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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 VARIATION  FAMILY  IN COLD HARDINESS OF  WESTERN WHITE PINE (Pinus  Dougl.  monticola  ex.  D.  Don)  by B a r b a r a R. Thomas B . S c . ( A g r . ) , The U n i v e r s i t y o f B r i t i s h  Columbia,  A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  OF  in THE FACULTY (Forest  We  Science, Faculty  OF GRADUATE  STUDIES  F o r e s t Tree of F o r e s t r y  Improvement)  a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d standard  THE UNIVERSITY  OF BRITISH COLUMBIA  October ©Barbara  1990  R u t h Thomas,  1990  1986  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department  this or  thesis by  for scholarly  his  or  publication of this thesis  her  Department of The University of British Columbia Vancouver, Canada  DE-6  (2/88)  Qd  /2  the  I agree  requirements  for  an  /*ft£>  advanced  that the Library shall make it  I further agree that permission for extensive  purposes  may  representatives.  It  be is  granted  by the  understood  that  for financial gain shall not be allowed without  permission.  Date  of  head of  my  copying  or  my written  ABSTRACT  T h i r t y - s e v e n s e e d l o t s of western white pine (Pi nus mont  i col  a  Doug. ex. D. Don) were t e s t e d f o r f r o s t h a r d i n e s s  to determine how t r a n s f e r a b l e seed would be from  different  seed sources w i t h i n white p i n e ' s coast and i n t e r i o r in B r i t i s h Columbia.  Twenty-nine  s e e d l o t s r e p r e s e n t e d the  coast and i n t e r i o r of B r i t i s h Columbia c o a s t a l United States  (BC), two were from  (US), three were from i n t e r i o r US and  three were h y b r i d s between i n t e r i o r US and i n t e r i o r parents.  ranges  BC  Detached needles were exposed to a s e r i e s of  f r e e z i n g temperatures i n a programable  f r e e z e r and  relative  h a r d i n e s s was c a l c u l a t e d as the l e n g t h of i n j u r e d needle expressed as a percentage of t o t a l needle l e n g t h after  freezing.  10 days  Seasonal p r o g r e s s i n hardening was t e s t e d  u s i n g f i v e dates i n the autumn of 1989.  S e e d l i n g s were  maintained at the U n i v e r s i t y of B r i t i s h Columbia nursery.  T e s t i n g a l s o was c a r r i e d out from samples c o l l e c t e d on separate dates from Nakusp i n the BC i n t e r i o r and from Ladysmith, a c o a s t a l BC s i t e . significant  (p<0.0l) r e g i o n a l d i f f e r e n c e between the BC  coast and BC i n t e r i o r the  There was a s t a t i s t i c a l l y  sources i n a l l t e s t  f i r s t UBC run and the Ladysmith run.  regions d i f f e r e d  significantly,  runs, e x c l u d i n g In the runs where  the d i f f e r e n c e i n percent  damage response of needles t o f r e e z i n g was approximately 20%.  /" / /'  Measurements  of shoot growth phenology were planned as  an a d d i t i o n a l component uncontrolled evaluation  freezing forced a  of genetic  Those e v a l u a t i o n s recovery.  of growth rhythm.  I n j u r y from  change of o b j e c t i v e to  d i f f e r e n c e s i n recovery from f r e e z i n g .  d i d not r e v e a l genetic  differences in  /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 2.7  Methods of E v a l u a t i o n  COLD HARDINESS, PHENOTYPIC PLASTICITY AND Pw  22 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  48  COLD HARDINESS OVER TIME AT ONE LOCATION 6.1.1  A l l dates combined, a l l r e g i o n s , Hypothesis  1 (Appendix C)  6.1.2  I n d i v i d u a l dates, a l l r e g i o n s  6.1.3  A l l dates, BC r e g i o n s ,  6.1.4  49 51  Hypothesis 2 (Appendix C)  52  I n d i v i d u a l dates, BC r e g i o n s  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  I n d i v i d u a l l o c a t i o n s , a l l regions  55  6.2.3  A l l l o c a t i o n s , BC regions,-  6.2.4  H y p o t h e s i s 4, (Appendix C)  56  I n d i v i d u a l l o c a t i o n s , BC r e g i o n s  57  Section I I I .  58  6.3  58  HYBRID COLD HARDINESS, 5 DATES  S e c t i o n 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/ /  L I S T OF  Table  1  Descriptive information for Pi  Table 2  TABLES  nus  monticola  seed sources  ..76  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  Table 3  78  Number of s e e d l i n g s a v a i l a b l e , by provenance, which were i n c l u d e d i n the damage assessment analysis  Table 4  79  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 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  using 2-year-old s e e d l i n g s grown a t UBC (See Table 2 f o r t e s t temperature  b)  Table 5  Multiple-comparison  a & b) foliar  and dates)  t e s t i n g of r e g i o n a l  i n j u r y and l a t i t u d e or e l e v a t i o n of Data on f o l i a r  from combined dates of c o n t r o l l e d  i n j u r y were freezing  using 2-year-old s e e d l i n g s grown a t UBC  c)  R e 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  foliar  means..80  R e 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  seedling o r i g i n .  Table 5  80  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  seedling o r i g i n .  Data on f o l i a r  i n j u r y were  81  V/ / /  from combined dates of c o n t r o l l e d  freezing  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...  Table 6  82  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  freezing  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 (See Table 2 f o r t e s t temperatures)  83  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 o r t h o g o n a l c o n t r a s t s among d i f f e r e n t combinations  of r e g i o n s at f i v e  different  dates of t e s t i n g  Table 7  a)  84  A n a l y s i s i n c l u d i n g highest order  i n t e r a c t i o n and e x c l u d i n g P(R), R x D, R x T ( D ) , P(R) x D, P(R) x T(D). The e x c l u s i o n s were imposed by l i m i t a t i o n s on computing c a p a c i t y . . . 8 5  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-year-old s e e d l i n g s grown a t UBC (BC m a t e r i a l only, H 2) (See Q  Table 2 f o r t e s t  temperatures  and dates)  ( a n a l y s i s excludes highest order i n t e r a c t i o n T x F(D P R)) .  86  / X  Table 8  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  freezing  using 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) (BC m a t e r i a l only, H 2)  Table 9  87  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 combined data from c o n t r o l l e d  freezing  using 2 - y e a r - o l d s e e d l i n g s from UBC  (day 66) 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)  89  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 o l d s e e d l i n g s from UBC s e e d l i n g s from  freezing using  2-year-  (day 45) and 3 - y e a r - o l d  Ladysmith and Nakusp (See Table  2 f o r t e s t temperature and dates)  89  Table 10 a) R e s u l t s of a n a l y s i s of v a r i a n c e T a b l e s f o r s i n g l e t e s t dates and l o c a t i o n s controlled UBC  from  freezing using 2-year-old seedlings  from  (days 45 and 66) 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 temperatures and dates)  90  X  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 o r t h o g o n a l c o n t r a s t s among d i f f e r e n t  combinations  of r e g i o n s at d i f f e r e n t t e s t dates and l o c a t i o n s . . . . 91  Table  11 a) R e s u l t s f o r 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 using  2-year-  o l d s e e d l i n g s from UBC (day 66) and 3-year-old s e e d l i n g s from o n l y , H 4) Q  Ladysmith and Nakusp (BC m a t e r i a l  (See Table 2 f o r t e s t  temperatures  and d a t e s )  b)  92  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  using  2 - y e a r - o l d s e e d l i n g s from UBC (day 45) and 3-yearold  s e e d l i n g s from Ladysmith and Nakusp (BC  m a t e r i a l o n l y , H 4) (See Table 2 f o r t e s t Q  temperatures and dates)  Table  92  12 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 d a t e s and l o c a t i o n s from c o n t r o l l e d  freezing  u s i n g 2 - y e a r - o l d s e e d l i n g s from UBC (days 45 and 66) and 3 - y e a r - o l d s e e d l i n g s from  Ladysmith and  Nakusp (BC m a t e r i a l only) (See Table 2 f o r t e s t 94  temperature and dates)  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  96  xi  Table  13 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 combined data of the Galena-Moscow h y b r i d , Galena-Arrow provenance and the US i n t e r i o r controlled  s e e d l o t s from  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 (See Table 1 for l o c a t i o n d e s c r i p t i o n s , F i g . 7 f o r p o l l e n source and Table 2 f o r t e s t temperatures  and  dates)  b)  97  Multiple-comparison  t e s t i n g of  provenance means  Table  97  14 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 needle e l o n g a t i o n phenology  (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 block grown a t UBC (Day  Table  0 i s March 3/89)  98  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 length  (cm) 2 - y e a r - o l d s e e d l i n g s  per s e e d l o t per block grown a t UBC (Day  0 i s March 3/89)  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  99  xi i  combinations of regions at two measurement dates  f o r shoot  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.  Table  length (cm) on f i v e  (Day 0 i s March 3/89)  101  16 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 j u r y assessment by a q u a n t i t a t i v e measure of % i n j u r y of t o t a l needles  scored on a l l  s e e d l i n g s i n both blocks exposed t o the u n c o n t r o l l e d f r e e z i n g i n Jan./Feb. 1989  102  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 i n j u r y assessment by shoot  length measurements on a l l  s e e d l i n g s i n both blocks exposed to the u n c o n t r o l l e d f r e e z i n g i n Jan./Feb. 1989  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 May  Table  18  102  length d i f f e r e n c e between  17, 1989 and J u l y 19, 1989  103  Shoot l e n g t h (mm) measurement of undamaged and damaged and  128  s e e d l i n g s i n blocks  1 and 2 f o r day 65 104  xi i i  L I S T OF FIGURES  Map of s e e d l o t o r i g i n s western  Fig. 2  white pine  (•)>, n a t u r a l range of  ( s t i p p l e d area) and r e g i o n  delineation  (numbers),  Mean needle  injury  ( m o d i f i e d from F o w e l l s  1965). 105  (%) and s t a n d a r d e r r o r by r e g i o n  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 a t UBC  Fig. 3  a)  Mean needle  injury  106  (%) and s t a n d a r d e r r o r of 7  c o a s t a l provenances by l a t i t u d e u s i n g combined from  5 dates of c o n t r o l l e d  freezing using  data  2-year-old  s e e d l i n g s grown at UBC...  b)  Mean needle  injury  107  (%) and s t a n d a r d e r r o r of 8  i n t e r i o r provenances by l a t i t u d e u s i n g combined from  5 dates of c o n t r o l l e d  freezing using  data  2-year-old  s e e d l i n g s grown at U B C .  Fig. 4  a)  Mean needle  region  injury  107  (%) and s t a n d a r d e r r o r by  f o r 5 dates of 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 grown a t UBC  Fig. 4  b)  Mean needle  injury  (%) and s t a n d a r d e r r o r  108  showing  v  xi  significant  region x temperature i n t e r a c t i o n  f o r the  i n d i v i d u a l t e s t day 20 f o r regions 1 and 2 only  Fig. 5  Mean needle i n j u r y from c o n t r o l l e d  (%) and standard e r r o r  freezing  109  by region  using 2-year o l d s e e d l i n g s  from UBC (day 66) and 3-year-old s e e d l i n g s from Ladysmith and Nakusp  Fig. 6  a)  Mean needle i n j u r y  r e g i o n from c o n t r o l l e d  110  (%) and standard e r r o r by freezing  using 2-year o l d  s e e d l i n g s from UBC (day 45) and 3-year-old s e e d l i n g s from Ladysmith and Nakusp  Fig. 6  b)  Mean needle i n j u r y  significant the  showing  regions 1 and 2 only  Fig. 7  L o c a t i o n of p o l l e n  Fig. 8  a)  112  source from Moscow, Idaho  Mean needle i n j u r y  freezing  (%) and standard e r r o r  region x temperature i n t e r a c t i o n f o r  Ladysmith  provenance  111  113  (%) and standard e r r o r by  f o r combined data from  controlled  on 5 t e s t dates using 2-year-old s e e d l i n g s  grown a t UBC.  BC-I = Galena-Arrow  provenance,  H y b r i d = Galena-Moscow c r o s s , US-I = Flower, C r y s t a l and Beaver Creek provenances combined  114  Fig. 8  b)  Mean needle i n j u r y  provenance f o r 5 dates 2-year-old  (%) and standard  e r r o r by  of c o n t r o l l e d f r e e z i n g using  s e e d l i n g s grown at UBC.  Arrow provenance, H y b r i d  BC-I = Galena-  = Galena-Moscow c r o s s , US-I  = Flower, C r y s t a l and Beaver Creek provenances combined  Fig. 9  a)  Mean s i z e and standard  elongation in  Fig. 9  b)  e r r o r of needle  (mm) by r e g i o n over 5 measurement  1989 on 5 s e e d l i n g s per s e e d l o t  Mean s i z e and standard  elongation in  115  i n block  dates 1  116  e r r o r of needle  (mm) by r e g i o n over 5 measurement  1989 on 5 s e e d l i n g s per s e e d l o t  i n block  dates 2  117  xv i  ACKNOWLEDGEMENTS  I would l i k e t o thank Dr. Don L e s t e r f o r h i s t h o u g h t f u l a d v i s i n g and c a r e f u l r e v i s i n g of t h i s t h e s i s ,  Dr. Mike  Meagher f o r h i s time and information p r o v i d e d and Dr. John Worrall for h i s helpful discussions.  Many thanks t o Dr.  Judy Loo-Dinkins f o r her a s s i s t a n c e with the computing, data a n a l y s i s and encouragement.  In a d d i t i o n , thank you t o my  f e l l o w graduate students, John R u s s e l l , David K o l o t e l o , Greg O ' N e i l l , M a r i l y n Cherry, George Harper and Donna Robertson for  t h e i r h e l p i n data c o l l e c t i o n .  I would a l s o l i k e to  thank Dr. Stan Boutin f o r h i s support, encouragement and skilful  e d i t i n g of s e c t i o n s of t h i s  thesis.  PREFACE  T h i s study was funded by the Canadian F o r e s t r y under the C a n a d a - B r i t i s h  Service  Columbia F o r e s t Resource Development  Agreement (1985-1990) and by the NSERC/Industrial Chair i n F o r e s t Genetics  and Tree  Improvement.  1  1.0  INTRODUCTION  Western white pine  (Pinus  (Pw) has a wide e c o l o g i c a l d i s t r i b u t i o n both a c o a s t a l  Dougl. ex. D. Don)  monticola  (Powells 1965), with  ( d r i e r c o a s t a l western hemlock zone) and an  interior  range ( i n t e r i o r cedar hemlock zone) i n B r i t i s h  Columbia  (BC)  ( K r a j i n a et al.  States (US)(Fig.  1982) and western U n i t e d  1). Although Pw has been d e v a s t a t e d by the  white pine b l i s t e r  rust  F i s c h . ex. Rabenh.),  fungus  (Cronartium  r i b i c o l a  i n t r o d u c e d to Vancouver  about  J.C. 1910,  it  has r e c e n t l y been r e i n s t a t e d f o r commercial use i n BC (Hunt 1988, Muir 1988) as i t has many d e s i r a b l e  characteristics  f o r use as a commercial s p e c i e s . Favourable response to g e n e t i c s e l e c t i o n f o r r u s t r e s i s t a n c e has reduced the r i s k of l o s s from b l i s t e r  rust  (Bingham  1983) as have s i l v i c u l t u r a l  et al.  techniques i n c l u d i n g pruning and  hazard r a t i n g of p o t e n t i a l p l a n t i n g s i t e s 1983, Hungerford et al. to the r e i n t r o d u c t i o n  1972, Bingham  1982).  (Hunt 1982, Hunt  At p r e s e n t , a c o n s i d e r a t i o n  i s how t r a n s f e r a b l e , r e g a r d i n g c o l d  h a r d i n e s s , seed from d i f f e r e n t Pw's coast and i n t e r i o r  seed sources w i l l be w i t h i n  ranges i n BC.  In g e n e r a l , Pw, r e l a t i v e t o many other c o n i f e r s , has shown r e l a t i v e l y l i t t l e  genetic v a r i a t i o n  c o l d h a r d i n e s s a s s o c i a t e d with provenance Hanover  1972, S t e i n h o f f  1979, R e h f e l d t  1984, Campbell & Sugano 1989).  i n phenology and (Townsend &  1979, R e h f e l d t et  Both D o u g l a s - f i r  al.  {Pseudotsuga (Abies  2 (Mirb.) Franco) (Df) and grand f i r  menziesii  [Dougl.] L i n d l ) , which a l s o have c o a s t a l and  grandis  i n t e r i o r d i s t r i b u t i o n s , have shown marked d i f f e r e n c e s i n s u r v i v a l and height growth when t r a n s f e r r e d two zones.  Rehfeldt  between these  (1977), found that c o a s t a l Df grew  f a s t e r than i n t e r i o r Idaho sources i n Idaho, but winter s u r v i v a l was very poor.  S i m i l a r l y , Steinhoff  (1980), found  c o a s t a l grand f i r to grow twice as f a s t as i n t e r i o r grand fir  i n Idaho.  The degree of winter s u r v i v a l was  however, than that of the Df. to the i n t e r i o r  (Steinhoff  higher,  Pw t r a n s f e r r e d from the coast  1981),  (12-year-old Olympic  P e n i n s u l a s t o c k ) , and from the i n t e r i o r to the coast of BC (Bower 1987)  (11-year-old Idaho s t o c k ) , showed no  s i g n i f i c a n t d i f f e r e n c e s i n e i t h e r height growth or winter survival.  Isozyme a n a l y s i s , by S t e i n h o f f that the major genetic v a r i a t i o n two l a t i t u d i n a l l y d i s t i n c t  et  i n Pw  al .  (1983) showed  i s a s s o c i a t e d with  subgroups. The Rocky Mountain  northern Cascade ranges form the major  subgroup of Pw,  a smaller subgroup i n the S i e r r a Nevada Mountain p o p u l a t i o n of Pw  range.  and  with The  i n the southern range of the Cascade  Mountains appears to be i n t e r m e d i a t e between the two subgroups.  The small amount of v a r i a t i o n which does occur  w i t h i n the p o p u l a t i o n s north of 44° l a t i t u d e appears to be u n r e l a t e d to geographic v a r i a b l e s Steinhoff  et  al .  1983, R e h f e l d t  et  (Rehfeldt al.  1984).  1979, Further,  v a r i a t i o n predominantly has been found to be among  3 (Townsend & Hanover 1972,  individuals  Campbell & Sugano 1989).  S q u i l l a c e and  reported e c o t y p i c v a r i a t i o n s e e d l i n g establishment. provide  evidence  findings  in Pw  Bingham  (1958),  f o r both growth r a t e  c o n t r a r y to S q u i l l a c e and  (Rehfeldt  1979,  and  R e s u l t s d i s c u s s e d above however, Bingham's (1958)  1979).  These isozyme and support  Steinhoff  s u r v i v a l s t u d i e s d i s c u s s e d above  the view that Pw  repopulated  i t s northern  range from  a s i n g l e refugium, p o s s i b l y i n southwestern Oregon, during the l a s t  (Wisconsin) g l a c i a t i o n  F u r t h e r , the north-south  split  (Critchfield  i n the degree of  found i n t h i s s p e c i e s i s a l s o present  refugium  (Critchfield  R e h f e l d t and et  al .  1984).  1984), suggest that the a b i l i t y of Pw  these very d i f f e r e n t environments (coast and  genetic v a r i a t i o n . phenotypic  The  by phenotypic direct  a  a l s o s u b j e c t e d to an  S t e i n h o f f (1970, Rehfeldt  governed predominately  variation  in Df which occupies  s i m i l a r geographic range and which was ice-age  1984).  1979,  Rehfeldt  to adapt to interior) is  plasticity  r a t h e r than  r e l a t i o n s h i p between  p l a s t i c i t y and c o l d h a r d i n e s s however, i s only  speculative.  T h i s study, p r o v i d e s a p r e l i m i n a r y  examination of t h i s r e l a t i o n s h i p .  The question  o b j e c t i v e of t h i s study was of Pw  hardiness,  seed t r a n s f e r a b i l i t y ,  between the coast and  to address  the  i n terms of c o l d  i n t e r i o r of BC.  Controlled  c o l d  hardiness  d i f f e r e n c e s seed  t e s t i n g  in  c o l d  c o l l e c t e d  ( F i g .  1)  in  l a t i t u d e  c o n d i t i o n s . r e g i o n a l ,  which  a d d r e s s i n g h a r d i n e s s  i t  s i g n i f i c a n t  of  et  al  .  in  damage  at  a  f r o s t at  development.  in  f r o s t  is  2  complex g e n e t i c  f a c t o r  in  r e s u l t s  the  a  s e e d l i n g s BC,  (Glerum  u s e f u l  t o o l  when  F r o s t  understanding  the  have  both  t r e e  breeders  1986).  been  only  coast  Meagher  1985.  at  b a s i s  w i l l  for  had  time  the  44°  p h y s i o l o g i c a l  t r a n s f e r a b i l i t y .  r e s u l t s  B a r r i e r e , in  a  (Bongarten  one  of  f i e l d  e s t i m a t e d  t e s t i n g  h a r d i n e s s  between  F  were  and  from  l e v e l s .  The  The  F u r t h e r , Idaho  nursery  i m p l i c a t i o n s  p l a n n e r s  d i f f e r e n c e  under  important  c o n d i t i o n s  s p r i n g  n o r t h  seed  s p e c i e s .  p l a n t a t i o n  severe  of  management  to  l o c a t i o n s  hardiness  of  1984).  d i f f e r e n t  u n d e r l y i n g  an  P r e v i o u s l y ,  l i n e  an  r e l a t i v e  grown  h a r d i n e s s  has  the  s e e d l i n g s  f a m i l y  a l s o  r e f o r e s t a t i o n  h a r d i n e s s  and  e s t i m a t e  using  grown  f r o s t  a  to  d i f f e r e n c e s  q u e s t i o n s is  c o n t r o l l e d  and  f r o s t  makes  d i s t r i b u t i o n  and  s e v e r a l  provenance  c h a r a c t e r i s t i c , 1975)  hardiness  G e n e t i c  Although  4 used  was  in  t e s t e d the  e a r l y  i d e n t i f i e d and  (1988), r e l a t i v e which  under  a  phase  t e n t a t i v e  i n t e r i o r c i t e d to  was  an  l o c a l  of  (Rehfeldt g r e a t e r s e e d l i n g s ,  s u b j e c t e d  to  a  5  2.0  LITERATURE  2.1  REVIEW  PHENOTYPIC P L A S T I C I T Y  The d i s t r i b u t i o n interactions, disease),  (e.g.:  of a s p e c i e s  competition,  i s d e t e r m i n e d by  predation,  and p h y s i o l o g i c a l t o l e r a n c e  biotic  parasitism,  to a b i o t i c  conditions  (eg:drought, c o l d , h e a t ) .  Physiological tolerance i s  determined  of g e n e t i c  species  by a c o m b i n a t i o n  and/or phenotypic  contributions difficult Bradshaw is  of these  to assess. (1965),  the a b i l i t y  morphological measure  but  individual  i s straight-forward  very  as d e s c r i b e d  of the i n d i v i d u a l .  t o p r o d u c e more t h a n  by It  one  with  be  grown  ( n o r m a l and s t r e s s e d ) .  This  for species other  from a p o p u l a t i o n  or h a l f - s i b  instances,  i s often  t h e same g e n o t y p e s h o u l d  environments  i s more d i f f i c u l t  full  relative  plasticity,  is a characteristic  i t s importance,  individuals of  two components  The  in a  a l t e r n a t i v e , o r p h y s i o l o g i c a l s t a t e and t o  under d i f f e r e n t process  plasticity.  Phenotypic  o f an  variation  species  must  w h i c h c a n be where  be u s e d .  different  Large  numbers  i n d i v i d u a l s a r e needed i n these  t o compare  responses  (means) a c r o s s  environments  (treatments).  The c l a s s i c  cloned  phenotypic  plasticity  experiment i n  6 p l a n t s  i n v o l v e s  f a m i l i e s ,  growing  p o p u l a t i o n s  environments  c h a r a c t e r s  of  e t c . ,  t y p i c a l l y l e v e l  of  p r o v i d e s  ( l e a f  by  a  separate  and  t o  the  In  a  i n d i c a t e s  genotype one-way  t h a t  measured  d e t e r m i n e d  i s  s i g n i f i c a n t  one  environment  s i g n i f i c a n t  genotype  i n d i c a t e s  t h a t  d i f f e r e n t  environments  d i f f e r e n t ) . of  d i f f e r e n t  versus  the  Among  same  the  to a  s i g n i f i c a n t  d e v i a t i o n  of  for  each  an  in  the  and  In  a a  a l l  the  c o n t r i b u t i o n  of  genotype  two-way  ANOVA  p l a s t i c  a  response  i n t e r a c t i o n d i f f e r e n t l y  i s  under  are  not  a  s a t i s f a c t o r y  p a t t e r n s when  f o r  a  of  genotype  (e.g.: from  f u r t h e r  ecovalence=the the  e x p e c t e d  c o m b i n a t i o n ,  p a i r w i s e a  being  environments.  i n t e r a c t i o n s ,  p e r f o r m i n g  the  t r e a t m e n t  c h a r a c t e r  a v a i l a b l e  environment  and  respond  response  d i f f e r e n t  then  ANOVA  responses  t h e r e  are  i n t e r a c t i o n  respond  p l a s t i c  s i z e  e n v i r o n m e n t a l  environment  g e n e t i c  of  s i g n i f i c a n t  i n d i c a t e s  t e c h n i q u e s  r e g r e s s i o n , average  and  genotypes  however,  i n d i v i d u a l  genotype  a  d i f f e r e n t  (ANOVA)  d i f f e r e n t i a t e between  way  a n a l y z i n g  d e t e r m i n e d .  genotypes  f i x e d  many  i s  p l a s t i c .  X  (the  p r e s e n t  a n a l y s i s  p l a s t i c i t y responds  At  v a r i a n c e  and  e f f e c t  of  f l o w e r s ,  of  environment  environment be  of  genotypes,  environment)  g e n e t i c X  s e r i e s  number  given  ANOVA,  to  a  ( c l o n e s ,  Measurements  d i f f e r e n t  in  method  a  a n a l y s i s  d i f f e r e n t l y  a  in  taxa  in  s i z e ,  p l a s t i c i t y  means  more  1986).  phenotypic  components.  and  or  s p e c i e s )  produced  a n a l y s e d  components  e f f e c t  or  ( S c h l i c h t i n g  phenotypic f l o w e r s  two  ANOVA's  genotype  to  j o i n t  and  the  means  e s t i m a t i n g  o v e r a l l  7 interaction),  a n a l y s e s can  (compares p a t t e r n s connecting  of  the  treatments  character  by  p l a s t i c response. with  variation compared  forest  and to  1986). of  the  (standard deviation,  (Bradshaw  The changes  the  variance,  1989).  The  the  phenotype  1965).  and  not  direction,  of  direction  l e v e l s among  genetic  can  be  taxa  samples  m e a s u r e s of  increases  variability  c o e f f i c i e n t of  variation),  genetic  to  by  variation  i s that The to  fluctations 1965,  which  altered be  in i t s  Bradshaw for  &  the  controls physiological  environmentally  i n the  1989)  physiological  responsible  character  is specific  (Bradshaw & H a r d w i c h  is  (Bradshaw  i s thought  change  of  environmental  clonal  1986).  a random s h i f t  A l t h o u g h the  a  (deviations  interactions  system  changes  r e s p o n s e or  amount and  individual  genetic  (Schlichting  means f o r  analysis  plasticity  (stress)  development  of  1986).  phenotypic  i n r e s p o n s e of  or  (species,  within-treatment  Schlichting  of  of  calculated  observed p h y s i o l o g i c a l  specific  use  amount of  1965,  basis  The  i n rank  treatment  estimates  analysis  segments  classic quantitative  X environment  immediate e n v i r o n m e n t Hardwich  the  determine p l a s t i c i t y  precision  controlling  For  trees,  family  (Schlichting the  changes  genotypes  determine  profile  environments),  p r i n c i p a l component to  by  line  g e n o t y p e s ) of  between two  from r e g r e s s i o n ) ,  studies  individual  (recording  between two  p o p u l a t i o n s ) or  the  performed  g e n o t y p e means i n d i f f e r e n t  Spearmans c o r r e l a t i o n  single  be  (Bradshaw  in pattern  t h i s d o e s not  and imply  that  8 a d a p t i v e and t h a t  i t i s necessarily  fitness  (Smith-Gill  thought  to a f f o r d  changes  i n environment  genotype  1983).  plants  (Bradshaw  i t increases  However, p l a s t i c i t y  an a d a p t i v e  experienced  1965).  basis  by an  Expression  i s usually  to deal with the individual  of p l a s t i c i t y or  changes  i n t h e p h e n o t y p e , do n o t have an a c c o m p a n y i n g  genetic  change  generally  fixed)  phenotypic is,  (since  selection  associated  plasticity  therefore,  the genetic  with that  (Jain  i n somatic  expression.  i s under a d d i t i v e  heritable  (Bradshaw  system  genetic  Finally, c o n t r o l and  1978) and a f f e c t e d  1965, S t e a r n s  cells is  by  1983, Bradshaw & H a r d w i c k  1989) .  In desire  much o f t h e l i t e r a t u r e ,  to link  phenotypic  heterozygosity. population selection are  plasticity  i s b a s e d on t h e h y p o t h e s i s  pressures will  fluctuate,  survive.  individuals  Some s t u d i e s  phenotypic Silander (Scheiner  only  Highly  those  i fa  individuals  which  f i t or s p e c i f i c a l l y  for natural  have f o u n d g e n e t i c  plasticity  that  v a r i a t i o n and  (e.g.: with s p e c i f i c  c o m p l e x e s ) may n o t be p r e s e n t upon.  t o l e v e l s of  o r s p e c i e s h a s low g e n e t i c  plastic,  adapted  This  t h e r e a p p e a r s t o be a  t o be i n v e r s e l y  adaptive  gene  selection  to act  v a r i a t i o n and related  ( J a i n 1979,  1985), b u t o t h e r s have f o u n d no c o r r e l a t i o n and G o o d n i g h t  1984, S c h l i c h t i n g  Schlichting  1986, M a c d o n a l d  correlation  (Wilken  & Chinnappa  1977, S c h l i c h t i n g  and L e v i n 1984,  1989), o r a  1986).  positive  The g e n e t i c  b u f f e r i n g  suggested dampen would the  the  and  high  b u f f e r e d of  suggests s t a b i l i t y  or  r e s p o n s i b l e g e o l o g i c a l  of  both  f o r time  in  in  The  mean  of  given  t h a t  r a p i d l y  given  another,  and  Wright  (1931),  in  thus  in  a  may  given  (Bradshaw  t r a i t slowing  1965).  m o s q u i t o f i s h ,  However, t r a i t  t r a i t  thereby  e v o l v i n g  may  appearing  m o r p h o l o g i c a l s p e c i e s .  a  f o r  s e l e c t i o n  s t u d i e d . a  p l a s t i c i t y  one.  p l a s t i c i t y  shown  most  t r a i t  lack a  of  c l e a r  d i f f e r e n t i a t i o n  has  the  in  the  a  d i r e c t i o n a l  p l a s t i c i t y s t a s i s  phenotypic  s e l e c t i o n .  from  p l a s t i c  t h a t  not  amounts  however,  was  most  a l s o  p o p u l a t i o n  (1983)  m a t u r i t y the  is  e f f e c t s  process  Stearns at  t h a t  be  9 between  r e l a t i o n s h i p  t r a i t  s t u d i e d  Stearns c o n f e r to  changes  age  be over  (1983)  10  2.2  P L A S T I C I T Y AND COLD HARDINESS  Phenotypic characters within  plasticity  i s specific for individual  a n d may a p p l y t o o n l y a few o r many c h a r a c t e r s  a given i n d i v i d u a l .  As w e l l ,  t h e degree of  plasticity  may v a r y between c h a r a c t e r i s t i c s a n d t h e  plasticity  o f one c h a r a c t e r i s t i c may r e s u l t  of  (Schlichting  another  1986).  on p h e n o t y p i c p l a s t i c i t y between d i r e c t  a direct 1989).  environmental cues  response  How  Most p h y s i o l o g i c a l  h a s f o c u s e d on  r e s p o n s e . An e n v i r o n m e n t a l  this  stress  varies  environments  research  relationships  and t h e a s s o c i a t e d such as drought,  i n stomatal closure  response  i n the s t a b i l i t y  can cause  (Bradshaw & H a r d w i c k  f o r a g i v e n genotype i n  different  drought  would be examined t o  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  t o an i n d i r e c t cue (Bradshaw & H a r d w i c k  Physiological  changes a s s o c i a t e d  stress  with c o l d  r e s i s t a n c e mechanism t o t h e s t r e s s  response  t o t h e e n v i r o n m e n t a l cues  temperature  respectively,  (cold)  (Bradshaw  stress  itself  plasticity  In  1989).  addition,  of c o l d ) ,  are a  of impending  stress  The c u e s a r e n o t t h e  i n t h i s case and t h e r o l e  in a cold  h a r d i n e s s (a  of p h o t o p e r i o d and  i n d i r e c t cues  & Hardwick  1989).  h a r d i n e s s response  of p h e n o t y p i c i s unknown. .  how p h o t o p e r i o d a f f e c t s  traits  such as  dormancy, a p o s s i b l e the  11 key component  development of c o l d  research  on p h e n o t y p i c  morphologically (Caswell poorly  plasticity  i s unknown.  in relation However,  Caswell  Since  altered  characteristics  responses  remain  ( 1 9 8 3 ) , d i s c u s s e s a model of  to p l a s t i c i t y  whereby  fitness is  t h i s model h a s y e t t o be t e s t e d  i n terms of the l i f e  most  h a s f o c u s e d on t h e d i r e c t  1983), i n d i r e c t p h y s i o l o g i c a l  increased. studied  hardiness,  and p h y s i o l o g i c a l l y  understood.  dormancy  or p a r a l l e l p r o c e s s i n  history  of a s p e c i f i c  or species.  1 2  2.3  COLD HARDINESS AND  Timing tolerate the  of a c c l i m a t i o n , t h e i n c r e a s i n g  cold,  temperate  well  is critical regions.  Blum  a secondary  and/or p h o t o p e r i o d  1988 and o t h e r s ) .  dormancy and f r o s t to  to the s u r v i v a l  be s p e c i e s - a n d  hardiness  process  f o r many n o r t h e r n  (2):  first  (usually winter for  stage  induced  a prolonged  (Weiser  resulting  resulting  from  Weiser  Stage ( 1 ) :  photoperiod, from  low  -30°C t o -50°C  shows t h a t two woody s p e c i e s ,  Viburnum  plicatum  independently  appears  stage  temperatures  ( 3 ) : a c c l i m a t i o n t o a phase of  p e r i o d . Work by I r v i n g  tomentosum  between  one and  species.  by a s h o r t e n e d  and s t a g e  1980,  a three-stage acclimation  however,  hardiness  t o changes i n  1970, L e v i t t  i s not a c l e a r  deciduous  of hardening  a freeze),  hardiness  reaction  h a s been  sometimes p o p u l a t i o n - s p e c i f i c .  describes in detail,  o f dormancy  o f woody p l a n t s i n  However, t h e r e l a t i o n s h i p  (1970),  onset  a b i l i t y to  This acclimation process  documented a s b e i n g  temperature  DORMANCY  and L a n p h e a r Acer  (1970)  (1967) L. and  negundo  Thunb., d e v e l o p e d  of Weiser's  temperatures  frost  'first  stage'  dormancy.  The  dormancy p r o c e s s  or c e s s a t i o n i n growth o f t e n  13 w i t h bud s e t i n d e t e r m i n a n t  associated stages.  The f i r s t  described  stage  i s usually  a s t h e p h a s e when p l a n t s  conifers,  the r e s t i n g will  by a p e r i o d  requirement  phase, c a l l e d occurs.  quiescence  In t h i s  second grow  phase,  (Glerum  temperate  and b o r e a l f o r e s t  increase  in cold  (Glerum  1975).  conditions  of  frost  1975, W e i s e r trees  break  i t appears  t o growth,  i s a very rapid  typically growth.  Glerum  i n blue spruce  December from they  Although  t o development  larch  still  showed  (1977) have  shown  Engelm.) s e e d l i n g s , t h e  pungens  i s the mediator  A new n o m e n c l a t u r e  trees  (1975) f o u n d bud  when t h e new n e e d l e s  c u e and i n d u c t i o n  while the  found  In e a s t e r n  that  photoperiodic  often  lags behind the  h a r d i n e s s t o - 1 7 ° C . Young and Hanover  phytochrome system  removed  grow.  cold  (Picea  that,  i t i s often  readily  (DuRoi) K. K o c h . ) ,  occurred i n A p r i l  second  For north-  by e a r l y  process r e l a t i v e  hardiness, i t s t i l l  when  i n the f a l l ,  F u r t h e r , i f t r e e s a r e then  l a r i c i n a  The  1970).  h a r d i n e s s t o some maximum  change t o q u i e s c e n c e and l a t e r (Larix  i s met.  i s being s a t i s f i e d  not conducive  i s broken  i f conditions are favourable,  have e n t e r e d q u i e s c e n c e and w i l l dehardening  i s sometimes  o r imposed dormancy, t h e n  will  requirement  stage  of the p l a n t  the p l a n t  chilling  stage  o f q u i e s c e n c e , d e s c r i b e d below, when  lammas g r o w t h may o c c u r . The r e s t i n g the c h i l l i n g  stage  n o t grow even i f  c o n d i t i o n s a r e f a v o u r a b l e . However, t h i s preceded  h a s two  between t h e e x t e r n a l  o f dormancy.  has e v o l v e d f o r t h e d e s c r i p t i o n o f  dormancy, b u t i s n o t y e t w i d e l y u s e d  i n the l i t e r a t u r e .  The  terms used  physiology-oriented increasing dormancy  versus  the p r e c i s i o n  i s defined  g r o w t h o f any p l a n t 1987). are  Within  into  (of a m o r p h o l o g i c a l  affected  perceived  suspension  structure  containing  structure  i s solely within  and t h e s i g n a l c o u l d  growth  (Lang  1987).  o f dormancy when d i s c u s s i n g  than  be due t o a  t h i s phenomenon.  of f r o s t h a r d i n e s s or f r e e z i n g  on a s i n g l e  factor.  sequences d e s c r i b e d  tolerance  p r o c e s s which i s  Thus, the m u l t i t u d e of  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  o f a l t e r n a t i v e p r o c e s s e s w h i c h have e v o l v e d t o temperatures.  (1980) s u g g e s t s t h a t  multiple  hardening  temperature c o n t i n u a l l y  induction,  Further,  Levitt  s t a g e s may e x i s t a s  drops over a p e r i o d  t h e same e n v i r o n m e n t a l c u e s p l a y  dormancy  other  use t h e c l a s s i c a l  freezing  Since  (of a  For the purposes of  overcome a n d s u r v i v e  the  the a f f e c t e d  are not s u i t a b l e f o r  1980) i s a complex p h y s i o l o g i c a l  dependent  variety  above  o r due t o t h e c o n t i n u a l  i n the growth environment  Development  possible  (Lang  induction  in a structure  l i t e r a t u r e r e v i e w however, I w i l l  (Levitt  of v i s i b l e  a meristem'  1) endodormancy, when t h e response)  system,  the phases d e s c r i b e d  response) o r i g i n a t e s  metabolic  descriptions  not  a s , 'the temporary  thereby  o f an i n h i b i t o r , 3) ecodormancy, when one o r  more f a c t o r s  this  With t h i s  e n v i r o n m e n t a l cue  production  overall  of the terms.  2) p a r a d o r m a n c y , when t h e i n d u c t i o n  morphological the  season- or p h a s e - o r i e n t e d ,  this description,  b r o k e n down  structure,  14 t h e p r o c e s s o f dormancy a r e  to describe  of time.  a significant role in  t h e r e l a t i o n s h i p between dormancy and  frost  h a r d i n e s s remains  15 blurred.  showed t h a t  the f r o s t  localized.  By e x p o s i n g d i f f e r e n t  seedling  h a r d i n e s s mechanism  to different  hardiness could  be c o n t r o l l e d  two-stage  (equal  discussed  above) sequence  transition  approximately (1975), enter  branches  i n each  o f t h e same of f r o s t  branch.  s p e c i e s , Glerum  to Weiser's  (1974),  i n Df s e e d l i n g s i s  environments, development  In s e v e n c o n i f e r o u s  The  Timmis and W o r r a l l  (1973) o b s e r v e d a  (1970) s t a g e s one and two  i n development  of f r o s t  hardiness.  from one s t a g e t o t h e n e x t o c c u r r e d a t -18°C over a p e r i o d  suggests that  o f a few d a y s .  most n o r t h e r n - t e m p e r a t e - z o n e  Glerum conifers  dormancy b e f o r e maximal w i n t e r h a r d i n e s s i s a t t a i n e d .  Studying  10 p r o v e n a n c e s  found  that  first  frost,  seedling  of Df, Campbell  f o r each a d d i t i o n a l  week o f bud s e t p r i o r  t h e r e was a p r o p o r t i o n a l  frost  injury.  and S o r e n s e n  (1973), to the  d e c r e a s e o f 25% i n  16  2.4  COLD HARDINESS AND  THE  ASSOCIATED  PHYSIOLOGICAL CHANGES  Although induction unifying  the  of c o l d concepts  sequence  of e v e n t s  hardiness i s varied,  of  freezing  do  supercooling' freezing.  fall  below t h i s  of l i v i n g  cells  & S t e s h n o f f 1979).  Burke  nucleating  use  'deep  al.  1976)  to  where  to  avoid  i n r e g i o n s where  to remain  1976,  At  et  growing  i s an a v o i d a n c e mechanism and  temperature  1979).  Plants  plants  t h r e s h o l d must employ  surrounding  supercool  The  freeze-  mechanisms.  ability  nucleating  hardiness.  herbaceous  (Burke  grow  and  various tolerance  below -40°C may  (-20°C t o - 4 5 ° C )  Supercooling the  fall  P o p u l a t i o n s which  temperatures tolerance  not  and  s h r u b s and  temperate-climate winters.  temperatures  t h e r e a r e some  with cold  avoidance  mechanisms a l l o w s t r e e s , survive  to the  r e g a r d i n g the p h y s i o l o g i c a l  b i o c h e m i c a l changes a s s o c i a t e d combination  leading  substance t o -38°C this  i s below  to i n i t i a t e  (Burke  et  temperature,  substance, pure  al.  In t h e a b s e n c e freezing,  et  (Burke  pure  Burke  al.  of a water  will  & Steshnoff  r e g a r d l e s s of the p r e s e n c e  water  will  to  u n f r o z e n when t h e  freezing  1976,  refers  crystalize.  The  of a limit  to deep s u p e r c o o l i n g freezing  i s -40°C when s p o n t a n e o u s  appears t o occur  through mechanical  In c o l d e r  and c e l l s  l o s s of c e l l  boreal  a v o i d a n c e mechanism  are k i l l e d ,  climates,  t h e deep  i s not employed.  supercooling  Tolerance  with in  this  effective  al.  membranes.  membrane p e r m e a b i l i t y cell  1976, L e v i t t  There  i s an i n c r e a s e  allowing  water  (Burke  et al.  t h a w i n g c a n be c r i t i c a l .  Concomitant  in cell-  t o more r e a d i l y  leave  relatively  be r e t u r n e d  hour  1985).  I f thawing  (Glerum  osmoregulation across  a t i s s u e , t h e r a t e of  When f r e e z e  should  in  occurs  1976).  temperatures  the c e l l  increase  in cell  i n phospholipids  the c e l l  membrane  woody s p e c i e s  testing,  i s too rapid, the membrane c a n n o t  accomodate  t i s s u e damage.  permeability  has been l i n k e d t o an  and u n s a t u r a t e d  (Graham & P a t t e r s o n  the degree of u n s a t u r a t i o n  seems v a r i a b l e a n d l i k e l y  starting  t o a t a r a t e o f 10-20°C p e r  change q u i c k l y enough t o p r e v e n t  increase  i n t o the  mechanical b a r r i e r t o e x t r a c e l l u l a r i c e  When i c e h a s f o r m e d w i t h i n  The  1980).  b u t a t t h e same t i m e c r e a t i n g a  penetration  the  cells  r e - a l l o c a t i o n o f f r e e , unbound w a t e r , a r e c h a n g e s  the c e l l  the  develops  s p a c e s a n d t h e stem, where f r e e z i n g  1970, Burke et  (Weiser  probably  integrity.  t h r o u g h movement o f w a t e r o u t o f t h e l i v i n g extracellular  intracellular  does n o t p l a y  fatty  acid  1982). of f a t t y  chains  However, i n acids  as b i g a r o l e as i t  18 i n non-woody s p e c i e s ( G l e r u m  does  1982).  Correlations  h a r d i n e s s and acids and  increases et  (Burke  others).  of p i n e  (Pi  Karst.) day  al .  nus  et  s i l v e s t r i s  lowered  by  less clear that  Levitt  1980,  al .  (1976),  L.)  and  temperatures,  hardiness.  f o u n d between  hardiness  (Picea  may  development  reflect  L . ) , with a p a r a l l e l l e d et  (Aronsson  low  al.  decrease  ' w i n t e r h a r d i n e s s ' , i t i s unknown w h e t h e r  death  i s due  temperatures,  to spontaneous  when p l a n t s a r e  intracellular  1970,  et  cells is  1976,  temperatures  of t h e t o t a l and  o f bound  damage t o t h e membrane from i c e  or extreme d e h y d r a t i o n of the c e l l s  40%  in a  tissue  freezing  penetration  winter  nus  1976).  of  al.  (Pi  in cold  state  Burke  levels  in pine  extremely  mechanical  fact  and  Carbohydrate  however, t o d e c r e a s e v e r y r a p i d l y  are f a r  the  than hardening  i s more d i f f i c u l t .  short-  increases in  At  water,  (L.)  abies  sucrose content, with  which  1988,  seedlings  In g e n e r a l , t h e s e r e l a t i o n s h i p s  analysis  s i l v e s t r i s  found t h a t  showed l a r g e  during dehardening,  were f o u n d ,  nucleic  Singh & Laroche  spruce  t h e p r o c e s s i s much more r a p i d  therefore  and  cold  s h o r t - d a y t r e a t m e n t s o r combined  carbohydrates, e s p e c i a l l y of c o l d  been  Graham & P a t t e r s o n  in sugars, proteins  1976,  Aronsson  hardened  and  have a l s o  1975,  Levitt  1980).  When t h e  lowest  a r e b e i n g e x p e r i e n c e d , between  water  of a p l a n t  a l l f r e e water  i s frozen  i s bound w i t h i n  and  the degree  between  30  the  extracellularly.  no e v i d e n c e , however, o f a r e l a t i o n s h i p  amount of bound water  (Weiser  to living  There the  of h a r d i n e s s which  can  19 be a t t a i n e d  At  each of t h r e e  exothermic Under  (Childers  reaction  slow f r e e z i n g  1983).  stages  when  the water  conditions,  measured  through d i f f e r e n t i a l  allowing  the progress  to  freezing  in freezing,  protoplasm  After  As f u r t h e r  of the l i v i n g  'deep  different  pattern  described  above  exhibit  cold  exotherm  i s the k i l l i n g  freezes.  the f i r s t Burr  et  exotherm  al.  (1985),  spruces which s u p e r c o o l  two e x o t h e r m s w i t h DTA.  They  e x o t h e r m w i t h no low o r l e t h a l  T h i s method would  h a r d i n e s s assessment  Those  show a  p i n e s do n o t s u p e r c o o l and t h e r e f o r e  show t h e i n i t i a l exotherm.  supercooling  a second  t o - 4 0 ° C would  not being p r e s e n t .  supercool), that  and f r e e z e s ,  of exotherms w i t h  Df and s e v e r a l  tolerance  -2 t o - 8 ° C ( W e i s e r 1970),  The t h i r d  supercool'  showed t h a t  state  the i n i t i a l  a t which a l l r e m a i n i n g water  s p e c i e s which  (DTA),  w a t e r moves o u t o f t h e  cells  e x o t h e r m c a n be m e a s u r e d . temperature  t h e s e e x o t h e r m s c a n be  o f i c e f o r m a t i o n and t i s s u e  t o be r e c o r d e d .  begins.  i s an  ( W e i s e r 1970).  thermal a n a l y s i s  has r e a c h e d i t s maximum, o f t e n freezing  freezes  there  therefore  in pines,  (deep further  would  temperature  be o f l i t t l e  including  only  Pw.  use i n  20  2.5  GENETICS OF  Breeding  for cold hardiness  crop plants  (Blum  b r e e d e r s as  t o whether  in  COLD HARDINESS  1988). the  h a r d y v a r i e t i e s can  breeding,  we  are  far  There  present  continue  from h a v i n g  improvement  a tree  typically  Today's u n d e r s t a n d i n g  of  components of c o l d h a r d i n e s s has  fields  (Blum  (1988),  biology  state that  extracellular  and  W i t h monosomic wheat 70%  of  t y p e of and  freeze  r e s u l t has  isolate  this  lead  polypeptides  was to  examples of  winter  tree  genetic 1973,  Blum  for s e l e c t i o n in  c o n t r o l of  remarkably  1988).  S i n g h and  is likely  on  cereal  Laroche  to  i t was  freezing  found  chromosome 5A.  those  sequences.  i n many  to  that This  identify  sections  Blum  studies  is  multigenic.  attempting  analyze  the  limited  studied  wheat t o l e r a n c e  studies  and  In  This  genetic  located  genome f o r u n i q u e h a r d e n i n g discusses  the  trait  hardiness  1975).  substitution lines,  tolerance  the  been e x t e n s i v e l y  for winter that  winter  & Weiser  a priority  is s t i l l  although cold hardiness plant  (Sakai  (Glerum  among  improved.  in cold hardiness.  improvement p r o g r a m  of  of  exhausted  1988),  i s not  level  t o be  which appears to e x i s t  characteristic  in a g r i c u l t u r a l  i s disagreement  potential, for  i s common  of  the  (1988) where  additive  21 gene a c t i o n genetic action  basis of  genetic fruit  and  non-additive  for cold hardiness  genomes showed no  components  trees  and  due  to  combining  ability  year  X year  however, have shown t h a t raspberry  et  autumn f r o s t  of  the  was  al .  large,  studied  Pi  nus  component. that  The  (1977), observed and  varieties  of  The  literature  c e r e a l s and types,  variation The  the  Isolating  the  general Some  studies  peach  of  seedlings.  genetic  components  f o r the  s e l e c t i o n for this  major  trait  4-year o l d  coast  and  to part  variation could  seedlings,  weak a d d i t i v e e f f e c t s f o r of  and  1988).  within-population  in hybrids  very  L.  accounted  In  in  proved  inheritance  suggests that  the  frost  interior  differences  s u r v i v i n g c o l d e m p l o y e d by  the  different evolutionary  could  available  genetics  the  Df.  mechanisms of  plant  bud-set  where  in apple,  s y l v e s t r i s  p r o d u c e a more h a r d y p o p u l a t i o n .  hardiness  large  (Blum  and  the  cold hardiness  cold hardiness  gene a c t i o n  suggesting  studies  interaction effects.  in  t o be  blueberry),  (e.g.:  environmental  Additive  genetic  Rehfeldt  of  e f f e c t s and  (1986),  hardiness  showed t h e  equal.  other  is quantitatively inherited  Norell  be  studies  fruits  large  and  additive effects.  in f i e l d  small  difficult  Results  gene a c t i o n a p p e a r  of  account  f o r the  in trees  supercooling  trees versus  h i s t o r i e s of  fact  that  i s much g r e a t e r in trees  in  the  these  genetic  (Blum  i s a l s o not  winter  1988). known.  22  2.6  TECHNIQUES TO MEASURE HARDINESS  T h e r e a r e many t e c h n i q u e s injury tree  a s an e x p r e s s i o n  breeders  natural  have a s s e s s e d  climatic  time  years  o n l y once e v e r y  controlled  identified.  (severe  frost  Traditionally,  in field  p l a n t i n g s under hardiness.  temperatures)  t h i s method  i n t e n s i v e (Glerum  of s e e d l i n g s h o o t s ,  environments  differences  hardiness.  injury  10 y e a r s ,  c o n s u m i n g and l a b o u r  Freezing  t o measure  c o n d i t i o n s t o measure f r o s t  However, s i n c e t e s t occur  of c o l d  available  typically  i s extremely 1975).  branches or leaves i n  i s now common p r a c t i c e a n d  in populations  o r g e n o t y p e s c a n be r e a d i l y  Further, controlled  freezing  allows  a large  number o f samples t o be s c r e e n e d  and t e s t e d over  temperatures  F r e e z i n g c a n be done i n a  controlled with the  a t any g i v e n chamber  time.  i n the laboratory or, less  a portable freezer unit field  2.6.1  (Glerum  a range o f  typically,  where t e s t s a r e c o n d u c t e d  1985).  METHODS OF EVALUATION  in  23 evaluating  A common method of loss of  of  integrity  of  the  can  be  electrical tests.  i n j u r y i s based  p l a s m a membrane and  e l e c t r o l y t e s into surrounding  This  frost  water  impedance t e s t s , l e a c h 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  seedlings  (Glerum  recently,  chlorophyll  assess  injury since  membrane, decrease Glerum  1975)  (twig  or  relative  (supercooling seedlings  substance,  since  the  w h i c h may  temperatures nucleation water  than  sites  likely  Flint of  (Singh  suggests  that  since  provide  freezing  Electrolytes  to  et  long  due  lenticels  spontaneously  are  freeze  (1967), d e s c r i b e d  measured  longer  ornamental  a 1988).  provide  whole  t e s t i n g of of  a  there  are  the  lower more  external  al.  more 1976).  electrolytic  s h r u b s and  from u n f r o z e n and  with  Short  or wounds) and (Burke et  the  nucleating  in contact  to  a  hardier  initial.  supercool  needles since  injury using  lack  a nucleating  shown t o  (stomata,  al.  will  they appear  t o the no  to  plasma  detached-tissue  t e m p e r a t u r e ) t h a n do  be  to  the  & Laroche  in p a r t i c u l a r , only  needles are  s p r u c e n e e d l e s have been  More  a l s o been u s e d  when compared w i t h whole s e e d l i n g T h i s may  freezing  freezing, leading  differences  to a lower  same s e e d l i n g .  plant  by  ability  further  needle)  involve  t h y l a k o i d membrane, l i k e  in photosynthetic  measure of  the  the  has  1967).  stain test,  growth p o t e n t i a l .  fluorescence  i s also disrupted  (1973,  samples  root  loss  plasmolysis  evaluation  assessing  or  al .  a vital  t e s t s or  O t h e r q u a l i t a t i v e methods of  1975)  et  (Flint  via  measured q u a n t i t a t i v e l y  subsequent  on  berry  frozen  method crops.  tissue  as  well  as  total of  from  heat-killed  electrolytes.  injury'  with  heat-killed advantage results  tissue  T h i s method  obtained i s used  a measure of  stress  1975,  1985).  Glerum  equations  to  100.  widely  to v i s u a l  to assess  T h i s i s then  s e e d l i n g s to minimize  used  stress  with  assessment  r e s i s t a n c e in nursery  which  methods.  hardiness seedlings  as a g u i d e  and  and  significant  rapidity  frost  of  'index  to zero  One  methods i s t h e  relative  f o r an  samples e q u a l  samples e q u a l  of e l e c t r o l y t i c  are  lifting  They p r o d u c e d  unfrozen  tissue  24 samples w h i c h p r o v i d e a r e a d i n g  as (Timmis  for  maximize  field  survival.  J o h n s o n and  Gagnon  (1988) compared measurement  ethane p r o d u c t i o n to e l e c t r o l y t e frozen  loblolly  results  pine  w h i c h d i d not  p r o d u c t i o n method has tissue, plant  less  tissue.  testing  and  trends  in frost  first  with a  s i n c e i t can suggests  1 kHz  be  h a r d i n e s s of steel  w h o r l ) and  bridge.  The  artificially  two  methods gave  The  ethane-  requiring  less  used  any  with  that e l e c t r o l y t e by  the  leakage  ethane-  method of  choice.  t h e e l e c t r i c a l - i m p e d a n c e method  i n a s e e d l i n g (root branch  advantages,  the q u a n t i t a t i v e  (1973) u s e d  apart  statistically.  further  Glerum  using The  replaced operationally  W i t h t h i s method, two cm  differ  T h i s study  p r o d u c t i o n method as  study  seedling needles.  h a n d l i n g and  c o u l d be  leakage  of  pins  seven  coniferous species.  (electrodes) are placed 1  collar  o r between t h e  electical  first  to  impedance  reading  i s taken  terminal  i s measured prior  to  exposure to  25 f r e e z i n g t e m p e r a t u r e s and  again  f r e e z i n g (Glerum  after  1973),  from a l l s p e c i e s L.,  strobus  spruce,  P.  P.  (DuRoi) K.  B.S.P., and  method  and  the  subsequent  i s i n f l u e n c e d by  frequency reading  of  the  i s between 20  measurement, t h i s method  injury  yet  The  not  are  (Simpson  no  examined  1985).  pine  samples.  T h i s method  (Pinus  chamber  electrolytic  reduced  only  of  of  measurements frost  second  3)  impedance  pre-frozen  difficult.  injury,  versus  this  temperature,  from t h e  at a gross  of  Therefore,  level but  for  the  degree  of  derived.  visual  evaluation  f o r browning a f t e r  Rehfeldt  lodgepole  Preparation  i f the  P.  l a r i c i n a  However, a c c u r a c y 2)  nus  P.  spruce,  impedance  i n n u r s e r i e s where n e e d l e ,  for population  freezing  50%  Pi  Karst.,  Larix  assessment  Further,  injury  be  larch,  1) t i s s u e s i z e ,  to  qualitative,  operationally tissues  growth.  is informative of  (L.)  abies  electical  i n t e r p r e t a t i o n i s very  interpretation can  Picea  visual  current.  (Glerum  A i t . , jack pine,  eastern  K o c h . ) , were t h a t  i s taken  results  (Moench) V o s s , b l a c k  corresponded well with hardiness  The  second  (eastern white pine,  resinosa  glauca  (Mill.)  mariana  method  tested  Lamb., Norway s p r u c e ,  banksiana  white  red pine,  1985).  the  (1980,  s t u d i e s of contort  a  et  only  and  tissue is relatively  cambial  controlled freezing 1984)  D o u g l . ) and  has  Pw  used  with  using  inexpensive  equipment  methods, making a  bud  cold hardiness  is relatively  i s the  al.  i s used  both needle  since  required.  simple  compared  l a r g e volume of  this  to  samples  a  26 manageable. make  a  Since  v i s u a l  s u b j e c t i v i t y The  v i s u a l  t h i s  assessment which  method  is was  method of  r e q u i r e s  the  removed employed  i n j u r y ,  w i t h in  the i t  r e s e a r c h e r is  not  e l e c t r o l y t i c my  study.  f r e e  to from  methods.  27  2.7  COLD HARDINESS, PHENOTYPIC PLASTICITY AND  Most c o n i f e r s whose r a n g e interior  distribution,  show a c o a s t ,  specificity  (e.g.  an  gene c o m p l e x ,  adaptive  Individuals typically  fall  Steinhoff  i n j u r e d or  to  wide l a t i t u d i n a l  differences  i n response  mode  individuals well  ( n o r t h of exploit  from c o a s t  44°  the  ecological  (Campbell  cueing  and  to a loss  & Sugano  et  al.  p l a n t i n g s i n the  a d v a n t a g e on  to the  Although  killed  Species  tend  are  individuals  i n j u r e d or  to  et  (Ekberg  Pw  1984),  in  with  evolve  al .  1979).  in that  populations northern has  can  grow  species  the  range  flexibility  to  continuum w i t h i n i t s  a p p e a r s t o have a  better sites  of genomic  1989), but  coast  w h i c h show marked  interior  Pw  response.  a g e n e r a l i s t in i t s adaptive  Rehfeldt  latitude).  1977), p o s s i b l y  responses.  to photoperiod  distribution,  due  are  and  region  spring while  distributions  warm-dry c o o l - w e t  competitive possibly  i n the  been d e s c r i b e d as  in reciprocal  interior  populations  ( S t e i n h o f f 1979,  interior  Rehfeldt  interior  geographic  distinct  has  to  incorrect  genetically  a coastal  for cold-hardiness  killed  from c o a s t  due  Pw  1980,  t r a n s f e r r e d from t h e  transferred the  i n c l u d e s both  Pw  on  the  stability  reduced coast,  (specialization)  competes much more  successfully (Tsuga  het  erophylI  Washington) Empire  one  species the  is  o r no  slower.  role  reflect  results  earlier al.  143  the a d a p t i v e d i f f e r e n c e s variation  locations.  work on Pw  Campbell  hypothesis  i s that  et  & Sugano  this  genetic  w h i t e p i n e may  amount o f s o m a t i c p l a s t i c i t y .  plasticity  growth  and  that  Pw's  potential  to the n i c h e s i t i s  (1989),  locations They  to e n t e r t a i n  i s inconsistent  174  measured  Their and  structure. be e x h i b i t i n g  with high  The an  Sugano  exploiting  individual  most  from  Steinhoff  (1989) d e v e l o p e d  o f Pw  growth  expression) to  (1984) and  and  Pw  were grown i n  supports findings  Campbell  the idea  species.  among g e n o t y p e s .  al.  play  in general,  between l o c a t i o n s  This  by R e h f e l d t  to explain  that  sites  (two components o f g e n e t i c  hypotheses  suggest  Sugano  to  a role in  from m o r e - a g g r e s s i v e s e r a i  nursery environments.  showed l i t t l e  slow  grow up  play  argue  restricted  from  (1983).  since  Inland  codominant  i t s physiological  ( s e e d l i n g progeny)  within  or may  (1989)  and  variation  i t may  coastal  sites  i t i s merely  and v i g o r  evaluate  better  a s t u d y by C a m p b e l l  controlled  rhythms  pattern  and Sugano  i n by c o m p e t i t i o n  families two  not  time  zone  western  is relatively  which  growth  in interior  Campbell  Pw  and  stands i n the  t h e dominant  d i s a d v a n t a g e on  r a n g e may  In  et  15 y e a r s , a f t e r  suggest that  found  1983).  in a stand. This  present and  (Bingham  p e r y e a r becoming  relative  little  of n o r t h e r n Idaho  where i t c a n o c c u r i n p u r e  t o age  meter  zones  a  o f Idaho  growing  28 s i t e s of t h e same v e g e t a t i o n  in interior  three first unusual (1989)  heterozygosity. the case.  29 discussed e a r l i e r ,  As  et  Rehfeldt  heterozygosity  which  al.  t h i s may  (1984) s u g g e s t  i s present  i n Pw  not  that  be  the  p r o v i d e s a means  by  w h i c h m u l t i p l e b i o c h e m i c a l p a t h w a y s c o u l d have e v o l v e d that  selection  tolerances be  for a l l e l e s  that  Bower  second  occupying  sites  different  from  a r g u e t h a t Pw  dominant  hypothesis  site  as  such  of t h e  these  sites,  from  cm  level  similar  of p r e c i p i t a t i o n  and  w i t h a 60-160 day  only a  & Dyrness  1973).  elevational  r a n g e as on  i t grows w i t h  (1989) sites  small However,  on  which i t  the c o a s t , with  100-225 f r o s t - f r e e can  up  growing vary  from  g r o w i n g p e r i o d and  the c o a s t  (Wellner  performance  t h a t of a n o t h e r  the  environmental  sites  m on  frost-free  i s i m p o s s i b l e t o compare Pw's  where  comprising  1,500  be  r e p r o d u c t i o n of  range the  to  earlier  greatly  to unfavourable  I n t h e Rocky M o u n t a i n s , p r e c i p i t a t i o n  75-150 cm  It  sea  argue  also  stated  Sugano  i s considerable  northern  are  p i n e may  C a m p b e l l and  (Franklin  there  as  t h a t a r e not  s i n c e g r o w t h or  stand  grows v a r y  days.  i s that white  to s i t e .  further  found  Again,  to  (Steinhoff  (1989), they  appeared  true.  i s often r e s t r i c t e d  In Pw's  280  be  with environments  diversity.  to  not  species i s restricted)  component between  a l s o may  has  survival  & Sugano  with high p l a s t i c i t y .  1978), t h i s  (identified  Campbell  environmental  f a r , Pw  (0.40) of h e r i t a b i l i t i e s  inconsistent  The  So  i n g r o w t h r a t e and  1987).  values  (Jain  broad  c o u l d have o c c u r r e d .  quite plastic  1981,  with  and  i n the  1962). niches  s p e c i e s or p o p u l a t i o n  a  with  30 genetic pattern.  a similar  the  sites  r e q u i r e Pw  the  conditions present,  Hypothesis emphasising  Therefore,  to  identify i f  t o have wide o r narrow t o l e r a n c e s f o r  three  i s a l s o not p o s s i b l e .  i s that white pine's  flexibility  (plasticity)  the  expense  stability  (specialization).  For  accepted,  Campbell  (1989) a r g u e t h a t t h e r e  & Sugano  this  at  genome may  u n i q u e t r a d e - o f f between p l a s t i c i t y Plasticity, broad  suggest,  environmental  phenotypic pressures shifting  base  i s being  s t r e s s e s by  from g e n e r a t i o n  a c t i n g on  the  gained  i n one  Therefore,  w h a t e v e r new  sites  the  highly  adapted  for  the  reject  next this  generation. hypothesis  physiological (Franklin are  for a given  Campbell  & Dyrness  1973)  ( R e h f e l d t et  Campbell  and  and  cold  the concept  t h a t the a l l e l e s  tolerances  be  lost  al.  Sugano  present  selection  by  are any  genetic  to s u r v i v e  via  stability  find  becoming  which might  & Sugano  because, they  tolerate  argue,  g e n e r a t i o n might  environment  is a  Subsequently,  flexibility  over  be  flexible  they  not  exist  (1989) however,  a r g u e , Pw's  t o l e r a n c e s f o r s t r e s s e s such  i n c o n s i s t e n t with  requires  may  of  variation.  t o g e n e r a t i o n . The  subsequent  favoured  to  s e l e c t e d f o r to  providing a  generation  next.  i n , would be  genetic  to generation.  i n the  itself  and  genetic v a r i a t i o n ,  from g e n e r a t i o n  stability drift  they  hypothesis  be  as  (Bingham  et  present  shade, al.  that phenotypic have b r o a d  drought  1972), plasticity  environmental  1984).  (1989) c o n c l u d e  that t h e i r  study  31  does not supply  the evidence  these  three hypotheses.  in  i s n e i t h e r agreed  Pw  researched. and  cold  study,  i n Pw  i n the process  transferability BC, may  The r o l e  of phenotypic  between p h e n o t y p i c  i s even l e s s c l e a r .  of a d d r e s s i n g  of s e e d  plasticity  plasticity  However,  above.  my  t h e q u e s t i o n s of  between t h e c o a s t and i n t e r i o r  h e l p t o e l u c i d a t e some of t h e q u e s t i o n s and  discussed  between  upon nor h a s i t been e x h a u s t i v e l y  The r e l a t i o n s h i p  hardiness  required to distinguish  of  concerns  32  3 . 0  M A T E R I A L S  The  s e e d l i n g s used  collected  between  distribution Columbia  and  and Seed  people  in  Franc,  Service,  coastal  s a m p l e s and  Population  s a m p l e s were  rust-free trees i n infested  trees to exhibit  United  White P i n e  Intermountain  States  Species  acceptable  communication  Leader, United  program  criteria).  latitude  Center,  Forest  Station  i n the Inland  ( s e e H o f f and Seed  from t h e Gansel,  Umpgua N a t i o n a l F o r e s t .  The  i s bounded by 4 4 ° t o 5 3 ° n o r t h  and 119° t o 1 2 7 ° west  meter s e l e v a t i o n .  1990).  States  U.S. was made a v a i l a b l e t h r o u g h C h a r l e s Improvement  form  (U.S.) was p r o v i d e d by  These t r e e s a r e p r e s e n t l y  of c o l l e c t i o n s  stem  F o r e s t and Range E x p e r i m e n t  (1980) f o r s e l e c t i o n  Dorena Tree region  1).  rust r e s i s t a n c e breeding  McDonald  the northern  of p o p u l a t i o n  (Meagher, p e r s o n a l  situ  Moscow, I d a h o . Empire  seeds  on t h e b a s i s o f a v a i l a b l e c o n e s  to select  from t h e i n t e r i o r  Jerry  from  t r e e s were s e l e c t e d b a s e d on t h e judgement o f  and f o r t h e s e  growth  were grown  Collections in British  s e l e c t i o n s (Table  experienced stands  of the s p e c i e s .  from t r e e s c h o s e n  parent  study  1983 and 1986 t h r o u g h o u t  (B.C.) were a m i x t u r e  parent-tree obtained  in this  l o n g i t u d e and 300- t o 1220-  The four  B.C. c o a s t  families  interior  was  was  33 r e p r e s e n t e d by 20 s e e d l o t s  i n each of f i v e  provenances.  the  from f i v e hybrid  U.S.  seedlots  location  Idaho  was  included.  seed  f a m i l i e s of B.C.  females  (See T a b l e 1  Two  hundred  seeds  followed  from each  Pacific  of Moscow,  seedlot  were  by m a i n t e n a n c e  immediately a f t e r  a t room t e m p e r a t u r e  germination.  t o a c h i e v e 50 s e e d l i n g s p e r s e e d l o t randomized  within  15, 1988).  five Due  f o r 30  standard nursery T r a y s were  per t r a y .  seeded  S e e d l o t s were  months o f g r o w t h  on t h e (July  5,  t o u n e x p e c t e d l y low  g e r m i n a t i o n , many s e e d l o t s had l e s s the o r i g i n a l  f o r 48  i n Leach f i r  t r a y s and t r a y s were r a n d o m i z e d  bench t w i c e o v e r t h e f i r s t 1988 and A u g u s t  stratified  1988, by s o a k i n g i n w a t e r  i n a p e a t : v e r m i c u l i t e medium under  practice  Forest  i n the s p r i n g of  d a y s , t h e n a t 2°C f o r 60 d a y s . They were grown cells  and  pollen.)  g e r m i n a t i o n i n May  hours  four  and F i g . 7 f o r l o c a t i o n  P r o d u c t s L t d . (CP) i n S a a n i c h t o n , B.C.  for  Four  between i n t e r i o r  were a l s o  descriptions  i n e a c h o f two  of w i n d - p o l l i n a t e d  separated trees.  from c r o s s e s pollen  families  (13 - 20 t r e e s p e r s e e d l o t ) ,  S e e d l i n g s were grown a t C a n a d i a n  1988.  B.C.  r e p r e s e n t e d by  sample c o n s i s t e d  geographically  and Moscow, Idaho for  and two f a m i l i e s  The U.S. c o a s t of b u l k e d s e e d  interior  The  r e p r e s e n t e d by 20 s e e d l o t s w i t h f o u r  in each of four provenances  provenances  provenances.  with  e x p e r i m e n t a l d e s i g n was  t h a n 200 s e e d l i n g s a n d reduced from  four  34 blocks  t o two  blocks.  Seedlings nursery  were t r a n s f e r r e d t o t h e UBC  on O c t o b e r  25,  1988 and November  were t r e a t e d f o r f u n g a l Fusarium compounds  29,  28,  and t r a y s  styrofoam  trays using mix  their  and p l a c e d  cells  t h e medium were  was  throughout  into a p a r t i a l l y  staff  and h e l p  the growing  per 6 l i t e r s  recommendations. rust  C a p t a n on A u g u s t  to retain  commenced  per 6 l i t e r s  from b l i s t e r  topped  May  of water  plug  season  until  In o r d e r  infection,  When a l l  3 gms  o f 20-  t i m e s p e r week  August  20, 1989.  b a s e d on PFC to protect  FeSO^  1989 a t a  rate  greenhouse  t h e new  growth  s e e d l i n g s were s p r a y e d  19, 1989. T h r o u g h o u t  hole,  moisture.  1989 a n d J u l y 5,  o f water  from  oh t o p t o r e d u c e  1, 1989 u s i n g 2-3  20  (PFC)  were removed filled  17,  up and a medium-grade  q u a l i t y g r a v e l was p l a c e d  was a l s o a p p l i e d on J u n e 2, .93 gms  into Styro  F o r e s t r y Center  Seedlings  t h e mix was  establishment  20-20 N-P-K  the P a c i f i c  on F e b r u a r y  tamped a r o u n d e a c h s e e d l i n g .  Fertilization  of  1988 and  1 t o 26 were  were r e p o t t e d  (Appendix B ) .  filled,  horticultural weed  Seedlings  (See A p p e n d i x A f o r  27 t o 88 were c o m p l e t e d  recommended  cells  8,  1989, t r a y s numbered  18 and 1 9 t h , 1989. S e e d l i n g s  and  1988  1988.  applied).  On J a n u a r y repotted  3,  g n a t s on November  s p p . on November  S o u t h Campus  the growing  with  s e a s o n and  early  fall,  35 were m a i n t a i n e d  seedlings  s u p p o r t e d by 45 cm cement b l o c k s . seedlings  were p l a c e d  probability  The with  of r o o t  original  On November  bars 30,  1989  on t h e g r o u n d t o r e d u c e t h e  freezing.  experimental  t h e 53 s e e d l o t s  following  on m e t a l  design  per block  contained  two  randomly a r r a n g e d w i t h  blocks the  structure:  1)  5 seedlings  per s e e d l o t  per block  f o r phenology  measurements 2)  15 o r 20 s e e d l i n g s hardiness  3)  Due  During  o f whole  hardiness  i t was n o t a few d a y s .  t h e minimum t e m p e r a t u r e -4.3°C  f o r approximately  dropped two  ( C l i m a t a l o g i c a l S t a t i o n R e p o r t , Vane. UBC). A l l were on t h e g r o u n d , however, where t h e t e m p e r a t u r e from t h a t  recorded  I t became o b v i o u s  repotted  subjected and  for cold  seedlings  the r e p o t t i n g period,  difference  not  for cold  t o complete a l l the r e p o t t i n g w i t h i n  seedlings  lower.  per block  t o time and weather c o n s t r a i n t s  t o - 1 0 ° C but on a v e r a g e was weeks  per block  of n e e d l e s  0 o r 28 s e e d l i n g s testing  possible  testing  per s e e d l o t  before  to harsher  were s e v e r e l y  could  range  i n e a r l y May  the temperature conditions  damaged,  f r o m 4 t o 8°C  that  drop  those  seedlings  i n January,  i n the uninsulated  delayed  i n bud f l u s h ,  or  were  f i r cells killed  36 by  root  f r e e z i n g .  To  p r o v i d e  i n f e r e n c e s , stock  to  p l a n t e d  at  two  the  d i f f e r e n t  i n t e r i o r  the  which  expanded  to  from  make  only  g e n e t i c  the  p o p u l a t i o n s  and  f a m i l i e s  l o c a t i o n s .  The  a d d i t i o n  e x p e r i m e n t a l  design  comparable  BC  l o c a t i o n s  ( l a t i t u d e  p l a n t a t i o n  5 0 ° 1 5 ' ,  l o n g i t u d e  as  of  Program.  the  ( l a t i t u d e  s e e d l i n g s , c o n t a i n e r  as  of  to  to  m a t e r i a l  UBC  from  t e s t  f i e l d of  i n c l u d e  t h i s the  t h r e e  nursery  at  p l a n t a t i o n  l o n g i t u d e  123°49')  Nakusp,  Both  s i t e s  Rust  sample  the  The  4 9 ° 1 6 ' ,  stock  c o a s t a l  Canada  PFC.  d i s c u s s e d  a  4 8 ° 5 9 ' ,  l o c a t e d  F o r e s t r y  Meagher  were  1 1 7 ° 4 8 ' ) .  P e r m i s s i o n  Mike  nursery  same  g e n e t i c a l l y  f i e l d  L a d y s m i t h ,  Dr.  was  from  environments.  The  p a r t  base  a d d i t i o n a l  a l l o w e d of  wider  p r o j e c t  i n c l u d e  m a t e r i a l t e s t i n g  the  a  UBC  (1+1).  were  stock  l o c a t i o n  were  and  an  ( l a t i t u d e p l a n t e d  R e s i s t a n c e 1+2  l o n g i t u d e  above,  BC  in  given  the  1 2 3 ° 1 5 ' ) ,  where  grown  r a i s e d  and  1988  Improvement  was  was  in  by  UBC the as  37  4.0  METHODS AND EXPERIMENTAL DESIGN  4.1  COLD HARDINESS  The  f i e l d  approximate for at  each t h e  t h e  commences r e l a t i v e  F o r  i t s  o c c u r r e d  on  f r e e z i n g  was  t e s t i n g  a t  p r e v i o u s  30  date  O c t .  delayed high  and  was  6,  s t a t i o n f i r s t  1989, t o of  temperatures  of  run  were  p r e v i o u s  run  UBC f o r  t h e  (Oct.  2 2 ) ,  Ladysmith  Nov.  r a p i d  was  t o  however, m a t e r i a l ,  hardening  t h e  (Rehfeldt  and  needles  The  date  et  al.  were  of does  Bay  not  S t a t i o n  14.  Sampling  f o r  t h e  c o n t r o l l e d  The  d e l a y  d e t e c t  1989. on  samples  Nov.  date  1,  based  of  Departure  t o  average)  Ladysmith  1989.  temperatures on  that  t h e  14,  out  3  1989.  f r o s t  and  Nov.  samples  t h e  chosen  needle  e s t i m a t e  1989.  and  date  (30-year  when  O c t .  1,  a  mature  and  an  was  O c t .  O c t .  of  of  between  date  on  f r o s t  expected  c a r r i e d  i n d i c a t o r  run  31,  f i r s t  p r o v i d e s  t h i s  weather  t o o  i s  once  t e s t i n g  v a r i a t i o n  f r e e z i n g  average  the  1970)  Sept.  own  of  sampled  Freeze  Nakusp,  on  were  f r o s t  g e n e t i c  c o n t r o l l e d  30-year  when  (Weiser  c o l l e c t e d  have  date  l o c a t i o n .  time  1984).  t e s t s  The  r e s u l t s  t h i s  was  p o s s i b l y  was  due  i n j u r y  on  t o a  t e s t from not due  a  t h e good t o  t h e  38  timing  of f r o s t  i n e a c h a r e a and d i f f e r e n c e  i n sampling  days.  For  t h e UBC s t o c k , h a r d i n e s s was t e s t e d  different  d a t e s chosen  hardening  to early-winter  lowered  f o r each  sampling desired  t o sample t h e r a n g e hardiness. Test  successive c o l l e c t i o n  and t e s t  dates).  Pre-testing,  was  targeted  50%  injury  the  range  of o r i g i n s .  of  5  0  Q )  -  to i d e n t i f y the 1 0 seedlots  resulting  range  injury  (Rehfeldt  1 9 8 0 ) .  h a r d i n e s s throughout  samples  were c o l l e c t e d  and t e s t e d  On e a c h  test  date, four  s h o o t , were c o l l e c t e d  Except  collection  each of four  and  one c o n t r o l ) were d i v i d e d  were  needle  freezer.  seedlings  and mixed t o form a b u l k c o l l e c t i o n .  f o r the f i r s t  and p l a c e d  i n a programable  from e a c h o f 1 0 d i f f e r e n t  for  five  the f a l l ,  n e e d l e s from t h e t o p 1 / 3 of t h e  o f 4 0 n e e d l e s was c o l l e c t e d  total  for testing  w i t h a maximum r a n g e o f 1 0 ° C t o c a p t u r e 100%  seedlot  from a  i n an a v e r a g e o f  To a s s e s s c o l d  from each  were  (see Table 2 f o r  The t e m p e r a t u r e  a t the temperature  ( L T  from p r e temperatures  t e m p e r a t u r e s , was c o n d u c t e d u s i n g  cross-section  at five  temperatures  per seedlot  per run.  a t UBC, t h e 1 0 n e e d l e s u s e d  (three  freezing  randomly  into prelabelled  A  into  plastic  temperatures  eight  ziplock  l o t s of bags.  Bags  taped t o cardboard sheets f o r suspension i n the  freezer.  T h i s method a l l o w e d f o r maximum u n r e s t r i c t e d a i r  movement a r o u n d  each bag.  A l l samples  were t h e n p l a c e d  in a  cold  chamber  (0°C t o + 5 ° C )  day,  control  needles remained  i n the c o l d  chamber w h i l e s i x  bags  were p l a c e d  i n the  of  the e i g h t  w i t h bags initial  per  randomized  30 m i n u t e s  then  seedlot within  One  After  were i m m e d i a t e l y p l a c e d h o u r s t o r e d u c e shock all  samples  expression  complete  removal  of i n j u r y .  t h e t i m e of a l l o c a t i o n  following  freezer After  period  the c o l d  of r e c o v e r y from  test was  more  test  a l l samples  chamber  freezing  f o r 24  and  to allow  t o t h e same c o n d i t i o n s p r i o r  N e e d l e s were kept into  t h e bags  i n the dark  until  post-testing  i n the c o l d  room. A f t e r  removal  from t h e  room, s a m p l e s  were p l a c e d  on a t a b l e ,  exposed  to  held  a t a p p r o x i m a t e l y 18°C.  over a t f i v e subsequent after  t o p r o m o t e even  expression  freezing,  severity  of  Injury tissue  days  injury  after  A t one scored  1980,  by  expression  by  the t o t a l  a p p r o x i m a t e l y 10 d a y s .  injury See  was  and days  f o r presence et  al.  and 1984).  (length  o f brown  l e n g t h of  100). S u p p l e m e n t a r y of  daylight  week t o 10  Rehfeldt  cold  were t u r n e d  exposure  m e a s u r e d a s p e r c e n t damage  on e a c h n e e d l e d i v i d e d  shown t h a t  samples  light  injury.  (Rehfeldt  n e e d l e and m u l t i p l i e d had  of  e a c h n e e d l e was  was  The  to  from  equilibration  and  an  3°C,  s e t o f samples  r e p e a t e d f o r two  into  at  to a pre-set  from t h e f r e e z e r ,  back  to e q u i l i b r a t e  The  temperature.  stabilization  T h i s p r o c e s s was  temperatures.  hours.  l o w e r e d 5°C p e r hour  f o r 1 hour.  removed.  a test  t o 1 hour  t h e t e m p e r a t u r e was temperature  f o r 12-15  the  freezing  most e a s i l y  Table 2 f o r the  tests  observed  test  temperatures  40 employed at each t e s t  date.  At Ladysmith and Nakusp, two i n t a c t needle  fascicles  were c o l l e c t e d from each of ten s e e d l i n g s per s e e d l o t ; one fascicle  f o r t e s t i n g and one f o r reserve m a t e r i a l .  c o l l e c t i o n s of ten were bulked  i n t o two  These  c o l l e c t i o n bags per  s e e d l o t . Samples were then t r a n s p o r t e d i n a p o r t a b l e c o o l e r containing a freezer  pack, styrofoam  i n s u l a t i o n s h e l f and a  -20° to 100°C thermometer. Samples were maintained at approximately  10°C f o r a maximum of 36 hours before  placement i n t o the 0-5°C c o l d room. divided  Collections  were then  i n t o the a p p r o p r i a t e bags f o r f r e e z e t e s t i n g as  d e s c r i b e d above.  A f t e r measuring i n j u r y on each needle, a mean value was c a l c u l a t e d  per bag.  This resulted  i n two values f o r  each t e s t temperature and seedlot to be entered  i n t o the  r e s p e c t i v e l i n e a r model (see Appendix C f o r hypotheses, l i n e a r models and expected  mean s q u a r e s ) .  average values f o r i n j u r y f e l l i n s t a n c e s , an angular (Sokal & Rohlf  4.2  Since the  between 25% - 75% i n most  t r a n s f o r m a t i o n was not necessary  1981) .  PHENOLOGY AND DAMAGED MATERIAL  41 Phenology data c o n s i s t e d of both shoot  elongation  measurements and needle e l o n g a t i o n measurements on f i v e seedlings  i n each of two b l o c k s .  A b a s e l i n e bud l e n g t h  was  taken on March 12, 1989, with measurements of e l o n g a t i n g shoots and needle f a s c i c l e s commencing on A p r i l continuing  every 10 days u n t i l May 5, 1989.  were taken to the nearest  mm.  apparent that d i f f e r e n t i a l  18, 1989 and  Measurements  At t h i s time i t became  f r o s t damage had o c c u r r e d  across  the two blocks and a l l s e e d l o t s .  Due t o high m o r t a l i t y , d i f f e r e n t i a l exposure to an u n c o n t r o l l e d decided  recovery  and  ' f r o s t treatment', i t was  that c o n t r o l l e d f r o s t - h a r d i n e s s treatments should be  r e s t r i c t e d to the remaining Thirty-seven  'undamaged'  seedlings  only.  s e e d l o t s had a minimum of 20 u n i n j u r e d  s e e d l i n g s . These s e e d l o t s c o n s t i t u t e d the core c o l d hardiness  testing.  from u n c o n t r o l l e d f r o s t ,  stock  f o r the  However, t o get a measure of i n j u r y (although  unquantifiable), a  complete measurement of bud and shoot lengths and a s c o r i n g f o r needle damage was c a r r i e d out on a l l s e e d l i n g s and s e e d l o t s May  17, 1989.  A second measurement of shoot  lengths was c a r r i e d out on J u l y 19, 1989, to assess recovery.  Since experimental  there were too few s e e d l i n g s to meet design  requirements f o r whole-seedling  t h i s p o r t i o n of the p r o j e c t was e l i m i n a t e d .  To  testing,  facilitate  the c o l l e c t i o n of needle samples on the remaining 37 s e e d l o t s , s e e d l i n g s were r e o r g a n i z e d 20 s e e d l i n g s per seedlot s e e d l o t s i n Table hardiness  i n t o a s i n g l e block of  i n l a t e Aug. 1989.  Unbracketted  1 are the 37 s e e d l o t s used f o r c o l d -  t e s t i n g ; F i g . 1 shows seedlot o r i g i n s .  43  5.0  DATA  5.1  CONTROLLED  ANALYSIS  FREEZING  Data a n a l y s i s from needle t e s t i n g c o n s i s t e d main s e c t i o n s .  Section  one included  a l l UBC grown samples  t e s t e d over time and s e c t i o n two i n c l u d e d sample and the two f i e l d divided according describes  samples.  a s i n g l e UBC  Each s e c t i o n was f u r t h e r  to the t e s t i n g s t r u c t u r e . Appendix C  the hypotheses and gives  models followed  of two  the r e s p e c t i v e l i n e a r  by EMS t a b l e s and estimates f o r each  hypothesis.  For  s e c t i o n one,, r e g i o n a l - l e v e l a n a l y s i s i n c l u d e d a l l  samples using combined data from a l l t e s t temperatures. T h i s was c a r r i e d out i n i t i a l l y then f u r t h e r analyzed using dates.  with a l l t e s t dates combined,  only data from s i n g l e t e s t  Regional means f o r the UBC stock  were compared by a  Duncan's New M u l t i p l e Range Test when a l l t e s t dates were included analysis.  and by orthogonal c o n t r a s t s  for individual-date  Orthogonal c o n t r a s t s were a l s o used to compare  r e g i o n a l means and l o c a t i o n means i n s e c t i o n two.  44 Only B.C. s e e d l o t s were i n c l u d e d at the provenancew i t h i n - r e g i o n and family-within-provenance a n a l y s e s f o r both s e c t i o n s .  T h i s was due to the small  sample s i z e and lack of comparable sampling U.S. m a t e r i a l .  l e v e l s of  design i n the  These analyses were i n i t i a l l y  done again  with a l l t e s t dates i n c l u d e d and then with s i n g l e t e s t  dates  only.  For s e c t i o n two, data from Nakusp and Ladysmith p l a n t a t i o n s were analyzed with data from the day 66 t e s t and day  45 t e s t on stock grown at UBC.  average  first  The  See Table 2 f o r 30-year  f r o s t v a l u e s , t e s t days and t e s t  expected mean square  each hypothesis  (Appendix  temperatures.  (EMS) v a l u e s , c a l c u l a t e d f o r  C ) , were used t o program the  c o r r e c t denominator i n the a n a l y s i s of v a r i a n c e t e s t s . a l l a n a l y s e s , r e g i o n , date, and temperature  In  were f i x e d  f a c t o r s and l o c a t i o n , provenance and f a m i l y were c o n s i d e r e d random f a c t o r s . for  Temperature was nested i n date or l o c a t i o n  the a p p r o p r i a t e model and f a m i l y was nested i n  provenance which was nested i n r e g i o n .  Regression a n a l y s i s was done on the provenance means (%needle i n j u r y ) f o r the combined UBC data by l a t i t u d e and by e l e v a t i o n  i n d i v i d u a l l y and with both f a c t o r s i n c l u d e d i n  the model, f o r the c o a s t a l and i n t e r i o r r e g i o n s ,  respectively.  45 Those s e e d l o t s which spanned an e l e v a t i o n a l  range and overlapped other s e e d l o t s were l e f t out of the a n a l y s i s . See Table  The SAS  1 f o r e l e v a t i o n of seed  statistical  source.  analyses was c a r r i e d out using  (1985) on the Michigan  Terminal System (MTS) mainframe  computing f a c i l i t i e s at UBC.  The type I I I sums of squares  were most a p p r o p r i a t e with the Proc GLM SAS procedure unequal  due to  sample s i z e s and m i s s i n g v a l u e s .  5.2  HYBRIDS  The h y b r i d s e e d l o t s were analyzed with the US i n t e r i o r s e e d l o t r e s u l t s , which represented the p o l l e n source, and the Galena-Arrow s e e d l o t s , which represented the provenance from which the cone parent o r i g i n a t e d .  A n a l y s i s was c a r r i e d  out at the provenance l e v e l only with three s e e d l o t s per provenance and data were combined over the f i v e f a l l  test  dates.  5.3  PHENOLOGY  A n a l y s i s of the phenology data was r e s t r i c t e d to the  damaged m a t e r i a l o n l y .  46 S i x s e e d l o t s were not i n c l u d e d i n  the a n a l y s i s s i n c e they were no longer in both blocks having  been subjected  represented  equally  to d i f f e r e n t  c o n d i t i o n s i n Jan./Feb. 1989. The s i x s e e d l o t s a r e marked i n Table  1 with  analyzed.  # s i g n s with the remaining 47 s e e d l o t s  being  Due to i n j u r y of the s e e d l i n g s used f o r  phenology a n a l y s i s , the o b j e c t i v e of e v a l u a t i o n changed to e v a l u a t i o n of genetic d i f f e r e n c e s i n recovery  from f r e e z i n g .  T h i s e v a l u a t i o n was expanded i n s e c t i o n 5.4.  Since  r e g i o n a l d i f f e r e n c e s i n shoot l e n g t h were  p o t e n t i a l l y confounded with  regional differences in frost  i n j u r y , means by region were c a l c u l a t e d f o r the shoot  length  of those s e e d l i n g s which were not damaged (Table 18).  5.4  UNCONTROLLED FREEZING  Estimation  of g e n e t i c d i f f e r e n c e s i n response t o  u n c o n t r o l l e d f r e e z i n g was r e s t r i c t e d only.  For block  1  t o damaged m a t e r i a l  of the experimental  maximum of f i v e s e e d l i n g s  design,  there was a  f o r 27 s e e d l o t s , a maximum of 28  s e e d l i n g s f o r s i x s e e d l o t s and a maximum of 53 s e e d l i n g s f o r the remaining 20 s e e d l o t s .  In block  2, one of the above 27  s e e d l o t s had no damaged s e e d l i n g s and the remaining 26 s e e d l o t s had a maximum of f i v e s e e d l i n g s .  One seedlot had a  47 maximum of 33 s e e d l i n g s and the remaining 25 s e e d l o t s had maximum of 53 s e e d l i n g s  a  for analysis.  For the data c o l l e c t e d on May 17, 1989, values f o r percent  injury  were c a l c u l a t e d f o r each s e e d l o t .  Measurement of i n j u r y was based on a q u a n t i t a t i v e s c a l e of 1-4 with  1 = 0-25% i n j u r y , 2 = 26-50% i n j u r y , 3 = 51-75%  i n j u r y and 4 = 76-100% i n j u r y .  Shoot l e n g t h measurements  were a l s o taken f o r r e g i o n a l a n a l y s i s at one date and f o r comparison with shoot l e n g t h measurements of the same s e e d l i n g s on J u l y 19, 1989.  T h i s comparison was used f o r a  damage-recovery assessment to see i f s e e d l o t s different  regions  recover.  May values were s u b t r a c t e d  from the  showed any d i f f e r e n t i a l a b i l i t y t o from the J u l y 19, 1989  shoot measurement and a n a l y s i s was based on the d i f f e r e n c e in shoot l e n g t h rather than a b s o l u t e  shoot  length.  A n a l y s i s of v a r i a n c e was done f o r both shoot and  needle damage with  the May data.  r e g i o n a l e f f e c t s were analysed.  length  Only block and  An ANOVA on d i f f e r e n c e s i n  shoot l e n g t h was done f o r comparison of May to J u l y measurements. l e v e l s only.  All standard  Again, t h i s was at the block and r e g i o n a l See Table  standard  3c f o r damage a n a l y s i s sample s i z e s .  e r r o r s presented  i n t h i s t h e s i s are  e r r o r s of the means f o r % needle i n j u r y .  48 6.0  RESULTS  The  r e s u l t s from c o l d - h a r d i n e s s t e s t i n g showed a  difference sources  (p<0.0l), between the BC coast and BC i n t e r i o r  i n a l l t e s t runs, e x c l u d i n g the f i r s t UBC run and  the Ladysmith run. Where regions d i f f e r e d the d i f f e r e n c e i n percent damage response f r e e z i n g was approximately  20%.  significantly, of needles to  Test temperatures  used on  UBC-grown stock dropped from a mean of -9°C i n e a r l y September, 1989, to a mean of -30°C i n l a t e November 2).  (Table  Over the course of the f i v e t e s t dates, f o r the UBC  stock, mean i n j u r y by region v a r i e d from 38 to 84% f o r day 0, 10 to 78% f o r day 20,  32 t o 68% f o r day 45, 40 to 67%  f o r day 66 (two t e s t temperatures day  80.  o n l y ) , and 36 to 79% f o r  The r e s u l t s from the u n c o n t r o l l e d f r e e z i n g are not  c l e a r , but l a r g e l y support  the general trends r e g a r d i n g  r e g i o n a l d i f f e r e n c e s found with the c o n t r o l l e d - f r e e z i n g data.  S e c t i o n 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)  A n a l y s i s of data combined from f i v e t e s t dates f o r s e e d l i n g s grown at UBC, showed that regions (p<0.00) i n response t o c o n t r o l l e d 4a).  Values presented  percent  dates.  f r e e z i n g ( F i g . 2, Table  i n F i g u r e 2 are means of  damage f o r combined t e s t dates.  temperatures v a r i e d f o r each date, values  differed  Since  test  the a b s o l u t e damage  f o r each t e s t date c o u l d not be compared a c r o s s Region 1 represents  represents  test  the BC c o a s t , region 2  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 r e s p e c t i v e l y . For a l l dates,  regional  comparisons were performed both l a t i t u d i n a l l y and longitudinally.  Within  BC, l o n g i t u d i n a l  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 and  (p<0.0l),  w i t h i n 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). sample  The US r e s u l t s may be a consequence of small  size.  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 coast and i n t e r i o r regions combined to the small sample s i z e the o b s e r v a t i o n s  from the US  (again t h i s may be due  f o r the US).  This result  supports  by Meagher (1988) that Idaho stock i n  i n t e r i o r BC may be l e s s hardy than the l o c a l s e e d l i n g s .  50 d i f f e r e n c e s a s s o c i a t e d with l a t i t u d e were  Although  found, no c l e a r l a t i t u d i n a l t r e n d i n hardiness was evident ( F i g . 3 a,b).  Regression  a n a l y s i s by region  (general model  Y^. = a + bX^. + e^. , where: Y. =% i n j u r y , a=estimate of the f  i n t e r c e p t parameter, b=estimate of slope parameter, X^. =value for l a t i t u d e or e l e v a t i o n , e^. =unexplained v a r i a n c e a s s o c i a t e d with Y^. (random e r r o r ) ) f o r l a t i t u d e showed a 2 very poor f i t f o r both coast (R =0.19) and i n t e r i o r regions 2 (R =0.02) (Table 5 a ( i ) & b ( i ) ) .  S i m i l a r l y , p l o t s of i n j u r y  by e l e v a t i o n of provenance d i d not r e v e a l any trends 5a(ii) & b ( i i ) ) .  F u r t h e r , the r e s u l t s from r e g r e s s i o n  performed with both e l e v a t i o n and l a t i t u d e  i n c l u d e d i n the  model d i d not show any r e l a t i o n s h i p t o percent 5 c ( i ) and ( i i ) .  (Table  injury  (Table  For the c o a s t a l r e g r e s s i o n on e l e v a t i o n and  2 l a t i t u d e R =0.11 and f o r the i n t e r i o r 2 e l e v a t i o n and l a t i t u d e R =-0.17.  r e g r e s s i o n on  Since values f o r n e i t h e r  l a t i t u d e nor e l e v a t i o n i n c l u d e zero, the non-zero i n t e r c e p t value present et  al.  1985).  i n a l l a n a l y s i s has no i n t r i n s i c value  (Neter  The Salmo provenance (Table 1), was not  i n c l u d e d i n the e l e v a t i o n r e g r e s s i o n s i n c e the c o l l e c t i o n spanned 1000 t o 1870 meters which i n c l u d e d two other provenances (Table 1). The poor f i t of c o l d i n j u r y geographic  v a r i a b l e s of seed o r i g i n  reported e a r l i e r  f o r other t r a i t s  Campbell & Sugano 1989).  with  i s s i m i l a r to r e s u l t s  (Townsend & Hanover 1972,  51  6.1.2  The  Individual dates, a l l regions  r e g i o n a l d i f f e r e n c e between BC coast and  i n t e r i o r was  shown to be c o n s i s t e n t ( F i g . 4, Table  BC 6a). A  more d e t a i l e d r e g i o n a l a n a l y s i s shows that days 20,  45,  and  80 a l l show d i f f e r e n c e s (p<0.0l) between the BC  coast  and  BC  interior  (Table 6b(3)) with no s i g n i f i c a n t d i f f e r e n c e  between BC and US  seedlots  (Table 6 b ( 2 ) ) .  Days 20 and  a l s o show a d i f f e r e n c e (p<0.05) between coast interior  versus  (BC and US)  (Table  Although r e s u l t s from day t h i s t r e n d , needle samples had r e p l i c a t e s f o r t e s t i n g and  p r e c i s i o n of t e s t i n g on day  b). to  6b(1)).  support  not been d i v i d e d i n t o  thus only one  mean value  two was  The  t h e r e f o r e reduced; no  s i g n i f i c a n t d i f f e r e n c e s were found (Table 6a &  F u r t h e r , t h i s t e s t was  conducted September 7/89,  the time of expected maximal e x p r e s s i o n  d i f f e r e n c e s for c o l d hardiness. day  US)  0 ( F i g . 4) appear to  0 was  80  (BC and  a v a i l a b l e f o r a n a l y s i s f o r each t e s t temperature.  statistically  66  prior  of genetic  ( D i f f e r e n c e i n the  0 a n a l y s i s should be kept in mind f o r the f o l l o w i n g  s e c t i o n s ) . However, a p r o b a b i l i t y of 0.12 BC  versus  f o r BC  coast  i n t e r i o r suggests that the d i f f e r e n c e s are  (Table 6b(3)).  The  two  real  d i f f e r e n c e i n the mean r e g i o n a l value  for  the BC coast  versus  for  day  20% on average f o r the subsequent 4 t e s t  days.  0 versus  the BC  interior  i s approximately  9%  52  6.1.3  A l l dates,  BC r e g i o n s ,  This analysis required  Hypothesis  2 ( A p p e n d i x C)  more computing power than was  a v a i l a b l e f o r SAS on the MTS mainframe system at UBC. T h e r e f o r e , to examine a l l components i n the model, was d i v i d e d  i n t o two s e c t i o n s .  Table 7a g i v e s  from the a n a l y s i s with the highest included.  analysis  the r e s u l t s  order i n t e r a c t i o n term  For t h i s term, the p r o b a b i l i t y of r e a l e f f e c t s  was 0.15 and t h i s source of v a r i a t i o n was then added t o the e r r o r term, reducing the complexity of the a n a l y s i s and allowing  complete a n a l y s i s of a l l other components i n the  model to be done (Table run with the highest to be l e f t  7b).  I  n  order f o r the program to  order i n t e r a c t i o n term, f i v e terms had  out from d i r e c t a n a l y s i s , being added t o the sums  of squares and degrees of freedom f o r other terms. doing, F - t e s t the  In so  a n a l y s i s denominators (EMS's) changed between  two a n a l y s e s .  mean s i g n i f i c a n c e .  T h i s had a d i r e c t e f f e c t only  on region  In the f i r s t a n a l y s i s , P=0.03 f o r region  but  was P=0.11 f o r region  i n the second a n a l y s i s .  F(R  P) component may have been an i n f l a t e d term f o r t e s t i n g  of R s i n c e the P(R) component was i n c o r p o r a t e d  The  into i t s  2 value.  The assumption was P ( R ) =0, meaning no c o n t r i b u t i o n 6  to the ' e r r o r ' term f o r R. Provenances-within-coast and - i n t e r i o r  regions d i d not  differ  53 (p=0.1l), although c o a s t a l provenances  statistically  d i f f e r e d by up to 28% 35%  (30% to 65%).  (45% to 73%)  and  i n t e r i o r provenances by  Families-within-provenances d i d d i f f e r  (p<0.0l) (with both a n a l y s e s ) , ranging between 4 and coast and by  15 and  42.5%  f o r the i n t e r i o r .  The minimal  of f a m i l i e s - w i t h i n - p r o v e n a n c e s and v a r i a b i l i t y of within-provenances statistically  c o u l d account  41% f o r the number  families-  f o r f a i l u r e to d e t e c t  s i g n i f i c a n t d i f f e r e n c e s among provenances  d e s p i t e the s u b s t a n t i a l range i n provenance means.  6.1.4  Individual dates,  BC  With a n a l y s i s by i n d i v i d u a l differed differ  f o r day 80  f o r days 0 and  (p<0.0l) and 66  regions  f r e e z i n g date, regions 45  (Table 8 ) .  (p<0.05),  but d i d not  I t i s l i k e l y that day  r e s u l t s made a s u b s t a n t i a l c o n t r i b u t i o n  0  i n reducing the  s i g n i f i c a n c e of o v e r a l l r e g i o n a l e f f e c t s d i s c u s s e d above. For day  66, temperature  t e s t temperatures  was  not s i g n i f i c a n t due  to only  (Table 2) being analyzed and t h i s  two  likely  c o n t r i b u t e d to the l a c k of s i g n i f i c a n c e being observed f o r r e g i o n a l d i f f e r e n c e s f o r day temperatures  66.  The d i f f e r e n c e s i n t e s t  appear to have been i n s u f f i c i e n t  d i f f e r e n c e s i n r e g i o n a l performance. Only day d i f f e r e n c e s f o r provenance-within-region within-provenance  differed  i n showing 0 data showed  (p<0.05).  Family-  (p<0.00) f o r a l l days (Table 8 ) .  Data from day 20  54 (Table 8) a l s o showed a 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 term, shown i n F i g . 4b. Region  1  (coast) shows r e l a t i v e l y more i n j u r y at the  temperatures than does region 2 ( i n t e r i o r ) , but consistently temperatures.  colder  exhibits  more i n j u r y than region 2 ( F i g 4b) at a l l With a s i g n i f i c a n t i n t e r a c t i o n , the  s i g n i f i c a n t main e f f e c t f o r day 20 has no meaning.  Section I I .  6.2  COLD HARDINESS AT THREE LOCATIONS.  6.2.1  A l l locations, a l l regions, Hypothesis 3, (Appendix C)  R e s u l t s from the two f i e l d c o l l e c t i o n at UBC in F i g s .  test locations  ( F i g s . 5 & 6) f u r t h e r  and  one  support the  2 and 4. Data from day 66 at UBC  findings  were chosen  because they most c l o s e l y matched the c r i t e r i o n f o r choosing c o l l e c t i o n dates at the two f i e l d date of f i r s t included, 9a).  frost).  s i t e s (i.e.  historical  With a l l l o c a t i o n and r e g i o n a l  there were r e g i o n a l  There was however, a  differences  data  (p<0.00, Table  region x l o c a t i o n  interaction  55 (p<0.05) ( F i g . 5, Table 9a).  effect  When the US data were  omitted, t h i s i n t e r a c t i o n was no longer present  Although  sampling  (Table 11a).  dates were chosen to c o i n c i d e with  the expected maximal e x p r e s s i o n of g e n e t i c v a r i a t i o n , obvious  i t is  from Table 2 that i t was not p o s s i b l e to show  d i f f e r e n c e s i n response  among s e e d l o t s through  at the same temperatures.  t e s t i n g them  With day 45 (UBC), used  i n the  combined a n a l y s i s , r e p l a c i n g day 66, regions d i f f e r e d (p<0.00) and the r e g i o n x l o c a t i o n significant  i n t e r a c t i o n term  (p<0.0l, F i g . 6a, Table 9b).  This  was  interaction  was not s i g n i f i c a n t however, (p<0.lO) with the US m a t e r i a l 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 at Ladysmith,  10a).  Region  d i d not d i f f e r  significantly  however, the same t r e n d i n r e g i o n a l  d i f f e r e n c e s between the BC coast and BC i n t e r i o r was present (Figs. 5 & 6).  Orthogonal  c o n t r a s t s f o r r e g i o n e f f e c t s showed no  s i g n i f i c a n t d i f f e r e n c e between s e e d l o t s 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 d i f f e r e n c e s f o r the BC coast  versus  BC i n t e r i o r  56 regions (p<0.0l, Table 10b ( 3 ) ) .  Ladysmith d i d not show a s i g n i f i c a n t d i f f e r e n c e but d i d show the  same trend  ( F i g . 5 ) . No a n a l y s i s  s i g n i f i c a n t coast  (US & BC)  versus  showed a s t a t i s t i c a l l y  interior  (US & BC)  d i f f e r e n c e although p=0.06 f o r Nakusp.  6.2.3  A l l locations, BC regions, Hypothesis 4 , (Appendix C)  Again, f o r a l l l o c a t i o n s with the BC data regional  only,  e f f e c t s remained s i g n i f i c a n t (p<0.05) using  45 but not f o r region  using  UBC day 66 (Table  Provenances-within-region d i d not d i f f e r  UBC day  11a & b ) .  statistically  f o r e i t h e r a n a l y s i s although the range of i n j u r y f o r provenance means was 18.5% (61 to 79.5%) f o r c o a s t a l seedlots  and 29% (48 to 77%) f o r i n t e r i o r  seedlots.  F a m i l i e s - w i t h i n - p r o v e n a n c e d i f f e r e d (p<0.00), by 8 t o 25% on the coast  and 24 t o 32% f o r f a m i l i e s - w i t h i n - i n t e r i o r  provenances. Steinhoff  This  et al.  6.2.4  i s i n keeping with the f i n d i n g s of  1983.  Individual locations, BC regions  57 For a n a l y s i s of i n d i v i d u a l l o c a t i o n s , data only from UBC  day 45 showed r e g i o n a l d i f f e r e n c e s  not s t a t i s t i c a l l y  significant  (p<0.05).  i n data from UBC  Region  day  was  66,  p=0.16, Ladysmith, p=0.55, or Nakusp, p=0.10, ( F i g s . 5 & 6, regions 1 and 2 only, Table 12a). w i t h i n - r e g i o n s d i d not d i f f e r  As w e l l ,  on any date, however, f a m i l i e s -  within-provenances d i f f e r e d on a l l dates Ladysmith effect.  showed a s i g n i f i c a n t  provenances-  (p<0.01).  (p<0.05) region x  temperature  F i g . 6b shows that at -22°C (the warmest t e s t  temperature), r e g i o n 2 ( i n t e r i o r ) shows r e l a t i v e l y more i n j u r y than r e g i o n 1 ( c o a s t ) .  At both -26°C and -30°C  however, the r e l a t i v e i n j u r y f o r the two reversed.  As noted above, at Ladysmith  regions i s regions d i d not  differ.  Table  12b p r o v i d e s a summary of the 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 d i s c u s s e d i n the above two  f o r a l l four  sections.  hypotheses  58 Section I I I .  6.3  HYBRID COLD HARDINESS, 5 DATES.  Response  of the Galena-Moscow h y b r i d was compared with  that of Galena-Arrow Galena-Bay seedlots  ( c l o s e s t BC i n t e r i o r provenance to  the cone parent o r i g i n ) and of the US  interior  (See F i g . 1 f o r s e e d l o t o r i g i n s and F i g . 7 f o r  d e p i c t i o n of the p o l l e n source a r e a ) .  Each set of data  represented f r e e z e - t e s t i n g r e s u l t s from three t r e e s on the same f i v e dates as the above a n a l y s i s . "provenance" w i l l of  data.  The term  be used to d e s c r i b e each of the three sets  Provenance A=Galena-Arrow, B=Galena-Moscow h y b r i d  and C=US i n t e r i o r w i l l  be used f o r f u t u r e  reference.  R e s u l t s showed a provenance d i f f e r e n c e  (p<0.0l, Table  13a) and a provenance x date i n t e r a c t i o n  (p<0.0l, Table  13a).  i n p o s i t i o n over  the  F i g . 8b shows the r e l a t i v e s h i f t s f i v e t e s t dates.  On days 0, 66 and 80,  Galena-Arrow  shows s i g n i f i c a n t l y more i n j u r y than the US i n t e r i o r with l i t t l e or no d i f f e r e n c e on days 20 and 45. the  The p o s i t i o n of  h y b r i d , r e l a t i v e to the other two provenances,  f l u c t u a t e d c o n s i d e r a b l y a c r o s s the f i v e t e s t d a t e s . Duncan's New  M u l t i p l e Range t e s t showed that provenance A  (Galena-Arrow) was  significantly different  from provenance C  59 (US i n t e r i o r ) and that n e i t h e r was d i f f e r e n t B (Galena-Moscow h y b r i d ) (Table 13b).  from provenance  60 Section  IV.  The r e s u l t s presented  i n the remainder of the R e s u l t s  s e c t i o n w i l l p e r t a i n only t o the m a t e r i a l which was damaged by an u n c o n t r o l l e d  frost  i n Jan./Feb. 1989.  were i n a randomized block design  a random e f f e c t .  seedlings  at the time the i n j u r y was  n o t i c e d and a n a l y s i s i n c l u d e s t h i s f a c t o r . considered  The  Blocks  were  Phenology r e s u l t s i n c l u d e both  needle e l o n g a t i o n measurements over f i v e measurement dates and  shoot e l o n g a t i o n measurements over seven dates.  Day 0 =  March 3/89 f o r the shoot measurements; needle measurements commenced on day 36.  6.4  PHENOLOGY  Regions d i d not d i f f e r on any measurement day although block but  x region  i n t e r a c t i o n was s i g n i f i c a n t  (p<0.05) on a l l  the l a s t measurement day (day 128) (Table  the s i g n i f i c a n t  14a).  Due to  i n t e r a c t i o n e f f e c t on day 36, the  s i g n i f i c a n t block e f f e c t  f o r needle l e n g t h has no meaning.  F i g s . 9a and 9b show e l o n g a t i o n p a t t e r n s over the f i v e measurement days f o r the two b l o c k s .  Data are missing i n  these f i g u r e s as r e g u l a r measurements ceased a f t e r the e f f e c t s of the u n c o n t r o l l e d  f r e e z i n g became apparent and the  study  61 o b j e c t i v e changed to e s t i m a t i o n of recovery  by u n c o n t r o l l e d  For differ  injury  freezing.  shoot-elongation  measurements, blocks d i d not  f o r any of the seven measurement days.  differed  from  Regions  (p<0.05) on days 30 and 36 only, although  average f o r days 43, 53, 65 and 128 (Table  15a).  from day 53 showed a s i g n i f i c a n t block x region  p=0.15 on Only  data  interaction  (p<0.05) however, p=0.09, on average, f o r days 43, 65 and 128. Regions 1 and 3 ranked the lowest  f o r growth i n both b l o c k s  with the two i n t e r i o r regions showing the most growth. Orthogonal c o n t r a s t s f o r the s i g n i f i c a n t region e f f e c t s f o r days 30 and 36 both show s i g n i f i c a n t coast versus  i n t e r i o r (US and BC) e f f e c t s  BC coast  versus  6.5  The  BC i n t e r i o r e f f e c t s  (p<0.05) and s i g n i f i c a n t (p<0.0l, Table 15b).  DAMAGE ASSESSMENT  means f o r needle damage by region were a l l very  c l o s e t o 50% i n j u r y mean i n j u r y  f o r both b l o c k s .  f o r region  f o r region  For block two, the mean  1 was 48%, r e g i o n 2 was 45%, region 3 was  45% and region 4 was 41%. block x region e f f e c t block e f f e c t  For block one, the  1 was 48%, region 2 was 51%, region 3  was 46% and region 4 was 47%. injury  (US and BC)  The r e s u l t s  (p=0.05, Table  showed a s i g n i f i c a n t  16). The s i g n i f i c a n t  (p<0.00) t h e r e f o r e has no meaning. There was  no r e g i o n a l e f f e c t .  62 Shoot height was  s e e d l i n g s at t h i s time. block and  block x region e f f e c t s  significant  region e f f e c t  Recovery r e s u l t s the measurement dates d i d not  These r e s u l t s  show any  interactions.  (Table  a l s o measured on a l l showed  significant  (p<0.00), but again a non16b).  from comparing shoot l e n g t h between (May  and J u l y ) by block and  significant  d i f f e r e n c e s (Table  region,  17)  or  63  7.0  DISCUSSION  The  r e s u l t s i n d i c a t e that seed o r i g i n of Pw i s  important when c o n s i d e r i n g seed t r a n s f e r between the coast and  i n t e r i o r of BC.  Inferences  based on the performance of  the US m a t e r i a l are much l e s s c l e a r . these c o n c l u s i o n s  The i m p l i c a t i o n s of  l e d t o the f o l l o w i n g s e r i e s of  recommendations f o r t r e e breeders and f o r e s t e r s .  7.1  RECOMMENDATIONS BASED ON FREEZE TESTING  When c o l l e c t i n g w i l d - s t a n d s e e d l o t s should  seed, coast and i n t e r i o r  be kept separate.  provenances i n a region  Since the v a r i a t i o n among  i s not s t a t i s t i c a l l y  significant,  seed c o u l d be combined from more than one provenance w i t h i n a region.  Combining provenances w i t h i n a region would make  seed c o l l e c t i o n and/or p a r e n t - t r e e purposes l e s s complicated  s e l e c t i o n s f o r breeding  s i n c e i t would allow  f o r more-  rigorous selection within a region.  Selection within  f o r t a i t s other  w i l l result in  than f r o s t h a r d i n e s s  region  64 p o p u l a t i o n s which have wide v a r i a t i o n  T h i s study suggests that two  in frost hardiness.  seed p l a n n i n g zones  be necessary to reduce the l i k e l i h o o d of r e s t o c k i n g due to f r o s t damage or k i l l were to be made.  may  failures  i f c o a s t - i n t e r i o r seed t r a n s f e r s  I t must be emphasized  however, that  this  recommendation i s based s o l e l y on the response of detached needles to f r e e z e t e s t i n g .  Although t h i s method of t e s t i n g  g i v e s a good measure of r e l a t i v e d i f f e r e n c e s i n performance, i t does not p r o v i d e a measure of a b s o l u t e d i f f e r e n c e s . 4 shows the average d i f f e r e n c e i n the % i n j u r y between the BC coast and BC  Fig.  response  i n t e r i o r to be approximately 20%  (not i n c l u d i n g day 0 ) .  Due  to the sample s i z e c o n s t r a i n t s i n t h i s study, the  variability  i n response observed and i l l u s t r a t e d  4, 5 and 6, makes i n t e r p r e t a t i o n  in Figs.  f o r the u t i l i z a t i o n  and parents from the US breeding program  2,  of seed  difficult.  G e n e r a l l y , however, the t r e n d i s of c o a s t a l m a t e r i a l e x p r e s s i n g g r e a t e r damage than i n t e r i o r  seedlots.  N e v e r t h e l e s s , the i n t e r i o r US s e e d l o t s c o n s i s t e n t l y show more damage than the i n t e r i o r BC s e e d l o t s .  F i g . 2 suggests  that US stock i s g e n e r a l l y i n t e r m e d i a t e i n c o n t r o l l e d f r e e z i n g response between BC coast and BC i n t e r i o r  seedlots.  Work by Bower (1987), i n d i c a t e s that the r u s t resistant  second-generation s e e d l i n g s from the  Idaho  breeding program c o u l d be used i n c o a s t a l BC f o r i n c r e a s e d  65  disease  resistance.  the Idaho stock the  i n t e r i o r due  Meagher (1988) however, suggests that  should  be r e s t r i c t e d to s p e c i f i c  to poorer performance of the Idaho stock  l o c a l sources at a s i n g l e s i t e . The  versus  (1987), was question  zones in  study by Bower  r e s t r i c t e d to t e s t i n g on the coast and  of r e s t r i c t e d r e g i o n a l use  i n v e s t i g a t i o n at present  i s under more  (Meagher and  the extensive  Hunt 1987).  A more-  comprehensive set of US m a t e r i a l should be r e t e s t e d f o r response to f r e e z e t e s t i n g before material d i f f e r s  in cold-hardiness  recommending whether response from  US  BC  material.  Given that two  seed planning  zones are recommended, i t  i s c l e a r that p l a n t i n g stock d e r i v e d from sources w i t h i n a c o a s t a l or i n t e r i o r  region should  be r e s t r i c t e d  d i s t r i b u t i o n w i t h i n the r e s p e c t i v e r e g i o n . however, seed l i k e l y c o u l d be bulked a  As  to s t a t e d above,  from provenances w i t h i n  region.  The  anomalous r e s u l t s from t e s t i n g of the Ladysmith  m a t e r i a l c o u l d have a number of e x p l a n a t i o n s .  Since  the date  of f i r s t  f r o s t at the c l o s e s t weather s t a t i o n (Nanaimo A)  was  31,  Oct.  experienced  1989,  i t is likely  that the Ladysmith s i t e  f r o s t p r i o r to c o l l e c t i n g .  The  had  temperature at  which the samples showed i n j u r y f u r t h e r suggests that  the  r a p i d development of f r o s t hardiness  by a  frost.  had  been induced  However, the date of c o l l e c t i o n was  the expected date of f i r s t  f r o s t and  very c l o s e to  d i f f e r e n c e s may  not  66  have expressed themselves y e t .  Due to missing  temperatures r e q u i r e d to detect  i n j u r y on the c o l l e c t i o n  date  (Nov.  1, 1989),  s u r p l u s samples were h e l d f o r two  weeks p r i o r to a second t e s t i n g .  These samples were h e l d i n  the dark i n a 0-5°C c o l d room which may samples t o a standard (Pers. Comm.)  the t e s t i n g  have s t a b i l i z e d the  l e v e l of h a r d i n e s s .  However, R e h f e l d t  suggested that the samples c o u l d be s t o r e d  f o r a two-week p e r i o d without a l t e r i n g the s t a t e of f r o s t hardiness.  In a d d i t i o n t h i s s i t e was  not c o n s i s t e n t l y w e l l  brushed and the environmental v a r i a t i o n due to cover  may  have prevented d e t e c t i o n of r e g i o n a l genetic v a r i a t i o n .  The r e s u l t s from a n a l y s i s of the h y b r i d s e e d l o t do not show a c l e a r r e l a t i o n s h i p with parent region However, the o v e r a l l means d e p i c t e d h y b r i d to be intermediate t h i s small study, the US  performance.  i n F i g . 8a show the  between p a r e n t a l provenances.  In  i n t e r i o r shows l e s s i n j u r y than the  BC i n t e r i o r which c o n t r a d i c t s the f i n d i n g s of Meagher (1988).  I, however, had only three t r e e s to t e s t  from each  of the c o n t r i b u t i n g provenances.  7.2  PHENOLOGY  Data from the phenology measurements and assessment of i n j u r y from unplanned f r e e z i n g do not show any  striking  67 Because, at the time of u n c o n t r o l l e d f r e e z i n g ,  results.  some s e e d l i n g s were i n the greenhouse and some were o u t s i d e , I am very h e s i s t a n t to draw strong results.  i n f e r e n c e s from  The high l e v e l of block x region  in the needle and shoot e l o n g a t i o n data,  interaction  i n j u r y among the  s e e d l i n g s rather than the blocks themselves being  different  inherently  However, r e g i o n a l shoot l e n g t h means (but s e e d l o t s i n most i n s t a n c e s ) on undamaged  showed a s i m i l a r dates  found  I would suggest,  was more l i k e l y the r e s u l t of d i f f e r e n t i a l  different.  these  f o r block  ranking  seedlings  f o r the May and J u l y measurement  1 but not region 4 i n block 2.  This  suggests that the r e g i o n a l d i f f e r e n c e s apparent f o r shoot e l o n g a t i o n may be r e a l , although by the d i f f e r e n t i a l  the r e s u l t s  f r e e z i n g response (Table  are compounded 18).  Further,  the number of undamaged s e e d l i n g s measured was not consistent  from region to r e g i o n .  s e e d l i n g s were from d i f f e r e n t  In a d d i t i o n , the  s e e d l o t s i n most i n s t a n c e s and  t h e r e f o r e a s t a t i s t i c a l comparison of shoot l e n g t h was not performed on damaged  The  versus  undamaged  seedlings.  s i g n i f i c a n t block and block x region  terms (Table  interaction  16 a & b ) , i n the i n j u r y assessment  analyses,  i n d i c a t e that the s e e d l i n g s were i n j u r e d d i f f e r e n t i a l l y p r i o r to r e p o t t i n g . of these analyses  Region was not s i g n i f i c a n t  (Table  16 a & b ) .  for either  The recovery  assessment  through d i f f e r e n c e i n height between J u l y 19, 1989 and May 17,  1989 showed no s i g n i f i c a n c e f o r block,  region.  region or block x  T h i s suggests that no regions s e e d l i n g s  recovered  68 other  s i g n i f i c a n t l y more than any burst appeared to be delayed Seedlings degree.  region's  although  bud  i n damaged s e e d l i n g s .  i n a l l regions must have been delayed  to an  equal  Although these r e s u l t s appear to c o n t r a d i c t the  c o n t r o l l e d freeze t e s t i n g r e s u l t s , several f a c t o r s could account f o r t h i s d i f f e r e n c e . c a r r i e d out  C o n t r o l l e d f r e e z i n g was  i n January or February so a d i r e c t comparison of  r e g i o n a l d i f f e r e n c e s i n p a t t e r n of i n j u r y i n the two material  i s not p o s s i b l e .  patterns  therefore  not  seedling  by region can not be compared.  f r e e z i n g / i n j u r y may  sets of  Whole-seedling t e s t i n g was  done with the c o n t r o l l e d f r e e z i n g and recovery  not  Root  have c o n t r i b u t e d s i g n i f i c a n t l y to the  subsequent death or reduced growth of the damaged s e e d l i n g s (Table  18).  However, i f c o n t r o l l e d whole-seedling  were done, roots would be freezing.  i n s u l a t e d to p r o t e c t  testing  from  Thus, the phenology r e s u l t s are d i f f i c u l t  i n t e r p r e t and cold-hardiness  to  hold l e s s s i g n i f i c a n c e than the r e s u l t s from testing.  69  7.3  The  Pw, COLD HARDINESS AND PHENOTYPIC PLASTICITY  implications  of phenotypic p l a s t i c i t y  l i v e d s p e c i e s such as c o n i f e r s , a r e q u i t e annuals or s h o r t - l i v e d p e r e n n i a l s .  d i f f e r e n t than f o r  When the l i f e - c y c l e i s  longer than the d u r a t i o n of the environmental (fine-grained  f o r long-  fluctuation  s p a t i a l v a r i a t i o n ) , a s e r i e s 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 variation  i n a p o p u l a t i o n , while appearing homogeneous  (Schlichting the  (Bradshaw 1965), masking g e n e t i c  1986).  Species whose l i f e c y c l e  same length as the f l u c t u a t i o n  (coarse-grained  v a r i a t i o n ) , can evolve through d i s r u p t i v e (Bradshaw 1965), s i n c e  selection  temporal i n time  the next generation i s adapted to an  environment which may no longer e x i s t T h e r e f o r e , a d a p t a t i o n occurs p r i m a r i l y not  i s shorter or  (Schlichting  1986).  through p l a s t i c i t y ,  through genetic change (Bradshaw 1965). Campbell &  Sugano's (1989) h y p o t h e s i s that genomic f l e x i b i l i t y  the s p e c i e s emphasizes  a t the expense of genomic s t a b i l i t y (as  d i s c u s s e d i n the l i t e r a t u r e review), under d i s r u p t i v e  selection  genetic v a r i a t i o n playing  Pw i s  i n time f o r s i t e v a r i a b l e s  with  a minor r o l e i n p r o v i d i n g the  means t o t o l e r a t e and s u r v i v e variation.  suggests that  coarse-grained  temporal  70 Critchfield age,  (1984), s t a t e s that during the l a s t i c e -  Pw was reduced i n i t s northern  refugium north. effect  range to a s i n g l e  i n Oregon, from which r e p o p u l a t i o n occurred  i n the  T h i s r e d u c t i o n c o u l d have p r o v i d e d a b o t t l e n e c k from which recovery  was dependent on Pw  d i v e r s e c o n d i t i o n s with l i m i t e d  tolerating  genetic d i v e r s i t y .  Plastic  genomes c o u l d have been s e l e c t e d f o r a t t h i s time. Using isozyme data, S t e i n h o f f et al. d i v e r s i t y estimates G =0.148 T  f o r the e n t i r e  s p e c i e s range of Pw t o be  ( c o e f f i c i e n t of g e n e t i c d i v e r s i t y G  ST  where D g  (1983) c a l c u l a t e d g e n e t i c  = d i v e r s i t y among p o p u l a t i o n s and H  T  S T  = D  S T  /H  T  = total  g e n e t i c d i v e r s i t y ) . T h i s suggests that 85% of the t o t a l variation and  can be e x p l a i n e d as v a r i a t i o n  S t e i n h o f f et al.  15% among p o p u l a t i o n s .  show that the p o p u l a t i o n s of 44° l a t i t u d e  exhibit  populations  (1983), f u r t h e r  sampled from the coast and north  the g r e a t e s t c o e f f i c i e n t of g e n e t i c  d i v e r s i t y at 0.124 and the l e a s t regions.  within  t o t a l d i v e r s i t y of a l l  T h i s suggests that Pw w i t h i n the c o a s t a l range  r e t a i n s a r e l a t i v e l y high degree of d i v e r s i t y populations  but e x h i b i t  r e l a t i v e to other  an o v e r a l l  regions.  between  low degree of v a r i a b i l i t y  The i n t e r i o r region s t u d i e d by  Steinhoff  et al.  diversity  of 0.07 i l l u s t r a t i n g that 93% of the v a r i a t i o n  present  (1983), had a c o e f f i c i e n t of genetic  occurred w i t h i n p o p u l a t i o n s .  Having experienced  b o t t l e n e c k , however, i t i s p l a u s i b l e that Pw, p r e s e n t l y having a r e l a t i v e l y high o v e r a l l (H =0.l5) ( S t e i n h o f f et al. m  a  while  heterozygosity  1983) i n i t s northern  71 p o p u l a t i o n s , c o u l d have s u f f e r e d a l o s s i n adaptive  genetic  v a r i a t i o n or that isozymes do not i n d i c a t e g e n e t i c diversity  in frost hardiness.  C o n i f e r s are thought t o c a r r y  a high genetic l o a d , r e l a t i v e t o other  organisms, which  c o u l d c o n t r i b u t e t o the o v e r a l l h e t e r o z y g o s i t y little  c o n t r i b u t i o n to the adaptive  genetic  As d i s c u s s e d e a r l i e r , phenotypic  but make  variation.  plasticity  i s not  only 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 but a l s o i s s p e c i f i c for i n d i v i d u a l c h a r a c t e r i s t i c s Phenotypic p l a s t i c i t y may apply  ( S c h l i c h t i n g 1986). to only a few or many  c h a r a c t e r s w i t h i n a given genome. c o u l d be a c t i n g  Therefore, s e l e c t i o n  d i f f e r e n t l y on d i f f e r e n t  characteristics.  Over time one would expect to see genetic v a r i a t i o n between p o p u l a t i o n s constant  constant  for particular characteristics i f a  s e l e c t i o n pressure  adaptive  (e.g.:  evolve  was p r e s e n t .  If specialization  gene complexes) i s the consequence of a  s e l e c t i o n pressure,  then i t i s l i k e l y  that a h i g h l y  adapted i n d i v i d u a l w i l l be more f i t than a g e n e r a l i s t . Given that Pw has d i s c r e t e c o a s t a l and i n t e r i o r gross d i f f e r e n c e s i n cues f o r c o l d - h a r d i n e s s (photoperiod generation  and temperature),  to generation,  ranges, the  induction  which are constant  from  c o u l d have given r i s e to the  r e g i o n a l d i f f e r e n c e s i n f r e e z i n g i n j u r y observed i n t h i s study; lost  o r , t o l e r a n c e f o r lower temperatures c o u l d have been  i n the c o a s t a l r e g i o n .  Campbell and Sugano (1989) suggest that the l a c k of  72 p r e d i c t a b i l i t y of s i t e s from g e n e r a t i o n to g e n e r a t i o n c o u l d s u s t a i n the high degree of phenotypic growth c h a r a c t e r s i n the Pw  genome.  p l a s t i c i t y of  However, they do  not  accept t h i s hypothesis as very p l a u s i b l e because they  can  not a s c e r t a i n i f the s i t e s are v a r i a b l e enough to provide a s e l e c t i o n pressure which would r e s u l t  i n s e l e c t i o n of genes  p r o v i d i n g wide t o l e r a n c e s to be s e l e c t e d .  I would argue  t h a t , with any pioneer s p e c i e s , c o n d i t i o n s w i l l  be  p r e d i c t a b l y v a r i a b l e from g e n e r a t i o n to generation throughout  i t s range and  of any one  s i t e are u n p r e d i c t a b l e .  account  that the environmental  These f a c t o r s c o u l d  f o r the high degree of phenotypic  growth c h a r a c t e r s i n the Pw  genome.  p l a s t i c i t y for  I would argue f u r t h e r  that s e l e c t i o n pressures f o r c o l d hardiness s t a b l e g e n e r a t i o n to g e n e r a t i o n genetic v a r i a t i o n  conditions  and,  are  relatively  t h e r e f o r e , geographic  (specialization) for this character could  be developing between r e g i o n a l p o p u l a t i o n s , as has in other c o n i f e r s  (Rehfeldt 1978,  occurred  S t e i n h o f f 1980).  These p o s s i b i l i t i e s are not n e c e s s a r i l y i n c o n s i s t e n t with the f i n d i n g s of S t e i n h o f f (1981) and Bower r e g a r d i n g ease of t r a n s f e r of Pw interior  d i f f e r e n c e s expressed Although  in f r e e z i n g  temperature  not examined i n my  with severe temperatures  study.  between the coast  s i n c e the a b s o l u t e k i l l i n g  a given p o p u l a t i o n was  (1987),  study.  and  ( L T ^ Q )  for  A year  however, might expose the r e g i o n a l  by the c o n t r o l l e d - f r e e z i n g t e s t s of  there were s i g n i f i c a n t d i f f e r e n c e s  i n j u r y found between r e g i o n s , s u p p o r t i n g  the  my  et  f i n d i n g s of Rehfeldt  73 (1984) the amount of  al.  v a r i a t i o n which i s present may material  i f the d i f f e r e n c e s  not  r e s t r i c t movement of  in c o l d hardiness are below  lowest winter temperatures in each l o c a t i o n . process of dehardening should be chamber s t u d i e s on Pw  seedlings  q u e s t i o n s of r e g i o n a l cue temperature t o l e r a n c e s  genetic  Further,  looked at i n Pw.  the the  Growth  c o u l d help to e l u c i d a t e  responses and  for a s p e c i f i c  the  look at minimum  ( c o n t r o l l e d ) growing  environment.  The  r e s u l t s of t h i s research  others (Townsend & Hanover 1972, Rehfeldt  et  al.  1984,  support the  provenance l e v e l , with c o n s i d e r a b l y provenance-within-region l e v e l .  indicating  al.  Pw  family-within-  l e s s d i f f e r e n c e at  For c o l d h a r d i n e s s ,  f i n d i n g s of Rehfeldt  et  al.  and  phenotypic p l a s t i c i t y  i n Pw,  The  the  The  of c o r r e l a t i o n of i n j u r y with e l e v a t i o n supports the Hanover (1972).  the  (1984),  c o a s t - i n t e r i o r regional differences.  of Townsend and  lack findings  issues: cold hardiness  are d i f f i c u l t  when the processes themselves are d i v e r s e and  to  resolve  dynamic.  i s a f a s c i n a t i n g system on which to study phenotypic plasticity. contribute  A greater  understanding of both  to management d e c i s i o n s  c l e a r e r understanding of the e v o l u t i o n , c o l d h a r d i n e s s and  of  1983,  and ,Campbell & Sugano 1989).  showed s i g n i f i c a n t d i f f e r e n c e s at the  r e s u l t s support the  et  Steinhoff  findings  f o r Pw  and  could l e a d to a  r e l a t i o n s h i p s between phenotypic  plasticity.  Pw  74  7.4  FUTURE RESEARCH  Information on s p r i n g dehardening  and  usable  phenology data would be u s e f u l to s u b s t a n t i a t e or r e f u t e the recommendations r e g a r d i n g 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  implementation.  The  ( d i s c u s s e d i n s e c t i o n 7.0)  r e l a t i o n s h i p between response  before to f r e e z e  t e s t i n g of needles versus whole s e e d l i n g s should be investigated.  Work by N i l s s o n and E r i k s s o n (1986) on  Scots  pine i n northern Sweden showed that a r t i f i c i a l f r e e z e t e s t i n g of whole s e e d l i n g s can represent f i e l d at  the p o p u l a t i o n l e v e l .  o f t e n appear more hardy  However,  performance  d e t a c h e d - t i s s u e samples  than whole-seedling  samples when  t e s t e d under c o n t r o l l e d - f r e e z i n g c o n d i t i o n s (Glerum  I a l s o suggest  that f u r t h e r r e s e a r c h be done on  a v a i l a b l e US m a t e r i a l .  The  size  ( i . e . :  the  sample s i z e s f o r US m a t e r i a l i n  t h i s study d i d not p r o v i d e f o r a powerful performance.  1973).  t e s t of  A balanced design with s u f f i c i e n t  sample  20-40 p o p u l a t i o n s with 30-50 s e e d l i n g s per  p o p u l a t i o n ) would more r e a d i l y allow the between US and BC m a t e r i a l to be  relationship  addressed.  C o l d - h a r d i n e s s t e s t i n g of a d d i t i o n a l f i e l d p l a n t i n g s of Pw  site  ( p r e s e n t l y i n the Rust R e s i s t a n c e program),  75 during the p e r i o d of c o l d h a r d i n e s s development c o u l d h e l p to e l u c i d a t e questions d e s c r i b e d above. of phenotypic  Further, questions  p l a s t i c i t y c o u l d a l s o be addressed  i f the same  genotypes were grown i n d i f f e r e n t environments and were researched and analyzed  i n the a p p r o p r i a t e manner.  REGION  LOCATION  B.C.  Ladysmith(PS)  Coast  Woss(PT)  SEEDLOT  1  •3  2183  LATITUDE °N  2191,2209 (2205) 2263 2265 (2244) 2410 2525 (2409) 2251 2252 (2248) 2276 2347 (2274) Z  2245  Sechelt(PT)  2407  Whistler(PS)  2250  Manning(PS) Park  2275  B.C.  Valemont(PS)  2293  Interior  Raft River(PS) Barriere(PS) Mount Revelstoke(PS) Salmo(PT) GalenaArrow(PT) GalenaMoscow  CODE  2295 2648 (2649) 2640 (2643) 2632 (2633) 2279 2280 2282 (2281) 2591 2592 2594 (2595) 2483 2484 2485 (2567) 2569 2570 F* 2571 (2572) M D  LONGITUDE W  ELEVATION METERS  49°01'  124°04'  800  50°11'  126°28'  300  49°35'  123°42'  700  50°07'  122°55'  850  49°08'  120°55'  1100  52°45' 51°48' 51°18'  119°18' 119°41' 119°55'  900 1300 750  51°02'  118°01'  600  49°12'  117°17'  1000 -1870  50°37' 117°53' 50°37' 117°53 see map 1  720 720 838 -1219  1  Table  1 cont.  U.S. Coast  U.S. Interior  1 2 3  4 5 6 7  White P a s s Willamette O l y m p i a NF Mount Hood  2 0 NF 1 3 12 NF 1 1  Flower Creek(PT) C r y s t a l Creek(PT) Beaver Creek(PT) Hungry H o r s e ( P T ) Elk River(PT)  2 6 8 9  4 6 ° 2 6 '  1 2 1 ° 3 4 '  9 1 5 - 1 2 2 0  2 6 9 0  4 4 ° 2 6 '  1 2 2 ° 0 2 '  9 1 5 - 1 2 2 0  ( 2 6 8 7 )  4 7 ° 2 3 '  1 2 8 ° 1 8 '  3 0 5 - 6 1 0  ( 2 6 8 8 )  4 5  3 8 '  1 2 1 ° 4 5 '  9 1 5 - 1 2 2 0  2 6 8 3  4 8 ° 2 3 '  1 1 5 ° 3 4 '  670  2 6 8 5  4 7 ° 0 8 '  1 1 6  9 1 5 9 1 5 1 1 2 8  U  0  2 2 '  2 6 8 6  4 8 ° 4 4 '  1 1 6 ° 5 2 '  ( 2 6 8 2 )  4 8 ° 1 9 '  1 1 3 ° 5 9 '  ( 2 6 8 4 )  4 6 ° 4 9 '  I ^ I O '  9 1 5  Seedlots not included i n the phenology a n a l y s i s . G a l e n a Bay t r e e s c r o s s e d w i t h Moscow, I d a h o p o l l e n . 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 : p r e s e n c e o f c o n e s . ( ) 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 t h e p h e n o l o g y a n d damage assessment a n a l y s i s o n l y . PT Parent tree - 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 s t a n d s , show a c c e p t a b l e s t e m f o r m a n d g r o w t h in s i t u (Meagher, p e r s o n a l communication 1 9 9 0 ) . For US PT s e l e c t i o n c r i t e r i a see Hoff & McDonald 1980. F = female M = male [] = number of t r e e s i n bulked s e e d l o t s NF = n a t i o n a l f o r e s t  78  Table 2  L o c a t i o n o f sampled t r e e s , t e s t d a t e , d a y ( f r o m S e p t . 7, 1989) and t e s t t e m p e r a t u r e s .  LOCATION  DAY  UBC  0  UBC UBC UBC Nov. 1 6 l (Dec . 11 ) UBC  TEST DATE Sept .  TEST TEMPERATURES °C  7/89  3,  "7,  "9, -1 1  20  Sept . 27/89  3,  "9, -11, -13  45  Oct.  22/89  3, -11, -13, -15  66  Nov.  12/89  3,  80  Nov.  26/89  3, -25, -30, -35  -28  3  LOCATION  NAKUSP  FROST ]DATES  TEST DATE  TEST TEMPERATURES °C  Oct. (Oct. 3 )  Oct.  6/89  3, -12, -14, -16  Nov. 3 (Oct. 3 0 )  Nov.  1 4/89  3, -22, -26, -30  3  LADYSMITH  1 4  J  1 Average date  (30-years) of f i r s t  frost.  2 These samples were not useable due to a t e c h n i c a l 3 Approximate date of f i r s t  frost  in f a l l ,  1989.  problem.  79 Table 3 a)  Number of seedlings a v a i l a b l e , by provenance, which were included in the damage assessment analysis.  Block 1  Block 2  Ladysmith  38  63  Woss  76  11 6  Sechelt  71  11 6  Whistler  71  1 16  Manning Park  43  63  Valemount  71  11 6  Raft River  53  53  Barriere  58  58  Mt. Revelstoke  38  63  Galena-Arrow  71  11 6  Salmo  38  63  US Coast  White Pass  17  17  US I n t e r i o r  Flower Creek  17  17  Crystal  17  17  17  17  BC Coast  BC I n t e r i o r  Creek  Beaver Creek  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 of Variation  Degrees of Freedom  1  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  12  9825  0.29  R x T(D)  27  7605  1 .00  791  544679  E 1  b)  See Appendix  C f o r d e f i n i t i o n s of terms and EMS's f o r H 1. o  Multiple-comparison testing of regional Duncan's New Multiple Range Test.  Region  1  Mean  N  Grouping  1  61 .73  375  A  3  52.02  50  B  4  51 .89  74  B  2  42.71  348  C  means by  2  1 = BC c o a s t , 2 = BC i n t e r i o r , 3 = US c o a s t , 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)  a) C o a s t a l  Results from analysis of regression for f o l i a r injury and l a t 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 c o n t r o l l e d freezing using 2-year-old seedlings grown at UBC.  provenances  ( i ) % i n j u r y by Variable  Df  Latitude error  1 5  ( i i ) % injury Elevat. error b)  1 5  latitude,  Parameter Estimate  Latitude error  1 6  ( i i ) % injury Elevat. error  1 6  0.19  Standard Error  T f o r Ho: Parameter=0  2  prob >  1 .566  0.18  -0.004 0.003 -1.146 Sums of Squares = 604.8  0.30  2 by e l e v a t i o n ,  ( i ) % i n j u r y by Df  =  2.991 1.910 Sums of Squares = 512.4  I n t e r i o r provenance  Variable  R  =  0.05  regressions  latitude,  Parameter Estimate  R  T"  R  = 0 .02  2  Standard Error  T f o r Ho: Parameter=0  -3.029 2.855 -1 .061 Sums of Squares = 1273.1 2 by e l e v a t i o n ,  R  prob > T  0.33  .07  0.004 0.006 0.746 Sums of Squares = 1 383.6  0.48  Measure of how much v a r i a t i o n i n the dependent v a r i a b l e can be accounted f o r by the model. Student's t value f o r t e s t i n g the n u l l h y p o t h e s i s that the parameter equals zero. P r o b a b i l i t y of a l a r g e r value of t i f the parameter i s t r u l y equal to zero. A very small v a l u e leads to the c o n c l u s i o n that the independent v a r i a b l e c o n t r i b u t e s s i g n i f i c a n t l y to the model.  82 Table  5 c)  R e s u l t s from a n a l y s i s o f 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 w i t h l a t i t u d e and e l e v a t i o n o f seedling origin. D a t a on f o l i a r i n j u r y were from combined d a t e s o f 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 grown a t UBC.  (i) Coastal R  provenances  = 0.11  2  Variable  Df  Latitude Elevation error  1 1 4  (ii)  Parameter Est imate  Standard Error  T f o r Ho: „ Parameter=0  1 . 172 7.526 6.424 0.008 0.743 0.010 Sums of Squares = 450.3  prob > T  0.31 0.50  I n t e r i o r provenances R  2  -  0. 1 7  Variable  Df  Latitude E l e v a t ion error  1 1 5  Parameter Est imate  Standard Error  T f o r Ho: ~ Parameter=0  -0.702 -4.232 6.026 -0.003 0.011 -0.233 Sums of Squares = 1259.9  prob > T  0.51 0.82  83 Table  6 a)  Source of Var i a t ion  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 d a t e s 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 grown a t UBC (See T a b l e 2 f o r t e s t t e m p e r a t u r e s )  Degrees of Freedom  Type I I I Sums of Squares  PR > F  Day=0, Test 1Date=Sept. 7/89 R 3  1969  0.38  T  2  5991  0.01  R x T  6  1112  0.94  89  56250  E  Day=20, Test Date=Sept. 27/89 R 3  28478  0.00  T  2  25249  0.00  R x T  6  4087  0.34  191  1 14507  E  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  1 92  Day=66, Test Date=Nov. 1 2/89 R 3  133613 10919  0.01  T  1  517  0.45  R x T  3  380  0.93  E  1 27  Day=80, Test Date=Nov. 26/89 R 3 T  2  R x T  6  E  1 92  1 13486 9442  0.00  22882  0.00  1 501 126821  0.89  84  b) Probability of a larger value for F in orthogonal contrasts among different combinations of regions at f i v e d i f f e r e n t dates of testing. Orthogonal Contrast  Degrees of Freedom  Test 0  Day  20  45  66  80  (1)  coast  vs i n t e r i o r  1  0 .32  0.00  0.16  0.47  0.04  (2)  north  vs south  1  0 .45  1 .00  0.61  0.13  0.85  1  0 .12  0.00  0.00  0.00  0.00  BC c o a s t v s BC i n t e r i o r (US m a t e r i a l not (3)  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 l i m i t a t i o n s on computing capacity.  R  F(R  1  38435  0.03  4  43284  0.00  E  9  94921  0.00  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  297  92206  D  DxF(R P)  T(D) F(R  E  P)  P)  1  2  Since P(R) i s i n c l u d e d i n the denominator F(R P) for„this t e s t , t h i s r e s u l t i s only v a l i d i f one assumes P ( R ) =0. Based on these r e s u l t s , i t was determined that the highest order i n t e r a c t i o n c o u l d be pooled f o r more r i g o r o u s a n a l y s i s of the other components i n the model. 6  NB:  F(R P) i n c l u d e s : DxF(R P) i n c l u d e s : E includes:  F(R P) + P(R) DxF(R P) + RxD + P(R)xD 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 d a t a f r o m c o n t r o l l e d f r e e z i n g on 5 test dates using 2-year-old seedlings grown a t UBC (BC m a t e r i a l o n l y , H 2) (See T a b l e 2 f o r t e s t t e m p e r a t u r e s and d a t e s ) (analysis excludes highest order i n t e r a c t i o n T(D) x F ( P R ) )  2  Source of Variation 1  Error Term Used  Degrees of Freedom  Type I I I 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  18  105099  0. 00  28  30294  0. 19  F(R  D)  E  P(R) x D  DxF(R P)  P(R) x T(D)  E  63  1 91 04  0. 67  D x F(R  E  72  60106  0. 00  458  152865  E  P)  See Appendix C f o r d e f i n i t i o n s of terms and H 2 model. See Appendix C f o r d e r i v a t i o n of e r r o r terms By EMS's.  87  Table  8  Source of Variation  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 - y e a r - o l d s e e d l i n g s grown at UBC (See Table 2 f o r t e s t temperatures) (BC m a t e r i a l o n l y , H 2)  Error Term Used  Degrees of Freedom  Type I I I 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  P(R)  F(R P)  9  20297  0.05  1 39  0.83  TxP(R)  E  18  6467  0.24  F(R P)  E  18  16117  0.00  35  9621  E  1  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  0.02  P(R)  F(R P)  9  201 75  0.48  18  5596  0.84  TxP(R)  TxF(R  P)  F(R P)  E  18  40765  0.00  TxF(R P)  E  36  1 7540  0.00  86  1 8337  E  For day=0, E= T x F(P R). See F i g . 4b f o r graph of s i g n i f i c a n t i n t e r a c t i o n .  2  88 Source of Variation Day=45, R  Error Term Used  Degrees of Freedom  Test Date=Oct . 22/89 1 P(R)  Type I I I Sums of Squares  PR > F  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  TxF(R P)  18  6695  0.38  TxP(R) F(R P)  E  18  41 327  0.00  TxF(R P)  E  36  1 2004  0.49  87  291 98  E Day=66, R  Test Date=Nov . 12/89 1 P(R)  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  TxF(R P)  9  2041  0.51  TxP(R) F(R P)  E  18  35738  0.00  TxF(R P)  E  18  4307  0.82  58  20379  E Day=80, R  Test Date=Nov . 26/89 1 P(R)  1 3276  0.05  26034  0.00  T  TxP(R)  2  RxT  TxP(R)  2  P(R)  F(R P)  9  22764  0.40  TxF(R P)  18  4459  0.93  TxP(R)  21 9  0.65  F(R P)  E  18  41 023  0.00  TxF(R P)  E  36  17185  0.13  87  30597  E  89  Table  9 a)  Source of Variation 1  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 d a t a 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 f r o m UBC ( d a y 6 6 ) and 3 - y e a r - o l d s e e d l i n g s from L a d y s m i t h and N a k u s p . (See T a b l e 2 f o r t e s t t e m p e r a t u r e a n d dates)  Degrees of Freedom  Type I I I Sums of Squares  PR > F  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  1 5  6109  0.80  R x T(L) E  51 1  594  b) R e s u l t s f r o m a n a l y s i s o f v a r i a n c e f o r combined d a t a f r o m 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 f r o m UBC (day 4 5 ) a n d 3 - y e a r - o l d s e e d l i n g s f r o m L a d y s m i t h a n d N a k u s p . (See T a b l e 2 f o r t e s t t e m p e r a t u r e and d a t e s ) R  3  25479  0.00  '  2  48061  0.00  T(L)  6  1 551 4  0.00  R x L  6  9740  0.01  18  6253  0.89  576  562  L  R x T(L) E  See Appendix  C for definitions  of terms and EMS's f o r H 3.  90  Table 10 a)  Source of Variation  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) Degrees of Freedom  Type I I I Sums of Squares  PR > F  UBC (Day=45), Test Date=Oct. 22/89 R  3  24564  0.00  T  2  5032  0.03  RxT  6  524  0.99  192  133613  E  UBC (Day=66), Test Date=Nov. 12/89 R  3  10919  0.01  T  1  517  0.45  RxT  3  380  0.93  127  113486  E  Nakusp, Test Date=Oct. 3/89 R  3  9595  0.00  T  2  8580  0.00  RxT  6  1950  0.80  192  121634  E  Ladysmith, Test Date=Nov. 14/89 R  3  T  2  1901  0.07  RxT  6  3779  0. 11  192  84342  E  }  1058  0.40  91  b) Probability of a larger value for F in orthogonal contrasts among d i f f e r e n t combinations of regions at d i f f e r e n t test dates and locations. Orthogonal Contrast  Degrees of Freedom  45  UBC  Test Day/Location 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) BC c o a s t vs BC i n t e r i o r (US m a t e r i a l not used)  1  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-yearold seedlings from UBC (day 6 6 ) and 3-year-old seedlings from Ladysmith and Nakusp. (BC material only, H 4 ) (See Table 2 for test temperatures and dates) Q  Source of Variation  2  1  R  Error Term Used  Degrees of Freedom  P(R)  Type I I I Sums of Squares  PR > F  1  1 4874  0. 1 0  L  LxF(R P)  2  3271 5  0. 00  RxL  LxP(R)  2  4201  0. 50 0. 18  T(L)  F(P R)xT(L)  5  25105  RxT(L)  T(L)xP(R)  5  271 0  0. 1 0  9  36428  0. 1 5  18  41849  0. 00  18  38241  0. 13  36  49349  0. 00  45  1 1866  0. 97  90  39580  0. 00  232  641 94  P(R) F(R P) LxP(R) LxF(R P) P(R)xT(L) F(P R)xT(L) E  F(R P) E LxF(R P) E F(P R)xT(L) E  93  b)  Results from analysis of variance for combined data from c o n t r o l l e d 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 Variation  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  RxT(L)  T(L)xP(R)  LxP(R) P)  PxT(R  L)  E  R)xT(L)  F(P  0. 00 0. 50  9  29585  0. 37  18  50679  0. 00  18  31518  0. 19  E  36  44841  0. 00  R)xT(L)  54  1 6520  0. 93  108  47277  0. 00  261  7301 2  LxF(R P)  LxF(R  31751 2784  E  F(R P)  6 6  F(R P)  P(R)  F(P  R)xT(L)  E  See Appendix C f o r d e f i n i t i o n s of terms and H 4 model. See Appendix C f o r d e r i v a t i o n of e r r o r 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 of Variation UBC  Error Term Used  (Day=45),  Degrees of Freedom  Type I I I Sums of Squares  PR > F  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  TxF(R P)  18  6695  0.38  E  18  41 327  0.00  E  36  12004  0.49  87  29198  TxP(R) F(R  P)  TxF(R P) E 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  TxF(R P)  9  2041  0.51  E  18  35738  0.00  E  18  4307  0.82  58  20379  TxP(R) F(R  P)  TxF(R'P) E  95  Table  12 a) cont.  Source of Variation Nakusp,  Error Term Used  Degrees of Freedom  Type I I I Sums of Squares  PR > F  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  TxF(R P)  18  6507  0.91  TxP(R) F(R P)  E  18  41863  0.00  TxF(R P)  E  36  23435  0.00  87  21746  E  Ladysmith, Test Date=Nov. 14/89 R  P(R)  1  T  TxP(R)  2  1 1 562  0.00  RxT  TxP(R)  2  1 354  0.05  P(R)  F(R P)  9  1 2527  0.10  TxF(R P)  18  331 7  0.90  TxP(R)  534  0.55  F(R P)  E  18  1 2330  0.00  TxF(R P)  E  36  1 1 837  0.16  87  22067  E  See F i g . 6b f o r graph of s i g n i f i c a n t  interaction.  1  96  Table  12 b)  H  Summary o f p r o b a b i l i t y o f d i f f e r e n c e s by s o u r c e of v a r i a t i o n f o r a l l f o u r h y p o t h e s e s .  Q  1 2 2 3 4 4 3 4 4 2 4 4 2 2 4 4  See Appendix  Source of Var i a t ion R R R R R R L L L P(R) P(R) P(R) F(PR) F(PR) F(PR) F(PR)  C for descriptions  PR > F  0.00 0.11 0.03 0.00 0.10 0.03 0.00 0.00 0.00 0.11 0.15 0.37 0.00 0.00 0.00 0.00  of H^'s.  Table  4a 7a 7b 9a&b 11a 1 1b 9a&b 1 1a 1 lb 7a 1 1a 1 1b 7a 7b 1 1a 1 1b  97 Table 13 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 combined data of the Galena-Moscow h y b r i d , Galena-Arrow provenance and the US i n t e r i o r s e e d l o t s 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 (See Table 1 for location descriptions, F i g . 7 for pollen source and Table 2 f o r t e s t temperatures and dates)  Source of Var i a t ion  Degrees of Freedom  1  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  0.00  18  7771  P x T(D) E  181  2  0.68  95286  See Appendix C f o r d e f i n i t i o n s of terms. NB: P=provenance i s not nested i n region f o r t h i s a n a l y s i s . See F i g . 8b f o r 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 t e s t i n g of provenance means Mean  N  A  60 .90  75  A  B  55 .45  74  A  C  51 .89  74  Provenance  1  Group ing  2 (Duncan's New  MRT)  B B  A = Galena-Arrow provenance B = Galena-Moscow h y b r i d C = US i n t e r i o r provenances combined (each provenance represented by 3 t r e e s o n l y ) . 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 = M u l t i p l e range t e s t .  3  98 Table  14  Source of Variation 1  Day = 36 B R BxR  Results from analysis of variance for i n d i v i d u a l measurement dates for needle elongation phenology (cm) on f i v e 2-year-old seedlings per seedlot per block grown at UBC. (Day 0 i s March 3/89) Error Term Used  R BxR  0.121  0.01  BxR  3  0.123  0.59  E  3  0. 1 62  0.03  R BxR  1  1 . 196  0.15  BxR  3  4.205  0.75  E  3  9.741  0.00  380  223.440  E  1  2.885  0.62  BxR  3  98.233  0.61  E  3  138.512  0.01  326  3814.793  E  1  0.697  0.91  BxR  3  936.227  0.38  E  3  641.352  0.01  380  223.440  E  1  378.416  0.22  BxR  3  2639.154  0.13  E  3  592.635  0.50  242  60625.530  E Day = 65 B R BxR E Day = 1 28 B R BxR  6.628  E  E Day = 53 B  PR > F  1  382  Day = 43 B  Type I I I Sums of Squares  E  E  E  Degrees of Freedom  Block (B) i s a random e f f e c t , region (R) i s a f i x e d  effect.  99  Table  15 a)  Source of Var i a t ion 1  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 d a t e s f o r s h o o t l e n g t h (cm) 2 - y e a r - o l d s e e d l i n g s p e r s e e d l o t p e r b l o c k grown a t UBC. (Day 0 i s M a r c h 3/89)  Error Term Used  Degrees of Freedom  Type I I I Sums of Squares  PR > F  Day = 0 B  E  1  7.734  0.08  R  BxR  3  14.865  0.50  E  3  14.722  0.13  386  995.289  BxR E Day = 30 B  E  1  R  BxR  3  56.684  0.03  E  3  4.342  0.81  385  1764.803  BxR E  1 . 1 79  0.61  Day = 3 6 B  E  1  0.003  0.98  R  BxR  3  148.591  0.03  E  3  10.437  0.69  376  2694.981  BxR E  100  Table  15 a) cont.  Source of Var i a t ion  Error Term Used  Degrees of Freedom  Type I I I Sums of Squares  PR > F  Day = 43 B  E  1  5.168  0.65  R  BxR  3  629.649  0.17  E  3  182.644  0.07  376  9674.786  BxR E Day = 53 B  E  1  59.119  0.35  R  BxR  3  2335.515  0.13  E  3  549.528  0.04  369  24677.018  BxR E Day = 65 B  E  1  18.915  0.63  R  BxR  3  2037.262  0.15  E  3  529.935  0.09  376  9674.786  BxR E Day = 128 B  E  1  12.887  0.68  R  BxR  3  1794.015  0.15  E  3  473.733  0. 11  246  19154.396  BxR E 1  Block (B) i s a random e f f e c t , region (R) i s a f i x e d  effect.  101  Table  15 b)  P r o b a b i l i t y of a l a r g e r v a l u e f o r F i n o r t h o g o n a l c o n t r a s t s amoung d i f f e r e n t c o m b i n a t i o n s o f r e g i o n s a t two measurement d a t e s f o r s h o o t l e n g t h (cm) on f i v e 2 - y e a r - o l d s e e d l i n g s p e r s e e d l o t p e r b l o c k grown a t UBC. (Day 0 i s M a r c h 3/89)  Orthogonal Contrast  Degrees of Freedom  Test  Day  30  36  (1) coast vs  interior  1  0.02  0.00  (2) n o r t h vs  south  1  0.14  0.14  1  0.00  0.00  (3) BC BC  coast vs interior  102  Table  16 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 j u r y assessment by a q u a n t i t a t i v e measure of i n j u r y of t o t a l needles scored on a l l s e e d l i n g s i n both blocks exposed t o the u n c o n t r o l l e d f r e e z i n g i n Jan./Feb. 1989.  Source of Variation  Error Term Used  Degrees of Freedom  Type I I I Sums of Squares  PR > F  B  E  1  9.568  0.00  R  BxR  3  4.723  >0.50  E  3  6.674  0.05  BxR E  2561  2200.400  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 i n j u r y assessment by shoot measurements on a l l s e e d l i n g s i n both b l o c k s exposed to the u n c o n t r o l l e d f r e e z i n g i n Jan./Feb. 1989.  Source of Variation  Error Term Used  Degrees of Freedom  Type I I I Sums of Squares  PR > F  B  E  1  2762.45  0.00  R  BxR  3  4827.53  0.1<p<0.5  E  3  2088.69  0.00  1858  187148.25  BxR E  1 03  Table  17  Source of Variation  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 r e c o v e r y a s s e s s m e n t by s h o o t 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.  Error Term Used  Degrees of Freedom  Type I I I Sums of Squares  PR > F  B  E  1  0.370  0.19  R  BxR  3  0.278  >0.50  E  3  0.371  0.62  32  6.643  BxR E  1 04  Table  18  Shoot l e n g t h (mm) measurement of undamaged and damaged s e e d l i n g s i n b l o c k s 1 and 2 f o r day 65 and 128.  ( i ) Block Day 65 65 65 65  1, undamaged  Region 1 2 3 4  Mean + SE  N  Day Region Mean + SE  N  40. 19 46.63 44.87 48.49  299 242 29 44  1 28 1 28 128 1 28  1 ' 2 3 4  41 .78 49.30 45.68 48.91  0.90 0.96 2.25 2.94  318 290 29 44  5 5 5 5  1 28 1 28 1 28 1 28  1 2 3 4  14.71 19.60 18.31 20.61  0.89 1 .27 1.71 2.20  5 5 5 5  1 28 128 1 28 . 1 28  1 2 3 4  34.72 45.79 29.77 32.87  1 .77 1 .37 3.18 2.88  1 28 1 28 1 28 1 28  1 2 3 4  1 6.60 22.24 1 1 .46 21 .60  1 .22 1 .56 1 .45 2.69  0.94 1 .04 2.15 2.88  ( i i ) ]Block 1, damaged 65 65 65 65  (iii) 65 65 65 65  1 2 3 4  1 4.53 0.89 18.60 1 .26 17.69 1 .85 1 9.53 2.31  Block 2 , undamaged 1 2 3 4  39.50 45.52 30.20 33.24  1 .36 1 .32 2.87 2.59  161 1 47 27 42  1 40 1 45 25 39  ( i v ) :Block 2, damaged 65 65 65 65  1 2 3 4  1 4.80 21.19 1 0.62 21 .45  1.13 1 .55 1 .35 2.71  5 5 5 5  5 5 5 5  105  Fig.  1  Map of s e e d l o t o r i g i n s (•), n a t u r a l range of western white pine ( s t i p p l e d area) and r e g i o n d e l i n e a t i o n (numbers), ( m o d i f i e d from F o w e l l s 1965)  1 06  Fig. 2  70  Mean needle i n j u r y (%) and s t a n d a r d e r r o r by r e g i o n 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.  % INJURY (needles) 5 prov's 15 SL's  2 bulked prov's  3  SL's  — T -  60  6 prov's 14 SL's  50  F  40 30 V.-VS>ss>W///.-SS/SS?S.'tY  <  20 /'ism  10 0  REGIONS I •  SL's  STD  ERROR  US-COAST  = seedlots  BC-INTERIOR  ill  BC-COAST  111  US-INTERIOR  prov's =  provenances  1 07  Fig. 3  a) Mean needle i n j u r y (%) and standard e r r o r of 7 c o a s t a l provenances by l a t i t u d e u s i n g combined data from 5 d a t e s of c o n t r o l l e d f r e e z i n g of needles from 2y e a r - o l d s e e d l i n g s grown a t UBC. % INJURY (NEEDLES)  LATITUDES (N-S) OF COASTAL SEEDLOTS I  STD ERROR  b) Mean needle i n j u r y (%) and s t a n d a r d e r r o r of 8 i n t e r i o r provenances by l a t i t u d e u s i n g combined data from 5 d a t e s of c o n t r o l l e d f r e e z i n g of needles from 2y e a r - o l d s e e d l i n g s grown a t UBC.  % INJURY (NEEDLES)  80  i  *  60 -  40  1  *  \  20  1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1  62°  <8<  LATITUDES (N-S) OF INTERIOR SEEDLOTS I  STD ERROR  108  Fig. 4  a) Mean needle i n j u r y (%) and s t a n d a r d e r r o r by r e g i o n f o r 5 dates of c o n t r o l l e d f r e e z i n g u s i n g 2y e a r - o l d s e e d l i n g s grown a t 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  STND  ERROR  DAY 4 5  DAY 0 & 8 0 DAY 6 6  1=80-0, 2=BC-I, 3=US-C, 4=US-I-  Islltl  DAY 2 0  1 09  Fig. 4  100  b) Mean needle i n j u r y (%) and standard e r r o r showing s i g n i f i c a n t r e g i o n x temperature i n t e r a c t i o n f o r the i n d i v i d u a l t e s t day 20 f o r r e g i o n s 1 and 2 o n l y .  % INJURY (needles)  -9  -11  -13  TEST TEMPERATURES (C) I  STD ERROR  X  REGION 1  *  REGION 2  1 10  Fig. 5  100  Mean needle i n j u r y (%) and s t a n d a r d e r r o r by r e g i o n 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 66) and 3 - y e a r - o l d s e e d l i n g s from Ladysmith and Nakusp.  % INJURY (needles) 1 + 2 stock  1 +2 stock  i +i stock  80  i 60  40  20  0  1;;;;e;;;I  li-i-l  1 2  knmi  li-ui  3  4  1  2  3  4  REGIONS I  STD ERROR LADYSMITH  1=BC-C, 2=BC-I, 3=US-C, 4=US-I  i l l  UBC NAKUSP  111  Fig.  100  6  a) Mean needle i n j u r y (%) and standard e r r o r by r e g i o n from c o n t r o l l e d f r e e z i n g using 2-and 3-yearo l d s e e d l i n g s from UBC (day 45), Ladysmith and Nakusp.  % INJURY (needles) 1  1 + 2 stock  + 1  stock  1 + 2 stock  80  i 60  40  20  0  1  2  3  4  1 2  3  4  REGIONS I  STD ERROR  ~J  LADYSMITH  1=BC-C, 2=BC-I, 3=US-C, 4=US-I  UBC %m  NAKUSP  11 2  Fig. 6  100  b) Mean needle i n j u r y (%) and s t a n d a r d e r r o r showing s i g n i f i c a n t r e g i o n x temperature i n t e r a c t i o n f o r the Ladysmith r e g i o n s 1 and 2 o n l y .  % INJURY (needles)  -22  -26  -30  TEST TEMPERATURES (C) I  STD ERROR  X  REGION 1  *  REGION 2  11 3  Fig.  7  L o c a t i o n of p o l l e n source from Moscow, Idaho.  •0  1 14  Fig. 8  70  a) Mean needle i n j u r y (%) and s t a n d a r d e r r o r by provenance f o r combined d a t a 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 d a t e s u s i n g 2 - y e a r - o l d s e e d l i n g s grown a t UBC. BC-I = Galena-Arrow provenance, H y b r i d = Galena-Moscow c r o s s , US-I = Flower, C r y s t a l and Beaver Creek provenances combined.  % INJURY (needles)  60  50 •^yyyy/^yy'>-^'--y^^y:y'A^>y:'^  40  30  W0 KtyyXj  20 y/yyyyyyy/y'X-yyyy'^yyyyyyy/y yy'yyy£yyyyyiw  10  0  Hill  BC - I  HYBRID I  STD E R R O R  US - I  1 15  Fig. 8  100  b) Mean needle i n j u r y (%) and s t a n d a r d e r r o r by provenance f o r 5 dates of 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 grown at UBC. BC-I = G a l e n a Arrow provenance, H y b r i d = Galena-Moscow c r o s s , US-I = Flower, C r y s t a l and Beaver Creek provenances combined.  % I N J U R Y (needles)  C  I  STD ERROR  DAY 0 & 8 0  DAY 4 5  DAY 6 6  A = B C - I , B = H Y B R I D , C=US-I  A  B  C  A  DAY 2 0  B  C  1 16  Fig. 9  a) Mean s i z e and s t a n d a r d e r r o r of needle l e n g t h (mm) by r e g i o n over 5 measurement dates i n 1989 on 5 s e e d l i n g s per s e e d l o t i n block 1.  LENGTH (mm) OF 1989 NEEDLES 80  36  43  53  65  128  DAY OF MEASUREMENT (OMARCH 3/89) I  STD ERROR  X  REGION 1  *  REGION 3  &  REGION 4  •  REGION 2  11 7  Fig.  9  b) Mean s i z e and standard e r r o r of needle l e n g t h (mm) by region over 5 measurement d a t e s i n 1989 on 5 s e e d l i n g s per s e e d l o t i n block 2.  LENGTH (mm) OF 1989 NEEDLES 80  I 60  40  20  0  X-  X  >KA  36  43  ^  53  65  128  DAY OF MEASUREMENT (OMARCH 3/89) I  STD ERROR  *  REGION 3  X  REGION 1  A  REGION 4  REGION 2  1 18 8.0  LITERATURE CITED  Aronsson, A., T. Ingestad, and L.Loof. 1976. Carbohydrate metabolism and f r o s t h a r d i n e s s i n pine and spruce s e e d l i n g s grown a t d i f f e r e n t photoperiods and thermoperiods. P h y s i o l . P l a n t . 36:127-132. Bingham, R.T. 1983. B l i s t e r r u s t r e s i s t a n t western white pine of the Inland Empire. USDA F o r e s t S e r v i c e Gen. Tech. Rpt. INT-146. 45pp. Bingham, R.T., R.J. Hoff and R.J. S t e i n h o f f . 1972. G e n e t i c s of western white p i n e . USDA F o r e s t S e r v i c e Research Paper WO-12. 18pp. Blum, A. 1988. Chapter 5, Cold r e s i s t a n c e , pp.99-132. P l a n t breeding f o r s t r e s s environments. CRC P r e s s , I n c . Bower, R.C. 1987. 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Plant P h y s i o l . 60:271-273.  1 24  APPENDIX A  Diazinon s o i l  drench:  Captan f o l i a r  spray  :  50 girts/ 4 l i t e r s ,  8 gms/  4 liters,  12.5%  10%  WP  WP  125  APPENDIX B  Seedling  mix f o r 1.5 c u b i c  yards:  675  gms micromax  1800  gms dolomite  1575  gms gypsum  7.5  kgs n u t r i c o a t balanced N-P-K  (micronutrients)  (slow  release  fertilizer)  6  b a i l s of Sunshine peat  3  bags of v e r m i c u l i t e  126  APPENDIX C  HYPOTHESES  LEGEND Y = % injury u = estimate of the mean of % i n j u r y i L-: R  D  n m m(n)  =  ^th 9 = o., l o c a t i o n r e  =  n  th th  t  e  s  i o n  t  d  a  t  e  P ( s i n g l e date) temperature on n date ^ (temperature nested i n £gte) m(o) th P ested in o location k(j) jfth P Y f ,iR 3 region _ l(ki) th ^ ^y ^ provenance xn j region E = p n e e d l e - e r r o r w i t h i n samples, p l o t e r r o r T T  =  m  =  m  =  m  t  e  m  e  r  a  t  u  r  e  t  e  m  e  r  a  t  u  r  e  t  T  P  Ho1  =  r o  =  a m i  n  a n c e i  n  - For s e e d l i n g s grown at the UBC Nursery, there i s no evidence of g e n e t i c v a r i a t i o n i n f r o s t hardiness a s s o c i a t e d with region of seed o r i g i n during the hardening p e r i o d . Y ,. x = u + R. + D + T , + R.D + R.T , \ + p(^mn) j n m(n) ] n 3 m(n) N  E  p(jmn)  H1a  - There i s evidence of r e g i o n a l g e n e t i c v a r i a t i o n c o n d i t i o n a l upon date of t e s t i n g .  Ho2  - For s e e d l i n g s grown at the UBC Nursery, there i s no evidence of g e n e t i c v a r i a t i o n i n f r o s t hardiness a s s o c i a t e d with r e g i o n , provenance w i t h i n r e g i o n , or f a m i l y w i t h i n provenance. (BC only) Y  p(jklmn) "  u  +  +  T  H2a  R  D  j  +  D  n  +  n k(j) P  R  +  j n T  m(n) l(kj) F  m(n)  +  m(n) k(j)  +  D  +  T  P  +  E  R  F  j m(n) T  l(kj)  +  +  P  D  being  k(j)  n l(kj) F  +  p(jklmn)  - There i s evidence of provenance and/or f a m i l y w i t h i n provenance genetic v a r i a t i o n being c o n d i t i o n a l upon date of t e s t i n g .  127  Ho3  - At one " p h y s i o l o g i c a l l y s i m i l a r " sampling time, there i s no evidence of g e n e t i c v a r i a t i o n i n f r o s t h a r d i n e s s a s s o c i a t e d with r e g i o n of seed o r i g i n when s e e d l i n g s are grown i n three d i f f e r e n t l o c a t i o n s . Y , .. x = u + R. + L + T / x + L R . + R.T , + E , ".. v p(o3t) ] o m(o) 0 3 ] m(o) p(o]t) x  H3a  - There i s evidence of r e g i o n a l g e n e t i c v a r i a t i o n being c o n d i t i o n a l upon l o c a t i o n .  Ho4  - At one " p h y s i o l o g i c a l l y s i m i l a r " sampling time, there i s no evidence of g e n e t i c v a r i a t i o n i n f r o s t h a r d i n e s s a s s o c i a t e d with r e g i o n , provenance w i t h i n r e g i o n or f a m i l y w i t h i n provenance where s e e d l i n g s are grown i n three d i f f e r e n t l o c a t i o n s . (BC only) Y , . , = u pijmkl)  + R. + L + T , + R . L + R .T , x + P. , . v ] o m(o) j o 3 m(o) k(;j)  M  X  +  T  H4a  T  m(o) k(j) P  m(o) l(jk) F  +  +  o k(j)  L  L  P  o l(jk) F  +  +  F  E  l(jk)  +  p(jmkl)  - There i s evidence of provenance and/or f a m i l y w i t h i n provenance g e n e t i c v a r i a t i o n being c o n d i t i o n a l upon locat ion.  128  Ho1 : Source of Variation  df  REGION DATE REGION*DATE TEMP(date) REGION*TEMP(date) ERROR  r-1 d-1 (r-1)(d-1) d(t-1) d(t-1)(r-1) rdt(p-l)  TOTAL  5 F n  3 F m  2 R p  Rj  0  5  3  2  6  n  4  0  3  2  6  m(n)  4  1  0  2  6  R,-D„ j"n  0 "  0 "  3 "  2 -  j m(n)  0  1  p(jmn)  1  T  R  E  T  1  3 4 12 10 30 60  rdtp-1  4 F j  D  df(actual)  0 1  2 1  119  EMS + 30  2 e  2 e  +  2 e  +  +  2  6  8  R  2 6  D  2 6  T ( D )  2 2 + 6 e RD  6 b  6  24  2 6  e  +  6 s  6  2  2 6  R T ( D )  e  F - t e s t s : A l l EMS's w i l l be t e s t e d a g a i n s t  2 6  .  129  H 2: o Source of Variation  df  df  coding  REGION  r-1  1  DATE  d-1  4  R x D TEMP(date) R x T(d) PROV(region) D x P(r) T(d) x P ( r ) F(p(r)) D x F(p(r)) T(d) x F ( p ( r ) ) Error  R . D D n R .D D n  (r-1)(d-1)  4  d(t-1 )  9  (r-1)d(t-1 )  9  r(p-1)  7  P  k ( j)  28  D  n k(j)  d(t-1)r(p-l)  63  T  m(n) k(j)  rp(f-l)  18  F  l(kj)  72  D  n l(kj)  T  m(n) l(kj)  (d-1)r(p-1 )  (d-1)rp(f-l) d(t-1)rp(f-l) rpftd(n-1)  243  T  m(n)  R .T / x 3 m(n)  P  P  F  F  E p( jklmn)  Total  rpftdn-1  458  130  R . D  2 F j  5 F n  3 F m  a R k  b R 1  2 R P  #  0  5  3  a  b  2  1  EMS  1  + 30b  2  D  0  3  a  b  2  2  n  2 6  e  3 0  6  2 F ( R  p ( R )  +  0  0  3  a  b  2  3  6  +  2 DF(RP)  6 e  +  +  2  m(n)  1  0  a  b  2  4  e  +  1  k(j)  3  5  1  b  2  5  e  6  2 ( 5  +  30b R  j m(n) T  0  1  0  a  b  2  6  2 6  e  n k(j) P  1  3  0  1  2  b  7  6  +  6 a b  T  m(n) k(j) P  1  1  0  1  b  2  8  6  l(kj)  D  n l(kj)  T  m(n) l(kj)  F  F  E p( jklmri)  1  5  3  1  1  2  9  1  0  3  1  1  2  10  1  1  0  1  1  2  1 1  1  1  1  1  1  1  12  a = between b = between  2  +  F(RP)  +  4ab  +  p ( R )  2 T(D)F(RP)  2 e  +  +  2 6  2 6  2  T ( D ) 6  +  +  2 RT(D)  2 a b f i  6 e  +  DP(R) 2 e T(D)F(RP)  6  6  D  2  +  2  2  T(D)F(RP)  3 0 ( 5  6  2 f i  +  T(D)P(R)  2 b e  F  b  * RD  2 T(D)P(R) 2 2 e DF(RP)  6 b e  R  +  2 b f i  D  a  T(D)P(R)  2 b e  P  2  6 f i  DP(R) 2 2  6  1  2 6  +  DP(R) * 2 2 e DF(RP)  6 b f i 2  T  +  30ab  2 6  6 b f i 2  R .D D n  p)  e  +  3  °  e  +  6 e  e  +  2 g  6  F(RP) 2 DF(RP) 2 T(D)F(RP)  e  5-6 1-3  F - t e s t s : # 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 t o program F - t e s t s , they do not n e c e s s a r i l y represent the r i g h t c o e f f i c i e n t s f o r the v a r i a n c e components. T h i s p e r t a i n s to H 4's EMS as w e l l .  131  H 3: o Source of Variation LOCATION REGION TEMP(location) L x R R x T(l) Error  o R. D m(o) L R. o D R  E  j m(o) T  p(ojt)  df  1-1  2 3 6 6  r-1 Kt-1)  (1-1)(r-1 )  Total  L  df  (r-OKt-l) lrt(p-1)  18 36  lrtp-1  71  3 F o  4 F J  3 F m  2 R P  0  4  3  2  « e  3  0  3  2  s  3  4  0  2  6  0  0  3  2  3  0  0  2  1  1  1  F-tests: A l l tested  coding  1 against  R  v  R°T^ , D m(o) p(ojt) E  EMS 2 2 e  2  e 2  e  6  2 6  6  e  2 e  +  2  4  2 L  6  + 18 + 24  R  2  T(L)  2 4 ( 5  + fi  2  + fi  2  LR  6  b s  2 6  RT(L)  132  H 4: df  A  REGION  r-1  LOCATION  1-1  df  * B  coding  1  1  2  2  R. D  Kt-1 )  5  6  R x L  (r-1)(1-1)  2  2  R x T(o)  (r-1)l(t-1 )  5  6  R  j m(o)  9  9  P  k(j)  Kt-1 )r(p-1 )  45  54  T  m(o) k(j)  (1-1)r(p-1)  18  18  L  o k(j)  rp(f-1)  18  18  F  l(jk)  l(t-1)rp(f-1)  90  108  T  m(o) l(jk)  (1-1)rp(f-l)  36  36  L  o l(jk)  lrpft(n-1)  232  261  E  p(jmkl)  lrpftn-1  463  521  Source of Variation  TEMP(location)  PROV(r) T(o)  x P(r)  L x P(r) F(p(r)) T(o)  x F(p(r))  L x F(p(r)) Error Total  r(p-1)  A: a n a l y s i s i n c l u d e s UBC day 4, B: a n a l y s i s i n c l u d e s UBC day 3. (See Tables 11a and 11b)  L  T  o m(o)  R .L. D © T  P  P  F  F  133  R. 3  T  3 F o  3 F m  a R k  b R 1  2 R P  #  0  3  3  a  b  2  1  EMS  6  2  +  E  l8b 2  o  L  2 F j  2  m(o)  0  1  3  0  a  a  b  b  2  2  2  3  0  0  3  a  b  2  4  6  8  +  2  e  6  +  0  j m(o) T  1  0  a  b  2  5  6  T  1  k(j)  1  m(o) k(j) P  3  1  3  0  1  1  b  b  2  2  6  7  R  P  )  +  2  6  2 LF(RP)  6 s  +  2 6  L  2 T(L)F(RP)  2 6  ' T(L) 2 2 e LF(RP) 2  +  6 f i  +  LP(R) R] 2 2 e T(L)F(RP) +  +  2 b e 2  P  (  + I8ab j  6 b f i 2  R  F  p ( R )  e 12ab 6  2  2 6  2  6  4 a b  R .L 3 o  1  I8b 2 e  6  +  p  +  (  R  +  2  F(RP)  1 8 6  2 6  a  b  +  )  2 T(L)F(RP)  2 f i  T(L)P(R) 2 2 e LF(RP)  2 b s  L  1  o k(j) P  0  3  1  b  2  8  6  +  3  3  1  1  2  9  1  0  1  1  2  10  1  0  3  1  1  2  1 1  1  1  1  1  1  1  12  1  F  l(jk)  T  m(o) l(jk)  L  o l(jk)  F  F  E  1  p(joklm) F-tests:  e  6  2 6  2 6  6  6 f i  +  LP(R)  6 b e  1 b 6  e e  2 e  F(RP)  , 2 T(L)F(RP) 2 LF(RP) 6  +  +  2 f i  T(L)P(R)  e  6  6 a b f i 2  6 e  11/12 10/12, 9/12, 8/11, 7/10, 6/9, 5/7, 4/8, 3/10, 2/11, 1/6.  2 6  RT(L)  

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