the r e l a t i o n s h i p between dormancy and f r o s t h a r d i n e s s i s not a c l e a r one and appears to be species-and sometimes p o p u l a t i o n - s p e c i f i c . Weiser (1970), d e s c r i b e s i n d e t a i l , a three-stage a c c l i m a t i o n process f o r many northern deciduous s p e c i e s . Stage ( 1 ) : onset of dormancy induced by a shortened photoperiod, stage (2): f i r s t stage of hardening r e s u l t i n g from low temperatures ( u s u a l l y a f r e e z e ) , and stage ( 3 ) : a c c l i m a t i o n to a phase of winter h a r d i n e s s r e s u l t i n g from -30°C to -50°C temperatures fo r a prolonged p e r i o d . Work by I r v i n g and Lanphear (1967) however, shows that two woody s p e c i e s , Acer negundo L. and Viburnum plicatum tomentosum Thunb., developed f r o s t h a r d i n e s s independently of Weiser's (1970) ' f i r s t stage' dormancy. The dormancy process or c e s s a t i o n i n growth o f t e n 13 a s s o c i a t e d with bud set i n determinant c o n i f e r s , has two stages. The f i r s t stage i s u s u a l l y the r e s t i n g stage d e s c r i b e d as the phase when p l a n t s w i l l not grow even i f c o n d i t i o n s are f a v o u r a b l e . However, t h i s stage i s sometimes preceded by a p e r i o d of quiescence, d e s c r i b e d below, when lammas growth may occur. The r e s t i n g stage i s broken when the c h i l l i n g requirement of the p l a n t i s met. The second phase, c a l l e d quiescence or imposed dormancy, then o f t e n o c c u r s . In t h i s second phase, i f c o n d i t i o n s are f a v o u r a b l e , the p l a n t w i l l grow (Glerum 1975, Weiser 1970). For n o r t h -temperate and b o r e a l f o r e s t t r e e s i t appears t h a t , while the c h i l l i n g requirement i s being s a t i s f i e d i n the f a l l , t r e e s i n c r e a s e i n c o l d h a r d i n e s s to some maximum by e a r l y December (Glerum 1975). F u r t h e r , i f t r e e s are then removed from c o n d i t i o n s not conducive to growth, i t i s o f t e n found they have entered quiescence and w i l l r e a d i l y grow. Although dehardening i s a very r a p i d process r e l a t i v e to development of f r o s t h a r d i n e s s , i t s t i l l t y p i c a l l y l a g s behind the change to quiescence and l a t e r growth. In e a s t e r n l a r c h (Larix l a r i c i n a (DuRoi) K. Koch.), Glerum (1975) found bud break o c c u r r e d i n A p r i l when the new needles s t i l l showed c o l d h a r d i n e s s to -17°C. Young and Hanover (1977) have shown that i n blue spruce (Picea pungens Engelm.) s e e d l i n g s , the phytochrome system i s the mediator between the e x t e r n a l p h o t o p e r i o d i c cue and i n d u c t i o n of dormancy. A new nomenclature has evolved f o r the d e s c r i p t i o n of dormancy, but i s not yet widely used i n the l i t e r a t u r e . The 14 terms used to d e s c r i b e the process of dormancy are p h y s i o l o g y - o r i e n t e d versus season- or phase-o r i e n t e d , thereby i n c r e a s i n g the p r e c i s i o n of the terms. With t h i s system, dormancy i s d e f i n e d as, 'the temporary suspension of v i s i b l e growth of any p l a n t s t r u c t u r e c o n t a i n i n g a meristem' (Lang 1987). Within t h i s d e s c r i p t i o n , the phases d e s c r i b e d above are broken down i n t o 1) endodormancy, when the i n d u c t i o n (of a morphological response) i s s o l e l y w i t h i n the a f f e c t e d s t r u c t u r e , 2) paradormancy, when the i n d u c t i o n (of a morphological response) o r i g i n a t e s i n a s t r u c t u r e other than the a f f e c t e d s t r u c t u r e and the s i g n a l c o u l d be due to a p e r c e i v e d environmental cue or due to the c o n t i n u a l p r o d u c t i o n of an i n h i b i t o r , 3) ecodormancy, when one or more f a c t o r s i n the growth environment are not s u i t a b l e f o r o v e r a l l metabolic growth (Lang 1987). For the purposes of t h i s l i t e r a t u r e review however, I w i l l use the c l a s s i c a l d e s c r i p t i o n s of dormancy when d i s c u s s i n g t h i s phenomenon. Development of f r o s t hardiness or f r e e z i n g t o l e r a n c e ( L e v i t t 1980) i s a complex p h y s i o l o g i c a l process which i s not dependent on a s i n g l e f a c t o r . Thus, the multitude of p o s s i b l e sequences d e s c r i b e d i n the l i t e r a t u r e i l l u s t r a t e s a v a r i e t y of a l t e r n a t i v e processes which have evolved to overcome and s u r v i v e f r e e z i n g temperatures. F u r t h e r , L e v i t t (1980) suggests that m u l t i p l e hardening stages may e x i s t as the temperature c o n t i n u a l l y drops over a p e r i o d of time. Since the same environmental cues p l a y a s i g n i f i c a n t r o l e i n dormancy i n d u c t i o n , the r e l a t i o n s h i p between dormancy and 15 f r o s t h a rdiness remains b l u r r e d . Timmis and W o r r a l l (1974), showed that the f r o s t h a r d i n e s s mechanism i n Df s e e d l i n g s i s l o c a l i z e d . By exposing d i f f e r e n t branches of the same s e e d l i n g to d i f f e r e n t environments, development of f r o s t h a r d i n e s s c o u l d be c o n t r o l l e d i n each branch. In seven c o n i f e r o u s s p e c i e s , Glerum (1973) observed a two-stage (equal to Weiser's (1970) stages one and two d i s c u s s e d above) sequence i n development of f r o s t h a r d i n e s s . The t r a n s i t i o n from one stage to the next occ u r r e d at approximately -18°C over a p e r i o d of a few days. Glerum (1975), suggests that most northern-temperate-zone c o n i f e r s enter dormancy before maximal winter h a r d i n e s s i s a t t a i n e d . Studying 10 provenances of Df, Campbell and Sorensen (1973), found that f o r each a d d i t i o n a l week of bud set p r i o r to the f i r s t f r o s t , there was a p r o p o r t i o n a l decrease of 25% i n s e e d l i n g f r o s t i n j u r y . 16 2 . 4 COLD HARDINESS AND THE ASSOCIATED PHYSIOLOGICAL CHANGES Although the sequence of events l e a d i n g to the i n d u c t i o n of c o l d h a r d i n e s s i s v a r i e d , there are some u n i f y i n g concepts r e g a r d i n g the p h y s i o l o g i c a l and b i o c h e m i c a l changes a s s o c i a t e d with c o l d h a r d i n e s s . The combination of f r e e z i n g avoidance and v a r i o u s t o l e r a n c e mechanisms allows t r e e s , shrubs and herbaceous p l a n t s to s u r v i v e temperate-climate w i n t e r s . P l a n t s growing where temperatures do not f a l l below -40°C may use 'deep s u p e r c o o l i n g ' (-20°C to -45°C) (Burke et al. 1976) to a v o i d f r e e z i n g . P o p u l a t i o n s which grow in regions where temperatures f a l l below t h i s t h r e s h o l d must employ f r e e z e -t o l e r a n c e mechanisms. Su p e r c o o l i n g i s an avoidance mechanism and r e f e r s to the a b i l i t y of l i v i n g c e l l s to remain unfrozen when the surrounding temperature i s below f r e e z i n g (Burke et al. 1976, Burke & Steshnoff 1979). In the absence of a n u c l e a t i n g substance to i n i t i a t e f r e e z i n g , pure water w i l l s u p e rcool to -38°C (Burke et al. 1976, Burke & Steshnoff 1979). At t h i s temperature, r e g a r d l e s s of the presence of a n u c l e a t i n g substance, pure water w i l l c r y s t a l i z e . The l i m i t to deep s u p e r c o o l i n g i s -40°C when spontaneous i n t r a c e l l u l a r f r e e z i n g appears to occur and c e l l s are k i l l e d , probably through mechanical l o s s of c e l l i n t e g r i t y . In c o l d e r b o r e a l c l i m a t e s , the deep s u p e r c o o l i n g avoidance mechanism i s not employed. Tolerance develops through movement of water out of the l i v i n g c e l l s i n t o the e x t r a c e l l u l a r spaces and the stem, where f r e e z i n g occurs (Weiser 1970, Burke et al. 1976, L e v i t t 1980). Concomitant with t h i s r e - a l l o c a t i o n of f r e e , unbound water, are changes in the c e l l membranes. There i s an inc r e a s e i n c e l l -membrane p e r m e a b i l i t y a l l o w i n g water to more r e a d i l y leave the c e l l but at the same time c r e a t i n g a r e l a t i v e l y e f f e c t i v e mechanical b a r r i e r to e x t r a c e l l u l a r i c e p e n e t r a t i o n (Burke et al. 1976). When i c e has formed w i t h i n a t i s s u e , the r a t e of thawing can be c r i t i c a l . When fr e e z e t e s t i n g , s t a r t i n g temperatures should be retu r n e d to at a ra t e of 10-20°C per hour (Glerum 1985). I f thawing i s too r a p i d , the osmoregulation across the c e l l membrane cannot accomodate the change q u i c k l y enough to prevent t i s s u e damage. The i n c r e a s e i n c e l l p e r m e a b i l i t y has been l i n k e d to an in c r e a s e i n p h o s p h o l i p i d s and unsaturated f a t t y a c i d chains i n the c e l l membrane (Graham & Pa t t e r s o n 1982). However, i n woody s p e c i e s the degree of u n s a t u r a t i o n of f a t t y a c i d s seems v a r i a b l e and l i k e l y does not p l a y as b i g a r o l e as i t 18 does i n non-woody s p e c i e s (Glerum 1975, Graham & P a t t e r s o n 1982). C o r r e l a t i o n s have a l s o been found between c o l d h a rdiness and i n c r e a s e s i n sugars, p r o t e i n s and n u c l e i c a c i d s (Burke et al . 1976, L e v i t t 1980, Singh & Laroche 1988, and o t h e r s ) . Aronsson et al . (1976), found that s e e d l i n g s of pine (Pi nus s i l v e s t r i s L.) and spruce (Picea abies (L.) Karst.) hardened by short-day treatments or combined s h o r t -day and lowered temperatures, showed l a r g e i n c r e a s e s i n carbohydrates, e s p e c i a l l y sucrose content, with development of c o l d h a r d i n e s s . In g e n e r a l , these r e l a t i o n s h i p s are f a r l e s s c l e a r d u r i n g dehardening, which may r e f l e c t the f a c t that the process i s much more r a p i d than hardening and t h e r e f o r e a n a l y s i s i s more d i f f i c u l t . Carbohydrate l e v e l s were found, however, to decrease very r a p i d l y i n pine (Pi nus s i l v e s t r i s L . ) , with a p a r a l l e l l e d decrease i n c o l d h a r d i n e s s (Aronsson et al. 1976). At extremely low temperatures, when p l a n t s are i n a s t a t e of 'winter h a r d i n e s s ' , i t i s unknown whether t i s s u e death i s due to spontaneous i n t r a c e l l u l a r f r e e z i n g of bound water, mechanical damage to the membrane from i c e p e n e t r a t i o n or extreme dehydration of the c e l l s (Weiser 1970, Burke et al. 1976, L e v i t t 1980). When the lowest winter temperatures are being experienced, between 30 to 40% of the t o t a l water of a p l a n t i s bound w i t h i n the l i v i n g c e l l s and a l l f r e e water i s f r o z e n e x t r a c e l l u l a r l y . There i s no evidence, however, of a r e l a t i o n s h i p between the amount of bound water and the degree of h a r d i n e s s which can 19 be a t t a i n e d ( C h i l d e r s 1983). At each of three stages i n f r e e z i n g , there i s an exothermic r e a c t i o n when the water f r e e z e s (Weiser 1970). Under slow f r e e z i n g c o n d i t i o n s , these exotherms can be measured through d i f f e r e n t i a l thermal a n a l y s i s (DTA), a l l o w i n g the progress of i c e formation and t i s s u e t o l e r a n c e to f r e e z i n g to be recorded. A f t e r the i n i t i a l s u p e r c o o l i n g has reached i t s maximum, o f t e n -2 to -8°C (Weiser 1970), f r e e z i n g begins. As f u r t h e r water moves out of the protoplasm of the l i v i n g c e l l s and f r e e z e s , a second exotherm can be measured. The t h i r d exotherm i s the k i l l i n g temperature at which a l l remaining water f r e e z e s . Those s p e c i e s which 'deep s u p e r c o o l ' to -40°C would show a d i f f e r e n t p a t t e r n of exotherms with the f i r s t exotherm d e s c r i b e d above not being p r e s e n t . Burr et al. (1985), showed t h a t Df and s e v e r a l spruces which supercool (deep s u p e r c o o l ) , e x h i b i t two exotherms with DTA. They f u r t h e r s t a t e that pines do not supercool and t h e r e f o r e would only show the i n i t i a l exotherm with no low or l e t h a l temperature exotherm. T h i s method would t h e r e f o r e be of l i t t l e use i n c o l d h a r d i n e s s assessment i n p i n e s , i n c l u d i n g Pw. 20 2.5 GENETICS OF COLD HARDINESS Breeding f o r c o l d h a r d i n e s s i s common in a g r i c u l t u r a l crop p l a n t s (Blum 1988). There i s disagreement among breeders as to whether the present l e v e l of winter h a r d i n e s s in hardy v a r i e t i e s can continue to be improved. In t r e e breeding, we are f a r from having exhausted the g e n e t i c p o t e n t i a l , which appears to e x i s t (Sakai & Weiser 1973, Blum 1988), f o r improvement i n c o l d h a r d i n e s s . T h i s c h a r a c t e r i s t i c i s not t y p i c a l l y a p r i o r i t y f o r s e l e c t i o n i n a t r e e improvement program (Glerum 1975). Today's understanding of the g e n e t i c c o n t r o l of the components of c o l d h a r d i n e s s i s s t i l l remarkably l i m i t e d although c o l d h a r d i n e s s has been e x t e n s i v e l y s t u d i e d i n many f i e l d s of p l a n t b i o l o g y (Blum 1988). Singh and Laroche (1988), s t a t e that f o r winter wheat t o l e r a n c e to f r e e z i n g i s e x t r a c e l l u l a r and that t h i s t r a i t i s l i k e l y m u l t i g e n i c . With monosomic wheat s u b s t i t u t i o n l i n e s , i t was found that 70% of f r e e z e t o l e r a n c e was l o c a t e d on chromosome 5A. T h i s type of r e s u l t has l e a d to s t u d i e s attempting to i d e n t i f y and i s o l a t e p o l y p e p t i d e s and analyze those s e c t i o n s of the genome f o r unique hardening sequences. Blum (1988) d i s c u s s e s examples of winter c e r e a l s t u d i e s where a d d i t i v e 21 gene a c t i o n and n o n - a d d i t i v e gene a c t i o n appear to be the g e n e t i c b a s i s f o r c o l d h a r d i n e s s and other s t u d i e s where the a c t i o n of genomes showed no a d d i t i v e e f f e c t s . I s o l a t i n g the g e n e t i c components in f i e l d s t u d i e s of c o l d h a r d i n e s s i n f r u i t t r e e s and small f r u i t s ( e . g . : b l u e b e r r y ) , proved very d i f f i c u l t due to l a r g e year e f f e c t s and l a r g e g e n e r a l combining a b i l i t y X year i n t e r a c t i o n e f f e c t s . Some s t u d i e s however, have shown that c o l d h a r d i n e s s i n apple, peach and raspberry i s q u a n t i t a t i v e l y i n h e r i t e d (Blum 1988). N o r e l l et al . (1986), s t u d i e d the i n h e r i t a n c e of autumn f r o s t h a r d i n e s s i n Pi nus s y l v e s t r i s L. s e e d l i n g s . R e s u l t s showed the environmental and g e n e t i c components to be equal. A d d i t i v e gene a c t i o n accounted f o r the major p a r t of the g e n e t i c component. The w i t h i n - p o p u l a t i o n v a r i a t i o n was l a r g e , suggesting that s e l e c t i o n f o r t h i s t r a i t c o u l d produce a more hardy p o p u l a t i o n . In 4-year o l d s e e d l i n g s , R e h f e l d t (1977), observed weak a d d i t i v e e f f e c t s f o r f r o s t h a r d i n e s s and bud-set i n h y b r i d s of coast and i n t e r i o r v a r i e t i e s of Df. The l i t e r a t u r e suggests that the d i f f e r e n c e s i n mechanisms of s u r v i v i n g c o l d employed by t r e e s versus winter c e r e a l s and the d i f f e r e n t e v o l u t i o n a r y h i s t o r i e s of these p l a n t types, c o u l d account f o r the f a c t that the g e n e t i c v a r i a t i o n a v a i l a b l e i n t r e e s i s much g r e a t e r (Blum 1988). The g e n e t i c s of s u p e r c o o l i n g i n t r e e s i s a l s o not known. 22 2.6 TECHNIQUES TO MEASURE HARDINESS There are many techniques a v a i l a b l e to measure f r o s t i n j u r y as an expre s s i o n of c o l d h a r d i n e s s . T r a d i t i o n a l l y , t r e e breeders have assessed i n j u r y i n f i e l d p l a n t i n g s under n a t u r a l c l i m a t i c c o n d i t i o n s to measure f r o s t h a r d i n e s s . However, s i n c e t e s t years (severe temperatures) t y p i c a l l y occur only once every 10 years, t h i s method i s extremely time consuming and labour i n t e n s i v e (Glerum 1975). F r e e z i n g of s e e d l i n g shoots, branches or leaves i n c o n t r o l l e d environments i s now common p r a c t i c e and d i f f e r e n c e s i n p o p u l a t i o n s or genotypes can be r e a d i l y i d e n t i f i e d . F u r t h e r , c o n t r o l l e d f r e e z i n g a l l o w s a l a r g e number of samples to be screened and t e s t e d over a range of temperatures at any given time. F r e e z i n g can be done i n a c o n t r o l l e d chamber i n the l a b o r a t o r y or, l e s s t y p i c a l l y , with a p o r t a b l e f r e e z e r u n i t where t e s t s are conducted i n the f i e l d (Glerum 1985). 2.6.1 METHODS OF EVALUATION 23 A common method of e v a l u a t i n g f r o s t i n j u r y i s based on l o s s of i n t e g r i t y of the plasma membrane and subsequent l o s s of e l e c t r o l y t e s i n t o surrounding water ( F l i n t et al . 1967). T h i s can be measured q u a n t i t a t i v e l y via a v i t a l s t a i n t e s t , e l e c t r i c a l impedance t e s t s , l e a c h i n g t e s t s or p l a s m o l y s i s t e s t s . Other q u a l i t a t i v e methods of e v a l u a t i o n i n v o l v e a s s e s s i n g t i s s u e d i s c o l o u r a t i o n , growth a f t e r f r e e z i n g s e e d l i n g s (Glerum 1975) or root growth p o t e n t i a l . More r e c e n t l y , c h l o r o p h y l l f l u o r e s c e n c e has a l s o been used to assess i n j u r y s i n c e the t h y l a k o i d membrane, l i k e the plasma membrane, i s a l s o d i s r u p t e d by f r e e z i n g , l e a d i n g to a decrease i n p h o t o s y n t h e t i c a b i l i t y (Singh & Laroche 1988). Glerum (1973, 1975) f u r t h e r suggests that d e t a c h e d - t i s s u e samples (twig or needle) i n p a r t i c u l a r , only w i l l p r o v i d e a measure of r e l a t i v e d i f f e r e n c e s s i n c e they appear h a r d i e r ( s u p e r c o o l i n g to a lower temperature) than do whole s e e d l i n g s when compared with whole s e e d l i n g t e s t i n g of the same s e e d l i n g . T h i s may be due to the l a c k of a n u c l e a t i n g substance, s i n c e the needles are no longer i n c o n t a c t with the p l a n t which may p r o v i d e a n u c l e a t i n g i n i t i a l . Short spruce needles have been shown to supercool to lower temperatures than long needles s i n c e there are more e x t e r n a l n u c l e a t i o n s i t e s (stomata, l e n t i c e l s or wounds) and more water l i k e l y to spontaneously f r e e z e (Burke et al. 1976). F l i n t et al. (1967), d e s c r i b e d the e l e c t r o l y t i c method of f r e e z i n g i n j u r y using ornamental shrubs and b e r r y c r o p s . E l e c t r o l y t e s are measured from unfrozen and f r o z e n t i s s u e as 24 w e l l as from h e a t - k i l l e d samples which provide a reading of t o t a l e l e c t r o l y t e s . They produced equations f o r an 'index of i n j u r y ' with unfrozen t i s s u e samples equal to zero and h e a t - k i l l e d t i s s u e samples equal to 100. One s i g n i f i c a n t advantage of e l e c t r o l y t i c methods i s the r a p i d i t y with which r e s u l t s are obtained r e l a t i v e to v i s u a l assessment methods. T h i s method i s used widely to assess f r o s t h a r d i n e s s as a measure of s t r e s s r e s i s t a n c e i n nursery s e e d l i n g s (Timmis 1975, Glerum 1985). T h i s i s then used as a guide f o r l i f t i n g s e e d l i n g s to minimize s t r e s s and maximize f i e l d s u r v i v a l . Johnson and Gagnon (1988) compared measurement of ethane p r o d u c t i o n to e l e c t r o l y t e leakage using a r t i f i c i a l l y f r o z e n l o b l o l l y pine s e e d l i n g needles. The two methods gave r e s u l t s which d i d not d i f f e r s t a t i s t i c a l l y . The ethane-p r o d u c t i o n method has f u r t h e r advantages, r e q u i r i n g l e s s t i s s u e , l e s s h a n d l i n g and s i n c e i t can be used with any p l a n t t i s s u e . T h i s study suggests that e l e c t r o l y t e leakage t e s t i n g c o u l d be r e p l a c e d o p e r a t i o n a l l y by the ethane-p r o d u c t i o n method as the q u a n t i t a t i v e method of c h o i c e . Glerum (1973) used the e l e c t r i c a l - i m p e d a n c e method to study trends i n f r o s t h a r d i n e s s of seven c o n i f e r o u s s p e c i e s . With t h i s method, two s t e e l p i n s ( e l e c t r o d e s ) are p l a c e d 1 cm apart i n a s e e d l i n g (root c o l l a r or between the t e r m i n a l and f i r s t branch whorl) and e l e c t i c a l impedance i s measured with a 1 kHz b r i d g e . The f i r s t r eading i s taken p r i o r to 25 exposure to f r e e z i n g temperatures and the second i s taken again a f t e r f r e e z i n g (Glerum 1985). The r e s u l t s (Glerum 1973), from a l l s p e c i e s t e s t e d ( e a s t e r n white pine, Pi nus strobus L., red pine, P. resinosa A i t . , jack pine, P. banksiana Lamb., Norway spruce, Picea abies (L.) K a r s t . , white spruce, P. glauca (Moench) Voss, black spruce, P. mariana ( M i l l . ) B.S.P., and e a s t e r n l a r c h , Larix l a r i c i n a (DuRoi) K. Koch.), were that e l e c t i c a l impedance measurements corresponded w e l l with the v i s u a l assessment of f r o s t h a r d i n e s s and subsequent growth. However, accuracy of t h i s method i s i n f l u e n c e d by 1) t i s s u e s i z e , 2) temperature, 3) frequency of the c u r r e n t . F u r t h e r , i f the second impedance reading i s between 20 to 50% reduced from the p r e - f r o z e n measurement, i n t e r p r e t a t i o n i s very d i f f i c u l t . T h e r e f o r e , t h i s method i s i n f o r m a t i v e only at a gross l e v e l f o r i n t e r p r e t a t i o n of no i n j u r y versus i n j u r y , but the degree of i n j u r y can not yet be d e r i v e d . The q u a l i t a t i v e , v i s u a l e v a l u a t i o n i s used o p e r a t i o n a l l y i n n u r s e r i e s where needle, bud and cambial t i s s u e s are examined f o r browning a f t e r c o n t r o l l e d f r e e z i n g (Simpson 1985). R e h f e l d t (1980, et al. 1984) has used t h i s method f o r p o p u l a t i o n s t u d i e s of c o l d h a r d i n e s s with both lodgepole pine (Pinus contort a Dougl.) and Pw u s i n g needle samples. T h i s method i s r e l a t i v e l y inexpensive s i n c e a f r e e z i n g chamber i s the only equipment r e q u i r e d . P r e p a r a t i o n of t i s s u e i s r e l a t i v e l y simple compared to e l e c t r o l y t i c methods, making a l a r g e volume of samples 26 m a n a g e a b l e . S i n c e t h i s m e t h o d r e q u i r e s t h e r e s e a r c h e r t o m a k e a v i s u a l a s s e s s m e n t o f t h e i n j u r y , i t i s n o t f r e e f r o m s u b j e c t i v i t y w h i c h i s r e m o v e d w i t h e l e c t r o l y t i c m e t h o d s . T h e v i s u a l m e t h o d w a s e m p l o y e d i n m y s t u d y . 27 2 . 7 COLD HARDINESS, PHENOTYPIC PLASTICITY AND Pw Most c o n i f e r s whose range i n c l u d e s both a c o a s t a l and i n t e r i o r d i s t r i b u t i o n , show a c o a s t , i n t e r i o r r e g i o n s p e c i f i c i t y (e.g. S t e i n h o f f 1980, R e h f e l d t 1977), p o s s i b l y an a d a p t i v e gene complex, f o r c o l d - h a r d i n e s s response. I n d i v i d u a l s t r a n s f e r r e d from the i n t e r i o r to the coast are t y p i c a l l y i n j u r e d or k i l l e d i n the s p r i n g while i n d i v i d u a l s t r a n s f e r r e d from coast to i n t e r i o r are i n j u r e d or k i l l e d i n the f a l l due to i n c o r r e c t cueing responses. Species with wide l a t i t u d i n a l geographic d i s t r i b u t i o n s tend to evolve g e n e t i c a l l y d i s t i n c t p o p u l a t i o n s which show marked d i f f e r e n c e s i n response to photoperiod (Ekberg et al . 1979). Pw has been d e s c r i b e d as a g e n e r a l i s t i n i t s a d a p t i v e mode ( S t e i n h o f f 1979, R e h f e l d t et al. 1984), i n that i n d i v i d u a l s from coast and i n t e r i o r p o p u l a t i o n s can grow w e l l i n r e c i p r o c a l p l a n t i n g s i n the northern s p e c i e s range (north of 44° l a t i t u d e ) . Although Pw has the f l e x i b i l i t y to e x p l o i t the warm-dry cool-wet continuum w i t h i n i t s e c o l o g i c a l d i s t r i b u t i o n , Pw appears to have a reduced c o m p e t i t i v e advantage on b e t t e r s i t e s on the c o a s t , p o s s i b l y due to a l o s s of genomic s t a b i l i t y ( s p e c i a l i z a t i o n ) (Campbell & Sugano 1989), but competes much more 28 s u c c e s s f u l l y i n i n t e r i o r s i t e s of the same v e g e t a t i o n zone (Tsuga het erophylI a zones of northern Idaho and western Washington) where i t can occur i n pure stands i n the Inland Empire of Idaho (Bingham 1983). Pw i s r e l a t i v e l y slow growing to age 15 years, a f t e r which time i t may grow up to one meter per year becoming the dominant or codominant s p e c i e s i n a stand. T h i s growth p a t t e r n may p l a y a r o l e i n the r e l a t i v e disadvantage on b e t t e r c o a s t a l s i t e s and p l a y l i t t l e or no r o l e i n i n t e r i o r s i t e s s i n c e growth i n g e n e r a l , i s slower. Campbell and Sugano (1989) argue that Pw's present range may not r e f l e c t i t s p h y s i o l o g i c a l p o t e n t i a l and suggest that i t i s merely r e s t r i c t e d to the niches i t i s found i n by competition from more-aggressive s e r a i s p e c i e s . In a study by Campbell and Sugano (1989), 174 Pw f a m i l i e s ( s e e d l i n g progeny) from 143 l o c a t i o n s were grown i n two c o n t r o l l e d nursery environments. They measured growth rhythms and v i g o r (two components of g e n e t i c e xpression) to e v a l u a t e the a d a p t i v e d i f f e r e n c e s among genotypes. T h e i r r e s u l t s showed l i t t l e v a r i a t i o n between l o c a t i o n s and most v a r i a t i o n w i t h i n l o c a t i o n s . T h i s supports f i n d i n g s from e a r l i e r work on Pw by R e h f e l d t et al. (1984) and S t e i n h o f f et al. (1983). Campbell & Sugano (1989) developed three hypotheses to e x p l a i n t h i s g e n e t i c s t r u c t u r e . The f i r s t h y p o t h e s i s i s that white pine may be e x h i b i t i n g an unusual amount of somatic p l a s t i c i t y . Campbell and Sugano (1989) suggest that to e n t e r t a i n the idea of Pw e x p l o i t i n g p l a s t i c i t y i s i n c o n s i s t e n t with h i g h i n d i v i d u a l 29 h e t e r o z y g o s i t y . As d i s c u s s e d e a r l i e r , t h i s may not be the case. R e h f e l d t et al. (1984) suggest that the h e t e r o z y g o s i t y which i s present i n Pw p r o v i d e s a means by which m u l t i p l e b i o c h e m i c a l pathways c o u l d have evolved and that s e l e c t i o n f o r a l l e l e s with broad environmental t o l e r a n c e s c o u l d have o c c u r r e d . So f a r , Pw has appeared to be q u i t e p l a s t i c i n growth r a t e and s u r v i v a l ( S t e i n h o f f 1981, Bower 1987). Campbell & Sugano (1989), f u r t h e r argue that v a l u e s (0.40) of h e r i t a b i l i t i e s they found are a l s o i n c o n s i s t e n t with high p l a s t i c i t y . Again, as s t a t e d e a r l i e r ( J a i n 1978), t h i s a l s o may not be t r u e . The second hypothesis i s that white pine may be occupying s i t e s with environments that are not g r e a t l y d i f f e r e n t from s i t e to s i t e . Campbell and Sugano (1989) argue that Pw i s o f t e n r e s t r i c t e d to unfavourable s i t e s ( i d e n t i f i e d as such s i n c e growth or r e p r o d u c t i o n of the dominant s p e c i e s i s r e s t r i c t e d ) comprising only a small component of the stand ( F r a n k l i n & Dyrness 1973). However, between these s i t e s , t here i s c o n s i d e r a b l e environmental d i v e r s i t y . In Pw's northern range the s i t e s on which i t grows vary from sea l e v e l to 1,500 m on the c o a s t , with up to 280 cm of p r e c i p i t a t i o n and 100-225 f r o s t - f r e e growing days. In the Rocky Mountains, p r e c i p i t a t i o n can vary from 75-150 cm with a 60-160 day f r o s t - f r e e growing p e r i o d and a s i m i l a r e l e v a t i o n a l range as on the coast (Wellner 1962). I t i s impossible to compare Pw's performance i n the niches where i t grows with that of another s p e c i e s or p o p u l a t i o n 30 with a s i m i l a r g e n e t i c p a t t e r n . T h e r e f o r e , to i d e n t i f y i f the s i t e s r e q u i r e Pw to have wide or narrow t o l e r a n c e s f o r the c o n d i t i o n s present, i s a l s o not p o s s i b l e . Hypothesis three i s that white p i n e ' s genome may be emphasising f l e x i b i l i t y ( p l a s t i c i t y ) at the expense of s t a b i l i t y ( s p e c i a l i z a t i o n ) . For t h i s hypothesis to be accepted, Campbell & Sugano (1989) argue that there i s a unique t r a d e - o f f between p l a s t i c i t y and g e n e t i c v a r i a t i o n . P l a s t i c i t y , they suggest, i s being s e l e c t e d f o r to t o l e r a t e broad environmental s t r e s s e s by p r o v i d i n g a f l e x i b l e phenotypic base from g e n e r a t i o n to g e n e r a t i o n . The s e l e c t i o n p r e s s u r e s a c t i n g on the g e n e t i c v a r i a t i o n , they argue, are s h i f t i n g from g e n e r a t i o n to g e n e r a t i o n . Subsequently, any s t a b i l i t y gained i n one g e n e r a t i o n may be l o s t by g e n e t i c d r i f t i n the next. T h e r e f o r e , f l e x i b i l i t y to s u r v i v e whatever new s i t e s the subsequent g e n e r a t i o n might f i n d i t s e l f i n , would be favoured over s t a b i l i t y via becoming h i g h l y adapted f o r a given environment which might not e x i s t f o r the next g e n e r a t i o n . Campbell & Sugano (1989) however, r e j e c t t h i s h y p o t h e s i s because, they argue, Pw's present p h y s i o l o g i c a l t o l e r a n c e s f o r s t r e s s e s such as shade, drought ( F r a n k l i n & Dyrness 1973) and c o l d (Bingham et al. 1972), are i n c o n s i s t e n t with the concept that phenotypic p l a s t i c i t y r e q u i r e s t hat the a l l e l e s present have broad environmental t o l e r a n c e s (Rehfeldt et al. 1984). Campbell and Sugano (1989) conclude that t h e i r study 31 does not supply the evidence r e q u i r e d to d i s t i n g u i s h between these three hypotheses. The r o l e of phenotypic p l a s t i c i t y i n Pw i s n e i t h e r agreed upon nor has i t been e x h a u s t i v e l y researched. The r e l a t i o n s h i p between phenotypic p l a s t i c i t y and c o l d h a r d i n e s s i n Pw i s even l e s s c l e a r . However, my study, i n the process of a d d r e s s i n g the q u e s t i o n s of t r a n s f e r a b i l i t y of seed between the coast and i n t e r i o r of BC, may h e l p to e l u c i d a t e some of the q u e s t i o n s and concerns d i s c u s s e d above. 32 3 . 0 M A T E R I A L S The s e e d l i n g s used i n t h i s study were grown from seeds c o l l e c t e d between 1983 and 1986 throughout the northern d i s t r i b u t i o n of the s p e c i e s . C o l l e c t i o n s i n B r i t i s h Columbia (B.C.) were a mixture of p o p u l a t i o n samples and p a r e n t - t r e e s e l e c t i o n s (Table 1). P o p u l a t i o n samples were obtained from t r e e s chosen on the b a s i s of a v a i l a b l e cones and parent t r e e s were s e l e c t e d based on the judgement of experienced people to s e l e c t r u s t - f r e e t r e e s i n i n f e s t e d stands and f o r these t r e e s to e x h i b i t a c c e p t a b l e stem form and growth in situ (Meagher, p e r s o n a l communication 1990). Seed from the i n t e r i o r U n ited S t a t e s (U.S.) was pro v i d e d by J e r r y Franc, White Pine Species Leader, U n i t e d S t a t e s F o r e s t S e r v i c e , Intermountain F o r e s t and Range Experiment S t a t i o n Moscow, Idaho. These t r e e s are p r e s e n t l y i n the Inland Empire r u s t r e s i s t a n c e breeding program (see Hoff and McDonald (1980) f o r s e l e c t i o n c r i t e r i a ) . Seed from the c o a s t a l U.S. was made a v a i l a b l e through C h a r l e s Gansel, Dorena Tree Improvement Center, Umpgua N a t i o n a l F o r e s t . The re g i o n of c o l l e c t i o n s i s bounded by 44° to 53° nort h l a t i t u d e and 119° to 127° west l o n g i t u d e and 300- to 1220-meter s e l e v a t i o n . 33 The B.C. coast was represented by 20 s e e d l o t s with four f a m i l i e s i n each of f i v e provenances. The B.C. i n t e r i o r was represented by 20 s e e d l o t s with four f a m i l i e s i n each of four provenances and two f a m i l i e s i n each of two provenances. The U.S. coast was represented by four provenances of bulked seed (13 - 20 t r e e s per s e e d l o t ) , and the i n t e r i o r U.S. sample c o n s i s t e d of w i n d - p o l l i n a t e d seed from f i v e g e o g r a p h i c a l l y separated t r e e s . Four f a m i l i e s of h y b r i d s e e d l o t s from c r o s s e s between i n t e r i o r B.C. females and Moscow, Idaho p o l l e n were a l s o i n c l u d e d . (See Table 1 f o r l o c a t i o n d e s c r i p t i o n s and F i g . 7 f o r l o c a t i o n of Moscow, Idaho p o l l e n . ) S e e d l i n g s were grown at Canadian P a c i f i c F o r e s t Products L t d . (CP) i n Saanichton, B.C. i n the s p r i n g of 1988. Two hundred seeds from each se e d l o t were s t r a t i f i e d f o r germination i n May 1988, by soaking i n water f o r 48 hours f o l l o w e d by maintenance at room temperature f o r 30 days, then at 2°C f o r 60 days. They were grown i n Leach f i r c e l l s i n a p e a t : v e r m i c u l i t e medium under standard nursery p r a c t i c e immediately a f t e r g ermination. Trays were seeded to achieve 50 s e e d l i n g s per s e e d l o t per t r a y . S e e d l o t s were randomized w i t h i n t r a y s and t r a y s were randomized on the bench twice over the f i r s t f i v e months of growth ( J u l y 5, 1988 and August 15, 1988). Due to unexpectedly low germination, many s e e d l o t s had l e s s than 200 s e e d l i n g s and the o r i g i n a l experimental design was reduced from four 34 blocks to two b l o c k s . S e e d l i n g s were t r a n s f e r r e d to the UBC South Campus nursery on October 25, 1988 and November 3, 1988. Se e d l i n g s were t r e a t e d f o r fungal gnats on November 8, 1988 and Fusarium spp. on November 29, 1988 (See Appendix A f o r compounds a p p l i e d ) . On January 28, 1989, t r a y s numbered 1 to 26 were re p o t t e d and t r a y s 27 to 88 were completed on February 17, 18 and 19th, 1989. S e e d l i n g s were r e p o t t e d i n t o Styro 20 styrofoam t r a y s using the P a c i f i c F o r e s t r y Center (PFC) recommended mix (Appendix B). S e e d l i n g s were removed from t h e i r c e l l s and p l a c e d i n t o a p a r t i a l l y f i l l e d p l u g h o l e , and the medium was tamped around each s e e d l i n g . When a l l c e l l s were f i l l e d , the mix was topped up and a medium-grade h o r t i c u l t u r a l q u a l i t y g r a v e l was p l a c e d oh top to reduce weed establishment and h e l p to r e t a i n moisture. F e r t i l i z a t i o n commenced May 1, 1989 using 3 gms of 20-20-20 N-P-K per 6 l i t e r s of water 2-3 times per week throughout the growing season u n t i l August 20, 1989. FeSO^ was a l s o a p p l i e d on June 2, 1989 and J u l y 5, 1989 at a rate of .93 gms per 6 l i t e r s of water based on PFC greenhouse s t a f f recommendations. In order to p r o t e c t the new growth from b l i s t e r r u s t i n f e c t i o n , s e e d l i n g s were sprayed with Captan on August 19, 1989. Throughout the growing season and 35 e a r l y f a l l , s e e d l i n g s were maintained on metal bars supported by 45 cm cement b l o c k s . On November 30, 1989 s e e d l i n g s were p l a c e d on the ground to reduce the p r o b a b i l i t y of root f r e e z i n g . The o r i g i n a l experimental design c o n t a i n e d two bl o c k s with the 53 s e e d l o t s per block randomly arranged with the f o l l o w i n g s t r u c t u r e : 1) 5 s e e d l i n g s per s e e d l o t per block f o r phenology measurements 2) 15 or 20 s e e d l i n g s per s e e d l o t per block f o r c o l d hardiness t e s t i n g of needles 3) 0 or 28 s e e d l i n g s per block f o r c o l d h a r d i n e s s t e s t i n g of whole s e e d l i n g s Due to time and weather c o n s t r a i n t s i t was not p o s s i b l e to complete a l l the r e p o t t i n g w i t h i n a few days. During the r e p o t t i n g p e r i o d , the minimum temperature dropped to -10°C but on average was -4.3°C f o r approximately two weeks ( C l i m a t a l o g i c a l S t a t i o n Report, Vane. UBC). A l l s e e d l i n g s were on the ground, however, where the temperature d i f f e r e n c e from that recorded c o u l d range from 4 to 8°C lower. I t became obvious i n e a r l y May that those s e e d l i n g s not r e p o t t e d before the temperature drop i n January, were su b j e c t e d to harsher c o n d i t i o n s i n the u n i n s u l a t e d f i r c e l l s and were s e v e r e l y damaged, delayed i n bud f l u s h , or k i l l e d b y r o o t f r e e z i n g . 3 6 T o p r o v i d e a w i d e r b a s e f r o m w h i c h t o m a k e g e n e t i c i n f e r e n c e s , t h e p r o j e c t w a s e x p a n d e d f r o m o n l y t h e U B C t e s t s t o c k t o i n c l u d e t h e s a m e p o p u l a t i o n s a n d f a m i l i e s f i e l d p l a n t e d a t t w o a d d i t i o n a l l o c a t i o n s . T h e a d d i t i o n o f t h i s m a t e r i a l a l l o w e d t h e e x p e r i m e n t a l d e s i g n t o i n c l u d e t h e t e s t i n g o f g e n e t i c a l l y c o m p a r a b l e m a t e r i a l f r o m t h r e e d i f f e r e n t e n v i r o n m e n t s . T h e f i e l d l o c a t i o n s w e r e a c o a s t a l p l a n t a t i o n i n L a d y s m i t h , B C ( l a t i t u d e 4 8 ° 5 9 ' , l o n g i t u d e 1 2 3 ° 4 9 ' ) a n d a n i n t e r i o r p l a n t a t i o n l o c a t e d a t N a k u s p , B C ( l a t i t u d e 5 0 ° 1 5 ' , l o n g i t u d e 1 1 7 ° 4 8 ' ) . B o t h s i t e s w e r e p l a n t e d i n 1 9 8 8 a s p a r t o f t h e F o r e s t r y C a n a d a R u s t R e s i s t a n c e I m p r o v e m e n t P r o g r a m . P e r m i s s i o n t o s a m p l e t h e 1+2 s t o c k w a s g i v e n b y D r . M i k e M e a g h e r o f P F C . T h e U B C l o c a t i o n w a s t h e U B C n u r s e r y ( l a t i t u d e 4 9 ° 1 6 ' , l o n g i t u d e 1 2 3 ° 1 5 ' ) , w h e r e t h e s e e d l i n g s , a s d i s c u s s e d a b o v e , w e r e g r o w n a n d r a i s e d a s c o n t a i n e r n u r s e r y s t o c k ( 1 + 1 ) . 3 7 4.0 METHODS AND EXPERIMENTAL DESIGN 4.1 COLD HARDINESS T h e f i e l d t e s t s w e r e s a m p l e d o n c e o n a d a t e c h o s e n t o a p p r o x i m a t e t h e d a t e o f t h e f i r s t f r o s t ( 3 0 - y e a r a v e r a g e ) f o r e a c h l o c a t i o n . F r e e z e t e s t i n g o f m a t u r e n e e d l e s a m p l e s a t t h e t i m e w h e n f r o s t i s e x p e c t e d a n d w h e n r a p i d h a r d e n i n g c o m m e n c e s ( W e i s e r 1 9 7 0 ) p r o v i d e s a n e s t i m a t e o f t h e r e l a t i v e g e n e t i c v a r i a t i o n b e t w e e n s a m p l e s ( R e h f e l d t et al. 1 9 8 4 ) . F o r N a k u s p , t h i s d a t e w a s O c t . 3 a n d n e e d l e s w e r e c o l l e c t e d o n S e p t . 3 0 a n d O c t . 1 , 1 9 8 9 . T h e d a t e o f c o n t r o l l e d f r e e z i n g w a s O c t . 6 , 1 9 8 9 . L a d y s m i t h d o e s n o t h a v e i t s o w n w e a t h e r s t a t i o n a n d t h e D e p a r t u r e B a y S t a t i o n 3 0 - y e a r a v e r a g e d a t e o f f i r s t f r o s t w a s N o v . 1 4 . S a m p l i n g o c c u r r e d o n O c t . 3 1 , 1 9 8 9 , a n d t h e d a t e f o r t h e c o n t r o l l e d f r e e z i n g w a s d e l a y e d t o N o v . 1 4 , 1 9 8 9 . T h e d e l a y w a s d u e t o t e s t i n g a t t o o h i g h o f t e m p e r a t u r e s t o d e t e c t i n j u r y o n a p r e v i o u s r u n c a r r i e d o u t o n N o v . 1 , 1 9 8 9 . T h e t e s t t e m p e r a t u r e s o f t h a t r u n w e r e b a s e d o n r e s u l t s f r o m t h e p r e v i o u s U B C r u n ( O c t . 2 2 ) , h o w e v e r , t h i s w a s n o t a g o o d i n d i c a t o r f o r t h e L a d y s m i t h m a t e r i a l , p o s s i b l y d u e t o t h e 3 8 t i m i n g of f r o s t i n each area and d i f f e r e n c e i n sampling days. For the UBC stock, h a r d i n e s s was t e s t e d at f i v e d i f f e r e n t dates chosen to sample the range from pre-hardening to e a r l y - w i n t e r h a r d i n e s s . Test temperatures were lowered f o r each s u c c e s s i v e c o l l e c t i o n (see Table 2 f o r sampling and t e s t d a t e s ) . P r e - t e s t i n g , to i d e n t i f y the d e s i r e d temperatures, was conducted using 1 0 s e e d l o t s from a c r o s s - s e c t i o n of o r i g i n s . The temperature range f o r t e s t i n g was t a r g e t e d at the temperature r e s u l t i n g i n an average of 5 0 % i n j u r y ( L T 5 Q ) with a maximum range of 1 0 ° C to capture the range of 0 - 1 0 0 % i n j u r y ( Rehfeldt 1 9 8 0 ) . To assess c o l d h a r d i n e s s throughout the f a l l , needle samples were c o l l e c t e d and t e s t e d i n a programable f r e e z e r . On each t e s t date, four needles from the top 1 / 3 of the shoot, were c o l l e c t e d from each of 1 0 d i f f e r e n t s e e d l i n g s from each s e e d l o t and mixed to form a bulk c o l l e c t i o n . A t o t a l of 4 0 needles was c o l l e c t e d per se e d l o t per run. Except f o r the f i r s t c o l l e c t i o n at UBC, the 1 0 needles used f o r each of four temperatures (three f r e e z i n g temperatures and one c o n t r o l ) were d i v i d e d randomly i n t o e i g h t l o t s of f i v e and p l a c e d i n t o p r e l a b e l l e d p l a s t i c z i p l o c k bags. Bags were taped to cardboard sheets f o r suspension i n the f r e e z e r . T h i s method allowed f o r maximum u n r e s t r i c t e d a i r movement around each bag. A l l samples were then p l a c e d i n a c o l d chamber (0°C to +5°C) f o r 12-15 hours. The f o l l o w i n g day, c o n t r o l needles remained i n the c o l d chamber while s i x of the e i g h t bags per s e e d l o t were p l a c e d i n the f r e e z e r with bags randomized w i t h i n a t e s t temperature. A f t e r an i n i t i a l 30 minutes to 1 hour s t a b i l i z a t i o n p e r i o d at 3°C, the temperature was lowered 5°C per hour to a p r e - s e t t e s t temperature f o r 1 hour. One complete set of samples was then removed. T h i s process was repeated f o r two more t e s t temperatures. A f t e r removal from the f r e e z e r , a l l samples were immediately p l a c e d back i n t o the c o l d chamber f o r 24 hours to reduce shock of recovery from f r e e z i n g and to allow a l l samples to e q u i l i b r a t e to the same c o n d i t i o n s p r i o r to e x p r e s s i o n of i n j u r y . Needles were kept i n the dark from the time of a l l o c a t i o n i n t o the bags u n t i l p o s t - t e s t i n g e q u i l i b r a t i o n i n the c o l d room. A f t e r removal from the c o l d room, samples were p l a c e d on a t a b l e , exposed to d a y l i g h t and h e l d at approximately 18°C. The samples were turned over at f i v e days to promote even l i g h t exposure and subsequent e x p r e s s i o n of i n j u r y . At one week to 10 days a f t e r f r e e z i n g , each needle was scored f o r presence and s e v e r i t y of i n j u r y ( R e h f e l d t 1980, R e h f e l d t et al. 1984). I n j u r y was measured as percent damage ( l e n g t h of brown t i s s u e on each needle d i v i d e d by the t o t a l l e n g t h of the needle and m u l t i p l i e d by 100). Supplementary f r e e z i n g t e s t s had shown that e x p r e s s i o n of i n j u r y was most e a s i l y observed a f t e r approximately 10 days. See Table 2 f o r the t e s t 40 temperatures employed at each test date. At Ladysmith and Nakusp, two intact needle f a s c i c l e s were c o l l e c t e d from each of ten seedlings per seedlot; one f a s c i c l e for testing and one for reserve material. These c o l l e c t i o n s of ten were bulked into two c o l l e c t i o n bags per seedlot. Samples were then transported in a portable cooler containing a freezer pack, styrofoam insulation shelf and a -20° to 100°C thermometer. Samples were maintained at approximately 10°C for a maximum of 36 hours before placement into the 0-5°C cold room. Collections were then divided into the appropriate bags for freeze testing as described above. After measuring injury on each needle, a mean value was calculated per bag. This resulted in two values for each test temperature and seedlot to be entered into the respective linear model (see Appendix C for hypotheses, linear models and expected mean squares). Since the average values for injury f e l l between 25% - 75% in most instances, an angular transformation was not necessary (Sokal & Rohlf 1981) . 4.2 PHENOLOGY AND DAMAGED MATERIAL 41 Phenology data consisted of both shoot elongation measurements and needle elongation measurements on five seedlings in each of two blocks. A baseline bud length was taken on March 12, 1989, with measurements of elongating shoots and needle f a s c i c l e s commencing on A p r i l 18, 1989 and continuing every 10 days u n t i l May 5, 1989. Measurements were taken to the nearest mm. At th i s time i t became apparent that d i f f e r e n t i a l frost damage had occurred across the two blocks and a l l seedlots. Due to high mortality, d i f f e r e n t i a l recovery and exposure to an uncontrolled 'frost treatment', i t was decided that controlled frost-hardiness treatments should be r e s t r i c t e d to the remaining 'undamaged' seedlings only. Thirty-seven seedlots had a minimum of 20 uninjured seedlings. These seedlots constituted the core stock for the cold hardiness t e s t i n g . However, to get a measure of injury from uncontrolled f r o s t , (although unquantifiable), a complete measurement of bud and shoot lengths and a scoring for needle damage was carr i e d out on a l l seedlings and seedlots May 17, 1989. A second measurement of shoot lengths was carried out on July 19, 1989, to assess recovery. Since there were too few seedlings to meet experimental design requirements for whole-seedling testing, t h i s portion of the project was eliminated. To f a c i l i t a t e the c o l l e c t i o n of needle samples on the remaining 37 seedlots, seedlings were reorganized into a single block of 20 seedlings per seedlot in late Aug. 1989. Unbracketted seedlots in Table 1 are the 37 seedlots used for cold-hardiness testing; F i g . 1 shows seedlot o r i g i n s . 43 5.0 DATA ANALYSIS 5.1 CONTROLLED FREEZING Data analysis from needle testing consisted of two main sections. Section one included a l l UBC grown samples tested over time and section two included a single UBC sample and the two f i e l d samples. Each section was further divided according to the testing structure. Appendix C describes the hypotheses and gives the respective linear models followed by EMS tables and estimates for each hypothesis. For section one,, regional-level analysis included a l l samples using combined data from a l l test temperatures. This was carried out i n i t i a l l y with a l l test dates combined, then further analyzed using only data from single test dates. Regional means for the UBC stock were compared by a Duncan's New Multiple Range Test when a l l test dates were included and by orthogonal contrasts for individual-date analysis. Orthogonal contrasts were also used to compare regional means and location means in section two. 44 Only B.C. seedlots were included at the provenance-within-region and family-within-provenance lev e l s of analyses for both sections. This was due to the small sample size and lack of comparable sampling design in the U.S. material. These analyses were i n i t i a l l y done again with a l l test dates included and then with single test dates only. For section two, data from Nakusp and Ladysmith plantations were analyzed with data from the day 66 test and day 45 test on stock grown at UBC. See Table 2 for 30-year average f i r s t frost values, test days and test temperatures. The expected mean square (EMS) values, calculated for each hypothesis (Appendix C), were used to program the correct denominator in the analysis of variance t e s t s . In a l l analyses, region, date, and temperature were fixed factors and location, provenance and family were considered random factors. Temperature was nested in date or location for the appropriate model and family was nested in provenance which was nested in region. Regression analysis was done on the provenance means (%needle injury) for the combined UBC data by l a t i t u d e and by elevation i n d i v i d u a l l y and with both factors included in the model, for the coastal and i n t e r i o r regions, 45 respectively. Those seedlots which spanned an elevational range and overlapped other seedlots were l e f t out of the analysis. See Table 1 for elevation of seed source. The s t a t i s t i c a l analyses was carried out using SAS (1985) on the Michigan Terminal System (MTS) mainframe computing f a c i l i t i e s at UBC. The type III sums of squares were most appropriate with the Proc GLM SAS procedure due to unequal sample sizes and missing values. 5.2 HYBRIDS The hybrid seedlots were analyzed with the US i n t e r i o r seedlot r e s u l t s , which represented the pollen source, and the Galena-Arrow seedlots, which represented the provenance from which the cone parent originated. Analysis was carried out at the provenance l e v e l only with three seedlots per provenance and data were combined over the five f a l l test dates. 5.3 PHENOLOGY Analysis of the phenology data was r e s t r i c t e d to the 46 damaged material only. Six seedlots were not included in the analysis since they were no longer represented equally in both blocks having been subjected to d i f f e r e n t conditions in Jan./Feb. 1989. The six seedlots are marked in Table 1 with # signs with the remaining 47 seedlots being analyzed. Due to injury of the seedlings used for phenology analysis, the objective of evaluation changed to evaluation of genetic differences in recovery from freezing. This evaluation was expanded in section 5.4. Since regional differences in shoot length were po t e n t i a l l y confounded with regional differences in frost injury, means by region were calculated for the shoot length of those seedlings which were not damaged (Table 18). 5.4 UNCONTROLLED FREEZING Estimation of genetic differences in response to uncontrolled freezing was r e s t r i c t e d to damaged material only. For block 1 of the experimental design, there was a maximum of fi v e seedlings for 27 seedlots, a maximum of 28 seedlings for six seedlots and a maximum of 53 seedlings for the remaining 20 seedlots. In block 2, one of the above 27 seedlots had no damaged seedlings and the remaining 26 seedlots had a maximum of five seedlings. One seedlot had a 47 maximum of 33 seedlings and the remaining 25 seedlots had a maximum of 53 seedlings for analysis. For the data c o l l e c t e d on May 17, 1989, values for percent injury were calculated for each seedlot. Measurement of injury was based on a quantitative scale of 1-4 with 1 = 0-25% injury, 2 = 26-50% injury, 3 = 51-75% injury and 4 = 76-100% injury. Shoot length measurements were also taken for regional analysis at one date and for comparison with shoot length measurements of the same seedlings on July 19, 1989. This comparison was used for a damage-recovery assessment to see i f seedlots from the di f f e r e n t regions showed any d i f f e r e n t i a l a b i l i t y to recover. May values were subtracted from the July 19, 1989 shoot measurement and analysis was based on the difference in shoot length rather than absolute shoot length. Analysis of variance was done for both shoot length and needle damage with the May data. Only block and regional effects were analysed. An ANOVA on differences in shoot length was done for comparison of May to July measurements. Again, t h i s was at the block and regional lev e l s only. See Table 3c for damage analysis sample size s . A l l standard errors presented in thi s thesis are standard errors of the means for % needle injury. 48 6.0 RESULTS The results from cold-hardiness testing showed a difference (p<0.0l), between the BC coast and BC i n t e r i o r sources in a l l test runs, excluding the f i r s t UBC run and the Ladysmith run. Where regions d i f f e r e d s i g n i f i c a n t l y , the difference in percent damage response of needles to freezing was approximately 20%. Test temperatures used on UBC-grown stock dropped from a mean of -9°C in early September, 1989, to a mean of -30°C in late November (Table 2). Over the course of the five test dates, for the UBC stock, mean injury by region varied from 38 to 84% for day 0, 10 to 78% for day 20, 32 to 68% for day 45, 40 to 67% for day 66 (two test temperatures only), and 36 to 79% for day 80. The results from the uncontrolled freezing are not clear, but largely support the general trends regarding regional differences found with the controlled-freezing data. Section I. 6.1 COLD HARDINESS OVER TIME AT ONE LOCATION. 49 6.1.1 A l l dates combined, a l l r e g i o n s , Hypothesis 1 (Appendix C) Analysis of data combined from five test dates for seedlings grown at UBC, showed that regions d i f f e r e d (p<0.00) in response to controlled freezing (Fig. 2, Table 4a). Values presented in Figure 2 are means of percent damage for combined test dates. Since test temperatures varied for each date, the absolute damage values for each test date could not be compared across test dates. Region 1 represents the BC coast, region 2 represents the BC i n t e r i o r and regions 3 and 4 represent the US coast and i n t e r i o r respectively. For a l l dates, regional comparisons were performed both l a t i t u d i n a l l y and lo n g i t u d i n a l l y . Within BC, longitudinal comparisons of coast and i n t e r i o r were s i g n i f i c a n t l y d i f f e r e n t (p<0.0l), and within the US, coast and i n t e r i o r were not d i f f e r e n t (Table 4b). The US results may be a consequence of small sample s i z e . Further, l a t i t u d i n a l comparisons showed that the BC coast and i n t e r i o r provenances were d i f f e r e n t from the US coast and i n t e r i o r regions combined (again t h i s may be due to the small sample size for the US). This result supports the observations by Meagher (1988) that Idaho stock in i n t e r i o r BC may be less hardy than the l o c a l seedlings. 50 Although differences associated with latitude were found, no clear l a t i t u d i n a l trend in hardiness was evident (Fig. 3 a,b). Regression analysis by region (general model Y^. = a + bX^ . + e^ . , where: Yf. =% injury, a=estimate of the intercept parameter, b=estimate of slope parameter, X^. =value for l a t i t u d e or elevation, e^ . =unexplained variance associated with Y^. (random error)) for latitude showed a 2 very poor f i t for both coast (R =0.19) and i n t e r i o r regions 2 (R =0.02) (Table 5a(i) & b ( i ) ) . S i m i l a r l y , plots of injury by elevation of provenance did not reveal any trends (Table 5 a ( i i ) & b ( i i ) ) . Further, the results from regression performed with both elevation and lati t u d e included in the model did not show any relationship to percent injury (Table 5c(i) and ( i i ) . For the coastal regression on elevation and 2 latit u d e R =0.11 and for the i n t e r i o r regression on 2 elevation and lati t u d e R =-0.17. Since values for neither lat i t u d e nor elevation include zero, the non-zero intercept value present in a l l analysis has no i n t r i n s i c value (Neter et al. 1985). The Salmo provenance (Table 1), was not included in the elevation regression since the c o l l e c t i o n spanned 1000 to 1870 meters which included two other provenances (Table 1). The poor f i t of cold injury with geographic variables of seed o r i g i n i s similar to results reported e a r l i e r for other t r a i t s (Townsend & Hanover 1972, Campbell & Sugano 1989). 51 6.1.2 Individual dates, a l l regions The regional difference between BC coast and BC i n t e r i o r was shown to be consistent (Fig. 4, Table 6a). A more detailed regional analysis shows that days 20, 45, 66 and 80 a l l show differences (p<0.0l) between the BC coast and BC i n t e r i o r (Table 6b(3)) with no s i g n i f i c a n t difference between BC and US seedlots (Table 6b(2)). Days 20 and 80 also show a difference (p<0.05) between coast (BC and US) versus i n t e r i o r (BC and US) (Table 6b(1)). Although results from day 0 (Fig. 4) appear to support t h i s trend, needle samples had not been divided into two replicates for testing and thus only one mean value was available for analysis for each test temperature. The precision of testing on day 0 was therefore reduced; no s t a t i s t i c a l l y s i g n i f i c a n t differences were found (Table 6a & b). Further, t h i s test was conducted September 7/89, prior to the time of expected maximal expression of genetic differences for cold hardiness. (Difference in the day 0 analysis should be kept in mind for the following two sections). However, a p r o b a b i l i t y of 0.12 for BC coast versus BC i n t e r i o r suggests that the differences are real (Table 6b(3)). The difference in the mean regional value for the BC coast versus the BC i n t e r i o r i s approximately 9% for day 0 versus 20% on average for the subsequent 4 test days. 52 6.1.3 A l l dates, BC re g i o n s , Hypothesis 2 (Appendix C) This analysis required more computing power than was available for SAS on the MTS mainframe system at UBC. Therefore, to examine a l l components in the model, analysis was divided into two sections. Table 7a gives the results from the analysis with the highest order interaction term included. For th i s term, the probability of real effects was 0.15 and t h i s source of variation was then added to the error term, reducing the complexity of the analysis and allowing complete analysis of a l l other components in the model to be done (Table 7 b ) . I n order for the program to run with the highest order interaction term, f i v e terms had to be l e f t out from d i r e c t analysis, being added to the sums of squares and degrees of freedom for other terms. In so doing, F-test analysis denominators (EMS's) changed between the two analyses. This had a direct effect only on region mean si g n i f i c a n c e . In the f i r s t analysis, P=0.03 for region but was P=0.11 for region in the second analysis. The F(R P) component may have been an i n f l a t e d term for testing of R since the P(R) component was incorporated into i t s 2 value. The assumption was P(R) 6 =0, meaning no contribution to the 'error' term for R. Provenances-within-coast and - i n t e r i o r regions did not 53 d i f f e r s t a t i s t i c a l l y (p=0.1l), although coastal provenances d i f f e r e d by up to 28% (45% to 73%) and i n t e r i o r provenances by 35% (30% to 65%). Families-within-provenances did d i f f e r (p<0.0l) (with both analyses), ranging between 4 and 41% for the coast and by 15 and 42.5% for the i n t e r i o r . The minimal number of families-within-provenances and v a r i a b i l i t y of families-within-provenances could account for f a i l u r e to detect s t a t i s t i c a l l y s i g n i f i c a n t differences among provenances despite the substantial range in provenance means. 6.1.4 I n d i v i d u a l dates, BC r e g i o n s With analysis by individual freezing date, regions d i f f e r e d for day 80 (p<0.0l) and 45 (p<0.05), but did not d i f f e r for days 0 and 66 (Table 8). It i s l i k e l y that day 0 results made a substantial contribution in reducing the significance of o v e r a l l regional e f f e c t s discussed above. For day 66, temperature was not s i g n i f i c a n t due to only two test temperatures (Table 2) being analyzed and t h i s l i k e l y contributed to the lack of significance being observed for regional differences for day 66. The differences in test temperatures appear to have been i n s u f f i c i e n t in showing differences in regional performance. Only day 0 data showed differences for provenance-within-region (p<0.05). Family-within-provenance d i f f e r e d (p<0.00) for a l l days (Table 8). 54 Data from day 20 (Table 8) also showed a s i g n i f i c a n t region x temperature interaction term, shown in F i g . 4b. Region 1 (coast) shows r e l a t i v e l y more injury at the colder temperatures than does region 2 ( i n t e r i o r ) , but exhibits consistently more injury than region 2 (Fig 4b) at a l l temperatures. With a s i g n i f i c a n t interaction, the s i g n i f i c a n t main ef f e c t for day 20 has no meaning. Section II. 6.2 COLD HARDINESS AT THREE LOCATIONS. 6.2.1 A l l locations, a l l regions, Hypothesis 3, (Appendix C) Results from the two f i e l d test locations and one c o l l e c t i o n at UBC (Figs. 5 & 6) further support the findings in Figs. 2 and 4. Data from day 66 at UBC were chosen because they most cl o s e l y matched the c r i t e r i o n for choosing c o l l e c t i o n dates at the two f i e l d s i t e s (i.e. h i s t o r i c a l date of f i r s t f r o s t ) . With a l l location and regional data included, there were regional differences (p<0.00, Table 9a). There was however, a region x location interaction 55 effe c t (p<0.05) (Fig. 5, Table 9a). When the US data were omitted, t h i s interaction was no longer present (Table 11a). Although sampling dates were chosen to coincide with the expected maximal expression of genetic v a r i a t i o n , i t is obvious from Table 2 that i t was not possible to show differences in response among seedlots through testing them at the same temperatures. With day 45 (UBC), used in the combined analysis, replacing day 66, regions d i f f e r e d (p<0.00) and the region x location interaction term was s i g n i f i c a n t (p<0.0l, F i g . 6a, Table 9b). This interaction was not s i g n i f i c a n t however, (p<0.lO) with the US material removed (Table 11b). 6.2.2 Individual locations, a l l regions UBC and Nakusp data showed that regions d i f f e r e d (P<0.00, Table 10a). Region did not d i f f e r s i g n i f i c a n t l y at Ladysmith, however, the same trend in regional differences between the BC coast and BC i n t e r i o r was present (Figs. 5 & 6). Orthogonal contrasts for region e f f e c t s showed no s i g n i f i c a n t difference between seedlots from BC and the US (Table 10b (2)). However, the UBC (days 45 and 66) and Nakusp data showed s i g n i f i c a n t differences for the BC coast 56 versus BC i n t e r i o r regions (p<0.0l, Table 10b (3)). Ladysmith did not show a s i g n i f i c a n t difference but did show the same trend (Fig. 5). No analysis showed a s t a t i s t i c a l l y s i g n i f i c a n t coast (US & BC) versus i n t e r i o r (US & BC) difference although p=0.06 for Nakusp. 6.2.3 A l l locations, BC regions, Hypothesis 4 , (Appendix C) Again, for a l l locations with the BC data only, regional effects remained s i g n i f i c a n t (p<0.05) using UBC day 45 but not for region using UBC day 66 (Table 11a & b). Provenances-within-region did not d i f f e r s t a t i s t i c a l l y for either analysis although the range of injury for provenance means was 18.5% (61 to 79.5%) for coastal seedlots and 29% (48 to 77%) for i n t e r i o r seedlots. Families-within-provenance d i f f e r e d (p<0.00), by 8 to 25% on the coast and 24 to 32% for fa m i l i e s - w i t h i n - i n t e r i o r provenances. This i s in keeping with the findings of Steinhoff et al. 1983. 6.2.4 Individual locations, BC regions 57 For analysis of individual locations, data only from UBC day 45 showed regional differences (p<0.05). Region was not s t a t i s t i c a l l y s i g n i f i c a n t in data from UBC day 66, p=0.16, Ladysmith, p=0.55, or Nakusp, p=0.10, (Figs. 5 & 6, regions 1 and 2 only, Table 12a). As well, provenances-within-regions did not d i f f e r on any date, however, families-within-provenances d i f f e r e d on a l l dates (p<0.01). Ladysmith showed a s i g n i f i c a n t (p<0.05) region x temperature e f f e c t . F i g . 6b shows that at -22°C (the warmest test temperature), region 2 ( i n t e r i o r ) shows r e l a t i v e l y more injury than region 1 (coast). At both -26°C and -30°C however, the r e l a t i v e injury for the two regions i s reversed. As noted above, at Ladysmith regions did not d i f f e r . Table 12b provides a summary of the p r o b a b i l i t y of differences by source of variation for a l l four hypotheses discussed in the above two sections. 58 S e c t i o n I I I . 6.3 HYBRID COLD HARDINESS, 5 DATES. Response of the Galena-Moscow hybrid was compared with that of Galena-Arrow (closest BC i n t e r i o r provenance to Galena-Bay the cone parent origin) and of the US i n t e r i o r seedlots (See F i g . 1 for seedlot origins and F i g . 7 for depiction of the pollen source area). Each set of data represented freeze-testing results from three trees on the same f i v e dates as the above analysis. The term "provenance" w i l l be used to describe each of the three sets of data. Provenance A=Galena-Arrow, B=Galena-Moscow hybrid and C=US i n t e r i o r w i l l be used for future reference. Results showed a provenance difference (p<0.0l, Table 13a) and a provenance x date interaction (p<0.0l, Table 13a). F i g . 8b shows the r e l a t i v e s h i f t s in position over the f i v e test dates. On days 0, 66 and 80, Galena-Arrow shows s i g n i f i c a n t l y more injury than the US i n t e r i o r with l i t t l e or no difference on days 20 and 45. The position of the hybrid, r e l a t i v e to the other two provenances, fluctuated considerably across the five test dates. Duncan's New Multiple Range test showed that provenance A (Galena-Arrow) was s i g n i f i c a n t l y d i f f e r e n t from provenance C 59 (US i n t e r i o r ) and that neither was d i f f e r e n t from provenance B (Galena-Moscow hybrid) (Table 13b). 60 S e c t i o n IV. The results presented in the remainder of the Results section w i l l pertain only to the material which was damaged by an uncontrolled frost in Jan./Feb. 1989. The seedlings were in a randomized block design at the time the injury was noticed and analysis includes t h i s factor. Blocks were considered a random e f f e c t . Phenology results include both needle elongation measurements over five measurement dates and shoot elongation measurements over seven dates. Day 0 = March 3/89 for the shoot measurements; needle measurements commenced on day 36. 6 . 4 PHENOLOGY Regions did not d i f f e r on any measurement day although block x region interaction was s i g n i f i c a n t (p<0.05) on a l l but the la s t measurement day (day 128) (Table 14a). Due to the s i g n i f i c a n t interaction effect on day 36, the s i g n i f i c a n t block e f f e c t for needle length has no meaning. Figs. 9a and 9b show elongation patterns over the f i v e measurement days for the two blocks. Data are missing in these figures as regular measurements ceased after the ef f e c t s of the uncontrolled freezing became apparent and the 61 study objective changed to estimation of recovery from injury by uncontrolled freezing. For shoot-elongation measurements, blocks did not d i f f e r for any of the seven measurement days. Regions d i f f e r e d (p<0.05) on days 30 and 36 only, although p=0.15 on average for days 43, 53, 65 and 128 (Table 15a). Only data from day 53 showed a s i g n i f i c a n t block x region interaction (p<0.05) however, p=0.09, on average, for days 43, 65 and 128. Regions 1 and 3 ranked the lowest for growth in both blocks with the two i n t e r i o r regions showing the most growth. Orthogonal contrasts for the s i g n i f i c a n t region e f f e c t s for days 30 and 36 both show s i g n i f i c a n t coast (US and BC) versus i n t e r i o r (US and BC) ef f e c t s (p<0.05) and s i g n i f i c a n t BC coast versus BC i n t e r i o r e f f e c t s (p<0.0l, Table 15b). 6.5 DAMAGE ASSESSMENT The means for needle damage by region were a l l very close to 50% injury for both blocks. For block one, the mean injury for region 1 was 48%, region 2 was 51%, region 3 was 46% and region 4 was 47%. For block two, the mean injury for region 1 was 48%, region 2 was 45%, region 3 was 45% and region 4 was 41%. The results showed a s i g n i f i c a n t block x region effect (p=0.05, Table 16). The s i g n i f i c a n t block e f f e c t (p<0.00) therefore has no meaning. There was 62 no regional e f f e c t . Shoot height was also measured on a l l seedlings at t h i s time. These results showed s i g n i f i c a n t block and block x region effects (p<0.00), but again a non-s i g n i f i c a n t region e f f e c t (Table 16b). Recovery results from comparing shoot length between the measurement dates (May and July) by block and region, did not show any s i g n i f i c a n t differences (Table 17) or interactions. 6 3 7.0 DISCUSSION The results indicate that seed o r i g i n of Pw i s important when considering seed transfer between the coast and i n t e r i o r of BC. Inferences based on the performance of the US material are much less c l e a r . The implications of these conclusions led to the following series of recommendations for tree breeders and foresters. 7.1 RECOMMENDATIONS BASED ON FREEZE TESTING When c o l l e c t i n g wild-stand seed, coast and i n t e r i o r seedlots should be kept separate. Since the var i a t i o n among provenances in a region i s not s t a t i s t i c a l l y s i g n i f i c a n t , seed could be combined from more than one provenance within a region. Combining provenances within a region would make seed c o l l e c t i o n and/or parent-tree selections for breeding purposes less complicated since i t would allow for more-rigorous selection within a region. Selection within region for t a i t s other than frost hardiness w i l l result in 64 populations which have wide va r i a t i o n in frost hardiness. This study suggests that two seed planning zones may be necessary to reduce the l i k e l i h o o d of restocking f a i l u r e s due to frost damage or k i l l i f c o a s t - i n t e r i o r seed transfers were to be made. It must be emphasized however, that t h i s recommendation is based solely on the response of detached needles to freeze testing. Although t h i s method of testing gives a good measure of r e l a t i v e differences in performance, i t does not provide a measure of absolute differences. F i g . 4 shows the average difference in the % injury response between the BC coast and BC i n t e r i o r to be approximately 20% (not including day 0). Due to the sample size constraints in t h i s study, the v a r i a b i l i t y in response observed and i l l u s t r a t e d in Figs. 2, 4, 5 and 6, makes interpretation for the u t i l i z a t i o n of seed and parents from the US breeding program d i f f i c u l t . Generally, however, the trend i s of coastal material expressing greater damage than i n t e r i o r seedlots. Nevertheless, the i n t e r i o r US seedlots consistently show more damage than the i n t e r i o r BC seedlots. F i g . 2 suggests that US stock i s generally intermediate in controlled-freezing response between BC coast and BC i n t e r i o r seedlots. Work by Bower (1987), indicates that the rust-resistant second-generation seedlings from the Idaho breeding program could be used in coastal BC for increased 65 disease resistance. Meagher (1988) however, suggests that the Idaho stock should be r e s t r i c t e d to s p e c i f i c zones in the i n t e r i o r due to poorer performance of the Idaho stock versus l o c a l sources at a single s i t e . The study by Bower (1987), was r e s t r i c t e d to testing on the coast and the question of r e s t r i c t e d regional use i s under more extensive investigation at present (Meagher and Hunt 1987). A more-comprehensive set of US material should be retested for response to freeze testing before recommending whether US material d i f f e r s in cold-hardiness response from BC material. Given that two seed planning zones are recommended, i t i s clear that planting stock derived from sources within a coastal or i n t e r i o r region should be r e s t r i c t e d to d i s t r i b u t i o n within the respective region. As stated above, however, seed l i k e l y could be bulked from provenances within a region. The anomalous results from testing of the Ladysmith material could have a number of explanations. Since the date of f i r s t frost at the closest weather station (Nanaimo A) was Oct. 31, 1989, i t i s l i k e l y that the Ladysmith s i t e had experienced frost prior to c o l l e c t i n g . The temperature at which the samples showed injury further suggests that the rapid development of fr o s t hardiness had been induced by a f r o s t . However, the date of c o l l e c t i o n was very close to the expected date of f i r s t frost and differences may not 6 6 have expressed themselves yet. Due to missing the testing temperatures required to detect injury on the c o l l e c t i o n date (Nov. 1, 1989), surplus samples were held for two weeks prior to a second t e s t i n g . These samples were held in the dark in a 0-5°C cold room which may have s t a b i l i z e d the samples to a standard l e v e l of hardiness. However, Rehfeldt (Pers. Comm.) suggested that the samples could be stored for a two-week period without a l t e r i n g the state of frost hardiness. In addition t h i s s i t e was not consistently well brushed and the environmental variation due to cover may have prevented detection of regional genetic v a r i a t i o n . The results from analysis of the hybrid seedlot do not show a clear relationship with parent region performance. However, the ov e r a l l means depicted in F i g . 8a show the hybrid to be intermediate between parental provenances. In thi s small study, the US i n t e r i o r shows less injury than the BC i n t e r i o r which contradicts the findings of Meagher (1988). I, however, had only three trees to test from each of the contributing provenances. 7.2 PHENOLOGY Data from the phenology measurements and assessment of injury from unplanned freezing do not show any s t r i k i n g 67 res u l t s . Because, at the time of uncontrolled freezing, some seedlings were in the greenhouse and some were outside, I am very hesistant to draw strong inferences from these r e s u l t s . The high l e v e l of block x region interaction found in the needle and shoot elongation data, I would suggest, was more l i k e l y the result of d i f f e r e n t i a l injury among the seedlings rather than the blocks themselves being inherently d i f f e r e n t . However, regional shoot length means (but di f f e r e n t seedlots in most instances) on undamaged seedlings showed a similar ranking for the May and July measurement dates for block 1 but not region 4 in block 2. This suggests that the regional differences apparent for shoot elongation may be r e a l , although the results are compounded by the d i f f e r e n t i a l freezing response (Table 18). Further, the number of undamaged seedlings measured was not consistent from region to region. In addition, the seedlings were from d i f f e r e n t seedlots in most instances and therefore a s t a t i s t i c a l comparison of shoot length was not performed on damaged versus undamaged seedlings. The s i g n i f i c a n t block and block x region interaction terms (Table 16 a & b), in the injury assessment analyses, indicate that the seedlings were injured d i f f e r e n t i a l l y p rior to repotting. Region was not s i g n i f i c a n t for either of these analyses (Table 16 a & b). The recovery assessment through difference in height between July 19, 1989 and May 17, 1989 showed no significance for block, region or block x region. This suggests that no regions seedlings recovered 68 s i g n i f i c a n t l y more than any other region's although bud burst appeared to be delayed in damaged seedlings. Seedlings in a l l regions must have been delayed to an equal degree. Although these results appear to contradict the controlled freeze testing re s u l t s , several factors could account for t h i s difference. Controlled freezing was not car r i e d out in January or February so a di r e c t comparison of regional differences in pattern of injury in the two sets of material i s not possible. Whole-seedling testing was not done with the controlled freezing and therefore seedling recovery patterns by region can not be compared. Root freezing/injury may have contributed s i g n i f i c a n t l y to the subsequent death or reduced growth of the damaged seedlings (Table 18). However, i f controlled whole-seedling testing were done, roots would be insulated to protect from freezing. Thus, the phenology results are d i f f i c u l t to interpret and hold less significance than the results from cold-hardiness testing. 69 7 . 3 Pw, COLD HARDINESS AND PHENOTYPIC PLASTICITY The implications of phenotypic p l a s t i c i t y for long-l i v e d species such as conifers, are quite d i f f e r e n t than for annuals or short-lived perennials. When the l i f e - c y c l e i s longer than the duration of the environmental fluctuation (fine-grained s p a t i a l v a r i a t i o n ) , a series of temporary phenotypes or responses can occur. Single genotypes can have d i f f e r e n t phenotypes and/or d i f f e r e n t genotypes can have the same phenotype (Bradshaw 1965), masking genetic va r i a t i o n in a population, while appearing homogeneous (Schlichting 1986). Species whose l i f e cycle i s shorter or the same length as the fluctuation (coarse-grained temporal v a r i a t i o n ) , can evolve through disruptive selection in time (Bradshaw 1965), since the next generation i s adapted to an environment which may no longer exist (Schlichting 1986). Therefore, adaptation occurs primarily through p l a s t i c i t y , not through genetic change (Bradshaw 1965). Campbell & Sugano's (1989) hypothesis that the species emphasizes genomic f l e x i b i l i t y at the expense of genomic s t a b i l i t y (as discussed in the l i t e r a t u r e review), suggests that Pw i s under disruptive selection in time for s i t e variables with genetic variation playing a minor role in providing the means to tolerate and survive coarse-grained temporal v a r i a t i o n . 70 C r i t c h f i e l d (1984), states that during the la s t i c e -age, Pw was reduced in i t s northern range to a single refugium in Oregon, from which repopulation occurred in the north. This reduction could have provided a bottleneck ef f e c t from which recovery was dependent on Pw to l e r a t i n g diverse conditions with limited genetic d i v e r s i t y . P l a s t i c genomes could have been selected for at thi s time. Using isozyme data, Steinhoff et al. (1983) calculated genetic d i v e r s i t y estimates for the entire species range of Pw to be GST=0.148 (c o e f f i c i e n t of genetic d i v e r s i t y G S T = D S T/H T where Dg T = d i v e r s i t y among populations and H T = t o t a l genetic d i v e r s i t y ) . This suggests that 85% of the t o t a l v a r i a t i o n can be explained as va r i a t i o n within populations and 15% among populations. Steinhoff et al. (1983), further show that the populations sampled from the coast and north of 44° latitude exhibit the greatest c o e f f i c i e n t of genetic d i v e r s i t y at 0.124 and the least t o t a l d i v e r s i t y of a l l regions. This suggests that Pw within the coastal range retains a r e l a t i v e l y high degree of d i v e r s i t y between populations but exhibit an ov e r a l l low degree of v a r i a b i l i t y r e l a t i v e to other regions. The i n t e r i o r region studied by Steinhoff et al. (1983), had a c o e f f i c i e n t of genetic d i v e r s i t y of 0.07 i l l u s t r a t i n g that 93% of the variation present occurred within populations. Having experienced a bottleneck, however, i t i s plausible that Pw, while presently having a r e l a t i v e l y high o v e r a l l heterozygosity (H m=0.l5) (Steinhoff et al. 1983) in i t s northern 71 populations, could have suffered a loss in adaptive genetic v a r i a t i o n or that isozymes do not indicate genetic d i v e r s i t y in frost hardiness. Conifers are thought to carry a high genetic load, r e l a t i v e to other organisms, which could contribute to the ov e r a l l heterozygosity but make l i t t l e contribution to the adaptive genetic v a r i a t i o n . As discussed e a r l i e r , phenotypic p l a s t i c i t y i s not only a c h a r a c t e r i s t i c of the individual but also i s s p e c i f i c for individual c h a r a c t e r i s t i c s (Schlichting 1986). Phenotypic p l a s t i c i t y may apply to only a few or many characters within a given genome. Therefore, selection could be acting d i f f e r e n t l y on d i f f e r e n t c h a r a c t e r i s t i c s . Over time one would expect to see genetic variation evolve between populations for p a r t i c u l a r c h a r a c t e r i s t i c s i f a constant selection pressure was present. If s p e c i a l i z a t i o n ( e . g . : adaptive gene complexes) i s the consequence of a constant selection pressure, then i t i s l i k e l y that a highly adapted individual w i l l be more f i t than a generalist. Given that Pw has discrete coastal and i n t e r i o r ranges, the gross differences in cues for cold-hardiness induction (photoperiod and temperature), which are constant from generation to generation, could have given r i s e to the regional differences in freezing injury observed in t h i s study; or, tolerance for lower temperatures could have been los t in the coastal region. Campbell and Sugano (1989) suggest that the lack of 72 p r e d i c t a b i l i t y of s i t e s from generation to generation could sustain the high degree of phenotypic p l a s t i c i t y of growth characters in the Pw genome. However, they do not accept t h i s hypothesis as very plausible because they can not ascertain i f the s i t e s are variable enough to provide a selection pressure which would result in selection of genes providing wide tolerances to be selected. I would argue that, with any pioneer species, conditions w i l l be predictably variable from generation to generation throughout i t s range and that the environmental conditions of any one s i t e are unpredictable. These factors could account for the high degree of phenotypic p l a s t i c i t y for growth characters in the Pw genome. I would argue further that selection pressures for cold hardiness are r e l a t i v e l y stable generation to generation and, therefore, geographic genetic variation (specialization) for this character could be developing between regional populations, as has occurred in other conifers (Rehfeldt 1978, Steinhoff 1980). These p o s s i b i l i t i e s are not necessarily inconsistent with the findings of Steinhoff (1981) and Bower (1987), regarding ease of transfer of Pw between the coast and i n t e r i o r since the absolute k i l l i n g temperature ( L T ^ Q ) for a given population was not examined in my study. A year with severe temperatures however, might expose the regional differences expressed by the controlled-freezing tests of my study. Although there were s i g n i f i c a n t differences in freezing injury found between regions, supporting the 73 findings of Rehfeldt et al. (1984) the amount of genetic variation which i s present may not r e s t r i c t movement of material i f the differences in cold hardiness are below the lowest winter temperatures in each location. Further, the process of dehardening should be looked at in Pw. Growth chamber studies on Pw seedlings could help to elucidate the questions of regional cue responses and look at minimum temperature tolerances for a s p e c i f i c (controlled) growing environment. The results of t h i s research support the findings of others (Townsend & Hanover 1972, Steinhoff et al. 1983, Rehfeldt et al. 1984, and ,Campbell & Sugano 1989). Pw showed s i g n i f i c a n t differences at the family-within-provenance l e v e l , with considerably less difference at the provenance-within-region l e v e l . For cold hardiness, the results support the findings of Rehfeldt et al. (1984), indicating c o a s t - i n t e r i o r regional differences. The lack of c o r r e l a t i o n of injury with elevation supports the findings of Townsend and Hanover (1972). The issues: cold hardiness and phenotypic p l a s t i c i t y in Pw, are d i f f i c u l t to resolve when the processes themselves are diverse and dynamic. Pw is a fascinating system on which to study phenotypic p l a s t i c i t y . A greater understanding of both could contribute to management decisions for Pw and lead to a clearer understanding of the relationships between evolution, cold hardiness and phenotypic p l a s t i c i t y . 74 7.4 FUTURE RESEARCH Information on spring dehardening and usable phenology data would be useful to substantiate or refute the recommendations regarding t r a n s f e r a b i l i t y of Pw between the coast and i n t e r i o r of BC (discussed in section 7.0) before implementation. The rel a t i o n s h i p between response to freeze testing of needles versus whole seedlings should be investigated. Work by Nilsson and Eriksson (1986) on Scots pine in northern Sweden showed that a r t i f i c i a l freeze testing of whole seedlings can represent f i e l d performance at the population l e v e l . However, detached-tissue samples often appear more hardy than whole-seedling samples when tested under controlled-freezing conditions (Glerum 1973). I also suggest that further research be done on the available US material. The sample sizes for US material in t h i s study did not provide for a powerful test of performance. A balanced design with s u f f i c i e n t sample size ( i . e . : 20-40 populations with 30-50 seedlings per population) would more readily allow the relationship between US and BC material to be addressed. Cold-hardiness testing of additional f i e l d s i t e plantings of Pw (presently in the Rust Resistance program), 75 during the period of cold hardiness development could help to elucidate questions described above. Further, questions of phenotypic p l a s t i c i t y could also be addressed i f the same genotypes were grown in d i f f e r e n t environments and were researched and analyzed in the appropriate manner. REGION LOCATION SEEDLOT CODE LATITUDE LONGITUDE ELEVATION °N W METERS B.C. L a d y s m i t h ( P S ) 1 2183 2191,2209 •3 ( 2 2 0 5 ) Z 49°01' 124°04' 800 Coast Woss(PT) 2245 2263 2265 (2244) 50°11' 126°28' 300 Se c h e l t ( P T ) 2407 2410 2525 (2409) 49°35' 123°42' 700 W h i s t l e r ( P S ) 2250 2251 2252 (2248) 50°07' 122°55' 850 Manning(PS) 2275 2276 2347 Park (2274) 49°08' 120°55' 1100 B.C. Valemont(PS) 2293 2295 2648 (2649) 52°45' 119°18' 900 I n t e r i o r R a f t R i v e r ( P S ) 2640 (2643) 51°48' 119°41' 1300 B a r r i e r e ( P S ) 2632 (2633) 51°18' 119°55' 750 Mount 2279 2280 2282 Revelstoke(PS) (2281) 51°02' 118°01' 600 Salmo(PT) 2591 2592 2594 (2595) 49°12' 117°17' 1000 -1870 Galena- 2483 2484 2485 Arrow(PT) (2567) 50°37' 117°53' 720 Galena- 2569 2570 F* 50°37' 117°53 1 720 Moscow 2571 (2572) M D see map 1 838 -1219 T a b l e 1 cont. U.S. Coast White Pass 2 0 -Willamette NF 1 3 Olympia NF 1 2 Mount Hood NF 1 1 2 6 8 9 2 6 9 0 ( 2 6 8 7 ) ( 2 6 8 8 ) 4 6 ° 2 6 ' 4 4 ° 2 6 ' 4 7 ° 2 3 ' 4 5 U 3 8 ' 1 2 1 ° 3 4 ' 1 2 2 ° 0 2 ' 1 2 8 ° 1 8 ' 1 2 1 ° 4 5 ' 9 1 5 - 1 2 2 0 9 1 5 - 1 2 2 0 3 0 5 - 6 1 0 9 1 5 - 1 2 2 0 U.S. Flower Creek(PT) 2 6 8 3 4 8 ° 2 3 ' 1 1 5 ° 3 4 ' 6 7 0 I n t e r i o r C r y s t a l Creek(PT) 2 6 8 5 4 7 ° 0 8 ' 1 1 6 0 2 2 ' 9 1 5 Beaver Creek(PT) 2 6 8 6 4 8 ° 4 4 ' 1 1 6 ° 5 2 ' 9 1 5 Hungry Horse(PT) ( 2 6 8 2 ) 4 8 ° 1 9 ' 1 1 3 ° 5 9 ' 1 1 2 8 E l k R iver(PT) ( 2 6 8 4 ) 4 6 ° 4 9 ' I ^ I O ' 9 1 5 Seedlots not i n c l u d e d i n the phenology a n a l y s i s . Galena Bay t r e e s c r o s s e d with Moscow, Idaho p o l l e n . 1 PS P o p u l a t i o n sample - c o l l e c t i o n c r i t e r i o n : presence of cones. 2 ( ) A d d i t i o n a l s e e d l o t s i n c l u d e d i n the phenology and damage assessment a n a l y s i s o n l y . 3 PT Parent t r e e - c o l l e c t i o n c r i t e r i a : r u s t - f r e e i n i n f e s t e d stands, show a c c e p t a b l e stem form and growth in s i t u (Meagher, personal communication 1990). For US PT selection c r i t e r i a see Hoff & McDonald 1980. 4 F = female 5 M = male 6 [] = number of trees in bulked seedlots 7 NF = national forest 78 Table 2 L o c a t i o n of sampled t r e e s , t e s t date, day (from Sept. 7, 1989) and t e s t temperatures. LOCATION DAY TEST DATE TEST TEMPERATURES °C UBC 0 Sept . 7/89 3, "7, "9, -1 1 UBC 20 Sept . 27/89 3, "9, -11, -13 UBC 45 Oct. 22/89 3, -11, -13, -15 UBC Nov. 16l 66 Nov. 12/89 3, -28 (Dec . 11 3) UBC 80 Nov. 26/89 3, -25, -30, -35 LOCATION FROST ] DATES TEST DATE TEST TEMPERATURES °C NAKUSP Oct. Oct. 6/89 3, -12, -14, -16 (Oct. 3 3) LADYSMITH Nov. 1 4 3 Nov. 1 4/89 3, -22, -26, -30 (Oct. 30 J) 1 Average date (30-years) of f i r s t f r o s t . 2 These samples were not useable due to a technical problem. 3 Approximate date of f i r s t frost in f a l l , 1989. 79 Table 3 a) Number of seedlings available, by provenance, which were included in the damage assessment analysis. Block 1 Block 2 BC Coast Ladysmith 38 Woss 76 Sechelt 71 Whistler 71 Manning Park 43 63 1 1 6 1 1 6 1 16 63 BC Interior Valemount 71 Raft River 53 Barriere 58 Mt. Revelstoke 38 Galena-Arrow 71 Salmo 38 1 1 6 53 58 63 1 1 6 63 US Coast White Pass 1 7 1 7 US Interior Flower Creek Crystal Creek Beaver Creek 1 7 1 7 1 7 1 7 1 7 1 7 80 Table 4 a) Results from analysis of variance for combined data from controlled freezing on 5 test dates using 2-year-old seedlings grown at UBC (See Table 2 for test temperature and dates) Source 1 of Variation Degrees of Freedom Type III Sums of Squares PR > F R 3 53533 0.00 D 4 27967 0.00 T(D) 9 59673 0.00 R x D 1 2 9825 0.29 R x T(D) 27 7605 1 .00 E 791 544679 1 See Appendix C for d e f i n i t i o n s of terms and EMS's for H 1. o b) Multiple-comparison testing of regional Duncan's New Multiple Range Test. means by Region 1 Mean N Grouping 2 1 61 .73 375 A 3 52.02 50 B 4 51 .89 74 B 2 42.71 348 C 1 = BC coast, 2 = BC i n t e r i o r , 3 = US coast, 4 = US i n t e r i o r . Means with the same l e t t e r s are not s i g n i f i c a n t l y di f ferent. 81 Table 5 a & b) Results from analysis of regression for fo l i a r injury and latitude or elevation of seedling origin. Data on f o l i a r injury were from combined dates of controlled freezing using 2-year-old seedlings grown at UBC. a) Coastal provenances (i) % injury by l a t i t u d e , R = 0.19 Variable Df Parameter Standard Estimate Error T for Ho: 2 Parameter=0 prob > T" Latitude 1 error 5 2.991 1.910 Sums of Squares = 1 .566 512.4 0.18 ( i i ) % injury 2 by elevation, R = 0.05 Elevat. 1 error 5 -0.004 0.003 -1.146 Sums of Squares = 604.8 0.30 b) Interior provenance regressions (i) % injury by l a t i t u d e , R 2 = 0 .02 Variable Df Parameter Standard Estimate Error T for Ho: Parameter=0 prob > T Latitude 1 error 6 -3.029 2.855 Sums of Squares = -1 .061 1273.1 0.33 ( i i ) % injury 2 by elevation, R .07 Elevat. 1 error 6 0.004 0.006 Sums of Squares = 0.746 1 383.6 0.48 Measure of how much va r i a t i o n in the dependent variable can be accounted for by the model. Student's t value for testing the n u l l hypothesis that the parameter equals zero. Probability of a larger value of t i f the parameter is t r u l y equal to zero. A very small value leads to the conclusion that the independent variable contributes s i g n i f i c a n t l y to the model. 82 Table 5 c) Re s u l t s from a n a l y s i s of r e g r e s s i o n f o r f o l i a r i n j u r y with l a t i t u d e and e l e v a t i o n of s e e d l i n g o r i g i n . Data on f o l i a r i n j u r y were from combined dates of c o n t r o l l e d f r e e z i n g using 2-year-old s e e d l i n g s grown at UBC. ( i ) Coastal provenances R 2 = 0.11 Variable Df Parameter Est imate Standard T for Ho: „ Error Parameter=0 prob > T Latitude Elevation error 1 1 4 7.526 0.008 Sums of 6.424 0.010 Squares = 1 . 172 0.743 450.3 0.31 0.50 ( i i ) I n t e r i o r provenances R 2 - 0. 1 7 Variable Df Parameter Est imate Standard T for Ho: ~ Error Parameter=0 prob > T Latitude Elevat ion error 1 1 5 -4.232 -0.003 Sums of 6.026 0.011 Squares = -0.702 -0.233 1259.9 0.51 0.82 83 Table 6 a) R e s u l t s from a n a l y s i s of v a r i a n c e f o r s i n g l e t e s t dates from c o n t r o l l e d f r e e z i n g u s i n g 2-year-old s e e d l i n g s grown at UBC (See Table 2 f o r t e s t temperatures) Source of Var iat ion Degrees of Freedom Type III Sums of Squares PR > F Day=0, R Test 1 Date=Sept. 3 7/89 1969 0.38 T 2 5991 0.01 R x T 6 1112 0.94 E 89 56250 Day=20, R Test Date=Sept. 3 27/89 28478 0.00 T 2 25249 0.00 R x T 6 4087 0.34 E 191 1 14507 Day=45, R Test Date=Oct. 3 22/89 24564 0.00 T 2 5032 0.03 R x T 6 524 0.99 E 1 92 133613 Day=66, R Test Date=Nov. 3 1 2/89 10919 0.01 T 1 517 0.45 R x T 3 380 0.93 E 1 27 1 13486 Day=80, R Test Date=Nov. 3 26/89 9442 0.00 T 2 22882 0.00 R x T 6 1 501 0.89 E 1 92 126821 84 b) Probability of a larger value for F in orthogonal contrasts among different combinations of regions at five different dates of testing. Orthogonal Degrees Test Day C o n t r a s t of Freedom 0 20 45 66 80 (1) c o a s t vs i n t e r i o r 1 0 .32 0.00 0.16 0.47 0.04 (2) n o r t h vs south 1 0 .45 1 .00 0.61 0.13 0.85 (3) BC coast vs 1 0 .12 0.00 0.00 0.00 0.00 BC i n t e r i o r (US m a t e r i a l not used) 85 Table 7 a) Analysis including highest order interaction and excluding P(R), R x D, R x T(D), P(R) x D, P(R) x T. The exclusions were imposed by limitations on computing capacity. R F(R P) 1 38435 0.031 D DxF(R P) 4 43284 0.00 T(D) E 9 94921 0.00 F(R P) E 25 189071 0.00 DxF(R P) E 1 04 981 64 0.00 F(P R)xT(D) E 233 82096 0. 15 2 E 297 92206 Since P(R) i s included in the denominator F(R P) for„this test, t h i s r e s u l t i s only v a l i d i f one assumes P(R) 6 =0. Based on these r e s u l t s , i t was determined that the highest order in t e r a c t i o n could be pooled for more rigorous analysis of the other components in the model. NB: F(R P) includes: F(R P) + P(R) DxF(R P) includes: DxF(R P) + RxD + P(R)xD E includes: E + P(R)xT(D) + RxT(D) 86 Table 7 b) R e s u l t s from a n a l y s i s of v a r i a n c e f o r combined data from c o n t r o l l e d f r e e z i n g on 5 t e s t dates u s i n g 2 - y e a r - o l d s e e d l i n g s grown at UBC (BC m a t e r i a l o n l y , H 2) (See Table 2 f o r t e s t temperatures and dates) ( a n a l y s i s excludes h i g h e s t order i n t e r a c t i o n T(D) x F(P R)) Source 1 of Variation 2 Error Degrees Term of Used Freedom Type III Sums of Squares PR > F R P(R) 1 38407 0. 1 1 D DxP(R) 4 45795 0. 00 R x D DxP(R) 4 7320 0. 18 P(R) F(R P) 7 82871 0. 1 1 T(D) P(R)xT(D) 9 91811 0. 00 R x T(D) P(R)xT(D) 9 2331 0. 57 F(R D) E 18 105099 0. 00 P(R) x D DxF(R P) 28 30294 0. 19 P(R) x T(D) E 63 1 91 04 0. 67 D x F(R P) E 72 60106 0. 00 E 458 152865 See Appendix C for d e f i n i t i o n s of terms and H 2 model. See Appendix C for derivation of error terms By EMS's. 87 Table 8 Results from analysis of variance for single test dates from controlled freezing using 2-year-old seedlings grown at UBC (See Table 2 for test temperatures) (BC material only, H 2) Source of Variation Error Term Used Degrees of Freedom Type III Sums of Squares PR > F Day=0, Test Date=Sept. 7/89 R P(R) 1 2254 0.34 T TxP(R) 2 7510 0.00 RxT TxP(R) 2 1 39 0.83 P(R) F(R P) 9 20297 0.05 TxP(R) E 18 6467 0.24 F(R P) E 18 16117 0.00 E 1 35 9621 Day=20, Test Date=Sept . 27/89 R P(R) 1 27708 0.01 T TxP(R) 2 41 635 0.00 RxT TxP(R) 2 3222 0.022 P(R) F(R P) 9 201 75 0.48 TxP(R) TxF(R P) 18 5596 0.84 F(R P) E 18 40765 0.00 TxF(R P) E 36 1 7540 0.00 E 86 1 8337 For day=0, E= T x F(P R). See F i g . 4b for graph of s i g n i f i c a n t interaction. 88 Source of Variation Error Term Used Degrees of Freedom Type III Sums of Squares PR > F Day=45, R Test Date=Oct P(R) . 22/89 1 23348 0.02 T TxP(R) 2 7414 0.00 RxT TxP(R) 2 536 0.50 P(R) F(R P) 9 27661 0.29 TxP(R) TxF(R P) 18 6695 0.38 F(R P) E 18 41 327 0.00 TxF(R P) E 36 1 2004 0.49 E 87 291 98 Day=66, R Test Date=Nov P(R) . 12/89 1 29707 0.16 T TxP(R) 1 768 0.10 RxT TxP(R) 1 462 0.19 P(R) F(R P) 9 37504 0.09 TxP(R) TxF(R P) 9 2041 0.51 F(R P) E 18 35738 0.00 TxF(R P) E 18 4307 0.82 E 58 20379 Day=80, R Test Date=Nov P(R) . 26/89 1 1 3276 0.05 T TxP(R) 2 26034 0.00 RxT TxP(R) 2 21 9 0.65 P(R) F(R P) 9 22764 0.40 TxP(R) TxF(R P) 18 4459 0.93 F(R P) E 18 41 023 0.00 TxF(R P) E 36 17185 0.13 E 87 30597 89 Table 9 a) R e s u l t s from a n a l y s i s of va r i a n c e f o r combined data from c o n t r o l l e d f r e e z i n g u s i n g 2 - y e a r - o l d s e e d l i n g s from UBC (day 6 6 ) and 3 - y e a r - o l d s e e d l i n g s from Ladysmith and Nakusp. (See Table 2 f o r t e s t temperature and dates) Source 1 Degrees Type III PR > F of of Sums of Vari a t i o n Freedom Squares R 3 1 5033 0.00 L 2 35451 0.00 T(L) 5 1 0999 0.00 R x L 6 7621 0.05 R x T(L) 1 5 6109 0.80 E 51 1 594 b) R e s u l t s from a n a l y s i s of va r i a n c e f o r combined data from c o n t r o l l e d f r e e z i n g u s i n g 2 - y e a r - o l d s e e d l i n g s from UBC (day 4 5 ) and 3 - y e a r - o l d s e e d l i n g s from Ladysmith and Nakusp. (See Table 2 f o r t e s t temperature and dates) R 3 25479 0.00 L ' 2 48061 0.00 T(L) 6 1 551 4 0.00 R x L 6 9740 0.01 R x T(L) 18 6253 0.89 E 576 562 See Appendix C for d e f i n i t i o n s of terms and EMS's for H 3. 90 Table 10 a) Results of analysis of variance Tables for single test dates and locations from controlled freezing using 2-year-old seedlings from UBC (days 4 5 and 6 6 ) and 3 -year-old seedlings from Ladysmith and Nakusp. (See Table 2 for test temperatures and dates) Source Degrees Type III PR > F of of Sums of Variation Freedom Squares UBC (Day=45), Test Date=Oct. 22/89 R 3 24564 0.00 T 2 5032 0.03 R x T 6 524 0.99 E 192 133613 UBC (Day=66), Test Date=Nov. 12/89 R 3 10919 0.01 T 1 517 0.45 R x T 3 380 0.93 E 127 113486 Nakusp, Test Date=Oct. 3/89 R 3 9595 0.00 T 2 8580 0.00 R x T 6 1950 0.80 E 192 121634 Ladysmith, Test Date=Nov. 14/89 R 3 } 1058 0.40 T 2 1901 0.07 R x T 6 3779 0. 11 E 192 84342 91 b) Probability of a larger value for F in orthogonal contrasts among different combinations of regions at different test dates and locations. Orthogonal Degrees Test Day/Location Contrast of UBC  Freedom 45 66 Nak. Ladysm. (1) coast vs i n t e r i o r 1 0. 1 6 0. 47 0. 06 0. 54 (2) north vs south 1 0. 61 0. 1 3 0. 19 0. 89 (3) (US BC coast vs 1 BC i n t e r i o r material not used) 0. 00 0. 00 0. 00 0. 22 92 Table 11 a) Results from analysis of variance for combined data from controlled freezing using 2-year-old seedlings from UBC (day 6 6 ) and 3-year-old seedlings from Ladysmith and Nakusp. (BC material only, H Q 4 ) (See Table 2 for test temperatures and dates) Source 1 of Variation 2 Error Term Used Degrees of Freedom Type III Sums of Squares PR > F R P(R) 1 1 4874 0. 1 0 L LxF(R P) 2 3271 5 0. 00 RxL LxP(R) 2 4201 0. 50 T(L) F(P R)xT(L) 5 25105 0. 18 RxT(L) T(L)xP(R) 5 271 0 0. 1 0 P(R) F(R P) 9 36428 0. 1 5 F(R P) E 18 41849 0. 00 LxP(R) LxF(R P) 18 38241 0. 13 LxF(R P) E 36 49349 0. 00 P(R)xT(L) F(P R)xT(L) 45 1 1866 0. 97 F(P R)xT(L) E 90 39580 0. 00 E 232 641 94 93 b) Results from analysis of variance for combined data from controlled freezing using 2-year-old seedlings from UBC (day 4 5 ) and 3-year-old seedlings from Ladysmith and Nakusp. (BC material only, H 4 ) (See Table 2 for test temperatures and dates) ° Source of Va r i a t i o n Error Term Used Degrees of Freedom Type III Sums of . Squares PR > F R P(R) 1 23092 0. 03 L LxF(R P) 2 75462 0. 00 RxL LxP(R) 2 8409 0. 10 T(L) F(P R)xT(L) 6 31751 0. 00 RxT(L) T(L)xP(R) 6 2784 0. 50 P(R) F(R P) 9 29585 0. 37 F(R P) E 18 50679 0. 00 LxP(R) LxF(R P) 18 31518 0. 19 LxF(R P) E 36 44841 0. 00 PxT(R L) F(P R)xT(L) 54 1 6520 0. 93 F(P R)xT(L) E 108 47277 0. 00 E 261 7301 2 See Appendix C for d e f i n i t i o n s of terms and H 4 model. See Appendix C for derivation of error terms By EMS's. 94 Table 12 a) Results from analysis of variance for single test dates and locations from controlled freezing using 2-year-old seedlings from UBC (days 4 5 and 6 6 ) and 3-year-old seedlings from Ladysmith and Nakusp. (BC material only) (See Table 2 for test temperature and dates) Source Error Degrees Type III PR > F of Term of Sums of Variation Used Freedom Squares UBC (Day=45), Test Date=Oct. 22/89 R P(R) 1 23348 0.02 T TxP(R) 2 7414 0.00 RxT TxP(R) 2 536 0.50 P(R) F(R P) 9 27661 0.29 TxP(R) TxF(R P) 18 6695 0.38 F(R P) E 18 41 327 0.00 TxF(R P) E 36 12004 0.49 E 87 29198 UBC (Day=66), Test Date=Nov. 12/89 R P(R) 1 29707 0.16 T TxP(R) 1 768 0.10 RxT TxP(R) 1 462 0.19 P(R) F(R P) 9 37504 0.09 TxP(R) TxF(R P) 9 2041 0.51 F(R P) E 18 35738 0.00 TxF(R'P) E 18 4307 0.82 E 58 20379 95 Table 12 a) cont. Source of V a r i a t i o n Error Term Used Degrees of Freedom Type III Sums of Squares PR > F Nakusp, Test Date=Oct . 6/89 R P(R) 1 7618 0.10 T TxP(R) 2 1 2775 0.00 RxT TxP(R) 2 893 0.31 P(R) F(R P) 9 20914 0.47 TxP(R) TxF(R P) 18 6507 0.91 F(R P) E 18 41863 0.00 TxF(R P) E 36 23435 0.00 E 87 21746 Ladysmith, Test Date=Nov. 14/89 R P(R) 1 534 0.55 T TxP(R) 2 1 1 562 0.00 RxT TxP(R) 2 1 354 0.051 P(R) F(R P) 9 1 2527 0.10 TxP(R) TxF(R P) 18 331 7 0.90 F(R P) E 18 1 2330 0.00 TxF(R P) E 36 1 1 837 0.16 E 87 22067 See F i g . 6b for graph of s i g n i f i c a n t i n t e r a c t i o n . 96 Table 12 b) Summary of p r o b a b i l i t y of d i f f e r e n c e s by source of v a r i a t i o n f o r a l l four hypotheses. H Q Source of PR > F Table Var iat ion 1 R 0.00 4a 2 R 0.11 7a 2 R 0.03 7b 3 R 0.00 9a&b 4 R 0.10 11a 4 R 0.03 1 1b 3 L 0.00 9a&b 4 L 0.00 1 1a 4 L 0.00 1 lb 2 P(R) 0.11 7a 4 P(R) 0.15 1 1a 4 P(R) 0.37 1 1b 2 F(PR) 0.00 7a 2 F(PR) 0.00 7b 4 F(PR) 0.00 1 1a 4 F(PR) 0.00 1 1b See Appendix C for descriptions of H^'s. 97 Table 13 a) Results from analysis of variance for combined data of the Galena-Moscow hybrid, Galena-Arrow provenance and the US i n t e r i o r seedlots from controlled freezing on 5 test dates using 2 -year-old seedlings grown at UBC (See Table 1 for location descriptions, F i g . 7 for pollen source and Table 2 for test temperatures and dates) Source 1 of Var iat ion Degrees of Freedom Type III Sums of Squares PR > F P 2 51 38 0.01 D 4 20777 0.00 T(D) 9 33151 0.00 P x D 8 1 4387 0.002 P x T(D) 18 7771 0.68 E 181 95286 See Appendix C for d e f i n i t i o n s of terms. NB: P=provenance is not nested in region for th i s a n a l y s i s . See F i g . 8b for graph of s i g n i f i c a n t i n t e r a c t i o n . b) Multiple-comparison testing of provenance means Provenance 1 Mean N Group 2 ing (Duncan's New MRT) A 60 .90 75 A B 55 .45 74 A B C 51 .89 74 B 3 A = Galena-Arrow provenance B = Galena-Moscow hybrid C = US i n t e r i o r provenances combined (each provenance represented by 3 trees only). Means with the same l e t t e r s are not s i g n i f i c a n t l y di fferent. MRT = Multiple range test. 98 Table 14 Results from analysis of variance for individual measurement dates for needle elongation phenology (cm) on five 2-year-old seedlings per seedlot per block grown at UBC. (Day 0 is March 3/89) Source 1 of Variation Error Term Used Degrees of Freedom Type III Sums of Squares PR > F Day = B 36 E 1 0.121 0.01 R BxR 3 0.123 0.59 BxR E 3 0. 1 62 0.03 E 382 6.628 Day = B 43 E 1 1 . 196 0.15 R BxR 3 4.205 0.75 BxR E 3 9.741 0.00 E 380 223.440 Day = B 53 E 1 2.885 0.62 R BxR 3 98.233 0.61 BxR E 3 138.512 0.01 E 326 3814.793 Day = B 65 E 1 0.697 0.91 R BxR 3 936.227 0.38 BxR E 3 641.352 0.01 E 380 223.440 Day = B 1 28 E 1 378.416 0.22 R BxR 3 2639.154 0.13 BxR E 3 592.635 0.50 E 242 60625.530 Block (B) i s a random e f f e c t , region (R) is a fixed e f f e c t . 99 Table 15 a) R e s u l t s from a n a l y s i s of v a r i a n c e f o r i n d i v i d u a l measurement dates f o r shoot l e n g t h (cm) 2-year-old s e e d l i n g s per s e e d l o t per block grown at UBC. (Day 0 i s March 3/89) Source 1 of Var iat ion Error Term Used Degrees of Freedom Type III Sums of Squares PR > F Day = 0 B E 1 7.734 0.08 R BxR 3 14.865 0.50 BxR E 3 14.722 0.13 E 386 995.289 Day = 30 B E 1 1 . 1 79 0.61 R BxR 3 56.684 0.03 BxR E 3 4.342 0.81 E 385 1764.803 Day = 3 6 B E 1 0.003 0.98 R BxR 3 148.591 0.03 BxR E 3 10.437 0.69 E 376 2694.981 100 Table 15 a) cont. Source of Var iat ion Error Term Used Degrees of Freedom Type III Sums of Squares PR > F Day = 43 B E 1 5.168 0.65 R BxR 3 629.649 0.17 BxR E 3 182.644 0.07 E 376 9674.786 Day = 53 B E 1 59.119 0.35 R BxR 3 2335.515 0.13 BxR E 3 549.528 0.04 E 369 24677.018 Day = 65 B E 1 18.915 0.63 R BxR 3 2037.262 0.15 BxR E 3 529.935 0.09 E 376 9674.786 Day = 128 B E 1 12.887 0.68 R BxR 3 1794.015 0.15 BxR E 3 473.733 0. 11 E 246 19154.396 1 Block (B) is a random e f f e c t , region (R) i s a fixed e f f e c t . 101 Table 15 b) P r o b a b i l i t y of a l a r g e r value f o r F i n orthogonal c o n t r a s t s amoung d i f f e r e n t combinations of regions at two measurement dates f o r shoot l e n g t h (cm) on f i v e 2 -year-old s e e d l i n g s per s e e d l o t per b l o c k grown at UBC. (Day 0 i s March 3/89) Orthogonal Degrees Test Day Contrast of Freedom 30 36 (1) coast vs i n t e r i o r 1 0.02 0.00 (2) north vs south 1 0.14 0.14 (3) BC coast vs 1 0.00 0.00 BC i n t e r i o r 102 Table 16 a) Results from analysis of variance for injury assessment by a quantitative measure of injury of t o t a l needles scored on a l l seedlings in both blocks exposed to the uncontrolled freezing in Jan./Feb. 1989. Source Error Degrees Type III PR > F of Term of Sums of Variation Used Freedom Squares B E 1 9.568 0.00 R BxR 3 4.723 >0.50 BxR E 3 6.674 0.05 E 2561 2200.400 b) Results from analysis of variance for injury assessment by shoot measurements on a l l seedlings in both blocks exposed to the uncontrolled freezing in Jan./Feb. 1989. Source Error Degrees Type III PR > F of Term of Sums of Variation Used Freedom Squares B E 1 2762.45 0.00 R BxR 3 4827.53 0.1<p<0.5 BxR E 3 2088.69 0.00 E 1858 187148.25 1 03 Table 17 R e s u l t s from a n a l y s i s of v a r i a n c e f o r recovery assessment by shoot l e n g t h d i f f e r e n c e between May 17, 1989 and J u l y 19, 1989. Source Error Degrees Type III PR > F of Term of Sums of Variation Used Freedom Squares B E 1 R BxR 3 BxR E 3 E 32 0.370 0.19 0.278 >0.50 0.371 0.62 6.643 1 04 Table 18 Shoot length (mm) measurement of undamaged and damaged seedlings in blocks 1 and 2 for day 65 and 128. (i) Block 1, undamaged Day Region Mean + SE N Day Region Mean + SE N 65 1 40. 19 0.94 299 1 28 1 ' 41 .78 0.90 318 65 2 46.63 1 .04 242 1 28 2 49.30 0.96 290 65 3 44.87 2.15 29 128 3 45.68 2.25 29 65 4 48.49 2.88 44 1 28 4 48.91 2.94 44 ( i i ) ] Block 1, damaged 65 1 1 4.53 0.89 5 1 28 1 14.71 0.89 5 65 2 18.60 1 .26 5 1 28 2 19.60 1 .27 5 65 3 17.69 1 .85 5 1 28 3 18.31 1.71 5 65 4 1 9.53 2.31 5 1 28 4 20.61 2.20 5 ( i i i ) Block 2 , undamaged 65 1 39.50 1 .36 161 1 28 1 34.72 1 .77 1 40 65 2 45.52 1 .32 1 47 128 2 45.79 1 .37 1 45 65 3 30.20 2.87 27 1 28 3 29.77 3.18 25 65 4 33.24 2.59 42 . 1 28 4 32.87 2.88 39 (iv) : Block 2, damaged 65 1 1 4.80 1.13 5 1 28 1 1 6.60 1 .22 5 65 2 21.19 1 .55 5 1 28 2 22.24 1 .56 5 65 3 1 0.62 1 .35 5 1 28 3 1 1 .46 1 .45 5 65 4 21 .45 2.71 5 1 28 4 21 .60 2.69 5 105 F i g . 1 Map of seedlot o r i g i n s (•), natural range of western white pine ( s t i p p l e d area) and region delineation (numbers), (modified from Fowells 1965) 1 0 6 F i g . 2 Mean needle injury (%) and standard error by region for combined data from c o n t r o l l e d freezing on 5 test dates using 2-year-old seedlings grown at UBC. 70 60 50 40 30 20 10 0 % INJURY (needles) 5 prov's 15 SL's — T -6 prov's 14 SL's 2 bulked prov's F V.-VS>ss>W///.-SS/SS?S.'tY< /'ism 3 SL's REGIONS I S T D E R R O R ill B C - C O A S T • U S - C O A S T 111 U S - I N T E R I O R B C - I N T E R I O R SL's = seedlots prov's = provenances 1 07 F i g . 3 a) Mean needle injury (%) and standard error of 7 coastal provenances by l a t i t u d e using combined data from 5 dates of c o n t r o l l e d freezing of needles from 2-year-old seedlings grown at UBC. % INJURY (NEEDLES) LATITUDES (N-S) OF COASTAL SEEDLOTS I STD ERROR b) Mean needle injury (%) and standard error of 8 i n t e r i o r provenances by l a t i t u d e using combined data from 5 dates of c o n t r o l l e d freezing of needles from 2-year-old seedlings grown at UBC. % INJURY (NEEDLES) 80 60 -* i 1 \ * 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 40 20 62° <8< LATITUDES (N-S) OF INTERIOR SEEDLOTS I STD ERROR 108 F i g . 4 a) Mean needle injury (%) and standard error by region for 5 dates of c o n t r o l l e d freezing using 2-year-old seedlings grown at UBC. % INJURY (needles) 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 REGIONS I S T N D E R R O R DAY 4 5 DAY 0 & 8 0 DAY 66 Islltl DAY 20 1=80-0, 2=BC-I, 3=US-C, 4=US-I-1 09 F i g . 4 b) Mean needle i n j u r y (%) and standard error showing s i g n i f i c a n t region x temperature interaction for the i n d i v i d u a l test day 20 for regions 1 and 2 only. 100 % INJURY (needles) - 9 -11 -13 TEST TEMPERATURES (C) I S T D E R R O R X R E G I O N 1 * R E G I O N 2 1 10 F i g . 5 Mean needle injury (%) and standard error by region from c o n t r o l l e d freezing using 2-year-old seedlings from UBC (day 66) and 3-year-old seedlings from Ladysmith and Nakusp. 100 % INJURY (needles) 80 60 40 20 0 i + i stock 1 + 2 stock li-i-l 1;;;;e;;;I knmi li-ui i 1 + 2 stock 1 2 3 4 1 2 3 4 REGIONS I S T D E R R O R L A D Y S M I T H i l l U B C N A K U S P 1=BC-C, 2=BC-I, 3=US-C, 4=US-I 111 F i g . 6 a) Mean needle injury (%) and standard error by region from c o n t r o l l e d freezing using 2-and 3-year-old seedlings from UBC (day 45), Ladysmith and Nakusp. 100 % INJURY (needles) 80 60 40 20 0 1 + 1 stock 1 + 2 stock i 1 + 2 stock 1 2 3 4 1 2 3 4 REGIONS I S T D E R R O R ~J L A D Y S M I T H U B C %m N A K U S P 1=BC-C, 2=BC-I, 3=US-C, 4=US-I 1 1 2 F i g . 6 b) Mean needle injury (%) and standard error showing s i g n i f i c a n t region x temperature i n t e r a c t i o n for the Ladysmith regions 1 and 2 only. 100 % INJURY (needles) - 22 - 26 - 3 0 TEST TEMPERATURES (C) I STD E R R O R X R E G I O N 1 * R E G I O N 2 1 1 3 F i g . 7 Location of pollen source from Moscow, Idaho. •0 1 1 4 F i g . 8 a) Mean needle injury (%) and standard error by provenance for combined data from c o n t r o l l e d freezing on 5 test dates using 2-year-old seedlings grown at UBC. BC-I = Galena-Arrow provenance, Hybrid = Galena-Moscow cross, US-I = Flower, Crys t a l and Beaver Creek provenances combined. 70 60 50 40 30 20 10 0 % INJURY (needles) Hill •^yyyy/^yy'>-^'--y^^y:y'A^>y:'^ W0 KtyyXj y/yyyyyyy/y'X-yyyy'^yyyyyyy/y yy'yyy£yyyyyiw BC - I HYBRID US - I I STD ERROR 1 15 F i g . 8 b) Mean needle injury (%) and standard error by provenance for 5 dates of c o n t r o l l e d freezing using 2-year-old seedlings grown at UBC. BC-I = Galena-Arrow provenance, Hybrid = Galena-Moscow cross, US-I = Flower, Crys t a l and Beaver Creek provenances combined. % INJURY (needles) 100 C A B C A B C I S T D E R R O R DAY 4 5 DAY 0 & 80 DAY 20 DAY 66 A=BC-I , B=HYBRID , C=US-I 1 16 F i g . 9 a) Mean siz e and standard error of needle length (mm) by region over 5 measurement dates in 1989 on 5 seedlings per seedlot in block 1. LENGTH (mm) OF 1989 NEEDLES 80 36 43 53 65 128 DAY OF MEASUREMENT (OMARCH 3/89) I S T D E R R O R X R E G I O N 1 • R E G I O N 2 * R E G I O N 3 & R E G I O N 4 1 1 7 F i g . 9 b) Mean size and standard error of needle length (mm) by region over 5 measurement dates in 1989 on 5 seedlings per seedlot in block 2. LENGTH (mm) OF 1989 NEEDLES 80 60 40 20 0 X - >KA I X ^ 36 43 53 65 128 DAY OF MEASUREMENT (OMARCH 3/89) I STD E R R O R * R E G I O N 3 X R E G I O N 1 A R E G I O N 4 R E G I O N 2 1 18 8.0 LITERATURE CITED Aronsson, A., T. Ingestad, and L.Loof. 1976. Carbohydrate metabolism and frost hardiness in pine and spruce seedlings grown at di f f e r e n t photoperiods and thermoperiods. Physiol. Plant. 36:127-132. Bingham, R.T. 1983. B l i s t e r rust resistant western white pine of the Inland Empire. USDA Forest Service Gen. Tech. Rpt. INT-146. 45pp. Bingham, R.T., R.J. Hoff and R.J. Steinhoff. 1972. Genetics of western white pine. 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Geographic patterns of genetic v a r i a t i o n in Pi nus 122 monticola. Bot. Gaz. 145(2):229-239. Sakai, A. and C.J. Weiser. 1973. Freezing resistance of trees in Northern America with reference to tree regions. Ecology 54:118-126. SAS Institute Inc. 1985. SAS User's Guide:Basics, Version 5 E d i t i o n . Cary, NC:SAS Institute Inc. 921pp. SAS Institute Inc. 1985. SAS R User's Guide:Statistics, Version 5 E d i t i o n . Cary, NC:SAS Institute Inc. 956pp. Schlichting C D . 1986. The evolution of phenotypic p l a s t i c i t y in plants. Ann. Rev. Ecol. Syst. 17:667-693. Schlichting CD. and D.L. Levin. 1984. Phenotypic p l a s t i c i t y of annual phlox: tests of some hypotheses. Amer. J. Bot. 71(2):252-260. Scheiner S.M. and C J . Goodnight. 1984. The comparison of phenotypic p l a s t i c i t y and genetic variation in populations of the grass Danthonia spi cat a. Evolution 38(4):845-855. Silander, J r . , J.A. 1985. The genetic basis of the ecological amplitude of Sparlina patens. I I . Variance and c o r r e l a t i o n analysis. Evolution 39(5):1034-1052. Simpson, D.G. 1985. Measuring cold hardiness. Presented at the Forest Nursery Association of B.C. Annual Meeting, Sept. 23-26, 1985, Duncan, B.C. Singh J. and A. Laroche. 1988. Freezing tolerance in plants: a biochemical overview. Biochem. C e l l B i o l . 66:650-657. Smith-Gill, S.J. 1983. Developmental p l a s t i c i t y : Developmental conversion versus phenotypic modulation. Amer. Zool. 23:47-55. Sokal R. and F. Rohlf. 1981. Biometry. Second Ed i t i o n . W. H. Freeman and Company, pp.427-428. Squillace, A.E. and R.T. Bingham. 1958. Localized ecotypic v a r i a t i o n in western white pine. Forest Science 4(1):20-34. Stearns S.C 1983. The evolution of l i f e - h i s t o r y t r a i t s in mosquitofish since their introduction to Hawaii in 1905: Rates of evolution, h e r i t a b i l i t i e s , and developmental p l a s t i c i t y . 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Plant Physiol. 60:271-273. 1 24 APPENDIX A Diazinon s o i l drench: 50 girts/ 4 l i t e r s , 12.5% WP Captan f o l i a r spray : 8 gms/ 4 l i t e r s , 10% WP 125 APPENDIX B Seedling mix for 1.5 cubic yards: 675 gms micromax (micronutrients) 1800 gms dolomite 1575 gms gypsum 7.5 kgs nutricoat (slow release balanced N-P-K f e r t i l i z e r ) 6 b a i l s of Sunshine peat 3 bags of vermiculite 126 APPENDIX C HYPOTHESES LEGEND Y = % injury u = estimate of the mean of % injury R i = ^th r e 9 i o n L-: = o., location D n = n t h t e s t d a t e Tm = m t h t e m P e r a t u r e (single date) Tm(n) = m temperature on n date t ^ (temperature nested in £gte) Tm(o) = m t h t e m P e r a t u r e nested in o location P k ( j ) = jfth P r o Y f a n c e , i R 3 region _ l ( k i ) = th ^ a m i ^ y i n ^ provenance xn j region E = p needle-error within samples, plot error Ho1 - For seedlings grown at the UBC Nursery, there i s no evidence of genetic variation in frost hardiness associated with region of seed o r i g i n during the hardening period. Y ,. x = u + R. + D + T , N + R.D + R.T , \ + p(^mn) j n m(n) ] n 3 m(n) Ep(jmn) H1a - There i s evidence of regional genetic variation being conditional upon date of tes t i n g . Ho2 - For seedlings grown at the UBC Nursery, there i s no evidence of genetic variation in frost hardiness associated with region, provenance within region, or family within provenance. (BC only) Yp(jklmn) " u + R j + D n + R j D n + Tm(n) + Rj Tm(n) + P k ( j ) + D n P k ( j ) + Tm(n) Pk(j) + F l ( k j ) + D n F l ( k j ) + T m ( n ) F l ( k j ) + Ep(jklmn) H2a - There i s evidence of provenance and/or family within provenance genetic variation being conditional upon date of testing. 127 Ho3 - At one "physiologically s i m i l a r " sampling time, there is no evidence of genetic v a r i a t i o n in frost hardiness associated with region of seed o r i g i n when seedlings are grown in three d i f f e r e n t locations. Y , .. x = u + R. + L + T / x + L R . + R.T , x + E , ".. v p ( o 3 t ) ] o m(o) 0 3 ] m(o) p(o]t) H3a - There i s evidence of regional genetic v a r i a t i o n being conditional upon locat i o n . Ho4 - At one "physiologically s i m i l a r " sampling time, there i s no evidence of genetic v a r i a t i o n in frost hardiness associated with region, provenance within region or family within provenance where seedlings are grown in three d i f f e r e n t locations. (BC only) Y , . M , = u + R. + L + T , X + R . L + R .T , x + P. , . v pijmkl) ] o m(o) j o 3 m(o) k(;j) + Tm(o) Pk(j) + L o P k ( j ) + F l ( j k ) + T m ( o ) F l ( j k ) + L o F l ( j k ) + Ep(jmkl) H4a - There i s evidence of provenance and/or family within provenance genetic v a r i a t i o n being conditional upon locat ion. 128 Ho1 : Source of df df(actual) Variation REGION DATE REGION*DATE TEMP(date) REGION*TEMP(date) ERROR TOTAL r-1 3 d-1 4 (r-1)(d-1) 12 d(t-1) 10 d(t-1)(r-1) 30 rdt(p-l) 60 rdtp-1 119 4 5 3 2 F F F R EMS j n m p Rj 0 5 3 2 6 2 e + 3 0 6 2 R D n 4 0 3 2 6 2 e + 2 4 6 2 D Tm(n) 4 1 0 2 6 2 e + 8 6 2 T ( D ) 2 2 j"n " " " - b e + 6 s RD R,-D„ 0 0 3 2 6 + 6 6 Rj Tm(n) 0 1 0 2 6 2 e + 2 6 2 R T ( D ) Ep(jmn) 1 1 1 1 6 e 2 F-tests: A l l EMS's w i l l be tested against 6 . 129 H 2: o Source of Variation df df coding REGION r-1 1 R . D DATE d-1 4 D n R x D (r-1)(d-1) 4 R .D D n TEMP(date) d(t-1 ) 9 Tm(n) R x T(d) (r-1)d(t-1 ) 9 R .T / x 3 m(n) PROV(region) r(p-1) 7 P k ( j) D x P(r) (d-1)r(p-1 ) 28 D n P k ( j ) T(d) x P(r) d ( t - 1 ) r ( p - l ) 63 Tm(n) Pk(j) F(p(r)) r p ( f - l ) 18 F l ( k j ) D x F(p(r)) ( d - 1 ) r p ( f - l ) 72 D n F l ( k j ) T(d) x F(p(r)) d ( t - 1 ) r p ( f - l ) T m ( n ) F l ( k j ) Error rpftd(n-1) 243 E p( jklmn) Total rpftdn-1 458 130 2 F j 5 F n 3 F m a R k b R 1 2 R P # EMS1 R . D 0 5 3 a b 2 1 + 3 0 6 2 F ( R p ) + 3 0 b 6 2 p ( R ) + 3 0 a b 6 2 R D n 2 0 3 a b 2 2 2 2 6 e + 6 e DF(RP) + 6 b f i 2DP(R) + 1 2 a b * 2 D R .D D n 0 0 3 a b 2 3 2 2 6 e + 6 f i DF(RP) + 6 b f i 2DP(R) + 6 a b* 2RD Tm(n) 2 1 0 a b 2 4 2 2 6 e + 2 ( 5 T(D)F (RP) + 2 b e T(D)P(R) + 4 a b 6 T ( D ) P k ( j ) 1 5 3 1 b 2 5 6 e + 3 0 ( 5 F(RP) + 3 0 b 6 2 p ( R ) Rj Tm(n) 0 1 0 a b 2 6 2 2 6 e + 2 e T(D)F(RP) + 2 2 2 b f i T(D)P(R) + 2 a b f i RT(D) D n P k ( j ) 1 0 3 1 b 2 7 2 2 6 e + 6 e DF(RP) + 6 b e DP(R) Tm(n) Pk(j) 1 1 0 1 b 2 8 2 2 6 e + 2 f i T(D)F(RP) + 2 b e T(D)P(R) F l ( k j ) 1 5 3 1 1 2 9 6 e + 3 ° 6 F(RP) D n F l ( k j ) 1 0 3 1 1 2 10 2 2 6 e + 6 e DF(RP) T m ( n ) F l ( k j ) 1 1 0 1 1 2 1 1 2 2 6 e + 2 g T(D)F(RP) E p( jklmri) 1 1 1 1 1 1 12 2 6 e a = between 5-6 b = between 1-3 F-tests: # 11/12 10/12, 9/12, 8/11, 7/10, 6/8, 5/9, 4/8, 3/7, 2/7, 1/5. EMS's were used to program F-tests, they do not necessarily represent the right c o e f f i c i e n t s for the variance components. This pertains to H 4's EMS as well. 131 H 3: o Source of Variation df df coding LOCATION REGION TEMP(location) L x R R x T(l) Error Total 1-1 2 r-1 3 Kt-1) 6 (1-1)(r-1 ) 6 ( r - O K t - l ) 18 lrt(p-1) 36 lrtp-1 71 Rv R ° T ^ , ED m(o) p(ojt) 3 4 3 2 F F F R EMS o J m P L o 0 4 3 2 2 2 « e + 2 4 6 L R . D 3 0 3 2 s2e + 1 8 6 2 R m(o) 3 4 0 2 2 + 24 2  6 e 2 4 ( 5 T(L) L R. o D 0 0 3 2 2 + fi 2 6 e 6 LR Rj Tm(o) 3 0 0 2 2 + fi 2 6 e b s RT(L) E p ( o j t ) 1 1 1 1 2 6 e F-tests: A l l tested against 132 H 4: * df Source of df A B coding Variation REGION r-1 1 1 R . D LOCATION 1-1 2 2 L o TEMP(location) Kt-1 ) 5 6 Tm(o) R x L (r-1)(1-1) 2 2 R .L. D © R x T(o) (r-1)l(t-1 ) 5 6 Rj Tm(o) PROV(r) r(p-1) 9 9 P k ( j ) T(o) x P(r) Kt-1 )r(p-1 ) 45 54 Tm(o) Pk(j) L x P(r) (1-1)r(p-1) 18 18 L o P k ( j ) F(p(r)) rp(f-1) 18 18 F l ( j k ) T(o) x F(p(r)) l ( t - 1 ) r p ( f - 1 ) 90 108 T m ( o ) F l ( j k ) L x F(p(r)) (1-1)rp(f-l) 36 36 L o F l ( j k ) Error lrpft(n-1) 232 261 Ep(jmkl) Total lrpftn-1 463 521 A: analysis includes UBC day 4, B: analysis includes UBC day 3. (See Tables 11a and 11b) 133 2 3 3 a b 2 F F F R R R EMS j o m k 1 P # R . 3 0 3 3 a b 2 1 6 2 E + 1 8 6 2 F ( R P ) + l 8 b 6 2 p ( R ) + I8ab 6 2j L o 2 0 3 a b 2 2 2 2 6 e + 6 s LF(RP) + 12ab 6 2 L Tm(o) 2 1 0 a b 2 3 2 2 6 e + 2 6 T(L)F(RP) 4 a b ' 2 T ( L ) R .L 3 o 0 0 3 a b 2 4 2 2 6 e + 6 f i LF(RP) + 6 b f i 2LP(R) + 6 a b f i 2R] Rj Tm(o) 0 1 0 a b 2 5 2 2 6 e + 2 f i T(L)F(RP) 2 b e 2 T ( L ) P ( R ) + 2 a b P k ( j ) 1 3 3 1 b 2 6 6 e + 1 8 6 F(RP) + 2 I8b 6 p ( R ) Tm(o) Pk(j) 1 1 0 1 b 2 7 2 2 6 e + 2 f i T(L)F(RP) 2 b s T(L)P(R) L o P k ( j ) 1 0 3 1 b 2 8 2 2 6 e + 6 f i LF(RP) + 6 b e LP(R) F l ( j k ) 1 3 3 1 1 2 9 6 e 1 b 6 F(RP) T m ( o ) F l ( j k ) 1 1 0 1 1 2 10 2 , 2 6 e 6 T(L)F(RP) L o F l ( j k ) 1 0 3 1 1 2 1 1 2 2 6 e + 6 e LF(RP) E p(joklm) 1 1 1 1 1 1 12 2 6 e + 2 6 RT(L) F-tests: 11/12 10/12, 9/12, 8/11, 7/10, 6/9, 5/7, 4/8, 3/10, 2/11, 1/6. 

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